Gene Expression Profile of Hemocytes of Kuruma Shrimp, Marsupenaeus japonicus Following Peptidoglycan Stimulation

Shrimps are believed to lack an adaptive immune system and therefore rely heavily on their innate immune mechanisms to ward off pathogens. Moreover, their innate defense reactions are triggered by bacterial and fungal cell wall components such as lipopolysaccharides, peptidoglycan and β-glucans. In this study, we used microarray to examine the gene expression profile of kuruma shrimp, Marsupenaeus japonicus, after stimulation with peptidoglycan. Subsequent results show that the number of upregulated genes and percentage of differential expression (21%) was highest at day 1 poststimulation. Differentially expressed genes in day 7 and day 14, on the other hand, were 3.25% and 11.21%, respectively. Sixty-one (61) genes of unknown function were found to have responded outright to peptidoglycan (PG) stimulation. Administration of PG also caused increases in the expressions of crustin, lysozyme, and a few antibacterial peptides, all of which are known to be involved in crustacean immune response. Taken together, our results suggest that innate response in shrimp is triggered instantaneously upon exposure to a bacterial component. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Haemolymph parameters of Pacific white shrimp (Litopenaeus vannamei) infected with Taura syndrome virus

Pacific white shrimp (Litopenaeus vannamei) were injected with Taura syndrome virus (TSV) to assess shrimp immune responses and survival. TSV-infected shrimp suffered high mortality, but mock-infected and untreated shrimp experienced no mortality. Moribund shrimp were a pale, reddish colour and were lethargic and soft-shelled. Their haemolymph was clear red and coagulated poorly. In TSV-infected shrimp, the total haemocyte count (THC), hyalinocyte and granulocyte counts, and total plasma protein decreased significantly to 21%, 24%, 17% and 56% of untreated control values, respectively. Haemocyanin decreased to 67%, and clottable proteins to 80% of control values (P< 0.01). Copper and calcium ions, haemocytic transglutaminase (TGase) activity and plasma growth inhibitory activity against Vibrio harveyi also decreased significantly. Generation of intrahaemocytic superoxide anion, O(-2), in TSV-infected shrimp was significantly greater (P< 0.05) than in both control groups, no matter whether glucan stimulated or unstimulated. But the relative increase of intrahaemocytic O(-2) generation in TSV-infected shrimp response to glucan stimulation was lower in both controls. Plasma phenoloxidase (PO) activity increased significantly in TSV-infected shrimp. The plasma bacterial agglutinin titre against E. coli and V. harveyi, growth inhibition of E. coli and the concentration of magnesium ions in TSV-infected shrimp did not change significantly.In conclusion, ten of thirteen haemolymph parameters changed significantly during the host-TSV interaction. These parameters might be valuable references of shrimp health status.     

Double-stranded RNA induces sequence-specific antiviral silencing in addition to nonspecific immunity in a marine shrimp: convergence of RNA interference and innate immunity in the invertebrate antiviral response?

Double-stranded RNA (dsRNA) is a common by-product of viral infections and a potent inducer of innate antiviral immune responses in vertebrates. In the marine shrimp Litopenaeus vannamei, innate antiviral immunity is also induced by dsRNA in a sequence-independent manner. In this study, the hypothesis that dsRNA can evoke not only innate antiviral immunity but also a sequence-specific antiviral response in shrimp was tested. It was found that viral sequence-specific dsRNA affords potent antiviral immunity in vivo, implying the involvement of RNA interference (RNAi)-like mechanisms in the antiviral response of the shrimp. Consistent with the activation of RNAi by virus-specific dsRNA, endogenous shrimp genes could be silenced in a systemic fashion by the administration of cognate long dsRNA. While innate antiviral immunity, sequence-dependent antiviral protection, and gene silencing could all be induced by injection of long dsRNA molecules, injection of short interfering RNAs failed to induce similar responses, suggesting a size requirement for extracellular dsRNA to engage antiviral mechanisms and gene silencing. We propose a model of antiviral immunity in shrimp by which viral dsRNA engages not only innate immune pathways but also an RNAi-like mechanism to induce potent antiviral responses in vivo.     

Uncovering the Mechanisms of Shrimp Innate Immune Response by RNA Interference

Because of the importance of shrimp in world aquaculture, there is much interest in understanding their immune system in order to improve their resistance to pathogenic microorganisms. An effective tool in studying genes involved in the immune response in shrimp is RNA interference (RNAi). RNAi, first recognized as an antiviral response against RNA viruses, is a cellular mechanism that is triggered by double-stranded RNAs and results in the degradation of homologous genes. In this review, we describe the current studies of genes in shrimp that employed RNAi technology to elucidate or confirm their functions. We also review the potential of RNAi to elicit antiviral response in shrimp.     

Comparing antibiotic resistance in commensal and pathogenic bacteria isolated from wild-caught South Carolina shrimps vs. farm-raised imported shrimps

The objective of this study was to assess and differentiate wild-caught South Carolina (SC) shrimps from imported shrimps on the basis of microbiological analysis. Seven wild-caught SC shrimp and 13 farm-raised imported shrimp samples were analyzed. Total plate counts from wild-caught shrimp samples ranged from 4.3 to 7.0 log10 CFU/g, whereas counts from imported shrimp samples ranged from 3.2 to 5.7 log10 CFU/g. There was no difference (P > 0.05) between total bacterial counts of wild-caught SC shrimp and farm-raised imported shrimp. However, the percentages of bacteria with reduced susceptibility towards ceftriaxone and tetracycline were higher (P < 0.05) for farm-raised shrimp than for wild-caught samples. Salmonella spp. detected only in one farm-raised sample was resistant to ampicillin, ceftriaxone, gentamicin, streptomycin, and trimethoprim. Vibrio vulnificus was detected in both wild-caught and farm-raised shrimp samples; however, only the isolate from farm-raised shrimp was resistant to nalidixic acid and trimethoprim. Escherichia coli detected in one wild-caught sample was resistant to ampicillin. Both Listeria spp. and Salmonella spp. were absent with wild-caught SC samples. Therefore, the presence of more ceftriaxone- and tetracycline-resistant bacteria and the observed antimicrobial resistance phenotypes of isolates from the imported shrimp may reflect the possible use of antibiotics in raising shrimp in those countries.     

Comparison of Gene Expression Profiles of Fenneropenaeus chinensis Challenged with WSSV and Vibrio

Microarray technique was used to analyze the gene expression profiles of shrimp when they were challenged by WSSV and heat-inactivated Vibrio anguillarum, respectively. At 6 h post challenge (HPC), a total of 806 clones showed differential expression profile in WSSV-challenged samples, but not in Vibrio-challenged samples. The genes coding energy metabolism enzyme and structure protein were the most downregulated elements in 6 h post WSSV-challenged (HPC-WSSV) tissues. However, a total of 155 clones showed differential expression in the Vibrio-challenged samples, but not in WSSV-challenged samples. Serine-type endopeptidase and lysosome-related genes were the most upregulated elements in tissues 6 h post Vibrio challenge (HPC-Vibrio). Totally, 188 clones showed differential expression in both 6 and 12 HPC-WSSV and HPC-Vibrio samples. Most of the differentially expressed genes (185/188) were downregulated in the samples of 12 HPC-WSSV, whereas upregulated in the samples at 6 and 12 HPC-Vibrio and 6 HPC-WSSV. The expression profiles of three differentially expressed genes identified in microarray hybridization were analyzed in hemocytes, lymphoid organ, and hepatopancreas of shrimp challenged by WSSV or Vibrio through real-time PCR. The results further confirmed the microarray hybridization results. The data will provide great help for us in understanding the immune mechanism of shrimp responding to WSSV or Vibrio. Wang and Li contributed equally to this work.     

Microbial Flora of Pond-Reared Brown Shrimp (Penaeus aztecus)

Agar plate counts and microbial types are reported for brown shrimp reared in 2-acre natural marshland and in 0.5-acre artificial ponds during June to October 1970. Bacterial counts of pond-reared shrimp ranged from 5 × 10⁴ to 5.5 × 10⁶ per g. At final harvest in October, bacterial counts ranged from 2 × 10⁵ to 5.5 × 10⁶ per g. In marsh ponds, bacterial counts of shrimp and pond water were lowest in August when both water temperature and salinity were high. Coryneform bacteria and to a lesser extent Vibrio were the predominant isolates from fresh pond shrimp. Shrimp stored at 3 to 5 C for 7 days were acceptable as judged by appearance and odor. Between 7 and 14 days of refrigerated storage, bacterial counts increased sharply and about 50% of the samples became unacceptable. Refrigerated storage of pond shrimp caused increases in coryneform bacteria and micrococci and decreases in Vibrio, Flavobacterium, Moraxella, and Bacillus species. Pseudomonas species were not significant in fresh or stored pond shrimp. The microbial flora of pond water usually was dominated by coryneform bacteria, Flavobacterium, Moraxella, and Bacillus species.     

Impaired cellular immune response to injected bacteria after knockdown of ferritin genes in the hard tick Haemaphysalis longicornis

Iron is an indispensable element for most microorganisms, including many pathogenic bacteria. Iron-withholding is a known component of the innate immunity, particularly of vertebrate hosts. Ticks are vectors of multiple pathogens and reports have shown that they naturally harbor several bacterial species. Thus, tick innate immunity must be crucial in limiting bacterial population to tolerable level that will not cause adverse effects. We have previously characterized two types of the iron-binding protein ferritin (HlFER) in the hard tick Haemaphysalis longicornis, known to be a vector of some protozoan parasites and rickettsiae, and showed their antioxidant function and importance in blood feeding and reproduction. Here we examined the possible role of HlFERs in tick immunity against bacterial infection. After silencing Hlfer genes, adult ticks were injected with live enhanced green fluorescence protein-expressing Escherichia coli, and then monitored for survival rate. Hemolymph that included hemocytes was collected for microscopic examination to observe cellular immune response, and for E. coli culture to determine bacterial viability after injection in the ticks. The expression of some antimicrobial peptides in whole ticks was also analyzed by RT-PCR. Hlfer-silenced ticks had a significantly lower survival rate than control ticks after E. coli injection. Greater number of bacteria inside and outside the hemocytes and higher bacterial colony counts after culture with hemolymph were also observed in Hlfer-silenced ticks. However, no difference on the expression of antimicrobial peptides was observed. These results suggest that ferritin molecules might be important in the cellular immune response of ticks to some bacteria.     

A functional genomics approach to the study of avian innate immunity

A second-generation 4,959 element cDNA microarray has been created and evaluated for its potential use in examining the avian innate immune response. The elements in this array were obtained from EST libraries of stimulated avian PMNC-derived monocytes/macrophages and supplemented by genes of interest from several specific innate immune pathways. The elements are spotted in triplicate resulting in 14,877 total spots per slide. The avian innate immunity microarray (AIIM) contains 25 avian interleukin, chemokine, and cytokine elements. The array also contains elements for several innate immune pathways, including genes involved in the Toll-like receptor (TLR) pathway (including six of the currently known avian TLR receptors), avian interferon/antiviral response pathway genes, and genes involved in apoptosis, antigen presentation and the oxidative burst. The AIIM can be used to evaluate global gene expression patterns in a number of immunologically relevant tissues and in chickens, turkeys and ducks. The array has also been evaluated for its ability to monitor the avian immune response to both bacterial (avian pathogenic Escherichia coli) and viral (avian influenza) avian pathogens.     

Mapping markers linked to porcine salmonellosis susceptibility

The goal of this study was to identify pig chromosomal regions associated with susceptibility to salmonellosis. Genomic DNA from pig reference populations with differences in susceptibility to Salmonella enterica serovar Choleraesuis as quantified by spleen and liver bacterial colonization at day 7 post-infection (dpi; Van Diemen et al. 2002 ) was used. These samples belonged to the offspring of a sire thought to be heterozygous for genes involved in susceptibility to salmonellosis. Amplified fragment length polymorphism (AFLP) markers were created and used to determine associations with spleen or bacterial counts at 7 dpi. To position linked markers, two mapping populations, the Roslin and Uppsala PiGMaP pedigrees were used to create an integrated map which included the AFLP markers associated with salmonellosis. Twenty-six AFLP markers located in 14 different chromosomal regions in the porcine genome were found to be significantly associated with susceptibility (Chi-square P  < 0.05). More than one linked marker was found on chromosomes 1, 7, 13, 14 and 18. It is likely that these regions contain genes involved in Salmonella susceptibility. Regions on chromosomes 1, 7 and 14 were significantly associated with Salmonella counts in the liver and regions on chromosomes 11, 13 and 18 with counts in spleen. The identification of these chromosomal regions highlights specific areas to search for candidate genes that may be involved in innate or adaptive immunity. Further investigation into these chromosomal regions would be useful to improve our understanding of host responses to infection with this widespread pathogen.     

Suppression of Shrimp Melanization during White Spot Syndrome Virus Infection

The melanization cascade, activated by the prophenoloxidase (proPO) system, plays a key role in the production of cytotoxic intermediates, as well as melanin products for microbial sequestration in invertebrates. Here, we show that the proPO system is an important component of the Penaeus monodon shrimp immune defense toward a major viral pathogen, white spot syndrome virus (WSSV). Gene silencing of PmproPO(s) resulted in increased cumulative shrimp mortality after WSSV infection, whereas incubation of WSSV with an in vitro melanization reaction prior to injection into shrimp significantly increased the shrimp survival rate. The hemolymph phenoloxidase (PO) activity of WSSV-infected shrimp was extremely reduced at days 2 and 3 post-injection compared with uninfected shrimp but was fully restored after the addition of exogenous trypsin, suggesting that WSSV probably inhibits the activity of some proteinases in the proPO cascade. Using yeast two-hybrid screening and co-immunoprecipitation assays, the viral protein WSSV453 was found to interact with the proPO-activating enzyme 2 (PmPPAE2) of P. monodon. Gene silencing of WSSV453 showed a significant increase of PO activity in WSSV-infected shrimp, whereas co-silencing of WSSV453 and PmPPAE2 did not, suggesting that silencing of WSSV453 partially restored the PO activity via PmPPAE2 in WSSV-infected shrimp. Moreover, the activation of PO activity in shrimp plasma by PmPPAE2 was significantly decreased by preincubation with recombinant WSSV453. These results suggest that the inhibition of the shrimp proPO system by WSSV partly occurs via the PmPPAE2-inhibiting activity of WSSV453.