REVIEW: Evidence supporting the 'preparation for oxidative stress' (POS) strategy in animals in their natural environment.

Hypometabolism is a common strategy employed by resilient species to withstand environmental stressors that would be life-threatening for other organisms. Under conditions such as hypoxia/anoxia, temperature and salinity stress, or seasonal changes (e.g. hibernation, estivation), stress-tolerant species down-regulate pathways to decrease energy expenditures until the return of less challenging conditions. However, it is with the return of these more favorable conditions and the reactivation of basal metabolic rates that a strong increase of reactive oxygen and nitrogen species (RONS) occurs, leading to oxidative stress. Over the last few decades, cases of species capable of enhancing antioxidant defenses during hypometabolic states have been reported across taxa and in response to a variety of stressors. Interpreted as an adaptive mechanism to counteract RONS formation during tissue hypometabolism and reactivation, this strategy was coined "Preparation for Oxidative Stress" (POS). Laboratory experiments have confirmed that over 100 species, spanning 9 animal phyla, apply this strategy to endure harsh environments. However, the challenge remains to confirm its occurrence in the natural environment and its wide applicability as a key survival element, through controlled experimentation in field and in natural conditions. Under such conditions, numerous confounding factors may complicate data interpretation, but this remains the only approach to provide an integrative look at the evolutionary aspects of ecophysiological adaptations. In this review, we provide an overview of representative cases where the POS strategy has been demonstrated among diverse species in natural environmental conditions, discussing the strengths and weaknesses of these results and conclusions.

Wastewater sequencing as a powerful tool to reveal SARS-CoV-2 variant introduction and spread in French Guiana, South America.

The origin of introduction of a new pathogen in a country, the evolutionary dynamics of an epidemic within a country, and the role of cross-border areas on pathogen dynamics remain complex to disentangle and are often poorly understood. For instance, cross-border areas represent the ideal location for the sharing of viral variants between countries, with international air travel, land travel and waterways playing an important role in the cross-border spread of infectious diseases. Unfortunately, monitoring the point of entry and the evolutionary dynamics of viruses in space and time within local populations remain challenging. Here we tested the efficiency of wastewater-based epidemiology and genotyping in monitoring Covid-19 epidemiology and SARS-CoV-2 variant dynamics in French Guiana, a tropical country located in South America. Our results suggest that wastewater-based epidemiology and genotyping are powerful tools to monitor variant introduction and disease evolution within a tropical country but the inclusion of both clinical and wastewater samples could still improve our understanding of genetic diversity co-circulating. Wastewater sequencing also revealed the cryptic transmission of SARS-CoV-2 variants within the country. Interestingly, we found some amino acid changes specific to the variants co-circulating in French Guiana, suggesting a local evolution of the SARS-CoV-2 variants after their introduction. More importantly, our results showed that the proximity to bordering countries was not the origin of the emergence of the French Guianese B.1.160.25 variant, but rather that this variant emerged from an ancestor B.1.160 variant introduced by European air plane travelers, suggesting thus that air travel remains a significant risk for cross-border spread of infectious diseases. Overall, we suggest that wastewater-based epidemiology and genotyping provides a cost effective and non-invasive approach for pathogen monitoring and an early-warning tool for disease emergence and spread within a tropical country.

Salinity stress in the black-chinned tilapia Sarotherodon melanotheron.

Physiological and morphological acclimation capacities of black-chinned tilapia, Sarotherodon melanotheron were studied from fish to gill cell level when fish are maintained in freshwater, seawater, and hypersaline conditions. Fish osmoregulatory capacity, gill ionocyte morphology, osmo-respiratory compromise, O2 consumption rate, branchial antioxidative defense, and cell apoptosis were considered. Captive juvenile tilapias were maintained in controlled freshwater conditions (FW: 0.4 ppt; 12 mOsm kg-1 ) or gradually transferred to seawater (SW: 32 ppt; 958 mOsm kg-1 ) and concentrated SW (cSW: 65 ppt; 1920 mOsm kg-1 ). After 15 days in these conditions, blood osmolality and chloride ion concentration were determined. Gill ionocyte density and morphology were measured using immunolabelled histological sections to specifically detect the sodium pump (NKA). Gill osmo-respiratory compromise was also calculated along with oxygen consumption rates from normoxic to hypoxic conditions from excised gills (indirect respirometry). Finally, catalase and caspase 3/7activities were recorded from gill extracts. Results indicate that elevated salinity induces an osmotic imbalance and a profound morphological change with proliferating and hypertrophied ionocytes. This thickening of the gill interlamellar cell mass and the shortening of the lamellae induce a reduced osmo-respiratory ratio and reduced respiratory capacity under both normoxic and hypoxic conditions. Although salinity changes do not affect one of the major antioxidative defense mechanism, it strongly affects apoptosis that appears the most elevated in SW. However, in freshwater condition, fish can maintain their osmotic balance with a low ionocyte density, a low apoptotic level and a drastically reduced O2 consumption in normoxic condition that is maintained in hypoxia. Therefore, S. melanotheron presents the typical functional remodeling due to environmental salinity changes ranging from FW to SW. However, elevated seawater induces major cellular stress inducing a profound gill morphofunctional dysfunctioning. While cell apoptosis is reduced, ionocyte proliferation is massively increased with impaired osmotic regulation and reduced O2 consumption both in normoxic and hypoxic conditions.

ONTdeCIPHER: an amplicon-based nanopore sequencing pipeline for tracking pathogen variants.

MOTIVATION: Amplicon-based nanopore sequencing is increasingly used for molecular surveillance during epidemics (e.g. ZIKA, EBOLA) or pandemics (e.g. SARS-CoV-2). However, there is still a lack of versatile and easy-to-use tools that allow users with minimal bioinformatics skills to perform the main steps of downstream analysis, from quality testing to SNPs effect to phylogenetic analysis. RESULTS: Here, we present ONTdeCIPHER, an amplicon-based Oxford Nanopore Technology sequencing pipeline to analyze the genetic diversity of SARS-CoV-2 and other pathogens. Our pipeline integrates 13 bioinformatics tools. With a single command line and a simple configuration file, users can pre-process their data and obtain the sequencing statistics, reconstruct the consensus genome, identify variants and their effects for each viral isolate, infer lineage and, finally perform multi-sequence alignments and phylogenetic analyses. AVAILABILITY AND IMPLEMENTATION: ONTdeCIPHER is available at https://github.com/emiracherif/ONTdeCIPHER. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Metabolic Cost of the Immune Response During Early Ontogeny of the Scallop Argopecten purpuratus.

The scallop Argopecten purpuratus is an important resource for Chilean and Peruvian aquaculture. Seed availability from commercial hatcheries is critical due to recurrent massive mortalities associated with bacterial infections, especially during the veliger larval stage. The immune response plays a crucial role in counteracting the effects of such infections, but being energetically costly, it potentially competes with the physiological and morphological changes that occur during early development, which are equally expensive. Consequently, in this study, energy metabolism parameters at the individual and cellular levels, under routine-basal status and after the exposure to the pathogenic strain bacteria (Vibrio splendidus VPAP18), were evaluated during early ontogeny (trochophore, D-veliger, veliger, pediveliger, and early juveniles) of A. purpuratus. The parameters measured were as follows: (1) metabolic demand, determined as oxygen consumption rate and (2) ATP supplying capacity measured by key mitochondrial enzymes activities [citrate synthase (CS), electron transport system (ETS), and ETS/CS ratio, indicative of ATP supplying efficiency], mitochondrial membrane potential (ΔΨm), and mitochondrial density (ρ m) using an in vivo image analysis. Data revealed that metabolic demand/capacity varies significantly throughout early development, with trochophores being the most efficient in terms of energy supplying capacity under basal conditions. ATP supplying efficiency decreased linearly with larval development, attaining its lowest level at the pediveliger stage, and increasing markedly in early juveniles. Veliger larvae at basal conditions were inefficient in terms of energy production vs. energy demand (with low ρ m, ΔΨm, enzyme activities, and ETS:CS). Post-challenged results suggest that both trochophore and D-veliger would have the necessary energy to support the immune response. However, due to an immature immune system, the immunity of these stages would rely mainly on molecules of parental origin, as suggested by previous studies. On the other hand, post-challenged veliger maintained their metabolic demand but decreased their ATP supplying capacity, whereas pediveliger increased CS activity. Overall, results suggest that veliger larvae exhibit the lowest metabolic capacity to overcome a bacterial challenge, coinciding with previous works, showing a reduced capacity to express immune-related genes. This would result in a higher susceptibility to pathogen infection, potentially explaining the higher mortality rates occurring during A. purpuratus farming.

Effects of temperature and salinity on antioxidant responses in livers of temperate (Dicentrarchus labrax) and tropical (Chanos Chanos) marine euryhaline fish.

Temperature and salinity are abiotic factors that affect physiological responses in aquaculture species. The European sea bass (Dicentrarchus labrax) is a temperate species that is generally farmed at 18 °C in seawater (SW). In the wild, its incursions in shallow habitats such as lagoons may result in hyperthermal damage despite its high thermal tolerance. Meanwhile, the milkfish (Chanos chanos), a tropical species, is generally reared at 28 °C, and in winter, high mortality usually occurs under hypothermal stress such as cold snaps. This study compared changes in hepatic antioxidant enzymes (superoxide dismutase, SOD; and catalase, CAT) in these two important marine euryhaline aquaculture species in Europe and Southeast Asia, respectively, under temperature challenge combined with hypo-osmotic (fresh water, FW) stress. After a four-week hyper- or hypo-thermal treatment, hepatic SOD activity was upregulated in both species reared in SW and FW, indicating enhanced oxidative stress in European sea bass and milkfish. The expression profiles of sod isoforms suggested that in milkfish, the increase in reactive oxygen species (ROS) was mainly at the cytosol level, leading to increased sod1 expression. In European sea bass, however, no obvious difference was found between the expression of sod isoforms at different temperatures. A lower expression of sod2 was observed in FW compared to SW in the latter species. Moreover, no significant change was observed in the mRNA expression and activity of CAT in the livers of these two species under the different temperature treatments, with the exception of the lower CAT activity in milkfish challenged with SW at 18 °C. Taken together, our results indicated that the antioxidant responses were not changed under long-term hypoosmotic challenge but were enhanced during the four-week temperature treatments in livers of both the temperate and tropical euryhaline species.

The hepatopancreas of the mangrove crab Neosarmatium africanum: a possible key to understanding the effects of wastewater exposure (Mayotte Island, Indian Ocean).

Mangrove crabs are ecosystem engineers through their bioturbation activity. On Mayotte Island, the abundance of Neosarmatium africanum decreased in wastewater-impacted areas. Previous analyses showed that global crab metabolism is impacted by wastewater, with a burst in O2 consumption that may be caused by osmo-respiratory trade-offs since gill functioning was impacted. As the hepatopancreas is a key metabolic organ, the purpose of this study was to investigate the physiological effects of wastewater and ammonia-N 5-h exposure on crabs to better understand the potential trade-offs underlying the global metabolic state. Catalase, superoxide dismutase, glutathione S-transferase, total digestive protease, and serine protease (trypsin and chymotrypsin) activities were assessed. Histological analyses were performed to determine structural modifications. No effect of short-term wastewater and ammonia-N exposure was found in antioxidant defenses or digestive enzyme activity. However, histological changes of B-cells indicate an increase in intracellular digestive activity through higher vacuolization processes and tubule dilation in wastewater-exposed crabs.

Environmental stress responses in sympatric congeneric crustaceans: Explaining and predicting the context-dependencies of invader impacts.

The role of ecophysiology in mediating marine biological pollution is poorly known. Here we explore how physiological plasticity to environmental stress can explain and predict the context-dependencies of invasive species impacts. We use the case of two sympatric skeleton shrimps, the invader Caprella scaura and its congener C. equilibra, which is currently replaced by the former on the South European coast. We compare their physiological responses to hyposalinity stress under suboptimal low and high temperature, while inferring on hypoxia tolerance. We use an energy-redox approach, analyzing mortality rate, the energetic balance and the consequent effects on the oxidative homeostasis. We found that decreased seawater salinity and/or oxygen levels can weaken biotic resistance, especially in females of C. equilibra, leading to periods of heightened vulnerability to invasion. Our approach provides mechanistic insights towards understanding the factors promoting invader impacts, highlighting the potential of ecophysiology for improving invasive species management.

The use of an in vitro approach to assess marine invertebrate carboxylesterase responses to chemicals of environmental concern.

Carboxylesterases (CEs) are key enzymes which catalyse the hydrolysis reactions of multiple xenobiotics and endogenous ester moieties. Given their growing interest in the context of marine pollution and biomonitoring, this study focused on the in vitro sensitivity of marine invertebrate CEs to some pesticides, pharmaceuticals, personal care products and plastic additives to assess their potential interaction on this enzymatic system and its suitability as biomarkers. Three bivalves, one gastropod and two crustaceans were used and CEs were quantified following current protocols set for mammalian models. Four substrates were screened for CEs determination and to test their adequacy in the hepatic fraction measures of the selected invertebrates. Two commercial recombinant human isoforms (hCE1 and hCE2) were also included for methodological validation. Among the invertebrates, mussels were revealed as the most sensitive to xenobiotic exposures while gastropods were the least as well as with particular substrate-specific preferences. Among chemicals of environmental concern, the plastic additive tetrabromobisphenol A displayed the highest CE-inhibitory capacity in all species. Since plastic additives easily breakdown from the polymer and may accumulate and metabolise in marine biota, their interaction with the CE key metabolic/detoxification processes may have consequences in invertebrate's physiology, affect bioaccumulation and therefore trophic web transfer and, ultimately, human health as shellfish consumers.

Are we neglecting earth while conquering space? Effects of aluminized solid rocket fuel combustion on the physiology of a tropical freshwater invertebrate.

Space launchers often use aluminized-solid fuel ("propergol") as propellant and its combustion releases tons of Al2O3 and HCl that sink in terrestrial and aquatic environments, polluting and decreasing water pH. We studied the impact of these events on the biochemical/physiological performance of the freshwater shrimp Macrobrachium jelskii, with wild specimens collected from a non-impacted site in French Guiana. In the laboratory, shrimps were exposed for one week to: i) undisturbed conditions; ii) Al2O3 exposure (0.5 mg L-1) at normal pH (6.6); iii) decreased pH (4.5) (mimicking HCl release in the environment) with no Al2O3; or iv) Al2O3 0.5 mg L-1 and pH 4.5, representing the average conditions found in the water bodies around the Ariane 5 launch pad. Results showed that shrimps bioaccumulated aluminium (Al) regardless of water pH. The combined effect of Al2O3 and low pH caused the most impact: acetylcholinesterase and carboxylesterase activities decreased, indicating neurotoxicity and reduced detoxification capacity, respectively. Animal respiration was enhanced with Al2O3 and pH variations alone, but the synergic interaction of both stressors caused respiration to decrease, suggesting metabolic depression. Oxidative damage followed a similar pattern to respiration rates across conditions, suggesting free radical-mediation in Al toxicity. Antioxidant activities varied among enzymes, with glutathione reductase being the most impacted by Al2O3 exposure. This study shows the importance of addressing space ports' impact on the environment, setting the bases for selecting the most appropriate biomarkers for future monitoring programs using a widespread and sensitive crustacean in the context of an increasing space-oriented activity across the world.

How do life-history traits influence the fate of intertidal and subtidal Mytilus galloprovincialis in a changing climate?

Coastal organisms (i.e. intertidal or upper subtidal species) live in between the terrestrial and aquatic realms, making them particularly vulnerable to climate change. In this context, intertidal organisms may suffer from the predicted sea level rise (increasing their submerged time) while subtidal organisms may suffer from anthropically-induced hypoxia and its consequences. Although there is some knowledge on how coastal organisms adapt to environmental changes, the biochemical and physiological consequences of prolonged submergence periods have not yet been well characterized. Thus, the present study aimed to assess the biochemical alterations experienced by intertidal organisms maintained always under tidal exposure (IT); intertidal organisms maintained submersed (IS); subtidal organisms maintained always submersed (SS); subtidal organisms under tidal exposure (ST). For this, Mytilus galloprovincialis specimens from contiguous intertidal and subtidal populations were exposed to the above mentioned conditions for twenty-eight days. Results indicated that both intertidal and subtidal mussels are adapted to the oxidative stress pressure caused by tidal and submerged conditions tested. Intertidal mussels did not seem to be negatively affected by submergence while ST specimens were energetically challenged by tidal exposure. Both IT and ST mussels consumed glycogen to fuel up mechanisms aiming to maintain redox homeostasis. Overall, both intertidal and subtidal populations were capable of coping with tidal exposure, although the strategies employed differed between them. These findings indicate that although IT mussels may not significantly suffer from the longer-term submergence, hypoxic events occurring in the context of global warming and other anthropogenic impacts may have consequences on both IT and ST populations. Altogether, it is important to highlight that tides may act as a confounding factor in experiments concerning coastal organisms, as it causes additional physiological and biochemical perturbations.

Biomarker considerations in monitoring petrogenic pollution using the mussel Mytilus galloprovincialis.

Mussels are worldwide bioindicators in pollution monitoring since they fulfil the requirements for being good sentinels. However, some methodological concerns arise in the use of particular biomarkers, particularly those displaying low enzymatic rates and/or limited responsiveness to chemicals and biological-related variability. In the present study, the suitability of oxidative stress and detoxification parameters when using mussels as sentinels of polycyclic aromatic hydrocarbon (PAH) pollution is addressed. Present results show that the S9 subcellular fraction of the digestive gland in mussels is an adequate and convenient matrix where to measure most pollution-related biomarkers. Furthermore, this work constitutes the first evidence of the potential suitability of using particular carboxylesterase (CE) activities in determining PAHs exposure in mussels. This fact could imply the replacement of more controversial cytochrome P450 components (phase I oxidation), which are only measurable in microsomal fractions, by CEs (measured in S9 fractions) as good alternatives for phase I reactions in PAH-exposed mussels. Some methodological considerations, such as the need of including commercial purified proteins in biomarker determinations for quality assurance, are evaluated.

Copper and cadmium administration induce toxicity and oxidative stress in the marine flatworm Macrostomum lignano.

The contamination of coastal regions with different toxicants, including heavy metal ions such as copper and cadmium jeopardize health and survival of organisms exposed to this habitat. To study the effects of high copper and cadmium concentrations in these marine environments, we used the flatworm Macrostomum lignano as a model. This platyhelminth lives in shallow coastal water and is exposed to high concentrations of all toxicants that accumulate in these sea floors. We could show that both, cadmium and copper show toxicity at higher concentrations, with copper being more toxic than cadmium. At concentrations below acute toxicity, a reduced long-term survival was observed for both metal ions. The effects of sublethal doses comprise reduced physical activities, an increase in ROS levels within the worms, and alterations of the mitochondrial biology. Moreover, cell death events were substantially increased in response to sublethal concentrations of both metal ions and stem cell activity was reduced following exposure to higher cadmium concentrations. Finally, the expression of several genes involved in xenobiotic metabolism was substantially altered by this intervention. Taken together, M. lignano has been identified as a suitable model for marine toxicological studies as it allows to quantify several relevant life-history traits as well as of physiological and behavioral read-outs.

Wastewater bioremediation by mangrove ecosystems impacts crab ecophysiology: In-situ caging experiment.

Mangroves are tidal wetlands that are often under strong anthropogenic pressures, despite the numerous ecosystem services they provide. Pollution from urban runoffs is one such threats, yet some mangroves are used as a bioremediation tool for wastewater (WW) treatment. This practice can impact mangrove crabs, which are key engineer species of the ecosystem. Using an experimental area with controlled WW releases, this study aimed to determine from an ecological and ecotoxicological perspective, the effects of WW on the red mangrove crab Neosarmatium africanum. Burrow density and salinity levels (used as a proxy of WW dispersion) were recorded, and a 3-week caging experiment was performed. Hemolymph osmolality, gill Na+/K+-ATPase (NKA) activity and gill redox balance were assessed in anterior and posterior gills of N. africanum. Burrow density decreased according to salinity decreases around the discharged area. Crabs from the impacted area had a lower osmoregulatory capacity despite gill NKA activity remaining undisturbed. The decrease of the superoxide dismutase activity indicates changes in redox metabolism. However, both catalase activity and oxidative damage remained unchanged in both areas but were higher in posterior gills. These results indicate that WW release may induce osmoregulatory and redox imbalances, potentially explaining the decrease in crab density. Based on these results we conclude that WW release should be carefully monitored as crabs are key players involved in the bioremediation process.

Twenty years of the 'Preparation for Oxidative Stress' (POS) theory: Ecophysiological advantages and molecular strategies.

Freezing, dehydration, salinity variations, hypoxia or anoxia are some of the environmental constraints that many organisms must frequently endure. Organisms adapted to these stressors often reduce their metabolic rates to maximize their chances of survival. However, upon recovery of environmental conditions and basal metabolic rates, cells are affected by an oxidative burst that, if uncontrolled, leads to (oxidative) cell damage and eventually death. Thus, a number of adapted organisms are able to increase their antioxidant defenses during an environmental/functional hypoxic transgression; a strategy that was interpreted in the 1990s as a "preparation for oxidative stress" (POS). Since that time, POS mechanisms have been identified in at least 83 animal species representing different phyla including Cnidaria, Nematoda, Annelida, Tardigrada, Echinodermata, Arthropoda, Mollusca and Chordata. Coinciding with the 20th anniversary of the postulation of the POS hypothesis, we compiled this review where we analyze a selection of examples of species showing POS-mechanisms and review the most recent advances in understanding the underlying molecular mechanisms behind those strategies that allow animals to survive in harsh environments.

Reproduction Immunity Trade-Off in a Mollusk: Hemocyte Energy Metabolism Underlies Cellular and Molecular Immune Responses.

Immune responses, as well as reproduction, are energy-hungry processes, particularly in broadcast spawners such as scallops. Thus, we aimed to explore the potential reproduction-immunity trade-off in Argopecten purpuratus, a species with great economic importance for Chile and Peru. Hemocytes, key immunological cells in mollusks, were the center of this study, where we addressed for the first time the relation between reproductive stage, hemocyte metabolic energetics and their capacity to support immune responses at cellular and molecular levels. Hemocyte metabolic capacity was assessed by their respiration rates, mitochondrial membrane potential and citrate synthase (CS) activity. Cellular immune parameters such as the number of circulating and tissue-infiltrating hemocytes and their reactive oxygen species (ROS) production capacity were considered. Molecular immune responses were examined through the transcriptional levels of two pattern recognition receptors (ApCLec and ApTLR) and two anti-microbial effectors (ferritin and big defensin). Their expressions were measured in hemocytes from immature, matured and spawned scallops under basal, and one of the following challenges: (i) in vitro, where hemocytes were challenged with the ß glucan zymosan, to determine the immune potentiality under standardized conditions; or (ii) in vivo challenge, using hemocytes from scallops injected with the pathogenic bacteria Vibrio splendidus. Results indicate a post-spawning decrease in the structural components of the immune system (hemocyte number/quality) and their potential capacity of performing immune functions (with reduced ATP-producing machinery and exhaustion of energy reserves). Both in vitro and in vivo challenges demonstrate that hemocytes from immature scallops have, in most cases, the best metabolic potential (increased CS activity) and immune performances, with for example, over threefold higher ROS production and tissue-infiltration capacity than those from mature and spawned scallops after the bacterial challenge. Agreeing with cellular responses, hemocytes from immature individuals induced the highest levels of immune receptors and antimicrobial effectors after the bacterial challenge, while spawned scallops presented the lowest values. Overall, results suggest a trade-off between resource allocation in reproduction and the immune responses in A. purpuratus, with hemocyte energy metabolic capacity potentially underlying cellular and molecular immune responses. Further research would be necessary to explore regulatory mechanisms such as signaling pleiotropy which may potentially be underlying this trade-off.

Exploring alternative biomarkers of pesticide pollution in clams.

Acetylcholinesterase (AChE) is a reliable biomarker of pesticide exposure although in clams this activity is often very low or undetectable. Carboxylesterases (CEs) exhort several physiological roles, but also respond to pesticides. Searching for an AChE alternative, baseline CE activities were characterised in Ruditapes decussatus gills and digestive glands using five substrates suggestive of different isozymes. The long chain p-nitrophenyl butyrate and 1-naphthyl butyrate were the most sensitive. In the digestive gland, their kinetic parameters (Vmax and Km) and in vitro sensitivity to the organophosphorus metabolite chlorpyrifos oxon (CPX) were calculated. IC50 values, in the pM-nM range, suggest a high protection efficiency of CE-related enzymes towards CPX neurotoxicity. Other targeted enzymes were: activities of glutathione reductase, glutathione peroxidase, catalase, glutathione S-transferases (GSTs) and lactate dehydrogenase in gills and digestive glands. The high GSTs activity and CE/AChE ratio suggests that R. decussatus has a great capacity for enduring pesticide exposure.

The use of carboxylesterases as biomarkers of pesticide exposure in bivalves: A methodological approach.

Bivalves are worldwide sentinels of anthropogenic pollution. The inclusion of biomarker responses in chemical monitoring is a recommended practise that has to overcome some difficulties. One of them is the time frame between sample collection and sample processing in order to ensure the preservation of enzymatic activities. In the present study, three bivalve species of commercial interest (mussel, Mytilus galloprovincialis, razor shell, Solen marginatus, and cockle, Cerastoderma edule) were processed within <2 h after being retrieved from their natural habitat, and 24 h after being transported in air under cold conditions (6-8 °C) to laboratory facilities. The enzymatic activities were compared in the three species submitted to both conditions revealing no differences in terms of carboxylesterase dependent activities (CEs) using different substrates: p-nitrophenyl acetate (pNPA), p-nitrophenyl butyrate (pNPB), 1-naphthyl acetate (1-NA), 1-naphthyl butyrate (1-NB) and 2-naphthyl acetate (2-NA). In mussels, three tissues were selected (haemolymph, gills and digestive gland). For comparative purposes, in razor shell and cockle only digestive gland was considered as it is the main metabolic organ. Baseline enzymatic activities for CEs were characterised in the digestive gland of the three bivalves using four out of the five selected CE substrates as well as the kinetic parameters (Vmax and Km) and catalytic efficiency. The in vitro sensitivity to the organophosphorus metabolite chlorpyrifos oxon was also calculated. IC50 values (pM-nM range) were lower than those obtained for vertebrate groups which suggest that bivalves have high protection efficiency against this pesticide as well as species dependent particularities.

Effects of domestic effluent discharges on mangrove crab physiology: Integrated energetic, osmoregulatory and redox balances of a key engineer species.

Mangroves are increasingly used as biofiltering systems of (pre-treated) domestic effluents. However, these wastewater discharges may affect local macrofauna. This laboratory study investigates the effects of wastewater exposure on the mangrove spider crab Neosarmatium meinerti, a key engineering species which is known to be affected by waste waters in effluent-impacted areas. These effects were quantified by monitoring biological markers of physiological state, namely oxygen consumption, the branchial cavity ventilation rate, gill physiology and morphology, and osmoregulatory and redox balance. Adults acclimated to clean seawater (SW, 32 ppt) and freshwater (FW, ∼0 ppt) were compared to crabs exposed to wastewater for 5 h (WW, ∼0 ppt). Spider crabs exposed to WW increased their ventilation and whole-animal respiration rates by 2- and 3-fold respectively, while isolated gill respiration increased in the animals exposed to FW (from 0.5 to 2.3 and 1.1 nmol O2 min-1 mg DW-1 for anterior and posterior gills, respectively) but was not modified in WW-exposed individuals. WW exposure also impaired crab osmoregulatory capacity; an 80 mOsm kg-1 decrease was observed compared to FW, likely due to decreased branchial NKA activity. ROS production (DCF fluorescence in hemolymph), antioxidant defenses (superoxide dismutase and catalase activities) and oxidative damage (malondialdehyde concentration) responses varied according to animal gender. Overall, this study demonstrates that specific physiological parameters must be considered when focusing on crabs with bimodal breathing capacities. We conclude that spider crabs exposed to WW face osmoregulatory imbalances due to functional and morphological gill remodeling, which must rapidly exhaust energy reserves. These physiological disruptions could explain the ecological changes observed in the field.

Hypoxically Induced Nitric Oxide: Potential Role as a Vasodilator in Mytilus edulis Gills.

Intertidal Mytilus edulis experience rapid transgression to hypoxia when they close their valves during low tide. This induces a physiological stress response aiming to stabilize tissue perfusion against declining oxygen partial pressure in shell water. We hypothesized that nitric oxide (NO) accumulation supports blood vessel opening in hypoxia and used live imaging techniques to measure NO and superoxide anion ( O 2 ∙ - ) formation in hypoxia-exposed gill filaments. Thirty minutes of moderate (7 kPa pO2) and severe hypoxia (1 kPa pO2) caused 1.6- and 2.4-fold increase, respectively, of NO accumulation in the endothelial muscle cells of the hemolymphatic vessels of the gill filaments. This led to a dilatation of blood vessel diameter by 43% (7 kPa) and 56% (1 kPa), which facilitates blood flow. Experiments in which we applied the chemical NO-donor Spermine NONOate (concentrations ranging from 1 to 6 mM) under normoxic conditions corroborate the dilatational effect of NO on the blood vessel. The formation of O 2 ∙ - within the filament epithelial cells increased 1.5 (7 kPa) and 2-fold (1 kPa) upon treatment. Biochemical analysis of mitochondrial electron transport complexes in hypoxia-exposed gill tissue indicates decreased activity of complexes I and III in both hypoxic conditions; whereas complex IV (cytochrome-c oxidase) activity increased at 7 kPa and decreased at 1 kPa compared to normoxic exposure conditions. This corresponds to the pattern of pO2-dependent gill respiration rates recorded in ex-vivo experiments. Severe hypoxia (1 kPa) appears to have a stabilizing effect on NO accumulation in gill cells, since less O2 is available for NO oxidation to nitrite/nitrate. Hypoxia thus supports the NO dependent inhibition of complex IV activity, a mechanism that could fine tune mitochondrial respiration to the local O2 availability in a tissue. Our study highlights a basal function of NO in improving perfusion of hypoxic invertebrate tissues, which could be a key mechanism of tolerance toward environmental O2 variations.