2009 NORTHEASTERN NATURALIST 16(1):45–52 Distribution and First Records of Branchiobdellida (Annelida: Clitellata) from Crayfishes (Crustacea: Decapoda) in the Maritime Provinces of Canada Stuart R. Gelder1,*, Lana McCurry1, and Donald F. McAlpine2 Abstract - Preserved specimens of the crayfish Cambarus bartonii, Orconectes limosus, and Orconectes virilis from 27 locations in New Brunswick, and O. limosus from Freshwater Lake, Cape Breton Highlands National Park, Cape Breton Island, NS, Canada, were examined for branchiobdellidans. Three species of branchiobdellidans— Bdellodrilus illuminatus, Cambarincola fallax, and Xironogiton instabilis— were identified on C. bartonii. Only branchiobdellidan cocoons were recorded on O. limosus, and only X. instabilis was recovered from O. virilis in New Brunswick. The introduction of O. limosus and O. virilis into New Brunswick does not appear to have simultaneously introduced non-native branchiobdellidans. No branchiobdellidans were found on introduced O. limosus from Freshwater Lake, NS. This work provides the first records of branchiobdellidans occurring in the Maritime Provinces of Canada, and establishes the eastern limit for these ectosymbionts in North America. In addition, one specimen of C. bartonii was found to have a total of 39 B. illuminatus in the branchial chambers, the largest infestation of this species yet recorded. Introduction The current status of the geographical distribution of ectosymbiotic branchiobdellidans (Brinkhurst and Gelder 2001) on freshwater crayfish on the North American continent has been reviewed by Gelder et al. (2002). It can be expected that the current distributional limits will be expanded following studies aimed at finding new endemic branchiobdellidan sites and through the examination of exotic crayfishes imported for aquaculture, sport fishing, and the pet industry. Hoffman (1963) reported the only record of a branchiobdellidan in New England to be in western Massachusetts. Gelder and Smith (1987) followed by reporting three species of branchiobdellidans in northern Maine. Subsequently, and predictably, branchiobdellidans were found to be widespread across New England and north to the St Lawrence drainage in Ontario and western Quebec, Canada (Gelder et al. 2001). Although freshwater crayfish are well known in New Brunswick, Canada (Martin 1997; McAlpine et al. 1991, 2007), there have been no reports from the region of branchiobdellidans on these potential hosts. Thus, the question of the eastern limit of these ectosymbionts’ distributions in North America remained unanswered. 1Department of Science, University of Maine at Presque Isle, 181 Main Street, Presque Isle, ME 04769. 2Natural Science Department, New Brunswick Museum, 277 Douglas Avenue, Saint John, NB E2K 1E5, Canada. *Corresponding author - stuart. gelder@umpi.edu. 46 Northeastern Naturalist Vol. 16, No. 1 Cambarus bartonii (Fabricus) (Appalachian Brook Crayfish) is widely distributed across New Brunswick, while Orconectes virilis (Hagen) (Northern Crayfish) is believed to be restricted to northeastern and northwestern areas of the province (McAlpine et al. 1999, 2007). In New Brunswick, Orconectes limosus (Rafinesque) (Spinycheek Crayfish) has been reported from a single river system in the southwestern part of the province (McAlpine et al. 1991). Only C. bartonii is believed to be native to the province, and to date, there have not been any native crayfish reported from the provinces of Nova Scotia, Prince Edward Island, or Newfoundland (Ganong 1898, Hobbs 1989). However, a recent introduction of O. virilis into Nova Scotia has extended the eastern range of crayfish in the Maritime Provinces (Lambert et al. 2007) and raised the possibility of exotic branchiobdellidan species being introduced as well (Gelder 2004). The current study was designed to establish the presence, species, and geographical distribution of branchiobdellidans in the Maritime Provinces of Canada by examining crayfish specimens that had been collected during previous freshwater surveys in the region, and deposited as vouchers in the New Brunswick Museum, Saint John, NB, Canada. These examinations also provided the opportunity to report microhabitat or unusual infestations should the ectosymbionts be found. Material and Methods Preserved specimens of C. bartonii, O. limosus, and O. virilis from 27 locations encompassing the widest distribution of sites across New Brunswick were examined for branchiobdellidans. Details from the New Brunswick Museum— reference numbers, geographical locations, and crayfish species—are given in Table 1. In addition, two collections of O. limosus from Freshwater Lake (46.6453°N, 060.3966°W), Cape Breton Highlands National Park, Cape Breton Island, NS, Canada, were also examined. Most crayfish from the museum’s collection had been preserved using one of several methods: placed in 10% formalin and subsequently transferred to 70% ethanol, placed directly into 90% ethanol and then moved to either 70% ethanol or 10% formalin, or frozen and then transferred to one of these two solutions. A small number of crayfish collected specifically for this study were placed directly into 70% ethanol, without subsequent transfer to any other solution; these collections are shown with a suffix letter (a or b) after the site number in Table 1. A maximum of five crayfish were examined from each New Brunswick collection site; however, 33 were examined from the Nova Scotia site. The external surfaces of the crayfish were visually examined under a dissection microscope, during which a probe was gently run through setae and cavities to dislodge any trapped branchiobdellidans. In addition, the carapace covering the branchial chambers was removed so that a thorough study of the gills could be made; approximately half the number of crayfish from a particular site was given a gill examination. The detritus and other residue 2009 S.R. Gelder, L. McCurry, and D.F. McAlpine 47 Table 1. Distribution of crayfish and their branchiobdellidans in New Brunswick, Canada. Site numbers in this table correspond to those indicated on Figure 1. Reference # = catalog number of crayfish specimens examined during this study and deposited in the New Brunswick Museum. The number of crayfish examined from each site is given under the abbreviation for the respective species, with the number of crayfish receiving a gill examination indicated in parentheses: Cambarus bartonii (C.b.), Orconectes limosus (O.l.), and Orconectes virilis (O.v.). The total number of branchiobdellidans obtained per site appears under the respective species: Bdellodrilus illuminatus (B.i.), Cambarus fallax (C.f.), and Xironogiton instablius (X.i.); the presence of cocoons is indicated by an “X”. Site Crayfish species and #s Branchiobdellidans and #s # Reference # Coordinates Location C.b. O.l. O.v. B.i. C.f. X.i. Cocoons 1 NBM-007094 47.183°N, 068.890°W St. Francois River 5(3) - - 0 0 0 0 2 NBM-001824 47.239°N, 068.742°W Mouth of Riviere de Crocs - - 14(3) 0 0 1 0 3 NBM-001817 47.300°N, 068.480°W Saint John River, Couturier Siding - - 5(3) 0 0 0 0 4 NBM-007059 47.288°N, 068.378°W Saint John River, Saint Hilair 5(3) - - 0 2 1 0 5 NBM-001825 47.357°N, 068.259°W Iroquois, near Saint John River - - 3(1) 0 0 0 0 5a NBM-001132 47.361°N, 068.326°W Saint John River, Edmunston - - 3(3) 0 0 0 0 5b NBM-001133 47.358°N, 068.309°W Saint John River, Edmunston 1(1) - - 2 0 2 X 6 NBM-000275 47.417°N, 068.250°W West Branch Mill Brook 3(1) - - 0 0 0 0 7 NBM-007079 48.016°N, 066.545°W Unamed beaver pond Highway 280 1(1) - - 0 0 0 0 8 NBM-007066 47.550°N, 066.516°W Eighteen Mile Brook 2(1) - - 5 0 0 0 9 NBM-007070 47.305°N, 066.187°W Quisibis River 5(3) - - 9 4 5 0 10 NBM-008241 46.969°N, 065.328°W Black River, Highway 11 - - 2(1) 0 0 0 0 11 NBM-007067 46.820°N, 066.860°W Clearwater Brook 5(3) - - 45 8 5 0 11a NBM-001797 46.392°N, 066.461°W Clearwater Brook 1(1) - - 0 16 37 X 11b NBM-001798 46.375°N, 066.401°W Sister Brook 1(1) - - 2 7 20 X 12 NBM-001131 46.520°N, 067.600°W Monquart River 1(1) - - 0 0 0 0 13 NBM-000052 46.520°N, 066.520°W Hayesville 2(1) - - 0 0 0 0 14 NBM-000054 45.970°N, 067.250°W South Hampton 2(1) - - 0 0 0 0 15 NBM-000238 46.230°N, 066.820°W Tay Creek 2(1) - - 0 0 0 0 16 NBM-000230 45.970°N, 066.700°W Garden Creek 6(3) - - 0 0 0 X 17 NBM-000232 45.970°N, 066.700°W Garden Creek 10(3) - - 0 0 0 X 18 NBM-000873 45.850°N, 066.900°W East Branch Longs Creek 5(3) - - 2 0 4 0 19 NBM-000273 45.950°N, 066.620°W Nashwaakis River 2(1) - - 0 0 0 0 20 NBM-000053 45.980°N, 066.650°W Nashwaakis 2(1) - - 0 0 0 0 21 NBM-008236 45.380°N, 067.350°W Canoose Stream - 5(3) - 0 0 0 X 22 NBM-000050 45.470°N, 066.130°W West Brown’s Flat 1(1) - - 0 0 0 0 23 NBM-000051 45.470°N, 066.130°W Brown’s Flat 1(1) - - 0 0 0 0 48 Northeastern Naturalist Vol. 16, No. 1 at the bottom of each collection jar were thoroughly searched for detached branchiobdellidans. All ectosymbionts from a site were counted, transferred to a specimen jar, and subsequently identified based on species descriptions in Hobbs et al. (1967) and Gelder and Hall (1990). Representative specimens were prepared as permanent microscope preparations following the protocol in Brinkhurst and Gelder (2001) and have been deposited in the collections of the New Brunswick Museum (NBM). Results The sites sampled for crayfish, along with the distribution of branchiobdellidans and their cocoons, are shown in Figure 1 and Table 1. Three species of branchiobdellidans, Bdellodrilus illuminatus (Moore) (NBM 002596, 002601), Cambarincola fallax Hoffman (NBM 002598), and Xironogiton instabilis (Moore) (NBM 002597, 002599, 002600, 002062) were identified in the province. Triple-species infestations of branchiobdellidans were found at sites 9 and 11, while other collections showed only one or two species present. The presence of branchiobdellidan cocoons on a host demonstrated that adult worms had been present. The cocoon was an important indicator given that O. limosus was only found at site 21. Specimens of O. limosus from an introduced population in Cape Breton Highlands National Park, NS, were collected in July–November 2006 (NBM 001991–001996) and again in August–September 2007 (NBM 002603– 002607). Twenty-two crayfish from the 2006 collection were examined, of which 11 were subjected to a gill examination; no branchiobdellidans or cocoons were observed. From the 2007 collection, 11 specimens were Figure 1. Map of New Brunswick, Canada (eastern portion truncated) showing the locations of crayfish collection sites with ▲ indicating the presence, or ● the absence of branchiobdellidans or their cocoons. Map scale has required that some site numbers refer to multiple locations (see Table 1). 2009 S.R. Gelder, L. McCurry, and D.F. McAlpine 49 examined, including their branchial chambers, and again no evidence of ectosymbionts was found. Most of the specimens of C. fallax and X. instabilis living on the exposed surfaces of the host in life were recovered from the debris on the bottom of the collecting jars. However, a small number of specimens did retain their attachment to the host, allowing their microhabitats to be confirmed. Adult X. instabilis were observed on the ventral surfaces of the claws, along with juveniles and cocoons. Cambarincola fallax were observed under the rostrum, on an eye, and around the mouth. Unlike the other two species, B. illuminatus are only known from the branchial chambers, and so specimens in this protected microhabitat were retained in situ despite the trauma of the collection process. Infestations of B. illuminatus were not common among the collections, but when they were present (Table 1), numbers were small. Therefore, finding 39 B. illuminatus in the branchial chambers of one C. bartonii from Clearwater Brook (Fig. 1, site 11; NBM-007067) was exceptional and is the highest infestation of this species ever reported on a single host. During the removal of the carapace, most of the branchiobdellidans were dislodged and fell to the bottom of the examination bowl, and so it is not known how many individuals were in each branchial chamber; however, a few did remain in situ between the gills and plates. In the right chamber, 50 cocoons were attached to the basal half of the facing surfaces of the outer and median gills above the third and fourth walking legs; 29 cocoons contained embryos. Cocoons in the left chamber were similarly located, but on the third and second walking legs, with 46 cocoons (28 with embryos). Crayfish collected from the same area in 2007 (NBM 001797 and 001798) had been placed directly into 70% ethanol on collection and not handled until examined in the laboratory. Although this reduced the trauma to the crayfish significantly, and more external branchiobdellidans were observed and collected in situ (Table 1), the numbers of B. illuminatus recovered were zero for one specimen and two for the other. Discussion The use of archived crayfish specimens as a source of branchiobdellidans and other ectosymbionts is not common. However, as demonstrated here, such studies can provide new information on the distribution of branchiobdellidans from regions that may be difficult to access and may provide temporal data on past branchiobdellidan status. The disadvantages of using archived crayfish is that the methods of collection and preservation in common use frequently causes symbionts to become detached from their host, and because of their soft-bodied nature, branchiobdellidans can suffer significant damage. In addition, specimens are commonly (and unknowingly) discarded with bottom debris when the ethanol is replaced. Nonetheless, the present study has shown that crayfish transferred directly into jars with 70% ethanol result in adequately preserved ectosymbionts and hosts. 50 Northeastern Naturalist Vol. 16, No. 1 Furthermore, the results of this present study have confirmed the expectation that branchiobdellidans occur on endemic crayfish in New Brunswick, Canada. That C. bartonii carried the same three species of branchiobdellidans found in adjacent Maine (Gelder and Smith 1987) supports the statement that New Brunswick is part of the endemic range of both host and ectosymbionts. Although C. fallax, X. instabilis, and B. illuminatus have well-known and spatially separate microhabitats, confirmation of their presence at the limit of their range is important. The three crayfish from site 11 that underwent a branchial chamber examination produced 45 B. illuminatus, with 39 taken from a single host—one that also supported a total of 96 cocoons in the branchial chambers, 39 of which were empty. It is interesting to speculate how many of the remaining embryos would have hatched, and what the impact of such a large number of juveniles would have had on the host. The introductions of O. limosus and O. virilis into New Brunswick (McAlpine et al. 1991, 1999) from unknown locations do not appear to have occurred simultaneously along with non-native branchiobdellidans. It is speculated that the single known occurrence of X. instabilis adults observed on Orconectes in New Brunswick represents a case of host-switching from an endemic C. bartonii. Gelder and Smith (1987) collected five O. virilis from two sites in northern Maine and found the only branchiobdellidan species they carried was B. illuminatus. The positive report from the Walker Siding, Squa Pan Lake site was incorrectly given as a negative in Gelder et al. (2001: Table 1). The Gelder et al. (2001) study of branchiobdellidans in New England also showed that O. limosus occurred south of southern Maine, and that when this host species carried any branchiobdellidans, it was infested with both Cambarincola mesochoreus Hoffman and Cambarincola philadelphicus (Leidy). Because the information in Gelder et al. (2001) was obtained only from museum material, no estimate of infestation abundance can be inferred from it. The absence of crayfish in the remainder of the Maritime Provinces has enabled the eastern limit of endemic branchiobdellidans in North America to be established. The introduction of O. limosus into Nova Scotia sometime between 2001 and 2005, and the species’ successful colonization of Freshwater Lake (Lambert et al. 2007) illustrate yet again the ease with which an invasive crayfish species can become established in a suitable habitat (Gherardi and Holdich 1999). Often such crayfish carry with them ectosymbionts and pathogenic organisms (Evans and Edgerton 2002). Although no branchiobdellidans were found on the Nova Scotia crayfish in this study, four species of ectosymbionts have been reported on O. limosus from sites in eastern North America (Gelder 2004). It appears from our examinations that branchiobdellidans did not survive the transportation and introduction of the crayfish into Nova Scotia, yet their absence during our study does not preclude their occurrence in the Province; small surviving populations of ectosymbionts in localized areas 2009 S.R. Gelder, L. McCurry, and D.F. McAlpine 51 likely would require a number of years to increase to levels where they would be readily detectable. Acknowledgments Our thanks to the University of Maine at Presque Isle, ME, for a student research grant to Lana McCurry, and to the New Brunswick Wildlife Trust, Canada, for a supporting grant to D.F. McAlpine. 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