Strong, Recent Selective Sweeps Reshape Genetic Diversity in Freshwater Bivalve Megalonaias nervosa.

Freshwater Unionid bivalves have recently faced ecological upheaval through pollution, barriers to dispersal, harvesting, and changes in fish-host prevalence. Currently, over 70% of species in North America are threatened, endangered or extinct. To characterize the genetic response to recent selective pressures, we collected population genetic data for one successful bivalve species, Megalonaias nervosa. We identify megabase-sized regions that are nearly monomorphic across the population, signals of strong, recent selection reshaping diversity across 73 Mb total. These signatures of selection are greater than is commonly seen in population genetic models. We observe 102 duplicate genes with high dN/dS on terminal branches among regions with sweeps, suggesting that gene duplication is a causative mechanism of recent adaptation in M. nervosa. Genes in sweeps reflect functional classes important for Unionid survival, including anticoagulation genes important for fish host parasitization, detox genes, mitochondria management, and shell formation. We identify sweeps in regions with no known functional impacts, suggesting mechanisms of adaptation that deserve greater attention in future work on species survival. In contrast, polymorphic transposable elements (TEs) appear to be detrimental and underrepresented among regions with sweeps. TE site frequency spectra are skewed toward singleton variants, and TEs among regions with sweeps are present at low frequency. Our work suggests that duplicate genes are an essential source of genetic novelty that has helped this species succeed in environments where others have struggled. These results suggest that gene duplications deserve greater attention in non-model population genomics, especially in species that have recently faced sudden environmental challenges.

Toxicological study on ibuprofen and selenium in freshwater mussel Lamellidens marginalis and exploring the microbial cytochrome through modelling and quantum mechanics approaches for its toxicity degradation in contaminated environment.

Toxicological stress in aquatic organisms is caused by the discharge of hundreds of toxic pollutants and contaminants among which the current study concentrates on the toxic effect of non-steroidal anti-inflammatory drug ibuprofen (IBF) and the trace element selenium (Se). In this study, IBF and Se toxicity on freshwater mussel Lamellidens marginalis was studied for 14 days, and in silico predictions for their degradation were made using Molecular modelling and Quantum Mechanical approaches. The degrading propensity of cytochrome c oxidase proteins from Trametes verticillatus and Thauera selenatis (Turkey tail fungi and Gram-negative bacteria) is examined into atom level. The results of molecular modelling study indicate that ionic interactions occur in the T. selenatis-HEME bound complex by Se interacting directly with HEME, and in the T. versicolor-HEME bound complex by IBF bound to a nearby region of HEME. Experimental and theoretical findings suggest that, the toxicological effects of Se and IBF pollution can be reduced by bioremediation with special emphasis on T. versicolor, and T. selenatis, which can effectively interact with Se and IBF present in the environment and degrade them. Besides, this is the first time in freshwater mussel L. marginalis that ibuprofen and selenium toxicity have been studied utilizing both experimental and computational methodologies for their bioremediation study.

Integrated analysis of physiological, transcriptome, and metabolome analyses of the gills in Solenaia oleivora under ammonia exposure.

Ammonia is a common toxicant in aquatic systems and one of the key factors affecting aquaculture. However, data on mollusks' toxic response and coping mechanisms to ammonia nitrogen, especially freshwater mollusks, are still lacking. In this study, we evaluated the tolerance of a freshwater mollusk Solenaia oleivora to ammonia and investigated its coping mechanisms by combining physiological, metabolic, and transcriptomic analyses in the gills. The acute toxicity test revealed that the LC50-96 h (temperature-20 â„ƒ, pH-7.4) of ammonia in S. oleivora was 63.29 mg/L. The physiological and TUNEL results showed that although 10 mg/L ammonia exposure increased the activities of antioxidant, immune and ammonia detoxification-related enzymes, it still caused oxidative damage and cell apoptosis of gill tissues. A total of 97 differential metabolites (DMs) and 3431 differential expressed genes (DEGs) were identified after ammonia stress. Among them, most DMs and DEGs were involved in immune response, antioxidant, cell apoptosis, carbohydrate metabolism, amino acid metabolism, and lipid metabolism. The enhancement of glycolysis and lipid metabolisms may provide energy for immune response and ammonia detoxification. In addition, glutamine synthesis, alanine synthesis and urea cycle were involved in ammonia nitrogen detoxification in the gill tissue of S. oleivora. Our results indicate that ammonia leads to individual death in S. oleivora, as wells as oxidative damage, cell apoptosis, immune response, and metabolic changes of gill tissues. The findings will provide valuable information to assess the potential ecological risk of environmental ammonia to freshwater mollusks and theoretical guidance for the healthy aquaculture of S. oleivora.

The First High-Quality Genome Assembly of Freshwater Pearl Mussel Sinohyriopsis cumingii: New Insights into Pearl Biomineralization.

China leads the world in freshwater pearl production, an industry in which the triangle sail mussel (Sinohyriopsis cumingii) plays a pivotal role. In this paper, we report a high-quality chromosome-level genome assembly of S. cumingii with a size of 2.90 Gb-the largest yet reported among bivalves-and 89.92% anchorage onto 19 linkage groups. The assembled genome has 37,696 protein-coding genes and 50.86% repeat elements. A comparative genomic analysis revealed expansions of 752 gene families, mostly associated with biomineralization, and 237 genes under strong positive selection. Notably, the fibrillin gene family exhibited gene family expansion and positive selection simultaneously, and it also exhibited multiple high expressions after mantle implantation by transcriptome analysis. Furthermore, RNA silencing and an in vitro calcium carbonate crystallization assay highlighted the pivotal role played by one fibrillin gene in calcium carbonate deposition and aragonite transformation. This study provides a valuable genomic resource and offers new insights into the mechanism of pearl biomineralization.

Copper Stress Causes Shell Morphology Changes in Early Juvenile Anodonta woodiana Based on Geometric-Morphometric Analysis.

In this study, the morphological characteristics of early juvenile shells of Anodonta woodiana, which were exposed to different concentrations of aqueous copper, were analyzed using 10 landmarks to determine morphological changes in the shells. Morphological changes mainly occurred at the top of the shell and front and back ends of the central axis. Stepwise discriminant analysis proved shape differences among experimental and control groups. The results of this study demonstrate for the first time that environmentally relevant copper concentrations cause stress-related morphological changes in A. woodiana in the vulnerable early juvenile stage.

Investigating biodiversity patterns across scales in freshwater mussels.

Hypotheses relating genomic diversity to community attributes such as abundance and species diversity attract attention from a wide and varied audience because their applications are twofold. First, testing such hypotheses can further a theoretical-and hopefully generalizable-understanding of the forces that assemble communities and create observed patterns of biodiversity. Second, relationships that hold true could ease the burden of data collection for conservation or other urgent applications; for example, a strong correlation between species diversity and genetic diversity could make it possible to use one as a proxy for the other, and focus limited resources on measuring the easier of the two without sacrificing information gained. In a From the Cover article in this issue of Molecular Ecology, Bucholz et al. (2023) explore the relationships between within-species genomic diversity, community relative abundance and community species richness, testing three types of ecological hypotheses in the freshwater mussel communities of the southeastern United States. They find positive relationships between mussel density and species richness, and between genomic diversity within a species and density of that species, but no robust support for the expectation of correlated genomic and species diversity. Their analyses highlight the among-species variability in relationships among these different levels of organization and also the complex ways in which interactions with the broader ecosystem (i.e. unionid mussels require fish hosts for maturation) affect these quantitative relationships, nonetheless pushing forward into the important frontier of community-wide genomic assessment for theoretical and conservation applications.

Community-wide correlations between species richness, abundance and population genomic diversity in a freshwater biodiversity hotspot.

Understanding patterns of diversity across macro (e.g. species-level) and micro (e.g. molecular-level) scales can shed light on community function and stability by elucidating the abiotic and biotic drivers of diversity within ecological communities. We examined the relationships among taxonomic and genetic metrics of diversity in freshwater mussels (Bivalvia: Unionidae), an ecologically important and species-rich group in the southeastern United States. Using quantitative community surveys and reduced-representation genome sequencing across 22 sites in seven rivers and two river basins, we surveyed 68 mussel species and sequenced 23 of these species to characterize intrapopulation genetic variation. We tested for the presence of species diversity-abundance correlations (i.e. the more-individuals hypothesis, MIH), species-genetic diversity correlations (SGDCs) and abundance-genetic diversity correlations (AGDCs) across all sites to evaluate relationships between different metrics of diversity. Sites with greater cumulative multispecies density (a standardized metric of abundance) had a greater number of species, consistent with the MIH hypothesis. Intrapopulation genetic diversity was strongly associated with the density of most species, indicating the presence of AGDCs. However, there was no consistent evidence for SGDCs. Although sites with greater overall densities of mussels had greater species richness, sites with higher genetic diversity did not always exhibit positive correlations with species richness, suggesting that there are spatial and evolutionary scales at which the processes influencing community-level diversity and intraspecific diversity differ. Our work reveals the importance of local abundance as indicator (and possibly a driver) of intrapopulation genetic diversity.

'Phototaxis' in the absence of light? Locomotory patterns in unionid mussels.

Locomotion in benthic invertebrates can strongly affect habitat selection and ecosystem nutrient cycling. In the case of freshwater mussels, the drivers of locomotion are largely unresolved. Our aim was to assess the influence of light presence and intensity on the locomotory behaviour of freshwater mussels in controlled laboratory experiments. The species investigated in our study were Anodonta anatina and Unio pictorum, two widely distributed mussels in European lentic and lotic inland waters. At low algal concentrations, known to be associated with more frequent locomotory activities, we found that both species moved primarily in the absence of light (72.7% of all movements across experiments). However, the movements of both species were directed towards the light source, resembling a net-positive 'phototactic' response but in the absence of light. The distance to the light source, which was negatively correlated to light intensity, had a positive effect on the distance covered in locomotory activities by A. anatina but not by U. pictorum. Intraspecific variation in shell size had no impact on movement distance, indicating that the energetic costs of movement were not a limiting factor. We suggest that the observed movement towards brighter locations helps to enhance food quantity and quality, whilst movement in darkness mitigates predation risks.

Analysis of the immune function of Caspase-3 in Cristaria plicata.

Caspase-3 is generally considered to be the most important terminal shear enzyme in the process of apoptosis, as well as an important part of cytotoxic T lymphocytes (CTL) killing mechanism, which is confirmed to play an important role in vertebrate cell apoptosis and immune system, and is poorly reported in invertebrates. In this paper, we used bioinformatics to perform amino acid multiple sequence alignment and protein structural domain analysis, and constructed a phylogenetic tree to identify the full-length cDNA of the cloned caspase-3 of Cristaria plicata (Named CpCaspase-3). The expression of caspase-1, caspase-7, caspase-8, and caspase-9 was found to be down-regulated by double-stranded RNA interference of CpCaspase-3 in C. plicata. Some degree of disruption of the caspase signaling pathway occurs. The expression of CpCaspase-3 was affected after injection of Lipopolysaccharide (LPS), Peptidoglycan (PGN), polyinosinic-polycytidylic acid (poly(I:C)), and Aeromonas hydrophila. These results were suggested that CpCaspase-3 was involved in the immune response of C. plicata. The wound recovery process of C. plicata was simulated and CpCaspase-3 was found to promote wound recovery. An autophagy inhibition and autophagy activation model of mussels was constructed, where apoptosis and autophagy undergo crosstalk, and inhibition of autophagy induces the onset of apoptosis, and similarly autophagy activation inhibits the process of apoptosis instead. In addition, a recombinant CpCaspase-3-pEGFP-C1 plasmid was constructed for subcellular localization experiments and found that CpCaspase-3 was distributed in both the nucleus and the cytoplasm. This paper aims to unveil the immune mechanism of C. plicata and provide a theoretical basis for the healthy culture of shellfish.

Prx5 of Cristaria plicata has antioxidant function and is regulated by Nrf2/ARE signaling pathway.

Cristaria plicata is one of the more important freshwater pearl bivalves in China, which is susceptible to pathogen infection, and greatly impacts the ability of breeding pearls. Nrf2/ARE signaling pathway and its downstream target gene Prx5 have endogenous antioxidant functions to protect cells from oxidative damage. The full-length cDNA of Prx5 was cloned from C. Plicata, which was 1420 bp, encoding a total of 189 amino acids and had two conserved cysteine residues (Cys78 and Cys179). The amino acid sequence of CpPrx5 was highly similar to Prx5 of other species. Real-time fluorescence quantitative PCR showed that CpPrx5 was distributed in various tissues of mussels, and the highest expression was in hepatopancreas. The expression of CpPrx5 up-regulated in hepatopancreas and gills after LPS, PGN and Poly:I:C stimulation. The recombinant plasmid DE3-PGEX-4T-1-CpPrx5 was expressed in Escherichia coli BL21 and showed antioxidant activity. With the increase of CpPrx5 protein concentration, the superhelical form of DNA was protected. The expression of CpPrx5 was up-regulated after interference CpKeap1 and down-regulated after interference CpNrf2. Gel block assay showed that CpNrf2 and CpMafK proteins blocked CpPrx5 promoter. Subcellular localization showed that CpPrx5 was located in 293T nucleus and cytoplasm and CpMafK was located in 293T nucleus. GST-Pull down verified that CpMafK and CpPrx5 could bind in vitro. These results indicated that Prx5 had antioxidant function and could protects DNA from oxidative damage, and participated in transcriptional regulation by combining with the transcription factor MafK. In addition, MafK could combine with Nrf2 to regulate the downstream target gene Prx5.

Histopathologic survey of free-living populations of 2 species of freshwater mussels in Indiana.

Freshwater mussels are one of the most endangered groups of animals in Indiana, with nearly half of the native species either extirpated or listed as "state endangered" or of "special concern." Nationally, numerous freshwater mussel species are considered threatened. Freshwater mussel diseases are not well understood and few published accounts of freshwater mussel diseases with detailed histological descriptions exist. Mass mortality events within mussel populations are increasingly recognized, often with undetermined etiology. Our objective was to determine baseline histopathology in free-living populations of freshwater mussels. One-hundred twenty individual mussels representing 2 species-plain pocketbook (Lampsilis cardium) and fatmucket (Lampsilis siliquoidea)-were collected from 3 different locations within the Wildcat Creek watershed in central Indiana during June and July 2019. A cross-section through the visceral mass was obtained and immersed in 10% neutral-buffered formalin, with routine processing and hematoxylin and eosin staining. Branchial acariasis occurred in 43/60 fatmuckets and 22/60 plain pocketbooks. Infection with a bucephalid trematode was recognized in 18/60 fatmuckets, while infection of the gonadal duct with an unidentified trematode species was identified in 4/60 fatmuckets and 18/59 plain pocketbooks. Additional changes associated with unidentified trematodes, bacteria, fungi or oomycetes, and ciliates were observed. Other miscellaneous changes included mineralization, neuronal lipofuscinosis, and gonadal atrophy/atresia. A range of histological changes were observed. These changes likely represented background lesions: incidental findings, spontaneous infectious or endosymbiotic conditions, or normal physiological changes that routinely occur in free-living wild populations. Awareness of baseline lesions should inform future diagnostic investigations of mussel mortality events.

Pathology and infectious agents of unionid mussels: A primer for pathologists in disease surveillance and investigation of mortality events.

Freshwater mussels are one of the most imperiled groups of organisms in the world, and more than 30 species have gone extinct in the last century. While habitat alteration and destruction have contributed to the declines, the role of disease in mortality events is unclear. In an effort to involve veterinary pathologists in disease surveillance and the investigation of freshwater mussel mortality events, we provide information on the conservation status of unionids, sample collection and processing techniques, and unique and confounding anatomical and physiological differences. We review the published accounts of pathology and infectious agents described in freshwater mussels including neoplasms, viruses, bacteria, fungi, fungal-like agents, ciliated protists, Aspidogastrea, Digenea, Nematoda, Acari, Diptera, and Odonata. Of the identified infectious agents, a single viral disease, Hyriopsis cumingii plague disease, that occurs only in cultured mussels is known to cause high mortality. Parasites including ciliates, trematodes, nematodes, mites, and insects may decrease host fitness, but are not known to cause mortality. Many of the published reports identify infectious agents at the light or ultrastructural microscopy level with no lesion or molecular characterization. Although metagenomic analyses provide sequence information for infectious agents, studies often fail to link the agents to tissue changes at the light or ultrastructural level or confirm their role in disease. Pathologists can bridge this gap between identification of infectious agents and confirmation of disease, participate in disease surveillance to ensure successful propagation programs necessary to restore decimated populations, and investigate mussel mortality events to document pathology and identify causality.

Alterations to unionid transformation during agricultural and urban contaminants of emerging concern exposures.

Highly imperiled unionids have a complex life cycle including the metamorphosis of an obligate parasite life stage, larval glochidia, to the juvenile stage. Despite the known vulnerabilities of both glochidia and juveniles to pollutants, little is known on how metamorphosis success may be affected by chemical stress. Disruption of the transformation process in which glochidia encyst on the gills of a host fish, could lead to lowered recruitment and population declines. Transformation rates of Lampsilis cardium on host fish Micropterus salmoides were empirically derived from experimental exposures to low, medium, or high concentrations of an agricultural or urban mixture of contaminants of emerging concern (CECs) over two exposure durations. Transformation was characterized by: (1) a zero-inflated Poisson general linear mixed effects model to compare difference in transformation between exposure durations and (2) time response curves to describe the transformation curve using long-term exposure data. Lampsilis cardium transformation was similar between exposure durations. When compared to controls, CEC stress significantly reduced juvenile production (p « 0.05) except for the agricultural medium treatment and tended to increased encapsulation duration which while statistically insignificant (p = 0.16) may have ecological relevancy. Combining the empirically derived reduction of transformation rates with parameters values from the literature, a Lefkovich stage-based population model predicted strong declines in population size of L. cardium for all treatments if these results hold in nature. Management focus on urban CECs may lead to best conservation efforts though agricultural CECs may also have a concentration dependent impact on transformation and therefore overall recruitment and conservation success.

Lethal and Sub-lethal Implications of Sodium Chloride Exposure for Adult Unionid Mussel Species: Eurynia dilatata and Lasmigona costata.

The elevated use of salt as a de-icing agent on roads in Canada is causing an increase in the chloride concentration of freshwater ecosystems. Freshwater Unionid mussels are a group of organisms that are sensitive to increases in chloride levels. Unionids have greater diversity in North America than anywhere else on Earth, but they are also one of the most imperiled groups of organisms. This underscores the importance of understanding the effect that increasing salt exposure has on these threatened species. There are more data on the acute toxicity of chloride to Unionids than on chronic toxicity. This study investigated the effect of chronic sodium chloride exposure on the survival and filtering activity of two Unionid species (Eurynia dilatata, and Lasmigona costata) and assessed the effect on the metabolome in L. costata hemolymph. The concentration causing mortality after 28 days of exposure was similar for E. dilatata (1893 mg Cl-/L) and L. costata (1903 mg Cl-/L). Significant changes in the metabolome of the L. costata hemolymph were observed for mussels exposed to non-lethal concentrations. For example, several phosphatidylethanolamines, several hydroxyeicosatetraenoic acids, pyropheophorbide-a, and alpha-linolenic acid were significantly upregulated in the hemolymph of mussels exposed to 1000 mg Cl-/L for 28 days. While no mortality occurred in the treatment, elevated metabolites in the hemolymph are an indicator of stress.

Resolving species-level diversity of Beringiana and Sinanodonta mussels (Bivalvia: Unionidae) in the Japanese archipelago using genome-wide data.

Accurate species identification is of primary importance in ecology and evolutionary biology. For a long time, the unionid mussels Beringiana and Sinanodonta have puzzled researchers trying to unravel their diversity because of their poorly discernible morphologies. A recent study conducted species delineation of unionid mussels based on mitochondrial DNA variation, opening up a new avenue to grasp species diversity of the mussels. However, mtDNA-based classification may not align with species boundaries because mtDNA is prone to introgression and incomplete lineage sorting that cause discordance between species affiliation and gene phylogeny. In this study, we evaluated the validity of the mtDNA-based classification of unionid mussels Beringiana and Sinanodonta in Japan using mitochondrial sequence data, double digest restriction site-associated DNA library (ddRAD) sequencing, and morphological data. We found significant inconsistencies in the mitochondrial and nuclear DNA phylogenies, casting doubt on the reliability of the mtDNA-based classification in this group. In addition, nuclear DNA phylogeny revealed that there are at least two unionid lineages hidden in the mtDNA phylogeny. Although molecular dating technique indicates that Beringiana and Sinanodonta diverged >35 million years ago, their shell morphologies are often indistinguishable. Specifically, morphological analyses exhibited the parallel appearance of nearly identical ball-like shell forms in the two genera in Lake Biwa, which further complicates species identification and the morphological evolution of unionid mussels. Our study adds to a growing body of literature that accurate species identification of unionid mussels is difficult when using morphological characters alone. Although mtDNA-based classification is a simple and convenient way to classify unionid mussels, considerable caution is warranted for its application in ecological and evolutionary studies.

A proteomic approach reveals biomineralization and immune response for mantle to pearl sac in the freshwater pearl mussel (Hyriopsis cumingii).

In the process of production of freshwater pearl, implanted mantle pieces undergo a series of complex physiological and biochemical processes to form pearl sac, which produce pearl. This is a very important site of occurrence due to immune-induced biomineralization, while its molecular regulatory mechanism is still unclear. Here, we use proteomics to identify differentially expressed proteins (DEPs) of the mantle and pearl sac and examine the biomineralization and immune response of the pearl sac formation process in Hyriopsis cumingii. Using iTRAQ technology and bioinformatics analysis, we obtained DEP profiles between the mantle and pearl sac. A total of 1871 proteins were identified. Of these, 74 DEPs were found between the pearl sac and outer mantle, 112 DEPs between the pearl sac and inner mantle, and 124 DEPs between the outer and inner mantles. Bioinformatics analysis revealed that the screened biomineralization-related DEPs were mainly enriched in signaling pathways associated with calcium signaling, regulation of the actin cytoskeleton and protein processing in the endoplasmic reticulum, while the immune-related DEPs were mainly enriched in the Notch, Hippo, nuclear factor kappa-B (NF-κB), and transforming growth factor-ß (TGF-ß) signaling pathways. In addition, the expression of six biomineralization-related and four immune-related proteins were verified at the transcriptional level using quantitative real-time PCR. Our findings contribute to furthering the understanding of the mechanisms of pearl formation and immune response, and have long-term implications for future studies on the production of high-quality freshwater pearls and development of the freshwater pearl industry.

ß-arrestin interacts with TRAF6 to negatively regulate the NF-κB pathway in triangle sail mussel Hyriopsis cumingii.

As members of arrestins family, ß-arrestins are widely expressed in monocytes, macrophages, neutrophils and other immune cells. They can regulate the immune response of bodies through various ways. In the present study, a ß-arrestin homolog named Hcß-arrestin was cloned and identified from Hyriopsis cumingii. Predicted Hcß-arrestin protein contained a conserved arrestin domain, which could be further divided into arrestin-N (39-192aa) and arrestin-C (211-365aa). Amino acid sequence alignment showed that it had the highest identity with Mytilus galloprovincialis and Mytilus edulis counterpart, which was 89.02% and 87.68%, respectively. Furthermore, real-time quantitative PCR analysis showed that the Hcß-arrestin gene was widely expressed in the detected tissues and with the highest expression in hepatopancreas. The transcription of Hcß-arrestin in hepatopancreas and gill of mussels was significantly up-regulated after stimulation with peptidoglycan, lipopolysaccharide (LPS) and polyinosinic polycytidylic acid. Knockdown of Hcß-arrestin gene significantly increased the expression of some antibacterial effector genes, such as lysozyme, LPS-binding protein/bactericidal permeability increasing protein and theromacin in hepatopancreas and gills of LPS stimulated mussels, but only had little effect on TLR pathway genes. In addition, GST pull-down assay confirmed that Hcß-arrestin can bind to HcTRAF6 protein in vitro. Dual luciferase reporter assay showed that the co-expression of HcTRAF6 and Hcß-arrestin inhibited the activation of NF-κB reporter by HcTRAF6. These findings indicated that Hcß-arrestins could interact with HcTRAF6 to negatively regulate the NF-κB pathway in H. cumingii.

Complete mitochondrial genome of freshwater pearl mussel Lamellidens marginalis (Lamarck, 1819) and its phylogenetic relation within unionidae family.

BACKGROUND: Freshwater mussels play a key role in ecology and are often considered as ecological indicators. Conversely, these molluscs are one of the most threatened groups due to several anthropogenic factors. Knowledge of phylogenetic diversity would assist in formulating effective management and conservation measures. Lamellidens marginalis is one of the most widely used freshwater mussel for pearl production in India. The genomic resources for investigating its evolutionary relationship within the Unionidae family are lacking. METHODS AND RESULTS: In this study, the f-type mitochondrial genome of L. marginalis was sequenced using the Illumina sequencing platform. The length of the mitochondrial genome was 15,732 bp consisting of 23 tRNAs, 2 rRNAs and 13 protein coding genes. The arrangement of genes was UF1 type and gene overlap was observed between trnG and nad1. Comparative analysis with other Unionidae species showed a high divergence rate in nad6 followed by nad2 atp8 and nad5. The phylogenetic tree supported monophyly of the Unioninae subfamily and L. marginalis (Parreysiinae) formed a sister branch to this subfamily. The divergence time of the Parreysiinae from its most recent common ancestor (MRCA) was placed in the Mesozoic era. CONCLUSION: This information will be useful for the understanding the evolutionary pattern of the species of Parreysiinae subfamily.

Gene identification and functional analysis of a D-type cyclin (CCND2) in freshwater pearl mussel (Hyriopsis cumingii).

BACKGROUND: Cyclin D (CCND) plays an important role in the cell cycle and is a rate-limiting factor that facilitates the G1/S transition. METHODS: In this study, the full-length cDNA of Hc-CCND2 was isolated from freshwater pearl mussel (Hyriopsis cumingii; Hc) and amplified using the 3´/5´ RACE system. The Hc-CCND2 expression profiles were analysed by quantitative real-time PCR. Functional analysis of the Hc-CCND2 genes was examined by both RNA interference (RNAi) and overexpression in H. cumingii. RESULTS: Hc-CCND2 protein sequences were 295 amino acids long, possessed D-type cyclin signature motifs and contained conserved cyclin box domains. Hc-CCND2 was expressed in all examined tissues (adductor, foot, visceral mass, gill, outer mantle, inner mantle and gonad), with the highest expression levels found in the gill (P < 0.05). During the different developmental periods of the embryo, the relative expression of Hc-CCND2 increased with embryonic development, peaking at the blastula stage and decreasing significantly in the gastrula stage. After knockdown of Hc-CCND2 by RNAi, a significant decrease in CDK6 expression levels was found, while the percentage of cells in the G0/G1 phase significantly increased. Overexpression of Hc-CCND2 in mantle cells led to increased proliferation of cultured cells (P < 0.05). CONCLUSIONS: Our results demonstrated that Hc-CCND2 may promote cell cycle progression in H. cumingii, and that overexpression of Hc-CCND2 promotes mantle cell proliferation. These findings may provide a novel approach for improving the slow proliferation rate of shellfish cells in in vitro cultures.

Effects of municipal wastewater effluents on the digestive gland microbiome of wild freshwater mussels (Lasmigona costata).

Gut microbial communities are vital for maintaining host health, and are sensitive to diet, environment, and chemical exposures. Wastewater treatment plants (WWTPs) release effluents containing antimicrobials, pharmaceuticals, and other contaminants that may negatively affect the gut microbiome of downstream organisms. This study investigated changes in the diversity and composition of the digestive gland microbiome of flutedshell mussels (Lasmigona costata) from upstream and downstream of two large (service >100,000) WWTPs. Mussel digestive gland microbiome was analyzed following the extraction, PCR amplification, and sequencing of bacterial DNA using the V3-V4 hypervariable regions of the 16 S rRNA gene. Bacterial alpha diversity decreased at sites downstream of the second WWTP and these sites were dissimilar in beta diversity from sites upstream and downstream of the first upstream WWTP. The microbiomes of mussels collected downstream of the first WWTP had increased relative abundances of Actinobacteria, Bacteroidetes, and Firmicutes, with a decrease in Cyanobacteria, compared to upstream mussels. Meanwhile, those collected downstream of the second WWTP increased in Proteobacteria and decreased in Actinobacteria, Bacteroidetes, and Tenericutes. Increased Proteobacteria has been linked to adverse effects in mammals, but their functions in mussels is currently unknown. Finally, effluent-derived bacteria were found in the microbiome of mussels downstream of both WWTPs but not in those from upstream. Overall, results show that the digestive gland microbiome of mussels collected upstream and downstream of WWTPs differed, which has implications for altered host health and the transport of WWTP-derived bacteria through aquatic ecosystems.