Carryover effects of embryonic hypoxia exposure on adult fitness of the Pacific abalone.

The widespread and severe drop in dissolved oxygen concentration in the open ocean and coastal waters has attracted much attention, but assessments of the impacts of environmental hypoxia on aquatic organisms have focused primarily on responses to current exposure. Past stress exposure might also affect the performance of aquatic organisms through carryover effects, and whether these effects scale from positive to negative based on exposure degree is unknown. We investigated the carryover effects of varying embryonic hypoxia levels (mediate hypoxia: 3.0-3.1 mg O2/L; severe hypoxia: 2.0-2.1 mg O2/L) on the fitness traits of adult Pacific abalone (Haliotis discus hannai), including growth, hypoxia tolerance, oxygen consumption, ammonia excretion rate, and biochemical responses to acute hypoxia. Moderate embryonic hypoxia exposure significantly improved the hypoxia tolerance of adult Pacific abalone without sacrificing growth and survival. Adult abalone exposed to embryonic hypoxia exhibited physiological plasticity, including decreased oxygen consumption rates under environmental stress, increased basal methylation levels, and a more active response to acute hypoxia, which might support their higher hypoxia tolerance. Thus, moderate oxygen declines in early life have persistent effects on the fitness of abalone even two years later, further affecting population dynamics. The results suggested that incorporating the carryover effects of embryonic hypoxia exposure into genetic breeding programs would be an important step toward rapidly improving the hypoxia tolerance of aquatic animals. The study also inspires the protection of endangered wild animals and other vulnerable species under global climate change.

Comparative cytogenetic analysis of diploid and triploid pacific abalone, Haliotis discus hannai.

Cytogenetic analysis of triploid Haliotis discus hannai larvae (produced by chemical treatment) and its diploids were performed. The results showed that triploid H. discus hannai had a chromosome number of 3n = 54, consisting of 30 metacentric (m) and 24 submetacentric (sm) chromosomes, while the diploids had a chromosome number of 2n = 36, consisting of 20 metacentric (m) and 16 submetacentric (sm) chromosomes. Notably, both triploids and diploids displayed variation in the number of NORs and/or their diameter. The average number of NORs was significantly higher in triploids than in diploids (P < 0.05), while there was no significant difference in the average diameter of NORs between the two groups (P > 0.05). Additionally, 5S rDNA localization to 3 submetacentric chromosomes was observed in triploids, compared to 2 submetacentric chromosomes in diploids. The number of 18S rDNA sites displayed positional conservancy and quantitative variability in both diploids and triploids. Specifically, 18S rDNA was found at the end of the chromosome in both groups, with triploids exhibiting a significantly higher number of loci than diploids (P < 0.01). This study provides valuable insights into the cytogenetic characteristics of triploid H. discus hannai, which could facilitate further research on the stability of the chromosome set in this species.

Transcriptome analysis reveals fluid shear stress (FSS) and atherosclerosis pathway as a candidate molecular mechanism of short-term low salinity stress tolerance in abalone.

BACKGROUND: Transcriptome sequencing is an effective tool to reveal the essential genes and pathways underlying countless biotic and abiotic stress adaptation mechanisms. Although severely challenged by diverse environmental conditions, the Pacific abalone Haliotis discus hannai remains a high-value aquaculture mollusk and a Chinese predominantly cultured abalone species. Salinity is one of such environmental factors whose fluctuation could significantly affect the abalone's cellular and molecular immune responses and result in high mortality and reduced growth rate during prolonged exposure. Meanwhile, hybrids have shown superiority in tolerating diverse environmental stresses over their purebred counterparts and have gained admiration in the Chinese abalone aquaculture industry. The objective of this study was to investigate the molecular and cellular mechanisms of low salinity adaptation in abalone. Therefore, this study used transcriptome analysis of the gill tissues and flow cytometric analysis of hemolymph of H. discus hannai (DD) and interspecific hybrid H. discus hannai ♀ x H. fulgens ♂ (DF) during low salinity exposure. Also, the survival and growth rate of the species under various salinities were assessed. RESULTS: The transcriptome data revealed that the differentially expressed genes (DEGs) were significantly enriched on the fluid shear stress and atherosclerosis (FSS) pathway. Meanwhile, the expression profiles of some essential genes involved in this pathway suggest that abalone significantly up-regulated calmodulin-4 (CaM-4) and heat-shock protein90 (HSP90), and significantly down-regulated tumor necrosis factor (TNF), bone morphogenetic protein-4 (BMP-4), and nuclear factor kappa B (NF-kB). Also, the hybrid DF showed significantly higher and sustained expression of CaM and HSP90, significantly higher phagocytosis, significantly lower hemocyte mortality, and significantly higher survival at low salinity, suggesting a more active molecular and hemocyte-mediated immune response and a more efficient capacity to tolerate low salinity than DD. CONCLUSIONS: Our study argues that the abalone CaM gene might be necessary to maintain ion equilibrium while HSP90 can offset the adverse changes caused by low salinity, thereby preventing damage to gill epithelial cells (ECs). The data reveal a potential molecular mechanism by which abalone responds to low salinity and confirms that hybridization could be a method for breeding more stress-resilient aquatic species.

Divergent Carry-Over Effects of Hypoxia during the Early Development of Abalone.

After being exposed to environmental stimuli during early developmental stages, some organisms may gain or weaken physiological regulating abilities, which would have long-lasting effects on their performance. Environmental hypoxia events can have significant effects on marine organisms, but for breeding programs and other practical applications, it is important to further explore the long-term physiological effects of early hypoxia exposure in economically significant species. In this study, the Pacific abalone Haliotis discus hannai was exposed to moderate hypoxia (∼4 mg/L) from zygote to trochophora, and the assessments of hypoxia tolerance were conducted on the grow-out stage. The results revealed that juvenile abalones exposed to hypoxia at the early development stages were more hypoxia-tolerant but with slower weight growth, a phenomenon called the trade-off between growth and survival. These phenotypic effects driven by the hypoxia exposure were explained by strong selection of genes involved in signal transduction, autophagy, apoptosis, and hormone regulation. Moreover, long non-coding RNA regulation plays an important role modulating carry-over effects by controlling DNA replication and repair, signal transduction, myocardial activity, and hormone regulation. This study revealed that the ability to create favorable phenotypic differentiation through genetic selection and/or epigenetic regulation is important for the survival and development of aquatic animals in the face of rapidly changing environmental conditions.

AMPK regulates behavior and physiological plasticity of Haliotis discus hannai under different spectral compositions.

In natural environments, the spectral composition of incident light is often subject to drastic changes due to the abundance of suspended particles, floating animals, and plants in coastal waters. In this study, after four months of culturing under blue light (NB), orange light (NY), dark environment (ND), and natural light (NN), the shell length and weight-specific growth rate in Pacific abalone, Haliotis discus hannai, were ranked in the following order: NY > NN > ND > NB. To understand the growth differences in abalone under these different light environments, we first performed 24-h video monitoring and found that the cumulative movement distance and duration were lowest in group NB, whereas the cumulative movement distance and duration were significantly higher in group ND than in any other group (P < 0.05). In group NB, the time spent hidden underneath the attachment substrate accounted for 81% of the resting time, but this ratio was lowest in group ND, at only 37% (P < 0.05). Next, LC-MS metabolomics identified 201 and 105 metabolites in NB vs. NN, ND vs. NN, and NY vs. NN under the positive and negative ion modes, respectively. According to the fold changes and annotations for differential metabolites in the KEGG enrichment pathways, adenosine, NAD+, cGMP, and arachidonic acid were used as differential metabolism markers, and the AMPK signaling pathway was enriched in every comparison group, and thus investigated further. The gene sequences of three subtypes of AMPK were obtained by cloning and we found that the expression levels of AMPKα and AMPKγ, and the AMP content were significantly higher in group NB than in any other group (P < 0.05). In addition, the ATP contents and adenylate energy charge values were ranked in the following order: NY > NN > ND > NB. According to in situ hybridization analysis, the three subtype genes were widely expressed in the hepatopancreas. Finally, the contents of many lipid metabolites differed significantly among groups and the expression levels of the triglyceride hydrolysis-related gene hormone sensitive lipase and fatty acid oxidation-related gene carnitine palmitoyltransferase 1 were higher in groups ND and NB than in groups NN and NY according to fluorescence quantification PCR (P < 0.05). The expression levels of fatty acid synthase and acetyl-CoA carboxylase were significantly lower in groups ND and NB than in groups NN and NY (P < 0.05). These findings indicated that differences in the spectral composition of incident light could reshape the behavior and physiological metabolism in abalone by influencing the "energy switch" AMPK, thereby providing some insights into the mechanisms that allow nocturnal marine organisms to adapt to different lighting environments.

Transcriptome analysis reveals the molecular mechanisms of heterosis on thermal resistance in hybrid abalone.

BACKGROUND: Heterosis has been exploited for decades in different animals and crops due to it resulting in dramatic increases in yield and adaptability. Hybridization is a classical breeding method that can effectively improve the genetic characteristics of organisms through heterosis. Abalone has become an increasingly economically important aquaculture resource with high commercial value. However, due to changing climate, abalone is now facing serious threats of high temperature in summer. Interspecific hybrid abalone (Haliotis gigantea ♀ × H. discus hannai ♂, SD) has been cultured at large scale in southern China and has been shown high survival rates under heat stress in summer. Therefore, SD has become a good model material for heterosis research, but the molecular basis of heterosis remains elusive. RESULTS: Heterosis in thermal tolerance of SD was verified through Arrhenius break temperatures (ABT) of cardiac performance in this study. Then RNA-Sequencing was conducted to obtain gene expression patterns and alternative splicing events at control temperature (20 °C) and heat stress temperature (30 °C). A total of 356 (317 genes), 476 (435genes), and 876 (726 genes) significantly diverged alternative splicing events were identified in H. discus hannai (DD), H. gigantea (SS), and SD in response to heat stress, respectively. In the heat stress groups, 93.37% (20,512 of 21,969) of the expressed genes showed non-additive expression patterns, and over-dominance expression patterns of genes account for the highest proportion (40.15%). KEGG pathway enrichment analysis showed that the overlapping genes among common DEGs and NAGs were significantly enriched in protein processing in the endoplasmic reticulum, mitophagy, and NF-κB signaling pathway. In addition, we found that among these overlap genes, 39 genes had undergone alternative splicing events in SD. These pathways and genes may play an important role in the thermal resistance of hybrid abalone. CONCLUSION: More alternative splicing events and non-additive expressed genes were detected in hybrid under heat stress and this may contribute to its thermal heterosis. These results might provide clues as to how hybrid abalone has a better physiological regulation ability than its parents under heat stress, to increase our understanding of heterosis in abalone.

Proteome of larval metamorphosis induced by epinephrine in the Fujian oyster Crassostrea angulata.

BACKGROUND: The Fujian oyster Crassostrea angulata is an economically important species that has typical settlement and metamorphosis stages. The development of the oyster involves complex morphological and physiological changes, the molecular mechanisms of which are as yet unclear. RESULTS: In this study, changes in proteins were investigated during larval settlement and metamorphosis of Crassostrea angulata using epinephrine induction. Protein abundance and identity were characterized using label-free quantitative proteomics, tandem mass spectrometry (MS/ MS), and Mascot methods. The results showed that more than 50% (764 out of 1471) of the quantified proteins were characterized as differentially expressed. Notably, more than two-thirds of the differentially expressed proteins were down-regulated in epinephrine-induced larvae. The results showed that "metabolic process" was closely related to the development of settlement and metamorphosis; 5 × 10- 4 M epinephrine induced direct metamorphosis of larvae and was non-toxic. Calmodulin and MAPK pathways were involved in the regulation of settlement of the oyster. Expression levels of immune-related proteins increased during metamorphosis. Hepatic lectin-like proteins, cadherins, calmodulin, calreticulin, and cytoskeletal proteins were involved in metamorphosis. The nervous system may be remodeled in larval metamorphosis induced by epinephrine. Expression levels of proteins that were enriched in the epinephrine signaling pathway may reflect the developmental stage of the larvae, that may reflect whether or not larvae were directly involved in metamorphosis when the larvae were treated with epinephrine. CONCLUSION: The study provides insight into proteins that function in energy metabolism, immune responses, settlement and metamorphosis, and shell formation in C. angulata. The results contribute valuable information for further research on larval settlement and metamorphosis.

Comparative immune response during the juvenile and adult stages of two abalones under Vibrio harveyi challenge.

Mass mortality of juvenile hybrid (Haliotis discus hannai â™€× H. fulgens ♂, DF) and adult H. discus hannai (DD) occurs in south China during the summer. This study showed that the juvenile DF and adult DD exhibited significantly lower survival rates than juvenile DD and adult DF under 72 h pathogenic bacteria (Vibrio harveyi) challenge at different temperatures (20 °C and 28 °C). Phenoloxidase (PO) and superoxide dismutase (SOD) activities were significantly higher in juvenile DD compared to juvenile DF, whereas that in adult abalone was the opposite. Juvenile DD and adult DF also exhibited advantages in terms of immune-related gene expression (TRAF, TLR, MIF, Lys, Spi, Cat, TNF, and SOD) compared to juvenile DF and adult DD. The data reveals immunocompetence differences in DD and DF at the juvenile and adult stages.

Bacterial Nucleobases Synergistically Induce Larval Settlement and Metamorphosis in the Invasive Mussel Mytilopsis sallei.

Marine bacterial biofilms have long been recognized as potential inducers of larval settlement and metamorphosis in marine invertebrates, but few chemical cues from bacteria have been identified. Here, we show that larval settlement and metamorphosis of an invasive fouling mussel, Mytilopsis sallei, could be induced by biofilms of bacteria isolated from its adult shells and other substrates from the natural environment. One of the strains isolated, Vibrio owensii MS-9, showed strong inducing activity which was attributed to the release of a mixture of nucleobases including uracil, thymine, xanthine, hypoxanthine, and guanine into seawater. In particular, the synergistic effect of hypoxanthine and guanine was sufficient for the inducing activity of V. owensii MS-9. The presence of two or three other nucleobases could enhance, to some extent, the activity of the mixture of hypoxanthine and guanine. Furthermore, we determined that bacteria producing higher concentrations of nucleobases were more likely to induce larval settlement and metamorphosis of M. sallei than were bacteria producing lower concentrations of nucleobases. The present study demonstrates that bacterial nucleobases play an important role in larval settlement and metamorphosis of marine invertebrates. This provides new insights into our understanding of the role of environmental bacteria in the colonization and aggregation of invasive fouling organisms and of the metabolites used as chemical mediators in cross-kingdom communication within aquatic systems.IMPORTANCE Invasive species are an increasingly serious problem globally. In aquatic ecosystems, invasive dreissenid mussels are well-known ecological and economic pests because they appear to effortlessly invade new environments and foul submerged structures with high-density aggregations. To efficiently control exotic mussel recruitment and colonization, the need to investigate the mechanisms of substrate selection for larval settlement and metamorphosis is apparent. Our work is one of very few to experimentally demonstrate that compounds produced by environmental bacteria play an important role in larval settlement and metamorphosis in marine invertebrates. Additionally, this study demonstrates that bacterial nucleobases can be used as chemical mediators in cross-kingdom communication within aquatic systems, which will enhance our understanding of how microbes induce larval settlement and metamorphosis of dreissenid mussels, and it furthermore may allow the development of new methods for application in antifouling.

The role of copper and zinc accumulation in defense against bacterial pathogen in the fujian oyster (Crassostrea angulata).

Cu and Zn are hyper-accumulated in oysters, and the accumulation of these metals increases host resistance to pathogens. However, the role of Cu/Zn in oyster immune defense remains unclear. In this study, Crassostrea angulata with different levels of Cu and Zn were obtained through metal exposure or selective breeding. Both in vivo and in vitro experiments showed that oysters accumulating more Cu/Zn exhibited stronger antibacterial abilities. Vibrio harveyi infection significantly promoted the metal redistribution in oysters: Cu and Zn concentrations decreased in the mantle, but increased in the plasma and hemocytes. This redistribution was accompanied by changes in the expression levels of Cu and Zn transporter genes (CTR1, ATP7A, ZIP1, and ZNT2), suggesting that the Cu/Zn burst observed in the hemocytes was likely due to the transfer of heavy metals from plasma (mediated by the metal importer proteins) or released from intracellular stores. The degree to which Cu/Zn concentration increased in the plasma and hemocytes was more dramatic in oysters with high levels of Cu/Zn accumulation. In vitro, Cu and Zn both inhibited the growth of V. harveyi, while Cu plus H2O2 was lethal to the bacteria. The strength of the growth-inhibition and lethal effects depended on the metal dose. In addition to these effects, increases in Cu concentration increased the activity levels of PO in the oyster plasma and hemocytes in vivo and in vitro. However, SOD activity was not affected by Cu or Zn accumulation. Thus, our results suggested that the Cu/Zn burst in the hemolymph was an important factor in the oyster immune reaction, creating a toxic internal environment for the pathogen, as well as catalyzing inorganic or enzymatic reactions to strengthen bacteriostasis. By determining the extent of Cu/Zn burst in the immune response, Cu/Zn accumulated levels could affect the resistance of oysters to pathogens.

Differential proteomic profiles and characterizations between hyalinocytes and granulocytes in ivory shell Babylonia areolata.

The haemocytes of the ivory shell, Babylonia areolata are classified by morphologic observation into the following types: hyalinocytes (H) and granulocytes (G). Haemocytes comprise diverse cell types with morphological and functional heterogene and play indispensable roles in immunological homeostasis of invertebrates. In the present study, two types of haemocytes were morphologically identified and separated as H and G by Percoll density gradient centrifugation. The differentially expressed proteins were investigated between H and G using mass spectrometry. The results showed that total quantitative proteins between H and G samples were 1644, the number of up-regulated proteins in G was 215, and the number of down-regulated proteins in G was 378. Among them, cathepsin, p38 MAPK, toll-interacting protein-like and beta-adrenergic receptor kinase 2-like were up-regulated in G; alpha-2-macroglobulin-like protein, C-type lectin, galectin-2-1, galectin-3, ß-1,3-glucan-binding protein, ferritin, mega-hemocyanin, mucin-17-like, mucin-5AC-like and catalytic subunit of protein kinase A were down-regulated in G. The results showed that the most significantly enriched KEGG pathways were the pathways related to ribosome, phagosome, endocytosis, carbon metabolism, protein processing in endoplasmic reticulum and oxidative phosphorylation. For phagosome and endocytosis pathway, the number of down-regulation proteins in G was more than that of up-regulation proteins. For lysosome pathway, the number of up-regulation proteins in G was more than that of down-regulation proteins. These results suggested that two sub-population haemocytes perform the different immune functions in B. areolata.

Identification and characteristics of muscle growth-related microRNA in the Pacific abalone, Haliotis discus hannai.

BACKGROUND: The Pacific abalone, Haliotis discus hannai, is the most important cultivated abalone in China. Improving abalone muscle growth and increasing the rate of growth are important genetic improvement programs in this industry. MicroRNAs are important small noncoding RNA molecules that regulate post-transcription gene expression. However, no miRNAs have been reported to regulate muscle growth in H. discus hannai. RESULTS: we profiled six small RNA libraries for three large abalone individuals (L_HD group) and three small individuals (S_HD group) using RNA sequencing technology. A total of 205 miRNAs, including 200 novel and 5 known miRNAs, were identified. In the L_HD group, 3 miRNAs were up-regulated and 7 were down-regulated compared to the S_HD specimens. Bioinformatics analysis of miRNA target genes revealed that miRNAs participated in the regulation of cellular metabolic processes, the regulation of biological processes, the Wnt signaling pathway, ECM-receptor interaction, and the MAPK signaling pathway, which are associated with regulating growth. Bone morphogenetic protein 7 (BMP7) was verified as a target gene of hdh-miR-1984 by a luciferase reporter assay and we examined the expression pattern in different developmental stages. CONCLUSION: This is the first study to demonstrate that miRNAs are related to the muscle growth of H. discus hannai. This information could be used to study the mechanisms of abalone muscle growth. These DE-miRNAs may be useful as molecular markers for functional genomics and breeding research in abalone and closely related species.

Hybridization improved bacteria resistance in abalone: Evidence from physiological and molecular responses.

Hybridization is an effective way of improving germplasm in abalone, as it often generates benign traits in the hybrids. The hybrids of Haliotis discus hannai and H. gigantea have shown heterosis in terms of disease resistance than one or both parental species. In the present study, to elucidate the physiological and molecular mechanism of this heterosis, we analyzed the dynamic changes of several immune indexes including survival rate, total circulating haemocyte count (THC), phagocytic activity, reactive oxygen species level (ROS) and phenoloxidase activity (PO) in two parental species, H. discus hannai (DD) and H. gigantea (GG), and their reciprocal hybrids H. discus hannai ♀ × H. gigantea ♂ (DG), H. gigantea ♀ × H. discus hannai ♂ (GD) challenged with a mixture of Vibrio harveyi, V. alginolyticus and V. parahaemolyticus (which have been demonstrated to be pathogenic to abalone). Besides, we cloned and analyzed three important immune genes: heat shock protein 70 (hsp70), ferritin and cold shock domain protein (csdp) in H. discus hannai and H. gigantea, then further investigated their mRNA level changes in the four abalone genotypes after bacterial challenge. Results showed that these physiological and molecular parameters were significantly induced by bacterial exposure, and their changing patterns were obviously different between the four genotypes: (1) Survival rates of the two hybrids were higher than both parental species after bacterial exposure; (2) DG had higher THC than the other three genotypes; (3) Phagocytosis responded slower in the hybrids than in the parental species; (4) DD's ROS level was lower than the other three genotypes at 48 h post infection; (5) Phenoloxidase activity was lower in DD during the infection compared to the other genotypes; (6) mRNA levels of hsp70 and csdp, were always lower in at least one parental species (DD) than in the hybrids after the bacterial exposure. Results from this study indicate that the hybrids are more active or efficient in immune system function, hence they could effectively defense against a bacterial invasion, leading to higher survival rates after challenge. This study provides physiological and molecular evidences for interpreting the disease resistant heterosis in this abalone hybrid system, which could help us in a better understanding and utilization of heterosis in abalone aquaculture.

Proteomic analysis of trochophore and veliger larvae development in the small abalone Haliotis diversicolor.

BACKGROUND: Haliotis diversicolor is commercially important species. The trochophore and veliger are distinct larval stages in gastropod development. Their development involves complex morphological and physiological changes. We studied protein changes during the embryonic development of H. diversicolor using two dimensional electrophoresis (2-DE) and label-free methods, tandem mass spectrometry (MS/ MS), and Mascot for protein identification. RESULTS: A total of 150 2-DE gel spots were identified. Protein spots showed upregulation of 15 proteins and downregulation of 28 proteins as H. diversicolor developed from trochophore to veliger larvae. Trochophore and veliger larvae were compared using a label-free quantitative proteomic approach. A total of 526 proteins were identified from both samples, and 104 proteins were differentially expressed (> 1.5 fold). Compared with trochophore larvae, veliger larvae had 55 proteins upregulated and 49 proteins downregulated. These differentially expressed proteins were involved in shell formation, energy metabolism, cellular and stress response processes, protein synthesis and folding, cell cycle, and cell fate determination. Compared with the 5 protein (fructose-bisphosphate aldolase, 14-3-3ε, profilin, actin-depolymerizing factor (ADF)/cofilin) and calreticulin) expression patterns, the mRNA expression exhibited similar patterns except gene of fructose-bisphosphate aldolase. CONCLUSION: Our results provide insight into novel aspects of protein function in shell formation, torsion, and nervous system development, and muscle system differentiation in H. diversicolor larvae. "Quality control" proteins were identified to be involved in abalone larval development.

iTRAQ-Based Identification of Proteins Related to Muscle Growth in the Pacific Abalone, Haliotis discus hannai.

The abalone Haliotis discus hannai is an important aquaculture species that is grown for human consumption. However, little is known of the genetic mechanisms governing muscle growth in this species, particularly with respect to proteomics. The isobaric tag for relative and absolute quantitation (iTRAQ) method allows for sensitive and accurate protein quantification. Our study was the first to use iTRAQ-based quantitative proteomics to investigate muscle growth regulation in H. discus hannai. Among the 1904 proteins identified from six samples, 125 proteins were differentially expressed in large specimens of H. discus hannai as compared to small specimens. In the large specimens, 47 proteins were upregulated and 78 were downregulated. Many of the significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including these differentially expressed proteins, were closely related to muscle growth, including apoptosis, thyroid hormone signaling, regulation of the actin cytoskeleton, and viral myocarditis (p < 0.05). Our quantitative real-time polymerase chain reaction (qRT-PCR) analyses suggested that the alterations in expression levels observed in the differentially expressed proteins were consistent with the alterations observed in the encoding mRNAs, indicating the repeatability of our proteomic approach. Our findings contribute to the knowledge of the molecular mechanisms of muscle growth in H. discus hannai.

Bioaccessibility and Health Risk Assessment of Cu, Cd, and Zn in "Colored" Oysters.

Bioaccessibility describes the fraction of contaminants released from the food matrix into the digestive tracts of humans, which is beneficial for improving the health risk assessment of contaminants. In this study, the bioaccessibilities of cadmium (Cd), copper (Cu), and zinc (Zn) in two severely contaminated green oyster (Crassostrea angulate) and blue oyster (Crassostrea hongkongensis) populations were investigated. A human health risk assessment of these metals was then performed based on bioaccessibility measurements. Among the three metals, the bioaccessibility was the highest for Cu (42-95%), and Cd and Zn had comparable bioaccessibility (13-58%). There was no major difference in the bioaccessibility between green and blue oysters. A significant correlation between the tissue Cu and Zn concentrations was found in these highly contaminated oysters. A health risk assessment showed that all three metals in both oyster species seriously exceeded the levels recommended by the United States Environmental Protection Agency. Thus, oysters from these locations, and the metals contained therein, presented quite high risks for human consumption, which should be a great cause of concern. A significant relationship was only found between metal bioaccessibility and its tissue concentration instead of between metal bioaccessibility and subcellular distribution. In addition, a significant relationship was only observed between metal health risks and its tissue concentration. The influence of metal bioaccessibilities on the health risks was limited. This may suggest that in the case of the colored oysters examined in this study, metal concentration instead of metal subcellular distribution could be the driving factor of the metal bioaccessibility, and metal concentration, instead of metal bioaccessibility, could be the driving factor of the metal health risks.

Cathepsin L is an immune-related protein in Pacific abalone (Haliotis discus hannai)--Purification and characterization.

Cathepsin L, an immune-related protein, was purified from the hepatopancreas of Pacific abalone (Haliotis discus hannai) by ammonium sulfate precipitation and column chromatographies of SP-Sepharose and Sephacryl S-200 HR. Purified cathepsin L appeared as two bands with molecular masses of 28.0 and 28.5 kDa (namely cathepsin La and Lb) on SDS-PAGE under reducing conditions, suggesting that it is a glycoprotein. Peptide mass fingerprinting (PMF) analysis revealed that peptide fragments of 95 amino acid residues was high similarity to cathepsin L of pearl oyster (Pinctada fucata). The optimal temperature and pH of cathepsin L were 35 °C and pH 5.5. Cathepsin L was particularly inhibited by cysteine proteinase inhibitors of E-64 and leupeptin, while it was activated by metalloproteinase inhibitors EDTA and EGTA. The full-length cathepsin L cDNA was further cloned from the hepatopancreas by rapid PCR amplification of cDNA ends (RACE). The open reading frame of the enzyme was 981 bp, encoding 327 amino acid residues, with a conserved catalytic triad (Cys134, His273 and Asn293), a potential N-glycosylation site and conserved ERFNIN, GNYD, and GCGG motifs, which are characteristics of cathepsin L. Western blot and proteinase activity analysis revealed that the expression and enzyme activity of cathepsin L were significantly up-regulated in hepatopancreas at 8 h following Vibrio parahaemolyticus infection, demonstrating that cathepsin L is involved in the innate immune system of abalone. Our present study for the first time reported the purification, characterization, molecular cloning, and tissue expression of cathepsin L in abalone.

Molecular cloning of two molluscan caspases and gene functional analysis during Crassostrea angulata (Fujian oyster) larval metamorphosis.

Caspases have been demonstrated to possess important functions in apoptosis and immune system in vertebrate. But there is less information reported on the oyster larval development. In the present work, two full-length molluscan caspase genes, named Cacaspase-2 and Cacaspase-3, were characterized for the first time from Fujian oyster, Crassostrea angulata. Which respectively encode two predicted proteins both containing two caspase domains of p20 and p10 including the cysteine active site pentapeptide "QACRG" and the histidine active site signature. Otherwise Cacaspase-2 also contains a caspase recruitment domain. Homology and phylogenetic analysis showed that Cacaspase-2 shared high similarity with initiator caspase-2 groups, but Cacaspase-3 clustered together with executioner caspase-3 groups. Cacaspase-2 and Cacaspase-3 mRNA were both highly expressed in gills and labial palp and were significantly expressed highly in larvae during settlement and metamorphosis. Through the whole mount in situ hybridization, the location of Cacaspase-2 is in the foot of the oyster larvae and the location of Cacaspase-3 is in both the foot and velum tissues. These results implied that Cacaspase-2 and Cacaspase-3 genes play a key role in the loss of foot and Cacaspase-3 gene has an important function in the loss of velum during larvae metamorphosis in C. angulata.

Proteomic profiling of eggs from a hybrid abalone and its parental lines: Haliotis discus hannai Ino and Haliotis gigantea.

Proteomic analysis was performed on the eggs of hybrid abalone and their corresponding parental lines. A total of 915 ± 19 stained protein spots were detected from Haliotis discus hannai♀ × H. discus hannai♂ (DD), 935 ± 16 from H. gigantea♀ × H. gigantea♂ (GG) and 923 ± 13 from H. gigantea♀ × H. discus hannai♂ (GD). The spots from DD and GD were clustered together. The distance between DD and GG was maximal by hierarchical cluster analysis. A total of 112 protein gel spots were identified; of these, 59 were abalone proteins. The proteins were involved in major biological processes including energy metabolism, proliferation, apoptosis, signal transduction, immunity, lipid metabolism, electron carrier proteins, protein biosynthesis and decomposition, and cytoskeletal structure. Three of 20 differential expression protein spots involved in energy metabolism exhibited as upregulated in GD, 13 spots exhibited additivity, and four spots exhibited as downregulated in the offspring. Eleven protein spots were expressed at the highest level in DD. The proteins involved in stress responses included superoxide dismutase, peroxiredoxin 6, thioredoxin peroxidase and glutathione-S-transferase. Two of seven differential expression protein spots involved in response to stress exhibited as upregulated in GD, three exhibited additivity, and two exhibited as downregulated. These results might suggest that proteomic approaches are suitable for the analysis of hybrids and the functional prediction of abalone hybridization.

SARP19 and vdg3 gene families are functionally related during abalone metamorphosis.

The transcriptional activity of the SARP19-I1 and vdg3-I1 genes increases over tenfold when Haliotis diversicolor larvae shift from the pelagic to benthic lifestyle, signifying the important role of these genes during abalone metamorphosis. In this study, eight paralogous SARP19 genes and six paralogous vdg3 genes were identified from H. diversicolor transcriptomes. Phylogenetic analyses were performed, and the spatio-temporal expression patterns of these genes were separately determined by quantitative polymerase chain reaction (qPCR) and whole mount in situ hybridization (WMISH). Five SARP19 paralogs and five vdg3 paralogs showed at least a tenfold increase in expression after settlement. Among these differentially expressed genes, three SARP19 paralogs and four vdg3 paralogs were verified as being spatially expressed in the digestive glands of newly settled postlarvae. We proposed that a hypothesis of coevolution between the two gene families might explain the similarities in their expression patterns and their phylogenetics.