Declines in freshwater mussel density, size and productivity in the River Thames over the past half century.

A pioneering, quantitative study published in Journal of Animal Ecology in 1966 on freshwater mussel populations in the River Thames, UK, continues to be cited extensively as evidence of the major contribution that mussels make to benthic biomass and ecosystem functioning in global river ecosystems. Ecological alteration, as well as declines in freshwater mussel populations elsewhere, suggest that changes to mussel populations in the River Thames are likely to have occurred over the half century since this study. We resurveyed the site reported in Negus (1966) and quantified the changes in mussel population density, species composition, growth patterns and productivity. We found large declines in population density for all unionid species. The duck mussel Anodonta anatina decreased to 1.1% of 1964 density. The painter's mussel Unio pictorum fell to 3.2% of 1964 density. The swollen river mussel Unio tumidus showed statistically nonsignificant declines. In contrast to 1964, in 2020 we found no living specimens of the depressed river mussel Pseudanodonta complanata (classified as Vulnerable by the IUCN Red List) but found new records of the invasive, nonnative zebra mussel Dreissena polymorpha and Asian clam Corbicula fluminea. Additionally, we found strong decreases in size-at-age for all species, which now grow to 65-90% of maximum lengths in 1964. As a result of reduced density and size, estimated annual biomass production fell to 7.5% of 1964 levels. Since mussels can be important to ecosystem functioning, providing key regulating and provisioning services, the declines we found imply substantial degradation of freshwater ecosystem services in the River Thames, one of the UK's largest rivers. Our study also highlights the importance to conservationists and ecologists of updating and validating assumptions and data about wild populations, which in the present era of anthropogenic ecosystem alteration are undergoing significant and rapid changes. Regular population surveys of key species are essential to maintain an accurate picture of ecosystem health and to guide management.

Anatomy and behavior of Laternula elliptica, a keystone species of the Antarctic benthos (Bivalvia: Anomalodesmata: Laternulidae).

Laternula elliptica (P. P. King, 1832) is the sole representative of the anomalodesmatan family Laternulidae and the largest bivalve in the Antarctic and Subantarctic. A keystone species of the regional benthic communities, it has reached model status, having been studied in hundreds of scientific works across many biological disciplines. In contrast, its anatomy has remained poorly known, with prior published data limited to partial descriptions based on chemically preserved specimens. Based on observations of aquarium-maintained living animals at the Brazilian Comandante Ferraz Antarctic Station, gross-morphological dissections, and histological sectioning, the comparative anatomy, functional morphology, and aspects of behavior of L. elliptica are described and discussed. Special focus is placed on the pallial organs (including elucidation of cleansing and feeding sorting mechanisms in the mantle cavity) and the musculature. Among the noteworthy findings are the presence of well-developed siphons furnished with sensory tentacles at its tips, some of which bearing eyes; large, folded gills and labial palps capable of sorting the material entering the mantle cavity; an inter-chamber communication in the posterior region of the mantle cavity; an ample ventral mantle fusion with an anterior pedal gape; the absence of a 4th pallial opening; and the absence of a ligamental lithodesma in adult specimens. This study reevaluates the available anatomical data in the literature, both supplementing and correcting previously published accounts.

Form and function of the mantle edge in Protobranchia (Mollusca: Bivalvia).

We analyzed, by optical and transmission electron microscopy, the morphology and function of the mantle edge, including the formation of the periostracum, of ten species of protobranchs. Five species from the order Nuculida, four species from the order Nuculanida and one species from the order Solemyida were studied. A second outer fold, which seems to function as a template for the internal marginal crenulations of the valves, is present in the crenulated species of Nucula. The minute non-crenulated Ennucula aegeensis shows the glandular basal cells displaced toward the periostracal groove, resembling a minute additional fold between the outer and middle folds. Intense secretion of glycocalyx, together with active uptake of particles, have been observed in the inner epithelium of the middle mantle fold and the whole epithelium of the inner mantle fold in all the studied species. Contrary to the rest of the bivalves, all the protobranchs analyzed have two basal cells involved in the formation of the external nanometric pellicle of the periostracum, a character that would support the monophyly of protobranchs. A three-layered pattern is the general rule for the periostracum in protobranchs, like for other bivalves. The presence of pouches of translucent layer inside the tanned dark layer under periostracal folds is characteristic of the species with a folded periostracum; its function is unclear but could give flexibility to the periostracum. The non-nacreous internal shell layer and the presence of translucent pouches under periostracal folds in Sarepta speciosa resemble those found in nuculanids. However, the free periostracum is rather similar to those of N. hanleyi and E. aegeensis, with a continuous vesicular layer. All the latter supports the inclusion of Sarepta in the order Nuculanida but could indicate either a basal lineage or that the translucent vesicular layer is an adaptive trait.

Specimen alignment with limited point-based homology: 3D morphometrics of disparate bivalve shells (Mollusca: Bivalvia).

Background: Comparative morphology fundamentally relies on the orientation and alignment of specimens. In the era of geometric morphometrics, point-based homologies are commonly deployed to register specimens and their landmarks in a shared coordinate system. However, the number of point-based homologies commonly diminishes with increasing phylogenetic breadth. These situations invite alternative, often conflicting, approaches to alignment. The bivalve shell (Mollusca: Bivalvia) exemplifies a homologous structure with few universally homologous points-only one can be identified across the Class, the shell 'beak'. Here, we develop an axis-based framework, grounded in the homology of shell features, to orient shells for landmark-based, comparative morphology. Methods: Using 3D scans of species that span the disparity of shell morphology across the Class, multiple modes of scaling, translation, and rotation were applied to test for differences in shell shape. Point-based homologies were used to define body axes, which were then standardized to facilitate specimen alignment via rotation. Resulting alignments were compared using pairwise distances between specimen shapes as defined by surface semilandmarks. Results: Analysis of 45 possible alignment schemes finds general conformity among the shape differences of 'typical' equilateral shells, but the shape differences among atypical shells can change considerably, particularly those with distinctive modes of growth. Each alignment corresponds to a hypothesis about the ecological, developmental, or evolutionary basis of morphological differences, but we suggest orientation via the hinge line for many analyses of shell shape across the Class, a formalization of the most common approach to morphometrics of shell form. This axis-based approach to aligning specimens facilitates the comparison of approximately continuous differences in shape among phylogenetically broad and morphologically disparate samples, not only within bivalves but across many other clades.

Localization and Bioreactivity of Cysteine-Rich Secretions in the Marine Gastropod Nucella lapillus.

Marine biodiversity has been yielding promising novel bioproducts from venomous animals. Despite the auspices of conotoxins, which originated the paradigmatic painkiller Prialt, the biotechnological potential of gastropod venoms remains to be explored. Marine bioprospecting is expanding towards temperate species like the dogwhelk Nucella lapillus, which is suspected to secrete immobilizing agents through its salivary glands with a relaxing effect on the musculature of its preferential prey, Mytilus sp. This work focused on detecting, localizing, and testing the bioreactivity of cysteine-rich proteins and peptides, whose presence is a signature of animal venoms and poisons. The highest content of thiols was found in crude protein extracts from the digestive gland, which is associated with digestion, followed by the peribuccal mass, where the salivary glands are located. Conversely, the foot and siphon (which the gastropod uses for feeding) are not the main organs involved in toxin secretion. Ex vivo bioassays with Mytilus gill tissue disclosed the differential bioreactivity of crude protein extracts. Secretions from the digestive gland and peribuccal mass caused the most significant molecular damage, with evidence for the induction of apoptosis. These early findings indicate that salivary glands are a promising target for the extraction and characterization of bioactive cysteine-rich proteinaceous toxins from the species.

Combining genotypic and phenotypic variation in a geospatial framework to identify sources of mussels in northern New Zealand.

The New Zealand green-lipped mussel aquaculture industry is largely dependent on the supply of young mussels that wash up on Ninety Mile Beach (so-called Kaitaia spat), which are collected and trucked to aquaculture farms. The locations of source populations of Kaitaia spat are unknown and this lack of knowledge represents a major problem because spat supply may be irregular. We combined genotypic (microsatellite) and phenotypic (shell geochemistry) data in a geospatial framework to determine if this new approach can help identify source populations of mussels collected from two spat-collecting and four non-spat-collecting sites further south. Genetic analyses resolved differentiated clusters (mostly three clusters), but no obvious source populations. Shell geochemistry analyses resolved six differentiated clusters, as did the combined genotypic and phenotypic data. Analyses revealed high levels of spatial and temporal variability in the geochemistry signal. Whilst we have not been able to identify the source site(s) of Kaitaia spat our analyses indicate that geospatial testing using combined genotypic and phenotypic data is a powerful approach. Next steps should employ analyses of single nucleotide polymorphism markers with shell geochemistry and in conjunction with high resolution physical oceanographic modelling to resolve the longstanding question of the origin of Kaitaia spat.

Brain mass explains prey size selection better than beak, gizzard and body size in a benthivorous duck species.

Prey size selection in some bird species is determined by the size of the beak. However, we assumed for bird species swallowing whole prey that a cognitive process may be involved. As cognitive feature, brain mass was used. We hypothesized that the mass of the brain was more strongly positively correlated with prey size than morphological features such as beak volume, gizzard mass and body mass. We tested this hypothesis on eiders Somateria mollissima that swallow the prey whole, by using mean and maximum size of nine prey categories. Eiders were collected at the main wintering grounds in Denmark. As index of brain mass we used head volume, which is positively correlated with brain mass (r2 = 0.73). Head volume of eiders was significantly, positive correlated with mean and maximum size of blue mussels Mytilus edulis, razor clams Ensis directus and all prey sizes combined and the maximum size of draft whelk Hinia reticulata and conch Buccinum undatum. Gizzard mass was also significantly positively correlated with maximum size of draft whelk and conch. Beak volume and body mass was not significantly correlated with the size of any of the nine food items. Analyses of effect size for organs showed that head volume was positively related to prey size, whereas beak volume, gizzard mass and body mass did not show a significant positive relationship. These results indicate that cognitive processes connected to brain mass may be involved in prey size selection by eiders.

Evo-devo of shell colour in gastropods and bivalves.

Recent technical innovations are revealing surprising patterns in mollusc shell pigmentation, such as an unexpectedly modest role for melanins and rapid divergences in the mix of pigments used to achieve similar colour patterns. The elucidation of the molecular genetic basis of shell pigmentation has been slow, probably because of the high genome complexity of gastropods and bivalves. Recent work within the old field of evolutionary ecology of shell pigmentation allows a greater role for the analysis of large-geographic-scale patterns (sometimes employing citizen-science data), as well as experimental field studies. However, the field remains dominated by land snails as model organisms, while colour pattern evolution in marine gastropods and bivalves, particularly those not exposed to visual predators, remains mysterious.

Effects of first intermediate host density, host size and salinity on trematode infections in mussels of the south-western Baltic Sea.

Trematode prevalence and abundance in hosts are known to be affected by biotic drivers as well as by abiotic drivers. In this study, we used the unique salinity gradient found in the south-western Baltic Sea to: (i) investigate patterns of trematode infections in the first intermediate host, the periwinkle Littorina littorea and in the downstream host, the mussel Mytilus edulis, along a regional salinity gradient (from 13 to 22) and (ii) evaluate the effects of first intermediate host (periwinkle) density, host size and salinity on trematode infections in mussels. Two species dominated the trematode community, Renicola roscovita and Himasthla elongata. Salinity, mussel size and density of infected periwinkles were significantly correlated with R. roscovita, and salinity and density correlated with H. elongata abundance. These results suggest that salinity, first intermediate host density and host size play an important role in determining infection levels in mussels, with salinity being the main major driver. Under expected global change scenarios, the predicted freshening of the Baltic Sea might lead to reduced trematode transmission, which may be further enhanced by a potential decrease in periwinkle density and mussel size.

Experimental tests of bivalve shell shape reveal potential tradeoffs between mechanical and behavioral defenses.

Bivalves protect themselves from predators using both mechanical and behavioral defenses. While their shells serve as mechanical armor, bivalve shells also enable evasive behaviors such as swimming and burrowing. Therefore, bivalve shell shape is a critical determinant of how successfully an organism can defend against attack. Shape is believed to be related to shell strength with bivalve shell shapes converging on a select few morphologies that correlate with life mode and motility. In this study, mathematical modeling and 3D printing were used to analyze the protective function of different shell shapes against vertebrate shell-crushing predators. Considering what life modes different shapes permit and analyzing the strength of these shapes in compression provides insight to evolutionary and ecological tradeoffs with respect to mechanical and behavioral defenses. These empirical tests are the first of their kind to isolate the influence of bivalve shell shape on strength and quantitatively demonstrate that shell strength is derived from multiple shape parameters. The findings of this theoretical study are consistent with examples of shell shapes that allow escape behaviors being mechanically weaker than those which do not. Additionally, shell elongation from the umbo, a metric often overlooked, is shown to have significant effects on shell strength.

Shells of the bivalve Astarte moerchi give new evidence of a strong pelagic-benthic coupling shift occurring since the late 1970s in the North Water polynya.

Climate changes in the Arctic may weaken the currently tight pelagic-benthic coupling. In response to decreasing sea ice cover, arctic marine systems are expected to shift from a 'sea-ice algae-benthos' to a 'phytoplankton-zooplankton' dominance. We used mollusc shells as bioarchives and fatty acid trophic markers to estimate the effects of the reduction of sea ice cover on the food exported to the seafloor. Bathyal bivalve Astarte moerchi living at 600 m depth in northern Baffin Bay reveals a clear shift in growth variations and Ba/Ca ratios since the late 1970s, which we relate to a change in food availability. Tissue fatty acid compositions show that this species feeds mainly on microalgae exported from the euphotic zone to the seabed. We, therefore, suggest that changes in pelagic-benthic coupling are likely due either to local changes in sea ice dynamics, mediated through bottom-up regulation exerted by sea ice on phytoplankton production, or to a mismatch between phytoplankton bloom and zooplankton grazing due to phenological change. Both possibilities allow a more regular and increased transfer of food to the seabed. This article is part of the theme issue 'The changing Arctic Ocean: consequences for biological communities, biogeochemical processes and ecosystem functioning'.

Neandertals on the beach: Use of marine resources at Grotta dei Moscerini (Latium, Italy).

Excavated in 1949, Grotta dei Moscerini, dated MIS 5 to early MIS 4, is one of two Italian Neandertal sites with a large assemblage of retouched shells (n = 171) from 21 layers. The other occurrence is from the broadly contemporaneous layer L of Grotta del Cavallo in southern Italy (n = 126). Eight other Mousterian sites in Italy and one in Greece also have shell tools but in a very small number. The shell tools are made on valves of the smooth clam Callista chione. The general idea that the valves of Callista chione were collected by Neandertals on the beach after the death of the mollusk is incomplete. At Moscerini 23.9% of the specimens were gathered directly from the sea floor as live animals by skin diving Neandertals. Archaeological data from sites in Italy, France and Spain confirm that shell fishing and fresh water fishing was a common activity of Neandertals, as indicated by anatomical studies recently published by E. Trinkaus. Lithic analysis provides data to show the relation between stone tools and shell tools. Several layers contain pumices derived from volcanic eruptions in the Ischia Island or the Campi Flegrei (prior to the Campanian Ignimbrite mega-eruption). Their rounded edges indicate that they were transported by sea currents to the beach at the base of the Moscerini sequence. Their presence in the occupation layers above the beach is discussed. The most plausible hypothesis is that they were collected by Neandertals. Incontrovertible evidence that Neandertals collected pumices is provided by a cave in Liguria. Use of pumices as abraders is well documented in the Upper Paleolithic. We prove that the exploitation of submerged aquatic resources and the collection of pumices common in the Upper Paleolithic were part of Neandertal behavior well before the arrival of modern humans in Western Europe.

Form and function of tentacles in pteriomorphian bivalves.

Tentacles are remarkable anatomical structures in invertebrates for their diversity of form and function. In bivalves, tentacular organs are commonly associated with protective, secretory, and sensory roles. However, anatomical details are available for only a few species, rendering the diversity and evolution of bivalve tentacles still obscure. In Pteriomorphia, a clade including oysters, scallops, pearl oysters, and relatives, tentacles are abundant and diverse. We investigated tentacle anatomy in the group to understand variation, infer functions, and investigate patterns in tentacle diversity. Six species from four pteriomorphian families (Ostreidae, Pinnidae, Pteriidae, and Spondylidae) were collected and thoroughly investigated with integrative microscopy techniques, including histology, scanning electron microscopy, and confocal microscopy. Tentacles can be classified as middle fold tentacles (MFT) and inner fold tentacles (IFT) according to their position with respect to the folds of the mantle margin. While MFT morphology indicates intense secretion of mucosubstances, no evidence for secretory activity was found for IFT. However, both tentacle types have appropriate ciliary distribution and length to promote mucus transportation for cleaning and lubrication. Protective and sensory functions are discussed based on different lines of evidence, including secretion, cilia distribution, musculature, and innervation. Our results support the homology of MFT and IFT only for Pterioidea and Ostreoidea, considering their morphology, the presence of ciliated receptors at the tips, and branched innervation pattern. This is in accordance with recent phylogenetic hypotheses that support the close relationship between these superfamilies. In contrast, major structural differences indicate that MFT and IFT are probably not homologous across all pteriomorphians. By applying integrative microscopy, we were able to reveal anatomical elements that are essential for the understanding of homology and function when dealing with such superficially similar structures.

Mechanics unlocks the morphogenetic puzzle of interlocking bivalved shells.

Brachiopods and mollusks are 2 shell-bearing phyla that diverged from a common shell-less ancestor more than 540 million years ago. Brachiopods and bivalve mollusks have also convergently evolved a bivalved shell that displays an apparently mundane, yet striking feature from a developmental point of view: When the shell is closed, the 2 valve edges meet each other in a commissure that forms a continuum with no gaps or overlaps despite the fact that each valve, secreted by 2 mantle lobes, may present antisymmetric ornamental patterns of varying regularity and size. Interlocking is maintained throughout the entirety of development, even when the shell edge exhibits significant irregularity due to injury or other environmental influences, which suggests a dynamic physical process of pattern formation that cannot be genetically specified. Here, we derive a mathematical framework, based on the physics of shell growth, to explain how this interlocking pattern is created and regulated by mechanical instabilities. By close consideration of the geometry and mechanics of 2 lobes of the mantle, constrained both by the rigid shell that they secrete and by each other, we uncover the mechanistic basis for the interlocking pattern. Our modeling framework recovers and explains a large diversity of shell forms and highlights how parametric variations in the growth process result in morphological variation. Beyond the basic interlocking mechanism, we also consider the intricate and striking multiscale-patterned edge in certain brachiopods. We show that this pattern can be explained as a secondary instability that matches morphological trends and data.

Spatial filters of function and phylogeny determine morphological disparity with latitude.

The drivers of latitudinal differences in the phylogenetic and ecological composition of communities are increasingly studied and understood, but still little is known about the factors underlying morphological differences. High-resolution, three-dimensional morphological data collected using computerized micro-tomography (micro-CT) allows comprehensive comparisons of morphological diversity across latitude. Using marine bivalves as a model system, this study combines 3D shape analysis (based on a new semi-automated procedure for placing landmarks and semilandmarks on shell surfaces) with non-shape traits: centroid size, proportion of shell to soft-tissue volume, and magnitude of shell ornamentation. Analyses conducted on the morphology of 95% of all marine bivalve species from two faunas along the Atlantic coast of North America, the tropical Florida Keys and the boreal Gulf of Maine, show that morphological shifts between these two faunas, and in phylogenetic and ecological subgroups shared between them, occur as changes in total variance with a bounded minimum rather than directional shifts. The dispersion of species in shell-shape morphospace is greater in the Gulf of Maine, which also shows a lower variance in ornamentation and size than the Florida Keys, but the faunas do not differ significantly in the ratio of shell to internal volume. Thus, regional differences conform to hypothesized effects of resource seasonality and predation intensity, but not to carbonate saturation or calcification costs. The overall morphological differences between the regional faunas is largely driven by the loss of ecological functional groups and family-level clades at high latitudes, rather than directional shifts in morphology within the shared groups with latitude. Latitudinal differences in morphology thus represent a complex integration of phylogenetic and ecological factors that are best captured in multivariate analyses across several hierarchical levels.

Eight new freshwater mussels (Unionidae) from tropical Asia.

Freshwater mussels are sensitive to habitat and water quality, revealing the fastest rates of human-mediated global extinction among aquatic animals. These animals are especially diverse in tropical Asia, the faunas of which are characterized by high levels of endemism. Here we describe four new species and four new subspecies of freshwater mussels from Myanmar. Leoparreysia whitteni sp. nov., the smallest representative of this genus, was discovered from the Ayeyarwady and Chindwin rivers. Radiatula myitthanensis sp. nov. and R. chindwinensis sp. nov. were recorded from the Chindwin Basin, and R. mouhoti haungthayawensis ssp. nov. has been discovered from the Haungthayaw River. Indochinella pugio has been revised with a description of three subspecies: I. pugio viridissima ssp. nov. from the Sittaung, Bilin and Bago rivers, I. pugio daweiensis ssp. nov. from the Dawei River, and I. pugio paradoxa ssp. nov. from the Haungthayaw River. Yaukthwa elongatula sp. nov., a peculiar species, conchologically resembling representatives of the genus Solenaia (Gonideinae) with ultra-elongated shell was found in the Chindwin Basin. Our records highlight that tropical Asia harbors numerous, but still overlooked local endemic lineages of freshwater bivalves, which may be on the brink of extinction due to the high anthropogenic and climate change impacts.

High-Throughput Segmentation of Tiled Biological Structures using Random-Walk Distance Transforms.

Various 3D imaging techniques are routinely used to examine biological materials, the results of which are usually a stack of grayscale images. In order to quantify structural aspects of the biological materials, however, they must first be extracted from the dataset in a process called segmentation. If the individual structures to be extracted are in contact or very close to each other, distance-based segmentation methods utilizing the Euclidean distance transform are commonly employed. Major disadvantages of the Euclidean distance transform, however, are its susceptibility to noise (very common in biological data), which often leads to incorrect segmentations (i.e., poor separation of objects of interest), and its limitation of being only effective for roundish objects. In the present work, we propose an alternative distance transform method, the random-walk distance transform, and demonstrate its effectiveness in high-throughput segmentation of three microCT datasets of biological tilings (i.e., structures composed of a large number of similar repeating units). In contrast to the Euclidean distance transform, the random-walk approach represents the global, rather than the local, geometric character of the objects to be segmented and, thus, is less susceptible to noise. In addition, it is directly applicable to structures with anisotropic shape characteristics. Using three case studies-tessellated cartilage from a stingray, the dermal endoskeleton of a starfish, and the prismatic layer of a bivalve mollusc shell-we provide a typical workflow for the segmentation of tiled structures, describe core image processing concepts that are underused in biological research, and show that for each study system, large amounts of biologically-relevant data can be rapidly segmented, visualized, and analyzed.

Molecules and morphology reveal 'new' widespread North American freshwater mussel species (Bivalvia: Unionidae).

In the Family Unionidae, the greatest radiation of freshwater mussels, malacologists have been misled by extreme intraspecific shell variation and conversely interspecific conchological stasis or convergence. We characterized the genetic and morphological diversity of two phenotypes of Lampsilis teres from specimens (n = 108) collected across its distribution using geometric and traditional morphometrics and multilocus molecular phylogenetics to test the hypothesis that phenotypes represent separate species. Results from our morphometric and molecular phylogenetic analyses unanimously indicate that L. teres sensu lato is made up of two divergent, widespread species with overlapping distributions. We describe a new species and provide a revised description of L. teres sensu stricto. We use morphometrics and machine-learning classification algorithms to test if shell morphology alone can be used to discriminate between these species. Classification percentages of 97.02% and 93.86% demonstrate that shell morphology is highly informative for species identification. This study highlights our lack of understanding of species diversity of freshwater mussels and the importance of multiple characters and quantitative approaches to species delimitation.

A new genus and two new species of freshwater mussels (Unionidae) from western Indochina.

The systematics of Oriental freshwater mussels (Bivalvia: Unionidae) is poorly known. Here, we present an integrative revision of the genus Trapezoideus Simpson, 1900 to further understanding of freshwater mussel diversity in the region. We demonstrate that Trapezoideus as currently circumscribed is non-monophyletic, with its former species belonging to six other genera, one of which is new to science and described here. We recognize Trapezoideus as a monotypic genus, comprised of the type species, T. foliaceus. Trapezoideus comptus, T. misellus, T. pallegoixi, and T. peninsularis are transferred to the genus Contradens, T. subclathratus is moved to Indonaia, and T. theca is transferred to Lamellidens. Trapezoideus prashadi is found to be a junior synonym of Arcidopsis footei. Trapezoideus dallianus, T. nesemanni, T. panhai, T. peguensis, and two species new to science are placed in Yaukthwa gen. nov. This genus appears to be endemic of the Western Indochina Subregion. The two new species, Yaukthwa paiensis sp. nov. and Y. inlenensis sp. nov., are both endemic to the Salween River basin. Our results highlight that Southeast Asia is a species-rich freshwater mussel diversity hotspot with numerous local endemic species, which are in need of special conservation efforts.

Enhanced discrimination of basophilic cells on mussel digestive gland tissue sections by means of toluidine-eosin staining.

Changes in the cell type composition of the digestive gland epithelium constitute a common and recognized biological response to stress in mussels. Usually, these changes are identified as alterations in the relative proportion of basophilic cells, determined in tissue sections stained with hematoxylin-eosin (H&E) and measured in terms of volume density of basophilic cells (VvBAS) after stereological quantification. However, the identification and discrimination of basophilic cells may be a difficult issue, even for a trained operator, especially when, in circumstances of environmental stress, basophilic cells lose their basophilia and the perinuclear area of digestive cells gains basophilia. Thus, the present study was aimed at exploring the best available practices (BAPs) to identify and discriminate basophilic cells on tissue sections of mussel digestive gland. In a first step, a thorough screening of potentially suitable staining methods was carried out; the final selection included several trichrome staining methods and some of their variants, as well as toluidine-based stains. Next, the sample processing (fixation/dehydration steps) was optimized. Toluidine-eosin (T&E) staining after fixation in 4% formaldehyde at 4 °C for 24 h was considered the BAP to identify and discriminate basophilic cells in the digestive gland of mussels. Using the mussel Mytilus galloprovincialis as a target organism, this approach was successfully applied to quantify VvBAS values after automated image analysis and compared with the conventional H&E staining in different field and laboratory tests. It is worth noting that VvBAS values were always higher after T&E staining than after H&E staining, apparently because discrimination of basophilic cells was enhanced. Thus, until more data are available, any comparison with VvBAS values obtained in previous studies using H&E staining must be done cautiously. Finally, the T&E staining was successfully used to discriminate basophilic cells in tissue sections of other marine molluscs of ecotoxicological interest, including Mytilus edulis, Mytilus trossulus, Crassostrea gigas and Littorina littorea.