Host fishes for the state-endangered Tennessee heelsplitter (Lasmigona holstonia) and state-threatened black sandshell (Ligumia recta) were identified through induced infestations of glochidia on potential hosts. Largemouth bass (Micropterus salmoides), yellow perch (Perca flavescens), convict cichlid (Cichlasoma nigrofasciatum), platy (Xiphophorus maculatus), green sunfish (Lepomis cyanellus), rock bass (Ambloplites rupestris), redbreast sunfish (Lepomis auritus), and white perch (Morone americana) were identified as suitable hosts for L. recta. The banded sculpin (Cottus carolinae) and rock bass were identified as hosts for L. holstonia; striped shiner (Luxilus chrysocephalus), central stoneroller (Campostoma anomalum), and warpaint shiner (Luxilus coccogenis) were identified as potential hosts. Additionally, rock bass and bluegill (Lepomis macrochirus) were identified as potential hosts for the state-endangered spectaclecase (Cumberlandia monodonta), with numerous encysted glochidia present at 11 days postinfestation when the fish died. Recirculating culture systems of different design were tested for suitability in juvenile mussel culture. In one system (high maintenance), juveniles of the wavy-rayed lampmussel, Lampsilis fasciola, were kept in culture dishes, and in the other system (low maintenance), the juveniles were kept in culture beds. At the end of the 16-wk culture period, the 31.3% (± 15.4) survival exhibited in the dish culture system was significantly greater than the 3.1% (± 2.8) survival in the bed culture system (P< 0.01, Tukey-Kramer). However, mussels grown in the bed system exhibited significantly greater growth (1.4 ± 0.50 mm height, 1.8 ± 0.76 mm length) than those grown in the dish system (0.86 ± 0.19 mm height, 1.1 ± 0.27 mm length) (P<0.01, Tukey-Kramer). Using the high maintenance dish culture system and juveniles of L. fasciola, the influence of high (4.1%) and low (2.5 %) organic content in substrate, and high (250 mg/L CaCO₃) and low (50 mg/L CaCO₃) water hardness levels were assessed on growth and survival. After 15 wk, juveniles in the high water hardness treatment exhibited significantly greater survival and growth (44.2 ± 9.3% survival, 1.5 ± 0.28 mm height, 2.1 ± 0.41 mm length) than those in the low water hardness treatment (9.0 ± 7.9% survival, 1.3 ± 0.25 mm height, 1.8 ± 0.37 mm length) (P<0.01, Tukey- Kramer). Juveniles in the high organic substrate exhibited similar growth (1.41 ± 0.24 mm height, 1.96 +- 0.37 mm length) to those in the low organic substrate (1.39 ± 0.28 mm height, 1.94 ± 0.42 mm length). Juveniles grown in high hardness and high organic substrate had similar survival (27.4 +- 9.2%) to those in low organic substrate (25.8 ± 8.1 %). For the culture of L. fasciola juveniles, I recommend using a culture system that is cleaned regularly (weekly), receives a consistent (daily) supply of algal food, has relatively even flow, and from which juveniles are easily sampled. I recommend culturing them in relatively hard water (~ 250 mg/L CaCO₃), in a substratum with some organic content. These recommendations may warrant modification for the culture of other freshwater mussel species. / Master of Science

Unionoid mussels are globally threatened and their conservation requires species-specific knowledge on their ecology and parasite-host interaction. Unio crassus is one of Europe’s most threatened unionoid species and has a temporary obligate parasitic life stage (glochidia) on fish. A lack of suitable hosts is probably a major limitation for mussel recruitment, but host species composition, suitability and availability in time and space have yet to be fully explored. This thesis examines different aspects of the host fish species, including their composition, suitability and ecological importance, in relation to U. crassus, using both field and laboratory studies. The effects of mussel and host density on mussel reproductive potential were considered, as were aspects of evolutionary adaptations between mussels and fish and how climate change may affect their interaction. The results show that U. crassus is a host generalist, parasitizing a variety of fish species. Host suitability and density, which varied among fish species and rivers, affected the level of glochidia encapsulation, hence mussel reproductive potential, more so than the density of mussels taking part in reproduction. Ecologically important hosts included both highly suitable primary hosts, and less suitable hosts that were highly abundant. Whether or not U. crassus has specific adaptations to its hosts to enhance juvenile transformation remains unclear. No distinct pattern of local adaptation was found, nor was there an effect of host fish presence on the timing of glochidia release by adult mussels. Instead, temperature played a major role, with results suggesting that changes in spring water temperature regimes can cause temporal and spatial mismatches in the mussel-host interaction. This thesis indicates that investigations of local mussel-host interactions help in identifying mechanisms important for unionoid conservation management and prioritization. / Många sötvattenmusslor har en komplex livscykel där larverna (glochidier) under sin utveckling till frilevande musslor parasiterar på gälarna hos lämpliga värdfiskar. Flera av våra musslor, såsom den tjockskaliga målarmusslan (Unio crassus), är globalt hotade och för att kunna bevara och förvalta dessa arter på bästa sätt behöver vi lära oss mer om deras ekologi och samspelet mellan musslan och dess värdfiskar. Avsaknaden av värdfiskar innebär förmodligen en stor begränsning för rekryteringen av juvenila musslor, men det finns trots detta en begränsad kunskap om hur artsammansättningen i fisksamhället och dess tillgänglighet påverkar musselpopulationer.  Min avhandling undersöker olika aspekter av interaktioner mellan U. crassus och dess värdfiskar, som hur värdfisksamhällen och fiskarters värdlämplighet påverkar musslans reproduktionspotential. Jag har även studerat hur tätheter av olika fiskarter och vuxna musslor påverkar rekryteringen, eventuella evolutionära anpassningar samt om en förhöjd temperatur skulle kunna påverka interaktionen mellan U. crassus och dess värdfiskar. Resultaten visar att U. crassus är en generalist som parasiterar på en mängd olika fiskarter. Jag fann dock en stor variation i dominerande fiskarter och lämpliga värdar mellan olika åar, vilket påverkade reproduktionspotentialen hos musslorna mer än vad tätheten vuxna musslor som deltog i reproduktionen gjorde. Som ekologiskt viktiga värdar fanns således både särskilt lämpliga, primära värdarter, men också mindre lämpliga arter som förekom i höga tätheter. Ingen tydlig lokal anpassning kunde observeras, och fiskens närvaro påverkade inte tidpunkten för när de vuxna musslorna släppte sina glochidielarver. Däremot fann jag att temperaturen spelade en viktig roll för musslans reproduktion, där ökad temperatur föreslås ha negativa effekter på interaktionen mellan musslan och dess värdfiskar. Avhandlingen visar på vikten av att studera interaktioner mellan den tjockskaliga målarmusslan och dess värdar på lokal skala för att bättre kunna identifiera och prioritera viktiga naturvårdsåtgärder. / UnioCrassusforLIFE (European LIFE+ project: LIFE10 NAT/SE/000046)

The dwarf wedgemussel (Alasmidonta heterodon) is a federally endangered freshwater mussel that once ranged from New Brunswick to North Carolina, but now only exists in isolated populations throughout its diminished range. Laboratory propagation in conjunction with augmentation or reintroduction is considered a critical component of dwarf wedgemussel restoration. My thesis aimed to I) develop in-vitro propagation techniques including methods to minimize microbial contamination for two Alasmidonta species and II) compile and assess critical fish assemblage information at existing dwarf wedgemussel presence and absence locations to inform future restoration activities. For the first objective (Chapter 2) I assessed three methods of contamination mitigation (media change frequency, concentration of the antifungal Amphotericin B, and method of antifungal replenishment). Across all experiments, higher levels of contamination severity had a negative impact on transformation success. In my experiments, dishes that had media changes every other day (vs. those changed daily and every 3 days) had the highest contamination and the lowest glochidia transformation success. Treatments with the lowest (0 µg/mL) concentration of the antifungal, Amphotericin B, and got a low-dose replenishment of Amphotericin B (vs frequent media changes) had the highest transformation success. The fungus was identified as Candida parapsilosis, a common fungus found in aquatic and human environments; future propagation efforts should use mitigation methods that are specific to the fungal contaminant. In the assessment of host fish near dwarf wedgemussel locations (Chapter 3), two fish repeatedly emerged as indicators of dwarf wedgemussel occurrence and abundance: the tessellated darter (Etheostoma olmstedi) and brown trout (Salmo trutta). Tessellated darter was positively related to dwarf wedgemussel which supports the importance of tessellated darter as a host fish in the wild. The negative relationship between dwarf wedgemussel and brown trout may indicate a difference in habitat between the species or brown trout’s predation upon wild hosts. Results from this project will be used in conjunction with habitat and genetic information to inform future restoration plans in the northeast and add to the growing body of literature on in-vitro propagation of freshwater mussels.

Pink heelsplitter (Potamilus alatus) mussels collected from Kentucky Lake, TN were held at two bottom locations (0.6 m, 2.5 m) and suspended in pocket nets (at depth about 1.0 - 1.5 m) in a pond at the Freshwater Mollusk Conservation Center (FMCC), Virginia Tech, for 1 yr. Survival of mussels after 1 yr was significantly different, with poorest survival (30 %) in the bottom of the deep end; and no difference between the shallow end (83.3 %) and the suspended pocket nets (63.3 %). Survival of mussels was inversely related to water temperature (r = - 0.72); lowest monthly survival occurred in summer, resulting in a significant difference among the three locations with a similar trend after 1 yr. The glycogen reserves of mussels in captivity for 1 yr differed by pond location, higher in mussels at the shallow end than those in suspended pocket nets and at the deep end. Therefore, the shallow end of pond was more suitable for holding mussels long-term, while the suspended pocket nets are an alternative site for holding captive mussels. Additionally, dissolved oxygen was very low at the deep end (1.9 mg/L) in summer, while it was adequate (range from 5.7 - 6.4 mg/L) at the location of suspended pocket nets, and 5.0 mg/L at the shallow end (24.7 °C). Data for 40 specimens indicated that sexual dimorphism in valve shape occurred in P. alatus. Female mussels had a significantly (p < 0.0001) greater ratio of height (H) to length (L) (52.3 %) and width (W) to length (31.8 %) than males (H/L: 48.4 %; W/L: 28.8 %), respectively. The posterior ends were somewhat round to oval in males and bluntly squared or truncated in females. Female mussels were more inflated than males. These morphological differences can be used to distinguish females from males during field collections. The red drum (Sciaenops ocellatus) was identified as a new fish host for P. alatus, as 48 active juveniles were transformed by this species, which is not a natural host. Four glochidia were observed on the fins versus 2,307 on the gills of five red drum. Freshwater drum also was verified as a suitable host fish, but black crappie (Pomoxis nigromaculatus), banded sculpin (Cottus carolinae), yellow perch (Perca flavescens) and nile tilapia (Oreochromis nilotica) did not support transformation of glochidia to juveniles. Survival and growth of propagated juveniles of P. alatus were assessed regarding the effects of algal diets (Nannochloropsis oculata and Neochloris oleoabundans) and substrate type (fine sediment and sand). Overall, survival of juveniles after 17 d ranged from 23.8 to 66.8 %, with mean of 48.5 %; however, survival dramatically declined during the next 2 wk period to only 5.8 % (range of 1.8 to 7.8 %). Survival rate of juveniles was significantly different (p = 0.027) between substrates, but not in diets (p = 0.520), with the lowest survival rate of 23.8 % in sand substrate and fed N. oculata. Juveniles grew faster in fine sediment (23.0 % increase in shell length) than in sand substrates (10.5 % increase) (p = 0.002). Moreover, mean growth rate of juveniles was 4.9 &#956;m/d during the first 2 wk, but decreased to 0.2 &#956;m/d in the remaining 2 wk. Therefore, fine sediments seemed more appropriate for juvenile culture compared to sands. Both species of algae, N. oculata and N. oleoabundans, can be used to feed juveniles in the laboratory. Adult pink heelsplitters were used to study feasibility of pearl production by using two surgical implants (non-nucleated implant = NNI, and image pearl implant = IPI) in two ponds of different nutrient levels (FMCC pond and Duck pond). NNI and IPI pearls with purple or purplish luster were successfully produced in P. alatus. Pearl weight was not significantly different (p = 0.562) between two ponds. No differences in monthly survival rates of mussels were observed in either pond (p = 0.051), or among mussels with surgical implants and the no-surgery control mussels (p = 0.881). Consequently, P. alatus can be considered a potential species for producing purple pearls in pearl culture. Additionally, mussels in the Duck pond had higher (p < 0.0001) glycogen levels, similar to those in wild collected mussels, than those in the FMCC pond, indicating that this pond environment may be more suited for holding implanted mussels in captivity. / Master of Science

The federally endangered tan riffleshell (<I>Epioblasma florentina walkeri</I>) is restricted to only one known reproducing population, in Indian Creek, Tazewell County, Virginia. Attempts to recover this species by augmenting relic populations throughout its historic range are aided through knowledge of its population biology and requirements in culture environments. Infestations of host fish (fantail darters, <I>Etheostoma flabellare</I>), obtained from four river drainages, with tan riffleshell glochidia showed that significantly more juveniles transformed per fish from infestations on fantail darters from Indian Creek (mean = 59.22 ± 10.01) than on fantail darters from the Roanoke River (mean = 9.45 ± 10.64) (p = 0.024). Number of juveniles from fantail darters collected from Elk Garden and the South Fork Holston River were not significantly different from those of either Indian Creek fish or Roanoke River fish. These results support the hypothesis that mussel-host fish relationships are likely mediated by fish immune responses. Furthermore, this study suggests that this compatibility has resulted from coadaptation between the tan riffleshell and fantail darter populations in Indian Creek. The tan riffleshell population in Indian Creek was estimated to be 1078 adults (95% CI= 760 - 1853), using Schumacher's modification of Schnabel's maximum likelihood estimator. The sex ratio and size distribution of males and females were approximately equal. Specimen ages, determined from thin-sections of shells, showed that mussels aged by external annuli on shells likely underestimates the true ages of individuals. Appropriate culture conditions for this species were examined using juveniles of the wavyrayed lampmussel (<I>Lampsilis fasciola</I>) as a surrogate. In the first experiment, juvenile growth and survival was compared between four substratum types (fine sediment, < 120μm; fine sand, 500 μm-800 μm; coarse sand, 1000 μm-1400 μm; and mixed sediment, < 1400 μm) and two light treatments in open versus covered recirculating troughs (2.8 m). Juveniles in fine sediment substratum and covered troughs fared poorest, with 7% survival and growth to only 0.86 mm in length after 16 wk. Juveniles in mixed sediment and open troughs fared best, with 26% survival and growth to 1.09 mm after 16 wk. Additionally, juveniles in fine sand in covered troughs had significantly higher survival (23.1%) than juveniles in fine sediment (p = 0.04), and juveniles in fine sand survived consistently better between light treatments than in the other substrata. There were no significant differences among the other treatments. A second experiment was performed to determine whether juveniles were responding directly to the presence of light or whether only the increased autochthonous production improved growth and survival. One-half of each of three 2.8 m troughs were covered with 50% shade cloth, while the other sides were left open to ambient light. Additionally, the best and worst sediments from the first experiment (fine sand and fine sediment) were used again to verify the results from the previous experiment. In this case, juveniles in both sides of the troughs grew equally well, but juveniles in the open sides had significantly poorer survival (open mean: 1.78%, sd = 5.01; covered mean: 7.4%, sd = 5.01) (p = 0.046). Fine sediment yielded significantly higher growth of juveniles than fine sand (p = 0.009), with shell lengths of 2.63 mm (sd = 0.075) in fine sediment and 1.94 mm (sd = 0.102) in fine sand. The differences in survival and growth between the two experiments were attributed to differential numbers of chironomids and platyhelminths, which are predators of young juveniles. Additionally, the fine sediment was more tightly packed in the first experiment than in the second, which may have restricted movement and subsequently reduced survival. Light alone likely did not affect juvenile survival and growth; rather, it was seemingly the greater abundance of aufwuchs available as food. This hypothesis was corroborated by a juvenile behavior experiment, which showed that juveniles did not act differently when in tanks not exposed to light versus those open to ambient light. / Master of Science

The Big South Fork National River and Recreation Area (NRRA), located in Tennessee and Kentucky, has prepared a management plan to include restoration of its mussel fauna to historic levels. Restoration activities include propagation of juvenile mussels and relocation of adults to suitable sites in the Big South Fork of the Cumberland River (BSF) and its tributaries. This study was conducted to identify host fish for Cumberland elktoe (Alasmidonta atropurpurea) and Cumberland bean (Villosa trabalis), to determine suitable juvenile culture conditions for Epioblasma brevidens and V. trabalis, and to locate sites important to future mussel restoration efforts in the NRRA. Host fish identifications and propagation techniques were determined for two of the endangered species in the NRRA, Cumberland elktoe (Alasmidonta atropurpurea) and Cumberland bean (Villosa trabalis). Of seven host species tested, banded sculpin (Cottus carolinae) was the most suitable host fish for propagation of A. atropurpurea. Of five host species tested, fantail darters (Etheostoma flabellare) were the most suitable host fish for propagation of V. trabalis. Culture techniques to raise juvenile mussels in captivity were evaluated, using newly metamorphosed juveniles of V. trabalis and E. brevidens in recirculating systems. No differences in juvenile growth or survival were detected among substrates used (fine sediment, coarse sand, and a mixture of the two). Recirculating system design seemed to affect juvenile growth and survival; however, variable condition of juveniles also seemed to affect results, making it difficult to determine effects from trial treatments. Finally, an assessment of potential sites in the NRRA for restoration activities was conducted using spatial analysis in a geographic information system (GIS) and several measures of conservation value. Mussel restoration sites were assessed for potential threats from adjacent land uses that may negatively affect mussels, including coal mines, oil and gas wells, transportation corridors, agriculture and urban development. Sites were also evaluated on their current conservation value to designate which sites are most important to long-term maintenance of mussel fauna. Several sites were identified that contain relatively few land-use threats, and are appropriate for mussel restoration activities, including Big Island, Station Camp Creek, and Parchcorn Creek sites on the mainstem BSF, as well as sites on Clear Fork and North White Oak Creek. Many of these sites also have high conservation values. Other sites had relatively high land-use threats that need to be addressed before restoration activities take place. Such sites include Leatherwood Ford, Rough Shoals Branch, Blue Heron, and Yamacraw on the mainstem BSF. The dominant threat to most sites came from transportation corridors, whereas some sites in southern and eastern portions of the watershed also were threatened by coal mines, and oil and gas wells. / Master of Science

Flodpärlmusslan (Margaritana margaritifera) är en starkt hotad mussla som är beroende av öring (Salmo trutta) för sin larvutveckling. Musslan är negativt påverkad av dagens användning av vattendrag och omgivande mark då de har höga krav på sin omgivning. Denna undersökning tittar på hur bottensubstrat, tillgång till värdfiskars habitat och kantzonernas sammansättning påverkar musslornas rekrytering. För detta användes biotopkarteringar, vilka relaterades till musslans rekrytering. Resultatet visade att hög förekomst av ståndplatser för värdfisken, strömmande vatten och buskskiktet på land var positivt för rekryteringen av flodpärlmusslan. Det visade sig däremot att det inte var någon skillnad i bottensubstratets sammansättning eller i antalet diken mellan vattendrag med och utan rekrytering. Slutsatsen för denna undersökning är att det behövs fördjupade studier på mindre skala och i akvarieexperiment med fokus på hur värdfiskens habitat, hydrologin och kantzonens utseende påverkar musslans rekrytering. / The freshwater pearl mussel (Margaritana margaritifera) is a highly endangered mussel that depends on trout (Salmo trutta) for its larval development. The mussels are negatively affected by the current use of watercourses and surrounding land as they have high demands on their surroundings. This study looks at how bottom substrates, access to host fish habitats and the composition of ecotone affect mussel recruitment. For this biotope mapping was used, which was related to the mussel's recruitment. The results showed that the high prevalence of sites for host fish, flowing water and the shrub layer on land was positive for the recruitment of freshwater pearl mussels. It turned out, however, that there was no difference in the composition of the bottom substrate or in the number of ditches between watercourses with and without recruitment. The conclusion of this study is that in-depth studies on a smaller scale and in aquarium experiments are needed with a focus on how the host fish's habitat, hydrology and the appearance of the edge zone affect the mussel's recruitment.

A freshwater mussel survey was conducted in Indian Creek, Tazewell County, Virginia, during 1996 and 1997. Fifteen species were identified, including the federally endangered <i>Epioblasma florentina walkeri<i>, <i>Villosa perpurpurea</i>, and <i>Quadrula cylindrica strigillata</i>. Population assessments and fish host identifications were completed for the tan riffleshell and purple bean populations. Host fish for <i>E. f. walkeri</i> were limited to the banded and mottled sculpin, greenside darter, redline darter, fantail darter, and snubnose darter. Fish hosts identified for <i>V. perpurpurea</i> also were the banded and mottled sculpin, greenside darter, and redline darter. Size class structure of the tan riffleshell population ranged from 19.9 to 53.3 mm, with the population estimated at nearly 700 individuals with a density of 0.015/m². Size class structure of the purple bean population ranged from 22.9 to 66.7 mm, with the population estimated at only 70 individuals with a density of 0.002/m². Host fish also were identified for <i>Dromus dromas</i> and <i>Lemiox rimosus</i>. The fantail darter was identified as a host for <i>D. dromas</i>, with the snubnose darter serving as a host for <i>L. rimosus</i>. Additional percids were implicated as hosts for both mussel species. A molecular genetic key for identifying host fishes of the upper Clinch River also was constructed. The key was constructed through the analysis of restriction fragment length polymorphisms from amplified regions of mussel DNA. Thirty-six unionid species were incorporated into the key. No host fishes were identified due to an unsolved problem with amplifying DNA from glochidia collected from wild fish. / Master of Science