Journal of Medical Entomology Advance Access published April 6, 2015

Journal of Medical Entomology Advance Access published April 6, 2015 DEVELOPMENT, LIFE HISTORY Ectoparasites of Propithecus diadema (Primates: Indriidae) With Notes on Unusual Attachment Site Selection by Haemaphysalis lemuris (Parasitiformes: Ixodidae) HANS KLOMPEN, 1,2 RANDALL E. JUNGE, 3 AND CATHY V. WILLIAMS 4 J. Med. Entomol. 1 5 (2015); DOI: 10.1093/jme/tjv032 ABSTRACT An examination of ectoparasite loads in two populations of wild diademed sifakas, Propithecus diadema Bennett, yielded seven species four mite species, a louse, a hippoboscid fly, and a leech. Prevalence of the tick Haemaphysalis lemuris Hoogstraal, the mites Liponyssella madagascariensis (Hirst) and Lemuralges propithecus Bochkov et al., and the louse Trichophilopterus babakotophilus Stobbe was quite high, at least 20%. H. lemuris was the most common ectoparasite in one population, while completely absent in a second one. When present, the most common attachment site for H. lemuris males was in the nares of their hosts. KEY WORDS Lemuroidea, ectoparasite, attachment site The Madagascar vertebrate fauna includes many endemics, including a large radiation of lemurs. These charismatic and well known organisms are in turn hosts to a moderate array of considerably less well known ectoparasites, ranging from lice and hippoboscid flies to a variety of mites in both Parasitiformes (Ixodidae, Laelapidae) and Acariformes (Atopomelidae, Psoroptidae, Sarcoptidae) (Klompen 2003, OConnor 2003). Much of our knowledge of these ectoparasites is limited to descriptions (Stobbe 1913; Hirst 1921; Hoogstraal 1953; Strandtmann and Camin 1956; Arthur 1958; Fain 1966, 1972; Blanco et al. 2013; Bochkov et al. 2015) and occasional new host or locality records, with relatively few observations on ecology or life history. As part of the Prosimian Biomedical Survey Project, a project surveying health conditions of lemurs in their natural environment, R.E.J. and C.V.W. opportunistically collected ectoparasites from a range of lemur species in a number of sites. The current study is based on ectoparasites collected at two sites, Tsinjoarivo (collections in June and July 2008) and Ambatovy (collections in September 2012, January 2013, August 2013, March 2014, and September 2014). These sites were selected because of availability of relatively large numbers of ectoparasite collections from one particular lemur species, the diademed sifaka, Propithecus diadema Bennett. This allows preliminary comparisons of the 1 Acarology Laboratory, Department of Evolution, Ecology and Organismal Biology, Ohio State University, Columbus, OH 43212. 2 Corresponding author, e-mail: klompen.1@osu.edu. 3 Department of Animal Health, Columbus Zoo and Aquarium, Columbus, OH 43065. 4 Duke Lemur Center, Duke University, Durham NC 27705. ectoparasite communities of this one host species across two localities. Most comments will focus on ticks, mainly because the records for these parasites are presumed to be more complete than for the other parasites. Of the two tick species commonly associated with Malagasy lemurs, Ixodes lemuris Arthur, has recently been redescribed for all instars, with notes on its distribution on mouse and dwarf lemurs (Cheirogaleidae) (Blanco et al. 2013). The other lemur associated tick species, Haemaphysalis lemuris Hoogstraal, is less well known, but was found to be quite common in this study. The new records allow some inferences on 1) ectoparasite diversity, prevalence, and intensity on the same host species across sites and 2) parasite diversity at the same site across host species, as well as preliminary notes on 3) attachment site selection of H. lemuris. Materials and Methods All lemurs were examined under Research permit #200/12/MEF/SG/DGF/DCB SAP/SCB, issued by the Secretary General, Department of Water and Forests, Republic of Madagascar. All animals underwent medical evaluations following the standard protocol used by the Prosimian Biomedical Survey Project while under anesthesia (see Junge et al. 2011). Ectoparasite presence was noted, relative abundance and location recorded, and representative individual ectoparasites were collected and placed in 90% ethanol. All hosts were released after examination. Mite specimens are deposited at the Ohio State University Acarology Collection (OSAL; http://osuc.bio sci.ohio-state.edu/acarology/; last accessed 20 March 2015), voucher specimens of the Hippoboscidae at the VC The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com

2 JOURNAL OF MEDICAL ENTOMOLOGY Triplehorn Insect Collection (OSUC), also at Ohio State University, Columbus, OH, lice at Georgia Southern University, Statesboro, GA, and leeches at the American Museum of Natural History (AMNH), New York, NY. Use of the terms prevalence and intensity follows Margolis et al. (1982). Site Description. The Tsinjoarivo site (19.6332 S, 47.6832 E) consists of high-altitude rain forest in eastcentral Madagascar. Tsinjoarivo forest is contiguous with the central plateau on its western side, an area that has been severely affected by human settlements and forest fragmentation. The eastern half, composed of lower-altitude continuous forest, has been minimally disturbed (Irwin 2008). Climate is seasonal at Tsinjoarivo, characterized by a rainy season between December and March and a dry season between April and November. Rainfall ranges from an average of 2,000 mm (in fragmented sites) to 2,500 mm in the continuous forest (Irwin 2008). TheAmbatovysite(18.8298S,48.3123E)consistsof mid-altitude forests, at the ecotone between an eastern rain forest known as the Ankeniheny-Zahamena corridor and montane Central Highland forests. The forest has undergone considerable human-induced pressures including hunting, selective logging, slash and burn agriculture, uncontrolled fires, and exploitation for commercial trade. The site currently includes a nickel and cobalt mine. Part of the current project is aimed at monitoring the effects of a biodiversity offset program designed to mitigate biodiversity loss (Dickinson and Berner 2010). Caveats. The project under which these parasites were collected was not set up as a parasite inventory, and quantitative results have to be interpreted with care. Collecting of larger ectoparasites is assumed to be fairly comprehensive (although e.g. ticks attached to hairy parts of the body are more likely to have escaped detection than those attached around the head), but smaller parasite taxa were often collected accidentally, or only when occurring in large numbers (e.g., Lemuralges). As a result, the total ectoparasite fauna of even the most commonly examined host species, P. diadema, may not be complete. For example, literature records suggest the possibility of Atopomelidae (Acariformes), a group of very small mites tightly attached to the hairs (Fain 1972, 1976). Atopomelidae were not recovered from P. diadema in this project, although three specimens were recovered from Eulemur macaco (L.) at a different locality. Given these caveats, prevalence listings should be interpreted as minimum estimates. Interpreting intensity data is more difficult: the opportunistic collecting regime may have missed (especially smaller) parasites on hosts carrying only small numbers, resulting in artificially high intensity numbers, or may not have recovered all parasites on confirmed infested hosts, resulting in artificially low numbers. We present intensity numbers solely as rough preliminary estimates for species or lineages for which no other data have been published previously. Moreover, it is unlikely that better data on ectoparasites of wild lemurs will become available any time soon, given the difficulty of working with lemurs in natural environments. As is, while insufficient for thorough statistical analyses, these collections should be sufficient to suggest general trends. Results A total of 26 individuals of P. diadema were examined at Tsinjoarivo, a total of 61 at Ambatovy. Several hosts at Ambatovy were examined twice (N ¼ 11) or even three times (N ¼ 1) for a total of 74 parasite collections. We treat all individual collections as independent. P. diadema was the only host species examined at Tsinjoarivo, while at Ambatovy hosts also included Avahi laniger (Gmelin) and Indri indri (Gmelin) (Indriidae), Eulemur fulvus (E. Geoffroy Saint-Hillaire) (Lemuridae), Lepilemur mustelinus I. Geoffroy Saint- Hillaire (Lepilemuridae), Cheirogaleus crossleyi Grandidier, and Microcebus sp. (Cheirogaleidae). No ectoparasites were collected from the three Microcebus sp. examined. Parasite Diversity of P. diadema. A total of seven species of ectoparasites were recovered from P. diadema: Haemaphysalis lemuris, Ixodes lemuris (both Parasitiformes: Ixodidae), Liponyssella madagascariensis (Hirst) (Parasitiformes: Laelapidae), Lemuralges propithecus Bochkov et al. (Acariformes: Psoroptidae), Trichophilopterus babakotophilus Stobbe (Phthirapthera : Philopteridae), louse flies (Diptera : Hippoboscidae) (probably Allobosca crassipes Speiser), and leeches. The two leeches collected at Ambatovy were not identified, and, given their very small number, will be ignored for the remainder of this study. Ectoparasite communities on P. diadema showed distinct differences between the two main sites (Table 1). All six common species were found at Ambatovy, but H. lemuris and L. madagascariensis were absent at Tsinjoarivo. This result is particularly striking for H. lemuris, which was the most commonly collected ectoparasite species at Ambatovy, and is likely to be recovered if present. In contrast, prevalence (and intensity) of the other tick species, I. lemuris, is similar in both localities (Tables 1 and 2). There may have been a modest outbreak of L. propithecus in the P. diadema population during the sampling period at Table 1. Number of lemurs carrying specific ectoparasite taxa at Tsinjoarivo and Ambatovy Species No. coll. Hae Ixo Lae Pso Lice Flies P. diadema (Tsinjoarivo) 26 4(15) 15(58) 12(46) 4(157) P. diadema (Ambatovy) 74 54(73) 7(9) 22(30) 16(22) 40(54) 11(15) Avahi laniger 33 5(17) 5(15) Cheirogaleus crossleyi 12 2 Eulemur fulvus 7 2 1 Indri indri 17 2(12) 2(12) 2(12) 2(12) Lepilemur mustelinus 16 7(44) 9(56) 4(25) Prevalence (%) listed for host taxa with >15 specimens examined (in brackets). Abbreviations: No. coll., total number of host individuals examined (includes repeat collections from the same host individual); Hae, Haemaphysalis lemuris; Ixo, Ixodes lemuris; Lae, Liponyssoides madagascariensis; Pso, Lemuralges propithecus.

2015 KLOMPEN ET AL.: ECTOPARASITES OF Propithecus diadema 3 Table 2. Total number of individual ectoparasites and intensity (in brackets) of parasites recovered from lemurs at Tsinjoarivo and Ambatovy Species Hae Ixo Lae Pso Lice Flies P. diadema (Tsinjoarivo) 4(1.0) 270(18.0) 187(15.6) 8(2.0) P. diadema (Ambatovy) 460(8.5) 9(1.3) 59(2.7) 53(3.3) 230(5.8) 16(1.5) Avahi laniger 14 (2.8) 6(1.2) Cheirogaleus crossleyi 2(1.0) Eulemur fulvus 4(2.0) 1(1.0) Indri indri 4 (2.0) 3(1.5) 7(3.5) 2(1.0) Lepilemur mustelinus 16(2.3) 31(3.4) 4(1.0) Tsinjoarivo, with both prevalence and intensity much higher than in Ambatovy. Prevalence and Intensity Across Host Species. (Minimum) prevalence of H. lemuris in Ambatovy on P. diadema (73%) is remarkably high (Table 1). High prevalence of this tick was also noted for Lepilemur mustelinus (44%). Given this, the complete absence of H. lemuris on I. indri (17 individuals examined) seems worth noting. Corresponding numbers for I. lemuris are much smaller. This species is relatively rare on P. diadema at both Ambatovy (9%) and Tsinjoarivo (17%), but recovered relatively frequently from L. mustelinus (56%). Among the other ectoparasites, L. madagascariensis appears to have a wide host range, although the far majority of our records were from P. diadema. Described from Eulemur albifrons Geoffroy (listed as Lemur mongoz albifrons), we have recorded this species at various localities from Propithecus edwardsi Grandidier, Eulemur coronatus Gray, E. fulvus (Geoffroy), E. macaco, Lemur catta L., and Indri indri. Similarly, the louse T. babakotophilus has been recorded during this project from a considerable range of hosts (Propithecus diadema, P. verreauxi Grandidier, Indri indri, Eulemur coronatus, E. macaco, ande. mongoz (L.)). In contrast, the makialgine psoroptid L. propithecus was found exclusively on P. diadema. Notably, it was present on that host at both Tsinjoarivo and Ambatovy. A related species, L. intermedius Fain has been recorded from a number of lemur species, specifically Lepilemur ruficaudatus Grandidier, Propithecus verreauxi, Eulemur fulvus, andhapalemur griseus (Link) (Fain 1963, 1966), but it is unclear whether it was established on all of these host taxa. Minimum prevalence of many of the nontick parasites on P. diadema from Ambotavy was quite high, 30% and 22%, respectively, for L. madagascariensis and L. propithecus, 54% for T. babakotophilus. Numbers for Tsinjoarivo are similar, although L. madagascariensis was not recovered from that site. As an aside, these are the first prevalence numbers for any lemurassociated Laelapidae or Psoroptidae. Attachment Site Selection. The considerable sample size for P. diadema at Ambatovy allowed a preliminary examination of attachment site preferences of H. lemuris. Specific attachment site data were available for 69 collections (Table 3). Ticks from four additional collections were mixed, with vials including material from both the nose and body regions (10 F, 20 M total). Table 3. Attachment site selection of H. lemuris on P. diadema from Ambatovy Attachment site No. coll. H. lemuris F no. ind. (inten.) H. lemuris M no. ind. (inten.) Body 16 23(1.4) 20(1.3) Ear 6 4(0.7) 4(0.7) Eye 9 0 10(1.1) Nose 38 0 295(7.8) Abbreviations: no. ind., number individual ticks collected; inten., intensity. In the majority of collections (55%), individual hosts had H. lemuris ticks attached in the external part of the nares, mostly within the first five millimeter of the nostril (Fig. 1). A total of 295 ticks were recovered from the nares, all of which were males. A few ticks were recovered from the eye region, especially the eyelids, also all males. Additional males were recovered from around the ears (4) and the body (20). Far fewer female ticks with site information (27) were recovered, with 23 of these attached to various parts of the body (chest, perianal region, groin), and the remaining four recovered from the ears. In many cases females were found associated with males, in one case attached in mating position. These numbers reflect the expected male bias in attached ticks (males will stay on the host longer), possibly reinforced by collecting bias, as tick recovery from the nose region is much easier than from the thickly furred regions of the body proper. H. lemuris was also recovered from several other lemur species, but we have limited attachment site information for those collections: one male each was recovered from the nose of a C. crossleyi and a L. mustelinus, another male from the neck of a L. mustelinus. None of the remaining ectoparasites showed comparable patterns in site preference. The louse T. babakotophilus (site data for 31 of 40 collections) was never confirmed from the head, and I. lemuris (mostly larvae and nymphs) were most common (7 of 9 collections) on the head, but both L. madagascariensis and L. propithecus were recovered from a range of sites on the head and body. Host Gender Preference. We examined whether there are any obvious differences in ectoparasite loads between male and female P. diadema (Table 4). This examination was limited to the more commonly recovered taxa (found in at least 10 collections). Records for

4 JOURNAL OF MEDICAL ENTOMOLOGY Fig. 1. Frontal view of P. diadema with ticks in both nostrils. Courtesy Lydia Green. Table 4. Prevalence and intensity (in brackets) of selected ectoparasites on P. diadema from Ambatovy by host gender Host gender No. coll. Hae Lae Pso Lice Female 35 74(6.4) 29(2.7) 17(5.2) 63(5.6) Male 39 72(10.5) 31(2.7) 26(2.2) 46(5.9) Table 5. Prevalence and intensity (in brackets) of selected ectoparasites on P. diadema from Ambatovy by season Host gender No. coll. Hae Lae Pso Lice Summer 17 82(9.6) 18(4.3) 18(2.0) 18(4.7) Winter 57 72(7.9) 33(2.3) 23(3.1) 56(5.8) H. lemuris show similar prevalence on male P. diadema vs. female hosts, but intensity is somewhat higher for male hosts. This difference is almost entirely due to higher numbers of male H. lemuris in the nares. Focusing on hosts with H. lemuris in the nares, the number of male and female hosts carrying these ticks in that position was identical (N ¼ 20; prevalence in females and males, respectively, 0.57 and 0.51). However, intensity in males was higher (respectively, 5.1 and 9.7). There are some differences between male and female hosts in terms of the numbers of associated L. madagascariensis, L. propithecus, or lice(table 4), but given the numbers of infested hosts, and the uncertainty in recovery efficiency, we prefer simply presenting the available data, and deferring on any conclusions based on these relatively small differences. Seasonal Differentiation. Infestation levels of especially ticks are often seasonal. In order to see whether unusual phenomena such as the attachment in the nose were seasonal, we compared numbers for local summer (January and March collections) vs. winter (August, September) collections (Table 5). Summer in Madagascar tends to be the wet season; winter is not only colder, it is also dryer. Differences in both prevalence and intensity for H. lemuris are quite small, providing no obvious indication of seasonal differentiation in parasite loads. The other parasites appear to show some differences, e.g., summer prevalence for L. madagascariensis, and T. babakotophilus is lower than in winter (although intensity is similar). As with differences across host gender, we prefer to simply report these numbers, in the hope that future research will allow improved evaluations of these possible trends. In conclusion, most ectoparasite taxa occurred in both main localities, but the most common ectoparasite of P. diadema in Ambatovy, H. lemuris, was completely absent in Tsinjoarivo, even though P. diadema was abundant. This result is consistent with results reported by Blanco et al. (2013) based on ticks recovered from mouse and dwarf lemurs (Cheirogaleidae). It reinforces the notion that studies of (especially nonpermanent) ectoparasites have to consider not only a diversity of hosts, but also a variety of localities. The general preference at Ambatovy of H. lemuris for P. diadema, and I. lemuris for L. mustelinus might seem to bolster arguments for some level of host specificity (Hoogstraal and Aeschlimann 1982), but available data could also be consistent with an alternative hypothesis of off-host site

2015 KLOMPEN ET AL.: ECTOPARASITES OF Propithecus diadema 5 specificity, with individual tick species using hosts in proportion to their occurrence in the preferred off-host habitat (Klompen et al. 1996). P. diadema, I. indri, and L. mustelinus most likely use their environment in different ways, resulting in different probabilities of attachment by the different tick species. Under that hypothesis, the absence of H. lemuris in Tsinjoarivo might be due to a lack of suitable off-host habitat. The most interesting observations deal with the attachment site selection of H. lemuris. There are anecdotal records of ticks attached in the noses of humans (e.g., Walton 1960, Aronsen and Robbins 2008, Hamer et al. 2013), and a report of frequent occurrence of ticks in the noses in a population of chimpanzees (Hamer et al. 2013), but the current observation, documenting such a prominent use of the nose as attachment site for ticks (64% of all H. lemuris recovered at Ambatovy), is unusual. It would be interesting to see if this site selection pattern is a local phenomenon or perhaps restricted to one host, P. diadema, or if H. lemuris prefers attaching in the nose of its hosts across its geographic and host range. The fact that all of the almost 300 ticks recovered from the nares were males, combined with the observation of similar numbers of males and females on the body and around the ears, might suggest an additional aspect, a gender bias in attachment sites. However, available numbers (only 27 females with site data), though suggestive, cannot exclude occasional presence of females in the nares. Still, some bias in attachment site selection appears likely. An explanation for such a bias might be related to social behavior of the hosts (e.g., frequent mutual sniffing), and the need for male ticks to find females. The presence of most females in the genital or perianal region of their hosts would be consistent with such an idea. Acknowledgments We thank Karine Mahefarisoa and Tsiky Rajaoarivelo for their assistance in collecting parasite samples; Andre Bochkov, Russian Academy of Sciences, St. Petersburg, and Lance Durden, Georgia Southern Univ., Statesboro, for identification of, respectively, the psoroptid mites and lice, and the Ambatovy Mining SA, Madagascar, for financial and logistical support to R.E.J. and C.V.W. References Cited Aronsen, G. P., and R. G. Robbins. 2008. An instance of tick feeding to repletion inside a human nostril. Bull. Peabody Mus. Nat. Hist. 49: 245 248. Arthur, D. R. 1958. The Ixodes schillingsi group: ticks of Africa and Madagascar, parasitic on primates, with descriptions of two new species (Ixodoidea, Ixodidae). Parasitology 47: 544 559. 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