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Changes in amino acids and lipids during embryogenesis of European lobster, Homarus gammarus (Crustacea: Decapoda).

We studied the amino acid and lipid dynamics during embryogenesis of Homarus gammarus. Major essential amino acids (EAA) in the last stage of embryonic development were arginine, lysine and leucine; major nonessential amino acids (NEAA) were glutamic acid, aspartic acid, valine and glycine. The highest percent of utilization occurred in respect to EAA (27.8%), mainly due to a significant decrease (p<0.05) of methionine (38.3%) and threonine (36.0%). NEAA also decreased significantly (p<0.05, 11.4%), namely serine (38.1%), tyrosine (26.4%) and glutamic acid (25.7%). In contrast, the free amino acid content increased significantly (p<0.05) during embryonic development, especially the free nonessential amino acids (FNEAA). In the last stage, the most abundant FNEAA were glycine, proline, alanine and taurine, and the major free essential amino acids (FEAA) were arginine, lysine and leucine. Lipid content decreased significantly (p<0.05) during embryonic development. A substantial decrease in all neutral lipid classes was observed (>80% of utilization). Major fatty acids were 16:0, 18:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:5n-3 and 22:6n-3. Unsaturated (UFA) and saturated fatty acids (SFA) were used up at similar rates (76.5% and 76.3%, respectively). Within UFA, monounsaturates (MUFA) were consumed more than polyunsaturates (PUFA) (82.9% and 67.5%, respectively).

Amino Acids↗

Effects of starvation on oxygen consumption, ammonia excretion and biochemical composition of the hepatopancreas on adult males of the False Southern King crab Paralomis granulosa (Crustacea, Decapoda).

Adults of the False Southern King crab, Paralomis granulosa, were starved between 0 and 12 days to evaluate the impact of fasting on the oxygen consumption, nitrogen excretion, O/N ratio and changes on biochemical composition of the hepatopancreas. During the experiment, no mortalities were recorded; physiological changes were detected after 6 days of fasting with an increase of nitrogen excretion (p<0.05). After 9 days of starvation, the crabs showed a maximum decrease in the lipid content (4.3+/-1.2%, p<0.05), accompanied by an increase in oxygen consumption (53.1+/-10.9 microg O2 h(-1) g(-1)). The lowest O/N ratio was detected after 6 days (6.4+/-4.8) and the highest after 12 days of fasting (38.1+/-20.4), indicating that initially crabs utilized proteins as source of energy , followed by lipids. Moreover, after 12 days, there was a significant increase in the hepatosomatic index (HI) and total lipid content (9.7+/-1.0%, p<0.05), which could be associated with the re-absorption of other tissues to the hepatopancreas. Our results provide new information on this species that shows a different pattern of adaptation for each period of starvation and a good correlation between physiological and biochemical parameters. The ability to withstand and recover from periods of nutritional stress is an important adaptation for survival of any organism that must sporadically endure periods of limited food supply.

Ammonia↗

Biochemical composition of ovary, embryo, and hepatopancreas in the grapsoid crabs Armases cinereum and Sesarma nr. reticulatum (Crustacea, Decapoda).

Biochemical composition of ovary, embryo, and hepatopancreas tissues in wild populations of Armases cinereum and Sesarma nr. reticulatum were monitored during the reproductive season. Total lipid, carbon, nitrogen, C:N ratio, and water concentration of the ovary, hepatopancreas and embryos were quantified over the course of ovarian maturation. Ovary nitrogen concentration decreased as ovaries matured. Ovary lipid and carbon concentration differed significantly over the course of ovarian maturation for both species, but there was no relationship between the concentration or total content of hepatopancreas lipid and the stage of ovarian development in females. Neither species showed a relationship between measures of hepatopancreas lipid and the gonadosomatic index. There was also no simultaneously measurable net decrease in mass of the females' hepatopancreas. Lipid demands of ovarian maturation thus appear to be met in large part by increased dietary intake rather than by substantial draw down of pre-existing lipid stores from the hepatopancreas. While these temperate grapsoid crabs live with putatively fluctuating quality and quantity of food resources, no evidence could be found to demonstrate depletion of lipid concentrations in the hepatopancreas concomitant with ovarian maturation.

Animals↗

Use of the multispecies freshwater biomonitor to assess behavioral changes of Corophium volutator (Pallas, 1766) (Crustacea, Amphipoda) in response to toxicant exposure in sediment.

Automated sediment toxicity testing and biomonitoring has grown rapidly. This study tested the suitability of the marine amphipod Corophium volutator (Pallas, 1766) for sediment biomonitoring using the Multispecies Freshwater Biomonitor (MFB). Two experiments were undertaken to (1) characterize individual behaviors of C. volutator using the MFB and (2) examine behavioral changes in response to sediment spiked with the pesticide Bioban. Four behaviors were visually identified (walking, swimming, grooming and falling) and characterized in the MFB as different patterns of locomotor activity (0-2 Hz range). Ventilation was not visually observed but was detected by the MFB (2-8 Hz). No clear diel activity patterns were detected. The MFB detected an overall increase in C. volutator locomotor activity after Bioban addition to the sediments (56, 100, 121 mg kg(-1)). C. volutator was more active (both locomotion and ventilation) in the water column than the spiked sediment. C. volutator appears a sensitive and appropriate species for behavioral sediment toxicity assessment and biomonitoring.

Amphipoda↗

Chronic effect of NaCl salinity on a freshwater strain of Daphnia magna Straus (Crustacea: Cladocera): a demographic study.

Daphnia magna is mainly recognized as a freshwater cladoceran, but there are some strains that grow in brackish waters. The tolerance to salinity of a freshwater strain was assessed at NaCl concentrations of 0, 2, 4, 6, and 7 g L(-1). The green microalga Ankistrodesmus falcatus was fed at optimal concentration (4 x 10(5)cells mL(-1)). Reproduction and survival were recorded in two experimental series: in the first one, 20 female neonates were individually studied for each treatment. In the second, cohorts of 10 female neonates were distributed in each of five replicates per treatment. In both cases, experiments were conducted over a full life-cycle. The determined 48-h LC(50) for NaCl was 5.48 g L(-1), but we recorded reproduction at up to 7 g NaCl L(-1). The average clutch size, total progeny, number of clutches, and longevity were significantly reduced by the NaCl concentration (P<0.01); total progeny ranged from 467 to 25 neonates as edge values for NaCl concentrations of 0-7 g L(-1). Inter-brood time was significantly higher for females grown at 7 g NaCl L(-1) (3.9 days). The Life Table analysis demonstrates that average lifespan, life expectancy at birth, net reproductive rate and intrinsic rate of growth were also significantly reduced according to NaCl concentration. Based on the results for the two highest NaCl concentrations (6 and 7 g L(-1)), we conclude that the used D. magna strain was acclimated to develop satisfactorily under concentrations of up to 6 g NaCl L(-1); however, the established salinity conditions reduced significantly reproduction and survival in this strain.

Animals↗

Heavy metal toxicity in Exosphaeroma gigas (Crustacea, Isopoda) from the coastal zone of Beagle Channel.

Acute toxicity of copper, cadmium, and zinc on isopod Exosphaeroma gigas was evaluated at 20 per thousand and 30 per thousand salinity. Six concentrations were assayed to estimate effective concentration of the toxicant that affects 50% of tested animal (EC50), while physiological responses and bioaccumulation were determined at 0.42 and 1.95 mg L(-1) of each metal. The following toxicity orders were obtained: Cd>or=Cu>Zn at 20 per thousand salinity and Cu>Zn>or=Cd at 30 per thousand salinity. Copper treatments showed a reduction in oxygen consumption at 30 per thousand salinity, while an opposite trend was observed at 20 per thousand salinity. Zinc caused dissimilar effects, while in cadmium significant reduction was only registered in 0.42(20 per thousand) mg L(-1). Ammonia excretion was generally higher in treatments than control at 20 per thousand salinity. At 30 per thousand salinity, excretion did not change in relation to control or diminished. In general terms, O:N atomic ratios indicated a preponderant protein metabolism. Bioaccumulation of assayed metals was higher at lowest salinity and increased with increasing toxic concentrations.

Ammonia↗

A novel Spiroplasma pathogen causing systemic infection in the crayfish Procambarus clarkii (Crustacea: Decapod), in China.

A novel disease of crayfish Procambarus clarkii appeared in the summer of 2004 in freshwater aquaculture in Jiangsu province of China. Light and transmission electron microscopy (TEM), molecular biological methods and in vitro culture were used to identify the pathogen. The agent was unique in having a helical morphology and rotary motility as observed by phase-contrast light microscopy and was found in haemolymph, muscles, nerves and connective tissues by smear method and TEM. Ultra-thin sections under TEM revealed the wall-free membrane of the microbe. The agent could pass through membrane filters with pores 220 nm in diameter and was cultivated in vitro in M1D medium. 16S rDNA of the crayfish pathogen was amplified by PCR using primers specific for Spiroplasma-specific 16S rDNA. The resultant 271bp PCR product showed 99% identity with Spiroplasma mirum 16S rDNA, having a close relationship with the spiroplasma from the Chinese mitten crab Eriocheir sinensis. This is the second time a spiroplasma has been found in a freshwater crustacean. The 271bp PCR product was also amplified from the bottom mud in the ponds associated with the disease. The PCR molecular method is an effective way to detect spiroplasma in freshwater environment. The results from this study are significant in expanding the host range of spiroplasma and freshwater ecology.

Animals↗

Large-scale gene rearrangements in the mitochondrial genomes of two calanoid copepods Eucalanus bungii and Neocalanus cristatus (Crustacea), with notes on new versatile primers for the srRNA and COI genes.

Partial sequences of the mitochondrial genomes were determined for two calanoid copepods, Eucalanus bungii [9530 bp (base pairs)] and Neocalanus cristatus (7965 bp), using an approach that employs a long polymerase chain reaction (PCR) technique and primer walking. In contrast to Tigriopus japonicus (Harpacticoida), in which the complete mitochondrial genome has been determined, genes are encoded on both strands of the genome. In spite of the close relationship between E. bungii and N. cristatus, the gene orders of these calanoid copepods differed greatly from each other. From the sequence we obtained, new copepod-specific versatile primers were designed for the cytochrome c oxidase I (COI) gene (1200 bp). These primer pairs, as well as the previously designed primer pairs for the small ribosomal RNA (srRNA) gene (500 bp), were applied for 20 species representing four orders of both calanoid and non-calanoid copepods and amplification of sequences from at least one species from each order was successful.

Animals↗

Organisation of trypsin genes in the salmon louse (Lepeophtheirus salmonis, Crustacea, copepoda) genome.

Trypsins constitute a subclass of the S1A family of serine peptidases found in all groups of animal and some bacteria. At present, no information about the genomic organisation of trypsins is available for copepods. The only data of copepod trypsins indicate several different trypsins in the marine parasitic copepod Lepeophtheirus salmonis. In the present study, 31.7 kbp of genomic DNA surrounding the previously described LsTryp1-5 sequences was sequenced. The sequenced regions contain nine full-length and three partial trypsin genes. A conservative estimate based on PCR analysis and genomic sequence indicated at least 22 different trypsin genes in L. salmonis, of which 18 are most similar to the previously described LsTryp1 and -2 cDNA sequences. Four of these genes are putative pseudogenes. In addition, a putative mariner like transposase gene was identified. The genomic sequences suggest that the L. salmonis trypsin genes reside within one or more gene clusters. Three different LsTryp intron exon structures were identified, and all three are different from the intron exon organisation previously reported for other S1A peptidases. This implies several intron loss and gain events in the evolution of the L. salmonis trypsin genes.

Animals↗

Genetic characterization of the mitochondrial DNA from Lepeophtheirus salmonis (Crustacea; Copepoda). A new gene organization revealed.

The mitochondrial DNA (mtDNA) from the salmon louse, Lepeophtheirus salmonis, is 15445 bp. It includes the genes coding for cytochrome B (Cyt B), ATPase subunit 6 and 8 (A6 and A8), NADH dehydrogenase subunits 1-6 and 4L (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6), cytochrome c oxidase subunits I-III (COI, COII and COIII), two rRNA genes (12S rRNA and 16S rRNA) and 22 tRNAs. Two copies of tRNA-Lys are present in the mtDNA of L. salmonis, while tRNA-Cys was not identified. Both DNA strands contain coding regions in the salmon louse, in contrast to the other copepod characterized Tigriopus japonicus, but only a few genes overlap. In vertebrates, ND4 and ND4L are transcribed as one bicistronic mRNA, and are therefore localized together. The same organization is also found in crustaceans, with the exceptions of T. japonicus, Neocalanus cristatus and L. salmonis that deviate from this pattern. Another exception of the L. salmonis mtDNA is that A6 and A8 do not overlap, but are separated by several genes. The protein-coding genes have a bias towards AT-rich codons. The mitochondrial gene order in L. salmonis differs significantly from the copepods T. japonicus, Eucalanus bungii, N. cristatus and the other 13 crustaceans previously characterized. Furthermore, the mitochondrial rRNA genes are encoded on opposite strands in L. salmonis. This has not been found in any other arthropods, but has been reported in two starfish species. In a phylogenetic analysis, using an alignment of mitochondrial protein sequences, L. salmonis groups together with T. japonicus, being distant relatives to the other crustaceans.

Animals↗

The mitochondrial genome of the Japanese freshwater crab, Geothelphusa dehaani (Crustacea: Brachyura): evidence for its evolution via gene duplication.

The translocation of tRNA genes has occurred frequently among multiple independent arthropod lineages. However, the mechanisms of such gene rearrangement are still unclear. In this study we sequenced the nearly complete mitochondrial genome of the Japanese freshwater crab, Geothelphusa dehaani (Decapoda, Brachyura, Potamidae). Its size is relatively large (app. 24 kb) for higher animal mtDNA and is due to the presence of many intergenic noncoding sequences (IGNs), one of which is a large repeat region (>7.0 kb). Excluding the repeat region and the putative control region (514 bp), the total size of IGNs (1953 bp) is still the largest reported in arthropods. In comparison to the proposed ancestral arrangement for the insect-crustacean clade, G. dehaani displays changes in the positional order for three tRNA genes (trnQ, trnL(uur) and trnH). At the putative former and current locations for trnQ and trnH, there are IGNs with high similarities to corresponding sequences in the ancestral arrangement. This suggests the occurrence of partial duplication, followed by a loss of function for one copy of these two duplicated genes in G. dehaani. Furthermore, three trnLs (one trnL(uur) and two trnL(cun)s) are found in G. dehaani; their sequences are highly similar to each other (70.8-82.5%) and similar to the trnL(cun) of other arthropods. This suggests that duplication of trnL(cun) occurred at least twice, and the current trnL(uur) of G. dehaani arose by a subsequent anticodon alternation. These observations provide direct evidence for the "duplication-random loss" model for mtDNA gene rearrangement. Comparisons with other available Brachyuran data(http://www.ncbi.nlm.nih.gov/genomes/ORGANELLES/6657.html) suggest that two of the three tRNA translocations are unique in G. dehaani. For G. dehaani, where duplications have occurred but deletion of extra sequences is incomplete, the IGNs found at the ancestral or trans-locations are likely to be footprints of relatively recent mitochondrial genome evolution.

Animals↗

Ultrastructural study on a novel microsporidian, Endoreticulatus eriocheir sp. nov. (Microsporidia, Encephalitozoonidae), parasite of Chinese mitten crab, Eriocheir sinensis (Crustacea, Decapoda).

A microsporidian pathogen, infecting the epithelial cells of the hepatopancreas of Chinese mitten crab, Eriocheir sinensis, was studied by electron microscopy. The detailed ultrastructure of life cycle of the pathogen including proliferative and sporogonic developmental stages are provided. All stages of the parasite are haplokaryotic and develop in a vacuole bounded by a single membrane in contact with host cell cytoplasm. Sporogenesis is synchronous with the same developmental stage in one vacuole. Sporogony shows a characteristic of multinucleate sporogonial plasmodia divided by rosette-like division, producing 4 or 8 sporoblasts. The mature spore is ellipsoidal, length (mean) 1.7 microm, width 1.0 microm, with a uninucleate in the center of the sporoplasm, 7 turns of the polar filament, a bell-like polaroplast of compact membranes and obliquely positioned posterior vacuole. The morphological characteristics of this novel microsporidian pathogen have led us to assign the parasite to a new species of Endoreticulatus, E. eriocheir sp. nov., that has not been reported previously from crab.

Animals↗

Architecture of the organic matrix in the sternal CaCO3 deposits of Porcellio scaber (Crustacea, Isopoda).

Before the molt terrestrial isopods resorb calcium from the posterior cuticle and store it in large deposits within the first four anterior sternites. In Porcellio scaber the deposits consist of three structurally distinct layers consisting of amorphous CaCO3 (ACC) and an organic matrix that consists of concentric and radial elements. It is thought that the organic matrix plays a role in the structural organization of deposits and in the stabilization of ACC, which is unstable in vitro. In this paper, we present a thorough analysis of the ultrastructure of the organic matrix in the CaCO3 deposits using high-resolution field-emission scanning electron microscopy. The spherules and the homogeneous layer contain an elaborate organic matrix with similar structural organization consisting of concentric reticules and radial strands. The decalcification experiments reveal an inhomogeneous solubility of ACC within the spherules probably caused by variations in the stabilizing properties of matrix components. The transition between the three layers can be explained by changes in the number of spherule nucleation sites.

Animals↗

Butyltin concentrations along the Japanese coast from 1997 to 1999 monitored by Caprella spp. (Crustacea: Amphipoda).

The concentrations of butyltins along the Japanese coastline were investigated from 1997 to 1999, 7 to 9 years after implementation of legislation limiting the use of tributyltin (TBT) in Japan. Seawater was sampled at 0.5 m depth, and Caprella spp. were collected from Sargassum spp. and aquaculture facilities from 18 areas within four broad areas along the coastline of Japan, i.e., the Pacific coast of northern Japan, the coast along the Sea of Japan, Tokyo Bay and adjacent areas, and western Japan. Butyltins (MBT, DBT and TBT) were detected in 32 of the 63 seawater samples with average concentrations of 4.6 ng MBT/l, 4.5 ng DBT/l and 6.8 ng TBT/l, respectively. Butyltin concentrations in seawater from western Japan indicate "hot spots" even in unpopulated areas. Butyltins (MBT, DBT and TBT) were detected in all samples of Caprella spp., varying from 2.3 ng BTs /g wet wt in C. penantis R-type from Tobishima Island in the Sea of Japan to 464 ng BTs /g wet wt in C. decipiens Mayer from Amakusa, western Kyushu. The BT concentrations in Caprella spp. form western Japan were significantly higher than those from other areas, including Tokyo Bay and adjacent areas, where large scale industry and international ports are located. These results indicate that butyltin contamination still remains even in unpopulated areas after the regulation on TBT usage, and that the regulation governing TBT usage since 1990 has not been effective enough to concede recovery of shallow water ecosystems around Japan.

Amphipoda↗

Detecting a field gradient of PAH exposure in decapod crustacea using a novel urinary biomarker.

Norwegian coastal waters are subject to PAH contamination from electrochemical industries such as aluminium smelters. Evidence of PAH exposure has been established in fish and bivalves. The present study tests the applicability of a novel crustacean PAH exposure biomarker to a PAH contamination gradient in the field (Karmsund Strait, SW Norway). Fluorescence analysis of urine samples collected from crabs at each site revealed 1-OH pyrene "equivalent" levels (indicative of pyrogenic PAH contamination) decreased with increasing distance from a point source of pyrogenic PAH (a large aluminium works). The assay was shown to be suitable for the detection of PAH exposure in wild crustacean populations, for discriminating between contaminated and clean sites and is also sufficiently sensitive to detect gradients of PAH contamination. The method provides a rapid, inexpensive and non-destructive measure of biologically available PAH in crustaceans.

Animals↗

Mediterranean Talitrus saltator (Crustacea, Amphipoda) as a biomonitor of heavy metals contamination.

The use of sandhoppers and beachfleas as biomonitors of heavy metals contamination is relatively recent. Using adult individuals of Talitrus saltator from nine localities on the northern Mediterranean Sea, we studied the concentrations of eight trace elements: Al, Cd, Cr, Fe, Hg, Pb, Cu, Zn, both in the substratum and in the individuals. We also carried out a preliminary investigation of the correspondence between the sandhoppers' genetic variability and heavy metal contamination at the sampling sites. T. saltator accumulated Cd, Cu, Zn and Hg (at higher concentrations than in the sand) and also Al and Fe (at lower concentrations than in the sand). It seems that Mediterranean sandhoppers do not accumulate Pb and Cr. An intraspecific comparison between northern European (Baltic) and Mediterranean populations of T. saltator was made. Finally, we observed a tendency to a positive correlation between the sandhoppers' genetic variability and heavy metals contamination.

Amphipoda↗

Analysis of 686 expressed sequence tags (ESTs) from the intertidal harpacticoid copepod Tigriopus japonicus (Crustacea, Copepoda).

The intertidal harpacticoid copepod Tigriopus japonicus is an important species in the study of marine pollution. To facilitate molecular biomonitoring using T. japonicus, we constructed a T. japonicus unidirectional cDNA library using lambdaZAP expression vector, excised to pBluescript vector with the aid of helper phage, and analyzed 686 randomly picked expressed sequence tags (ESTs) from this species. From the 686 ESTs sequenced, we found several functional genes such as vitellin, kinases and potential detoxification-related genes. We are now preparing a T. japonicus cDNA chip for molecular ecotoxicological studies. In this paper, we discuss the potential use of T. japonicus ESTs and their importance in ecotoxicology.

Animals↗

Toxicity of antifouling biocides to the intertidal harpacticoid copepod Tigriopus japonicus (Crustacea, Copepoda): effects of temperature and salinity.

Intertidal harpacticoid copepods are commonly used in eco-toxicity tests worldwide. They predominately live in mid-high shore rock pools and often experience a wide range of temperature and salinity fluctuation. Most eco-toxicity tests are conducted at fixed temperature and salinity and thus the influence of these environmental factors on chemical toxicity is largely unknown. This study investigated the combined effect of temperature and salinity on the acute toxicity of the copepod Tigriopus japonicus against two common biocides, copper (Cu) and tributyltin (TBT) using a 2 x 3 x 4 factorial design (i.e. two temperatures: 25 and 35 degrees C; three salinities: 15.0 per thousand, 34.5 per thousand and 45.0 per thousand; three levels of the biocide plus a control). Copper sulphate and tributyltin chloride were used as the test chemicals while distilled water and acetone were utilised as solvents for Cu and TBT respectively. 96 h-LC50s of Cu and TBT were 1024 and 0.149 microg l(-1) respectively (at 25 degrees C; 34.5 per thousand) and, based on these results, nominal biocide concentrations of LC0 (i.e. control), LC30, LC50 and LC70 were employed. Analysis of Covariance using 'concentration' as the covariate and both 'temperature' and 'salinity' as fixed factors, showed a significant interaction between temperature and salinity effects for Cu, mortality increasing with temperature but decreasing with elevated salinity. A similar result was revealed for TBT. Both temperature and salinity are, therefore, important factors affecting the results of acute eco-toxicity tests using these marine copepods. We recommend that such eco-toxicity tests should be conducted at a range of environmentally realistic temperature/salinity regimes, as this will enhance the sensitivity of the test and improve the safety margin in line with the precautionary principle.

Analysis of Variance↗