Seasonal variation in metabolism of cultured Pacific oyster, Crassostrea gigas, in Sanggou Bay, China

Rates of respiration and excretion of the Pacific oyster, Crassostrea gigas, were measured seasonally from June 2002 to July 2003 under ambient conditions of food, water temperature, pH, and salinity in Sanggou Bay, an important mariculture coast in north China. The aim of this study is to obtain fundamental data for further establishing an energy budget model and assessing the carrying capacity for cultivation of C. gigas in north China. Oysters were collected monthly or bimonthly from the integrated culture areas of bivalve and kelp in the bay. Oxygen consumption and ammonium and phosphorus excretion rates were measured, and ratios of O/N and NIP were calculated. One-way ANOVA was applied to determine differences among these parameters that act as a function of seasonal variation. All the physiological parameters yielded highly significant variations with season (P<0.01) The rate of respiration varied seasonally, with the highest oxygen consumption rate in July and the lowest rate in January, ranging from 0.07 to 2.13 mg O-2 h(-1) g(-1) dry tissue weight (DW). Maximum and minimum ammonium excretion rates were recorded in August and January, respectively, ranging from 0.51 to 5.40 mu mol NH4-N h(-1) g(-1) DW. Rates of phosphorus excretion varied from 0.11 (in January) to 0.64 (in July) mu mol PO4-P h(-1) g(-1) DW. The O/N and N/P ratios changed from 9.2 (in January) to 59.8 (in July) and from 4.6 (in January) to 10.9 (in August), respectively. For each season, the allometric relationship between the physiological response (e.g., rate of oxygen consumption, ammonium and phosphorus excretion) and DW of the animal was estimated using the formula: Y=a x DWb. (C) 2005 Elsevier B.V. All rights reserved.

FIRST RECORD OF THE GEBIIDEAN GENUS AXIANASSA SCHMITT, 1924 (CRUSTACEA: DECAPODA: GEBIIDEA: AXIANASSIDAE) IN THE WEST PACIFIC, WITH DESCRIPTION OF A NEW SPECIES FROM THE SOUTH CHINA SEA

A new species, Axianassa sinica, from the Beibu Gulf (Gulf of Tonkin), northern South China Sea, is described and illustrated. The new species is readily distinguished from A. australis Rodrigues & Shimizu, 1992, by its acute rostrum, merus of pereopod 1 with a tooth distally on lower margin and an elongated telson.

Further Indo-West Pacific palaemonoid shrimps (Crustacea : Decapoda : Palaemonoidea), principally from the New Caledonian region

Based on the material deposited in the Museum national d'Histoire naturelle, Paris, collected from the Indo-West Pacific, principally from the New Caledonian region, the present paper reports 117 palaemonoid shrimp species, which belong, respectively, to Anchistioididae ( one genus, one species), Gnathophyllidae ( one genus, one species), Palaemonidae Palaemoninae ( seven genera, nine species), and Palaemonidae Pontoniinae ( 30 genera, 106 species), including eight new species. The new species are all Pontoniinae: Mesopontonia brevicarpalis sp. nov., Palaemonella komaii sp. nov., Periclimenes crosnieri sp. nov., Periclimenes forgesi sp. nov., Periclimenes loyautensis sp. nov., Periclimenes paralcocki sp. nov., Periclimenes paraleator sp. nov., and Periclimenes pseudalcocki sp. nov. The last six new species are members of the deep-water "Periclimenes alcocki species complex'', which has more than two ( usually four) pairs of dorsolateral telson spines anterior to the posterior telson margin, the cornea is usually reduced, the dactyl of the major second chela is generally flanged and the chela is sometimes covered with small tubercles. The complex is usually found at more than 200m depth in the West Pacific. The species can be distinguished from each other by the armature of ambulatory propod and dactyl, diameter of cornea, rostrum shape and the number of pairs of dorsolateral telson spines. Mesopontonia brevicarpalis sp. nov., from the southeast coast of Africa, is the seventh species of the genus. Palaemonella komaii sp. nov. is very similar to Palaemonella dolichodactylus Bruce, 1991 and Palaemonella hachijo Okuno, 1999. These three species share the features of very long and slender ambulatory pereiopods with the dactyl more than eight times longer than its basal depth and with several long setae on the dorsal dactylar margin.

Heat-shock protein70 gene expression in four hatchery Pacific Abalone Haliotis discus hannai Ino populations using for marker-assisted selection

Heat shock proteins (Hsps) are molecular chaperones that help organisms cope with stressful conditions. Here, we report on the growth rates and Hsp70 expressions in inbred and hybrid populations of abalone Haliotis discus hannai Ino. In abalone, inbred populations expressed more Hsp70 than hybrid populations at all temperatures, except at very high temperatures close to the physiological limit. At benign temperatures, there was a clear trend towards higher Hsp70 expression in inbred than hybrid populations, whereas at higher temperatures, a trend in the opposite direction was observed. The temperature of maximal Hsp70 expression (T-peak) varied with the population type. The T-peak of inbred populations (26 degrees C) was lower than that of the hybrid populations (28 degrees C). The maximal inducible Hsp70 of inbred populations was higher than that of hybrid populations. The results showed a trend towards higher expression in inbred population at a lower temperature. These results provide direct experimental evidence that hybrids can cope with the intrinsic stress even at non-stressful temperatures. The constitutive Hsp70 may therefore be used for marker-assisted selection in a breeding programme.

Identification of quantitative trait loci for growth-related traits in the Pacific abalone Haliotis discus hannai Ino

The locations and effects of quantitative trait loci (QTL) were estimated for nine characters for growth-related traits in the Pacific abalone (Haliotis discus hannai Ino) using a randomly amplified polymorphic DNA (RAPD), amplification fragment length polymorphism (AFLP) and SSR genetic linkage map. Twenty-eight putatively significant QTLs (LOD > 2.4) were detected for nine traits (shell length, shell width, total weight, shell weight, weight of soft part, muscle weight, gonad and digestive gland weight, mantle weight and gill weight). The percentage of phenotypic variation explained by a single QTL ranged from 8.0% to 35.9%. The significant correlations (P < 0.001) were found among all the growth-related traits, and Pearson's correlation coefficients were more than 0.81. For the female map, the QTL for growth were concentrated on groups 1 and 4 linkage maps. On the male map, the QTL that influenced growth-related traits gathered on the groups 1 and 9 linkage maps. Genetic linkage map construction and QTL analysis for growth-related traits are the basis for the marker-assisted selection and will eventually improve production and quality of the Pacific abalone.

Characterization of 12 single nucleotide polymorphisms (SNPs) in Pacific abalone, Haliotis discus hannai

We report here for the first time 12 polymorphic single nucleotide polymorphisms (SNPs) in a commercially important gastropod, Pacific abalone (Haliotis discus hannai) that were identified by searching expressed sequence tag database. These SNP loci (seven nuclear and five mitochondrial SNPs) were polymorphic among 37 wild abalone individuals, based on a four-primer allele-specific polymerase chain reaction analysis. All loci had two alleles and the minor allele frequency ranged from 0.027 to 0.473. For the seven nuclear SNPs, the expected and observed heterozygosities ranged from 0.053 to 0.499 and from 0.054 to 0.811, respectively.

Mining expressed sequences for single nucleotide polymorphisms in Pacific abalone Haliotis discus hannai

Although single nucleotide polymorphisms (SNPs) are important resources for population genetics, pedigree analysis and genomic mapping, such loci have not been reported in Pacific abalone so far. In this study, a bioinformatics strategy was adopted to discover SNPs within the expressed sequences (ESTs) of Pacific abalone, Haliotis discus hannai, and furthermore, polymerase chain reaction direct sequencing (PCR-DS) and allele-specific PCR (AS-PCR) were used for SNPs detection and genotype scoring respectively. A total of 5893 ESTs were assembled and 302 putative SNPs were identified. The average density of SNPs in ESTs was 1%. Fifty-two sets of sequencing primers were designed from SNPs flanking ESTs to amplify the genomic DNA, and 13 could generate products of expected size. Polymerase chain reaction direct sequencing of the amplification products from pooled DNA samples revealed 40 polymorphic SNP loci. Using a modified tetra-primer AS-PCR, seven mitochondrial and six nuclear SNPs were typed and characterized among 37 wild abalones. In conclusion, it is feasible to discover SNPs from number limited ESTs and the AS-PCR as a simple, robust and reliable assay could be a primary method for small- and medium-scale SNPs detection in abalones as well as other non-model organisms.

Effects of the initial size, stocking density and sorting on the growth of juvenile Pacific abalone, Haliotis discus hannai Ino

During winter months, a novel overwintering mode of transferring juvenile abalones to open seawaters in southern China rather than keeping them in closed land-based nursery systems in northern China is a popular practice. The initial size, stocking density and sorting are among the first considerations when establishing an abalone culture system. This study aimed to investigate the effects of these factors on the growth of juvenile Pacific abalone, Haliotis discus hannai Ino, during overwintering. Juvenile abalones were reared in multi-tier basket form for overwintering in open seawaters in southern China for 106 days. The daily growth rates (DGRs) in the shell length of all experimental groups ranged from 67.08 to 135.75 mu m day(-1), while the specific growth rates (SGRs) were 0.2447-0.3259% day(-1). Variance analysis indicated that both DGRs and SGRs in shell length were significantly affected by the initial body size and stocking density. Furthermore, the effects of stocking density on DGRs and SGRs varied with the initial size. However, sorting abalones according to their initial sizes may not be necessary in practice as sorting did not alter growth significantly at all densities in this study. Factors potentially affecting abalone growth such as genetic control and intraspecific competition were discussed.

Inheritance of 15 microsatellites in the Pacific oyster Crassostrea gigas: segregation and null allele identification for linkage analysis

Microsatellites were screened in a backcross family of the Pacific oyster, Crassostrea gigas. Fifteen microsatellite loci were distinguishable and polymorphic with 6 types of allele-combinations. Null alleles were detected in 46.7% of loci, accounting for 11.7% of the total alleles. Four loci did not segregate in Mendelian Ratios. Three linkage groups were identified among 7 of the 15 segregating loci. Fluorescence-based automated capillary electrophoresis (ABI 310 Genetic Analyzer) that used to detect the microsatellite loci, has been proved a fast, precise, and reliable method in microsatellite genotyping.

Growth of eight Pacific abalone families at three temperatures

Growth rates, measured as shell length and body weight daily growth, were studied in the eight families of Pacific abalone, Haliotis discus hannai Ino, reared at 12, 16 and 20 degrees C for 40 d respectively. The results show that J(1)Rh family grew the best at 12 degrees C, with growth rates of (32.88 +/- 4.66) mu m/d and (5.24 +/- 1.84) mg/d. C(1)Jm family had the highest growth rates of (58.00 +/- 2.00) mu m/d and (9.71 +/- 1.21) mg/d at 16 degrees C. J(1)Jm family ranked the first at 20 degrees C, with growth rates of (66.00 +/- 1.76) mu m/d and (10.99 +/- 0.34) mg/d. RjRh family had the slowest growth rates at all three temperatures. Shell length growth rates were 18.25, 33.00 and 43.13 mu m/d respectively, while body weight growth rates were 2.47, 2.56 and 4.75 mg/d respectively. Both temperature and family had significant effect on growth rates (P<0.05). At 16 and 20 degrees C, maternal effects on growth rates were not significant (P>0.05), but paternal effects on growth rates were significant (P<0.05). Results of this study indicate genetic difference among the families and importance of selecting male breeders in the commercial hatchery.

AFLP-based genetic linkage maps of the Pacific oyster Crassostrea gigas Thunberg

Amplified fragment length polymorphisms (AFLPs) were used for genome mapping in the Pacific Oyster Crassostrea gigas Thunberg. Seventeen selected primer combinations produced 1106 peaks, of which 384 (34.7%) were polymorphic in a backcross family. Among the polymorphic markers, 349 were segregating through either the female or the male parent. Chi-square analysis indicated that 255 (73.1%) of the markers segregated in a Mendelian ratio, and 94 (26.9%) showed significant (P < 0.05) segregation distortion. Separate genetic linkage maps were constructed for the female and male parents. The female framework map consisted of 119 markers in 11 linkage groups, spanning 1030.7 cM, with an average interval of 9.5 cM per marker. The male map contained 96 markers in 10 linkage groups, covering 758.4 cM, with 8.8 cM per marker. The estimated genome length of the Pacific oyster was 1258 cM for the female and 933 cM for the male, and the observed coverage was 82.0% for the female map and 81.3% for the male map. Most distorted markers were deficient for homozygotes and closely linked to each other on the genetic map, suggesting the presence of major recessive deleterious genes in the Pacific oyster.

Chromosome segregation in fertilized eggs from triploid Pacific oysters, Crassostrea gigas (Thunberg), following inhibition of polar body 1

Chromosome segregation in fertilized eggs from triploid Pacific oysters, following inhibition of the first polar body (PB1), was studied with acetic orcein staining techniques. To block the release of PB1, fertilized eggs were treated with 0.5 mg/l of cytochalasin B (CB). Four types of segregation were observed, namely, ''tripolar segregation'' (54.5%), ''united bipolar segregation'' (12%), ''separated bipolar segregation'' (2.5%), and ''incomplete united bipolar segregation'' (4%). The remaining 23% could not be classified because of chromosome disorganization, but appeared to be variants of the above. It seemed clear that the predominant pattern that gave rise to tetraploids was united bipolar segregation, although certain separated bipolar segregations might also lead to the formation of tetraploids. The sequential events of meioses observed in CB-treated eggs are described. The asynchrony of meiotic events and possible mechanisms for the various types of chromosome segregation are discussed.

Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai)

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631 bp, consisting of a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 573 by with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968 bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96 h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12 h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response. (c) 2006 Elsevier Ltd. All rights reserved.