Long-term monitoring of two endangered freshwater mussels (Bivalvia: Unionidae) reveals how demographic vital rates are influenced by species life history traits.

To meet monitoring and recovery planning needs, demographic vital rates of two endangered freshwater mussels (Bivalvia: Unionidae)-the Cumberlandian Combshell (Epioblasma brevidens, Lea 1831) and Oyster Mussel (Epioblasma capsaeformis, Lea 1834), species endemic to the Tennessee and Cumberland river basins, U.S.A-were estimated and compared using census methodologies. Annual variation in population density and size, recruitment rate, mortality rate, sex ratios, and female fecundity of both species were observed from 2004-2014 at three fixed sites, spanning a 33.8 kilometer (KM) reach of the Clinch River, Hancock County, Tennessee. Mean population size of E. brevidens estimated from 11 censuses was 2,598 individuals at Swan Island (KM 277.1), 8,744 at Frost Ford (KM 291.8), and 879 at Wallen Bend (KM 309.6); collectively, these demes grew at an annual rate of 7% over the study period. Mean population size of E. capsaeformis was 7,846 individuals at Swan Island, 265,442 at Frost Ford, and 11,704 at Wallen Bend; collectively, these demes grew at an annual rate of 6%. Population size, variability in population growth, recruitment, and mortality of the shorter-lived E. capsaeformis (maximum age = 16 yrs, rarely >10 yrs) were higher than those of the longer-lived E. brevidens (maximum age = 25 yrs). Stream discharge was associated with realized per-capita population growth rate for both species when juvenile (Ages 1-3) data was included. Linear regression analysis showed that the growth rate of E. brevidens was negatively associated with median annual discharge (p = 0.0274) and that growth rate of E. capsaeformis was negatively associated with the number of days having extreme high discharge preceding a census (p = 0.0381). Fecundity of female E. brevidens averaged 34,947 (SE = 2,492) glochidia and ranged from 18,987 to 56,151, whereas fecundity of female E. capsaeformis averaged 9,558 (SE = 603) glochidia and ranged from 3,456 to 22,182. Estimated vital rates indicated that the two species are characterized by different life-history strategies, with E. brevidens exhibiting a periodic strategy (between K- and r-selected) and E. capsaeformis an opportunistic strategy (r-selected). These life history strategies are likely influenced by each species' longevity and habitat preference, in addition to the life histories and population dynamics of their primary fish hosts.

The temptin gene of the clade Lophotrochozoa is involved in formation of the prismatic layer during biomineralization in molluscs.

The biomineralization mechanism of mollusc shell has been studied for a long time, but there is a lack of understanding about the relationship between the shell formation in vitro and the signaling system in vivo. In this study, we cloned a novel shell matrix protein gene (hc-temptin), which only be characterized as a water-borne protein pheromone of molluscs in previous studies, from the freshwater mussel Hyriopsis cumingii. By bioinformatics analysis we found that temptin was a gene unique to the clade Lophotrochozoa, and it exists in all mollusc taxa except Cephalopoda. The current data supported the premise that temptin was generated in the early emergence of molluscs and that it maintained a high mutation rate to evolve relative independently. The specificity of hc-temptin expression in the mantle tissue suggests its potential to participate in biomineralization. Its sequence contained typical Ca2+ binding sites. Our experiments involving the pearl formation process, damaged shell repair process, and RNAi experiment showed that hc-temptin was a shell matrix protein that plays an important role in formation of the prismatic layer. The results of this study provided new insights about the origin of the temptin gene and its role in molluscs.

The kidney of the Nodularia freshwater mussel has a larger filtration-size and counter-current system with improved water excretion compared with the seawater mussel Mytilus.

Histological studies and magnetic resonance imaging were employed to analyze the kidney structure and function of the freshwater mussel, Nodularia douglasiae. The Nodularia kidney consists of proximal, intermediate and distal tubules. The epithelia of the renal tubules were composed of a single layer of cuboidal cells. The proximal and distal tubules run in opposite directions underneath the pericardial cavity. Molecular weight cut-off (MWCO) values for the kidney filtration were detected by MR tracer injections: gadolinium-diethylenetriaminepentaacetic acid (GdDTPA) at 0.55 kDa, an oligomer-based contrast agent (CH3-DTPA-Gd) at 2.2 kDa, as well as Gd-DTPA-polylysine at 10, 22, and 110 kDa. The T1w-MRI intensity and T1 relaxation rate (R1) of the pericardial cavity and renal tubules increased with tracers smaller than 10 kDa. The other tracers showed only minimal or no increase. Thus, we concluded that the MWCO of the kidney is 22 kDa, 50 times larger than that for the Mytilus living in seawater. Since the R1 values of the renal tubules were similar to those of the pericardial cavity, the kidney did not concentrate filtrated tracers. The slow decay of the magnetic resonance (MR) tracers from the renal tubules indicated a low filtration rate, suggesting that the counter-current system reabsorbs useful solutes without reabsorption of water. The higher MWCO may be beneficial to maintain the tubular oncotic pressure and allow excretion of excess water. In conclusion, a main renal function of the freshwater mussel is the excretion of water, opposite to that of the seawater mussel and vertebrates, which preserve water.

Reproductive aspects of freshwater unionid mussel sperm: Seasonal dynamics, male-to male variability, and cell quantification.

Freshwater unionid mussel diversity is decreasing because of species extirpation or extinction. While little can be done to recover lost species, there is an opportunity to develop techniques to save other species. This can be facilitated through gene banking and assisted reproduction. Unfortunately, limited information is available on mussel reproduction, especially relating to sperm quality. Objectives, therefore, were to quantify seasonal changes in sperm concentration and morphology for two unionid mussels, Ligumia subrostrata and Lampsilisstraminea, measure intraspecific heterogeneity for sperm morphometry, and develop an efficient method to quantify sperm concentration using a microspectrophotometer. There were no differences in sperm concentration when cells were extracted from the center or at a half centimeter on either side of the visceral mass, during the spawning season. There was a seasonal change in sperm concentration, such that concentration for L. subrostrata ranged from 1.1 × 109 to 19.60 × 109 cells/mL with there being the largest counts between 26 September to 7 November. L. straminea sperm concentration was greatest (20.0 × 109 cells/mL) on 13 September and subsequently decreased. Sperm were uniflagellated and SEM results for L. subrostrata and L. straminea showed mean head length and width (mid-spawning) were 3.38 ± 0.04 µm and 1.61 ± 0.01 µm and 3.37 ± 0.04 µm and 1.61 ± 0.01 µm, respectively. There were close (R2 ≥ 0.85) quadratic associations between hemocytometer counts and absorbance (300, 600, 700 nm). These results provide baseline information to further investigate sperm quality, fertilizing capacity, and cryopreservation for freshwater mussels.

Diversification dynamics of freshwater bivalves (Unionidae: Parreysiinae: Coelaturini) indicate historic hydrographic connections throughout the East African Rift System.

Invertebrates are exceptionally diverse, but many are in decline because of anthropogenic changes to their habitat. This situation is particularly problematic for taxa that are not well monitored or taxonomically poorly understood, because the lack of knowledge hampers conservation. Despite their important functional role in freshwater ecosystems, African bivalves of the family Unionidae remain poorly studied compared to their highly threatened relatives in Europe, the U.S.A. and Canada. To resolve relationships and to study diversification dynamics in space and time, we performed time-calibrated phylogenetic studies and biogeographical modeling on the unionids from the East African Rift System and surroundings, including representatives of all currently recognized Afrotropical genera except for Brazzaea (and Unio from southern Africa). Our analyses indicate that all sampled Afrotropical unionids belong to the tribe Coelaturini (subfamily Parreysiinae), as does the genus Moncetia from Lake Tanganyika, which is currently attributed to the family Iridinidae. Colonization of Africa from Eurasia by Parreysiinae occurred ~17 Ma ago, and the subsequent diversification of Coelaturini in Africa continued at a steady pace, although net diversification decreased over time as more niches and ecoregions became occupied. Clades in Coelaturini largely reflect drainage basins, with the oldest lineages and highest regional diversity occurring in Lake Tanganyika, followed by the Congo Basin watershed in general. The species assemblage of Lake Tanganyika reflects multiple independent events of colonization and intralacustrine diversification since the Late Miocene or Early Pliocene. The clades of other regions, including that containing the species from Lake Malawi, are comparatively young. Biogeographical analyses indicate that the colonization history was mainly driven by cladogenesis in sympatry, whereas few anagenetic events contributed to the modern distribution of Coelaturini. Ancestral range estimations demonstrate that Coelaturini originated in the Victoria and/or Tanganyika ecoregions, and that the Congo Basin played an essential role in the colonization of Africa by Coelaturini.

Antioxidant responses of triangle sail mussel Hyriopsis cumingii exposed to harmful algae Microcystis aeruginosa and high pH.

In lakes and reservoirs, harmful algal blooms and high pH have been deemed to be two important stressors related to eutrophication, especially in the case of CO2 depletion caused by dense blooms. However, the effects of these stressors on the economically important shellfish that inhabit these waters are still not well-understood. This study evaluated the combined effects of the harmful algae Microcystis aeruginosa (0%, 50%, and 100% of total dietary dry weight) and high pH (8.0, 8.5 and 9.0) on the antioxidant responses of the triangle sail mussel H. cumingii. The mussels were exposed to algae and high pH for 14 d, followed by a 7-day depuration period. Reactive oxygen species (ROS) in the mussel hemolymph, antioxidant and detoxifying enzymes, such as glutathione-S-transferase (GST), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) in the digestive glands were analyzed during the experimental period. GST, SOD and GPx activity levels and the content of GSH increased following exposure to toxic M. aeruginosa, whereas CAT activity was inhibited. pH showed no significant effects on the immune defense mechanisms and detoxification processes. However, a high pH could cause increased ROS and MDA levels, resulting in oxidative injury. After a 7-day depuration period, exposure to toxic M. aeruginosa or high pH resulted in latent effects for most of the examined parameters. The treatment group exposed to the highest pH (9.0) displayed an increased oxidation state compared with the other pH treatments (8.0 and 8.5) for the same concentrations of toxic M. aeruginosa. The trends observed for ROS, MDA, GPx, GST, SOD and GSH levels indicated that a high density of toxic algae could result in severe and continuous effects on mussel health.

Sensitivity of larval and juvenile freshwater mussels (unionidae) to ammonia, chloride, copper, potassium, and selected binary chemical mixtures.

In aquatic environments, organisms such as freshwater mussels are likely exposed to complex contaminant mixtures related to industrial, agricultural, and urban activities. With growing interest in understanding the risk that chemical mixtures pose to mussels, this investigation focused on the effects of various waterborne contaminants (ammonia, chloride, copper, and potassium) and selected binary mixtures of these chemicals following a fixed-ratio design to Villosa iris glochidia and juvenile Lampsilis fasciola. In individual exposures, 48-h EC50 values were determined for V. iris glochidia exposed to ammonia chloride (7.4 [95% confidence interval (CI) 6.6-8.2] mg N/L), ammonia sulfate (8.4 [7.6-9.1] mg N/L), copper sulfate (14.2 [12.9-15.4] µg Cu2+/L), potassium chloride (12.8 [11.9-13.7] mg K+/L), potassium sulfate (10.1 [8.9-11.2] mg K+/L), and sodium chloride (480.5 [435.5-525.5] mg Cl-/L). The 7-d LC50 values for juvenile L. fasciola were determined for potassium sulfate (45.0 [18.8-71.2] mg K+/L), and sodium chloride (1738.2 [1418.6-2057.8] mg Cl-/L). In Ontario these waterborne contaminants have been reported to co-occur, with concentrations exceeding the EC10 for both life stages at some locations. Data from binary mixture exposures for V. iris glochidia (chloride-ammonia, chloride-copper, and copper-ammonia) and juvenile L. fasciola (chloride-potassium) were analyzed using a regression-based, dose-response mixture analysis modeling framework. Results from the mixture analysis were used to determine if an additive model for mixture toxicity [concentration addition (CA) or independent action (IA)] best described the toxicity of each mixture and if deviation towards dose-ratio (DR) or dose-level (DL) synergism/antagonism (S/A) occurred. For all glochidia binary mixture exposures, CA was the best fit model with DL deviation reported for the chloride-copper mixture and DR deviation reported for the copper-ammonia mixture. Using the model deviation ratio (MDR), the observed toxicity in all three glochidia mixture exposures were adequately described by both CA (mean = 0.71) and IA (mean = 0.97) whereas the juvenile mixture exposure was only adequately described by CA (mean = 0.64; IA mean = 0.05).

Effects of suppressing Smads expression on wound healing in Hyriopsis cumingii.

As a specific pearl mussel in China, Hyriopsis cumingii has enormous economic value. However, the organism damage caused by pearl insertion is immeasurable. TGF-ß/Smad signal transduction pathways are involved in all phases of wound healing. We have previously reported on two cytoplasmic signal transduction factors, Smad3 and Smad5 in mussel H. cumingii (named HcSmads), suggesting their involvements in wound healing. Here, Smad4 was cloned and described. The full length cDNA of HcSmad4 was 2543 bp encoded 515 amino acids. Deduced HcSmad4 protein possessed conserved MH1 and MH2 domains, nuclear location signals (NLS), nuclear exput signals (NES) and Smad activation domain (SAD). Transcripts of Smad3, 4 and 5 were constitutively expressed in all detected tissues, at highest levels in muscles. Furthermore, HcSmad4 mRNA levels were significantly increased at incision site post wounding, and expression of downstream target genes of Smad4, such as HcMMP1, HcMMP19, HcTIMP1 and HcTIMP2 were upregulated to a certain extent. Whatever knocked down HcSmad3/4 or treated by specific inhibitors of Smad 3 (SIS3), expression levels of these genes displayed a significantly downregulated tendency compared with the wound group. In addition, histological evaluation suggested that Smad3 knockdown or SIS3 treatment was accelerated wound healing, and then Smad4 knockdown delayed the process of wound healing in mussels. These data implicate that Smad3/4 play an important role in tissue repair in mollusks.

Acute toxicity of lead in fresh water bivalves Lamellidens jenkinsianus obesa and Parreysia (Parreysia) corrugata with evaluation of sublethal effects on acetylcholinesterase and catalase activity, lipid peroxidation, and behavior.

The median lethal concentrations (LC50) of Pb at 96 h were 8.84, 192.14, and 3.22 mg L-1 for pre-reproductive and reproductive individuals of Lamellidens jenkinsianus obesa and reproductive individuals of Parreysia (Parreysia) corrugata, respectively. Thus, young L. j. obesa were much more sensitive to Pb than its adults, while P. (P.) corrugata was the most sensitive. However, all the three values were much higher than the Pb levels commonly found in natural waters, and indicative of the tolerance of these mollusks to acute, short-term Pb exposure. In contrary to these findings, acetylcholinesterase (AChE) and catalase (CAT) activities were affected and lipid peroxidation (LPO) elevated in young L. j. obesa and P. (P.) corrugata in 21-day sublethal toxicity tests at 26-68 ppb Pb concentrations, which might be considered environmentally realistic. Some behavioral patterns such as number of movement (Mov) and durations of foot mobilization together with siphon extensions (FSE) were reduced, and the durations of valves remaining completely closed without any extension of foot and siphons (VC) increased significantly at 25-69 ppb Pb as well. Thus, the study revealed significant interspecific differences as well as that between life stages of the same species, suggesting that apparently hardy species could be impacted by low Pb concentrations in their young stages. Further, a multi-biomarker approach involving biological effects, anti-oxidative enzyme activity and easy-to-measure behavioral elements could comprise a valuable tool in assessment of Pb-induced stress in freshwater bivalves.

Potential impacts of the invasive species Corbicula fluminea on the survival of glochidia.

Freshwater mussels (Bivalvia, Unionida) are one of the most imperilled faunal groups globally, being the introduction of invasive species a possible major mechanism of threat. The Asian clam Corbicula fluminea is a problematic invasive species in aquatic ecosystems and can impair the survival of parasitic larvae (glochidia) of native freshwater mussels. However, this possible mechanism of threat remains speculative and to date very few studies addressed quantitatively this issue. In order to cover this gap, we have performed a series of manipulative laboratory studies to assess how distinct densities of C. fluminea can affect the survival of glochidia after 6, 12, 24 and 48 h of exposure, using larvae of the native freshwater mussel Anodonta anatina. Our results suggest an increase in mortality of A. anatina glochidia with an increase in density of C. fluminea. Two main mechanisms may possibly explain our results: 1) the high filtration capacity of C. fluminea that can contribute to the mortality of glochidia due to the mechanical damage of their fragile shells when passing by siphons and/or digestive tract of C. fluminea and 2) the high excretion capacity of C. fluminea that can lead to mortality of glochidia due to increase in ammonia concentration. Mortality of glochidia was also time dependent with higher values registered after 48 h. This work is one of the first showing the influence of C. fluminea density on the survival of glochidia, being filtration (and consequent passage in the digestive tract) and biodeposition the main potential mechanisms explaining overall mortality. These results also suggest that sites with high densities of C. fluminea may be highly detrimental for the conservation of freshwater mussels, potentially impairing the survival of glochidia and negatively affecting the recruitment of juveniles.

Salinity tolerance of a rare and endangered unionid mussel, Popenaias popeii (Texas Hornshell) and its implications for conservation and water management.

Unionid mussels are considered sensitive to salinity and there is growing concern in arid and semi-arid regions that declining flows coupled with anthropogenic impacts are amplifying natural salinity levels. In this study, we tested the effects of varying salinity concentrations (3.0, 4.0, 5.0, 6.0, 7.0 and 10.0 ppt NaCl) on survival of adult Popenaias popeii, (Texas Hornshell). This species occurs in the Rio Grande basin of Texas and northern Mexico, an arid to semi-arid stream plagued by salinization, and was recently listed as Endangered under the U.S. Endangered Species Act. We performed 2, 4, and 10-day toxicity tests on individuals from two disjunct populations: Laredo, TX, and the Lower Canyons of the Rio Grande near Big Bend National Park. We found no significant differences in LC50 estimates between populations at 96-hrs or 10-days but significant differences in TUD50s at 5 ppt between populations, which indicates that tolerance does not vary but sensitivity may between these populations. Overlaying LC50 estimates at 10-days for both populations on plots of salinity (ppt) measured over time, we show parts of the Rio Grande periodically approach or exceed 4.0 ppt, indicating these reaches are becoming unsuitable for P. popeii and populations within them at risk.

Invasive crayfishes as a threat to freshwater bivalves: Interspecific differences and conservation implications.

Freshwater bivalves have suffered major global declines, being the introduction of invasive alien species (IAS) an important, but not well studied, mechanism of threat. This study assessed the predator-prey relationship between two non-native crayfish species (Procambarus clarkii and Pacifastacus leniusculus) and three native (Anodonta anatina, Potomida littoralis and Unio delphinus) and one non-native (Corbicula fluminea) freshwater bivalve species through experiments in laboratory and validation under natural conditions (Sabor River basin, Portugal). All native bivalve species were preyed both in laboratory and in the field; however, both crayfish species were unable to prey C. fluminea. Predation was dependent on crayfish and bivalve species but was not affected neither by crayfish nor bivalve sizes. In the laboratory, the most preyed species by both crayfishes was A. anatina. On average, this species was preyed at least 12% more than other species, when crayfishes had a choice. Similar results were found in the field. We also found signs of competition between both crayfishes, being P. clarkii more dominant and aggressive as this species, on average, manipulated the bivalves 63.6% more times and 24:33 min longer than P. leniusculus, and initiated 55.8% more agnostic bouts. Our results support the idea that P. clarkii and P. leniusculus can affect native freshwater bivalves, but clear interspecific differences were detected. Both crayfishes may have direct and indirect impacts on bivalve populations by increasing mortality or by reducing their fitness. In addition, since both crayfishes do not prey C. fluminea, they offer this IAS another advantage over native bivalves. Given the widespread distribution of both P. clarkii and P. leniusculus and the threatened status of many freshwater bivalves, the dynamics and impacts of this relationship should be taken in account in the implementation of management measures devoted to the conservation of native freshwater bivalves.

Reestablishing a host-affiliate relationship: migratory fish reintroduction increases native mussel recruitment.

Co-extirpation among host-affiliate species is thought to be a leading cause of biodiversity loss worldwide. Freshwater mussels (Unionida) are at risk globally and face many threats to survival, including limited access to viable host fish required to complete their life history. We examine the relationship between the common eastern elliptio mussel (Elliptio complanata) and its migratory host fish the American eel (Anguilla rostrata), whose distribution in the Chesapeake Bay watershed is limited, in part, by dams. We examined population demographics of E. complanata across locations in the Chesapeake Bay watershed, primarily in the Susquehanna River in the absence of American eels, and conducted experimental restocking of eels to assess potential impacts on mussel recruitment. Compared to surveys completed ~20 yr prior, E. complanata could be experiencing declines at several historically abundant sites. These sites also had extremely limited evidence of recruitment. Restoration of host fish improved recruitment, but results were not equivalent between stocking sites, indicating that host reintroduction alone may not be fully effective in reestablishing mussel populations. One site where eels were introduced (Pine Creek, Tioga County, Pennsylvania, USA) experienced an increase from 0 juveniles found during quantitative surveys prior to eel stocking to 151 (21% of individuals collected during quantitative surveys) E. complanata juveniles found four years after stocking. A second site (Buffalo Creek, Union County, Pennsylvania) experienced a more moderate increase from 2 to 7 juveniles found during 2010 and 2014 quantitative surveys, respectively. Continued examination of other potential interacting factors affecting recruitment, including water quality or habitat conditions, is necessary to target favorable sites for successful restoration.

Misidentification of sex for Lampsilis teres, Yellow Sandshell, and its implications for mussel conservation and wildlife management.

Correct identification of sex is an important component of wildlife management because changes in sex ratios can affect population viability. Identification of sex often relies on external morphology, which can be biased by intermediate or nondistinctive morphotypes and observer experience. For unionid mussels, research has demonstrated that species misidentification is common but less attention has been given to the reliability of sex identification. To evaluate whether this is an issue, we surveyed 117 researchers on their ability to correctly identify sex of Lampsilis teres (Yellow Sandshell), a wide ranging, sexually dimorphic species. Personal background information of each observer was analyzed to identify factors that may contribute to misidentification of sex. We found that median misidentification rates were ~20% across males and females and that observers falsely identified the number of female specimens more often (~23%) than males (~10%). Misidentification rates were partially explained by geographic region of prior mussel experience and where observers learned how to identify mussels, but there remained substantial variation among observers after controlling for these factors. We also used three morphometric methods (traditional, geometric, and Fourier) to investigate whether sex could be more correctly identified statistically and found that misidentification rates for the geometric and Fourier methods (which characterize shape) were less than 5% (on average 7% and 2% for females and males, respectively). Our results show that misidentification of sex is likely common for mussels if based solely on external morphology, which raises general questions, regardless of taxonomic group, about its reliability for conservation efforts.

Physical legacy of freshwater bivalves: Effects of habitat complexity on the taxonomical and functional diversity of invertebrates.

Bivalves may play a major role in structuring aquatic communities. This may be especially relevant in aquatic communities dominated by non-native invasive bivalves, which can contribute to the increase of habitat homogenization. In this study, we assess how habitat homogenization, through the reduction of empty bivalve shells identities, influences the macroinvertebrate assemblages. Towards this end, a manipulative experiment with the empty shells of two native (Potomida littoralis and Unio delphinus) and one non-native (Corbicula fluminea) species was performed. Seven treatments were prepared, three of them consisting of homogeneous substrates using shells of one species, and four of them consisting in heterogeneous substrates using more than one species. The associated fauna colonizing different treatments was analyzed through taxonomic and trait-based approaches. Our results showed that the substrate complexity influenced the density of macroinvertebrates, with the heterogeneous treatments significantly yielding more dense assemblages. Also, the trait patterns differed among the levels of habitat heterogeneity, influencing mainly organisms that feed on microphytes of both small and big sizes, that inhabit areas with slow to moderate water flow, and that have short and long live cycles. Further, the functional diversity was not influenced by the substrate heterogeneity. Therefore, the habitat homogenization, through the accumulation of non-native C. fluminea empty shells in the river bottom, did not affect the functional diversity of the macroinvertebrate assemblages.

A semi-arid river in distress: Contributing factors and recovery solutions for three imperiled freshwater mussels (Family Unionidae) endemic to the Rio Grande basin in North America.

Freshwater resources in arid and semi-arid regions are in extreme demand, which creates conflicts between needs of humans and aquatic ecosystems. The Rio Grande basin in the southwestern United States and northern Mexico exemplifies this issue, as much of its aquatic biodiversity is in peril as a result of human activities. Unionid mussels have been disproportionately impacted, though the specific factors responsible for their decline remain largely unknown. This is problematic because the Rio Grande basin harbors one federally endangered unionid mussel (Popenaias popeii, Texas Hornshell) plus two other mussel species (Potamilus metnecktayi, Salina Mucket; and Truncilla cognata, Mexican Fawnsfoot), which are also being considered for listing under the U.S. Endangered Species Act. To date, surveys for these species have not corrected for variability in detection so current range estimates may be inaccurate. Using single occupancy-modeling to estimate detection and occupancy at 115 sites along ~800 river kilometers of the Rio Grande in Texas, we found that detection probabilities were relatively high, indicating that our survey design was efficient. In contrast, the estimated occupancy was low, indicating that our focal species were likely rare within the Rio Grande drainage. In general, the predicted occupancy of our focal species was low throughout their respective ranges, indicating possible range declines. A comparison of currently occupied ranges to presumptive ranges underscores this point. The best-approximating models indicated that occupancy was influenced by habitat, water quantity and quality, and proximity to large-scale human activities, such as dams and major urban centers. We also discuss a series of conservation options that may not only improve the long-term prognosis of our focal species but also other aquatic taxa.

Vitro culture of axe-head glochidia in pink heelsplitter Potamilus alatus and mechanism of its high host specialists.

The basal media M199 or MEM was utilized in the classical method of vitro culture of glochidia where 1-5% CO2 was required to maintain stable physiological pH for completion of non-parasitic metamorphosis. The classical method encounters a great challenge to those glochidia which undergo development of visceral tissue but significantly increase in size during metamorphosis. The improved in vitro culture techniques and classical methods were firstly compared for non-parasitic metamorphosis and development of glochidia in pink heelsplitter. Based on the improved method, the optimal vitro culture media was further selected from 14 plasmas or sera, realizing the non-parasitic metamorphosis of axe-head glochidia for the first time. The results showed that addition of different plasma (serum) had significant effect on glochidial metamorphosis in pink heelsplitter. Only glochidia in the skewband grunt and red drum groups could complete metamorphosis, the metamorphosis rate in skewband grunt was 93.3±3.1% at 24±0.5°C, significantly higher than in marine and desalinated red drum. Heat-inactivated treatment on the plasma of yellow catfish and Barbus capito had significant effect on glochidia survival and shell growth. The metamorphosis rate also varied among different gravid period, and generally decreased with gravid time. Further comparison of free amino acid and fatty acid indicated that the taurine of high concentration was the only amino acid that might promote the rapid growth of glochidial shell, and the lack of adequate DPA and DHA might be an important reason leading to the abnormal foot and visceral development. Combined with our results of artificial selection of host fish, we tentatively established the mechanism of its host specialists in pink heelsplitter for the first time. This is the first report on non-parasite metamorphosis of axe-head glochidia based on our improved vitro culture method, which should provide important reference to fundamental theory research of glochidia metamorphosis and also benefit for better understand of mechanism of host specialists and generalists of Unionidae species.

Hematological and Biochemical Assessment of Two Species of Freshwater Mussels, Quadrula quadrula and Amblema plicata, Following Translocation.

Nondestructive, sublethal, and sensitive health monitoring tools are needed to assess the health of freshwater mussels (family Unionidae). Recent developments to standardize hemocyte characterization have assisted in the hematologic assessment of wild and captive freshwater mussels. In this study, preliminary baseline hematological reference ranges were established for wild mapleleaf mussels Quadrula quadrula (n = 14) and threeridge mussels Amblema plicata (n = 20) collected from the Muskingum River in Devola, Ohio. Mussels were collected from the wild, and hemolymph was sampled from each mussel in the field upon capture (baseline sample). They were then transported live to a propagation facility. Subsequent hemolymph samples were collected at 2 and 4 weeks and quarterly thereafter for 11 months following translocation. Hemocyte counts, hemocyte morphology, and hemolymph chemistry (Na+ , Cl- , Mg2+ , P3- , K+ , Ca2+ , glucose, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase]) were measured from each sample on each sampling occasion. Hemocyte counts were consistently greater in Q. quadrula than in A. plicata following transfer to captivity. Baseline hemocyte morphology and hemolymph chemistry varied between species. This study provides a foundation of reference ranges for hemocyte characterization for Q. quadrula, and A. plicata and a preliminary understanding of how hemocyte character might be expected to change when wild mussels are translocated into captivity, and thus be a useful technique for monitoring the health of freshwater mussels.

Suspended solid concentration reduces feeding in freshwater mussels.

We examined the effect of TSS concentration on the clearance rates (CR) of newly transformed juvenile and adult Lampsilis fasciola, L. siliquoidea, Ligumia nasuta, and Villosa iris, as increased total suspended solids (TSS) are thought to interfere with feeding processes. Mussel CR were measured in aerated (or swirled for juveniles) chambers at TSS concentrations up to 15mgL-1 for laboratory-transformed juveniles, and up to 100mgL-1 for adult mussels. The CR of one-week old animals increased with TSS concentration, likely due to ontological differences in feeding (pedal vs. suspension feeding) and gill development, but CR decreased monotonically with TSS concentration in older animals (two-, three- and four-week old juveniles). The CR of adult mussels were significantly lower at TSS concentrations ≥8mgL-1, which represented a threshold in CR. Although this threshold occurred at similar concentrations across the four species, the decline in CR was largest in L. fasciola (46% compared to no-TSS control), and smallest in V. iris (21%). Differences among species are likely related to differences in the TSS and substrate found in their source rivers given that greater decline occurred for species in rivers with relatively lower TSS. The decrease in CR as TSS increased is consistent across marine and freshwater bivalves, at both juvenile and adult stages. The decrease in feeding was five times greater in juvenile compared to adult bivalves, which indicates how the vulnerability to environmental stressors differ across life stages. These results demonstrate that TSS reduces suspension feeding rates in freshwater unionids, therefore TSS should be managed to ensure their survival.

Structural characteristics of gonadal development and hermaphroditic phenomenon in freshwater pearl mussel, Sinohyriopisis schlegelii (Hyriopisis schlegelii).

The period of gonads development was first studied from one to five years in the freshwater pearl mussel Hyriopisis schlegelii. It lasted for 36 months and was divided into three main stages: initiation of gonad formation, a stable growth phase, and a reproductive cell development phase. Each reproductive cycle consisted of five stages: proliferative stage (from late January to late February), growth stage (from late February to late March), maturation stage, spawning stage (from early April to late October) and recovery stage (from early November to late January). Interestingly, a hermaphroditic phenomenon was observed in this mussel for the first time, which appears during the development stage from 26 to 32 months. Male and female follicular tissues coexisted in hermaphrodite individuals with the male follicular tissue accounting for more than 90% of the whole gonad tissue. No hermaphroditic phenomenon was observed in matured gonad. We thus speculate that self-fertilization does not exist in H. schlegelii.