A Cambrian spiny stem mollusk and the deep homology of lophotrochozoan scleritomes.

Mollusks encompass enormous disparity, including familiar clams and snails alongside less familiar aculiferans (chitons and vermiform aplacophorans) with complex multicomponent skeletons. Paleozoic fossils trace crown mollusks to forms exhibiting a combination of biomineralized shells and sclerites (e.g., scales, spines, and spicules). We describe a shell-less, Cambrian stem mollusk, Shishania aculeata gen. et sp. nov., with conical, hollow chitinous sclerites and a smooth girdle, broad foot, and mantle cavity. The sclerites have a microstructure of narrow canals consistent with the impressions of chaetal microvilli found in annelids and brachiopods. Shishania sclerites provide a morphological stepping stone between typical chaetae (chitinous bristles) and the external organic part of aculiferan sclerites that encloses a mineralized body. This discovery reinforces a common origin of lophotrochozoan chaetae and the biomineralized aculiferan sclerites, suggesting that the mollusk ancestor was densely covered with hollow chitinous chaetae.

Remarkable shifts in the megabenthic community structure over four decades in Tokyo Bay, Japan, in relation to environmental variations.

We investigated long-term changes in the megabenthic community in Tokyo Bay, Japan, using data from fisheries-independent trawl surveys conducted from 1977 to 2023. In addition, we examined the potential relationship between changes in biotic communities and environmental conditions. The total abundance and biomass exhibited an increasing trend until 1987, followed by a substantial decline from the late 1980s to the 1990s due to a decrease in small to medium-sized fish and crustacean species. Meanwhile, a marked increase in the number of large fish (including elasmobranchs), mollusks, and echinoids, was observed in the 2000s. These shifts in the megabenthic community structure were correlated with an increase in water temperature and a decrease in nutrient concentrations and copepod densities. Cumulative evidence suggests that a remarkable shift in the megabenthic community structure occurred between the 1970s and the 2020s, which was possibly associated with variations in the environmental conditions in Tokyo Bay.

Molluscan pleiotropic FADD involved in innate immune signaling and induces apoptosis.

Fas-associated protein with death domain (FADD) was initially identified as a crucial adaptor protein in the apoptotic pathway mediated by death receptor (DR). Subsequently, many studies have confirmed that FADD plays a vital role in innate immunity and inflammatory responses in animals. However, the function of this pleiotropic molecule in mollusk species has not been well explored. In this study, we successfully verified the gene sequence of FADD in the Zhikong scallop (Chlamys farreri) and designated it as CfFADD. The CfFADD protein contains a conserved death effector and death domains. Phylogenetic analysis showed that CfFADD is a novel addition to the molluscan FADD family with a close evolutionary relationship with molluscan FADD subfamily proteins. CfFADD mRNA expression in various scallop tissues was significantly induced by challenge with pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, and poly(I:C)), suggesting its role in innate immunity in scallops. Co-immunoprecipitation showed that CfFADD interacted with the scallop DR (tumor necrosis factor receptor) and a signaling molecule involved in the Toll-like receptor pathway (interleukin-1 receptor-associated kinase), confirming that CfFADD may be involved in DR-mediated apoptosis and innate immune signaling pathways. Further studies showed that CfFADD interacted with CfCaspase-8 and activated caspase-3. HEK293T cells exhibited distinct apoptotic features after transfection with a CfFADD-expression plasmid, suggesting a functional DR-FADD-caspase apoptotic pathway in scallops. Overexpression of CfFADD led to a significant dose-dependent activation of interferon ß and nuclear factor-κB reporter genes, demonstrating the key role of CfFADD in innate immunity. In summary, our research has confirmed the critical roles of CfFADD in innate immunity and apoptosis and provides valuable information for developing comparative immunology theories.

New records of non-indigenous species from the eastern Mediterranean Sea (Crustacea, Mollusca), with a revision of genus Isognomon (Mollusca: Bivalvia).

We report new data on non-indigenous invertebrates from the Mediterranean Sea (four ostracods and 20 molluscs), including five new records for the basin: the ostracods Neomonoceratina iniqua, Neomonoceratina aff. mediterranea, Neomonoceratina cf. entomon, Loxoconcha cf. gisellae (Arthropoda: Crustacea)-the first records of non-indigenous ostracods in the Mediterranean-and the bivalve Striarca aff. symmetrica (Mollusca). Additionally, we report for the first time Electroma vexillum from Israel, and Euthymella colzumensis, Joculator problematicus, Hemiliostraca clandestina, Pyrgulina nana, Pyrgulina microtuber, Turbonilla cangeyrani, Musculus aff. viridulus and Isognomon bicolor from Cyprus. We also report the second record of Fossarus sp. and of Cerithiopsis sp. cf. pulvis in the Mediterranean Sea, the first live collected specimens of Oscilla galilae from Cyprus and the northernmost record of Gari pallida in Israel (and the Mediterranean). Moreover, we report the earliest records of Rugalucina angela, Ervilia scaliola and Alveinus miliaceus in the Mediterranean Sea, backdating their first occurrence in the basin by 3, 5 and 7 years, respectively. We provide new data on the presence of Spondylus nicobaricus and Nudiscintilla aff. glabra in Israel. Finally, yet importantly, we use both morphological and molecular approaches to revise the systematics of the non-indigenous genus Isognomon in the Mediterranean Sea, showing that two species currently co-occur in the basin: the Caribbean I. bicolor, distributed in the central and eastern Mediterranean, and the Indo-Pacific I. aff. legumen, at present reported only from the eastern Mediterranean and whose identity requires a more in-depth taxonomic study. Our work shows the need of taxonomic expertise and investigation, the necessity to avoid the unfounded sense of confidence given by names in closed nomenclature when the NIS belong to taxa that have not enjoyed ample taxonomic work, and the necessity to continue collecting samples-rather than relying on visual censuses and bio-blitzes-to enable accurate detection of non-indigenous species.

Performance of biological food processing interfaces: Perspectives on the science of mollusc radula.

The Mollusca comprises a diverse range of organisms, with the class Gastropoda alone boasting approximately 80 000 extant species. Their adaptability across various habitats is facilitated by the evolution of the radula, a key structure for food acquisition. The radula's composition and mechanical properties, including its chitinous membrane, teeth, and supporting structures, enable efficient food gathering and processing. Through adaptive tooth morphology and composition, an interplay between radular components is facilitated, which results in collective effects to withstand forces encountered during feeding and reduce structural failure, with the broad range of variations reflecting ecological niches. Furthermore, teeth consist of composite materials with sometimes high contents of iron, calcium, or silicon to reduce wear. During interaction with the food, the radula performs complex three-dimensional motions, challenging to document. Here, we provide a review on the morphology, the mechanical properties, the composition, and various other parameters that contribute to radular performance. Due to, e.g., the smallness of these structures, there are, however, limitations to radular research. However, numerical simulations and physical models tested on substrates offer avenues for further understanding radular function and performance during feeding. These studies not only advance our knowledge of molluscan biology and ecology but also provide inspirations for biomimetic design and further advances in materials engineering.

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To understand the macrozoobenthic community composition and spatial-temporal distribution characteristics of macrobenthos in the waters of Miaodao Archipelago, Yantai, Shandong and its response to habitat changes, we conducted surveys of macrobenthos and environmental elements in the waters of Miaodao Islands in May (spring), August (summer), and October (autumn) in 2022. Results showed that a total of 127 macrozoobenthic species were recorded, with Mollusca and Annelida (Polychaeta) as the dominant taxa, consisting of 47 and 45 species, respectively. The key dominant species included Sternaspis chinensis, Glycinde bonhourei, Moerella hilaris, and Amphioplus (Lymanella) japonicus. The average annual density and biomass of macrozoobenthos were 190 ind·m-2 and 28.69 g·m-2, respectively. There was no significant seasonal differences in density and biomass. The Shannon diversity index (H), evenness index (J), and richness index (D) averaged 3.10, 0.90, and 2.40, respectively. Cluster analysis results showed low similarity coefficients of community among the three seasons, suggesting a distinct distribution pattern. Factors such as bottom seawater temperature, chlorophyll a, nutrient, sediment grain size, and organic matter content could significantly influence the structure and diversity of macrozoobenthic community. Compared with historical research data, the Changdao National Wetland Nature Reserve and the implementation of enclosure aquaculture have led to notable changes in the dominant species of macrobenthos. Specifically, there was a noticeable decline in both density and H, and an increase in biomass and J. Additionally, body size of benthic fauna was transitioning from small to big.

Molluscs generate preferred crystallographic orientation of biominerals by organic templates, the texture and microstructure of Caudofoveata (Aplacophora) shells.

Caudofoveata are molluscs that protect their vermiform body with a scleritome, a mosaic of unconnected blade/lanceolate-shaped aragonite sclerites. For the species Falcidens gutturosus and Scutopus ventrolineatus we studied the crystallographic constitution and crystal orientation texture of the sclerites and the scleritome with electron-backscatter-diffraction (EBSD), laser-confocal-microscopy (LCM) and field-emission electron microscopy (FE-SEM) imaging. Each sclerite is an aragonite single crystal that is completely enveloped by an organic sheath. Adjacent sclerites overlap laterally and vertically are, however, not connected to each other. Sclerites are thickened in their central portion, relative to their periphery. Thickening increases also from sclerite tip towards its base. Accordingly, cross-sections through a sclerite are straight at its tip, curved and bent towards the sclerite base. Irrespective of curved sclerite morphologies, the aragonite lattice within the sclerite is coherent. Sclerite aragonite is not twinned. For each sclerite the crystallographic c-axis is parallel to the morphological long axis of the sclerite, the a-axis is perpendicular to its width and the b-axis is within the width of the sclerite. The single-crystalinity of the sclerites and their mode of organization in the scleritome is outstanding. Sclerite and aragonite arrangement in the scleritome is not given by a specific crystal growth mode, it is inherent to the secreting cells. We discuss that morphological characteristics of the sclerites and crystallographic preferred orientation (texture) of sclerite aragonite is not the result of competitive growth selection. It is generated by the templating effect of the organic substance of the secreting cells and associated extracellular biopolymers.

Zoonotic human liver flukes, a type 1 biocarcinogen, in freshwater fishes: genetic analysis and confirmation of molluscan vectors and reservoir hosts in Bangladesh.

BACKGROUND: Opisthorchiid flukes, particularly Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, and Metorchis spp. are the most common fish-borne zoonotic human liver flukes (hLFs). Liver fluke infections are more prevalent in resource-deprived and underprivileged areas. We herein estimated the prevalence of the metacercariae (MC) of major hLFs in common large freshwater fishes (lFWF) marketed for human consumption from some selected areas of Bangladesh along with detection of their molluscan vectors and reservoirs. METHODS: The current status of fish-borne zoonotic hLF infections in lFWF was investigated along with their molluscan vectors and mammalian reservoir hosts in Mymensingh and Kishoreganj in Bangladesh from July 2018-June 2022 using conventional and multiple molecular techniques, such as PCR, PCR-restriction fragment length polymorphism (RFLP), sequencing, and bioinformatic analyses. The infection rate of fishes was analyzed using the Z-test and the loads of MC were compared using the chi-squared (χ2) test. RESULTS: The MC of C. sinensis, Opisthorchis spp., and Metorchis spp. were detected in 11 species of common and popular lFWF. In lFWF, the estimated prevalence was 18.7% and the mean load was 137.4 ± 149.8 MC per 100 g of fish. The prevalence was the highest (P < 0.05) in spotted snakehead fishes (Channa punctata, 63.6%). The highest rate of infection (P < 0.05) was observed with the MC of C. sinensis (11.8%). Metacercariae were almost equally (P > 0.05) distributed between the head and body of fishes. The infection rate was slightly higher in cultured (19.6%) fishes. The MC of C. sinensis, O. felineus, O. viverrini, and Metorchis orientalis in fishes were confirmed using PCR, PCR-RFLP and bioinformatics. The cercariae of opisthorchiid (Pleurolophocercus cercariae) flukes were only recovered from Bithynia spp. (3.9%, 42 out of 1089). The ova of hLFs from dogs (4.3%, 5 out of 116) and cats (6.0%, 6 out of 100), and adult flukes (M. orientalis) from ducks (41.1% 113 out of 275) were detected. CONCLUSIONS: The MC of hLFs are highly prevalent in fresh water fishes in Bangladesh. Reservoir hosts, such as street dogs, cats, and ducks carried the patent infection, and residents of Bangladesh are at risk.

Diversification of von Willebrand Factor A and Chitin-Binding Domains in Pif/BMSPs Among Mollusks.

Pif is a shell matrix protein (SMP) identified in the nacreous layer of Pinctada fucata (Pfu) comprised two proteins, Pif97 and Pif 80. Pif97 contains a von Willebrand factor A (VWA) and chitin-binding domains, whereas Pif80 can bind calcium carbonate crystals. The VWA domain is conserved in the SMPs of various mollusk species; however, their phylogenetic relationship remains obscure. Furthermore, although the VWA domain participates in protein-protein interactions, its role in shell formation has not been established. Accordingly, in the current study, we investigate the phylogenetic relationship between PfuPif and other VWA domain-containing proteins in major mollusk species. The shell-related proteins containing VWA domains formed a large clade (the Pif/BMSP family) and were classified into eight subfamilies with unique sequential features, expression patterns, and taxa diversity. Furthermore, a pull-down assay using recombinant proteins containing the VWA domain of PfuPif 97 revealed that the VWA domain interacts with five nacreous layer-related SMPs of P. fucata, including Pif 80 and nacrein. Collectively, these results suggest that the VWA domain is important in the formation of organic complexes and participates in shell mineralisation.

Hierarchical diversity partitioning of microscopic epibiont community on intertidal molluscan shells and inert surfaces over three geographic regions in Japan.

Microscopic epibionts on molluscan shells are a component of the biodiversity of intertidal coastal areas. Because molluscan shells are discrete habitats for the epibiont community, and the molluscan basibionts belong to the local community, epibiont diversity can be evaluated hierarchically by basibiont categories including species. To evaluate the structure of epibiont diversity and effects of taxonomic resolution on the evaluation, epibionts on molluscan shells and inert surfaces were investigated at three geographically distant sites in Japan. In total, 94 species-level taxonomic units of epibionts were obtained from 31 basibiont molluscan species and inert surfaces (plastics and rock chips). The density and the species richness at the site of the lowest latitude were significantly lower than those at the other sites. The epibiont community differed between the three sites, although the major portion of the epibionts were diatoms. Between-site diversity contributed most of the total diversity of the species richness and Simpson diversity in the five levels of the hierarchical partitioning: sample (individual basibiont), basibiont species (molluscan species), surface group (bivalves, chitons + limpets, and globose gastropods), site, and the total. The taxonomic resolution did not markedly affect the variability of communities between the three sites, although the taxon richness was reduced to 51 in the genus-level analysis. The lower taxonomic resolution (genus level); however, increased the contribution of the within-sample and decreased the contribution of ß diversities at the higher hierarchies, leading to a possible overestimation of biotic homogenization between the communities.

Development of environmental DNA metabarcoding primers for marine mollusks and comparison with published primers.

Monitoring mollusk biodiversity is a great challenge due to their large diversity and broad distribution. Environmental DNA (eDNA) technology is increasingly applied for biodiversity monitoring, but relevant studies on marine mollusks are still limited. Although previous studies have developed several pairs of primers for mollusk eDNA analyses, most of them targeted only a small group of mollusks. In this study, seven primers were designed for the mollusk community and validated and compared with eight pairs of published primers to select the best candidates. After in silico test, MollCOI154 and MollCOI255 primers showed non-specific amplification, and same results were also obtained in published primers (COI204, Sepi, and veneroida). Moll12S100, Moll12S195 and Moll16S primers failed to amplify across all genomic DNA from selected mollusk. Except Moll16S, all developed and two published (unionoida and veneroida) primers were successfully amplified on four eDNA samples from Yangtze River estuary. After annotation of the amplified sequences, MollCOI253 showed higher annotation of the amplification results than the other primers. In conclusion, MollCOI253 had better performance in terms of amplification success and specificity, and can provide technical support for eDNA-based research, which will be beneficial for molluscan biodiversity investigation and conservation.

A systematic review on microplastic contamination in marine Crustacea and Mollusca of Asia: Current scenario, concentration, characterization, polymeric risk assessment, and future Prospectives.

Microplastics (MPs) pollution has wreaked havoc on biodiversity and food safety globally. The false ingestion of MPs causes harmful effects on organisms, resulting in a decline in biodiversity. The present review comprehended the current knowledge of MP contamination in Crustacea and Mollusca from 75 peer-reviewed articles published in Asia between 2015 and 2023. A total of 79 species (27 Crustacea and 52 Mollusca) have been recorded to be contaminated with MPs. Out of the total 27 species of Crustacea, Metopograpsus quadridentatus (327.56 MPs/individual) and Balanus albicostatus (0.42 MPs/individual) showed the highest and lowest contamination, respectively. Out of the total 52 species of Mollusca, Dolabella auricularia (2325 MPs/individual) and Crassostrea gigas and Mytilus edulis (0.2 MPs/individual) showed the highest and lowest contamination, respectively. In terms of country-wise MP contamination, China has the highest number of contaminated species in both phylums among Asia. Findings of pollution indices revealed a very high risk of MP contamination in all the countries. Fiber was reported predominantly in both groups. Blue and black-colored MPs having <500 µm and <500 µm-1 mm size were found dominantly in Crustacea and Mollusca, respectively. Polypropylene was recorded as the dominant plastic polymer in both Crustacea and Mollusca. In essence, this review has provided a comprehensive insight into MP concentration in Crustacea and Mollusca of Asia, highlighting variations among species and geographic locations. This understanding is crucial for tackling urgent environmental challenges, safeguarding human health, and promoting global sustainability initiatives amid the escalating issue of plastic pollution. PRACTITIONER POINTS: Microplastic pollution has created havoc on biodiversity and food safety. A total of 27 and 52 species of crustaceans and Mollusca have been recorded to be contaminated with MPs. Metopograpsus quadridentate and Dolabella auricularia have shown higher MPs contamination. Polypropylene was recorded as the dominant plastic polymer in both crustacean and Mollusca. Findings of pollution indices revealed a very high risk of MP contamination in all the countries.

Immunotherapeutic Potential of Mollusk Hemocyanins in Murine Model of Melanoma.

The development of antitumor drugs and therapy requires new approaches and molecules, and products of natural origin provide intriguing alternatives for antitumor research. Gastropodan hemocyanins-multimeric copper-containing glycoproteins have been used in therapeutic vaccines and antitumor agents in many cancer models. MATERIALS AND METHODS: We established a murine model of melanoma by challenging C57BL/6 mice with a B16F10 cell line for solid tumor formation in experimental animals. The anticancer properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) were evaluated in this melanoma model using various schemes of therapy. Flow cytometry, ELISA, proliferation, and cytotoxicity assays, as well as histology investigations, were also performed. RESULTS: Beneficial effects on tumor growth, tumor incidence, and survival of tumor-bearing C57BL/6 mice after administration of the RtH or HaH were observed. The generation of high titers of melanoma-specific IgM antibodies, pro-inflammatory cytokines, and tumor-specific CTLs, and high levels of tumor-infiltrated M1 macrophages enhanced the immune reaction and tumor suppression. DISCUSSION: Both RtH and HaH exhibited promising properties for applications as antitumor therapeutic agents and future experiments with humans.

Paralytic Shellfish Toxins in Mollusks from Galicia Analyzed by a Fast Refined AOAC 2005.06 Method: Toxicity, Toxin Profile, and Inter-Specific, Spatial, and Seasonal Variations.

Paralytic shellfish poisoning is an important concern for mollusk fisheries, aquaculture, and public health. In Galicia, NW Iberian Peninsula, such toxicity has been monitored for a long time using mouse bioassay. Therefore, little information exists about the precise toxin analogues and their possible transformations in diverse mollusk species and environments. After the change in the European PSP reference method, a refinement of the Lawrence method was developed, achieving a 75% reduction in chromatogram run time. Since the beginning of 2021, when this refinement Lawrence method was accredited under the norm UNE-EN ISO/IEC 17025, it has been used in the area to determine the toxin profiles and to estimate PSP toxicity in more than 4500 samples. In this study, we have summarized three years of monitoring results, including interspecific, seasonal, and geographical variability of PSP toxicity and toxin profile. PSP was detected in more than half of the samples analyzed (55%), but only 4.4% of the determinations were above the EU regulatory limit. GTX1,4 was the pair of STX analogs that produced the highest toxicities, but GTX2,3 was found in most samples, mainly due to the reduction of GTX1,4 but also by the higher sensitivity of the method for this pair of analogs. STX seems to be mainly a product of biotransformation from GTX2,3. The studied species (twelve bivalves and one gastropod) accumulated and transformed PSP toxins to a different extent, with most of them showing similar profiles except for Spisula solida and Haliotis tuberculata. Two seasonal peaks of toxicity were found: one in spring-early summer and another in autumn, with slightly different toxin profiles during outbreaks in relation to the toxicity during valleys. In general, both the total toxicity and toxin profiles of the southernmost locations were different from those in the northern part of the Atlantic coast and the Cantabrian Sea, but this general pattern is modified by the PSP history of some specific locations.

Biomineral crystallographic preferred orientation in Solenogastres molluscs (Aplacophora) is controlled by organic templating.

Aplacophoran molluscs are shell-less and have a worm-like body which is covered by biomineralized sclerites. We investigated sclerite crystallography and the sclerite mosaic of the Solenogastres species Dorymenia sarsii, Anamenia gorgonophila, and Simrothiella margaritacea with electron-backscattered-diffraction (EBSD), laser-confocal-microscopy and FE-SEM imaging. The soft tissue of the molluscs is covered by spicule-shaped, aragonitic sclerites. These are sub-parallel to the soft body of the organism. We find, for all three species, that individual sclerites are untwinned aragonite single crystals. For individual sclerites, aragonite c-axis is parallel to the morphological, long axis of the sclerite. Aragonite a- and b-axes are perpendicular to sclerite aragonite c-axis. For the scleritomes of the investigated species we find different sclerite and aragonite crystal arrangement patterns. For the A. gorgonophila scleritome, sclerite assembly is disordered such that sclerites with their morphological, long axis (always the aragonite c-axis) are pointing in many different directions, being, more or less, tangential to cuticle surface. For D. sarsii, the sclerite axes (equal to aragonite c-axes) show a stronger tendency to parallel arrangement, while for S. margaritacea, sclerite and aragonite organization is strongly structured into sequential rows of orthogonally alternating sclerite directions. The different arrangements are well reflected in the structured orientational distributions of aragonite a-, b-, c-axes across the EBSD-mapped parts of the scleritomes. We discuss that morphological and crystallographic preferred orientation (texture) is not generated by competitive growth selection (the crystals are not in contact), but is determined by templating on organic matter of the sclerite-secreting epithelial cells and associated papillae.

Taxonomic, phylogenetic, and functional diversity of mollusk death assemblages in coral reef and seagrass sediments from two shallow gulfs in Western Cuban Archipelago.

Mollusk death assemblages are formed by shell remnants deposited in the surficial mixed layer of the seabed. Diversity patterns in tropical marine habitats still are understudied; therefore, we aimed to investigate the taxonomic, phylogenetic, and functional diversity of mollusk death assemblages at regional and local scales in coral reef sands and seagrass meadows. We collected sediment samples at 11 sites within two shallow gulfs in the Northwestern Caribbean Sea and Southeastern Gulf of Mexico. All the shells were counted and identified to species level and classified into biological traits. We identified 7113 individuals belonging to 393 species (290 gastropods, 94 bivalves, and nine scaphopods). Diversity and assemblage structure showed many similarities between gulfs given their geological and biogeographical commonalities. Reef sands had higher richness than seagrasses likely because of a more favorable balance productivity-disturbance. Reef sands were dominated by epifaunal herbivores likely feeding on microphytobenthos and bysally attached bivalves adapted to intense hydrodynamic regime. In seagrass meadows, suspension feeders dominated in exposed sites and chemosynthetic infaunal bivalves dominated where oxygen replenishment was limited. Time averaging of death assemblages was likely in the order of 100 years, with stronger effects in reef sands compared to seagrass meadows. Our research provides evidence of the high taxonomic, phylogenetic, and functional diversity of mollusk death assemblages in tropical coastal sediments as result of the influence of scale-related processes and habitat type. Our study highlights the convenience of including phylogenetic and functional traits, as well as dead shells, for a more complete assessment of mollusk biodiversity.

Laetoli, Tanzania: Extant terrestrial mollusc faunas shed new light on climate and palaeoecology at a Pliocene hominin site.

Laetoli, Tanzania is one of the most important palaeontological and palaeoanthropological localities in Africa. We report on a survey of the extant terrestrial gastropod faunas of the Laetoli-Endulen area, examine their ecological associations and re-examine the utility of Pliocene fossil molluscs in palaeoenvironmental reconstruction. Standardised collecting at 15 sites yielded 7302 individuals representing 58 mollusc species. Significant dissimilarities were found among the faunas of three broad habitat types: forest, woodland/bushland and open (grassland and scattered, xeric shrubland). Overall, more species were recorded in the woodland/bushland sites than in the forest sites. Open sites were less diverse. Environmental factors contributing most strongly to the separation of habitat types were aridity index and elevation. The results are supplemented with new mollusc data from the Mbulu Plateau south of Lake Eyasi, and compared to the list of species cumulatively recorded from the Ngorongoro area. Some regional variation is apparent and historical factors may explain the absence of some fossil taxa from Laetoli today. Differences in seasonality separated upland forest sites on the Mbulu plateau from those at Lemagurut at Laetoli. Indicator species were identified for each habitat. These included several large-bodied species analogous to the Laetoli Pliocene fossil species that were then used for palaeoenvironmental reconstruction. Based on the estimated aridity index, and adopting the widely used United Nations Environment Programme (UNEP) global climate classification, the four stratigraphic subunits of the Upper Laetolil Beds (3.6-3.85 Ma) would be placed in either the UNEP's Dry Sub-humid or Semi-arid climate classes, whereas the Upper Ndolanya Beds (2.66 Ma) and Lower Laetolil Beds (3.85-<4.36 Ma) would be assigned to the Humid and Semi-arid climate classes respectively. Pliocene precipitation at Laetoli is estimated as 847-965 mm per year, refining previous estimates. This is close or slightly higher than the present mean annual precipitation, and is likely to have corresponded to a mosaic of forest, woodland and bushland within a grassland matrix consistent with other reconstructions.

Advancing responsible genomic analyses of ancient mollusc shells.

The analysis of the DNA entrapped in ancient shells of molluscs has the potential to shed light on the evolution and ecology of this very diverse phylum. Ancient genomics could help reconstruct the responses of molluscs to past climate change, pollution, and human subsistence practices at unprecedented temporal resolutions. Applications are however still in their infancy, partly due to our limited knowledge of DNA preservation in calcium carbonate shells and the need for optimized methods for responsible genomic data generation. To improve ancient shell genomic analyses, we applied high-throughput DNA sequencing to 27 Mytilus mussel shells dated to ~111-6500 years Before Present, and investigated the impact, on DNA recovery, of shell imaging, DNA extraction protocols and shell sub-sampling strategies. First, we detected no quantitative or qualitative deleterious effect of micro-computed tomography for recording shell 3D morphological information prior to sub-sampling. Then, we showed that double-digestion and bleach treatment of shell powder prior to silica-based DNA extraction improves shell DNA recovery, also suggesting that DNA is protected in preservation niches within ancient shells. Finally, all layers that compose Mytilus shells, i.e., the nacreous (aragonite) and prismatic (calcite) carbonate layers, with or without the outer organic layer (periostracum) proved to be valuable DNA reservoirs, with aragonite appearing as the best substrate for genomic analyses. Our work contributes to the understanding of long-term molecular preservation in biominerals and we anticipate that resulting recommendations will be helpful for future efficient and responsible genomic analyses of ancient mollusc shells.

Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs.

Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.

Elastic constants of biogenic calcium carbonate.

Living organisms form complex mineralized composite architectures that perform a variety of essential functions. These materials are commonly utilized for load-bearing purposes such as structural stability and mechanical strength in combination with high toughness and deformability, which are well demonstrated in various highly mineralized molluscan shell ultrastructures. Here, the mineral components provide the general stiffness to the composites, and the organic interfaces play a key role in providing these biogenic architectures with mechanical superiority. Although numerous studies employed state-of-the-art methods to measure and/or model and/or simulate the mechanical behavior of molluscan shells, our understanding of their performance is limited. This is partially due to the lack of the most fundamental knowledge of their mechanical characteristics, particularly, the anisotropic elastic properties of the mineral components and of the tissues they form. In fact, elastic constants of biogenic calcium carbonate, one of the most common biominerals in nature, is unknown for any organism. In this work, we employ the ultrasonic pulse-echo method to report the elasticity tensor of two common ultrastructural motifs in molluscan shells: the prismatic and the nacreous architectures made of biogenic calcite and aragonite, respectively. The outcome of this research not only provides information necessary for fundamental understanding of biological materials formation and performance, but also yields textbook knowledge on biogenic calcium carbonate required for future structural/crystallographic, theoretical and computational studies.