Fuhua Li

Recent advances in researches on the innate immunity of shrimp in China

The annual production of shrimp culture in mainland of China has been over one million tons for several years. The major cultivated penaeidae species are Litopenaeus vannamei, Fenneropenaeus chinensis, Penaeus monodon and Marsupenaeus japonicus. Due to the importance of shrimp aquaculture in China, researchers have paid more attention to the molecular mechanism of shrimp disease occurrence and tried to develop an efficient control strategy for disease. This paper summarizes the research progress related to innate immunity of penaeid shrimp made in the last decade in Mainland China. Several pattern recognition receptors, such as lectin, toll, lipopolysaccharide and β-1,3-glucan binding protein (LGBP) and tetraspanin were identified. The major signal transduction pathways, including Toll pathway, IMD pathway, which might be involved in the immune response of shrimp, were focused on and most of the components in Toll pathway were identified. Also, cellular immune responses such as phagocytosis and apoptosis were regarded playing very important roles in anti-WSSV infection to shrimp. The molecules involved in the maintenance of the immune homeostasis of shrimp and the progress on molecular structure and pathogenic mechanism of WSSV were summarized. Therefore, the brief outline about the immune system of shrimp is drawn based on the recent data which will help us to understand the immune responses of shrimp to different pathogens.

A Toll receptor from Chinese shrimp Fenneropenaeus chinensis is responsive to Vibrio anguillarum infection.

Toll-like receptors (TLRs) are an evolutionarily ancient family of pattern recognition receptors (PRRs), playing a crucial role in innate immune responses. Here we present a Toll homolog from Chinese shrimp Fenneropenaeus chinensis, designated FcToll. The full-length cDNA of FcToll is 4115 bp including a poly A-tail of 16 bp, encoding a putative protein of 931 amino acids. The predicted protein consists of an extracellular domain with a potential signal peptide, 16 leucine-rich repeats (LRR), two LRR-C-terminal (LRR-CT) motifs, and two LRR-N-terminal (LRR-NT) motifs, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic Toll/Interleukin-1R (TIR) domain of 139 residues. Genomic structure of FcToll gene contains five exons and four introns. Phylogenetic analysis revealed that it belongs to insect-type invertebrate Toll family. Transcripts of FcToll gene were constitutively expressed in various tissues, with predominant level in lymphoid organ. Real-time PCR assays demonstrated that expression patterns of FcToll were distinctly modulated after bacterial or viral stimulation, with significant enhancement after 5h post-Vibrio anguillarum challenge but markedly reduced levels immediately after white spot syndrome virus (WSSV) exposure. These results suggest that FcToll might be involved in innate host defense, especially against the pathogen V. anguillarum.

Signaling pathways regulating innate immune responses in shrimp.

The first line of defense against microbial infections in animals is innate immune response which triggers diverse humoral and cellular activities via signal transduction pathways. Toll, IMD and JAK/STAT pathways are regarded as the main pathways regulating the immune response of invertebrates. This paper reviews the main progress of the investigation on the immune response to pathogens infection in shrimp and supposes that these three signal pathways exist in shrimp. Most of the components (proteins or genes) involved in Toll pathway of Drosophila have been cloned also in shrimp which suggested the existence of Toll pathway in shrimp. The data update shows that the Toll pathway of shrimp is responsive not only to Gram-positive bacteria, Gram-negative bacteria, but also to WSSV. Challenge of WSSV can lead to the variation of transcription level of all identified components in shrimp Toll pathway, which supported that Toll pathway in shrimp played important roles during WSSV infection. Two major homologs to the components of IMD pathway of Drosophila, IMD and Relish, have been identified in shrimp, which indicated that IMD pathway should be existed in shrimp and might play important roles in regulating the immune response of shrimp to bacteria and virus infection. Relish in IMD pathway and dorsal in Toll pathway of shrimp were both involved in the immune response of shrimp to bacteria and virus infection, which implied that these two pathways are not completely separated during the immune response of shrimp. The transcription of STAT in shrimp was modulated after WSSV infection, which suggested that a putative JAK/STAT pathway might exist in shrimp and be very important to virus infection. Study on the signaling pathway regulating the immune response in shrimp could help us to understand the innate immune system, and would provide instructions to shrimp disease control. Obviously, to get more clear ideas about the innate immunological pathways in shrimp, more solid functional studies should be done in the future.

Molecular cloning and expression profile of putative antilipopolysaccharide factor in Chinese shrimp (Fenneropenaeus chinensis)

A new antimicrobial protein gene of the anti-lipopolysaccharide factor family (tentatively named as ALFFc) has been cloned from hemocytes of the Chinese shrimp Fenneropenaeus chinensis by rapid amplification of 3′ and 5′ complementary DNA ends with polymerase chain reaction. The full-length complementary DNA of ALFFc consists of 600 bp with a 369-bp open reading frame, encoding 123 amino acids. The deduced peptide contains a putative signal peptide of 25 amino acids and mature peptide of 98 amino acids. The molecular mass of the deduced mature peptide is 13799.16 Da. It is highly cationic, with a theoretical pI of 10.3. The deduced amino acid sequence of ALFFc showed 56% homology with sequences of Tachypleustridentatus and L. polyhemus. The tissue expression profile of this gene was studied by Northern blot, and ALFFc transcripts were mainly detected in hemocytes, gill, and intestine. RNA in situ hybridization showed that ALFFc was constitutively expressed in hemocytes. Capillary electrophoresis reverse transcriptase PCR was used to quantify the variation of messenger RNA transcription level during the artificial infection process with Vibrio anguillarum. Significant enhancement of ALFFc transcription appeared during the first 24 hours in response to Vibrio infection. These results provide useful information for understanding the function of ALFFc in shrimp.

Transcriptome analysis on Chinese shrimp Fenneropenaeus chinensis during WSSV acute infection.

Previous studies have discovered a lot of immune-related genes responding to white spot syndrome virus (WSSV) infection in crustacean. However, little information is available in relation to underlying mechanisms of host responses during the WSSV acute infection stage in naturally infected shrimp. In this study, we employed next-generation sequencing and bioinformatic techniques to observe the transcriptome differences of the shrimp between latent infection stage and acute infection stage. A total of 64,188,426 Illumina reads, including 31,685,758 reads from the latent infection group and 32,502,668 reads from the acute infection group, were generated and assembled into 46,676 unigenes (mean length: 676 bp; range: 200–15,094 bp). Approximately 24,000 peptides were predicted and classified based on homology searches, gene ontology, clusters of orthologous groups of proteins, and biological pathway mapping. Among which, 805 differentially expressed genes were identified and categorized into 11 groups based on their possible function. Genes in the Toll and IMD pathways, the Ras-activated endocytosis process, the RNA interference pathway, anti-lipopolysaccharide factors and many other genes, were found to be activated in shrimp from latent infection stage to acute infection stage. The anti-bacterially proPO-activating cascade was firstly uncovered to be probably participated in antiviral process. These genes contain not only members playing function in host defense against WSSV, but also genes utilized by WSSV for its rapid proliferation. In addition, the transcriptome data provides detail information for identifying novel genes in absence of the genome database of shrimp.

Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to hypoxic stress

Hypoxia, as one suboptimal environmental condition, can affect the physiological state of shrimp during pond aquaculture. To better understand the mechanism of response to hypoxic stress in Chinese shrimp Fenneropenaeus chinensis, proteome research approach was utilized. Differentially expressed proteins of hepatopancreas in adult Chinese shrimp between the control and hypoxia‐stressed groups were screened. By 2‐DE analysis, 67 spots showed obvious changes after hypoxia. Using LC‐ESI‐MS/MS, 51 spots representing 33 proteins were identified including preamylase, arginine kinase, phosphopyruvate hydratase, citrate synthase, ATP synthase alpha subunit, chymotrypsin BI, chitinase, ferritin, C‐type lectin receptors, transketolase, formylglutathione hydrolase, formyltetrahydrofolate dehydrogenase, aldehyde dehydrogenase, glutathione peroxidase, cytosolic manganese superoxide dismutase, protein disulfide isomerase, β‐actin, oncoprotein nm23, crustacyanin‐C1 and so on. These proteins could be functionally classified into several groups such as proteins related to energy production, metabolism‐related proteins, immune‐related proteins, antioxidant proteins, chaperones, cytoskeleton proteins and ungrouped proteins. The transcription levels of ten selected genes encode the identified proteins were analyzed by real‐time PCR at different sampling times of hypoxia. This study is the first analysis of differentially expressed proteins in the hepatopancreas of shrimp after hypoxia and provides a new insight for further study in hypoxic stress response of shrimp at the protein level.

Multiple forms of alpha-2 macroglobulin in shrimp Fenneropenaeus chinesis and their transcriptional response to WSSV or Vibrio pathogen infection.

Alpha-2 macroglobulin (A2M) is a non-specific protease inhibitor involved in host defense. By full length cloning and sequencing we identified three distinct cDNAs for A2M in Chinese shrimp Fenneropenaeus chinesis, designated FcA2M-1, FcA2M-2 and FcA2M-3, respectively. Expression profiles in normal tissues as well as tissues after challenge by white spot syndrome virus (WSSV) and Vibrio pathogen were conducted for FcA2M-1 and FcA2M-2. The FcA2M-1 and FcA2M-2 cDNAs encode proteins with 1501 or 1502 amino acids, respectively, containing the typical conserved domain architecture of A2M. Similar to complement component C3, FcA2M-2 has a catalytic histidine, which may confer opsonic properties on this shrimp A2M. Six variants in the bait region were found in FcA2M-2 responding differently to Vibrio challenge, thereby widening the spectrum of inhibition and the diversity of immune recognition. FcA2M-1 and FcA2M-3, as well as most other protostomia invertebrate A2Ms identified so far, have a serine residue in the catalytic histidine position instead of the conserved asparagine residue found in vertebrate A2Ms. This, as inferred from a carp C3 molecule in which the catalytic histidine is substituted by a serine, suggests A2Ms in lower invertebrates possibly bear C3-like opsonic activity. These FcA2Ms showed much lower similarity to each other than to the A2Ms in other shrimp species, further supported by pylogenetic analysis. FcA2M-1 was found to be expressed most highly in hemocytes and lymphoid organ, while FcA2M-2 was expressed most highly in the heart and lymphoid organ, with the lowest expression in hemocytes. Challenge by WSSV or Vibrio pathogen increased the FcA2M-1 mRNA level in both hemocytes and lymphoid organ. After challenge, FcA2M-2 showed up-regulation in lymphoid organ but not in hemocytes. These expression features indicate that the different types of A2M in F. chinesis carry out different functions and that they are not simply functionally redundant.

A Dorsal homolog (FcDorsal) in the Chinese shrimp Fenneropenaeus chinensis is responsive to both bacteria and WSSV challenge

Rel/NFkappaB is a family of transcription factors. In the present study, a Rel/NFkappaB family member, Dorsal homolog (FcDorsal) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcDorsal consists of 1627bp, revealed a 1071bp open reading frame encoding 357 aa. The predicted molecular weight (MW) of the deduced amino acid sequence of FcDorsal was 39.78kDa, and its theoretical pI was 8.85. Amino acid sequence analysis showed that FcDorsal contains a Rel homolog domain (RHD) and an IPT/TIG (Ig-like, plexins and transcriptions factors) domain. The signature sequence of dorsal protein existed in the deduced amino acid sequence. Spatial expression profiles showed that FcDorsal had the highest expression level in the hemocytes and lymphoid organ (Oka). The expression profiles in the hemocytes and lymphoid organ were apparently modulated when shrimp were stimulated by bacteria or WSSV. Both Gram-positive (G(+)) bacteria (Micrococcus lysodeikticus) and Gram-negative (G(-)) bacteria (Vibrio anguillarium) injection to shrimp caused the up-regulation of FcDorsal at the transcription level. DsRNA approach was used to study the function of FcDorsal and the data showed that FcDorsal was related to the transcription of Penaeidin 5 in shrimp. The present data provide clues that FcDorsal might play potential important roles in the innate immunity of shrimp. Through comparison of the expression profiles between FcDorsal and another identified Rel/NFkappaB member (FcRelish) in shrimp responsive to WSSV challenge, we speculate that FcDorsal and FcRelish might play different roles in shrimp immunity.

Identification of a novel relish homolog in Chinese shrimp Fenneropenaeus chinensis and its function in regulating the transcription of antimicrobial peptides

Penaeid shrimp, as an invertebrate, relies on the innate immunity to oppose the microbial invaders. Antimicrobial peptides (AMP) are an integral component of the innate immune system in most organisms and function as an early first line of defense against pathogens, but the knowledge about the pathways to regulate the shrimp AMP gene expression is still absent up to date. In the current study, a Relish homolog (FcRelish) was cloned from Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcRelish consists of 2157 bp, including 1512 bp open reading frame, encoding 504 amino acids. The predicted molecular weight of FcRelish is 57 kDa, and the theoretical PI is 7.00. Spatial expression profiles showed that FcRelish had the highest expression levels in the hemocytes and lymphoid organ. Both Vibrio anguillarium and Micrococcus lysodeikticus stimulation to shrimp can affect the transcription profile of FcRelish. Silencing of FcRelish through DsRNA interference can greatly change the transcription profile of AMP. Therefore, we suggest that FcRelish identified in the present study is closely related to the transcription of AMP, and then we inferred that Imd pathway might exist in shrimp.

Molecular characterization and effect of RNA interference of retinoid X receptor (RXR) on E75 and chitinase gene expression in Chinese shrimp Fenneropenaeus chinensis

Retinoid X receptor (RXR)/ultraspiracle (USP) is the heterodimeric partner of ecdysteroid receptor and is required for the molting process of arthropods. To better understand the molecular aspects governing the process of molting in shrimp, the full-length cDNA of two RXRs, named as FcRXR-1 and FcRXR-2 were obtained from Chinese shrimp Fenneropenaeus chinensis which were of 1715 and 1700 bp long, revealed a 1315 and 1300 bp open reading frame (ORF) respectively. Quantitative Real time PCR analysis showed a marked tissue-specific difference in the expression of FcRXR transcript, which revealed that the expression of FcRXR could be regulated in a tissue-specific manner. Moreover, high expression of FcRXR mRNAs was observed in late pre-molt period (D3) and post molt stages (A-B) of shrimp. Among the two isoforms, FcRXR-2 appeared in a considerably high level in all the stages compared to the FcRXR-1. In addition, we examined the temporal expression of two chitinase genes: FcChitinase (FcChi) and FcChitinase-1 (FcChi-1) during the molt cycle of F. chinensis. Both the FcChi and FcChi-1 transcripts were detected in all stages of molting, although considerable fluctuations observed through the molt cycle. Injection of double stranded RXR (dsRXR) into juvenile shrimp resulted in a maximum silencing effect at 48 h post injection. We analyzed the expression levels of FcChi, FcChi-1 and the ecdysone inducible gene E75 (FcE75) in samples of dsRXR injected shrimp. Significant reduction in levels of both FcE75, FcChi and FcChi-1 transcripts occurred in the silenced shrimp. This correlation suggested that RXR might involve in the downstream regulation of E75 and chitinase gene transcription in the ecdysone signaling pathway of decapod crustaceans.