A Review

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Title: A Review Incorporating Entomopathogenic Fungi into Integrated Vector Management Programs to Diminish Tropical Disease
Physical Description: Book
Language: English
Creator: Cooper, Carli Janelle
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2009
Publication Date: 2009


Subjects / Keywords: Entomopathogens
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation


Abstract: Tropical diseases sicken millions of people annually. Insect vector control is imperative for improving the quality of life for those at risk of contracting tropical diseases by reducing incidences of tropical disease. However, current vector control programs could be at the brink of failure because chemical insecticides provide a substantial portion of modern-day control measures, but incidence of insect resistance completely threatens their effectiveness (Kuile et al. 2003; Gullan and Cranston 2005; O'Reilly et al. 2006). New control methods and agents must be discovered, researched, and implemented, or the number of tropical disease cases will likely soar (Lacey et al. 2001; Mohanty et al. 2007. Biological control agents could be an essential part of these much needed new control methods. Fungal biological agents comprise one of many types of biological control agents, and these are explored and reviewed in this thesis for their effectiveness in reducing populations of mosquitoes, triatomines, and tsetse and sand flies. These insects vectors carry malaria, viral hemorrhagic fevers, arbovirus encephalitis, Chikungunya fever, O'nyong-nyong, lymphatic filariasis, Chagas disease, African sleeping sickness, leishmaniasis, Bartonellosis, and other less common and widespread diseases. Entomopathogenic fungi provide a multi-dimensional approach to insect control. They contain several destructive compounds that assist hyphal penetration of the insect cuticle. Two species in particular, Metarhizium anisopliae and Beauveria bassiana have been researched extensively and could significantly aid in vector control; however, many different entomopathogenic fungal species exist and with proper research, some of these could become crucial in vector control methods (Scholte et. al 2004; Lacey et. al 2001). Any use of pathogens must be undertaken with care to avoid unintended and unwanted effects on environmental and human health. Ultimately, evidence suggests that perhaps the most sensible use of entomopathogenic fungi would be in a multi-faceted control plan. Such a plan could use as many available control techniques as possible, including physically minimizing insect habitats, using several different chemical pesticides in rotation, using insect repellants and barriers such as mosquito netting, and applying an array of biological control agents including fungal agents. This is known as Integrative Vector Management and will likely provide the most successful tropical disease control measures possible, based upon current research.
Statement of Responsibility: by Carli Janelle Cooper
Thesis: Thesis (B.A.) -- New College of Florida, 2009
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Clore, Amy; McCord, Elzie

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Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2009 C7
System ID: NCFE004065:00001

Permanent Link:

Material Information

Title: A Review Incorporating Entomopathogenic Fungi into Integrated Vector Management Programs to Diminish Tropical Disease
Physical Description: Book
Language: English
Creator: Cooper, Carli Janelle
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2009
Publication Date: 2009


Subjects / Keywords: Entomopathogens
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation


Abstract: Tropical diseases sicken millions of people annually. Insect vector control is imperative for improving the quality of life for those at risk of contracting tropical diseases by reducing incidences of tropical disease. However, current vector control programs could be at the brink of failure because chemical insecticides provide a substantial portion of modern-day control measures, but incidence of insect resistance completely threatens their effectiveness (Kuile et al. 2003; Gullan and Cranston 2005; O'Reilly et al. 2006). New control methods and agents must be discovered, researched, and implemented, or the number of tropical disease cases will likely soar (Lacey et al. 2001; Mohanty et al. 2007. Biological control agents could be an essential part of these much needed new control methods. Fungal biological agents comprise one of many types of biological control agents, and these are explored and reviewed in this thesis for their effectiveness in reducing populations of mosquitoes, triatomines, and tsetse and sand flies. These insects vectors carry malaria, viral hemorrhagic fevers, arbovirus encephalitis, Chikungunya fever, O'nyong-nyong, lymphatic filariasis, Chagas disease, African sleeping sickness, leishmaniasis, Bartonellosis, and other less common and widespread diseases. Entomopathogenic fungi provide a multi-dimensional approach to insect control. They contain several destructive compounds that assist hyphal penetration of the insect cuticle. Two species in particular, Metarhizium anisopliae and Beauveria bassiana have been researched extensively and could significantly aid in vector control; however, many different entomopathogenic fungal species exist and with proper research, some of these could become crucial in vector control methods (Scholte et. al 2004; Lacey et. al 2001). Any use of pathogens must be undertaken with care to avoid unintended and unwanted effects on environmental and human health. Ultimately, evidence suggests that perhaps the most sensible use of entomopathogenic fungi would be in a multi-faceted control plan. Such a plan could use as many available control techniques as possible, including physically minimizing insect habitats, using several different chemical pesticides in rotation, using insect repellants and barriers such as mosquito netting, and applying an array of biological control agents including fungal agents. This is known as Integrative Vector Management and will likely provide the most successful tropical disease control measures possible, based upon current research.
Statement of Responsibility: by Carli Janelle Cooper
Thesis: Thesis (B.A.) -- New College of Florida, 2009
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Clore, Amy; McCord, Elzie

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2009 C7
System ID: NCFE004065:00001

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A REVIEW: INCORPORATING ENTOMOPATHOGENIC FUNGI INTO INTEGRATED VECTOR MANAGEMENT PROGRAMS TO DIMINISH TROPICAL DISEASE BY CARLI JANELLE COOPER A Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillm ent of the requirement for the degree Bachelor of Arts in Biology Under the sponsorship of Dr. Amy Clore and Dr. Elzie McCord Sarasota, FL April, 2009


i Acknowledgements I thank Dr. Amy Clore for her tireless effort critiquing and evaluating this t hesis and second for the knowledge and wisdom she has given me in both scientific and personal capacities while I have attended New College. I will sorely miss her as I enter my new academic setting. I also thank Dr. Elzie McCord for his entomology exper tise and for his pleasant sense of humor that enriched the process of creating this thesis. Additionally I extend my thanks to Dr. Alfred Beulig for agreeing to partake in my baccalaureate committee. I thank m y classmates and friends for the balance and comradery they provided me with while at New College. Last but not least, I thank my mother, Charlotte, my father Clarke, my two sisters Callie and Courtney, and my extended family for their con stant support and encouragements throughout my life as well as while writing this thesis.


ii Table of Contents P age 1. I ntroduction to Chemical and Biological Con trol of Tropical Disease Vectors --------1 1.1 General Overvi ew ----------------------------------------------------------------------------1 1.2 Chemical Insect Control: Dangers, Problems, and Limitations --------------------------2 1.3 Principles of Biol ogical Control ------------------------------------------------------------7 1.4 Entomopathogenic Fungi Biological Control ----------------------------------------------9 1.5 Overview -------------------------------------------------------------------------------------10 2. Devastating Insect borne Tropical Diseases -----------------------------------------------20 2.1 Mosquito borne Diseases -------------------------------------------------------------------20 2.1.1 Malaria --------------------------------------------------------------------------------------21 2.1.2 Viral Hemorrhagic Fevers --------------------------------------------------------25 Dengue Fever -----------------------------------------------------------25 Yellow F ever -----------------------------------------------------------27 2.1.3 Arbovirus Encephalitis -----------------------------------------------------------28 Japanese Encephalitis ----------------------------------------------------------29 Eastern Equine Encephalitis ------------------------------------------31 West Nile Fever ----------------------------------------------------------------31 Rocio Encephalitis --------------------------------------------------------------32 Rift Valley Fever -------------------------------------------------------33 2.1.4 Chikungunya Fev er (CF) and O'nyong nyong (ONN) ------------------------34 2.1.5 Lymphatic filariasis --------------------------------------------------------------34 2.2 Triatomine borne Disease: Chagas Disease -------------------------------------3 6 2.3 Fly borne tropical diseases -----------------------------------------------------------------40 2.3.1 Human African Trypanosomiasis (African Sleeping Sickness) ------------40 2.3.2 Leishmaniasis --------------------------------------------------------------------43 2.3.3 Bartonell osis ----------------------------------------------------------------------46 2.4 Conclusion and Remarks: Tropical Diseases --------------------------------------------47 3. General Overview of Insect Colonization by Entomopathogenic Fungi ----------------51 3.1 Mechanistic overview of fungal infections of insects ----------------------------------51 3.2 The Insect Cuticle ---------------------------------------------------------------------------53 3.3 Step by Step Process of Fungal I nvasion of Insect ------------------------------------55 3.4 Enzymes and Toxins ------------------------------------------------------------------------56 3.4.1 Chitinases -------------------------------------------------------------------------57 3.4.2 Proteases --------------------------------------------------------------------------58 3.4.3 Destruxins ------------------------------------------------------------------------59 3.5 Insect Death ----------------------------------------------------------------------------------59 3.6 Widely studied entomopathogenic fungal vector control agents --------------61 3.6.1 Metarhizium anisopliae ----------------------------------------------------------62 3.6.2 Beauveria bassia na --------------------------------------------------------------62 3.7 Summary -------------------------------------------------------------------------------------63 4. The Role of Mycoinsecticide in Mosquito Control ---------------------------------------67 4.1 Mosquito Biology, Ecology, and Distribution -------------------------------------------67


iii 4.2 Identification of Mosquito Species ------------------------------------------------69 4.3 Vector Qualifications and Requirements -----------------------------------------7 0 4.4 Fungi and mosquito control ----------------------------------------------------------------7 1 4.5 Adulticidal Entomopathogenic Fungi ------------------------------------------------------76 4.5.1 Beaveria bassiana --------------------------------------------------------------76 4.5.2 Metarhizium anisopliae ----------------------------------------------------------77 4.5.3 Fusarium pallidoroseum ---------------------------------------------------------81 4.6 Larvicidal and Ovicidal Fungal parasites of mosquitoes --------------------------------82 4.6.1 Langenidium giganteum Couch -------------------------------------------------82 4.6.2 Leptolegenia chapman ni Seymour ---------------------------------------------86 4.6.3 Metarhizium anisopliae (Metschnikoff) Sorokin ------------------------------86 4.8 Summary --------------------------------------------------------------------------------------86 4.9 The Future and Barriers to Entomopathogenic Fungal B io logical Control of Mosquitoes ----------------------------------------------------------------------------------------88 5. Mycoinsecticides and Vector Control of Triatomines -----------------------------------95 5.1 Biology, Ecology and Distribution of Triatomines --------------------------------------95 5.2 Entomopathogen ic fungi and triatomine control ---------------------------------------100 5.2.1 Determining the fungal pathogens of Triatomines --------------------------100 5.2.2 Control of Triatomines with Beauveria sp. and Metarhizium -------------102 Early studies on the requirements of B. bassiana and M. anisopliae -------------------------------------------------------102 Additional Studies of Beauveria sp. Metarhizium sp. --------108 Triatomine biocontrol: B. bassiana verses B. brogniartii --114 5.1.3 Additional Potential Entomopathogenic Fungal Species of Triatomines ---------115 Evlachovaea sp --------------------------------------------------115 Aspergillus gigante us and Penicillium corylophilum --------115 Paecilomyces lilacinus ------------------------------------------1 16 5.3 Conclusions ---------------------------------------------------------------------------------1 16 6. Biological control of Two Diptera: Tsetse Flies and Sand Flies ----------------------122 6.1 Tsetse Flies (Glossinidae) Biology and Ecology ---------------------------------------122 6.2 Curr ent Tsetse Fly Control Methods -----------------------------------------------------124 6.3 Tsetse Fly and Fungal Biological Control ----------------------------------------------125 6.4 Sand Fly Biology and Ecology -----------------------------------------------------------1 29 6.5 Current Sand Fly Control Methods ------------------------------------------------------131 6.6 Fungal Biological Control of Sand Flies ------------------------------------------------1 33 6.7 Summary and the Future of Tsetse Fly and S andfly Control --------------------------133 7. Perspectives and Ideas for the Future of Mycoinsecticide Use -------------------------1 37 7.1 Additional Research of Individual Entomopathogenic Fungi Species ---------------1 37 7.2 Mycoinsecticide Application, Formulation Innovation, and Commercialization --1 38 7.3 Continue d Research of Biochemistry and Biology of Entomopathogenic Fungi --141 7.4 The Economics and Education of Mycoinsecticide Use -------------------------------142 7.5 Concerns of Entomopathogenic Fungi use as BC agent -------------------------------143 7.6 The Bottom L ine ---------------------------------------------------------------------------145


iv 7.7 Conclusions ---------------------------------------------------------------------------------140


v Appendices Appendix A: Glossary -------------------------------------------------------------------------1 49 Appendix B: Evidence of Resistance --------------------------------------------------------154 Appendix C : Mosquito and Mosquito borne disease Distribution ------------------------1 58 Appendix D : CDC Explanations of Life Cycles of Select Parasites ----------------------1 60 Appendix E: Effects of L. chapmanii in target and non target species -------------------1 63 Appendix F : Isolates of B. bassiana and M. anisopliae used in Luz et. al (1998 a) ---165 Appendix G : Lethal Concentrations of Evlachovaea sp. for triatomine third instar nymphs -------------------------------------------------------------------------------------------1 66 Appendix H : Distribution and Examples of Major Vector Triatomines -----------------1 67


vi List of Tables and Figures Page Figure 2.1 Ae. agypti feeding on human arm -------------------------------------------------21 Figure 2.2 Sporoz o ites --------------------------------------------------------------------------22 Figure 2.3 P. falcipariums in human red blood cell ----------------------------------------24 Figure 2.4 T. cruzi in thin blood smear -------------------------------------------------------38 Figure 3.1 Infection of An. stephensi with B.bassiana : -------------------------------------52 Figure 3.2 Penetration of cuticle and haemocoel --------------------------------------------53 Figure 3.3 Insect cuticle anatomy --------------------------------------------------------------54 Figure 4.1 Engorg ed vector species of mosquitoes ------------------------------------------70 Table 4.2 Phylogenetic depiction of all the species of fungi known ----------------------74 Table 5.1 The significa nt triatomine v ector species of Chagas disease -------------------96 Figure 5.1 Triatoma infestans, dorsal view, side view --------------------------------------98 Figure 5.2 P.lilacinus sporulating on T. infestans during Marti et al (2006) experiment ---------------------------------------------------------------------------------------116 Figure 6.1 Glossiana morsitans --------------------------------------------------------------123 Figure 6.2 Phlebot omus papatasi feeding on a human ------------------------------------131


vii A REVIEW: INCORPORATING ENTOMOPATHOGENIC FUNGI INTO INTEGRATED VECTOR MANAGEMENT PROGRAMS TO DIMINISH TROPICAL DISEASE Carli Coop er New College of Florida, 2009 ABSTRACT Tropical diseases sicken mill ions of people annually. Insect vector control is imperative for improving the quality of life for those at risk of contracting tropical diseases by reducing incidences of tropical disease. However, current vector control programs could be at the brink o f failure because chemical insecticides provide a substantial portion of modern day control measures, but incidence of insect resistance completely threatens their effectiveness (Kuile et. al. 2003; Gullan and Cranston 2005; et. al 2006). New con trol methods and agents must be discovered, researched, and implemented, or the number of tropical disease cases will likely soar (Lacey et. al 2001; Mohanty et. al 2007). Biological control agents could be an essential part of these much needed new cont rol methods. Fungal biological agents comprise one of many types of biological control agents, and these are explored and reviewed in this thesis for their effectiveness in reducing populations of mosquitoes, triatomines, and tsetse and sand flies. These insects vectors carry malaria, viral hemorrhagic fevers, arbovirus encephalitis, Chikungunya


viii fever, O'nyong nyong lymphatic filariasis, Chagas disease, African sleeping sickness, leishmaniasis, Bartonellosis and other less common and widespread diseases Entomopathogenic fungi provide a multi dimensional approach to insect control. They contain several destructive compounds that assist hyphal penetration of the insect cuticle. Two species in particular, Metarhizium anisopliae and Beauveria bassiana have been researched extensively and could significantly aid in vector control; however, many different entomopathogenic fungal species exist and with proper research, some of these could become crucial in vector control methods (Scholte et. al 2004; Lace y et. al 2001). Any use of pathogens must be undertaken with care to avoid unintended and unwanted effects on environmental and human health. Ultimately, evidence suggests that perhaps the most sensible use of entomopathogenic fungi would be in a multi fa ceted control plan. Such a plan could use as many available control techniques as possible, including physically minimizing insect habitats, using several different chemical pesticides in rotation, using insect repellants and barriers such as mosquito net ting, and applying an array of biological control agents including fungal agents. This is known as Integrative Vector Management and will likely provide the most successful tropical disease control measures possible, based upon current research. ________ ________________________ Dr. Amy Clore Division of Natural Sciences ________________________________ Dr. Elzie McCord Division of Natural Sciences


1 All bolded words are defined in the glossary in Appendix A Chapter 1: Introduction to Chemical and Biological Control of Tropical Disease Vectors 1.1 General Overvi ew Entomopath ogenic fungi 1 were first considered as possible disease vector pathogens began many decades later, during the 1950s (Golkar et al. 1993; Lacey et al. 20 01; Mohanty et al. 2007). Entomopathogenic fungal vector control research began with the identification of naturally occurring pathogens, and the field has progressed to include mechanistic and biochemical studies of fungal colonization. Interest in this area insecticides (Lacey et al. 2001). Fungal biological control of vectors research has increased since then (Lacey et al. 2001; Mohanty et al. 2007). Future research will likely continue and will examine the benefits of using fungal pathogens in conjunction with other vector control techniques (Lacey et al. 2001; Mohanty et al. 2007). Currently, vector borne tropical disease control programs rely heavily on chemical insec t control. C hemical insecticide use expanded in the middle of the twentieth century and continues to be the most common form of insect control (Gullan and Cranston 2005). Unfortunately, vectors have developed resistances to these chemicals severely comp romising the effectiveness of these programs (Lacey et al. 2001; Scholte et al. 2004; Constant 2005; Scholte et al. 2005). However, tropical diseases pose a large threat to the health of many countries, and some insect borne tropical diseases are untreata ble; therefore, vect or control is essential. These v ector borne diseases include malaria, dengue fever, lymphatic filariasis, different encephalitises,


2 Chagas disease, leishmaniasis, and African sleeping sickness all of which will be further discussed in Chapter Two. These diseases are caused by parasites or microbes that use insects as secondary hosts which then transfer the microorganism to the human host. If one were able to target a particular vector through biological control methods, such as the use of entomopathogenic fungi insect borne illne sses would decrease without adverse environmental and human health problems associated with chemical insecticides which are discussed in the next section (Scholte et al. 2005; Thomas and Read 2007). 1.2 Chemical Insect Control: Dangers, Problems, and Limitations The first successful vector control programs involved chemical pesticides; however, they produced many unwanted side effects (Kuile et al. et al. 2006). The most significant dangers of che mical pesticides include destruction and harm of non target organisms, selection for pesticide resistant insect strains and subsequent pest resurgence (Gullan and Cranston 2005). Additionally, outbreaks of new insects filling the now empty niche can arise, environmental degradation from soil, water, and air contamination can occur, and human health can be compromised (Gullan and Cranston 2005). Chemical insecticides are divided into several classes and are typically either plant derived or synthetically d evised. The major classes used in vector control are plant based pyrethrins and their synthetic counterparts, pyrethroids as well as other synthetic insecticides, carbamates, organophosphates, and organochlorines (or chlorinated hydrocarbons) (Gullan and Cranston 2005). Carbamates and organophos phates act by inhibiting acetyl cholinesterase and organochlorines pyrethrins,


3 and pyrethroids act by changing the normal opening and closing of sodium channels (Daly et al. 1998; Kuile et al. et a l. 2006). Additionally, insects develop resistance to pesticides. Some populations achieve resistance by genetic selection of resistant organisims within the group. For example, m etabolic resistance occurs when insects gain the ablity to detoxify themse lves of the chemical. Target site resistance results from acquiring decreased sensitivity to the insecticide et al. 2006) Learned behaviors like avoid ance of insecticides are also type s of resistance that renders pesticides ineffective (Hoy et al. 1998) Appendix B demonstrates the enormity of insect resistance studies by outlining some recent studies that have been completed thus far. Pyrethrins were the first successful vector control agent used to co ntrol mosquito populations beginning in t he early 1900s (Kuile et al. 2003). Then, chlor inated hydrocarbon compounds such as Dichloro Diphenyl Trichloroethane ( DDT ) and lindane were discovered and used following World War II Due to the environmental and health problems of pesticides like DDT, sy nthetic pyrethrins, known as pyrethroids are the preferred chemical insecticides for vector control (Kuile et al. 2003; Waliszewski et al. 2008). However, d eveloping countries still use a limited amount of DDT in vector control programs because it is muc h more effective at killing pests than any other pesticide and can kill insects for up to six months after application (Hargreaves et al. 2000; Kuile et al. 2003; Pennetier et al. 2008; Gabianelle et al. 2009). Chlorinated hydrocarbon based pesticides li ke DDT, polychlorinated biphenyls, carbamates, carbinols, and polycyclic aromatic hydrocarbon insecticides have been linked to severe environmental degradation as well as numerous health problems (Beard


4 et al. 2006; Mills et al. 2006; Belpomme et al. 2007) DDT, in particular, provides a dramatic example of the ill effects of chemical insecticidal treatments. It accumulates overtim e in large quantities in animal fatty tissue which render even low doses harmful and animals can eventually die (Lundholm 1987 ; Beard et al. 2006). One well known effect of DDT is how it severely affects predatory birds' ability to form egg shells by slowing calcium metabolism (Cooke et al. 1970; Cooke et al. 1976; Lundholm 1987; Beard et al. 2006). DDT has been suggested as a cau sative agent of chronic fatigue syndrome (Racciatti et al. 2001), chronic renal failure (Wanigasuriyaa et al. 2007), decreased neurobehavioral performance (Eckerman et al. 2007), diminished sperm count (Wonga et al. 2003), essential tremor disorder (Loui s et al. 2006), Parkinson's disease (Menegon et al. 1998), cancer (Belpommea et al. 2007; Irigaray et al. 2007; Carozzaa et al. 2008; Fiorini et al. 2008) and intrauterine growth retardation (Levario Carrillo et al. 2004). Malathion could also have signifi cant detrimental health effects including intestinal problems ( Wali et al. 1994) leukemia (Reeves et al. 1981), kidney damage (Albright et al. 1983) and chromosomal damage ( Balaji 1993 ). These problems are not limited to those who directly work with the pesticide since DDT can accumulate in the water supply therefore contaminatio n can be widespread (Longnecker 1997). For additional information on the health effects of DDT, the reader is referred to a lengthy database of articles on the environmental and human health effects of DDT compiled by Cornell researcher, Suzanne Snedeker (Snedeker et al. 2003). DDT was banned from most developed countries by 1970, but is still a vital method of mosquito control in developing countries with high Entomological


5 In oculation Rates (EIR) (Beard et al. 2006). Houses in these countries are treated with DDT every six months (Matteson et al. 1998). These treatments are very vital to immediate human health of those within the region, but the aforementioned environmental an d health problems will continue to be an issue as long as DDT is used (Beard et al. 2006, Carlier et al. 2008). Malathion h as also correlated with environmental degradation, but currently is more widely used than DDT (Thompson et al. 2001 ; Relyea et al. 2004; Sinclair et al. 2009) In small doses, it causes low rates of toxicity; however, it can degrade within an organism into a more toxic compoun d, malaoxon (Sinclair et al. 2009). Nontarget organisms such as honey bees and amphibians can be killed when Malathion is applied in areas around or wit hin their habitats (Thompson et al. 2001 ; Relyea et al. 2004) Wayne Sinclair, University of South Florida researcher, created a database on Malathion similar Lindane, the Cyclodienes ( Endrin, Endosulf an, Aldrin, Dieldrin, Hepta chlor, Chlordane, and Toxaphene) Chlordecone, Kepone, Mirex, and Hexachlorobenzene have all been shown to be endocrine disrupters and/ or to be carcinogenic in animal models (Zaim and Gulliet 2002; Pennetier et al. 2008; Gabiane lle et al. 2009) Even pyrethroids, the only class of pesticides approved by the World Health Organization to treat mosquito nets, could potentially be detrimental to human health (Zaim and Gulliet 2002; Pennetier et al. 2008; Gabianelle et al. 2009). Ov er the short term, pyrethroids demonstrate low toxicity in mammals; however, the very long term effects of this pesticide are unknown (Gabianelle et al. 2009). Research demonstrated that older rats continually exposed to permethrin (a type of pyrethroid) can present with oxidative


6 damage in the brain, liver, erythrocytes and lymphocytes, phagocyte respiration burst, and DNA damage (Gabianelle et al. 2009). Human occupational exposure has lead to paresthesia and respiratory irritation, possibly due to cont inued firing of sensory nerve endings and massive exposure has resulted in human poisoning symptoms (Vij verberg and Bercken 1990). Studies have also demonstrated that low level exposure to pyrethroids has caused chronic illness es characterized by symptoms of fatigue, loss of memory, depression and reduced attention span in some individuals ( Kolaczinski and Curtis 2004). However, this concept is debated, and it is important to consider the advantages and disadvantages of use case by case. Pyrethroids can be invaluable when treating for malarial mosquitoes ( Kolaczinski and Curtis 2004). An opposing theory is that mammals are able to detoxify themselves following pyrethroid exposure by rapidly metabolizing the components of pyrethroids into much less harmfu l compounds ( Zain et al. 2000 ; Scollon et al. 2009). Studies in rat models have shown that certain enzymes degrade pyrethroids by hydrolyzing the active component into more benign substances (Scollon et al. 2009; Nakamura et al. 2007). C arboxylesterases wi thin the liver may be responsible for detoxification (Ross et al. 2006; Nishi et al. 2006) Regardless, pyrethroids will continue to be used because they are considered to be among the least harmful of the effective pesticides (Zain et al. 2000). Biologi cal control could reduce the amount of chemical insecticides used in countries with high EIR (Beard et al. 2006, Carlier et al. 2008). As previously mentioned pesticides are also losing effectiveness. Insects reproduce in vast numbers; therefore the incid ence of mutations is high. When mutations occur that reduce the binding or increase the metabolism of the chemical insecticide


7 typically uses to kill the insect the mutant individual s survive and subsequently, the mutation can flourish in the population r endering the pesticide useless. Studies show that such mutations have occurred in many species of mosquitoes increasing the population of mosquitoes that are resistant to DDT and pyrethroids (Hargreaves et a l 2000; Guessana et al. 2007; Alout et al. 2008; Alout and Weill 2008; Enayati et al. 2008; Hall et al. 2008; Morou et al. 2008; Okoyea 2008). This provides an additional reason for supporting research of new biological control agents over new chemical control agents. Vector control programs will becom e more effective when alternatives to chemical pesticides or more efficacious chemical pesticides are discovered (Zaim and Guillet 2002). Insect resistance to insecticides severely compromises vector control efforts. Additionally, very few new cost effec tive vector control chemical pesticides have 2). Biological control methods have a low er likelihood of insect resistance developement than chemical methods (Zaim and Guillet 2002). Fu r thermore, insect icides are expensive and the countries where vector control is needed most are some of the poorest nations in the world (Zaim and Guillet 2002; Torr et al. 2007). 1.3 Principles of Biological Control Biological control, integrated disease management (IDM) and integrated vector management (IVM), ( types of integrated pest management or IPM ) could provide healthy solutions for vector control. Biological control encompasses the regulation of pest populations using biological agents (Khan et al. 2007; Rhagu et al. 2007; Blair et al. 2008). Ideally, IVM, IDM, and IPM use many strategies to create a comprehensive, multi faceted control program using insecticides only after biological control,


8 environmental management and cultural methods have failed (Matteson et a l. 1993). When vectors are involved, the management plan is known as IVM a form of IDM (Matteson et al. 1993). For example, a mosquito control IVM would include techniques such as the release of biological control agents like bacteria, fungi and predatory fish, diminish ing the amounts of standing water that serve as a breeding ground for mosquitoes, and lastly, application of chemical insecticides. Additional helpful measures include window screens and insect repellents (Matteson et al. 1993). Biological c ontrol systems generally utilize naturally occurring interactions to control and kill unwanted vector populations. There are three different types: classical, augmentative, and conservative. The classical approach involves introducing a non native species or pathogen into an environment to control an unwanted organism, whereas augmentative control simply increases the numbers or viability of native species or pathogens that alter the undesired population (Hoffmann and Frodsham 1993; Federici 1999). Finally, conservative control pro tects, stimulates or supports existing populations of biological agent s (Hoffmann and Frodsham 1993; Federici 1999). Classical control requires careful evaluation because introducing exotic species could potentially wreak havoc on non target populations and destroy entire ecosystems (Berner et al. 2005). Augmentative control works best in combination with low risk pesticides (Colliera et al. 2004 ). The potential of naturally occurring control organisims such as nematodes, bacteria, predatory insects, viruses, protozoa, predatory fish, and fungi to kill vectors has been explored with some success (Lacey et al. 2001, Scholte et al. 2004; Hajek et al. 2007). The first successful microbial biological pathogen used to control vector


9 popu lations was Bacillus thuringiensis Berliner (Bt), although scientists have tried to use fungi to ward off insects since the 19th century (Prentiss 1880; Luxananil et al. 2001; Bravo and Sobern 2008). Bt is a bacterium that produces Crystal Protein Recepto r (Cry) toxins that are effective insect pathogens in the orders Lepidoptera, Diptera, and Coleoptera. Bt has been used to co ntrol Dipteran mosquito and black fly populations Lepidopteran a gricultural pests and many different larvae of Coleoptera have als o been controlled (Schnepf et al. 1998). Unfortunately, resistance to the Cry toxins has developed in some insect species (Luxananil et al 2001; Bravo and Sobern 2008). Release of a variety of different predatory fish and the nematode Romanomermis cul icivorax Ross and Smith (Mermithidae) which prey upon insect larva has been explored (Scholte et al. 2004). Inundations of mosquito habitats with the nematode cause d reductions in the mosquito populations (Shamseldean and Platzer 1989; Hajek et al. 2007). Densoviruses (viruses that infect insects) are an additional method being explored (Cook et al. 2008). Use of these pathogens could reduce chemical reliance, protecting the environment and safety of humans. Unfortunately, biological entomopathogenic inte ractions are complex and rather unpredictable because the pathogens often involve several chemical and physical methods to induce insect deaths. Furthermore, environmental conditions required for successful use of biological control agents can be very spe cific. The extent of the complexities involved will become clearer in the discussions of entomopathogenic fungi in subsequent chapters. 1.4 Entomopathogenic Fungi Biological Control


10 Entomopathogenic fungal vector control is an expanding field. Continued research is justified because greater success with entomopathogenic fungi in controlling insects that cause lethal disease would save many lives. To be an effective, successful mycoinsecticide, mortality levels of 50% or higher must be established (Da Cos ta 2003). Perhaps the most challenging aspect of fungal entomopathogenic biological control is ensuring that the spores come into contact with the targeted insect (Scholte et al. 2004 ). Both physical and chemical methods are used by these fung i dur ing the process of fungal colonization (Scholte et al. 2004). In Chapters Three, Four, Five, and Six, these methods will first be described generally and then individually for each specific fungus used to control tropical disease vectors The intended ou tcome of entomopathogenic fungal biocontrol is eradicating the secondary host insect thereby reducing incidenc e of tropical disease infection (Scholte et al. 2004). 1.5 Overview The main goal of subsequent chapters is to describe existing research that has explore d fungal entomopathogens and their potential in reducing incidences of insect borne tropical diseases. In order to convey the significance of this topic, the pathology and epidemiology of major, life threatening insect borne tropical diseases, a nd the relationship between the disease causing microbe s the secondary insect host, and the primary human host will also be describ ed. In the conclusion chapter, ideas for improving research and application of biological control will be offered. The vecto rs investigated in this thesis are mosquitoes, tsetse flies, and triatomines which were selected on the basis of available research on the fungal entomopathogenic


11 o f the se insect s as significant vector s of medically important tropical diseases played a role in the selection process. The next chapter will discuss these diseases and the ir relationship with the corresponding vector.


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20 Chapter 2: Devastating Insect borne Tropical Diseases Tropical diseases are frequently horrific and often difficult or impos sible to treat. M any of these are spr ead by prolific insect vectors including malaria, dengue fever, lymphatic filariasis, different encephalitises, Chagas disease, leishmaniasis, and African sleeping sickness all of which will b e discussed in this chapter Vector control is currently the most effective way to manage several of these diseases because treatments for many do not exist or are difficult to access ( Cook and Zumla 2003; Hunter et al. 2003; Kwan Gett et al 2006). How ever, vector control is a multifaceted problem and requires careful consideration so that unwanted side effects are avoided Keating (2001) asserts that e fficien t use of chemical control along with insect repelling techniques that reduce contact between i nsects and humans ( such as mosquito repellants, specialized clothing, insect netting, bed nets, and screens ) would reduce disease related illne sses and deaths The addition of biological control to these methods may further reduce incidence of tropical di sease. The following discussion will explain the nature of these diseases and their effects to elucidate the need for vector control. 2.1 Mosquito borne Diseases Mosquitoes transmit many deleterious diseases including malaria, dengue fever, lymphatic fil ariasis, O'nyong nyong virus, and a variety of tropical encephalitises, which collectively kill and sicken millions every year (Strickland et al. 2000; Eddleston et al. 2005; Sachs 2005; Kwan Gett et al. 2006 ). The most common mosquitoes species that carry tropical diseases are Aedes aegypti L which carries yellow fever and dengue (Venzanni et al. 2005; Kwan Gett et al. 2006; Charlwood JD 1997); Aedes aegypti


21 simpsaloni L, Anopheles africanus Meigen and Anopheles gambiae Giles which transmit malaria and dengue fever (Strickland et al. 2000; Eddleston et al. 2005; Kwan Gett et al. 2006; CDC 2008); and Aedes polynesiensis Marks, Aedes albo pictus Skuse and Anopheles funestus Giles (Scholte et. al 2008) which carry dengue fever as well (Strickland et al. 20 00; Eddleston et al. 2005; Kwan Gett et al. 2006; CDC 2008). Figure 2.1 shows one example of a vector, Ae. agypti. Figure 2.1 Ae. agypti feeding on human arm ( CDC 2008 ). 2.1.1 Malaria As m any as 41% ( 2.5 billion people ) of the world population exists in areas suscepti ble to malaria which includes nin e ty different countries in parts of Africa, Asia, the Middle East, Central and South America, Hispaniola, and Oceania as shown in the distribution map in figure d o f Appendix C (Strickland et al. 2000; Cook an d Zumla 2003; Yasuoaka et al. 2006 ). Between 300 and 500 hundred million cases of malaria occur yearly, and it claims as many as three million lives annually (CDC 2008 ; WHO 2008 ). The majority of deaths occur in children of sub Saharan Africa ( Marsh et al. 1995; Sachs 2005). Prevention and control programs are a part of U nited N Millenium


22 Development Goals, but eradicating malaria from mosquito infested ecosystems is an enormously difficult task. Mal aria infections are on the rise due to wars, natur al disasters, climate change, and human migrations that disturb control programs (Matteson et al. 1998; Hume et al. 2003 ; Sachs and Malaney 2002 ). However, malaria is not a new problem. Fossil and evolutionary evidence shows that malaria has plagued the hu man species for as long as 100,000 years (Hume et al. 2003). Malaria is caused by a parasite called a plasmodium that is transmitted by mosquitoes to humans. In order for infection to be transmitted a mosquito must first bite a n infected human Then, the plasmodia must develop in the mosquito's gut for fourteen days and migrate into the salivary gland at which stage, the y are known as sporoz o ite s (depicted in figure 2.2) ( James et al. 1936; James and Tate 1937; Strickland et al. 2000; Miller et al. 2002; Kwan Gett et al. 2006; Babiker et al. 2008; CDC 2008). Finally the mosquito has to bite a different human, and then the sporozoite is released into the human s locate a re d blood cell and then replicate in vast numbers (Strickland et al. 2000; Miller et al. 2002; Babiker et al. 2008; CDC 2008). Figure 2. 2 Sporoz o ites (CDC 2008)


23 Malaria can be diagnosed microscopically by examining a blood smear for plasmodia; however, microsco pic diagnosis is not always reliable because the number of sporoz o ites in the blood fluctuates, and the sporoz o ites are difficult to distinguish (Babiker et al. 2008, CDC 2008 ; Tolle 2009) Antigen detection tests can also be used in diagnosis ( Strickland et al. 2000; Eddleston et al. 2005; Kwan Gett et al. 2006). For a more thorough and visual description of the life cycle of the plasmodium, the Center for Disease Control's description of mala ria is illustrated in figure a of Appendix D Symptoms of human malaria take seven to 30 days to emerge following plasmodia l invasion of the circulatory system. C lassical malaria lasts six to ten hours, first causing shivering, fever, vomiting, and in small children, seizures and ending in sweats and fatigue ( Strickl and et al. 2000; Eddleston et al. 2005; Kwan Gett et al. 2006; CDC 2008 ) Anemia, jaundice, headaches, and body aches can also ensue. Plasmodium falciparum Welch, Plasmodium vivax Grassi and Feletti P lasmodium ovale Stephans and P lasmodium malariae Fele tti and Grassi can cause classic malaria Fu r thermore, P.vivax and P. ovale can enter a dormant stage by hiding in liver cells to later cause relapses of malaria long after the initial infection (Strickland et al. 2000; Eddleston et al. 2005 ; CDC 2008 ; To lle 2009). Severe malaria is rarer than classical malaria and is only caused by P. falcipariums shown in Figure 2.4 Typically only young, old, and immune compromised individuals develop severe malaria (Strickland et al. 2000; Eddleston et al. 2005; Tolle 2009). The illness begins as


24 Figure 2. 3 P. falciparium sporozoites in human red blood cell ( CDC 2008 ) classical malaria and progresses to the more malign type. This form of malaria involves the destruction of many red blood cells and can cause neurol ogical disorders, hemorrhaging, serious anemia, bloody urine, respiratory problems, bacterial infections, thrombocytopenia, enlargement of spleen, renal failure, gastrointestinal and liver dysfunction, other internal organ failures, acidosis (often the cau se of death) hypoglycemia, and cardiovascular collapse coma, and shock ( Brewster et al. 1990; Cook and Zumla 2003; Eddleston et al. 2005; CDC 2008 ; Tolle 2009 ). Malaria treatments are limited to anti malarial drugs. Several are available, but many have unpleasant side effects. Combinations of these drugs are sometimes used to treat malaria. Anti malarial drugs include quinine, quinidine, chloroquine, artesunate, artemether, tafenoquine, mefloquinine, halofantrine, lumefantrine, pyrimethanine and a tovaquone (G reenwood 2004; Mutabingwa 2005 ; Miller et al. 2006 ; Kwan Gett et al. 2006) Preventative anti malarial drugs include proquanil, primaquine, chlorguanide, and pyrimethamime (G reenwood 2004; Mutabingwa 2005 ; Miller et al. 2006 ; Kwan Gett et al 2006 ; Tolle 2009 ) Untreated malaria has a high rate of fatality, especially in those prone to malaria. Treated classic malaria patients regularly recover from the illness


25 ( Strickland et al. 2000; Tolle 2009) Resistance to malaria drugs is common and m any of the listed drugs are no longer effective alone, therefore combinations are used (G reenwood 2004; Mutabingwa 2005 ; Miller et al. 2006 ). 2.1.2 Viral Hemorrhagic Fevers Viral hemorrhagic fevers are a class of viral diseases that include several debili tating mosquito borne illnesses such as Dengue Fever, Rift Valley Fever and Yellow Fever. In general, viral hemorrhagic fevers cause hemorrhaging and vascular damage in their human hosts (Strickland et al. 2000; Eddleston et al. 2005; Tolle 2009 ). Dengue Fever Dengue Fever (DF) is a viral mosquito borne disease endemic to semi urban and urban tropical regions that infects between 50 and 100 million people annually (Keating 2001; Zaim and Guillet 2002; Halstead 2007; Jain et al. 2008). An estimated 150 thousand people per year a re killed by DF and it is the tenth leading in fectious disease (Strickland et al. 2000; Sachs et al. 2005 ; Kwan Gett et al. 2006 ; CDC 2008 ). DF is endemic world wide in subtropical and tropical regions and approximately 40% o f the in areas in which DF is located (see figure c of A ppendix C ) Mosquitoes transmit the disease through their salivary secretions because the and thereby contaminates th e Then when the mosquitoes insert their mouthparts into the host becomes infected with the virus. The two main vectors of DF are Ae. ae gypti and Ae. albpictus ( Halstead 2007; Jain et al. 2008). In the simplest form of DF, the virus incubates for three to eight days and the illness lasts for 48 hours upon which the patient


26 fully recovers Clinical manifestations include high fever, chills, headache, eye pain, bleeding, sore throat, changes in taste, nausea, vomiting, diarrhea, anorexia, depression, severe aching, myalgia arthralgia petechiae and a flush like rash on face neck and chest ( Cook and Zumla 2003; Tolle 2009 ). DF is rarely fata l; h owever, it can persist for three to seven days and depression, and long termed debility can result ( Cook and Zumla 2003; Kwan Gett et al. 2006; Strickland et al. 2000). In complex cases, DF causes shock and hemorrhaging which can lead to death (CDC 200 8 ; WHO 2008; Tolle 2009 ). In this stage, the malady is known as Dengue Hemorrhagic Fever (DHF) which can lead to Dengue Shock Syndrome (DSS). The most accepted theory of how DSS leads to mortalities is essentially that the cytokines released when T cells a ttack dengue infected cells cause rapid increases in vascul ar permeability (Halstead 2007 ). DHF patients present impaired h emostasis thrombocytopenia and liver damage. Shock occurs following extensive plasma leakage (Strickland et al. 2000; Kwan Gett et al. 2006; Jain et al. 2008). This leads to bleeding (typically gastrointestinal) and eventually loss of blood pressure. Of those who contract DHF, 5% die (CDC 2008; Jain et al. 2008). DF is considered an arbovirus and is included in the group of arbovirus encephalitises ( Halstead 2007 ). There are four different Dengue virus serotypes (1, 2, 3, and 4) and all were derived from one common ancestor. These viruses are of the family Fla vivirid a e and the genus Flavivirus (Halstead 2007 ; Jain et al. 2008). Appro ximately 500 years ago, the viruses evolved a human urban transmission cycle (McBride 2000). When humans began living in closer proximity to one another the dengue viruses were able to spread more rapidly because mosquitoes like A e aegypti adapted to li ving in


27 hu man made stagnant water sources. This adaptation enabled mosquito es to bite a n infected person and then bite another pers on within the next five days as is required for transmission of virus to occur from mosquito to human (McBride 2000). Labora tory findings can aid in diagnosis. The virus can be cultured. In addition DF patients present with neutropenia elevated levels of lymphocytes thrombocytopenia metabolic acidosis decreased fibrinogen levels, and increased fibrin degradation products ( Kwan Gett et al. 2006 ). Enzyme linked immunosorbent a ssay (ELISA) testing can also be used to determine if antibodies against the DF virus are present in the patient (Kwan Gett et al. 2006; Strickland et al. 2000 ; Tolle 2009 ). Treatment is primitive becau se effective drugs to combat DF do not exist. Rest, fluids, and administering fever reducing medications compri se the current DF treatment plan ( Cook and Zumla 2003; Halstead 2007). Ultrasounds can be used to diagnose DHF by detecting abnormal fluid accumu lat ion (Halstead 2007). B lood transfusions may be administered to DHF patients (Strickland et al. 2000 ; Cook and Zumla 2003 ). Mosquito control is currently the most effective way to reduce DF and DHF's deleterious effects on populations (Jain et al. 2008) Yellow Fever Yellow fever is a related viral disease in the viral genus Flavivirus (like many of the viral arthropod borne diseases) carried mainly by Ae. a egypti An effective vac cine against the malady exists, so it is less of a threat to the world population (Monath 2001; Barrett and Higgs 2007) An estimated 200 thousand people contract yellow fever per year with 30 thousand cases resulting in death ( Monath 2001; Barrett and Higgs 2007; CDC 2008 ; WHO 2008 ) T he yellow fever yearly case load has increased over the last


28 25 years therefore it is considered a reemerging disease ( Barrett and Higgs 2007). Affordable v accines and access to medical care are not available for many who reside in the areas in which yellow fever is endemic (Monath 2 001), so improved mosquito contro l c ould d iminish the yellow fever death toll The Flavi virus sp. that causes yellow fever ( indigenous to sub Saharan Africa and South America ) requires three to six days to incubate in a human host. A mosquito must consum e a minimum threshold of the virons and the viro ns then bind to the mosquito gut epithelial cell membranes and multiply. Next the virons pass from the epithelial cells into the b asement membrane and enter the hemocoel. T he then virons must migrate into t he sal ivary glands of the mosquito, and t he virons are released into the human host with the next mosquito feeding ( Cook and Zumla 2002; Barrett and Higgs 2007). The illness is typically biphasic. The first s ymptoms of yellow fever include fever, chills, myalgia, lumbosacral pain, headache, conjunctival injection, viremia, and neutropenia The sec ond stage occurs a few days to two weeks after the first phase and is characterized by nausea, vomiting, lower back pain, slight proteinuria, subclinical infecti on, bradycardia jaundice, renal failure, severe asthenia epigastric pain cardiovascular instabilit y, and shock and hemorrhagic complications. The fever is severe in 15% of cases. Of those, 50% die if they do not receive hospital care, and 20% die with h ospital care ( Monath 2008 ; WHO 2008 ). Hospital care is largely supportive similar to the care given to DF patients ( Kwan Gett Monath 2008) 2.1.3 Arbovirus Encephalitis There are 500 different known arboviruses world wide and 20 of these are known to cause encephalitis ( Gunn and Mansourian 2005 ). Arboviruses typically require a


29 particular species that serves as a host for the insect, known reservoir or maintenance host, such as a bird, a rodent, or a pig. The reservoir develops viremia the mosqui to vector bites this host and then transmits the disease to other animals including humans. Once in the human, the viruses enter the bloodstream, divide and reproduce, and then invade the central nervous system ( WHO 2008; Tolle 2009 ). Most arbovirus en cephalitises are asymptomatic or cause nonspecific flu like symptoms ( Kwan Gett et al. 2006) Severe symptoms typically affect children and the elderly more often than others. These symptoms include fever, vomiting, photophobia, tremors, meningismus con vulsions, mental status changes, and progressing stupor that can lead to coma and death ( Tunkel et al. 2008 ) Physicians can diagnose patients with any of the arbovirus encephalitises by s specific antibodies, althoug h suspected encephalitis should be treated immediately ( Soni b and Polhill a 2007 ; Tunkel et al. 2008 ) Unfortunately encephalitis treatments available for encephalitis are mostly supportive, including attention to fluid and electro lyte levels, prevention of secondary bacterial infections, and managing seizures with sedation, if necessary ( Soni b and Polhill a 2007 ; Tunkel et al. 2008 ) Below are specific descriptions of the most dangerous insect borne tropical arbovirus encephalitises; however, Japanese Encephalitis causes substantially more harm to humans than the others discussed Japanese Encephalitis


30 Japanese encephalitis (JE) is endemic to Asia and the Pacific region and is caused by a flaviviru s transmitted by Culex tritaeniorynchus complex mosquitoes (Strictland et. al 2000; Kwan Gett et. al 2006). This particular arbovirus encephalitis is the only one with an available vaccination ( Tunkel et al. 2008) Between 30,000 and 50,000 people contra ct JE annually ( Diagana et al. 2007; CDC 2008 ; WHO 2008 ). Children are most affected, and as many as ten thousand per year die from this disease ( Diagana et al. 2007; WHO 2008 ). T he fa tality rate is approximately 30% and 30% of the survivors suffer from sequelae, developmental delays, and behavioral abnormalities ( Tunkel et al. 2008 ). The incubation period for JE is four to 16 d ays and the most common disease amplifying reservoirs are pigs and wading birds. Rice paddies provide breeding grounds for Cu lex tritaeniorynchus mosquitoes and significantly contribute to the transmission of the disease ( Diagana et al. 2007 ). This encephalitis begins with malaise, fever, headache, myalgia, nausea, and vomiting followed by abrupt onset of encephalitis ( Diagana et al. 2007 ; Tunkel et al. 2008 ) This neuroinvasion is distinguished by severe fever, headache, involuntary movements, change in consciousness, ataxia, slurred speech, and often convulsions and poliomyelitis ( Kwan Gett et al. 2006 ; Diagana et al. 2007; T unkel et al. 2008 ). Ultimately paralysis, seizures, coma, and death can ensue following infection ( Diagana et al. 2007; CDC 2008). However, many are infected with flavivirus es but do not develop clinical illness, s ince immunity to the virus is acquired in areas with high infection rates (Strickland et al. 2000; CDC 2008). Concrete evidence based diagnosis is difficult after a few days because the viruses are no longer in the bloodstream and only i n the first few days can blood tests detect the viruse s. Furthermore, treatments are limited to use of m annitol to control


31 intra cranial pressure and anti convulsant therapy as well as supportive care Vaccinations are the best option to avoiding transmission; however, these are expensive and sometimes react ogenic (Strickland et al. 2000; CDC 2008 ; Tunkel 2008 ). Eastern Equine Encephalitis Eastern Equine Encephalitis (EEE) is found in the Americas and t he Caribbean and is caused by alphaviruses in the family Togaviridae (Strickland et al. 2000; Tu nkel 2008 ; CDC 2008 ; Tolle 2009 ) EEE is transmitted by Culiseta melanura Coquillett mosquitoes to birds, humans, horses, and a few other mammals. The transmission cycle is not fully understood and diagnosis is difficult Case numbers peak during the s ummer and migratory birds may be involved in maintenance of the virus (CDC 2008). The disease requires seven to 21 days to incubate and first manifests itself as a fever that lasts for over a week. Most patients suffer from solely the fever ( Long et al. 2 002; Kwan Gett et al. 2006) while 5% of those infected with this type of alphaviruses develop encephalitis. However, 50% of those who do develop encephalitis die, and the remainder of the survivors often suffer from neurological disorders ( Deresiewicz et al. 1997; Kwan Gett et al. 2006 ; Tunket et al. 2008 ). Diagnosis is accomplished through nd symptoms, and analyses by Computerized Axial Tomography (CAT) scans and magnetic resonance imaging used to search for cranial lesions (Piliero et al. 1994; Long et al. 2002) Treatment for this encephalitis is also supportive ( Long et al. 2002 ). West Nile Fever West Nile Fever is caused by a flavivirus that is prevalent in Africa, the Middle East, Eur ope, Russia, parts of former USSR, Southeast Asi a, and now North America


32 ( Cook and Zumla 2003; Kramer et al. 2006 ) The disease is thought to be one of the most wide spread diseases in the world and researchers be live that migratory birds play a central r ole in transmitting West Nile Fever from one region to another (Kramer et al. 2006). Transmission of West Nile is normally between mosquitoes and birds but humans can be infected when bitten by an infected mosquito Less than 1% of patients infected with West Nile become severely ill and 80% do not experience symptoms of infection ( Hayes and Gublar 2006 ) This virus requires a seven to 10 day incubation period within the human ( Hayes and Gublar 2006; Kwan Gett et al. 2006). In addition to encephalitis, West Nile Fever can present poliomyelitis like general muscle weakness that can lead to respiratory failure and coma in 15% of patients ( Kwan Gett et al. 2006). Nausea, headaches, ras h, fever, myalgia, backache, anorexia, lymphadenopathy, confusion, and generalized weakness are also often common symptoms. Ultimately, death can be the final outcome since currently neither specific treatments beyond supportive care nor vaccines for West Nile Fever are available ( Cook and Zumla 2003; Hayes and Goblar 2006; CDC 2008). Survivors can have lasting neurological problems following infection (Carson et al. 2006). Rocio Encephalitis Rocio encephalitis (RE) is caused by a flavivirus endemic to Brazil, and transmitted by Psorophora ferox Humboldt and possi bly Aedes scapularis Rondani mosquitoes (Strickland et al. 2000; Mullen and Durden 2003). Chickens and wild birds may provide the viruses with a reservoir in which amplification of the pathogen can occur; however the exact nature of transmission is not w ell known (Strickland et al.


33 2000; Mullen and Durden 200 2; Cook and Zumla 2003 ). RE outbreaks are found mostly in the coastal areas of Brazil and are small and sporadic. Only 5% of cases are fatal; however rates are higher in children and the elderly (Str ickland et al. 2000; Mullen and Durden 2003 ). Additionally 20% of survivors suffer from motor impairment, loss of sphincter control, vision, and hearing as well as bulbar palsy (Strickland et al. 2000). The virus incubates for seven to fourteen days withi n the human host (Strickland et al. 2000). The first symptoms of RE are typical of encephalitis and include fever, headache, malaise, neck stiffness, nausea, vomiting, and abdominal distention. Mental confusion, meningeal irritation, meningeal inflammati on and congestion, focal brain hemorrhages and neuronal degeneration can also accompany RE (Strickland et al. 2000 ; Cook and Zumla 2003 ; Tunket et al. 2008 ). Treatments beyond supportive care are not yet available ( Tunket et al. 2008 ). 2.1.3 .3 Rift Valle y Fever Rift Valley Fever (RVF) is found throughout much of Africa and the Middle East and is caused by virus es in the family Bunyaviridae ( Kwan Gett et al. 2006). The disease affects both humans and domestic animals (CDC 2008). RVF is diagnosed by isola ting the virus from the blood in the first seven days of infection or by Polymerase Chain Reaction ( PCR ) and antigen detection methods. RVF causes encephalitis in addition to hemorrhagi c fever, though many who are infected with the virus never develop RVF ( Kwan Gett et al. 2006). Hemorrhagi c fever occurs in less than 1% of those who contract RVF (Kwan Gett et al. 2006; CDC 2008). Jaundice, pain, fever, headache, joint and muscle pain, photophobia, retinitis, and sometimes blindness can also occur in thos e with this disease. As of yet, no treatment is available to cure RVF ; however most RVF


34 patients make full recoveries. High level su pportive care must be provided in serious cases (Cook and Zumla 2003; Kwan Gett et al. 2006). 2.1.4 Chikungunya Fever (C F) and O'nyong nyong (ONN) O'nyong nyong Fever (ONN) and Chikungunya Fever are mosquito born arboviruses that cause severe illness in humans and some animals endemic to Asia, India, the Middle East, and Africa. ONN and Chikungunya are caused by related A lphaviruses of the family Togaviridae. In India and Asia, the mosquito species Ae. albopictus Ae aegypti and Anopheles mosquito vectors of Chikungunya (Pialoux et al. 2007). The virus incubates one to 12 days (Cook and Zumla 2003). Diagnosis can be mad e by isolating the virus from serums three to four days aft er onset of illness and by anti body tests. These diseases are distinguished by fever, headaches, joint pain, arthropathy arthralgia, conjunctivitis, sore throat, petichae, myalgia lymphadenopath y pharyngitis and rashes (Strickland et al. 2000; Eddleston et al. 2005; Kwan Gett et al. 2006; Pialoux et al. 2007). Bleeding often occurs in the mucosal membranes as well as in the gastrointestinal system. Fortunately, fatalities are rare with CF and O NN but the illness can last for as much as one month and some patients have chronic joint pain for life. Available treatments are supportive, typical to tropical disease treatments (Eddleston et al. 2005; Kwan Gett et al. 2006 ; Pardigon 2009 ). 2.1.5 Lymph atic filariasis Lymphatic filariasis (LF) is another mosquito borne tropical disease caused by particular species of nematodes endemic to 80 different countries in the tropics and subtropics with concentrations of the disease in Africa, India, Southeast As ia, the Pacific


35 islands and the Americas. Approximately 120 million people living in tropical and subtropical regions are afflicted by this disease (Strickland et al. 2000; Eddleston et al. 2005; Kwan Gett et al. 2006; CDC 2008). Although, t here are eight different species of nematodes that cause this sickness Wuchereria bancrofti Cobbold is the most common lymphatic filarial nematode. This microscopic worm is transmitted similarly to malaria, through mosquito bites. The mosquito bites an infected human or other host and ingests the filarial worm while consuming a blood meal. The worm then penetrates through the stomach wall muscles to the thoracic muscles. There, the mother w mosquito for the first two instars and th en nearby for the third instar. The larvae next migrate to the mouthparts and are finally transmitted to the next host the mosquito bites. The adult worms can live in various parts of the human body but often live in the lymph system They require four to 12 months to incubate and can live in the human for as long as 20 years. For tunately, they cannot produce fully developed offspring because the complete development cycle requires a mosquito host; however, they do produce embryos (microfilaria) which c an survive within the human for up to a year (Strickland et al. 2000; Cook and Zumla 2003; Eddleston et al. 2005; Kwan Gett et al. 2006; CDC, 2008). Initial symptoms of LF include localized inflammation, fever, chills headache, and malaise, although 50% o f individuals recently infected with the worm will not experience definitive symptoms, as the disease can be asymptomatic in early stages After a few years, some people develop malaise, fever, chills, lymphedema elephantiasis and eosinophilia syndrome. Lymphedema and elephantiatis are caused by fluid collection


36 and swelling in the legs, arms, genitalia, and breasts resulting in decreased function of lymph system (Strickland et al. 2000; Cook and Zumla 2003; Eddleston et al. 2005; Kwan Gett et al. 2006; C DC 2008). The diminished function of the lymphatic system can lead to many different secondary health problems. When the worm dies, the body can produce an inflammatory response. Diagnosing LF can be difficult because the worms are nocturnal. Blood must b e drawn at night and then examined microscopically. Symptom based diagnosis is more practical but less definitive. Currently effective treatments that kill the adult worm living in the lymph system do not exist. Diethylcarbamazine can be used to kill the worms in the blood, but those in the lymph system persist (Strickland et al. 2000; Eddleston et al. 2005; Kwan Gett et al. 2006; CDC 2008). 2.2 Triatomine borne Disease: Chagas Disease Chagas Disease is another vector borne tropical disease indigenous to parts of Central and South America, and it is now emerging in North America (Cook and Zumla 2003). Chagas is caused Trypanosoma cruzi Chagas (Kinetoplastida: Trypanosomatidae) that is transmitted to humans through a group of i nsects from the family Reduvi id ae (Cook and Zumla 2003; Kwan Gett 2006; CDC 2008). The T. cruzi parasite is a protozoa n that reproduces by binary fission. The insect vectors have many different common names, but will be referred to as triatomines in this thesis because this is gener ally the name used by the scientific community. The most common triatomine that transmits Chagas Disease to humans is Triatoma infestans (Klug) (Cook and Zumla 2003; Kwan Gett 2006; CDC 2008).


37 Chagas disease represents a serious threat to many throughout Latin America as it presently affects eight to 11 million people (CDC 2008). The majority of those who are affected are impoverished and live in rurally located, poorly constructed housing. Cracks and gaps provide the triatomines with both an entry into the home as well as hiding places when not feeding (Mullen and Durden 2002; Cook and Zumla 2003). Thousands of triatomines can hide in one home causing occupants to be bitten several times in one night. Serological studies have indicated that as many as 18 million people may carry T. cruzi and at some po int in the future, as many as 90 million people could be exposed to Chagas Disease ( Cook and Zumla 2003 ) The studies also project that up to 10% of those infected will die in from the acute infection a nd of the remainin g survivors, 30% will eventually die during the chronic stage of the disease (Cook and Zumla 2003). Chagas disease is spread through the feces of triatomines that contain T. cruzi The triatomine bites a T. cruzi infected human and lat er bites a different uninfected human (CDC 2008). The parasite reproduces in the gut lumen and attaches to its relatively unscathed. Then, the triatomine bit es an uninfected human host (often on the face) and defecates around the bite, depositing the T. cruzi near the new wound. If the human scratches the itchy insect bite, the feces containing the parasite is pushed into the open wound created by the insect bite and scratching (Kwan Gett et al. 2006; CDC 2008). The parasite incubates for five to fourteen days and is then able to invade red blood cells and spread throughout the body (Kwan Gett et al. 2006; CDC 2008). On the cell ular level, the protozoa inva de nonphagocytotic cells, especially muscle cells, as flagellated trypomastigotes move to the cytoplasm, lose their flagella


38 becoming amastigote s and divide forming new amastigotes in pseudocysts. The amastigotes mature into trypomastigotes and the pseu docysts eventually rupture releasing new trypomastigotes into the bloodstream (Cook and Zumla 2003). Alternate modes of infection include oral transmission through consuming food contaminated with triatomine feces, eating an infected reservoir species (i.e wild birds), or receiving infected blood transfusions (Cook and Zumla 2003). Figure b in Appendix D depicts the life cycle of T. cruzi in greater detail. Figure 2.4 depicts a T. cruzi infected human red blood cell (CDC 2008). Figure 2.4 T. cruzi in t hin blood smear (CDC 2008) When the parasite first invades, the disease is asymptomatic in most people, although mild illness can accomp any the invasion and 50% of patients develop an edematous nodule known as a chagoma (Kwan Gett sign, unilateral periophthalmic edema and conjunctivitis at the parasite entry site, is also an indicator of Chagas Disease. Fever, general enlargement of the lymph nodes, enlarged liver and spleen, myalgia, headache, hepatosplenomegaly vomiting, diarrh ea, anorexia, and skin rashes can be symptoms of the acute stage of the disease. At this stage, the


39 disease is treatable through two different drugs, nifurtimox and benznidazole that kill T. cruzi (Strickland 2000; Cook and Zumla 2003; CDC 2008). Many p atients ( 5 15% ) who develop acute Chagas Disease die and typically the cause of death is myocarditis and meningoencephalitis (Mullen and Durden 2002 ; Cook and Zumla 2003 ). This stage lasts four to eight weeks (Kwan Gett 2006). An indeterminate sta ge follows the acute stage which can last from a few years to decades and is also asymptomatic (Cook and Zumla 2003; Kwan Gett et al. 2005; CDC 2008). Low levels of the trypomastigotes remain in the blood ; therefore, infected humans remain potential carri ers of the disease (Cook and Zumla 2003). Eventually the disease can progress into the chronic phase. Onset of the chronic stage is typically between 35 and 45 years of age and typically affects the heart. This stage of Chagas Disease is incurable since do ctors can treat the symptoms, but cannot remove the T. cruzi (CDC 2008). Symptoms include conduction abnormalities like arrhythmias and palpitations edema generalized chest pain, dizziness, syncope dysponea apical aneurysm thrombus formation, cardi omyopathy dilated colon and esophagus, loss of peristalsis, dysphagia constipation, fecaloma congestive heart failure, thromboembolism increases (Strickland 2000; Mullen and Durden 2002; Cook and Zumla 20 03; Kwan Gett 2006; CDC 2008). 20 40 of those who develop chronic forms of the disease experience some of these symptoms (CDC 2008). Diagnosis at the acute stage involves microscopic examination of blood smears for the parasites. In the late a cute stage and subsequent phases of the disease, a technique called x enodiagnosis can be used (Cook and Zumla 2003). In this process, laboratory


40 reared triatomines are allowed to feed on patients suspected of having Chagas Disease and then after a month, the feces of the triatomine is examined to determine presence of flagellates. Animal inoculation can also be used by transfusing blood into susceptible laboratory animals and examining their blood for trypomastigotes five to fifteen days later. Serologi cal diagnosis for antibodies is most used method for determining if a patient has Chagas (Cook and Zumla 2003). 2.3 Fly borne tropical diseases 2.3.1 Human African Trypanosomiasis (African S leeping S ickness) Human African Trypanosomiasis (HAT) is caused by a protozoa n carried by the tsetse fly. The disease is commonly known as African Sleeping Sickness because of the drowsy and comatose behaviors exhibited by acute ly ill patients. Endemic to 36 countries in sub Saharan Africa north of the Kalahari desert there are two different species that infect thousands of Africans every year (Strickland 2000; Mullen and Durden 2002; Cook and Zumla 2003; Kwan Gett 2006; CDC 2008) From 1990 2004 epidemiological studies by the World Health Organization determined th at between 11 thousand and 37 thousand people were infected with HA T yearly and thousands of these people died (Mullen and Durden 2002 ; WHO 2006). 60 million cows and other livestock are additionally at risk for the disease (Maniania et al. 2006) Trypano soma brucei Plimmer and Bradford (Sarcmastigophora: Kinetoplastida: Trypansomatida) is the particular species that causes African T rypanosomiasis (related to T. cruzi, the disease agent of Chagas Disease) and is transmitted by riverine tsetse flies, Glossi na sp. The combinations of the protozoa n subspecies an d fly species involved in transmitting the human disease are T. brucei gambiense Plimmer and Bradford


41 transmitted by Glossina palpalis Vanderplank and T. brucei rhodesiense Bruce transmitted by Glossi na morsitans Wiedmann (Strickland 2000; Mullen and Durden 2002; Cook and Zumla 2003; Kwan Gett 2006; CDC 2008). B oth modes of transmission cause a three phased disease that is often fatal The tsetse fly serves as the intermediate host of T. brucei and the human is the definitive host. Within human s the protozoa live and replicate by binary fission in the blood, spinal fluid, and lymph as trypomastigotes ( Kwan Gett 2006) These trypomastigotes are ingested by the fly when it takes a human blood meal. Within the fly, the trypomastigotes multiply in the midgut. The protozoa then migrate to the salivary gland as epimastigotes. In the salivary gland the epimastigotes change into infective metacyclic trypomastigotes and are then transmitted to the next h ost the tsetse fly bites. Maturation of the epimastigotes into trypomastigotes takes three weeks ( Kwan Gett 2006; CDC 2008) The two sub species of protozoa produce two similar phasic diseases both of which are types of HAT Western Trypanosomiasis is c aused by T. brucei gambiense and requires an incubation time between two and three weeks. The first signs of infection with this parasite are irregular, cyclical fevers, lymphadenopathy, headache, myalgia, fatigue, hepatosplenomegaly, pruritus, transient facial edema, faint popular cutaneous eruption and malaise (Cook and Zumla 2003; Kwan Gett 2006) In later stages, the T. brucei become harder to locate in the blood, and endocrine dysfunction loss of libido, and spontaneous abortion can occur, along wi th severe anemia (Cook and Zumla; Kwan Gett 2006 )


42 Months to years after infection, the second stage of disease sets in as the trypanosomes attack the central nervous system ( Kwan Gett 2006) This stage is characterized by severe intractable headaches, slurred speech, abnormal Parkinsonian movements, impaired motor functions, somnolence, personality changes, and other psychotic tendancies. The patient can also develop chronic encephalitis and suffer from weight loss and chronic wasting (Strickland 2000; Cook and Zumla 2003; Kwan Gett et al. 2006). The first stage is characterized by development of a chancre at the site of invasion. The final meningoencephalitic stage causes the patient to experience headache, somnolence abnormal behavior, lo ss of consc iousness, and coma (Strickland 2000; Mullen and Durden 2002; Cook and Zumla 2003; Kwan Gett 2006; CDC 2008) The symptoms of Eastern African Trypanosomiasis caused by T. brucei rhodesiense appear almost immediately following the chancre between hours to d ays after the bite and are similar to the trypanosomiasis caused by T. brucei gambiense However, this form of disease progresses very rapidly. All three stages of the disease develop within a few weeks and the progression to death can occur in one to th ree months. In the last stages of disease, secondary infections, wasting, and malnutrition are dangerous complications that can arise ( Kwan Gett et al. 2006) The major difference between the two forms of trypanosomiasis is that T. brucei rhodesiense cau ses a much more acute disease than T. brucei gambiense (Strickland 2000; Mullen and Durden 2002; Cook and Zumla 2003; Kwan Gett 2006; CDC 2008) Diagnosis is accomplished by the card agglutination test for trypanosomiasis which tests for agglutination reac tions of trypanosomes on a plastic card or other similar tests in addition to looking for parasites in the blood l ymph or cerebrospinal fluid using


43 such methods as blood films, sm ears, miniature anion exchange centrifugation, wet preparations, inoculation of biological material from humans in animal host or vectors, and in vitro cultures (Cook and Zumla 2003; Kwan Ghett 2006). Anti trypanosome drugs can be used to treat HAT in the early stages of the illness. The first part of T. brucei gambiense induced trypanosomiasis is treated with intravenous doses of pentamidine or dimen azene, and the intermediate stages are treated with eflor nithine (Cook and Zumla 2003; Kwan Gett et al. 2006) T. brucei rhodesiense induced trypanosomiasis is first treated with i ntravenous doses of suramin and t he subsequent stage of the disease is treated with intravenous doses of melarsopr ol. However, once the cerebro spinal fluid becomes i nfected with trypanosomes, drugs are in effective and patients usually die These dru gs ex hibit significant toxicity; therefore their usage should be limited when possible (Cook and Zumla 2003). Relapses of these diseases are common and without treatment death can ensue ( Kwan Gett et al. 2006; CDC 2008) 2.3.2 Leishmaniasis Leishmaniasis is caused by a parasite species, Leishmania and spread by female phlebotomine sand flie s. It is endemic worldwide in 88 different countries in parts of East and North Africa, the Middle East, Southern Europe, Central, South, and East Asia, South America, and Southern Mexico (Strickland et al. 2000; Cook and Zumla 2003; Kwan Gett et al. 2006) An estimated twelve million people are inf ected with leishmaniasis and 350 million people are e xposed to the disease. As many as two million new cases occur each year (Cook and Zumla 2003). There are three different forms of Leishmaniasis and each type is caused by a different species of protozoa n also in the Trypanosomatidae family Visceral


44 Leishmaniasis is mainly caused by Leishmania donovani Laveran and Mesnil Lei shmania chagasi Da Cunha & Chagas and Leishmania infantum Russell Cutaneous leishmaniasis is mostly caused by Leishmania major Cunningham Leishmania aethiopica Cunningham Leishmania Mexicana Cunningham Leishmania braziliensis Cunningham and Leishman ia tropica Cunningham L. braziliensis is also responsible for mucocutaneous leishmaniasis ( Kwan Gett et al. 2006) Together, approximately 20 different leishmanial species are transmitted by about 30 different species of sand flies. Visceral Leishmanias is, the most dangerous of the leishmaniases, is found in 47 countries and an estimated 500,000 people are newly infected with this disease annually (Cook and Zumla 2003). One of the worst leishmaniasis epidemics in modern times occurred in an Upper Nile Province of Sudan. In this population of less than one million, 100 thousand people died between 1989 and 1994 (Cook and Zumla 2003). Leishmania are dimorphic parasites that invade and live in mammalian macrophages and in the insect intestinal tract. As with the other trypanosome caused diseases, t here are two morphological forms that the parasite assumes but in the case of Leishmaniasis the two forms are amastigotes and promastigotes The amastigotes are round or oval with a body 2 6 m in diameter a nd t hey have a nucleus kinetoplast and an internal fla gellum Promastigotes have slender bodies, an anterior flagellum, a kinetoplast, and a central nucleus (Cook and Zumla 2003). The parasites are spread when the sand fly bites an infected host. With in the fly, the ingested amastigotes transform into promastigotes which reproduce rapidly within the intestinal tract and move to anterior part of the midgut (Cook and Zumla 2003 ). When the fly bites its next host, the promastigotes are deposited The pro mastigotes are then phagocytosed by macrophages


45 where the promastigotes transform to the amastigotes, which are resistant to intracellular digestion and reproduce by rapid mitosis (Cook and Zumla 2003). Leishmania can incubate in the human host for two to six months; however, th ese organism s can hide and cause relapsing illness for up to 10 years after the first illness (Cook and Zumla 2003; Kwan Gett et al. 2006). The first sig n of visceral leishmaniasis is fever. Later, anemia, weight loss, asthenia hep atomegaly adenopathy and splenomegaly can ensue. The patient also can have a protuberant abdomen and muscle wasting in the limbs. Without treatment, visceral leishmaniasis is nearly always fatal since p atients die from progressive wasting, secondary inf ections, or bleeding. Presence of pancytopenia hypergammaglobulinemia and liver enzyme elevation are all laboratory findings that can be used to diagnose patients with leishmaniasis (Cook and Zumla 2003 ; Kwan Gett et al. 2006) Treatment of visceral le ishmaniasis includes both supportive care and antimicrobial treatment. The best available visceral leishmaniasis anti trypanosomal drugs are miltefosine and if this drug fails, sodium stibogluconate although the latter is not approved by the FDA ( Kwan Get t et al. 2006) Other drugs that can be used include amphotericin B, l ipid associated amphotericin B, and pentamidine. Supportive treatment is comprised of good nutrition and treatment of secondary infections (Strickland et al. 2000; Cook and Zumla 2003; Kwan Gett et al. 2006) Those infected with cutaneous leishmaniasis show cutaneous lesions at the site of the insect bite. These lesions can remain localized or spread across the body. Mucocutaneous leishmaniasis presents similar lesions; however these l esions are at


46 mucosal sites, commonly in the mouth and the nose ( Kwan Gett et al. 2006) Tissue destruction these lesions cause can eventually obstruct the airway and kill the patient. Cutaneous leishmaniasis typically heals in six to 12 months without a ny sort of treatment although s odium stibogluconate, ketoconazole, and methybenzethonium can be used as treat ments he aling process is lengthy and the disease may turn into visceral lei shmaniasis (Strickland et al. 2000; Cook and Zumla 2003; Kwan Gett et al. 2006) 2.3.3 Bartonellosis Bartonellosis is a disease carried by the New World sand fly, Lutzomyia sp. and is caused by a bacterium, Bartonella bacilliformis Barton The disease is most prevalent in the mountainous valleys of Peru, Ecuador, and Columbia and presents a 90% case fatality rate in untreated patients (Strickland et al. 2000; Cook and Zumla 2003; Kwan Gett et al. 2006) while t reatment reduces this rate to 5% These bact eria typically require an incubation period in the host for between sixteen and 22 days and attack erythrocytes at the conclusion of the incubation period ( Kwan Gett et al. 2006). This disease first manifests itself with symptoms of fever and malaise and i rregular fever, headache, myalgia, and arthralgia follow In extreme cases, the onset is quick and induces high fever, chills, and altered mentation (Kwan Gett et al. 2006). Weakness, vertigo, nausea and vomiting, syncope, prostration, lesions, and del irium can also accompany the afore mentioned symptoms (Kwan Gett et al. 2006). Laboratory diagnosis can determine Bartonellosis. Fortunately antibiotics can be used to combat it Chloramphenicol is administered for three weeks or more the rid the body o f the parasite (Strickland 2000; Cook and Zumla 2003; Kwan Gett et al. 2006).


47 2.4 Conclusion and Remarks: Tropical Diseases The tropical diseases discussed in this chapter affect millions of people and are often lethal. At the very least, these diseases are quite unpleasant and reduce the quality of life of those who contract them. Many of these diseases are classified by the World Health Organization as neglected tropical diseases, indicating that the vast majority of those affected by are impoverished. These diseases are understudied and undertreated most likely because the people who contract these illnesses live in rural areas, in poor housing, or do not readily have access to adequate medical care. Every dise ase discussed in this chapter i s caused by a microorganism and transmitted from human to human by an insect. Reducing these insect vector populations has the potential to drastically decrease the case load of tropical diseases. The next chapters will explore exactl y how fungal biological contro l can be used to control t hese vectors


48 Bibliography: Barrett A, Higgs S (2007). Yellow Fever: A disease that has yet to be conquered Annu al Rev iew Entomology 2007. 52:209 29 Bessaud M, Peyrefitte CN Pastorino BMA, Gravie r P, Tock F, BoeteF, Tolou, HJ, Grandadam M (2006). Dispatch, O'nyong, nyong virus, Chad. Emerging Infectious Diseases 12(8). Accessed Nov 2008. 0119.htm. Brewster DR, Kwiatkowski, White NJ (1990). Neurological sequelae of cerebral malar ia in children. The Lancet 336, 1039 1043. st ed. Elsevier Science Limited. London, England. Center for Disease Control (CDC) (2003). Global Anopheles Distribution. Accessed Nov 2008 Availab le from Center for Disease Control (CDC) (2004). Malaria Distribution. Accessed Nov 2008 Available from Center for Disease Control (CDC) (2008). A Z Disease Listings. Accessed Feb 2009 Available from Diagana M, Preux PM, Dumas M (2007). Japanese encephalitis revisited. Journal of the Neurological Sciences 262: 165 170 Eddleston M, Davidson R, Pierin i S (2005). Oxford handbook of Tropical Medicine. Oxford. University Press. Oxford, England. Greenwood B, Mutabingwa T (2002). Malaria in 2002. Nature 415: 670 672. Greenwood B, (2004). The use of anti malarial drugs to prevent malaria in the p opulation of malaria endemic areas. Am. J. Trop. Med. Hyg. 70(1): 1 7 Gunn SWA, Mansourian PB (2005). Understanding the Global Dimensions of Health Springer US. Hotez PJ, Molyneux DH, Fenwick A, Kumaresan J, Sachs SE, Sachs, JD, Savioli L(2007). Current Concepts: Control of Neglected Tropical Diseases New Eng land Jo urnal of Med icine 357(6): 1018 1027 Hume JC, Lyons EJ, and Day KP (2003). Human migration, mosquitoes and the evolution of falciparum. in Parasitology (3).


49 Kwan Gett TS, Kemp C Kovarik C (2006). Infectious and Tropical Diseases: Handbook for Primary Care. Elsevier Mosby. St. Louis, MO. Ichiro, Kurane (2007). Dengue hemorrhagic fever with special emphasis on immunopathogenesis. Comparative Immunology, Microbiology & Inf ectious Diseases 30: 329 340. Jain M, Ganjua L, Katiyal A, Padwad Y, Mishra KP, Chanda S, Karana D, Yogendra KMS, Sawhneya RC (2008). of rhamnoides extract against Dengue virus in human blood derived macrophages. Phytomedicine 15: 793 799. James S P, Nicol WD, Shute PG (1936). Clinical and Parasito logical Observations on Induced Malaria (Section of Trop ical Diseases and Parasitology). Proc edings of the R oyal Soc iety of Medicine 29(8): 879 894. James SP, Tate P (1937). New knowledge of the life cyc le of malaria parasites. Nature 139: 545. Maniania NG, Ekesi S, Odulaja A, Okech MA, Nadel DJ (2006). Prospects of a fungus contamination device for control of tsetse fly Glossina fuscipes fuscipes Biological Science and Technology 26(2): 129 1 39. Marsh K, Forster D, Waruiru C, Mwangi I, Winstanley M, Marsh V, C, Peter Winstanley P, Warn P, Norbert Peshu N, Geoffrey Pasvol G, Snow R (1995). Indicators of Life Threatening Malaria in African Children. New Eng land Journ al Med icine 332(21): 1399 1404. Miller LH, Baruch DI, Marsh K. Doumbo OK (2002). The pathogenic basis of malaria. Nature 415: 673 679. Miller SR, Wongsrichanalai C, Buathong N, Mcdaniel P, Walsh DS, Knirsch C, Ohrt C (2006). Effective treatment of uncomplicated plasmodiu m Falciparum malaria with azithromycin quinine combinations: a randomized, dose rangi ng study. American Journal of Tropical Medicine and Hyg iene 74(3): 401 406 Monath TP (2001). Yellow fever: an update. The Lancet: Infectious Diseases 1(1): 11 20. Mon ath TP (2008). Review: Treatment of yellow fever. Antiviral Research 78(1): 116 124. Mullen and Durden (2003). Medical and Veterinary Entomology 1 st ed. Academic P ress. St. Louis, MO. Mutabingwa AK (2005). Artemisinin based combination therapies (ACTs): Best hope for malaria treatment but inaccessible to the needy! Acta Tropica, 95(3): 305 315


50 Myint a KSA, Gibbons a RV, Perng a GC, Solomon b T ( 2007 ). Unravelling the neuropathogenesis of Japanese encephalitis Transactions of the Royal Society of Tropical Medicine and Hygiene 101 (10): 955 956 Pialoux G, Gausere BA, Jaureguiberry S, Strobel M (2007). Chikungunya,an epidemic. arbovirosis. Lancet Infect ious Dis ease 7: 319 327. Polhill a ,S, Soni b N (2007). Encephalitis in the ICU setting. Current Anaesthes ia & Critical Care 18(2): 107 116 Remme JHF, Erik Blas E, Chitsulo L, Desjeux PMP, Engers HD, Kanyok TP, Kengeya Kayondo JF, Kioy DW, Kumaraswami V, Lazdins JK, Nunn PP, Oduola A, Ridley RG, Toure YT, Zicker F, Morel CM (2002). Strategic emphases f or tropical diseases research: a TDR perspective. TRENDS in Parasitology 18(10). Sachs J, Malaney P (2002). The economic and social burden of malaria Nature 415: 680 689 Strickland TG et al. (2000). d Emerging Infectiou s Diseases 8 th ed. WB Saunders Company. Philadelphia, PN. Tolle MA (2009). Mosquito borne Diseases Current Probl ems in Pediatric and Adolescent Health Care 39(4 ): 97 140. Tunkel AR, Glaser CA, Bloch KC, Sejvar JJ, Marra CM, Roos KL, Hartman BJ, Kaplan SL, Scheld WM, Whitley RJ (2008). The Management of Encephalitis: Clinical Practice Guidelines by the Infectious Diseases Society of America Clinical Infect ions Diseases 47(3): 303 327 World Health Organization (WHO) (2006). Human African Trypanosomiasis. Weekly ep idemiological Record 8: 71 80. Accessed Feb 2009. World Health Organization (WHO) (2008). Neglect ed Tropical Diseases. Accessed March 2009


51 Chapter 3: General Overview of Insect Colonization by Entomopathogenic Fungi An understanding of how entomopathogenic fungi invade and colonize insects is fundamental t o our knowledge of biological control agents. While many of the small nuances are not fully understood and vary from species to species, the general process of infection is reasonably well known (Lacey et al. 2001; Scholte et al. 2004; Crespo et al. 2008). 700 fungal species could potentially become biological insect control agents since e ntomopathogenic fungal infections are one main method of natural in sect population control (Lacey et al. 2001). However, most research within this field has focused on a core group of approximately 10 fungi leaving many under studied and underrepresented (Lacey et al. 2001; Scholte et al. 2004). This chapter will discuss the general process of fungal invasions of insects. 3.1 Mechanistic overview of fungal infections of insects Insects can acquire fungal infections in slightly different ways, but the general mode of infection involves attachment of the conidia to the insect cuticle. Then through a combination of enzymes (specifically, lipases, chitinases, and proteases), toxins, and mechanical force, the fungi can break through. Since i nsects' cuticles are roughly 60 % chitin, and the insect body also contains significant quantities of proteins and lipids, these enzymes are important in this process (Hegedus et al. 1995 ; Scholte et al. 2004; Kanzok et al. 2006; Pedrini et al. 2007). Figure 3.1 depicts the process in cuticular penetration of Anopheles stephensi Liston (Asian malaria mosquito), by Bea u veria bassiana (Bals. Criv) Vuill


52 Figure 3 .1: Infection of An stephe ns i with B.bassiana a) freshly infected mosquito b) after 12 hours c) after 24 hours (Kanzok et al. 2006) The fungus proliferates within the hemocoel using reproductive units called blastophores (Hegedus et al. 1995 ; Scholte et al. 2004; Kanzok et al. 2006; Pedrini et al. 2007). The insect is first weakened and then killed as the fungus fills the entire body with mycelium giving the insect a whitish green mummified appearance. At this point, the fungi also produces lipases that degrade the lipids in the interior of the insect (Hegedus et al. 1995 ; Scholte et al. 2004; Kanzok et al. 2006; Pedrini et al. 2007). Once the fungi has depleted its supply of nutrients, the fungus breaks through the cuticle and sporulates in efforts to colonize a new host. In na ture, the released spores can remain


53 viable for several years (Hegedus et al. 1995 ; Scholte et al. 2004; Kanzok et al. 2006; Pedrini et al. 2007). Figure 3.2 depicts the progressive fungal infection of the insect cuticle. However, the anatomy of the inse ct cutic le must be addressed before further details of fungal infections can be discussed. Figure 3 .2 Cuticle& haemocoel penetration (Clarkson & Charnley 1996) 3.2 The Insect Cuticle The insect cuticle provides support and protection from foreign pathog ens, like fungi. It is made of two different layers the epicuticle and the procuticle, a s shown in figures 3.2 and 3.3. The cuticle is the first line of defense of the insect immune system.


54 Each layer has different physical and chemical properties that p ose a slightly different challenge for the fungus (Daly et al. 1998; Pedrini et al. 2007). Figure 3 .3 Insect cuticle anatomy Modified from Nation et al. ( 2003 ) & Jurez and Fernndez, (2006 ) The epicuticle is very thin and multilayered measuring 0.1 to 3 micrometers in thickness. This layer is most resistant to the degrading fungal enzymes and it contains two layers; a wax layer and a cement layer The outermost wax layer of the epicuticle is comprised of wax blooms as shown in figure 3.3 and this la yer protects the insect from dessication. In fact, most degradative enzymes cannot penetrate the cuticle unless the wax layer is physically broken. T he inner cement layer is hardened and it protects the insect from physical trauma (Daly et al. 1998). The procuticle is the thickest cuticular layer made of the exocuticle and the endocuticle and predominantly comprises protein. It lies underneath the epicuticle and contains chitin embe dded in the protein matrix that additionally contains lipids and quinones (Daly et al. 1998; Nation 2008). The hardened and dark exocuticle is the outer la yer of the procuticle and t he lighter colored and flexible endocuticle is the inner layer of the procuticle (Daly et al. 1998 Nation 2008).


55 Spiracles are the trachea opening s located on the integument that permit gas exchange between the interior and exterior of the insect. The simplest spiracle is comprised of folds of the integument that muscles can open and close (Nation 2008). Some insects have more elaborate spiracles t hat open with valves or integument flaps The spiracles can be both unguarded or guarded by entities such as sclerotized cuticle (Nation 2008). Familiarity with the insect cuticle is critical for re search o n entomopathogenic fungal insect control, as norm ally, the cuticle must be penetrated by the fungi before successful colonization can ensue (Nation 2008). Penetration of the gut and spiracles does occur, but cuticular fungal infections are by far the most common form of fungal infections of insects (Nn akumusana 1985). However, fungal infections of mosquito larvae can obstruct their modified spiracle (known as a siphon) (Balaraman et al. 2006). C onidia have limited mobility and contact with the insect cuticle is more probable than contact with a spirac le, although in theory, combinatorial attacks could occur. 3.3 Step by Step Process of Fungal Invasion of Insect At the start of the fungal invasion, the conidium must adhere to the cuticle for suc surface structur e and chemical composition play a role in this process (Pedrini et al. 2007). It is thought that the fungus may be able to detect the host through chemical signs and that static force between the spore and the cuticle may also contribute to the process. However, these ideas are mostly speculations (Pedrini et al. 2007). After conidia adhesion occurs, mechanical pressure allows fungal hyphae to invade the cuticle and penetrate the hemocoel. First, the fungi germinate on the exterior


56 of the cuticle using t he cuti cular chitin and fatty acids to satisfy their nutrient requirements and then they form appresoria that enable contact with cuticular cell walls (Leger et al. 1986; Pedrini et al. 2007). Some fungi are able to kill an insect by simply harming the c uticle whereas others further penetrate the hemocoel to acquire more nutrients (Leger et al. 1986; Pedrini et al. 2007). It is important to note that m ost entomopathogenic fungi require relatively high humidity levels to germinate (Clarkson and Charnley 1 996). Subsequently, penetration pegs extend into the hemocoel and further propagate hyphal growth laterally (Pedrini et al. 2007). The secondary messengers Ca 2+ and cAMP are likely involved in the formation of appresoria and other hyphal growth bodies (Cl arkson and Charnley 1996). After penetrating the hard, protective exoskeleton, the fungus has to further glycoproteins to combat pathogens (Wang et al. 2007). Studies sho w that following fungal infection, the protein profile dramatically changes. For example, a locust infected with Metarhizium anisopliae was shown to produce 13 new glycoproteins to combat the invasion but the exact role of these glycoproteins is still und er investigation (Wang et al. 2007). 3.4 Enzymes and Toxins After the cuticle has been physic ally broken, different enzymes and toxins degrade specific components of the insect cuticle. The major ones to be discussed are chitinase, proteases, lipase, and destruxins. Chitinase and N acetylglucosaminease (NAGase), are two of the most abundant fungal enzymes produced within the first few


57 days of the infection process In contrast, lipase is produced over five days after the initial fungal infection (Leger e t al. 2005). 3.4.1 Chitinases Chitinases are enzymes that degrade chitin and are involved in fungal morphogenesis, insect parasitism, and plant defense (Sahai and Manocha 1993; Guthrie et al. 2005). These enzymes play an important role in biological contro l of insects (Guthrie et al. 2005). There are three types and five classes. Classes I, II, and IV are all structurally similar and found in plants. Class III is found in plants and fungi and Class V primarily consists of bacterial chitinases (Guthrie et al 2005). Each class contains the 4 N acetylglucosaminidases, endochitinases, and 4 N acetylglucosaminidases are essentially exochitinases that cleave glycosidic bonds of the chitin polymer yielding N acetylglucosamine (GlcNAc) monomers. Endochitinases arbitrarily cleave chitin polymer at internal locations. Chitobiosidases cleaves disaccharides of N acetylglucosamine from the ends of chitin polymers (Guthrie et al. 2005). The specific involvement of chitinases in entomopathogenic fungi is unclear. Researchers are trying to determine if they are required for fungal penetration of the insect cuticle. However, it has been shown that in the case of many entomopathogenic fungi, chitinases are present and do appear to at least aid in hyphal penetration (Sahai and Manocha 1993). Fungal chitinases are located both extracellularly in periplasmic spaces and in the plasma membranes of the fungi (Sahai and Manocha 1993). Detecting and quantifying chitinases and t heir activities can be difficult and complicated. Basically, electrophoretic separation detects specific chitinases, and


58 spectrophotometry can also be used to measure chitinase activity but neither method simultaneously detects and quantifies; therefore, t he exact amount of chitinases and their activities cannot be precisely measured. These techniques are the primary tools available to determine the amount of chitinases within fungi. A complex process was devised as outlined in Guthrie et al. 2005 for conc omitantly measuring the aforementioned enzymatic activities In gel chitinase assays, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) is used to first determine the masses of denatured enzymes. Approximate n ondenatured enzyme masses are then determined using non denaturing PAGE and a non denatured protein molecular weight marker kit. Specific chitinolytic activity of proteins in the gel is quantified by measuring released fluorescent product 4 methylumbelliferone (4 MU) (Guthrie et al 2005). In this way, chitinase activity can be assigned to specific bands in the gels which is more convenient than performing separate SDS PAGE and spectrophotometry analyses. Such tools that illuminate the constituents involved in this process are esse ntial to determining how fungi kill insects. 3.4.2 Proteases Proteases are another class of fungal enzymes involved in fungal colonization. Fungi produce large quantities of proteases which are the first enzymes to be secreted by fungi upon contact with t et al. 1986; Qazi and Khachatourians 2007). Enzyme potencies determine the virulence of the fungi (Qazi and Khachatourians 2007). One common protease is PR1 which is produced in all tested ascomyc etes and deuteromycetes. The appressoria and other penetrating hyphae sec rete PR1, a serine endoprotease (Clarkson and Charnley 1998).


59 Some insects, like certain locusts, are able to produce protease inhibitors (PI) decreasing the effectiveness of proteas es (Franssens et al. 2008). Howe ver, fungi produce many different isozymes and are able to circumvent this defense mechanism (Qazi and Khachatourians 2007). 3.4.3 Destruxins Destruxins (DTXs) are another group of compounds involved in insect fungal infect ions. These are depsipeptides, peptides made of amino and hy droxyl carboxylic acid residues (Clarkston and Charnley 1996; Kershaw et al. 1999) DTXs cause lethal effects when injected or ingested by Lepidopteran and Orthopterans Inhibition of ATPase allo ws the DTXs to unbalance the homeostasis of eukaryotic cells, and therefore DTXs are potentially toxic to humans in large quantities (Clarkston and Charnley 1996). Ultimately, studies have shown that DTXs simply speed up the time of death because fungi wi were able to cause fatalities in insects, although more time is required (Kershaw et al. 1999). While many DTXs have been found in M. anisopliae these could be more widespread among other entomopathogenic fungi ; however, more studies of a larger group of entomopathogenic fungi must be undertaken to see if this is the case 3.5 Insect Death Ultimately, death ensues following the above mentioned events involved in fungal colonization. Different entomopathogenic fungi colonize an d kill insects in different ways. Those that exude toxins, like DTXs, are much more virulent than those that do not Most kill the insect before reaching internal organs because th ey digest the


60 nutrients in the haemolymph (Kershaw et al. 1999). In subsequ ent chapters, the lethality rates of different fungal biological control agents will be discussed. 3.6 An overview of w idely studied entomopathogenic fungal vector control agents As mentioned in chapter one, Beauveria bassiana (Bals. Criv) Vuill. and M etarhizium anisopliae (Metchnikoff) Sorokin are two of the most well researched entomopathogenic fungi because they nonspecifically kill a large number of insects and have been used with reasonable success in both vector and pest control. Considerable ove rlap in usage exists between these two species. Both of these fungi are ubiquitous world wide. (Lacey et al. 2001; Shah et al. 2003; Scholte et al. 2004). In particular, studies have indicated that B. bassiana and M. anisopliae c an potentially be used to combat vectors including mosquitoes, flies, kissing bugs, and ticks (Arachnida) (Kaaya 1989; Kershaw et al. 1999; Samish 2000; Ivie et al. 2002; Luiz et al. 2004; Scholte et al. 2004; Thomas et al. 2004; Blanford et al. ; 2005; Scholte et al. 2005; Kanzok et al. 2006). This is significant be cause these insects are all secondary hosts for disease causing microbes. M. anisopliae and B. bassiana are also routinely used in controlling some agricultural pests, including locusts, cockroaches, and several others (Ivie et al. 2002; Thomas and Kooyman 2004; Clarkson et al. 2008). M. Anisopliae and B. bassiana conidia, zoospores, or oospores are suspended in oil formulations that are sprayed onto surfaces frequented by the target insect (Lacey et al. 2001; Scholte et al. 2004). The insect lands, contacts the formulation, the conidia adhere and the fungal invasion begins (Lacey et al. 2001; Scholte et al. 2004). Different formulas specific for individual insect species are routinely studied and will be discussed


61 th roughout the remainder of this thesis. Adherence agents and the type of oil can affect the formula productivity. Other fungal species researched for their potential in fungal insect and these will be further discussed in subsequent chapters. Generally, a fungus o r mycoinsecticide is considered successful when it induces insect mortalities rates of 50% or more. It is unlikely that fungal species with l ower mortality rates will provide long term insect control benefits except when these fungi are used in conjunction wi th other insect control methods (Scholte et al. 2005; Luz and Fargues 1999 b). 3.6.1 Metarhizium anisopliae M. anisopliae is a well known entomopathogenic fungus and a Deuteromycete commonly known as the green muscardine fungus. Its life cyc le begins with asexual haploid conidia (Driver et al. 2000, Hughes 2004). Oil formulations of M. anisopliae are relatively easy to make into viable biopesticides, and as mentioned, such biopesticides have been used with marked success in controlling locust populations (Driver et al. 2000; Ivie et al. 2002; Thomas et al. 2004; Rangel et al. 2008). producing M. anisopliae (Driver et al. 2000); however, M. anisopliae grows rapidly when cultured on insect cuticle containing media (Leger et al. 1986). The optimal growing temperature for M.anisopliae is 25 C, but this fungal species can survive lower and higher temperatures, from approximately 20 C to 30 C. B. bassiana grows in wider temperature range; therefore it is more regularly used than M. anisopliae (Fernandez et al. 2008).


62 There are many different variants of M. anisopliae and these variants tend to differ in terms of how they specifically kill their host. Some produce more virulent toxins that kill the host more quickly than others (Rangel et al. 2008). Using fungi that induce rapid death can be both detrimental and beneficial. It means that the pest population is controlled more quickly; however, the probability of the fu ngus causing coevolution leading to mutations in the targ eted insect population increases (Lacey et al. 2001; Scholte et al. 2004; Rangel et al. 2008). Such mutations could render the insect resistant to the fungus (Lacey et al. 2001; Scholte et al. 200 4; Rangel et al. 2008). 3.6.2 Beauveria bassiana B. bassiana is phylogenetically similar to M. anisopliae and shares many physical characteristics. Researchers rout inely use these two fungi simul taneously in the same experiments because they require simila r growin g conditions. Researchers use t he same formulation methods with B. bassiana and grow and distribute it in the same manner as M. anisopliae (Alexopoulos et al. 1996; Pathan et al. 2007). B. bassiana is also a Deuteromycete, a generalist and an e ndemic entomopathogenic fungus. It can infect over 700 different species of insects, and it is an opportunistic saprophyte (Alexopoulos et al. 1996; Fernandez et al. 2008. It grows well at a broad range of temperatures from 8C to 35 C although the dormant conidia can survive temperatures of up to 50 C. This is broader than the normal o ptimal temperature range (25 35 C) of most entomopathogenic fungi (Fernandez et al. 2008). However, the optimal germination range is 20 30 C and humidity in addition to te mperature affect s germination rates. Well designed bioinsecticides can avoid these germination problems by creating a suitable microclimate for the fungus (Lecuona et al. 2001). Examples of


63 successful applicative studies of these two fungi in the context of specific insect vectors will be discussed in the next three chapters. 3.7 Summary Fungal colonization of insects is a complex process. Many different enzymes and chemicals play important roles in the infection process in addition to the mechanical pres sure that provides cuticular entry so that the enzymes can enter the cuticle and hemocoel. Further understanding of the enzymes and chemicals involved will permit a more advanced conceptualization of how these fungi can be used for biological control. Fut ure research should examine the processes involved in entomopathogenic fungi colonization in order to build upon the current knowledgebase.


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65 Malagasy Coleoptera and Anti Lo cus t Entomopathogenic Fu ngi. Journa l Economical Entomology 95(4): 651 660. Jurez MP and Fernndez GC (2007). Cuticular hydrocarbons of triatomines Comparative Biochem istry and Physiology Part A: Molecular & Integrative Physiology. 147(3): 711 730. Kanzok SM, Jacobs Lo rena M (2006). Entomopathogenic fungi as biological insecticides to control malaria. TRENDS in Parasitology 22(2): 49 52. Kershaw MJ, Morehouse ER, Bateman R, Reynolds SE, Charnley AK (1996). The role of destruxins in the pathogenicity of Metarhizium an isopliae for three species of insects. Journal of Invertebrate Pathology 74: 213 223. Lacey LA, Frutos R, Kaya HK, Vails P (2001). Insect Pathogens Do they have a Future. Biological Control 21: 230 248. Leger RJ, Charnley AK, Cooper RM (1986). Cuticle Degrading Enzymes of Entomopathogenic Fungi: Synthesis in Culture on Cuticle. Journal of Invertebrate Pathology 48: 85 95. Luz C, Rocha FN, Nery GV (2004). Public Health: Detection of Entomopathoge nic Fungi in Peridomestic Triatomine Infested Areas in Central Brazil and Fungal Activity Against Triatoma infestans (Klug) (Hemiptera: Reduviidae) Neotropical Entomology 33(6): 783 791. Luz C, Fargues J (1997). Temperature and moisture requirements for conidial germination of an isolate of Beauveria ba ssiana pathogenic to Rhodnius prolixus Mycopathologia 138: 117 125 .Nation JL (2008). Insect physiology & biochemistry. 2 nd ed. CRC Press Florida. Boca Raton, FL. Nnakumusana ES (1985). Laboratory infection of mosquito larvae by entomopathogenic f ungi with particular reference to Aspergillus parasiticus and its effects on fecundity and longevity of mosquitoes exposed to conidial infections in larval stages. Current Science 54(23): 1221 1228 Pathan AAK, Devi KU, Vogel H, Reineke A (2007). Analy sis of differential gene expression in the generalist entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin grown on different insect cuticular extracts and synthetic medium through cDNA AFLPs Fungal Genetics and Biology 44: 1231 1241. Pedrini N, Crespo R, M. Patricia Jurez (2007). Biochemistry of insect epicuticle degradation by entomopathogenic fungi. Comparative Biochemistry and Physiology, Part C 146: 124 137.


66 Qazi SS, Khachatourians GG (2007). Hydrated conidia of Metarhizium anisopliae release a family of metalloproteases. Journal of Invertebrate Pathology 95(1): 48 59. Rangel DEN, Alston DG, Roberts DW (2009). Effects of physical and nutritional stress conditions during culture on conidial germination speed, adhesion to host cuti cle, and virulence of Metarhizium anisopliae an entomopathogen ic fungus. Mycological Research: In press. Sahai AS and Manocha MS (1993). Chitnases of fungi and plants: their involvement in morphogenesis and host parasite interaction. FEMS Microbiolog y Reviews 11: 317 338. Samish M (2000). Biological Control of Ticks. Annals of New York Academy of Science 916: 172 8. Scholte, E.J. Knols BGJ, Samson RA, and Takken W (2004). Entomopathogenic fungi for mosquito control: a review. Journal of Insect Sc ience 4: 1 24. Knols BGJ (2005). Entomopathogenic Fungus Control of Adult African Mosquitoes. Science; 308 (10) : 1642. Thomas M, Kooyman C (2004). Locust Biopesticides: a T ale of Two Continents. Biocontrol news and information 25(3): 47N 63N. Wang C, Yueqing Cao, Zhongkang Wang, Youping Yin, Guoxio ng Peng, Zhenlun Li, Hua Zhao, Yuxian Xia (2007). Differentially expressed glycoproteins in Locusta migratoria hemolymph infe cted with Metarhizium anisopliae Journal of Invertebrate Pathology 96: 230 236.


67 Chapter 4 : The Role of Mycoinsecticide in Mosquito Control Sinc e entomopathogenic fungi cause insect deaths, they could provide a promising new bioinsecticide to control m osquito borne tropical diseases that cause illness and fatalities of millions annually. Recent research with two species of fungi, Beauveria bassiana and Metarhizium anisopliae could open new doors in the field of mycoinsecticides mosquito control. The fol lowing sections will explain the need for improved mosquito control methods and how entomopathogenic fungi may fulfill this need. 4.1 Mosquito Biology, Ecology, and Distribution Mosquitoes (Diptera: Culicidae) are hematophagous insects found in most areas of the world. Over 2500 mosquitoes species occur in a variety of climates, but only some are capable of transmitting human diseases (Mullen and Durden 2002; Cutwa Francis et al. ). Appendix C shows the world wide distribution of mosquitoes (Cutwa Francis et al. 2008; Daly et al. 1998; Rey 2008). Mosquitoes have sucking mouth parts and their salivary secretions contain a mixture of chemicals including anesthetics and anti coagulants. This fluid combination provides t he mosquito with stealth and unimpeded bloo d flow. Pathogenic agents enter and exit the mosquito through these mouth parts. Mosquitoes must bite an infected human and subsequently bite an uninfecte d human to transmit the disease (Mullen and Durden 2002; Cutwa Francis et al. 2008). Mosquitoes have holometabolous meta morphosis with four life stages: egg, larvae, pupae, and adult (Cutwa Francis et al. 2008; Daly et al. 1998). Stagnant water


68 affords immature stages some degree of protection because the low oxygen levels deter aquatic predators (Daly e t al. 1998). Eggs are laid singly or in rafts depending on the species. Larvae are legless aquatic filter feeders often known as wigglers or wrigglers because of the lashing motions they make with their abdomen. They feed on decaying organic matter with in the standing water and breathe through their siphons (Mullen and Durden 2002). The larvae undergo four instars each of which is formed underneath the old cuticle before the exuviae is shed Molting enables the insect to grow (M ullen and Durden 2002; Da ly et al. 1998 ). The pupal stage is also aquatic but non feeding. Winged adults emerge around four days afte r entering the pupal stage. Generally t he winged adults feed on plant saps and the females take blood meals only to provide their eggs with prote in (Mullen and Durden 2002; Rey 2008; Sweeny 2008). Their life cycle can take as few as six days during the summer (Mullen and Durden 2002). Female mosquitoes live an average of three to five weeks, although they can live as long as five months (Rey 2008). Mosquitoes feed on a variety of sources, but many are specific and some such as Aedes aegypti feed solely on humans (Mullen and Durden 2002; Sweeny 2008). To ensure human blood meals, mosquitoes are capable of recognizing specific compounds called kairomo ne s As many as 400 different constituents may comprise human kairomones (Constantini et al. 1999; Mullen and Durden 2002; Bernier et al. 2007). Additionally, the mosquito can respond to levels of carbon dioxide to determine the presence of a human host (C onstantini et al. 1999; Bernier et al. 2007). Furthermore,


69 bacteria on human skin can release semiochemicals that vector mosquitoes can detect (Constantini et al. 1999; Mullen and Durden 2002; Bernier et al. 2007). Knowledge of mosquito biology is imperat ive to our understanding of how vector borne diseases persist. This understanding could indirectly lead to better vector management programs and ultimately reduce the number of mosquito borne disease incidents. Sri Lanka and other countries that cultivat e rice have an especially high number of malaria cases because stagnant water, which is part of the agricultural process, provides mosquito larvae with excellent growing conditions (Yasuoaka et al. 2006). Use of chemical insecticides to reduce mosquito pop ulations has been increasingly costly and ineffective due to the ir high price and the development of insect resistance to the pesticides (Yasuoaka et al. 2006). C ontrol through community based vector control programs and mosquito proofing structures have b een attempted with mixed success (Yasuoaka et al. 2006), but a long lasting and inexpensive technique s are needed to reduce populations. Biological control will hopefully soon fulfill this need. 4.2 Identification of Mosquito Species Identification of mos quitoes is an important part of vector control and can be accomplished by macroscopic, microscopic, and molecular biological techniques (Temu et al. 2007). Entomologists can examine individual specimens for defining physical characteristics (Darsie et al. 2005). The shape of the proboscis, the length of palpi length of antennae, width of frons and shape of tarsal claws are some of the attributes used to determine species. Additionally, the pattern s and coloration as well as the location, and number of sc ales and the segmentation of the abdomen are anatomical


70 features that help d etermine species (Darsie et al. 2005). Some examples of different pathogenic species are shown in figure 4.1 Aedes aegypti L. (Gathany 2003) Aedes albopictus S kuse (Gathany 2006) Anopheles minimus L (Gathany 2005) Armigeres subalbatus (Gathany 2003) Figure 4. 1 Engorged vector species of mosquitoes Reprinted with permission from CDC PHIL. PCR is another technique that is frequently used in i dentification. PCR amplifies DNA prot ein components. Knowledge of mosquito proteins helps identify the species and determine the p roteins of the blood meal allowing researc hers to deduce the mosquito's hosts. Ultimately, use of these techniques allows investigators to determine which species specifically have human hosts (Temu et al. 2007). 4.3 Vector Qualifications and Requirements To be a vector of human disease the mosqu ito must consistently feed on humans, ( anthropophily ) Humans are hairl ess which renders them an easier food source than very furry animals; however human defense mechanisms do substantially deter mosquitoes.


71 Mosquitoes must adopt specific behavioral patt erns to successfully feed on humans The mos t important of these is endophagy, which is the ability to enter structures to feed on humans. Moreover, the mosquito species must feed more selectively on humans than other animals (Constantini et. al 1999). Ad ditionally, u nderstanding and identifying the disease parasite is crucial. A reasonable database on the parasite and which species transmit the parasite is important to control programs. Some mosquito species are unable to transmit disease causing paras ite s ; therefore, targeting these species would be useless. Control programs using fungi must consider the parasite and the insect vector as well as the fungal agent. 4.4 Fungi and mosquito control As discussed earlier, entomopathogenic fungi can attack b y penetrating the cuticle, proliferating in the hemocoel, and then r upturing through the cuticle forming fruiting bodies (Kanzok et al. 2006; Scholte et al. 2004; Blanford et al. 2005; Scholte et al. 2007). Fungal biological control can b e used against eac h different stage in the mosquito life cycle (egg, larva, and adult) (Lacey et al. 2001). Thus far, mosquito larvae control has been cited as one of the most successful type of mosquito fungal biological control (Scholte et al. 2004). Langenidium giganteum Couch is available commercially for control of mosquitoes ; however unfortunately this fungus is ineffective at substantially controlling a major malaria vector, Anopheles gambiae (Golkar et al. 1993 ; Scholte et al. 2004). Lagenidium, Colelomomyces, Cul icinomyces, Entomophthora, Beauveria, and Metarhizium are genera of fungi that have demonstrated or have the potential to demonstrate success as a commercial mycoinsecticide (Scholte et al. 2004).


72 Important aspects of the pathogenic relationship between mo squitoes and fungi include the effects of the fungi on the adult mosquito populations, the effectiveness of the spore delivery to the mosquito, and the spores viability over time. Perhaps most importantly, the complete vector populations must be targeted by potential pathogens. Many la rvicidal fungi fail because they can not overcome the immune system of Anopheles gambiae complex mosquitoes (Scholte et al. 2004). The genera Conidiobolus (Zygomycota: Zygomycetes : Entomophthorales : Ancylistaceae ), Entomopht hora (Zygomycota: Zygomycetes : Entomophthorales : Entomophthoraceae ) Erynia (Zygomycota: Zygomycetes : Entomophthorales : Entomophthoraceae ) and Smittium (Zygomycota: Zygomycetes : Entomophthorales : Entomophthoraceae ) have been researched for their potentia l at killing adult mosquitoes ( Scholte et al. 2004 ). H owever these fungi can also infect other animals. Entomophthora species could be useful at killing hibernating mosquitoes ( Scholte et al. 2004 ) Colidiobolus species are effective agents, but infects h umans and horses ( Scholte et al. 2004 ) Erynia species can induce 100% infection, but kills frogs (Scholte et al. 2004). Smittium species have demonstrated laboratory success in killing adult mosquitoes but failed in field setting s (Scholte et al. 2004). A comprehensive chart by Scholte et al. ( 2004) details the known fungi that could potentially bec ome biological control agents and is shown in Table 4.4 ( Scholte et al. 2004 ) The d euteromycetes Culicinomyces, Beauveria, Metarhizium, and Tolypocladium a re other fung i genera that have been explored as mosquito control agents. Culicinomyces is inconsistently successful (Debenham et al. 1977; Cooper et al. 1982; Campbell et al. 2002) but Bea u veria and Metarhizium have demonstrated siz e able


73 success es in the laboratory and field setting in controlling adult mosquito populations (Scholte et al. 2003; Blanford et al. 2005; Scholte et al. 2005) Tolypocladium has a very narrow range of growing conditions ; therefore it has limited potential as a biological contro l agent ( Gardener and Pillai 1987; Weiser et al. 1991;


(lf) indicates species that have demonstrated success in both laboratory and field settings, (x ) denotes species that have s hown no success, (l) indicate species that demonstrated success in only laboratory settings, (v)indicates success in combating larv ae and species marked with (c) indicate species that were successful in the laboratory and the field and are now commericially available as biocontrol agents. 74 Table 4.2 Entomopathogenic Fungi used in Mosquito Control Phylogenetic depiction of all the species of fungi known to have been explored as mosquito biocontrol agents (Scholte et al. 2004 a). 1)Kin gdom 2) Phylum 3)Class 4) Order 5) Family 6) Genus CHROMISTA 1) Oomycota 2) Oomycetes 3) Saprolegniales 4) Saprolegniaceae 5) Leptolegnia 6) (v) Pythiales Pythiaceae Pythium Lagenidium (v, c) Myzocytiopsidales Crypticolaceae Crypticola (l) FUNGI Chytridiomycota Chytridiomycetes Blastocladiales Coelomomycetaceae Zygomycota Zygomycetes Entomophthorales Ancylistaceae Conidiobolus (lf) Entomophthoraceae Entomop hthora Erynia (lf,kills frogs) Trichomycetes Harpellales Legeriomycetaceae Smittium (l) Deuteromycetes ( Hyphomycetes ) Culicinomyces (l) Beauveria (lf) Metarhizium (lf) Tolypocladium (l)


75 Nadeau and Boisvert 1994; Scholte et al. 2004). Coelomomyces is also successful at colonizing mosquito larvae ( Chytridiomycota : Chytridiomyctes : Blastocla diales : Coelomomycetaceae ) Coleomomyces sp. However, mass production techniques rely on a mosquito substrate ; therefore mosquitoes must be reared to obtain the fungus which renders the process costly (Couch 1972; Buchanan et al. 1990 ; Rueda 1990; Lu carotti et al. 2000) As introduced in Chapter Three, Metarhizium anisopliae and Beauveria bassiana are currently being thoroughly explored as mosquito control agents because hyp h omyceteous fungi (a class of Deuteromycetes) are endemic around the world and are considered harmless to humans, mammals, reptiles, birds, and fish M ost scientists be lieve their spores cannot survive temperatures found within animals However, rare cases of B. bassiana infecting immunocompromised human patients have occurred, and a team under Ward has shown the potential for M. anisopliae to induce an allergic response in a rodent model (Ward et al. 1998; Genthener 1998; Ward et al. 2000 a ; Ward et al. 2000 b ). Also, M. anisopliae has been shown to cause deleterious effects in som e marine life forms and B. bassiana is known to kill frogs. M. anisopliae is currently registered in the US to combat cockroaches and flies (Ward et al. 1998; Genthener 1998; Ward et al. 2000 a ; Ward et al. 2000 b ) and M. anisopliae and B. bassiana are com mercially available around the globe for combating an array of insect pests from locusts to cochroaches to flies (Faria et al. 2007). Leptolegnia, Pythium, Lagenidium, and Crypticola are fungi like organisms of the kingdom Chromista and phylum Oomycota th at have also been explored as pos sible biological control agents These are water molds that are facultative parasites of mosquito


76 larvae (Scholte et. al 2004). Of the four genera, Lagenidium has demonstrated success in the field and was first made com m erc ially available in the 1990s (Golkar et al. 1993). The other three only exhibited success in laboratory settings (Scholte et al. 2004). 4.5 Adulticidal Entomopathogenic Fungi Within the last 15 years, ground breaking work with entomopathogenic fungi and m osquito control of adults has materialized. Two separate research groups including a team led by Read and Blandford of the Center of Infectious Disease Dynamics, Pennsylvania State University and another team led by Scholte at Wageningen University, Holla nd, have explored the efficacy of using fungi to biologically control adult mosquitoes with some success (Kanzok et al. 2006; Scholte et al. 2004; Blanford et al. 2005 ; Scholte et al. 2007; Thomas and Read 2007). 4.5 .1 Bea u veria bassiana The Blanford tea m used fungal spores infused in an oil formulation to test the effectiveness of the fungi (Blanford et al. 2005) They screened eight different strains of Beauveria bassiana although, ultimately, the strain selected was chosen on the basis of availability (Blanford et al. 200 5). The fact that this strain i s already used in agricu lture c ould open up the possibility of using pre existing formulations sold by companies such as Mycotrol, Bioblast, Green Guard, and Green Muscle (Kanzok 2006; Thomas and Read 2007 ). The team further asserted that if B. bassiana is used, authorizing these biopesticides for use as mosquito control agents would be easier than creating an entire new product (Blanford et al. 2005)


77 Mammalian mouse models were used to determine infec tion rates of mosquitoes that were inoculated with B. bassiana The study demonstrated a n 80% reduction rate in incidenc es of malaria in a rodent model (Blanford et al. 2005). The team also suggested that the oil formulations could b e used in residential dwel lings since t he y c ould be sprayed on ceilings and walls where mosquito rest. Unfortunately the spores remain viable for only a thre e week period in commercially available oil suspension s therefore regular applications would be required (Scholte 2004 et a l. ; Blanford 2005 et al. ; Scholte 2007 et al. ; Thomas and Read 2007). However, application innovations are underway and may result in formulas with longer residual shelf life (Thomas and Read 2007). 4.5 .2 Metarhizium anisopliae The team from Wageningen Uni versity, Holland has been researching entomopathogenic fungi for mosquito control for at least five years. They are working to determine the efficacy of mycopesticides using M. anisopliae and to establish an effective fungal application method. Their stud ies thus far have examined the proclivity of fungally infected mosquitoes to take a blood meal, the effects of augmenting formulas with m osquito attractants (Brak et al. 20 02), and the overall effectiveness of using M. anisopliae to control mosquito popula tions (Braks et al. 2002; Scholte et al. 2003; Scholte a et al. 2004; Scholte b et al. 2005; Scholte et al. 2006; Scholte et al. 2007; Farenhorst et al. 2008). The team's work with mosquito attractants is significant because addition of these to mycoinsect icides could increase contact between spores and insects The researchers found that human sweat is indeed a mosquito attractant ( Braks et al. 2002 ) Microbes


78 feeding off the sweat release byproducts that were shown to attract mosquitoes. Ammonia, a maj or constituent of sweat, was found to be a significant kairomone (Braks et al. 2002). In 2003, the Wageningen team examined M. anisopliae fungal infections of mosquitoes A n gambiae and Culex quinquefasciatus Say vectors of malaria and filariasis respect ively. The y determined that both Anopheles and Culex are affected by M. anisopliae and the degree to which the populations are affected varies depending on fungal dose. The Lethal Time ( LT 50 ) values varied from 9.69 +/ 1.24 days for the lowest dose to 5. 89+/ 0.35 days for the highest dose applied. The researchers also established that by encapsulation of blastospores and melanization mosquitoes can evade low grade fungal infections (Scholte et al. 2003). A ctivation of prophenoloxidase provides the insec t with some protection against pathogens and leads to melanization ( Dimopoulos et al. 1998) The next study by this team examined the propensity of autodissemination natural dispersal of fungal spores between mosquitoes. Autodissemination is important to th e functionality of mosquito fungal biological control because it ensures perpetuation o f the biological control agent which would reduce the need for reapplication of sp ores (Scholte et al. 2004 b ). I n order to study this phenomenon, i nvestigators exposed virgin female Anopheles mosquitoes to papers impregnated with fungal spores and then placed these mosquitoes into a closed environment with male mos quitoes. There were three experimental variations In one, 30 infected females were placed with 30 uncontam inated males, in another, one infected female was placed w ith one uninfected male, and in the last, one


79 infected female was placed with ten uninfected females. Between 10.7 + 3.2 % and 33 + 3.8 % males were infected. This demonstrates that autodissemenat ion of spores could occur to some degree in a laboratory setting, although, the exact factors that vary the results require determination (Scholte et al. 2004 b ). However, field studies could indica te different results because some natural variabl es were not r eplicated in the laboratory. Furthermore, infection from males to females has not yet been shown The same experiment completed in reverse c ould further test this possibility Additionally, the experiment could be carried one step further by placing the ex posed males with a new set of uninfec ted mosquitoes and examining if any autodissemenation has taken place. In a more recent laboratory study, a Wageningen team infected mosquitoes with M. anisopliae to determine the effects of the fungal infection on mos quito fecundity and propensi ty to take blood meals (Scholte et al. 2006 ). Mortality rates were determined by applying 1.6 x 10 10 conidia/m 2 on fabric The team reported that 87.1 + 2.65% of Ae aegypti and 8 0.3 + 0.2 % of Ae. albopictus acquired fungal infe ction s The LT 50 was determined to be 3.10.2 days for male Ae. aegypti 4.10.3 days for female Ae. aegypti 17.70.4 days for female Ae. a lbopictus and 19.70.6 days for male Ae. albopictus Therefore, v ariation between species was demonstrated and f unga l treatment of Ae. albopictus was deemed less successful based on these data (Scholte et al. 2006 ) To test the propensity of the insects to take blood meals, human arms were offered to fungally infected and parasitically infected mosquitoes in cups with netting over them. The r esults were as follows. For the second blood meal, 87.5 + 10.5% of the control group took the meal versus only 44.8 + 6.8 % of the group treated with M. anisopliae By the fourth blood meal 93.5 + 1/5% of the control group fe d ver sus only


80 67.1 + 8.0% of the treated group (Scholte et al. 2006). Based on the data, it was concluded that fungal infections of mosquitoes do reduce propensity to feed. Even a small reduction in mosquito bites may have a large effect on the end number of c ases of mosquito borne tropical diseases because of the enormity of the disease problem. For this reason, even though some mosquitoes will evade fungal infection, this method would have far reaching effects if a practical cost effect ive way to implement t his mycoinsect icide was discovered (Scholte et al. 2006). T he next study by the Wagenigen University group was the first direct field study of the effectiveness of entomopathogenic fungi mosquito control by this group. In this study, bed nets and black she ets inoculated with M. anisopliae were hung from the ceiling so that t he mosquitoes would acquire a M. anisopliae fungal infection after taking a human blood meal Three to eleven days later the mosquito typically died. Ultimately this study indicated tha t the EIR rate could be reduced by at least 75% which would greatly reduce the number of disease transmissions (Scholte et al. 2005). If just 50% of individuals in the mosquito population acquired fungal infections, the EIR rate would be reduced by 96%, dr astically reducing the incidence of mosquito borne tropical diseases (Scholte et al. 2005). Farenhorst et al. (2008) of Wagenigen University published work proposing the use of clay pots as a fungal application method. Based on the prior work of this team, the main barrier to M. anisopliae use is establishing contact between mosquito and conidia. In this laboratory study the team demonstrated that clay pots can be used to infect mosquitoes and that mosquitoes chose clay pots as resting sites. The pots were impregnated with conidia of M. anisopliae Of 858 An. gambiae mosquitoes exposed to


81 conidial dose of 4 x 10 10 95.0 +/ 1.2% were infected by the fungi. At a lower conidial dose of 1x 10 10 91.5 +/ .6% became infected Of the 233 An. f unestus Gilles and S mith exposed, 91.8 +/ 1.2 % displayed fungal infections (Farenhorst et al. 2008). In the attractiveness experiments, an experimental cage was designed comparing the preference of mosquitoes for resting places. The cage contained PVC pipe, terra cotta pot s, wet clay pots, and dry clay pots These experiments showed that of the 219 male An. g ambiae mosquitoes, 47.4 +/ 7.2% chose to rest inside the clay pots and of the 245 females, 81.8 +/ 2.0% selected the clay pots as resting points. The moistness of the pot did not affect the selection of mosquitoes for one pot over th e other. The team also tested fungally infected pot s versus a control and was able to determine that the impregnated pots were equally as attractive to the mosquitoes as the control. The te am also proposed that mosquito attractan ts like kairomones and CO 2 could increase attractiveness of clay pots if the pots were treated with these substances ( Braks et al. 2002 ; Farenhorst et al. 2008). Based on these preliminary laboratory data, the team p lans to pursue field studies to determine if these laboratory results persist in the field ( Scholte et al. 2004; Scholte et al. 2006; Scholte et al. 2007; Farenhorst et al. 2008 ) This application method could prove to be an effective method to curb mosqui to populations. The Wagenigen researchers suggest that this method could potentially be used as an important part of an integrated vector management program, but that practical issues such as increasing viability of conidia, finding the perfect location a nd material to attract mosquitoes to the mycoinsecticide, and formulating a long lasting effective mycoinsecticide containing M.


82 anisopliae are the major challenges they must face (Scholte et al. 2004; Scholte et al. 2006; Scholte et al. 2007; Farenhorst e t al. 2008). 4.5 .3 Fusarium pallidoroseum Even more recently, a group from India brought forth an additional fungus Fusarium pallidoroseum (Cooke) Saccardo to the list of fungi with m osquito killing capabilities The group b egan by isolating the naturall y occurring fungi that colonize lymphatic filariasis vector mosquito Culex quinquefasciatus The investigators isolated a few different species of Aspergillus and Fusarium and then determined the LT 50 values of these species. Of these, only the LT 50 valu es of F. pallidoroseum were high enough to indicate possible viability as a mosquito biocontrol agent. The LT 50 value was 2.08 days following a four hour exposure to 1.11 x 10 10 conidia/m 2 (Mohanty et al. 2008). 4. 6 Larvicidal and Ovicidal Fungal parasite s of mosquitoes Larvicidal fungi were the first entomopathogenic fungi to be researched and the first to become commercially available for mosquito control ( Scholte et al. 2004; Mohanty et al. 2008 ) Ovicidal fungi were the last of the fungi to be research ed, with investigations beginning only in the last five years (Golkar et al. 1993; Scholte et al. 2004; Mohanty et al. 2008). 4.6 .1 Langenidium giganteum Couch Golkar et al. (1993) conducted a laboratory study with mosquito larvacidal fungi by examining th e potential of Lagenidium giganteum to be an effective biological control agent. L. giganteum is a facultative oomycete that colonizes mosquito larvae (Golkar et al. 1993). In nature, it can persist in a mosquito population for as long as an entire mosquit o season (Scholte et al. 2004; Vandergheynst et al. 2006). Golkar et al. (1993)


83 specifically studied the factors that render mosquitoes susceptible to fungal invasion including phototaxis, aerotaxis, and chemotaxis ( Golkar et al. 1993 ) The team conclude d that the amount of time the larvae spent on the surface of the water correlated with the highest rates of fungal infection. Additionally they suggested that an unknown water soluble chemical released by L. giganteum zoospores caused other zoospores to ag gregate to the infected mosquito larvae intensifying rates of infection (Golkar et al. 1993). Of the three species examined, A n gambies, A e aegypti, and C. pipens, A n gambies w as the most resistant to infection. Approximately 56% of A n gambies exposed to higher than natural doses of zoospores survived infection because this particular species was able to encyst hyphae through its melanization process. Hyphae invading A e aegypti and C. pipens were able to attack the h emocoel without difficult y because t hese two species lacked the melanization immune response of A n gambies (Golkar et al. 1993). Another group determined that L. giganteum can deleterious ly affect Culex quinquefasciatus larvae, vectors of Japanese encephalitis (Suh et al. 1999). The LC 50 va lue of zoospores was 26 zoospores per ml and larval mortality was greatest at 25 C in unpolluted freshwater (Suh et al. 1998). Experimentation with individual chemical components of L. giganteum is an additional emerging area of study. Teng et al. (2005) determined that two particular constituents designated by the researchers as Lg product A and Lg product T, reduced Culex mosquito populations by 49.7% and 21.9%, respectively, and diminished An. sinensis by 8.6% and 44.6%, respectively, within the first w eek of exposu re. However, six weeks after exposure, a negligible decrease in mos quito populations was observed


84 becaus e the populations had recovered ; therefore, these products would not be very effective mosquito control agents alone (Teng et al. 2005). L giganteum is one of few registered mycoinsecticides in the US as a biological agent. However, it is infrequently used because the formulas in which the delicate zoospores are typically suspended keep the spores viable for a mere one to three weeks (Vand ergheynst et al. 2006). Additionally, the mycoinsecticide requires refrigeration and contamination is common (Vandergheynst et al. 2006) In order for L. giganteum based mycoinsecticides to be embraced by the commercial mosquito control industry, shelf l ife must increase, cost must decrease, and incidences of contamination must diminish (Vandergheynst et al. 2006). According to Vandergheynst et al. 2006, three main strategies are being pursued. One strategy is to experiment with new formula ingredients ( but using the same zoospores), another is to strengthen fungal strains to produce heartier zoospores, and the third is to make more effective formulas from L. giganteum oospores (Vandergheynst et al. 2006). Oospores survive desiccation and remain viable e ven following several years of storage, contrasting against the fragile zoospore counterparts (Vandergheynst et al. 2006). In 2006, the team le d by Vandergheynst researched formulation methods in an attempt to extend the viablility time of L. giganteum i n the field. This group devised a invert emulsions with silica that extended the shelf life and spore viability from three weeks to over twelve weeks. In this formula, special e quipment is used to encapsulate each i ndividual spore with water and then oil, thereby providing protection against desiccation (Vandergheynst et al. 2006). This oil


85 formula also distributes spores more evenly, increasing the probability of an insect coming into contact with a spore. Ultimat ely, storage ti me drastically increased so the number of applications that would be required to apply this mycoinsecticide sh ould be dramatically reduced (Vandergheynst et al. 2006). A study exploring a formula made from both zoospores and oospores using et al. (2006) strategy for creating L. giganteum formulas might pro duce interesting result s if a group were to perform such a study. Potentially, the zoospores could initially infect the mosquito p opulations while the oospores matured. 4.6.2 Leptolegenia chapmanni Seymour An additional fungus like organism, Leptolegenia chapmanni was examined for its Ae. aegypti larvicidal capabilities by a grou p of Argentinian researchers le d by Pelizza, adding to the arsenal of fungal biological contr ol ag ents. This mosquito pathogen inhabits a vari ety of locations including tree holes, artificial containers, freshwate r and brackish water flood plain s, and woodland ponds ( Mullens and Durden 2002; Pelizza et al. 2008). The researchers determined that L. chapmanni infects by two methods, providing evidence for the potential efficacy of the agent in inducing mosquito mortalit y The more lethal process involves the encystment of motile zoospores on the larval cuticle, but death can result from germination o f ingested zoospores (Pelizza et al. 2008). L. chapmanni was able to infect larvae for 51 days when kept at 25 C, 12 days at 35 C, and five days at 10 C, signifying a temperature dependent relationship The maximum zoospore production was 9.6 + 1.4 x 10 4 z oospores/larvae after 48 hours at 25 C. Spores remain viabile for an average of five weeks when at 25 C.


86 Pelizza et al. ( 2008 ) indicated that one of the reasons they explored this fungus was that currently only one oomycete is widely recognized as a mosqu ito pathogen, L. giganteum ; however data comparing the two water mold species, L. giganteum and L. chapmanni were not provided. Perhaps a useful future study would systematically compare the se two watermolds' ( L. giganteum and L. chapmanni ) mosquito ki lling potentials (Pelizza et al. 2008). Another group of Argentinian scientists tested the capacity for L. chapmanii to harm twelve different species of mosquitoes. Additionally this group examined L. chapmanii for possible adverse non target effects (Lpe z et al. 2004). As outlined in Appendix E, fe w if any adverse effects were recorded on non target species and except for Isostomyia paranensis Brethes Culex renatoi Lane & Remaalho and C ulex castroi Casal & Garcia all of the mosquitoes tested acquired fungal infections upon exposure to the fungal spores. Anophe les sp. and A e gambiae were among those tested and determined to be capable of acquiring infections of L. chapmanii (Lpez et al. 2004) C harts in Appendix E show both the infections of L. chap manii in non target species and the infections in target species. 4.6.3 Metarhizium anisopliae (Metschnikoff) Sorokin In addition to larvicidal species, fungal species with ovicidal capabilities are now being researched. For example, species already inve stigated for its adulticidal effects, M. anisopliae, is being studied for its ovicidal potential in Ae. aegypti eggs. Luz et al. (2008) determined in the laboratory that the fungus was effective in reducing eclosion rates but only when the eggs were satura ted with water (Luz et al. 2008 ). 4.8 Summary


87 Researchers at Wagenigen University are diligently working to determine if M. anisopliae could be used as a successful biological control agent. To sum marize the researchers have determined that both sheets a nd clay pots impregnated with spores can provide a n adequate surface in which spores can be transmitted to mosquitoes, that attractants found in human sweat caused by microbial growth can enhance mosquito attraction to the reservoir of spores, and most imp ortantly, that mosquito vectors can acquire M. anisopliae fungal infections (Scholte a et al. 2004 a; Scholte et al. 2005, Scholte et al. 2007, Farenhorst et al. 2008). Moreover, Read and Wagenigen research groups determined that following fungal in fection the mosquito s first beco me sluggish and less likely to take a blood meal, and then died, despite the fact that the g roups used two different fungi ( The Read and Blanford group used B. bassiana and the Wagenigen group used M. anisopliae ) This is a two ph ased pathway that could reduce in cidences of disease transmission (Blandford et al. 2005; Scholte et al. 2005; Kanzok et al. 2006; Scholte et al. 2007, Farenhorst et al. 2008). Using fungal biopesticides to control mosquito populations could be advantageo us for several reasons. First, Blanford et al. (2005) and Scholte et al. (2005) have shown that mosquitoes al ready weakened by plasmodia infection are attacked by fungi more frequently than others This indicates that the mycoinsecticide can target the de sired infected organism with some regularity (if malaria control is the goal ) ( Blanford et al. 2005; Scholte et al. 2006). Chance mutations that transfer resistance are also diminished due to incubation timin g becau se by the time the mosquito is a ffected by the fung us they will have produced offspring without mutations ( Kileen et al. 2000; Blanford et al. 2005; Scholte et al. 2005; Ghett et al. 2006)


88 4.9 The Future and Barriers to Entomopathogenic Fungal Biological Control of Mosquitoes. The use of ento mopathogenic fungi to control mosquito populations requires more research before commercial use s will be possible. In particular, techniques to increase efficacy need to be discovered the control method needs to be proven to be economically sound, and eco logical concerns need to be considered. Some researchers suggest that genetic ally altered fungi strains may be more viable thereby decreasing the time to death (Thomas and Read 2007) However, in the case of malaria, the less virulent strains could stil l effect transmission. If the spore adheres to the mosquito early in its life, either as larvae or a young adult, more virulent strain s would not be needed because the fungi takes 4 11 days to kill the adult and the plasmodium needs 14 days to develop. Wh ile increased virulence would assure rapid death of the insect and potentially reduce incidence of diseases like dengue incidences of resistance could increase.


89 Bibliography: Babiker HA Schneider P, Reece SE (2008) Gametocytes: insights gained durin g a decade of molecular monitoring. Trends in Parasitology: Article in Press. Blanford S, Chan BHK, Jenkins N, Sim D, Turner RJ Read AF, Thomas MB (2005). Fungal Pathogen Reduces Potential for Malaria Transmission Science 38: 1639 1641. Bernier UR, Kline DL, Allan SA, Barnard DR (2007). Laboratory comparison of Ae des aegypti attraction to human odors and to synthetic human odor compounds and blends. J Am Mosq Control Assoc, 23(3): 288 93. Braks MAH, S cholte EJ, Takken W, Dekker T (2000). Microbia l growth enhances the attractiveness of human sweat for the malaria mosquito, Anopheles gambiae sensu stricto (Diptera: Culi cidae). Chemoecology 10: 129 134. Buchanan FC, Pillai JS. 1990. Coelomomyces psorophorae var tasmaniensis Couch + La ird (1988) (Coelomomycetaeceae: Blastocladiales), a fungal pathogen of the mosquito Aedesaustralis Mycopathologia 111: 25 32. transformation of the mosquito pathogenic fungus Culicinomyces clavisporus Biocontrol Science and Technology 12: 395 399. Charlwood JD (1997). Vectorial capacity, species diversity and population cycles of anopheline mosquitoes (Diptera: Culicidae) from indoor light trap collections in a house in southeastern Ta n zania. African Entomology 5(1): 93 101. Cooper R, Sweeney AW (1982). The comparative activity of the Australian and United States strains of Culicinomyces clavosporus bioassayed in mos quito larvae of three different genera. Journal of Invertebrate Path ology 40 383 387. Couch JN (1972). Mass Production of Coelomomyces a Fungus That Kills Mosquitoes. Proc. Nat. Acad. Sci. USA 69(8): 2043 2047. Cutwa Francis MM, Nishimura N, and O'Meara GF (2008). University of Florida Florida Medical Entomology Labo ratory. Identification Guide to Common Mosquitoes of Florida. Debenham ML, Russell RC ( 1977 ) The insect pathogenic fungus Culicinomyces in adults of the mosquitoes Anopheles amictis hilli. Journal of Australian Entomolog ical Society 16: 46. de Faria MR, Wraight SP (2007). Perspective Mycoinsecticides and Mycoacaricides: A comprehensive list with worldwide coverage and international classification of formulati on types. Biological Control 43: 237 256.


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92 Leptolegnia chapmanii Seymour (Oomycota: Peronosporomycetes). Journ al o f Invertebrate Pathology 98: 314 319. Pialoux G, Gausere BA, Jaureguiberry S, Strobel M (2007). Chikungunya, an epidemic a rbovirosis. Lancet Infect ious Dis ease 7: 319 327. Read AF (2008). The Center of Infectious Disease Dynamics. http://www.there Accessed Dec 2008. Rey J (2008). The Mosquito. University of Florida IFAS Extension. Rueda LM, Patel KJ, Axtell RC. 1990. Efficacy of encapsuled Coelomomyces giganteum (Oomycetes: Lagenidiales) against Culex quinquefasciatus and Aedes aegypti larvae in artificial containers. Journal of the American Mosquito Control Association 6: 694 699. Sahai AS and Manocha MS (1993). Chitinases of fungi and plants: their involvement in morphogenesis and host parasite inter action. Microbiology Re views 11: 317 338. Scholte EJ, Njiru BN, Smallegange RC, Takken W, and Knols BGJ (2003). Infection of malaria ( Anopheles gambiae s.s.) and filariasis ( Culex quinquefasciatus ) vectors with the entomopathogenic fungus Metarhizium anisopliae Malaria J ournal 2 (29). Scholte EJ, Knols BGJ, Samson RA, and Takken W (2004) (a). Entomopathogenic fungi or mosquito control: a review. Journal of Insect Science 4(19): 1 24 Scholte EJ, Knols BGJ, Takken W (2004) (b). Autodissemination of the entomopathogeni c fungus Metarhizium anisopliae adults of the malaria vector Anopheles gambiae s.s. Malaria Journal 3(45) : 1 6. Knols BGJ (2005). Entomopathogenic Fungus Control of Ad ult Af rican Mosquitoes. Science: 308 (10) : 1642. Scholte EJ, Knols BGJ, Takken W (2006). Infection of the malaria mosquito Anopheles gambiae with the entomopathogenic fungus Metarhizium anisopliae reduces blood feeding and fecundity. Journ al of Invertebr ate Pathology 91: 43 49. Suh CP, Axtell RC (1999). Lagenidium giganteum Zoospores: Effects of Concentration, Movement, Light, and Temperature on Infection of Mosquito Larvae. Biological Control 15: 33 38.

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93 Temua EA, Minjas JN Tunoa N, Kawadaa H, Takag i M (2007). Identification of four members of the Anopheles funestus (Diptera: Culicidae) group and their role in Plasmodium falciparum transmission in Bag amoyo coastal. Acta Tropica 102: 119 125. Teng HJ, Lu LC, Wu YL, Fang JG (2005). Evaluation of va rious control agents against mosquito larvae in rice paddies in Taiwan. J ournal of Vector Ecol ogy 30(1): 126 32. Thomas MB, Read AF (2007). Can Biopesticides Control Malaria? Nature Reviews: Microbiology 5: 377 383. Tucker DL, Beresford CH, Sigler L, Rogers K (2004). Disseminated Beauveria bassiana Infection in a Patient with Acute Lymphoblastic Leukemia Journal of Clinical Microbiology 42(11): 5412 5414. Vandergheynst J, Scher H, Guo H, Schultz D (2007). Water in oil emulsions that improve the storage and delivery of the biolarvacide Lagenidium giganteum. BioControl 52: 207 229. Vezzani D, Rubio A, Velazquez SM, S chweigmanna N, Wiegand T (2005) Detailed assessment of microhabitat suitability for Aedes aegypti (Diptera: Culicidae) in Buen os Ai res, Argentina. Acta Tropica 95: 123 131. Ward MDW Sailstad DM and Selgrade MJK (1998) Allergic Responses to the Biopesticide Metarhizium anisopliae in Balb/c Mice. Tox icological Sciences 45: 195 203. Ward MDW, Madison SL, Andrews DL Sailstad DM, Gavett SH, Selgrade MJK (2000). Comparison of respiratory responses to Metarhizium anisopliae extract using two different sensitization protocols. Toxicology 147 : 133 145. Ward MDW, Madison SL, Sailstad DM, Gavett SH, Selgrade MJK (2000). Allergen t riggered airway hyperresponsiveness and lung pathology in mice sensitized with the biopesticide Metarhizium anisopliae Toxicology 143 : 141 154. Ward MDW, Selgrade MJK (2005). Letters Benefits and Risks in Malaria Control. Science 310: 49 51 Weiser J, Matha V, Jegrov A (1991) Tolypocladium terricola sp.n., a new mosquito killing species of the genus Tolypocladium Gams (Hyphomycetes). Folia Parasitology 38: 363 369. Yasuokaa J, Thomas RL, Mangioneb W, Spielmanc A (2006). Community based rice

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94 ecos ystem management for suppressing vector anophelines in Sri Lanka Transactions of the Royal Society of Tropical Medic ine and Hygiene 100: 995 1006 Zaim M and Guillet P (2002). Alternative insecticides: an urgent need TRENDS in Parasitology 18(4) : 161 163. Zimmerman PA, Mehlotra RK, Kasehagen LJ, Kazura JW (2004). Why do we need to know more about mixed Plasmodium species infections in humans? TRENDS in Parasitology 20 (9) : 441 447.

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95 Chapter 5: Mycoinsecticides and Vector Control of Triatomines As discussed in Chapter 2, triatomines endemic to Mexico and Central and South America are the medically important vector s of Chagas Disease, an illness affecting millions. While some synthetic pesticides effectively control triatomines indoors, these pesticides degrade too rapidly to provide effective pest control outside ( Daly et al. 1998; Hargreaves 2000; Zaim and Guillet 2002 ; Luz et al. 2004) Additionally the same basic pesticides are used in triatomine control as other vector control; therefore the same problems occur as discussed in prior chapters ( Daly et al. 1998; Hargreaves 2000; Zaim and Guillet 2002 ; Luz et al. 2004). Furthermore, the risk of triatomine resistance is also a factor that compromises control efforts (Daly et al. 1998; Hargr eaves 2000; Zaim and Guillet 2002 ; Luz et al. 2004) More eff ective and safe control measures are necessary to prevent continued loss of lives and again, e ntomopathogenic fungi could aid in such measures ( Daly et al. 1998; Hargreaves 2000; Zaim and Guille t 2002 ; Luz et al. 2004) Triatoma infestans Klug is the most important triatomine vector of Chagas diseas e and the potential for this vector and its parasite T. cruzi to s pread to new areas could place many more Latin Americans at risk (Da Costa e 2003) Until fully effective treatments and cures for Chagas disease become available, control of the triatomine vectors will continue to be the best way to reduce Chagas disease infections and subsequent fatalities (Da Costa 2003). This chap ter will discuss the biology of triatomines and the possibility of using entomopathogenic fungi as triatomine biological control agents. 5.1 Biology, Ecology and Distribution of Triatomines

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96 Triatomines are found in a variety of ecological niche but those that t ypically tr ansmit Chagas Disease to humans flourish in rural areas around mud huts with thatched roofs and feed on human blood ( Mullen and Durden 2002; Cook and Zumla 2003; CDC 2008) However, they are nocturnal relatively slow moving and typic ally bite w hile the human host is sleeping; often on the face (Mullen and Durden 2002; Cook and Zumla 2003; CDC 2008). All triatomine species (Hemiptera: Reduviidae ) have the potential to transmit T. cruzi ; however, T. infestans is by far the most comm on Chagas d isease vector (Mullen and Durden 2002). Table 5.1 lists the well established human vector species. Table 5.1 : The significant triatomine vector species of Chagas d isease and their regional location (Mullen and Durden 2002) Rhodnius proli xus Southern Mexico, Guetamala, El Salvador, Honduras, Nicaragua, Costa Rica, Colombia, Venezuela Triatoma infestans Peru, Bolivia, Brazil, Paraguay, Argentina, Uruguay, and Chile Triatoma dimidiata Mexico south to Ecuador and Peru Triatoma pallidipenni s Mexico Triatoma phyllosoma Mexico Rhodnius pallescens Panama, Columbia Triatoma maculate Colombia, Venezuela, Netherlands Antilles, Guyana, Suriname Triatoma brasiliensis Northeastern Brazil Panstronglylus herreri Northern Peru Panstronglyus megai stus Brazil, Paraguay, Argentina, Uruguay Triatoma guasayana Bolivia, Paraguay, Argentina Triatoma sordida Bolivia, Brazil, Paraguay, Uruguay, Argentina These blood sucking insects have acquired many different common names including kissing bug, coneno ses, barbeiro, bicudo, chupao, vinchicca, bush chinch, chipo, pito, chinchorro, chirimacho, iquipito, and chupon reflective of the large ar ea in which these bugs reside (Mullen and Durden 2002). Interestin gly, human behavior may have inadvertently elevat ed populations of triatomines in certain instances For example,

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97 Romaa et al. ( 2003 ) hypothesizes that Panamanian deforestation between the 1940s and the 1970s led to a substantial increase in the population of the palm, Attalea butyracea (Mutis ex Lf) W ess. Boer This palm regularly serves as a habitat of R hodnius pallescens Barber a Chagas disease vector increase led to an increase in R. pallescens populations ( Romaa et al. 2003) Triatomines live in close association with differe nt reptiles, mammals, and birds residing in the location wher e these animals nest or burrow, including palm trees, hollow trees, burrows, and rock crevices (Mullen and Durden 2002; Cook and Zumla 2003). Triatomines are betw een five and 45 mm long, though the majority of them are 20 28 mm. They are typically black or dark brown and often have yellow orange or red patches on their abdomen (Mullen and Durden 2002 ; Cook and Zumla 2003 ). They have a distinct neck and two pro minent compound eyes, and o celli are also present The part of the head in front of the eyes is cylindrical to coned, providing the explanation for one the common names of the bug s conenoses. The antennae are filiform and four segmented, and the rostrum has three segments. The hemelytra or forewings are leathery on the basal portion, whereas the hind wings are membranous. The abdomen has eleven segments (Mullen and Durden 2002 ; Cook and Zumla 2003 ). Appendix I contains triatomine photographs and distrib ution maps.

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98 Figure 5 .1 Triatoma infestans, From left to right, dorsal view, side view (CDC 1976 a; CDC 1976 b) Reprinted with permission from the CDC. Triatomines exhibit hemimetabolous metamorphosis with five instars. A ll the instar s require blood to survive and grow. Females lay an average of 200 eggs and are ready to mate once they have taken at least one blood meal one to three days following the last molt (Mullen and Durden 2002 ; Cook and Zumla 2003 ). In relation to humans and animals there a re three different categories of triatomines, namely sylvatic, peridomestic, and domestic which host preference. Sylvatic triatomines feed on wild animals and l ive in their nest or burrows. Peridomestic triatomines live in close association with domestic animals. Domestic triatomines live inside human dwellings. Peridomestic and domestic triatomines are of the most concern to humans (Mullen and Durden 2002 ; Cook and Zumla 2003 ). When hosts are available, triatomines feed every four to nine days, although they can live without a blood meal for months. The bug consumes blood by using serrated mandibular stylets to cut through the epidermis to reach the blood vessel and then it uses its rostrum to suck the blood (Mullen and Durd en 2002). Upon finishing the meal, most triatomines defecate immediately on the host The feces contain t he Chagas d isease

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99 parasite, and this parasite can survive without a host for up to 30 days. Triatomine adults can consume as much as three times its body weight in blood, and nymphs can consume six to twelve times its body weight in blood (Mullen and Durden 2002 ; Cook and Zumla 2003 ). H undreds of triatomine species exist and all are capable of transmitting Chagas disease, should they feed on humans; however, the majority of triatomines feed on other organisms N onetheless, it has been shown that sylvatic triatomines can live in peridomestic or domestic habitats, thereby taking human blood meals, especially if a natural disaster were to alter the ava ilability of their normal hosts (Mullens and Durden 2002; Cook and Zumla 2003). Additionally, if the normal human hemophagous species (typically T. infestans ) were to be completely eradicated, a sylvatic triatomine could move in and fill an unoccupied ecol ogical niche. A study in Argentina has shown that in this country some sylvatic species could become significant vectors of human Chagas disease by such a mechanism, complicating control measures (Canale et al. 2000). Chagas disease control programs are faced with the additional problem of chemical insecticide resistance. Studies such as those by Zerba et al. (1999), Vassena et al. (2000), Sfara et al. (2006) have demonstrated that triatomines can acquire resistance to insecticides. In t h ese stud ies, th e researchers show that both T. infestans and R. prolixus could d evelop resistance to pyrethroid deltamethrin (Vassena et al. 2000). For this reason, it is imperative that IVM practices be implemented, new biological and physical control methods be develo ped, and specifically that multiple chemical insecticides are used in rotat ion and conserved when possible (Sfara et al. 2006) The

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100 next section will discuss how fungal biological control can also be used against T. infestans 5 .2 Entomopathogenic f ungi and triatomine control Although Chagas disease control could potentially benefit from incorporation of fungal biological control agents t his field is relatively understudied However, with the advent of triatomine chemical insecticide resistance, altern ative pest control options must be pursued with vigor. Currently, most of the research has been with B. bassiana under teams led by Luz and Fargues (Luz et al 1997 ; Luz et al 1998; Luz et al. 1999a; Luz et al. 1999b; Luz and Fargues 1999c; Luz et al. 200 0; Fargues and Luz 2000; Luz et al 2003a; Luz et al 2003b; Luz et al 2004a; Luz et al 2004b; Luz et al 2004c; Luz et al 2005) 5.2. 1 Determining the fungal pathogens of Triatomines Inventorying the common and prolific triatomine fungal pathogens is an important undertaking. Luz et al. (2004 a) of the Instituto de Patologia Tropical e Sade Pblica, in Goiana, Brazil took soil samples from the typical habitats of triatomines from a central Brazilian farm. The researchers collected and cultured 148 soil samples. Many different fungal species were located in the cultures; however these researchers did not have the resources to identify and test all of the species. The team quantitatively selected M. anisopliae and B. bassiana from the fungi identif ied in the soil samples. Thirty one isolates of M. anisopliae and fifteen isolates of B. bassiana were cultured from the soil samples and tested on T. infestans to determine pathogenicity. All isolates wer e determined to be virulent; therefore potential control agents (Luz et al. 2004 a).

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101 While researchers only studied the B.bassiana and M.anisopliae strains, they indicated that there were other fungal species on the cadavers (Luz et. al 2004a). Studies of these entomopathogenic fungi could potentially e xpand the arsenal of effective triatomine fungal biological control agents. In 2004 the Luz team also publis hed the first triatomine biological control study o n the fungus, Evlachovaea (Luz et al. 2004 b). Hopefully, future research will include more exp loratory studies like this one. Another group under Marti (2005) of the Centro Regional de Investigaciones Cient ficas y Transferencias Tecnol gicas in La Plata, Argentina sought to determine the native prevalence of B. bassiana in a n Argentinean populati on of T. infestans They determined that i n a survey of 301 adults and 274 nymphs, 3.3% of the adults and 1.5% of the fifth instar nymphs were infected with B. bassiana isolates (Marti et al. 2005). These small results are s ignificant because they indica te that this fungus does naturally infect T. infestans (at least in Argentina). If B. bassiana populations were sustained at a higher level in nature T. infestans populations could possibly be reduced Two studies by Moraes et al. (2001 and 2006) of the Instituto Oswaldo Cruz/FIOCRUZ of Rio de Janeiro RJ, Brazil on the fungi found in the gastrointestinal system of triatomines should also be briefly mentioned; although further studies on their implications have not yet been completed. T he first stud y pu blished in 2001 identified 3 93 different isolates of fungi from 699 triatomines belonging to the species T.infestans, Triatoma brasiliensis Nieva, Triatoma sordida Stal Triatom a pseudomaculata Corra e Espnola and T riatoma vitticeps Stal (Moraes et al. 2001) In their 2004 study they reported obtaining 365 fungal isolates from 515 triatomines belonging to the species Rhodnius prolixus Stal, Rhodnius neglectus Lent

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102 Dipet lanogaster maximus Uhler, and Panstrongylus megistus Burmeister, (Moraes et al. 2004) The fungi identified during both studies known to be toxic or detrimental to insects or other animals were Penicillium corylophilum Dierckx Aspergillus nigers Tieghem, Aspergillus flavus Link Aspergillus ochraceus Wilhelm Aspergillus parasiticus Fusarum sp. Penicillium citrinum Thom Penicillium implicatum Biourge, Penicillium viridicatum, and A n fumigates It is important to note that the first two species on this list were among the most abundant species found in this group (Moraes 2001; Mor aes 2006). Before any biological control implica tions can be made based on the overall findings, pathogenicity studies of these fungi on triatomine species are required. 5.2.2 Control of Triatomines with Beauveria sp. and Metarhizium Early studi es on the requirements of B. bassiana and M. anisopliae B. bassiana and M. anisopliae have received the most attention from fungal biological of insects; however due to greater cold tolerance, it has received more attention than M. anisopli ae The majority of triatomine fungal biological control research has been completed by groups lead by Luz, Fargues, and Romaa. This section will discuss the current findings pertaining to triatomine fungal biological control Romaa and Fargues (1992) we re some of the first to examine B. bassiana and its potential to control Chagas disease vectors. In their joint study, they examined the susceptibility of different stages of triatomines to B. bassiana. By spraying various concentrations of B. bassiana m ycoinsecticides directly on R. prolixus they determined tha t all stages were susceptible T he two oldest stages, fifth instar and adult, were the

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103 most susceptible while the first instar was the least susceptible since it required a mycoinsecticide concen trated 700 times more than the concentration used for the adult (Romaa and Fargues 1992 ). This group also determined that the fungal infections depended on temperature and moisture levels. The ideal temperature was determined to be 25 C and the moistur e levels should be close to saturation (Romaa and Fargues 1992). Th e Luz and Fargues team began to study B. bassiana to determine its virulence to R. prolixus and conidial germination requirements (Luz and Fargues 1997 ). They determined that B. bassiana w as virulent to R. prolixus and that in saturated environments, the conidia germinated at temperatures from 15 35 C at a rate of 95% or more However, conidial germination was delayed in the most extreme temperatures tested, namely, 15 C and 35 C (Luz and Fargues 1997). Lower relative humidity (RH) levels also delayed the process. For example, at 25 C and 95.5% RH, the conidia too k 20 hours to germinate, while at the same temperature but with a reduction to 90% RH, the conidia required 72 hours to germinat e (Luz and Fargues 1997). Luz et al. (1998 a) later examined the potential of several strains (which are listed in Appendix F ) of M. anisopliae and B. bassiana as to be used as biological control for T. infestans The investigators sought to determine if M. anisopliae germination was possible at 50% humidity. To test this p ossibility the y cultured the fungus and made it into a conidial suspension (Luz et al. 1998 a). The T. infestans were placed in this suspension for six seconds and then put in a labor atory environment with a 50% humidity level. The outcome of this procedure was a r anged from seven to eleven days with 50% survival of triatomines and a mortality rate of 45 90% depending on the mycoinsecticide

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104 concentration and strain used The most vi rulent of the strains tested were CG144 and CG491 (Luz et al. 1998 a). It is important to note however, that attaining contact with control test may vary drastical ly even within a single strain Luz et al. (1998a) also explored T. infestans control potential by B. bassiana Using the same procedures, the group determined that at 50% RH, th is fungus exhibited a 17.5 97.5% mortality rate on the T. infestans and a 50% survival time of seven to eleven days concentration They found that v irulence levels greatly di minished with less than 50% RH and that temperatures of 20 25 C (the average temperat ure range of most of the regions inhabited by T. infestans ) produced the highest virul ence rate. The effect of the strain used varied in virulence with strain CG306 being the most virulent, closely followed by strains CG14 and CG24 ( Luz et al. 1998 a) In short, this study showed that different humidity levels play an enormous role in the ability of B. bassiana to infect T. infestans The temperatures and the particular B. bassiana strain s used are also important variables but to a lesser degree (Luz et a l. 1998 a ). Recycling or natural propagation of B. bassiana within triatomine populations has not yet been shown to occur in field studies. Since the Marti et al. 2005 study reported the existence of B. bassiana in naturally occurring triatomine pop ulations ; it is thought that recycling must occur in these population s Recycling is important because fewer applications of the mycoinsecticide would be needed. Luz et al. (1998 b) set out to determin e if recycling could occur in controlled laboratory se ttings. The group analyzed the abiotic and biotic factors of B. bassiana

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105 recycling by sporulation on R hodnius prolixus They also sought to understand whether or not recent blood meals affected sporulation rates, but they did not observe signifi cant vari ations on the sporulation rate between recently fed triatomines and those that had not been recently fed. They determined that under optimal conditions (near 100% humidity and 25 o C), sporulation occurs four to five days after the death of the R. prolixus Using four different isolates of B. bassiana ( INRA 297, INRA 252, Bb INRA G, and T4 ) at 97% RH and 25 C, 5.3 x 10 6 to 1.7 x 10 8 conidia per insect were produced ( Luz and Fargues 1998 b ) When temperatures were raised to 28 C, sporulation significa ntly decreased and at 35 C, sporulation ceased. Sporulation also ceased at RH levels lower than 96.8%. The researchers concluded that high humidity requirements are the most restricting ecological necessity of B. bassiana and speculated that recycling m ost likely occur s only during the rainy season because of this humidity requirement (Luz and Fargues 1998 b). In 1999, Luz et al. (1999 a ) further examined the potential of B. bassiana (isolate CG 306) as a triatomine control agent in a combined laboratory and field study They studied the effects of a mineral emul sifier on germination rates, the effects of exposure time on infection rates, the mortality rates of T. infestans in both laboratory and the field settings, and the effects of particular applicati on techniques (Luz et al. 1999 a ). An indirect application method was used to infect T. infestans by placing filter paper treated with B. bassiana conidial suspensions of varying concentrations through vacuum filtration. These papers were then placed in g auze cups in the laboratory and in homes in the field study. For each concentration one group of T. infestans was exposed to the spore inoculated filter paper for one hour and another set was exposed

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106 con tinuously to the filter paper (Luz et al. 1999 a ). The researchers determined that to reach ing a 50% survival rate in the laboratory setting required between 15 21 days of continuous exposure to 3x10 6 conidia/cm 2 Increased conidia concentrations of 3x10 7 conidia/cm 2 did not increase infection levels. Con tinuous exposure to conidia was more effective than one hour of insect exposure to the mycoinsecticide treated paper s since t he LC 50 increased to 2.0x10 7 conidia/cm 2 w hen exposure was merely one hour (Luz et al. 1999 a ). The 2% mine ral oil based emulsifier u sed in the experiment was shown to have no effect on germination of conidia in vitro though h igher levels did significantly reduce germination rates (Luz et al. 1999 a ). Formulas with 2% emulsifier required fewer conidia than unformulated conidia suspensi ons to achieve the same results (Luz et al. 1999 a ). In the field experiment, B. bassiana filter papers sprayed with 10 7 conidia/cm 2 dosages were placed in houses and the third instar nymphs of T. infestans were released (Luz et al. 1999 a ) 25 days after i noculation, the insect population recovery was much lower than in the control house. Triatomine mortality rates in the treated ho uses were 38.1 93.8% while t he control group had a negligible mortality rate during the course of the experiment. It is impo rtant to note that t he field studies required higher concentrations of conidia for the same results as the laboratory studies (Luz et al. 1999 a ). The last important consideration of this study is that the conidi a in the formulas remained over 98% viable f or the duration of the 25 day field and laboratory study (Luz et al. 1999 a ).

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107 Another study by Luz et al. (1999 b) determined the extent of sporulation and its effect on recycling B. bassiana in T. infestans populations. They used the isolates CG14, CG24, C G306 and CG474 and incubated T. infestans cadavers at 15 C, 20 C, 25 C, and 30 C. Isolate CG474 produced a significantly higher yield of conidia than the rest of the isolates, and over all the temperatures tested all the isolates produced substantially fe wer conidia at temperatures 15 C and 30 C. Universally, the highest conidia production levels resulted when the T. infestans cadavers were incubated at 25 C and 97% humidity (or greater) (Luz et al. 1999 b). Juarez et al. (2000) of the Instituto de Invest igaciones Bioqu i micas de La Plata, Facultad de Ciencias M dicas, investigated the lethality of B. bassiana and M. anisopliae to T. infestans attempting t o determine why Mean Lethal Time (MLT) rates vary even under the same optimal conditions (25 C and RH above 97%) (Jurez et al. 2000). The strains Bb10, Bb5, and Ma6 were used and fungi were grown in alkane or glucose medium to determine if culture medium influences the infection and mortality rates of triatomines (Jurez et al. 2000). Both culture media were identical exc ept that the glucose medium contained 10 g glucose and 5 g yeast extract while the alkane media contained n octacosane ( Jurez et al. 2000) The glucose medium produced the fastest Mean Lethal Time (MLT). A full 100% mortality was achie ved in 5.8 days for Ma6, 7.7 days for Bb5, and 11.1 days in Bb10 (Jurez et al. 2000). The glucose and alkane culture media did influence the lipid constituents of the fungi. Th e fungi cultured on alkane medium mostly had triacylglycerols and the fungi gr own on glucose medium had a lipid composition

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108 dominated by sterols (Jurez et al. 2000). This study demonstrated that different fungi, fungal strains, a nd culture medium can influence triatomines MLT of (Jurez et al. 2000). In 1999 and 2000, Fargues and Luz further examined potential in controlling R. prolixus populations ( Luz and Fargues 1999 b; Fargues and Luz 2000) Similar to their 1998 study with T. infestans they reported in both of these subsequent studies that high humidity levels and specifically the initial RH were crucial to B. ability to induce death Temperature was also an important aspect; however, more moderate temperatures tested, these between 15 C and 35 C, did not significantly affect the results Only at 15 C and above 30 C showed significantly lower mortality rates (Luz and Fargues 1999 b; Fargues and Luz 2000). The 1999 b study tested R. prolixus 1 st instars at RH levels between 75% and 100% at the ideal growing temperature of B. bassiana 25 C. The initial humidity was the important factor in this process. As long as the initial humidity was above 96% or more, 100% mortality rates were observed. The RH levels tested that induced approximately 100% mortality were those above 96.5%: 96.5%, 98 .5%, and 100%. Below 95.5% initial RH, mortality rates were less than 50% ( Luz and Fargues 1999 b ) The 2000 study expanded upon the latter study by examining the effects of fluctuating RH levels and temperatures under a constant dose of 3 x10 5 conidia/ c m 2 of B. bassiana In order for mortality rate to be greater than 50%, the RH had to be more than 96.5% for at least two days and at least eight hours per day on all subsequent days. Fargues and Luz state d that the weather determined windows of opportunity must be recognized for B. bassiana to be best used (Fargues and Luz 2000). Additional Studies of Beauveria sp. Metarhizium sp.

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109 A more recent study by Luz et al. (2003 b) further developed knowledge of R. prolixus by investigating t he effects of blood meals and different development stages on susceptibility to B. bassiana infections because both of these entities result ed in triatomine molting. They examined the difference s in the number of fungal infections in starv ed and engorged triatomines and between different instars and adult T. infestans development phases (Luz et al. 2003 b). Their results indicated that both engorged and starved T. infestans acquired B. bassiana infections at the same rate; however, different stages of development did effect fungal infections. Since T. infestans nymphs were more susceptible to B.bassiana before and after molting (Luz et al. 2003 b). A separate group, Lecu o na et al. (2001) corroborated Luz and Fargues findings in a laboratory s tudy. Lecuona et al. found that B. bassiana could have lethal effect s in over 95% of the population of T. infestans (Lecu o na et al. 2001). Several stains of B. bassiana isolated from Hemiptera and Lepidoptera hosts were cultured on complete agar medium ( CAM) which included glucose agar, and yeast extract, or wheat bra n and rice husk medium (WH). The cultures were then used to treat T. infestans that were placed in chambers under varying temperatures and humidities The effects at 26 C at both 35 % and 9 0% humidity were that nearly all of the tested T. infestans samples were infected and between 96% and 98% T. infestans were killed This temperature also yielded the largest number of fungal colonies (Lecuona et al. 2001). Slight temperature variation sig nificantly affected the mortality rate. At 22 C, only 27 73% of fungi were killed and at 30 C, just 74 79% of the T. infestans died (Lecuona et al. 2001). Th ese data further demonstrate the temperature limitations of B. bassiana biological control, but a re not

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110 consistent with previously described studies in terms of humidity since significant death occurred at 35% RH. It is unclear whether this discrepancy exists. The virulence and lethal properties of B. bassiana strains varied significantly as well. The strain Bb10 was determined to be the most virulent The MLT for this strain was 4.8 days in adults and 7.1 in nymphs with 100% mortality on both CAM and WH media (Lecuona et al. 2001). The MLT of other strains ranged from 5.4 7.5 days in adults to 6. 5 7.3 days in nymphs. The group also reported that B. bassiana adversely affected eggs of T. infestans but did not provide specific evidence to support this claim (Lecuona et al. 2001). Overall, the percent mortality values of B.bassiana strains were 100 % for the nymphs grown on CAM, 97.5% for nymphs grown on WH, 75 100% for adults grown on CAM, a nd 93 95% of adults grown on WH (Lecuona et al. 2001). The LC 50 ranged from 2.10 x 10 6 (confidence interval (CI) 1.3 3.2) to 21.7 x 10 6 (CI 12 53) conidia/m l (L ecuona et al. 2001). In the same study, investigators also examined the effects of using two different pesticides Deltamethrin ( K Othrina FW 0.75% Aventis, Argentina; fi eld rate: 25 mg [AI]/m 2 ) and Beta Cypermethrin (Sipertrin FW5%, Chemotecnica, Argent ina; Field rate: 50 mg [AI]/m 2 ) in conjunction with B. bassiana (Lecuona et al. 2001). The researchers examined the combinator ial effects of 10, 50, and 100 percent concentrations of these two p yrethroids and determined that Deltamethrin did not demonstr ate inhibitory effects on B. bassiana growth whereas Beta Cypermethrin significantly affected B. bassiana growt h starting at 50% concentration and halting all growth at 100% concentration (Lecuona et al. 2001).

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111 Another group under Luz et al. (2005) examin ed the effects of how the constituents of different 10% oil mycoinsecticide formulations affect ed T. infestans and B. bassiana. Oil form ula tion s typically have three parts: surfactants, oil, and water (Luz et al. 2005). Surfactants, which are wetting age nts and oils which protect the spores from desiccation are important to the mycoinsecticide formulas. The oils tested were linseed, Linum usitatissimum L. (Linaceae) (Naturata, Germany); soybean, Glycine max L. ; groundnut, Arachis hypogaea L. (Papiliona ceae); rapeseed, Brassica rapa L. (Cruciferae); thistle, Carduus sp.; sunflower, Helianthus annuus L. (Compositae); olive, Olea europaea L. (Oleaceae); sesame, Sesamum indicum L. (Pedaliaceae); corn, Zea mays L. (Gramineae) and castor, Ricinus communis L (Euphorbiaceae) ; and Orbygnia speciosa (Palmaceae) (Luz et al. 2005) None of the o ils detrimentally affect ed germination rates. Corn oil yielded the highest germination rate which was 92.5%, 64.3% higher than the control. Two other oils soybean and thistle, elevated germination rates but only by approximately 10% (Luz et al. 2005) The oils did not adversely affect the survival rates of any of the triatomine nymphs; however, oils did affect the settling behaviors of the nymphs because the nymphs fo und the oils unattractive and avoided the mycoinsecticide Since the oil helps adhere the conidia to the triatomine oil formulas must be used; although, formulas with minimal oil concentrations were preferable (Luz et al. 2005). Several surfactants were s hown to inhibit B. bassiana growth. The surfactants tested were either non ionic or anionic in vol/vol concentrations of 0.1%, 0.5%, 1%, 2.5%, 5%, and 10% (Luz et al. 2005). The non ionic surfactants studied were MP 6400 (monoleate glycol polyethylene) MP 600 Renex 60 (nonyl phenol), Renex 95 or

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112 Span 80 Tween 20 (polyethylene sorbitan monolaureate) and Tween 80 (polyoxyethylene sorbitan monoleate) (Luz et al. 2005) The anionic surfactants reviewed were DOS 75 (dioctyl sodium sulfosuccinate), Hostapaval BVQ 9 (nonyl phenol 9 EO sodium sulphate) and Surfax 220 (sodium lauryl sulphate) (Luz et al. 2005) In higher concentrations, above 2.5% most surfactants retarded B. bassiana growth to below a 2% rate. Surfax 220 and DOS 075 even reduced germination rates to 0 2% at only 0.1% concentration (Luz et al. 2005) On the other hand, B. bassiana treated with Hostapaval maintained a significant germination rate and only at 5% concentration did the rate fall below the control to 15%. Even at 10% concentrations, the surfactant reduced the rate to 9% but did not eliminate germination Based on th ese data, surfactants can be used at low concentrat ions in mycoinsecticide formulations (Luz et al. 2005) The Luz group also examined the techniques of applying the formula tion As other researchers have reported, B.bassiana requires humidity levels near saturation in order to germinate However, this could be accomplished using o il water formulas to create high humidity microclimates such that high env ironmental RH levels are not required (Luz et al. 2005) Luz et al. 2005 showed that s praying the mycoinsecticide directly on the insect is also more effective than spraying around the habitat, as greater adherence between spore and cuticle occurs but ac knowledged that this is not a practical technique (Luz et al. 2005) In another field study, Luz et al. (2004 c) examined how spraying B. bassiana myc oinsecticide inside hen houses a ffected T sordida populations. B. bassiana isolate CG14 was used in this study and the mycoinsecticide was sprayed on the walls and the

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113 floor of the hen house. The results were assayed 25 days following the initial exposure. Despite the RH levels being lower than 97% for fourteen or more hours per day, a significant number o f triatomine deaths occurred (Luz et al. 2004c). The r esearchers determined that 7.3% of the triatomines remained in the mycoinsecticide treated hen houses compared to the 42.9% remain ing in the control hen house. These results demonstrated that r ecycling did occur, and T. sordida populations were reduced with B. bassiana The next step would be to conduct studies that integrate B. bassiana into comprehensive IVM programs. One important consideration of this study is that triatomines are easily camouflag ed and are therefore hard to find and identify which could have slightly skewed the results of this study (Luz et al. 2004 c). A later study by Lazzarini et al. (2006) tested the efficacy of M. anisopliae and B. bassiana in controlling T. infestan popula tions. This study tested 11 different strains of M. anisopliae IP 220, IP 221, IP 222, IP225, IP 226, IP 227, IP 230, IP 232, IP 233, IP 234, and IP 235, and eleven strains of B. bassiana IP 155, IP 157, IP 161, IP 165, IP 170, IP 171, IP 180, IP 224, I P 228, IP 229, and IP 231 Every strain tested produced a 100% mortality rate at RH levels greater than 98% and mortality rates of 25% or less at RH levels under 75%, confirming that humidity is a strong factor in germination of B. bassiana and M. anisopl iae To summarize, B. bassiana and M. anisopliae research to date demonstrates that neither fungus has the ability to control triatomine populations alone They could be integrated into IVM programs, especially during the rainy season and any other time whe n humidity levels are high. Research has uncovered many significant findings including that B. bassiana and M. anisopliae are best used when humidity levels are

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114 greater than 97% and temperatures are around 25 C (Roma a et al. 1992; Luz et al. 1997; Luz et al. 1998a; Luz and Fargues 1998 b; Lecuona et al. 2001). Additionally, large variations of MLT, recycling rates, and germination rates exist between different fungal species and fungal strains. Invert emulsion formulations appear to be most effective and could be used for triatomine control. The culture media also influences the efficacy of the fungus. The search for a fungus or fungal strain potentially capable of controlling triatomines alone should continue. The ideal species would flourish and re cycle at a high rate in relatively low RH While most of the triatomine fungal biological control work has centered around B. bassiana and M. anisopliae a few other fungi have been researched and will be discussed in these next few sections. Tri atomine biocontrol: B. bassiana verses B. brogniartii Gutirrez et al. (2003) explored the differences between B. bassiana and its relative, B eauveria brongniartii (Sacc.) Petch to determine if one was more lethal to triatomines than the other. They app lied 1 x 10 7 spores on canvas and infected fifth instar triatomines by allowing them to walk on the canvas. After 22 days at 25 C and near saturated humidity, 100% of the B. brongniartii infected triatomines and 77.5% of the B. bassiana infected triatomin es were dead. These results demonstrate that B. brongniartii could be even more eff ective in triatomine control than B. bassiana (Gutirrez et al. 2003). This study highlights the need for expanding knowledge of additional entomopathogenic fungi. While B. bassiana and M. anisopliae are ubiquitous fungi found world wide, many other fungi that could produce comparable or better results than

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115 these two frequently researched examples could exist The next section will discuss a few more potential fungal spe cies which have been researched to a lesser extent. 5.1.3 Additional Potential Entomopathogenic Fungal Species of Triatomines Evlachovaea sp. The Luz research group (2003) determined that Evlachovaea sp. is an entomopathogenic fungus that colonize s T. sordida a discovery that could lead to the use of Evlachovaea as a biological control agent (Luz et al. 2003). (2004 b) expan ded upon this discovery by studying the pathogenicity of existing Evlachovaea sp in 13 different wild species of triatomines They determined that overall Evlachovaea sp. inoculated triatomines experienced a 50.8% infection of Evlachovaea sp. alone, 18.7% had both Evlachovaea and other saprophytic fungal infections, 25% were infected with only unidentified saproph ytic fungi, and 5% did not acquire fungal infections (Luz et al. 2004 b). 5.1.3 .2 Aspergillus giganteus and Penicillium corylophilum Da Costa (2003) examined the lethal effects of Aspergillus giganteus Wehmer on triatomine nymphs. This fungal spe cies has been determined to be one of the most common species found on native triatomines ( Da Costa 2003 ) The group determined that this fungal species was capable of inducing an average 50% mortality rate in 1 st and 4 th instar T. infestans nymphs but was less effective at controlling Panstrongylus megistu s Burmeister (Da Costa 2003). Da Costa et al. (2003) also studied the potential of Penicillium corylophilum Dierckx another fungal species found on triatomines, to kill the nymphs of two different

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116 triatomine species. The group determined that P. corylophilum was able to kill above 50% first instar (not the fourth instar) triatomine nymphs P. megistus and T. infestans (Da Costa 2003). 5.1.3 .3 Paecilomyces lilacinus Marti et al. (2006) discovered an a dditional entomopathogenic fungus native to Argentina, called Paecilomyces lilacinus (Thom) Samson (Ascomycota: Hypocreales) (Marti et al. 2006). The researchers collected 570 T. infestans specimens from many different triatomine endemic areas of Argentina. Three of the specimens were infected with P. lilacinus This was the first scientific record of P. lilacinus triatomine infections (Marti et al. 2006). Figure 5.1 depicts P. lilacinus sporulating on T. infestans The resea rchers conducted pathogenicity tests of this fungus on T. infestans and determined that at a dose of 1 x 10 8 P. lilacinus conidia /ml a temperature of 25 C, and a nearly saturated humidity level, the MLT was 12.8 days (Marti et al. 2006). While the MLT w as two times long er than B. bassiana the fungus is still effective and could be used in mycoinsecticides (Marti et al. 2006 ) Additional research into the ecological requirements of P. lilacinus the nontarget effects of P. lilacinus and additional effi cacy studies should be pursued to determine whether P. lilacinus can be added to the arsenal of control agents (Marti et al. 2006) 5.3 Conclusions Figure 5.1 P.lilacinus sporulating on T. infestan s ( Marti et a l. 2006).

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117 The work to date on triatomine fungal biological control has centered on B. bassiana and to a lesser extent M. anisopliae both hyphomycetes fungi that invade triatomines provided that the humidity levels are high and the temperatures are around 25C; however, water in oil inver t emulsion formula tion s reduce the need for high humidity levels. The less studied fungi like Evlachovaea sp. P. lilacinus B. brogniartii A giganteus and P corylophilum discussed in this chapter should be further explored. The Moraes et al. (2000; 2006) studies on fungi within the gastrointestinal tract indicate that further research of the biological control potential of orally ingested fungi is warranted. Unknown hyphomycetes species capable of triatomine control should continue to be explored al ong with other potentially entomopathogenic fungi. Finally, additional field studies incorporating B. bassiana and M. anisopliae into existing vector control programs should also be pursued.

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118 Bibliography: Bastien J (1998). Kiss of Death: Chagas Disease in the Americas. Accessed: Feb 20 09 Canale DM, Cecere MC, Huit R, Curtler RE (2000). Peridomestic distribution of Triatoma gardiabesi and Triatoma guasayana in Northwest Argentina. Medical and Veterinary Entomology 14: 383 390. Center for Disease Control (1976 a ). 6282 Triatoma infestans. CDC Public Health Image Library. Accessed: Feb 2009. Center for Disease Control (1976 b ). 2538 Triatoma infestans. CDC Public Health I mage Library.Accessed : Feb 2009. st ed. Elsevier Science Limited. London, England. Center for Disease Control (CDC) (2008). A Z Disease Listings. Accessed Fe b 2009. Available from Da Costa GL, de Moraes, AML, Galvao C (2003). Entomopathogenic effect of Aspergillus giganteus and Penicillium corylophilum on tw o triatomine vectors of Chagas dise ase. Journal Ba sic Microbiol ogy 43(1): 3 7. Daly HV, Doyen JT, Purcell AH (1998). Introduction to Insect Biology and Diversity. Oxford Press 2nd ed. New York, NY : 124 153. Fargues J, Luz C (2000). Effects of fluctuating moisture on infection potential of B. bassia na and R. prolixus Journal of Invertebr ate Pathology 75: 202 211. Gutirrez FP, Osorio YS, Osorno JC, Soto SR (2003). Susceptibility of Rhodnius pallescens (Hemiptera: Reduviidae) of fifth instar nymph to the action of Beauveria spp. Entomotropica 1 8(3): 1 10 Hargreaves K, Koekemoer LL, Brooke BD, Hunt RH, Mtembu J, Coetzee M (2000). Anopheles funestus resistant to pyrethroid insecticides in South Africa. Medical and Veterinary entomology 14: 181 189. Juarez MP Crespo R, Fernandez GC, Lecuona R ,Cafferata LFR (2000). Characterization and Carbon Metabolism in Fungi Pathogenic to Triatoma infestans, a Chagas Disease Vector. Journal of Invertebrate Pathology 76: 198 207. Lazzarini GMJ, Roncha LFN, Luz C(2006). Impact of moisture on in vitro ge rmination of Metarhizium anisopliae and Beauveria bassiana and their activity on Triatoma infestans. Mycological research 110: 485 492.

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119 Lecuona RE, Edelstein JD, Berretta MF, La Rossa FR, and Arcas JA (2001). Evaluation of Beauveria bassiana (Hyp homycetes) Strains as potential agents for control of Triatoma infestans (Hemiptera: Reduviidae). Journal of Medical Entomology 38(2): 172 179. Luz C, Fargues J (1997). Temperature and moisture requirements for conidial germination of an isolate of Beauveria bassiana pathogenic to Rhodnius prolixus Mycopathologia 138: 117 125 Luz C, Tigano MS, Silva IG, Cordeiro CMT, Aljanabi SM (1998) (a). Selection of Beauveria bassiana and Metarhizium anisopliae isolates to control Triatoma infestans Mem rias Inst ituto do Oswaldo Cruz, Rio de Janeiro 93(6): 839 846. Luz C, Fargues J (1998). Factors Affecting Conidial Production of Beauveria bassiana from Fungus killed cadavers of Rhodnius prolixus Journal of Invertebrate Pathology 72: 97 103. Luz C, Silva I, Magalhaes BP, Cordeiro CMT, Tigano MS (1999 a). Control of Triatoma infestans (Klug) (Reduviidae: Triatominae) with Beauveria bassiana (Bals.) Vuill.: Preliminary Assays on Formulation an d Application in the Field. Annals of the Society of Entomology Brasil 28(1): 101 110. Luz C, Fargues J (1999 b). Dependence of the entomopathogenic fungus, Beauveria bassiana on high humidity for infection of Rhodnius prolixus. Mycopathologia 146: 33 41. Luz C, Silva IL, Cordeiro CMT Tigano MS (19 99 c). NOTE Sporulation of Beauveria bassiana on Cadavers of Triatoma infestans after Infection at Different Temperatures and Doses of Inoculum. Journal of Invertebrate Pathology 73: 223 225. Luz C, Rocha LFN, Humber RA (2003 a). Record of Evlachovae a sp. (Hyphomycetes) on Triatoma sordida in the State of Goias, Brazil, and Its Activity Against Triatoma infestans (Reduviidae, Triatominae) Journal Medical Entomol ogy 40(4): 451 454. Luz C, Fargues J, Roma a C (2003 b). Influence of starvation and blood meal induced moult on the susceptibility of nymphs of Rhodnius prolixus : Stal (Hemiptera: Triatominae) to Beau veria bassiana (Blas.) Vuill. infection. Journal of Applied Entomology 127: 153 156. Luz C, Luiz FN, Rocha, Nery GV (2004 a). Detection of Entomopathogenic Fungi in Peridomestic Triatomine Infested Areas in Centra l Brazil and Fungal Activity Against Triatoma infestans (Klug) (Hemiptera: Reduviidae) Neotropical Entomology 33(6): 783 791.

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120 Luz C, Rocha LFN, Garcia Silva IG (2004 b). Pathogenicity of Evlachovaea sp (Hyphomycetes), a new species isolated from Triatoma sordida disease vectors. Revista da Sociedade Brasileira de Medicina Tropical 37(2): 189 191. Luz C, Rocha LFN, Nery GV, Magalhes BP, Tigano MS (200 4 c). Activity of Oil formulated Beauveria bassiana against Triatoma sordida in Peridomestic Areas in Central Brazil. Mem Inst Oswaldo Cruz, Rio de Janeiro (2): 211 218. Luz C, Batagin I (2005). Potential of oil based formulations of Beauveria bassiana to control Triatoma infestans Mycopathologia 160: 51 62. Marti GA, Scorsetti AS, Lastra CCL (2005). Isolation of Bea u veria bassiana (Blas.) Vuill. (Deuteromycotina: Hyphomycetes) from the Chagas disease vector Triatoma ingestans (Hemiptera: Reduvii dae) in Argentina. Mycopathologia 159: 398 391. Marti GA, Lastra CCL, Pelizza SA, Garcia JJ (2006). Isol ation of Paecilomyces lilacinus (Thom) Samson (Ascomycota: Hypocreales) from the Chagas dise ase vector, Triatoma infestans Klug (Hemiptera: Reduv iidae) in an endemic area in A rgentina. Mycopathologia 162: 369 372. Moraes AML, Junquiera ACV, Costa GL, CElano V, Oliveira PC, Coura JR (2000). Fungal flora of the digestive tract of five species of triatomine vectors of Trypsanosoma cruzi Chagas 1 909. Mycopathologia 151:41 48. Moraes AML, Vieira Junqueira ACV, Celano V, da Costa GL, Coura JR (2004). Fungal flora of the digestive tract of Rhodnius prolixus,Rhodnius neglectus, Diptelanogaster maximus and Panstrongylus megistus Vectors of T rypa nosoma cruzi chagas, 1909. Brazilian Journal of Microbiology 35: 288 291 Mullen G, Durden L (2002). Medical and Veterinary Entomology. Academic Press. San Diego, CA. Romaa CA, Fargues JF (1992). Relative susceptibility of different stages of Rho dnius prolixus to Entomopathogenic hyphomycete Beauveria bassiana Mem rias Inst ituto do Oswaldo Cruz, Rio de Janiero 87(3): 363 368. Romaa CA, Brunstein A, Collin Delavaud A, Sousse O, Ortege Barria E (2003). Public policies of development in La tin America and Chagas disease. Lancet 362: 579. Sfara V, Eduardo N Zerba EN Alzogaray RA ( 2006 ). Toxicity o f pyrethroids and repellency of diethyltoluamide in two deltamethrin resistant colonies of Triatoma infestans Klug, 1834 (Hemiptera: Reduviidae ) Mem rias Inst ituto do Oswaldo Cruz 101( 1 ): 89 94.

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121 Vassen CV, Picollo MI, Zerba En (2000). Insecticide resistance in Brazilian Triatoma infestans and Venezuelan Rhodnius prolixus Medical and Veterinary Entomology 14: 51 55. Zaim M, Guillet P ( 20 02 ). Alternative insecticides: an urgent need TRENDS in Parasitology ( 18 )4: 161 163. Zerba EN (1999). Susceptibility and resistance to insecticides of Chagas disease vectors. Medicina (Buenos Aires) 1999; 59 : 41 46.

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122 Chapter 6: Biological contr ol of Two Diptera: Tsetse Flies and Sand F lies Both sand flies and tsetse flies carr y often fatal diseases in addition to being painful and annoying blood suckin g pests. Controlling these diptera ns helps manage the diseases they carry. Currently, most tsetse and sand fly control efforts rely heavily on pesticides, traps, and habitat alteration (Kaaya et al. 1994; Alexander and Mavoli 2003). Modern research on fungal biological control of tsetse flies and sand flies is relatively limited, despite a few p romising reports of success (Kaaya et al. 1994; Maniania et al. 2006). However, tsetse fly control research using entomopathogenic fungi began as early as 1930 (Fenwick 1930). Based on the triumphs of the very few studies completed, much more research is required so that control programs can reduce dependency on chemical insecticides and sand and tsetse fly borne diseases can be eradicated without the environmental and health da mage that accompanies extensive pesticide use 6.1 Tsetse Flies (Glossinidae) Biology and Ecology Tsetse flies are hematophagous insects that include a number of species in the genus Glossina which inhabit tropical and subtropical regions of Africa and transmit one of the most dangerous tropical diseases, African sleeping sicknes s. These flies prefer humid and shaded habitats including rai nforests, swamps, mangroves, vegetation around lakes, rivers, and streams, and open country areas like dry thickets, scrub, and savanna woodlands ( Mullen and Durden 2002; Cook and Zumla 2003). Glossina sp. (Diptera: Glossinidae) are typically brown, black, or tan and are 6 14 mm in length. Their proboscis consists of elongated stylet like mouthparts (labrum and hypopharny x) and their antennae are three segmented. Their eyes are large and brown or reddish (Mullen

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123 and Durden 2002; Cook and Zumla 2003) The wings are hyaline to dusky and are folded scissor like across the back. One of the discal wing cells is distinctly hatchet shaped. The base of the abdomen is approximately the same width of the head and thorax ( Mullen and Durden 2002; Cook and Zumla 2003). Figure 6.1 Glossiana morsitans (Rockefeller University) Tsetse flies are able to reproduce one to three days after eclosion The female fly needs to mate only once because sperm is tran sferred in a spermatophore and stored in g at a time and the egg develop and hatch within the abdomen The first two larval instars are subsequently and then sh e deposits the larvae in sandy soil The larva e then burrow into the ground and pupate for 30 days after which the adult s emerges (Mullen and Durden 2002; Cook and Zumla 2003 ) Adults live for an average of 20 40 days and develop young one by one in quic k succession This reproductive strategy provides the tsetse fly with an increased likelihood of reaching adulthood; however, the strategy also means that the loss of an immature fly has a large impact on the next generation ( Mullen and Durden 2002; Cook and Zumla 2003).

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124 Blood serves as the only food source for the tsetse fly. To obtain the blood meal, labellum and the labium, the fly penetrates the skin, causing hemorrhaging at the site of the bite (Mullen and Durden 2002; Cook and Zumla 2003 ) The labrum sucks the blood into the alimentary canal while the hypopharnyx pumps saliva with digestive fluids and analgesics into the wound. After one to ten minutes, the fly has consumed its blood meal, and weighs two to three times its original body weight (Mullen and Durden 2002; Cook and Zumla 2003 ) Finally, the fly slowly and clumsily flies to a nearby tre e to rest and to digest the meal. Within the first 30 minutes of digestion comprised of mostly water, allowing the tsetse fly to quickly re gain its normal mobility. T setse flies rely on blood as its sole f ood source; therefore protein is this insects' main source of energy ( Mullen and Durden 2002; Cook and Zumla 2003). 6.2 Current Tsetse Fly Control Methods Major tsetse fly control methods currently used include insecticide spraying, bush clearing, host (such as domestic dogs) relocation or destruction, use of devices that attr act tsetse flies to insecticide treated traps, and sterile insect technique (Maniania and Odulaja 1998b). Spraying insecticides on cows and other domestic animals prone to contract ing T. brucei is another control method used to control tsetse flies and the diseases they carry. For example a recent study demonstrated that spraying just the belly and legs of the cow every two weeks as opposed to the entire cow once a month reduces t he amount of pesticide used and is more efficacious (Torr et al. 2007). Minimizing chemical insecticides used in tsetse fly control is ideal and could be accomplished in part

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125 through biological control. C hemical insecticides and fungi are often compatibl e ; therefore integrating the two together while reducing the amonts of chemical inputs would create a safer control plan without necessarily reducing efficacy (Kaaya and Munyinyi 1995). 6.3 Tsetse Fly and Fungal Biological Control Most research on fungal biological control of tsetse flies has been completed by two groups, one lead by Kaaya and another by Maniania. Additionally, there are two main tsetse fly fungal control methods that have been explored One method involves applying dry fungal spores to the ground where tsetse fly larvae develop, whereas the other involves infecting adults with fungi through baited tsetse fly traps. As c ited for biological control of other vectors, the application method is also thought to be the most difficult aspect of fungal tsetse fly control (Maniania 1998a). Kaaya et al. (1989) determined that the tsetse fly, Glossina morsitans morsitans Westwood was susceptible to B. bassiana M. anisopliae and Paecilomyces fumosoroseus (W ize ) Brown and Smith (Deuteromycotina: Hyphomycetes) as well as Paecilomyces farinosus (Holmski) Brown and Smith This group reported mortality rates of 60 95% in G. m. morsitans using B. bassiana and M. anisopliae and mortality rates of 29 36% for the two Paecilomyces sp., although the pupae were determined to be naturally protected from fungal infection (Kaaya et al. 1989). The researchers point out that their experimentation was conducte d at 70% relative humidity (RH) (Kaaya et al. 1989). In con trast, the RH near lakes and bodies of water where tsetse flies live near is often close to 100% which was subsequently determined to enhance fungal infections in at least other bugs, such as the triatomines

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126 discussed in Chapter five (Luz and Fargues 1998; Luz et al. 1999). Tsetse fly specific st udies at lower RH levels may be needed if the fungi are to be used in areas where the humidity is substantially less than saturation. A subsequent study by Kaaya et al. (1990) explored the potential of horizontal transmission of fungi in adult tsetse fly populations. G. m. morsitans were exposed to wet conidia of B. bassiana and within two weeks of exposure, 90 100% of the tsetse flies died. The group determined that female to male transmissio n occur red Specifically, i n their laboratory setting, the re searchers determined that B. bassiana caused a 62% mortality in males exposed to fungi through the females and a 48% mortality rate occurred in a similar manner when M. anisopliae was used. This study also confirmed that pupae were not infected by fungi d ue to scleritization (Kaaya et al. 1990). More recently, Kaaya and Munyini (1995) examined the effects of mixing fungal spores in with the sands that comprise tsetse fly larvae habitat. The researchers mixed a gram or a half gram of B. bassiana or M. ani sopliae in the sands of the tsetse fly larvae habitat Their experiment achieved high mortality rates, although lower spore concentrations resulted in lower mortalities (Kaaya and Munyini 1995). The highest mortality rate achieved in this larval control experiment was 97% for B. bassiana and 80% for M. anisopliae Confirming findings in previous studies, pupae mortalities were low and equivalent to the mortalities of the control group, 2 8%. Most mortalities occur red between days two and ten of exposure To confirm infection, d ead tsetse flies were incubated in order to developed fungal growth. The authors state that because larviposition sites are relatively small areas, this technique of mixing sand and spores could be feasible (Kaaya and Munyini 199 5).

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127 Maniania (1998a) researched infecting chambers for Glossina pallidipes Austen, G. longipennis Corti, and G. fuscipes fuscipes Newstead. This idea is a modification of ead of killing the tsetse flies with chemical insecticides, the flies are infected with fungi and released back into the wild (Mani a nia 1998a) In the experiment, the goal was to attract the tsetse flies to traps baited with cow urine, have them enter the infection chamber, and become contaminate d with fungus. The experiment was conducted at 20 26 C with RH of between 50 and 60%. Additionally the tsetse flies were sustained on rabbits. It was thought that, u ltimately, these infected flies could be releas ed into the wild and hopefully transmit the fungi to other tsetse flies when mating, as was demonstrated in a later study (Mani a nia 2006 ). Mortality rates for G. pallidipes G. fuscipes fuscipes and G. lonigpennis were around 0 80%. As discussed in Cha pter Five, contact between spore and in sect cuticle is quite difficult; therefore a dramatic range of results can occur between trials within a given fly species as was the case for this study The dimensions of the infection chamber or trap also signifi cantly affected the outcome. Maniania (1998a) examined eight different chambers that varied in height, width, length, and amount of conidia. Three of the chambers were completely ineffective, producing mortality rates of 0%. The chambers with the largest dimensions and containing the largest amount of conidia produced the greatest mortality rates (Maniania 1998a). In another study, Maniania and Odulaja (1998b), investigated the effect of sex and age on the susceptibility of Glossina m orsitans morsitans and Glossina morsitans centralus to M. anisopliae fungal infections They determined that females are more

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128 susceptible than males (Maniania and Odulaja 1998b). On average, male and female flies 40 days old were killed in seven or eight days. Twenty day old G. m centraluses reached a 100% mortality rate by day seven whereas G. m. morsitanses took ten days to reach 100% mortality rate (Maniania and Odulaja 1998b ). The researchers suggest that M. anisopliae could be used to control residual populations of t setse flies following initial suppression by other means (Maniania and Odulaja 1998b). Another recent study of tsetse fly control with entomopathogenic fungi was completed by Maniania et al. (2006). The researchers conducted a field study that applied Man islands in Lake Victoria were used to compare the differences in effectiveness between fungi was to determine whether autodissemination (horizontal transfer) occurs in tsetse populations using the ICIPE30 isolate of M. anisopliae and if fungi could control tsetse fly populations more effectively than treated and baited traps (Maniania et al. 200 6). Their res ults showed that if given sufficient time M. anisopliae traps reduce d Initially the fungus method reduced populations by 95.8% (Maniania et al. 2006). However, five months after all the traps were removed from the islands, the fungus treated island had fewer tsetse flies. Using survelliance traps, method had an average of 175 flies per trap per day whereas the fungus treated island only had an average of 29 flies per trap per day (Maniania et al. 2006).

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129 However, t he researchers note that important factors could affect results including density of traps, type of vegetation present, and damage and theft to traps. In this experiment the trap density was one trap for every 300 400 meters, the vegetation was an theft levels were 8% per month (Maniania et al. 2006) 6.4 Sand Fly Biology and Ecology Sand flies, the vector of Leishmaniasis are found in the order Diptera and the subfamily Phlebot o minae. There are 800 different species of sa nd flies (Cook a nd Zumla 2003), of which 70 are vectors of Leishmaniasis (Alexander and Mavoli 2003). They begin their life cycle as an egg, have four larval inst ars, and ultimately become winged adults. During the day, the flies rest in dark areas. They feed at dusk and some are active during the night. Sand flies feed mostly on plants, although females need blood for oviposition (Cook and Zumla 2003) These blood meals are the events that lead to the transfer of diseases Although a substantial amount of information is know n about the biology of sand flies, a significant amount of information remains unknown compared to what is known about the previously discussed insect disease. This lack of information combined with the behavior of sand flies severely hinder s control effo rts (Cook and Zumla 2003). Female sand flies feed on reptiles, amphibian s birds, and mammals, and typically each species has a preference for a particular reservoir. Female lays 50 100 eggs and the larvae take 50 100 days to develop. Interestingly, the e xact breeding location is not known but r esearchers do know that the breeding areas are humid terrestrial areas (Cook and Zumla 2003).

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130 Sand fly eggs are 400 m long, elongate, and dark brown (Mullen and Durden 2002; Cook and Zumla 2003) The larvae are 5 mm long, elongate, white, and le gless. Additionally, the larvae have short antennae, eyespots, dark setae, and heavy, toothed mandibles, and the abdom en has nine segments. Pupae erectly attach to substrate and have clavate bodies (Mullen and Durden 2002; Cook and Zumla 2003). Adult sand flies are less than 5 mm long, are haematophagous, have densely hairy, gray ish, brownish, or yellowish bodies, and have small hypog nathous head s Sand flies lack occeli but do have large compound eyes (Mullen and Durden 2002 ; Alexander and Mavoli 2003 ). The long, slender antennae have 12 16 segments. The thorax is humped and wings are large, broadly ovate or elliptical and pointed, densely hairy and do not contain cross veins. The abdomen has six to eight segments and the mouthparts include a short proboscis, long, recurved, five segmented palps with scattered setae, and distally toothed mandibles and maxillae (Mullen and Durden 2002). Adults live in dark humid places like forest litter, tree trunks, tree hollows, plan t leaves caves, burrows, nests, and livestock pens. The adults can fly as far as 500 meters to 2.5 kilometers (Mullen and Durden 2002). Once females have taken a blood meal and mated with a male, they can lay their eggs in four different cycles of 30 60 e ggs each (Mullen and Durden 2002) These eggs hatch into larvae four to 20 days later. The larvae feed on decaying matter and fungi for 30 60 days and have four instars (Mullen and Durden 2002 ) L arvae become pupae that pupate for seven to eight days bef ore the adult emerges (Mullen and Durden 2002).

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131 Figure 6.2 Phlebotomus papatasi feeding on a human ( Gathany 2006 ) 6.5 Current Sand Fly Control Methods Sand fly control remains a difficult problem because the usual techniques of applying control agents are ineffective. For instance, a pplying larvicides is difficult because the larvae are terrestrial and tricky to locate ( Alexander and Mavoli 2003) Since the breeding grounds are unknown, insecticides cannot be sprayed where the insects breed. Addition ally, the flies feed at dusk and are typically exophagous, therefore netting and indoor insecticides are ineffective for reducing sand fly populations and sand fly bites. Using protective clothing and repellants are the few methods available to avoid sand fly bites. Applying insecticides to clothin g is also occasionally used to prevent bites although this entails direct contact with insecticides ( Alexander and Mavoli 2003; Cook and Zumla 2003)

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132 Control of sand flies is important b ecause as discussed in Chapter T wo, these insects transmit one of the most dangerous groups of tropical diseases, leishmaniasis, along with bartonelloses, and arboviruses ( Alexander and Mavoli 2003; Cook and Zumla 2003) Chemical insecticides are used for control when possib le. However, resistance has already been shown in three sand flies P hlebotomus papatasi (Scopoli) Phlebotomus argentipe s Annandale and Brunetti and S ergentomyia shorttii Adler and Thedor A erial spraying is the most effective method to control the flie s, covering large regions with insecticides ( Alexander and Mavoli 2003) Tactics like barrier spraying have been used with limited success ( Alexander and Mavoli 2003; Cook and Zumla 2003) This technique creates a pe sticide barrier between sand flies an d human habitats. The edge of a forest is commonly sprayed to keep the sand flies away fr om villages. Placing pesticide impregnated dog collars on village dogs is another control technique that is regularly used ( Alexander and Mavoli 2003) Destroying san d fly resting sites can also assist in control ( Alexander and Mavoli 2003) Orally fed pesticides like avermectins have been given to livestock to control sand fly populations (Mascari et al. 2008 ; Alexander and Mavoli 2003) Planting plants such as Sol anum jasminoides Paxton, Ricinus communis L, or Bougainvillea glabra Choisy that contain sugars toxic to sand fl ies can also act as a barrier (Schlein et al. 2001). Pheromone infused chemical insect growth inhibitors are another control method and Bti and B. sphaericus are bacterial control agents sometimes used ( Alexander and Mavoli 2003) Lastly B. bassiana has been explored as a fungal biological control agent as will be discussed in subsequent sections ( Alexander and Mavoli 2003)

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133 6.6 Fungal Biologi cal Control of Sand Flies One of the first of only a few studies of sand fly biological control was completed by Warburg et al. in 1991. He tested the lethality of B. bassiana on the sand fly species Phlebotomus papatasi Scopoli and Lutzomyia longipalpis Lutz and Neiva He attempted to infect the flies orally, but this technique did not produce significant results. He then infected the flies by placing filter papers inoculated with B. bassiana in the same area as the flies. This technique produced 100% mortality in the flies within five days. Warburg also noted that the flies had other fungal species on them. The Colombian native, L. longipalpis had an Entomophthorales species on it, and some of the lab bred P. papatasi harbored A. flavus B. bassiana and S. marcescens (Warburg et al. 2001) Reithinger et al. (1997) also explored the effects of B. bassiana in sand f ly populations. This study arose from an already implemented and successful biological control method for the coffee berry borer, Hypoth enemus hampei (Coleoptera: S colytidae) (Haraprasad et al. 2001). Since coffee berry borer populations are in areas also occupied by the sand fly, P. papatasi Reithinger et al. (1997) wanted to determine if sand fly populations were also reduced by applica tions of this mycoinsecticide. They discovered that B. bassiana reduce d the survival time of P. papatasi by 50 75% (Reithinger et al. 1997). 6.7 Summary and the Future of Tsetse Fly and Sandfly Control In comparison to mosquito and triatomines, substanti ally less research has been completed on fungal biological control of their populations. The studies discussed above demonstrate d that tsetse flies and sand flies can be killed by fungi, especially B. bassiana and M. anisopliae, in such a way that the fung i could significantly reduce tsetse fly

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134 populations. Additional field studi es that manipulate the means of fungal trapping as discussed above should be completed and if this method is deemed cost effective and efficacious, incorporation into an IVM progr am would be useful. The larvicidal study by Kaaya and Munyini (1995) seems to indicate that this method is less likely to succeed because treating the tsetse fly habitat with the fungal spores appears to be labor intensive and difficult. Even if as the r esearchers assert the area to be treated is small the task is large Future studies should be focused on adult tsetse fly trapping rather than treatment of sand with larvacidal fungi Research on the fungal biological control of sand flies will be relati vely futile until a method of targeting the flies is established. Widespread application of mycoinsecticides would be too cost ly and many non target species could suffer. Sand fly control efforts must be centered around determining how to target these sp ecies in a more specific manner

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135 Bibliography: Alexander B, Mavoli M (2003). Control of Phlebotomine Sand flies. Med. & Vet. Ent. 17: 1 18. st ed. Elsevier Science Limited. London, England. F enwick, B (1930). Commentary on tsetse research. The British Journal of Nursing 78:263. Gathany J (2003). 10274 P. papatasi. Center for Disease Control. Haraprasad N Niranjana S R Prakash H S Shetty H S Wahab S ( 2001 ). Beauveria bassiana A Potentia l Mycopesticide for the Efficient Control of Coffee Berry Borer, Hypothenemus hampei (Ferrari) in India Biocontrol Scie nce and Technology 11(2): 251 260. Kaaya GP. Glossina marsitans morsitans : Mortalities caused in adults by experimental infection with entomopathogenic fungi. Acta Tropica 46: 107 114. Luz C, Fargues J (1999 b). Dependence of the entomopathogenic fungus, Beauveria bassiana on high humidity for infection of Rhodnius prolixus. Mycopathologia 146: 33 41. Luz C, Fargues J (1998). Factors Affecting Conidial Production of Beauveria bassiana from Fungus killed cadavers of Rhodnius prolixus Journ al of Invertebrate Pathology 72: 97 103. Maniania NK (1998 a ) A device for infecting adult tsetse f lies, Glossina spp., with an entomopathogenic fungus in the f ield. Biological Control 11: 248 254 Maniania NK, Odulaja A (1998b). Effect of species, age, and sex of tsetse on response to infection by Metarhizium anisopliae Bio logical Control 43: 311 323 Maniania NG, Ekesi S, Odulaja A, Okech MA, Nadel DJ (2006). Prospects of a fungus contamination device for control of tsetse fly Glossina fuscipes fuscipes Biolo gical Science and Technology 26(2): 129 139 Mascari TM, Mitchell MA, Rowton ED, Foil FD (2008). Ivermectin as a Rodent Feed Through Insecticide for Control of Immature Sand Flies (Diptera: Psychodidae). Journal of the American Mos quito Control Association 24(2):323 326. Mullen and Durden (2003). Medical and Veterinary Entomology 1 st ed. Academic Press. St. Louis, MO.

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136 Reithinger R, Davies CR, Cadena H, Alexander BA (1997). Evaluation of the Fungus Beauveria bassiana as a Poten tial Biological Control Agent against Phlebotomine Sand Flies in Colombian Coffee Plantations Journal of Invertebrate Pathology 70(2): 131 135 Schlein Y, Jacobson RL, and Muller GC (20 01). Sand fly feeding on noxious plants: a potential method for t he control of leishmaniasis. American Journal of Tropical Medicine and Hygiene 65(4): 300 303 Strickland TG et al. (2000). Diseases 8 th ed. WB Saunders Company. Philadelphia, PN. Torr SJ, Maudlin I, Vale GA (2007). Less is more: restricted application of insecticide to cattle to improve the cost and efficacy of tsetse control Medical and Veterinary Entomology 21: 53 64 Warburg A (1991). Entomopathogens of phlebotomine sand flies: Laboratory experiments and natural infections. Journal of Invertebrate Pathology 58: 189 202

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137 Chapter 7 : Perspectives and Ideas for the Future of Mycoinsecticide Use in Tropical Disease Vector Control Programs While entomopathogenic fungi are not the panacea for mosquito control or an agent that will end all insect borne tropical diseases, incorporating mycoinsecticides into mult ifaceted IPM insect control programs could have a dramatic effect. Research like the previously discussed study by Scholte et al. (2005) indicating that 50% fungal infection rate of mosquitoes could reduce the EIR by 96% suggests that these biological cont rol agents have potential and should be examined in depth. Many questions remain unanswered but future investigations may clarify exactly how entomopathogenic fungi can be integrated into control programs. 7 .1 Research of Additional I ndividual Entomopatho genic Fungal Species As mentioned before, only a handful of the known entomopathogenic fungi of vectors have been researched as potential biological control agents (Lacey et al. 2001). Further study of a variety of fungal species could illuminate more eff ective species. One specific aspect in need of further understanding is how and if the fungi of question recycle within the insect population. Fungi that spread their spores to other target insects following colonization of hosts are more effective than others because of continued persistence within the particular vector population. This is an important concept for mycoinsecticide research because fungi with this characteristic require fewer applications of the mycoinsecticide, resulting in cost savings and possible increases in efficacy.

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138 New fungi could be investigated by examining the endemic entomopathogenic fungi of the targeted insect. Fungi related to the known successful fungi, like M. anisopliae and B. bassiana could also be researched. 7 .2 Mycoi nsecticide Application, Formulation Innovation, and Commercialization Additionally, the available methods and techniques of formulating and applying mycoinsecticides must expand. Commercializing is also an important aspect of using entomopathogenic fungi for vector control. Growing and harvesting the fungi, devising an acceptable formulas and application process, and registering the fungal agent with the proper governmental agency are essential parts of developing viable mycoinsecticides with commercial po tential. Typically, entomopathogenic fungi are grown on a liquid or solid culture substrate. When harvested, they are purified to 90% (de Faria et al. 2007). The fungi are sold as either conidia and hyphae alone, hyphae, zoospores or spores (de Faria et al. 2007). A variety of formulations are available. The types of formulas available include fungus colonized (FC), wettable powder (WP), granules (GR), ready to use baits (RB), water dispersible granules (WG), contact powders (CPs), suspension concentrate s (SC), oil miscible flowable concentrates (OF), and oil dispersions (OD) (de Faria et al. 2007). FC is simply a solid substrate on which fungal spores have been grown. WPs are sold as a powder form of the fungi to be used in a water suspension (de Faria e t al. 2007). GRs are sold in conjunction with an active ingredient that adheres the fungal reproductive unit to the target insect (de Faria et al. 2007). RBs are traditionally formulas that encourage the pests to eat the control agent, however because fu ngi require

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139 simply contact and adherence of the spore or conidia to the insect cuticle, baits are often used as solely attractants for the insect to the fungi (de Faria et al. 2007). WGs are similar to GRs except the agent is suspended in water at the time of activation (de Faria et al. 2007). CPs are currently not commercially used with fungal agents, but in concept, the powder would be dusted over an area. CPs will probably never be used because of their high costs and the different biological needs of e ntomopathogenic fungi, such (de Faria et al. 2007). SCs are sold as spores or conidia suspended in water and diluted before use (de Faria et al. 2007). In OFs, the conidia or spores float in oil. In ODs, the fungus is infused in a mixture of oil and water (de Faria et al. 2007). To date, twelve different species of fungi have been used commercially to control pest populations in general (Lacey et al. 2001; Scholte et al. 2004; de Faria et al. 2007). Some of these could be used for biological control of ve ctors given current research, especially B. bassiana and M. anisopliae There are over 171 different mycoinsecticide formulas used and commercially produced by 80 different companies worldwide, though only a few of these were designed specifically for vect or control (de Faria et al. 2007). Research into practical aspects of mycoinsecticide application is the newest area of mycoinsecticide research; however, this area is essential to the success of ble studies were reviewed for this thesis. Below is a discussion of common problems associated with using mycoinsecticides. To begin, the ideal commercial mycoinsecticides used for insect vector control would have to meet many of the following criteria. T he mycoinsecticide should have a long shelf life and remain viable following the a pplication for at least a few months,

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140 requiring few yearly applications. DDT remains as an active pesticide for approximately six months ( Kuile et al. 2003; Pennetier et al. 2008; Gabianelle et al. 2009 ). Preferably, the desired mycoinsecticide would have a similar time period of effectiveness. Species of fungi that naturally cycle in the targeted insect population could potentially produce this life extending effect. The mycoinsecticide would need to be easily integrated into the lives of people of developing countries and be relatively cheap. Those most affected by tropical diseases live in poor nations, and it seems unlikely that they would have the time or resources to buy and apply expensive and complicated insecticides. Improved technologies are needed to have formulations that preserve the viability of spores for much longer are needed. Currently, many formulas require refrigeration and expire within a few weeks of creation. Furthermore, after application, the present commercially available mycoinsecticides are often efficacious for less than one or two months (Lacey et al. 2001; Vandergheynst et al. 2006). The water in oil invert emulsion formulation of M. anisopl iae devised by Vandergheynst et al. (2006) discussed in Chapter Three is a decent example of the types of innovations needed. This new formula successfully extended the shelf life of the mycoinsecticide from three weeks to twelve weeks (Vandergheynst et a l. 2006). This issue must be resolved before mycoinsecticides can effectively be used to combat tropical disease carrying insects. Formulas that confer greater adherence of conidia to insect cuticles are needed and could be found through research of other oils and formula additives. The specific methods used to apply the mycoinsecticide are another important facet of research. As discussed in prior chapters, application techniques undertaken so far include applying the formula to standing water, clay wate r storage pots, and to bed

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141 netting and screens hung inside homes. Methods of application must consider the types of insects and life stages. For example, L. giganteum requires too much water to be an effective biological pathogen of non aquatic life stag es. (Golkar et al. 1993; Scholte et al. 2004; Blanford et al. 2005; Scholte et al. 2005; Scholte et al. 2007. Research of different application techniques should continue, such as applying mycoinsecticides to humans. Since the vectors seek contact with humans unaided, they could potentially transfer the fungi to the vector with relative ease. This idea has probably been avoided to date because of potential complications and liability concerns. While human infections are unlikely, immunocompromised indiv iduals have been shown to acquire opportunistic fungal infections from entomopathogenic fungi. Humans also move around and spores would readily come off the human. In the case of mosquitoes, this system would only target female mosquitoes diminishing eff orts to reduce vector populations. Nevertheless, application to humans might help produce positive results. Clearly, would lessen. Studies of the potential haza rds of applying the particular fungus would definitely be required before attempting a human application study, to rule out the possibility opportunistic invasion. Additionally, the destruxin levels would need to be examined for human toxicity levels. 7 .3 Continued Research of Biochemistry and Biology of Entomopathogenic Fungi Continued study of lethal fungal metabolites and biochemistry in general would further benefit the field. Although, the process in which fungi invade insects is both

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142 physical and ch emical, isolation of the lethal chemical (such as destruxins) involved could lead to new and effective chemical insecticides. Additional research of the interrelations between fungi, humans, and the vectors of tropical disease are also parts of mycoinsect icide research that should continue. Generally speaking, mycoinsecticides are understudied and many gaps exist within the research. 7 .4 The Economics and Education of Mycoinsecticide Use Economics must be considered when evaluating methodology, commercial ization, and practical use of mycoinsecticides as vector control agents. However, very few economic analyses of biological control programs of have been conducted (Cullen 2008). Presently economic studies of vector control through entomopathogenic fungi cannot be fully conducted, in part because basic research is still underway. As more entomopathogenic fungal biological vector control research surfaces; however, economics must be considered. One of the few studies of fungal agricultural biological contr ol economics (Cullen 2008) proposed that some basic principles be considered when attempting to enhance cost effectiveness of biological control programs. These researchers have also developed mathematical tools and models to evaluate and measure the utili ty of biological control. This study suggests that enhanced yield through diminution of pest damage, savings from not using insecticides, and improved market access are entities to be considered when accessing utility (Cullen 2008). Successful vector contr ol will result in reduced medical care needs, prevented deaths,diminished pesticide use, and perhaps even improved market access through an increase in tourism that could accompany decreased prevalence of tropical disease (Cullen 2008). However these benef its may be offset by additional

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143 costs such as equipment needed i.e. bed nets, spray equipment, spore production facilities, as well as labor forces (Cullen 2008). Further cost benefit analysis of mycoinsecticide is crucial. If biocontrol treatments are ec onomically viable and effective, if the procedure is poorly perceived by the public and governments, the biocontrol agent will not be used. Fungi are living entities and community members will observe the effects of the mycoinsectide on the insects. As Cullen (2008 ) points out, this could incite fear or other emotional responses from individuals. Community education of fungi treatments must be supplied and networks and institutions must provide support for the mycoinsecticides (Cullen 2008). 7 .5 Concern s of Entomopathogenic Fungi use as BC agent Finally, ecological and health concerns must be fully considered when manipulating natural ecosystems. Host specificity and the effects of inundating a region with a large quantity of fungi must be considered bef ore implementing biological control methods because the ecological balance could be disturbed. Some studies have shown evidence counter to the accepted idea that M. anisopliae is not toxic to organisms other than insects (Genthner et al. 1998). For example a study under Genthner has shown that the secondary metabolites of M. anisopliae are toxic and/or mutanogenic to some species of developing fish, mysids, shrimp, and amphibians (Genthner et al. 1998). Resistance is still possibile with biological contro l methods, although it is unlikely because of the multi faceted complexities involved in fungal infections (Thomas and Read 2007; Scholte et al. 2005). Some scientists are wary because the Wageningen mortality in these studies

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144 (Scholte et. al 2005). These fungi could render insects resistant to them because mortality (and the selection pressure) is high and mutants could quickly dominate the gene pool if resistant species were to develop (Hutchinson a nd Cunningham 2005; Read and Thomas 2007). There are fewer health concerns with biological control agents compared to chemical insecticides; however, biological control is not necessarily without risks. B. bassiana and M. anisopliae are considered to be safe agents; however, some researchers have suggested that the two species can invoke allergic responses. For example, a team under Ward demonstrated that in a mouse model, M. anisopliae can trigger such a response (Ward et al. 1998; Ward et al. 2000 a; W ard et al. 2000 b). Additionally, cases of opportunistic B. bassiana infections in immunocompromised patients do exist, although they are rare (Tucker et al. 2004). L. giganteum has received similar scrutiny by some investigators. A recent clinical stud y revealed mycosis in six dogs that was caused by another species of Lagenidium (Grooters et al. 2003). The potential for any of the entomopathogenic fungi to cause mycosis must be considered when the intent is to place the fungus near humans. Because HI V/AIDS has rendered large numbers of people immunocompromised in many tropically located nations, it is imperative that studies be conducted on the potential for the fungus to cause opportunistic mycosis. While research of vector biological control is und erway, application of insecticides must be used cautiously but judiciously in order to reduce further developments of resistance. Targeting use to times of outbreaks, using the insecticides

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145 safely and effectively, and employing multiple agencies in the dec ision process would be helpful for vector control (Zaim and Guillet 2006 ; Thomas and Read ). 7 .6 The bottom line Based on current research, mycoinsecticides designed for tropical disease carrying insects would be best used in conjunction with other insect control methods. Success rates greater than 50% mortalities have been attained using certain fungi with every insect explored in this thesis; however, a multifaceted control plan would be more efficacious. Studies exploring the potential of fungi use in I PM programs could produce meaningful results diminishing both the reliance on increasingly ineffective chemical insecticides and the number of tropical disease cases. Future studies devising comprehensive IPM programs of tropical disease carrying insects could be extremely beneficial. Ideally, such programs would employ multiple biological control agents, use rotating chemical insecticide techniques, physically remove insect vectors and prevent vector growth. Even different fungi could be used together, since they often act in different ways and affect different life stages. Bed nets, repellants, and protective clothing are useful ways to prevent disease. Altering the habitat of vectors is another helpful control method by pouring out standing water in potential mosquito habitats. Additionally, available vaccinations should be better distributed and vaccine research should continue. Treatments should also be quickly dispersed and research of treatments and drugs to treat these diseases needs to continu e. Ultimately, research must continue on all levels, and insect borne tropical disease control programs must combine

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146 available methods described above in a manner that is current, feasible, health conscious, environmentally sound, and cost effective. 7 .7 C onclusions Mycoinsecticides cannot be used alone in insect vector control programs. However, if incorporated into IVM programs that employ several different control tactics, entomopathogenic fungi could bring forth qualities lacked by other control agents thereby aiding in the reduction of insect bites and the caseload of tropical diseases. Fungi like B. bassiana and M. anisopliae have demonstrated significant efficacy, yet to date, the results are simply not sufficient to replace other forms of control. In the future, discoveries might be made rendering mycoinsecticides so much more effective that they are able to be used as the main vector control agent; however it is much more likely that the fungi will be used in successful combination with other vec tor control techniques and agents.

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147 Bibliography: de Faria MR, Wraight SP (2007). Perspective Mycoinsecticides and Mycoacaricides: A comprehensive list with worldwide coverage and international classification of formulati on types. Biological Control 43 : 237 256. Genthner FJ, Chancy CA, Couch JA, Foss SS, Middaugh DP, George SE, Warren MA, Bantle JA (1998). Toxicity and Pathogeni city Testing of the Insect Pest Control Fungus Metarhizium anisopliae. Arc hi ves Environmental Contamination and Toxicology 35 : 317 324. Grooters AM, Hodgin EC, Bauer RW, Detrisac CJ, Znajda NR, Thomas RC (2003). Clinicopathologic findings associated with Lagenidium sp. infection in 6 dogs: initial description of an emerging oom ycosis. Journal of Veternary Internal Medicine 17(5): 607 8. Hutchinson OC, and Cunningham AA (2005). Letters: Benefits and Risks in Malaria Control. Science 310. Lacey LA, Frutos R, Kaya HK, Vails P (2001). Insect Pathogens Do they have a Future. Biological Control 21: 230 248. Rockefeller Uni versity (2009). Glossina morsitans Accessed Feb 2009 Scholte EJ, Knols BGJ, Samson RA, and Takken W (2004) (a). Entomopathogenic fungi or mosquito control: a re view. Journal of Insect Science 4(19 ). Knols BGJ (2005). Entomopathogenic Fungus Control of Adult African Mosquitoes. Science 308 (10) : 1642. Thomas MB, Read AF (2007). Can Biopesticides Control Malaria? Nat ure Reviews: Microbiology (5), 377 383. Tucker DL, Beresford CH, Sigler L, Rogers K (2004). Disseminated Beauveria bassiana Infection in a Patient with Acute Lymphoblastic Leukemia Journal of Clinical Microbiology 42(11): 5412 5414. Vandergheynst J, Scher H, Guo H, Schultz D (2007). Water in oil emulsions that improve the storage and delivery of the biolarvacide Lagenidium giganteum BioControl 52: 207 229. Ward MDW Sailstad DM and Selgrade MJK (1998) Allergic Responses to the Biopesticide M etarhizium anisopliae in Balb/c Mice. Tox icological Sciences 45: 195 203.

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148 Ward MDW, Madison SL, Andrews DL Sailstad DM, Gavett SH, Selgrade MJK (2000). Comparison of respiratory responses to Metarhizium anisopliae extract using two different sensitizat ion protocols. Toxicology 147 : 133 145. Ward MDW, Madison SL, Sailstad DM, Gavett SH, Selgrade MJK (2000). Allergen triggered airway hyperresponsiveness and lung pathology in mice sensitized with the biopesticide Metarhizium anisopliae Toxicology 14 3 : 141 154. Zaim M and Guillet P (2002). Alternative insecticides: an urgent need TRENDS in Parasitology 18(4) : 5 10

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149 Appendix A: Glossary Adenopathia: Glandular tissue enlargement Aneurysm: Bulging blood filled vessel due to weakened state Appress oria: Tip of hyphal branch that parasitic fungi use to attach to host Arrhythmias: A change in the heartbeat Arthralgia : Pain in the joints Arthritis: Inflammation in the joints Asthenia: Loss of strength Biocontrol : The use of natural enemies suc h as predators, parasitoids, or pathogens to control pest insects, weeds or diseases. The normal ambition is that the introduced organ ism will be self sustaining but can also incldue inundative approaches whish need not be self sustaining. Biopesticides : A pesticide that is biological in orgin (i.e. Viruses, fungi, bacteria). Repeated applications are necessary. Blastospores : Fungal spore made by budding B radycardia: Slow action in the heart Cardiomyopathy: Structural or functional heart diseases disting uished by hypertrophy and obstructive damage. Chancre: Sore or ulcer at site of entry of a pathogen Conidia : An asexual fungal spore produced on the conidiaphore Conjunctivitis: conjunctiva inflammation Dysphagia: Difficulty swallowing Dyspnoea: Diff iculty breathing

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150 Edema: Improper filtration of fluids resulting in accumulation of serous fluids in connective tissue. Encephalitis: Inflammation or swelling of the brain Endomyocardial fibrosis: Fibrous degeneration of the heart Entomological Inoculat ion Rates (EIR): A measure of the frequency in which a human is bitten by an infectious mosquito (Thomas and Read 2007). Entomopathogenic fungi: Fungi parasite that can kill insects. Eosinophilia: Increased number of eosinophils in the blood Erythema: Cap illary congestion resulting in abnormal redness Faecaloma: An accumulation of feces in the colon giving the appearance of a protuberant tumor Fibrinogen : Plasma protein converted into Fibrin needed for clot formation Flaviviruses : A genus of arthropod b orne viruses Frons : Glomerulonephritis : Inflammation of the renal glomeruli capillaries H emostasis : Stopping the flow of blood. Hematophagous : A blood based diet Hematuria: Blood in urine Hemocoel : The body cavity of an inse ct where most the major organs are found (Thomas and Read 2007). Hepatomegaly: Swelling of the liver Hepatosplenomegaly: Enlargement of both the spleen and the liver Hydroceles: An accumulation of serous fluid in a sacculated cell

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151 Hypergammaglobulinemi a: Too many gamma globulins in the blood Hyphomycetes: A class of fungi in the order Deuteromycetes Integrated Disease Management (IDM) : Disease control using a variety of techniques to combat disease. Integrated Pest Management (IPM): A comprehensive a pproach of pest management using biological, cultural, and genetic pest control methods using pesticides as a last resort. Integrated Vector Management (IVM): Vector control program that employs several methods to control disease causing insects. Isozyme: A generic term referring to forms of a particular enzyme. Kairomones : A blend of volatiles released by humans along with their metabolism products Keratitis: Inflammation of the cornea LC 50 : Lethal concentration for 50% of exposed population to die afte r exposure to an agent Lichenified: Condition of having hard leathery skin due to chronic irritation LT 50 : Lethal time 50% time to kill 50% of population exposed to a particular toxin Lymphangitis: Lymphatic vessel inflammation Lymphocytes : White bloo d cells originating from the stem cells of bone marrow Malaise: A general feeling of illness Melanization: I nfiltrate with melanin Meningismus: Meningoencephalitis : Inflammation of the meninges and the brain

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152 Metabolic acidosis : To o much acid in the body Microfilariae: Minute larval nematode Myalgia : Pain in muscles Myocarditis: Inflammation of the myocardium Neovascularization: Excessive formation of blood vessels Neutropeni a : Abnormal levels of white blood cells, particularly neutrophils Oedema: Fluid collection in the circulatory system Oospore : The sexual reproductive unit of sexual fungi Oviposition : to lay eggs (especially in insects) Palpitations: Rapid and abnorma l beating of the heart Palpi: Insect mouthpart, normally tactile or gustatory Pancytopenia : Low levels of erythrocytes, blood platelets, and white blood cells in the blood Peridomestic : In or around human habitations Periophthalmic: Around the eye Pet echiae : Localized hemorrhage demarked by small red or purple spots on the skin. Proboscis: A sucking, tubular process of an insect used for feeding Proteinuria: Protein in the blood Pruritus : Irritation of sensory nerve endings causing itching Semioche micals : Biologically produced chemicals that carry a message Sequelae: A negative after effect Serotype : Related group of microorganisms that present similar antigens Splenomegaly: Enlargement of the spleen

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153 Sterile Insect technique: A technique used to control or eradicat e insect pests or vectors utiliz ing induction by irradiation of dominant lethality in the chromosomes of the released insects Syncope: Loss of consciousness because of insufficient blood flow to the brain. Syncope: Loss of consciousness due to loss of blood flow to the brain Thrombocytopenia : Low platelet count. Thromboembolism: Block age of a blood vessel due to part of a blood clot that has broken away. Thrombus: A clot formed that remains at its site of formation Vascular permeability : measure of a molecules propensity to go from the blood vessel into the tis sue Viremia: Viruses in the blood Zoospore : The asexual reproductive unit of the fungi Sources : merriam www.biology

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154 Appendix B: Evidence of Resistance Studies published citin g resistance in target vector insects from 1998 2008. Vector of interest Agent of Resistance Title of Publication or Summary of Findings and Year Mosquito Pyrethroids West Africa: Pyrethroid Resistance in Culex quinquefasciatus (Chandre et al. 1998) Mosquito DDT (Contra evid ence to carbamate, organophosphorus, and pyrethroid Mexico: Low carbamate, organophosphorus, and pyrethroid resistance but high DDT resistance in Anopheles albimanus (Penilla et al. 1998) Mosquito Contra evidence to iAChE and es terase based insecticides Trinidad: iAChE and esterase based insecticides still without resistance in Trinidad (Vaughan et al. 1998) Mosquito Carboxylesterases Review: Mosquito carboxylesterases: a review of the molecular biology and biochemistry of a major insecticide resistance mechanism (Hemingway 1998) Mosquito Malathion Cuba: Cross resistance to malathion in Cuban Culex quinquefasciatus induced by larval selection with deltamethrin (Bisset et al. 1998) Mosquito Pyrethroid Mosquitoes: Correlat ion between developing resistance at high temperatures over low temperatures (Hodjati et al. 1999) Triatomine Pyrethins Triatomine resistance to pyrethrins (Zerba 1999) Mosquito Pyrethroid South Africa: Anopheles funestus resistant to ( Hargreaves et al. 2000) Mosquito Dieldrin + fipronil Resistance to dieldrin + fipronil of malaria vector Anopheles gambiae (Brooke 2000) Triatomine Deltametrin, Beta Cyflurthrin, cypermethrin, not Beta cybermethrin or Beta cyahalothrin Insecticide resistance in Brazilian Triatoma infestans and Venezuelan Rhodnius prolixus (Vassena et al. 2000). Mosquito Propoxur (a Carbamate) A. funestus from Mozambique are shown to demonstrate resistance to Propoxur (Brooke et al. 2001) Mosquito Pyrethroid Nigeria and Ghana : Pyrethroi d resistance/susceptibility and differential urban/rural distribution of Anopheles arabiensis and An. gambiae s.s. malaria vectors (Kristan et al. 2003) Mosquito Acetylcholinesterase Ivory Coast: Resistance to carbosulfan in Anopheles gambiae Reduced se nsitivity of acetylcholinesterase (N'Guessan et al. 2003). Mosquito Pyrethroid and DDT Pyrethroid and DDT cross resistance in Aedes

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155 aegypti is correlated with novel mutations in the voltage gated sodium channel gene (Brengues et al. 2003). Mosquito Bac illus sphaericus (contra, B. thuringien sisisraelensis) Cross resistance between strains of Bacillus sphaericus but not B. thuringiensisisraelensis in colonies of the mosquito Culex quinquefasciatus (Yuan et al. 2003). Mosquito Carbamates and Organophos phates Mosquito resistance to carbamates and organophosphates (Weill 2003) Mosquito Dieldrin Ghana: Dieldrin resistance in the malaria vector Resistance in Anopheles gambiae (Brooke et al. 2003). Sand flies Malathion Sand fly resistance to malathion i n Sri Lanka (Surendrana 2006). Mosquito Pyrethroids kdr mutations conferring resistance in A n gambiae in African mosquitoes (Pinto 2006). Mosquito Carbamate, Chlorinated hydrocarbons, Organophosphates Resistance found to several insecticides in Chinese mosquito(Cui 2006) Mosquito Pyrethroids, Carbamate, chlorinated hydrocarbons, organophosphates Determining Mozambique A n funestus resistance to several insecticides(Casimiro2006) Mosquito Genes resistance in th e mosquito Culex pipiens. (Labbe 2007) Mosquito Carbamates, pyrethrins, chlorinated hydrocarbons, organophosphates Resistance in all four pesticide classes demonstrated in A n arabiensis (Matambo 2007) Bibliography: Bisset J, Rodriguez M, Soca A (1998 ). Cross resistance to malathion in Cuban Culex quinquefasciatus induced by larval selection with deltamethrin Medical and Veterinary Entomology 12(1): 109 112. Brengues C, Hawkes NJ, Chandre F, Mccarroll L, Duchon S, Guillet P, Manguin S, Morgan J C, Hemingway J (2003). Pyrethroid and DDT cross resistance in Aedes aegypti is correlated with novel mutations in the voltage gated sodium channel gene. Medical and Veterinary Entomology 17(1): 87 94. Brooke BD, Hunt RH, Coetzee M (2000) Resistance to d ieldrin + fipronil assorts with chromosome inversion in the malaria vector Anopheles gambiae Medical & Veterinary Entomology 14(2): 190 194.

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156 Brooke BD, Kloke G, Hunt RH, Koekemoer LL, Tem EA, Taylor ME Small G, Hemingway J, Coetzee M (2001). Bioassa y and biochemical analyses of insecticide resistance in southern African Anopheles funestus (Diptera: Culicidae). Bulletin of Entomological Research 91 :265 272. Brooke BD, Hunt RH, Matambo TS, Koekemoer LL, Van Wyk P, Coetzee M (2006) Dieldrin resista nce in the malaria vector Anopheles gambiae in Ghana Medical and Veterinary Entomology 20(3): 294 299. Casimiro S, Coleman M, Moahlaoa P, Hemingway J, Sharp B (2006). Insecticide Resistance in Anopheles funestus (Diptera: Culicidae) from Mo zambiqu e. Journal of Medical Entomol ogy. 43(2): 267 275. Chandre F, Darriet F, Darder M, Cuany A, Doannio JMC, Pasteur N, Guillet P (1998). Pyrethroid resistance in Culex quinquefasciatus from West Africa. Medical and Veterinary Entomology 12: 359 356. Cu i F, Lin LF, Qiao CL, Xu Y, Marquine M, Weill M, Raymond M (2006). Insecticide resistance in Chinese populations of the Culex pipiens complex through esterase overproduction Entomologia Experimentalis et Applicata 120: 211 220. Hargreaves K, Koekemoer LL, Brooke BD Hunt RH Coetzee MM (2000). Anopheles funestus resistant to pyrethroid insecticides in South Africa Medical & Veterinary Entomology 14(2): 181 189. Hemingway K (1998). Mosquito carboxylesterases: a review of the molecular biology and bi ochemistry of a major insecticide resistance mechanism Medical and Veterinary Entomology 12(1): 1 12. Hodjati MH, Curtis CF (1999). Effects of permethrin at different temperatures on pyrethroid resistant and susceptible strains of Anopheles Medical & Veterinary Entomology 13(4): 415 422. Jawara M, McBeath J, Lines JD, Pinder M, Sanyang F, Greenwood BM (1998). Comparison of bednets treated with alphacypermethrin, permethrin or lambdacyhalothrin against Anopheles gambiae in the Gambia. Journal o f Medical and Veterinar y Entomology 12(1): 60 66. Kristan M, Fleischmann H, Della Torre A, Stich A, Curtis CF (2003). Pyrethroid resistance/susceptibility and differential urban/rural distribution of Anopheles arabiensis and An. gambiae s.s. malaria vectors in Nigeria and Ghana Medical and Veterinary Entomology 17(3): 326 332. Labbe P, Berthomieu A, Berticat, Alout H, Raymond M, Lenorm T, Weill M (2007). Independent Duplications of the Acetylcholinesterase Gene Conferring

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157 Insecticide Resistance i n the Mosquito Culex pipiens Molecular biology and evolution 24 (4): 1056 1068. Matambo TS, Abdalla H, Brooke BD, Koekemoer LL, Mnzava A, Hunt RH, Coetzee M (2007). Insecticide resistance in the malarial mosquito Anopheles arabiensis and association with the kdr mutation Medical and Veterinary Entomology 21: 97 102. N'Guessan R, Darriet F, Guillet P, Carnevale P, Traore Lamizana, M, Corbel V,. Koffi A, Chandre F (2003). Resistance to carbosulfan in Anopheles gambiae from Ivory Coast, based on red uced sensitivity of acetylcholinesterase. Medical and Veterinary Entomology 17(1): 19 25. Penilla RP, Rodriguez AD, Hemingway J, Torres JL, Arrendondo Jimez, Rodriguez MH (1998). Resistance management strategies in malaria vector mosquito control. Baseline for a large scale field trial against Anopheles albimanus in Mexico. Journal of Medical & Veterinary Entomology 12: 217 233. Pinto J, Lynd A, Elissa N, Donnelly MJ, Costa C, Gentile G, Caccone A, Rosrio VEDO (2006). Co occurrence of East and West African kdr mutations suggests high levels of resistance to pyrethroid insecticides in Anopheles gambiae from Libreville, Gabon. Medical & Veterinary Entomology 20(1): 27 32. Surendrana SN, Karunaratnea SHPP, Adams S, Hemingway J, Hawkes NJ (2005). Molecular and biochemical characterization of a sand fly population from Sri Lanka: evidence for insecticide resist ance due to altered esterases and insensitive acetylcholinesterase. Bulletin of Entomological Research 95:371 380. Vassena CV, Picollo MI, Zerba EN (2000). Insecticide resistance in Brazilian Triatoma infestans and Venezuelan Rhodnius prolixus Medic al & Veterinary Entomology 14(1): 51 55. Vaughan A, Chadee DD, Ffrench Constant R (1998). Biochemical monitoring of organophosphorus and carbamate insecticide resistance in Aedes aegypti mosquitoes from Trinidad. Journal of Medical and Veterinary Ento mology 12: 138 141. Weill M, Lutfalla G, Mogensen K, Chandre, FBerthomieu A, Berticat C, Pasteur N, Philips A, Fort P, Raymond M (2003). Insecticide resistance in mosquito vectors Nature 423: 136 137. Zerba EN (1999). Susceptibility and resistance t o insecticides of Chagas disease vectors Medicina (Buenos Aires) 59 : 41 46.

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158 Appendix C: Mosquito and Mosquito borne disease Distribution Mosquito range Figure a Range of mosquito s worldwide (National Geographic website, 2008) Figure b. Range of a particular genus of mosquitoes, Anopheles (lighter color) Mosquito range (National Geographic website, 2008).

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159 Figure c Malaria distribution Map (CDC 2004) Countries that have experienced DHF outbreaks Imported cases (probable or confirmed) Figure d Geographic distribution of Dengue Fever (WHO 2003)

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160 Appendix D : CDC Explanations of Life Cycles of Select Parasites a. Malaria Figure a. The lifecycle of plasmodiums "The malaria parasite life cycle involves two hosts. During blood meal, a malaria inf ected female Anopheles mosquito inoculates sporozoites into the human host Sporozoites infect liver cells and mature into schizonts which rupture and release merozoites (Of note, in P. vivax and P. ovale a dormant stage [hypnozoites] can persis t in the liver and cause relapses by invading the bloodstream weeks, or even years later.) After this initial replication in the liver (exo erythrocytic schizogony ), the parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizog ony ). Merozoites infect red blood cells The ring stage trophozoites mature into schizonts, which rupture releasing merozoites Some parasites differentiate into sexual erythrocytic stages (gametocytes) Blood stage parasites are responsible for the clinical manifestations of the disease. The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by an Anopheles mosquito during a blood meal sporogonic cyc le While in the mosquito's stomach, the microgametes penetrate the macrogametes generating zygotes The zygotes in turn become motile and elongated (ookinetes) which invade the midgut wall of the mosquito where they develop into oocysts The ooc ysts grow, rupture, and release sporozoites which make their way to the mosquito's salivary glands. Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle ." ( CDC 2008; DPDx 2008) b. Chagas Disease

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161 Figure b. T he life cycle of a triatomine its feces near the site of the bite wound. Trypomastigotes enter the host through the wound or through intact mucosal membranes, such as the conjunctiva Common triatomine vector species for trypanosomiasis belong to the genera Triatoma Rhodinius and Panstrongylus Inside the host, the trypomastigotes invade cells near the site of inoculation, where they differenti ate into intracellular amastigotes The amastigotes multiply by binary fission and differentiate into trypomastigotes, and then are released into the circulation as bloodstream trypomastigotes Trypomastigotes infect cells from a variety of tissues and transform into intracellular amastigotes in new infection sites. Clinical manifestations can result from this infective cycle. The bloodstream trypomastigotes do not replicate (different from the African trypanosomes). Replication resumes only when the parasites enter another cell or are ingested by another vector. blood that contains circulating parasites The ingested trypomastigotes transform into epimastigotes in dgut The parasites multiply and differentiate in the midgut and differentiate into infective metacyclic trypomastigotes in the hindgut (CDC 2008; DPDx 2008) c. African Sleeping Sickness

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162 (CDC 2008; DPDx 2008 )

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163 Appendix E : Effect s of L. chapmanii in target and non target species Taken from Lpez et al. 2004 Table E .a. Testing of Leptolegina chapmanii response in Non target aquatic organisms Non target Fungal Infections of L. chapmanii Host species No. exposed Replicates % Infection (SD) Crustacea: Cladocera Daphnia sp. 30 2 0 Amphipoda Hyalella curvispina 10 1 0 Cyclopoida Mesocyclops annulatus 20 2 0 Insecta Odonata: Zygoptera Fam. Coenagrionidae Diptera: Fam. Psychodidae 15 1 0 Fam. Ceratopogonidae Dasyhelea necrophila 85 3 0 Fam. Chironomidae 10 1 0 Sp. 1 65 3 16.9(2.1) Sp. 2 35 3 5.7(0.8) Coleoptera: Fam. Hydrophyllidae 8 1 0 Nematoda: Fam. Mermithidae Strelkovimermis spiculatus 10 1 0 Vert ebrata: Pisces Cnesterodon decenmaculatus 40 4 0 Amphibia Bufo arenarum (larvae) 8 1 0

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164 Target Fungal Infections of L. chapmanii Table E .b Infections of twelve species of mosquitoes to Leptolegnia chapmanii Hosts species Instar No. Expo sed Replicates % Infection (SD) Aedes aegypti I 40 4 100 II 40 4 100 III 40 4 100 IV 40 2 85 Anopheles sp. III 20 2 100 Culex apicinus II 60 3 13(4.7) III 130 3 0 Cx. castroi IV 30 2 10 Cx. dolosus III 40 4 200 Cx. pipiens II 70 7 92.8(6.9 9) III 60 3 95(7.07) Cx. renatoi III 20 3 0 Ochlerotatus albifasciatus II 20 2 100 III 10 2 80 IV 100 5 59(26.1) Oc. crinifer II 60 6 68.3(6.87) III 80 3 96(5.65) Psorophora cyanescens III 20 2 90 Ps. ferox II 80 4 86.2(9.6) III 70 7 77 .1(8.8) Isostomyia paranensis II 20 2 0 III 20 2 0 Taken from Lpez et al. 2004

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165 Appendix F : Isolates of B. bassiana and M. anisopliae used in Luz et al. (1998 a) study

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166 Appendix G : Evlachovaea Lethal Concentration: triat omine 3rd instar nymph Table 1 Lethal concentrations (LC50 and LC90) (CFU/cm2) and respective confidence intervals (95% C.I.) of Evlachovaea sp calculated for triatomine third instar nymphs, 15 and 20 days after exposure at 75% and >98% relative humidi ty and 25C1. ( Taken directly from Luz et al. Time after inoculation (days) 15 20 Humidity LC 50 LC 90 LC 50 LC 90 Triatoma delpontei Triatoma lecticularia Triatoma picturata Triatoma sor dida Triatoma vitticeps Panstrongyl us herreri Rhodnius ecuadoriensi s Rhodnius nasutus Rhodnius neglectus Rhodnius prolixus Rhodnius robustus 75 98 75 98 75 98 75 98 75 98 75 98 75 98 75 98 75 98 75 98 75 98 75 98 0.20a ,b (0.05 0.55) 0.89b,c (0.32 2.28) 1.66c (0.84 8.30) 4.34c (1.74 30) 1.37c (0.69 5.26) 0.06a (0.02 0.13) 3.11c (1.29 27.6) 0.33a,b (0.18 0.55) 0.04a (0.02 0.12 0.03a (0.01 0.06) 0.06a (0.01 0.19) 0.35b (0.22 0.58) 0.09a (0.03 0.19) 5.02b,c (1.36 402.0) 1.54b,c (1.07 2.61x104) c (3.27 796.0) 67.40c (13.80 6.91x103) 8.77b,c (2.95 235.0) 0.63a,b (0.28 3.89) 59,30c (11,0 3.4x104 0.93b (0.56 4.62) 0.64 a,b(0.22 4.45) 0.11 a(0.06 0.55 ) 0.57 a,b(0.19 6.34) 0.78 a,b(0.5 2.76) 0.94 a,b(0.43 4.10) ** 0.20 c,d(0.12 1.06) 1.44 d(0.74 5.73) 1.08 d(0.60 2.69) 1.12d (0.51 5.36) 0.05a,b (0.02 0.09) 1.68d (0.70 6.66) 0.13b,c (0.07 0.21) ** ** 0.03a (0.01 0.05) 0.22b,c (0.13 0.44) 3.71d (1.02 6.59x102) ** ** 0.52a,b (0.27 279.0) 8.44a,b (2.93 290.0) 6.97 ab (2.79 51.20) 12.3 ab (3.21 682.0) 0.27a (0.13 1.64) 33.50 ab (7.85 429.0) 0.37a (0.22 1.38) ** ** 0.08 a (.05 11.1) 0.38a 0.26 15.1) (17.0 1016)

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167 Appendix H : Distribution & Examples of Major Vector Triatomines Figure a: Distribution of the most prominent vectors of Chagas Disease ( OPS 2009) Figure b: Distribution of all the major vectors of Ch agas Disease ( Organizacion Panamerica de Salud 2009) Bibliography: Organizacion Panamerica de Salud (OPS). Situacin de enfermedad de Chagas: Amrica Latina. http://www.mex.ops Accessed Feb 2009.