Bioaccumulation and metabolism of polybrominated diphenyl ethers (PBDEs) in coenobitid hermit crabs from marine litter-polluted beaches in remote islands.

Plastic litter containing additives is potentially a major source of chemical contamination in remote areas. We investigated polybrominated diphenyl ethers (PBDEs) and microplastics in crustaceans and sand from beaches with high and low litter volumes on remote islands that were relatively free of other anthropogenic contaminants. Significant numbers of microplastics in the digestive tracts, and sporadically higher concentrations of rare congeners of PBDEs in the hepatopancreases were observed in coenobitid hermit crabs from the polluted beaches than in those from the control beaches. PBDEs and microplastics were detected in high amounts in one contaminated beach sand sample, but not in other beaches. Using BDE209 exposure experiments, similar debrominated products of BDE209 in field samples were detected in the hermit crabs. The results showed that when hermit crabs ingest microplastics containing BDE209, BDE209 leaches out and migrates to other tissues where it is metabolized.

King crab gills as a new source of chitin/chitosan and protein hydrolysates.

This is the first report on the physicochemical properties of chitin obtained from gills of the king crab Paralithodes camtschaticus. In the present study, we investigated the chemical composition of red king crab gills and considered methods of its complex processing to obtain chitin and enzymatic protein hydrolysates. The gills contained approximately 21 % chitin in terms of dry matter. For the first time, the gills of the king crab were investigated as a source of chitin and chitosan. Chitin was isolated from crab gills using chemical and enzymatic methods. The physicochemical properties of chitin and chitosan from the gills were investigated. By performing infrared spectroscopy and X-ray phase analyses, the chitin present in the gills was established to be α-chitin. The physical and chemical properties (degree of deacetylation, molecular weight and crystal structure) of gill chitin and chitosan were absolutely similar to those of crab shell. Crab gills can be used as an additional source of chitin in the integrated processing of king crabs. The yield of chitin from the gills is up to 45 % of the yield of chitin from the crab carapace.

Metabolism Interactions Promote the Overall Functioning of the Episymbiotic Chemosynthetic Community of Shinkaia crosnieri of Cold Seeps.

Remarkably diverse bacteria have been observed as biofilm aggregates on the surface of deep-sea invertebrates that support the growth of hosts through chemosynthetic carbon fixation. Growing evidence also indicates that community-wide interactions, and especially cooperation among symbionts, contribute to overall community productivity. Here, metagenome-guided metatranscriptomic and metabolic analyses were conducted to investigate the taxonomic composition, functions, and potential interactions of symbionts dwelling on the seta of Shinkaia crosnieri lobsters in a methane cold seep. Methylococcales and Thiotrichales dominated the community, followed by the Campylobacteriales, Nitrosococcales, Flavobacteriales, and Chitinophagales Metabolic interactions may be common among the episymbionts since many separate taxon genomes encoded complementary genes within metabolic pathways. Specifically, Thiotrichales could contribute to detoxification of hydroxylamine that is a metabolic by-product of Methylococcales. Further, Nitrosococcales may rely on methanol leaked from Methylococcales cells that efficiently oxidize methane. Elemental sulfur may also serve as a community good that enhances sulfur utilization that benefits the overall community, as evidenced by confocal Raman microscopy. Stable intermediates may connect symbiont metabolic activities in cyclical oxic-hypoxic fluctuating environments, which then enhance overall community functioning. This hypothesis was partially confirmed via in situ experiments. These results highlight the importance of microbe-microbe interactions in symbiosis and deep-sea adaptation. IMPORTANCE Symbioses between chemosynthetic bacteria and marine invertebrates are common in deep-sea chemosynthetic ecosystems and are considered critical foundations for deep-sea colonization. Episymbiotic microorganisms tend to form condensed biofilms that may facilitate metabolite sharing among biofilm populations. However, the prevalence of metabolic interactions among deep-sea episymbionts and their contributions to deep-sea adaptations are not well understood due to sampling and cultivation difficulties associated with deep-sea environments. Here, we investigated metabolic interactions among the episymbionts of Shinkaia crosnieri, a dominant chemosynthetic ecosystem lobster species in the Northwest Pacific Ocean. Meta-omics characterizations were conducted alongside in situ experiments to validate interaction hypotheses. Furthermore, imaging analysis was conducted, including electron microscopy, fluorescent in situ hybridization (FISH), and confocal Raman microscopy (CRM), to provide direct evidence of metabolic interactions. The results support the Black Queen Hypothesis, wherein leaked public goods are shared among cohabitating microorganisms to enhance the overall adaptability of the community via cooperation.

Latitudinal changes in the lipid content and fatty acid profiles of juvenile female red squat lobsters (Pleuroncodes monodon) in breeding areas of the Humboldt Current System.

The red squat lobster Pleuroncodes monodon is a species of high commercial value that inhabits the Humboldt Current System. Along the Chilean coast, two populations are exploited by the fishing industry, one located off the coast of Coquimbo and the other off the coast of Concepción. Yet, it is unknown whether there are differences in the "bioenergetic fuel" (measured as lipid content and fatty acid profile) of juvenile populations of these two fishing units and whether these bioenergetic compounds can be modulated by differences in the environmental parameters (such as temperature or chlorophyll-a) of their breeding areas. To shed some light on this, we measured the lipid content and fatty acid profiles of the viscera and muscle of juvenile female red squat lobsters from these two fishing units, specifically from breeding areas near long-exploited fishing grounds: a) the northern fishing unit (NFU, from 26°S to 30°S) and b) the southern fishing unit (SFU, from 32°S to 37°S). We found differences in the lipid content, fatty acid profiles, and ratios of saturated fatty acids (C16:0/C18:0) of juvenile females from these two locations. In addition, the essential fatty acids (DHA/EPA) found in the viscera versus the muscle of these lobsters varied significantly. Juvenile females from the SFU (i.e. Concepción) showed a higher lipid content compared to the juvenile females from the NFU (i.e. Coquimbo). Consistently, individuals from the SFU had a higher content of fatty acids, which also proved to be richer in saturated and monounsaturated fatty acids compared to those from the NFU. Our results are important for the fisheries in both areas because these juvenile populations are the source of new recruits for the adult populations that are exploited by the fishing industry. Our study also aids in determining which populations are healthier or of better quality in bioenergetic terms. Furthermore, increasing the incorporation of bioenergetic parameters in fishery models is essential for the recruitment and stock assessment within an ecosystem approach, since it allows for the evaluation of the nutritional condition of different fishing populations.

Influence of pesticides and abiotic conditions on biochemical biomarkers in Aegla aff. longirostri (crustacea, anomura): Implications for conservation.

Freshwater ecosystems are constantly threatened by the advance of agricultural activities. Abiotic variables (such as temperature, ammonia, and nitrite) and contaminants (e.g. pesticides) can potentially interact, increasing metabolism and the absorption of toxic substances, which can alter the ability of organisms to establish adequate stress responses. This study aimed to verify which pesticides were most frequently found and in the greatest quantities in low-order streams, and whether the combination of these pesticides with the abiotic variables altered the biological metabolism of aeglids. These freshwater crustaceans are important shredders that inhabit low-order streams and are sensitive to disturbances and/or abrupt environmental variations. The animals were exposed in situ in four streams (reference site and sites 1, 2, and 3). The reference site is a preserved stream with no apparent anthropogenic interference where aeglids still occur, while the other sites no longer exhibit populations of these animals and are influenced by agricultural activities. The exposure was performed bimonthly from November 2017 to September 2018 and lasted 96 h. Measured abiotic data and water samples were collected through all days of exposure. The analyzed biochemical parameters were acetylcholinesterase activity in muscle; and glutathione S-transferase, lipid peroxidation, protein carbonylation, non-protein thiols, antioxidant capacity against peroxides, and reactive oxygen species (ROS) in muscle, gills, and hepatopancreas. We found 24 active principles of pesticides, the most frequently being clomazone, atrazine, and propoxur. Bentazone was present at the highest amounts. The parameters evaluated in this study, including biochemical biomarkers and abiotic factors measured from the water, provided a separation of the months as a function of environmental conditions. There was a difference in activity and biomarker levels throughout the year within the same site and in some months between sites. The greater concentration or variety of pesticides associated with extreme abiotic (very high temperatures) data generated increased oxidative stress, with high levels of protein damage and considerable lipid damage in all tissues, as well as elevation in ROS, even with high levels of antioxidant capacity and non-protein thiols. With these data, we intend to warn about the risks of exposure to these environmental conditions by trying to contribute to the preservation of limnic fauna, especially aeglid crabs, because most species are under some degree of threat.

Transcriptomic analysis reveals insights into deep-sea adaptations of the dominant species, Shinkaia crosnieri (Crustacea: Decapoda: Anomura), inhabiting both hydrothermal vents and cold seeps.

BACKGROUND: Hydrothermal vents and cold seeps are typical deep-sea chemosynthetically-driven ecosystems that allow high abundance of specialized macro-benthos. To gather knowledge about the genetic basis of adaptation to these extreme environments, species shared between different habitats, especially for the dominant species, are of particular interest. The galatheid squat lobster, Shinkaia crosnieri Baba and Williams, 1998, is one of the few dominant species inhabiting both deep-sea hydrothermal vents and cold seeps. In this study, we performed transcriptome analyses of S. crosnieri collected from the Iheya North hydrothermal vent (HV) and a cold seep in the South China Sea (CS) to provide insights into how this species has evolved to thrive in different deep-sea chemosynthetic ecosystems. RESULTS: We analyzed 5347 orthologs between HV and CS to identify genes under positive selection through the maximum likelihood approach. A total of 82 genes were identified to be positively selected and covered diverse functional categories, potentially indicating their importance for S. crosnieri to cope with environmental heterogeneity between deep-sea vents and seeps. Among 39,806 annotated unigenes, a large number of differentially expressed genes (DEGs) were identified between HV and CS, including 339 and 206 genes significantly up-regulated in HV and CS, respectively. Most of the DEGs associated with stress response and immunity were up-regulated in HV, possibly allowing S. crosnieri to increase its capability to manage more environmental stresses in the hydrothermal vents. CONCLUSIONS: We provide the first comprehensive transcriptomic resource for the deep-sea squat lobster, S. crosnieri, inhabiting both hydrothermal vents and cold seeps. A number of stress response and immune-related genes were positively selected and/or differentially expressed, potentially indicating their important roles for S. crosnieri to thrive in both deep-sea vents and cold seeps. Our results indicated that genetic adaptation of S. crosnieri to different deep-sea chemosynthetic environments might be mediated by adaptive evolution of functional genes related to stress response and immunity, and alterations in their gene expression that lead to different stress resistance. However, further work is required to test these proposed hypotheses. All results can constitute important baseline data for further studies towards elucidating the adaptive mechanisms in deep-sea crustaceans.

Decreased Metabolic Rate in the Mole Crabs, Emerita analoga, Infected with the Acanthocephalan Profilicollis altmani.

The relationship between a parasite and its host will have an energy cost for the host at some point; however, this basic hypothesis has seldom been evaluated. Using Emerita analoga as a model species, we investigated the aerobic metabolism, ammonia excretion rate, and locomotor performance patterns of crabs both uninfected and infected with the acanthocephalan Profilicollis altmani. Our results show that infected Emerita had a lower metabolic rate compared to uninfected ones. Whether or not this decrease is a result of the pathology of the parasite infection or due to manipulation of the host by the parasite is still unknown. We discuss the importance of using anaerobic and aerobic metabolism and more-complex scenarios in order to understand the effects on host fitness and not only on the immediate response of the host.

What are the sympatric mechanisms for three species of terrestrial hermit crab (Coenobita rugosus, C. brevimanus, and C. cavipes) in coastal forests?

Terrestrial hermit crabs play a significant role in coastal ecology. For example, as seed dispersers and debris scavengers in coastal forests, they accelerate the decomposition of organic substances. In the Indo-Pacific Ocean, Coenobita rugosus, C. brevimanus, and C. cavipes are the three most common species of terrestrial hermit crab. Because the mechanisms that contribute to the sympatry of these three species of crab have not been identified, this study investigated the three most likely explanations: niche differences, competition, and predation. The results showed that the three species displayed niche differences in terms of seasonal activity, habitat, utilization of shells, and food preference, suggesting that competition for resources is avoided. The habitat of terrestrial hermit crabs in Taiwan is closely associated with that of humans. Our study helps improve our understanding of the ecology of terrestrial hermit crabs and their conservation.

Effect of Cd and Pb Pollutions on Physiological Growth: Wavelet Neural Network (WNN) as a New Approach on Age Determination of Coenobita scaevola.

Environmental pollution of aquatic ecosystems leads to an interference in several fundamental biochemical and physiological functions. In this study the interference of Cd and Pb pollutions on the physiological growth and subsequently on the age determination was investigated. The hermit crab, Coenobita scaevola (C.s) was selected as a bioaccumulator in this study. The direct and indirect age determination methods were carried out using annual band counts and carapace length, respectively. The results showed that, there was very low correlation (R2 < 0.5) between direct and indirect age determination. Wavelet Neural Network (WNN) was applied to take into account the environmental effects such as the accumulation of Cd and Pb elements in the C.s' tissues. It was observed that WNN successfully enhanced the growth rate model and estimated the C.s' age (R2 > 0.95). In addition, it was concluded that the environmental pollution had interaction with the growth physiology such as weight and length.

Metabolic suppression in the pelagic crab, Pleuroncodes planipes, in oxygen minimum zones.

The pelagic red crab, Pleuroncodes planipes, is abundant throughout the Eastern Tropical Pacific in both benthic and pelagic environments to depths of several hundred meters. The oxygen minimum zones in this region reaches oxygen levels as low as 0.1kPa at depths within the crabs vertical range. Crabs maintain aerobic metabolism to a critical PO2 of ~0.27±0.2kPa (10°C), in part by increasing ventilation as oxygen declines. At subcritical oxygen levels, they enhance anaerobic ATP production slightly as indicated by modest increases in lactate levels. However, hypoxia tolerance is primarily mediated via a pronounced suppression of aerobic metabolism (~70%). Metabolic suppression is achieved, primarily, via reduced protein synthesis, which is a major sink for metabolic energy. Posttranslational modifications on histone H3 suggest a condensed chromatin state and, hence, decreased transcription. Under hypoxia, p-H3S10, Ac-H3K9, Ac-H3K14 were 39, 68, and 36% of control values, respectively. We also report a net decrease in protein translation. In particular, eEF2 activity is reduced due to a ~5-fold increase in inhibitory phosphorylation and a significant decrease in protein level. Elevated heat shock proteins suggest that, despite impressive tolerance, the cellular stress response is triggered during hypoxia. We discuss the implications for pelagic ecology and biogeochemical cycles.

The proteomic response of cheliped myofibril tissue in the eurythermal porcelain crab Petrolisthes cinctipes to heat shock following acclimation to daily temperature fluctuations.

The porcelain crab Petrolisthes cinctipes lives under rocks and in mussel beds in the mid-intertidal zone where it experiences immersion during high tide and saturating humid conditions in air during low tide, which can increase habitat temperature by up to 20°C. To identify the biochemical changes affected by increasing temperature fluctuations and subsequent heat shock, we acclimated P. cinctipes for 30 days to one of three temperature regimes: (1) constant 10°C, (2) daily temperature fluctuations between 10 and 20°C (5 h up-ramp to 20°C, 1 h down-ramp to 10°C) and (3) 10-30°C (up-ramp to 30°C). After acclimation, animals were exposed to either 10°C or a 30°C heat shock to analyze the proteomic changes in claw muscle tissue. Following acclimation to 10-30°C (measured at 10°C), enolase and ATP synthase increased in abundance. Following heat shock, isoforms of arginine kinase and glycolytic enzymes such as aldolase, triose phosphate isomerase and glyceraldehyde 3-phosphate dehydrogenase increased across all acclimation regimes. Full-length isoforms of hemocyanin increased abundance following acclimation to 10-30°C, but hemocyanin fragments increased after heat shock following constant 10°C and fluctuating 10-20°C, possibly playing a role as antimicrobial peptides. Following constant 10°C and fluctuating 10-20°C, paramyosin and myosin heavy chain type-B increased in abundance, respectively, whereas myosin light and heavy chain decreased with heat shock. Actin-binding proteins, which stabilize actin filaments (filamin and tropomyosin), increased during heat shock following 10-30°C; however, actin severing and depolymerization proteins (gelsolin and cofilin) increased during heat shock following 10-20°C, possibly promoting muscle fiber restructuring. RAF kinase inhibitor protein and prostaglandin reductase increased during heat shock following constant 10°C and fluctuating 10-20°C, possibly inhibiting an immune response during heat shock. The results suggest that ATP supply, muscle fiber restructuring and immune responses are all affected by temperature fluctuations and subsequent acute heat shock in muscle tissue. Furthermore, although heat shock after acclimation to constant 10°C and fluctuating 10-30°C showed the greatest effects on the proteome, moderately fluctuating temperatures (10-20°C) broadened the temperature range over which claw muscle was able to respond to an acute heat shock with limited changes in the muscle proteome.

Trace and major elements distribution and transfer within a benthic system: polychaete Chaetopterus variopedatus, commensal crab Polyonyx gibbesi, worm tube, and sediments.

Samples of the polychaete Chaetopterus variopedatus, worm tubes, commensal crab Polyonyx gibbesi and sediments were collected in eight sites in Todos os Santos Bay, Brazil, in order to evaluate the potential use of the polychaetes and crabs as biomonitors and to assess the relationships and accumulation of trace and major elements in different benthic compartments. Trace and major elements were determined by ICP OES. Organic carbon, total nitrogen and sulfur were determined by CNS elemental analyser. Tubes, crabs and polychaetes were important in the retention of trace and major elements. Metals that presented the highest accumulation in polychaetes (i.e. Mg>Al>Fe>Zn>Mn>Co>Cu>Ba>Cr) where the same for crabs (i.e. Mg>Al>Fe>Mn>Co>Zn>Cu>Ba>Cr). High concentrations of Al, Ba, Cr, Mn and Fe, from terrigenous sources, were observed in tubes, which presented accumulation factors up to 81.5 for Mn. Sedentary polychaetes are seen as good biomonitor alternatives for metal contamination studies, because they are one of the most abundant taxon in the benthic system, live in direct contact with sediments, are present in broad distributions and can also handle relatively high concentrations of metals ensuring chronic exposition. The possibility to work with not only the polychaete but also its tube offers advantages compared to bivalves that generally do not accumulate certain metals in very high levels.

Neuropeptide complexity in the crustacean central olfactory pathway: immunolocalization of A-type allatostatins and RFamide-like peptides in the brain of a terrestrial hermit crab.

BACKGROUND: In the olfactory system of malacostracan crustaceans, axonal input from olfactory receptor neurons associated with aesthetascs on the animal's first pair of antennae target primary processing centers in the median brain, the olfactory lobes. The olfactory lobes are divided into cone-shaped synaptic areas, the olfactory glomeruli where afferents interact with local olfactory interneurons and olfactory projection neurons. The local olfactory interneurons display a large diversity of neurotransmitter phenotypes including biogenic amines and neuropeptides. Furthermore, the malacostracan olfactory glomeruli are regionalized into cap, subcap, and base regions and these compartments are defined by the projection patterns of the afferent olfactory receptor neurons, the local olfactory interneurons, and the olfactory projection neurons. We wanted to know how neurons expressing A-type allatostatins (A-ASTs; synonym dip-allatostatins) integrate into this system, a large family of neuropeptides that share the C-terminal motif -YXFGLamide. RESULTS: We used an antiserum that was raised against the A-type Diploptera punctata (Dip)-allatostatin I to analyse the distribution of this peptide in the brain of a terrestrial hermit crab, Coenobita clypeatus (Anomura, Coenobitidae). Allatostatin A-like immunoreactivity (ASTir) was widely distributed in the animal's brain, including the visual system, central complex and olfactory system. We focussed our analysis on the central olfactory pathway in which ASTir was abundant in the primary processing centers, the olfactory lobes, and also in the secondary centers, the hemiellipsoid bodies. In the olfactory lobes, we further explored the spatial relationship of olfactory interneurons with ASTir to interneurons that synthesize RFamide-like peptides. We found that these two peptides are present in distinct populations of local olfactory interneurons and that their synaptic fields within the olfactory glomeruli are also mostly distinct. CONCLUSIONS: We discuss our findings against the background of the known neurotransmitter complexity in the crustacean olfactory pathway and summarize what is now about the neuronal connectivity in the olfactory glomeruli. A-type allatostatins, in addition to their localization in protocerebral brain areas, seem to be involved in modulating the olfactory signal at the level of the deutocerebrum. They contribute to the complex local circuits within the crustacean olfactory glomeruli the connectivity within which as yet is completely unclear. Because the glomeruli of C. clypeatus display a distinct pattern of regionalization, their olfactory systems form an ideal model to explore the functional relevance of glomerular compartments and diversity of local olfactory interneurons for olfactory processing in crustaceans.

[The distribution of catecholamine-containing neurons in the brain of Pagurus middendorffii and Paralithodes camtschaticus (Anomura, Decapoda)].

Immunocytochemical methods using an antibody raised against tyrosine hydroxylase (TH) were investigated the distribution of catecholamine-containing neurons in the brain of two species of crustacean Pagurus middendorffii and Paralithodes camtschaticus. Morphologically different types of TH-immunoreactive neurons are found in the cell clusters in the protocerebrum, deutocerebrum, and tritocerebrum of divisions the brain in both species studied crustaceans. The similarities and differences in topography, numbers and morphology of TH-positive neurons in the different structures of the brain in the hermit crab and king crab are discussed.

Heavy metal concentrations in some macrobenthic fauna of the Sundarbans mangrove forest, south west coast of Bangladesh.

Heavy metal concentrations in some macrobenthic fauna have been reported for the first time from the Sundarbans mangrove forest, south west coast of Bangladesh, in the northern part of Bay of Bengal. The concentration of Fe, Cu, Zn, Cd and Pb in macrobenthos ranged from 235 ± 10.11 to 1,051 ± 38.42, 3.66 ± 0.89 to 7.55 ± 1.29, 76.8 ± 8.55 to 98.5 ± 6.49, 0.46 ± 0.11 to 0.859 ± 0.2 and 4.66 ± 1.17 to 6.77 ± 2.1 µg/g, respectively. Significant variations (p ≤ 0.05) in heavy metal concentrations have been observed among the mud crab, mudskipper and gastropod. However, heavy metal burdens did not vary significantly among the hermit and horseshoe crabs. In mud crab, horseshoe crab and gastropod, heavy metal concentrations were recorded in the sequence: Fe > Zn > Pb > Cu > Cd. Hermit crab and mudskipper contained heavy metals in the order of Fe > Zn > Cu > Pb > Cd. Fe and Zn concentrations were found significantly (p ≤ 0.05) higher in macrobenthos. The lead (Pb) concentration found in the edible portion of macrobenthos exceeded the international permissible limits certified by the WHO. Bioconcentration factors >1.00 obtained for Fe (17.05 in mudskipper) and Cd (1.87 in gastropod) indicated that these metals were highly bioaccumulated and biomagnified in benthic fauna of Sundarbans. The findings of this study refer to the potential impact of heavy metals in the mangrove ecosystem of Bangladesh.

DSN depletion is a simple method to remove selected transcripts from cDNA populations.

A novel DSN-depletion method allows elimination of selected sequences from full-length-enriched cDNA libraries. Depleted cDNA can be applied for subsequent EST sequencing, expression cloning, and functional screening approaches. The method employs specific features of the kamchatka crab duplex-specific nuclease (DSN). This thermostable enzyme is specific for double-stranded (ds) DNA, and is thus used for selective degradation of ds DNA in complex nucleic acids. DSN depletion is performed prior to library cloning, and includes the following steps: target cDNA is mixed with excess driver DNA (representing fragments of the genes to be eliminated), denatured, and allowed to hybridize. During hybridization, driver molecules form hybrids with the target sequences, leading to their removal from the ss DNA fraction. Next, the ds DNA fraction is hydrolyzed by DSN, and the ss fraction is amplified using long-distance PCR. DSN depletion has been tested in model experiments.

Isolation and properties of collagenolytic serine proteinase isoenzyme from King Crab Paralithodes camtschatica.

An electrophoretically homogeneous isoenzyme CSP-2 of collagenolytic serine proteinase has been isolated from the total preparation of king crab digestive enzymes. The molecular mass of the proteinase is 24.8 +/- 0.3 kD, pH optimum for activity is 8.5, the temperature optimum for activity is 38-40 degrees C, and the pH range of stability is 7-10. The enzyme has dual substrate specificity, but preference for positively charged amino acid residues in P(1)-position is more pronounced than in the case of the major isoenzyme. The temperature dependence of kinetic constants for synthetic substrate hydrolysis by CSP-2 has been investigated. Inhibition specificity of the enzyme is characteristic of serine proteinases but more like that of crab trypsin than that of the major CSP isoenzyme. The isolated collagenolytic proteinase also cleaves fibrinogen and fibrin and activates plasminogen. The amino acid sequence of the CSP-2 proteinase, which has been partially determined by tandem mass spectrometry, displays some similarity to the sequence of the major CSP isoenzyme.

[Change of carotenoid composition in crabs during embryogenesis].

Changes of the qualitative and quantitative compositions of carotenoids are studied at various development stages of the external egg, determined based on color differences, for the species C. opilio, P. camtschaticus, and P. platypus. It has been established that the main carotenoids of the new egg are astaxanthin and beta-carotene. Intermediate products of transformation of beta-carotene into astaxanthin are identified: echinenon, cantaxanthin, and phoenicoxanthin. The carotenoid content per embryo for the new egg of C. opilio (the orange egg) amounted to 22.7 ng, of P. camtschaticus and P. platypus (the violet egg)--to 49.2 and 23.3 ng, respectively. In the egg at the later development stage (the brown egg) the carotenoid content was decreased to 13.1 ng in C. opilio and to 20.1 ng in P. camtschaticus. Development of embryos is accompanied by accumulation of esterified carotenoids and a decrease of beta-carotene and astaxanthin concentrations in all studied species.

New insights into evolution of crustacean hyperglycaemic hormone in decapods--first characterization in Anomura.

The neuropeptides of the crustacean hyperglycaemic hormone (CHH) family are encoded by a multigene family and are involved in a wide spectrum of essential functions. In order to characterize CHH family peptides in one of the last groups of decapods not yet investigated, CHH was studied in two anomurans: the hermit crab Pagurus bernhardus and the squat lobster Galathea strigosa. Using RT-PCR and 3' and 5' RACE methods, a preproCHH cDNA was cloned from the major neuroendocrine organs (X-organs) of these two species. Hormone precursors deduced from these cDNAs in P. bernhardus and G. strigosa are composed of signal peptides of 29 and 31 amino acids, respectively, and CHH precursor-related peptides (CPRPs) of 50 and 40 amino acids, respectively, followed by a mature hormone of 72 amino acids. The presence of these predicted CHHs and their related CPRPs was confirmed by performing MALDI-TOF mass spectrometry on sinus glands, the main neurohaemal organs of decapods. These analyses also suggest the presence, in sinus glands of both species, of a peptide related to the moult-inhibiting hormone (MIH), another member of the CHH family. Accordingly, immunostaining of the X-organ/sinus gland complex of P. bernhardus with heterologous anti-CHH and anti-MIH sera showed the presence of distinct cells producing CHH and MIH-like proteins. A phylogenetic analysis of CHHs, including anomuran sequences, based on maximum-likelihood methods, was performed. The phylogenetic position of this taxon, as a sister group to Brachyura, is in agreement with previously reported results, and confirms the utility of CHH as a molecular model for understanding inter-taxa relationships. Finally, the paraphyly of penaeid CHHs and the structural diversity of CPRPs are discussed.