Chaoqun Hu

Chemical composition and nutritional quality of sea cucumbers

BACKGROUND The dried form of sea cucumbers has been a seafood and medicinal cure for Asians over many centuries. In this study the chemical composition and nutritional quality of eight common sea cucumbers (Stichopus herrmanni, Thelenota ananas, Thelenota anax, Holothuria fuscogilva, Holothuria fuscopunctata, Actinopyga mauritiana, Actinopyga caerulea and Bohadschia argus) were determined. RESULTS All species except T. anax and A. caerulea had higher protein and lower fat levels. A. mauritiana and B. argus had less ash content. Glycine was the dominant amino acid found in all species, and content ranged from 126 to 216 mg g(-1) of crude protein. All species exhibited low lysine:arginine ratio and higher essential amino acid scores were obtained by threonine and phenylalanine + tyrosine. A. mauritiana had proportionally less saturated fatty acids (31.23%), and more monounsaturated fatty acids (45.64%) and polyunsaturated fatty acids (PUFA, 23.13%) than other species. Arachidonic acid (C20:4n-6) was the major PUFA in all species. T. ananas, A. mauritiana and A. caerulea contained more n-3 PUFA. The n-3/n-6 ratios of eight sea cucumbers species ranged from 0.25 to 0.61. CONCLUSION Sea cucumbers are a seafood with high protein and low fat levels. The amino acid contents were similar but fatty acid profiles were different among species. The comparison showed that T. ananas, A. mauritiana and B. argus possessed higher nutritional values than other sea cucumber species.

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.

Pacific White Shrimp (Litopenaeus vannamei) Vitellogenesis-Inhibiting Hormone (VIH) Is Predominantly Expressed in the Brain and Negatively Regulates Hepatopancreatic Vitellogenin (VTG) Gene Expression

ABSTRACT 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.

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.

Autophagy is induced by the type III secretion system of Vibrio alginolyticus in several mammalian cell lines

Vibrio alginolyticus is a gram-negative bacterium and has been recognized as an opportunistic pathogen in marine animals as well as humans. Here, we further characterized a cell death mechanism caused by this bacterium in several mammalian cell lines. The T3SS of V. alginolyticus killed HeLa cells by a very similar cell cytolysis mechanism in fish cells, as evidenced by cell rounding and LDH release; however, DNA fragmentation was not observed. Further studies showed that caspase-1 and caspase-3 were not activated during the T3SS-mediated cell death, indicating that the death mechanism is completely independent of pyroptosis and apoptosis in HeLa cells. Conversely, autophagy was detected during the T3SS-mediated cell death by the appearance of MDC-labeled punctate fluorescence and accumulation of autophagic vesicles. Moreover, western blot analysis revealed increase in conversion of LC3-I to LC3-II in infected mammalian cell lines, confirming that autophagy occurs during the process. Together, these data demonstrate that the death process used by V. alginolyticus in mammalian cells is different from that in fish cells, including induction of autophagy, cell rounding and osmotic lysis. This study provides some evidences hinting that differences in death mechanism in responses to V. alginolyticus infection may be attributed to the species of infected cells from which it was derived.

Elemental composition of commercial sea cucumbers (holothurians)

Toxic and essential elements in 11 different sea cucumber species were determined and compared with daily intake recommendations and maximum allowed levels. The contents of macro-elements contents in dried sea cucumber samples were found to be 25,000–152,000 mg kg–1 for Na, 4000–8600 mg kg−1 for Mg, 1100–5200 mg kg−1 for K, 15,000–68,000 mg kg−1 and 36,300–251,000 mg kg−1 for Cl. Trace element concentrations in dried sea cucumber samples were found to be 11–100 mg kg−1 for Zn, 41–660 mg kg−1 for Fe, 3–74 mg kg−1 for Cu, 1.1–16 mg kg−1 for Mn, 1.4–3.7 mg kg−1 for Se, 1.1–9.6 mg kg−1 for Cr, and 0.3–5.1 mg kg−1 for Ni. All sea cucumber species were rich sources of Na, Cl, Mg, Ca, Fe, Cu, Se and Cr for human consumption. Regarding contaminants, As, Cd and Pb concentrations in dried sea cucumbers were in the ranges of 1.1–6.1, 0.03–0.06 and 0.11–0.69 mg kg−1, respectively. Moreover, Hg values of 11 sea cucumbers were below the detection limit (0.01 mg kg−1).

Differential regulation of hepatopancreatic vitellogenin (VTG) gene expression by two putative molt-inhibiting hormones (MIH1/2) in Pacific white shrimp (Litopenaeus vannamei).

Molt-inhibiting hormone (MIH), a peptide member of the crustacean hyperglycemic hormone (CHH) family, is commonly considered as a negative regulator during the molt cycle in crustaceans. Phylogenetic analysis of CHH family peptides in penaeidae shrimps suggested that there is no significant differentiation between MIH and vitellogenesis-inhibiting hormone (VIH, another peptide member of CHH family), by far the most potent negative regulator of crustacean vitellogenesis known. Thus, MIH may also play a role in regulating vitellogenesis. In this study, two previously reported putative MIHs (LivMIH1 and LivMIH2) in the Pacific white shrimp (Litopenaeus vannamei) were expressed in Escherichia coli, purified by immobilized metal ion affinity chromatography (IMAC) and further confirmed by western blot. Regulation of vitellogenin (VTG) mRNA expression by recombinant LivMIH1 and LivMIH2 challenge was performed by both in vitro hepatopancreatic primary cells culture and in vivo injection approaches. In in vitro primary culture of shrimp hepatopancreatic cells, only LivMIH2 but not LivMIH1 administration could improve the mRNA expression of VTG. In in vivo injection experiments, similarly, only LivMIH2 but not LivMIH1 could stimulate hepatopancreatic VTG gene expression and induce ovary maturation. Our study may provide evidence for one isoform of MIH (MIH2 in L. vannamei) may serve as one of the mediators of the physiological progress of molting and vitellogenesis. Our study may also give new insight in CHH family peptides regulating reproduction in crustaceans, in particular penaeidae shrimps.

Prevalence of mobile genetic elements and transposase genes in Vibrio alginolyticus from the southern coastal region of China and their role in horizontal gene transfer.

Vibrio alginolyticus has high genetic diversity, but little is known about the means by which it has been acquired. In this study, the distributions of mobile genetic elements (MGEs), including integrating conjugative elements (ICEs), superintegron-like cassettes (SICs), insertion sequences (ISs), and two types of transposase genes (valT1 and valT2), in 192 strains of V. alginolyticus were investigated. ICE, SIC, and IS elements, valT1, and valT2 were detected in 8.9%, 13.0%, 4.7%, 9.4%, and 2.6% of the strains, respectively. Blast searches and phylogenetic analysis of the acquired sequences of the ICE, SIC, IS elements and transposase genes showed that the corresponding homologues were bacterial and derived from extensive sources. The high prevalences of these MGEs in V. alginolyticus implied the extensive and frequent exchange of genes with environmental bacteria and that these elements strongly contribute to the genetic and phenotypic diversity of the bacterium. To our knowledge, this is the first report of V. alginolyticus harboring ICE and SIC elements.

The expression of Na, K-ATPase in Litopenaeus vannamei under salinity stress

Abstract The purpose of this study was to explore the mRNA expression of Na, K-ATPase alpha subunit in Litopenaeus vannamei (Boone, 1931) in response to salinity stress. L. vannamei acclimated to 30 ppt, was transferred to 30 (control), 15, 7.5 and 1 ppt for 0.5, 1, 3, 6, 12 and 24 h. Partial Na, K-ATPase alpha subunit expression in both gills and hepatopancreas was examined using quantitative real-time PCR, showing that there was no significant difference within 3 h after treatment (P>0.05). However, there was a suddenly increase to a peak value at 6 h (P<0.05), then the expression level decreased from 12 h to 24 h. Similar results were obtained for the activities of the Na, K-ATPase. These results suggest that L. vannamei Na, K-ATPase expression is stimulated by salinity stress and that it may play important roles in regulation of the ion density in vivo. To our knowledge, this is the first report on the mRNA expression of Na, K-ATPase alpha subunit in L. vannamei in response to salinity stress.

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.