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OLFACTORY COMMUNICATION AND INDIVIDUAL RECOGNITION IN EULEMUR MONGOZ THE MONGOOSE LEMUR BY KATHERINE SMITH 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 With A Concentration In Biology/Chemistry Under The Sponsorship Of Dr. Sandra Gilchrist Sarasota, Florida April, 2011
ii Dedication This thesis is dedicated to: My family and friends who supported me throughout this process and my entire academic career. To my Mom and Dad and Andrew who have always been there for me. To Katie, Karen, Jasmine, and Bryant wh o gave me the motivation to finish and enough Thank you.
iii Acknowledgments I would like to thank my sponsor, Dr. Sandra Gilchrist, and the rest of my committee for guiding me through this process. To Monica Mo gilewsky and the rest of the team at the Lemur Conservation Foundation who made this project possible and for the support and encouragement they provided. To Samantha Ortiz who spent countless hours and got up at the crack of dawn to help me run trials. To Katharine Dean who helped code my data. And last but not least, a thank you to my lemurs, without whom this project really would not have happened. This is LCF Publication #17.
iv Table of Contents Title Page i Dedication ii Acknowledgements iii List of Tables and Figures vi Abstract vii Preface viii 1. The Biology and Behavior of Olfactory Communication 1 1.1. Olfactory Communication An Overview 1 1.2. Olfactory Communication in Lemurs 4 1.3. Behavioral Experiments into Olfactory Communication 6 1.4. Chemical Analysis of Scent Secretions 11 1.5. Importance of Understanding Lemur Olfactory Communication 14 2. Methods 20 2.1. Study Subject: Eulemur mongoz 20 2.2. Study Site: Lemur Conservation Foundation Myakka City, Florida 22 2.3. Behavioral Methods 23 3. Results and Discussion 29 3.1. Results 29 3.2. Discussion and Future Directions 31 Works Cited 40
v Appendix I: E. mongoz used in experiment 49 Appendix II: Behavioral Trials 51 Experiment I, Group 1 51 Experiment I, Group 2 52 Experiment II 53 Appendix III: Investigation time for each trial 54 Experiment I, Group 1 54 Experiment I, Group 2 57 Experiment II 60 Appendix IV: IACUC Approvals 63
vi List of Tables and Figures Figure 1.1. Vomeronasal gland of a mouse 1 Figure 1.2. L. catta glands 5 Figure 1.3. E. mongoz glands 6 Table 1.1. Milestone behavioral studies 7 Table 1.2. Milestone chemical analysis experiments 12 Figure 2.1. Sexual dichromatism in Eulemur mongoz 20 Figure 2.2. Map of E. mongoz distribution. 21 Figure 2.3. Aerial map of Lemur Reserve 22 Figure 2.4. Lemur enclosures at Lemur Reserve 23 Figure 2.5. Scent collection set up 24 Figure 2.6. Presenter running trials 28 Figure 3.1. Average Investigation Time for Experiment I, Group 1 29 Figure 3.2. Average Investigation Time for Experiment I, Group 2 30 Figure 3.3.Presenter with Clarissa on her back 32 Figure 3.4. Eulemur mongoz investigation of pad placed under her nose 34 Figure 3.5. Emilia and Fred fighting during a trial 35
vii Abstract Olfactory communication is a major aspect in lemur biology and behavior. Since it was first studied, research and experiments have focused on Lemur catta the ring tailed lemur. The purpose of this thesis was to begin to bridge the information gap that existed between L. catta and most other lemurs, by trying to determine whether Eulemur mongoz the mongoose lemur, could distinguish between individuals ba sed solely on olfactory communication. A habituation/dishabituation experiment was done using a total of eight E. mongoz from the Lemur Conservation Foundation in Myakka City, ries to E. mongoz individuals. Overall, the lemurs were very uninterested in the trials, hardly investigating the pads. It remains unknown whether E. mongoz are able to differentiate between individuals based on scent, however, these findings indicate th at E. mongoz may use olfactory communication very differently from L. catta Future experiments are needed to explore use of scent by the mongoose lemur. ____ __________________________ Approved by S. Gilchrist Division of Natural Sciences
viii Preface No one really knows where lemurs came from. Isolated on the island of Madagascar, lemurs have become a diverse group with 8 different families and 32 species currently known today. At least 15 additional species have already gone extinct since humans first arrived on Madagascar 2,000 years ago (Mittermeier et al. 1994). At the time of colonization the eastern rainforest covered 11.2 million hectares in 1950 only 7.6 million hectares remained and in 1985 only 3.8 million hec tares remained. This means the rainforest is being destroyed at a rate of 111,000 hectares a year (Sussman et al. 1994). If this rate continues the rainforests will be completely destroyed by 2020 (Irwin et al. 2005). This destruction of native landcove r is being seen across Madagascar, threatening the survival of lemurs. How this loss of habitat will affect lemur social behavior, as more territories overlap, is yet to be seen. New conservation and education measures need to be enacted to save the lemu late.
1 1. The Biology and Behavior of Olfactory Communication 1.1. Ol factory Communication An Overview Olfactory communication is important in many animal interactions. Evidence of olfactory communication can be fou nd in everything from reptiles to rodents, bats to primates (Safi and Kerth 2003; Arakawa et al. 2008; Houck 2009; Setchell et al. 2010). Olfactory information is processed by dual olfactory organs: the main olfactory epithelium and the vomeronasal gland (figure 1.1) (Swaney and Keverne 2009). At first these systems were thought to be two distinct pathways: the vomeronasal olfactory system, closely connected with the hypothalamus, processed pheromones that controlled reproductive functions, while the main olfactory system processed general volatile chemosignals controlling social communication (Keller et al. 2009, Keller et al. 2010). However, it is now believed that these two processes work in tandem allowing chemical signals to govern everything from in dividual recognition to delineating territories to mate choice (Safi and Ke rth 2003; Keller et al. 2009). Figure 1.1. Vomeronasal gland of a mouse (Lloyd Thomas and Keverne 1982) Though most research into olfactory communication focuses on mammals, num erous experiments show that reptiles and amphibians also use chemical cues to
2 mediate behavior (Houck 2009). While snakes use tongue flicking to send chemical cues to their vomeronasal gland (Marinez Marcos et al. 2002), some lizards use femoral exocrine glands to excrete lipid protein secretions that code for sex and individuality (Alberts et al. 1993). Amphibians have been found to express odorants through cloacal glands and possibly from steroid hormone metab olites (Park et al. 2004). A ll these forms of chemical communication can be used in mate selection and attraction, territory defense, and general interaction with conspecifics (Houck 2009). In mice, urinary scent marks are used in conspecific interactions including sexual selection and social domi nance. Studies have shown that urinary scents contain information on species, sex, individual identity, social dominance, health, and reproductive status via protein ligand complexes as well as the volatiles brevicomin and thiazole ( Humphries et al. 1999; Arakawa et al. 2008). Interestingly, it has also been shown that mice can convey fear and stress in their scent markings and the conspecifics that encounter these markings show avoidance behaviors (Eisenberg and Kleiman 1972). In ferrets, Mustela putori us f furo males are much more interested in female scent secretions from the urogenital and body glands, than females are in male scent secretions from the same glands. However, males and females are equally interested in between familiar and unfamili ar individuals (Berzins and Helder 2008). It is believed that scent secretions are used both in sex attraction and territorial defense (Clapperton et al. 1988). Olfactory communication is important in bats based on their nocturnal and social way of life (Safi and Kerth 2003). It is common knowledge that bats rely heavily on
3 acoustic signals, yet olfactory signals also play a role in bat communication. Some species of bats are able to discriminate between sexes, and possibly between individuals (Bouchard 2001). Gas chromatograpy olfactometry (GC O) showed that roost mates have a chemical signature more similar to each other than to non roost mates (Bloss et al. 2002). Safi and Kerth (2003) found similar results using gas chromatography/mass spectrometry (GC MS), but added that no evidence of genetic relatedness was found to be encoded in the scent secretions. A general belief is that primates are microsmatic that their sense of smell is relatively weak, with the animal relying on its well developed se nse of vision instead (Setchell et al. 2010). However, this theory is being contradicted by many recent studies that have found that olfactory communication may play a larger role in primat e interaction tha n was previously imagined. A field study of Sagu inus mystax the moustached tamarin, was conducted focusing on its scent marking behavior (Heymann 1998, 2000). Due to a higher incidence and intensity of scent marking by females, with males were more likely to investigate the scent marks, it was conclud ed that olfactory communication helps to govern sexual selection (Heymann 1998). No evidence was found that S. mystax uses olfactory communication as a form of territory defense (Heymann 2000). A field study of squirrel monkeys, Simia sciureus showed th at investigation times of scent marks seemed to be linked to the females repr oductive status, indicating olfactory communication in mating behavior (Hennessy et al. 1978). A conditioning paradigm was used to show that S. sciureus has a very high level of olfactory sensitivity,
4 being able to significantly discriminate dilutions as low as 1:300,000 of 1,8 cineole 1 These findings showed that in addition to visual cues, olfactory communication could be very important in primate behavior (Laska et al. 2000). The presumption of microsmatic primates is most prevalent in Old World monkeys and apes, otherwise known as catarrhines (Setchell et al. 2010). However, emerging research is challenging this assumption. It has been shown that macaques are able to detect scents at very low concentrations nearly as well as dogs and rats (Hbener and Laska 2001; Laksa and Seibt 2002). In baboons, males depend partially on olfactory investigation of the females to determine their reproductive status (Clarke et al. 2009). Setchell and colleagues (2010) used GC MS to analyze the secretions of the sternal gland in male and female Mandrillus sphinx the mandrill. The analysis showed th at the sternal gland encodes age, sex, and individuality in both males and females, and for dominance in males only. These studies illustrate that the general notion of Old World monkeys being microsmatic is misguided, and that olfactory communication is a greater part of their social lives than previously imagined. 1.2. Olfactory Communicatio n in Lemurs Lemurs demonstrate a complex system of olfactory communication (Macedonia and Stanger 1994). The types of scent marking seen for lemurs depends on the species. 1 The structure of 1,8 cineole: (Chen et al. 2004)
5 Ring tailed lemurs, L emur catta for example, scent mark using their anogenital g lands 2 (both sexes) as well as their brachial and antebrachial glands 3 (males only) (Scordato et al. 2007) Mongoose lemurs, E ulemur mongoz however, only scent mark using their anogenital glands (male and female) and the top s of their head s (male only) ( Tattersall and Sussman 1975) Figures 1.2 and 1.3 illustrate the glandular differences between L. catta and E. mongoz T he marking behavior of Lemur catta ring tailed lemurs, and their use of olfactory communication has been well researched, while resea rch into Eulemur mongoz has been lacking. While the messages encoded in the scent secretions of L. catta have been slowly translated, the purpose of olfactory communication in E. mongoz remains unknown. Figure 1.2. Male L. catta exhibiting genital, bra chial, and antebrachial glands. ( photo from http://opencage.info) 2 Both males and females possess sebaceous glands in the anogenital region. In males the glands are located on the scrotum, while in females they are found on the labia (Evans and Goy 1968). 3 Identified by Montagna and Yun (1962), the antebrachial gland i s a collection of sebaceous glands on the forearm of L. catta which has an accompanying spur, while the brachial gland is a single sebaceous gland located above each clavicle. Both are found only in males.
6 Figure 1.3. Female E. mongoz exhibiting genital, palm, and scalp glands. Note that the palm and scalp glands are only found on males. (Photo by K. Smith). 1.3. Behavioral Experiments into Olfactory Communication Research into the meaning of olfactory communication in lemurs began in the mid to late 1970s (Mertl 1975; Harrington 1976, 1977, 1979). These early behavioral studies focused on Lemur catta and Eulemur fulvus (brown lemurs), but by the 1980s research was focused exclusively on Lemur catta Due to the high number in captivity, L. catta have been the lemur species of choice to study. These studies have shown that olfactory communication is an important aspect in lemur behavior, being used to govern everything from territory defense to individual recognition to mate selection (Table 1.1).
7 Table 1.1. Milestone behavioral studies of lemur olfactory communication. Year Species Gland Methods Results Discussion Reference 1975 L. catta (captive) Brachial & Antebrachial Habituation Dishabituation using scented pads Could distinguish individuals based solely on scent secretions. Possible that L. catta use scent marking in territory demarcation. (Mertl 1975) 1976 E. fulvus (captive) Unknown Habituation Dishabituation using scented pads Could distinguish individuals based solely on scent secretions Scent marking possibly used in group social behavior such as mating, dominance hierarchies, etc. (Harrington 1976) 1977 E. fulvus (captive) Unknown Habituation Dishabituation using scented pads Males could distinguish between male and female scent Different messages are encoded in the chemical make up of the scent secretions. The meaning of the messages remains uncertain. (Harring to n 1977) 1979 E. fulvus (captive) Unknown Habituation Dishabi tu ation using scented pads E. fulvus could discriminate between scents from conspecifics other Lemuriformes but not between subspecies of E. fulvus Scent marking could be used in territory demarcation and to lessen the degree of interspecies confusion of scent marks in overlapping territories. (Harrington 1979) 1984 L. catta (captive) Females: Genital Males: Antebrachial Discrimination experiment using scented wooden dowels Male L. catta were able to distinguish between the brachial/antebrachial scent marks of males and the genital scent marks of females This could be due to the short breeding season observed in L. catta The males may be investigating the females scent mark to assess her reproductive status in order to determine the narrow window in which she would be receptive. (Dugmore et al. 1984)
8 1990 L. catta (captive) Female: Genital Male: Genital & antebrachial Discrimination experiment using scented wooden dowels Females showed great interest in the markings from strange females, and little interest in their own labial markings and male scrotal or antebrachial markings This could be due to the femal e dominance observed in L. catta S cent marks may contain information about the dominance of the individual which would be of interest to all other group members (Dugmore and Evans 1990) 1996 L. catta (captive) Females: genital Males: antebrachial Discrimination experiment using scented sticks. Scents from unknown lemurs individuals with whom the subject lemurs have never interacted invoked greater investigation time and more instances of scent marking behavior. Possible explanation given for th ese findings was the concept of group scent when all members of the troop have a common, or same, scent and any outsider has a different scent. (Ramsay and Giller 1996) 1998 L. catta (captive) All Natural scent marking behavior of semi free ranging L. c atta observed. Olfactory communication in L. catta over 60% of markings investigated by a group member within a few minutes. Most signals were also quickly counter marked, marking over an existing mark, by the investigating lemu r. A mount of male scent marking is directly correlated to rank; male marking peaks just before mating season; male scents are most often investigated by other males; and males are observed to investigate female markings more often than females investigate Females may prefer to mate with the counter marking male instead of the original marker. Scent marking could also govern sexual selection and mating behavior (Kappeler 1998)
9 2002 E. fulvus rufus & L. catta (wild) All Observation of natural marking behavior in the wild Similar rates of scent marking were seen between the two species. In L. catta instances of scent marking increased during mating and migration season, while no seasonal differences were seen in E. f. rufus In L. catta the m not influence marking quantity. Individuals seen to mark more had greater reproductive success during mating season. Scent markings are used as a form of indirect mating competition and in response to intergroup interactions (Gould a nd Overdorff 2002) 2006 L. catta (captive) Male brachial gland Habituation Dishabituation & Bioassy L. catta could perceive the scents derived from the male brachial gland. L. catta would preferentially investigate scents from an unfamiliar conspecific over scents from a familiar conspecific. However, if the lemur had a choice between investigating the scent of a known competing male (from a different group with whom ited interaction) and an unknown scent (from a male they had never interacted with), the lemurs would preferentially investigate the scent from the competing male. Territorial competition and mating behavior. (Palagi and Dapporto 2006) 2007 L. catta (captive) Females: genital Discrimination experiment Male and female L. catta respond differ ently to the Scent marking behavior of both male and female L. catta (Scordato and Drea
10 Male: genital, brachial, & antebrachial using scented dowels. Samples were taken from conspecifics varying in sex, age, social status, and reproductive condition. presence of odors males showed higher amounts of investigation of presented scents, while females demonstrated greater occurrences of genital countermarking are highly correlated to rep roductive status and mating. 2007) 2008 L. catta (semi free ranging) Females: genital Male: genital, brachial, & antebrachial Observation of semi free ranging L. catta and a discrimination experiment. During mating season (November through February) males and females are more aggressive and show higher rates of scent marking; dominate males marking more frequently then subordinate males. Responses by females to presented odors were highest to other females and were dependent on thei r reproductive state and the reproductive state of the female donor. Males were interested in all scent secretions, regardless of sex. Additionally, males were found to discriminate based on social status if the odorant sample came from a known donor, bu t not if it came from an unknown donor. L. catta use information encoded in scent secretions during mating season to help determine their mating behavior (Drea and Scordato 2008)
11 1.4. Chemical Analysis of Scent Secretions the messages in lemur scent secretions. The use of gas chromatography mass spectrometry (GC MS) 4 became a useful and popular tool when analyzing the differe nt components of scent secretions (Table 1.2). 4 The GC portion of the instrument separates and analyzes the various volatile components, producing a separate peak for each component. The size of the peak corresponds to the quantity of the substance. The MS portion indentifies each substance by electrically charging the molecules and then passing t hem through a magnetic field, fragmenting the molecules and then detecting the different charges. These data can then be used to identify the different compounds in the sample. So together, a GC MS separates out the compounds in a sample giving a quantit ative information (GC) and then identifies each compound (MS). (Douglas 2011).
12 Table 1.2. Milestone experiments in the chemical analysis of lemur olfactory communication. Year Species Gland Methods Results Discussion Reference 2004 Lemur catta and Propithecus verreauxi coquereli Anogenital glands Gas chromatography mass spectrometry (GC MS) Anogenital gland secretions chain hydrocarbons with alcohol and aldehyde possibly age and reproductive status could be differentiated in the case of L. catta while not of P. v. coquereli GC MS can be used to determine the chemical composition of scent secretions. Showed that Lemur catta and P. v. coqueri li most likely use scent marking very differently. (Hayes et al. 2004) 2006 Propithecus edwardsi Anogenital glands Gas chromatography mass spectrometry (GC MS) After analyzing the compounds for differences between social group, age class, sex, and season, no significant differences were found except based on season. In the case of seasonal variation the same volatile components were found, but a large difference in the quantity of these components was observed. Information on sex may still be found in the secretions but may be in the form of nonvolatile components that would not be found on the GC MS, such as proteins. (Hayes et al. 2006)
13 2006 Lemur catta Brachial glands (males only) Gas chromatograph/flame ionization detector (GC/FID) Analysis of the che mical signatures showed that nearly all of the males had similar compounds present, yet they were seen in different quantities, providing each individual with a unique chemical profile. While seasonal variation was also seen in the chemical analysis, domi nance, age, and group membership were not. Believe that L. catta representation of a specific individual by its L. catta probably use olfactory communication in sexual selection and mating. (Palagi and Dappor to 2006) 2007 Lemur catta Labial, scrotal, and brachial Gas chromatography mass spectrometry (GC MS) Different glands produce different volatile compounds the labial and scrotal glands produce organic acids and esters, while the male brachial secretions contained squalene and cholesterol. Differences between individuals and season were found, but no variation due to social status Olfactory communication im portant in intrasexual competition (for the males) and as a means to advertise a females reproductive state. (Scordato et al. 2007) 2008 Lemur catta Labial, scrotal, and brachial Gas chromatography mass spectrometry (GC MS) Seasonal variation was evident in the scent secretions from the labial, scrotal and brachial glands. I ndividual signatures occur in the genital glands, but not in brachial glands. No evidence was found to suggest social status is en coded in the chemical composition of scent marks Genital and brachial secretions contain compounds with high molecular weight, which may help increase stability and longevity of signals, while the antebrachial gland secretions are composed of lower molecular weight compounds an d less long lasting. Shows that form follows function. (Drea and Scordato 2008)
14 1.5. Importance of Understanding Lemur Olfactory Communication Olfactory communication has been implicated for governing many aspects of lemur social life, primarily based on observations of L. catta from mating to territory demarcation and protection. Much of the research done on olfactory communication in lemurs h as suggested that it is involved in mating behavior and sexual selection. The higher rates of male marking before mating season (Drea and Scordato 2008; Gould and Overdorff 2002; Kappeler 1998) indicate that male scent marking functions in intrasexual com petition (Kappeler 1998, Scordato and Drea 2007). During the mating season, males with higher rates of scent marking have better mating success (Gould et al. 2002). Female L. catta have a very short breeding season and are only receptive to the male for a limited time. The genital secretions of the females may function in intragroup communication, to convey their reproductive status to the males (Dugmore et al. 1984; Kappeler 1998). Because female L. catta do not display any interest in the odors of mal es, olfactory communication does not function as a form of female mate choice (Dugmore and Evans 1990; Scordato and Drea 2007). However, some researchers stress the importance of olfactory communication in female mate choice (Charpentier et al. 2008). Ol factory cues from male scrotal glands may provide information on genetic quality and relatedness (Boulet et al. 2010; Charpentier et al. 2010). Chemical analysis of scrotal secretions shows a decrease in compound diversity, with in bred males losing more diversity tha n out bred males giving female L. catta the ability to determine how genetically variant a potential mate is by examining the complexity of his scent secretions (Charpentier et al. 2008). Information
15 on kin relationships was also found, meani ng that L. catta may also use information encoded in scrotal and labial marks to avoid incest and facilitate nepotism by favoring and protecting kin ( Chapais et al. 1997; Boulet et al. 2009). Similar chemical findings have been reported for the labial sec retions of female L. catta (Boulet et al. 2010). In fact, it has been shown experimentally that male L. catta can determine their relatedness to other male and female conspecifics and they may be able to discern the genetic variability of females; female L. catta are able to determine their relatedness to other males and the genetic variability of other females and possibly of males (Charpentier et al. 2010). These findings suggest that both male and female L. catta use information from genital secretions offs between selection genes Unfortunately, a new study has shown that the use of hormonal birth control in captive L. catta dramatically alters the chemical make al secretions (Crawford et al. 2011). The following is a summary of these findings: Medroxyprogesterone acetate (MPA) was found to decrease the diversity of compounds as well as change the quantity of volatile components normally found in labial secretion chemical signature. Contraception was also found to degrade chemical information L. catta may use in determining the genetic diversity of another lemur and their potential relatedness. Males will preferentially choose to investigate females who were not on contraceptives, showing they were able to distinguish between lemurs who could or could not reproduce. Hormonal contraception, therefore, has the ability to disturb intraspecific interactions by radically changing th e chemical make up of female labial secretions. Olfactory communication is also important for territory protection and intergroup interactions. Mertl (1977) worked with a troop of wild L. catta to show that habituation to foreign scent is highly positi on specific and could explain why L. catta mark more near
16 the edges of their territories. By marking the periphery of their territory, L. catta troops are habituating adjacent troops to their home range and perhaps reducing the number of aggressive intera ctions between groups (Mertl 1977). This demarcation and protection of their territory also protects the resources within it, allowing for a healthier troop (Mertl Millhollen 2006). Territories are constantly being maintained with scent marks in order to protect it from competitors (Jolly et al. 2006), with many of the scent marks being left in areas of overlap with adjacent troops (Mertl Millhollen 1988). In addition to scent marking to protect their territories, L. catta also use urine to demarcate the ir boundaries (Palagi et al. 2005). Ramsay and Giller (1996) believe that L. catta could have the ability to remember individual scents and are able to discriminate between scents left by group mates and scents left by intruders. Another possibility is t hat all L. catta in the same group have the same scent termed group scent. Therefore when a scent is found in a group scent to determine if an unknown lemur has entered their home r ange (Ramsay and Giller 1996). The use of GC MS has allowed researchers to identify the chemical composition of the different glands which gives insight into the possible functionality of each gland. While the genital (labial and scrotal) and brachial gl ands produce high molecular weight compounds, such as organic acids, esters, squalene, and cholesterol, the antebrachial glands were found to secrete low molecular weight compounds (Drea and Scordato 2008). This is a case of form follows function: the gen ital and brachial secretions are longer lasting and used to signal identity and resource ownership, while secretions from the antebrachial glands are meant to be short lived, such as when they are used in social dominance displays (Scordato et al. 2007).
17 While this research has helped to understand the marking behavior seen in L. catta there is a major gap in the research in regard to Eulemur mongoz Although no direct research into olfactory communication has been done on E. mongoz scent marking behavio r has been noted in other observational studies (Tattersall and Sussman 1975; Curtis and Zaramody 1998, 1999). Due to the many behavioral differences seen between L. catta and E. mongoz there is no guarantee that the chemical compounds of the two species are similar or that they encode similar messages. Many differences are seen between the social structure of L. catta and E. mongoz While L. catta are observed to live in a multi male, multi female group (Jolly 1998), E. mongoz are considered to be nearly monogamous, and to live in small family groups consisting of an adult male, female, and their young offspring 5 (Curtis and Zaramody 1998). While L. catta are known to be quite promiscuous, where females mate with many males, E. mongoz form pair bonds between one adult male and female (Curtis and Zaramody 1999). In L. catta olfactory communication is used extensively during the mating season and may help drive sexual selection (Dugmore et al. 1984; Kappeler 1998; Gould and Over dorff 2002; Scordato and Drea 2007; Drea and Scordato 2008; Charpentier et al. 2008; Charpentier et al. 2010; Boulet et al. 2010). Scent marking by E. mongoz is still presumed to influence mating behavior, as it may act to reinforce the pair bond male and female E. mongoz have been seen rubbing their genitals against each other, perhaps ensuring that each is marked with the scent of its mate (Curtis and 5 While observations have been made of stable, pair bonding in E. mongoz (Curtis and Zaramody 1998, 1999), others have seen seasonal variation, with monogamy seen when E. mongoz are nocturnal, with small polygamous groups existing when E. mongoz are diurnal (Jolly 1998). Although initial observation of wild E. mongoz reported the species to be nocturnal (Tattersall and Sussman 1975), more recent observations regard the species t o be cathemeral throughout the year, shifting toward diurnal behavior during the wet season and nocturnal behavior during the dry season (Harrington 1978; Curtis and Zaramody 1998; Curtis et al. 1999).
18 Zaramody 1999). Due to extreme differences in the mating styles of L. catta and E. mongoz it is pre sumable that the two species use olfactory communication very differently, and that the chemical make up of secretions varies greatly. Home ranges of E. mongoz are relatively small: home ranges of L. catta range from 5 to 9 hectares (Sussman 1974), while those of E .mongoz are less than 3 hectares (Curtis and Zaramody 1998). Unfortunately very few studies have been done in the wild focusing on E. mongoz The following is a summary of the three main sources that could be found on the subject (Tattersall a nd Sussman 1975; Curtis and Zaramody 1998, 1999). The home ranges of E. mongoz correspond with the location of resources and seem to overlap extensively with other E. mongoz families (Tattersall and Sussman 1975; Curtis and Zaramody 1998, 1999). There is some disagreement on whether E. mongoz actively defend the borders of their home range Tattersall and Sussman (1975) claim they do not, while Curtis and Zaramody (1998, 1999) claim that they do. Both agree that anogenital scent marking increases during encounters with other E. mongoz Curtis and Zaramody (1999) believe that another of the functions of scent marking in E. mongoz is territorial. After one group had displaced another following a territorial dispute, the victors were seen to anogenitally c ountermark in the area that the other group had previously been. In addition to territorial conflicts, scent marking was also observed during traveling, with a high occurrence of scent marking in feeding trees (Curtis and Zaramody 1999). Unlike in L. cat ta very few instances of intragroup aggression or conflict were observed If active defense of home ranges does occur in E. mongoz then scent marking could be used in a similar way to L. catta
19 Experiments must be done to determine if there are any sim ilarities between the behavioral reaction to presented marks and in the chemical composition of glandular secretions between the two species before informed comparisons can be made. In a more practical sense, a better understanding of olfactory communicati on in E. mongoz could lead to effective olfactory enrichment in captivity. Enrichment is used to help encourage natural behaviors that the animal would exhibit in the wild (Carlstead et al. 1991). If it is determined how E. mongoz use olfactory communica tion in the wild, enrichment can be created in captivity to help mimic these behaviors. This thesis is testing whether or not E. mongoz can distinguish between individuals based on olfactory communication. It is assumed, due to the well developed olfacto ry behavior of L. catta that E. mongoz will be able to differentiate between individuals based on genital gland secretions.
20 2. Methods 2.1. Study Subject: Eulemur mongoz (Tattersall 1982). Eulemur mongoz are a medium sized lemur with an average weight of 2 kg (Mitter meier et al. 1994). The body of the E. mongoz can be up to 35 cm with a tall length of up to 48 cm. Born with white beards, E. mongoz becomes sexually dichromatic around six weeks of age: the females retain the white beard while the males develop red che eks and beard (Figure 2.1). Figure 2.1. S exual dichromatism of E. mongoz Felix, on the left, sports the male characteristic of red cheeks and beard. Kikeli, on the right, shows the white beard and cheeks characteristic of females. Note the similar si zes of the male and female E. mongoz do not show size related sexual dimorphism.
21 Eulemur mongoz is native to the northwestern part of Madagascar (Figure 2.2) where it lives in dry deciduous forests, with introduced species found in the humid forests of the Comores islands (Mittermeier et al. 1994). Figure 2.2 H ome range of E. mongoz The dark red hash marks on the mainland of Madagascar is the native population, while the light red hash marks on the northern islands of the Comores are an introduced population. The inset map shows Madagascar in relation to the rest of the globe ( An drainarivo et al. 2010) Unfortunately, the IUCN Red List of Threatened Species lists E. mongoz as a vulnerable species due to a decreasing population and habitat loss ( Andrainarivo et al. 2010). Eulemur mongoz was chosen for this study because of high n umbers present at the Lemur Conservation Foundation and because there is so little research on them. At the
22 time of this study the Myakka City Lemur Reserve (figure 2.2) had 12 E. mongoz the largest population in captivity. Because of their pair bonded social structure, E. mongoz are incredibly difficult to keep in captivity, one of the reasons that so little research has been previously done on them. 2.2. Study Site: Lemur Conservatio n Foundation Myakka City, Florida The Myakka City Lemur Reserve is a 100 acre reserve, which is home to about forty lemurs of differing genera and species. Some are semi free ranging, living in two different forests both of which are 13+ acres in size. The rest are housed in two different shelters, Toomey and North. Figure 2.3 Aerial map of the Lemur Reserve. The red outline shows the approximate size of the two free ranging forests. The yellow outline shows the two shelters: Toomey to the North (marked with an Enclosures Free ranging forests (approximate) A B
23 Both shelters consist of indoor/outdoor enclosures that allow the lemurs continual access to either. The indoor rooms are constructed of cinderblock, while the outdoor ones are chain link, giving the lemurs limited access (visual and auditory) to the surrounding lemur s. A door connects the outdoor and indoor enclosures, allowing the lemurs to be confined to either one, if needed, or to separate mates from each other. Figure 2.4 Pictures showing examples of indoor (left) and outdoor (right) enclosures. In the phot o on the right, Felix can be seen scent marking a branch with his anogenital gland s 2.3. Behavioral Methods A habituation dishabituation study was done to determine whether E. mongoz could differentiate between individuals based solely on olfactory com munication. Scent secretions were collected from the anogenital glands of caged male E. mongoz specimens and presented to both male and female conspecifics in a manner similar to Mertl (1975). Only the males were used for scent collection to eliminate the confounding variable that birth control has on female scent secretions (Crawford et al. 2011). Most of the female E. mongoz are currently on birth contro l ( Depot medroxyprogesterone acetate also known as depo provera) to prevent unwanted pregnancy at th e Reserve. Originally four male E. mongoz were used for scent collection: Bimbini, Felix, Merced, and Miguel
24 (for a table of individuals used see Appendix I). Unfortunately, Miguel is geriatric and arthritic and did not appear physically able to assume t he posture required for scent marking. He was never observed to mark anogenitally, so he was dropped as one of the lemurs being used for scent collection. Dowels measuring 0.95 cm in diameter and 20.3 cm in length were affixed with zip ties to the branch 6 cage. The target lemur was not separated from its mate. However, the lemurs were not interested in the dowels and would not mark on them. After learning that wooden dowels were not a good marking substrate, a switch to PVC pipe was made (figure 2.4). Three sections of PVC pipe, all varying in diameter ties. This method worked well and was maintained for the rest of the study. Figure 2.5 Pieces of PV C pipe were attached to the branching in the enclosures using zip ties. 6 Donor lemur refers to the male whose anogenital glan d scent secretions were being collected.
25 ties were wiped down with liberal amounts of 70% isopropyl alcohol and allowed to dry in an area that had also been wiped down with isopropyl alcohol. contaminated and no samples were collected from it. Once the donor male had marked on an uncontaminated piece of PVC a pad of sterile gauze was used to collect the sample and was then immediately placed in a pint sized Ziploc freezer bag. To try to remain consistent in the quantity of scent secretion on each piece of gauze, one piece of gauze was used to collect each marking multiple samples were not collected from the same marking. Unfortunately, some variability will still exist, because markings would vary in amount of secretion left. However, multiple samples would be collected from the same piece of PVC if only the male had marked it. Up to five or six samples could be collected in a single session 7 If the lemur was not cooperating or seemed agitated the session was stopped. On numerous oc casions no samples were collected. before the males had the opportunity. On a regular basis no samples were collected from the donor lemur because all the PVC was quickly conta minated by the female. In these cases, the male and female were sometimes separated with one being confined in the outside cage and the other being confined in the inside room. In some instances, the male was inside, in which case PVC was attached to t he limited amount of branching and the 7 A session refers to each time a donor lemur was presented with the PVC pipe in its cage for the purpose of scent secretion collection. A total of 89 sessions were done to collect all of the samples.
26 tables present. This did not seem to have an effect on the samples collected. If either lemur seemed agitated or anxious due to the separation the session was aborted and the lemurs reunited. Once the decision had been made to stop the session, the individual samples were numbered based on the order they were collected and moved to the freezer. The PVC and zip ties were quickly wiped down with isopropyl alcohol and allowed to dry in a non sterile area. Preparation s were then made for the next session with a different donor lemur. Once everything else was ready gauze, bags, etc. the PVC was wiped down once more and allowed to dry in an area that had just been wiped down with isopropyl alcohol. Gloves were also changed between each session to prevent cross contamination of scents. Sample collection occurred between January 21 st 2011 and March 8 th 2011. In all 32 samples were collected from Merced, 52 from Felix, and 76 from Bimbini. Number of samples needed was determined by the number of behavioral trials needed for observations. For the behavioral trials six E. mongoz lemurs were used: Bimbini, Felix, Miguel, Clarissa, Emilia and Kikeli. Merced was unavailable to use as a subject lemur due to his mate ( Estella) being very pregnant and it was impossible to separate them. Estella was also not used. No lemur was ever presented with their own scent. Bimbini and Kikeli are siblings, so to avoid the variable of relatedness, Kikeli was never presented with B not present a lemur with the scent of its mate. However, the lemurs have switched mates and cages a number of times, and as an enrichment exercise the Lemur Reserve do es
27 rotate items between cages, so all of the E. mongoz lemurs should be familiar with each other to some degree. dishabituation experiment, five different sets of trials were conducted. In Experiment I, Group 1, seven pads were presented to the subject lemur 8 all from the same donor male. Each pad was presented for 30 seconds in quick succession. In Experiment I, Group 2, a to tal of seven pads was presented again, the first four from one donor lemur (Lemur A) and the last three from a different donor lemur (Lemur B). Again, the pads were presented for 30 seconds, each in quick succession. In Experiment II, three pads were pre sented, each from a different donor male. Pads were again presented for 30 seconds in quick succession. In Experiment III, Group 1, three pads, all from the same donor male, were presented in 3.5 minute intervals. Each pad was presented for 30 seconds, but there was a three minute break between the presentations. In Experiment III, Group 2, three pads were presented, each from a different donor lemur. Pads were again presented in 3.5 minute intervals. See Appendix II for a full list of every trial run showing who the subject lemur was and which donor samples were used. The pads were placed in screen envelopes. Two sides of the envelope were stapled together and then duct tape was placed over the staples. This kept the staples from potentially falli the presenter to hold the envelopes by the tape and avoid any cross contamination that would have occurred if the pads had been touched. Figure 2.6 shows presention of a scented pad hel d in the screen envelope. 8 The subject lemur is the lemur that the gauze pads are being presented to. They are therefore being presented with the scents of the different donor lemurs.
28 At first the pads were presented through the enclosure at 90 second intervals. However, the lemurs had very little interest in them and could not be convinced to come over and investigate the pads. Food was used as a lure, but the lemurs would then just look for additional food on the ground instead of investigating the pad. If the pads were presented in the enclosure with the lemur, lemurs were much more interested. This method was used for the remainder of the trials. The l ength of presentation was also decreased from 90 seconds to 30 seconds due to the short attention span of E. mongoz The time the subject lemur spent investigating (i.e. sniffing) each sample was recorded. Control pads, pads on which no sample was presen t, were presented first and last for every trial. These control pads were also frozen to remove any factor freezing may have had. Figure 2.6 P resenter holding a screen envolope with a scented pad inside. The presenter is holding the taped sides of th e envelope to avoid cross contamination between samples. She is also whistling to try to get the subject lemurs attention.
29 3. Results and Discussion 3.1. Results For each trial, investigation time was recorded for each pad presented. Appendix III sho amount of time a lemur would spend sniffing and/or licking the pad. Other responses, such as scent marking were rarely seen and not recorded. No instance of overmarking oc curred. Average investigation time was also determined for Experiment I, Groups 1 and 2 and for Experiment II. As evident in the following figures, the E. mongoz were not very interested in investigating the presented pads. Figure 3.1 A verage investigation time for Experiment I, Group 1. For this experiment all of the seven pads came from the same male. The first and last pads were controls with no scent on them. They were presented for 30 seconds each in quick succession. 0.2 0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Control 1 2 3 4 5 6 7 Control Investigation Time (sec.) Pads Experiment I, Group 1 Average Investigation Time
30 Figure 3.2 A ver age investigation time for Experiment I, Group 2. For this experiment the first four (1 4) pads came from one male (Male A) and the last three (5 7) came from a different male (Male B). The first and last pads were controls with no scent on them. They w ere presented for 30 seconds each in quick succession. No investigation time was recorded in any of the trials for Experiment II average investigation time for each of the controls was zero seconds. Because of a lack of investigation time, no further analysis could be done that would have any significant meaning. It is interesting to note, however, that of the five scented pads that were investigated (not including the control pads), Felix was the donor for all of them. This seems to be a coincidence though, seeing as many other pads were investigated. 1 0 1 2 3 4 5 6 Control 1 2 3 4 5 6 7 Control Investigation Time (sec.) Pads Experiment I, Group 2 Average Investigation Time
31 3.2. Discussion and Future Directions There are a number of possible reasons for the lack of interest E. mongoz showed towards the pads. 1) The samples were frozen for too long and lost the scent 2) The lemurs were scared of the presenter and/or the presentation of the pads 3) The lemurs were too stressed from being separated to participate in the trials 4) There was not enoug h scent on each pad 5) The lemurs were too interested in/concerned about other factors 6) The social structure of E. mongoz may not stress olfactory communication as much 7) The samples were not taken and the trials were not run during breeding season Various steps were taken to determine whether any of these reasons were related to the low amount of investigation seen during all of the trials. It was a concern that the samples had been frozen for a long time and this could degrade the chemical components of the se cretions. Perhaps the volatile components in the anogenital gland secretions of E. mongoz were not stable enough and degraded over time, even while frozen. However, when looking at the five scented pads that were investigated (see Appendix III), one of t hem was collected in January with the newest being collected in March. So it did not seem as if the lemurs were only interested in investigating newer samples. This is not a very reliable conclusion due to the low number of samples that were investigated To show that freezing did not in fact degrade the samples, fresh samples were taken from Bimbini. The same procedure was used PVC pipe in branching, collected
32 on sterile gauze and the pad was then placed in a screen envelope and presented as before Two samples were taken and presented to Clarissa and Miguel within half an hour of collection. Clarissa and Miguel were both separated and the trials were done as before. Again, no investigation was seen. Freezing the samples did not appear to influe nce the reactions of E. mongoz they were scared of the presenter or the presentation of the pads. This, however, seems unlikely since the presenter has been working with the lemurs for nearly three years and the lemurs are habituated to her presence inside the enclosures. Even the newest arrival, Clarissa, seemed comfortable around her. Figure 3.3 Presenter with Clarissa, the newest E. mongoz to arrive at the Reserve, on her back during one of the trials. The lemur was obviously comfortable around the presenter. Although the lemurs show surprise at sudden movement, this should not have been the case with the trials. They may have been frightened when pads were first being put up, or put away, but they were held still for thirty seconds in which the lemur would have had plenty of time to come investigate it.
33 At the Reserve E. mongoz are housed in pairs. For the trials to be conducted the pairs had to be separated. Perhaps the lemurs were too stressed due to this separation to participate in the trials. It was observed that they became a little agitated and would go up to the window, looking into the inside room where their mate was being kept, and look for their ma te while the trials were being performed. In the wild the male and female pair is usually found in close contact with each other, especially while resting (Tattersall and Sussman 1975). This same behavior is observed at the Reserve, with pairs rarely see n out of contact with each other. To test this hypothesis an extra trial was done with a pair (Bimbini and Emilia) that was allowed to stay together. The two seemed more at ease, but still ignored the presented pad. Another possible explanation for the observed results was that there was not enough scent on the pad and the E. mongoz could not smell it. Each pad had the scent secretion from one anogenital gland marking on it. This did lead to some inconsistency, since the quantity of secretion varied wi th each marking. It was assumed that one marking would be enough since this method was used in other experiments (Ramsay and Giller 1996). Even though the lemurs would rarely approach the pads and investigate them, if the pad was placed directly under th eir nose they would spend several seconds eagerly investigating it, showing there was enough scent placed on it.
34 Figure 3.4 Series showing Emilia investigating a scented pad that was placed under her nose. This was done following a trial in which she did not investigate a single pad. Pictures were taken over several seconds in which Emilia continuously and eagerly sniffed a nd licked the presented pad. Pictures are ordered chronologically, 1 4. This was seen for both Emilia and Bimbini (while the two were separated) when they were directly presented with scented pads. Unfortunately, it would not be possible to run all of t he trials this way. If the presenter was to follow around all of the lemurs and stick the pads directly under their noses, possibly forcing them to investigate the pads, the results would have had a great positive skew. In a future study perhaps the lemu rs could be crated with the pads presented through the front of the crate. This would keep the lemur in close proximity to the pad, but still give them the option of investigating it or not. This method would have the side effect of adding more stress to the lemurs if they were not crate trained and comfortable in their crates. 1 4 3 2
35 The E. mongoz are caged, but the fencing of the enclosures allows them to have auditory, visual, and olfactory contact with the lemurs around them. If lemurs caged next to each o ther have great animosity towards each other hair, toes, and fingers are sometimes lost as the lemurs fight through the caging. Possibly, the E. mongoz were too interested in the other lemurs around them (not always E. mongoz sometimes a different species) to pay attention to the trials. In particular, Bimbini and Emilia were caged next Eulemur sandfordi ) with whom they regularly fought. Figure 3.5 Emilia fighting with Fred during one o f the trials. Emili a is seen on top of the blue pipe while Fred can be seen through the caging. Besides the threat of other lemurs, the subjects risked being scared by birds, environmental sounds, and human induced sounds such as planes and trucks. How ever, it
36 is not believed that these distractions caused the lack of interest seen. Even for E. mongoz that had empty cages around them, such as Felix and Kikeli or Miguel and Clarissa, no increase in interest was seen. One of the trials (Experiment I, Gr oup 2 with Kikeli as the subject) was also run indoors, where most of the aforementioned distractions were removed, yet no investigation of pads occurred. The E. mongoz at the Reserve have been rotated between cages and between mates. In addition, the Re serve rotates certain items, such as toys, between cages as a form of olfactory enrichment. It is possible that the lemurs were already too familiar with however, becaus e Clarissa also did not show any interest in investigating the pads. Clarissa is the newest addition to the Reserve, arriving 17 January 2011. She also spent over a month in quarantine, separated from the rest of the lemurs, before being caged with Migue l. Therefore, she was not very familiar with the scents of the other E. mongoz at the Reserve, and still seemed uninterested in the pads that were presented. Another study that should be done is to present the lemurs with unknown scents scents that hav e been collected from E. mongoz with which the subjects have never interacted with before. This would remove the potential confounding variable of scents presented. For L catta it has been shown that individuals are much more likely to investigate scents that have come from unknown rather than known conspecifics ( Ramsay and Giller 1996). Another possible explanation for lack of interest is that only the scent markings from male E. mongoz were used. Possibly if female scent had been used, more interest
37 would have been shown, since female L. catta show little interest in the scent marks of males, and much greater interest in the scent markings of other females ( Dugmore and Evans 1990 ). Using female scent was not possible in this experiment since the females were all on birth control shots, which has been shown to change the chemical composi tion of scent secretions in L. catta ( Crawford et al. 2011 ). Although this has not been shown in E. mongoz it was considered a possibility that birth control would have the same effect as it did in L. catta To eliminate this possible variable, females were simply not used as donors for this research. A study that uses females as the scent donors could yield more investigation times and should be attempted in the future. It is also possible that without the visual cue accompanying the scent secretion the physical act of leaving the mark the subject lemurs did not realize what was being presented to them. Perhaps the visual cue is what calls attention to group members that the scent mark is being left and stimulates investigation. However, if E. mo ngoz use olfactory communication similar to that of L. catta it would also be used for territory demarcation ( Mertl 1977; Mertl Millhollen 2006 ). In that case, it seems unlikely that the visual cues would be needed since scent markings would be left behin d for other lemurs to find and investigate. Most likely, however, is that the social structure of E. mongoz does not stress the importance of olfactory communication the way that L. catta does. While L. catta live in large social groups, E. mongoz live in small pair bonded family groups consisting of a mature male and female and their offspring ( Jolly 1998 ). It has been shown that L. catta use olfactory communication in sexual selection to ensure that they pick a mate with good genetic quality and outbreeding ( Charpentier et al. 2008; Charpentier et al. 2010 ).
38 Because E. mongoz pair bond, perhaps this is not as much of a concern, as individuals Lemur catta also use scent marking in sexual competit ion ( Dugmore et al. 1984; Kappeler 1998; Gould and Overdorff 2002; Scordato and Drea 2007; Drea and Scordato 2008; Charpentier et al. 2008; Charpentier et al. 2010; Boulet et al. 2010 ) something that E. mongoz may not do. If E. mongoz does still use olf actory communication in sexual selection a possible explanation for the results seen is that samples were taken, and trials conducted, towards the end of mating season. Mating season at the Reserve is from October to February, with the most intense mating activity seen in December. Perhaps if the samples were both collected and presented at the height of mating season, mor e interest would have been observed. Lemur catta are considered to have one of the most complex olfactory systems of primates (Drea and Scordato 2008), so it may simply be possible that E. mongoz do not have the same interest and sensitivity to scent marks as seen in L. catta To see if the scent secretions of E. mongoz are at all similar to those of L. catta GC MS should be used. The u se of GC MS would allow researchers to determine what compounds are present in the scent secretions of E. mongoz Are the anogenital gland secretions composed of stable long lasting organic acids and esters like in L. catta (Scordato et al. 2007)? Is it possible to see individual differences in the quantity of different compounds in the secretions that could correspond to an individual signature? This sort of analysis could answer many questions that still remain unsolved about the chemical make up of sc ent secretions and the function of olfactory communication in E. mongoz
39 In conclusion, it has been shown that E. mongoz individuals do not show interest in investigating scent marks that are presented to them. Various follow up studies need to be done t o determine possible causes for this disinterest. It still remains unknown if E. mongoz are able to distinguish between individuals based on olfactory communication, but with the use of different experimental methods and GC MS analysis this question may o ne day be answered.
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49 Appendix I: E. mongoz used in experiment. Name Picture Donor or Subject DOB (age) Relationships Date of arrival at Reserve Bimbini Donor and Subject 12 March 2003 (8 years old) Full sibling of Kikeli Currently caged with Emilia 24 September 2007 Merced Donor and Subject 18 April 1997 (almost 14 years old) No relations at Reserve Currently caged with Estella 05 June 2008 Felix Donor and Subject 16 April 1996 (almost 15 years old) Sire of Catherina (no longer at Reserve) Currently caged with Kikeli 12 October 2006 Miguel Subject 10 May 1991 (almost 20 years old) No relations at Reserve Currently caged with Clarissa 05 June 2008 Emilia Subject 04 May 2003 (almost 8 years old) Offspring of Christina (not included in the study) Currently caged with Bimbini Born at Reserve Estella Subject 27 April 1992 (Almost 19 years old) Dam of Catherina (no longer at Reserve) Currently caged with 12 October 2006
50 Merced (Not used due to pregnancy) Kikeli Subject 05 May 2001 (Almost 10 years old) Full sibling of Bimbini Currently caged with Felix 24 September 2007 Clarissa Subject 13 April 1994 (Almost 17 years old) No relations at Reserve Currently caged with Miguel 17 January 2011
51 Appendix II: Behavioral Trials Experiment I, Group 1 Seven samples all from the same donor. Thirty second presentation of pads in quick succession. Trial Number Subject Samples # of Samples from Donor 1 Bimbini Merced 7 2 Felix Bimbini 7 3 Miguel Bimbini 7 4 Emilia Bimbini 7 5 Clarissa Bimbini 7 6 Kikeli Felix 7
52 Experiment I, Group 2 Seven samples, the first four from one donor, and the last three from a different donor. Thirty second presentation of pads in quick succession. Trial Number Subject Samples # of Samples from Donor 1 Bimbini Felix 4 Merced 3 2 Felix Bimbini 4 Merced 3 3 Miguel Bimbini 4 Felix 3 4 Emilia Bimbini 4 Felix 3 5 Clarissa Bimbini 4 Felix 3 6 Kikeli Felix 4 Merced 3
53 Experiment II Three samples, each from a different donor. Thirty second presentation of pads in quick succession. Trial Number Subject Samples # of Samples from Donor 1 Emilia Merced 1 Bimbini 1 Felix 1 2 Estella Merced 1 Bimbini 1 Felix 1 3 Miguel Merced 1 Felix 1 Bimbini 1 4 Emilia Felix 1 Merced 1 Bimbini 1 5 Clarissa Merced 1 Bimbini 1 Felix 1 6 Miguel Felix 1 Merced 1 Bimbini 1
54 Appendix III: Investigation time for each trial ran. The title of the chart corresponds with the subject of the trial. For information on who the donor lemur was for each pad refer to Appendix II. Experiment I, Group 1 7 samples, all from the same male. Presente d in quick succession for 30 seconds each. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Investigation Time (sec.) Pads Bimbini 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Investigation Time (sec.) Pads Felix
55 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Investigation Time (sec.) Pads Miguel 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Investigation Time (sec.) Pads Emilia
56 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Investigation Time (sec.) Pads Clarissa 0 0.5 1 1.5 2 2.5 3 3.5 4 Investigation Time (sec.) Pads Kikeli
57 Experiment I, Group 2 7 samples, pads 1 4 from the one male (Male A) and pads 4 7 from another male (Male B). Presented in quick succession for 30 seconds each. 0 5 10 15 20 25 30 Investigation Time (sec.) Pads Bimbini 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Investigation Time (sec.) Pads Felix
58 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Investigation Time (sec.) Pads Miguel 0 1 2 3 4 5 6 Investigation Time (sec.) Pads Emilia
59 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Investigation Time (sec.) Pads Clarissa 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Investigation Time (sec.) Pads Kikeli
60 Experiment II 3 samples, pads all from different males. Presented in quick succession for 30 seconds each. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Control 1 2 3 Control Investigation Time (sec.) Pads Emilia 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Control 1 2 3 Control Investigation Time (sec.) Pads Clarissa
61 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Control 1 2 3 Control Investigation Time (sec.) Pads Miguel 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Control 1 2 3 Control Investigation Time (sec.) Pads Emilia (2)
62 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Control 1 2 3 Control Investigation Time (sec.) Pads Clarissa (2) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Control 1 2 3 Control Investigation Time (sec.) Pads Miguel (2)
63 Appendix IV: IACUC Approvals for all research done at the Lemur Conservation Foundation.