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OBJECTIVES: The two oyster species studied hold considerable economic importance for artisanal harvest (Crassostrea rhizophorae) and aquaculture (Crassostrea gasar). Their draft genomes will play an important role in the application of genomic methods such as RNAseq, population-based genomic scans aiming at addressing expression responses to pollution stress, adaptation to salinity and temperature variation, and will also permit investigating the genetic bases and enable marker-assisted selection of economically important traits like shell and mantle coloration and resistance to temperature and disease. DATA DESCRIPTION: The draft assembly size of Crassostrea gasar is 506 Mbp, and of Crassostrea rhizophorae is 584 Mbp with scaffolds N50 of 11,3 Mbp and 4,9 Mbp, respectively. The general masked bases by RepeatMasker in both genomes were highly similar using different datasets. The masked bases varied from 9.41% in C. gasar to 10.05% in C. rhizophorae and 42.85% in C. gasar to 44.44% in C. rhizophorae using Dfam and RepeatModeler datasets, respectively. Functional annotation with eggNog resulted in 34,693 annotated proteins in C. rhizophorae and 26,328 in C. gasar. BUSCO analysis shows that almost 99% of genes (5,295) are complete in relation to the mollusk orthologous genes dataset (mollusca_odb10).

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The activities of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), and glutathione-S-transferase (GST) were evaluated in the gills (GI) and digestive gland (DG) of Magallana gigas oysters exposed to tamoxifen (TAM) at environmental concentrations of 10 and 100 ng L-1 for 1 and 4 days. A higher CAT activity in the GI and DG and higher GPx activity only in the DG was observed of oysters exposed to both concentrations after 1 day. Furthermore, a significant increase in GR and G6PDH, was detected in the DG after 1 day of exposure to 10 ng L-1 and only G6PDH activity increase after 1 day of exposure to 10 ng L-1 in the GI. This suggests that the DG is a tissue more sensitive to TAM exposure and was confirmed with the individual Integrated Biomarker Response version 2 index (IBRv2i), highlighting the acute stress caused by TAM and a cellular adaptation.

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This study assessed the effects of pollutants on Magallana gigas along a coastal zone with different levels of human activity: a highly impacted zone in the Bahía Blanca Estuary and a less impacted zone on the adjacent sandy beaches. Oysters collected in 2021 were analyzed for various factors, including metals, polycyclic aromatic hydrocarbons (PAHs), organochlorine pesticides, microplastics, oxidative stress and histology. Oysters of both environments exhibited detectable concentrations of all these pollutants in their tissues. However, the estuarine oysters showed higher concentrations of Zn, Cu and As and total PAHs than the beach oysters. Banned organochlorine pesticides were detected only in beach oysters. Estuarine oysters displayed morphological changes in their digestive gland including a reduction in the mean epithelial thickness of the tubule and elevated lipid peroxidation levels, indicating cellular damage. This study underscores the widespread presence of pollutants in M. gigas, indicating the need for effective strategies to safeguard coastal ecosystem health.

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Contamination of aquatic environments has been steadily increasing due to human activities. The Pacific oyster Crassostrea gigas has been used as a key species in studies assessing the impacts of contaminants on human health and the aquatic biome. In this context, cytochrome P450 (CYPs) play a crucial role in xenobiotic metabolism. In vertebrates many of these CYPs are regulated by nuclear receptors (NRs) and little is known about the NRs role in C. gigas. Particularly, the CgNR5A represents a homologue of SF1 and LRH-1 found in vertebrates. Members of this group can regulate genes of CYPs involved in lipid/steroid metabolism, with their activity regulated by other NR, called as DAX-1, generating a NR complex on DNA response elements (REs). As C. gigas does not exhibit steroid biosynthesis pathways, CgNR5A may play other physiological roles. To clarify this issue, we conducted an in silico investigation of the interaction between CgNR5A and DNA to identify potential C. gigas CYP target genes. Using molecular docking and dynamics simulations of the CgNR5A on DNA molecules, we identified a monomeric interaction with extended REs. This RE was found in the promoter region of 30 CYP genes and also the NR CgDAX. When the upstream regulatory region was analyzed, CYP2C39, CYP3A11, CYP4C21, CYP7A1, CYP17A1, and CYP27C1 were mapped as the main genes regulated by CgNR5A. These identified CYPs belong to families known for their involvement in xenobiotic and lipid/steroid metabolism. Furthermore, we reconstructed a trimeric complex, previously proposed for vertebrates, with CgNR5A:CgDAX and subjected it to molecular dynamics simulations analysis. Heterotrimeric complex remained stable during the simulations, suggesting that CgDAX may modulate CgNR5A transcriptional activity. This study provides insights into the potential physiological processes involving these NRs in the regulation of CYPs associated with xenobiotic and steroid/lipid metabolism.

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Marine mollusk production is increasing worldwide, and this trend is being evidenced in South American countries, where several species of bivalves are produced, exploited, and traded. This activity brings benefits either for the ecosystem, as it is a less impactful and polluting than other aquaculture practices, and to coastal human communities, as it provides food and income. However, emergence of outbreaks by pathogens is a major concern and can put an entire developing sector at risk. Perkinsosis is a disease caused by Perkinsus spp. protozoans that affect mollusks worldwide. In this review we provide information on Perkinsus spp. among bivalves from South America. Infections by these parasites were only reported to date among coastal Atlantic bivalves of Argentina, Uruguay, and Brazil. The vast majority of cases and studies are reported from Brazil. We comprehensively review those results here. Finally, we suggest some considerations for future investigations that may expand our knowledge of these parasites.

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This study aimed to carry out a general diagnosis of the contamination of the coastal marine environment of the Santa Catarina state (SC, Brazil) by different classes of environmental pollutants, as well as to evaluate possible adverse effects of the contaminants on biochemical biomarkers of oysters, Crassostrea gasar and Crassostrea rhizophorae. 107 chemicals were evaluated in water, sediment and oyster samples from nine sites along the coastline of SC. We also examined various biochemical biomarkers in the oysters' gills and digestive glands to assess potential effects of contaminants. In general, the northern and central regions of the littoral of SC presented higher occurrences and magnitudes of contaminants than the southern region, which is probably related to higher urbanization of center and northern areas of the littoral. The biomarker analysis in the oysters reflected these contamination patterns, with more significant alterations observed in regions with higher levels of pollutants. Our results may serve as a first baseline for future and more extensive monitoring actions and follow-up of the degree of contamination in the state, allowing for inspection actions and management of areas most affected by marine pollutants.

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In biomonitoring and laboratory studies, it is typical to measure a battery of molecular, biochemical, and cytogenetic biomarkers to evaluate the effects of xenobiotics in biota. However, summarizing the results of several biomarkers to inform laypersons and environmental agencies is still a challenge for researchers and environmental specialists. To address this issue, researchers have developed indexes such as the Integrated Biomarker Responses (IBR) and Integrated Biomarker Response version 2 (IBRv2) to summarize all biomarkers responses into a single value. Unfortunately, these indexes do not preserve the original biological variability, which hampers subsequent statistical analyses. In this study, we created new versions of IBR and IBRv2, which preserve individual data variability and can be used in typical statistical analyses. The new Integrated Biomarker Responses individual (IBRi), Integrated Biomarker Responses version 2 individual (IBRv2i) and Weighted Integrated Biomarker Responses version 2 individual (Weighted IBRv2i) indexes correlated with the original IBR and IBRv2 indexes and were able to detect differences among experimental groups in a simulated and case studies. Using the IBRi, IBRv2i, or Weighted IBRv2i indexes is advantageous because they maintain the data variability of the experimental groups and can be analyzed using hypothesis testing statistics like any other parameter. Additionally, this approach can help translate technical scientific terminology into a more accessible language suitable for environmental governmental agencies and decision-makers.

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The present study aimed to evaluate concentrations of 16 priority PAHs in the mussel Perna perna and oyster Crassostrea rhizophorae of the Santos Estuary (Brazil) and the potential risk to human health associated with the consumption of these species. The levels of ΣPAHs in the tissues of bivalves ranged from 96.94 to 988.76 µg/kg for mussels and from 88.38 to 138.62 µg/kg for oysters. A general trend of higher concentrations of PAHs was observed according to the increase in log Kow values in both species. The estimated amount of ΣPAHs via mussel and oyster intake ranged from 1.0 to 3.2 ng/kg and from 0.4 to 1.2 ng/kg of body weight per day, respectively. The calculated THQs, HI and CRs for the regular consumption of raw bivalves followed the internationally acceptable limits and represent a low risk to human health.

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The increasing presence of anthropogenic contaminants in aquatic environments poses challenges for species inhabiting contaminated sites. Due to their structural binding characteristics to ligands that inhibit or activate gene transcription, these xenobiotic compounds frequently target the nuclear receptor superfamily. The present work aims to understand the potential interaction between the hormone 17-ß-estradiol, an environmental contaminant, and the nuclear receptors of Crassostrea gigas, the Pacific oyster. This filter-feeding, sessile oyster species is subject to environmental changes and exposure to contaminants. In the Pacific oyster, the estrogen-binding nuclear receptor is not able to bind this hormone as it does in vertebrates. However, another receptor may exhibit responsiveness to estrogen-like molecules and derivatives. We employed high-performance in silico methodologies, including three-dimensional modeling, molecular docking and atomistic molecular dynamics to identify likely binding candidates with the target moecule. Our approach revealed that among the C. gigas nuclear receptor superfamily, candidates with the most favorable interaction with the molecule of interest belonged to the NR1D, NR1H, NR1P, NR2E, NHR42, and NR0B groups. Interestingly, NR1H and NR0B were associated with planktonic/larval life cycle stages, while NR1P, NR2E, and NR0B were associated with sessile/adult life stages. The application of this computational methodological strategy demonstrated high performance in the virtual screening of candidates for binding with the target xenobiotic molecule and can be employed in other studies in the field of ecotoxicology in non-model organisms.

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Pollutant exposure is considered an important factor responsible for the decline in marine biodiversity of Latin American coastal ecosystems. This threat has been detected in an estuarine system in southern Brazil, which prompted an investigation into the long-term biological effects of a chronic metal contamination on resident oysters from the Laguna Estuarine System (LES). Here, we present the species- and size-specific variations of biomarker responses (catalase, glucose-6-phosphate dehydrogenase, glutathione S-transferase, and protein carbonylation) in the gills and digestive gland of Crassostrea gigas and Crassostrea gasar. In parallel, concentrations of eight metals (Al, Cd, Cr, Cu, Fe, Mn, Pb, Zn) in soft tissues were measured. Our analyses revealed that the metal levels exhibited decreasing order in both species: Zn > Fe > Al > Cu > Mn > Cd. Except for Cu and Al, metal concentrations did not differ between oyster species. Biomarker results highlighted that C. gasar presented higher antioxidant responses, whereas C. gigas showed increased biotransformation upon exposure to LES pollutants, which varied according to the tissue. However, C. gasar showed a significant higher content of protein carbonylation but was not related to metals. In our research approach, the observation of metals presence and biomarkers-related responses are considered biologically relevant from an ecotoxicological perspective and serve as a baseline for future pollution studies in estuaries of Latin America. Finally, we recommend adopting a suite of biomarkers in both C. gasar and C. gigas, regardless their size and weight, as sentinel organisms in future regional biomonitoring studies in southern Brazil.

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A pathogen with high virulence potential in some host species, Perkinsus marinus remains a challenge for the ecological integrity of marine ecosystems and the health of bivalve molluscs. This study investigates the occurrence of P. marinus in Crassostrea sp. in estuaries of the Potengi River and the Guaraíras lagoon in Rio Grande do Norte, Brazil. A total of 203 oyster samples that tested positive for Perkinsus sp. in Ray's fluid thioglycollate medium (RFTM) were subjected to species-specific quantitiative PCR, where 61 animals (30.05 %) presented amplification graphs with a melting temperature of 80.1 ± 0.6 °C matching the positive control. This was the first record of P. marinus in oysters in these estuaries using qPCR as a diagnostic tool.

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Hemocytes are the circulating cells of the hemolymph of oysters and are responsible for numerous physiological functions, including immune defense. The oyster Crassostrea gasar is a native species inhabiting mangrove habitat and is of great commercial interest, cultured throughout the Brazilian coast, mainly in the north and northeast. Despite its commercial importance, little is known about its immunological aspects and defense cells, the hemocytes. This work aimed to morphologically characterize hemocytes of the oyster C. gasar and to study one of the main cellular defense response, phagocytosis, using light microscopy and flow cytometry. The results showed the presence of six hemocyte populations in C. gasar hemolymph. These comprise of large and small granulocytes, large and small hyalinocytes, blast-like cells and a rare type classified as vesicular or serous hemocytes. Hyalinocytes were highly abundant and the most heterogeneous cell population, while small granulocytes, along with vesicular hemocytes were the less abundant population. Hemocytes of C. gasar oysters demonstrated capabilities to phagocytose three different types of particles tested: zymosan A, latex particles and Escherichia coli, indicating a broad defense capacity. The zymosan A were the most engulfed particles, followed by beads, mainly phagocytized by granulocytes, the most phagocytic cells, and finally E. coli, which were the least phagocytized. This study is the first characterization of C. gasar oyster hemocytes and will support future studies that aim to understand the participation of different hemocyte types in defense responses against pathogens and/or environmental changes.

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Organochlorine pesticides (OCPs) have been intensively used without proper regulation and control in Latin America due to the prevalence of diseases and pests, thus posing potential risks to nontarget organisms. Initiatives for ecosystem preservation, such as to designate protected areas, may not be enough to avoid contamination by OCPs, considering that protected areas tend to be permeable to diffuse sources. Here, we investigate multi-level responses of the oyster Crassostrea virginica to OCPs in Laguna de Términos, a RAMSAR coastal lagoon in the southern Gulf of Mexico. For this aim, OCPs occurrence and concentrations in the water, sediment, and in oysters from 3 settlement banks were assessed. Enzymatic and non-enzymatic biochemical biomarkers were quantified in the oysters' mantle and digestive gland, and the human health risk due to oyster consumption was also evaluated. OCPs in water were below detection limits. Fourteen OCPs were detected in sediments (∑OCPs mean of 49 ngg-1) and 7 in oyster tissues (∑OCPs mean of 121 ngg-1). The occurrence of OCPs was related to the land uses along the watersheds of the rivers that drain into the lagoon. Biochemical responses were correlated with OCPs (∑HCH, ∑DDT, heptachlor and endosulfan) in sediment, and oyster tissues. OCPs in oyster tissues showed a strong association with pro-oxidant forces and oxidative stress responses (Superoxide dismutase, Catalase, Glutathione Peroxidase, and lipid peroxidation), and neurotoxicity (Acetylcholinesterase), suggesting that the current OCPs contamination exerts significant stress. Our study also shows that the consumption of oysters from the lagoon increases the potential human health risk. Considering that Laguna de Términos is a protected Ramsar site, we suggest that environmental protection measures should be increased and that a monitoring program for OCPs exposure is necessary to assess the effects on this ecosystem.

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Aquatic organisms that inhabit coastal areas are often exposed to several contaminants. It is known that the bioaccumulation of contaminants can be amplified according to the species feeding habits and contaminant properties. As a consequence, species can experience different effects to contaminant exposure even if they inhabit the same area. The present study aimed to investigate the activities of carbonic anhydrase (CA), Ca2+-ATPase, and Mg2+-ATPase in different tissues (soft tissue, mantle, and gill) of three mollusk species (Lottia subrugosa, Stramonita brasiliensis, and Crassostrea brasiliana) with different feeding habits (herbivore, carnivore, and filter-feeder, respectively) which were sampled within a known contamination gradient at Santos Estuarine System (Southeastern Brazil). From the three enzymes tested, only CA was affected by the presence of contaminants within the contamination gradient evaluated. In general, the CA activity from the three species were lower in contaminated sites when compared to the reference site. The contrasting CA activity response observed in S. brasiliensis compared to L. subrugosa and C. brasiliana could be related to the tissue-specificity of this enzyme activity and species feeding habits (filter-feeders can accumulate more contaminants than herbivores and even carnivores). Results indicated that C. brasiliana mantle is the most suitable tissue for the use of CA analysis as a biomarker.

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The eastern oyster, Crassostrea virginica, is divided into four populations along the western North Atlantic, however, the only published mitochondrial genome sequence was assembled using one individual in Delaware. This study aimed to (1) assemble C. virginica mitochondrial genomes from Texas with pooled restriction-site-associated DNA sequencing (ezRAD), (2) evaluate the validity of the mitochondrial genome assemblies including comparison with Sanger sequencing data, and (3) evaluate genetic differentiation both between the Delaware and Texas genomes, as well as among three bays in Texas. The pooled-genome-assembled-genomes (PAGs) from Texas exhibited several characteristics indicating that they were valid, including elevated nucleotide diversity in non-coding and the third position of codons, placement as the sister haplotype of the genome from Delaware in a phylogenetic reconstruction of Crassostrea mitochondrial genomes, and a lack of genetic structure in the ND4 gene among the three Texas bays as was found with Sanger amplicons in samples from the same bays several years prior. In the comparison between the Delaware and Texas genome, 27 of 38 coding regions exhibited variability between the two populations, which were differentiated by 273 mutations, versus 1-13 mutations among the Texas samples. Using the full PAGs, there was no additional evidence for population structure among the three Texas bays. While population genetics is rapidly moving towards larger high-density datasets, studies of mitochondrial DNA (and genomes) can be particularly useful for comparing historic data prior to the modern era of genomics. As such, being able to reliably compile mitochondrial genomes from genomic data can improve the ability to compare results across studies.

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The eastern oyster, Crassostrea virginica, provides critical ecosystem services and supports valuable fishery and aquaculture industries in northern Gulf of Mexico (nGoM) subtropical estuaries where it is grown subtidally. Its upper critical thermal limit is not well defined, especially when combined with extreme salinities. The cumulative mortalities of the progenies of wild C. virginica from four nGoM estuaries differing in mean annual salinity, acclimated to low (4.0), moderate (20.0), and high (36.0) salinities at 28.9 °C (84 °F) and exposed to increasing target temperatures of 33.3 °C (92 °F), 35.6 °C (96 °F) or 37.8 °C (100 °F), were measured over a three-week period. Oysters of all stocks were the most sensitive to increasing temperatures at low salinity, dying quicker (i.e., lower median lethal time, LT50) than at the moderate and high salinities and resulting in high cumulative mortalities at all target temperatures. Oysters of all stocks at moderate salinity died the slowest with high cumulative mortalities only at the two highest temperatures. The F1 oysters from the more southern and hypersaline Upper Laguna Madre estuary were generally more tolerant to prolonged higher temperatures (higher LT50) than stocks originating from lower salinity estuaries, most notably at the highest salinity. Using the measured temperatures oysters were exposed to, 3-day median lethal Celsius degrees (LD50) were estimated for each stock at each salinity. The lowest 3-day LD50 (35.1-36.0 °C) for all stocks was calculated at a salinity of 4.0, while the highest 3-day LD50 (40.1-44.0 °C) was calculated at a salinity of 20.0.

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ABSTRACT: Desiccation is a routine farming practice used in off-bottom oyster aquaculture to reduce biofouling organisms and improve shell quality. This practice can increase Vibrio parahaemolyticus and Vibrio vulnificus levels, leading to increased risk of illness for raw oyster consumers. Previous resubmersion studies were performed in geographic proximity to one another, so to better understand the broader applicability of resubmersion, the next step was to perform concurrent studies in multiple geographic locations within a region. This study evaluated the effect of variations in geographic location on the recovery time needed for elevated vibrio levels to return to ambient levels in desiccated oysters after resubmersion at Gulf Coast farms. Two trials were performed between May and August 2019 at sites spanning ∼100 km: three in Alabama and one in Florida. Oysters were deployed in OysterGro cages at each location, 2 weeks before each trial, and then either were desiccated for 24 h or remained submersed as controls. Triplicate samples were taken before and immediately following the desiccation period, as well as 7 and 14 days after resubmersion. Total and pathogenic V. parahaemolyticus and V. vulnificus levels were determined using most-probable-number (MPN) real-time PCR. Vibrio levels increased by 0.23 to 3.50 log MPN/g after desiccation. Recovery times varied among geographic locations by trial and Vibrio spp., with all vibrio counts recovering to levels not significantly higher than those in control oysters within 7 to 14 days of resubmersion (P ≥ 0.06). These results suggest a 14-day resubmersion period of cultured oysters allowed vibrio levels, elevated because of routine handling, to return to ambient levels at all farm sites studied.

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This paper assesses the effects of exposure to toxic concentrations (1200 to 6000 cells/mL) of the dinoflagellates Prorocentrum lima, Prorocentrum minimum, and Prorocentrum rhathymum and several concentrations of aqueous and organic extracts obtained from the same species (0 to 20 parts per thousand) on the Crassostrea gigas (5-7 mm) proteomic profile. Through comparative proteomic map analyses, several protein spots were detected with different expression levels, of which eight were selected to be identified by liquid chromatography-mass spectrometry (LC-MS/MS) analyses. The proteomic response suggests that, after 72 h of exposure to whole cells, the biological functions of C. gigas affected proteins in the immune system, stress response, contractile systems and cytoskeletal activities. The exposure to organic and aqueous extracts mainly showed effects on protein expressions in muscle contraction and cytoskeleton morphology. These results enrich the knowledge on early bivalve developmental stages. Therefore, they may be considered a solid base for new bioassays and/or generation of specific analytical tools that allow for some of the main effects of algal proliferation phenomena on bivalve mollusk development to be monitored, characterized and elucidated.

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The eastern oyster, Crassostrea virginica, is a foundation species within US Gulf of Mexico (GoM) estuaries that has experienced substantial population declines. As changes from management and climate are expected to continue to impact estuarine salinity, understanding how local oyster populations might respond and identifying populations with adaptations to more extreme changes in salinity could inform resource management, including restoration and aquaculture programs. Wild oysters were collected from four estuarine sites from Texas [Packery Channel (PC): 35.5, annual mean salinity, Aransas Bay (AB): 23.0] and Louisiana [Calcasieu Lake (CL): 16.2, Vermilion Bay (VB): 7.4] and spawned. The progeny were compared in field and laboratory studies under different salinity regimes. For the field study, F1 oysters were deployed at low (6.4) and intermediate (16.5) salinity sites in Alabama. Growth and mortality were measured monthly. Condition index and Perkinsus marinus infection intensity were measured quarterly. For the laboratory studies, mortality was recorded in F1 oysters that were exposed to salinities of 2.0, 4.0, 20.0/22.0, 38.0 and 44.0 with and without acclimation. The results of the field study and laboratory study with acclimation indicated that PC oysters are adapted to high-salinity conditions and do not tolerate very low salinities. The AB stock had the highest plasticity as it performed as well as the PC stock at high salinities and as well as Louisiana stocks at the lowest salinity. Louisiana stocks did not perform as well as the Texas stocks at high salinities. Results from the laboratory studies without salinity acclimation showed that all F1 stocks experiencing rapid mortality at low salinities when 3-month oysters collected at a salinity of 24 were used and at both low and high salinities when 7-month oysters collected at a salinity of 14.5 were used.

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The estuarine channel of Vitória Bay was evaluated regarding bioaccumulation of metals (Al, Ba, Mn, Fe, Zn, Cu, Cr, Pb, Ni, Cd, Hg) and As in mollusks. Mussels from an aquaculture farm and transplanted into the estuary, whereas oysters were collected in situ in the same area. Concentrations of Al, Mn, Fe, Cr and As were higher in P. perna, whereas C. rhizophorae bioaccumulated more Ba, Zn and Cu. Arsenic concentrations in P. perna exceeded the limit of the Brazilian legislation in the outer estuary. Salinity seemed to influence metal uptake differently for each bivalve, with P. perna absorbing more metal at higher salinities and C. rhizophorae in areas of lower salinity. Hazard index (HI) >1 revealed risk for both bivalves for high level consumers. Target Cancer Risk (TCR) for As revealed threat for human health associated with the consumption of mussels and oysters from the study area.

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