Impaired reproduction, energy reserves and dysbiosis: The overlooked consequences of heatwaves in a bivalve mollusc.

Extreme events like Marine Heatwaves (MHWs) are becoming more intense, severe, and frequent, threatening benthic communities, specifically bivalves. However, the consequences of non-lethal MHWs on animals are still poorly understood. Here, we exposed the Manila clam Ruditapes philippinarum to non-lethal MHW for 30 days and provided an integrative view of its effects. Our result indicated that albeit non-lethal, MHW reduced clam's energy reserves (by reducing their hepato-somatic index), triggered antioxidant defenses (particularly in males), impaired reproduction (via the production of smaller oocytes in females), triggered dysbiosis in the digestive gland microbiota and altered animals' behaviour (by impacting their burying capacity) and filtration rate. Such effects were seen also at RNA-seq (i.e. many down-regulated genes belonged to reproduction) and metabolome level. Interestingly, negative effects were more pronounced in males than in females. Our results show that MHWs influence animal physiology at multiple levels, likely impacting its fitness and its ecosystem services.

MRF: a tool to overcome the barrier of inconsistent genome annotations and perform comparative genomics studies for the largest animal DNA virus.

BACKGROUND: The genome of the largest known animal virus, the white spot syndrome virus (WSSV) responsible for huge economic losses and loss of employment in aquaculture, suffers from inconsistent annotation nomenclature. Novel genome sequence, circular genome and variable genome length led to nomenclature inconsistencies. Since vast knowledge has already accumulated in the past two decades with inconsistent nomenclature, the insights gained on a genome could not be easily extendable to other genomes. Therefore, the present study aims to perform comparative genomics studies in WSSV on uniform nomenclature. METHODS: We have combined the standard mummer tool with custom scripts to develop missing regions finder (MRF) that documents the missing genome regions and coding sequences in virus genomes in comparison to a reference genome and in its annotation nomenclature. The procedure was implemented as web tool and in command-line interface. Using MRF, we have documented the missing coding sequences in WSSV and explored their role in virulence through application of phylogenomics, machine learning models and homologous genes. RESULTS: We have tabulated and depicted the missing genome regions, missing coding sequences and deletion hotspots in WSSV on a common annotation nomenclature and attempted to link them to virus virulence. It was observed that the ubiquitination, transcription regulation and nucleotide metabolism might be essentially required for WSSV pathogenesis; and the structural proteins, VP19, VP26 and VP28 are essential for virus assembly. Few minor structural proteins in WSSV would act as envelope glycoproteins. We have also demonstrated the advantage of MRF in providing detailed graphic/tabular output in less time and also in handling of low-complexity, repeat-rich and highly similar regions of the genomes using other virus cases. CONCLUSIONS: Pathogenic virus research benefits from tools that could directly indicate the missing genomic regions and coding sequences between isolates/strains. In virus research, the analyses performed in this study provides an advancement to find the differences between genomes and to quickly identify the important coding sequences/genomes that require early attention from researchers. To conclude, the approach implemented in MRF complements similarity-based tools in comparative genomics involving large, highly-similar, length-varying and/or inconsistently annotated viral genomes.

Toxicity of tributyltin to the European flat oyster Ostrea edulis: Metabolomic responses indicate impacts to energy metabolism, biochemical composition and reproductive maturation.

Tri-Butyl Tin (TBT) remains as a legacy pollutant in the benthic environments. Although the toxic impacts and endocrine disruption caused by TBT to gastropod molluscs have been established, the changes in energy reserves allocated to maintenance, growth, reproduction and survival of European oysters Ostrea edulis, a target species of concerted benthic habitat restoration projects, have not been explored. This study was designed to evaluate the effect of TBT chloride (TBTCl) on potential ions and relevant metabolomic pathways and its association with changes in physiological, biochemical and reproductive parameters in O. edulis exposed to environmental relevant concentrations of TBTCl. Oysters were exposed to TBTCl 20 ng/L (n = 30), 200 ng/L (n = 30) and 2000 ng/L (n = 30) for nine weeks. At the end of the exposure, gametogenic stage, sex, energy reserve content and metabolomic profiling analysis were conducted to elucidate the metabolic alterations that occur in individuals exposed to those compounds. Metabolite analysis showed significant changes in the digestive gland biochemistry in oysters exposed to TBTCl, decreasing tissue ATP concentrations through a combination of the disruption of the TCA cycle and other important molecular pathways involved in homeostasis, mitochondrial metabolism and antioxidant response. TBTCl exposure increased mortality and caused changes in the gametogenesis with cycle arrest in stages G0 and G1. Sex determination was affected by TBTCl exposure, increasing the proportion of oysters identified as males in O. edulis treated at 20ng/l TBTCl, and with an increased proportion of inactive stages in oysters treated with 2000 ng/l TBTCl. The presence and persistence of environmental pollutants, such as TBT, could represent an additional threat to the declining O. edulis populations and related taxa around the world, by increasing mortality, changing reproductive maturation, and disrupting metabolism. Our findings identify the need to consider additional factors (e.g. legacy pollution) when identifying coastal locations for shellfish restoration.

An integrated eco-evolutionary framework to predict population-level responses of climate-sensitive pathogens.

It is critical to gain insight into how climate change impacts evolutionary responses within climate-sensitive pathogen populations, such as increased resilience, opportunistic responses and the emergence of dominant variants from highly variable genomic backgrounds and subsequent global dispersal. This review proposes a framework to support such analysis, by combining genomic evolutionary analysis with climate time-series data in a novel spatiotemporal dataframe for use within machine learning applications, to understand past and future evolutionary pathogen responses to climate change. Recommendations are presented to increase the feasibility of interdisciplinary applications, including the importance of robust spatiotemporal metadata accompanying genome submission to databases. Such workflows will inform accessible public health tools and early-warning systems, to aid decision-making and mitigate future human health threats.

Spatial variation in the gastrointestinal microbiome, diet, and nutritional condition of a juvenile flatfish among coastal habitats.

Gut microbiota are important for the health, fitness and development of animal hosts, but little is known about these assemblages in wild populations of fish. Such knowledge is particularly important for juvenile life stages where nutritional intake critically determines early development, growth, and ultimately recruitment. We characterise the microbiome inhabiting the gut of young-of-the-year European plaice ('YOY plaice') on sandy beaches, their key juvenile habitat, and examine how these microbial communities vary spatially in relation to diet and nutritional condition of their plaice hosts. Body size, diet (stomach fullness and eukaryotic 18S ribosomal sequencing), nutritional condition (RNA:DNA) and gut microbiota (16S prokaryotic ribosomal sequencing) were compared in fish at two spatial scales: between beaches separated by 10s of kilometres and between sites at different depths on the same beach, separated by 10s of metres. The main microbial phyla in YOY plaice guts were Proteobacteria, Spirochaetes, Tenericutes and Verrucomicrobiae. Within the Proteobacteria there was an unusual dominance of Alphaproteobacteria. Differences in body size, diet and nutritional condition of YOY plaice between beaches were accompanied by differences in gut microbial assemblage structure. Notably, substantially reduced nutritional condition and size at one of the beaches was associated with lower stomach fullness, reduced consumption of annelids and differences in the abundance and presence of specific microbial taxa. Differences were also detected in microbial assemblages, body size, and diet between depths within the same nursery beach, although stomach fullness and nutritional condition did not vary significantly. The functional links between the environment, gut microbiota, and their hosts are potentially important mediators of the development of young fish through critical life stages. Our study indicates that these links need to be addressed at 10 km and even 10 m scales to capture the variability observed in wild populations of juvenile fish.

Acclimation to cyclic hypoxia improves thermal tolerance and copper survival in the caridean shrimp Palaemon varians.

In response to the continuous variation of environmental parameters, species must be able to adjust their physiology to overcome stressful conditions, a process known as acclimatization. Numerous laboratory studies have been conducted to understand and describe the mechanisms of acclimation to one environmental stressor (e.g. cyclic hypoxia), but currently our understanding of how acclimation to one stressor can change tolerance to a subsequent stressor is limited. Here, in two different experiments, we used the shrimp Palaemon varians to test how, following 28-days acclimation to cyclic hypoxia (mimicking a cyclic hypoxic regime currently found in its natural habitat), critical thermal maximum (CTmax) and sensitivity to copper (Cu2+) exposure (30 mgL-1) changed in comparison to shrimp acclimated to normoxic conditions and then exposed to thermal stress or Cu2+. Acclimation to cyclic hypoxia improved both CTmax (~1 °C higher than controls) and survival to acute Cu2+ exposure (~30% higher than controls) and induced significant gene expression changes (i.e. up-regulation of heat shock protein 70 - HSP70, hypoxia inducible factor - HIF, phosphoenolpyruvate carboxykinase - PEPCK, glucose 6-P transporter - G6Pt, metallothionein - Mt, and down-regulation of hemocyanin - Hem) in animals acclimated to cyclic hypoxia. Our results demonstrate how acclimation to cyclic hypoxia improved tolerance to subsequent stressors, highlighting the complexity of predicting organismal performance in variable (i.e. where multiple parameters can simultaneously change during the day) environments.

What do the terms resistance, tolerance, and resilience mean in the case of Ostrea edulis infected by the haplosporidian parasite Bonamia ostreae.

The decline of the European flat oyster Ostrea edulis represents a loss to European coastal economies both in terms of food security and by affecting the Good Environmental Status of the marine environment as set out by the European Council's Marine Strategy Framework Directive (2008/56/EC). Restoration of O. edulis habitat is being widely discussed across Europe, addressing key challenges such as the devastating impact of the haplosporidian parasite Bonamia ostreae. The use of resistant, tolerant, or resilient oysters as restoration broodstock has been proposed by restoration practitioners, but the definitions and implications of these superficially familiar terms have yet to be defined and agreed by all stakeholders. This opinion piece considers the challenges of differentiating Bonamia resistance, tolerance, and resilience; challenges which impede the adoption of robust definitions. We argue that, disease-resistance is reduced susceptibility to infection by the parasite, or active suppression of the parasites ability to multiply and proliferate. Disease-tolerance is the retention of fitness and an ability to neutralise the virulence of the parasite. Disease-resilience is the ability to recover from illness and, at population level, tolerance could be interpreted as resilience. We concede that further work is required to resolve practical uncertainty in applying these definitions, and argue for a collaboration of experts to achieve consensus. Failure to act now might result in the future dispersal of this disease into new locations and populations, because robust definitions are important components of regulatory mechanisms that underpin marine management.

Silencing of prophenoloxidase (proPO) gene in freshwater prawn, Macrobrachium rosenbergii, makes them susceptible to white spot syndrome virus (WSSV).

Prophenoloxidase (proPO) is very important to protect the invertebrates from microbial infections. Our previous studies revealed that proPO was up-regulated in WSSV-injected Macrobrachium rosenbergii and is responsible for protecting M. rosenbergii from WSSV. In order to prove this mechanism, an attempt was made in the present study to silence the proPO gene in freshwater prawn by injection of dsRNA-proPO followed by WSSV challenge. Two partial fragments of proPO with the size of 251 and 331 bp were used to synthesize dsRNA using LITMUS38i vector and E. coli. The bacterially synthesized dsRNA-proPO was used to silence proPO gene to determine its involvement in developing resistance in prawn against WSSV. In proPO gene-silenced prawn, 100% mortality was observed after WSSV challenge whereas no mortality was observed in prawn injected with WSSV alone. The WSSV infection in gene-silenced prawn was confirmed by PCR, and its propagation was quantified by ELISA and real-time PCR at different time intervals. Real-time PCR assay revealed a significant reduction in the expression of proPO gene in WSSV-challenged proPO-silenced prawn when compared to normal prawn. Level of proPO was reduced significantly in the haemolymph of proPO-silenced prawn when compared to prawn injected with PBS.

Transcriptome analysis reveals immune pathways underlying resistance in the common carp Cyprinus carpio against the oomycete Aphanomyces invadans.

Infection with Aphanomyces invadans is a serious fish disease with major global impacts. Despite affecting over 160 fish species, some of the species like the common carp Cyprinus carpio are resistant to A. invadans infection. In the present study, we investigated the transcriptomes of head kidney of common carp experimentally infected with A. invadans. In time course analysis, 5288 genes were found to be differentially expressed (DEGs), of which 731 were involved in 21 immune pathways. The analysis of immune-related DEGs suggested that efficient processing and presentation of A. invadans antigens, enhanced phagocytosis, recognition of pathogen-associated molecular patterns, and increased recruitment of leukocytes to the sites of infection contribute to resistance of common carp against A. invadans. Herein, we provide a systematic understanding of the disease resistance mechanisms in common carp at molecular level as a valuable resource for developing disease management strategies for this devastating fish-pathogenic oomycete.

Molecular insights into the mechanisms of susceptibility of Labeo rohita against oomycete Aphanomyces invadans.

Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome, is one of the most destructive pathogens of freshwater fishes. To date, the disease has been reported from over 160 fish species in 20 countries and notably, this is the first non-salmonid disease that has resulted in major impacts globally. In particular, Indian major carps (IMCs) are highly susceptible to this disease. To increase our knowledge particularly with regards to host immune response against A. invadans infection in a susceptible host, the gene expression profile in head kidney of A. invadans-infected and control rohu, Labeo rohita was investigated using RNA sequencing. Time course analysis of RNA-Seq data revealed 5608 differentially expressed genes, involved among others in Antigen processing and presentation, Leukocyte transendothelial migration, IL-17 signaling, Chemokine signaling, C-type lectin receptor signaling and Toll-like receptor signaling pathways. In the affected pathways, a number of immune genes were found to be downregulated, suggesting an immune evasion strategy of A. invadans in establishing the infection. The information generated in this study offers first systematic mechanistic understanding of the host-pathogen interaction that might underpin the development of new management strategies for this economically devastating fish-pathogenic oomycete A. invadans.

Intraspecific plasticity and trans-generational adaptation of reproductive traits and early development in a temperate marine neogastropod.

Climate warming is altering the distribution of species, producing range shifts and promoting local extinctions. There is an urgent need to understand the underlying mechanisms that influence the persistence of populations across a species' distribution range in the face of global warming. Ocenebra erinaceus is a marine gastropod that exhibits high intraspecific variability in maternal investment and physiological capacity during early stages, which suggests local adaptation to natal environmental conditions. In this study, reproductive traits and trans-generational adaptation were measured in two subtidal populations: one from the middle (the Solent, UK) and another towards the southern end of their geographic distribution (Arcachon, France). Local adaptation was evaluated with a transfer experiment (i.e. Arcachon females transferred to Solent thermal conditions) and trans-generational adaptation was evaluated in the thermal tolerance response of embryos exposed to temperatures between 10 and 20 °C. This study shows that both populations have similar fitness; however, there are adaptive costs to live under their natal location, resulting in trade-offs between reproductive traits. Transferred females show lower reproductive output, which suggests that females are maladapted to live under a new environment. The trans-generational experiment demonstrates contrasting thermal tolerance ranges between populations. Adaptation to local thermal conditions was observed in transferred embryos, showing poor performance and high mortalities under the new environment. Our results provide a better understanding of intraspecific differences and adaptations across a species' distribution range and provide insights into how climate warming will impact encapsulated species exhibiting location-specific adaptation.

Ephemeral detection of Bonamia exitiosa (Haplosporida) in adult and larval European flat oysters Ostrea edulis in the Solent, United Kingdom.

The haplosporidian parasite Bonamia exitiosa was detected using PCR in four adult and six larval brood samples of the European flat oyster Ostrea edulis from the Solent, UK. This represents the second reported detection of this parasite along the south coast of England. Adult oysters were collected and preserved from seabed populations or restoration broodstock cages between 2015 and 2018. The larvae within brooding adults sampled during 2017 and 2018 were also preserved. Molecular analysis of all samples was performed in 2019. The DNA of B. exitiosa was confirmed to be present within the gill tissue of one oyster within the Portsmouth wild fishery seabed population (n = 48), sampled in November 2015; the congeneric parasite Bonamia ostreae was not detected in this individual. This is the earliest record of B. exitiosa in the Solent. Concurrent presence of both B. ostreae and B. exitiosa, determined by DNA presence, was confirmed in the gill and heart tissue of three mature individuals from broodstock cages sampled in October 2017 (n = 99), two from a location on the River Hamble and one from the Camber Dock in Portsmouth Harbour. B. exitiosa was not detected in the November 2018 broodstock populations. A total of six larval broods were positive for B. exitiosa, with five also positive for B. ostreae. None of the brooding adults were positive for B. exitiosa suggesting that horizontal transmission from the surrounding environment to the brooding larvae is occurring. Further sampling of broodstock populations conducted by the Fish Health Inspectorate at the Centre for Environment, Fisheries and Aquaculture Science in June 2019 did not detect infection of O. edulis by B. exitiosa. These findings together suggest that the pathogen has not currently established in the area.

The role of temperature on the aerobic response of encapsulated embryos of Ocenebra erinaceus (Neogastropoda, Muricidae): A comparative study between two populations.

Climate warming can affect the developmental rate and embryonic survival of ectothermic species. However, it is largely unknown if the embryos of populations from different thermal regimes will respond differently to increased warming, potentially due to adaptations to natal environmental conditions. The effects of temperature on respiration rates and oxygen content of the intracapsular fluid were studied during the intracapsular development of Ocenebra erinaceus in two subtidal populations, one from the middle of their geographic distribution, the Solent, UK and another towards the southern portion: Arcachon, France. In this laboratory study, embryos were exposed to temperatures in the range of 14-20 °C. The encapsulation period for both populations was shorter at higher temperatures and intracapsular oxygen availability decreased as development progressed. However, the embryonic aerobic response differed between populations. Encapsulated embryos from the southern population (Arcachon) showed higher respiration rates and metabolic adjustment to elevated temperatures; however, encapsulated embryos from the Solent showed no metabolic adjustment, high capsular mortalities and limited acclimation to high temperatures. Our results suggest that aerobic response of encapsulated embryos is locally adapted to the temperature history of their natal environment and illustrates the importance of local environmental history in determining the fate of key life stages in response to a changing marine climate.

Temporal changes in transcriptome profile provide insights of White Spot Syndrome Virus infection in Litopenaeus vannamei.

Shrimp aquaculture is severely affected by WSSV. Despite an increasing effort to understand host/virus interaction by characterizing changes in gene expression (GE) following WSSV infection, the majority of published studies have focussed on a single time-point, providing limited insight on the development of host-pathogen interaction over the infection cycle. Using RNA-seq, we contrasted GE in gills of Litopenaeus vannamei at 1.5, 18 and 56 hours-post-infection (hpi), between WSSV-challenged and control shrimps. Time course analysis revealed 5097 differentially expressed genes: 63 DEGs were viral genes and their expression in WSSV group either peaked at 18 hpi (and decreased at 56 hpi) or increased linearly up to 56 hpi, suggesting a different role played by these genes during the course of infection. The remaining DEGs showed that WSSV altered the expression of metabolic, immune, apoptotic and cytoskeletal genes and was able to inhibit NF-κB and JAK/STAT pathways. Interestingly, GE changes were not consistent through the course of infection but were dynamic with time, suggesting the complexity of host-pathogen interaction. These data offer novel insights into the cellular functions that are affected during the course of infection and ultimately provide a valuable resource towards our understanding of the host-pathogen dynamics and its variation with time.

Comparative analysis of shrimp (Penaeus vannamei) miRNAs expression profiles during WSSV infection under experimental conditions and in pond culture.

In recent years, the importance of viral and host microRNAs (miRNAs) in mediating viral replication and control of host cellular machinery, has been realised and increasing efforts have been taken in order to understand the interactions of miRNAs from host and pathogen during infection. However, all existing studies has thus far been conducted in controlled experimental conditions and the veracity of these data for field conditions are yet to be established. In this framework, small RNA sequencing was performed to identify the miRNAs involved in shrimp (Penaeus vannamei) immune responses under two different WSSV infection conditions of natural infection and experimentally challenged conditions. The expression profiles of miRNAs of shrimp infected with WSSV under two contrasting conditions were compared and as a result, 23365 known miRNAs and 481 novel miRNAs were identified. Amongst the most abundantly expressed miRNAs, the hypoxia related miR-210 and immune pathway related miR-29b were expressed only in infected shrimps of both conditions. miR-8-5p, having a functional role in modulation of chitin biosynthesis was exclusively represented in higher numbers in the WSSV -infected shrimps under natural conditions whilst four of the miRNAs (mja-miR-6493-5p, mja-miR-6492, mmu-miR-3968, tcf-miR-9b-5p) identified from shrimps collected from pond culture targeted chitinase, an important enzyme involved in growth and moulting in shrimps, indicating an interaction between WSSV infection and moult cycle under culture conditions. Some of the miRNAs (tca-miR-87b-3p, cte-miR-277a) and miRNAs belonging to class miR-9, miR-981 that were identified only in WSSV infected shrimps under experimental conditions, are known to respond against WSSV infection in shrimps. Moreover, the miRNA target prediction revealed several immune-related gene targets such as cathepsin, c-type lectin, haemocyanin and ubiquitin protein ligase were commonly identified under both the conditions. However, the miRNAs identified from challenge experiment had wide number of gene targets as compared to the miRNAs of natural infection. The shrimp miRNA mja-miR-6489-3p, was also found to target early virus gene wsv001 of WSSV. Our study, therefore, provides the comparative analysis of miRNA expression from shrimp during WSSV infection in two different conditions.

Effects of the interaction between temperature and steroid hormones on gametogenesis and sex ratio in the European flat oyster (Ostrea edulis).

Throughout Europe, populations of Ostrea edulis have been in decline since the 1970s. Temperature has an important influence on physiological, biochemical and reproductive attributes of oysters. It is also the most easily modulated environmental factor in hatcheries, so it is useful to understand the implications of temperature variation in driving gametogenesis and sex development in a protandrous sequential hermaphrodites such as O. edulis. To understand the effect of temperature on gametogenesis and sex ratio, as well as the potential mechanism of sex determination through the role of steroid hormone homologues, oysters were exposed to three temperatures (10, 14, and 18 °C) for four months. Gametogenic stage and sex ratio were assessed histologically for each treatment. In parallel, concentrations of estradiol (E2)- and testosterone (T)- were determined in developing gonads. Our data show that by some biometric parameters, gametogenesis and sex ratio were significantly influenced by temperature during the experiment. There was a weak but significant correlation between E2 and T concentration during the treatments. However, and importantly, a direct relation between gonadal maturation, sex determination and hormones concentration was not found. These results suggest that gametogenesis and sex determination are predominantly affected by temperature in this species, and that steroids may not be actively involved as endogenous modulators in sex determination. Rising sea water temperatures and warmer condition through the year could cause an accelerated gametogenesis and skewed sex ratios in natural populations of O. edulis.

Sensitivity to near-future CO2 conditions in marine crabs depends on their compensatory capacities for salinity change.

Marine crabs inhabit shallow coastal/estuarine habitats particularly sensitive to climate change, and yet we know very little about the diversity of their responses to environmental change. We report the effects of a rarely studied, but increasingly prevalent, combination of environmental factors, that of near-future pCO2 (~1000 µatm) and a physiologically relevant 20% reduction in salinity. We focused on two crab species with differing abilities to cope with natural salinity change, and revealed via physiological and molecular studies that salinity had an overriding effect on ion exchange in the osmoregulating shore crab, Carcinus maenas. This species was unaffected by elevated CO2, and was able to hyper-osmoregulate and maintain haemolymph pH homeostasis for at least one year. By contrast, the commercially important edible crab, Cancer pagurus, an osmoconformer, had limited ion-transporting capacities, which were unresponsive to dilute seawater. Elevated CO2 disrupted haemolymph pH homeostasis, but there was some respite in dilute seawater due to a salinity-induced metabolic alkalosis (increase in HCO3- at constant pCO2). Ultimately, Cancer pagurus was poorly equipped to compensate for change, and exposures were limited to 9 months. Failure to understand the full spectrum of species-related vulnerabilities could lead to erroneous predictions of the impacts of a changing marine climate.

Metabolic rates are significantly lower in abyssal Holothuroidea than in shallow-water Holothuroidea.

Recent analyses of metabolic rates in fishes, echinoderms, crustaceans and cephalopods have concluded that bathymetric declines in temperature- and mass-normalized metabolic rate do not result from resource-limitation (e.g. oxygen or food/chemical energy), decreasing temperature or increasing hydrostatic pressure. Instead, based on contrasting bathymetric patterns reported in the metabolic rates of visual and non-visual taxa, declining metabolic rate with depth is proposed to result from relaxation of selection for high locomotory capacity in visual predators as light diminishes. Here, we present metabolic rates of Holothuroidea, a non-visual benthic and benthopelagic echinoderm class, determined in situ at abyssal depths (greater than 4000 m depth). Mean temperature- and mass-normalized metabolic rate did not differ significantly between shallow-water (less than 200 m depth) and bathyal (200-4000 m depth) holothurians, but was significantly lower in abyssal (greater than 4000 m depth) holothurians than in shallow-water holothurians. These results support the dominance of the visual interactions hypothesis at bathyal depths, but indicate that ecological or evolutionary pressures other than biotic visual interactions contribute to bathymetric variation in holothurian metabolic rates. Multiple nonlinear regression assuming power or exponential models indicates that in situ hydrostatic pressure and/or food/chemical energy availability are responsible for variation in holothurian metabolic rates. Consequently, these results have implications for modelling deep-sea energetics and processes.