Crustin Defense against Vibrio parahaemolyticus Infection by Regulating Intestinal Microbial Balance in Litopenaeus vannamei.

Crustins are a kind of antimicrobial peptide (AMP) that exist in crustaceans. Some crustins do not have direct antimicrobial activity but exhibit in vivo defense functions against Vibrio. However, the underlying molecular mechanism is not clear. Here, the regulatory mechanism was partially revealed along with the characterization of the immune function of a type I crustin, LvCrustin I-2, from Litopenaeus vannamei. LvCrustin I-2 was mainly detected in hemocytes, intestines and gills and was apparently up-regulated after Vibrio parahaemolyticus infection. Although the recombinant LvCrustin I-2 protein possessed neither antibacterial activity nor agglutinating activity, the knockdown of LvCrustin I-2 accelerated the in vivo proliferation of V. parahaemolyticus. Microbiome analysis showed that the balance of intestinal microbiota was impaired after LvCrustin I-2 knockdown. Further transcriptome analysis showed that the intestinal epithelial barrier and immune function were impaired in shrimp after LvCrustin I-2 knockdown. After removing the intestinal bacteria via antibiotic treatment, the phenomenon of impaired intestinal epithelial barrier and immune function disappeared in shrimp after LvCrustin I-2 knockdown. This indicated that the impairment of the shrimp intestine after LvCrustin I-2 knockdown was caused by the dysbiosis of the intestinal microbiota. The present data suggest that crustins could resist pathogen infection through regulating the intestinal microbiota balance, which provides new insights into the functional mechanisms of antimicrobial peptides during pathogen infection.

LvCD14L Acts as a Novel Pattern Recognition Receptor and a Regulator of the Toll Signaling Pathway in Shrimp.

Leucine-rich repeat (LRR) is a structural motif has important recognition function in immune receptors, such as Tolls and NOD-like receptors (NLRs). The immune-related LRR proteins can be divided into two categories, LRR-containing proteins and LRR-only proteins. The latter contain LRR motifs while they are without other functional domains. However, the functional mechanisms of the LRR-only proteins were still unclear in invertebrates. Here, we identified a gene encoding a secretory LRR-only protein, which possessed similarity with vertebrate CD14 and was designated as LvCD14L, from the Pacific whiteleg shrimp Litopenaeus vannamei. Its transcripts in shrimp hemocytes were apparently responsive to the infection of Vibrio parahaemolyticus. Knockdown of LvCD14L with dsRNA resulted in significant increase of the viable bacteria in the hepatopancreas of shrimp upon V. parahaemolyticus infection. Further functional studies revealed that LvCD14L could bind to microorganisms' PAMPs, showed interaction with LvToll1 and LvToll2, and regulated the expression of LvDorsal and LvALF2 in hemocytes. These results suggest that LvCD14L functions as a pattern recognition receptor and activates the NF-κB pathway through interaction with LvTolls. The present study reveals a shrimp LvCD14L-Tolls-NF-κB signaling pathway like the CD14/TLR4/NF-κB signaling pathway in mammalians, which enriches the functional mechanism of secretory LRR-only immune receptors during pathogens infection in invertebrates.

A Lymphoid Organ Specific Anti-Lipopolysaccharide Factor from Litopenaeus vannamei Exhibits Strong Antimicrobial Activities.

Different shrimp species are known to possess apparent distinct resistance to different pathogens in aquaculture. However, the molecular mechanism underlying this finding still remains unknown. One kind of important antimicrobial peptides, anti-lipopolysaccharide factors (ALF), exhibit broad-spectrum antimicrobial activities. Here, we reported a newly identified ALF from the shrimp Litopenaeus vannamei and compared the immune function with its counterpart in the shrimp Fenneropenaeus chinensis. The ALF, designated as LvALF8, was specifically expressed in the lymphoid organ of L. vannamei. The expression level of LvALF8 was apparently changed after white spot syndrome virus (WSSV) or Vibrio parahaemolyticus challenges. The synthetic LBD peptide of LvALF8 (LvALF8-LBD) showed strong antibacterial activities against most tested Gram-negative and Gram-positive bacteria. LvALF8-LBD could also inhibit the in vivo propagation of WSSV similar as FcALF8-LBD, the LBD of LvALF8 counterpart in F. chinensis. However, LvALF8-LBD and FcALF8-LBD exhibited apparently different antibacterial activity against V. parahaemolyticus, the main pathogen causing acute hepatopancreatic necrosis disease (AHPND) of affected shrimp. A structural analysis showed that the positive net charge and amphipathicity characteristics of LvALF8-LBD peptide were speculated as two important components for its enhanced antimicrobial activity compared to those of FcALF8-LBD. These new findings may not only provide some evidence to explain the distinct disease resistance among different shrimp species, but also lay out new research ground for the testing and development of LBD-originated antimicrobial peptides to control of shrimp diseases.

Characterization of a gill-abundant crustin with microbiota modulating function in Litopenaeus vannamei.

Gills as the respiratory tissue of crustacean encounter various threats in the environment. The immune defense in gills is essential to the health of shrimp. In the present study, we identified a novel gill-abundant crustin, LvCrustin Ⅱ-1, from the shrimp Litopenaeus vannamei. The full-length open reading frame of LvCrustin Ⅱ-1 is 522 bp, which encodes 173 amino acid residues. The amino acid sequence of LvCrustin Ⅱ-1 contained a signal peptide, a glycine rich region, a cysteine rich region and a whey acidic protein (WAP) domain. The expression level of LvCrustin Ⅱ-1 was significantly up-regulated at different time points after Vibrio parahaemolyticus immersion. When LvCrustin Ⅱ-1 was silenced by dsRNA interference, the count of bacteria colonies increased significantly in the hepatopancreas of shrimp after V. parahaemolyticus immersion, which indicated that the infection progress of pathogenic bacteria was accelerated after LvCrustin Ⅱ-1 knockdown. Compared with the microbiota of seawater, the lower proportion of aquatic bacteria and higher proportion of symbiont in the gills microbiota of shrimp indicated the bacterial colonization was modulated by the host. Knockdown of LvCrustin Ⅱ-1 changed the proportion of some potential pathogens and aquatic bacteria, which supported the idea that the new identified crustin in the gills played important roles in modulation of the microbiota community in shrimp. The present data provided new insights into the multiple functions of crustin in the immunity of shrimp.

Molecular and Functional Diversity of Crustin-Like Genes in the Shrimp Litopenaeus vannamei.

Crustins are crustacean cationic cysteine-rich antimicrobial peptides that contain one or two whey acidic protein (WAP) domain(s) at the carboxyl terminus and mainly show antimicrobial and/or proteinase inhibitory activities. Here, we performed genome and transcriptome screening and identified 34 full-length crustin-like encoding genes in Litopenaeus vannamei. Multiple sequence analysis of the deduced mature peptides revealed that these putative crustins included 10 type Ia, two type Ib, one type Ic, 11 type IIa, three type IIb, four type III, one type IV, one type VI, and one type VII. These putative crustins were clustered into different groups. Phylogenetic analysis, considering their domain composition, showed that different types of crustin-like genes in crustaceans might be originated from the WAP core region, along with sequence insertion, duplication, deletion, and amino acid substitution. Tissue distribution analysis suggested that most crustin-like genes were mainly detected in immune-related tissues while several crustin-like genes exhibited tissue-specific expression patterns. Quantitative PCR analysis on 15 selected crustin-like genes showed that most of them were apparently upregulated after Vibrio parahaemolyticus or white spot syndrome virus (WSSV) infection. One type Ib crustin-like gene, mainly expressed in the ovary, showed the highest expression levels before the gastrula stage and was hardly detected after the limb bud stage, suggesting that it was a maternal immune effector. Collectively, the present data revealed the molecular and functional diversity of crustins and their potential evolutionary routes in crustaceans.

Multiple Isoforms of Anti-Lipopolysaccharide Factors and Their Antimicrobial Functions in the Ridgetail Prawn Exopalaemon carinicauda.

As a kind of antimicrobial peptides (AMP) in crustacean, anti-lipopolysaccharide factors (ALFs) have broad spectrum antimicrobial activities. In the present study, we identified four ALF genes, EcALF2-5, from the ridgetail prawn Exopalaemon carinicauda. Tissue distribution analysis showed that EcALF2 and EcALF4 transcripts were mainly located in gill, epidermis, and stomach, while EcALF3 and EcALF5 were mainly in hemocytes. Peptides corresponding to the LPS binding domain (LBD) of EcALFs were synthesized for analyzing their antimicrobial activities. Minimal inhibitory concentration (MIC) analysis showed that the synthetic LBD peptides of EcALF3 and EcALF4 could inhibit the growth of Gram-positive and Gram-negative bacteria, while the synthetic LBD peptides of EcALF2 and EcALF5 showed antibacterial activity against Vibrio. Incubation of white spot syndrome virus (WSSV) with the synthetic LBD peptides of EcALF3, EcALF4, and EcALF5 could reduce the in vivo viral copy number in WSSV-infected prawns. After silencing of EcALFs, Vibrio exhibited a rapid proliferation in the hepatopancreas of the prawn. The present data showed the important function of different EcALFs in modulating the in vivo bacterial and viral propagation in E. carinicauda. This study will provide new clues into the disease control in aquaculture.

An invertebrate gene encoding a Mab21-containing protein involves in antiviral response through regulating the STING pathway.

The cGAS-STING pathway plays essential roles in detecting cytosolic dsDNA and initiating antiviral and antibacterial responses in vertebrates. However, knowledge about its function in antiviral response of invertebrates is very limited. In the present study, a gene encoding a Mab21-containing protein, a cGAS homologue, was identified from a decapod crustacean Litopenaeus vannamei and designated as LvMab21cp. LvMab21cp was mainly distributed in intestine and hepatopancreas, showing similar expression profile with other genes in the cGAS-STING pathway, such as LvSTING and LvIRF. The expression levels of LvMab21cp, LvSTING and LvIRF were up-regulated in intestine and hepatopancreas of shrimp after white spot syndrome virus (WSSV) infection. Knockdown of LvMab21cp by dsRNA-mediated RNA interference could decrease the expression levels of its putative downstream genes, including LvSTING, LvIRF, LvVago4 and LvVago5, and enhance the in vivo propagation of WSSV in shrimp. Overexpression of LvMab21cp and LvSTING in HEK 293T cells activated the expression of mammalian IFNs upon simulation with interferon stimulatory DNA (ISD). These data suggest that LvMab21cp was a cGAS homologue, a member of the shrimp cGAS-STING pathway, and play an important role during WSSV infection. To our knowledge, this is the first report to show the role of the cGAS-STING pathway in the antiviral response of invertebrates, which will provide new insights into the innate immunity of invertebrates.

Corrigendum: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei.

[This corrects the article DOI: 10.3389/fphys.2019.01525.].

The immune function of a novel crustin with an atypical WAP domain in regulating intestinal microbiota homeostasis in Litopenaeus vannamei.

Crustins are a family of antimicrobial peptides (AMP) with multiple functions, including antimicrobial activity, capability of protease inhibition, phagocytosis promotion, and wound healing in crustaceans. Till present, several members of crustins have been identified and their activities were studied. However, there are still less investigations on how they play functions in vivo. Here, we identified a novel crustin with an atypical WAP domain, LvCrustin Ⅰ-1, which is mainly distributed in tissues, including intestine, gill, epidermis and stomach of the shrimp Litopenaeus vannamei. The expression level of LvCrustin Ⅰ-1 was significantly up-regulated at 3 h, 6 h, 12 h, and 24 h after Vibrio parahaemolyticus infection. Knockdown of LvCrustin Ⅰ-1 with dsRNA resulted in a significant increase of the bacteria number in hepatopancreas of shrimp upon V. parahaemolyticus infection, showing that LvCrustin Ⅰ-1 participated in pathogen infection process. Recombinant LvCrustin Ⅰ-1 protein showed microorganism-binding activity rather than antibacterial activity against tested bacteria. Furthermore, significant difference existed between the intestinal microbiota in shrimp before and after LvCrustin Ⅰ-1 knockdown based on the result of alpha and NMDS analyses. Knockdown of LvCrustin Ⅰ-1 increased the proportion of Demequina, Nautella, Propionibacterium, Anaerospora and decreased the proportion of Bacteroidia and Vibrio. These data suggest that LvCrustin Ⅰ-1 might perform its immunological function through modulation of the intestinal microbiota homeostasis rather than direct inhibition of bacterial growth in shrimp.

Characterization of a Lymphoid Organ Specific Anti-lipopolysaccharide Factor From Shrimp Reveals Structure-Activity Relationship of the LPS-Binding Domain.

Anti-lipopolysaccharide factor (ALF) is a kind of important antimicrobial peptides with broad-spectrum antimicrobial activities. The LPS-binding domain (LBD) contributes to the major antimicrobial activity of ALF. However, LBDs from different ALFs share low sequence similarity. The general character of LBDs needs to be elucidated to understand the molecular mechanism of their function and facilitate LBD-original drug design. Here we identified a lymphoid organ specifically expressed ALF, designated as FcALF8, from the Chinese shrimp Fenneropenaeus chinensis. The synthetic LBD peptide of FcALF8 (LBD8) showed strong antibacterial activities to the pathogenic Vibrio, such as Vibrio alginolyticus, Vibrio harveyi, and Photobacterium damselae with a MIC value of 0.5-1, 1-2, and 1-2 µM, respectively. FcALF8 knock-down using dsRNA led to significant increase of the viable bacteria in the lymphoid organ and hepatopancreas of shrimp upon V. harveyi infection. On the contrary, the proliferation of V. harveyi in the shrimp lymphoid organ and hepatopancreas significantly decreased after infected by LBD8 pre-incubated V. harveyi. Sequence alignments showed that the LBDs from 39 ALFs shared only two identical cysteine residues. However, 17 of the total 22 LBD residues showed high similarity when the amino acids were classified into hydrophobic and hydrophilic ones. A further activity analysis on modified LBD8 peptides showed that the antibacterial activity of LBD8 was lost after linearization and apparently weakened after changing the amino acid property at certain positions. The data indicated that the disulfide bond and amino acid property contributed to the conservation of the functional domain. To the best of our knowledge, this is the first identified ALFs specifically expressed in the lymphoid organ of shrimp with strong antibacterial activity. The present data will give creative instructions for the design of LBD-originated antimicrobial agents.

Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei.

The "eyestalk-androgenic gland (AG)-testis" endocrine axis is involved in male sexual differentiation of crustaceans. The insulin-like androgenic gland hormone (IAG), secreted from the AG, plays a central role in this axis, however key factors upstream the IAG are still poorly understood. Here, two crustacean hyperglycemic hormone (CHH) genes (LvCHH1 and LvCHH2) and their putative receptor guanylate cyclase (LvGC) were identified in Litopenaeus vannamei. LvCHH1 and LvCHH2 belonged to CHH subfamily I members and LvGC was a membrane-bound guanylate cyclase. They were all differentially expressed in eyestalks and gonads of males and females. RNA interference (RNAi) of either LvCHH1 or LvCHH2 increased LvIAG expression, while injection of their recombinant protein decreased LvIAG expression, indicating that LvCHH1 and LvCHH2 are inhibitory factors of LvIAG expression. Yeast two-hybrid assay showed that both LvCHH1 and LvCHH2 interacted with LvGC and their RNAi and recombinant protein injection exerted opposite regulatory effects on the transcriptional expression of LvGC. Meanwhile, knockdown of LvGC increased LvIAG expression. These results suggest that LvGC is the receptor of LvCHH1 and LvCHH2 and they are all involved in male sexual development by regulating LvIAG expression. The present study unveils missing upstream elements in the "eyestalk-AG-testis" endocrine axis in crustacean.

Penaeid shrimp genome provides insights into benthic adaptation and frequent molting.

Crustacea, the subphylum of Arthropoda which dominates the aquatic environment, is of major importance in ecology and fisheries. Here we report the genome sequence of the Pacific white shrimp Litopenaeus vannamei, covering ~1.66 Gb (scaffold N50 605.56 Kb) with 25,596 protein-coding genes and a high proportion of simple sequence repeats (>23.93%). The expansion of genes related to vision and locomotion is probably central to its benthic adaptation. Frequent molting of the shrimp may be explained by an intensified ecdysone signal pathway through gene expansion and positive selection. As an important aquaculture organism, L. vannamei has been subjected to high selection pressure during the past 30 years of breeding, and this has had a considerable impact on its genome. Decoding the L. vannamei genome not only provides an insight into the genetic underpinnings of specific biological processes, but also provides valuable information for enhancing crustacean aquaculture.

A Putative Insulin-like Androgenic Gland Hormone Receptor Gene Specifically Expressed in Male Chinese Shrimp.

The insulin-like androgenic gland hormone (IAG) is the key regulator in crustacean male sexual differentiation. As a secreted peptide hormone, IAG might perform its biological function through interacting with the membrane receptor. However, the receptor of IAG remains unclear. In the current study, a putative IAG receptor gene (FcIAGR) was identified in Fenneropenaeus chinensis. The deduced amino acid sequence of FcIAGR contained several conserved domains of insulin-like receptor proteins, including two L domains (L1 and L2), a cysteine-rich domain, three fibronectin III domains, a transmembrane domain, and an intracellular tyrosine kinase domain. Tissue distribution and in situ hybridization analysis showed that FcIAGR was predominantly expressed in the androgenic gland and testis in male F. chinensis. Protein colocalization analysis in HEK293 cells showed that FcIAGR could colocalize with both FcIAG1 and FcIAG2, respectively. Yeast two-hybrid assay further confirmed the interactions between FcIAGR and FcIAGs. After a long-term silencing of FcIAGR with double-stranded RNA, most of the germ cells in the testis were arrested at the secondary spermatocytes, whereas those in the control developed into sperm cells. The data indicated that FcIAGR was the receptor of FcIAGs in F. chinensis. The current study provides insight into the mechanism that the insulin-like signaling pathway regulates the male sexual differentiation in Decapoda crustaceans.

Identification and function analysis of an anti-lipopolysaccharide factor from the ridgetail prawn Exopalaemon carinicauda.

Anti-lipopolysaccharide factor is a kind of antimicrobial peptide (AMP) with broad-spectrum activities against bacteria, virus, and fungi in crustacean. Different isoforms of ALFs showed different activities to virus or bacteria. Therefore, discovery of more ALFs will provide new insights into drug development and disease control. Here we reported an ALF gene, EcALF1, isolated from the ridgetail prawn Exopalaemon carinicauda. Its transcripts was mainly detected in hemocytes. Silencing of EcALF1 caused a lesion of hepatopancreas and finally led to death of the prawn. In vivo bacteria detection to the hepatopancreas showed that the colonies of thiosulphate citrate bile salts (TCBS) cultured bacteria in EcALF1-silenced prawn increased dramatically. We identified the dominant bacteria cultured in TCBS and found that Vibrio alginolyticus and Vibrio parahaemolyticus were dominant bacteria which were pathogenic species to prawn. We further synthesized the functional domain, LPS binding domain (LBD) of EcALF1, and its antibacterial and antiviral activities were detected. The synthetic EcLBD1 peptide showed very strong antibacterial activity against Micrococcus luteus and V. alginolyticus. Pre-incubation of WSSV with EcLBD1 peptide reduced the pathogenicity of WSSV infection to the prawn. The present data showed the important roles of an ALF gene in modulating the in vivo bacterial proliferation in E. carinicauda and provided new insight in disease control in aquaculture.

Recombinant expression and functional analysis of an isoform of anti-lipopolysaccharide factors (FcALF5) from Chinese shrimp Fenneropenaeus chinensis.

Antimicrobial peptides (AMPs) have a great potential to be used as a substitute for antibiotics since AMPs don't lead to bacteria's drug resistance. Anti-lipopolysaccharide factors (ALFs) are one type of AMPs and exist in crustaceans. In the present study, we produced a recombinant protein (rFcALF5) of an ALF isoform (FcALF5) from Chinese shrimp Fenneropenaeus chinensis through a prokaryotic expression system. The rFcALF5 exhibited varied antibacterial activities against different bacteria. Besides its antibacterial activities, it could also inhibit the infection of white spot syndrome virus (WSSV) to shrimp after pre-incubation with this virus. In order to learn the antiviral mechanism on how rFcALF5 influences WSSV infection, the interaction between the total proteins of WSSV and rFcALF5 was analyzed and the data showed that rFcALF5 had direct interaction with the envelope protein VP24 of WSSV. The LPS binding domain (LBD) of FcALF5 also showed direct interaction with VP24 of WSSV. Therefore we inferred that the antiviral activity of FcALF5 might be achieved through the binding of its LBD to VP24 of WSSV. These findings provided more information to develop new strategies for the control of shrimp disease in aquaculture.