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Abstract: Haliotis diversicolor (small abalone) is an economic seafood found off the Southern coast of China. Since 1999, the cultured abalone yields in China have been affected severely by continual outbreaks of a fatal epidemic disease caused by abalone shriveling syndrome associated virus (AbSV), a double-stranded DNA virus. Although the pathogenicity and genome of AbSV have been ascertained, the epidemiology of AbSV infection remains to be investigated. In the present study, four pairs of AbSV-specific primers were designed on the basis of open reading frame (ORF)24 and ORF25 sequences in the AbSV genome. Two nested PCR detection methods were established by optimization of the annealing temperatures of primers. The results showed that the specificity of primers for AbSV detection could not be interfered with by the host genome and other aquaculture species or viruses. The detection limits of the two methods were about 10 copies of recombinant plasmid containing AbSV genes in 20μL reaction mixture. The results of detection of the AbSV epidemic showed that AbSV was still present in juvenile abalones in some farms along the Southern coast of China (Fujian and Guangdong). [Copyright &y& Elsevier]

Abstract: Haliotis diversicolor (small abalone) is an important seafood found along the southern coast of China. Since 1999, the yields of cultured abalone in China have been severely affected by an epidemic of continuous outbreaks of a fatal disease. A novel double-stranded DNA virus, abalone shriveling syndrome-associated virus (AbSV), was proven to be one of the main causative agent. Although the pathogenicity and genome of AbSV has been ascertained, the epidemiology of AbSV remains to be investigated. In this study, four pairs of AbSV-specific primers were designed on the basis of the AbSV genome, and were tested for their specificities and sensitivities in quantitative real-time PCRs (qPCRs) after optimization of the annealing temperature. The 3F3/3B3 primer pair was finally chosen with a good specificity and high efficiency of amplification, with a detection limit of about 10 copies of recombinant plasmid containing AbSV genes in a 20-μL reaction mixture. In the detection of AbSV in abalone samples along the southern coast of China, most of the diseased samples had more than 80 virus copies in 1ng host genome DNA. AbSV was also demonstrated in mature hybrid (LY) and juvenile (JH) abalones from assays of healthy animals collected in recent years. [Copyright &y& Elsevier]

Abstract: The complete mitochondrial coding regions of three small abalones Haliotis diversicolor Reeve, two collected from Vietnam and one from southern China, were successfully sequenced. The molecular feature of the mitochondrial genome is identical with the general description of the family Haliotidae mtDNAs that have been sequenced so far. The sequenced nucleotides are 16,186–16,266bp in length. The mitogenome encodes 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. Between adjacent genes trnH and nad5 there is an overlapping region. Comparison of the ratios of Ka and Ks among four species of Haliotis (H. diversicolor, H. discus hannai, H. rubra and H. tuberculata tuberculata) reveals that values of Ka/Ks in some NADH dehydrogenase and ATPase genes such as nad2, nad6 and atp8 are higher than those in other mitochondrial genes. Genome-wide gene arrangement among four species of Haliotis has been compared. Although all gene arrangement is the same in H. diversicolor, H. discus hannai and H. rubra, the location of trnS 2 and trnF in H. tuberculata tuberculata are inter-exchanged. Both gene arrangement and phylogenetic analysis support that the family Haliotidae is at a relatively primordial phylogenetic position in Gastropoda. Through alignment between Vietnam and southern China individuals, 111 SNPs are detected, most SNPs are synonymous mutations, and 7, 94 and 10 SNPs are observed at NCR, protein-coding region and RNA region, respectively. The result of SNP analysis also demonstrates that the difference is mainly in some NADH dehydrogenase genes between the Vietnam and southern China individuals. [Copyright &y& Elsevier]

Abstract: The small abalone, Haliotis diversicolor, is a widely distributed and cultured species in the subtropical coastal area of China. To identify and classify functional genes of this important species, a normalized expressed sequence tag (EST) library, including 7069 high quality ESTs from the total body of H. diversicolor, was analyzed. A total of 4781 unigenes were assembled and 2991 novel abalone genes were identified. The GC content, codon and amino acid usage of the transcriptome were analyzed. For the accurate annotation of the abalone library, different influencing factors were evaluated. The gene ontology (GO) database provided a higher annotation rate (69.6%), and sequences longer than 800bp were easily subjected to a BLAST search. The taxonomy of the BLAST results showed that lancelet and invertebrates are most closely related to abalone. Sixty-seven identified plant-like genes were further examined by reverse transcription–polymerase chain reaction (RT–PCR) and sequencing, only seven of these were real transcripts in abalone. Phylogenic trees were also constructed to illustrate the positions of two Cystatin sequences and one Calmodulin protein sequence identified in abalone. To perform functional classification, three different databases (GO, KEGG and COG) were used and 60 immune or disease-related unigenes were determined. This work has greatly enlarged the known gene pool of H. diversicolor and will have important implications for future molecular and genetic analyses in this organism. [Copyright &y& Elsevier]

The article discusses research on the occurrence of Withering syndrome-Rickettsia-like organism (WS-RLO) infection in the abalone Haliotis diversicolor supertexta found in Thailand, Taiwan and the People's Republic of China (PRC). It was noted that approximately 50% of the abalone that were studied from each nation was infected with RLO. It is inferred that the RLO infection in abalone had occurred for at least 9 months, but remained asymptomatic. The authors suggest that California is the source of WS-RLO, according to sequence homology and transport history. The World Animal Health Organization (OIE) recommended an extensive health examinations of abalone prior to transfer within and outside Thailand to avoid the spread of the disease.

To determine whether genetic improvement can be attained through a selective breeding programme, divergent selection for shell length was applied to two stocks of Haliotis diversicolor. Stock A was descended from the cross between males from a Japanese wild population and females from a Taiwan aquacultured population and Stock B was from the Taiwan cultured population, which had been successively cultured in mainland China for about 10 generations. The 10% largest and 10% smallest abalones for each of these two stocks were selected as parents for the large-selected and small-selected lines respectively. Equal numbers of abalone were randomly chosen from the two stocks to serve as parents for the control lines before the selection. The selected and control lines were reared under the same conditions at early juvenile, later juvenile and grow-out stages. Stock A showed a significantly higher response to selection and realized heritability than Stock B ( P<0.01). The large-selected line of Stock A and Stock B grew 12.79% and 4.58% faster than their control lines on shell length respectively. The average realized heritability for shell length was 0.441±0.064 for Stock A and 0.113±0.013 for Stock B. Responses to selection were different at different ages in each stock and the body weights of the selected lines were significantly different from the control lines in both stocks at the grow-out stage. Asymmetric responses to selection in the two directions were also observed in both stocks. Differences in response to selection and realized heritability between the two stocks are presumably due to genetic variability. [ABSTRACT FROM AUTHOR]

Abstract  Outbreaks of mass mortality of postlarval abalone, Haliotis diversicolor supertexta, have occurred in south China since 2002 and have forced many abalone farms to close. About 30 representative bacterial strains were isolated from a sample of five diseased postlarval abalone, taken 25 days post-fertilization during an outbreak of postlarval disease in Shenzhen, China, in October 2006. Bacterial challenge tests showed that the predominant strain, designated as strain 22, was highly virulent to postlarvae with an LD50 value of 7.8 × 104 colony forming units (CFU) ml−1, while four of the other isolates were weakly virulent with LD50 values ranging from 1 × 106 to 1 × 107 CFU ml−1, and the remaining 25 isolates were classified as avirulent with LD50 values greater than 1 × 108 CFU ml−1. By means of API 20NE and 16S rDNA and ITS sequencing analyses, strain 22 was identified as Pseudomonas aeruginosa. Antibiotic susceptibility tests showed that strain 22 exhibited around 75% of susceptibility to 16 various antibiotics tested. The results of this study show P. aeruginosa as one of the bacteria involved in the mortality of abalone postlarvae in Shenzhen, China. [ABSTRACT FROM AUTHOR]

The aquaculture industry of small abalone Haliotis diversicolor in southern China is now close to collapse because of high mortality at the grow-out stage. To examine if survival and growth (shell length increase) could be improved by cross breeding among populations, a 3 × 3 complete diallel cross was conducted among three populations from Japan (A), Taiwan (B) and Vietnam (C). Performances in growth rate and survival at early juvenile, later juvenile and grow-out stages were compared among the six reciprocal cross lines and three parental lines. Magnitude of heterosis for survival and growth varied among cross lines in the three growing stages. As to mid-parent heterosis, HAB was significantly ( P<0.01) higher than HAC and HBC at all three growing stages for both growth and survival, and all mid-parent heterosis fell between the two corresponding single-parent heterosis. HAB was 6.84% for shell length and 69.09% for survival at Day 420. Our results of high survival heterosis in line AB and line BA show that crossbreeding between different populations can benefit the small abalone breeding programs and the industry. [ABSTRACT FROM AUTHOR]

Background: The effects of diverse stresses ultimately alter the structures and functions of proteins. As molecular chaperones, heat shock proteins (HSPs) are a group of highly conserved proteins that help in the refolding of misfolded proteins and the elimination of irreversibly damaged proteins. They are mediated by a family of transcription factors called heat shock factors (HSFs). The small abalone Haliotis diversicolor is a species naturally distributed along the southern coast of China. In this study, the expression of HdHSF1 was inhibited by RNAi in hemocytes in order to further elucidate the regulatory roles of HdHSF1 on heat shock responsive genes in abalone. Meanwhile, to understand the transcriptional regulation of the HdHSF1 gene, the 5′-upstream regulatory region of HdHSF1 was characterized, and the relative promoter activity was examined by dual-luciferase reporter gene assay system in HEK293T cell lines. Results: After the inhibition of the H. diversicolor HSF1 gene (HdHSF1) by dsRNA (double-stranded RNA), the expression of most heat shock related-genes was down-regulated (p < 0.05). It indicated the importance of HdHSF1 in the heat shock response of H. diversicolor. Meanwhile, 5′-flanking region sequence (2633 bp) of the HdHSF1 gene was cloned; it contained a putative core promoter region, TATA box, CAAT box, CpG island, and many transcription elements. In HEK293T cells, the 5′-flanking region sequence can drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. Exposure of cells to the high-temperature (39 °C and 42 °C) resulted in the activation of HdHSF1 promoter activity, which may explain why the expression of the HdHSF1 gene participates in heat shock response. Luciferase activity of different recombinant plasmids, which contained different truncated promoter fragments of the HdHSF1 gene in HEK293T cells, revealed the possible active regions of the promoter. To further identify the binding site of the critical transcription factor in the region, an expression vector with the site-directed mutation was constructed. After being mutated on the GATA-1 binding site, we found that the luciferase activity was significantly increased, which suggested that the GATA-1 binding site has a certain weakening effect on the activity of the HdHSF1 promoter. Conclusions: These findings suggest that GATA-1 may be one of the transcription factors of HdHSF1, and a possible signaling pathway mediated by HdHSF1 may exist in H. diversicolor to counteract the adverse effects of heat shock stress. [ABSTRACT FROM AUTHOR]

Inhibitor of NF-κB (IκB), nuclear factor-κB (NF-κB), and Akirin2 are all important members of Rel/NF-κB signaling pathway, which plays a pivotal role in regulating the innate immune response of vertebrates and invertebrates. In this study, the IκB (SaIκB) and Akirin2 (SaAkirin2) cDNAs of small abalone Haliotis diversicolor were cloned and characterized. The full length cDNA of SaIκB and SaAkirin2 were 1748 bp and 1452 bp respectively, encoding a protein of 401 aa and 187 aa respectively. A conserved degradation motif (DS56GIYS60) and six ankyrin repeats were identified in the SaIκB by SMART analysis. Meanwhile, a typical nuclear localization signal (NLS) was found at the N-terminal region of the SaAkirin2 protein. Also, the mRNA expression level of SaIκB, SaAkirin2, and AbNF-κB were detected by quantitative real-time PCR. The results revealed that all these three genes were ubiquitously expressed in 7 selected tissues. The expression level of SaIκB in gills was higher than that in other tissues (P < 0.05) while the expression level of AbNF-κB was significantly higher in hepatopancreas and haemocytes. The highest expression level of SaAkirin2 was detected in hepatopancreas, followed by mantle. The mRNA expression levels in either gills or haemocytes of SaIκB, SaAkirin2, and AbNF-κB were significantly up-regulated (P < 0.05) post thermal stress, hypoxia exposure, thermal plus hypoxia stress and the injection of Vibrio parahaemolyticus. These results indicated that these three NF-κB signaling pathway-related genes are involved in response to bacterial infection and play essential roles in response to thermal and hypoxia stress., (Copyright © 2014 Elsevier Ltd. All rights reserved.)