Recent and old duplications in crustaceans "Internal Transcribed Spacer 1″: structural and phylogenetic implications.

Internal Transcribed Spacer structures are important in preserving accessibility to specific enzymes for the maturation of rRNAs. ITS1 sequences reported in the literature in Crustaceans range between 182 and 820 bp and are characterized by the absence of repeats or the presence of only a limited number of microsatellites. Here, we sequenced ITS1 for a range of shrimp families (infraorder Caridea) and show that most taxa have much larger ITS1 sequences. We find a high number of microsatellites in Alpheus hebes and Crangon crangon and we report repeat units in Pandalidae, Palaemonidae and mainly in Alpheidae species. Up to four repeats were found in A. vanderbilti (1915 bp), A. rostratus (1635 bp) and A. lottini (1625 bp). In general, four helices were found in ITS1. Repeat units led to extra hairpins and loops. No conserved positions occurred except in helix 4. Three clades were defined in A. lottini for the first time. We estimated the ITS1 divergence rate for the three clades of A. lottini collected in French Polynesia using existing calibrations of substitution rates. Rates of sequence evolution are largely influenced by repeat units, which likely evolve separately. By comparison with COI marker, we estimated the divergence rate of the whole ITS1 sequence to range from 0.5 to 1.4% Pmy and between 0.12 and 0.5% for the 3' end of ITS1 located outside the repeat units. Given the degree of identity between repeats, we suggest that a duplication event recently occurred in A. floridanus (98% identity) whereas an ancient duplication happened in A. sulcatus (50% identity) early at the origination of the group Alpheidae, approximately 50 mya ago. In conclusion, our results highlight an over representation of shorter ITS1 sequences in public repositories, and underlines the importance to further understand patterns of molecular evolution of this functionally important gene.

The Effect of Pleistocene Climate Fluctuations on Distribution of European Abalone (Haliotis tuberculata), Revealed by Combined Mitochondrial and Nuclear Marker Analyses.

The genetic differentiation among the populations of the European abalone Haliotis tuberculata was investigated using different markers to better understand the evolutionary history and exchanges between populations. Three markers were used: mitochondrial cytochrome oxidase I (COI), the sperm lysin nuclear gene, and eight nuclear microsatellites. These markers present different characteristics concerning mutation rate and inheritance, which provided complementary information about abalone history and gene diversity. Genetic diversity and relationships among subspecies were calculated from a sample of approximately 500 individuals, collected from 17 different locations in the north-eastern Atlantic Ocean, Macaronesia, and Mediterranean Sea. COI marker was used to explore the phylogeny of the species with a network analysis and two phylogenetic methods. The analysis revealed 18 major haplotypes grouped into two distinct clades with a pairwise sequence divergence up to 3.5 %. These clades do not correspond to subspecies but revealed many contacts along Atlantic coast during the Pleistocene interglaciations. The sperm lysin gene analysis separated two different subtaxa: one associated to Macaronesian islands, and the other to all other populations. Moreover, a small population of the northern subtaxon was isolated in the Adriatic Sea-probably before the separation of the two lineages-and evolved independently. Microsatellites were analyzed by different genetics methods, including the Bayesian clustering method and migration patterns analysis. It revealed genetically distinct microsatellite patterns among populations from Mediterranean Sea, Brittany and Normandy, Morocco, and Canary and Balearic islands. Gene flow is asymmetric among the regions; the Azores and the Canary Islands are particularly isolated and have low effective population sizes. Our results support the hypothesis that climate changes since the Pleistocene glaciations have played a major role in the geographic distribution of the European abalone. Traces of these events related to maternal inheritance were shown on COI marker.

Seasonal and Cyclical Changes in Genetic Composition of the Marine Intertidal Rock Pool Copepod Tigriopus brevicornis.

Tigriopus brevicornis is a marine rock pool copepod widely distributed along Atlantic coasts. Due to the absence of a known dispersal mechanism by free swimming stages, exchanges between populations over long distances are questionable. In order to analyse the evolution of an isolated supralittoral rock pool population, sampling of the copepod was performed monthly during 1 year and compared to samplings over 5 years in the same rock pool, as well as from other rock pools. Using ITS1 analysis, cyclical changes in genetic composition were detected. Our results give clear indications concerning the segregation of the rock pool population and a lack of gene flow among outside populations. A network analysis shows the presence of several shared dominant haplotypes and also singletons differing by one mutation point. F(st) analyses indicate that the main changes occur in autumn and winter. The few analogies of ITS1 sequences with nearby populations may indicate that new migrants must re-colonise the pools from surrounding rock crevices in the intertidal habitat where they may have found a refuge after bad weather conditions.

Characterization and expression of glutamate dehydrogenase in response to acute salinity stress in the Chinese mitten crab, Eriocheir sinensis.

BACKGROUND: Glutamate dehydrogenase (GDH) is a key enzyme for the synthesis and catabolism of glutamic acid, proline and alanine, which are important osmolytes in aquatic animals. However, the response of GDH gene expression to salinity alterations has not yet been determined in macro-crustacean species. METHODOLOGY/PRINCIPAL FINDINGS: GDH cDNA was isolated from Eriocheir sinensis. Then, GDH gene expression was analyzed in different tissues from normal crabs and the muscle of crabs following transfer from freshwater (control) directly to water with salinities of 16‰ and 30‰, respectively. Full-length GDH cDNA is 2,349 bp, consisting of a 76 bp 5'- untranslated region, a 1,695 bp open reading frame encoding 564 amino acids and a 578 bp 3'- untranslated region. E. sinensis GDH showed 64-90% identity with protein sequences of mammalian and crustacean species. Muscle was the dominant expression source among all tissues tested. Compared with the control, GDH expression significantly increased at 6 h in crabs transferred to 16‰ and 30‰ salinity, and GDH expression peaked at 48 h and 12 h, respectively, with levels approximately 7.9 and 8.5 fold higher than the control. The free amino acid (FAA) changes in muscle, under acute salinity stress (16‰ and 30‰ salinities), correlated with GDH expression levels. Total FAA content in the muscle, which was based on specific changes in arginine, proline, glycine, alanine, taurine, serine and glutamic acid, tended to increase in crabs following transfer to salt water. Among these, arginine, proline and alanine increased significantly during salinity acclimation and accounted for the highest proportion of total FAA. CONCLUSIONS: E. sinensis GDH is a conserved protein that serves important functions in controlling osmoregulation. We observed that higher GDH expression after ambient salinity increase led to higher FAA metabolism, especially the synthesis of glutamic acid, which increased the synthesis of proline and alanine to meet the demand of osmoregulation at hyperosmotic conditions.

Allozyme variation and population genetic structure in the carpet shell clam Ruditapes decussatus across the Siculo-Tunisian Strait.

This study reports on the polymorphism of 15 allozyme loci in Ruditapes decussatus clams collected from 11 locations along the Tunisian coasts. We concentrated our sampling effort around the Siculo-Tunisian region to verify if any population structuring exists in this region and to identify the factors that have shaped this structure. Measurements of genetic diversity were quantified both within and between populations, and the geographic variability of gene frequencies was analyzed. Our study shows that the Siculo-Tunisian Strait is an important genetic boundary between eastern and western regions, which agrees with findings for a variety of other species. We suggest that vicariance is a predominant factor shaping the current distribution of genetic diversity of R. decussatus, and the mixing of divergent gene pools from the eastern and western regions still seems to be limited by some physical and/or biological factors.

Mitochondrial DNA Introgression in the European abalone Haliotis tuberculata tuberculata: evidence for experimental mtDNA paternal inheritance and a natural hybrid sequence.

Two subspecies of the European abalone have been morphologically recognized: Haliotis tuberculata tuberculata, present in the North Atlantic, and Haliotis tuberculata coccinea, present in the Canary Islands. Among the different nuclear markers used to differentiate these two subspecies, the sperm lysin gene was the most reliable, leading to a 2.2% divergence. Concerning the subunit I of the mitochondrial cytochrome oxydase gene (COI), we observed a difference of 3.3% between the two subspecies. In the North Atlantic, an introgression of mitochondrial DNA from H. tuberculata coccinea to H. tuberculata tuberculata was evident in around 30% of individuals. Due to this difference, we were able to experimentally detect the transfer of paternal mitochondrial DNA (mtDNA) by specific quantitative polymerase chain reaction measurements. The presence of the two mtDNA signatures was also detected in 20% of individuals tested in the field. Moreover, one mt DNA hybrid sequence was identified. The sequencing of this mitochondrial DNA hybrid revealed a mosaic structure with many specific mutations. The origin of this hybrid sequence is discussed.

Two different and functional nuclear rDNA genes in the abalone Haliotis tuberculata: tissue differential expression.

Analysis of the 18S rDNA sequences of Haliotis tuberculata tuberculata and H. t. coccinea subtaxa identified two different types of 18S rDNA genes and ITS1 regions. These two different genes were also detected in H. marmorata, H. rugosa and H. diversicolor that are separated from H. tuberculata by 5-65 mya. The mean divergence value between type I and type II sequences ranged from 7.25% for 18S to 80% for ITS1. ITS1 type II is homologous with the ITS1 consensus sequences published for many abalone species, whereas ITS1 type I presented only minor homology with a unique database entry for H. iris ITS1. A phylogenetic analysis makes a clear separation between type I and type II ITS1 sequences and supports grouping H. t. tuberculata, H. t. coccinea and H. marmorata together. The two subtaxa do not show any significant differences between the homologous 18S rDNA sequences. A general structure of the ITS1 transcript was proposed, with four major helices for the two types. The two genes were expressed and, for the first time, a putative differential expression of ITS1 type I was detected in the gills, digestive gland and gonads whereas ITS1 type II was expressed in all tissues.

Transcriptional regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase in the adductor muscle of the oyster Crassostrea gigas during prolonged hypoxia.

The response of Crassostrea gigas to prolonged hypoxia was investigated for the first time by analyzing the metabolic branch point formed by pyruvate kinase (PK) and hosphoenolpyruvate carboxykinase (PEPCK). PK and PEPCK cDNAs were cloned and sequenced. The main functional domains of the PK sequence, such as the binding sites for ADP/ATP and phosphoenolpyruvate (PEP), were identified whereas the PEPCK sequence showed the specific domain to bind PEP in addition to the kinase-1 and kinase-2 motifs to bind guanosine triphosphate (GTP) and Mg(2+), specific for all PEPCKs. A C-terminal extension was detected for the first time in eukaryota PK. Separation of mitochondrial and cytosolic fraction showed that more than 92% of the PEPCK enzyme activity was cytosolic in gills, digestive gland, mantle and muscle. PK and PEPCK mRNAs and enzyme activities have been measured in muscle during prolonged hypoxia for 20 days. Adaptation of PK in hypoxic muscle at transcriptional level occurred lately by decreasing significantly the PK mRNA level at day 20 while PK enzyme activity was inhibited by the high content of alanine. The PEPCK mRNA ratio in hypoxic muscle significantly increased at day 10 simultaneously to the PEPCK enzyme activity. Succinate accumulation observed at day 10 and day 20 confirmed the anaerobic pathway of muscle metabolism in oyster subjected to hypoxia. Regulation of C. gigas PEPCK in muscle occurred at gene transcription level while PK was first regulated at enzyme level with alanine as allosteric inhibitor, and then at molecular level under a fast effect of hypoxia.

Effect of hyper or hypo-osmotic conditions on neutral amino acid uptake and oxidation in tissues of the crab Chasmagnathus granulata.

We investigated the transport of (14)C-methylaminoisobutyric acid ((14)C-MeAIB) and (14)C-alanine oxidation in hepatopancreas and jaw muscle of Chasmagnathus granulata submitted to 24, 72, and 144 h of hypo- or hyperosmotic stress. While (14)C-MeAIB uptake increased in jaw muscle and hepatopancreas from crabs submitted to hyperosmotic stress, it did not change in tissues from animals submitted to hypo-osmotic stress. Incubation of jaw muscle and hepatopancreas from control groups with 1 mM ouabain did not decrease (14)C-MeAIB uptake. However, ouabain prevented (14)C-MeAIB uptake in hepatopancreas at 24 h of hyperosmotic stress. In contrast, in jaw muscle from crabs submitted to the same conditions, (14)C-MeAIB uptake was not prevented by ouabain in the incubation medium. Jaw muscle from the control group produced four times more (14)CO(2) from (14)C-alanine than the hepatopancreas. During hypo-osmotic stress, amino acid oxidation does not seem to be one of the pathways implicated in the decrease of the amino acid pools in hepatopancreas and jaw muscle. In contrast, during hyperosmotic stress the reduction in (14)C-alanine oxidation appears to be one of the mechanisms involved in the increase of the amino acid pool in the hepatopancreas.

Factorial effects of salinity, dietary carbohydrate and moult cycle on digestive carbohydrases and hexokinases in Litopenaeus vannamei (Boone, 1931).

Litopenaeus vannamei were reared in close cycle over seven generations and tested for their capacity to digest starch and to metabolise glucose at different stages of the moulting cycle. After acclimation with 42.3% of carbohydrates (HCBH) or 2.3% carbohydrates (LCBH) diets and at high salinity (40 g kg(-1)) or low salinity (15 g kg(-1)), shrimp were sampled and hepatopancreas (HP) were stored. Total soluble protein in HP was affected by the interaction between salinity and moult stages (p<0.05). Specific activity of alpha-amylase ranged from 44 to 241 U mg protein(-1) and a significant interaction between salinity and moult stages was observed (p<0.05), resulting in highest values at stage C for low salinity (mean value 196.4 U mg protein(-1)), and at D0 in high salinity (mean value 175.7 U mg protein(-1)). Specific activity of alpha-glucosidase ranged between 0.09 and 0.63 U mg protein(-1), an interaction between dietary CBH and salinity was observed for the alpha-glucosidase (p<0.05) and highest mean value was found in low salinity-LCBH diet treatment (0.329 U mg protein(-1)). Hexokinase specific activity (range 9-113 mU mg protein(-1)) showed no significant differences when measured at 5 mM glucose (p>0.05). Total hexokinase specific activity (range 17-215 mU mg protein(-1)) showed a significant interaction between dietary CBH and salinity (p<0.05) with highest value (mean value 78.5 mU mg protein(-1)) found in HCBH-high salinity treatment, whereas in the other treatments the activity was not significantly different (mean value 35.93 mU mg protein(-1)). A synergistic effect of dietary CBH, salinity and moult stages over hexokinase IV-like specific activity was also observed (p<0.05). As result of this interaction, the highest value (135.5+/-81 mU mg protein(-1)) was observed in HCBH, high salinity at D0 moult stage. Digestive enzymes activity is enhanced in the presence of high starch diet (HCBH) and hexokinase can be induced at certain moulting stages under the influence of blood glucose level. Perspectives are opened to add more carbohydrates in a growing diet, exemplifying the potential approach for less-polluting feed.

Effect of hyperosmotic shock on phosphoenolpyruvate carboxykinase gene expression and gluconeogenic activity in the crab muscle.

Chasmagnathus granulata phosphoenolpyruvate carboxykinase (PEPCK) cDNA from jaw muscle was cloned and sequenced, showing a specific domain to bind phosphoenolpyruvate in addition to the kinase-1 and kinase-2 motifs to bind guanosine triphosphate (GTP) and Mg(2+), respectively, specific for all PEPCKs. In the kinase-1 motifs the GK was changed to RK. The first 19 amino acids of the putative enzyme contain hydrophobic amino acids and hydroxylated residues specific to a mitochondrial type signal. The PEPCK is expressed in hepatopancreas, muscles, nervous system, heart, and gills. Hyperosmotic stress for 24 h increased the PEPCK mRNA level, gluconeogenic and PEPCK activities in muscle.

Secretagogue activities in cod (Gadus morhua) and shrimp (Penaeus aztecus) extracts and alcalase hydrolysates determined in AR4-2J pancreatic tumour cells.

Peptides with gastrin immunoreactivity were measured in cod muscle (Gadus morhua) and shrimp heads (Penaeus aztecus) extracts and alcalase hydrolysates and separated by two chromatographic steps. Secretagogue activities present in crude extracts fractions were examined with or without specific antagonists of CCK receptors in AR4-2J cells. Several sub-fractions significantly stimulate amylase release, up to 110%. These stimulatory effects could be completely inhibited by the presence of L 365, 260 specific antagonist of CCKB receptors. After hydrolysis of the raw material, the samples were partially fractionated by two chromatographic steps and potential active fractions detected by a gastrin-CCK radioimmunoassay. The molecular masses of the active fractions were lower than for the extracts. Stimulation of amylase release was higher than with extracts, and the inhibition by L 365, 260, less pronounced. These results show that some peptides remaining after hydrolysis or extraction still exert biological activities and have to be tested in nutritional studies.

Highly variable polymorphism of the alpha-amylase gene family in Litopenaeus vannamei (Crustacea Decapoda).

Alpha-amylase from the tropical shrimp Litopenaeus vannamei presents a high degree of polymorphism and at least eight different electromorphs are detected by electrophoresis. Based on nucleotide sequences, three cDNAs have been previously characterized. In this paper we report on the organization and the evolution of corresponding alpha-amylase genes, determined after PCR amplification. Three AMY genes have been characterized, spanning over 3.3 kb and encoding mature proteins of 495 amino acids (aa), which are all expressed in the digestive gland. The existence of nine short introns, ranging from 86 to 454 bp, located at the same positions for each of the different genes, and presenting no similarity between them, is reported. Between 11 and 15% of changes are observed in the coding aa sequences of genes II and III compared to the gene I sequence respectively. One 5' putative promoter sequence has been sequenced and shows no classical TATA box upstream to the coding sequence. Based on the intron size difference, a single PCR (producing the S-R fragments) allows the separation of a partial gene I (750 bp), corresponding to cDNA 20, from the others (650-680 bp). Sequencing different S-R PCR fragments from one shrimp shows at least eight different haplotypes. A complex microsatellite repeat is present in intron 6 of gene II. Using size and sequence differences in this repeated portion, it is possible to characterize two gene subfamilies (IIa and IIb) encoding previously described cDNAs 28 and 37, respectively. For the gene II family, two to four alleles are present in one shrimp corresponding to these two genes. Within the Panama natural population, 35 different alleles are shown at this locus. Regarding alpha-amylase gene structure in the shrimp, many recombinants are present from a set of individuals and constitute an important mechanism of evolution of alpha-amylase function.

Immunocytochemical study of crustacean hyperglycemic hormone in the eyestalks of the prawn Palaemon serratus (Pennant) and some other Palaemonidae, in relation to variations in the blood glucose level.

With the use of rabbit antisera against crustacean hyperglycemic hormone (CHH), it is possible to describe a distinct immunopositive reaction in a group of neurosecretory cells in the medulla terminalis ganglionic X-organ2 (MTGX2 ), in the MTGX-sinus gland tract, and in a considerable part of the sinus gland from several species of prawns belonging to the Palaemonidae. By introductory studies on the CHH system in Palaemon serratus, we can postulate a sequence in the activity cycle of the CHH-producing cells on the basis of differences in staining intensity of the immunoreaction and such morphometric parameters as cellular and nuclear diameter. By studying the CHH-producing system in combination with variations in the glucose level of the blood, an "inverse relationship" is observed between the number of immunoreactive cells and the blood glucose level during different periods of the year as well as during different stages of the molting cycle. A "shift in phase" of this correlation during the diurnal cycle suggests that several rhythmical phenomena may play a role in the regulation of glycemia in Crustacea.