Ruditapes Philippinarum Research Articles

Metabolic variation and oxidative stress responses of clams (Ruditapes philippinarum) perturbed by ofloxacin exposure

Ofloxacin (OFL), one of the most widely used fluoroquinolone antibiotics, has been frequently detected in marine environments. Nonetheless, researchers are yet to focus on the effects of OFL on the benthos. In the present study, marine clams (Ruditapes philippinarum) were exposed to OFL (0.5, 50, and 500 μg/L) for 14 d, followed by a 7 d depuration period. The accumulation of OFL, antioxidative defense responses, neurotoxicity, burrowing behavior, and metabolomic changes in clams were evaluated. The results indicated that OFL could accumulate in clams, albeit with a low bioaccumulation capacity. The intermediate (50 μg/L) and high (500 μg/L) levels of OFL induced significant antioxidative responses in the gills and digestive glands of clams, mainly manifesting as the inhibition of catalase activities and the induction of superoxide dismutase and glutathione S-transferase activities, which ultimately elevated the content of malondialdehyde, causing oxidative damage. Furthermore, the significant induction of acetylcholinesterase activities was observed, coinciding with a significant increase in burrowing rates of clams. The high level of OFL affected glycerophospholipid, arachidonic acid, steroid hormone biosynthesis, unsaturated fatty acids biosynthesis, and glycolysis/glycogenesis metabolism. In conclusion, this study has contributed to the understanding of the physiological and biochemical effects and molecular toxicity mechanisms of OFL to marine bivalves.

Geographic patterns and ecological adaptations of Manila clam (Ruditapes philippinarum) along the Chinese coast: Insights from migration dynamics and gene flow

The Manila Clam (Ruditapes Philippinarum) is one of the most economically significant marine bivalves along the China coast. In recent years, due to the escalating scale of aquaculture, the artificial activities of "cross-regional introduction" have to some extent exerted an influence on the genetic diversity and structure of clam larvae, leading to an ongoing occurrence of genetically mixed clam larvae populations and ambiguous genetic foundation of germplasm resources in China. Therefore, there is an imperative need to gain profound insights into the lineage, genetic structure, and assess the adaptability potential of clams originating from both northern and southern regions. We use Specific Locus Amplified Framents technology to simplify genome sequencing of wild clams along the coast of China. Through genetic diversity, genetic structure, gene flow, and selection elimination analysis, the results show that clams can be divided into distinct north-south lineages, with Wafangdian in Liaoning (RPLW), Zhangzi Island in Liaoning (RPLZ), and Laizhou in Shandong (RPSL) comprise the northern lineage, while Lianjiang in Fujian (RPFL) constitutes the southern lineage. The artificial relocation of clams in China has resulted in a blended population structure among local clams. The genetic functional differences between clams from the north and south are primarily manifest in energy metabolism, and the adaptive evolution of clams is mainly associated with temperature. This integrative research not only furnishes valuable insights into the genetic structure of clam populations, thereby contributing to the advancement of natural conservation and genetic breeding efforts for clams but also establishes the groundwork for a comprehensive exploration of clam adaptive evolution.

Preventing illegal seafood trade using machine-learning assisted microbiome analysis

BackgroundSeafood is increasingly traded worldwide, but its supply chain is particularly prone to frauds. To increase consumer confidence, prevent illegal trade, and provide independent validation for eco-labelling, accurate tools for seafood traceability are needed. Here we show that the use of microbiome profiling (MP) coupled with machine learning (ML) allows precise tracing the origin of Manila clams harvested in areas separated by small geographic distances. The study was designed to represent a real-world scenario. Clams were collected in different seasons across the most important production area in Europe (lagoons along the northern Adriatic coast) to cover the known seasonal variation in microbiome composition for the species. DNA extracted from samples underwent the same depuration process as commercial products (i.e. at least 12 h in open flow systems).ResultsMachine learning-based analysis of microbiome profiles was carried out using two completely independent sets of data (collected at the same locations but in different years), one for training the algorithm, and the other for testing its accuracy and assessing the temporal stability signal. Briefly, gills (GI) and digestive gland (DG) of clams were collected in summer and winter over two different years (i.e. from 2018 to 2020) in one banned area and four farming sites. 16S DNA metabarcoding was performed on clam tissues and the obtained amplicon sequence variants (ASVs) table was used as input for ML MP. The best-predicting performances were obtained using the combined information of GI and DG (consensus analysis), showing a Cohen K-score > 0.95 when the target was the classification of samples collected from the banned area and those harvested at farming sites. Classification of the four different farming areas showed slightly lower accuracy with a 0.76 score.ConclusionsWe show here that MP coupled with ML is an effective tool to trace the origin of shellfish products. The tool is extremely robust against seasonal and inter-annual variability, as well as product depuration, and is ready for implementation in routine assessment to prevent the trade of illegally harvested or mislabeled shellfish.

Manila clams Ruditapes philippinarum spreading north and establishing in the European Wadden Sea

In an era of accelerating biological globalization and climatic warming, it is vital to understand how introduced species integrate. Pacific Manila clams Ruditapes philippinarum were introduced for aquaculture to Europe in the 1970s, spread and became harvested from the Mediterranean Sea to British coasts. From the Dutch Rhine Delta, Manila clams immigrated stepwise into the southern Wadden Sea, and by long distance jump dispersal they arrived in the northern Wadden Sea in the 2010s. Encounters remained few until 2021, when live clams were often found in intertidal seagrass beds and around mixed beds of Pacific oysters with native mussels. Shell lengths reached up to 74 mm with a longevity of 7–8 years. Adult abundances remained low (<10 clams m−2), although larval numbers substantially increased in 2022 and 2023. Strong predation on spat may constitute ecological resistance to the immigrant. Haplotype diversity at mtDNA COI gene fragments is high and its composition is mixed from dominant haplotypes of other European sites, suggesting multiple introductions or an unknown source with an already mixed population. Currently, this is the most northern (55° N) population of R. philippinarum in continental Europe, but with the rapid expansion of this genetically variable population, further northward spread can be expected.

Assessing the Effects of a Diet of BPA Analogue-Exposed Microalgae in the Clam Ruditapes philippinarum.

In our previous study, we demonstrated that the microalgae Phaeodactylum tricornutum can bioaccumulate bisphenol A analogues. Since this microalgae species is part of the diet of marine filter-feeding organisms, such as bivalves, in this study we tested the hypothesis that a diet based on exposed microalgae can exert negative effects on the clam Ruditapes philippinarum. Microalgae were exposed for 7 days to 300 ng/L of bisphenol AF (BPAF), bisphenol F (BPF), and bisphenol S (BPS), alone or as a mixture (MIX), to allow bioaccumulation. Microalgae were then supplied as food to bivalves. After 7 and 14 days of diet, the effects of exposed microalgae were evaluated on a battery of biomarkers measured in haemolymph/haemocytes, gills and digestive glands of clams. In addition, bioaccumulation of the three bisphenols was investigated in clams by UHPLC-HRMS. The results obtained demonstrated that total haemocyte count (THC) increased in clams following ingestion for 7 days of BPAF- and BPF-exposed microalgae, while BPS-exposed microalgae significantly reduced THC after 14 days of diet. MIX- and BPS-exposed microalgae increased haemocyte proliferation. The diet of exposed microalgae affected acid and alkaline phosphatase activity in clams, with an opposite response between haemolymph and haemocytes. Regarding antioxidants, an increase in catalase activity was observed in clams after ingestion of BPA analogue-exposed microalgae. The results also demonstrated marked oxidative stress in gills, the first tissue playing an important role in the feeding process. Oxidative damage was recorded in both the gills and digestive glands of clams fed BPA analogue-exposed microalgae. Alterations in epigenetic-involved enzyme activity were also found, demonstrating for the first time that BPA analogue-exposed food can alter epigenetic mechanisms in marine invertebrates. No bioaccumulation of BPA analogues was detected in clam soft tissues. Overall, this study demonstrated that a diet of BPA analogue-exposed microalgae can induce significant alterations of some important biological responses of R. philippinarum. To our knowledge, this is the first study demonstrating the effects of ingestion of BPA analogue-exposed microalgae in the clam R. philippinarum, suggesting a potential ecotoxicological risk for the marine food chain, at least at the first levels.

SmithHunter: a workflow for the identification of candidate smithRNAs and their targets

BackgroundSmithRNAs (Small MITochondrial Highly-transcribed RNAs) are a novel class of small RNA molecules that are encoded in the mitochondrial genome and regulate the expression of nuclear transcripts. Initial evidence for their existence came from the Manila clam Ruditapes philippinarum, where they have been described and whose activity has been biologically validated through RNA injection experiments. Current evidence on the existence of these RNAs in other species is based only on small RNA sequencing. As a preliminary step to characterize smithRNAs across different metazoan lineages, a dedicated, unified, analytical workflow is needed.ResultsWe propose a novel workflow specifically designed for smithRNAs. Sequence data (from small RNA sequencing) uniquely mapping to the mitochondrial genome are clustered into putative smithRNAs and prefiltered based on their abundance, presence in replicate libraries and 5′ and 3′ transcription boundary conservation. The surviving sequences are subsequently compared to the untranslated regions of nuclear transcripts based on seed pairing, overall match and thermodynamic stability to identify possible targets. Ample collateral information and graphics are produced to help characterize these molecules in the species of choice and guide the operator through the analysis. The workflow was tested on the original Manila clam data. Under basic settings, the results of the original study are largely replicated. The effect of additional parameter customization (clustering threshold, stringency, minimum number of replicates, seed matching) was further evaluated.ConclusionsThe study of smithRNAs is still in its infancy and no dedicated analytical workflow is currently available. At its core, the SmithHunter workflow builds over the bioinformatic procedure originally applied to identify candidate smithRNAs in the Manila clam. In fact, this is currently the only evidence for smithRNAs that has been biologically validated and, therefore, the elective starting point for characterizing smithRNAs in other species. The original analysis was readapted using current software implementations and some minor issues were solved. Moreover, the workflow was improved by allowing the customization of different analytical parameters, mostly focusing on stringency and the possibility of accounting for a minimal level of genetic differentiation among samples.

Combinatorial accumulation, stress response, detoxification and synaptic transmission effects of cadmium and selenium in clams Ruditapes philippinarum

This study investigated the toxicological effects and mechanisms of cadmium (Cd) (5 and 50 μg/L) and selenium (Se) (3 and 30 μg/L) at environmentally relevant concentrations on the gills and digestive glands of clams Ruditapes philippinarum. Results indicated that Cd and Se could tissue-specifically impact osmoregulation, energy metabolism, and synaptic transmission in the gills and digestive glands of clams. After exposure to 50 μg/L Cd, the digestive glands of clams up-regulated the expression of methionine-gamma-lyase and metallothionein for detoxification. Clam digestive glands exposed to 3 μg/L Se up-regulated the expression of catalase and glutathione peroxidase to alleviate oxidative stress, and down-regulated the expression of selenide-water dikinase to reduce the conversion of inorganic Se. Additionally, the interaction mode between Cd and Se largely depended on their molar ratio, with a ratio of 11.71 (50 μg/L Cd + 3 μg/L Se) demonstrated to be particularly harmful, as manifested by significantly more lesions, oxidative stress, and detoxification demand in clams than those exposed to Cd or Se alone. Collectively, this study revealed the complex interaction patterns and mechanisms of Cd and Se on clams, providing a reference for exploring their single and combined toxicity.

Studies on the characteristics of polycyclic aromatic hydrocarbons accumulation in lipids and the disturbance of lipid metabolism of Ruditapes philippinarum

Polycyclic aromatic hydrocarbons (PAHs) constitute a class of persistent organic pollutants with strong lipophilicity, which readily accumulate within organisms and have the effect to induce disorders in lipid metabolism. The present study aimed to investigate the accumulation localization and pattern of PAHs in Ruditapes philippinarum, and to reveal the association between PAHs and lipids metabolism. The 21-day exposure experiment was conducted using a mixture of phenanthrene, chrysene, and benzo[a]pyrene (the proportion is 1:1:1) at concentrations of 0.4 μg/L, 2 μg/L, and 10 μg/L. The tissue distribution of PAHs indicated that the digestive gland was the primary site of PAHs accumulation. Meanwhile, fluorescence colocalization suggested that PAHs primarily accumulated within the lipid droplets of digestive gland cells. This study further determined the transcriptomic and lipidomic profiles of the digestive gland to analyze the key genes involved in disrupted lipid metabolism and the major lipids affected. Lipidomic analysis identified the key differential metabolites as triglycerides (TGs). Furthermore, TGs were upregulated in the digestive gland had a total carbon atom number of 50–64 and a total number of 3–9 double bonds in the acyl side chains. Biochemical analysis experiments and oil red O stained frozen sections confirmed that the content of TGs steadily increased in various tissues during the experiment, leading to an elevated digestive gland index. Changes of lipid metabolism associated genes expression level also indicated that the synthesis of lipid in digestive gland were up-regulated while the decomposition was down-regulated. This study is the first to demonstrate the cellular localization of PAHs accumulation in bivalves and confirms the pattern of variation in TGs, providing new insights into the mechanisms of PAHs bioaccumulation and lipid metabolism disruption.

Heat hardening enhances the energy metabolism activity and oxidative defense ability of Manila clam Ruditapes philippinarum under different thermal stress

Heat-hardening treatment can improve the thermal tolerance of aquatic organisms, likely due to enhanced antioxidant capacity and energy metabolism. In this study, the Heat-hardened Manila clam exhibited an increase in survival rate ranging from 15.5% to 80% compared to Non-hardened Manila clam under acute heat stress. Under chronic heat stress, following sub-lethal heat shock treatment, the activities of antioxidant enzymes-Catalase (CAT), Glutathione peroxidase (GSH-PX), Superoxide dismutase (SOD) in Heat-hardened Manila clam was significantly elevated in comparison to the control group (P < 0.05). Compared with the non-hardened Manila clam, the activity of energy metabolism enzymes - Na+/K+-ATPase (NKA), Ca2+/Mg2+-ATPase (CMA) of heat-hardened Manila clam decreased after the first sublethal heat shock treatment, and increased during recovery treatment, and the total ATPase activity significantly increased. Within 7 days of chronic heat stress, the activities of CAT and SOD in the Heat-hardened group higher than that in the non-hardened group. The balance between antioxidant ability and energy metabolism may be critical for the enhanced thermal tolerance observed in the Heat-hardened Manila clam. These findings indicated that Heat-hardening treatment is likely to enhance the resistance of Manila clam to high temperature in the natural environment. Such insights could be advantageous in aquaculture to protect Manila clam from the detrimental effects of heat stress.

Characterization and expression patterns of the Fox gene family under heat and cold stress in Manila clam Ruditapes philippinarum based on genome-wide identification

In this study, the Fox gene family of Ruditapes philippinarum was identified by bioinformatics analysis and genome data. The results showed that a total of 21 Fox genes were identified in R. philippinarum, which were divided into 16 subfamilies, including two members of Foxa subfamily (Foxa1, Foxa2), three members of Foxl subfamily (Foxl1b, Foxl1a, FOXL2), three members of Foxn subfamily (FOXN3, FOX4A, Foxn4b) and one member of other families. The chromosome distribution, domains, conserved motifs, introns, exons and protein tertiary structures of these 21 Fox genes were predicted. By analyzing the RNA-seq data of R. philippinarum, it was found that the Fox gene family was differentially expressed in different tissues, different developmental stages and under heat and cold stress. Most of Fox genes were highly expressed in four tissues: labial palp, gonad, gill and foot. Most of the Fox genes were highly expressed in blastula stage. Most of the Fox genes were highly expressed in high temperature group of two populations, and Foxo, FOXG1 were highly expressed in low temperature group. In addition, qPCR showed that the expression levels of Foxo and Foxj1b genes increased significantly under acute cold stress. Therefore, we speculate that Fox genes may play important roles in embryo development and the temperature stress of R. philippinarum, and this study provides a basis for further exploring the molecular mechanism of low temperature tolerance mediated by Fox.

Discover the secrets of salty taste in Manila clam (Ruditapes philippinarum): MALDI-TOF MS and flavor fingerprint

In this work, the ultrasound-assisted enzymatic (UAE) conditions for salty compounds from Manila clam were optimized using a combination of fractional factorial design (FFD) and Box-Behnken design (BBD). The optimum protein extraction rate (40.13 ± 0.37), DH (25.92 ± 0.78), and salty score (4.01 ± 0.02) of salty extract were achieved with a solid-liquid ratio of 1:4 (w/w), hydrolysis temperature of 49 °C, and ultrasound time of 10 min. Sensory results showed that the UAE hydrolysates exhibited pronounced saltiness (6.25 ± 0.03) and umami (6.62 ± 0.03), with an impressively low detection threshold for salty taste at 69 mmol/L. The electronic tongue analysis revealed that the UAE hydrolysate had a distinct saltiness (3.73 ± 0.05), umami (8.96 ± 0.02) and richness (0.83 ± 0.00). The molecular weights of the peptides in the UAE solution were 84.497 Da, 211.710 Da, 380.530 Da, 568.644 Da and 830.696 Da, respectively. It was rich in alcohols, ketones and furans, and total of 79 volatile compounds (VOCs) were identified. After ultrasonic pretreatment, the content of organic acids was significantly increased, the content of succinic acid was 8.5 ± 0.24, and the content of malic acid was 4.68 ± 0.07. In conclusion, ultrasound-assisted enzymatic is an effective treatment for the extraction of salty compounds.

Prefacing the challenge – Assessment of the environmental efficiency of Manila clam (Ruditapes philippinarum) production based on hatchery-produced and wild seed

In 2021, Italy produced 23 thousand tons of Manila clam (Ruditapes philippinarum, Adam & Reeve 1850) from aquaculture, worth € 212 million, ranking as the world's second-largest producer. Production volumes of Manila clam have started to decline over the past 10 years due to, among other reasons, a decrease in the availability of wild seed on which clam farmers rely. This has led Italian clam farmers to buy seed from both national and foreign hatcheries. The Manila clam supply chain based on hatchery seed brings benefits to farmers, ensuring the continuity of production, but, on the other hand, production and environmental costs increase. As wild seed supply is expected to further decline due to climate change effects and habitat degradation, it is important to assess the environmental impacts of the actual production chains (conventional-wild seed and hatchery-based) to identify the hotspots and possible mitigation strategies. To this aim, this work applies the Life Cycle Assessment methodology to two case studies: a production chain based on wild seed, and, for the first time, a production chain based exclusively on seed produced in hatcheries. The results confirmed the general good environmental performance of mollusk aquaculture. For example, the Global Warming potential ranged from 0.85 to 1.05 kg CO2 eq kg−1 of clams, where the worst results refer to the production chain based on seed from the hatchery, as expected. Some production chain hotspots were highlighted: especially regarding the Global Warming potential, electricity consumption for the hatchery and depuration phases, and the use of fuels during the growth-out phase. Finally, scenario analysis has been applied to the worst-performing case study to understand the magnitude of the environmental benefits deriving from the adoption of some improvement strategies.

Diving into the metabolic interactions of titanium dioxide nanoparticles in “Sparus aurata” and “Ruditapes philippinarum”

The biological response to nanomaterials exposure depends on their properties, route of exposure, or model organism. Titanium dioxide nanoparticles (TiO2 NPs) are among the most used nanomaterials; however, concerns related to oxidative stress and metabolic effects resulting from their ingestion are rising. Therefore, in the present work, we addressed the metabolic effects of citrate-coated 45 nm TiO2 NPs combining bioaccumulation, tissue ultrastructure, and proteomics approaches on gilthead seabream, Sparus aurata and Japanese carpet shell, Ruditapes philippinarum. Sparus aurata was exposed through artificially contaminated feeds, while R. philippinarum was exposed using TiO2 NPs-doped microalgae solutions. The accumulation of titanium and TiO2 NPs in fish liver is associated with alterations in hepatic tissue structure, and alteration to the expression of proteins related to lipid and fatty acid metabolism, lipid breakdown for energy, lipid transport, and homeostasis. While cellular structure alterations and the expression of proteins were less affected than in gilthead seabream, atypical gill cilia and microvilli and alterations in metabolic-related proteins were also observed in the bivalve. Overall, the effects of TiO2 NPs exposure through feeding appear to stem from various interactions with cells, involving alterations in key metabolic proteins, and changes in cell membranes, their structures, and organelles. The possible appearance of metabolic disorders and the environmental risks to aquatic organisms posed by TiO2 NPs deserve further study.

Survey of trematodes in Manila clam Ruditapes philippinarum on the west coast of Korea: A preliminary study

The Manila clam Ruditapes philippinarum on the west coast of Korea harbors several digenetic trematodes. However, most studies in this region have been restricted to a few sampling sites and the current species designation of some trematodes is largely based on morphology, leaving the molecular phylogenetic position among the Digenea unsolved. Thus, we first provide both morphology and molecular phylogeny of some components in the trematodes community in the Manila clam based on a large-scale survey of 26 sites on the west coast, where well-developed tidal flats serve as large commercial clam culture beds. Our study revealed that the trematodes community in the clams consisted of at least 5 species that belong to 3 families (Himasthlidae, Gymnophallidae, Baccigeridae) and 1 superfamily (Monorchioidea). The life mode of the 5 different species included the metacercaria and sporocyst, with one species (Parvatrema duboisi) utilizing the clams as both the first and/or second intermediate host. Trematode infection prevalences were not evenly distributed among the study sites, although the reasons behind this are yet to be determined. Morphological identification was confirmed with the molecular analyses based on ITS and 28S rDNA; phylogenetic analysis also revealed that Cercaria pectinata infecting the clam gonad should be referred to as Bacciger bacciger hereafter. The present preliminary study provides a crucial baseline that could be further developed in a future study on the digenean trematodes community in the Manila clam.

Genetic diversity of the Ruditapes decussatus and evidence of its hybridization with the alien R. philippinarum in the Western Mediterranean Sea

The introduction of alien species in marine ecosystems is often driven by the increasing demand of fishery resources. This is the case of the Manila clam (R. philippinarum), imported in Europe from Japan since the 1970s, to meet the growing demand for clams that the native species, the grooved carpet shell clam (Ruditapes decussatus), could not satisfy. Alien species introduction could threaten the genetic diversity and integrity of the native clam, also causing hybridization (i.e., gene flow from one species into the gene pool of another). Since R. philippinarum recently spread in a few important Mediterranean coastal areas, a combined approach based on morphological characteristics, length differences of two nuclear species-specific markers (ITS2, 5SrDNA) and the sequence of the mitochondrial gene cytochrome c oxidase subunit I (COI), was used to investigate the presence of hybrids in six Mediterranean wetlands (Sardinia, Italy). Eight individuals morphologically identified as R. decussatus were hybrids, having sequences specific to both R. decussatus and R. philippinarum in their nuclear DNA (ITS2 and 5SrDNA). Most of these individuals were found to be post-first generation (F1) hybrids indicating that F1-hybrids may be fertile. Secondly, to study the genetic diversity of R. decussatus in the Sardinian wetlands as well as in its whole distribution area, >380 new COI sequences from the eastern Atlantic Ocean and Mediterranean Sea were analysed along with those available from public databases. Mitochondrial COI data revealed variable haplotype and nucleotide diversities in different areas, which were not dependent on sample sizes. The aquaculture breeding activities and clam transplantation between different countries, along with the long pelagic larval dispersal and the commercial import of other bivalve species might have promoted gene exchange between different sites and thus higher diversity levels in a few wild populations. Our research, evaluating the genetic makeup of wild and hatchery stocks and clarifying the degree of hybridization, can contribute to develop further recommendations for conserving the genetic integrity of R. decussatus.

From a validated targeted method to a retrospective UHPLC-HRMS non-targeted analysis unveiling COVID-19-related contaminants in clams. Have we bias in marine model organisms for ecotoxicological studies?

This study explores a retrospective non-targeted analysis (NTA), based on Ultra High-Performance Liquid Chromatography coupled to High-Resolution Mass Spectrometry (UHPLC-HRMS), to assess hidden chemicals of emerging concern (CECs) in marine model organisms. Conventional ecotoxicological studies do not include evaluating the natural habitats of the collected organisms, missing the possibility of highlighting unexpected pollutants, and thus compromising the correctness and reliability of the experimental results. In this paper we reprocessed samples previously collected from the Venice Lagoon for ecotoxicological studies and used for targeted analysis of three bisphenols-related compounds (i.e. BPS, BPF and BPAF) on seawater and specimens of the clam Ruditapes philippinarum. Results from the validation were the following: accuracy, expressed as percentage recoveries (R%), in the range 80%<R%<120% for all the considered compounds and matrices, and precision, expressed as relative standard deviation of the absolute areas of IS, was <20% for clams (N = 24) and <15% for seawater (N = 30). LODs ranged from 5 to 50 ng/L for seawater and 5–12 ng/g for clam tissues.After validation, a retrospective NTA was carried out on control samples, showing the presence of some CECs, i.e. Lopinavir, Ritonavir, DEHS, and DEHA, two antiviral drugs used during the COVID-19 pandemic and two plasticizers, respectively, reported here for the first time in the Mediterranean Sea. CEC identifications were confirmed by matching MS/MS spectra with libraries. The present study emphasizes the importance of retrospective investigations to describe the contamination scenario of environmental matrices and the related effect in biota, to correctly address studies on model animals, also including possible “cocktail effects”.

Extraction of melanin from the shell and mantle and activities of enzymes related to the melanin biosynthesis in the Ruditapes philippinarum

The shell surface of Manila clam (Ruditapes philippinarum) has a variety of colors and stripes, with diverse shell color polymorphism. As an important trait of economic shellfish, shell color plays an important role in the selection breeding of shellfish species. In this study, we extracted melanin from the shell and mantle of R. philippinarum by the hydrochloric acid method and measured the melanin content by spectrophotometry. The absorption spectrum of melanin and its absorption peak at 350 nm absorbance was determined. We found significant differences in the melanin content of clams with dark strain and light strain, as well as four different shell colors. Moreover, we further detected the activities of enzymes related to melanin synthesis in R. philippinarum mantle, such as tyrosinase (TYR), tyrosine hydroxylase (TH), dopa decarboxylase (DDC), N-acetyltransferase (NAT), and N-β- alanyldopamine (NBAD) in the mantle of R. philippinarum. The results showed that the activities of TYR, TH, DDC and NAT were positively correlated with the content of melanin. However, the activity of NBAD was negatively correlated with the content of melanin. Altogether, these results indicating that these enzymes are involved in the regulation of the melanogenesis pathway of R. philippinarum. This study provides valuable information for further research on shell color genetic mechanism and breeding of R. philippinarum.

MiRNA-mRNA joint analysis reveals the response mechanism of Ruditapes philippinarum to CO2-driven ocean acidification

To explore the response mechanism of Ruditapes philippinarum to CO2-driven ocean acidification, 540 clams with healthy physique and consistent specifications were randomly divided into 3 groups, with 3 repetitions in each group, and 60 specimens in each repetition. Stress tests were then conducted under different pH conditions (8.0, 7.2, 6.4). After 96 hours of stress, gill tissues were collected for mRNA-seq and miRNA-seq analysis. The results show that when Philippine clams are stimulated by seawater acidification, the gene expression levels in the glycine, serine and threonine metabolism and arachidonic acid metabolism pathways in their gill tissue are increased, thereby inducing the occurrence of inflammation in the body and reducing the body's immunity and disease resistance. Philippine clams can also alleviate the adverse effects of seawater acidification on the body by regulating the expression of genes such as miR-184–3p. And when Philippine clams are stimulated by seawater acidification, they can also regulate the interferon-inducible GTPase (IIGP) by regulating the expression of Novel-m0002–3p, which ultimately affects the cellular defense mechanism, material transport ability, and ion exchange ability with the external environment.

Estrogen receptor knockdown suggests its role in gonadal development regulation in Manila clam Ruditapes philippinarum

The estrogen receptor (ER), a ligand-dependent transcription factor, is critical for vertebrate reproduction. However, its role in bivalves is not well understood, with ongoing debates regarding its function in regulating reproduction similarly to vertebrates. To investigate ER's function, we conducted a 21-day RNA interference experiment focusing on its role in gonadal development in bivalves. Histological analyses revealed that ER inhibition significantly suppressed ovarian development in females and, conversely, promoted gonadal development in males. Additionally, levels of 17β-estrogen (E2) were markedly reduced in the gonads of both sexes following ER suppression. Transcriptomic analysis from RNA-seq of testes and ovaries after ER interference showed changes in the expression of key genes such as Vtg, CYP17, 3β-HSD, and 17β-HSD. These genes are involved in the estrogen signaling pathway and steroid hormone biosynthesis. Furthermore, ER suppression significantly affected the expression of genes linked to gametogenesis and the reproductive cycle. Our findings highlight ER's crucial, yet complex and sex-specific roles in gonadal development in bivalves, emphasizing the need for further detailed studies.

Environmental concentration of ammonia nitrogen induced marked changes in proteome of clam Ruditapes philippinarum in dose- and time-dependent manner

Environmental concentration of ammonia nitrogen induced marked changes in proteome of clam Ruditapes philippinarum in dose- and time-dependent manner