The combined analysis of transcriptomics and metabolomics reveals the mechanisms by which dietary quercetin regulates growth and immunity in Penaeus vannamei.

As a potent antioxidant, the flavonoid compound quercetin (QUE) has been widely used in the farming of aquatic animals. However, there are fewer reports of the beneficial effects, especially in improving immunity of Penaeus vannamei by QUE. The aim of this study was to investigate the effects of dietary QUE on growth, apoptosis, antioxidant and immunity of P. vannamei. It also explored the potential mechanisms of QUE in improving the growth and immunity of P. vannamei. P. vannamei were fed diets with QUE for 60 days. The results revealed that QUE (0.5 or 1.0 g/kg) ameliorated the growth, and the expressions of genes related to apoptosis, antioxidant, and immunity. The differentially expressed genes (DEGs) and differential metabolites (DMs) obtained through transcriptomics and metabolomics, respectively, enriched in pathways related to nutritional metabolism such as lipid metabolism, amino acid metabolism, and carbohydrate metabolism. After QUE addition, especially at 0.5 g/kg, DEGs were enriched into the functions of response to stimulus and antioxidant activity, and the pathways of HIF-1 signaling pathway, C-type lectin receptor signaling pathway, Toll-like receptor signaling pathway, and FoxO signaling pathway. In conclusion, dietary QUE can ameliorate growth, apoptosis, antioxidant and immunity of P. vannamei, the appropriate addition amount was 0.5 g/kg rather than 1.0 g/kg. Regulations of QUE on nutrient metabolism and immune-related pathways, and bioactive metabolites, were important factors for improving the aforementioned abilities in P. vannamei.

Comparative analysis of transcriptomics and metabolomics provides insights into the mechanisms of VPAHPND invasion and hepatopancreatic damage in Litopenaeus vannamei.

Acute hepatopancreatic necrosis disease (AHPND) poses significant threats to the global shrimp farming industry; however, its molecular mechanisms remain largely unknown. Previous research has primarily focused on comparisons between infected and non-infected states, limiting our understanding of VPAHPND mechanisms. We integrated transcriptomic and metabolomic analyses to investigate the pathogenic mechanism underpinning AHPND in highly vulnerable post-larvae (PL) stage shrimp. By comparing shrimp infected with VPAHPND, those infected with non-VPAHPND, and uninfected shrimp (controls), we identified different VPAHPND infection responses, including significant cytoskeleton and metabolic reprogramming changes. Specifically, VPAHPND infection disturbed lipid, glutathione, and bile acid metabolism, while a key regulatory factor Farnesoid X Receptor (FXR) in these pathways was down-regulated. These findings suggest that VPAHPND manipulates host metabolism to enhance infectivity, leading to severe and irreparable hepatopancreas damage. Our study highlights the molecular interactions between VPAHPND and shrimp, and provides potential targets to mitigate the impact of AHPND in aquaculture.

A comparative transcriptomic analysis in late embryogenesis of the red claw crayfish Cherax quadricarinatus.

The red claw crayfish (Cherax quadricarinatus) is an emerging and important commercial species in several countries, and is also a potential biological model in crustacean biology. However, its molecular embryonic development mechanism remains largely unknown because of a lack of genomic resources and systematic research. A comprehensive and integrated transcriptomic analysis is necessary to reveal the cell biological function, gene expression profiles, and embryo patterning that occur during embryogenesis. In the present study, transcriptomic profiles of C. quadricarinatus embryos during three developmental stages were investigated by high-throughput Illumina sequencing technology, and the genes related to development were further analyzed. In total, 49,436 unigenes were assembled and clustered, in which 13,727 were annotated in the Nonredundant database, 5087 were classified based on Gene Ontology annotations, and 2735 were associated with 189 Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, gene expression differences among the embryos stages were analyzed, and 6658 differentially expressed genes (DEGs) were identified. In total, 3300, 5211, and 1262 DEGs were identified between the eye pigments forming stage (EP) and prepare-hatching stage (PH), EP and larvae (L), as well as PH and L; meanwhile, 1595, 2540 and 680 DEGs were annotated, respectively. The fundamental developmental genes related to apoptosis, neurogenesis, and segmentation, as well as signaling pathways related to Hedgehog, MAPK, Wnt, TGF-ß and Notch, showed higher expression during the EP stage than in other two stages, indicating that the EP stage has more active biological processes than the latter stages. This transcriptome studies gene expression at different stages of embryonic development and the datasets provide a basis for understanding crustacean developmental biology and guiding seedling production.

Response of the Litopenaeus vananmei intestinal bacteria and antioxidant system to rearing density and exposure to Vibrio paraheamolyticus E1.

Intestinal bacteria and bacterial metabolic products are indispensable components of both invertebrate and vertebrate physiology, directly influencing many functions including host energy absorption and metabolism, intestinal barrier integrity and immune function. To investigate the influence of rearing density on shrimp intestinal health, antioxidant responses and disease susceptibility, we simultaneously monitored the dynamic changes of intestinal bacteria and antioxidant enzymes activities in Litopenaeus vananmei under two different rearing densities (400 and 800 shrimp/m3) and further investigated the difference in response to Vibrio paraheamolyticus E1 (VPE1) challenge. We showed that the phyla Proteobacteria, Bacteroidetes, Planctomycetes and Firmicutes were the predominant microflora in all treatment groups. Rearing L. vannamei at high density for 15 days resulted in the reduction of Bacteroidetes and Firmicutes and increase of Planctomycetes. At the genus level, high rearing density induced reduction of Pseudoalteromonas and Blastopirellula, and an increase of Photobacterium and Vibrio. Notably, the relative abundance of Ascidiaceihabitans and Flavobacteria NS10_marine_group increased in the low rearing density groups after VPE1 challenge, suggesting that these two types of bacteria might have an important role in resisting to VPE1 infection. High density stress caused suppression of total superoxide dismutase (T-SOD), catalase (CAT), and glutathione peroxidase (GPX) activities in shrimp. Hence, high density stress altered the functional composition of shrimp intestinal bacteria and damaged the antioxidant system, which increased pathogen susceptibility.

Comparative study of ß-thymosin in two scallop species Argopecten irradians and Chlamys farreri.

The ß-thymosin (Tß) proteins participate in numerous biological processes, such as cell proliferation and differentiation, anti-inflammatory and antimicrobial mechanism. To date, Tß proteins have been well studied in vertebrates, especially mammals. While limited Tß or Tß-like proteins have been reported in invertebrates. Moreover, rare information of Tß or Tß-like proteins is available in scallop species yet. In the present study, two Tß homologues, AiTß and CfTß, were identified and characterized from two scallop species bay scallop Argopecten irradians and Zhikong scallop Chlamys farreri. They were both 41 amino acid peptide and contained one THY domain, a highly conserved actin-binding motif and two conserved helix forming regions. Tissue distribution and expression profiles of their mRNA transcripts were roughly similar yet different in detail, while their recombinant proteins exhibited different immunomodulation activity on the downstream immune parameters. These results collectively indicated that the function of Tß family in scallop were functionally differentiated.

Exploring the influence of the surface proteins on probiotic effects performed by Lactobacillus pentosus HC-2 using transcriptome analysis in Litopenaeus vannamei midgut.

In order to understand the mediation function of surface proteins in probiotic effects executed by Lactobacillus pentosus HC-2 in midgut of Litopenaeus vannamei, the immune and digestion related enzymes and the transcriptome expression were analyzed after shrimp fed with normal HC-2 or with stripped surface proteins HC-2 by lithium chloride (LiCl) treatment. The results showed that the shrimp fed with normal HC-2 produced much higher immune and digestion related enzymes than the control group or LiCl-treated HC-2 group to defense the Vibrio parahaemolyticus E1 infection. We obtained total over 275,099 unigenes from L. vannamei midgut, 981 genes were significant differentially expressed in normal HC-2 group compared with control, 1314 genes were significant differentially expressed in LiCl-treated HC-2 group compared with control, and 1689 genes were significant differentially expressed in LiCl-treated HC-2 group compared with normal HC-2 group. The GO/KEGG enrichment analysis of the significantly different genes demonstrated that L. vannamei fed with normal HC-2 induced immune-related, signal transduction, ion homeostasis, cell-cell adhesion, response stress/stimulus, vascular endothelial growth factor and peritrophin genes up-regulation, which were important genes involved in improving the shrimp intestine immune response, nutrition and growth performance, and bacteria adhesion and colonization, but these genes were suppressed in the midgut of shrimp fed with deprived surface proteins bacteria. Taken together, these results indicated that the surface proteins were essential for HC-2 executing probiotic effects in midgut of shrimp. Our data contribute to improve the current understanding of host - Lactobacillus interaction and the probiotic mechanisms in shrimps.

A preliminary attempt to explore the potential functions of a tetraspanin gene (MmTSPAN) in the innate immunity of hard clam Meretrix meretrix: Sequence features and expression profiles.

Tetraspanins belong to the transmembrane 4 superfamily (TM4SF), and play crucial roles in immune responses. In the present study, a novel tetraspanin gene (designated MmTSPAN) was cloned and characterized from the hard clam Meretrix meretrix. The complete cDNA sequence of MmTSPAN contained an open reading frame (ORF) of 816 bp, which encoded a protein of 271 amino acids. MmTSPAN exhibited highly similarity with previously identified tetraspanins from other species. It contained four transmembrane domains (12-35 aa, 69-92 aa, 99-123 aa and 238-261 aa), characteristic CCG motif and four conservative cysteine residues. The mRNA transcripts of MmTSPAN were ubiquitously detectable in all the tested tissues, with the highest expression level in hepatopancreas. Temporal transcriptional levels in the hepatopancreas revealed significant up-regulation of MmTSPAN by Vibrio splendidus stimulation, with a 3.14-fold increase at 6 h compared to the control, and reaching 32.98-fold at 24 h. These results provide useful information for further study of the function of tetraspanin in the innate immune system of M. meretrix, and may offer a new therapeutic target for diseases of M. meretrix.

Understanding the roles of surface proteins in regulation of Lactobacillus pentosus HC-2 to immune response and bacterial diversity in midgut of Litopenaeus vannamei.

The interactions of microbiota in the intestines play an important role in promoting or maintaining the health of hosts. The present study aim to investigate the effects of the surface proteins of Lactobacillus pentosus HC-2 on the immune response and the bacterial composition of Litopenaeus vannamei, thus, the immune-related genes, surface condition, HC-2 numbers and the bacteria diversity in midgut were explored after shrimp feeding the normal HC-2 and 5 M -  lithium chloride (LiCl) treated HC-2 for four weeks. Obvious improvements in the intestinal surface were observed in R group than the control group and L group. qPCR analysis demonstrated that the selected immune-related genes of lysozyme, proPO, LGBP, PEN-3α, crustin, and lvLec were significantly up-regulated in group R than in group L. Meanwhile, in the challenge test, shrimp in R group received 72% relative percent survival, which was significantly higher than the L group (RPS = 9%). The bacteria composition analysis showed that the abundance of Proteobacteria were significantly higher in group R and L than in group C, and the Bacteroidetes were significantly higher in group C than in group R and L, whereas the numbers of Chloroflexi were significantly higher in group R than in group C and L. The bacterial community difference analysis revealed that the harmful bacteria such as genus of Vibrio, Tenacibaculu and Thalassobius were decreased and the beneficial bacterium as Ruegeria and Lactobacillus were increased in R group, whereas this phenomenon were not found in L group. Taken together, above results indicating that the surface proteins were indispensable for L. pentosus HC-2 adhesion and colonization in shrimp intestines to improve intestine condition, enhance immune response, competitively exclude the pathogens, and promote the beneficial bacteria growth to protect the shrimp from pathogens infection. The findings in this work will help to promote the understanding of the roles of probiotics in shrimp intestines displaying probiotic-function by regulating the intestinal bacteria.

The influence of surface proteins on the probiotic effects of Lactobacillus pentosus HC-2 in the Litopenaeus vannamei hepatopancreas.

Our previous work showed that Lactobacillus pentosus HC-2 has high antibacterial and adhesion activity, and as a probiotic could improve the nutrients and immunomodulatory effects in the Litopenaeus vannamei farming. In order to further investigate the influence of HC-2 surface protein on its probiotic effects, the immune and digestion related genes expression and enzymes activities, the colonization numbers of HC-2, and the histologic characteristics were analysis in shrimp hepatopancreas after feeding either the intact surface proteins, the probiotic treated with lithium chloride (LiCl) to remove noncovalently bound surface proteins or no probiotic for four weeks. The results showed that the immune genes expression of lysozyme, proPO, LGBP, Penaeidins-3α, crustin and C-type lectin, the immune enzymes activities of superoxide dismutase, catalase and Alkaline phosphatase, and the digestion enzymes of Trypsin, Lipase and α-Amylase were significantly higher in hepatopancreas of shrimp fed with intact HC-2 than that in shrimp fed with base diet or striped surface proteins HC-2 post feeding and challenge. In addition, the shrimp fed with intact HC-2 leads to the bacteria cells adhesion to hepatopancreas was significantly higher than that in shrimp fed with no surface proteins HC-2. Furthermore, the tissue damages of hepatopancreas caused by pathogenic vibrio were obviously observed in shrimp fed with base diet or no surface proteins HC-2, but no signs of damages were found in shrimp fed with intact HC-2. These results demonstrate that surface proteins are important components for HC-2 to execute probiotic effect that improve hepatopancreas immune response and nutrition digestion to protect shrimp against pathogen damage.

Exploration of the influence of surface proteins on the probiotic activity of Lactobacillus pentosus HC-2 in the Litopenaeus vannamei midgut via label-free quantitative proteomic analysis.

Our previous work showed that using Lactobacillus pentosus HC-2 as a probiotic could improve the growth performance, immune response, gut bacterial diversity and disease resistance of Litopenaeus vannamei. However, the probiotic mechanism had not been fully characterized. In the present study, histology and proteomic analysis were performed to explore the influence of HC-2 surface protein on its probiotic effects on L. vannamei after feeding either the intact surface proteins, the probiotic treated with lithium chloride (LiCl) to remove noncovalently bound surface proteins or no probiotic for four weeks. Histological observation found that feeding with normal HC-2 obviously improved the intestinal histology and enhanced the protective effect against pathogen damage, but feeding with LiCl-treated HC-2 did not improve the intestinal environment. A total of over 2764 peptides and 1118 uniproteins were identified from the L. vannamei midgut; 211 proteins were significantly differentially expressed in the normal HC-2 group compared with the control group; 510 proteins were significantly differentially expressed in the LiCl-treated HC-2 group compared with the control group, and 458 proteins were significantly differentially expressed in the LiCl-treated HC-2 group compared with the normal HC-2 group. GO/KEGG enrichment analysis of the significantly different proteins demonstrated that feeding normal HC-2 mainly induced immune response, metabolic, cell adhesion and cell-cell signaling-related protein upregulation, which contributed to bacterial adhesion and colonization in the midgut to improve the shrimp immune system and growth, but these proteins were suppressed after the shrimp were fed bacteria deprived of surface proteins. Taken together, these results indicate that the surface proteins were indispensable for HC-2 to execute probiotic effects in the shrimp midgut.

A mitochondrial manganese superoxide dismutase involved in innate immunity is essential for the survival of Chlamys farreri.

Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri. The complete cDNA sequence of CfmtMnSOD contained a 681 bp open reading frame (ORF), encoding a peptide of 226 amino acids. A SOD_Fe_N domain and a SOD_Fe_C domain were found in the deduced amino acid sequence of CfmtMnSOD. The mRNA transcripts of CfmtMnSOD were constitutively expressed in all the tested tissues, including gill, gonad, hepatopancreas, hemocytes, mantle and muscle, with the highest expression level in hemocytes. After the stimulation of Vibrio splendidus, Staphylococcus aureus and Yarrowia lipolytica, the mRNA transcripts of CfmtMnSOD in hemocytes all significantly increased. The purified rCfmtMnSOD protein exhibited Mn2+ dependent specific and low stable enzymatic activities. After Vibrio challenge, the cumulative mortality of CfmtMnSOD-suppressed scallops was significantly higher than those of control groups and the semi-lethal time for CfmtMnSOD-suppressed scallops was rather shorter than those of control groups either. Moreover, the final mortality rate of CfmtMnSOD-suppressed group was significant higher than those of control groups, even without Vibrio challenge. All these results indicated that CfmtMnSOD was efficient antioxidant enzyme involved in the innate immunity, and also essential for the survival of C. farreri.

Transcriptomic analysis of exosomal shuttle mRNA in Pacific oyster Crassostrea gigas during bacterial stimulation.

As marine invertebrates, oysters lack adaptive immunity and employ innate immunity as the front line and almost the solo defense mechanism to protect them against invaders. Accumulating research achievements demonstrated that exosomes could act as innate immune effectors that contribute to host defense mechanism. To better understand the immune functions of exosomes in Crassostrea gigas against bacterial stimulation, RNA-Seq was applied to explore the global expression changes of exosomes in oyster after Staphylococcus aureus and Vibrio splendidus stimulation. Totally 171573691 single end raw reads were yielded via Ion Torrent Proton sequencing, which were trimmed into 121988325 clean reads, and then 1505 abundant exosomal shuttle mRNAs (esmRNAs) were identified. Gene ontology (GO) analysis revealed that these abundant esmRNAs could be categorized into 15 cellular components, 12 molecular functions and 21 biological processes, and these abundant esmRNAs were mapped onto 62 biological signaling pathways by KEGG. In total, 68 significant differentially expressed genes (DEGs, Fold change ≥ 2, Q-value < 0.05) were identified between S. aureus stimulated group and control group, including 21 up-regulated and 47 down-regulated ones. While 99 significant DEGs between V. splendidus challenged group and control group were identified, including 42 up-regulated and 57 down-regulated ones. To validate the transcriptomic data, 24 DEGs were randomly selected and confirmed via quantitative real-time PCR (qRT-PCR) and the results showed that their expression patterns agreed well with the RNA-Seq analysis. This study would enrich the C. gigas transcriptome database and provide insight into the immune functions of oyster exosomes against bacterial infection.

A comparative study on oxidative stress response in the hepatopancreas and midgut of the white shrimp Litopenaeus vannamei under gradual changes to low or high pH environment.

White shrimp Litopenaeus vannamei were reared under conditions of gradual changes to a low pH (gradual-low pH, 6.65-8.20) or a high pH (gradual-high pH, 8.20-9.81) versus a normal pH environment (8.14-8.31) during a 28-day period. Survival of shrimp, and ROS production, antioxidant responses and oxidative damage in the hepatopancreas and midgut were investigated. Consequently, shrimp enhanced MnSOD, GPx, and Hsp70 transcripts as early defense mechanism in the hepatopancreas and midgut to scavenge excessive ROS during short-term (≤ 7 days) gradual-low and high pH stress. Meanwhile, the hepatopancreas was more sensitive to ROS than midgut because of earlier ROS production increase, antioxidant response and oxidative damage. Then, suppressed antioxidant response in the hepatopancreas and midgut of shrimp suggested a loss of antioxidant regulatory capacity caused by aggravated oxidative damage after long-term (≥ 14 days) gradual-high pH stress, leading to continuous death. However, enhanced GPx, GST, and Hsp70 transcripts in the hepatopancreas and midgut might be long-term(≥ 14 days) antioxidant adaptation mechanism of shrimp to gradual-low pH stress, which could prevent further ROS perturbation and weaken oxidative damage to achieve a new immune homeostasis, contributing to stable survival rate. Therefore, we have a few insights that it is necessary to protect hepatopancreas for controlling shrimp death under gradual-high pH stress.

Adaptation of the white shrimp Litopenaeus vannamei to gradual changes to a low-pH environment.

pH variation could cause a stress response in euryhaline penaeids, we evaluated the mortality, growth performance, osmoregulation gene expression, digestive enzyme activity, histology, and resistance against Vibrio parahemolyticus of white shrimp Litopenaeus vannamei reared under conditions of gradual changes to a low-pH environment (gradual-low pH, 6.65-8.20) or a high-pH environment (gradual-high pH, 8.20-9.81) versus a normal pH environment (8.14-8.31) during a 28-d experiment. Consequently, under gradual-high pH, the cumulative mortality rate (CMR) rose with time until 39.9% on days 28; the weight gain percentage (WGP) and length gain percentage (LGP) decreased continuously. However, under gradual-low pH, the CMR of shrimp stabilized at 6.67% during 7-28 d; the WGP and LGP decreased first and then returned to normal. These results indicated that L. vannamei displayed a moderate tolerance to gradual-low pH, compared with gradual-high pH. Under gradual-low pH, the Na+/K+-ATPase, cytoplasmic carbonic anydrase (CAc), and glycosyl-phosphatidylinositol-linked carbonic anhydrase (CAg) transcripts of shrimp increased continuously or then back to normal; the amylase, lipase, and trypsin activities decreased first and then returned to normal or increased; the hepatopancreases and midguts showed histopathological lesions first and then got remission. Thus, the major adaptation mechanism of shrimp to gradual-low pH might be its high osmoregulation ability, which made shrimp achieve a new, balanced steady-state, then promoted longer intestinal villi and recuperative hepatopancreases of shrimp with enhanced digestive enzyme activities to increase nutrient absorption after long-term exposure. Meanwhile, the enhanced resistance against V. parahemolyticus under gradual-low pH would probably inhibit disease outbreak in the shrimp farming.

Transcriptomic and morphological analyses of Litopenaeus vannamei intestinal barrier in response to Vibrio paraheamolyticus infection reveals immune response signatures and structural disruption.

The white shrimp Litopenaeus vannamei has been greatly impacted by Vibrio infection. In this study, we investigated the intestinal barrier response of L vannamei following challenge with Vibrio parahaemolyticus E1, by examining morphological changes and transcriptome expression levels. A total of 16,4420 unigenes were obtained from RNAseq data after quality control and assembly, and 4646 differentially expressed genes (DEGs) were identified following Vibrio challenge, of which 2469 unigenes were significantly up-regulated and 2177 were significantly down-regulated. DEGs were determined to be involved in various physical, chemical and immunological intestinal barrier functions, including peritrophin, cytoskeleton and cell junction, pattern recognition receptors, antimicrobial peptide and immune signaling pathways, serine protease/protease inhibitor and prophenoloxidase system, apoptosis and phagocytosis, and antioxidant systems. Fifteen DEGs were randomly selected for validation by real-time quantitative PCR (RT-qPCR) and showed results consistent with the RNA-seq data. Intestinal epithelial cell morphology was also affected by Vibrio challenge, showing epithelial detachment, nuclear pyknosis, and destruction of cell junctions. These results improve our current understanding of the intestinal barrier function in the shrimp response to bacterial infection.

A new method to evaluate the effects of bacterial dosage, infection route and Vibrio strain in experimental challenges of Litopenaeus vannamei, based on the Cox proportional hazard model.

In the shrimp challenge test the Vibrio dosage, infection route, and strain are considered as risk factors that result in mortality. Assessment of Vibrio/shrimp interactions, and disease dynamics following infection by Vibrio, are useful techniques needed for detailed studies on the control of risk factors. In this paper we used an application of the Cox proportional hazard model to assess relative survival probability, estimate mortality risk, and construct a prognostic model to assess predictions of estimated time to death. Results indicate that infection route was the most important prognostic factor contributing to mortality in the challenge test (ß = 3.698, P < 0.000). The shrimp infection rate following injection was found to be 40.4 times greater than that following immersion (hazard ratio (HR) = 40.4; p = 0.000). Our results also indicated that the HR resulting in shrimp mortality following a high dose of 10(8) cfu/shrimp was significantly greater (HR = 5.9, P < 0.000), than that following a baseline dosage of 10(7) cfu/shrimp. Strain Vh was found to be more virulent than Strain Vp (HR = 4.8; P < 0.000). The prognostic index also indicated that the infection route is the most important prognostic factor contributing to mortality in the challenge test.

The transcriptomic response to copper exposure in the digestive gland of Japanese scallops (Mizuhopecten yessoensis).

The present study was conducted to elucidate the effects of copper exposure on the immune system and lipid metabolism of the Japanese scallop, Mizuhopecten yessoensis. Transcriptional levels of differentially expressed genes (DEGs)in M. yessoensis digestive gland tissue were analyzed using the deep-sequencing platform Illumina HiSeq™ 2000. In total, 841 and 877 genes were identified as significantly up- or down-regulated, respectively. In addition, significant enrichment analysis identified 3 gene ontology terms and 15 pathways involved in the response to copper exposure. Analysis of transcripts related to the immune response revealed a complex pattern of innate recognition receptors, including toll-like receptors, NOD-like receptors and downstream pathway effectors, including those involved in apoptosis. Furthermore, genomic analysis revealed that genes involved in extracellular matrix (ECM)-receptor interactions were enriched in Cu-exposed scallop glands. These results will provide a resource for subsequent gene expression studies regarding heavy metal exposure and the identification of copper-sensitive biomarkers for the aquaculture of M. yessoensis.

The transcriptomic response to copper exposure by the gill tissue of Japanese scallops (Mizuhopecten yessoensis) using deep-sequencing technology.

The bivalve Mizuhopecten yessoensis has been greatly impacted by marine pollutants in northern China. To elucidate the toxicological mechanism of copper exposure on the immune system, we investigated differentially expressed genes (DEGs) and transcript abundance in M. yessoensis gill tissue using the deep-sequencing platform Illumina HiSeq™ 2000. In total, 1312 and 2237 genes were identified as significantly up- or down-regulated, respectively. In addition, significant enrichment analysis identified 9 GO terms and 38 pathways involved in the response to copper exposure. The analysis of immune-related transcripts revealed a complex repertoire of innate recognition receptors, including toll-like receptors, NOD-like receptors and RIG-like receptors. Downstream pathway effectors, such as apoptotic, lysosomal and C-type lectin transcripts, were also analyzed. These results will provide a resource for subsequent gene expression studies regarding heavy metal exposure and the identification of copper-sensitive biomarkers to monitor the aquaculture of M. yessoensis.

Identification and Expression Analysis of an Interacting Protein (LvFABP) that Mediates Vibrio parahaemolyticus AHPND Toxin Action.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus causing AHPND (VPAHPND) is the most serious disease affecting shrimp farming. The PirAvp and PirBvp toxins of VPAHPND are known virulence factors. However, the corresponding target protein in shrimp that mediates their action has not been identified. By screening yeast two-hybrid cDNA libraries from intestine, stomach, and hepatopancreas of Litopenaeus vannamei, the protein with the largest increase in gene expression in shrimp hepatopancreas in response to VPAHPND challenge was identified and designated LvFABP. Analysis revealed high sequence homology of the LvFABP gene and a lipocalin/cytosolic fatty acid binding gene. Yeast two-hybrid pairwise analysis, GST-pull down assay, and far-western blot assay were performed to determine the interaction between LvFABP and PirBvp. LvFABP was able to directly bind to PirBvp. The expression of LvFABP in the hepatopancreas was significantly higher at P23 and P27 developmental stages of L. vannamei. RNA interference (RNAi) of LvFABP reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the intestine, stomach, and hepatopancreas after VPAHPND challenge. We concluded that the LvFABP was involved in AHPND pathogenesis and acted as a VPAHPND toxin interacting protein. This is the first identification of VPAHPND toxin interacting protein from the shrimp digestive system by yeast two-hybrid library screening and were confirmed by in vitro protein interaction verification and in vivo challenge experiments. This study provides novel insight into the contributions of LvFABP towards AHPND pathogenesis in shrimp. The findings could inform AHPND preventative measures in shrimp farming.

Characterization and function analysis of a Kazal-type serine proteinase inhibitor in the red claw crayfish Cherax quadricarinatus.

Kazal-type serine proteinase inhibitors (KPIs) function in physiological and immunological processes requiring proteinase action. In the present study, the first Cherax quadricarinatus KPI gene (designated CqKPI) was identified and characterized. The open reading frame of CqKPI contains 405 nucleotides and encodes a protein of 134 amino acids. CqKPI has two Kazal domains comprising 44 amino acid residues with the conserved amino acid sequence C-X3-C-X7-C-X6-Y-X3-C-X6-C-X12-C. Each Kazal domain has six conserved cysteine residues, which can form a structural conformation of three pairs of disulfide bonds stabilizing the Kazal domain. CqKPI exhibited high similarity with previously identified KPIs from crayfish hemocytes. The results of tissue distribution showed that CqKPI had the highest expression level in hemocytes, and this was in agreement with phylogenic relationships. Recombinant CqKPI (rCqKPI) was heterologously expressed in Escherichia coli and purified for further study. The proteinase inhibition assays suggested that rCqKPI could potently inhibit elastase and weakly inhibit trypsin, subtilisin A, and proteinase K, but not α-chymotrypsin. It can firmly bind to Bacillus hwajinpoensis, Staphylococcus aureus, and Vibrio parahaemolyticus, with weak binding to Candida albicans. In addition, CqKPI inhibited bacterial secretory proteinase activity and inhibited the growth of B. hwajinpoensis and C. albicans. These data suggest that CqKPI might be involved in anti-bacterial immunity, acting as an inhibitor of the proteinase cascade in the resistance to invasion of pathogens.