Freshwater mussels, comprising the families of Unionidae and Margaritiferidae, are a diverse group comprising approximately 300 species distributed throughout North America (Haag 2012; Williams et al. 2017). However, widespread declines have been observed over the last century, largely attributed to anthropogenic impacts (Master et al. 2000). Freshwater mussels are among the most imperiled groups of organisms in North America, with ~10% (29) of known taxa considered extinct and ~65% (195) listed as endangered, threatened, or of special concern at the state and federal level (Williams et al. 1993, Neves 1999, Master et al. 2000, Haag 2012). Further declines are projected over the next century, resulting in up to 50% of at-risk taxa going extinct (Ricciardi and Rasmussen 1999). Losses in freshwater mussel diversity and abundance can result in long-term changes to primary and secondary production in freshwater ecosystems (Allen et al. 2012, Spooner et al. 2012, Atkinson et al. 2013). In addition, freshwater mussels are known to provide the following ecosystem services: nutrient cycling (Vaughn et al. 2008), filtering suspended sediments (Spooner and Vaughn 2008), stabilizing substrates (Vaughn and Hakenkamp 2001), and providing microhabitats for aquatic macroinvertebrates (Vaughn and Spooner 2006). Freshwater mussels demonstrate a unique reproductive life history among bivalves, in which parasitic larvae (glochidia) must attach and metamorphose on a host, which is almost always a fish (Waters and O'Dee 1998). Mussel-host relationships vary by species, ranging from generalist (e.g. many suitable hosts) to specialists (e.g. a single host species)(Haag 2012). Freshwater mussels attract hosts via modified mantle tissue, specialized larval packages (i.e. conglutinates), or by releasing glochidia into the water column (Barnhart et al. 2008, Sietman et al. 2012). Successful reproduction relies on adequate flows, water conditions (e.g. temperature), food availability, and host availability (Roe et al. 1997, Galbraith and Vaughn 2009). The fate of mussel populations is often tied to host availability and host dispersal ability (Bogan 1993, Barnhart et al. 2008, Horky et al. 2014), thus understanding mussel-host relationships can provide necessary information for developing conservation and restoration plans (Kelner and Sietman 2000, Jones et al. 2004). Western Pearlshell (Margaritifera falcata) and Winged Floater (Anodonta californiensis/nuttalliana) are the only freshwater mussels present in Utah. Both species have broad historic and current distributions west of the Rocky Mountains (Xerces Society 2010a, 2010b). As seen in other freshwater mussel species, populations have been declining throughout their respective ranges (summarized in Xerces Society 2010a, 2010b). Freshwater mussels require a host fish to complete their life cycle, and unfortunately, the hosts required for these two mussels are not clearly defined throughout their ranges. Furthermore, investigations of host relationships have occurred only on populations outside of the Bonneville Basin, in genetically isolated populations (Mock et al. 2010). Host fish requirements for mussels in Utah may be different than those for the same mussel species in Oregon. Therefore, conducting host fish research for Utah mussel populations will be critical to their conservation. In Utah Western Pearlshell historically occurred at 11 sites in northern Utah (Oliver and Bosworth 1999), but is currently only found in Beaver Creek, a tributary of the Weber River, in Summit County (Tait 2010). This species is considered vulnerable, but is not likely to face an immediate risk of extinction (NatureServe Explorer 2008). Western Pearlshell demonstrates a tachytictic reproductive strategy, in which spawning occurs in the spring followed by a short brooding period (Murphy 1942, Toy 1998). Brooding has been observed in May, when average water temperature reaches 12.5 Celsius (O'Brien et al. 2013). Glochidia are small (~55 micrometers), hookless, white, and sub-round shape. Glochidia are released in white, irregular conglutinates that appear to resemble food items in drift (O'Brien et al. 2013). Conglutinate morphology such as this suggests that the host attempts to ingest the conglutinate, facilitating attachment to the gills (Haag and Warren 1997, O'Brien and Brim Box 1999). Throughout their range, native trout are considered the most suitable hosts for Western Pearlshells. However, other less suitable hosts include: non-native trout, mountain whitefish, Tahoe sucker, and speckled dace (Stagliano et al. 2020). Of additional interest as host species are managed sportfish present in Utah reservoirs. Winged Floaters (Anodonta californiensis/nuttalliana) are known to occur at up to 24 sites throughout the state of Utah, and are extirpated from at least 6 sites (Mock et al. 2004, Utah DNR 2007). A study by Mock et al (2010) suggests that the populations are genetically unique due to long-term isolation. Furthermore, 8 of the 24 extant sites sampled demonstrated recent genetic bottlenecking, with two populations (Bear River and Redden Springs) having such low genetic diversity that they may not be viable (Mock et al. 2004). Winged Floaters are currently ranked as S2/N3 in Utah. Like Western Pearlshell, Winged Floater is a tachytictic breeder, spawning in the spring and brooding into the summer (O'Brien et al. 2013). Gravid females have been observed in early June through late July (O'Brien et al. 2013). Glochidia for this species have been described as hooked, rust-colored, sub-triangulate, and averaged 276 micrometers (O'Brien et al. 2013). Glochidia are released directly into the water column in a mucus web (O'Brien et al. 2013). Similar to other species in Anodontinae, this species appears to be a host generalist, with a glochidia morphology that facilitates external attachment to a fish host (Watters 1994, Haag and Warren 1997, O'Brien et al. 2013). Prior host studies in California have identified 12 fish species as hosts (Spring River 2007). Identifying host fish for Utah's mussels will be important for understanding their conservation needs. If the identified hosts are not present where mussel populations are declining, introducing host fish or removing barriers will help restore mussel populations by improving reproductive success. If identified hosts are present where mussel populations are declining, this may suggest that mussel habitat is degraded and restoration is needed. Also, identifying host fish will allow the Division to effectively propagate these mussels if it is needed in the future. Proper conservation of these species will help prevent listing of these species under the Endangered Species Act. If these species are federally listed, there could be impacts to water development, water management, and fishing regulations (since fish are required for mussel reproduction) across the northern and central parts of the state. Adair, B., G. S. Silver, T. A. Whitesel, and K. Kittell. 2008. Preliminary evidence that sculpin species native to the Pacific northwest do not serve as a host in the reproductive cycle of the western pearlshell mussel (Margaritifera falcata). US Fish and Wildlife Service Annual Report. Allen, D. C., C. C. Vaughn, J. F. Kelly, J. T. Cooper, and M. H. Engel. 2012. Bottom-up biodiversity effects increase resource subsidy flux between ecosystems. Ecology 93:2165-2174. Atkinson, C. L., C. C. Vaughn, K. J. Forshay, and J. T. Cooper. 2013. Aggregated filter-feeding consumers alter nutrient limitation: Consequences for ecosystem and community dynamics. Ecology 94:1359-1369. Barnhart, M.C., W.R. Haag, and W.N. Roston. 2008. Adaptations to host infection and larval parasitism in Unionoida. Journal of the North American Benthological Society 27:370-394. Bogan, A.E. 1993. Freshwater Bilvalve Extinctions (Mollusca: Unionoida): A Search for Causes. American Zoologist 33: 599-609. Brim-Box, Jayne, David Wolf, Jeanette Howard, Christine O'Brien, Donna Nez, David Close, "Distribution and Status of Freshwater Mussels in the Umatilla River System", 2002-2003 Annual Report, Project No. 200203700, 74 electronic pages, (BPA Report DOE/BP-00011402- 1). D'Eliscu, P.N. 1972. Observation of the Glochidium, Metamorphosis, and Juvenile of Anodonta californiensis Lea, 1857. The Veliger 15(1): 57-58. Galbraith, H. S., and C. C. Vaughn. 2009. Temperature and food interact to influence gamete development in freshwater mussels. Hydrobiologia 636:35-47. Haag, W. R. 2012. North American freshwater mussels: Natural history, ecology, and conservation. Cambridge University Press, Cambridge, United Kingdom. Horky, P., K. Douda, M. Maciak, L. Zavorka, and O. Slavik. 2014. Parasite-induced alterations of host behaviour in a riverine fish: The effects of glochidia on host dispersal. Freshwater Biology 59:1452-1461. Hovingh, P. 2004. Intermountain freshwater mollusks, USA (Margaritifera, Anodonta, Gonidea, Valvata, Ferrissia): geography, conservation, and fish management implications. Monographs of the Western North American Naturalist 2(1):109-135. Jones et al. 2004 Jones, J. W., R. J. Neves, S. A. Ahlstedt, and R. A. Mair. 2004. Life history and propagation of the endangered Dromedary Pearlymussel (Dromus dromas) (Bivalvia: Unionidae). Journal of the North American Benthological Society 23:515-525. Karna, D.W., and R.E. Millemann. 1978. Glochidiosis of salmonid fishes. III. Comparative susceptibility to natural infection with Margaritifera margaritifera (L.) (Pelecypoda: Margaritanidae) and associated histopathology. The Journal of Parasitology 64:528-537. Kelner, D. E., and B. E. Sietman. 2000. Relic populations of the Ebony Shell, Fusconaia ebena (Bivalvia: Unionidae), in the upper Mississippi River drainage. Journal of Freshwater Ecology 15:371-377. Lang, B.Z. 1998. Anodonta californiensis from Curlew Lake, Washington. WDFW Contract Number 53081050. 7 pages. Master, L. L., B. A. Stein, L. S. Kutner, and G. A. Hammerson. 2000. Vanishing assets: Conservation status of US species. Pages 93--118 in B. A. Stein, L. S. Kutner, and J. S. Adams (editors). Precious heritage: The status of biodiversity in the United States. Oxford University Press, Oxford, United Kingdom. Meyers, T.R., and R.E. Millemann. 1977. Glochidiosis of salmonid fishes. I. Comparative susceptibility to experimental infection with Margaritifera margaritifera (L.) (Pelecypoda: Margaritanidae). The Journal of Parasitology 63:728-733. Mock, K.E., J.C. Brim-Box, M.P. Miller, M.E. Downing, and W.R. Hoeh. 2004. Genetic diversity and divergence among freshwater mussel (Anodonta) populations in the Bonneville Basin of Utah. Molecular Ecology 13: 1085-1098. Mock, K. E., J. C. Brim-Box, J. P. Chong, J. K. Howard, D. A. Nez, D. Wolf, and R. S. Gardner. 2010. Genetic structuring in the freshwater mussel Anodonta corresponds with major hydrologic basins in the western United States. Molecular Ecology 19:569-591. Murphy, G. 1942. Relationship of the fresh water mussel to trout in the Truckee River. California Fish Game 28: 89-102. NatureServe Explorer 2008. NatureServe Explorer: An online encyclopedia of life [web application]. Version 7.1. NatureServe, Arlington, Virginia. Available http://www.natureserve.org/explorer. (Accessed: 19 March, 2010). Neves, R. J. 1999. Conservation and commerce: Management of freshwater mussel (Bivalvia: Unionoidae) resources in the United States. Malacologia 41:461-474. O'Brien, C. A., and J. Brim Box. 1999. Reproductive biology and juvenile recruitment of the shinyrayed pocketbook, Lampsilis subangulata (Bivalvia: Unionidae) in the Gulf Coastal Plain. American Midland Naturalist 142:129-140. O'Brien, C., D. Nez, D. Wolf, and J. Brim Box. 2013. Reproductive Biology of Anodonta californiensis, Gonidea angulate, and Margaritifera falcata (Bivalvia: Unionoida) in the Middle Fork John Day River, Oregon. Northwest Science, 87(1):59-72. Oliver, G.W., and W.R. Bosworth. 1999. Rare, Imperiled and Recently Extinct or Extirpated Mollusks of Utah: A Literature Review. Utah Division of Wildlife Resources Publication 99--29. Utah Division of Wildlife Resources, Salt Lake City, Utah. 236 pp. Ricciardi, A., and J. B. Rasmussen. 1999. Extinction rates of North American freshwater fauna. Conservation Biology 13:1220-1222. Roe, K. J., A. M. Simons, and P. Hartfield. 1997. Identification of a fish host of the Inflated Heelsplitter Potamilus inflatus (Bivalvia: Unionidae) with a description of its glochidium. American Midland Naturalist 138:48-54. Sietman, B. E., J. M. Davis, and M. C. Hove. 2012. Mantle display and glochidia release behaviors of five quadruline freshwater mussel species (Bivalvia: Unionidae). American Malacological Bulletin 30:39-46. Spooner, D. E., M. A. Xenopoulos, C. Schneider, and D. A. Woolnough. 2012. Coextirpation of host-affiliate relationships in rivers: The role of climate change, water withdrawal, and host-specificity. Global Change Biology 17:1720-1732. Spooner, D. E., and C. C. Vaughn. 2008. A trait-based approach to species' roles in stream ecosystems: Climate change, community structure, and material cycling. Oecologia 158:307-317. Spring Rivers. 2007. Reproductive Timing of Freshwater Mussels and Potential Impacts of Pulsed Flows on Reproductive Success. California Energy Commission. PIER Energy-Related Environmental Research Program. CEC-500-2007-097. Stagliano, D. M. Anderson, and K. Cook. 2020. Western Pearlshell mussel (WEPE) reproduction and life history study in five watersheds of Montana: Aquatic SWG Implementation. Vaughn, C. C., and C. C. Hakenkamp. 2001. The functional role of burrowing bivalves in freshwater ecosystems. Freshwater Biology 46:1431-1446. Vaughn, C.C. and D.E. Spooner. 2006. Scale-dependent associations between native freshwater mussels and invasive Corbicula. Hydrobiologia 568:331-339. Vaughn, C.C., S.J. Nichols, and D.E. Spooner. 2008. Community and foodweb ecology of freshwater mussels. Journal of the North American Benthological Society 27(2): 409-423. Watters, G.T. 1994. An Annotated Bibliography of the Reproduction and Propagation of the Unionoidea (Primarily of North America). Ohio Biological Survey Miscellaneous Contributions No. 1. 158 pp. Watters, G. T., and S. H. O'Dee. 1998. Metamorphosis of freshwater mussel glochidia (Bivalvia: Unionidae) on amphibians and exotic fishes. American Midland Naturalist 139:49-57. Williams, J.D., M.L. Warren, K.S. Cummings, J.L. Harris, and R.J. Neves. 1993. Conservation status of freshwater mussels of the United States and Canada. Fisheries 18(9): 6-22. Williams, J. D., A. E. Bogan, R. S. Butler, K. S. Cummings, J. T. Garner, J. L. Harris, N. A. Johnson, and G. T. Watters. 2017. A revised list of the freshwater mussels (Mollusca: Bivalvia: Unionida) of the United States and Canada. Freshwater Mollusk Biology and Conservation 20:33-58. Workman, G.W., R.A. Valdez, W.F. Sigler, and J.M. Henderson. 1979. Studies on the least chub in geothermal active areas of western Utah. U.S. Bureau of Land Management Xerces Society. 2010a. Margaritifera falcata (Gould, 1850). Profile prepared by Sarina Jepsen, Caitlin LaBar, and Jennifer Zarnoch. The Xerces Society for Invertebrate Conservation. Xerces Society 2010b. Anodonta californiensis (Lea, 1852)/Anodonta nuttalliana (Lea, 1832). Profile prepared by Sarina Jepsen, Caitlin LaBar, and Jennifer Zarnoch. The Xerces Society for Invertebrate Conservation. Zale, A.V., and R.J. Neves. 1982. Fish hosts of four species of lampsiline mussels (Mollusca: Unionidae) in Big Moccasin Creek, Virginia. Canadian Journal of Zoology 60(11):2535-2542.

1) Identify fish hosts for Western Pearlshell (Margaritifera falcata) mussels in Beaver Creek, Summit County, UT 2) Identify fish hosts for Winged Floater (Anodonta californiensis/nuttalliana) at several locations in Utah: Raft River (Raft River drainage), Bear River ( Bear River drainage, Rich County), Sevier River (Piute Reservoir), Mona (Jordan River drainage)

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Western Pearlshell (S1, N4) and Winged Floater (S2, N3) are both included in the 2015 Utah Wildlife Action Plan, a document that provides guidance on protecting species and their habitats and managing threats to prevent the need for species listings under the ESA. Implementing this proposed project will fill a WAP data gap - Inadequate Understanding Ecology and Life History - for both mussels. This data gap is not identified for either mussel in the WAP, but there is a lack of scientific literature on these topics for western freshwater mussels. A lack of confirmed host fish needs for Utah's mussels impedes effective conservation efforts for these species. Although some host fish research has been conducted for these mussels in other parts of their range, the results of those studies cannot be automatically applied to mussel populations in Utah. Assuming host fish needs for these mussel species are similar across their whole species range is risky (host fish needs could be drainage-specific for these mussel species; (Mock et al 2010) and could lead to poor conservation decisions. Confirming host fish needs for mussels in Utah will provide critical information for future propagation efforts, host fish reintroductions, and habitat restoration efforts. This project also meets the goals of the UDWR Statewide Mollusk Conservation Strategy -- the goals of this strategy are to fill crucial data gaps identified in the UWAP and to mitigate threats to SGCN mollusks. Aside from addressing these goals, this proposed project will specifically execute Objective 3 (Action 1.1, 1.2, and 1.3). 1. Objective 3 -- Conduct laboratory experiments to identify local host-fish requirements for Western Pearlshell and Anodonta nuttalliana/californiensis, preferably for each major drainage or isolade water body occupied by each freshwater mussel species. a. Action 1.1 -- Conduct laboratory experiments to identify host-fish requirements for Western Pearlshell from Beaver Creek (Summit County) and at least one other Utah population, if possible. b. Action 1.2 -- Conduct laboratory experiments to identify local host-fish requirements for A. nuttalliana/californiensis from one population in each of the following drainages: Raft River, Bear River, Sevier River, and Jordan River. c. Action 1.3 -- Publish results of host-fish research for Western Pearlshell and A. nuttalliana/californiensis. Mock, K. E., J. C. Brim Box, J. P. Chong, J. K. Howard, D. A. Nez, D. Wolf, and R. S. Gardner. 2010. Genetic structuring in the freshwater mussel Anodonta corresponds with major hydrologic basins in the western United States. Molecular Ecology 19:569-591.

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If these species are federally listed, there could be impacts to water development, water management, and fishing regulations (since fish are required for mussel reproduction) across the northern and central parts of the state.

Fish health/disease certifications will be conducted on fish populations for which no disease certification or proxy certification exists. This will only be done for fish species/populations that do not already have a disease certification and will be collected for host trials. To prevent introduction to neighboring water bodies around the Fisheries Experiment Station (FES), outflow water will be sieved to prevent dispersal of untransformed glochidia and juvenile mussels. 55 micron mesh sieves will be used to catch prematurely released glochidia and conglutinates from gravid females in the coldwater holding system. Transformed glochidia (i.e., juveniles) will be captured in 55 micron mesh sieves from a recirculating system. As such the probability of escapement and establishment in Cutler Marsh are extremely low.

Hosts for freshwater mussels can be determined by infecting fish with glochidia (larval mussels) in a laboratory setting and waiting to see which fish allow the metamorphosis of glochidia into juvenile mussels (Zale and Neves 1982).2020). The standard laboratory setting requires a flow-through or recirculating aquarium system to hold the infected fish. The aquarium system includes approximately 30 Aquatic Habitat Units (AHAB units) arranged on a shelving unit. AHAB units are self-cleaning. A filter cup (PVC section with fine mesh on one end) is placed at the outflow of each AHAB to catch sloughed glochidia and metamorphosed juvenile mussels as they fall off the fish. Potential host-fish used in the study are preferably collected from a location where mussels are not present. This helps ensure that the fish have not developed prior immunity to glochidial infestations (Rogers and Dimock 2003). Fish hatcheries can be a good source of fish for host-fish studies. Brood mussels that provide glochidia for the host-fish studies will be collected prior to the study and held in captivity until glochidia are mature. It is important to ensure that the brood mussels do not abort their glochidia prematurely, and this can usually be achieved by keeping the mussels cool during transport and in the laboratory. Mussels will be held in aerated containers of non-chlorinated water in the laboratory. Fish hosts are infected by placing them into a known volume of water with a known number of glochidia for approximately 30 minutes. It is important to ensure the glochidia stay suspended in the water to ensure they are able to attach to the gills or fins of the fish. The target concentration of glochidia is 2,000-4,000 glochidia/L water (Dudding et al. 2019; Holcomb et al 2020). Only fish of the same species should be infected together. Mixing fish species could increase cortisol levels in the fish and increase the chance of glochidia infestation (bias) (Holcomb et al. 2020). It is also important to ensure the viability of glochidia used in the study using the salt test method (Zale and Neves 1982). Once fish have been infected with glochidia, they are returned to the AHAB system. Filter cups should be checked every-other-day to count the number of glochidia and metamorphosed juveniles from each fish. A fish is considered a host if it produces viable juvenile mussels. Metamorphosis rate will also be calculated for each fish [(number of juvenile mussels produced/(number of juvenile mussels produced + number of sloughed glochidia)) *100] (Dudding et al 2019; Holcomb et al 2020). Dudding, J., M. Hart, J. Khan, C. R. Robertson, R. Lopez, and C. R. Randklev. 2019. Host fish associations for two highly imperiled mussel species from the Southwestern United States: Cyclonaias necki (Guadalupe Orb) and Fusconaia mitchelli (False Spike). Freshwater Mollusk Biology and Conservation 22:12-19. Holcomb, K. M., J. M. Holcomb, S. C. Pursifull, and J. R. Knight. 2020. Distribution, period of gravidity, and host identification for the Narrow Pigtoe mussel. Journal of Fish and Wildlife Management 11:1-12. Rogers, C. L., and R. V. Dimock Jr. 2003. Acquired resistance of bluegill sunfish Lepomis macrochirus to glochidia larvae of the freshwater mussel Utterbackia imbecillis (Bivalvia: Unionidae) after multiple infections. Journal of Parasitology 89:51--56. Zale AV, Neves RJ. 1982. Fish hosts of four species of Lamsiline mussels (Mollusca: Unionidae) in Big Moccasin Creek, Virginia. Canadian Journal of Zoology 60:2535--2542

UDWR research biologist Connor Schwepe will be heading the host fish trial at FES. Connor has extensive experience building and maintaining recirculating aquaculture systems, as well as raising and caring for a variety of fish housed within them. In addition, a seasonal tech experienced in malacology is expected to be hired to assist in all aspects of the trial. If issues arise, project leads will be able to determine appropriate solutions to allow the project to proceed successfully. Potential host fish health will be monitored prior to initiating the study to ensure they are healthy enough to survive the duration of the study. Viability of glochidia, and an appropriate concentration of glochidia for host fish infections, will be assessed to ensure the trials will be successful and comparable to other studies. Filter cups at the outflow of each fish tank will be monitored at least every other day to determine which fish are hosts and which are not (presence of juvenile mussels = host).

The Utah Division of Wildlife Resources partners with or is supported by: Utah Geological Survey, Utah State University, all signatories of the Conservation Agreement and Strategy for Springsnails of UT and NV, the Utah Native Mollusk Workgroup, Utah Division of Water Quality, U.S. Forest Service, and Bureau of Land Management.

This project will require 2-3 years to identify fish hosts for both mussel species. Once the project is completed, hosts will be known for each mussel species. Conducting this study in a laboratory study will allow us to determine the primary hosts (highest metamorphosis rate) and any marginal hosts (lower metamorphosis rate). This information will help UDWR determine which host fish are most appropriate for rearing large numbers of juvenile mussels in captivity. This study will also help UDWR determine why mussel populations are declining. If the necessary host fish are not present or they cannot access mussel beds due to a barrier, the mussels are not able to reproduce. Introducing host fish populations or removing barriers could improve reproductive success for Utah mussel populations. Also, if appropriate host fish are present but mussel populations are still declining, this may suggest that mussel habitat is degraded.

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