New microsatellite markers for the snow crab Chionoecetes opilio (Brachyura: Majidae)

Five microsatellite markers were developed for the snow crab (Chionoecetes opilio) and polymerase chain reaction conditions and/or primer sequences of three previously developed microsatellites were adapted for fluorescent labelling analysis. All loci were analysed in 449 individuals from 11 sampling sites in the northwest Atlantic. The high degree of polymorphism exhibited by these microsatellites (mean of 34 alleles per locus and mean observed heterozygosity of 0.76) suggests that they will be suitable for spatial and temporal genetic analysis of C. opilio populations.     

Prevalence of bacteria in the spermathecae of female snow crab, Chionoecetes opilio (Brachyura: Majidae)

The spermathecal contents of primiparous, multiparous and barren female snow crabs were observed by a scanning electron microscope. Bacteria colonies were observed with a significantly higher frequency in the spermathecae of primiparous and old barren females. Bacteria infect the spermathecae and destroy the spermatophores and spermatozoa inside. These observations suggest that bacteria in the spermathecae do not exclude opportunistic microbes by modifying pH of the medium as suggested in the literature. The prevalence of bacteria in primiparous and old barren females suggests that they infect individuals with a weak anti-microbial protection. The absence of bacteria in the highly acidic seminal fluid derived from males upon copulation suggests that it may provide anti-microbial protection. Bacteria do not seem to be able to survive in an anaerobic environment for a long period.     

Isolation and characterization of polymorphic microsatellite markers in the red snow crab (Chionoecetes japonicus)

A total of 12 polymorphic microsatellite DNA loci were isolated from the red snow crab, Chionoecetes japonicus (Brachyura: Majidae), one of important fisheries resources in the Far East. The number of alleles observed at each locus ranged from two to 19, with the observed and expected heterozygosities of 0.125-0.875 and 0.156-0.949, respectively, suggesting these loci to be a useful molecular marker for population analysis in this species. Of the 12 loci, seven also were available for genotyping of the snow crab, Chionoecetes opilio, implying these loci as a useful molecular marker in the genus Chionoecetes.     

Evidence for a permanent establishment of the snow crab (Chionoecetes opilio) in the Barents Sea

In the Atlantic the snow crab (Chionoecetes opilio) is naturally distributed on the northwestern side, i.e. eastern Canada and west Greenland. Until recently, there have been no observations of snow crab in eastern Atlantic. However, in 1990s single and occasional reports were made of crabs captured in the eastern part of the Barents Sea, presumably introduced through ballast water. Special attention during the annual bottom-trawl surveys in the Barents Sea during February 2004–2006 were given to include recordings of snow crab to evaluate if the introduced species has succeeded to establish a self-sustaining population in this region. Recordings of snow crabs were systematically noted and biological measurements carried out. The results confirm previous Russian observations of snow crabs in the northern region of Gåsebanken. In addition, a significant number of crabs were also found in the central region of the Barents Sea, mainly in deeper waters from 180 to 350 m depth. The sizes ranged from 14 to 136 mm carapace width. All females above 70 mm were berried with fertilised eggs. A major fraction (31% in 2005; 76% in 2006) of the crabs consisted of juveniles below 50 mm CW, providing evidence for successful recruitment. The small-sized crabs were exclusively found in Gåsebanken, identifying the main recruiting area at present for snow crab in the Barents Sea. The results obtained show that the snow crab is now adapted to the northeast Atlantic.     

Population genetic analysis and origin discrimination of snow crab (Chionoecetes opilio) using microsatellite markers

Major habitats for the snow crab Chionoecetes opilio are mostly found within the northwest Atlantic and North Pacific Oceans. However, the East Sea populations of C. opilio, along with its relative the red snow crab (C. japonicas), are two of the most important commercial crustacean species for fisheries on the east coast of the Korean Peninsula. The East Sea populations of C. opilio are facing declining resources due to overfishing and global climate change. Thus, an analysis of population structure is necessary for future management. Five Korean and one Russian group of C. opilio were analyzed using nine microsatellite markers that were recently developed using next-generation sequencing. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The number of alleles per locus varied from 4 to 18 with a mean of 12, and allelic richness per locus ranged from 4.0 to 17.1 across all populations with a mean of 9.7. The Hardy–Weinberg equilibrium test revealed significant deviation in three out of nine loci in some populations after sequential Bonferroni correction and all of them had higher expected heterozygosity than observed heterozygosity. Null alleles were presumed in four loci, which explained the homozygosity in three loci. The pairwise fixation index (F _ST ) values among the five Korean snow crab populations did not differ significantly, but all of the pairwise F _ST values between each of the Korean snow crab populations and the Russian snow crab population differed significantly. An UPGMA dendrogram revealed clear separation of the Russian snow crab population from the Korean snow crab populations. Assignment tests based on the allele distribution discriminated between Korean and Russian origins with 93 % accuracy. Therefore, the snow crab populations around the Korean Peninsula need to be managed separately from the populations in Bering Sea in global scale resource management. Also, this information can be used for identification of snow crab origin which is problematic in worldwide crab trade.     

Polymorphic microsatellite DNA markers in the mangrove crab Ucides cordatus (Brachyura: Ocypodidae)

The isolation and characterization of the first polymorphic microsatellite markers for the mangrove crab Ucides cordatus are described. The number of alleles at each locus ranged from three to 25, mean of nine alleles, in 46 crabs captured in two Brazilian mangroves. The markers averaged high levels of observed (0.709 ± 0.183) and expected (0.716 ± 0.170) heterozygosities. Departures from Hardy-Weinberg equilibrium were observed at two loci. Linkage disequilibrium tests were not significant and no evidence of null alleles was detected. All these microsatellite loci are expected to be useful in estimating fine-scale population processes of this valuable mangrove species currently subjected to excessive fishing efforts.     

Ultrastructure of the Spermatozoa and Spermatophores of the Zuwai Crab,Chionoecetes opilio (Majidae,Brachyura)

The fine structure of the spermatozoa and spermatophores of the zuwai crab, Chionoecetes opilio, was examined electron microscopically. The spermatophores embedded in the secretory droplets within the vasa deferentia showed a spherical structure with an extremely wrinkled envelope and contained numerous spermatozoa. The mature spermatozoa of this crab, similar to those of other brachyurans, were stellate in shape and had a globular acrosome surrounded by a cup-like nucleus with several radiating processes. The acrosome was ultrastructurally complex and its apical part was characterized by an electron-dense discoid structure, whereas its innermost part was occupied by an electron-lucent cylindrical structure containing assemblies of thin tubules and a reticular formation of electron-dense material. The cytoplasm interposed between the nucleus and acrosome was remarkably reduced in volume and displayed a membranous lamellar complex, few mitochondria, and a centriole. The nuclear chromatin was not condensed but represented by finely flocculent material. The morphological aspects of the zuwai crab spermatozoa are discussed in comparison with those of other decapod crustaceans.     

Pathology of Hematodinium infections in snow crabs (Chionoecetes opilio) from Newfoundland, Canada

Bitter crab disease (BCD) of snow crabs, Chionoecetes opilio, is caused by a parasitic dinoflagellate, Hematodinium sp. The disease has shown an alarming increase in prevalence in the commercial fishery in eastern and northeastern areas of Newfoundland and Labrador since it was first recorded there in the early 1990s. We documented histopathological alterations to the tissues in snow crabs with heavy infections of Hematodinium sp. and during sporulation of the parasite. Pressure necrosis was evident in the spongy connective tissues of the hepatopancreas and the blood vessels in most organs. In heavy infections, little remained of the spongy connective tissues around the hepatopancreas. Damage to the gills varied; in some cases it was severe, particularly during sporulation, involving apparent thinning of the cuticle, loss of epithelial cells, and fusion of the membranous layers of adjacent gill lamellae. Affected lamellae exhibited varying degrees of distention with a loss of trabecular cells, hemocyte infiltrations, and swelling or "clubbing" along the distal margins. Large numbers of zoospores were located along the distal margins of affected lamellae suggesting that sporulation may cause a lysis or bursting of the thin lamellar cuticle, releasing spores. Pressure necrosis, due to the build up of high densities of parasites, was the primary histopathological alteration in most tissues. Hematodinium infections in the snow crab are chronic, long-term infections that end in host death, during sporulation of the parasite.     

First record of the decapod-egg predator Ovicides paralithodis (Nemertea,Carcinonemertidae) from the snow crab Chionoecetes opilio (Decapoda,Brachyura)

The carcinonemertid monostiliferan Ovicides paralithodis Kajihara and Kuris, 2013 was originally described as an egg predator of the red-king crab Paralithodes camtschaticus (Tilesius, 1815) in the Sea of Okhotsk, the Bering Sea, and the Gulf of Alaska. In the present study, several carcinonemertid specimens were obtained from the egg mass of the snow crab Chionoecetes opilio (O. Fabricius, 1788) in the Sea of Japan. Partial sequences of the mitochondrial cytochrome c oxidase subunit I gene (COI) determined from two specimens of the carcinonemertid were identical with a barcode sequence from the holotype of O. paralithodis, indicating that the host range of the species covers at least the two decapod species, P. camtschaticus and C. opilio.     

The moulting cycle and changes in body density in larvae of the snow crab Chionoecetes opilio (Brachyura: Majoidea) under laboratory conditions

The moulting cycle and the time course of changes in body density from hatching to the end of the megalopal stage in snow crab (Chionoecetes opilio) larvae were investigated in laboratory-reared specimens. Morphological changes in the epidermis and cuticle were photographically documented to characterize the moult-cycle stages: A–B (postmoult), C (intermoult), D (premoult) and E (ecdysis). Moult-stage characteristics were based on a microscopical examination of integumental modifications, particularly of the telson. During stages A–C, the larval cuticle changed from a spongy structure to become conspicuously thicker and more solid in appearance. In stage D, the epidermis retracted from the cuticle and new setae and appendages were formed. The body densities of larval snow crabs were lowest just after moulting; they increased greatly during stage C, and then gradually increased to reach a plateau at 1.0897–1.0931 g cm^?3 during stage D. Over the whole larval period, they have a density greater than that of seawater. These observations will assist in understanding of larval distribution and transport in snow crabs in their natural habitat, and provide a useful tool to determine the developmental stages of larvae sampled from the plankton and from larval cultures.     

Highly polymorphic microsatellite markers for Radix balthica (Linnaeus 1758)

We present data for eight polymorphic microsatellite markers isolated from a microsatellite-enriched DNA library for the freshwater snail Radix balthica. Three of them were specific for R. balthica while five also amplified polymorphic products in two congeneric species. Test application on populations from all over the species range has shown that these loci are highly informative for analysing population structure and estimating migration rates. Observed deviations from Hardy-Weinberg equilibrium are attributed to a mixed mating system.     

Isolation and characteristics of 10 microsatellite markers from the endangered coconut crab (Birgus latro)

The coconut crab (Birgus latro), an endangered marine-dispersed crustacean, is facing severe and probably accelerating population extinction worldwide, but biological information on its conservation remains deficient. In order to reveal the genetic structure of B. latro, 10 microsatellite loci were developed. A high degree of polymorphism was observed with a mean number of alleles per locus of 16.9. The mean expected heterozygosities were also high, ranging from 0.742 to 0.965. The observed heterozygosities ranged from 0.210 to 0.925. Departures from Hardy-Weinberg equilibrium were observed at five loci after the Bonferroni correction. These hypervariable markers will be utilized to study the genetic diversity and conservation of B. latro throughout its distribution range in the Pacific and Indian Oceans.     

Biochemical indicators of muscle growth in the snow crab Chionoecetes opilio (O. Fabricius)

This study examined the relationships between muscle growth rate, the activity of metabolic enzymes and the RNA:DNA ratio, in adult snow crabs Chionoecetes opilio. After moulting, crabs were assigned to three feeding rations to attain a range of tissue growth rates. Muscle growth rate, estimated by the variation in dry tissue content per ml of merus of the first walking leg, was positively correlated with changes in muscle cell number, as evaluated by the DNA content per ml of merus. However, no significant correlation was detected between growth rate and the variation in muscle cell size, the latter being estimated by the change in the protein:DNA ratio. This is due to the fact that, in starved crabs, a reduction in the number of cells is partly compensated by a size increment of the remaining ones. This phenomenon also weakened the overall relationship between muscle growth rate and the phosphofructokinase (PFK) capacity per ml of merus. The simple correlation between those two variables was significantly positive for animals which increased their mass of muscle but insignificant for those which were loosing muscle mass. The lactate dehydrogenase (LDH), citrate synthase (CS) and cytochrome c oxidase (CCO) capacity per ml of merus did not match growth rate. The significant simple correlations that were detected between growth rate and the various enzyme activity expressed per g of protein, per μg of DNA and per g of dry mass did not hold when partial correlations were computed. Variations in muscle cell size were related to adjustments in the quantity of RNA per cell, as depicted by the RNA:DNA ratio. Since muscle growth was not correlated with the variation in muscle cell size, it was not correlated with the RNA:DNA ratio either.     

A select panel of polymorphic microsatellite loci for individual identification of snow leopards (Panthera uncia)

Snow leopards (Panthera uncia) are elusive endangered carnivores found in remote mountain regions of Central Asia. New methods for identifying and counting snow leopards are needed for conservation and management efforts. To develop molecular genetic tools for individual identification of hair and faecal samples, we screened 50 microsatellite loci developed for the domestic cat (Felis catus) in 19 captive snow leopards. Forty‐eight loci were polymorphic with numbers of alleles per locus ranging from two to 11. The probability of observing matching genotypes for unrelated individuals (2.1 × 10^?11) and siblings (7.5 × 10^?5) using the 10 most polymorphic loci was low, suggesting that this panel would easily discriminate among individuals in the wild.     

Novel polymorphic microsatellite markers in section Caulorrhizae (Arachis,Fabaceae)

This work reports the characterization of 11 polymorphic microsatellite loci in section Caulorrhizae. The primer pairs were designed from Arachis pintoi and showed full transferability to Arachis repens species. These new markers were used to evaluate the genetic diversity in germplasm (accessions and cultivars) of section Caulorrhizae. This new set of markers detected greater gene diversity than morphological and molecular markers such as AFLP (amplified fragment length polymorphism) and RAPD (rapid analysis of polymorphic DNA) previously used in this germplasm.     

Isolation of 10 microsatellite markers for mongoose lemurs (Eulemur mongoz)

We present here 10 new microsatellite markers for the mongoose lemur (Eulemur mongoz), nine of which were isolated from E. mongoz and one from Lemur catta. At least 60 individuals were genotyped for each of the 10 loci. Mendelian inheritance at each locus was tested by genotyping five captive families with known pedigrees. All loci were polymorphic and demonstrated simple Mendelian inheritance. The microsatellite markers presented here will be useful for genetic surveys of wild E. mongoz, to gain insights into the genetic background of the captive population, and to aid in the conservation of the species’ genetic diversity.     

Microsatellite DNA markers for the study of horseshoe crab (Limulus polyphemus) population structure

Twenty‐two microsatellite DNA loci were identified and characterized for horseshoe crabs (Limulus polyphemus) collected from two Atlantic coast and one Gulf of Mexico site. These markers revealed a high degree of genetic diversity (8–35 alleles per locus), heterozygosity (25.0% to 100.0%), and allelic heterogeneity (69.8% of comparisons). Considerable regional differentiation was observed as genetic distances (chord) ranged between 0.25 and 0.45, and all F_ST values (0.014–0.092) were significant. These preliminary findings are consistent with patterns of regional differentiation observed using allozyme variation and contradictory to findings of limited gene flow reported for sequence variation at the mitochondrial DNA COI region.     

Characterization of microsatellite markers derived from sequence databases for the European flounder (Platichthys flesus)

The European flounder (Platichthys flesus; Pleuronectidae) is an important commercial fish species. Nevertheless, no microsatellites for this species have been published to date. The sequence of flounder genes present in the public database was searched for short tandem repeats. Out of 12 microsatellite pairs derived from Sequenced Databases, five amplified successfully with an expected heterozygosity ranging from 0.43 to 0.75. The average allele number of these microsatellites was 4.8 per locus with a range between two and nine.     

Identification of polymorphic microsatellite loci in the mud crab Scylla serrata (Brachyura: Portunidae)

Five microsatellite loci are identified and characterized from the genome of Scylla serrata, a widespread and commercially important species of coastal marine crab. The loci were detected by randomly screening for di‐ and tri‐nucleotide repeat units within a partial genomic library developed for the species. The five loci consist of dinucleotide repeats and are both co‐dominant and polymorphic within the species. A sample (N = 36) of S. serrata from one Australian population has an average observed heterozygosity of 0.875 and provides no evidence of either linkage among loci or significant deviation from random mating expectations across loci. PCR products for each of the five loci were also observed from a small sample of three other species within the Scylla genus. These markers may provide genetic information that will be useful for both aquaculture and studies of natural populations of the genus.     

New microsatellite markers in turbot (Scophthalmus maximus) derived from an enriched genomic library and sequence databases

Scophthalmus maximus is an important commercially aquaculture fish species. We tackle the search for new microsatellites using two different approaches: an enriched partial genomic library and a screening of all turbot DNA sequences deposited in GenBank. Out of 15 genomic library derived loci, five gave working primer pairs, with expected heterozygosities (H_E) ranging from 0.13 to 0.91. Out of seven loci derived from database sequences, two amplified successfully with an H_E ranging from 0.56 to 0.63. The average allele number of these microsatellites was 5.7 per locus with a range between two and 15.