Stochastic determination of the spatial variation of potentially pathogenic bacteria communities in a large subtropical river.

Understanding the composition and assembly mechanism of waterborne pathogen is essential for preventing the pathogenic infection and protecting the human health. Here, based on 16S rRNA sequencing, we investigated the composition and spatial variation of potentially pathogenic bacteria from different sections of the Pearl River, the most important source of water for human in Southern China. The results showed that the potential pathogen communities consisted of 6 phyla and 64 genera, covering 11 categories of potential pathogens mainly involving animal parasites or symbionts (AniP), human pathogens all (HumPA), and intracellular parasites (IntCelP). Proteobacteria (75.87%) and Chlamydiae (20.56%) were dominant at the phylum level, and Acinetobacter (35.01%) and Roseomonas (8.24%) were dominant at the genus level. Multivariate analysis showed that the potential pathogenic bacterial community was significantly different among the four sections in the Pearl River. Both physicochemical factors (e.g., NO3-N, and suspended solids) and land use (e.g., urban land and forest) significantly shaped the pathogen community structure. However, spatial effects contributed more to the variation of pathogen community based on variation partitioning and path analysis. Null model based normalized stochasticity ratio analysis further indicated that the stochastic process rather than deterministic process dominated the assembly mechanisms by controlling the spatial patterns of potential pathogens. In conclusion, high-throughput sequencing shows great potential for monitoring the potential pathogens, and provided more comprehensive information on the potentially pathogenic community. Our study highlighted the importance of considering the influences of dispersal-related processes in future risk assessments for the prevention and control of pathogenic bacteria.

Deep Insights into Gut Microbiota in Four Carnivorous Coral Reef Fishes from the South China Sea.

Investigations of gut microbial diversity among fish to provide baseline data for wild marine fish, especially the carnivorous coral reef fishes of the South China Sea, are lacking. The present study investigated the gut microbiota of four carnivorous coral reef fishes, including Oxycheilinus unifasciatus, Cephalopholis urodeta, Lutjanus kasmira, and Gnathodentex aurolineatus, from the South China Sea for the first time using high-throughput Illumina sequencing. Proteobacteria, Firmicutes, and Bacteroidetes constituted 98% of the gut microbiota of the four fishes, and 20 of the gut microbial genera recovered in this study represent new reports from marine fishes. Comparative analysis indicated that the four fishes shared a similar microbial community, suggesting that diet type (carnivorous) might play a more important role in shaping the gut microbiota of coral reef fishes than the species of fish. Furthermore, the genera Psychrobacter, Escherichia-Shigella, and Vibrio constituted the core microbial community of the four fishes, accounting for 61-91% of the total sequences in each fish. The lack of the genus Epulopiscium in the four fishes was in sharp contrast to what has been found in coral reef fishes from the Red Sea, in which Epulopiscium was shown to be the most dominant gut microbial genus in seven herbivorous coral reef fishes. In addition, while unique gut microbial genera accounted for a small proportion (8-13%) of the total sequences, many such genera were distributed in each coral reef fish species, including several genera (Endozoicomonas, Clostridium, and Staphylococcus) that are frequently found in marine fishes and 11 new reports of gut microbes in marine fishes. The present study expands our knowledge of the diversity and specificity of gut microbes associated with coral reef fishes.

Transcriptome analysis revealed changes of multiple genes involved in muscle hardness in grass carp (Ctenopharyngodon idellus) fed with faba bean meal.

An 8-week feeding trial and transcriptome analysis were conducted to investigate the potential mechanism of muscle-hardening caused by faba bean in grass carp (Ctenopharyngodon idellus). Ordinary grass carp (fed with practical diet) and crisp grass carp (fed with faba bean meal) groups were designed. Lower water holding capacity and higher some texture parameters were observed in the muscle of crisp grass carp compared with another group. 19.62 GB clean reads were generated, and total 1354 genes exhibiting differentially expression were identified (FDR < 0.05). Genes function enrichment revealed up-regulated genes in crisp grass carp mainly in response to myofibroblast proliferation, while down-regulated genes in response to immune regulation. Consistent with this, the tight junction pathway and the NF-κB signaling pathway were likewise significantly enriched. In summary, this study identified several candidate genes and putative signaling pathways deserving further investigation to the mechanism of muscle-hardening in fish fed with faba bean.

Intestinal Microbiota of Grass Carp Fed Faba Beans: A Comparative Study.

Many reports of the intestinal microbiota of grass carp have addressed the microbial response to diet or starvation or the effect of microbes on metabolism; however, the intestinal microbiota of crisp grass carp has yet to be elucidated. Moreover, the specific bacteria that play a role in the crispiness of grass carp fed faba beans have not been elucidated. In the present study, 16S sequencing was carried out to compare the intestinal microbiota in the fore-, mid- and hind-intestine segments of grass carp following feeding with either faba beans or formula feed. Our results showed that (1) the hind-intestine presented significant differences in diversity relative to the fore- or midintestine and (2) faba beans significantly increased the diversity of intestinal microbiota, changed the intestinal microbiota structure (Fusobacteria was reduced from 64.26% to 18.24%, while Proteobacteria was significantly increased from 17.75% to 51.99%), and decreased the metabolism of energy, cofactors and vitamins in grass carp. Furthermore, at the genus and species levels, Acinetobacter accounted for 15.09% of the microbiota, and Acinetobacter johnsonii and Acinetobacter radioresistens constituted 3.41% and 2.99%, respectively, which indicated that Acinetobacter of the family Moraxellaceae contributed to changes in the intestinal microbiota structure and could be used as a potential biomarker. These results may provide clues at the intestinal microbiota level to understanding the mechanism underlying the crispiness of grass carp fed faba beans.

Cross protection against the watermelon strain of Papaya ringspot virus through modification of viral RNA silencing suppressor.

Papaya ringspot virus watermelon strain (PRSV-W) causes huge economic losses to cucurbits production. Here, we constructed an infectious clone of PRSV-W, pCamPRSV-W, which can induce similar symptoms and accumulate to same levels as wild type virus in plants of Cucurbita pepo, Cucumis melo, Citrullus lanatus and Cucumis sativus. The green fluorescence protein gene gfp was cloned into pCamPRSV-W to produce pCamPRSV-W-GFP, which produced strong green fluorescence in systemic leaves of inoculated Cucurbita pepo, Cucumis melo, Citrullus lanatus and Cucumis sativus plants, indicating that pCamPRSV-W can be used to express foreign genes. Ten mutants of PRSV-W, obtained by site-directed mutagenesis in the RNA silencing suppressor helper-component proteinase encoding region, produced dramatically attenuated symptoms in plants of Cucumis melo. The Cucumis melo plants pre-infected with mutants K125D and G317 K showed effective protection against the challenge inoculation of wild type PRSV-W. The attenuated mutants generated in this study will be helpful for the eco-friendly control of PRSV-W.

Regulation of IL-17 by lncRNA of IRF-2 in the pearl oyster.

Long noncoding RNAs (lncRNAs), once thought to be nonfunctional, have recently been shown to participate in the multilevel regulation of transcriptional, posttranscriptional and epigenetic modifications and to play important roles in various biological processes, including immune responses. However, the expression and roles of lncRNAs in invertebrates, especially nonmodel organisms, remain poorly understood. In this study, by comparing a transcriptome to the PfIRF-2 genomic structure, we identified lncIRF-2 in the PfIRF-2 genomic intron. The results of the RNA interference (RNAi) and the nucleus grafting experiments indicated that PfIRF-2 might have a negative regulatory effect on lncIRF-2, and PfIRF-2 and lncIRF-2 may have a positive regulatory effect on PfIL-17. Additionally, lncIRF-2, PfIRF-2 and PfIL-17 were involved in responses to the nucleus graft. These results will enhance the knowledge of lncIRF-2, IRF-2, and IL-17 functions in both pearl oysters and other invertebrates.

Phylogenetic analysis and antifouling potentials of culturable fungi in mangrove sediments from Techeng Isle, China.

To search for more microbial resources for screening environment-friendly antifoulants, we investigated the phylogenetic diversity and antifouling potentials of culturable fungi in mangrove sediments from Techeng Isle, China. A total of 176 isolates belonging to 57 fungal taxa were recovered and identified. The high levels of diversity and abundance of mangrove fungi from Techeng Isle were in accordance with previous studies on fungi from other mangrove ecosystems. Fifteen of the 176 isolates demonstrated high divergence (87-93%) from the known fungal taxa in GenBank. Moreover, 26 isolates recorded in mangrove ecosystems for the first time. These results suggested that mangrove sediments from Techeng Isle harbored some new fungal communities compared with other mangrove ecosystems. The antifouling activity of 57 representative isolates (belonging to 57 different fungal taxa) was tested against three marine bacteria (Loktanella hongkongensis, Micrococcus luteus and Pseudoalteromonas piscida) and two marine macrofoulers (bryozoan Bugula neritina and barnacle Balanus amphitrite). Approximately 40% of the tested isolates displayed distinct antifouling activity. Furthermore, 17 fungal isolates were found to display strong or a wide spectrum of antifouling activity in this study, suggesting that these isolates deserve further study as potential sources of novel antifouling metabolites. To our knowledge, this is the first report on the investigation of the phylogenetic diversity and antifouling potential of culturable fungi in mangrove sediments from Techeng Isle, China. These results contribute to our knowledge of mangrove fungi and further increases the pool of fungi available for natural bioactive product screening.

A PDGF/VEGF homologue provides new insights into the nucleus grafting operation and immune response in the pearl oyster Pinctada fucata.

The platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF, PVF) family of proteins have been implicated in a wide range of biological functions in vertebrates, including cell proliferation, cell differentiation, cell migration, neural development and especially angiogenesis/vasculogenesis. In this study, a PVF gene, belonging to the PDGF/VEGF family, was cloned and characterized from Pinctada fucata. It contained an ORF of 1110bp encoding a putative protein of 369 amino acids. The deduced amino acid sequence presented the typical structural features of PDGF family members and the N-terminal signal peptide for secretion. Comparative phylogenetic analysis revealed that PfPVF shows relatively high identity with other invertebrate PVF homologues. Furthermore, gene expression analysis revealed that PfPVF is involved in not only the nucleus grafting operation and but also the response to immune stimulation. The study may help to increase understanding of the functions of molluscan PVF.

Cloning and gene expression of signal transducers and activators of transcription (STAT) homologue provide new insights into the immune response and nucleus graft of the pearl oyster Pinctada fucata.

The signal transducers and activators of the transcription (STAT) family play an important role in regulatory and cellular functions by regulating the expression of a variety of genes, including cytokines and growth factors. In the present study, a Pinctada fucata STAT protein, termed PfSTAT, was described. The deduced amino acid sequence of PfSTAT contains the conserved STAT_bind domain and the SH2 domain, and the additional Bin/Amphiphysin/Rvs (BAR) domain, but does not have STAT_alpha and STAT_int domains. Multiple sequence alignments revealed that PfSTAT showed relatively low identity with vertebrate and other invertebrate STATs, and phylogenetic analysis indicated that the evolution of STAT may have been more complex and ancient. Gene expression analysis revealed that PfSTAT is involved in the immune response to polyinosinic-polycytidylic acid (poly I:C) stimulation and in the nucleus insertion operation. This study contributes to a better understanding of PfSTAT in protecting the pearl oyster from disease or injury caused by grafting.

Comparative and Evolutionary Analysis of the Interleukin 17 Gene Family in Invertebrates.

Interleukin 17 (IL-17) is an important pro-inflammatory cytokine and plays critical roles in the immune response to pathogens and in the pathogenesis of inflammatory and autoimmune diseases. Despite its important functions, the origin and evolution of IL-17 in animal phyla have not been characterized. As determined in this study, the distribution of the IL-17 family among 10 invertebrate species and 7 vertebrate species suggests that the IL-17 gene may have originated from Nematoda but is absent from Saccoglossus kowalevskii (Hemichordata) and Insecta. Moreover, the gene number, protein length and domain number of IL-17 differ widely. A comparison of IL-17-containing domains and conserved motifs indicated somewhat low amino acid sequence similarity but high conservation at the motif level, although some motifs were lost in certain species. The third disulfide bond for the cystine knot fold is formed by two cysteine residues in invertebrates, but these have been replaced by two serine residues in Chordata and vertebrates. One third of invertebrate IL-17 proteins were found to have no predicted signal peptide. Furthermore, an analysis of phylogenetic trees and exon-intron structures indicated that the IL-17 family lacks conservation and displays high divergence. These results suggest that invertebrate IL-17 proteins have undergone complex differentiation and that their members may have developed novel functions during evolution.

Nuclear factor of activated T cells (NFAT) in pearl oyster Pinctada fucata: molecular cloning and functional characterization.

Nuclear factor of activated T cells (NFAT) plays an important role in nonimmune cells and also in T cells and many other cells of the immune system, by regulating the expression of a variety of genes involved in the immune response, organ development, developmental apoptosis and angiogenesis. In the present study, the NFAT homology gene, PfNFAT, from the pearl oyster Pinctada fucata was cloned and its genomic structure and promoter were analyzed. PfNFAT encodes a putative protein of 1226 amino acids, and contains a highly conserved Rel homology region (RHR) with DNA-binding specificity, and a regulatory domain (NFAT homology region, NHR) containing a potent transactivation domain (TAD). The PfNFAT gene consists of 12 exons and 11 introns, and its promoter contains potential binding sites for transcription factors such as NF-κB (Nuclear factor κB), STATx (signal transducer and activator of transcription), AP-1 (activator protein-1) and Sox-5/9 (SRY type HMG box-5/9), MyoD (Myogenic Differentiation Antigen) and IRF (Interferon regulatory factor). Comparison and phylogenetic analysis revealed that PfNFAT shows high identity with other invertebrate NFAT, and clusters with the NFAT5 subgroup. Furthermore, gene expression analysis revealed that PfNFAT is involved in the immune response to lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (poly I:C) stimulation and in the nucleus inserting operation. The study of PfNFAT may increase understanding of molluscan innate immunity.

The shrimp IKK-NF-κB signaling pathway regulates antimicrobial peptide expression and may be subverted by white spot syndrome virus to facilitate viral gene expression.

The IκB kinases IKKα and IKKß and the IKK-related kinases TANK-binding kinase 1 (TBK1) and IKKε are the master regulators of the NF-κB signaling pathway. Although this pathway has been extensively studied in mammals, less attention has been paid in crustaceans, which have significant economic value. Here, we report the cloning and functional studies of two IKK homologs, LvIKKß and LvIKKε, from Pacific white shrimp, Litopenaeus vannamei. LvIKKß and LvIKKε mRNAs are widely expressed in different tissues and are responsive to white spot syndrome virus (WSSV) infection. When overexpressed in Drosophila S2 cells, LvIKKß but not LvIKKε activates the promoters of NF-κB pathway-controlled antimicrobial peptide genes (AMPs), such as the Penaeidins (PENs). In HEK 293T cells, both LvIKKß and LvIKKε activate an NF-κB reporter. The silencing of LvIKKß or LvIKKε using double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) decreases the expression of L. vannamei AMPs, including PENs, lysozyme and crustins. Intriguingly, LvIKKß- or LvIKKε-silenced L. vannamei are resistant to WSSV infection. We hypothesized that successful infection with WSSV requires the activation of the IKK-NF-κB signaling pathway to modulate viral gene expression. We constructed luciferase reporters for 147 WSSV genes. By screening, we found that the WSV051, WSV059, WSV069, WSV083, WSV090, WSV107, WSV244, WSV303, WSV371 and WSV445 promoters can be activated by LvIKKß or LvIKKε in Drosophila S2 cells. Taken together, our results reveal that LvIKKß and LvIKKε may participate in the regulation of shrimp AMPs and that WSSV may subvert the L. vannamei IKK-NF-κB signaling pathway to facilitate viral gene expression.

Molecular characterization of interferon regulatory factor 2 (IRF-2) homolog in pearl oyster Pinctada fucata.

Interferon regulatory factors (IRFs) control many facets of the innate and adaptive immune responses, regulate the development of the immune system itself and involve in reproduction and morphogenesis. In the present study, the IRF-2 homology gene, PfIRF-2 from pearl oyster Pinctada fucata was cloned and its genomic structure and promoter were analyzed. PfIRF-2 encodes a putative protein of 350 amino acids, and contains a highly conserved N-terminal DNA-binding domain and a variable C-terminal regulatory domain. Comparison and phylogenetic analysis revealed that PfIRF-2 shared a relatively higher identity with other mollusk but relatively lower identity with vertebrate IRF-2, and was clustered with IRF-1 subfamily composed of IRF-2 and IRF-1. Furthermore, gene expression analysis revealed that PfIRF-2 involved in the immune response to LPS and poly(I:C) stimulation. Immunofluorescence assay showed that the expressed PfIRF-2 was translocated into the nucleus and dual-luciferase reporter assays indicated that PfIRF-2 could involved and activate interferon signaling or NF-κB signal pathway in HEK293 cells. The study of PfIRF-2 may help better understand the innate immune in mollusk.

Interleukin-17 in pearl oyster (Pinctada fucata): molecular cloning and functional characterization.

IL-17 from pearl oyster Pinctada fucata, one of mollusk, was identified and characterized, and its genomic structure and promoter were analyzed. The full-length cDNA of P. fucata IL-17 (PfIL-17) is 907 bp with an open reading frame of 585 bp encoding a putative protein of 194 amino acids. The deduced PfIL-17 contains a 19 amino acid signal peptide and a conserved IL-17 domain. Multiple sequence alignments and phylogenetic analysis revealed that PfIL-17 has lower similarity with other invertebrate IL-17 and was clustered with CgIL-17, but not clustered with other invertebrate IL-17. Gene expression analysis indicated that PfIL-17 took part in the immune response to LPS and poly(I:C) stimulation, and dual-luciferase reporter assays showed that PfIL-17 could active vertebrate target genes containing the NF-κB binding site and involve NF-κB signal pathway in HEK293 cells. Combined with the results mentioned above, it is suggested that PfIL-17 might involve and activate NF-κB signal pathway against extracellular pathogens.

Gene cloning and function analysis of cytokine-induced suppressor of cytokine signaling (SOCS) from pearl oyster Pinctada fucata.

Cytokine-induced suppressor of cytokine signaling (SOCS) family acts as a negative regulator of cytokine receptor signaling to control excessive cytokine effects and inhibit a variety of signal transduction pathways, particularly the Janus kinases/signal transducers and activators of transcription (JAK/STAT) pathway. In present study, SOCS-2 homolog (PfSOCS-2) from pearl oyster Pinctada fucata was cloned and its gene has no intron. Multiple sequence alignments and phylogenetic analysis showed that PfSOCS-2 was clustered with other mollusk SOCS-2. LPS or polyI:C challenge and gene expression analysis revealed that PfSOCS-2 involved the innate immune response against bacterial and viral infections and that induction of PfSOCS-2 was varied with the different challenge stimulations. Furthermore, Dual-luciferase reporter assays showed that PfSOCS-2 involved in the regulation of vertebrate target genes containing the IFN-stimulated response element or NF-κB binding site in vitro. These results indicated that SOCS-2 from P. fucata plays a regulatory role against the stimulation.

Gigabase-scale transcriptome analysis on four species of pearl oysters.

Pearl oysters have been found to secrete nacre and form pearls with good quality and significant commercial interest. However, the transcriptomic and genomic resources for pearl oysters are still limited. To improve this situation, transcriptome sequencing was conducted from four species of pearl oysters with Illumina HiSeq™ 2000. There were four gigabase-scale transcriptomes for four species of pearl oysters, ∼26.3 million reads with ∼2.37 gigabase base pairs (Gbp) in Pinctada fucata, ∼26.5 million reads with ∼2.39 Gbp in Pinctada margaritifera, ∼27.0 million reads with ∼2.43 Gbp in Pinctada maxima, and ∼25.9 million reads with ∼2.33 Gbp in Pteria penguin, respectively. After sequence assembly and blastx alignment, the numbers of annotated unigenes ≥200 bp were 33,882 in P. fucata, 30,666 in P. margaritifera, 26,420 in P. maxima, and 29,928 in P. penguin. Based on these annotated unigenes among four species of pearl oysters, CDSs were extracted and predicted and furthermore, analyses of GO and KEGG assignments were performed. In addition, 60 putative genes of growth factors and their receptors from four species of pearl oysters were predicted. This study established an excellent resource for gene discovery and expression in pearl oysters, but also offered a significant platform for functional genomics and comparative genomic studies for mollusks.

Molecular cloning, characterization and expression analysis of tumor necrosis factor receptor-associated factor 3 (TRAF3) from pearl oyster Pinctada fucata.

TRAF3 is a highly versatile regulator that negatively regulates JNK and alternative nuclear factor-κB signalling, but positively controls type I interferon production. To investigate TRAF3 function in innate immune responses among invertebrate especially mollusk, we characterized TRAF3 (PfTRAF3) from pearl oyster Pinctada fucata, one of the most important bivalve mollusks for seawater pearl production. PfTRAF3 cDNA is 2261 bp with an open reading frame of 1623 bp encoding a putative protein of 541 amino acids. The deduced PfTRAF3 contains a RING finger domain, two TRAF domains with zinc finger domains and a conserved C-terminal meprin and TRAF homology (MATH) domain. Comparison and phylogenetic analysis revealed that PfTRAF3 from mollusk shared a higher identity with Ciona intestinalis TRAF3 from urochordata, Branchiostoma belcheri TRAF3 from cephalochordate, and even TRAF3 from vertebrate than with insect homologues. Furthermore, gene expression analyses suggested that PfTRAF3 was involved in the immune response to Vibrio alginolyticus.

Molecular cloning and characterization of class I NF-κB transcription factor from pearl oyster (Pinctada fucata).

NF-κB transcription factors play central roles in many important physiological and pathological processes including innate immune responses. Here we report the cloning of an NF-κB transcription factor, PfRelish from pearl oyster Pinctada fucata, one of the most important bivalve mollusks for seawater pearl production. PfRelish full-length cDNA is 3916 bp with an open reading frame of 3558 bp encoding a putative protein of 1186 amino acids. The deduced PfRelish contains a N-terminal RHD, a nucleus localization signal, an IκB-like domain with six ankyrin repeats and a death domain at the C-terminus, which is similar to class I NF-κB transcription factors. Comparison and phylogenetic analysis revealed that class I NF-κBs in mollusks including PfRelish might have most distant relationship to the arthropod Relish. Further expression analysis showed that PfRelish was apparently upregulated after Vibrio alginolyticus injection, which suggested that PfRelish was involved in the immune response to V. alginolyticus.

Cloning and characterization of a shrimp ML superfamily protein.

ML superfamily proteins, including MD-1, MD-2, Niemann-Pick type C2 (Npc2) protein, GM2 activator protein, phosphatidylinositol/phosphatidylglycerol transfer protein (PG/PI-TP) and mite allergen Der p 2, bind to specific lipids and play important roles in lipid-recognition and metabolism. Among these ML (MD-2-related lipid-recognition) proteins, MD-2 is essential for lipopolysaccharide (LPS) signaling and the following secretion of proinflammatory factors. In this report, we identified the cDNA and gene of an ML protein from an important white shrimp Litopenaeus vannamei and named it LvML. The gene consists of four exons and three introns. The putative LvML contains 6 cysteines which may form 3 disulfide bonds that are conserved in ML proteins. Reverse transcription PCR analysis showed that in the examined tissues LvML mRNA is only expressed in the hepatopancreas, while not in hemocytes, eyestalk, gill, heart, stomach, intestine, nerve core, muscle or pyloric caecum. Its expression is positively regulated after injection of LPS. Then enzyme-linked immunosorbent assay showed that the recombinant LvML possessed activity of binding to LPS, and that the binding was inhibited by pre-incubation with LPS. We suggested that the LvML may play roles in the shrimp innate immunity.

Two Litopenaeus vannamei HMGB proteins interact with transcription factors LvSTAT and LvDorsal to activate the promoter of white spot syndrome virus immediate-early gene ie1.

White spot syndrome virus (WSSV) has caused great economic damage to shrimp aquaculture. Previous studies have shown that WSSV successfully usurps the immunity system of the host for its own gene regulation. To investigate the role of shrimp high mobility group box (HMGB) proteins in WSSV gene regulation, two Litopenaeus vannamei HMGB genes, LvHMGBa and LvHMGBb, were isolated by rapid amplification of cDNA ends (RACE). Recombinant LvHMGBa/b proteins were present in the nucleus of transfected Drosophila Schneider 2 (S2) cells. Luciferase reporter assays revealed that LvHMGBa/b upregulated the WSSV immediate-early (IE) gene (ie1) in a NF-κB and STAT binding site-dependent manner. GST pull-down assays demonstrated that LvHMGBa/b interacted with L. vannamei Dorsal (LvDorsal) and L. vannamei STAT (LvSTAT), respectively. LvHMGBa was highly expressed in hepatopancreas while HMGBb was highly expressed in stomach, intestine, heart, antennal gland, and epidermis. Moreover, an immune challenge assay demonstrated that the expression of LvHMGBa/b was upregulated by WSSV infection and that both mRNAs reached peak values at 24 h post-infection. To our knowledge, this is the first report that invertebrate HMGB proteins participates in viral gene regulation.