Search

The TGF-β signaling pathway plays an important role in wound healing and immune response. In this study, a TGF-β type I receptor (TGF-βRI) homolog was cloned and characterized from freshwater mussel Hyriopsis cumingii . The full-length cDNA of the TGF-β RI gene was 2017 bp, with a 1554 bp open reading frame (ORF), and encoded 517 amino acids. The predictive analysis further identified distinct regions within the TGF-βRI protein: a signal peptide, a membrane outer region, a transmembrane region, and an intracellular region. Real-time quantitative PCR results showed that the TGF-β RI gene was expressed in all tissues of healthy mussels. The transcripts of TGF-β RI in hemocytes and hepatopancreas were significantly up-regulated at different periods after stimulation with Aeromonas hydrophila and peptidoglycan (PGN) ( P < 0.05). The mRNA expression of TGF-β RI progressively increased from day 1 to day 10 after trauma ( P < 0.05), and it returned to the initial level by day 15. The expression levels of TGF-β , Smad5 , MMP1/19 , and TIMP1/2 , but not Smad3/4 , were significantly up-regulated at different time points after trauma. However, the expression levels of TGF-β , MMP1/19 , and TIMP2 were decreased after treatment with the inhibitor SB431542. Furthermore, the recombinant TGF-βRI proteins were expressed in vitro and existed in the form of inclusion bodies. Western blotting results showed that TGF-βRI proteins were expressed constitutively in various tissues of mussels, and their expression was up-regulated after trauma, which was consistent with the mRNA expression trend. These results indicate that TGF-β RI is involved in the process of wound repair and immune response.

Accurate identification and precise classification of freshwater mussel species that are among the most threatened freshwater taxa in the world, play a crucial role in informing conservation and management efforts for these organisms. However, due to the variability in shell morphology, relying solely on shell characteristics for species taxonomy poses significant challenges, thereby impeding effective conservation planning and management. The freshwater mussel genus Ptychorhynchus Simpson, 1900 is one such group in need of study. We integrate molecular phylogeny, shell morphology and soft-body anatomy to examine the classification of Ptychorhynchus denserugata (Haas, 1910) and Ptychorhynchus resupinatus (von Martens, 1902). The COI barcoding data support the clustering of P. denserugata and Nodularia douglasiae within a single clade, and P. denserugata shares the diagnostic feature of the genus Nodularia , i.e. knobs or bumps on the inner mantle surface in the excurrent aperture. Therefore, by integrating molecular data and anatomical characteristics, we confirm that the nominal species P. denserugata syn. nov. is a new synonym for N. douglasiae . The multi-locus (COI + ND1 + 16S rRNA + 18S rRNA + 28S rRNA ) phylogeny and mitochondrial phylogenomics support the transfer of P. resupinatus from Ptychorhynchus to the newly elevated genus Cosmopseudodon stat. rev., as Cosmopseudodon resupinatus stat. rev. that is still considered the designated type species. We also describe a new species based on integrative taxonomy, i.e. Cosmopseudodon wenshanensis sp. nov. The comprehensive understanding of the taxonomy and diversity of the revised Cosmopseudodon species, and shell heteromorphism of N. douglasiae (=P. denserugata syn. nov.), will serve as a crucial foundation for further scientific assessment and conservation strategies pertaining to these taxa. ZooBank: urn:lsid:zoobank.org:pub:E48968B1-DF0F-42AD-8F31-B8C95F23CE57.

Sinosolenaia oleivora (Bivalve, Unionida, Unionidae), is a near-endangered edible mussel. In 2022, it was selected by the Ministry of Agriculture and Rural Affairs as a top-ten aquatic germplasm resource, with potential for industrial development. Using Illumina, PacBio, and Hi-C technology, a high-quality chromosome-level genome of S. oleivora was assembled. The assembled S. oleivora genome spanned 2052.29 Mb with a contig N50 size of 20.36 Mb and a scaffold N50 size of 103.57 Mb. The 302 contigs, accounting for 98.41% of the total assembled genome, were anchored into 19 chromosomes using Hi-C scaffolding. A total of 1171.78 Mb repeat sequences were annotated and 22,971 protein-coding genes were predicted. Compared with the nearest ancestor, a total of 603 expanded and 1767 contracted gene families were found. This study provides important genomic resources for conservation, evolutionary research, and genetic improvements of many economic traits like growth performance., (© 2024. The Author(s).)

Unionoid freshwater mussels (Bivalvia: Unionidae) are free-living apart from a brief, obligately parasitic, larval stage that infects fish hosts, and gravid female mussels have evolved a spectrum of strategies to infect fish hosts with their larvae. In many North American species, this involves displaying a mantle lure: a pigmented fleshy extension that acts as an aggressive mimic of a host fish prey, thereby eliciting a feeding response that results in host infection. The mantle lure of Lampsilis fasciola is of particular interest because it is apparently polymorphic, with two distinct primary lure phenotypes. One, described as "darter-like", has "eyespots", a mottled body coloration, prominent marginal extensions, and a distinct "tail". The other, described as "worm-like", lacks those features and has an orange and black coloration. We investigated this phenomenon using genomics, captive rearing, biogeographic, and behavioral analyses. Within-brood lure variation and within-population phylogenomic (ddRAD-seq) analyses of individuals bearing different lures confirmed that this phenomenon is a true polymorphism. The relative abundance of the two morphs appears stable over ecological timeframes: the ratio of the two lure phenotypes in a River Raisin (MI) population in 2017 was consistent with that of museum samples collected at the same site six decades earlier. Within the River Raisin, four main "darter-like" lure motifs visually approximated four co-occurring darter species ( Etheostoma blennioides, E. exile, E. microperca , and Percina maculata ), and the "worm-like" lure resembled a widespread common leech, Macrobdella decora . Darters and leeches are typical prey of Micropterus dolomieui (smallmouth bass), the primary fish host of L. fasciola . In situ field recordings of the L. fasciola "darter" and "leech" lure display behaviors, and the lure display of co-occurring congener L. cardium , were captured. Despite having putative models in distinct phyla, both L. fasciola lure morphs have largely similar display behaviors that differ significantly from that of sympatric L. cardium individuals. Some minor differences in the behavior between the two L. fasciola morphs were observed, but we found no clear evidence for a behavioral component of the polymorphism given the criteria measured. Discovery of discrete within-brood inheritance of the lure polymorphism implies potential control by a single genetic locus and identifies L. fasciola as a promising study system to identify regulatory genes controlling a key adaptive trait of freshwater mussels., Competing Interests: The authors declare that they have no competing interests., (© 2024 Hewitt et al.)

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.

Serine protease inhibitors (SPIs) are abundantly reported for its inhibition against specific proteases involved in the immune responses, but SPI data related to calcareous shells are scarce. Previously, our research group has reported the proteome analysis of non-nucleated pearl powder, and a candidate matrix protein containing two Kunitz domains in the acid soluble fraction caught our attention. In the present study, the full-length cDNA sequence of HcKuSPI was obtained from Hyriopsis cumingii. HcKuSPI was specifically expressed in the mantle, with hybridization signals mainly concentrated to dorsal epithelial cells at the mantle edge and weak signals at the mantle pallium, suggesting HcKuSPI was involved in shell formation. HcKuSPI expression in the mantle was upregulated after Aeromonas hydrophila and Staphylococcus aureus challenge to extrapallial fluids (EPFs). A glutathione S transferase (GST)-HcKuSPI recombinant protein showed strong inhibitory activity against the proteases, trypsin and chymotrypsin. Moreover, HcKuSPI expression in an experimental group was significantly higher when compared with a control group during pellicle growth and crystal deposition in shell regeneration processes, while the organic shell framework of newborn prisms and nacre tablets was completely destroyed after HcKuSPI RNA interference (RNAi). Therefore, HcKuSPI secreted by the mantle may effectively neutralize excess proteases and bacterial proteases in the EPF during bacterial infection and could prevent matrix protein extracellular degradation by suppressing protease proteolytic activity, thereby ensuring a smooth shell biomineralization. In addition, GST-HcKuSPI was also crucial for crystal morphology regulation. These results have important implications for our understanding of the potential roles of SPIs during shell biomineralization., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Shells and pearls are the products of biomineralization of shellfish after ingesting external mineral ions. Bone morphogenetic proteins (BMPs) play a role in a variety of biological function, and the genes that encode them, are considered important shell-forming genes in mollusks and are associated with shell and pearl formation, embryonic development, and other functions, but bone morphogenetic protein 10 (BMP10) is poorly understood in Hyriopsis cumingii. In this study, we cloned Hc-BMP10 and obtained a 2477 bp full-length sequence encoding 460 amino acids with a conserved TGF-β structural domain. During the embryonic developmental stages, the cleavage stage had the highest expression of Hc-BMP10, followed by juvenile clams; the expression in the mantle gradually decreased with increasing mussel age. A strong signal was detected on epidermal cells on the mantle edge by in situ hybridization. In both the shell notching and inserting operations of the pearl fragment assay, we found that the expression of Hc-BMP10 increased after the above treatments. RNA interference assays showed that the silencing of Hc-BMP10 resulted in a change in the morphology of the prismatic layer and nacreous layer, with the prismatic layer less closely aligned and the disordered aragonite flakes in the nacreous layer. These findings indicate that Hc-BMP10 is involved in the growth and development of H. cumingii, as well as the formation of shells and pearls. Therefore, this study provides some reference for selecting superior species for growth and pearl breeding of H. cumingii at a molecular level and further investigation of the molecular mechanism for biomineralization of Hc-BMP10., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The United States of America has a diverse collection of freshwater mussels comprising 301 species distributed among 59 genera and two families (Margaritiferidae and Unionidae), each having a unique suite of traits. Mussels are among the most imperilled animals and are critical components of their ecosystems, and successful management, conservation and research requires a cohesive and widely accessible data source. Although trait-based analysis for mussels has increased, only a small proportion of traits reflecting mussel diversity in this region has been collated. Decentralized and non-standardized trait information impedes large-scale analysis. Assembling trait data in a synthetic dataset enables comparison across species and lineages and identification of data gaps. We collated data from the primary literature, books, state and federal reports, theses and dissertations, and museum collections into a centralized dataset covering information on taxonomy, morphology, reproductive ecology and life history, fish hosts, habitats, thermal tolerance, geographic distribution, available genetic information, and conservation status. By collating these traits, we aid researchers in assessing variation in mussel traits and modelling ecosystem change., (© 2023. The Author(s).)

In response to stress, cells can utilize several processes, such as the activation of the Nrf2/Keap1 pathway as a critical regulator of oxidative stress to protect against oxidative damage. C-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, is involved in regulating the NF-E2-related nuclear factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway. NAD(P)H quinone redox enzyme-1 (NQO1), a downstream target gene of the Nrf2 pathway, plays a vital role in removing peroxide and providing resistance to oxidative injury. We found that microcystins (MCs) stimulated CpNrf2 to express and increase anti-oxidative enzyme activities in a previous experiment. In our current study, the full-length cDNAs of JNK and NQO1 from Cristaria plicata (designated CpJNK and CpNQO1) were cloned. The relative levels of CpJNK and CpNQO1 were high in hepatopancreas. Upon MCs induction, the relative level of CpNQO1 was increased, whereas that of CpJNK was decreased significantly. In contrast, CpNrf2 knockdown upregulated the expression of CpJNK mRNA and phosphorylation of CpJNK protein (Cpp-JNK), but inhibited CpNQO1 expression. Additionally, we found that JNK inhibitor SP600125 stimulated expression of CpNQO1 and CpNrf2 upon exposure to MCs, and we further confirmed that CpNrf2 protein combined with the ARE element in CpNQO1 gene promoter in vitro, and increased CpNQO1-ARE-luciferase activity in a CpNrf2-dependent manner. These findings indicated C. plicata effectively alleviated MC-induced oxidative injury through JNK participated in regulating the Nrf2/NQO1-ARE pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Unionid mussels are among the most imperiled group of organisms in North America, and Pustulosa pustulosa is a freshwater species with a relatively wide latitudinal distribution that extends from southern Ontario, Canada, to Texas, USA. Considerable morphological and geographic variation in the genus Pustulosa (formerly Cyclonaias ) has led to uncertainty over species boundaries, and recent studies have suggested revisions to species-level classifications by synonymizing C. aurea , C. houstonensis , C. mortoni , and C. refulgens with C. pustulosa (currently P. pustulosa ). Owing to its wide range and shallow phylogenetic differentiation, we analyzed individuals of P. pustulosa using mitochondrial DNA sequence data under a population genetics framework. We included 496 individuals, which were comprised of 166 samples collected during this study and 330 additional sequences retrieved from GenBank. Pairwise Φ ST measures based on ND1 data suggested there may be up to five major geographic groups present within P. pustulosa . Genetic differentiation between regions within Texas was higher compared to populations from the Mississippi and Great Lakes populations, which may reflect differences in historical connectivity. Mitochondrial sequence data also revealed varying demographic histories for each major group suggesting each geographic region has also experienced differential population dynamics in the past. Future surveys should consider exploring variation within species after phylogeographic delimitation has been performed. In this study, we begin to address this need for freshwater mussels via the P. pustulosa system., Competing Interests: The authors declare that they have no competing interests., (© 2023 Rodriguez et al.)

In bivalves, the heterogeneity of mitochondrial DNA and its unique mode of transmission have been the focus of attention, which is called doubly uniparental inheritance (DUI). Prohibitin-2 (phb2) is a mitochondrial inner membrane protein that is a key mitophagy receptor for parental mitochondrial removal. Hyriopsis cumingii is a freshwater bivalve in China, the full-length cDNA of H. cumingii phb2 (named Hcphb2) is 2917 bp and encodes a total of 300 amino acids, a highly conserved sequence. Hcphb2 was highly expressed in the ovary. In the gonadal tissues of 5- to 8-month-old female mussels, the expression level of Hcphb2 continued to significantly increase. After Hcphb2 siRNA interference in 6-month-old female mussels, the expression of M-COII, a marker gene on M-type mitochondria, showed a considerable increase (p < 0.05). In contrast, the expression of autophagosome formation and maturation-related genes, atg4b, atg5, atg12, and atg16l, in the ATG family genes was significantly decreased (p < 0.01). Subcellular localization showed that Hcphb2 appeared in spermatogonia, spermatocyte, spermatid, and sperm, and its location changes synchronize with the behavior of M-type mitochondria location changes in DUI species. And it was found that miR-184 negatively regulated Hcphb2. The above results suggest that the mitochondrial autophagy receptor gene Hcphb2 may be associated with the degradation of M-type mitochondria in the freshwater mussel. This process requires multiple genes to participate, of which Hcphb2 and autophagy genes are only some of those that may play a role., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Thioredoxin plays an important role in inhibiting apoptosis and protecting cells from oxidative stress. This study was aimed to clarify how the expression of Trx from Cristaria plicata is regulated by Nrf2/ARE pathway. The expression of CpTrx mRNA was significantly up-regulated in gill and kidney tissues under microcystin stress. The Nrf2 gene of Cristaria plicata was identified to possess an auto active domain bit. While CpNrf2 was knocked down by specific small RNA, CpTrx mRNA expression was significantly down-regulated. The promoter of CpTrx gene had high transcriptional activity, and this basic transcriptional activity persisted after ARE element mutation. The region of promoter -206 to +217 bp was a core promoter region and had forward regulatory elements. Gel shift Assay exhibited that the CpTrx promoter could bind to the purified proteins CpNrf2 and CpMafK in vitro. The binding phenomenon disappeared after the ARE element mutation in promoter region. Subcellular localization experiments displayed that fluorescence overlap between CpNrf2 and Trx promoter increased under microcystin toxin stress. These results suggested that Trx expression was regulated by Nrf2/ARE pathway under oxidative stress., Competing Interests: Declaration of competing interest All authors have no potential financial conflicts of interest related to this manuscript., (Copyright © 2023 Elsevier B.V. All rights reserved.)

C-Mos , a proto-oncogene, regulates oocyte maturation by activating the classical MAPK pathway in cells. To examine the function of C-Mos in Hyriopsis cumingii , C-Mos was identified in this study. The full-length cDNA of C-Mos was 2213 bp, including 144 bp in the 5' UTR, 923 bp in 3' the UTR, and 1146 bp in the open reading frame (ORF) region. During early gonad development, the expression of C-Mos from 4 to 6 months of age in H. cumingii was significantly higher than that in other months, with the highest expression in 6-month-old H. cumingii , suggesting that C-Mos may be involved in early gonadal development in H. cumingii . Clear hybridization signals were found by in situ hybridization in the oocytes, oocyte nucleus and oogonium, and a small number of hybridization signals were found in the follicular wall of the male gonads. In addition, the C-Mos RNA interference (RNAi) assay results showed that the knockdown of C-Mos caused a down-regulation of ERK and P90rsk . In summary, these results indicate that C-Mos has a crucial part to play in gonadal development in H. cumingii .

Limiting harm to organisms caused by genetic sampling is an important consideration for rare species, and a number of non-destructive sampling techniques have been developed to address this issue in freshwater mussels. Two methods, visceral swabbing and tissue biopsies, have proven to be effective for DNA sampling, though it is unclear as to which method is preferable for genotyping-by-sequencing (GBS). Tissue biopsies may cause undue stress and damage to organisms, while visceral swabbing potentially reduces the chance of such harm. Our study compared the efficacy of these two DNA sampling methods for generating GBS data for the unionid freshwater mussel, the Texas pigtoe ( Fusconaia askewi ). Our results find both methods generate quality sequence data, though some considerations are in order. Tissue biopsies produced significantly higher DNA concentrations and larger numbers of reads when compared with swabs, though there was no significant association between starting DNA concentration and number of reads generated. Swabbing produced greater sequence depth (more reads per sequence), while tissue biopsies revealed greater coverage across the genome (at lower sequence depth). Patterns of genomic variation as characterized in principal component analyses were similar regardless of the sampling method, suggesting that the less invasive swabbing is a viable option for producing quality GBS data in these organisms.

The nacre layer is composed of sheet-like aragonite crystals, with often loosely arranged polycrystal aragonite sheets which may induce poor mechanical properties in shells. In this study, a full-length low-complexity domain-containing protein (LCDP) cDNA from the triangle sail mussel Hyriopsis cumingii was generated and its role in shell formation investigated. The full-length cDNA was 1058 bp; it had an open reading frame (ORF) of 714 bp encoding 237 amino acids and contained a 20-amino acid signal peptide at the N-terminus and two low-complexity domains. H. cumingii LCDP was not homologous with other species. Tissue expression analyses showed that LCDP was specifically expressed in the mantle. In shell repair assays, significantly higher LCDP expression was observed in the shell repair group from days 12-21 (p < 0.01). After LCDP silencing, aragonite flake shapes in pearl layers became irregular with disordered deposition, while calcium carbonate (CaCO 3 ) crystal surfaces in prismatic layers became rougher and organic matrices between crystals appeared skeletonized, indicating the importance of biomineralization. Our in vitro CaCO 3 crystallization assays showed that LCDP induced single crystals to polycrystals, probably via loose arrangement between aragonite flakes. These results provide new insights on freshwater mollusk biomineralization and a theoretical basis for improved pearl quality., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The use of environmental DNA (eDNA) to assess aquatic biodiversity is a growing field with great potential for monitoring and managing threatened species, like freshwater mussel (Unionidae) populations. Freshwater mussels are globally imperiled and serve essential roles in aquatic systems as a food source and as a natural water filter making their management essential for ecosystem health. Unfortunately, mussel populations are often understudied, and challenges exist to accurately and efficiently describe the full suite of species present. Multispecies eDNA approaches may also be more challenging where freshwater mussel populations are most diverse due to ongoing and significant taxonomic restructuring that has been further complicated by molecular phylogenies using mitochondrial genes. For this study, we developed a microfluidic metabarcoding array that targets a wide range of species, from invertebrates to fishes, with an emphasis on detecting unionid mussels known to be present in the Sipsey River, Alabama. We compared mussel species diversity across six sites with well-studied mussel assemblages using eDNA surveys and traditional quadrat surveys in 2016. We examined how factors such as mussel population density, biomass and location in the river substrate impacted our ability to detect certain species; and investigated unexpected eDNA detections through phylogenetic analysis. Our eDNA results for fish and mussel species were broadly consistent with the data from traditional electrofishing and quadrat-based field surveys, although both community eDNA and conventional sampling detected species unique to that method. Our phylogenetic analysis agreed with other studies that treat Pleurobema decisum and P. chattanoogaense as synonymous species; however, they are still listed as unique species in molecular databases which complicates their identity in a metabarcoding assay. We also found that Fusconaia flava and F. cerina are indistinguishable from one another using a portion of the NADH dehydrogenase Subunit 1 (ND1) marker, which may warrant further investigation into whether or not they are synonymous. Our results show that many factors impacted our ability to detect and correctly identify Unionidae mussel species. Here we describe the obstacles we faced, including the murky phylogeny of Unionidae mussels and turbid river conditions, and our development of a potentially impactful freshwater mussel monitoring eDNA assay., Competing Interests: Jessica A. Homyack, Clay Mangum, Jamie Nettles, Jamie E. Thornton-Frost and Miranda J. Fix are employed by Weyerhaeuser Company.

Microcystins (MC) are one of the most abundant and widely distributed cyanotoxins in aquatic systems. MC inhibits the functions of protein phosphatase 1 and 2A (PP1/2A), which can seriously affect ecosystem integrity. The NF-E2-related nuclear factor 2 (Nrf2)/Kelch-like epichlorohydrin-related protein-1 (Keap1) signaling pathway protects against oxidative damage by activating phase II detoxification/antioxidant enzymes. Our previous study revealed that MC upregulates the expression and enhances the activities of the antioxidant enzymes by stimulating the CpNrf2 signaling pathway. In the current study, to further clarify the regulatory role of Keap1 in response to MC-induced oxidative stress in shellfish, we cloned the full-length cDNA of Keap1a and Keap1b from Cristaria plicata (designated CpKeap1a and CpKeap1b), which are 2952 and 3710 bp peptides, respectively. The amino acid sequence of CpKeap1a and CpKeap1b contained Tram-track and Bric-a-brac (BTB), Intervening region (IVR), and Double glycine repeat (DGR) domain. Additionally, CpKeap1a contained two cysteine residues analogous to Cys-273 and -288 in zebrafish, but CpKeap1b did not. Moreover, CpKeap1a and -1b formed a homodimer and heterodimer, respectively, and also formed a heterodimer with CpNrf2. In the hepatopancreas, the expression levels of CpKeap1a and -1b were the highest, but MC treatment down-regulated the expression of these proteins. Moreover, the transcription of antioxidant enzymes with antioxidant response element (ARE-driven enzymes), including CpMnSOD, CpCu/ZnSOD, CpTRX, CpPrx, CpSe-GPx, and Cpsigma-GST was upregulated by CpNrf2 in the hepatopancreas. Compared with the MC-induced group, CpKeap1a-siRNA1117 injection significantly increased the transcription of mRNAs for ARE-driven enzymes and Nrf2. CpKeap1a-siRNA1117 also enhanced the activities of antioxidation enzymes. These findings demonstrated that Keap1a negatively regulated the expression of Nrf2 protein and MC-induced oxidative stress response in C. plicata. Therefore, we speculated that CpKeap1a promoted CpNrf2 by recognizing and binding MC. These events then protected molluscs from MC-induced oxidative damage., Competing Interests: Declaration of competing interest The authors report no declarations of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The body of knowledge regarding the classification and evolution of freshwater mussels in the family Unionidae (Bivalvia) in Indochina has recently increased. However, the taxonomic revision of all extant taxa in the region is still ongoing. In this study, the genus Pilsbryoconcha was revised based on an integrative analysis of shell morphology, biogeography, and molecular data. Multi-locus phylogeny indicated the availability of eight species within the genus. Four previously recognized species are P. exilis (Lea, 1838), P. schomburgki (Martens, 1860) stat. rev., P. linguaeformis (Morelet, 1875), and P. carinifera (Conrad, 1837), while four other species are described herein as P. acuta sp. nov., P. mekongiana sp. nov., P. kittitati sp. nov., and P. hoikaab sp. nov. In addition, the neotype of P. carinifera is also designated to clarify its long taxonomic ambiguity. Divergent time estimation and historical biogeography analysis revealed that Pilsbryoconcha originated in the area now called the Khorat Plateau around the middle of the Eocene (mean age = 43.12 Mya), before its range was expanded across Indochina through a series of complex geomorphological changes of river systems, which also led to diversification of the genus., (© 2022. The Author(s).)

MAPKAPK2 ( MK2 ) is an important regulator of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, which is involved in a plethora of cellular processes concluding the development of gamete cells in meiosis and resisting pathogenic bacterial infestation. Hyriopsis cumingii is a significant mussel resource in China and a good material for pearl breeding. To explore the role of MK2 in H. cumingii, MK2 was identified and cloned, whose full-length cDNA was 1568 bp, including 87 bp in 5' UTR, 398 bp in 3' UTR, and 1083 bp in the open reading frame (ORF) region, encoding 360 amino acids. The expression of MK2 was the highest in the gills. Meanwhile, there was a significant difference in the gonads. After Aeromonas hydrophila and Lipopolysaccharide (LPS) infestation, the transcript level of the MK2 was upregulated in the gills. It indicated that MK2 might be involved in the innate immune response of H. cumingii after a pathogenic attack. After quantifying H. cumingii of different ages, it was found that the expression of MK2 was highest at 1 year old. In situ hybridization (ISH) results showed that the blue-purple hybridization signal was very significant in the oocytes and egg membranes of the female gonads of H. cumingii . The expression of MK2 increased gradually at the age of 1 to 5 months and showed a downward trend at the age of 5 to 8 months. It was suggested that MK2 might play an important role in the formation of primitive germ cells in H. cumingii . To sum up, MK2 might not only be involved in the immune response against pathogenic bacterial infection but also might play an important role in the development of the gonads in H. cumingii .

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Protein kinase C (PKC) is a multifunctional serine and PKC can phosphorylate serine residues in the cytoplasmic domain of tyrosinase, thereby regulating the activity of tyrosinase. Activated PKC is bound to the melanosome membrane, and unactivated PKC is free in the cytoplasm of melanocytes. In this study, we study the role of PKC gene in the melanin synthesis pathway and its effect on the color of the nacre of H. cumingii., Results: In this study, a HcPKC gene in H. cumingii was cloned and its effects on melanin synthesis and nacre color were studied. HcPKC was expressed in both purple and white mussels, and the level of mRNA expression was higher in the purple mussels than in white mussels. Strong and specific mRNA signals were detected in the dorsal epithelial cells of the mantle pallial layer, indicating that HcPKC may be involved in nacre formation. After SNP association with inner shell color related traits, according to the principle that 0.25 < PIC < 0.5 is medium polymorphism and PIC < 0.25 is low polymorphism, the A + 332G site on the HcPKC gene was a site of moderate polymorphism, and the other four sites were low polymorphism sex sites. There was strong linkage disequilibrium among the five loci. A haplotype was constructed and it was found that the frequency of T1 (AGGAA)in the white population was significantly higher than that in the purple population (P < 0.05)., Conclusion: The study found that HcPKC of H. cumingii can be used as a candidate gene related to inner shell color, and some of the SNP sites can be used for molecular-assisted breeding in the spinnaker mussel, providing a reference for cultivating high-quality freshwater pearls., (© 2022. The Author(s).)

Shell acidic matrix proteins are widely considered to be essential for shell formation given their low affinity and high loading for calcium ion. In the present study, a novel matrix protein, hic12, was isolated from the mantle of Hyriopsis cumingii. High expression in tissue and positive signals with in situ hybridization were detected in the mantle center and mantle pallium, indicating that hic12 mainly participated in the biomineralization of the shell nacreous layer. The expression pattern of hic12 in the pearl sac during early pearl formation indicated that it was involved in pearl biomineralization. Moreover, the recombinant protein, rGST-Hic12, was successfully expressed and purified. The addition of rGST-Hic12 could accelerate the calcium carbonate deposition rate, change the morphology of crystals, and promote the conversion of calcite to vaterite. These results may provide new insights into the molecular mechanisms of aragonite mollusk shell formation., (Copyright © 2022. Published by Elsevier Inc.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Pearl color is an important factor influencing pearl value, and is affected by the nacre color of the shell in Hyriopsis cumingii. Coproporphyrinogen-III oxidase (CPOX) is a key enzyme in porphyrin synthesis, and porphyrins are involved in color formation in different organisms, including in the nacre color of mussels. In this study, a CPOX gene (HcCPOX) was identified from H. cumingii, and its amino acid sequence was found to contain a coprogen-oxidase domain. HcCPOX mRNA was expressed widely in the tissues of white and purple mussels, and the highest expression was found in the gill, followed by the fringe mantle. The expression of HcCPOX in all tissues of purple mussels (except in the middle mantle) was higher than that of white mussels. Strong hybridization signals for HcCPOX were observed in the dorsal epithelial cells of the outer fold of the mantle. The activity of CPOX in the gill, fringe mantle, and foot of purple mussels was significantly higher than that in white mussels. Moreover, the expression of HcCPOX and CPOX activity were decreased in RNA interference experiments. The findings indicate that HcCPOX might contributes to nacre color formation in H. cumingii by being involved in porphyrin synthesis., Competing Interests: The authors have declared that no competing interests exist.

Metalloproteinase tissue inhibitors (TIMPs) have the activity of inhibiting matrix metalloproteinases (MMPs), which can promote cell growth, bind to the matrix, inhibit angiogenesis, and play a key role in extracellular matrix (ECM) metabolism regulation. In this study, TIMP-1, 2 from Hyriopsis cumingii (designated as HcTIMP-1, 2) were cloned and identified. Full-length cDNA of HcTIMP-1, 2 was 1160 bp and 729 bp, encoding 235 and 150 amino acid residues, respectively. The predicted molecular weight of HcTIMP-1 and 2 protein was 27.26 and 16.58 kDa, with isoelectric points of 8.89 and 8.72, respectively. HcTIMP-2 contained only one netrin (NTR) domain at the N-terminal but lacked a C-terminal domain. The mRNA of HcTIMP-1, 2 was expressed in hepatopancreas, gills, muscles, hemocytes, and mantles, which had the highest expression in hemocytes and muscles. The expression of HcTIMP-1, 2 had increased remarkably in hemocytes after bacterial challenge. After trauma, HcTIMP-1, 2 genes had the highest expression level in the first day. This indicated that HcTIMP-1 and 2 were involved in the immune response of H. cumingii. The soluble recombinant proteins HcTIMP-1, 2 were expressed efficiently in Escherichia coli BL21 (DE3) by constructing pET32a-TIMP1, 2 recombinant plasmids. The concentration of the recombinant was 0.14 and 0.31 mg/mL, respectively. The recombinant HcTIMP-1, 2 proteins were shown to inhibit human MMP2 activity and promoted the growth of NBL-7 and HUVE cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Bacterial disease outbreaks in filter feeder bivalve Hyriopsis cumingii as water contamination become more frequent in the water ecosystem, especially in intensive aquaculture habitats. To characterize host-pathogen interactions between H. cumingii and bacterial infection, we investigated the effects of Stenotrophomonas maltophilia HOP3 and Aeromonas veronii GL1 on the antioxidant response, tissue invasion and transcriptome expression of H. cumingii by infectivity trials. We showed that bacterial infections resulted in tubular necrosis of the hepatopancreas and induced the acute immune response in H. cumingii. The transcriptomic study identified a total of 5957 differentially expressed genes (DEGs) after A. veronii challenge. These DEGs were implicated in 302 KEGG pathways, notably in Apoptosis, Phagosome and Lysosome. The results showed that the relative expressions of all six immune-related DEGs were effectively stimulated with A. veronii, accompanied by tissue differences. Overall, these findings will contribute to an analysis of the immune response of H. cumingii to bacterial infection at the transcriptomic level and its genomic resource for research., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Wnt2 is a significant factor in the Wnt signaling pathway, which is associated with a variety of physiological activities, including inflammatory response, cell apoptosis, reproductive system development, and cell differentiation. Hyriopsis cumingii is the main pearl breeding mussel in China. However, the role of wnt2 in this species remains unclear. In this study, wnt2 from H. cumingii was cloned and identified. The full-length cDNA of wnt2 is 1524 bp, containing a 963 bp open reading frame (ORF), encoding 320 amino acid residues. The tissue distribution of H. cumingii indicated that wnt2 was predominantly highly expressed in the ovary and gill. And the expression profile after Aeromonas hydrophila or LPS injection indicated that wnt2 was up-regulated in gill, suggesting its role in the innate immune response. The expression of wnt2 was high at 4-month-old of early gonadal development and throughout ovarian development. In situ hybridization (ISH) showed significant hybridization signals on the gills and mature eggs of female gonads. In addition, miR-1988b-5p was found to negatively regulate wnt2 to affect the expression of key genes (frizzled-5, ctnnb1, and tcf7l) in the Wnt signaling pathway. Thus, these findings suggest a key role for wnt2 in immune regulation and gonadal development in H. cumingii., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 Ca 2+ 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., (Copyright © 2021. Published by Elsevier B.V.)

Located at the junction between Europe, Africa, and Asia, with distinct evolutionary origins and varied ecological and geographical settings, together with a marked history of changes in orogeny and configuration of the main river basins, turned the Eastern Mediterranean into a region of high diversity and endemism of freshwater taxa. Freshwater mussels (Bivalvia, Unionidae) from the Western Palearctic have been widely studied in their European range, but little attention has been dedicated to these taxa in the Eastern Mediterranean region and their diversity and phylogeography are still poorly understood. The present study aims to resolve the diversity, biogeography, and evolutionary relationships of the Eastern Mediterranean freshwater mussels. To that end, we performed multiple field surveys, phylogenetic analyses, and a thorough taxonomic revaluation. We reassessed the systematics of all Unionidae species in the region, including newly collected specimens across Turkey, Israel, and Iran, combining COI + 16S + 28S and COI phylogenies with molecular species delineation methods. Phylogeographical patterns were characterized based on published molecular data, newly sequenced specimens, and species distribution data, as well as ancestral range estimations. We reveal that Unionidae species richness in the Eastern Mediterranean is over 70% higher than previously assumed, counting 19 species within two subfamilies, the Unioninae (14) and Gonideinae (5). We propose two new species, Anodonta seddonisp. nov. and Leguminaia anatolicasp. nov. Six additional taxa, Unio delicatusstat. rev., Unio eucirrusstat. rev., Unio huetistat. rev., Unio sesirmensisstat. rev., Unio terminalisstat. rev. removed from the synonymy of Unio tigridis, as well as Unio damascensisstat. rev. removed from the synonymy of Unio crassus, are re-described. The nominal taxa Unio rothi var. komarowi O. Boettger, 1880 and Unio armeniacus Kobelt, 1911 are proposed as new synonyms of Unio bruguierianus, and Anodonta cyrea Drouët, 1881 and Anodonta cilicica Kobelt & Rolle, 1895 as new synonyms of Anodonta anatina. Also, the presence of Unio tumidus in the Maritza River is confirmed. The phylogeographic patterns described here are interpreted concerning major past geological events. Conservation needs and implications are presented, together with populations and species conservation priorities., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Shell matrix proteins have important roles in the biomineralization of shells. In this study, we isolated and identified a novel shell matrix protein gene, hic7, from the mussel Hyriopsis cumingii. The cDNA of hic7 was 459 bp long, including a 240-bp open reading frame. It encoded a 79 amino acid-long protein, with amino acids 1-19 constituting the signal peptide. The resulting hic7 is rich in cysteine (16.5%). After removing the signal peptide, the molecular weight was 8.85 kDa and the theoretical isoelectric point was 6.34, indicating that hic7 is a weakly acidic shell matrix protein. Hic7 is mainly expressed in the mantle tissue of H. cumingii. In situ hybridization showed hic7 signals at the edge and dorsal region of the mantle outer fold, indicating that it is related to the formation of the prismatic and nacreous layer of the shell. RNA interference indicated that when hic7 was inhibited by 80%, the crystal morphology of the prism and nacre layers of the shell were irregular and disordered. In addition, the expression of hic7 during the early development of the pearl sac indicated that it has an important role in the transformation of calcium carbonate crystals from a disordered to an orderly deposition pattern. These results suggest that matrix protein hic7 take part in constructing the framework of crystal nucleation and regulating the calcium carbonate crystal morphology of the nacreous and prismatic layers of shells and pearls., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Nitric oxide synthase (NOS) is a critical enzyme that catalyzes nitric oxide biosynthesis and orchestrates various immunological responses mediated by nitric oxide (NO) in host animals. In this study, the NOS gene was identified in the triangle shell mussel (Hyriopsis cumingii) (HcNOS). HcNOS was highly conserved in the characteristic gene structures of NOS. Phylogenetic analysis suggested that HcNOS was a typical invertebrate NOS. Further gene expression analysis, NOS activity assays and nitric oxide content measurements demonstrated the inducibility of HcNOS in responses to lipopolysaccharide (LPS) challenge and during tissue transplantation. Of note, mantle grafting induced a prolonged HcNOS/NO response, suggesting that through the HcNOS/NO system, multiple immunomodulators may play decisive roles in tissue grafting in triangle shell mussels. Thus, HcNOS appears to be a crucial player in responding to both bacterial infection and tissue transplantation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Dorsal is a Rel/NF-κB transcription factor, which forms a key part of the Toll pathway. Lysozyme is a ubiquitous enzyme that degrades bacterial cell walls. In this study, a Dorsal homolog was cloned and characterized from triangle sail mussel Hyriopsis cumingii, namely, HcDorsal. Dorsal consisted of 3041 bp, including a 1938 bp open reading frame encoding a 645 amino acid protein. The deduced HcDorsal protein contained a Rel homology domain and an Ig-like, plexin, transcription factor domain. Analysis of expression patterns showed that HcDorsal was highly expressed in the hepatopancreas of H. cumingii. The expression level of HcDorsal continuously increased after Vibrio parahaemolyticus stimulation. When HcDorsal was knocked down by siRNA interference, two phage lysozyme genes (HcLyso1 and HcLyso2) obtained by horizontal gene transfer were significantly downregulated in hemocytes of mussels. Furthermore, knockdown of HcLyso1 and HcLyso2 could weaken V. parahaemolyticus clearance ability. Recombinant HcLyso1 and HcLyso2 proteins accelerated the bacterial clearance in vivo in mussels and evidently inhibited the growth of V. parahaemolyticus. These results suggested that HcDorsal could be activated after V. parahaemolyticus stimulation and then modulate the immune response through the transcriptional regulation of HcLyso1 and HcLyso2, thereby playing a protective role in mussels., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 × 10 9 to 19.60 × 10 9 cells/mL with there being the largest counts between 26 September to 7 November. L. straminea sperm concentration was greatest (20.0 × 10 9 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 (R 2 ≥ 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Correct species identification and delineation are crucial for effective conservation and management. However, species delineation can be problematic in the presence of morphological ambiguities due to phenotypic plasticity, convergence, and/or interspecific hybridization. Here, we investigated the degree of hybridization between two closely related freshwater mussel species [Bivalvia: Unionidae; Lampsilis siliquoidea (Barnes) and L. radiata (Gmelin)] that present intermediate forms in areas of sympatry. Unionids have a distinct form of mitochondrial DNA (mtDNA) inheritance, termed doubly uniparental inheritance (DUI) where female mtDNA (F-type) is transmitted to all progeny but male mtDNA (M-type) is mostly inherited by the males resulting in mostly homoplasmic females and heteroplasmic males. An individual was identified as hybrid when F-type and M-type mtDNA of the two different species were found in the same individual. Twelve out of 116 sequenced males were identified as hybrids indicating that these species hybridize where their geographic range overlaps in the lower Great Lakes and St. Lawrence basins. Microsatellite analyses further support the occurrence of hybridization but at a larger spatial scale than indicated by the mitochondrial analyses. We also found that strong within-species population genetic structure affects the detection of purebred individuals overestimating the number of hybrids. Given the large geographic scale and proportion of hybrids found in this study, natural hybridization and introgression need to be considered when implementing local biodiversity inventories, identifying waterbodies as source of organisms for relocation and restoration projects and when setting appropriate conservation policies.

Detailed information on species delineation and population genetic structure is a prerequisite for designing effective restoration and conservation strategies for imperiled organisms. Phylogenomic and population genomic analyses based on genome-wide double digest restriction-site associated DNA sequencing (ddRAD-Seq) data has identified three allopatric lineages in the North American freshwater mussel genus Cyprogenia. Cyprogenia stegaria is restricted to the Eastern Highlands and displays little genetic structuring within this region. However, two allopatric lineages of C. aberti in the Ozark and Ouachita highlands exhibit substantial levels (mean uncorrected F ST  = 0.368) of genetic differentiation and each warrants recognition as a distinct evolutionary lineage. Lineages of Cyprogenia in the Ouachita and Ozark highlands are further subdivided reflecting structuring at the level of river systems. Species tree inference and species delimitation in a Bayesian framework using single nucleotide polymorphisms (SNP) data supported results from phylogenetic analyses, and supports three species of Cyprogenia over the currently recognized two species. A comparison of SNPs generated from both destructively and non-destructively collected samples revealed no significant difference in the SNP error rate, quality and amount of ddRAD sequence reads, indicating that nondestructive or trace samples can be effectively utilized to generate SNP data for organisms for which destructive sampling is not permitted.

Venustaconcha ellipsiformis (Unionidae) is a freshwater mussel species inhabiting small to medium streams of the Midwestern United States; however, its occurrence is rather sporadic and populations are often isolated. Due to anthropogenic habitat degradation and water pollution, this species is designated as some sort of conservation status in many states. To prioritize conservation strategies, highly variable genetic markers are necessary to assess population genetic structure and potential genetic erosion of V. ellipsiformis. Using whole genome sequence data, we developed and characterized microsatellite markers for V. ellipsiformis. Among 23 tetranucleotide loci tested, 14 loci were consistently amplified and showed polymorphism. Analyses performed on three populations in the upper Mississippi River basin showed that the number of alleles per locus ranged from 2 to 11 and the observed heterozygosity varied from 0.15 to 0.75. Based on genotypic and allelic rarefaction curves, these loci had adequate statistical power to genetically discriminate between individuals and the sample size was large enough to capture most alleles available in the populations at most loci. Finally, cross-species screening of the loci successfully amplified and showed polymorphism in six species in the tribe Lampsilini. The microsatellite loci developed in this study provide a valuable addition to extant genetic markers for freshwater mussels and can be useful to provide high-level resolution of population genetic parameters for V. ellipsiformis. Such information will be of great value for resource managers developing and prioritizing conservation strategies for imperiled mussel species.

Freshwater unionid bivalves currently face severe anthropogenic challenges. Over 70% of species in the United States are threatened, endangered or extinct due to pollution, damming of waterways and overfishing. These species are notable for their unusual life history strategy, parasite-host co-evolution and biparental mitochondrial inheritance. Among this clade, the washboard mussel Megalonaias nervosa is one species that remains prevalent across the Southeastern United States, with robust population sizes. We have created a reference genome for M. nervosa to determine how genome content has evolved in the face of these widespread environmental challenges. We observe dynamic changes in genome content, with a burst of recent transposable element proliferation causing a 382 Mb expansion in genome content. Birth-death models suggest rapid expansions among gene families, with a mutation rate of 1.16 × 10 -8 duplications per gene per generation. Cytochrome P450 gene families have experienced exceptional recent amplification beyond expectations based on genome-wide birth-death processes. These genes are associated with increased rates of amino acid changes, a signature of selection driving evolution of detox genes. Fitting evolutionary models of adaptation from standing genetic variation, we can compare adaptive potential across species and mutation types. The large population size in M. nervosa suggests a 4.7-fold advantage in the ability to adapt from standing genetic variation compared with a low diversity endemic E. hopetonensis. Estimates suggest that gene family evolution may offer an exceptional substrate of genetic variation in M. nervosa, with P sgv  = 0.185 compared with P sgv  = 0.067 for single nucleotide changes. Hence, we suggest that gene family evolution is a source of 'hopeful monsters' within the genome that may facilitate adaptation when selective pressures shift. These results suggest that gene family expansion is a key driver of adaptive evolution in this key species of freshwater Unionidae that is currently facing widespread environmental challenges. This work has clear implications for conservation genomics on freshwater bivalves as well as evolutionary theory. This genome represents a first step to facilitate reverse ecological genomics in Unionidae and identify the genetic underpinnings of phenotypic diversity., (© 2020 John Wiley & Sons Ltd.)

From a genomics perspective, bivalves (Mollusca: Bivalvia) have been poorly explored with the exception for those of high economic value. The bivalve order Unionida, or freshwater mussels, has been of interest in recent genomic studies due to their unique mitochondrial biology and peculiar life cycle. However, genomic studies have been hindered by the lack of a high-quality reference genome. Here, I present a genome assembly of Potamilus streckersoni using Pacific Bioscience single-molecule real-time long reads and 10X Genomics-linked read sequencing. Further, I use RNA sequencing from multiple tissue types and life stages to annotate the reference genome. The final assembly was far superior to any previously published freshwater mussel genome and was represented by 2,368 scaffolds (2,472 contigs) and 1,776,755,624 bp, with a scaffold N50 of 2,051,244 bp. A high proportion of the assembly was comprised of repetitive elements (51.03%), aligning with genomic characteristics of other bivalves. The functional annotation returned 52,407 gene models (41,065 protein, 11,342 tRNAs), which was concordant with the estimated number of genes in other freshwater mussel species. This genetic resource, along with future studies developing high-quality genome assemblies and annotations, will be integral toward unraveling the genomic bases of ecologically and evolutionarily important traits in this hyper-diverse group., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Hyriopsis cumingii is an important species for freshwater pearl cultivation in China. In terms of pearl production, males have larger pearls and better glossiness than females, but there are few reports focusing on the sex of H. cumingii. In this study, six mRNA and six microRNA (miRNA) libraries were prepared from ovaries and testes. Additionally, 28,502 differentially expressed genes (DEGs) and 32 differentially expressed miRNAs (DEMs) were identified. Compared with testis, 14,360 mRNAs and 20 miRNAs were up-regulated in ovary, 14,142 mRNAs and 12 miRNAs were down-regulated. In DEGs, the known genes related to sex determinism and/or differentiation were also identified, such as DMRT1, SOX9, SF1 for males, FOXL2 for females, and other potentially significant candidate genes. Three sex-related pathways have also been identified, which are Wnt, Notch, and TGF-beta. In 32 DEMs, the three miRNAs (miR-9-5p, miR-92, miR-184) were paid more attention, they predicted 28 target genes, which may also be candidates for sex-related miRNAs and genes. Differential miRNAs target genes analysis reveals the pathway associated with oocyte meiosis and spermatogenesis. Overall, the findings of the study provide significant insights to enhance our understanding of sex differentiation and/or sex determination mechanisms for H. cumingii.

Freshwater mussels belonging to the genus Nodularia (Family Unionidae) are known to be widely distributed in East Asia. Although phylogenetic and population genetic studies have been performed for these species, there still remain unresolved questions in their taxonomic status and biogeographic distribution pathways. Here, the nucleotide sequences of CO1 and 16S rRNA were newly determined from 86 N. douglasiae and 83 N. breviconcha individuals collected on the Korean Peninsula. Based on these data, we revealed the following results: (1) N. douglasiae can be divided into the three genetic clades of A (only found in Korean Peninsula), B (widely distributed in East Asia), and C (only found in the west of China and Russia), (2) the clade A is not an independent species but a concrete member of N. douglasiae given the lack of genetic differences between the clades A and B, and (3) N. breviconcha is not a subspecies of N. douglasiae but an independent species apart from N. douglasiae. In addition, we suggested the plausible scenarios of biogeographic distribution events and demographic history of Nodularia species.

Microcystin (MC) is a cyclic heptapeptide toxin. Nuclear factor erythocyte 2-related factor 2 (Nrf2) can enhance cellular survival by mediating phase 2 detoxification and antioxidant genes. In this study, CpNrf2 cDNA sequences were cloned from freshwater bivalve Cristaria plicata. The full-length CpNrf2 cDNA sequence was 4259 bp, and its homology was the highest with Mizuhopecten yessoensis, reaching 46%. CpNrf2 transcription levels were examined in all tested tissues, and the highest level was in hepatopancreas from C. plicata. The recombinant protein pET32-CpNrf2 was purified with the content of 1.375 mg/mL. The expression levels of CpNrf2 mRNA were raised in hepatopancreas after MC stimulation. After CpNrf2 knockdown, CpNrf2 mRNA levels were significantly down-regulated after 24 h. Compared with control group, the expression levels of ARE-driven enzymes (CpMnSOD, CpCuZnSOD, CpTRX, CpPrx, CpSe-GPx and Cpsigma-GST) were significantly increased, and those enzyme activities were also significantly up-regulated in MC-stimulated group. However, in CpNrf2-iRNA group, they were significantly down-regulated. The results revealed that Nrf2/ARE pathway is very crucial to protect molluscs from MC., Competing Interests: Declaration of Competing Interest All authors have no potential financial conflicts of interest related to this manuscript., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this study, a novel matrix protein (teosin) was isolated from Hyriopsis cumingii. Gene expression analysis showed that teosin is mainly expressed in the mantle and blood, and a hybridization signal was found in dorsal epithelial cells of the mantle pallial by in situ hybridization. Moreover, teosin expression during pearl formation indicated its participation in initial nacreous layer biomineralization, and suppressing teosin expression resulted in irregular crystal morphology and disordered arrangement in RNAi assay. In vitro crystallization assays indicated teosin could increase the size of calcite. By turning the sample stage about 15°, we got the high-resolution TEM images of the crystals' edges. This is a novel method to observe the crystal which is over 200 nm under TEM. In the control experiment group, the calcite show the character of long range order. The calcite induced by teosin were composed of nano-grains, and the polycrystal character were confirmed by EDS. These results suggested that teosin is involved in regulating crystal morphology regulation and inducing polycrystal formation during nacreous-layer formation., Competing Interests: Author contribution Xiaojun Liu designed the experiments and developed the method for TEM observation; Can Jin and Xiaojun Liu conducted the experiments; Can Jin, Jiale Li and Xiaojun Liu processed the data and wrote the manuscript., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Freshwater mussels (Bivalvia: Unionidae) is a diverse family with around 700 species being widespread in the Northern Hemisphere and Africa. These animals fulfill key ecological functions and provide important services to humans. Unfortunately, populations have declined dramatically over the last century, rendering Unionidae one of the world's most imperiled taxonomic groups. In Far East Asia (comprising Japan, Korea, and Eastern Russia), conservation actions have been hindered by a lack of basic information on the number, identity, distribution and phylogenetic relationships of species. Available knowledge is restricted to studies on national and sub-national levels. The present study aims to resolve the diversity, biogeography and evolutionary relationships of the Far East Asian Unionidae in a globally comprehensive phylogenetic and systematic context. We reassessed the systematics of all Unionidae species in the region, including newly collected specimens from across Japan, South Korea, and Russia, based on molecular (including molecular species delineation and a COI + 28S phylogeny) and comparative morphological analyses. Biogeographical patterns were then assessed based on available species distribution data from the authors and previous reference works. We revealed that Unionidae species richness in Far East Asia is 30% higher than previously assumed, counting 43 species (41 native + 2 alien) within two Unionidae subfamilies, the Unioninae (32 + 1) and Gonideinae (9 + 1). Four of these species are new to science, i.e. Beringiana gosannensissp. nov., Beringiana fukuharaisp. nov., Buldowskia kamiyaisp. nov., and Koreosolenaia sitgyensisgen. & sp. nov. We also propose a replacement name for Nodularia sinulata, i.e. Nodularia breviconchanom. nov. and describe a new tribe (Middendorffinaiini tribe nov.) within the Unioninae subfamily. Biogeographical patterns indicate that this fauna is related to that from China south to Vietnam until the Mekong River basin. The Japanese islands of Honshu, Shikoku, Kyushu, Hokkaido, and the Korean Peninsula were identified as areas of particularly high conservation value, owing to high rates of endemism, diversity and habitat loss. The genetically unique species within the genera Amuranodonta, Obovalis, Koreosolenaiagen. nov., and Middendorffinaia are of high conservation concern., (Copyright © 2020 Elsevier Inc. All rights reserved.)

As a specific pearl mussel in China, Hyriopsis cumingii has enormous economic value. However, the organism damage caused by pearl insertion is immeasurable. TGF-β/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., Competing Interests: Declaration of competing interest We declare that we have no conflict of interest., (Copyright © 2019. Published by Elsevier Ltd.)

Using a new fossil-calibrated mitogenome-based approach, we identified macroevolutionary shifts in mitochondrial gene order among the freshwater mussels (Unionoidea). We show that the early Mesozoic divergence of the two Unionoidea clades, Margaritiferidae and Unionidae, was accompanied by a synchronous split in the gene arrangement in the female mitogenome (i.e., gene orders MF1 and UF1). Our results suggest that this macroevolutionary jump was completed within a relatively short time interval (95% HPD 201-226 Ma) that coincided with the Triassic-Jurassic mass extinction. Both gene orders have persisted within these clades for ~200 Ma. The monophyly of the so-called "problematic" Gonideinae taxa was supported by all the inferred phylogenies in this study using, for the first time, the M- and F-type mitogenomes either singly or combined. Within Gonideinae, two additional splits in the gene order (UF1 to UF2, UF2 to UF3) occurred in the Mesozoic and have persisted for ~150 and ~100 Ma, respectively. Finally, the mitogenomic results suggest ancient connections between freshwater basins of East Asia and Europe near the Cretaceous-Paleogene boundary, probably via a continuous paleo-river system or along the Tethys coastal line, which are well supported by at least three independent but almost synchronous divergence events.

Freshwater mussels are a species-rich group of aquatic invertebrates that are among the most endangered groups of fauna worldwide. As filter-feeders that are constantly exposed to new microbial inoculants, mussels represent an ideal system to investigate the effects of species or the environment on gut microbiome composition. In this study, we examined if host species or site exerts a greater influence on microbiome composition. Individuals of four co-occurring freshwater mussel species, Cyclonaias asperata, Fusconaia cerina, Lampsilis ornata, and Obovaria unicolor were collected from six sites along a 50 km stretch of the Sipsey River in Alabama, USA. High throughput 16S rRNA gene sequencing revealed that mussel gut bacterial microbiota were distinct from bacteria on seston suspended in the water column, and that the composition of the gut microbiota was influenced by both host species and site. Despite species and environmental variation, the most frequently detected sequences within the mussel microbiota were identified as members of the Clostridiales. Sequences identified as the nitrogen-fixing taxon Methylocystis sp. were also abundant in all mussel species, and sequences of both bacterial taxa were more abundant in mussels than in water. Site physicochemical conditions explained almost 45% of variation in seston bacterial communities but less than 8% of variation in the mussel bacterial microbiome. Together, these findings suggest selective retention of bacterial taxa by the freshwater mussel host, and that both species and the environment are important in determining mussel gut microbiome composition., Competing Interests: The authors have declared that no competing interests exist.

Complement component C3 is well recognized as the central mediator of complement system, whose activation is responsible for the immune surveillance and elimination of non-self-antigens. In this study, C3 gene (HcC3) from a pearl making mussel, Hyriopsis cumingii, was successfully identified. The putative HcC3 possessed the canonical domains and highly conserved functional residues of C3 family members. In phylogenetic analysis, HcC3 was also clustered into C3 subfamily and separated from α2 macroglobulin clade. HcC3 gene was constitutively expressed in a wide range of tissues of pearl mussels, among which the immune-related tissues like hemocytes got highest expression. After allograft surgery of mantle tissues for aquaculture pearl production, the gene expression of HcC3 exhibited a rapid upregulation on day 1, dropped back on day 3, peaked the value on day 7, and restored to the level similar to control samples on day 14 after mantle allograft. The biphasic expression within the two weeks post the surgery suggests the important roles for HcC3 in alloimmune responses and an intricate complement activation mechanism in mollusks during tissue allograft., (Copyright © 2019 Elsevier Ltd. All rights reserved.)