Extrapallial Fluid Research Articles

Vital effects and the fractionation of rare earth elements and yttrium during uptake by and transfer within freshwater bivalves and their shells

Mussels are commonly used as bioarchives in environmental monitoring, yet the impact of vital effects on the trace element or isotope ratios used as biogeochemical proxies is often only ill constrained. A prime example of such trace elements are the Rare Earth elements and Yttrium (REY) which have become (micro)contaminants in freshwater systems worldwide. We here report on the distribution of REY in different soft tissues and in the shells of freshwater bivalve A. anatina, commonly known as “duck mussel”, from the Danube River in Hungary and the Vistula River in Poland. Both rivers are contaminated with anthropogenic Gd from contrast agents used in magnetic resonance imaging (MRI). Regardless of the mussels' origin, all of their compartments show very similar shale-normalised REY patterns. None of the samples show any anthropogenic Gd anomaly, implying that in freshwater anthropogenic Gd from MRI contrast agents is either not bioavailable or that REY from ambient river water are insignificant for the REY budget of freshwater mussels. Compared to ambient water, the bivalves bioaccumulate the REY with preferential uptake of Ce and of light REY over heavy REY. However, REY concentrations in mussels are similar to or lower than those in their potential food source, with minor fractionation along the REY series besides slight preferential uptake of La and Y. Comparison of shells and tissues reveals the systematic oxidative decoupling of Ce from its REY neighbours, probably due to the presence of Ce(IV) solution-complexes in the mussels' extrapallial fluid. Despite possible REY fractionation during their initial uptake, vital effects do not impose any major control on REY fractionation during REY transfer within the mussels or during formation of their shells. Mussel shells may, therefore, conveniently be used for environmental monitoring of REY without major disturbance from vital effects.

Biologically driven isotope fractionation in ultrastructurally different shell portions of freshwater pearl mussels (Margaritifera margaritifera): Implications for stream water δ18O reconstructions

AbstractOxygen isotopes in stream water can serve as natural tracers of watershed dynamics. Freshwater pearl mussels provide δ18Owater estimates that overcome temporal and spatial limitations of instrumental records. The reliability of shell‐based δ18Owater reconstructions depends on understanding which shell layer biomineralizes closer to oxygen isotopic equilibrium with ambient water. To determine this, both the (outer) prismatic and (inner) nacreous sublayers of the outer shell layer were sampled. Over 2500 isotope values were obtained from shells collected from the Our River (Luxembourg) and from mussels cultured in tanks at constant temperature and monitored δ18Owater. Calculated δ18Owater from the prismatic portion was in excellent agreement with monitored δ18Owater, while δ18Oshell of the nacreous portion was systematically offset by +0.43‰, overestimating δ18Owater by +0.53‰. Although shell portions were formed simultaneously from the same extrapallial fluid, they underwent different fractionation mechanisms, presumably due to differences in carbonic anhydrase activity catalyzing mineralization processes.

Histological, biochemical and immunohistochemical assessments of Roundup®, atrazine, and 2,4-D mixtures on tissue architecture, body fluid conditions, nitrotyrosine protein and Na+/K+-ATPase expressions in the American oyster, Crassostera virginica

Pesticides are widely used to control weeds and pests in agricultural settings but harm non-target aquatic organisms. In this study, our objective was to evaluate the effect of short-term exposure (one week) to environmentally relevant concentrations of pesticides mixture (low concentration: 0.4 μg/l atrazine, 0.5 μg/l Roundup®, and 0.5 μg/l 2,4-D; high concentration: 0.8 μg/l atrazine, 1 μg/l Roundup®, and 1 μg/l 2,4-D) on tissue architecture, body fluid conditions, and 3-nitrotyrosine protein (NTP) and Na+/K+-ATPase, expressions in tissues of American oyster (Crassostrea virginica) under controlled laboratory conditions. Histological analysis demonstrated the atrophy in the gills and digestive glands of oysters exposed to pesticides mixture. Periodic acid-Schiff (PAS) staining showed the number of hemocytes in connective tissue increased in low- and high-concentration pesticides exposure groups. However, pesticides treatment significantly (P < 0.05) decreased the amount of mucous secretion in the gills and digestive glands of oysters. The extrapallial fluid (i.e., body fluid) protein concentrations and glucose levels were dropped significantly (P < 0.05) in oysters exposed to high-concentration pesticides exposure groups. Moreover, immunohistochemical analysis showed significant upregulations of NTP and Na+/K+-ATPase expressions in the gills and digestive glands in pesticides exposure groups. Our results suggest that exposure to environmentally relevant pesticides mixture causes morphological changes in tissues and alters body fluid conditions and NTP and Na+/K+-ATPase expressions in tissues, which may lead to impaired physiological functions in oysters.

A Novel Kunitz-Type Serine Protease Inhibitor (HcKuSPI) is Involved in Antibacterial Defense in Innate Immunity and Participates in Shell Formation of Hyriopsis cumingii.

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

Biologically driven isotopic fractionations in bivalves: from palaeoenvironmental problem to palaeophysiological proxy.

Traditional bulk stable isotope (δ18 O and δ13 C) and clumped isotope (Δ47 ) records from bivalve shells provide invaluable histories of Earth's local and global climate change. However, biologically driven isotopic fractionations (BioDIFs) can overprint primary environmental signals in the shell. Here, we explore how conventional measurements of δ18 O, δ13 C, and Δ47 in bivalve shells can be re-interpreted to investigate these physiological processes deliberately. Using intrashell Δ47 and δ18 O alignment as a proxy for equilibrium state, we separately examine fractionations and/or disequilibrium occurring in the two major stages of the biomineralisation process: the secretion of the extrapallial fluid (EPF) and the precipitation of shell material from the EPF. We measured δ18 O, δ13 C, and Δ47 in fossil shells representing five genera (Lahillia, Dozyia, Eselaevitrigonia, Nordenskjoldia, and Cucullaea) from the Maastrichtian age [66-69 million years ago (Ma)] López de Bertodano Formation on Seymour Island, Antarctica. Material was sampled from both the outer and inner shell layers (OSL and ISL, respectively), which precipitate from separate EPF reservoirs. We find consistent δ18 O values across the five taxa, indicating that the composition of the OSL can be a reliable palaeoclimate proxy. However, relative to the OSL baseline, ISLs of all taxa show BioDIFs in one or more isotopic parameters. We discuss/hypothesise potential origins of these BioDIFs by synthesising isotope systematics with the physiological processes underlying shell biomineralisation. We propose a generalised analytical and interpretive framework that maximises the amount of palaeoenvironmental and palaeobiological information that can be derived from the isotopic composition of fossil shell material, even in the presence of previously confounding 'vital effects'. Applying this framework in deep time can expand the utility of δ18 O, δ13 C, and Δ47 measurements from proxies of past environments to proxies for certain biomineralisation strategies across space, time, and phylogeny among Bivalvia and other calcifying organisms.

Proteomic and Transcriptomic Responses Enable Clams to Correct the pH of Calcifying Fluids and Sustain Biomineralization in Acidified Environments.

Seawater pH and carbonate saturation are predicted to decrease dramatically by the end of the century. This process, designated ocean acidification (OA), threatens economically and ecologically important marine calcifiers, including the northern quahog (Mercenaria mercenaria). While many studies have demonstrated the adverse impacts of OA on bivalves, much less is known about mechanisms of resilience and adaptive strategies. Here, we examined clam responses to OA by evaluating cellular (hemocyte activities) and molecular (high-throughput proteomics, RNASeq) changes in hemolymph and extrapallial fluid (EPF-the site of biomineralization located between the mantle and the shell) in M. mercenaria continuously exposed to acidified (pH ~7.3; pCO2 ~2700 ppm) and normal conditions (pH ~8.1; pCO2 ~600 ppm) for one year. The extracellular pH of EPF and hemolymph (~7.5) was significantly higher than that of the external acidified seawater (~7.3). Under OA conditions, granulocytes (a sub-population of hemocytes important for biomineralization) were able to increase intracellular pH (by 54% in EPF and 79% in hemolymph) and calcium content (by 56% in hemolymph). The increased pH of EPF and hemolymph from clams exposed to high pCO2 was associated with the overexpression of genes (at both the mRNA and protein levels) related to biomineralization, acid-base balance, and calcium homeostasis, suggesting that clams can use corrective mechanisms to mitigate the negative impact of OA.

Effects of elevated temperature on 8-OHdG expression in the American oyster (Crassostrea virginica): Induction of oxidative stress biomarkers, cellular apoptosis, DNA damage and γH2AX signaling pathways

Global temperature is increasing due to anthropogenic activities and the effects of elevated temperature on DNA lesions are not well documented in marine organisms. The American oyster (Crassostrea virginica, an edible and commercially important marine mollusk) is an ideal shellfish species to study oxidative DNA lesions during heat stress. In this study, we examined the effects of elevated temperatures (24, 28, and 32 °C for one-week exposure) on heat shock protein-70 (HSP70, a biomarker of heat stress), 8‑hydroxy-2′-deoxyguanosine (8-OHdG, a biomarker of pro-mutagenic DNA lesion), double-stranded DNA (dsDNA), γ-histone family member X (γH2AX, a molecular biomarker of DNA damage), caspase-3 (CAS-3, a key enzyme of apoptotic pathway) and Bcl-2-associated X (BAX, an apoptosis regulator) protein and/or mRNA expressions in the gills of American oysters. Immunohistochemical and qRT-PCR results showed that HSP70, 8-OHdG, dsDNA, and γH2AX expressions in gills were significantly increased at high temperatures (28 and 32 °C) compared with control (24°C). In situ TUNEL analysis showed that the apoptotic cells in gill tissues were increased in heat-exposed oysters. Interestingly, the enhanced apoptotic cells were associated with increased CAS-3 and BAX mRNA and/or protein expressions, along with 8-OHdG levels in gills after heat exposure. Moreover, the extrapallial (EP) fluid (i.e., extracellular body fluid) protein concentrations were lower; however, the EP glucose levels were higher in heat-exposed oysters. Taken together, these results suggest that heat shock-driven oxidative stress alters extracellular body fluid conditions and induces cellular apoptosis and DNA damage, which may lead to increased 8-OHdG levels in cells/tissues in oysters.

Molecular and biochemical responses to tributyltin (TBT) exposure in the American oyster: Triggers of stress-induced oxidative DNA damage and prooxidant-antioxidant imbalance in tissues by TBT

Environmental pollution increases due to anthropogenic activities. Toxic chemicals in the environment affect the health of aquatic organisms. Tributyltin (TBT) is a toxic chemical widely used as an antifouling paint on boats, hulls, and ships. The toxic effect of TBT is well documented in aquatic organisms; however, little is known about the effects of TBT on DNA lesions in shellfish. The American oyster (Crassostrea virginica, an edible and commercially important species) is an ideal marine mollusk to examine the effects of TBT exposure on DNA lesions and oxidative/nitrative stress. In this study, we investigated the effects of TBT on 8′-hydroxy-2′-deoxyguanosine (8-OHdG, a biomarker of pro-mutagenic DNA lesion), double-stranded DNA (dsDNA), dinitrophenyl protein (DNP, a biomarker on reactive oxygen species, ROS), 3-nitrotyrosine protein (NTP, a biomarker of reactive nitrogen species, RNS), catalase (CAT, an antioxidant), and acetylcholinesterase (AChE, a cholinergic enzyme) expressions in the gills and digestive glands of oysters. We also analyzed extrapallial (EF) fluid conditions. Immunohistochemical and qRT-PCR results showed that TBT exposure significantly increased 8-OHdG, dsDNA, DNP, NTP, and CAT mRNA and/or protein expressions in the gills and digestive glands. However, AChE mRNA and protein expressions, and EP fluid pH and protein concentrations were decreased in TBT-exposed oysters. Taken together, these results suggest that antifouling biocide-induced production of ROS/RNS results in DNA damage, which may lead to decreased cellular functions in oysters. To the best of our knowledge, the present study provides the first molecular/biochemical evidence that TBT exposure results in oxidative/nitrative stress and DNA lesions in oysters.

Can element chemical impurities in aragonitic shells of marine bivalves serve as proxies for environmental variability?

In many biogenic and geogenic materials, ion impurities can provide serviceable proxies for environmental conditions. However, the element/Ca ratios of bivalve shells are notoriously challenging to interpret. Due to strong vital effects, nonclassical nucleation and growth mechanisms, and/or kinetic processes, the concentration of trace and minor elements in marine shells typically remains below values observed in inorganic CaCO3 precipitated from a solution resembling seawater chemistry but above those expected for thermodynamic equilibrium. The interpretation is further complicated by non-lattice bound and microstructure-specific element content. If environmental conditions were still encoded in the shells, they should result in statistically significantly reproducible element/Ca chronologies between contemporaneous specimens from the same site. Here, we tested this hypothesis and exemplarily studied seven elements in twelve modern specimens of Arctica islandica collected from four different localities in the North Atlantic (Faroe Islands, NE Iceland, Isle of Man, Gulf of Maine). Age-detrended chronologies of weighted annual B, Mg, Sr and Ba/Ca ratios (Al, Zn and Pb largely remained below detection limit) measured in the shells were reproducible between most specimens from the same site, supporting the hypothesis that the incorporation of these elements was at least partly controlled by environmental forcings. Notably, the agreement (explored with linear regression analyses and sign tests) between shell element/Ca ratios and environmental quantities was weaker than the agreement of respective element/Ca ratios between specimens suggesting that the available information on temperature, food and water chemistry did not properly reflect the in-situ conditions to which the bivalves were exposed or other extrinsic factors were at work. As in inorganic aragonite – but in contrast to thermodynamic expectations –, annual Sr/Ca, Mg/Ca and B/Ca ratios were negatively correlated to water temperature (up to 40% explained variability). The link between Ba/Ca and bulk phytoplankton often remained below the significance threshold, but was otherwise positive. Quantitative environmental reconstructions based on ion impurities in bivalve shells will remain challenging or impossible unless the chemistry of the parent solution (= extrapallial fluid) from which the shell actually formed is known, including temporal changes thereof. This information is crucial to compute representative partition coefficients required to calibrate transfer functions.

Effects of elevated pCO2 and temperature on the calcification rate, survival, extrapallial fluid chemistry, and respiration of the Atlantic Sea scallop Placopecten magellanicus

AbstractAnthropogenic CO2‐emission is causing ocean warming and acidification. Understanding how basic physiological processes of marine organisms respond to these stressors is important for predicting their responses to future global change. We examined the effects of elevated pCO2 and temperature (pCO2 = 344–2199 ppm; temperature = 6°C, 9°C, and 12°C) on the calcification rate, extrapallial fluid (EPF) carbonate chemistry, respiration, and survivorship of Atlantic sea scallops (Placopecten magellanicus) in a fully crossed 143‐d experiment. Rates of calcification and respiration were inhibited by elevated pCO2, and mortality occurred when elevated pCO2 was accompanied by high‐temperature stress. Declines in growth and survivorship were likely caused by external shell dissolution, thermal stress, and hypercapnic reduction of metabolism under elevated pCO2. Concentrations of dissolved inorganic carbon (DIC) and total alkalinity in the EPF increased above seawater concentrations in response to elevated pCO2. EPF pH declined, but did not decline as much as seawater pH, indicating that scallops regulate EPF pH to support calcification. The combination of EPF pH regulation and DIC elevation yielded an increase in EPF [] under elevated pCO2 treatments. The combination of low respiration rates, high EPF [], and low calcification rates under elevated pCO2 suggests that the impaired calcification arises more from hypercapnic inhibition of metabolic activity and external shell dissolution than from chemically unfavorable conditions in the EPF. These results demonstrate the importance of EPF chemistry for bivalve biomineralization, but show that regulation efforts are insufficient to fully offset the deleterious effects of elevated pCO2 on scallop performance.

Molecular characterization, immunofluorescent localization, and expression levels of two bicarbonate anion transporters in the whitish mantle of the giant clam, Tridacna squamosa, and the implications for light-enhanced shell formation

Giant clams conduct light-enhanced shell formation, which requires the increased transport of Ca2+ and inorganic carbon (Ci) from the hemolymph through the shell-facing epithelium of the whitish inner mantle to the extrapallial fluid where CaCO3 deposition occurs. The major form of Ci in the hemolymph is HCO3−, but the mechanisms of HCO3− transport through the basolateral and apical membranes of the shell-facing epithelial cells remain unknown. This study aimed to clone from the inner mantle of Tridacna squamosa the complete coding cDNA sequences of electrogenic Na+-HCO3−cotransporter 1 homolog (NBCe1-like-b) and electrogenic Na+-HCO3−cotransporter 2 homolog (NBCe2-like). NBCe1-like-b comprised 3360 bp, encoding a 125.7 kDa protein with 1119 amino acids. NBCe1-like-b was slightly different from NBCe1-like-a of the ctenidium reported elsewhere, as it had a serine residue (Ser1025), which might undergo phosphorylation leading to the transport of Na+: HCO3− at a ratio of 1: 2 into the cell. NBCe1-like-b was localized at the basolateral membrane of the shell-facing epithelial cells, and its gene and protein expression levels increased significantly in the inner mantle during illumination, indicating a role in the light-enhanced uptake of HCO3− from the hemolymph. The sequence of NBCe2-like obtained from the inner mantle was identical to that reported previously for the outer mantle. In the inner mantle, NBCe2-like had an apical localization in the shell-facing epithelial cells, and its protein abundance was upregulated during illumination. Hence, NBCe2-like might take part in the light-enhanced transport of HCO3− through the apical membrane of these cells into the extrapallial fluid.

Transcriptomic, Proteomic, and Functional Assays Underline the Dual Role of Extrapallial Hemocytes in Immunity and Biomineralization in the Hard Clam Mercenaria mercenaria.

Circulating hemocytes in the hemolymph represent the backbone of innate immunity in bivalves. Hemocytes are also found in the extrapallial fluid (EPF), the space delimited between the shell and the mantle, which is the site of shell biomineralization. This study investigated the transcriptome, proteome, and function of EPF and hemolymph in the hard clam Mercenaria mercenaria. Total and differential hemocyte counts were similar between EPF and hemolymph. Overexpressed genes in the EPF were found to have domains previously identified as being part of the “biomineralization toolkit” and involved in bivalve shell formation. Biomineralization related genes included chitin-metabolism genes, carbonic anhydrase, perlucin, and insoluble shell matrix protein genes. Overexpressed genes in the EPF encoded proteins present at higher abundances in the EPF proteome, specifically those related to shell formation such as carbonic anhydrase and insoluble shell matrix proteins. Genes coding for bicarbonate and ion transporters were also overexpressed, suggesting that EPF hemocytes are involved in regulating the availability of ions critical for biomineralization. Functional assays also showed that Ca2+ content of hemocytes in the EPF were significantly higher than those in hemolymph, supporting the idea that hemocytes serve as a source of Ca2+ during biomineralization. Overexpressed genes and proteins also contained domains such as C1q that have dual functions in biomineralization and immune response. The percent of phagocytic granulocytes was not significantly different between EPF and hemolymph. Together, these findings suggest that hemocytes in EPF play a central role in both biomineralization and immunity.

Microbiota of the Digestive Glands and Extrapallial Fluids of Clams Evolve Differently Over Time Depending on the Intertidal Position.

The Manila clam (Ruditapes philippinarum) is the second most exploited bivalve in the world but remains threatened by diseases and global changes. Their associated microbiota play a key role in their fitness and acclimation capacities. This study aimed at better understanding the behavior of clam digestive glands and extrapallial fluids microbiota at small, but contrasting spatial and temporal scales. Results showed that environmental variations impacted clam microbiota differently according to the considered tissue. Each clam tissue presented its own microbiota and showed different dynamics according to the intertidal position and sampling period. Extrapallial fluids microbiota was modified more rapidly than digestive glands microbiota, for clams placed on the upper and lower intertidal position, respectively. Clam tissues could be considered as different microhabitats for bacteria as they presented different responses to small-scale temporal and spatial variabilities in natural conditions. These differences underlined a more stringent environmental filter capacity of the digestive glands.

Impacts of anthropogenic contaminants and elevated temperature on prevalence and proliferation of Escherichia coli in the wild-caught American oyster, Crassostrea virginica in the southern Gulf of Mexico coast

ABSTRACT Escherichia coli is one of the most common indicators of environmental contamination and health risk to aquatic organisms including shellfish. E. coli can adapt to various stress conditions (e.g. heat, pollution, acidification, etc.). American oyster, Crassostrea virginica, a marine bivalve mollusc, is a seafood popular due to its delicacy and high nutritional value. Based on increasing concern about global warming, coastal pollution and microbial contamination in live or raw seafood, the present study focused on the detection and enumeration of the bacterial pathogen E. coli in the American oyster in south Texas waters, and in controlled laboratory heat-exposure conditions. Immunohistochemical, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) analyses showed substantial bacterial pathogen presence in the gills and digestive glands of oysters collected from polluted sites in San Martin Lake and South Padre Island on the southern Gulf coast of Texas. Laboratory heat-exposure studies showed an increasing trend of E. coli protein and mRNA expression in gills and digestive glands in elevated temperatures (28°C and 32°C) compared with control (24°C) and reduced integrated protein density of E. coli in gills at high temperature. Biochemical analyses were also performed to examine the extrapallial fluid (EPF, an integral body fluid) conditions in oysters. EPF glucose levels, pH and protein concentration varied at high temperatures and appeared to be pertinent to pathogen intensity in oysters. Collectively, these results suggest that American oyster is prone to waterborne pathogen contamination in the southern Gulf of Mexico coast, and E. coli proliferation escalates at rising sea surface temperatures.

Ocean acidification alters the diversity and structure of oyster associated microbial communities

AbstractHost‐associated microbial communities are fundamental to host physiology, yet it is unclear how these communities will respond to environmental disturbances. Here, we disentangle the environment‐linked and host‐linked effects of ocean acidification on oyster‐associated microbial communities. We exposed adult oysters (Crassostrea virginica) to CO2‐induced ocean acidification (400 vs. 2800 ppm) for 80 d. We measured the oyster extrapallial fluid pH and sampled the gills for microbial analysis at six time points. We found that different subsets of microbes were linked to acidification (n = 34 amplicon sequence variants [ASVs]) and to host response (n = 20 ASVs) with little overlap (n = 8 ASVs), suggesting that some members of the oyster microbiome were more responsive to environmental conditions while others were more tightly linked to host condition. Our results provide insight into which members of the oyster microbiome may contribute to the health and resistance of their host, and which members are the most vulnerable to changing environmental conditions.

Elevated seasonal temperature disrupts prooxidant-antioxidant homeostasis and promotes cellular apoptosis in the American oyster, Crassostrea virginica, in the Gulf of Mexico: a field study.

One of the major impacts of climate change has been the marked rise in global temperature. Recently, we demonstrated that high temperatures (1-week exposure) disrupt prooxidant-antioxidant homeostasis and promote cellular apoptosis in the American oyster. In this study, we evaluated the effects of seasonal sea surface temperature (SST) on tissue morphology, extrapallial fluid (EPF) conditions, heat shock protein-70 (HSP70), dinitrophenyl protein (DNP, an indicator of reactive oxygen species, ROS), 3-nitrotyrosine protein (NTP, an indicator of RNS), catalase (CAT), superoxide dismutase (SOD) protein expressions, and cellular apoptosis in gills and digestive glands of oysters collected on the southern Texas coast during the winter (15°C), spring (24°C), summer (30°C), and fall (27°C). Histological observations of both tissues showed a notable increase in mucus production and an enlargement of the digestive gland lumen with seasonal temperature rise, whereas biochemical analyses exhibited a significant decrease in EPF pH and protein concentration. Immunohistochemical analyses showed higher expression of HSP70 along with the expression of DNP and NTP in oyster tissues during summer. Intriguingly, CAT and SOD protein expressions exhibited significant upregulation with rising seasonal temperatures (15 to 27°C), which decreased significantly in summer (30°C), leaving oysters vulnerable to oxidative and nitrative damage. qRT-PCR analysis revealed a significant increase in HSP70 mRNA levels in oyster tissues during the warmer seasons. In situ TUNNEL assay showed a significant increase in apoptotic cells in seasons with high temperature. These results suggest that elevated SST induces oxidative/nitrative stress through the overproduction of ROS/RNS and disrupts the antioxidant system which promotes cellular apoptosis in oysters.

Effects of extra feeding combined with ocean acidification and increased temperature on the carbon isotope values (δ13C) in the mussel shell

Ocean acidification (OA) and global warming present future challenges for shell producing organisms such as mussels through reduction in the carbonate available to produce shells in these and other valuable aquaculture species. Molluscs control their shell growth through biomineralisation, but the response of the mechanisms behind biomineralisation to OA conditions are relatively unknown. It is unclear how much carbon is taken into the shell from the environment compared to the uptake through the food source. Shell production is energetically costly to molluscs and metabolic processes and energetic partitioning may affect their ability to perform the underlying mechanisms of biomineralisation under OA. It is possible that additional food consumption might alleviate some impacts caused by acidification. We assessed the ability of extra feeding to alter the impacts of OA and increased temperatures on adult Mytilus edulis. Carbon isotopes (δ13C) were used to examine the change in biomineralisation pathway in mussels. OA did not alter the δ13C directly in separate analyses of the shell calcite and aragonite layers, mantle tissue and extrapallial fluid. However, ambient treatments with increased temperatures altered the mussel biomineralisation pathway in the shell calcite using CO32− instead of HCO3− as the main source of carbon. The proportion of metabolic carbon uptake into the mussel shell calcite layer increased under OA, with additive effects when exposed to increased temperatures and extra feeding. The proportion of metabolic carbon uptake is higher (7%–11%) in the shell aragonite layer compared to calcite, under ambient treatments. OA initially reduced the metabolic carbon uptake into the shell aragonite, but after a period of 4-months with extra feeding, the mussels were able to adjust their metabolic carbon uptake to a level experienced under ambient treatments. This indicates that an abundance of food resources may enable changes in mussel biomineralisation pathways to compensate for any decrease in seawater inorganic carbon associated with OA. The impact of OA on phytoplankton varies from species to species, changing the structure of the community which could provide sufficient food resources to maintain metabolic carbon uptake for mussel shell growth. This study of δ13C isotopic values has identified changes in biomineralisation pathway relating to the mussel metabolic carbon uptake from their food source, with varying results for the aragonite and calcite shell polymorphs. The implications of these findings suggest that some bivalve species with different shell composites may cope better under OA than others, demanding further study into species-specific biomineralisation pathways.

Morphometric parameters of erythroid hemocytes of alien mollusc &lt;i&gt;Anadara kagoshimensis &lt;/i&gt;(Bivalvia, Arcidae) under normoxia and anoxia

In the present work we investigated the influence of three days anoxia on hematological parameters, morphological and functional characteristics of eryhroid cells of alien bivalve Anadara kagoshimensis . Oxygen concentration in seawater was decreased by bubbling with nitrogen gas for 5 h. Temperature was maintained at 20±1oC and photoperiod was 12h day: 12h night. Extrapallial fluids were sampled by a puncture of extrapalial cavity. Three-day exposure to anoxia caused pronounced cellular responses. At the organismic level changes were not observed, as hemoglobin concentration the total number of erythroid cells and mean hemoglobin concentration ( МСН ) remained at the level of normoxia. We observed an increase of cellular anomalies, i.e. shistocytes and erythroid cells with polymorphic nuclei, and cells undergoing reactive amitotic division, which resulted in formation of binuclear cells. Nuclear volume ( V ) increased for more than 40 % compared to control level. This increase depended on the duration of anoxia. Changes in cellular volume ( V ) possessed a complicated manner. At the end of experimental period (3rd day of anoxia) nucleo- cytoplasmic ratio was 36% lower comparing to normoxia. Exposure to anoxia did not cause mortality of erythroid cells. The number or red blood cell shades observed on slides remained at control level.

Ocean Acidification Induces Subtle Shifts in Gene Expression and DNA Methylation in Mantle Tissue of the Eastern Oyster (Crassostrea virginica)

Early evidence suggests that DNA methylation can mediate phenotypic responses of marine calcifying species to ocean acidification (OA). Few studies, however, have explicitly studied DNA methylation in calcifying tissues through time. Here, we examined the phenotypic and molecular responses in the extrapallial fluid and mantle (fluid and tissue at the calcification site) in the Eastern oyster (Crassostrea virginica) exposed to experimental OA over 80 days. Oysters were reared under three experimental pCO2 treatments (‘control’, 580 μatm; ‘moderate OA’, 1000 uatm; ‘high OA’, 2800 μatm) and sampled at 6 time points (24 hours - 80 days). We found that high OA initially induced changes in the pH of the extrapallial fluid (pHEPF) relative to the external seawater, but the magnitude of this difference was highest at 9 days and diminished over time. Calcification rates were significantly lower in the high OA treatment compared to the other treatments. To explore how oysters regulate their extrapallial fluid, gene expression and DNA methylation were examined in the mantle-edge tissue of oysters from day 9 and 80 in the control and high OA treatments. Mantle tissue mounted a significant global molecular response (both in the transcriptome and methylome) to OA that shifted through time. Although we did not find individual genes that were significantly differentially expressed to OA, the pHEPF was correlated with the eigengene expression of several co-expressed gene clusters. A small number of OA-induced differentially methylated loci were discovered, which corresponded with a weak association between OA-induced changes in genome-wide gene body DNA methylation and gene expression. Gene body methylation, however, was not significantly correlated with the eigengene expression of pHEPF correlated gene clusters. These results suggest that in C. virginica, OA induces a subtle response in a large number of genes, but also indicates that plasticity at the molecular level may be limited. Our study highlights the need to re-assess the plasticity of tissue-specific molecular responses in marine calcifiers, as well as the role of DNA methylation and gene expression in mediating physiological and biomineralization responses to OA.

Oyster Calcifying Fluid Harbors Persistent and Dynamic Autochthonous Bacterial Populations That May Aid in Shell Formation.

The eastern oyster (Crassostrea virginica) is a keystone species in estuarine environments but faces threats to shell formation associated with warming temperatures and acidification. Extrapallial fluid (EF), which is responsible for shell formation, harbors diverse and abundant microbial communities. Commensal microbial communities are vital to host health and fitness, yet long-term studies investigating temporal responses of the EF microbiome and its function in oyster fitness are lacking. In this study, bacterial communities of oyster EF and the water column were characterized monthly from October 2010 to September 2011. We investigated the selection, composition, and dynamics of resident and transient community members, evaluated the impact of temperature on EF microbial communities, and examined the functional role of the EF microbiome. Oyster EF communities were significantly different from the water column and were enriched for several taxa, including the Deltaproteobacteria, Epsilonproteobacteria, and Gammaproteobacteria. Overall, 94 resident members were identified in oyster EF. These members were persistent and abundant, comprising on average 33% of EF communities. Resident EF communities formed high-temperature and low-temperature groups and were more abundant overall at colder temperatures. Oyster EF resident communities were predicted to be enriched for dissimilatory nitrate reduction, nitrogen fixation, nitrification, and sulfite reductase genes. Sulfate and nitrate reduction may have a synergistic effect on calcium carbonate precipitation and indirectly aid in shell formation. Therefore, the potential role of the oyster EF microbiome in shell formation warrants further investigation as oysters and other shellfish face the future impacts of ocean warming and acidification.