Identification, diversity, and evolution analysis of Commd gene family in Haliotis discus hannai and immune response to biotic and abiotic stresses.

The Commd (Copper Metabolism gene MURR1 Domain) family genes play crucial roles in various biological processes, including copper and sodium transport regulation, NF-κB activity, and cell cycle progression. Their function in Haliotis discus hannai, however, remains unclear. This study focused on identifying and analyzing the Commd genes in H. discus hannai, including their gene structure, phylogenetic relationships, expression profiles, sequence diversity, and alternative splicing. The results revealed significant homology between H. discus hannai's Commd genes and those of other mollusks. Both transcriptome quantitative analysis and qRT-PCR demonstrated the responsiveness of these genes to heat stress and Vibrio parahaemolyticus infection. Notably, alternative splicing analysis revealed that COMMD2, COMMD4, COMMD5, and COMMD7 produce multiple alternative splice variants. Furthermore, sequence diversity analysis uncovered numerous missense mutations, specifically 9 in COMMD5 and 14 in COMMD10. These findings contribute to expanding knowledge on the function and evolution of the Commd gene family and underscore the potential role of COMMD in the innate immune response of H. discus hannai. This research, therefore, offers a novel perspective on the molecular mechanisms underpinning the involvement of Commd genes in innate immunity, paving the way for further explorations in this field.

A metallothionein from disk abalone (Haliotis discus discus): Insights into its functional roles in immune response, metal tolerance, and oxidative stress.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins whose expression is induced by exposure to essential and non-essential metals, making them potential biological markers for assessing metal pollution in various biomonitoring programs. However, the functional properties of these proteins are yet to be comprehensively characterized in most marine invertebrates. In this study, we identified and characterized an MT homolog from the disk abalone (Haliotis discus discus), referred to as disk abalone MT (AbMT). AbMT exhibited the same primary structural features as MTs from other mollusks containing two ß-domains (ß2ß1-form). AbMT protein demonstrated metal-binding and detoxification abilities against Zn, Cu, and Cd, as evidenced by Escherichia coli growth kinetics, metal tolerance analysis, and UV absorption spectrum. Transcriptional analysis revealed that AbMT was ubiquitously expressed in all analyzed tissues and upregulated in gill tissue following challenge with Vibrio parahaemolyticus, Listeria monocytogenes, and viral hemorrhagic septicemia virus (VHSV). Additionally, overexpression of AbMT suppressed LPS-induced NO production in RAW264.7 macrophages, protected cells against H2O2-induced oxidative stress, and promoted macrophage polarization toward the M1 phase. Conclusively, these findings suggest an important role for AbMT in environmental stress protection and immune regulation in disk abalone.

Molecular characterization, expression and antibacterial function of a macin, HdMac, from Haliotis discus hannai.

Macins are a family of antimicrobial peptides, which play multiple roles in the elimination of invading pathogens. In the present study, a macin was cloned and characterized from Pacific abalone Haliotis discus hannai (Designated as HdMac). Analysis of the conserved domain suggested that HdMac was a new member of the macin family. In non-stimulated abalones, HdMac transcripts were constitutively expressed in all five tested tissues, especially in hemocytes. After Vibrio harveyi stimulation, the expression of HdMac mRNA in hemocytes was significantly up-regulated at 12 hr (P < 0.01). RNAi-mediated knockdown of HdMac transcripts affected the survival rates of abalone against V. harveyi. Moreover, recombinant protein of HdMac (rHdMac) exhibited high antibacterial activities against invading bacteria, especially for Vibrio anguillarum. In addition, rHdMac possessed binding activities towards glucan, lipopolysaccharides (LPS), and peptidoglycan (PGN), but not chitin in vitro. Membrane integrity analysis revealed that rHdMac could increase the membrane permeability of bacteria. Meanwhile, both the phagocytosis and chemotaxis ability of hemocytes could be significantly enhanced by rHdMac. Overall, the results showed that HdMac could function as a versatile molecule involved in immune responses of H. discus hannai.

Hemocyte migration and expression of four Sox genes during wound healing in Pacific abalone, Haliotis discus hannai.

In molluscs, migration of hemocytes and epithelial cells is believed to play central roles in wound healing. Here, we assessed cellular and molecular mechanisms of wound healing in Pacific abalone, a marine gastropod. Light and electron microscopy in the wounds showed early accumulation of putative hemocytes, collagen deposition by fibroblasts, and further coverage of this tissue by migration of adjacent epithelial cells. Cell labelling technique allowed us to track hemocytes, which migrated to wound surface within 24 h. The migrated cells first expressed PCNA and SoxF weakly, and then the epithelial cells expressed abundant PCNA and SoxB1, SoxB2, and SoxC. These findings imply that abalone SoxF is involved in hemocyte migration or their differentiation into fibroblasts, and suggest that the migrated epithelia acquire stem cell-like property and undergo active proliferation. This study is the first to show direct evidence of hemocyte migration to wounds and expression of Sox genes in molluscan wound healing.

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.

Immune response of mollusk Onchidium struma to extremely low-frequency electromagnetic fields (ELF-EMF, 50 Hz) exposure based on immune-related enzyme activity and De novo transcriptome analysis.

Along with rapid offshore and onshore wind power development in modern society, extremely low frequency electromagnetic fields (ELF-EMF) is produced extensively in the habits of aquatic organisms. However, the biological effects of ELF-EMF on aquatic organisms are almost sparse. In this study, Onchidium struma without shell was chosen to aim whether ELF-EMF can elicit immune response of mollusk based on immune-related enzyme activities and gene expression through high-throughput transcriptome sequencing. Three experimental groups, i.e. ELF-EMF unexposed control group (C), ELF-EMF (50 Hz, 100 µT) exposed E1 group, and ELF-EMF (50 Hz, 500 µT) exposed E2 group, were set, and coelomocytes were collected to analyze. The results showed that total coelomocyte and spherulocyte density in E1 group increased significantly compared to groups C and E2 (P < 0.05). There were no significant differences on amoebocyte and chromatocyte density among groups C, E1 and E2. ELF-EMF exposure could significantly increase immune-related enzyme activities in coelomic fluid of O. struma, including acidic phosphatase, alkaline phosphatase, antioxidative capacity, catalase, superoxide dismutase, and polyphenol oxidase (P < 0.05). A total of 54.32 Mb and 55.27 Mb raw reads with average length of 1520 bp were obtained from coelomocytes of O. struma in unexposed and exposed groups, respectively. There were 341 differentially expressed genes (DGEs) between unexposed and exposed groups, including 209 up-regulated and 132 down-regulated unigenes. All the DGEs were allocated to 14 Kyoto Encyclopedia of Genes and Genomes pathways, and five pathways were associated with immune response, including TLR/TNF/NOD-like receptor/MAPK/Fc epsilon RI signaling pathways. Altogether, short-term (to one week) exposure of O. struma to lower luxy density ELF-EMF (<500 µT) could elicit the immune response, and antioxidant system is recommended as indicators of immunological effects. Hopefully, this study will further provide insights into exploring biomarker for evaluation of the effect of ELF-EMF exposure on aquatic organisms regarding to field density, frequency and exposure duration, and provide good guidance for exploitation and utilization of renewable energy.

Molecular characterization, redox regulation, and immune responses of monothiol and dithiol glutaredoxins from disk abalone (Haliotis discus discus).

Glutaredoxins (Grxs) are well-known oxidoreductases involved in a wide range of redox activities in organisms. In this study, two invertebrate Grxs (AbGrx1-like and AbGrx2) from disk abalone were identified and characterized in an effort to gain a deeper understanding into their immune and redox regulatory roles. Both AbGrxs share typical thioredoxin/Grx structures. AbGrx1-like and AbGrx2 were identified as monothiol and diothiol Grxs, respectively. AbGrxs were significantly expressed at the egg and 16-cell stage of early abalone development. Although the expression of both AbGrxs demonstrated similar patterns, the expression of AbGrx1-like was higher than AbGrx2 during development stages. In contrast, AbGrx2 expression was significantly higher than that of AbGrx1-like in adult tissues. Highest AbGrx1-like expression was observed in the hepatopancreas and digestive tract, while highest AbGrx2 expression was found in the gills, followed by the mantle, in healthy adult abalone tissues. The highest expression of AbGrx1-like was observed in the gills at 12 h and 6 h post injection (p.i) of Vibrio parahemolyticus and other stimulants, respectively. The highest expression of AbGrx2 in the gills were observed at 120 h, 6 h, 24 h, and 12 h post injection of V. parahaemolyticus, Listeria monocytogenes, Viral hemorrhagic septicemia virus, and Polyinosinic:polycytidylic acid, respectively. AbGrxs possessed significant 2-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) reduction activity, but AbGrx2 exhibited higher redox activity than AbGrx1-like. Altogether, our results suggest an important role of AbGrx1-like and AbGrx2 in redox homeostasis, as well as in the invertebrate immune defense system. Our findings will aid the development of new disease management strategies for this economically valuable species.

Immune response in probiotic-fed New Zealand black-footed abalone (Haliotis iris) under Vibrio splendidus challenge.

Vibriosis disease is a major constraint for sustainable molluscan aquaculture. Development of strategies to enhance disease resistance during grow out would greatly reduce stock mortality and boost production yields. In this study, New Zealand black-footed abalone (Haliotis iris) were fed a commercial diet enhanced with multi-strain probiotics (Exiguobacterium JHEb1, Vibrio JH1 and Enterococcus JHLDc) for four months, then challenged with an injection of pathogenic Vibrio splendidus. Host immune responses in haemocytes were characterized using flow cytometry by measuring total haemocyte counts (THC) and viability, degree of apoptosis, and production of reactive oxygen species (ROS) 48 h post-challenge. Probiotic-fed abalone had significantly higher survival rates compared to control animals after the bacterial challenge. Infected probiotic-fed abalone also had significantly higher haemocyte viabilities, slightly lower proportions of haemocytes undergoing early apoptosis, and lower proportions of ROS-producing haemocytes compared to infected control-fed abalone. In addition, metabolite profiles of muscle tissues generated via gas chromatography-mass spectrometry (GC-MS) delivered complimentary evidence to support a perturbed ROS-regulatory system in infected abalone through changes in key metabolites associated with glutathione biosynthesis. The results of this study provide valuable information to assist in farm management practices, leading to enhance production and sustainability of the New Zealand abalone aquaculture industry.

Toll-like signaling pathway in the transcriptome of Littorina littorea.

The transcriptome of the caenogastropod mollusk Littorina littorea was scanned for the presence of sequences encoding Toll-like receptors (TLRs) and corresponding proteins involved in downstream TLR signaling pathway. In the transcriptomic snapshots of hemocytes and kidney tissues, 45 complete TLRs encoded by 35 genes were identified. Out of the 59 non-TLR molecules involved in a canonical TLR signaling pathway, 35 genes were classified as homologous and could be placed within the TLR-mediated MyD88-and MAPK-dependent circuitries. No reference vertebrate adapters TIRAP, TRIF and TRAM were identified in the transcriptome. The results of RNA-seq experiments with an immune challenge (rediae of the digenean Himasthla elongata) indicate that four TLRs (LlTLR1, 3, 5 and 8) and a set of upregulated genes involved in signal transduction (LlMyd88, LlTNFα, LlCASP8, LlFADD, LlNFKBIA (IkBα), LlIRAK1, LlSTAT1, LlMAPK14 (P38), LlMAP2K1 (MEK1/2), LlIRF3 and LlIRF5) may participate in the anti-digenean immune response of L. littorea.

Quadruple domain-containing galectin from marine invertebrate disk abalone (Haliotis discus discus): Molecular perspectives in early development, immune expression, and potent antiviral responses.

Galectins are well-known ß-galactoside-binding proteins, which play vital roles in innate immune responses of both vertebrates and invertebrates. However, knowledge regarding invertebrate galectins is still in its infancy. With the intention of filling the knowledge gap, here we identified a quadruple domain-containing galectin from marine invertebrate disk abalone, Haliotis discus discus (AbGalec), and characterized it. AbGalec consisted of four distinct carbohydrate-recognition domains (CRDs) and lacked a signal peptide. Expression analysis revealed AbGalec to be ubiquitously expressed in all the examined early embryonic stages of abalone, with highest expression in the 16-cell stage, suggesting the importance of AbGalec in early developmental processes. Tissue distribution analysis revealed the highest expression of AbGalec in abalone mantle, followed by that in gills and hemocytes. Immune challenge experiments revealed significant upregulation of AbGalec at 24 h and 48 h post injection (p.i.) with bacterial and viral components. These results suggested the possible involvement of AbGalec in host defense mechanisms. Polyinosinic: polycytidylic acid (Poly I:C) and viral hemorrhagic septicemia virus (VHSV) injections were capable of inducing AbGalec transcript expression more prominently than bacterial stimulants, thus providing evidence for its role in viral infections. We determined the virus-neutralizing ability of a quadruple domain-containing galectin for the first time, by analyzing the downregulation of VHSV transcripts during the overexpression of AbGalec. Significant downregulation of VHSV transcripts was observed after 24 h and 48 h of post infection. Collectively, our findings reveal the potent antiviral responses of molluscan quadruple domain-containing galectin, AbGalec, along with its involvement in innate immunity.

Over high or low dietary protein levels depressed the growth, TOR signaling, apoptosis, immune and anti-stress of abalone Haliotis discus hannai.

A 120-day feeding trial was conducted to investigate the effects of relative higher and lower dietary protein levels on the growth, immunity and anti-stress of abalone Haliotis discus hannai fed diets with 17.64% (low), 30.49% (normal) and 43.27% (high) of proteins, respectively. The results showed that compared with 30.49% of dietary protein, 17.64% and 43.27% of dietary protein levels significantly decreased the weight gain rate and the activities of α-amylase, trypsin, alanine aminotransferase and aspartate aminotransferase in the hepatopancreas and serum of abalone (P < 0.05). Abalone fed 30.49% of dietary protein had the highest activity of superoxidase, acid phosphatase, alkaline phosphatase, lysozyme and the total anti-oxidative capacity, and the lowest content of malondialdehyde in the serum and hepatopancreas (P < 0.05). The gene expressions of TOR, S6k, Bcl-2, IκB, NfκB, TNF-α and Nrf2 were significantly up-regulated in the group with 30.49% of dietary protein (P < 0.05). Pathological abnormalities in hepatocyte cells of abalone were found in the groups with 17.64% and 43.27% of dietary protein. Meanwhile, accumulative mortalities of abalone after the Vibrio parahaemolyticus challenge test and heat stress test were significantly increased within these two groups (P < 0.05). In conclusion, the excessive (43.27) or deficient (17.64) dietary protein levels depressed the growth and immunity of abalone. Combined with the stress tests results, 17.63% or 43.27% of dietary protein contents are not recommended to the abalone facing the stress of vibriosis or high-water temperature (≥28 °C).

iTRAQ-based quantitative proteomic profiling of the immune response of the South African abalone, Haliotis midae.

The South African abalone Haliotis midae is a commercially important species farmed at high densities in land-based aquaculture systems. Disease outbreaks have had a severe financial impact on the abalone industry yet the molecular mechanisms underlying the immune response of H. midae remain obscure. In this study, a comparative shotgun proteomics approach using iTRAQ coupled with LC-MS/MS was employed to investigate H. midae proteome changes in response to Vibrio anguillarum challenge. A total of 118 non-redundant, unique haemocyte proteins were identified and quantified, with 16 proteins significantly regulated. Hierarchical clustering and pathway analysis uncovered a coordinated response dominated by calcium and cAMP signalling via activation of MAPK cascades. Early up-regulated biological processes involve phagocytosis, nitric oxide production and ATP-synthesis, whilst down-regulated responses were predominantly involved in the regulation of apoptosis. The late up-regulated response involved protein kinase activity and detoxification processes. Expression of selected proteins was validated by Western blot. A putative allograft inflammatory factor-1 protein was further selected to establish its functional molecular role in haemocytes. Confocal imaging revealed that allograft inflammatory factor-1 regulates phagocytosis via a functional interaction with filamentous actin. This is the first time a high-throughput proteomics approach has been used to investigate the immune response of H. midae.

Effect of pathogenic bacteria on a novel C-type lectin, hemocyte and superoxide dismutase/ alkaline phosphatase activity in Onchidium reevesii.

Bacterial infection in the marine environment is a serious problem to maintain the stability of marine ecosystems. Nevertheless, there is little report so far for the biological effects of pathogenic bacteria in coastal ecosystems. Hence, we investigated the responses of shell-less Onchidium reevesii to resist against pathogenic bacterial infection. Analysis of data here could be used as fundamental information for assessment of innate immune response of O. reevesii. The full-length OrCTL cDNA was cloned and consists of 1849 base pair (bp) encoding protein of 192 amino acids. Constructing multiple alignments suggested that OrCTL has conserved carbohydrate recognition domain (CRD) of CTLs, containing an EPS (Glu-Pro-Ser) motif that may imply the function of recognition of carbohydrates like others invertebrate. OrCTL mRNAs were mainly detected in ganglion and hepatopancreas, and expression was highly up-regulated from 2 h after Vibrio harveyi challenge, rapidly decreased at 4 h, and significantly increased at 12 h. In addition, after challenge with Vibrio parahaemolytics, OrCTL gene expression was slightly up-regulated from 2 h, peaked at 12 h. Enzyme activity (in the hepatopancreas) and cell immune (in the hemolymph) were investigated along with Superoxide dismutase (SOD) activity, alkaline phosphatase (ALP) activity and cell cycle. SOD activities were significantly higher after V. harveyi and V. parahaemolytics challenge than that in the control group, respectively. By contrast, ALP activities were significantly inhibited after challenged with bacteria than that in the control group, respectively. Enzyme activities in the hepatopancreas obviously fluctuated, and ALP activity was more sensitive to bacteria. Cell responses illustrated that there were a significant higher percentage of cells in the S and G2/M phase in hemolymph after challenged with bacteria. Our results suggested that the immune response of O. reevesii could be activated by pathogenic bacteria, and the data will provide referent for the disease prevention of systematic investigation in aquatic animal.

Structural and functional analysis of three Iκb kinases (IKK) in disk abalone (Haliotis discus discus): Investigating their role in the innate immune responses.

The IκB kinases (IKK) are large multiprotein complexes that regulate the activation of the transcription factor NF-κB and are involved in a diverse range of biological processes, including innate immunity, inflammation, and development. To explore the potential roles of invertebrate IKKs on immunity, three IKK encoding genes have been identified from molluscan species disk abalone and designed as AbIKK1, AbIKK2 and AbIKK3 at the transcriptional level. Coding sequences of AbIKK1, AbIKK2 and AbIKK3 encode the peptides of 746, 751 and 713 amino acids with the predicted molecular mass of 86.16, 86.12 and 81.88 kDa respectively. All three AbIKKs were found to share conserved IKK family features including the kinase superfamily domain (KD), ubiquitin-like domain (ULD), and α-helical scaffold/dimerization domain (SDD), similar to their mammalian counterparts. Under normal physiological conditions, AbIKKs were ubiquitously detected in six different tissues, with the highest abundance in the digestive tract and gills. Temporal transcriptional profiles in abalone hemocytes revealed the induction of AbIKK1, AbIKK2, and AbIKK3 expression following exposure to Gram-negative (Vibrio parahemolyticus) and Gram-positive (Listeria monocytogenes) bacteria, viruses (viral hemorrhagic septicemia virus, VHSV), LPS, or poly I:C. The overexpression of AbIKKs in HEK293T or RAW264.7 murine macrophage cells induced NF-κB promoter activation independent of stimulation by TNF-α or LPS. Moreover, iNOS and COX2 expression was induced in AbIKK transfected RAW264.7 murine macrophage cells and the induced state was maintained post-LPS treatment. Furthermore, mRNA levels of three selected cytokine-encoding genes (IL-1ß, IL-6, and TNF-α) were found to be elevated in abalone IKK overexpressed RAW264.7 murine macrophage cells, both with and without LPS exposure. Overall, our findings demonstrated that AbIKKs identified in this study were positively involved in eliciting innate immune responses in abalone. In addition, the data revealed the presence of an evolutionarily conserved signaling mechanism for IKK mediated NF-κB activation in mollusks.

Effects of Bacillus lincheniformis feeding frequency on the growth, digestion and immunity of Haliotis discus hannai.

To study the effects of Bacillus lincheniformis feeding frequency on the survival and growth of Haliotis discus hannai abalone, we measured the expression levels of nonspecific immune genes and monitored the anti-Vibrio parahaemolyticus immune reaction. H. discus hannai (shell length: 32.75 ± 2.63 mm, body weight: 4.91 ± 0.34 g) was selected to perform a 70 d laboratory culture experiment including a 14 d V. parahaemolyticus artificial infection experiment. The control group (C) was fed normal commercial feed every day. The M1 experimental group was given experimental feed and basal feed on alternating days until the end of the experiment. The M2 experimental group was given experimental feed for 4 d and basal feed for 3 d, and this cycle was repeated every 7 d until the end of the experiment. The M3 experimental group was given experimental feed for 2 d and basal feed for 5 d, and this cycle was repeated every 7 d until the end of the experiment. The M4 group was continuously given experimental feed for the duration of the experiment. The concentration of added B. lincheniformis in each experimental group was 105 cfu/g (according to the quantity of viable bacteria). The specific growth rate (as measured by body weight) and the feed conversion efficiency of the abalone in M1 and M2 were significantly higher than those in M4 and C (P < 0.05). The cellulose and lipase activities of abalone in M1, M2 or M4 were significantly higher than those in M3 or C (P < 0.05). The acid phosphatase, superoxide dismutase, total haemocyte counts, O2- levels generated by respiratory bursts, and the expression levels of Mn-SOD, TPx, GSTs and GSTm in abalone in the M2 group were significantly higher than those in any other feeding frequency group (P < 0.05). At the end of the V. parahaemolyticus infection, the cumulative mortality of the abalone in M2 was significantly lower than that in any other group (P < 0.05). Consequently, given the growth advantages and the enhancement of immune function, the feeding plan in which B. lincheniformis was applied for 4 d per week, and basal feed was then applied for 3 d, did not lead to a high level of immune reaction, immune fatigue or waste of resources, but increased the growth rate of individuals and their resistance to V. parahaemolyticus infection.

Transcriptome analysis of air-breathing land slug, Incilaria fruhstorferi reveals functional insights into growth, immunity, and reproduction.

BACKGROUND: Incilaria (= Meghimatium) fruhstorferi is an air-breathing land slug found in restricted habitats of Japan, Taiwan and selected provinces of South Korea (Jeju, Chuncheon, Busan, and Deokjeokdo). The species is on a decline due to depletion of forest cover, predation by natural enemies, and collection. To facilitate the conservation of the species, it is important to decide on a number of traits related to growth, immunity and reproduction addressing fitness advantage of the species. RESULTS: The visceral mass transcriptome of I. fruhstorferi was enabled using the Illumina HiSeq 4000 sequencing platform. According to BUSCO (Benchmarking Universal Single-Copy Orthologs) method, the transcriptome was considered complete with 91.8% of ortholog genes present (Single: 70.7%; Duplicated: 21.1%). A total of 96.79% of the raw read sequences were processed as clean reads. TransDecoder identified 197,271 contigs that contained candidate-coding regions. Of a total of 50,230 unigenes, 34,470 (68.62% of the total unigenes) annotated to homologous proteins in the Protostome database (PANM-DB). The GO term and KEGG pathway analysis indicated genes involved in metabolism, phosphatidylinositol signalling system, aminobenzoate degradation, and T-cell receptor signalling pathway. Many genes associated with molluscan innate immunity were categorized under pathogen recognition receptor, TLR signalling pathway, MyD88 dependent pathway, endogenous ligands, immune effectors, antimicrobial peptides, apoptosis, and adaptation-related. The reproduction-associated unigenes showed homology to protein fem-1, spermatogenesis-associated protein, sperm associated antigen, and testis expressed sequences, among others. In addition, we identified key growth-related genes categorized under somatotrophic axis, muscle growth, chitinases and collagens. A total of 4822 Simple Sequence Repeats (SSRs) were also identified from the unigene sequences of I. fruhstorferi. CONCLUSIONS: This is the first available genomic information for non-model land slug, I. fruhstorferi focusing on genes related to growth, immunity, and reproduction, with additional focus on microsatellites and repeating elements. The transcriptome provides access to greater number of traits of unknown relevance in the species that could be exploited for in-depth analyses of evolutionary plasticity and making informed choices during conservation planning. This would be appropriate for understanding the dynamics of the species on a priority basis considering the ecological, health, and social benefits.

Sub-optimal or reduction in temperature and salinity decrease antioxidant activity and cellularity in the hemolymph of the Pacific abalone (Haliotis discus hannai).

This study investigated the oxidative stress and hemocyte responses of Pacific abalone exposed to various water temperatures (4, 6, 8, and 10 °C) and salinities (26, 30, and 34 psu) for 7 days, to identify their tolerance ranges of temperature and salinity. The survival rate of Pacific abalone ranged from 98.7 to 100% at 8 °C and 10 °C, but dropped to 25-55% at 4 °C at all levels of salinity. The levels of superoxide dismutase and glutathione in the hemolymph were significantly higher at 4 °C and 6 °C than in the controls in all salinity groups, indicating that these temperatures induced greater stress in the Pacific abalone. Total hemocyte count was lowest at 6 °C in the 26 psu group. The percentages of apoptotic and necrotic cells were higher in the 26 psu group than in the other salinity groups, and higher in the 4 °C and 6 °C groups than in the other temperature groups. These results indicate that the lowest tolerance to water temperature and salinity in the Pacific abalone was 8 °C and 30 psu, respectively.

Nucleus-translocated matrix metalloprotease 1 regulates innate immune response in Pacific abalone (Haliotis discus hannai).

As an important economical shellfish in coastal area of China, abalone is susceptible to bacterial infection, especially Vibiro parahemolyticus (V. parahemolyticus). Matrix metalloproteinases (MMPs) have been extensively investigated in the immune response of mammals. However, little is known about the involvement of MMP in abalone innate immune system against pathogen infection. In this study, the role of MMP-1 in the immune response of Pacific abalone (Haliotis discus hannai) was explored. The results showed that V. parahemolyticus infection induced significantly elevated expression of MMP-1 as well as immune related genes including allograft inflammatory factor 1 (AIF-1), macrophage expressed gene 1 (MPEG-1) and TPA-inducible sequence 11 family protein (Tis11FP). Notably, silencing of MMP-1 reduced the expression of these genes, suggesting that MMP-1 was an upstream regulatory factor in V. parahemolyticus infection. Further analysis showed that MMP-1 was engaged in the regulation of cellular (phagocytosis, apoptosis) and humoral [superoxide dismutase (SOD), alkaline phosphatase (ALP), acid phosphatase (ACP)] immunity. Interestingly, the extracellularly distributed MMP-1 could be translocated to the nuclei of hemocytes, thereby functioning as a transcriptional regulator or by selectively activating or inactivating other components through proteolysis. Hence, our study established an important role of MMP-1 in abalone innate immunity against V. parahemolyticus infection and it represented the first report on the investigation of MMP in abalone.

Integrative transcriptome analysis and discovery of genes involving in immune response of hypoxia/thermal challenges in the small abalone Haliotis diversicolor.

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.

Effects of the probiotic Bacillus amyloliquefaciens on the growth, immunity, and disease resistance of Haliotis discus hannai.

The effects of a diet containing the probiotic Bacillus amyloliquefaciens on the survival and growth of Haliotis discus hannai were evaluated by measuring growth and hematological parameters and the expression levels of nonspecific immune genes. In addition, the abalone's response to Vibrio parahaemolyticus infection was assessed. H. discus hannai (shell length: 29.35 ±â€¯1.81 mm, body weight: 4.28 ±â€¯0.23 g) were exposed to an 8-week culture experiment in indoor aquariums and a 2-week V. parahaemolyticus artificial infection experiment. In each experiment, the control group (C) was fed daily with the basal feed; the experimental groups were fed daily with the experimental feed, prepared by spraying B. amyloliquefaciens onto the basal feed at final concentrations of 103 (group A1), 105 (A2), and 107 (A3) cfu/g. The survival rate, body weight specific growth rate, and food conversion efficiency in A2 and A3 were significantly higher than those in A1 and C (P < 0.05). The total number of blood lymphocytes, the O2- and NO levels produced from respiratory burst, the activities of acid phosphatase, superoxide dismutase, and catalase, and the expression levels of catalase and thiol peroxidase in A2 were not significantly different from those in A3, but these factors were significantly higher in A2 compared to A1 and C (P < 0.05). The total antioxidant capacity and expression levels of glutathione S-transferase in A1, A2 and A3 were significantly higher than those in C (P < 0.05). At day 9 after infection with V. parahaemolyticus, all abalone in C were dead; at the end of the experiment, the cumulative mortality of abalone in A2 was significantly lower than that in any other group (P < 0.05). Thus, the experimental feed containing 105 cfu/g B. amyloliquefaciens not only facilitated the food intake and growth of abalone, but also effectively enhanced their non-specific immunity and resistance to V. parahaemolyticus infection. In this regard, B. amyloliquefaciens may be a useful probiotic strain for abalone aquaculture.