Transgenerational exposure to ocean acidification impacts the hepatic transcriptome of European sea bass (Dicentrarchus labrax).

Physiological effects of ocean acidification associated with elevated CO2 concentrations in seawater is the subject of numerous studies in teleost fish. While the short time within-generation impact of ocean acidification (OA) on acid-base exchange and energy metabolism is relatively well described, the effects associated with transgenerational exposure to OA are much less known. Yet, the impacts of OA can vary in time with the potential for acclimation or adaptation of a species. Previous studies in our lab demonstrated that transgenerational exposure to OA had extensive effects on the transcriptome of the olfactory epithelium of European sea bass (Dicentrarchus labrax), especially on genes related to ion balance, energy metabolism, immune system, synaptic plasticity, neuron excitability and wiring. In the present study, we complete the previous work by investigating the effect of transgenerational exposure to OA on the hepatic transcriptome of European sea bass. Differential gene expression analysis was performed by RNAseq technology on RNA extracted from the liver of two groups of 18 months F2 juveniles that had been exposed since spawning to the same AO conditions as their parents (F1) to either actual pH or end-of-century predicted pH levels (IPCC RCP8.5), respectively. Here we show that transgenerational exposure to OA significantly impacts the expression of 236 hepatic transcripts including genes mainly involved in inflammatory/immune responses but also in carbohydrate metabolism and cellular homeostasis. Even if this transcriptomic impact is relatively limited compared to what was shown in the olfactory system, this work confirmed that fish transgenerationally exposed to OA exhibit molecular regulation of processes related to metabolism and inflammation. Also, our data expand the up-regulation of a key gene involved in different physiological pathways including calcium homeostasis (i.e. pthr1), which we already observed in the olfactory epithelium, to the liver. Even if our experimental design does not allow to discriminate direct within F2 generation effects from transgenerational plasticity, these results offer the perspective of more functional analyses to determine the potential physiological impact of OA exposure on fish physiology with ecological relevance.

Draft genome assembly and transcriptome data of the icefish Chionodraco myersi reveal the key role of mitochondria for a life without hemoglobin at subzero temperatures.

Antarctic fish belonging to Notothenioidei represent an extraordinary example of radiation in the cold. In addition to the absence of hemoglobin, icefish show a number of other striking peculiarities including large-diameter blood vessels, high vascular densities, mitochondria-rich muscle cells, and unusual mitochondrial architecture. In order to investigate the bases of icefish adaptation to the extreme Southern Ocean conditions we sequenced the complete genome of the icefish Chionodraco myersi. Comparative analyses of the icefish genome with those of other teleost species, including two additional white-blooded and five red-blooded notothenioids, provided a new perspective on the evolutionary loss of globin genes. Muscle transcriptome comparative analyses against red-blooded notothenioids as well as temperate fish revealed the peculiar regulation of genes involved in mitochondrial function in icefish. Gene duplication and promoter sequence divergence were identified as genome-wide patterns that likely contributed to the broad transcriptional program underlying the unique features of icefish mitochondria.

Aerobic capacities and swimming performance of polar cod (Boreogadus saida) under ocean acidification and warming conditions.

Polar cod (Boreogadus saida) is an important prey species in the Arctic ecosystem, yet its habitat is changing rapidly: climate change, through rising seawater temperatures and CO2 concentrations, is projected to be most pronounced in Arctic waters. This study aimed to investigate the influence of ocean acidification and warming on maximum performance parameters of B. saida as indicators for the species' acclimation capacities under environmental conditions projected for the end of this century. After 4 months at four acclimation temperatures (0, 3, 6, 8°C) each combined with two PCO2  levels (390 and 1170 µatm), aerobic capacities and swimming performance of B. saida were recorded following a Ucrit protocol. At both CO2 levels, standard metabolic rate (SMR) was elevated at the highest acclimation temperature indicating thermal limitations. Maximum metabolic rate (MMR) increased continuously with temperature, suggesting an optimum temperature for aerobic scope for exercise (ASex) at 6°C. Aerobic swimming performance (Ugait) increased with acclimation temperature irrespective of CO2 levels, while critical swimming speed (Ucrit) did not reveal any clear trend with temperature. Hypercapnia evoked an increase in MMR (and thereby ASex). However, swimming performance (both Ugait and Ucrit) was impaired under elevated near-future PCO2  conditions, indicating reduced efficiencies of oxygen turnover. The contribution of anaerobic metabolism to swimming performance was very low overall, and further reduced under hypercapnia. Our results revealed high sensitivities of maximum performance parameters (MMR, Ugait, Ucrit) of B. saida to ocean acidification. Impaired swimming capacity under ocean acidification may reflect reduced future competitive strength of B. saida.

Gene expression profiling in gills of the great spider crab Hyas araneus in response to ocean acidification and warming.

BACKGROUND: Hypercapnia and elevated temperatures resulting from climate change may have adverse consequences for many marine organisms. While diverse physiological and ecological effects have been identified, changes in those molecular mechanisms, which shape the physiological phenotype of a species and limit its capacity to compensate, remain poorly understood. Here, we use global gene expression profiling through RNA-Sequencing to study the transcriptional responses to ocean acidification and warming in gills of the boreal spider crab Hyas araneus exposed medium-term (10 weeks) to intermediate (1,120 µatm) and high (1,960 µatm) PCO2 at different temperatures (5°C and 10°C). RESULTS: The analyses reveal shifts in steady state gene expression from control to intermediate and from intermediate to high CO2 exposures. At 5°C acid-base, energy metabolism and stress response related genes were upregulated at intermediate PCO2, whereas high PCO2 induced a relative reduction in expression to levels closer to controls. A similar pattern was found at elevated temperature (10°C). There was a strong coordination between acid-base, metabolic and stress-related processes. Hemolymph parameters at intermediate PCO2 indicate enhanced capacity in acid-base compensation potentially supported by upregulation of a V-ATPase. The likely enhanced energy demand might be met by the upregulation of the electron transport system (ETS), but may lead to increased oxidative stress reflected in upregulated antioxidant defense transcripts. These mechanisms were attenuated by high PCO2, possibly as a result of limited acid-base compensation and metabolic down-regulation. CONCLUSION: Our findings indicate a PCO2 dependent threshold beyond which compensation by acclimation fails progressively. They also indicate a limited ability of this stenoecious crustacean to compensate for the effects of ocean acidification with and without concomitant warming.

A new haemocyanin in cuttlefish (Sepia officinalis) eggs: sequence analysis and relevance during ontogeny.

BACKGROUND: Haemocyanin is the respiratory protein of most of the Mollusca. In cephalopods and gastropods at least two distinct isoforms are differentially expressed. However, their physiological purpose is unknown. For the common cuttlefish Sepia officinalis, three isoforms are known so far, whereas for only two of them the complete mRNA sequences are available. In this study, we sequenced the complete mRNA of the third haemocyanin isoform and measured the relative expression of all three isoforms during embryogenesis to reveal a potential ontogenetic relevance. RESULTS: The cDNA of isoform 3 clearly correlates to the known Sepia officinalis haemocyanin subunits consisting of eight functional units and an internal duplicated functional unit d. Our molecular phylogenetic analyses reveal the third isoform representing a potentially ancestral haemocyanin isoform, and the analyses of the expression of haemocyanin type 3 reveal that haemocyanin type 3 only can be observed within eggs and during early development. Isoforms 1 and 2 are absent at these stages. After hatching, isoform 3 is downregulated, and isoform 1 and 2 are upregulated. CONCLUSIONS: Our study clearly shows an embryonic relevance of the third isoform, which will be further discussed in the light of the changes in the physiological function of haemocyanin during ontogeny. Taken together with the fact that it could also be the isoform closest related to the common ancestor of cuttlefish haemocyanin, the phylogeny of cuttlefish haemocyanin may be recapitulated during its ontogeny.

Cephalopods in neuroscience: regulations, research and the 3Rs.

Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of "live cephalopods" became regulated within the European Union by Directive 2010/63/EU on the "Protection of Animals used for Scientific Purposes", giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce "guidelines" and the potential contribution of neuroscience research to cephalopod welfare.

Temperature tolerance of different larval stages of the spider crab Hyas araneus exposed to elevated seawater PCO2.

INTRODUCTION: Exposure to elevated seawater PCO2 limits the thermal tolerance of crustaceans but the underlying mechanisms have not been comprehensively explored. Larval stages of crustaceans are even more sensitive to environmental hypercapnia and possess narrower thermal windows than adults. RESULTS: In a mechanistic approach, we analysed the impact of high seawater CO2 on parameters at different levels of biological organization, from the molecular to the whole animal level. At the whole animal level we measured oxygen consumption, heart rate and activity during acute warming in zoea and megalopa larvae of the spider crab Hyas araneus exposed to different levels of seawater PCO2. Furthermore, the expression of genes responsible for acid-base regulation and mitochondrial energy metabolism, and cellular responses to thermal stress (e.g. the heat shock response) was analysed before and after larvae were heat shocked by rapidly raising the seawater temperature from 10°C rearing temperature to 20°C. Zoea larvae showed a high heat tolerance, which decreased at elevated seawater PCO2, while the already low heat tolerance of megalopa larvae was not limited further by hypercapnic exposure. There was a combined effect of elevated seawater CO2 and heat shock in zoea larvae causing elevated transcript levels of heat shock proteins. In all three larval stages, hypercapnic exposure elicited an up-regulation of genes involved in oxidative phosphorylation, which was, however, not accompanied by increased energetic demands. CONCLUSION: The combined effect of seawater CO2 and heat shock on the gene expression of heat shock proteins reflects the downward shift in thermal limits seen on the whole animal level and indicates an associated capacity to elicit passive thermal tolerance. The up-regulation of genes involved in oxidative phosphorylation might compensate for enzyme activities being lowered through bicarbonate inhibition and maintain larval standard metabolic rates at high seawater CO2 levels. The present study underlines the necessity to align transcriptomic data with physiological responses when addressing mechanisms affected by an interaction of elevated seawater PCO2 and temperature extremes.

Microsatellite DNA variation indicates low levels of genetic differentiation among cuttlefish (Sepia officinalis L.) populations in the English Channel and the Bay of Biscay.

Population substructure of the cuttlefish Sepia officinalis (Mollusca: Cephalopoda), as investigated by genetic variation of microsatellite loci, has been reported to be significantly extensive around the Iberian Peninsula with F(ST)=0.061 [Pérez-Losada, M., Guerra, A., Carvalho, G.R., Sanjuan, A., Shaw, P.W., 2002. Extensive population subdivision of the cuttlefish Sepia officinalis (Mollusca: Cephalopoda) around the Iberian Peninsula indicated by microsatellite DNA variation. Heredity 89, 417-424] and panmictic in the semi-enclosed Adriatic Sea with F(ST)=0.011 [Garoia, F., Guarniero, I., Ramsak, A., Ungaro, N., Landi, M., Piccinetti, C., Mannini, P., Tinti, F., 2004. Microsatellite DNA variation reveals high gene flow and panmictic populations in the Adriatic shared stocks of the European squid and cuttlefish (Cephalopoda). Heredity 93, 166-174]. Yet, no verified genetic information on population substructure existed for the northern distribution range of this species in the English Channel and the Bay of Biscay. So far, reproductive and migration behaviour and in vitro oxygen binding properties of haemocyanin have suggested the existence of separate populations in the English Channel and the Bay of Biscay. Examination of genetic variation at seven microsatellite loci within samples from the Bay of Biscay, the English Channel and the southern North Sea indicated low levels of genetic differentiation in this area but also a breakdown of free gene flow at highly significant average F(ST)=0.018. Although there is a considerable genetic exchange between populations of S. officinalis in the English Channel and the Bay of Biscay, they cannot be regarded as a single, freely interbreeding population. Earlier reported biological differences might thus be due to genetic variability between the populations.