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! THE DISTRIBUTION OF CYPHOMA GIBBOSUM (MOLLUSCA: GASTROPODA: CYPRAEOIDEA: OVULIDAE) (THE FLAMINGO TONGUE GASTROPOD ) IN RELATION TO THE PRESENCE OF THE FUNGAL DISEASE ASPERGILLOSIS ON GORGONIA SPP. (ANTHOZOA: CNIDARIA: OCTOCORALLIA: GORGONIIDAE) BY JULIE CHRISTINA KRZYKWA A Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Dr. Sandra Gilchrist Sarasota, Flori da May 2012
! "" Acknowledgments Thank you to the New College of Florida Alumni Association, the National Explorer's Grant program, and the Sarasota Shell Club for their assistance in ensuring that I was able to travel to Honduras to work on this research. T hank you to the Plantation Beach Resort staff for providing assistance with logistics during the research process. Thank you to my committee for their assistance and time. Special thanks to Dr. Sandra Gilchrist for her help in working on the research, for presenting me with the opportunity to travel to Honduras to work on my own research and for all of her support throughout my undergraduate experience Thank you to Kyle McC ormick and Rachel Perry for their assistance during the research process and to Dr Duff Cooper for his help with my data analysis. Thank you to my friends and family for all of their encouragement and their unyielding confidence that I will be su ccessful in all of my endeavors, especially my roommates Jakilah Mason and Lyssabeth Ped erson
! """ Table of contents: Acknowledgments ................................................................................. ............................. ................ii Table of Contents ................................................................. .................. ..........................................iii List of Figures.. ................................................................... ............................. ...................... .............iv List of Tables.. ......................... ....................................................................... ...................... ............. ..v Abstract ................................................................................................. ............................. ........... ...... v i Chapter 1: I ntroduction 1.1 Coral diseases in the Caribbean .......................................................................... ..1 1.2 Antifungal activity of Gorgonians ................................................ .......................2 1.3 Aspergi llosis epizootic ................................................ ............................................9 1.4 Effects of Aspergillosis on sea fan populations ................ .............................. 13 1.5 Origins of Aspergillus sydowii ................... ............................................... .............15 1.6 Transmission of Aspergillosis ............................................... ............................ 20 1.7 Cyphoma gibbosum as a likely vector ................................................ .. ................ 23 1.8 Gorgonian defense against predators ................................................ .............. 25 1.9 Effects of Cyphoma gibbosum grazing ................ .................................................27 1.10 Cyphoma gibbosum prey preference s ................................................ .................. 28 1.11 Uptake of gorgonian toxins .............................................. ................................. .. 31 1.12 Purpose and Hypothesis ................................................ ..... ..................................34 Chapter 2: M ethods 2.1 Cyphoma gibbosum survey................................................................................ .............35 2.2 Disease Prevalence survey............................................. ................................... ..........42 Chapter 3: R esults 3.1 Cyphoma gibbosum survey................................................................................ .............43 3.2 Disease Prevalence survey.................................. .............................................. ..........47 Chapter 4: D iscussion ........................................................................................ ...............................48
! "# List of Figures: Figure 1....................Purple Lesions indicating Aspergillosis infection......................5 Figure 2....................Purple and clear sclerites.................................................................8 Figure 3....................Diseased coral colony off shore of Cayo G rande......................10 Figure 4.................... Aspergillus sydowii .............................................................................11 Figure 5.................... Asexual reproduction of Aspergillus nidulans .... ............... ....... ..12 Figure 6.................... Cyphoma gibbosum feeding on gorgonians..................................23 Figure 7....................Overview of uptake of allelochemicals in marine herbivores................ .............................................. ........................ ....33 Figure 8....................Honduran Bay Islands....................................................................35 Figure 9....................Los Cayos Cochinos............................................................ ............36 Figure 10 .................. Map of location of surv ey sites from both years....... ....... .. ......37 Figure 11..................General location of all sites from the first year of surveying..................................................... ......................................38 Figure 12..................Infected sea fan off of Cayo Grande............................................39 Figure 13..................Demonstration of sea fan area measurements..........................39 Figure 1 4..................General locations of all sites from the second year of surveying.......................................................................................41 Figure 15..................Map of grid locations from the survey looking at disea se prevalence............................................................................42 Figure 16..................Area consumed in relation to total sea fan area ......... ..............44 Figure 17..................The average sea fan area in cm2...... ....... ............................ .........46 Figure 18.................. Histograms of sea fan size distribution ..... ..... ............. ...... ........47 Figure 19.................. Area consumed on a sea fan in relation to the presence of Aspergillosis a nd the size of the gastropod ...................... ..................................................... .... ...........48 Figure 20..................Comparison of results from both surveys .... ................ ............. 51
! # List of Tables Table 1......... .............Immune responses of coral species and how they were identified...............................................................................................3 Table 2......................Pathogenic strains of Aspergillus and the organisms aff ected................................................................................................19 Table 3......................Linear trendline equations for Figure 16........ ................... .......45 Table 4......................Breakdown of the number of gastropods on infected and uninfected sea fans................................... ...................... ............45 Table 5......................Observed values for different factors on infected and uninfected sea fans........................ ................................. .. ..................45 Table 6......................Sea fan size information for Figure 17........... ................... .........46
! #" THE DISTRIBUTION OF C YPHOMA GIBBOSUM (MOLLUSCA : GASTROPODA: CYPRAEOIDEA: O VULIDAE) (THE F LAMINGO TONGUE GASTROPOD) I N RELATION TO THE PRESENCE OF THE FUNGAL DISEASE ASPERGILLOSIS ON GORGONIA SPP. (ANTHOZOA: CNIDARIA: OCTOCORALLIA: G ORGONIIDAE) Julie Krzykwa New College of Florida, 2012 ABSTRACT The fungal disease Aspergillosis has been an ongoing epizootic in the Caribbean since its identification in 1995. Further investigation into the environmental or biological vectors of Aspergillosis is of great interest, as this disease has had a considerable impact on coral reefs in the Cari bbean. The gastropod Cyphoma gibbosum is able to pass viable spores of Aspergillus sydowii (Ascomycota: Eurotiomycetes: Eurotiales: Trichocomaceae) the fung us thought to cause the disease, and previous field studies noted an increased presence of C. gibbo sum during Aspergillosis outbreaks These observations suggest that C. gibbosum may be a biological vector of Aspergillosis This study looked at the distribution of C. gibbosum in relation to the presence of Aspergillosis on its gorgonian prey along a por tion of shallow (<3m) fringing reef off of Los Cayos Cochinos in the Honduran Bay Islands. The disease prevalence of Aspergillosis along the reef was also investigated. Gastropods were not found to feed preferentially on infected, or uninfected fans but t here is circumstantial evidence suggesting a preference for infected fans that
! #"" indicates the need for further investigation. Gastropod s did appear to consume less of infected sea fans, and large r sea fans were observed to be more likely to show signs of an Aspergillosis infection than smaller fans. Dr. Sandra Gilchrist Division of Natural Sciences
! $ Chapter 1: Introduction 1.1 Coral Diseases in the Caribbean Coral disease has been a prevalent cause for reef decline in the Caribbean Sea in addition to v arious environmental and anthropogenic factors (Aronson et al. 2003; Aronson and Precht 1997; Harvell et al. 1999; Porter and Meier 1992; Richardson 1998). Coral disease in general has been increasing (Ward and Lafferty 2004). This increase is especially d rastic in the Caribbean. As of 1997 only 8% of the coral reef area is found in the Caribbean Sea (Spalding and Grenfell 1997). An extensive review of coral disease by Green and Bruckner (2000) found that despite this small percentage of coral reef area, 66 % of the coral disease reports up to the year 2000 came from the Caribbean region and coral diseases originating from the Caribbean make up 76% of the reported coral diseases (Green and Bruckner 2000). Researchers suggested that this high level of disease might have some correlation with high level of human activity along most of the coral reefs in the Caribbean. This high density of coral disease -as well as the fast rate of disease emergence, high occurrence and high virulence of emerging coral disease s -has caused the region to be dubbed a "disease hotspot"(Weil 2004). T his hot spot effect may be a result of dust being carried over from the Sahara desert (Garrison et al. 2006; Garrison et al. 2003; Harvell et al. 2007), but recent studies have found this is probably not the case for all observed coral diseases (Rypien 2008a). It is also possible that the observed frequency of disease may be in part due to increased research in the Caribbean. The Caribbean Sea has a smaller area and is well populated, allowing diseases and coral reefs in the region to be studied with the same resources as would be needed to study a small percentage of reef in a larger area such as the Pacific
! % The evaluation of these diseases is hindered by difficulties studying diseas e pathology; etiology, or the relationship between the host and the pathogen ; and epizootiology, or distribution and transmission methods, of marine diseases (Weil 2004). Very rarely is the pathogen for coral disease identified. Out of the twenty one most commonly reported diseases in the Caribbean the pathogen is only known for less than half of them and Koch's postulates have only been fulfilled fo r a less than half of the pathogens that have been identified (Harvell et al. 2007; Weil and Gil Agudelo 20 06) While the reason for this increase in coral disease is unknown, some researchers have hypothesized that increased water temperatures effect biological and physiological properties, or the physiological equilibrium, between disease causing biotic agent s and coral species (Alker et al. 2001; Bruno et al. 2007; Harvell et al. 2002; Harvell et al. 2007; Lafferty 2009; Rosenberg and Ben Haim 2002; Weil 2004 ) 1.2 Antifungal Activity and Immune System of Sea Fans The coral immune system consists of a numbe r of innate and inducible responses (Table 1) Granular amoebocytes are important for the phagocytosis of foreign bodies and particles in corals, as well as for wound healing (Meszaros and Bigger1999; Olano and Bigger 2000) and allogeneic rejection (Olano and Bigger 2000). The production of prophenoloxidase within granular amoebocytes may contribute to the production of melanin as an immune response to infection (Mullen et al. 2004 ; see review by Sderhll and Cerenius 1998 ). Melanin develops in plates bet ween encroaching pathogens and healthy tissue to prevent the spread of pathogens throughout the sea fan (Mullen et al. 2004; Palmer et al. 2007; Petes et al. 2003). Gorgonian corals have also been observed to build proteinaceous capsules to surround and c ontain invading algal pathogens
! & (Goldberg et al. 1984; Morse et al. 1981). In Gorgonia ventalina the filaments of an algal pathogen were encased in endoskeleton gorgonin tubules, isolating the filaments, and these capsules were surrounded by healthy tissue (Morse 1981). In the gorgonian Pseudoplexaura fiagellosa similar observations were made of an algal pathogen being isolated by encapsulation in the host skeleton (Goldberg et al. 1984). Periodoxases (Mydlarz and Harvell 2007) and potentially homarine or a homarine analog (Shapo et al. 2007) have been identified as antifungal compounds that are a part of the go rgonian inducible immune system. In the gorgonians Subergoris suberosa and Scripearia gracillis subergorgic acid and pregn 4 ene 3,20 dione were ide ntified as the most potent antibacterial chemicals (Qi et al. 2008). Table 1 Immune responses of coral species and how they were identified. Immune System activity Species originally identified in How it was identified Source (s) Granular amoebocytes and prophenyloxidase (PPO) Swiftia exserta Cellular response to the introduction of foreign particles (India ink) was observed in vivo and observations of internalized ink were used to identify cells involved Goldberg et al. 1984 Mesz aros and Bigger 1999 Olano and Bigger 2000 Mullen et al. 2004 Mydlarz et al. 2008 Melanin Gorgonia ventalina Acropora millepora Slides were prepared of decalcified corals and the presence of melanin deposits were verified histochemically. Petes et al. 200 3 Mullen et al. 2004 Palmer et al. 2007 Mucus (and associated bacterial community) found in the surface mucopolysaccharide layer (SML) Acropora palmata Bacterial cultures were developed of bacteria isolated from A. palmate SML and the antimicrobial proper ties were investigated Ritchie 2006 Bactericidal and a ntifungal compounds and peptides Various species of scleractinian and gorgonian corals The a ntibacterial activity of crude extracts from a number of coral species was examined Kim 1994 Koh 1997 Geffen and Rosenberg 2005 Mydlrz and Harvell 2007 Gochfeld and Aeby 2008 Qi et al. 2008 Encapsulation Gorgonia ventalina and Pseudoplexaura fiagellosa Identification of tumors on gorgonians and spectroscopic analysis and chromatography of the contents of the t umors. Morse et al. 1981 Goldberg et al. 1984
! Sea fan antifungal activity appears to be an inducible response to the presence of fungal infection (Dube et al. 2002; Kim et al. 2000; Kim and Harvell 2002; Ward 2007; Ward et al. 2007) Jensen and collea gues (1996) noted a lack of broad spectrum antibiotics in gorgonians after observing that bacteria that are commonly associated with the surface of gorgonian corals do not suffer ill effects from gorgonian secondary metabolites. It has also been observed t here was an increase in antifungal activity in the central regions of infected sea fans (Kim et al. 2000). The main immune anti fungal responses used by gorgonian sea fans, specifically Gorgonia ventalina are melanin, lipid antifungal products amoebocyt es, and antibacterial products (Mydlarz et al. 2006 ; Mydlarz et al. 2008 ). Melanin develops in a layer between necrotic tissue and the axial skeleton in infected regions of gorgonians, forming a barrier between invading A. sydowii and the necrotic tissue b ordering the uninfected tissue (Mullen et al. 2004; Petes et al. 2003). T his increase in melanin may be related to an increase in acidophilic granular amoebocytes in the mesoglea of infected sea fans near sites of infection (Mydlarz et al. 2008). Peroxidas es were identified as one of the lipid antifungal responses of Gorgonia ventalina against Aspergillus sydowii infection, and the response was inducible (Mydlarz and Harvell 2007). Circumstantial evidence indicates that homarine or a homarine analog have an anti microbial role in the innate immune response in the common sea whip, Leptogorgia virgulata (Shapo et al. 2007). The anti microbial activity of sea fans appears to be concentrated in different ar eas of the sea fan Kim and colleagues (2000) observed in healthy gorgonian coral colonies that extracts from the tips of the sea fan were more effective at fighting off microbes, specifically Aspergillus sydowii, than extracts from other areas of the sea fan.
! ( Puglisi and colleagues (2002) had similar findings They observed an increased concentration of lipophilic crude extracts in the outer edges of in four out of the seven gorgonian species they examined. This might be why the characteristic lesions are usually observed near the base of infected sea fans (Ki m and Harvell 2004; Nagelkerken et al. 1997) ; (Figure 1) Figure 1 Purple Lesions indicating Aspergillosis infection Arrow indicates purple lesions near base of sea fan. Photo taken by Julie Krzykwa. There are high levels of variability concerning the antifungal activity of different sea fans of different sizes and on different coral reefs. For example, large sea fans seem less resistant to fungal infection than smaller sea fans (Dube et al. 2002; Kim et al. 2000; Kim and Harv ell 2002). T his is probably because the larger sea fans have been fending off pathogens for a longer period of time (essentially, the y become senescent) and also serve as a larger target to pathogens due to their larger size (Dube et al. 2002; Kim and
! ) Har vell 2004). This increased antifungal response by smaller gorgonians may also have to do with the faster growth rates observed in smaller sea fans (Grigg 1974) although other observations indicate that growth rates are not directly related to gorgonian si ze in Pseudopterogorgia (Yoshioka 1994) The increased antifungal activity near the edges of the sea fan also may have a role in the decreased resistance of large sea fans. In smaller sea fans the edge area makes up a larger portion of the overall sea fan surface, whereas larger sea fans have a larger center area that is less aptly defended. S ea fans that are already infected with Aspergillus sydowii have a reduced response to new infections (Ellner et al. 2007) and are less likely to recover from, or prev ent, an Aspergillosis infection It is possible that larger fans that are more likely to have a primary infection may have a harder time fending off new infections, making them more susceptible to a more devastating secondary infection. Ellner and colleagu es (2007) hypothesized that increased severity during cases where there were multiple points of infection resulted from increased hyphae load on the sea fans, and the need for the fan's immune response to work on multiple fronts to combat Aspergillosis at different locations on the sea fan. It has also been postulated that there may be a size specific defense, such as greater chemical resistance to disease in smaller fans (Dube et al. 2002). It is likely that the increased infection levels seen in large sea fans have more to do with the age of the sea fan than the size. In a study concerned with the effects of size on antifungal activity in Gorgonia ventalina Dube and colleagues (2002) observed that mature sea fans were more likely to become infected with A spergillosis when inoculated with A. sydowii. Toledo Hernndez and colleagues (2009) noted that smaller, younger sea fans have been observed to suffer greater tissue loss once an infection does set in but mature fans suffered from large r
! lesions, and more of them, than younger sea fans (Dube et al. 2002). The greater tissue loss may result from the sloughing off of sections of the colony, which would have increased repercussions in a gorgonian of a smaller size. A study by Couch and colleagues (2008) did n ot find significant variation in immune response between large and small sea fans, but this maybe a result of only sampling edge fragments where antifungal activity is known to be high. Rypien (2008b) investigated the responses of individual sea fans to t he presence of fungal treatments containing isolates of Aspergillosis sydowii from a diseased sea fan, and from a dried fish. P revious observations concerning antifungal response in individual sea fans found a high variation in individuals' antifungal immu ne respon se (Kim et al. 2000, Dube e t al. 2002, Mullen et al. 2006). Rypien (2008b) observed an antifungal response that was highly variable at the individual level and w hile most of the sea fans demonstrated anti fungal activity in response to the presen ce of fungal isolates neither treatment induced a universally high anti fungal response. These results confirmed previous observations (Kim et al. 2000, Dube et al. 2002, Mullen et al. 2006), and may indicate that differing genotypes in sea fan populations determine the effectiveness of gorgonian immune response to Aspergillosis. Smith and colleagues (1998) fou nd a higher concentration of pigmented sclerites in infected sea fans than in uninfected sea fans (Figure 2 and refer to Figure 1 for a sea fan show ing purpling ) ; Ward and colleagues (2007) reaffirmed these results. The abnormal coloring is common in compromised coral colony tissues (Palmer et al. 2009). Sclerites are calcareous structures that act as support structures and defense mechanisms. They ar e usually colorless and found in the internalized skeleton of gorgonian coral colonies. These pigmented sclerites, which cause the characteristic
! + purple lesions signifying an Aspergillosis infection (F igure 1) represent a relatively fast acting physical r esponse to t he presence of biotic agents through an increase in the concentration of sclerites around the infected tissue (Alker 2004). This is supported by observations that infected sea fans with less of a sclerite response to Aspergillosis infection suf fered from higher mortality rates (Smith et al. 1998). The purple areas an increased resistance to infection, suggesting they sequester the fungal isolates and preventing disease spread. Whether the purple coloring is a by product of the defense mechanism or a part of the defense mechanism is undetermined (Alker 2004). The only observed difference between purple and clear sclerites is the type of carotenoids present (Leverette et al. 2008). The exact role of the carotenoids is unknown, but certain carotenoi ds may have antifungal properties in plants (Hymete et al. 2005). The observation further suggests that the sclerites act as an immune response to infection. Figure 2 Purple and clear sclerites. Bright eld microscopic image of purple and clear sclerites. Purple arrow indicates purple scl erite and black arrows indicate clear sclerite s Image taken by Leverette and colleagues (2008).
! 1.3 Aspergillosis Epizootic Aspergillosis is an extensively studied coral disease in the Caribbea n, and one of the many diseases first identified during the 1990s (Smith and Weil 2004; Weil 2004). Aspergillosis is considered one of the only long term, widespread epizootics in the Caribbean (Weil 2004). The Aspergillosis outbreak had distressing e ffect s on reef populations resulting in a greater than 50% loss in sea fan tissue in the Florida Keys between 1997 and 2003 and affected at least 8 species of gorgonians (Kim and Harvell 2004 ; Ward et al. 2007 ). Nagelkerken and colleagues (1997) first identifi ed the disease in 1995. Aspergillosis may have also been a factor in the mass mortalities seen in Trinidad (Laydoo 1983) and off the coast of Costa Rica during the 1980s (Guzm and Corts 1984). The prevalence of Aspergillosis on coral reef s peaked between 1994 and 1997 (probably due to increased host resistance, changes in environmental conditions, or decreased virulence of the disease ) but there have been a number of smaller outbreaks noted over the years during May 1998, July 2000, and August 2002 ( Harve ll et al. 2007 ; Kim and Harvell 2004) M ore recent studies found a gradual increase in disease prevalence (Flynn and Weil 2009) Complete colony death as a result of an Aspergillosis infection has not been seen since the disease was first observed in 1995 (Nagelkerken et al. 1997). The disease was originally noted due to the presence of lesions, purpling, galls, and signs of necrotic tissue near the basal regions of Georgonia ventalina and Gorgonia flabellum sea fans (Figure 3 ) (Nagelkerken et al. 1997). T he disease observed in Costa Rica during the early 1980s was probably Aspergillosis due to the presence of tumors on infected fans (Guzm and Corts 1984).
! $! Figure 3 D iseased coral colony off shore of Cayo Grande. Arrow indicate s distinctive purple galling that is a symptom of Aspe rgillosis. Photo taken by Julie Krzykwa In 1998 the opportunistic fungal pathogen Aspergillus sydowii was identified by its 18s ribosomal RNA sequence and morphology as the pathogen for the disease (G eiser et al. 1998; Smith et al. 1996). Koch's postulates were fulfilled by isolating A. sydowii hyphae from an infected fan, inoculating healthy fans with the fungus, and then re isolating hyphae from the newly infected fans (Smith et al. 1996). Aspergillo sis sydowii is a terrestrial fungus typically found in soil worldwide ( Klich et al. 1992; Klich 2002). Aspergillus sydowii is a filamentous fungus with a large conidial head con taining asexual spores (conidia ) upon an erect conidiophore that arises from th e substratum the fungus settled upon (Figure 4) (Raper and Fennell 1965 ; Yager 1992 ). Strains of Aspergillus reproduce by asexual means, although there is genetic evidence of sexual reproduction in specific strains ( Dyer and Paoletti 2005; Horn et al. 2009 ; Raper and Fennell 1965) During asexual reproduction, or sporulation, the conidia develop after the growth of the coni diophore and are released to germinate (Figure 5 ) ( Horio 2007; Yager 1992).
! $$ Sclerotia, food reserves contained in compact masses of hard ened fungal mycelium that have been identified as survival structures used during times of hostile environmental conditions ( Coley Smith and Cooke 1971), are commonly produced by A. parasiticus and are found in other strains, (Horn et al. 1996) These scl erotia may be associated with sexual reproduction in Aspergillus but have not been conclusively tied to reproduction (Horn et al. 2009; Figure 4 Aspergillus sydowii Conidial head and part of conidiophore (x 8000). Photos taken by Klich ( 2002).
! $% F igure 4 A sexual reproduction of Aspergillus nidulans Conidia d evelop, are released, then germination and hyphal growth occurs. Image by Horio (2007). Since its original identification as the pathogen for As pergillosis there ha s been some observations that question whether A. sydowii is actually the pathogen of the disease. Toledo Hernndez and colleagues (2008) found fungi that are considered pathogenic, including Aspergillus sydowii on uninfected fans while t hey only isolate d A. sydowii from apparently uninfected fans. Zuluaga Montero and c olleagues (2010) concluded that Aspergillosis was caused by a number of different pathogens, rather than just A. sydowii as they did not collect A. sydowii spores from infected fans off of Puerto Rico However, most previous evidence indicates A. sydowii as the pathogen (Geiser et al. 1998; Kim and Harvell 2004; Nagelkerken et al. 1997; Rypien 2008b; Smith et al. 1996) and A. sydowii is commonly acknowledged as the pathogen of the disease (Harvell et al. 2006; Raghukumar and Ravindran 2012; Weil and Gil Agudelo 2006). This suggestion is bolstered by findings that A. sydowii may be the causative agent of an Aspergillosis outbreak in Annela spp. in Thailand after the 2004 t sunami (Phongpaichi t
! $& et al. 2006) and an Aspergillosis outbreak in the marine sponge Spongia obscura in the Caribbean (Ein Gil et al. 2009) Observations of Aspergillosis like symptoms after exposing healthy sea fans to A. sydowii also indicates that A. sydowii at least pla y an important role in the spread of Aspergillosis (Mydlarz et al. 2008). Despite how well studied Aspergillosis is many important aspects of understanding both the disease and sea fan resistance to the disease are still very unclear, including the method of transmission and the origins of pathogenic A. sydowii It is important to identify how A. sydowii was introduced and how it spreads to understand the far reaching impacts of Aspergillosis, and what may have caused the epizootic. Understanding coral dis ease is important, as coral epizootics are one of the primary causes of coral reef degradation and coral loss (Aronson et al. 2003; Aronson and Precht 1997; Porter and Meier 1992; Richardson 1998). 1.4 Effects of Aspergillosis on Sea Fan Populations The effects of Aspergillosis on sea fan populations in the Caribbean have been declining in recent years due to a general shift in the demographics of the sea fan population itself. Depending on the reef, differing susceptibility among species has resulted in a change of the dominant species of gorgonians along individual reefs. While Aspergillosis has been observed to cause partial or whole colony mortality (Alker 2004; Kim and Harvell 2004; Nagelkerken et al. 1997; Ward 2007), usually the coral colonies will shed portions of the sea fan as a result of infection and lesion growth near the base (Kim and Harvell 2004). When this defense mechanism of shedding is combined with the increased likelihood of infection in larger sea fans (Bruno et al. 2011; Dube et al. 2002; Kim et al. 2000; Kim and Harvell 2002; Kim and Harvell 2002; Kim and Harvell
! $' 2004), the result is a sea fan population that has been shifted towards more smaller, younger sea fans, and fewer larger, more mature sea fans (Bruno et al. 2011; Kim and Ha rvell 2004). Slower growth rates have been observed in infected sea fans, further reducing the return of larger sea fans during times of great disease prevalence (Toledo Hernndez et al. 2009) This skewing of the populat ion towards smaller sea fans effec ts the recovery of Gorgonian sea fans in the Caribbean, as larger sea fans have greater reproductive output (Beiring and Lasker 2000). This means that as the population in the Caribbean shifts towards smaller and less reproductively active sea fans, recove ry of the gorgonian population may be delayed by slow recruitment rates (Kim and Harvell 2004). Further reducing reproductive rates and potentially recruitment rates, is the effect of Aspergillosis infection on the reproductive system of gorgonian sea fan s. Petes and colleagues (2003) noted that in infected female gorgonian fans, there was reduced reproductive effort, and a larger percentage of infected fans were non reproductiv e when compared to healt hy fans. Kim and Harvell (2004) found a correlation bet ween disease prevalence and recruitment along reefs in the Florida Keys and observed decreased recruitment in periods of high disease prevalence There is also a reduction in the symbiont density in coral tissues in gorgonians infected with Aspergillus syd owii (Kirk et al. 2005; Ward et al. 2007). This may decrease the overall fitness of the colonies, decreasing their ability to recover from the disease. The decreased symbiont density might explain the reduction in reproductive effort, as more energy is nee ded for survival when the symbiont density is reduced. The size dependence of Aspergillosis might help explain the reduction in prevalence of Aspergillosis since the height of the epizootic in the late 1990s. Smaller
! $( sea fans that were resistant to the di sease most likely survived, allowing the population to become more resistant to Aspergillosis. Bruno and colleagues (2011) predicted that at least partly due to this selection process, and partly due to some more resistant genotypes in the population, curr ent populations are more likely to recover from an Aspergillosis infection and have a lower infection risk. This idea of some genotypes being more resistant to Aspergillosis than others coincides with the observed high variation in sea fan resistance to As pergillosis infection (Dube et al. 2002; Kim et al. 2000; Kim et al. 2006; Mullen et al. 2006). The reduction in disease prevalence may also be a result of evolution by the pathogen to cause reduced mortality rates of their host populations, as to prevent extinction of its host ( Ewald 1993; Nesse 2008) 1.5 Origins of Aspergillosis sydowii One of the major questions concerning Aspergillosis in sea fans is how the fungal disease was introduced to the marine environment. There are a series of hypotheses su ggesting how a terrestrial soil microbe developed into a virulent coral pathogen. The "endemic marine hypothesis" is based on the belief that the microbe has been present in the marine environment for an unknown period of time, but only recently developed into a coral disease (Rypien et al. 2008). Evidence supporting this hypothesis includes the identification of Aspergillus species in water samples collected before the outbreak (Roth et al. 1964; Sparrow 1937), including A. sydowii (Kendrick et al. 1982). Various environmental changes especially increasing water temperatures, could have facilitated the progression of A. sydowii from a n endemic microbe to a pathogen.
! $) Increasing water temperature has a detrimental effect on t he antifungal defenses of coral and may increase the severity of Aspergillosis infections. D uring hotter summer months there is an observed increase in coral disease (Kim and Harvell, unpublished data phide Alker et al. 2001). Increased water temperature (30¡C) increases growth and dise ase activity by Aspergillus sydowii when compared to a baseline water temperature around 27¡C (Alker et al. 2001; Ward et al. 2007). The efficiency of coral antifungal compounds is temperature dependent. Alker and colleagues (2001) concluded that the main effect of increased temperature better allows the fungus to overcome the coral defenses. The expulsion of zooxanthellae in many species around 30¡C (see review by Jokiel 2004) may contribute to the decreased immune capabilities of gorgonian sea fans. This link between disease activity and water temperature is also supported by the smaller outbreaks observed by Kim and colleagues (2004) during May 1998, July 2000, and August 2002, and similar observations by Flynn and Weil (2009). N utrient enrichment of offs hore waters may also have contributed to the severity of the observed Aspergillosis outbreaks (Bruno et al. 2003), but there has not been a significant correlation established between disease prevalence and distance from shore (Flynn and Weil 2009). While this evidence would suggest a link, conclusive evidence has not been found to support the endemic hypothesis. Another hypothesis is the "African Dust Hypothesis". This hypothesis suggests that a pathogenic strain of A. sydowii was introduced into the marin e environment via dust carried from the Sahara Sahel region of Africa (Garrison et al. 2006; Garrison et al. 2003; Shinn et al. 2000). Husar and colleagues (1997) established that the dust can be carried across the Atlan tic via wind and that in some cases pathogenic organisms including Aspergillus spores have been isolated from African dust samples (Kellogg et al.
! $* 2004; Shinn et al. 2000). While investigating the African Dust Hypothesis, Rypien and colleagues (2008a) proposed two versions of the hypothesis The single origin African Dust Hypothesis predicted that the terrestrial isolates collected from Africa would be closely related to the pathogenic marine isolates, and t he "multiple origin hypothesis" that predicted the pathogenic isolates would be close ly related to terrestrial isolates collected in Africa (Rypien et al. 2008 a ; Rypien 2008b). When samples of airborne dust from Africa and the Caribbean and terrestrial samples from Africa and the Cape Verde Islands were tested for A. sydowii no evidence w as found to suggest that the coral pathogen was present (Rypien 2008a). The lack of pathogens present in the airborne dust, as well as previous observations that terrestrial A. sydowii does not induce Aspergillosis in sea fans (Geiser 1998) make it unlike ly that the pathogenic strains of A. sydowii infecting Caribbean gorgonians originated in airborne dust blown over the Caribbean from Africa. The third main hypothesis on the potential origins of pathogenic Aspergillus sydowii is the "Terrestrial Run Off Hypothesis". Originally proposed by Smith and colleagues (1996), the Terrestrial Run Off Hypothesis states that pathogenic A. sydowii is entering marine ecosystems through terrestrial run off It is believed that A. sydowii may have become more prevalent i n coastal waters in recent years as a result of increased coastal building, leading to increased terrestrial run off (Burke and Maidens 2004). The finding by Geiser and colleagues (1998) that terrestrial strains of A. sydowii were not pathogenic to sea fan s, whereas strains isolated from infected sea fans were pathogenic, undermines this hypothesis Alker and colleagues (2001) suggest instead that coral disease causing A. sydowii originated as a terrestrial strain and underwent rapid evolution, resulting in the pathogenic A. sydowii observed during t he epizootic
! $+ One important aspect of the Terrestrial Run Off Hypothesis is determining how pathogenic Aspergillus sydowii isolated from sea fans could have evolved from common terrestrial A. sydowii In the rel ated A. fumigate which is pathogenic in immune compromised humans, dogs and wild birds (Table 2 ), it was found that there was no genetic difference between fungi isolated from environmental sources, and fungi isolated from clinical samples when a non hier archal principle component analysis was used (Debeaupuis et al. 1997) When a 485 base pair portion of the 5' non translated region of the trp C gene from terrestrial and marine pathogenic A. sydowii were compared, minimal difference was noted (a maximum of 1.1% sequence divergence was observed) (Geiser et al. 1998) These results indicate that a small genetic differentiation could have allowed A. sydowii to become a marine pathogen. However, A. sydowii isolated from infected sea fans has been shown to diffe r from terrestrial isolates metabolically (Alker et al. 2001) In an analysis of terrestrial clinical and saprotrophic A. sydowii genetic differences were observed in the nucleotide sequences of the TS1 5.8S ITS2 loci and in the D1/D2 regions of the 28S rD NA resulting in 2 different groups of A. sydowii but the groups did not correlate with the clinical or saprotrophic groups, indicating a lack of genetic difference in these regions between the clinical and saprotrophic A. sydowii (Marfenina et al. 2010) More research into the genetic differences between terrestrial and marine pathogenic A. sydowii is necessary to determine what mutations, if any, allowed the fungus to move to the marine environment and to become infectious to gorgonians.
! $, Table 2 Pathogenic strains of Aspergillus and the organisms affected. Strain Organism(s) Source(s) A. fumigatus A. flavus A. terreus A. niger A. beijingensis A. qizutongii A. wangduanlii. Humans Klich 2006 Li et al. 1998 Raper and Fennell 196 5 A. flavus Cattle Ticks Miranda Miranda et al 2012 A. ochraseus Dog Ticks Denning 1998 A. fumigatus Dogs Denning 1998 A. fumigatus Aspergillus spp. Horses Pace 1994 Denning 1998 A. fumigatus A. flavus Wild Birds (Including shorebirds, songbirds, wate rfowl, crows, and game birds) Pier and Richard 1992 Friend 1999 A. aruii A. fumigatus Dairy Cows Miller 1977 Knudston and Kirkbride 1992 Aho et al. 1994 A. flavus Tilapia Olufemi and Roberts 1986 A. avus Plants Munkvold 2003 A. fumigatus Rabbits Niy o et al. 1988 While there is evidence supporting some portions of the different theories, conclusive evidence has not been found to disprove any of them. It seems mostly likely that there was not a single origin of pathogenic A. sydowii but that ins tead A. sydowii is an opportunistic pathogen that was able to infiltrate the marine environment (Rypien et al. 2008). It is likely that A. sydowii has a high enough phenotypic plasticity and evolutionary potential that it is able to adapt readily to new en vironments and cause emergent infectious diseases (Rypien 2008b). Other strains of Aspergillus are opportunistic pathogens of humans as well as a number of terrestrial organisms (Table 2 ) (Denning 1998; Rinaldi 1983). Out of the 200 known strains of Asperg illus 40 of them cause Aspergillosis in humans (Klich 2006 If other strains of Aspergillus have been able to adapt as opportunist pathogens it is likely that the disease causing strain of
! %! Aspergillus observed in the Caribbean has done the same. A. sydowii is an especially salt tolerant strain, which would allow it to adapt to the marine environment more readily (Geiser et al. 1998). A molecular study of A. sydowii demonstrated that between pathogenic and environmental isolates there was little, if any, gen etic differentiation (Rypien et al. 2008; Rypien 2008b). Rypien (2008b) suggests that external stress is more likely to explain the variations observed in Aspergillosis prevalence, rather than the strain of A. sydowii present but further genetic analysis is required. 1.6 Transmission of Aspergillosis Another major aspect of the Aspergillosis epizootic that has yet to be determined is how Aspergillosis was spread once it successfully entered the marine environment. A number of potential hypotheses have b een proposed, but none of them have fully explained the observed disease spread in the Caribbean. The "Terrestrial Hypothesis" states that Aspergillosis spreads only via fungal hyphae or spores being carried from terrestrial sources, such as terrestrial ru n off or from airborne dust (Shinn et al. 2000; Smith et al. 1996). The "Direct Contact Hypothesis" is usually paired with the Terrestrial Hypothesis and suggests that as a secondary method of transmission Aspergillosis can be spread through infected sea fans coming into contact with uninfected sea fans during times of significant water movement (Smith et al. 1996). This hypothesis is supported by obser vations that in the Yucatan in areas where there was higher sea fan density also had higher disease preva lence (Mullen et al. 2006). This higher disease prevalence may also be because in cases of high sea fan density more closely related sea fans may be clustered together in an area as a result from reproduction via fragmentation ( Heyward and Collins 1985; H ighsmith 1982; Smith and
! %$ Hughes 1999) Kim and Harvell (2004), however, found that there was no relationship between the density of sea fans and the prevalence of Aspergillosis. The "Water borne Infection Hypothesis" proposes that fungal hyphae can be rele ased into the water column by infected sea fans, allowing the hyphae to come into contact with uninfected sea fans (Jolles et al. 2002). Because of the lack of conclusive evidence supporting any of these hypotheses, it has been suggested that there are sec ondary methods of transmission, such as coral predators that act as vectors for the disease. Jolles and colleagues (2002) investigated the spatial pattern of Aspergillosis disease transmission and observed that there were clusters of infected fans at a 1 3 m scale, indicating that vectors are a likely form of disease transmission in areas with high disease prevalence. It is also possible that in this close of an area the high rate of infection may result from similar genetic makeup. Because recruitment of go rgonian corals can occur asexually through fragmentation, it is possible that the gorgonians in such a small scale are clones of one another, and therefore have the same level of susceptibility to the disease (Heyward and Collins 1985; Highsmit h 1982; Smit h and Hughes 1999). Invertebrates have been linked to disease transmission for a number of coral diseases. In the Caribbean a species of corallivorous gastropod Coralliophila abbreviata was a vector for the White Band Disease outbreak that infected Acrop ora cervicornis corals in 2003. Williams and Miller (2005) suggested that in this particular instance it seemed likely that the gastropod also exacerbated the disease outbreak by exhibiting increased foraging on infected corals. In the Red Sea, White Band Disease was associated with feeding activities by the corallivorous gastropod Drupella conzus (Antonius and Riegl 1997), and in Mediterranean Sea, the marine fireworm Hermodice
! %% carunculata was a suspected vector for Vibrio shiloi a coral disease that res ults in coral bleaching (Sussman et al. 2003). While corallivorous vertebrates are also possible vectors for Aspergillosis, an invertebrate is a more likely candidate While corallivorous fish have a major role in disease ecology along coral reefs it has not been concluded whether they prevent or promote the spread of coral diseases (Chong Seng et al. 2011). In a study looking at the feeding preference of a number of different coral reef fish, obligate corallivorous fish were observed to feed on both heal thy and diseased tissue, but whether they spread the disease is unknown (Rogers 2008). Aeby and Santavy (2006) found that butterfly fish might be vectors for black band disease in Montastraea faveolata thus providing evidence that corallivorous fish may a ct as vectors for coral disease. However, r esults by Cole and colleagues (2008) suggest that feeding by corallivorous fishes may actually prevent the spread of coral diseases, specifically black band disease Raymundo and colleagues (2009) supported these results; they found decreased disease prevalence along reefs where there was an increase in predation on corals by corallivorous fish. These findings indicate that if there is a vector for Aspergillosis it is most likely a corallivorous invertebrate as th e role of corallivorous fish as disease vectors is much more uncertain.
! %& 1.7 Cyphoma gibbosum as a likely vector Figure 5 Cyphoma gibbosum feeding on g o rgonians. Photos taken by Julie Krzykwa The most common corallivorous i nvertebrate predator on gorgonians in the Caribbean and South Atlantic is Cyphoma gibbosum or Flamingo Tongue Gastropod (Figure 6 ) This species of gastropod is dependent on gorgonians for food, habitat, an d oviposition sites, making the gastropods a like ly vector for the disease as they move from coral colony to coral colony. When C. gibbosum was studied in a laboratory the gastropods were able to pass active Aspergillus sydowii spores and hyphae, and C. gibbosum consumed significantly more food when the artificial food contained diseased extracts (Rypien 2008b). H ow frequently this occurs in a natural setting and the level of mortality experienced by the fungus during digestion is also unknown. Further evidence that C. gibbosum is a vector for Aspergillo sis is that in cases where there was a high level of disease prevalence there appeared to be an increase in the C. gibbosum population, and C. gibbosum grazing increased on infected sea fans (Slattery 1999). Nagelkerken and colleagues (1997) noted that dur ing their research most of the C. gibbosum they encountere d were found on infected gorgonians This preference for feeding on infected sea fans demonstrates the potential impacts of Aspergillosis on non host species. If there is preferential feeding on inf ected sea fans, and the gastropod s
! %' frequently come into contact with Aspergillosis lesions along the bases of infected sea fans (Figures 1 and 3) it is possible that the gastropod s pick up Aspergillus sydowii spores and hyphae during their movements on inf ected sea fans, and can transmit the fungus to healthy sea fans. It is also possible that if the gastropod s do carry A. sydowii spores and hyphae between sea fans, the gastropod s might exasperate infections present in gorgonians that are already infected w ith Aspergillosis. It is possible that during the feeding and mating movements of Cyphoma gibbosum the gastropod s come into contact with Aspergillus sydowii and carry fungal hyphae and spores to other sea fans they travel to (Gerhart 1986; Lasker et al. 19 88; Nowlis 1993). Especially due to observations that C. gibbosum consume the coenenchym along axial regions of sea fans, right where Aspergillosis lesions are usually observed (Harvell and Fenical 1989). The gastropod s travel from fan to fan frequently; o n average gastropod s appear to move to a new sea fan every three days (Rypien, unpublished phide Rypien 2008b). This is very similar to the observed passage time of two days for ingested food. Rypien and Baker (2009) postulated the potential for a positive feedback loop where the gastropod s spread Aspergillosis once it is present in an area, leading to an increase in the prevalence of the disease. The increased prevalence of Aspergillosis then increases the likelihood of the gastropod s consuming diseased po lyps and encountering A. sydowii hyphae and spores. If this feedback loop is correct, reefs that are experiencing overfishing and low top predator densities may be especially vulnerable to Aspergillosis, as C. gibbosum populations have been known to increa se when there is a reduction in predators (Burkepile and Hay 2007). If there is a predator release of C. gibbosum along a reef, and C. gibbosum is indeed a vector for Aspergillosis, it is possible that the reef will be hit harder with the disease due to th e increased number
! %( of gastropod s acting as vectors for the disease (Rypien 2008b). Burkepile and Hay (2007) observed that in systems where top predators were removed from the ecosystem due to overfishing gorgonian defenses appeared to be ineffective agains t predators such as Cyphoma gibbosum This reduction in the effectiveness of gorgonian defenses might also increase the prevalence of Aspergillosis along reefs suffering from overfishing. Based on the intermittent distribution pattern of Cyphoma gibbosum that is usually observed in the field (Chiappone et al. 2003; Harvell and Suchanek 1987; Lasker and Coffroth 1988), it is highly improbable that they would act as the sole means of disease transmission. Even if Cyphoma gibbosum does not explicitly act as a vector for Aspergillosis, the effects of C. gibbosum grazing may make gorgonians more likely to contract Aspergillosis. Grazing by Cyphoma gibbosum is known t o accelerate colonization of sea fan s by fouling organisms by exposing the internal skeleton (Ge rhart 1990; Wahle 1985). By exposing the inert (non cellular) skeleton, C. gibbosum grazing can also increase the likelihood of fungal infection, as most A. sydowii hyphae have been observed to be located within the inert skeleton (Mullen et al. 2004; Smit h et al.1998). However, it has also been observed in a laboratory setting that Aspergillosis could be transmitted without the presence of scar r ing from C. gibbosum grazing (Smith et al. 1996). These observations imply that C. gibbosum may have a role in th e transmission of Aspergillosis, but there are most certainly other forms of disease transmission. 1.8 Gorgonian defense against predators One of the major interactions between Cyphoma gibbosum and their prey is through secondary metabolites (SMs) that s erve as a chemical defense mechanism against predators (Hay 1996; Hay and Kubanek 2002). These SMs are taken up when an
! %) animal consumes tissue that contains the metabolites. In the case of Cyphoma gibbosum the coral polyp and coen chyme it consumes contain these SMs, which are then absorbed, distributed (or detoxified), and excreted (Sotka et al. 2009). Organisms that are especially vulnerable to predators such as benthic, sessile, or highly conspicuous organisms are most likely to develop this type of chem ical defense against predation (Cimino and Ghiselin 1998; Paul and Puglisi 2004; Pawlik 1993). In the case of aquatic and marine invertebrates such as corals, these SMs are generally alkaloids, terpenoids, and compounds resulting from acetogenin pathways ( Harper et al. 2001). In a study investigating the effectiveness of lipophilic crude extracts as a chemical defense against predation in 7 species of gorgonians, researchers observed that crude extracts from the tips of 6 out of the 7 species successfully deterred predation by fish at natural concentrations (Puglisi et al. 2002). This may be in part due to increased concentrations of both chemical and physical defenses along the outer edges of the gorgonian sea fans (Harvell and Fenical 1989; Puglisi et al. 2002). These increased concentrations are most likely due to the addition of new polyps near the edges of the colony (Szmant Froelich 1974). Gerhart (1984) showed that in the gorgonian Plexaura homomalla prostaglandin A2 (PGA 2 ) was found in very high co ncentrations, specifically in the form of 15(R) PGA and 15(S) PGA. Both 15(R) PGA and 15(S) PGA when consumed orally in concentrations that could be consumed by predators, induced vomiting in killifish and yellowhead wrasse. Although when PGA s was looked a t as a chemical defense against predation in the field it was determined that the extract did not have a significant impact as a chemical defense (Pawlik et al. 1987).
! %* Gorgonians and other corals rely both on their chemical defenses as well as on physica l defenses. In gorgonians this physical defense appears to be mainly in the form of sclerites (Van Alstyne and Paul 1992). Koh and colleagues (2000) showed that certain species of gorgonians had sclerites that were more effective at deterring predators in field bioassays than other species. West (1998) observed that long sclerites, and a high concentration of sclerites, in artificially prepared food was a deterrent to feeding by Cyphoma gibbosum and Harvell and Suchanek (1987) noted a negative correlation between the foraging time of C. gibbosum and the proportion and size of sclerites. It is possible that longer sclerites hinder ingestion or egestion of food products, as sclerites have been noticed in Cyphoma gibbosum feces (Birkeland and Gregory 1975; Wes t 1998). O'Neal and Pawlik (2002) found that sclerites were not effective at deterring generalist predation. It was hypothesized that the corals relied more heavily on their chemical defenses. This supports conclusions from Puglisi and colleagues in a stu dy done in Guam on Viminella sp. that mainly relied on its physical defenses. In a review by Paul and Puglisi (2004) it was noted that in different regions, and in different species, the effectiveness of sclerites as a defense mechanism varied, perhaps par tly explaining why sclerite content alone did not provide an accurat e predictor of gastropod foraging. 1.9 Effects of Cyphoma gibbosum grazing The effects of Cyphoma gibbosum grazing on coral colonies does not usually end in whole colony mortality altho ugh there have been cases of Cyphoma gibbosum outbreaks that resulted in the loss of an estimated 90% of shallow (<40m) gorgonian colonies (Schrer and Nemeth 2010). In cases where there are not large outbreaks, the known effects of C. gibbosum grazing are various sub lethal effects such as increased
! %+ fouling on the coral colony (Gerhart 1990; Wahle 1983) or increased defense response by the sea fan such as increased sclerite length (West 1997). Grazing by predators such as Cyphoma gibbosum leaves the axial skeleton of a coral colony exposed. This exposure of the axial skeleton can result in the settlement of epibionts, resulting the fouling of the coral colony (Gerhart 1990; Wahle 1983). Because Aspergillus sydowii spores are usually observed in the axial sk eleton, it is also possible that sea fans that have exposed segments of axial skeleton are more likely to suffer from Aspergillosis (Mullen et al. 2004; Smith et al. 1998). Another effect of Cyphoma gibbosum grazing is an alteration in the physical defens es of the gorgonian. Sclerites are generally longer near the edges of sea fans, but w hen predator damage was simulated on the gorgonian Briareum asbestinum the sclerite morphology changed and resulted in longer sclerites in the central region of the fan ( West 1997) The simulated predator damage caused the morphology of the sclerites to better adapt to C. gibbosum feeding, resulting in longer sclerites in the central region of the fan where the gastropods typically feed 1.10 Cyphoma gibbosum Prey Prefer ence Distinct prey preferences have been observed in Cyphoma gibbosum (Nowlis 1993), and Chiappone and colleagues (2003) concluded that there are enough gorgonian hosts that C. gibbosum is not food limited, allowing gastropod s to select for certain host t raits. In a census of C. gibbosum distribution in Panama, C. gibbosum was seen to prefer specific species of gorgonians and strong individual host preferences were apparent (Lasker and Coffroth 1988). Nowlis (1993) looked at the movements of C. gibbosum an d noted that there was a distinct preference for specific hosts when analyzed by the
! %, frequency each host was occupied, and when those frequencies were cross classified by the species of the previous host. Gerhart (1986) suggested that this preference was b ased on the presence of mucus trails that allows gastropod s to influence the movements of other gastropod s. But what guides which hosts are selected? There are a variety of possibilities as to what factors influence C. gibbosum prey preferences. Researc h has shown that gorgonian defenses such as sclerites and secondary metabolites (Harvell and Suchanek 1987; Van Alstyne and Paul 1992), do not aptly explain the observed movements and distribution of Cyphoma gibbosum (Lasker et al. 1988; West 1997). Observ ations of C. gibbosum foraging patterns have indicated that gregariousness plays an important role in the host selection and distribution of C. gibbosum (Nowlis 1993). Some studies have suggested that the prey preference of Cyphoma gibbosum was depe ndent o n the presence of defense mechanisms such as the presence of secondary metabolites (Gerhart 1984; Hay et al. 1987; Lasker et al. 1988; Steinberg 1985) and sclerites (Harvell and Suchanek 1987; Harvell and Fenical 1989; West 1997), which may explain the pre ferences displayed by gastropod s for specific species and sea fans, but does not fully explain observed foraging patterns (Chiappone et al. 2003; Gerhart 1986; Lasker and Coffroth 1988). Lasker and colleagues (1988) observed very little correlation between the feeding patterns of C. gibbosum and the presence of chemical de fenses in its gorgonian prey. This might be because C. gibbosum is usually observed feeding along axial regions of sea fans where the defensive secondary metabolite concentration of sea fa ns is less (Harvell and Fenical 1989; Puglisi et al. 2002). Lasker and Coffroth (1988) determined that sclerite content was not an accurate predictor of Cyphoma gibbosum foraging behaviors; instead species preferences and various other processes appeared t o
! &! have a greater effect on the distribution Differences in the effectiveness of sclerites (Koh et al. 2000; Paul and Puglisi 2004) may explain the observed C. gibbosum preference for certain species of gorgonians (Birkeland and Gregory 1975; Chiappone et al. 2003; Gerhart 1986; Lasker and Coffroth 1988). It has also been hypothesized that ATP Binding Cassette (ABC) transporters may have a significant influence on the diet choices of organisms that absorb allelochemicals through their d iets, as ABC transpor ters transport and possibly absorb the allelochemicals (Sorensen and Dearing 2006; Sotka and Whalen 2008; Whalen et al. 2010a). Lasker and colleagues (1998) discussed the potential for gastropod s to graze preferentially for species that would provide the most organic content with the least effort. This hypothesis supported their observations of Cyphoma gibbosum preferring two species of gorgonian, but the third preferred gorgonian was predicted to be the poorest food choice (Lasker et al. 1988). While the ir results did not conclusively determine that C. gibbosum feeds preferentially for higher quality food, other researchers have found indications that it is possible that invertebrate predators might forage based on prey quality. Mayntz and colleagues (2 005) examined invertebrate foraging patterns specifically for indications of nutrient specific foraging. They determined that invertebrate predators might be able to rectify imbalances in their diet concerning proteins and lipids by adjusting what types of prey are consumed or by selectively extracting nutrients from wi thin individual prey items. The study mostly focused on insects and arthropods, but the results may suggest an alternative selection process behind the observed Cyphoma gibbosum foraging patt erns. Female ground beetles, Anchomenus dorsalis also showed this ability to regulate their nutrient uptake to
! &$ maximize fitness (Jensen et al. 2012), proving that this ability to selectively forage for nutrients is present in predatory organisms. Based on studies concerning food availability and foraging for nutrition in the forest tent caterpillar, Malacosoma disstria (Despland and Noseworthy 2006), locusts (Simpson et al. 2002; Simpson and Raubenheimer 2000), and the African armyworm, Spodoptera exempt a (Lee et al. 2003; Lee et al. 2004) it is likely that C. gibbosum is able to compensate for dietary imbalances through prey selection due to its wider dietary breadth. A variety of organisms have been identified as selectively foraging for needed nutrient s (Morales Ramos et al. 2011) resulting in the nonrandom foraging distribution like what is seen in the case of C. gibbosum (Houston et al. 2011). 1.11 Uptake of Gorgonian Toxins As predators of coral species, specifically gorgonian corals, that produce s econdary metabolites as a defense mechanism Cyphoma gibbosum has had to evolve to cope with the natural defenses of sea fans. Cyphoma gibbosum is not immune to these chemical and physical defenses, but instead has developed adaptations that reduce the effe cts (Van Alstyne and Paul 1992). The allelochemicals are taken up by Cyphoma gibbosum thr ough the coral polyps and coenchyme consumed by the gastropod s (Harvell and Fenical 1989). Once the allelochemicals are present in the gastropod 's gut they are slowly transformed into a hydrophilic compounds that are easier to excrete than the toxic lipophilic compounds ingested (Sotka and Whalen 2008). Because the ingested lipophilic compounds are excreted, they are not retained in the body chemistry of Cyphoma gibbosu m (Cronin et al. 1995).
! &% There are three described phases in the detoxification process concerning marine organisms that feed on chemically defended prey (Figure 7 ) Phase I of the process is accomplished by a group of heme thiolate enzymes that are collec tively referred to as cytochrome P450 monooxygenases (CYPs) (Parkinson 2001). There is evidence that some of these CYPs comprise an inducible response to the presence of gorgonian allelochecmicals in Cyphoma gibbosum (Whalen et al. 2010b). These enzymes he lp to detoxify the ingested allelochemicals through the addition of a polar group to the compound (Sotka and Whalen 2008). There are a number of different phase II reactions (Parkinson 2001), but the reaction most discussed concerning Cyphoma gibbosum is o ne involving Glutathione S transferases (GSTs). These GSTs are highly expressed in C. gibbosum (Whalen et al. 2010). GTSs help in detoxification by acting as a catalyst for a nucleophilic attack of reduced lipophilic substrates that result from phase I (So tka and Whalen 2008). This process is usually referred to as conjugation and through the attachment of compounds such as glutathione, glucuronic acid, or glycine (Gonzalez and Nebert 1990; Sheehan et al. 2001) to the polar groups attached during phase I th e xenobiotic allelochemicals are rendered hydrophilic and ready for transport out of the cell (Sotka and Whalen 2008). In the third phase of cellular detoxification of xenobiotic allelochemicals ATP Binding Cassette (ABC) transporters transport the product s of the Phase II reaction and other metabolites across the cellular membrane (Sotka and Whalen 2008) or into subcellular organelles for processing (Oude Elferink et al. 1993; van Luyn et al. 1998). It is also possible that ATP Binding Cassette (ABC) trans porters are also responsible for absorption of allelochemicals (Sorensen and Dearing 2006; Sotka and Whalen 2008; Whalen et al. 2010a).
! && Figure 6 Overview of uptake of allelochemicals in marine herbivores. Diagram by Sotka and Wh alen (2008). In Cyphoma gibbosum GSTs were not observed to vary with the concentration of allelochemicals in their diet (Whalen et al. 2010). This is unusual I n other mollusks whose GST levels were monitored in relation to the quantity of allelochemical s present in their diet, GST levels increased as the concentration of allelochemicals increased (DeBusk et al. 2000; Kuhajek and Schlenk 2003) Whalen and colleagues (2010) hypothesized that t he production of GSTs may be independent of the quantity of alle lochemicals present in the diet of C. gibbosum as a result of the large amount of secondary metabolites found in their go rgonian diet. In a study by Whalen and colleagues (2008) it was observed that C. gibbosum had especially high levels of GSTs present in their gut when compared to other organisms (Le Pennec and Le Pennec 2003; Vrolijk and Targett 1992), which may allow C. gibbosum to feed for longer periods of time without suffering ill effects from allelochemicals. The levels of GSTs found in C.
! &' gibbosum also varied depending on the sea fan species being consumed, suggesting that it is at least to an extent, an inducible response (Vrolijk and Targett 1992). In the army fallworm ( Spodoptera frugiperda ) GSTs were observed to have an important role in the d etoxification of defensive secondary metabolites taken up through diet. Wadleigh and Yu (1988) suggested that GSTs activity is induced by the presence of toxic secondary compounds, including its own substrates. This may indicate that it potentially has a p rominent role in the detoxification and excretion of toxic chemicals ingested through feeding. In contrast to the high levels of GST observed in Cyphoma gibbosum only low levels of CYPs are present in C. gibbosum digestive glands (Vrolijk and Targett 1992) 1.12 Purpose and hypotheses for the survey In the present study the distribution of Cyphoma gibbosum along a portion of fringing reef off of Cayos Cochinos Grande in the Honduras Bay Islands was examined. It was hypothesized that C. gibbosum would be found more frequently on fans infected with Aspergillosis due to observed foraging preferences in laboratory settings (Rypien 2008b; Rypien and Baker 2009), and from field observations taken during Aspergillosis surveys (Nagelkerken et al. 1997; Slattery 1 999). If Cyphoma gibbosum preferentially selects for sea fans infected with Aspergillosis, it is possible that the gastropod s may act as a vector for the disease, and may further reduce the fitness of the sea fan due to feeding activities exposing the axia l skeleton.
! &( Chapter 2: Methods 2.1 Cyphoma gibbosum distribution survey The survey was done off of the shore of Plantation Beach Resort on Cayo Grande in the Cayos Cochinos archipelago. The survey data were collected from July 14 to 29, 2010 during the fi rst year of the survey and from July 24 to 29, 2011 during the second year. This archipelago is a part of the Honduran Bay Islands and is located 19 mi les from the main land (Figure 8 ) (Ives [Date Unknown]) Cayos Cochinos consists of two main islands and thirteen coral cays (Figure 9 ) (Ives [Date Unknown]). In 2003 a 489.25km 2 area, including the Cayos Cochinos islands, was declared a Marine Natural Monument under Legislative decree 114 2003. The Honduran Coral Reef Foundation (http://www.cayoscochinos.org ) is the agency responsible for the conservation of the Marine Natural Monument. Figure 7 Honduran Bay Islands. Red box indicates Cayos Cochinos. Image retrieved from http://www.caribbeantravelweb.com/honduras/cayoscochinos_gui de.htm
! &) Figure 8 Los Cayos Cochinos, red arrow ind icates location of survey. Image retrieved from http://www.moon.com/maps?filter0=75362 The area surveyed was a shallow (<3m) fringing reef in the southwestern bay of Cayo Grande; most observations were made along the reef crest and reef flat. Observations were made using snorkeling techniques, limiting the depth of the survey. Sites were found using a method similar to the roving diver technique (Schmitt et al. 2002). Researcher s snorkeled along the reef looking for useable sites where Cyphoma gibbosum was present on a sea fan. As a result there is some overlap in the area of the reef that was surveyed during years one and two, as can be seen in Figure 10 During the first year 36 sites were survey ed and during the second year 35 sites were surveyed. All of the surveying took place during the daytime, usually between 9am 11am, and between 2pm 5pm. It is not known if the C. gibbosum are mainly nocturnal or diurnal so the timing of the survey may have had an effect on the observations made but most of the snails observed appeared to be active while data collection was performed For both years the weather was sunny, with an occasional overcast day. During the first year of surveying there was one day where the water was too rough out for sampling due to a
! &* storm, and a couple mornings observations were not made due to a large number of unidentified stinging organisms being present. During the second year there was one day of r ain, and a couple mornings observations were not made due to similar unidentified stinging organisms. Figure 9 L ocation of survey sites from both years. Measurements indicate length of each line. Original map was courtesy of Alberto F enix.
! &+ Figure 10 General location of all sites from the first year of surveying. Measurements indicate length of each line. Original map was courtesy of Alberto Fenix. Information collected each year of the survey overlapped, bu t there was some information that differed. During the first year of the survey, field observations were made mainly along the back reef area of the bay in one region (Figure 1 1 ). Data concerning the species of sea fan were collected, but this was not cont inued during the second year of the survey due to a lack of significance and lack of reliability in identification of species. The number of gastropod s, depth of the site, and presence of Aspergillosis was also noted. Depth was recorded in feet using the d epth function of a scuba diving computer and was then converted to meters The presence of Aspergillosis was determined by the presence of purple lesions around the base of the fan, as shown in Figure 1 Figure 3, and Figure 12 This has been determined a s a reliable method for identifying the presence of Aspergillosis in the field (Alker 2004; Jolles et al. 2002; Kim et al. 2000; Nagelkerken et al. 1997; Petes at al. 2003; Smith et al. 1996). Photos were
! &, also taken of each site. These photographs were use d to determine the approximate surface area of the sea fan using AnalyzingDigitalImages Software (http://mvh.sr.unh.edu/software/software.htm) (Figure 13 ). Figure 12. Infected sea fan off of Cayo Grande. A) Lesions caused by Aspergillosis. B) Purpling an d galls caused by Aspergillosis. Figure 13. Demonstration of sea fan area measurements. A) Determining the consumed area. B) Determining the overall fan area. The pencil (14.5cm), indicated by red arrow, was used as a reference for determining size.
! '! The second year of the survey some of the aspects being investigated were slightly changed. Observations concerning species were no longer taken, and depth was no longer measured for each site. One of the main reasons that depth was not included during th e second year of the survey was because there was very little change in the depth of the individual sites. The number of gastropod s, and presence of Aspergillosis was once again noted. As in the first year of the survey signs of purple lesions, galling, an d pox marks were taken to indicate the presence of Aspergillosis (Alker 2004; Jolles et al. 2002; Kim et al. 2000; Nagelkerken et al. 1997; Petes et al. 2003; Smith et al. 1996). Photos were taken of each site to determine the approximate surface area of t he sea fan using AnalyzingDigitalImages Software Additional photographs were taken of individual mantle patterns and the apparent area consumed by the gastropod (s) currently grazing on the sea fan. It was assumed that the area consumed was the visible fee ding trail left by gastropod grazing (Gerhart 1990). The AnalyzingDigitalImages Software was then used to determine the surface area of the fan that had been fed u pon ( Figure 13 ). These values were then used to determine the percentage of the overall surfa ce area of the sea fan that had been fed upon. Another change made during the second year was that a larger variety of habitats along the fringing reef were investigated, rather than just one portion of the bay area, as is seen in Figure 14
! '$ Figure 14. General locations of all sites from the second year of surveying. Measurements indicate length of each line. Original map was courtesy of Alberto Fenix. The final analysis of the data was performed using SAS. Kruskal Wallis and Spearman's rank correlation s were performed on the collected data to test for significance between variables. The goal of the statistical analysis was to determine if the re was a correlation between the presence of gastropod (s) on a fan and the pre sence of Aspergillosis symptoms; be tween the size of the fan and the pre sence of Aspergillosis symptoms; between the size of the fan and the presence of gastropod s; between the percentage of the surface area consumed by the gastropod and the presence of Aspergillosis symptoms; or between th e number of gastropod s presence on the sea fan and the presence of Aspergillosis symptoms. It was hypothesized that gastropod s would be found more frequently on infected fans. It was also hypothesized that a higher percentage of the overall surface area o f the infected fans would be consumed when
! '% compared to uninfected fans. Additional statistical analyses were performed to determine the frequency of observed phenomena. 2.2 Disease p revalence survey From July 12, 2011 to July 23, 2011 a survey was done us ing a series of grids to investigate the prevalence of Aspergillosis throughout the fringing reef investigated. This survey was also preformed off of the shore of Plantation Beach Resort on Cayo Grande. Six different grid systems (Figure 15 ) were construct ed along the reef; each contained 5 squares that were 1m x 1m square. Within each square the number of sea fans, and whether they were showing symptoms of Aspergillosis were noted. This survey was done as a baseline for disease prevalence in the area. The percentage of infected sea fans within the grid systems was compared against the percentage observed in the Cyphoma gibbosum survey. Fans that were monitored within these grid systems were not included in the C. gibbosum survey. Figure 15. Map of grid l ocations from the survey looking at disease prevalence. Each g r id had a total area of 1m x 5m. Measurements indicate length of each line. Original map was courtesy of Alberto Fenix.
! '& Chapter 3 Results 3.1 Cyphoma gibbosum Survey Over the two years of surve ying a total of 71 sites were examined. The number of gastropod s on each sea fan was noted as well as the total area of the sea fan, the approximate area consumed, and whether there were symptoms of Aspergillosis. During the first year, 67% of the 36 fans surveyed showed symptoms of Aspergillosis, and 54% of the 35 sea fans surveyed the second year showed symptoms of the disease. Out of the total 71 sites, 60% showed established signs of an Aspergillosis infection. A nonparametric Kruskal Wallis Test was u sed to determine correlation between the percentage of the overall sea fan area that was consumed at each site, the number of gastropod s present on each sea fan, and whether the sea fan displayed symptoms of an Aspergillosis infection. When the percentage of the overall surface area that was consumed by the gastropod inhabiting it at the time of the survey from year two data were analyzed it was found with statistical significance (chi square = 8.4765, pr>chi square=0.0036) that on infected sea fans a small er percentage of the overall sea fan area was consumed. When the overall sea fan area of infected and uninfected sea fans from year 2 was analyzed, it was found with statistical significance (chi square = 3.3779, pr>chi square=0.0661) that larger sea fans were more likely to be suffering from an Aspergillosis infection.
! "" Figure 16 Area consumed in relation to total sea fan area for data from year 2 Refer to Table 3 for trendline equations and R 2 values.
! "# Table 3 Linear trend line equations fo r data presented in Figure 16 1 Gastropod 2 Gastropod s Equation R 2 Equation R 2 Infected y = 0.0079x + 2.1703 R! = 0.65238 y = 0.0088x + 8.3266 R! = 0.24948 Uninfected y = 0.0181x + 8.5569 R! = 0.43462 y = 0.0043x + 8.9033 R! = 0.93 078 Table 4 Breakdown of the number of gastropods on infected and uninfected sea fans during year 2, with average percentage of the overall sea fan consumed. Average percentage of overall sea fan area consumed standard dev iation Number of sites with 1 gastropod Number of sites with 2 gastropod s Infected 2.1%2.1 13 6 Uninfected 5.4%3.4 11 5 Table 5 Observed values for different factors on infected and uninfected sea fans. Year 1 Year 2 Years 1 & 2 Average number of gastropod s on infected sea fans standard deviation 10.6 10.5 10.5 Average number of gastropod s on uninfected sea fans standard deviation 1 0.5 10.5 10.5 Average number of gastropod s on all sea fans standard deviation 1 0.5 10.5 1 0.5 Average sea fan area and standard deviation 640 cm 2 7 10 700 cm 2 710 670 cm 2 700 Average gastropod size on infected sea fans standard deviation 2.9cm0.7 Average gastropod size on uninfected sea fans standard deviation 2.9cm0 6
! "$ 623 679 819 565 701 612 !" #!!" $!!" %!!" &!!" '!!" (!!" )!!" *!!" +!!" Infected Uninfected Area [cm 2 ] Average sea fan area Year 1 Year 2 Years 1 & 2 Figure 17 The average sea fan area in cm 2 for each year of the survey and the data from both years combined. Data listed in Table 6. Table 6 Sea fa n size information for Figure 17 Sea Fan Area of Uninfected Sea Fans Year 1 Year 2 Years 1&2 Maximum 3771cm 2 2853cm 2 Minimum 117cm 2 125cm 2 AverageSt. Dev. 679cm 2 1039 565cm 2 692 612cm 2 833 Median Sea Fan Area of Infected Sea Fans Year 1 Year 2 Years 1&2 Maximum 2048cm 2 3250cm 2 Minimu m 31cm 2 119cm 2 AverageSt. Dev. 623cm 2 495 819cm 2 731 701cm 2 614 Median
! "% Figure 1 8 Histogra ms of sea fan size distribution
! "# Figure 19 Area consumed on a sea fan in relation to the presence of Aspergillosis and the size of the gastropod. Refe r to Table 5 for averages.
! "# 3.2 Disease prevalence s urvey One hundred and sixty one sea fans were examined for general signs of Aspergillosis. Only 16% of the sea fans showed signs of infection. Throughout the sampling period the grids were being monitore d (it varied from 10 days to 4 days depending on when the grid was constructed). Only 7 gastropod s were observed within the grid during the sampling time. Five were observed on gorgonians that showed symptoms of infection.
! $% Chapter 4: Discussion The ori ginal hypothesis was that Cyphoma gibbosum would be found more frequently on infected fans, and that larger areas would be consumed on infected sea fans, due to indications of preferential feeding in other surveys (Nagelkerken et al. 1997; Slattery 1999) a nd laboratory observations (Rypien 2008b; Rypien and Baker 2009). The observed distribution and feeding patterns from this survey showed that gastropod s did not seem to feed preferentially on infected sea fans, and on infected sea fans gastropod s appeared to consume a smaller percentage of the overall sea fan area. This survey did confirm previous observations that larger sea fans are more likely to become infected with Aspergillosis (Bruno et al. 2011; Dube et al. 2002; Kim et al. 2000; Kim and Harvell 200 2; Kim and Harvell 2004). When the size of gastropods on infected and uninfected sea fans were looked into there did not appear to be a relationship between snail size the area consumed. The lack of correlation between the presence of Cyphoma gibbosum and the presence of Aspergillosis appears to contradict previous field observations made by Slattery and colleagues (1999) and Nagelkerken and colleagues (1997). This also is contrary to laboratory observations made by Rypien (2008) and Rypien and Baker (2009 ). Instead of actively selecting for sea fans infected with the disease the gastropod s seem to be found on infected fans just as frequently as on uninfected sea fans, at least in this region. When this survey was compared to the survey on disease prevalen ce along the reef, a much higher percentage of the sea fans occupied by C. gibbosum displayed symptoms of Aspergillosis (60%) than in the disease prevalence survey (60%) (Figure 20 ) This may indicate preferential feeding, but is not conclusive.
! $& Figure 20 Comparison of percentage of sea fans infected in the survey investigating disease prevalence and the survey looking at Cyphoma gib bosum. There was a difference between infected sea fans and uninfected sea fans in how much was consume d by the gastropod occupying it at the time of the survey. When the percentage of the overall sea fan surface area consumed was investigated in relation to whether the sea fan was infected, there was a very significant correlation between the percentage co nsumed and whether Aspergillosis appeared to be present. Cyphoma gibbosum seemed to consume a significantly smaller portion of the overall sea fan area on infected sea fans. Because gastropod s consume less on infected sea fans, there may be a reduced chanc e of secondary infection. Secondary infections tend to be more severe than the primary infection due to the sea fan's immune response being compromised (Ellner
! $' et al. 2007). However, because observations were not taken daily for the two to three day period of feeding, the full extent of feeding on each sea fan is undetermined. It is possible that the some of the feeding scars used for measurements in this survey were taken after the gastropod s had just arrived on the sea fan, so estimates concerning total c onsumed area may be incomplete. This decreased consumption on infected sea fans may occur for a variety of reasons. The two hypotheses proposed in this study are that reduced consumption resulted from decreased nutritional quality of the sea fan and or i ncreased immune response by the sea fan. The possibility of decreased nutritional content in infected sea fans was suggested as a possible reason for the decreased feeding activity on infected sea fans related to the preferentially prey selection in other invertebrates. If other invertebrates make prey selections based on nutritional quality (Despland and Noseworthy 2006; Lee et al. 2003; Lee et al. 2004; Morales Ramos et al. 2011; Simpson et al. 2002; Simpson and Raubenheimer 2000), it is likely that Cypho ma gibbosum can also feed for superior nutritional quality. Mydlarz and Harvell (2007) noted that they observed less protein per gram of coral in infected sea fans. This may be a result of decreased polyp density in infected areas, as evidenced by decreas ed symbiont concentrations in infected fans (Kirk et al. 2005; Ward et al. 2007). As protein is a major factor in what invertebrates have been observed to select (Mayntz et al. 2005), this decreased protein content supports the theory that infected sea fan s have less nutritional content then uninfected fans, resulting in C. gibbosum feeding less on infected coral colonies. However, there have been no systematic studies of such preferences in the field or lab. A compounding issue is that there is degra ding a nd recession of the
! $( coen chyme observed in sea fans showing symptoms of Aspergillosis (Smith and Weil 2004). Because this is mainly what Cyphoma gibbosum consumes while grazing (Harvell and Fenical 1989), it seems likely that there might also be decreased g razing as a result of this degraded coenchyme on infected sea fans. The second hypothesis is that increased immune response by the gorgonians resulted in the toxicity of the coenenchym and coral polyps to be too great for Cyphoma gibbosum to continue fee ding on the sea fan. The increased immune response by sea fans during an Aspergillosis infection has been demonstrated in that there is increased antifungal activity in the central regions of infected sea fans (Kim et al. 2000), increased sclerite concentr ations in infected sea fans (Smith et al. 1998; Ward 2007), and increased concentrations of antifungal compounds found in the healthy tissues of infected sea fans (Mydlarz and Harvell 2007). It is also possible that the change in morphology that allows the purple sclerites to act as an antifungal defense may also decrease the palatability of gorgonians to C. gibbosum In a study concerning optimal toxicity in animals, Longson and Joss (2006) proposed that in cases where an organism is prey limited, they wi ll move to prey that has less defensive chemicals. It is possible that as there is increased antifungal activity in gorgonian sea fans as a result of Aspergillosis infection, Cyphoma gibbosum feeds more on uninfected fans to reduce the levels of defensive chemicals ingested. Although C. gibbosum has not been shown to be prey limited (Chiappone et al. 2003), it is possible that in cases of increased immune response by gorgonians, Cyphoma gibbosum will select for fans that are not demonstrating increased immu ne activity. Observations of C. gibbosum movements during prey selection may provide further information concerni ng
! $" the role of Aspergillosis in prey selection. If the gastropods "taste" the sea fan towards the base of the sea fan and then continue their s earch, it may indicate that Aspergillosis plays a role in C. gibbosum prey selection. A relatively high percentage of Aspergillosis was noted during the survey looking at Cyphoma gibbosum distribution (approximately 60% of fans examined), when compared to the percentage of infected sea fans observed in the survey considering dise ase prevalence along the reef. Both of these values are much higher than those observed by Kim and Harvell (2004). At the end of their survey in August 2003 only 5.9% of all sea fans looked at in the Florida Keys showed symptoms of Aspergillosis. There may be increased prevalence of Aspergillosis along this portion of reef, or the timing of the survey might have affected the results. All of these observations were made during the summer, specifically July, when water temperatures are at their highest. Because increased water temperature has been shown to have effects on the virulence of Aspergillus sydowii and on the antifungal extracts of gorgonians (Alker et al. 2001; Ward et al. 2007), it is possible that surveys at Cayos Cochinos observed an increased level of disease due to increased water temperatures. In other surveys this time of the year was associated with increased disease prevalence (Flynn and Weil 2009; Kim and Harvell 2004, Kim and Harvell unpublished data cited by Alker et al. 2001). There are plenty of areas for future study concerning the role of Cyphoma gibbosum as a potential vector for Aspergillosis. Cyphoma gibbosum was not observed to feed on infected areas o f gorgonians, tending to feed more towards the upper edges of the sea fans, and not near the base. Uptake of Aspergillus sydowii through ingestion seems unlikely due to the lack of observed feeding on areas of gorgonians showing
! $$ symptoms of Aspergillosis. This reduces the likelihood of C. gibbosum as a vector, but if the snails can pick up A. sydowii spores or hyphae during their movements up the sea fan blades, it is possible that they can still carry the fungus to uninfected sea fans. Investigation into w hether it is possible for C. gibbosum to carry A. sydowii by moving over infected regions of gorgonian containing A. sydowii spores and hyphae would provide valuable information about the potential for C. gibbosum to act as a vector for Aspergillosis. Thi s study looked mostly at the distribution of gastropod s on sea fans. To properly understand C. gibbosum 's role in the spread of Aspergillosis, understanding gastropod movements and prey preferences would provide important information. It is also important to observe foraging and feeding patterns over a longer period of time than in this study. More extensive study concerning the movements of C. gibbosum to investigate whether they frequently move how far they travel during movements between coral colonies, and how long they take to move between coral colonies would be useful for understanding if C. gibbosum may carried A. sydowii isolates from infected to uninfected areas. Because of observations that sea fans infected with Aspergillosis cluster at a 1 3m s cale (Jolles and colleagues 2002), understanding how far and how frequently C. gibbosum will travel from one sea fan to another may provide vital information about the potential for C. gibbosum as a vector for the disease. One interesting aspect that shou ld be taken under consideration for further study would be to compare the compounds used as defense against predators synthesized by gorgonians to the antifungal compounds synthesized by gorgonians. If the chemicals are similar and have similar effects it may explain the decreased feeding
! $) on infected sea fans. Investigating whether other corallivorous predators that feed on sea fans are also deterred from feeding on infected gorgonians would help with drawing conclusions about whether immune system toxins a re the reason for decreased feeding on infected sea fans by Cyphoma gibbosum
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