Description: In a mechanistic investigation of heat stress, heterosis (hybrid vigour), and underlying gene expression patterns, we assessed the thermal performance of inbred (selfings) and outbred (reciprocal crosses) sporophytes of the N-Atlantic kelp Laminaria digitata among clonal isolates from two divergent populations; one from the temperate North Sea (Helgoland) and one from the Arctic (Spitsbergen). First, we investigated the upper thermal tolerance of microscopic sporophytes in a 14-day experiment applying sublethal to lethal 20–23°C. We then subjected 4–7 cm long sporophytes to a control temperature (10°C), moderate (19°C) and sublethal to lethal heat stress (20.5°C) for 18 days to assess the physiological parameters growth and optimum quantum yield.

Description: Southern Ocean organisms are thought to be particularly vulnerable to ocean acidification, as they inhabit cold waters where calcite-aragonite saturation states are naturally low. It is also generally assumed that calcifying animals would be more affected by ocean acidification than non-calcifying ones. In this context, we aimed to study the impacts of reduced pH on the ascidia Cnemidocarpa verrucosa sp. A. Here, we used gene expression profiling and enzymatic activity to study the responses of that Antarctic benthic species to ocean acidification. We sampled Cnemidocarpa verrucosa sp. A. by scuba diving at approximately 15 m depth at Carlini station, Potter Cove, King George Island, Antarctica. Superoxide dismutase (SOD) activity was measured in the ascidia, samples (approximately 70 mg of brachial basket) were homogenized in 20 mM Tris-HCl, 1 mM EDTA, pH 7.6, with a ratio 1:4 w/v. Homogenates were centrifuged at 14,000 x g for 3 min at 4°C and the supernatant was used to measure SOD activity at 20°C following Livingstone et al. (1992) protocol. Supernatant was mixed with the measurement buffer (43 mM K₂HPO₄, 43 mM KH₂PO₄, 0.1 mM EDTA, pH 7.68), 5 mM Xanthina (Sigma X-0626), 100 µM Citocromo-C (Sigma C-2037), 0.3 mU/µl XOD (Xanthin-Oxidasa, Sigma X-4875) in 2 M (NH₄)2SO₄. The measurement was made in a photometer at 20°C, 550 nm wavelength, for 3 minutes, every 10 seconds. For the calculations, the total protein content of the samples was measured using the method of Bradford (1976). Superoxide dismutase activity was expressed in activity in the extract (mU) / amount of protein (mg). All measurements were made in triplicate.

Description: Southern Ocean organisms are thought to be particularly vulnerable to ocean acidification, as they inhabit cold waters where calcite-aragonite saturation states are naturally low. It is also generally assumed that calcifying animals would be more affected by ocean acidification than non-calcifying ones. In this context, we aimed to study the impacts of reduced pH on the ascidia Cnemidocarpa verrucosa sp. A. Here, we used gene expression profiling and enzymatic activity to study the responses of that Antarctic benthic species to ocean acidification. We sampled Cnemidocarpa verrucosa sp. A. by scuba diving at approximately 15 m depth at Carlini station, Potter Cove, King George Island, Antarctica. Caspases 3/7 activity as indicators of apoptosis intensity was measured using the Caspase-Glow 3/7 Assay kit (Promega, USA) following the manufacturer's instructions. Samples were homogenized (16-33 mg) in lysis buffer consisting in 25 mM HEPES, 5 mM MgCl₂·6H₂O, 1 mM EGTA, 1 μg/mL pepstatin, 1 μg/mL leupectin, and 1 μg/mL aprotinin at a ratio 1:100 (Rivera-Ingraham et al., 2013) using a Precellys homogenizer (2 cycles at 5,500 x g at 4°C for 20 s). Homogenates were centrifuged at 13,000 x g at 4°C for 15 min and the supernatant was used to measure luminescence using Tristar LB941 plate reader (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). The total protein content of the samples was measured using the method of Bradford (1976). Caspase/Apoptotic activity was expressed as relative light units (RLU) per μg of protein × 104.

Description: 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.

Description: Early life stages of marine crustaceans respond sensitively to elevated seawater P CO 2 . However, the underlying physiological mechanisms have not been studied well. We therefore investigated the effects of elevated seawater P CO 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 P CO 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 P CO 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 P CO 2 level

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

Description: Sensitivity of marine crustaceans to anthropogenic CO2 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 comprehensively been explored. Here we report the impact of elevated seawater PCO2 values (3100 µatm) on Hyas araneus during the last 2 weeks 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 pre-exposed to elevated PCO2 during maternal care, mortality increased about 60% under continued CO2 exposure during the zoea I phase. The larvae that moulted into zoea II, displayed a developmental delay by about 20 days compared to larvae exposed to control PCO2 during embryonic and zoeal phases. Elevated PCO2 caused a reduction in zoea I dry weight and feeding rates, while survival of the starved larvae was not affected by the seawater CO2 concentration. In conclusion, CO2 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 PCO2 effects on the larval stages.

Description: 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.

Description: Sea ice is a key factor for the functioning and services provided by polar marine ecosystems. However, ecosystem responses to sea-ice loss are largely unknown because time-series data are lacking. Here, we use shotgun metagenomics of marine sedimentary ancient DNA off Kamchatka (Western Bering Sea) covering the last ~20,000 years. We traced shifts from a sea ice-adapted late-glacial ecosystem, characterized by diatoms, copepods, and codfish to an ice-free Holocene characterized by cyanobacteria, salmon, and herring. By providing information about marine ecosystem dynamics across a broad taxonomic spectrum, our data show that ancient DNA will be an important new tool in identifying long-term ecosystem responses to climate transitions for improvements of ocean and cryosphere risk assessments. We conclude that continuing sea-ice decline on the northern Bering Sea shelf might impact on carbon export and disrupt benthic food supply and could allow for a northward expansion of salmon and Pacific herring.

Description: Current global warming results in rising sea-water temperatures, and the loss of sea ice in arctic and subarctic oceans impacts the community composition of primary producers with cascading effects on the food web and potentially on carbon export rates. This study analyzes metagenomic shotgun and diatom rbcL amplicon-sequencing data from sedimentary ancient DNA (sedaDNA) of the subarctic western Bering Sea that records phyto- and zooplankton community changes over the last glacial–interglacial cycle, including the last interglacial period (Eemian). Our data show that interglacial and glacial plankton communities differ, with distinct Eemian and Holocene plankton communities. The generally warm Holocene period is dominated by pico-sized cyanobacteria and bacteria-feeding heterotrophic protists, while the Eemian period is dominated by eukaryotic pico-sized chlorophytes and Triparmaceae. In contrast, the glacial period is characterized by micro-sized phototrophic protists, including sea-ice associated diatoms in the family Bacillariaceae and co-occurring diatom-feeding crustaceous zooplankton. Our deep-time record of plankton community changes reveals a long-term decrease in phytoplankton cell size coeval with increasing temperatures, and resembling community changes in the currently warming Bering Sea. The phytoplankton community in the warmer-than-present Eemian period is distinct from modern communities and limits the use of the Eemian as an analog for future climate scenarios. However, under enhanced future warming, the expected shift towards the dominance of small-sized phytoplankton and heterotrophic protists might result in an increased productivity, whereas the community’s potential of carbon export will be decreased, thereby weakening the subarctic Bering Sea’s function as an effective carbon sink.