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) PCO 2 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 CO 2 exposures. At 5°C acid-base, energy metabolism and stress response related genes were upregulated at intermediate PCO 2 , whereas high PCO 2 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 PCO 2 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 PCO 2 , possibly as a result of limited acid-base compensation and metabolic down-regulation. Conclusion:Our findings indicate a PCO 2 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.

Research investigating the genetic basis of physiological responses has significantly broadened our understand-28 ing of the mechanisms underlying organismic response to environmental change. However, genomic data are 29 currently available for few taxa only, thus excluding physiological model species from this approach. In this 30 study we report the transcriptome of the model organism Hyas araneus from Spitsbergen (Arctic). We generated 31 20,479 transcripts, using the 454 GS FLX sequencing technology in combination with an Illumina HiSeq sequenc-32 ing approach. Annotation by Blastx revealed 7159 blast hits Q4 in the NCBI non-redundant protein database. The 33 comparison between the spider crab H. araneus transcriptome and EST libraries of the European lobster Homarus 34 americanus and the porcelain crab Petrolisthes cinctipes yielded 3229/2581 sequences with a significant hit, 35 respectively. The clustering by the Markov Clustering Algorithm (MCL) revealed a common core of 1710 clusters 36 present in all three species and 5903 unique clusters for H. araneus. The combined sequencing approaches 37 generated transcripts that will greatly expand the limited genomic data available for crustaceans. We introduce 38 the MCL clustering for transcriptome comparisons as a simple approach to estimate similarities between 39 transcriptomic libraries of different size and quality and to analyze homologies within the selected group of 40 species. In particular, we identified a large variety of reverse transcriptase (RT) Q3 sequences not only in the 41 H. araneus transcriptome and other decapod crustaceans, but also sea urchin, supporting the hypothesis of a herita-42 ble, anti-viral immunity and the proposed viral fragment integration by host-derived RTs in marine invertebrates.43

IntroductionExposure 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.ResultsIn 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12983-014-0087-4) contains supplementary material, which is available to authorized users.

Early life stages of marine crustaceans respond sensitively to elevated seawater PCO 2 . However, the underlying physiological mechanisms have not been studied well. We therefore investigated the effects of elevated seawater PCO 2 on oxygen consumption, dry weight, elemental composition, median developmental time (MDT) and mortality in zoea I larvae of the spider crab Hyas araneus (Svalbard 79°N/11°E; collection, May 2009; hatch, December 2009). At the time of moulting, oxygen consumption rate had reached a steady state level under control conditions. In contrast, elevated seawater PCO 2 caused the metabolic rate to rise continuously leading to a maximum 1.5-fold increase beyond control level a few days before moulting into the second stage (zoea II), followed by a pronounced decrease. Dry weight of larvae reared under high CO 2 conditions was lower than in control larvae at the beginning of the moult cycle, yet this difference had disappeared at the time of moulting. MDT of zoea I varied between 45 ± 1 days under control conditions and 42 ± 2 days under the highest seawater CO 2 concentration. The present study indicates that larval development under elevated seawater PCO 2 levels results in higher metabolic costs during premoulting events in zoea I. However, H. araneus zoea I larvae seem to be able to compensate for higher metabolic costs as larval MDT and survival was not affected by elevated PCO 2 levels.

The sensitivity of marine crustaceans to anthropogenic CO 2 emissions and the associated acidification of the oceans may be less than that of other, especially lower, invertebrates. However, effects on critical transition phases or carry-over effects between life stages have not been comprehensively explored. Here we report the impact of elevated partial pressure of CO 2 (pCO 2) values (3100 µatm) in seawater on Hyas araneus during the last 2 wk of their embryonic development (pre-hatching phase) and during development while in the consecutive zoea I and zoea II larval stages (post-hatching phase). We measured oxygen consumption, dry weight, developmental time and mortality in zoea I to assess changes in performance. Feeding rates and survival under starvation were investigated at different temperatures to detect differences in thermal sensitivities of zoea I and zoea II larvae depending on pre-hatch history. When embryos were preexposed to elevated pCO 2 during maternal care, mortality increased about 60% under continued CO 2 exposure during the zoea I phase. The larvae that moulted into zoea II displayed a developmental delay by about 20 d compared to larvae exposed to control pCO 2 during embryonic and zoeal phases. Elevated pCO 2 caused a reduction in zoea I dry weight and feeding rates, while survival of the starved larvae was not affected by seawater CO 2 concentration. In conclusion, CO 2 effects on egg masses under maternal care carried over to the first larval stages of crustaceans and reduced their survival and development to levels below those previously reported in studies exclusively focussing on acute pCO 2 effects on the larval stages.