Cloning and Characterization of an Abalone （Haliotis discus hannai ） Actin Gene

1　Introduction　Actinisanimportantcontractileproteinineukary oticcells .Itisoneofthetwomajorcomponentsin volvedinthecontractionofmusclecells .Innon mus clecells ,itisthemajorpartofcytoskeletoninvolvedinmanyprocessessuchascellmotility ,endocytosis ,exocyt…     

Development and Characterization of Microsatellite Markers for the Pacific Abalone （Haliotis discus） via EST Database Mining

The EST database of the Pacific abalone (Haliotis discus) was mined for developing mierosatellite markers. A total of 1476 EST sequences were registered in GenBank when data mining was performed. Fifty sequences (approximately 3.4%) were found to contain one or more mierosatellites. Based on the length and GC content of the flanking regions, duster analysis and BLASTN, 13 microsatellite-containing ESTs were selected for PCR primer design. The results showed that 10 out of 13 primer pairs could amplify seorable PCR products and showed polymorphism. The number of alleles ranged from 2 to 13 and the values of Hoand He varied from 0.1222 to 0.8611 and 0.2449 to 0.9311, respectively. No significant linkage disequilibrium (LD) between any pairs of these loci was found, and 6 of 10 loci conformed to the Hardy-Weinberg equilibrium (HWE). These EST-SSRs are therefore potential tools for studies of intraspecies variation and hybrid identification.     

Sequences Characterization of Microsatellite DNA Sequences in Pacific Abalone(Haliotis discus hannai)

1 Introduction Microsatellites or simple sequence repeats (SSRs) are tandemly repeated motifs of one to six bases found in all prokaryotic and eukaryotic genomes analysed to date (Zane et al., 2002). Due to their hyper-variable and co-dominant nature, relatively high abundance and random distribution in the genome, microsatellites are among the most efficient class of molecular markers. Such repeats display high polymorphism because of variation in repeat length and can be rapidly analysed t…     

Effect of Dietary Lipid on the Growth,Fatty Acid Composition and Δ5 Fads Expression of Abalone(Haliotis discus hannai Ino) Hepatopancreas

This study investigated the effect of dietary lipid on the growth, fatty acid composition and Δ5 fatty acyl desaturase genes(Fads) expression of juvenile abalone(Haliotis discus hannai Ino) hepatopancreas. Six purified diets were formulated to contain tripalmitin(TP), olive oil(OO, 72.87% 18:1n-9), grape seed oil(GO, 68.67% 18:2n-6), linseed oil(LO, 70.48% 18:3n-3), ARA oil(AO, 41.81% ARA) or EPA oil(EO, 44.09% EPA and 23.67% DAH). No significant difference in survival rate was observed among abalone fed with different diets. Weight gain rate(WGR) and daily growth rate of shell length(DGRSL) were significantly increased in abalone fed with diets containing OO, AO and EO, but decreased in abalone fed with LO diet(P 0.05) in comparison with those fed with TP. High level of dietary 18:2n-6 resulted in higher content of n-6 polyunsaturated fatty acids(PUFAs) in abalone fed with GO than those fed with TP, OO, LO and EO(P 0.05). n-3 PUFAs in abalone fed with LO was significantly higher than those in abalone fed with TP, OO, GO and AO(P 0.05). The highest contents of 20:1n-9 and 22:1n-9 were observed in abalone fed with OO. The expression of Δ5 Fads in hepatopancreas of abalone was enhanced by high concentration of 18:3n-3 and suppressed by dietary LC-PUFAs; however it was not affected by dietary high concentration of 18:1n-9 or 18:2n-6. These results provided valuable information for understanding the synthesis of LC-PUFAs and nutritional regulation of Δ5 Fads expression in abalone.     

Descriptions of three new species of Mitridae （Mollusca： Gastropoda） from South China Sea

Three new species of Family Mitridae （Mollusca： Gastropoda） from the South China Sea are described in the present paper. They are Ziba aglais sp. nov. B. LI ＆ S. ZHANG, Neocancilla daidaleosa sp.nov. B. LI ＆ X. LI, and Mitra holkosa sp. nov. B. LI. Their systematic positions are also discussed.     

Assessment of South Pacific Albacore Stock （Thunnus alalunga ） by Improved Schaefer Model

Based on catch and effort data of tuna longline fishery operating in the South Pacific Ocean, the South Pacific al-bacore stock was assessed by an improved Schaefer model. The results revealed that the intrinsic growth rate was about 1.28374 and carrying capacities vareied in the range from 73734 to 266732 metric tons. The growth ability of this species is remarkable. Stock dynamics mainly depends on environmental conditions. The stock is still in good condition. However, the continuous decreasing of biomass in recent years should be noticed.     

Structural Changes and Rheological Properties of Dry Abalone Meat （Haliotis diversicolor） During the Process of Water Restoration

Changes in tissue structure, rheological property and water content of dry abalone meat in the process of water restora- tion were studied. The weight and volume of dry abalone meat increased with water restoration. When observed under a light micro- scope, structural change in myofibrils was obvious and a distinct network was found. When water restoration time increased from 24 h to 72 h, the instantaneous modulus E0 and viscosity η1 increased, whereas the rupture strength and relaxation time (τ1) were re- duced. There were no significant changes of rheological parameters (E0, η1, τ1, rupture strength) from 72 h to 96 h of water restoration. Therefore, the dry abalone meat was swollen enough at the time of 72 h. The rheological parameters were obviously influenced by the structural changes.     

The Effect of Three Culture Methods on Intensive Culture System of Pacific White Shrimp （Litopenaeus vannama）

Different culture methods may affect the intensive culture system of Pacific white shrimp （Litopenaeus vannamei） re- garding water quality and growth and economic performance. This study evaluated the potential effects of three culture methods through cultivation of juvenile shrimps under consistent tank management conditions for 84 d. The three methods involved shrimp cultivation in different tanks, i.e., outdoor tanks with cement bottom （mode-C）, greenhouse tanks with cement bottom （mode-G） and outdoor tanks with mud-substrate （mode-M）. Results showed that water temperature was significantly higher in mode-G than that in mode-C （P 〈 0.05）. In contrast to the other two treatments, mode-M had stable pH after 50 d cultivation of shrimps. In the mid-late period, the average concentrations of TAN, NO2-N, DIP and COD were significantly lower in mode-M and mode-G compared with those in mode-C （P 〈0.05）. Despite lack of differences in the final shrimp weight among different treatments （P 〉0.05）, mode-M had significantly higher shrimp yield, survival rate and feed conversion rate （P 〈 0.05） than other modes. There were significant dif- ferences in revenue and net return among different treatments （P 〈 0.05）. These demonstrated that the treatments of mode-G and mode-M were conductive to the intensive culture system ofL. vannamei.     

Genetic Variation and Differentiation in Wide Ranging Populations of Razor Clam （Sinonovacula constricta） Inferred from AFLP Markers

The genetic variation and differentiation of the razor clam Sinonovacula constricts distributed along the coast of China were studied through amplified fragment length polymorphism(AFLP)analysis. Six primer combinations generated 193 fragments. The He values varied from 0. 322 to 0. 463 and the percentage of polymorphic loci ranged from 74. 1% to 98. 4%, which indicates a high level of genetic diversity. Cluster analysis by Nei's pairwise distance grouped all specimens by geographical origins. AMOVA consistently showed that genetic variation among populations was 8. 71%, and most of the variation came from the genetic variation within populations(91.29%). Genetic differentiation among the six populations was moderate; pairwise FST ranged from 0. 0282 to0. 1480, which indicated that S. Constricts populations along the coast of China are genetically connected. Among all the six populations, the Beihai population is the mostly differentiated from the others, suggesting that Hainan Island and Leizhou Peninsula act as barriers to gene flow. All populations abide isolation by distance model as indicated by Mantel test, except for ZS(Zhoushan)and YQ(Yueqing)populations. Information obtained in this study will provide guidelines for conservation and fishery management of this species in the future.     

Characterization of defensin gene from abalone Haliotis discus hannai and its deduced protein

Defensin is one of preserved ancient host defensive materials formed in biological evolution. As a regulator and effector molecule, it is very important in animals' acquired immune system. This paper reports the defensin gene from the mixed liver and kidney cDNA library of abalone Haliotis discus hannai Ino. Sequence analysis shows that the gene sequence of full-length cDNA encodes 42 mature peptides (including six Cys), molecular weight of 4 323 Da, and pI of 8.02. Amino acid sequence homology analysis shows that the peptides are highly similar (70% in common) to other insects defensin. Because of a typical insect-defensin structural character of mature peptide in the secondary structure, the polypeptide named Haliotis discus defensin (hd-def), a novel of antimicrobial peptides, belongs to insects defensin subfamily. The RT-PCR result of Haliotis discus defensin shows that the gene can be expressed only in the hepatopancreas by Gram-negative and positive bacteria stimulation, which is ascribed to inducible expression. Therefore, it is revealed that the Haliotis discus defensin gene expression was related to the antibacterial infection of Haliotis discus hannai Ino.     

Characterization of genic microsatellite markers derived from expressed sequence tags in Pacific abalone (Haliotis discus hannai)

Simple sequence repeat (SSR) markers were developed from the expressed sequence tags (ESTs) of Pacific abalone (Haliotis discus hannai).Repeat motifs were found in 4.95% of the ESTs at a frequency of one repeat every 10.04 kb of EST sequences,after redundancy elimination.Seventeen polymorphic EST-SSRs were developed.The number of alleles per locus varied from 2-17,with an average of 6.8 alleles per locus.The expected and observed heterozygosities ranged from 0.159 to 0.928 and from 0.132 to 0.922,respective...     

Sequences characterization of microsatellite DNA sequences in Pacific abalone (Haliotis discus hannai)

The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the microsatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber(1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats(13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (＜ 20 repeats) were most abundant,accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatetlite isolation in other abalone species.     

Effects of Dietary Carbohydrates Sources on Lipids Compositions in Abalone, Haliotis discus hannai Ino

A study was conducted to evaluate the effects of dietary carbohydrates on triglyceride, cholesterol and fatty acid concentrations in abalone, Haliotis discus hannai Ino. Six semi-purified diets with different carbohydrates (dextrin, heat-treated wheat starch, wheat starch, corn starch, tapioca starch and potato starch, respectively), all containing a carbohydrate level of 33.5%, were fed to abalone (initial shell length: 29.98 mm±0.09 mm; initial weight. 3.42 g±0.02 g) for 24 weeks in a recirculation system. The results indicate that serum triglyceride concentrations were significantly (P<0.05) higher in the abalone fed with dextrin, heat-treated wheat starch and wheat starch than those fed with corn starch, and serum cholesterol concentrations were significantly (P<0.05)higher in the abalone fed with dextrin, heat-treated wheat starch than those fed with corn starch. Fatty acid C20:4n-6 in the foot muscles were significantly (P<0.05) lower in the abalone fed with dextrin than those fed with wheat starch, corn starch, tapioca starch and potato starch. Fatty acid C20:4n-6 in hepatopancreas was significantly (P<0.05) lower in abalone fed with heat-treated wheat starch than those fed with corn starch, tapioca starch and potato starch. Fatty acid C22:6n-3 in the foot muscles were significantly (P< 0.05) lower in the abalone fed with dextrin and heat-treated wheat starch than those fed with wheat starch and potato starch.     

Effects of dietary menadione on the activity of antioxidant enzymes in abalone, Haliotis discus hannai Ino

A 240-day growth experiment in a re-circulating water system was conducted to investigate the effects of dietary menadione on the growth and antioxidant responses of abalone Haliotis discus hannai Ino. Triplicate groups of juvenile abalone (initial weight: 1.19 ± 0.01 g; shell length: 19.23 ± 0.01 mm) were fed to satiation with 3 semi-purified diets containing 0, 10, and 1 000 mg menadione sodium bisulfite (MSB)/kg, respectively. Results show that there were no significant differences in the rate of weight gain or in the daily increment in shell length of abalone among different treatments. Activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) in viscera were significantly decreased with dietary menadione. However, activities of these enzymes except for GPX in muscle were increased. Therefore, antioxidant responses of abalone were increased in muscle and decreased in viscera by dietary menadione.     

Effects of dietary carbohydrates sources on lipids compositions in abalone, Haliotis discus hannai Ino

A study was conducted to evaluate the effects of dietary carbohydrates on triglyceride, cholesterol and fatty acid concentrations in abalone, Haliotis discus hannai Ino. Six semi-purified diets with different carbohydrates (dextrin, heat-treated wheat starch, wheat starch, corn starch, tapioca starch and potato starch, respectively), all containing a carbohydrate level of 33.5%, were fed to abalone (initial shell length: 29.98 mm ± 0.09 mm; initial weight: 3.42 g ± 0.02 g) for 24 weeks in a recirculation system. The results indicate that serum triglyceride concentrations were significantly (P < 0.05) higher in the abalone fed with dextrin, heat-treated wheat starch and wheat starch than those fed with corn starch, and serum cholesterol concentrations were significantly (P < 0.05) higher in the abalone fed with dextrin, heat-treated wheat starch than those fed with corn starch. Fatty acid C20:4n-6 in the foot muscles were significantly (P < 0.05) lower in the abalone fed with dextrin than those fed with wheat starch, corn starch, tapioca starch and potato starch. Fatty acid C20:4n-6 in hepatopancreas was significantly (P < 0.05) lower in abalone fed with heat-treated wheat starch than those fed with corn starch, tapioca starch and potato starch. Fatty acid C22:6n-3 in the foot muscles were significantly (P < 0.05) lower in the abalone fed with dextrin and heat-treated wheat starch than those fed with wheat starch and potato starch.     

Acclimation-dependent expression of heat shock protein 70 in Pacific abalone (Haliotis discus hannai Ino) and its acute response to thermal exposure

Heat shock protein 70 (Hsp70) is one important member of heat shock protein (Hsp) family that is responsible for various stresses, especially thermal stress. Here we examined the response of Hsp70 gene to both chronic and acute thermal exposure in Pacific abalone (Haliotis discus hannai Ino). For the chronic exposure, abalones were maintained at 8, 12, 20, and 30°C for four months and their mRNA levels were measured. The highest mRNA level of Hsp70 gene relative to actin gene was detected in the 30°C-acclimated group, followed by the 8°C-acclimated group and then the 12°C- and 20°C-acclimated groups. After the long-term acclimation, gills from each of the above acclimation groups were dissected and exposed to different temperatures between 8°C and 38°C for 30 min. Hsp70 expression in gills acclimated to different temperatures responded differentially to the same temperature exposure. The incubation temperature that induced maximum Hsp70 mRNA expression was higher in the higher temperature acclimation groups than lower temperature groups. Pacific abalones could alter the expression pattern of Hsp70 gene according to environmental thermal conditions, through which they deal with the stress of thermal variations.