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Ganglioneuritis was the primary pathologic change in infected abalone associated with Haliotid herpesvirus 1 (HaHV-1) infection, which eventually became known as abalone viral ganglioneuritis (AVG). However, the distribution of HaHV-1 in the other tissues and organs of infected abalone has not been systemically investigated. In the present study, the distribution of HaHV-1-CN2003 variant in different organs of small abalone, Haliotis diversicolor supertexta, collected at seven different time points post experimental infection, was investigated with histopathological examination and in situ hybridization (ISH) of HaHV-1 DNA. ISH signals were first observed in pedal ganglia at 48 h post injection, and were consistently observed in this tissue of challenged abalone. At the same time, increased cellularity accompanied by ISH signals was observed in some peripheral ganglia of mantle and kidney. At the end of infection period, lesions and co-localized ISH signals in infiltrated cells were detected occasionally in the mantle and hepatopancreas. Transmission electron microscope analysis revealed the presence of herpes-like viral particles in haemocyte nuclei of infected abalone. Our results indicated that, although HaHV-1-CN2003 was primarily neurotropic, it could infect other tissues including haemocytes.

Haliotid herpesvirus-1 (HaHV-1) is the first identified gastropod herpesvirus, causing a highly lethal neurologic disease of abalone species. The genome of HaHV-1 has been sequenced, but the functions of the putative genes and their roles during infection are still poorly understood. In the present study, transcriptomic profiles of Haliotis diversicolor supertexta at 0, 24 and 60 h post injection (hpi) with HaHV-1 were characterized through high-throughput RNA sequencing. A total of 448 M raw reads were obtained and assembled into 2.08 &times, 105 unigenes with a mean length of 1486 bp and an N50 of 2455 bp. Although we detected increased HaHV-1 DNA loads and active viral expression at 24 hpi, this evidence was not linked to significant changes of host transcriptomic profiles between 0 and 24 hpi, whereas a rich immune-related gene set was over-expressed at 60 hpi. These results indicate that, at least at the beginning of HaHV-1 infection, the virus can replicate with no activation of the host immune response. We propose that HaHV-1 may evolve more effective strategies to modulate the host immune response and hide during replication, so that it could evade the immune surveillance at the early stage of infection.

Heat-shock protein 70 (HSP70) is a molecular chaperone that plays critical roles in cell protein folding and metabolism, which helps to protect cells from unfavorable environmental stress. Haliotis diversicolor is one of the most important economic breeding species in the coastal provinces of south China. To date, the expression and transcriptional regulation of HSP70 in Haliotis diversicolor (HdHSP70) has not been well characterized. In this study, the expression levels of HdHSP70 gene in different tissues and different stress conditions were detected. The results showed that the HdHSP70 gene was ubiquitously expressed in sampled tissues and was the highest in hepatopancreas, followed by hemocytes. In hepatopancreas and hemocytes, the HdHSP70 gene was significantly up-regulated by Vibrio parahaemolyticus infection, thermal stress, and combined stress (Vibrio parahaemolyticus infection and thermal stress combination), indicating that HdHSP70 is involved in the stress response and the regulation of innate immunity. Furthermore, a 2383 bp of 5&prime, flanking region sequence of the HdHSP70 gene was cloned, and it contains a presumed core promoter region, a CpG island, a (TG)39 simple sequence repeat (SSR), and many potential transcription factor binding sites. The activity of HdHSP70 promoter was evaluated by driving the expression of luciferase gene in HEK293FT cells. A series of experimental results indicated that the core promoter region is located between &minus, 189 bp and +46 bp, and high-temperature stress can increase the activity of HdHSP70 promoter. Sequence-consecutive deletions of the luciferase reporter gene in HEK293FT cells revealed two possible promoter activity regions. To further identify the binding site of the key transcription factor in the two regions, two expression vectors with site-directed mutation were constructed. The results showed that the transcriptional activity of NF-1 site-directed mutation was significantly increased (p &lt, 0.05), whereas the transcriptional activity of NF-&kappa, B site-directed mutation was significantly reduced. These results suggest that NF-1 and NF-&kappa, B may be two important transcription factors that regulate the expression of HdHSP70 gene.

Gayadomonas joobiniege G7 is an agar-degrading bacterium, which produces various agarases that have been biochemically characterized recently. In this study, we biochemically characterized a new β-agarase AgaJ10 belonging to the glycoside hydrolase (GH) 42 family from G. joobiniege G7. AgaJ10 is composed of 762 amino acids (89 kDa) and has the highest similarity (63% identity) to a putative β-agarase from the agar-degrading bacterium Catenovulum sp. DS-2, which was obtained from the intestines of a Haliotis diversicolor. The optimal pH and temperature for AgaJ10 activity were determined to be 5.0 and 30 °C, respectively. AgaJ10 exhibited a cold tolerance, retaining more than 40% of its enzymatic activity at 5 °C. The Km and Vmax of AgaJ10 for agarose were 61.5 mg/mL and 294.1 U/mg, respectively. Notably, the activity of AgaJ10 was significantly enhanced by Mn2+ but was strongly inhibited by some metal ions, including Fe2+, Ni2+, and Cu2+. Agarose-liquefaction, mass spectrometry, and thin-layer chromatography analyses showed that AgaJ10 is an exo-type β-agarase that hydrolyzes agarose only into neoagarobiose. Therefore, this study is the first report of a GH42 β-agarase that catalyzes a neoagarobiose-producing exo-type reaction.

Hemocyanins (Hc), the main protein components of hemolymph in invertebrates, are not only involved in oxygen transport but also linked to non-specific immune responses. In this study, we used abalone (Haliotis diversicolor) Hc to study the basis of its diversified functions through gene, protein, peptides, and phenoloxidase (PO) activity levels. Three complete hemocyanin gene (HdH) sequences were cloned for the first time. By comparing the copies and location of HdH between abalone and other mollusks, we propose that Hc gene duplication and linkage is likely to be common during the evolution of mollusk respiratory proteins. We further demonstrate that all three genes could be expressed in abalone, with expression varying based on the developmental stages, tissue types, and different pathogen infections. However, HdH1 and HdH2 appear to be synthesized by the same cells by fluorescence in situ hybridization. Furthermore, the PO activity of HdH can be induced by trypsin, urea, and SDS in vitro. Viral infection can stimulate its PO activity in vivo by cleaving the protein into fragments. Consequently, we present a comprehensive study of abalone hemocyanin, providing important evidence for an in-depth understanding of the physiological and immune functions of Hc in mollusks.

Heat-shock protein 70 (HSP70) is a molecular chaperone that plays critical roles in cell protein folding and metabolism, which helps to protect cells from unfavorable environmental stress. Haliotis diversicolor is one of the most important economic breeding species in the coastal provinces of south China. To date, the expression and transcriptional regulation of HSP70 in Haliotis diversicolor (HdHSP70) has not been well characterized. In this study, the expression levels of HdHSP70 gene in different tissues and different stress conditions were detected. The results showed that the HdHSP70 gene was ubiquitously expressed in sampled tissues and was the highest in hepatopancreas, followed by hemocytes. In hepatopancreas and hemocytes, the HdHSP70 gene was significantly up-regulated by Vibrio parahaemolyticus infection, thermal stress, and combined stress (Vibrio parahaemolyticus infection and thermal stress combination), indicating that HdHSP70 is involved in the stress response and the regulation of innate immunity. Furthermore, a 2383 bp of 5′-flanking region sequence of the HdHSP70 gene was cloned, and it contains a presumed core promoter region, a CpG island, a (TG)39 simple sequence repeat (SSR), and many potential transcription factor binding sites. The activity of HdHSP70 promoter was evaluated by driving the expression of luciferase gene in HEK293FT cells. A series of experimental results indicated that the core promoter region is located between −189 bp and +46 bp, and high-temperature stress can increase the activity of HdHSP70 promoter. Sequence-consecutive deletions of the luciferase reporter gene in HEK293FT cells revealed two possible promoter activity regions. To further identify the binding site of the key transcription factor in the two regions, two expression vectors with site-directed mutation were constructed. The results showed that the transcriptional activity of NF-1 site-directed mutation was significantly increased (p < 0.05), whereas the transcriptional activity of NF-κB site-directed mutation was significantly reduced. These results suggest that NF-1 and NF-κB may be two important transcription factors that regulate the expression of HdHSP70 gene.

Abalone viral ganglioneuritis (AVG), caused by Haliotid herpesvirus-1 (HaHV-1) infection, has been reported as the main cause of mortality and heavy losses of wild and cultivated abalone in Taiwan and Australia since 2003. HaHV-1 DNA has also been reported in diseased abalone collected in early 2000s in China. However, no data is available about the susceptibility, disease process and pathological changes of HaHV-1 infection in the primary cultivated abalone species in China. In the present study, two cultivated abalone species, Haliotis diversicolor supertexta and Haliotis discus hannai, were challenged with HaHV-1-CN2003 collected in 2003 in China using three different methods. Results showed that H. diversicolor supertexta was highly susceptible to HaHV-1-CN2003 infection and suffered acute mortality using all three challenge methods. H. discus hannai was not susceptible to the viral infection. Histopathology combined with transmission electron microscopy and quantitative PCR analysis revealed that the tropism of HaHV-1-CN2003 includes both neural tissue and haemocytes.

In recent years, the abalone aquaculture industry has been threatened by the deteriorating environmental conditions, such as hypoxia and thermal stress in the hot summers. It is necessary to investigate the molecular mechanism in response to these environmental challenges, and subsequently understand the immune defense system. In this study, the transcriptome profiles by RNA-seq of hemocytes from the small abalone Haliotis diversicolor after exposure to hypoxia, thermal stress, and hypoxia plus thermal stress were established. A total of 103,703,074 clean reads were obtained and 99,774 unigenes were assembled. Of the 99,774 unigenes, 47,154 and 20,455 had homologous sequences in the Nr and Swiss-Prot protein databases, while 16,944 and 10,840 unigenes could be classified by COG or KEGG databases, respectively. RNAseq analysis revealed that the differentially expressed genes (DEGs) after challenges of hypoxia, thermal stress, or hypoxia plus thermal stress were 24,189, 29,165 and 23,665, among which more than 3000 genes involved in at least 230 pathways, including several classical immune-related pathways. The genes and pathways that were involved in immune response to hypoxia/thermal challenges were identified by transcriptome analysis and further validated by quantitative real-time PCR and RNAi technology. The findings in this study can provide information on H. diversicolor innate immunity to improve the abalone aquaculture industry, and the analysis of the potential immune-related genes in innate immunity signaling pathways and the obtained transcriptome data can provide an invaluable genetic resource for the study of the genome and functional genes.

Abalone herpesvirus (AbHV) infection of cultured abalones Haliotis diversicolor supertexta induced acute high mortality in 2003. Years later, sporadic mortality was noted for an extended period of months, resulting in high cumulative mortality. Moribund abalones were analyzed using PCR, in situ hybridization, and histopathology, because thus far no viral particles have been observed by transmission electron microscopy. PCR using 20 primer sets, specifically designed from sequences of acute AbHV infection, failed to amplify any products from abalones suffering from chronic mortality. Subsequently, a 1406-bp sequence was amplified from chronic moribund abalones, and this sequence showed a 92% (553 bp/602 bp) homology with the gene of an AbHV Taiwan isolate (NCBI serial no. KF537536.1), suggestive of an AbHV pathotype. Histopathology of AbHV pathotype infection showed hemocyte infiltration in the lamina propia of the digestive tract, and hemocytes of various stages were evident, as well as the loss of seminal tubules in the gonad. In situ hybridization revealed that in AbHV infection, positive signals were restricted to the neural ganglia, while in AbHV pathotype infection, positive signals were observed only in the hemocytes. It appeared that the tropism of AbHV shifted from mainly neurotropic in AbHV infection to mainly hemocytotropic in abalone suffering from chronic mortality. Abalone shriveling syndrome-associated virus co-infection was detected in some of AbHV pathotype infection events. Further studies are needed to better understand the pathogenesis of AbHV pathotype affecting H. diversicolor in Taiwan.

Metamorphosis is the most critical life stage for abalone. There are morphogenetic transformations and habitat selection with metamorphosis. A large amount of larvae often die during this stage. Insulin/insulin-like growth factor signaling pathway (IIS) plays a role in the development. However, there is little knowledge of IIS function in metamorphosis. IGFBP7, a component of IIS, was found to be involved in metamorphosis of small abalone Haliotis diversicolor . The expression of saIGFBP7 mRNA rapidly increased before metamorphosis, which were mainly localized in the velum and foot. There was a significant decrease of metamorphosis rate after competent larvae were exposed to 5 μg/mL dsRNA of saIGFBP7 (p = 2.50 × 10 − 10 , F = 389.05). The expressions of three downstream genes of IIS ( PI3K , ERKα and AKT ) as well as saIGFBP7 were decreased in the dsRNA exposure experiment. There was no significant difference of metamorphosis rate (p = 0.97, F = 0.071) after competent larvae were induced in different concentrations of insulin. The expression of saIGFBP7 , PI3K and ERKα also had no significant difference in insulin inducing assay. However, AKT expression level of 1 μmol/L insulin induced group was significantly higher than that of other concentrations (p = 3.41 × 10 − 7 , F = 26.68). These results suggested that IGFBP7 is involved in metamorphosis of abalone larvae. Our study provides insight on developing new methods for increasing the metamorphosis rate in abalone aquaculture. However, it does not work as a negative regulator of IIS activity, or plays its role in an IGF- and insulin-independent manner during metamorphosis of abalone larvae.

546 Vibrio isolates from rearing seawater (292 strains) and intestines of abalone (254 strains) were tested to ten antibiotics using Kirby-Bauer diffusion method. Resistant rates of abalone-derived Vibrio isolates to chloramphenicol (C), enrofloxacin (ENX) and norfloxacin (NOR) were28%, whereas those from seawater showed large fluctuations in resistance to each of the tested antibiotics. Many strains showed higher resistant rates (40%) to kanamycin (KNA), furazolidone (F), tetracycline (TE), gentamicin (GM) and rifampin (RA). 332 isolates from seawater (n=258) and abalone (n=74) were resistant to more than three antibiotics. Peaked resistant rates of seawater-derived isolates to multiple antibiotics were overlapped in May and August. Statistical analysis showed that pH had an important effect on resistant rates of abalone-derived Vibrio isolates to RA, NOR, and ENX. Salinity and dissolved oxygen were negatively correlated with resistant rates of seawater-derived Vibrio isolates to KNA, RA, and PG.

The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33%. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor.

Objectives Shewanella algae is an environmental marine bacterium and an emerging opportunistic human pathogen. Moreover, there are increasing reports of strains showing multidrug resistance, particularly carbapenem-resistant isolates. Although S. algae has been found in bivalve shellfish aquaculture, there is very little genome-wide data on resistance determinants in S. algae from shellfish. The aim of this study was to determine the whole genome sequence of carbapenem-resistant S. algae strain AC isolated from small abalone in Taiwan. Methods Bacterial genomic DNA was sequenced using an Illumina MiSeq platform with 250-bp paired-end reads. De novo genome assembly was performed using Velvet v.1.2.07. The whole genome was annotated and several candidate genes for antimicrobial resistance were identified. Results The genome size was calculated at 4 751 156 bp, with a mean G + C content of 53.09%. A total of 4164 protein-coding sequences, 7 rRNAs, 85 tRNAs and 5 non-coding RNAs were identified. The genome contains genes associated with resistance to β-lactams, trimethoprim, tetracycline, colistin and quinolones. Multiple efflux pump genes were also detected. Conclusion Small abalone is a potential source of foodborne drug-resistant S. algae. The genome sequence of carbapenem-resistant S. algae strain AC isolated from small abalone will provide valuable information for further study of the dissemination of resistance genes at the human–animal interface.

Increasing atmospheric CO2 can decrease seawater pH and carbonate ions, which may adversely affect the larval survival of calcareous animals. In this study, we simulated future atmospheric CO2 concentrations (800, 1500, 2000 and 3000 ppm) and examined the effects of ocean acidification on the early development of 3 mollusks (the abalones Haliotis diversicolor and H. discus hannai and the oyster Crassostrea angulata). We showed that fertilization rate, hatching rate, larval shell length, trochophore development, veliger survival and metamorphosis all decreased significantly at different pCO2 levels (except oyster hatching). H. discus hannai were more tolerant of high CO2 compared to H. diversicolor. At 2000 ppm CO2, 79.2% of H. discus hannai veliger larvae developed normally, but only 13.3% of H. diversicolor veliger larvae. Tolerance of C. angulata to ocean acidification was greater than the 2 abalone species; 50.5% of its D‑larvae developed normally at 3000 ppm CO2. This apparent resistance of C. angulata to ocean acidification may be attributed to their adaptability to estuarine environments. Mechanisms underlying the resistance to ocean acidification of both abalones requires further investigation. Our results suggest that ocean acidification may decrease the yield of these 3 economically important shellfish if increasing CO2 is a future trend.

Although a wide range of viruses have been reported in marine molluscs, most of these reports rely on ultrastructural examination and few of these viruses have been fully characterized. The lack of marine mollusc cell lines restricts virus isolation capacities and subsequent characterization works. Our current knowledge is mostly restricted to viruses affecting farmed species such as oysters Crassostrea gigas, abalone Haliotis diversicolor supertexta or the scallop Chlamys farreri. Molecular approaches which are needed to identify virus affiliation have been carried out for a small number of viruses, most of them belonging to the Herpesviridae and birnaviridae families. These last years, the use of New Generation Sequencing approach has allowed increasing the number of sequenced viral genomes and has improved our capacity to investigate the diversity of viruses infecting marine molluscs. This new information has in turn allowed designing more efficient diagnostic tools. Moreover, the development of experimental infection protocols has answered some questions regarding the pathogenesis of these viruses and their interactions with their hosts. Control and management of viral diseases in molluscs mostly involve active surveillance, implementation of effective bio security measures and development of breeding programs. However factors triggering pathogen development and the life cycle and status of the viruses outside their mollusc hosts still need further investigations.

The aquaculture techniques and operating management of production processes can influence the production performance of the overall aquaculture fishery. This study collected biological and economic data through of questionnaires from the production process of Taiwan's abalone culture industry and used the principles of economics and multivariate statistics to analyze the key for the profits of Taiwan's abalone industry. The results showed that the stocking density, production scale, and culture mode influenced the inputs and outputs of the abalone industry. Large-scale polyculture of Haliotis diversicolor supertexta and Haliotis discus hannai had the best profitability. Therefore, farmers who only culture Haliotis diversicolor supertexta are suggested to use polyculture model and moderately increase the stocking density to increase the economic benefit of cultures.

The organic matrix proteins of molluscan nacre are known to engage biocompatible and osteoinductive properties. In this study, we aimed to extract Haliotis diversicolor nacreous proteins and assayed their effect on pre-osteoblastic cell differentiation. We found that acid extracted nacreous proteins (AEP) consisted primarily of a major protein band of 25 kDa and three other minor proteins. AEP enhanced alkaline phosphatase (ALP) activity of MC3T3-E1 cells both in time- and concentration-dependent manners. Transcriptional up-regulation of osteogenic markers, including collagen type I (COL-I), osteopontin (OPN) and osteocalcin (OCN), was also apparent in days 7 and 14 upon AEP treatments. At the translational level, higher protein expression of COL-I was evident in AEP treated cells, and the protein was presumably laid down as extracellular matrix. Further de novo sequencing of an AEP major protein revealed a match with the abalone mantle protein sometsuke. Conclusively, we demonstrated that H. diversicolor AEP contains a factor, potentially the mantle protein sometsuke, which may impart in the osteoblastic cell differentiation.

Mass mortalities among cultured postlarvae of abalone, Haliotis diversicolor supertextaLischke of between 10 and 30 days post fertilization, characterized by a white appearance and falling of the diatom films on which they grow, has occurred since 2002 on the south coast of China, including Taiwan. However, its causes remain debated. In this paper, we reported our latest findings on two different types of virus-like particles that are potentially associated with mass mortalities of postlarvae of abalone. Fourteen-day-old moribund postlarvae collected during an outbreak of postlarval disease in Shenzhen, China on November 10th, 2010 were examined by transmission electron microscopy. Besides showing severe damages in the postlarval tissues, two different types of virus-like particles were detected. These two virus-like particles were detected within both nuclei and cytoplasms of diseased abalone tissues. As they were detected in the diseased postlarval tissues, it is therefore suggested that they were potentially pathogens for postlarvae in that particular outbreak. Key words: Postlarvae, abalone, Haliotis diversicolor supertexta, mass mortality, virus-like particles.

Small abalone (Haliotis diversicolor) is a commercially valuable species for both fisheries and aquaculture. The production of annual farmed small abalone in Taiwan, once the highest in the world, has dramatically decreased in the past 15 years, and currently, the industry is close to collapse. Understanding the genetic diversity of small abalone and developing stock identification methods will be useful for genetic breeding, restoring collapsed stocks, managing stocks, and preventing illegal trade. We investigated 307 cultured and wild individuals from Taiwan, Japan, and Bali Island (Indonesia) by using the mitochondrial cytochrome c oxidase subunit I (COI) gene. Network analysis of mtDNA COI gene sequences revealed that the individuals collected from Taiwan, Japan, and Indonesia could be identified, and showed significant genetic divergence. In addition, the Indonesian population (Haliotis diversicolor squamata) was significantly different from the other populations and might need to be considered a separate species. We discovered a single nucleotide polymorphism marker in the mtDNA COI gene that can be used to distinguish the Taiwan population from the Japan population. We also developed a polymerase chain reaction-restriction fragment length polymorphism method for rapid detection. Furthermore, we could identify the cultured stocks, wild population, and hybrid stocks by using 6 microsatellites and amplified fragment length polymorphism. This study contributes useful tools for stock identification and the production of high-disease resistant small abalone strains (Japan × Taiwan or Taiwan × Japan). Efforts should be made to avoid unintentional random genetic mixing of the Taiwan population with the Japan population and subsequent breakdown of population differentiation, which impair local adaptation of the Taiwan wild population. Molecular markers revealed a split between the Taiwan and Japan populations, and the existence of a possible barrier to the free dispersal of small abalone is discussed.

An ecosystem is a community comprising living and nonliving components of the environment. Microbes are ubiquitous elements in each of these components. The dynamics of microbiota formation in an ecosystem is important to elucidate, because how the different components of a system exchange microbes, and how the microbes control ecological processes remain unresolved. In this study, an abalone, Haliotis diversicolor, seed-nursing pond was used as a model system. We first examined changes in bacterial communities during the seedling cultivation of this herbivorous juvenile aquatic invertebrate animal. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to analyze bacterial community dynamics and spatio-temporal interactions of different system components: consumers (abalone), producers (algae or a substrate), and the environment (water). DGGE fingerprints revealed that the developmental stages of abalone influences bacterial communities of both the abalone and substrate. Although the communities in water fluctuated daily, they could be divided into two clusters that coincided with abalone stages, reflecting the transition from larva to juvenile at around day 21. Pyrosequencing showed that the microbiota in the abalone and substrate had more operational taxonomic units in common than that of either with water. The Bray-Curtis similarity index was used to quantify the formation dynamics of microbiota among the various components of the system. The bacterial communities in producers and consumers showed similar changes. These communities were unstable at the beginning and then slowly stabilized over time. The environmental bacterial community was more stable than the bacterial communities in consumers and producers, and may have been the basis for stability in the system. Our research provides insights into the dynamics of microbiota formation in various biotic elements of a system that will contribute to predictive systems modeling.

Vibrio parahemolyticus is a devastating bacterial pathogen that often causes outbreak of vibriosis in abalone Haliotis diversicolor. Elucidation of metabolic mechanisms of abalones in responding to V. parahemolyticus infection is essential for controlling the epidemic. In this work, 1H NMR-based metabolomic techniques along with correlation and network analyses are used to investigate characteristic metabolites, as well as corresponding disturbed pathways in hepatopancreas and gill of H. diversicolor after V. parahemolyticus infection for 48 h. Results indicate that obvious gender- and tissue-specific metabolic responses are induced. Metabolic responses in female abalones are more clearly observed than those in males, which are primarily manifested in the accumulation of branched-chain amino acids and the depletion of organic osmolytes (homarine, betaine and taurine) in the infected gills of female abalones, as well as in the depletion of glutamate, branched-chain and aromatic amino acids in the infected hepatopancreases of female abalones. Moreover, based on major metabolic functions of the characteristic metabolites, we have found that V. parahemolyticus infection not only cause the disturbance in energy metabolism, nucleotide metabolism and osmotic balance, but also induce oxidative stress, immune stress and neurotoxic effect in different tissues with various mechanisms. Our study provides details of metabolic responses of abalones to V. parahemolyticus infection and will shed light on biochemical defence mechanisms of male and female hosts against pathogen infection.

The embryos of a marine abalone, Haliotis diversicolor supertexta, were exposed to a typical environmental estrogen, 17α-ethynylestradiol (EE2), for 96 h, to examine the acute toxicity of EE2 to the embryogenesis of the abalone. A marine diatom, Navicula incerta, was used in the test media as settlement substrate and food for the abalone larvae. During the embryo culture, more than 30 % of EE2 could be removed from the test media by the diatom, mainly via biodegradation, leading to a decrease of water-borne exposure dose. Further, the exposure concentrations of EE2 around the living microenvironment of the abalone larvae could be magnified 350–468 times after the larvae settled on the diatom, as indicated by the bioconcentration factors of EE2 in the diatom. Increased bioaccumulation of EE2 in the diatom caused greater inhibition on the metamorphosis of the abalone larvae by enhancing the uptake of EE2 in the larvae via dietary exposure, while declined water-borne exposure dose did not affect the embryonic toxicity of EE2 and its uptake in the abalone larvae. The 96-h median effective concentration of EE2 to the metamorphosis of the abalone larvae was 10.01 μg L−1, when the exposure doses in both the test media and the diatom were controlled stable. The 96-h hazard concentration for 5 % of the species was 1.20 μg L−1, which was still higher than but close to the reported upper contamination level of EE2 and could be employed as the safety threshold for the metamorphosis of the abalone embryos.

Ferritin is an iron storage protein that plays a key role in the processes of physiology and pathology. In the present study, the authors reported the ferritin gene from abalone Haliotis diversicolor supertexta, which we named hds-ferritin. The full-length of hds-ferritin cDNA consisted of 879 bp with an ORF encoding a 171 amino acids. Amino acid sequence analysis revealed that hds-ferritin shared highly homology with other species. Real time PCR and western blot analysis showed that hds-ferritin was distributed ubiquitously in abalone tissues and had the highest expression level in digestive glands, but its transcripts are not modified remarkably by the stimulation with LPS. The recombinant protein was successfully expressed in Escherichia coli BL21 (DE3), and the titre of anti-ferritin antibody was about 1:14 000. The effects of ROS and RNS on ferritin were analyzed in the present study. The results showed that H2O2 played an important role in decreasing hds-ferritin, however NO cation appeared to have a protecting effect on H2O2-medied reduction of hds-ferritin.

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 trnS₂ 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.

The small abalone Haliotis diversicolor is an economically important shellfish species in southern China. Wild (Japanese, Guangdong, and Hainan) and cultured (Taiwanese and Vietnamese) subpopulations of small abalone were examined using seven microsatellite loci to assess the levels of genetic diversity and the degree of genetic differentiation among them. These seven microsatellite markers showed a high level of polymorphism. A total of 209 alleles were detected, with an average of 29.86 alleles per locus among the five subpopulations. The average observed heterozygosities per locus ranged from 0.733 in the Japanese subpopulation to 0.804 in the Hainan subpopulation, while the average expected heterozygosities per locus ranged from 0.909 in the Vietnamese subpopulation to 0.940 in the Guangdong subpopulation. Values for the differentiation index D est varied from 0.1071 to 0.4020 among different subpopulations. The values for genetic distance (D A) ranged from 0.244 to 0.409, with an average of ...

The effects and associated toxicological mechanisms of five phthalate esters (PAEs) on abalone embryonic development were investigated by exposing the embryos to a range of PAEs concentrations (0.05, 0.2, 2 and 10 μg/mL). The results showed that PAEs could significantly reduce embryo hatchability, increase developmental malformations, and suppress the metamorphosis of abalone larvae. The possible toxicological mechanisms of PAEs to abalone embryos included, affecting the Na+-K+-pump and Ca2+-Mg2+-pump activities, altering the peroxidase (POD) level and the malondialdehyde (MDA) production, damaging the extraembryonic membranes structure, as well as disrupting endocrine-related genes (gpx, cyp3a, and 17β-hsd 12) expression properties. Taken together, this work showed that PAEs adversely affected the embryonic ontogeny of abalone. The abilities of PAEs affecting the osmoregulation, inducing oxidative stress, damaging embryo envelope structure, and causing physiological homeostasis disorder, are likely to be a part of the common mechanisms responsible for their embryonic toxicity.

The increasing use of nanotechnology highlights the need to understand and clarify the environmental impacts of nanomaterials. In this study, the acute toxicity and oxidative stress of titanium dioxide nanoparticles (nTiO2) in mature marine abalone (Haliotis diversicolor supertexta) were assessed. No acute effect was found in any of the treatment groups with nTiO2 concentration gradients ranging from 0.1 to 10 mg/L. However, the activity of an antioxidant enzyme superoxide dismutase (SOD) significantly increased in the group that was exposed to 1.0 mg/L nTiO2. The content of a non-enzymatic antioxidant, glutathione (GSH), significantly decreased in the groups with an nTiO2 concentration ⩾1.0 mg/L. The level of lipid peroxidation (LPO) was found to increase as the nTiO2 dose increased. Furthermore, NO was produced in excess in abalone. These results demonstrated that, although nTiO2 is not acutely toxic to abalone, it does exert oxidative stress on abalone.

The abalone, Haliotis diversicolor, is one of the most important mariculture species in southern China. We developed 60 new polymorphic microsatellite markers for H. diversicolor and characterized them in 30 individuals from a cultured population in Sanya, China. All 60 markers were found to be polymorphic. The number of alleles ranged from two to nine per locus, with an average of 4.12/locus. The expected and observed heterozygosities ranged from 0.10 to 0.88 and from 0.07 to 0.87, respectively. Forty-four loci were in Hardy-Weinberg equilibrium. These 44 microsatellite markers should be useful for genome mapping and population genetic studies.

Rbx1 (RING box1) is an evolutionarily conserved RING-H2 finger protein and belongs to the RING-finger family of Ubiquitin ligase E3, which determines the substrate specificity of ubiquitination and regulates a variety of biological processes. We report here the identification and functional characterization of an Rbx1 homologue in abalone, which we named ab-Rbx1. Ab-Rbx1 contains conserved cysteine/histidine residues which are the characteristics of Rbx proteins. Phylogenetic tree analysis further demonstrated that ab-Rbx1 belongs to the Rbx1 family other than Rbx2 family. Real-time PCR analysis revealed that ab-Rbx1 was ubiquitously expressed in all examined tissues of abalone and the expression level of ab-Rbx1 was significantly induced by mitogenic situation. Immunohistochemical and immunofluorescent staining showed that the ab-Rbx1 was expressed predominantly in epithelial cells and localized both in the cytoplasmic and nuclear compartment. Ubiquitination assay demonstrated that ab-Rbx1 had ubiquitin ligase activity and could auto-ubiquitinated itself. These results suggest that ab-Rbx1 is an Rbx1 homologue and may be indirectly involved in the immune response of abalone through ubiquitination.

Genome size (C-value) and the percentage of adenine and thymine nucleotides in the genome (AT content) are fundamental characteristics of every species, and very important parameters in molecular cytogenetic and phylogenic studies, and for the progress of whole-genome sequencing. In this study, the C-value and AT content of nine abalone species inhabiting the Northwest Pacific (around Japan), Southeast Asia, South Africa, and Oceania were determined using flow cytometry. C-value ranged from 1.32 pg for Haliotis varia (Southeast Asia) to 2.01 pg for Haliotis laevigata (Oceania), and AT content ranged from 58.0% for Haliotis planata (Southeast Asia) to 66.3% for Haliotis diversicolor aquatilis (Southeast Asia). Reported chromosome numbers ranged from 2n = 32 to 36, and were higher in Oceanian, South African, and Northwest Pacific species (2n = 36) compared with the Southeast Asian group (2n = 32). This increase of chromosome number in the Oceanian species seems to be related to an increase in the DNA amount, unlike that of Haliotis midae (South African species). An increase of the C-value associated with an increase in DNA amount was also shown in the North Pacific group. These results suggest that increases in the DNA amount occurred independently in the ancestor of each of the different lineages.

To determine the genomic composition of the interspecific hybrid between Haliotis diversicolor supertexta ♀ and H. discus discus ♂ at an early developmental stage, veliger larvae produced from hybrid (SJ-5 and SJ-50) and pure species crosses (SS and JJ) were sampled and analyzed using standard karyological methods and genomic in situ hybridization. In hybrid metaphase spreads, chromosomes from both parents were detected, except one metaphase, which showed the H. diversicolor supertexta haploid karyotype. The genomic composition of the hybrid was also confirmed through preliminary genomic in situ hybridization results. Many more aneuploids and chromosome fragments were found in the hybrids than those in the control pure species crosses, indicating genome instability and chromosome loss in the hybrids. In the hybrid hypodiploid metaphase spreads, two intact sets of H. diversicolor supertexta chromosomes and several H. discus discus chromosomes were detected by pairing. Spontaneous diploidization of...

Twelve half-sib groups and 36 full-sib groups of small abalone Haliotis diversicolor were obtained by the unbalanced nest design using artificial fertilization of 3 females by each male. Heritabilities of growth-related traits were estimated from postlarva to market size at days 10, 40, 120, 220, 320, and 420. The estimated heritabilities based on sire components for shell length and shell width were 0.15–0.37 and 0.18–0.42, respectively. Heritability estimate for shell length and shell width based on dam component were larger than those based on sire component. The results in the current study indicate that genetic improvement through selective breeding conducted on small abalone might be available and would be an efficient method to obtain a positive response to selection.

A total of 241 samples of small abalone (Haliotis diversicolor) from 8 full-sib families of market size were used in this experiment. Shell length (X1), shell width (X2), shell height (X3), apex height (X4), body weight (Y1), muscle weight (Y2), and shell weight (Y3) were measured, and the correlation coefficient matrix was calculated. The shell shape traits were used as independent variables, then body weight and muscle weight were used as dependent variable for path analysis. Path coefficients, determination coefficients, and correlation index were calculated. The results showed that correlation coefficients between each shell shape trait and body weight, muscle weight, and shell weight were all significant (P < 0.01). For the 4 shell morphological traits, body weight (Y1) had the highest correlation coefficient with shell length (X1), and muscle weight (Y2) had the highest correlation coefficient with shell width (X2). The results of high correlation index would be useful for selecting importa...

Benthic diatoms constitute the primary diet of abalone during their early stages of development. To evaluate the dietary preferences of early post-larval abalone, Haliotis diversicolor supertexta, we analyzed the gut contents of post-larvae that settled on diatom films. We compared the abundance and species diversity of diatom assemblages in the gut to those of the epiphytic diatom assemblages on the attachment films, and identified 40 benthic diatom species in the gut contents of post-larvae 12 to 24 d after settlement. The most abundant taxa in the gut contents were Navicula spp., Amphora copulate, and Amphora coffeaeformis. Navicula spp. accounted for 64.0% of the cell density. In the attachment films, we identified 110 diatom species belonging to 38 genera. Pennate diatoms were the dominant members including the species Amphiprora alata, Cocconeis placentula var. euglypta, Cylindrotheca closterium, Navicula sp. 2, and A. coffeaeformis. Nano-diatoms (

Interspecific hybrid families of female Haliotis diversicolor × male H. discus discus were produced and analyzed using amplified fragment length polymorphism (AFLP) technology to reveal the genetic makeup of F1 progenies. The survival rates of the hybrid F1 were very low, ranging from 0–0.13%. Twenty hybrid F1 from 3 families along with 3 different female parents and their common male parent were analyzed with 3 AFLP primer combinations. In total, 266 markers were detected. Genetic relationships among the progenies and the parents were evaluated by generating a similarity and genetic distance matrix. The genetic divergence between Haliotis diversicolor and Haliotis discus was at a high level, with genetic distance ranging from 1.471–1.492. The AFLP band patterns of hybrid F1 progeny were similar to those of the female parents, but were quite different from that of the male parent. The mean genetic distance between hybrid F1 and their female parents were 0.024–0.039, slightly less than that among ...

National High Technology Research and Development Program of China [2006AA10A407]; Modern Agro-industry Technology Research System of China [nycytx-47]; Innovative Platform for Science and Technology, Fujian Province of China [2008N2004]

In this study, we examined the chronic waterborne and dietary exposure of silver (Ag) and cadmium (Cd) to the abalone Haliotis diversicolor using various endpoints such as growth and feeding rates, metal body burden, subcellular distribution, and metallothionein (MT) concentration over a period of 7 weeks of exposure. The growth and feeding rates of abalones were inhibited during the early stage of exposure to different extents, but then recovered to nearly the control levels. A large portion of Ag was redistributed to organelles and metal-rich granules from the cellular debris fraction, whereas cellular debris and metallothionein-like protein were the dominant pools for the storage of Cd, which remained comparable during the exposure period. The MT concentrations were significantly elevated (in a dose-dependent manner) within the first 2 weeks of exposure, after which the MT concentrations started to decrease. All these results implied that abalones respond rapidly to metal exposure, but apparently developed subsequent acclimation.

The abalone, Haliotis diversicolor supertexta, is an important aquaculture species in South China Sea. Inducing more rapid or greater degree of settlement of H. diversicolor supertexta larvae using chemical cues can significantly improve the commercial hatchery production of this valuable species. In this study, the effects of 11 chemicals on inducing larval settlement of the H. diversicolor supertexta were investigated in the laboratory. Among the chemicals tested, K+ (10 and 20 mM), Ca2+ (1 mM), NH4+ (0.1 mM), gamma-aminobutyric acid (at 10-7 to 10-2 M), 3-isobutyl-1-methylxanthine (10-4 M), 3-(3,4-dihydroxyphenyl)-l-alanine (at 10-6 to 10-5 M), and dopamine (10-6 M) induced high percentages of the larvae to settle, while Mg2+, choline, acetylcholine, and serotonin showed no obvious induction on larval settlement. Larval settlement was significantly induced after exposure to 20 mM KCl for only 2 h. KCl was not only an effective inducer, but also an inexpensive and easily available chemical, therefore it maybe a potentially economical inducer in the commercial production.

Haliotis diversicolor Reeve is one of the most important mollusks cultured in South China. Preliminary genetic linkage maps were constructed with amplified fragment length polymorphism (AFLP) markers. A total of 2 596 AFLP markers were obtained from 28 primer combinations in two parents and 78 offsprings. Among them, 412 markers (15.9%) were polymorphic and segregated in the mapping family. Chi-square tests showed that 151 (84.4%) markers segregated according to the expected 1:1 Mendelian ratio (P