Pikul Jiravanichpaisal

Antilipopolysaccharide factor interferes with white spot syndrome virus replication in vitro and in vivo in the crayfish Pacifastacus leniusculus.

ABSTRACT In a study of genes expressed differentially in the freshwater crayfish Pacifastacus leniusculus infected experimentally with the white spot syndrome virus (WSSV), one protein, known as antilipopolysaccharide factor (ALF), was chosen, among those whose transcript levels increased upon viral infection, for further studies. ALF RNA interference (RNAi) experiments in whole animals and in cell cultures indicated that ALF can protect against WSSV infection, since knockdown of ALF by RNAi specifically resulted in higher rates of viral propagation. In a cell culture of hematopoietic tissue (Hpt) from P. leniusculus, quantitative PCR showed that knockdown of ALF by RNAi resulted into WSSV levels that were about 10-fold higher than those treated with control double-stranded RNA (dsRNA). In addition, RNAi experiments with other crayfish genes that had been found to be up-regulated by a WSSV infection did not result in any changes of viral loads. Thus, the cell culture does not respond to dsRNA in a similar manner, as shown earlier for dsRNA injected into shrimp, which gave a higher degree of resistance to WSSV infection. If ALF transcription in whole animals was stimulated by the administration of UV-treated WSSV, a partial protection against a subsequent challenge with the active virus was conferred to the host. This is the first crustacean gene product identified with the capacity to interfere with replication of this important pathogen.

Phenoloxidase is an important component of the Defense against Aeromonas hydrophila infection in a crustacean, Pacifastacus leniusculus

The melanization cascade, in which phenoloxidase is the terminal enzyme, appears to play a key role in recognition of and defense against microbial infections in invertebrates. Here, we show that phenoloxidase activity and melanization are important for the immune defense toward a highly pathogenic bacterium, Aeromonas hydrophila, in the freshwater crayfish, Pacifastacus leniusculus. RNA interference-mediated depletion of crayfish prophenoloxidase leads to increased bacterial growth, lower phagocytosis, lower phenoloxidase activity, lower nodule formation, and higher mortality when infected with this bacterium. In contrast, if RNA interference of pacifastin, an inhibitor of the crayfish prophenoloxidase activation cascade, is performed, it results in lower bacterial growth, increased phagocytosis, increased nodule formation, higher phenoloxidase activity, and delayed mortality. Our data therefore suggest that phenoloxidase is required in crayfish defense against an infection by A. hydrophila, a highly virulent and pathogenic bacterium to crayfish.

Antiviral immunity in crustaceans.

Abstract Viral diseases of shrimp have caused negative effects on the economy in several countries in Asia, South America and America, where they have numerous shrimp culture industries. The studies on the immunity of shrimp and other crustaceans have mainly focused on general aspects of immunity and as a consequence little is known about the antiviral responses in crustaceans. The aim of this review is to update recent knowledge of innate immunity against viral infections in crustaceans. Several antiviral molecules have been isolated and characterized recently from decapods. Characterization and identification of these molecules might provide a promising strategy for protection and treatment of these viral diseases. In addition dsRNA-induced antiviral immunity is also included.

Bacteria-Induced Dscam Isoforms of the Crustacean, Pacifastacus leniusculus

The Down syndrome cell adhesion molecule, also known as Dscam, is a member of the immunoglobulin super family. Dscam plays an essential function in neuronal wiring and appears to be involved in innate immune reactions in insects. The deduced amino acid sequence of Dscam in the crustacean Pacifastacus leniusculus (PlDscam), encodes 9(Ig)-4(FNIII)-(Ig)-2(FNIII)-TM and it has variable regions in the N-terminal half of Ig2 and Ig3 and the complete Ig7 and in the transmembrane domain. The cytoplasmic tail can generate multiple isoforms. PlDscam can generate more than 22,000 different unique isoforms. Bacteria and LPS injection enhanced the expression of PlDscam, but no response in expression occurred after a white spot syndrome virus (WSSV) infection or injection with peptidoglycans. Furthermore, PlDscam silencing did not have any effect on the replication of the WSSV. Bacterial specific isoforms of PlDscam were shown to have a specific binding property to each tested bacteria, E. coli or S. aureus. The bacteria specific isoforms of PlDscam were shown to be associated with bacterial clearance and phagocytosis in crayfish.

Bacterial Population in Intestines of the Black Tiger Shrimp (Penaeus monodon) under Different Growth Stages

Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A total of 25,121 pyrosequencing reads (reading length = 442±24 bases) were obtained, which were categorized by barcode for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20 bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15 and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles. This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth development stages in rearing facilities.

Bacterial Community Associated with the Intestinal Tract of P. monodon in Commercial Farms

The potentially important roles of intestinal bacteria on immune response, disease resistance, and nutrition for the black tiger shrimp Penaeus monodon have been increasingly investigated. However, so far, little is known about the intestinal bacterial community of the shrimp in the commercial aquaculture settings. In this study, the intestinal bacterial communities of juvenile P. monodon (70 individuals) from eight commercial farms in Thailand were examined using 16S rDNA PCR-DGGE, and seven 16S rDNA clone libraries from representative DGGE profiles were constructed. Bacteria in the γ-Proteobacteria class were the only common bacteria group found in the intestinal tracts of shrimp from all farms. The dominant bacterial genera in the intestinal population of each shrimp varied among different farms, and these genera were Vibrio, Photobacterium, Aeromonas, or Propionigenium (phylum Fusobacteria). Other commonly found genera included Actinomyces, Anaerobaculum, Halospirulina, Pseudomonas, Mycoplasma, and Shewanella. Twelve phyla of bacteria including Proteobacteria, Firmicutes, Fusobacteria, Actinobacteria, Cyanobacteria, Tenericutes, Deinococcus-Thermus, Planctomycetes, Spirochaetes, Synergistetes, Thermotogae, and Verrucomicrobia were represented in the sequences. Additionally, strictly anaerobic bacteria such as Propionigenium and Fusibacter were found. These intestinal bacterial communities varied significantly among different commercial farms and were distinct from their rearing water. The results provide descriptive structures of the intestinal bacterial communities of P. monodon in commercial farms, which can further be applied to areas of research on the immunity, disease resistance, and nutrition of shrimp to improve aquaculture of the black tiger shrimp.

In vitro effects on bacterial growth of phenoloxidase reaction products

An active phenoloxidase preparation from the freshwater crayfish Pacifastacus leniusculus exhibited a strong antibacterial effect in vitro on the bacteria Aeromonas hydrophila, Escherichia coli, Streptococcus pneumoniae whereas a weaker but still significant effect against Bacillus cereus, Pseudomonas aeruginosa and Staphylococcus aureus. In most cases reduction of bacterial growth was stronger when dopamine was used as substrate as compared to L-dopa. The effect on bacteria was abolished if no substrate was available for the phenoloxidase or in the presence of the phenoloxidase inhibitor phenylthiourea.

A gC1qR Prevents White Spot Syndrome Virus Replication in the Freshwater Crayfish Pacifastacus leniusculus

ABSTRACT The gC1qR/p32 protein is a multiple receptor for several proteins and pathogens. We cloned a gC1qR homologue in a crustacean, Pacifastacus leniusculus, and analyzed the expression of P. leniusculus C1qR (PlgC1qR) in various tissues. The gC1qR/p32 transcript was significantly enhanced by white spot syndrome virus (WSSV) infection 6 h after viral infection both in vitro in a hematopoietic tissue cell culture (Hpt) and in vivo compared to appropriate controls. Moreover, PlgC1qR silencing in both the Hpt cell culture and live crayfish enhanced the WSSV replication. In addition, by making a recombinant PlgC1qR protein we could show that if this recombinant protein was injected in a crayfish, Pacifastacus leniusculus, followed by injection of WSSV, this significantly reduced viral replication in vivo. Furthermore, if the recombinant PlgC1qR was incubated with Hpt cells and then WSSV was added, this also reduced viral replication. These experiments clearly demonstrate that recombinant PlgC1qR reduce WSSV replication both in vivo and in vitro. The results from a far-Western overlay and glutathione S-transferase pull-down assays showed that PlgC1qR could bind to VP15, VP26, and VP28. Altogether, these results demonstrate a role for PlgC1qR in antiviral activity against WSSV.

Invertebrate Hematopoiesis: An Anterior Proliferation Center As a Link Between the Hematopoietic Tissue and the Brain

During evolution, the innate and adaptive immune systems were developed to protect organisms from non-self substances. The innate immune system is phylogenetically more ancient and is present in most multicellular organisms, whereas adaptive responses are restricted to vertebrates. Arthropods lack the blood cells of the lymphoid lineage and oxygen-carrying erythrocytes, making them suitable model animals for studying the regulation of the blood cells of the innate immune system. Many crustaceans have a long life span and need to continuously synthesize blood cells, in contrast to many insects. The hematopoietic tissue (HPT) of Pacifastacus leniusculus provides a simple model for studying hematopoiesis, because the tissue can be isolated, and the proliferation of stem cells and their differentiation can be studied both in vivo and in vitro. Here, we demonstrate new findings of a physical link between the HPT and the brain. Actively proliferating cells were localized to an anterior proliferation center (APC) in the anterior part of the tissue near the area linking the HPT to the brain, whereas more differentiated cells were detected in the posterior part. The central areas of HPT expand in response to lipopolysaccharide-induced blood loss. Cells isolated from the APC divide rapidly and form cell clusters in vitro; conversely, the cells from the remaining HPT form monolayers, and they can be induced to differentiate in vitro. Our findings offer an opportunity to learn more about invertebrate hematopoiesis and its connection to the central nervous system, thereby obtaining new information about the evolution of different blood and nerve cell lineages.

An ancient cytokine, astakine, mediates circadian regulation of invertebrate hematopoiesis

Invertebrate circulating hemocytes are key players in the innate immune defense and their continuous renewal from hematopoietic tissues is tightly regulated in crustaceans by astakine, a new family of cytokines sharing a prokineticin (PROK) domain. In vertebrates, brain PROKs function as transmitters of circadian rhythms and we present evidence that hemocyte release from hematopoietic tissues in crayfish is under circadian regulation, a direct result of rhythmic expression of astakine. We demonstrate that the observed variation in astakine expression has an impact on innate immunity assessed as susceptibility to a pathogenic Pseudomonas species. These findings enlighten the importance of comparing immune responses at fixed times not to neglect circadian regulation of innate immunity. Moreover, our results entail an evolutionary conserved function for prokineticins as mediators of circadian rhythm, and for the first time show a role for this domain in circadian regulation of hematopoiesis that may have implications also in vertebrates.