Chemistry and biological processes during experiments with spider crab Hyas araneus

The combined effects of ocean warming and acidification were compared in larvae from two popula- tions of the cold-eurythermal spider crab Hyas araneus, from one of its southernmost populations (around Helgo- land, southern North Sea, 54°N, habitat temperature 3-18°C; collection: January 2008, hatch: January-February 2008) and from one of its northernmost populations (Svalbard, North Atlantic, 79°N, habitat temperature 0-6°C; collection: July 2008, hatch: February-April 2009). Larvae were exposed to temperatures of 3, 9 and 15°C combined with present-day normocapnic (380 ppm CO2) and projected future CO2 concentrations (710 and 3,000 ppm CO2). Calcium content of whole larvae was measured in freshly hatched Zoea I and after 3, 7 and 14 days during the Megalopa stage. Significant differences between Helgoland and Svalbard Megalopae were observed at all investigated temperatures and CO2 condi- tions. Under 380 ppm CO2, the calcium content increased with rising temperature and age of the larvae. At 3 and 9°C, Helgoland Megalopae accumulated more calcium than Svalbard Megalopae. Elevated CO2 levels, especially 3,000 ppm, caused a reduction in larval calcium contents at 3 and 9°C in both populations. This effect set in early, at 710 ppm CO2 only in Svalbard Megalopae at 9°C. Fur- thermore, at 3 and 9°C Megalopae from Helgoland replenished their calcium content to normocapnic levels and more rapidly than Svalbard Megalopae. However, Svalbard Megalopae displayed higher calcium contents under 3,000 ppm CO2 at 15°C. The findings of a lower capacity for calcium incorporation in crab larvae living at the cold end of their distribution range suggests that they might be more sensitive to ocean acidification than those in temperate regions.

Seawater carbonate chemistry and biological processes during experiments with the spider crab Hyas araneus in Kongsfjorden, Svalbard, 2009

With global climate change, ocean warming and acidification occur concomitantly. In this study, we tested the hypothesis that increasing CO2 levels affect the acid-base balance and reduce the activity capacity of the Arctic spider crab Hyas araneus, especially at the limits of thermal tolerance. Crabs were acclimated to projected oceanic CO2 levels for 12 days (today: 380, towards the year 2100: 750 and 1,120 and beyond: 3,000 ?atm) and at two temperatures (1 and 4 °C). Effects of these treatments on the righting response (RR) were determined (1) at acclimation temperatures followed by (2) righting when exposed to an additional acute (15 min) heat stress at 12 °C. Prior to (resting) and after the consecutive stresses of combined righting activity and heat exposure, acid-base status and lactate contents were measured in the haemolymph. Under resting conditions, CO2 caused a decrease in haemolymph pH and an increase in oxygen partial pressure. Despite some buffering via an accumulation of bicarbonate, the extracellular acidosis remained uncompensated at 1 °C, a trend exacerbated when animals were acclimated to 4 °C. The additional combined exposure to activity and heat had only a slight effect on blood gas and acid-base status. Righting activity in all crabs incubated at 1 and 4 °C was unaffected by elevated CO2 levels or acute heat stress but was significantly reduced when both stressors acted synergistically. This impact was much stronger in the group acclimated at 1 °C where some individuals acclimated to high CO2 levels stopped responding. Lactate only accumulated in the haemolymph after combined righting and heat stress. In the group acclimated to 1 °C, lactate content was highest under normocapnia and lowest at the highest CO2 level in line with the finding that RR was largely reduced. In crabs acclimated to 4 °C, the RR was less affected by CO2 such that activity caused lactate to increase with rising CO2 levels. In line with the concept of oxygen and capacity limited thermal tolerance, all animals exposed to temperature extremes displayed a reduction in scope for performance, a trend exacerbated by increasing CO2 levels. Additionally, the differences seen between cold- and warm-acclimated H. araneus after heat stress indicate that a small shift to higher acclimation temperatures also alleviates the response to temperature extremes, indicating a shift in the thermal tolerance window which reduces susceptibility to additional CO2 exposure.

Spider crab Hyas araneus larval dry weight, C/N ratio and developmental time during experiments from different latitudes (54° vs 79°N), 2010

The combined impacts of future scenarios of ocean acidification and global warming on the larvae of a cold-eurythermal spider crab, Hyas araneus L., were investigated in one of its southernmost populations (living around Helgoland, southern North Sea, 54°N) and one of the northernmost populations (Svalbard, North Atlantic, 79°N). Larvae were exposed at temperatures of 3, 9 and 15°C to present day normocapnia (380 ppm CO2) and to CO2 conditions expected for the near or medium-term future (710 ppm by 2100 and 3000 ppm CO2 by 2300 and beyond). Larval development time and biochemical composition were studied in the larval stages Zoea I, II, and Megalopa. Permanent differences in instar duration between both populations were detected in all stages, likely as a result of evolutionary temperature adaptation. With the exception of Zoea II at 3°C and under all CO2 conditions, development in all instars from Svalbard was delayed compared to those from Helgoland, under all conditions. Most prominently, development was much longer and fewer specimens morphosed to the first crab instar in the Megalopa from Svalbard than from Helgoland. Enhanced CO2 levels (710 and particularly 3000 ppm), caused extended duration of larval development and reduced larval growth (measured as dry mass) and fitness (decreasing C/N ratio, a proxy of the lipid content). Such effects were strongest in the zoeal stages in Svalbard larvae, and during the Megalopa instar in Helgoland larvae.

Effects of pre-hatching pCO2 on development and survival of zoea stages of Arctic spider crab Hyas araneus

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.

Sequential origin in the high performance properties of spider dragline silk

Major ampullate (MA) dragline silk supports spider orb webs, combining strength and extensibility in the toughest biomaterial. MA silk evolved ~376 MYA and identifying how evolutionary changes in proteins influenced silk mechanics is crucial for biomimetics, but is hindered by high spinning plasticity. We use supercontraction to remove that variation and characterize MA silk across the spider phylogeny. We show that mechanical performance is conserved within, but divergent among, major lineages, evolving in correlation with discrete changes in proteins. Early MA silk tensile strength improved rapidly with the origin of GGX amino acid motifs and increased repetitiveness. Tensile strength then maximized in basal entelegyne spiders, ~230 MYA. Toughness subsequently improved through increased extensibility within orb spiders, coupled with the origin of a novel protein (MaSp2). Key changes in MA silk proteins therefore correlate with the sequential evolution high performance orb spider silk and could aid design of biomimetic fibers.

The variability and interdependence of spider viscid line tensile properties

True stress-true strain curves of naturally spun viscid line fibers retrieved directly from the spiral of orb-webs built by Argiope trifasciata spiders were measured using a novel methodology. This new procedure combines a method for removing the aqueous coating of the fibers and a technique that allows the accurate measurement of their cross sectional area. Comparison of the tensile behaviour of different samples indicates that naturally spun viscid lines show a large variability, comparable to that of other silks, such as major ampullate gland silk and silkworm silk. Nevertheless, application of a statistical analysis allowed identifying two independent parameters that underlie the variability and characterize the observed range of true stress-true strain curves. Combination of this result with previous mechanical and microstructural data suggested the assignment of these two independent effects to the degree of alignment of the protein chains and to the local relative humidity which, in turn, depends on the composition of the viscous coating and on the external environmental conditions.

A conserved mode of head segmentation in arthropods revealed by the expression pattern of Hox genes in a spider

Chelicerates constitute a basic arthropod group with fossil representatives from as early as the Cambrian period. Embryonic development and the subdivision of the segmented body region into a prosoma and an opisthosoma are very similar in all extant chelicerates. The mode of head segmentation, however, has long been controversial. Although all other arthropod groups show a subdivision of the head region into six segments, the chelicerates are thought to have the first antennal segment missing. To examine this problem on a molecular level, we have compared the expression pattern of Hox genes in the spider Cupiennius salei with the pattern known from insects. Surprisingly, we find that the anterior expression borders of the Hox genes are in the same register and the same relative segmental position as in Drosophila. This contradicts the view that the homologue of the first antennal segment is absent in the spider. Instead, our data suggest that the cheliceral segment is homologous to the first antennal segment and the pedipalpal segment is homologous to the second antennal (or intercalary) segment in arthropods. Our finding implies that chelicerates, myriapods, crustaceans, and insects share a single mode of head segmentation, reinforcing the argument for a monophyletic origin of the arthropods.

Posttranslational amino acid epimerization: enzyme-catalyzed isomerization of amino acid residues in peptide chains.

Since ribosomally mediated protein biosynthesis is confined to the L-amino acid pool, the presence of D-amino acids in peptides was considered for many years to be restricted to proteins of prokaryotic origin. Unicellular microorganisms have been responsible for the generation of a host of D-amino acid-containing peptide antibiotics (gramicidin, actinomycin, bacitracin, polymyxins). Recently, a series of mu and delta opioid receptor agonists [dermorphins and deltorphins] and neuroactive tetrapeptides containing a D-amino acid residue have been isolated from amphibian (frog) skin and mollusks. Amino acid sequences obtained from the cDNA libraries coincide with the observed dermorphin and deltorphin sequences, suggesting a stereospecific posttranslational amino acid isomerization of unknown mechanism. A cofactor-independent serine isomerase found in the venom of the Agelenopsis aperta spider provides the first major clue to explain how multicellular organisms are capable of incorporating single D-amino acid residues into these and other eukaryotic peptides. The enzyme is capable of isomerizing serine, cysteine, O-methylserine, and alanine residues in the middle of peptide chains, thereby providing a biochemical capability that, until now, had not been observed. Both D- and L-amino acid residues are susceptible to isomerization. The substrates share a common Leu-Xaa-Phe-Ala recognition site. Early in the reaction sequence, solvent-derived deuterium resides solely with the epimerized product (not substrate) in isomerizations carried out in 2H2O. Significant deuterium isotope effects are obtained in these reactions in addition to isomerizations of isotopically labeled substrates (2H at the epimerizeable serine alpha-carbon atom). The combined kinetic and structural data suggests a two-base mechanism in which abstraction of a proton from one face is concomitant with delivery from the opposite face by the conjugate acid of the second enzymic base.

Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes.

The nucleotide sequences of four genes encoding Trimeresurus gramineus (green habu snake, crotalinae) venom gland phospholipase A2 (PLA2; phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) isozymes were compared internally and externally with those of six genes encoding Trimeresurus flavoviridis (habu snake, crotalinae) venom gland PLA2 isozymes. The numbers of nucleotide substitutions per site (KN) for the noncoding regions including introns were one-third to one-eighth of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions of exons, indicating that the noncoding regions are much more conserved than the protein-coding regions. The KN values for the introns were found to be nearly equivalent to those of introns of T. gramineus and T. flavoviridis TATA box-binding protein genes, which are assumed to be a general (nonvenomous) gene. Thus, it is evident that the introns of venom gland PLA2 isozyme genes have evolved at a similar rate to those of nonvenomous genes. The numbers of nucleotide substitutions per nonsynonymous site (KA) were close to or larger than the KS values for the protein-coding regions in venom gland PLA2 isozyme genes. All of the data combined reveal that Darwinian-type accelerated evolution has universally occurred only in the protein-coding regions of crotalinae snake venom PLA2 isozyme genes.

Extraordinarily high spider densities on islands: flow of energy from the marine to terrestrial food webs and the absence of predation.

Some islands in the Gulf of California support very high densities of spiders. Spider density is negatively correlated with island size; many small islands support 50-200 spiders per m3 of cactus. Energy for these spiders comes primarily from the ocean and not from in situ productivity by land plants. We explicitly connect the marine and terrestrial systems to show that insular food webs represent one endpoint of the marine web. We describe two conduits for marine energy entering these islands: shore drift and seabird colonies. Both conduits are related to island area, having a much stronger effect on smaller islands. This asymmetric effect helps to explain the exceptionally high spider densities on small islands. Although productivity sets the maximal potential densities, predation (by scorpions) limits realized spider abundance. Thus, prey availability and predation act in concert to set insular spider abundance.