N7 2016 Southeastern Naturalist Notes Vol. 15, No. 1 D.J. Richardson, C.I. Hammond, and K.E. Richardson The Florida Ivory Millipede, Chicobolus spinigerus (Diplopoda: Spirobolidae): A Natural Intermediate Host of Macracanthorhynchus ingens (Acanthocephala: Oligacanthorhynchidae) Dennis J. Richardson1,*, Charlotte I. Hammond1, and Kristen E. Richardson1 Abstract - Eleven (55%) of 20 Chicobolus spinigerus (Florida Ivory Millipede) collected from Miami-Dade County, FL, were infected with 1–57 cystacanths of Macracanthorhynchus ingens, a common acanthocephalan of North American Procyon lotor (Raccoon), representing a new host record. The distribution of cystacanths among the millipedes exhibited the highly overdispersed negative binomial distribution that is characteristic of parasite populations. Little information exists in the scientific literature concerning the natural history of Chicobolus spinigerus (Wood) Chamberlin (Florida Ivory Millipede) and there is no information available concerning its parasite fauna. Chicobolus spinigerus has been reported from Florida, Georgia, South Carolina, and Alabama (Shelly and Floyd 2014). Shelly and Floyd (2014) suggested that the ultimate existence of the genus Chicobolus might be threatened because northern representatives of the genus are displaced by the expanding range of spirobolid millipedes of the genus Narceus. Currently, the southern-most region of Florida including Miami-Dade County constitutes the primary range of C. spinigerus and the only portion of the range distribution of C. spinigerus in which millipedes of the genus Narceus do not occur. In addition to incursion from the north by Narceus spp., the southern representatives of Chicobolus are threatened by rising sea levels in conjunction with proliferation of invasive species of millipedes representing the families Rhinocricidae and Trigoniulidae (Shelly and Floyd 2014). In view of these threats, it is important to amass as much information as possible concerning the natural history of this imperiled millipede species. During January 2015, we purchased from Backwater Reptiles® a total of 20 individual C. spinigerus collected from under fallen trees in a heavily forested area of about 8 ha in Miami-Dade County, FL. We examined them for the presence of helminth parasites. These examinations revealed the presence of cystacanths of the acanthocephalan Macracanthorhynchus ingens (von Linstow) Meyer. We calculated prevalence, mean intensity, and relative abundance according to Richardson (2012) as follows: prevalence of infection was determined by dividing the number of millipedes sampled by the number infected with M. ingens, mean intensity was determined by dividing the total number of cystacanths collected by the number of infected millipedes, and mean abundance was determined by dividing the total number of cystacanths collected by the total number of millipedes examined, including both infected and uninfected individuals. Of the 11 infected millipedes (55% of examined individuals), 7 individuals each contained a single cystacanth of M. ingens; other millipedes were infected with 2, 4, 7, and 57 cystacanths, respectively. The infections had a mean intensity (± SE) of 7.00 (± 5.03) and mean abundance (± SE) of 3.9 (± 2.82). Many of the cystacanths were surrounded by an envelope, and the proboscis of each cystacanth was fully invaginated, suggesting that the cystacanths were fully developed and infective (Moore 1946, Schmidt 1985). Size and morphology of cystacanths were consistent 1Quinnipiac University, 275 Mount Carmel Avenue, Hamden, CT 06518. *Corresponding author - Dennis.Richardson@quinnipiac.edu. Manuscript Editor: Jason Cryan Notes of the Southeastern Naturalist, Issue 15/1, 2016 2016 Southeastern Naturalist Notes Vol. 15, No. 1 N8 D.J. Richardson, C.I. Hammond, and K.E. Richardson with that of M. ingens according to Richardson (2005), who found the cystacanths of M. ingens to be substantially larger than those of the confamilial Oligacanthorhynchus microcephalus (Rudolphi) Schmidt; however, even after being placed in distilled water for 72 hr, the proboscides did not evaginate, necessitating confirmation of identification using molecular techniques. We extracted and purified genomic DNA from 4 of the cystacanths isolated in different hosts and subjected each sample to DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit I (CO-I) gene following techniques described by Richardson et al. (2010). Purified PCR products were sequenced using the LCO1490 primer from the CO-I gene by the W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University. Nucleotide sequences were aligned using ClustalW and compared with other acanthocephalan DNA sequences found in GenBank using nucleotide BLAST search. Molecular comparisons among the 4 specimens revealed differences of 1–1.7% (0–10 of 651 nucleotides). Molecular comparisons of 651 nucleotides of the CO-I gene between the 4 specimens and a morphologically confirmed specimen of M. ingens (YPMIZ.067503) (GenBank KT881244) collected from a Procyon lotor L. (Raccoon) in Drew County, AR (Richardson 2014) revealed differences of 0–2.0% (2–11 nucleotides) The CO-I DNA sequences of the C. spinigerus specimens were also compared to a CO-I sequence from a specimen of M. ingens obtained from GenBank (GenBank AF416997.2), revealing differences of 1.0% (4–7 of 629 nucleotides). Comparison of CO-I sequences from our specimens to CO-I sequences of Macracanthorhynchus hirudinaceus (Pallas) Travassos (GenBank FR856886.2; Weber et al. 2013), a common congener of M. ingens that occurs in swine, revealed differences of 25–26% (160–165 of 646 nucleotides). Comparison of CO-I sequences from our specimens to specimens of Oligacanthorhynchus microcephalus (Rudolphi) Schmidt, collected from a Didelphis virginiana Kerr (Virginia Opossum) from Faulkner County, AR (YPMIZ.077148; GenBank KT881245) revealed differences of 30–31% (149–155 of 499 nucleotides). Host voucher representatives of C. spinigerus were deposited in the Entomology Collection of the Yale Peabody Museum of Natural History at Yale University (New Haven, CT) and assigned collection number YPMENT.844833. Sequences of the CO-I gene for 4 cystacanths of M. ingens were deposited in GenBank and assigned accession numbers GenBank KT881246–KT881249. All remaining cystacanths of M. ingens collected in this study were deposited in the Invertebrate Zoology collection of the Yale Peabody Museum of Natural History and assigned collection numbers YPMIZ.077142–YPMIZ.077146. The primary definitive hosts of M. ingens are the Raccoon and Ursus americanus Pallas (Black Bear). Macracanthorhynchus ingens has been reported from throughout much of the eastern and midwestern United States, predominantly from Raccoons, and has also been reported from Racoons in Nicaragua and Bassariscus astutus (Lichtenstein) Coues (Ringtail) in Texas. Additionally, patent infections of M. ingens have been reported from Canis lupus familiaris L. (Domestic Dog) and humans in the United States (Richardson 2014). Macracanthorhynchus ingens has previously been reported from Raccoons and Black Bears from Florida (Conti et al. 1983, Crum et al. 1978, Forrester 1992, Foster et al. 2004, Harkema and Miller 1964, Schaffer et al. 1981). Macracanthorhynchus ingens apparently utilizes a wide range of both paratenic and intermediate hosts (Richardson 2014). Natural intermediate hosts previously reported for M. ingens include Odontotaenius disjunctus (Illiger) Kuwert (Bessbug) and Parcoblatta pennsylvanica (De Geer) Hebard (Woodroach) from Louisiana (Elkins and Nickol 1983, Richardson 2014). Beetles of the genera Phyllophaga and Ligyrus were demonstrated to be intermediate hosts by laboratory infection (Moore 1946). Previously documented N9 2016 Southeastern Naturalist Notes Vol. 15, No. 1 D.J. Richardson, C.I. Hammond, and K.E. Richardson natural infections from millipedes include Narceus americanus (Beauvois) Rafinesque from Louisiana (Richardson 2006) and Ohio (Crites 1964) and Narceus annularis Rafinesque from New Jersey (Fahnestock 1985a, b). In addition, Bowen (1967) successfully infected the spirobolid millipedes Floridobolus penneri Causey and Narceus gordanus (Chamberlin) Loomis with M. ingens by feeding eggs taken from adult M. ingens. The present report of M. ingens from C. spinigerus represents a new host record. Given the fact that Raccoons are omnivorous and exceedingly opportunistic, utilizing a wide variety of food sources (Whitaker and Hamilton 1998) including millipedes (Harman and Stains 1979, Johnson, 1970, Llewellyn and Uhler 1952), we postulate that spirobolid millipedes play an epizootiologically important role in the life cycle of M. ingens in nature. Additionally, the only known intermediate host (Richardson 2006, Richardson et al. 2014) for the confamilial acanthocephalan O. microcephalus is the millipede N. americanus. Richardson (2006) demonstrated the complete life cycle of O. microcephalus, a common acanthocephalan of the Virginia Opossum utilizing cystacanths taken from naturally infected N. americanus collected from Louisiana. Richardson (2006) found naturally occurring co-infections of O. microcephalus and M. ingens in the specimens of N. americanus collected from southern Louisiana. Such a high prevalence of infection of acanthocephalans in intermediate host populations, as observed in this study, is unusual. It has been our observation that such natural populations of millipedes exhibiting high prevalence of cystacanths are restricted to isolated areas of enhanced transmission, or epizootiological “hot spots”, apparently where there is a co-occurring high density of infected Raccoons and/or Opossums. Such settings may play an important role in maintaining the suprapopulation of these acanthocephalans. It seems likely, given the array of intermediate and paratenic hosts reported for M. ingens, that certain microhabitats could exhibit differing epizootiolgical profiles with different intermediate and paratenic hosts assuming paramount roles in different settings. If this assertion is correct, such hot spots provide excellent sites to conduct epizootiological investigations. Although relatively small, the available sample of 20 millipedes from the intermediate host population was adequate to facilitate analysis of the population structuring of M. ingens. The over-dispersion parameter k was calculated using Fisher’s maximum likelihood technique (Bliss and Fisher 1953). The negative binomial distribution (Fisher 1941) was fit to the data and goodness of fit was tested by comparison of observed and expected frequencies by chi-square analysis as described by Bliss and Fisher (1953). Cystacanths of M. ingens within individuals of C. spinigerus have exhibited a high degree of over-dispersion that is characteristic of parasite populations (Crofton 1971, Richardson 2012, Richardson et al. 2011). Similary in our study, the M. ingens cystacanth population among the 20 individuals of C. spinigerus examined exhibited pronounced overdispersion characteristic of parasite populations, with a variance-to-mean ratio (VMR) of 41.40 and k value of 0.285. The standard error variance of k was 0.0003. The data fit the negative binomial distribution (χ2 = 3.054, 2 d.f., critical value = 5.991). The most heavily infected millipede, constituting 5% of the host population sampled, accounted for 74% of all cystacanths collected from the 20 millipedes. Richardson and Barger (2005) reported a high level of over-dispersion of M. ingens in Raccoons examined from Ossabaw Island, GA, evidenced by a VMR of 411.9 with over 75% of the 1481 individuals of M. ingens collected occurring in 3 (7.0%) of the 43 raccoons examined. In view of the data collected in the present study, we postulate that the over-dispersion/negative binomial distribution of parasites in the intermediate host population may provide the infrastructure for overdispersion in the definitive host population when a few individual definitive hosts ingests 2016 Southeastern Naturalist Notes Vol. 15, No. 1 N10 D.J. Richardson, C.I. Hammond, and K.E. Richardson the very few heavily infected intermediate hosts. For further discussion on the implications of over-dispersion of parasite populations see Crofton (1971), Poulin (2007), Richardson et al. (2011), Richardson (2012), and references therein. Acknowledgments. Chris T. McAllister, Eastern Oklahoma State College, Idabel, OK, provided confirmation of the identification of Chicobolus spinigerus. Samuel Sundberg, Backwater Reptiles, Rocklin, CA, provided valuable information concerning specific information about the collection site. Lourdes Rojas, Division of Invertebrate Zoology, Yale Peabody Museum of Natural History, Yale University, New Haven, CT, assisted in specimen management. Literature Cited Bliss, C.I., and R.A. Fisher. 1953. Fitting the negative binomial distribution to biological data and note on the efficient fitting of the negative binomial. Biometric s 9:176–200. Bowen, R.C. 1967. Defense reactions of certain spirolbolid millipedes to larval Macracanthorhynchus ingens. Journal of Parasitology 53:1092–1095. Conti, J.A., D.J. Forrester, and J.R. Brady. 1983. Helminths of Black Bears in Florida. Proceedings of the Helminthological Society of Washington 50:252–256. Crites, J.L. 1964. A millipede, Narceus americanus, as a natural intermediate host of an acanthocephalan. Journal of Parasitology 50:293. Crofton, H.D. 1971. A quantitative approach to parasitism. Parasitology 62:179–193. Crum, J.M., V.F. Nettles, and W.R. Davidson. 1978. Studies on endoparasites of the Black Bear (Ursus americanus) in the southeastern United States. Journal of Wildlife Diseases 14:178–186. Elkins, C.A., and B.B. Nickol. 1983. The epizootiology of Macracanthorhynchus ingens in Louisiana. Journal of Parasitology 69:951–956. Fahnestock, G.R. 1985a. Macracanthorhynchiasis in dogs (Part 1). Modern Veterinary Practice 66:31–34. Fahnestock, G.R. 1985b. Macracanthorhynchiasis in dogs (Part 2). Modern Veterinary Practice 66:81–83. Fisher, R.A. 1941. The negative binomial distribution. Annals of Eugenics 11:182–187. Forrester , D.J. 1992. Parasites and Diseases of Wild Mammals in Florida. University Press of Florida, Gainesville, FL. 459 pp. Foster , G.W., M.W. Cunningham, J.M. Kinsella, and D.J. Forrester. 2004. Parasitic helminths of Black Bear cubs (Ursus americanus) from Florida. Journal of Parasitology 90:173–175. Harkema, R., and G.C. Miller. 1964. Helminth parasites of the Raccoon, Procyon lotor, in the southeastern United States. Journal of Parasitology 50:60–66. Harman, D.M., and H.J. Stains. 1979. The Raccoon on St. Catherines Island, Georgia. 5. Winter, spring, and summer food habits. American Museum Novitates 2679:1–24. Johnson, A.S. 1970. Biology of the Raccoon (Procyon lotor varius Nelson and Goldman) in Alabama. Auburn University Experiment Station Bulletin 402:1–148. Llewellyn, L.M., and F.M. Uhler. 1952. The foods of fur animals of the Patuxent Research Refuge, Maryland. American Midland Naturalist 48:193–203. Moore, D.V. 1946. Studies on the life history and development of Macracanthorhynchus ingens Meyer, 1933, with a re-description of the adult worm. Journal of Parasitology 32:387–399. Poulin, R. 2007. Evolutionary Ecology of Parasites, 2nd Edition. Princeton University Press, Princeton, NJ. 360 pp. Richardson D.J. 2005. Identification of cystacanths and adults of Oligacanthorhynchus tortuosa, Macracanthorhynchus ingens, and Macracanthorhynchus hirudinaceus based on proboscis and hook morphometrics. Journal of the Arkansas Academy of Science 59:205–209. Richardson, D.J. 2006. Life cycle of Oligacanthorhynchus tortuosa (Oligacanthorhynchidae), an acanthocephalan of the Virginia Opossum (Didelphis virginiana). Comparative Parasitology 73:1–6. N11 2016 Southeastern Naturalist Notes Vol. 15, No. 1 D.J. Richardson, C.I. Hammond, and K.E. Richardson Richardson, D.J. 2012. Population structuring and transmission dynamics of Gromphadorholaelaps schaferi: A symbiotic mite of the Madagascan Hissing-cockroach, Gromphadorhina portentosa. Journal of the Arkansas Academy of Science 66:125–136. Richardson, D.J. 2014. Acanthocephala of the Raccoon (Procyon lotor) with a faunal review of Macracanthorhynchus ingens (Archiacanthocephala: Oligacanthorhynchidae). Comparative Parasitology 81:44–52. Richardson, D.J., and M.A. Barger. 2005. Microhabitat specificity of Macracanthorhynchus ingens (Acanthocephala: Oligacanthorhynchidae) in the Raccoon (Procyon lotor). Comparative Parasitology 72:173–178. Richardson, D.J., W.E. Moser, C.I Hammond, A.C. Shevchenko, and E. Lazo-Wasem. 2010. New geographic distribution records and host specificity of Placobdella ali (Hirudinida: Glossiphoniidae). Comparative Parasitology 77:202–206. Richardson, D.J., K.R. Richardson, K.D. Callahan, J. Gross, P. Tsekeng, B. Dondji, and K.E. Richardson. 2011. Geohelminth infections in rural Cameroonian villages. Comparative Parasitology 78:161–179. Richardson, D.J., S.L. Gardner, and J.W. Allen, Jr. 2014. Redescription of Oligacanthorhynchus microcephalus (Rudolphi, 1819) Schmidt 1972 (syn. Oligacanthorhynchus tortuosa (Leidy, 1850) Schmidt, 1972) (Acanthocephala: Oligacanthorhynchidae). Comparative Parasitology 81:53–60. Schaffer, G.D., W.R. Davidson, V.F. Nettles, and E.A. Rollor III. 1981. Helminth parasites of translocated Raccoons (Procyon lotor) in the southeastern United States. Journal of Wildlife Diseases17: 217–227. Schmidt, G.D. 1985. Development and life cycles. Pp. 273–305, In D.W.T. Crompton and B.B. Nickol (Eds.). Biology of the Acanthocephala. Cambridge University Press, Cambridge, UK. 519 pp. Shelly, R.M., and S.D. Floyd. 2014. Expanded concept of the millipede family Spirobolidae (Diplopoda: Spirobolida: Spirobolidae): Proposals of Aztecolini n. tribe and Floridobolinae/ini and Tylobolini n. stats.; (re)descriptions of Floridobolus and F. penneri, both Causey, 1957, and F. orini n. sp.; hypotheses on origins and affinities. Insecta Mundi 0357 :1–50. Weber, M., A.R. Wey Fabrizius, L. Podsiadlowski, A. Witek, R.O. Schill, L. Sugár, H. Herlyn, and T. Hankeln. 2013. Phylogenetic analyses of endoparasitic Acanthocephala based on mitochondrial genomes suggest secondary loss of sensory organs. Molecular Phylogenetics and Evolution 66:182–189. Whitaker, J.O., Jr., and W.J. Hamilton Jr. 1998. Mammals of the eastern United States. Cornell University Press, Ithaca, NY. 583 pp.