N29 2017 Northeastern Naturalist Notes Vol. 24, No. 4 J.L. Robinson, M.J. Wetzel, and J.S. Tiemann Some Phoretic Associations of Macroinvertebrates on Transplanted Federally Endangered Freshwater Mussels Jason L. Robinson1,*, Mark J. Wetzel1, and Jeremy S. Tiemann1 Abstract - Benthic macroinvertebrates were washed from nearly 1000 federally endangered freshwater mussels that had been collected from Pennsylvania during a reintroduction project to 2 eastern Illinois streams. Most benthic macroinvertebrates collected were larvae of the Neophylax fuscus, but other caddisflies and segmented worms were also observed. No unoccupied caddisfly cases were observed on live mussels, leaving open the question as to the seasonal fate of these microhabitats after caddisflies pupate and emerge in the Autumn of each year. Unionid mussel shells might modify local-scale species diversity by influencing physical and hydrau lic properties of microhabitats. Life-history knowledge gaps. Basic attributes of macroinvertebrate life histories are often poorly known and under-reported in the scientific literature. Gaps in the information on the ecology of individual organisms are one of a number of knowledge gaps that systematically limit the effective management and conservation of species, as well as our understanding of the factors that constrain species diversity and the evolution of new traits and taxa (Cardoso et al. 2011, Hortal et al. 2015). The prospect of improving the management of species under special conservation protections provides an additional impetus for reporting basic life-history and ecological attributes of these species, and the other members of the ecological communities in which they persist. Relocation project. Beginning in 2005, biologists from the Illinois Natural History Survey (INHS) partnered with personnel from the US Fish and Wildlife Service and from several state resource management agencies in Ohio and Pennsylvania to rescue individuals of 2 federally endangered mussel species, Epioblasma rangiana (Lea) (Northern Riffleshell) and Pleurobema clava (Lamarck) (Clubshell). The mussels were collected from the footprint of a proposed bridge construction on the Allegheny River in Forest County, PA, in areas of swiftly flowing water with clean and stable sand, gravel and cobble substrates (Stodola et al. 2017, Tiemann 2014). Mussels were relocated to the Vermilion River basin in Champaign and Vermilion counties, IL, with the goal of re-establishing viable populations of these 2 species into areas where they were considered extirpated (Cummings and Mayer 1997, Tiemann 2014). This paper concerns macroinvertebrates collected from live mussels transplanted during 26–27 August 2013. Individual mussels (249 Northern Riffleshell and 758 Clubshell) were quarantined in a holding facility at the University of Illinois at Urbana-Champaign (UIUC), tagged with passive integrated transponder (PIT) tags, and then resituated at 8 different sites in the Middle Fork (5) and Salt Fork (3) of the Vermilion River (Stodola et al. 2017, Tiemann 2014). During tagging, the external shell of individual mussels was scrubbed and temporarily dried to facilitate the attachment of tags. This process rinsed and removed attached sediment and epibionts, including caddisfly cases and other aquatic macroinvertebrates. Most of this material was retained in 95% ethanol for later microscopic inspection and identification. We identified 152 individual macroinvertebrates, representing 4 species (Table 1). The macroinvertebrates we report herein are a nonrandom and limited subset of the complete 1Illinois Natural History Survey, Prairie Research Institute at the University of Illinois Champaign- Urbana, Champaign, IL 61820. *Corresponding author - jrob@illinois.edu. Manuscript Editor: Trip Krenz Notes of the Northeastern Naturalist, Issue 24/4, 2017 2017 Northeastern Naturalist Notes Vol. 24, No. 4 N30 J.L. Robinson, M.J. Wetzel, and J.S. Tiemann phoretic fauna associated with these 2 mussel species, because many organisms were certainly detached or disassociated during the process of removal and quarantine. Life history and local ecology. Both of the mussel species considered here are typically found in medium to large rivers in clean, stable sand, gravel, and cobble riffles, where they may live several inches beneath the streambed surface (Cummings and Mayer 1992, Watters et al. 2009). These 2 species vertically migrate to the streambed surface during their reproductive period; Northern Riffleshell are bradytictic (brooding from September to the following June), whereas Clubshells are tachytictic (brooding from early May to July). The macroinvertebrate assemblage associated with the shells of these 2 mussel species could be different during these reproductive periods compared to the rest of the year. Associations of macroinvertebrates and unionids within this interstitial microhabitat are likely to experience temporal progression within each year, as a function of mussel vertical migration and/ or macroinvertebrate life history (e.g., adult emergence of Neophylax sp. in autumn). Table 1. List of macroinvertebrates dislodged from 249 Epioblasma rangiana (Northern Riffleshell) and 758 Pleurobema clava (Clubshell) from the Allegheny River (Route 62 Bridge, 4.5 km SW Tionesta, Forest County, PA, 41.472348°N, 79.499838°W), collectors J.S. Tiemann, K.S. Cummings, S.A. Douglass, A.L. Price, et al. Phylum Class Order Family Species Count Arthropoda Insecta Trichoptera Thremmatidae Neophylax fuscus 113 Arthropoda Insecta Trichoptera Leptoceridae Oecetis inconspicua 2 Annelida Clitellata Rhynchobdellida Glossiphoniidae Helobdella papillata 35 (Hirudinea) Annelida Clitellata Tubificida Naididae Nais bretscheri 1 Annelida Clitellata Tubificida Naididae Unidentified Naidinae 1 Figure 1. Ventral view of Neophylax fuscus case, with larva enclosed. Remnants of mussel shell are visible at anterior and posterior attachment sites. N31 2017 Northeastern Naturalist Notes Vol. 24, No. 4 J.L. Robinson, M.J. Wetzel, and J.S. Tiemann Neophylax cases are tube-like, typically with large stones flanking the tube along the axis of the body of the organism (Fig. 1). The insect prepares for pupation by enclosing the tube and firmly attaching the larval case to some firm substrate (including mussel shells) with silk produced from glands located in the mouth of the larva (Sehnal and Akai 1990). Cases may persist, in some habitats, for several years after the emergence of adults. Larvae occupied all cases removed from mussels in this study, and no empty cases from previous seasons were observed. This finding is curious because Neophylax fuscus Banks cases were so firmly attached to mussels that removing the cases during our prepping procedure also removed small pieces of periostracum (the non-living outer layer of the shell) at the attachment sites (Fig. 2). Lawfield et al. (2014) suggested that Trichoptera case attachment might not harm or damage the shell of mussels because this attachment is confined to the periostracum. The aquatic annelids rinsed from external mussel shells included the leech Helobdella papillata (Moore) (5 brooding adults, 5 non-brooding adults, and 25+ young of the year that had detached from parents), 1 aquatic oligochaete (Nais bretscheri Michaelsen) and 1 other unidentified naidid oligochaete (Table 1). Several leech species in the family Glossiphoniidae (including H. papillata) are known associates of freshwater mollusks, feeding primarily if not exclusively on mollusks (Sawyer 1986). Several species in the oligochaete genus Chaetogaster (most commonly, Chaetogaster limnaei von Baer) are often collected from pulmonate snails (externally, from within the mantle cavities, around the apertures, and as parasites in the kidneys; Klemm 1985), from unionid bivalves (externally and from within the mantle cavities; Anderson and Holm 1987, Beckett et al. 1996, Kelly 1988), and occasionally from freshwater sponges, bryozoans, and crayfishes (Sawyer 1986; Stephenson 1930; Wetzel et al. 2009; M.J. Wetzel, pers. observ.). Surprisingly, no Chaetogaster specimens were present in the material washed from the mussels from our study, but as noted above, many organisms were certainly detached or Figure 2. Close up view of remnants of freshwater mussel shell remaining at the posterior attachment site on a Neophylax fuscus case. 2017 Northeastern Naturalist Notes Vol. 24, No. 4 N32 J.L. Robinson, M.J. Wetzel, and J.S. Tiemann disassociated during the process of removal and quarantine. Neither Nais bretscheri nor other congeners are known to have commensal or parasitic relationships with mollusks. It is possible that aquatic oligochaetes washed from the mussels were living within silt and sand on the shell of the mussels, and also possible that these individuals were only incidentally using areas around caddisfly cases as habitat or refugia. We note a litany of personal field and lab experiences on oligochaetes and other macroinvertebrate fauna in the contents of caddisfly cases or puparia, which frequently contain more macroinvertebrate species when examined in detail (Beckett et al. 1996; Bodis et al. 2014; Lawfield et al. 2014; J.L. Robinson and M.J. Wetzel, pers. observ.; pers. comm. from caddisfly taxonomists D. Denson [Reedy Creek Improvement District, Lake Buena Vista, FL], C. Parker [ret. USGS, Gatlinburg, TN], D. Etnier [ret. UT-Knoxville, Knoxville, TN], and D. Ruiter [ret. USEPA, Centennial, CO]) and sometimes provide a substrate for Podostemum ceratophyllum Michx. (Hornleaf Riverweed) (Vaughn et al. 2002). Among our observations, all cases we report were occupied by living caddis, and older cases from which caddis had previously emerged were completely absent. Little is known about the behaviors or ecological significance of burrowing mussels (Newton et al. 2015). Although mussels are known to vertically migrate to escape predation (Burlakova et al. 2000) and control zebra mussel infestation (Nichols and Wilcox 1997), we hesitate to speculate that vertical migration can remove spent cases. Regardless, caddisfly cases may help to create and maintain fine-scale structural and hydraulic and ecological diversity widely reported from freshwater mussel habitats (Commito and Rusignuolo 2000, Gutierrez et al. 2003, Lawfield et al. 2014, Taniguchi and Tokeshi 2004, Vaughn and Spooner 2006). We believe that this report is the first literature discussion of a phoretic association between living unionid mussels and any of the confirmed eastern North American species of Neophylax (Trichoptera: Thremmatidae) in the ecological literature. However, phoretic associations of Trichoptera with unionids have been reported from fossils dating to the Paleocene of North Dakota, where psychomyiid caddisfly retreats and net were preserved on a unionid (Erickson 1983). Trichoptera associations must be known or familiar to malacologists who observe organisms in the field, and associations with case-building Trichoptera have previously been suggested from photographs of organisms attached to dead shells (Lawfield et al. 2014), but not identified to genus or species. Images posted on the USFWS website for this specific project clearly depicted Neophylax cases on specimens in situ, and images published in Lawfield et al. (2014) suggest hydropsychids, hydroptilid, and glossosomatid caddisflies may successfully colonize the surface of unionids. Interestingly, Anderson and Vinikour (1984) reported the use of unionid mussels and viviparid snails as pupation sites for the leptocerid caddisfly Oecetis inconspicua (Walker), but no associations with other extant Trichoptera species have yet been reported. Interspersed among Neophylax cases were 2 very early instar larvae of some species of Oecetis, in the O. inconspicua group (Floyd 1995)—instars that could not be confidently associated with any of the morphologically distinguishable forms within this group. Although interesting as ecological trivia, this observation raises issues about quarantine and unionid reintroduction efforts. Our results are at best an underestimate of the fauna attached to or living on the mussels in situ but a great example of how organisms can unintentionally be transported great distances. Predicting which species might be most likely to be introduced might prove difficult, because numerous observations of epibiotic relations suggest that many different taxa can form these associations without specificity (Wahl and Mark 1999). Caddis cases, or macrophytes, might be obvious to N33 2017 Northeastern Naturalist Notes Vol. 24, No. 4 J.L. Robinson, M.J. Wetzel, and J.S. Tiemann most observers and frequently removed during basic quarantine. Oligochaetes or other invertebrates inhabiting the mantle cavities or internal organs of mollusks, as well as those capable of hiding in the crevices of the shells, may be more difficult or impossible to positively remove. The chance for accidental introduction of non-native macroinvertebrates obviously increases when hundreds, if not thousands, of mussels are translocated into new river basins across multiple years. To avoid potential contamination and unwanted introduction of macroinvertebrates, careful, stringent quarantine procedures should be considered when transporting freshwater mussels. Acknowledgments. This relocation project is being funded, in part, by a natural resource damage assessment settlement (Hegeler Zinc–Lyondell Basell Companies) to the US Fish and Wildlife Service (USFWS) and to the State of Illinois. USFWS, Pennsylvania Fish and Boat Commission (PFBC), and Illinois Department of Natural Resources (IDNR) issued collecting permits. Staff and volunteers from EnviroScience (Ohio), IDNR, Illinois Natural History Survey, Ohio State University, Columbus Zoo and Aquarium (Ohio), PFBC, Prairie Rivers Network, USFWS, and West Virginia Department of Natural Resources assisted with collecting and scrubbing mussels. Dead mussel specimens, trichopterans and annelid specimens identified during this study are deposited, respectively, in the INHS Mussel, Insect, and Annelid Collections (Epioblasma rangiana- INHS 46825, Pleurobema clava- INHS 46829, Neophylax fuscus- INHS 797483, Oecetis inconspicua group- INHS 797484, Helobdella papillata INHS_ANNELID_10269_431, Nais bretscheri INHS_ANNELID_10270_164, unidentified Naidinae INHS_ANNELID_10271_102). Literature Cited Anderson, R.V., and D.J. Holm. 1987. Chaetogaster limnaei (Oligochaeta: Naididae) infesting unionid mollusks (Pelecypoda: Unionidae) and Corbicula fluminea (Pelecypoda: Corbiculidae) in Pool 19, Mississippi River. Journal of Freshwater Ecology 4(1):61–64. Anderson, R.V., and W.S. Vinikour. 1984. Use of molluscs as pupation sites by Oecetis inconspicua (Trichoptera: Leptoceridae). Journal of Freshwater Ecology 2(5):4 17–422. Beckett, D.C., B.W. Green, S.A. Thomas, and A.C. Miller. 1996. Epizoic invertebrate communities on upper Mississippi River unionid bivalves. American Midland Naturalist 135(1):102–114. Bodis, E., B. Toth, J. Szekeres, P. Borza, and R. Sousa. 2014. Empty native and invasive bivalve shells as benthic habitat modifiers in a large river. Limnologica - Ecology and Management of Inland Waters 49:1–9. Burlakova, L.E., A.Y. Karateyev, and D.K. Padilla. 2000. The impact of Dreissena polymorpha (Pallas) invasion on unionid bivalves. International Review of Hydr obiology 85:529–541. Cardoso, P., T.L. Erwin, P.A.V. Borges, and T.R. New. 2011. The seven impediments in invertebrate conservation and how to overcome them. Biological Conservation 144(11):2647–2655. Commito, J.A., and B.R. Rusignuolo. 2000. Structural complexity in mussel beds: The fractal geometry of surface topography. Journal of Experimental Marine Biology and Ecology 255(2):133–152. Cummings, K.S., and C.A. Mayer. 1992. Field Guide to Freshwater Mussels of the Midwest. Illinois Natural History Survey Manual Vol. 5, Champaign, IL. Cummings, K.S., and C.A. Mayer. 1997. Distributional checklist and status of Illinois freshwater mussels (Mollusca: Unionacea). Pp. 129–145, In K.S. Cummings, A.C. Buchanan, C.A. Mayer, and T.J. Naimo (Eds.). Conservation and management of freshwater mussels II: Initiatives for the future. Proceedings of a UMRCC Symposium, 16-18 October 1995, St. Louis, Missouri. Upper Mississippi River Conservation Committee, Rock Island, IL. 293 pp. Erickson, J.M. 1983. Trichopterodomus leonardii, a new genus and species of psychomyiid caddisfly (Insecta: Trichoptera) represented by retreats from the Paleocene of North Dakota. Journal of Paleontology 57:560–567. Floyd, M. 1995. Larvae of the caddisfly genus Oecetis (Trichoptera: Leptoceridae) in North America. Bulletin of the Ohio Biological Survey 10(3):1–85. Gutierrez, J.L., C.G. Jones, D.L. Strayer, and O.O. Iribarne. 2003. Mollusks as ecosystem engineers: The role of shell production in aquatic habitats. Oikos 101(1):79–90. 2017 Northeastern Naturalist Notes Vol. 24, No. 4 N34 J.L. Robinson, M.J. Wetzel, and J.S. Tiemann Hortal, J., F. de Bello, J.A.F. Diniz-Filho, T.M. Lewinsohn, J.M. Lobo, and R.J. Ladle. 2015. Seven shortfalls that beset large-scale knowledge of biodiversity. Annual Review of Ecology and Evolutionary Systematics 46:523–549. Kelly, H.M. 1988. A statistical study of the parasites of the Unionidae. Bulletin of the Illinois State Laboratory of Natural History 5(8):399–418. Klemm, D.J. (Ed.). 1985. A Guide to the Freshwater Annelida (Polychaeta, Naidid and Tubificid Oligochaeta, and Hirudinea) of North America. Kendall/Hunt Publ. Co., Dubuque, IA. xiii + 198 pp. Lawfield, A.M.W., M.K. Gingras, S.G. Pemberton, J.M. Erickson. 2014. Freshwater unionid bivalve shells as substrata for Trichoptera attachment. Palaios 29:525–532. DOI:10.2110/palo.2013.126. Newton, T.J., S.J. Zigler, and B.R. Gray. 2015. Mortality, movement, and behaviour of native mussels during a planned water-level drawdown in the Upper Mississippi River. Freshwater Biology 60:1–15. Nichols, S.J., and D.A. Wilcox. 1997. Burrowing saves Lake Erie clams. Nature 389:921. Sawyer, R.T. 1986. Leech biology and behavior. Clarendon Press, Oxford, UK. ix + 1065 pp. Sehnal, F., and H. Akai. 1990. Insect silk glands: their types, development and function, and effects of environmental factors and morphogenetic hormones on them. International Journal of Insect Morphology and Embryology 19(2):79–132. Stephenson, J. 1930. The Oligochaeta. Clarendon Press, Oxford, UK. xii + 978 pp. Stodola, K.W., A.P. Stodola, and J.S. Tiemann. 2017. Survival of translocated Clubshell and Northern Riffleshell in Illinois. Freshwater Mollusk Biology and Conserva tion 20:89–102. Taniguchi, H., and M. Tokeshi. 2004. Effects of habitat complexity on benthic assemblages in a variable environment. Freshwater Biology 49(9):1164–1178. Tiemann, J. 2014. Monitoring translocated Northern Riffleshell and Clubshell in Illinois. Illinois Natural History Survey Technical Report (2). Champaign, IL. 27 pp. Vaughn, C.C., and D.E. Spooner. 2006. Unionid mussels influence macroinvertebrate assemblage structure in streams. Journal of the North American Benthological Society 25(3):691–700. Vaughn, C.C., D.E. Spooner, and B.W. Hoagland. 2002. River weed growing epizoically on freshwater mussels. Southwestern Naturalist 47(4):604. Wahl, M., and O. Mark. 1999. The predominantly facultative nature of epibiosis: Experimental and observational evidence. Marine Ecology Progress Series 187:59–6 6. Watters, G.T., M.A. Hoggarth, and D.H. Stansberry. 2009. The Freshwater Mussels of Ohio. The Ohio State University Press, Columbus, OH. 421 pp. Wetzel, M.J., S.V. Fend, K.A. Coates, R.D. Kathman, and S.R. Gelder. 2009. Taxonomy, systematics, and ecology of the freshwater oligochaetes and branchiobdellidans (Annelida, Clitellata) of North America. A workbook. Illinois Natural History Survey, Champaign, IL; now out of print, with revision in preparation. vi + 280 pp. + 4 color plates.