2009 NORTHEASTERN NATURALIST 16(1):125–130 First Record of Millipeds as Hosts for Horsehair Worms (Nematomorpha) in North America Andreas Schmidt-Rhaesa1,*, Monica A. Farfan2, and Ernest C. Bernard3 Abstract - We report several cases in which Gordionus lineatus (Horsehair Worm) (Nematomorpha) parasitizes the diplopod Cambala annulata (Myriapoda, Diplopoda) in Ohio. Diplopods as host for nematomorphs in North America have previously only briefly been mentioned. This report is the first host record for a Nearctic Gordionus species. Gordionus lineatus is also a new record for Ohio. Though the sample size is small, the prevalence may be high (50%). Myriapods appear to survive the parasitization. As Cambala annulata is likely to be herbivorous, pathways of infection with nematomorphs are discussed. Introduction Gordionus lineatus (Leidy) (Horsehair Worm) (Nematomorpha) occur free in water as adults and the early larval stage develops parasitically within hosts (Hanelt et al. 2005). In most cases, there appear to be intermediate (paratenic) hosts near the interface of the aquatic and terrestrial environments, where the majority of final hosts occur (Hanelt et al. 2005). Exceptions are the few marine nematomorphs and some cases of freshwater nematomorphs, in which the entire development takes place in freshwater (caddisflies or diving beetles as final hosts). All final hosts of freshwater nematomorphs (about 300 species of the taxon Gordiida) are arthropods, mainly insects (Hanelt et al. 2005). There are comparatively few reports of chelicerates or millipeds as hosts. Most reported cases of chelicerates as hosts are doubtful (see Poinar 2000), but there are some reliable records of millipeds as hosts (see Table 1). Cooper and Storck (1973) briefly report a diplopod (Narceus sp.) as host of an undetermined nematomorph species in North America. We report here the nematomorph Gordionus lineatus parasitizing the diplopod Cambala annulata (Say). This is the first host record for a species of the genus Gordionus in North America (see Schmidt-Rhaesa et al. 2003). Methods Nineteen specimens of Cambala annulata (Diplopoda, Spirostreptida) were collected in pitfall traps around Deep Wood Farm, Benton Township, Hocking County, OH (39°24.3'N 82°34.6'W) during 2006. The habitat is a mixed deciduous forest (Quercus rubra L. [Northern Red Oak], Liriodendron 1Biocentre Grindel and Zoological Museum, University Hamburg. Martin-Luther- King-Platz 3, 20146 Hamburg, Germany. 2Department of Entomology, Ohio State University, Columbus, Ohio 43210. 3Entomology and Plant Pathology, University of Tennessee, 2431 Joe Johnson Drive, 205 Plant Sciences, Knoxville, TN 37996-4560. *Corresponding author - andreas.schmidt-rhaesa@uni-hamburg.de. 126 Northeastern Naturalist Vol. 16, No. 1 tulipifera L. [Tulip Tree], ash, Tsuga canadensis (L.) Carr. [Eastern Hemlock], maple, hickory, beech). Pitfall traps were used and contained ethylene glycol or propylene glycol and therefore killed the millipeds. Parasitization with nematomorphs was observed by dissecting the host. One specimen of C. annulata was collected in Waterloo Wildlife Area, Athens County, Ohio (39°21'06"N 82°15'58"W) on 1 June 2006. This site has a forest of Quercus alba L. (White Oak), Northern Red Oak, and hickory on the ridge-tops and beech and maple in the ravine bottoms. The specimen was collected in a pitfall trap baited with carrion (rotten chicken wings) and was kept alive in the lab for six months. The emerged G. lineatus was fixed in ethanol and investigated by scanning electron microscope (SEM). Two of the Hocking County specimens were investigated in detail with SEM. Specimens were dehydrated in an increasing ethanol series, criticalpoint dried, gold-sputtered, and investigated with a LEO SEM-1525. Images were recorded digitally. A voucher specimen of host and parasite was deposited in the Cleveland Museum of Natural History in Cleveland, Ohio under the accession number CMNH 2008-18. Results Of 19 specimens of C. annulata collected at the Deep Wood Farm site, 9 (47.7%) were parasitized by nematomorphs (Figs. 1A–C). One specimen (Fig. 1A) was only partially dissected to preserve the spatial relationships of host and parasite. Another specimen of C. annulata was parasitized by one male of G. lineatus. Its body color was light brown, length = 265 mm, and diameter = 0.5 mm. Another specimen of C. annulata was parasitized by four specimens of G. lineatus. All these specimens were males; their body color was medium brown. The four specimens measured 170, 175, 180, and 205 mm (mean = 182.5 mm), and the diameter was 0.5 mm for each of the four. The specimen of C. annulata from the Waterloo Wildlife Area site was parasitized by a single female G. lineatus, which was white. While the posterior end was missing, the remaining length is 325 mm, and the diameter was 0.6 mm. The characters of these nematomorph specimens correspond with those known of G. lineatus (Schmidt-Rhaesa et al. 2003). The cuticle contained one type of areole (roundish elevations of the cuticular surface) (Figs. 1D and 2D,E). Areoles bordered each other directly; there were no further structures in the interareolar space. The posterior end was bifurcated (Fig. 2B), with distally branched cuticular bristles in a paired row anterolateral to the cloacal opening (Fig. 2C) and conical spines posterior to the cloacal opening. The anterior end was whitish in color, and a dark collar (which follows the white tip in many nematomorph species) was absent (Fig. 2A). The specimen of C. annulata collected alive at the Waterloo Wildlife Area site released its nematomorph approximately 12 hours after it was placed in a petri dish and misted with room temperature tap water. It emerged 2009 A. Schmidt-Rhaesa, M.A. Farfan, and E.C. Bernard 127 from the terminal end in the region of the anus. The host continued to live for seven months after the emergence. We did not check for internal damage to the host by the parasite. Discussion The specimens of G. lineatus correspond to the previous descriptions of this species (Schmidt-Rhaesa et al. 2003, Smith 1991). The only exception is that the previously reported specimens were all whitish, but in the Ohio specimens this species was also sometimes light or medium brown. However, it is assumed that coloration is variable in nematomorphs. Apart from probable intraspecific variation, there also is a developmental gradient, with worms inside the host being white; coloration of the cuticle starts late in the parasitic phase (Schmidt-Rhaesa 2005). Gordionus lineatus has been reported from Maryland, Massachusetts, Michigan, New York, Pennsylvania, and Tennessee (Schmidt-Rhaesa et al. 2003). Ohio is a new distribution record for this species, and it fits in well with the previously known range of the species. The host of G. lineatus was unknown until now; other hosts of other Gordionus species in North America remain unknown (see Schmidt-Rhaesa et al. 2003). In Europe, hosts of Gordionus species usually are beetles, but one case of an earwig host has been Figure 1. Cambala annulata (Diplopoda) infected with Gordionus lineatus (Horsehair Worm) (Nematomorpha). A–C show different specimens and stages of dissection, illustrating the position of G. lineatus in the body cavity of C. annulata. The diameter of C. annulata is approximately 2.5 mm. D. Light microscopical image of the cuticular surface, showing areole type. For scale compare with Figure 2E. 128 Northeastern Naturalist Vol. 16, No. 1 reported, and myriapods are hosts for G. alpestris (see Table 1). Most records of myriapods as hosts of nematomorphs come from Europe, Table 1: Reports of myriapods as hosts of nematomorphs. Nematomorph Myriapod species species Locality Reference Diplopoda Glomeris marginata Juvenile stages France Dorier 1929, 1930 Glomeris marginata Gordius aquaticus France Dorier 1929, 1930 Glomeris humbertiana Undetermined No data Dorier 1930 Julus sp. Gordionus alpestris France Dorier 1929 Julus sp. Gordionus alpestris Italy Sciacchitano 1932 Tachypodoiulus albipes Undetermined France Sahli 1972 Schizophyllum sabulosum Undetermined France Sahli 1972 Cylindroiulus teutonicus Undetermined France Sahli 1972 Ommatoiulus moreletii Undetermined Portugal Baker 1985 Polydesmus complanatus Gordionus alpestris Romania Căpuşe 1971 Narceus sp. Undetermined Ohio, USA Cooper and Storck 1973 Cambala annulata Gordionus lineatus Ohio, USA This report Chilopoda Lithobius forficatus Gordius aquaticus Probably France Villot 1886, Dorier 1930 Scolopendra sp. Gordius villoti Italy Camerano 1888 Scolopendra cingulata Gordius aquaticus Morocco Dorier 1929 Figure 2. Gordionus lineatus (Horsehair Worm). A. Anterior end, showing the coloration pattern. B. Bilobed male, posterior end, dorsal view. C. Magnification of bristles anterolateral of the ventral cloacal opening, showing apical branching (circle). D, E. Cuticular structure showing areoles. 2009 A. Schmidt-Rhaesa, M.A. Farfan, and E.C. Bernard 129 with one exception from northern Africa (Morocco; Dorier 1929) and the present one from North America (Ohio). Several of these nematomorphs are undetermined, including one record from Ohio. When species have been determined, they are Gordionis alpestris, Gordius aquaticus, and Gordius villoti (Table 1). Among myriapods, diplopods as well as chilopods are parasitized. Both taxa have fundamentally different food requirements. Chilopods are carnivorous, while diplopods are generally herbivorous or detritivorous. This difference probably accounts for the fact that C. annulata kept in the laboratory showed no interest in animal remains (although some were caught in a trap baited with carrion). Herbivorous hosts pose some problems to our current knowledge of the nematomorph life cycle. Most observations and experiments (see summaries in Hanelt et al. 2005, Schmidt-Rhaesa 2001) suggest that nematomorph larvae encyst in aquatic paratenic hosts such as insect larvae or snails, which are then consumed by carnivorous or omnivorous terrestrial hosts. Entirely herbivorous hosts cannot infect themselves in this way. However, at least two possibilities exist for a transition of nematomorph larvae into herbivores or detritivores. The first is by drinking water containing larvae, and the second is by feeding on vegetation close to water, on which nematomorph larvae are present as cysts. The formation of such cysts was observed by Dorier (summarized in 1930), but the formation of cysts by nematomorph larvae could not be confirmed in other studies. Dorier was also successful in dissolving nematomorph cysts by exposing them to digestive fluids from various potential hosts, including myriapods (Dorier 1930). Although the sample size is low (20 specimens of C. annulata, 10 infected with G. lineatus), the findings suggest that the prevalence is quite high (50%). There are few published data on prevalence. Baker (1985) found 28.7% of the milliped Ommatoiulus moreletii (Lucas) infected by undetermined nematomorphs in Portugal. Thorne (1940) found that parasitation of crickets with Gordius robustus decreased with increasing distance from a pond. As C. annulata can be kept in the lab, this is a potential system to study host-parasite interactions. Acknowledgments We thank George D. Keeney, who collected the C. annulata specimen from the Waterloo Wildlife Area site. 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