Molecular characterization of Pacific white shrimp (Litopenaeus vannamei) sodium bicarbonate cotransporter (NBC) and its role in response to pH stress.

The sodium bicarbonate cotransporter (NBC) is an integral membrane ion transporter that can transport HCO3- (or a related species, such as CO32-) across the plasma membrane. Previous researches revealed that NBC might play an important role in the regulation of intracellular pH in vertebrates. In the present study, an NBC cDNA was identified from Pacific white shrimp (Litopenaeus vannamei) and designated as Lv-NBC. The full-length Lv-NBC cDNA is 4479 bp in size, containing a 5'-untranslated region (UTR) of 59 bp, a 3'-UTR of 835 bp and an open reading frame (ORF) of 3585 bp that encodes a protein of 1194 amino acids with a deduced molecular weight of 134.34 kDa. The Lv-NBC protein contains two functional domains (Band_3_cyto and HCO3_cotransp) and twelve transmembrane (TM) domains. Expression of the Lv-NBC mRNA was ubiquitously detected in all selected tissues, with the highest level in the gill. By in situ hybridization (ISH) with Digoxigenin-labeled probe, the Lv-NBC positive cells were shown mainly located in the secondary gill filaments. After low or high pH challenge, the transcript levels of Lv-NBC in the gill were found to be up-regulated. After knockdown of the Lv-NBC level by siRNA, the mortality of shrimp significantly increased under pH stress. Our study, as a whole, may provide evidences for the role of NBC in shrimp responding to pH stress, and give a new insight of the acid/base homeostasis mechanism in crustaceans.

Goldfish Leptin-AI and Leptin-AII: Function and Central Mechanism in Feeding Control.

In mammals, leptin is a peripheral satiety factor that inhibits feeding by regulating a variety of appetite-related hormones in the brain. However, most of the previous studies examining leptin in fish feeding were performed with mammalian leptins, which share very low sequence homologies with fish leptins. To elucidate the function and mechanism of endogenous fish leptins in feeding regulation, recombinant goldfish leptin-AI and leptin-AII were expressed in methylotrophic yeast and purified by immobilized metal ion affinity chromatography (IMAC). By intraperitoneal (IP) injection, both leptin-AI and leptin-AII were shown to inhibit the feeding behavior and to reduce the food consumption of goldfish in 2 h. In addition, co-treatment of leptin-AI or leptin-AII could block the feeding behavior and reduce the food consumption induced by neuropeptide Y (NPY) injection. High levels of leptin receptor (lepR) mRNA were detected in the hypothalamus, telencephalon, optic tectum and cerebellum of the goldfish brain. The appetite inhibitory effects of leptins were mediated by downregulating the mRNA levels of orexigenic NPY, agouti-related peptide (AgRP) and orexin and upregulating the mRNA levels of anorexigenic cocaine-amphetamine-regulated transcript (CART), cholecystokinin (CCK), melanin-concentrating hormone (MCH) and proopiomelanocortin (POMC) in different areas of the goldfish brain. Our study, as a whole, provides new insights into the functions and mechanisms of leptins in appetite control in a fish model.

Pacific white shrimp (Litopenaeus vannamei) vitellogenesis-inhibiting hormone (VIH) is predominantly expressed in the brain and negatively regulates hepatopancreatic vitellogenin (VTG) gene expression.

Ovarian maturation in crustaceans is temporally orchestrated by two processes: oogenesis and vitellogenesis. The peptide hormone vitellogenesis-inhibiting hormone (VIH), by far the most potent negative regulator of crustacean reproduction known, critically modulates crustacean ovarian maturation by suppressing vitellogenin (VTG) synthesis. In this study, cDNA encoding VIH was cloned from the eyestalk of Pacific white shrimp, Litopenaeus vannamei, a highly significant commercial culture species. Phylogenetic analysis suggests that L. vannamei VIH (lvVIH) can be classified as a member of the type II crustacean hyperglycemic hormone family. Northern blot and RT-PCR results reveal that both the brain and eyestalk were the major sources for lvVIH mRNA expression. In in vitro experiments on primary culture of shrimp hepatopancreatic cells, it was confirmed that some endogenous inhibitory factors existed in L. vannamei hemolymph, brain, and eyestalk that suppressed hepatopancreatic VTG gene expression. Purified recombinant lvVIH protein was effective in inhibiting VTG mRNA expression in both in vitro primary hepatopancreatic cell culture and in vivo injection experiments. Injection of recombinant VIH could also reverse ovarian growth induced by eyestalk ablation. Furthermore, unilateral eyestalk ablation reduced the mRNA level of lvVIH in the brain but not in the remaining contralateral eyestalk. Our study, as a whole, provides new insights on VIH regulation of shrimp reproduction: 1) the brain and eyestalk are both important sites of VIH expression and therefore possible coregulators of hepatopancreatic VTG mRNA expression and 2) eyestalk ablation could increase hepatopancreatic VTG expression by transcriptionally abolishing eyestalk-derived VIH and diminishing brain-derived VIH.

Characterization of role of the toxR gene in the physiology and pathogenicity of Vibrio alginolyticus.

toxR, a conserved virulence-associated gene in vibrios, is identified in Vibrio alginolyticus ZJ51-O, a pathogenic strain isolated from diseased fish. To reveal the role of ToxR in the pathogenicity of V. alginolyticus, a deletion mutant was constructed by allelic exchange. The mutant showed the same level of growth in trypticase soy broth (TSB) and iron-limiting condition, as the wild type strain. However, deletion of toxR severely reduced resistance against bile salts and the capability of biofilm formation. Outer-membrane protein (OMP) analysis showed that a 37-kD protein was absent and a 43-kD protein was decreased in the mutant. By MS/MS, the two proteins are identified as the homologues of OmpT and OmpN, respectively. These data suggest that ToxR might have enhanced the bile resistance and biofilm formation through modulating the production of OMP without affecting the ability of iron acquisition and the virulence to the fish via injection. These results indicate that ToxR may assist V. alginolyticus to colonize on the surface of the fish intestine which is crucial for the initiation of the infection, though it may not be involved in the proliferation of the bacteria in the host tissue.

The type III secretion system of Vibrio alginolyticus induces rapid apoptosis, cell rounding and osmotic lysis of fish cells.

Vibrio alginolyticus is a Gram-negative bacterium and has been recognized as an opportunistic pathogen in humans as well as marine animals. However, the virulence mechanisms for this species of Vibrio have not been elucidated. This study characterized multiple mechanisms that induce cell death in fish cells upon infection with a V. alginolyticus strain, ZJO. The bacterium required its type III secretion system (T3SS) to cause rapid death of infected fish cells. Dying cells exhibited some features of apoptotic cells, such as membrane blebbing, nuclear condensation and DNA fragmentation. Further studies showed that caspase-3 was activated by the T3SS of the ZJO strain, confirming that infection with V. alginolyticus rapidly induces T3SS-dependent apoptosis in fish cells. Infection with the ZJO strain also led to membrane pore formation and release of cellular contents from infected fish cells, as evidenced by lactate dehydrogenase release and the uptake of a membrane-impermeable dye. Importantly, inhibition of apoptosis did not prevent ZJO-infected cells from releasing cellular contents and did not block cell rounding. Taken together, these data demonstrate that infection with V. alginolyticus may promote at least three different T3SS-dependent events, which lead to the death of fish cells. This study provides an important insight into the mechanism used by Vibrio species to cause host-cell death.

Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated isothermal amplification.

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid technique, which can be applied for disease diagnosis in aquaculture. Using the LAMP method, a highly specific and sensitive diagnostic system for infectious hypodermal and hematopoietic necrosis virus (IHHNV) detection was designed. A set of four primers was designed by targeting the IHHNV genome DNA. By the detection system, target DNA was amplified and visualized on agarose gel within 60min under isothermal condition at 64 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by the white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. The assay had a detection limit of 5-500 copies of DNA template with gel electrophoresis, SYBR Green I and white turbidity with naked-eye inspection. The detection sensitivity of LAMP was 100-fold higher than the PCR. A diagnostic procedure which is rapid and highly sensitive was developed for IHHNV detection.

ExsE Is a Negative Regulator for T3SS Gene Expression in Vibrio alginolyticus.

Type III secretion systems (T3SSs) contribute to microbial pathogenesis of Vibrio species, but the regulatory mechanisms are complex. We determined if the classic ExsACDE protein-protein regulatory model from Pseudomonas aeruginosa applies to Vibrio alginolyticus. Deletion mutants in V. alginolyticus demonstrated that, as expected, the T3SS is positively regulated by ExsA and ExsC and negatively regulated by ExsD and ExsE. Interestingly, deletion of exsE enhanced the ability of V. alginolyticus to induce host-cell death while cytotoxicity was inhibited by in trans complementation of this gene in a wild-type strain, a result that differs from a similar experiment with Vibrio parahaemolyticus ExsE. We further showed that ExsE is a secreted protein that does not contribute to adhesion to Fathead minnow epithelial cells. An in vitro co-immunoprecipitation assay confirmed that ExsE binds to ExsC to exert negative regulatory effect on T3SS genes. T3SS in V. alginolyticus can be activated in the absence of physical contact with host cells and a separate regulatory pathway appears to contribute to the regulation of ExsA. Consequently, like ExsE from P. aeruginosa, ExsE is a negative regulator for T3SS gene expression in V. alginolyticus. Unlike the V. parahaemolyticus orthologue, however, deletion of exsE from V. alginolyticus enhanced in vitro cytotoxicity.

Signal transduction mechanism for glucagon-induced leptin gene expression in goldfish liver.

Leptin is a peripheral satiety hormone that also plays important roles in energy homeostasis in vertebrates ranging from fish to mammals. In teleost fish, however, the regulatory mechanism for leptin gene expression still remains unclear. In this study, we found that glucagon, a key hormone in glucose homeostasis, was effective at elevating the leptin-AI and leptin-AII transcript levels in goldfish liver via both in vivo intraperitoneal injection and in vitro cells incubation approaches. The responses of leptin-AI and leptin-AII mRNA to glucagon treatment were highly comparable. In contrast, blockade of local glucagon action could reduce the basal and induced leptin-AI and leptin-AII mRNA expression. The stimulation of leptin levels by glucagon was caused by the activation of adenylate cyclase (AC)/cyclic-AMP (cAMP)/ protein kinase A (PKA), and probably cAMP response element-binding protein (CREB) cascades. Our study described the effect and signal transduction mechanism of glucagon on leptin gene expression in goldfish liver, and may also provide new insight into leptin as a mediator in the regulatory network of energy metabolism in the fish model.

Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification.

Vibrio vulnificus is a serious bacterial pathogen for humans and aquatic animals. We developed a rapid, sensitive and specific identification method for V. vulnificus using loop-mediated isothermal amplification (LAMP) technique. A set of primers, composed of two outer primers and two inner primers, was designed based on the cytolysin gene sequence of V. vulnificus. The LAMP reaction was processed in a heat block at 65 degrees C for 60 min. The amplification products were detected by visual inspection using SYBR Green I, as well as by electrophoresis on agarose gels. Our results showed that the LAMP reaction was highly specific to V. vulnificus. This method was 10-fold more sensitive than conventional PCR. In conclusion, the LAMP assay was extremely rapid, simple, cost-effective, sensitive and specific for the rapid identification of V. vulnificus.