Molecular cloning of a cDNA encoding vitellogenesis-inhibiting hormone in the whiteleg shrimp Litopenaeus vannamei and preparation of its recombinant peptide using an E. coli expression system

The cDNA encoding a precursor of Liv-SGP-G, the most abundant crustacean hyperglycemic hormone-family peptide in the sinus gland of the whiteleg shrimp Litopenaeus vannamei, was cloned by RT-PCR coupled with 5′- and 3′-RACE. The determined cDNA sequence consisted of 621 nucleotides comprising a 69-bp 5′-untranslated region, a 357-bp open reading frame, and a 195-bp 3′-untranslated region. The deduced sequence of 72 amino acid residues corresponding to mature Liv-SGP-G was completely identical to that of natural Liv-SGP-G, determined in our previous study. The recombinant Liv-SGP-G was thereafter heterologously expressed in Escherichia coli, and its vitellogenesis-inhibiting activity in ex vivo cultured ovarian fragments was examined. The recombinant peptides inhibited vitellogenin gene expression with almost the same efficacy as that of natural Liv-SGP-G purified from sinus glands.     

Changes in crustacean hyperglycemic hormones in Pacific whiteleg shrimp Litopenaeus vannamei subjected to air-exposure and low-salinity stresses

Changes in crustacean hyperglycemic hormone (CHH)-family peptides in response to stress were investigated in Litopenaeus vannamei. Stress treatments consisted of air exposure and low salinity. High-performance liquid chromatography was used to quantify CHH-family peptides in the X-organ?Csinus gland complex (XO?CSG) in the eyestalks. Among the CHH-family peptides analyzed, only the level of sinus gland peptide-G (SGP-G) in the XO?CSG was decreased. SGP-G was also detectable by Western blotting analysis in the hemolymph of animals subjected to stress. These results suggest that SGP-G was secreted from the XO?CSG into the hemolymph during stress. Glucose levels in the hemolymph increased under conditions during which SGP-G was detected in the hemolymph. Hyperglycemia was also observed when SGP-G was injected. SGP-G may function to shift energy use to deal with stress.     

Effect of recombinant crustacean hyperglycemic hormones rCHH‐B1 and rCHH‐B2 on lipid metabolism in the Pacific white shrimp Litopenaeus vannamei

The crustacean hyperglycemic hormones (CHH) are pleiotropic neuropeptides controlling diverse processes in the life cycle of crustaceans. In the Pacific white shrimp Litopenaeus vannamei, CHH‐B1 and CHH‐B2 are generated by alternative splicing of the same gene, and the recombinant versions of these peptides have shown differential effects on glucose metabolism and osmoregulation in this species. In this work, we explored the role of both recombinant variants, expressed in yeast Pichia pastoris, on lipid metabolism. The results showed that rCHH‐B1 and rCHH‐B2 injections into eyestalk‐ablated shrimp significantly increased the phospholipid and triglyceride levels in haemolymph and restored free fatty acid levels, suggesting that these CHH variants mobilize lipid reserves in shrimp. The effects on lipids were concomitant to its hyperglycemic activity, suggesting that both variants, in addition to their hyperglycemic activity, have hyperlipidemic functions.     

泛甲壳动物激素调控卵黄蛋白原合成的分子机制研究进展

昆虫和甲壳动物由于进化上亲缘关系近统称泛甲壳动物，大多数泛甲壳动物卵黄蛋白原为雌性特异性蛋白，是卵母细胞中储存的卵黄蛋白的前体，可为胚胎发育提供营养和能量，是决定繁殖性能的关键因素，在生殖发育起着十分重要作用。业已证明，调控泛甲壳动物卵黄蛋白原的主要激素有保幼激素、蜕皮激素、神经肽和胰岛素样肽等。昆虫保幼激素和蜕皮激素发挥促进作用，而神经肽和胰岛素样肽对卵黄蛋白原合成的调控作用因种类不同存在不同。除了以上激素，甲壳动物特有的眼柄高血糖激素家族以及促雄腺激素对卵黄蛋白原的合成起负调控作用。本文总结了近年来泛甲壳动物卵黄蛋白原合成的调控机制的相关研究，概括并比较了调控昆虫与甲壳动物的卵黄蛋白原合成的主要激素及调控机制，为养殖虾蟹甲壳动物生殖调控相关研究提供参考。     

中华绒螯蟹类浮舰蛋白基因Esflol的克隆和组织表达分析

甲壳动物高血糖激素家族目前仅被发现存在于节肢动物中,在血糖水平调节、蜕皮、应激反应等多种生理代谢活动中发挥着重要的作用。为了解这个家族中的重要一员--甲壳动物高血糖激素(crustacean hyperglycemic hormone, CHH)如何调节河蟹体内的血糖水平,我们前期进行了转录组和表达谱分析,最终选取Esflol(Eriocheir sinensis flotillin-1 like,Esflol)作为我们的研究对象。根据转录组数据提示,结合RACE方法首次从中华绒螯蟹肝胰腺组织中克隆得到Esflol基因并进行了序列及组织表达分析。结果显示Esflol基因开放阅读框(ORF)1281bp,编码426个氨基酸,具有典型的SPFH超家族和PHB结构域,不含信号肽序列。多重序列比对分析表明,Esflol氨基酸序列与中华绒螯蟹flotillin-1相似度最高,达到57%。荧光定量PCR结果显示成熟中华绒螯蟹Esflol基因在肝胰腺组织中的表达量最高,其次为肌肉,另外在心,鳃,肠,胸神经节和胃中均有一定量表达,在脑中表达量较低。上述实验结果为下一步继续研究中华绒螯蟹糖代谢的生理机制奠定了基础,也为其他养殖类甲壳动物糖类代谢的研究提供了重要参考。     

Isolation and characterization of an additional crustacean hyperglycemic hormone from the greasyback shrimp <Emphasis Type="Italic">Metapenaeus ensis</Emphasis>

Crustacean hyperglycemic hormone (CHH) is released from the X-organ/sinus gland complex located in the eyestalks. In this study, the most abundant CHH in the sinus gland of the greasyback shrimp Metapenaeus ensis was purified by reversed-phase HPLC and identified by N-terminal amino acid sequencing. Although two CHH molecules (Mee-CHH-A and Mee-CHH-B) have already been identified from M. ensis by cDNA cloning, this study revealed the presence of an additional CHH peptide based on differences in the N-terminal amino acid sequences of the CHH-A and CHH-B. Therefore, this novel CHH was designated as Mee-CHH-C. A cDNA encoding the Mee-CHH-C precursor was cloned by RT-PCR coupled with 5′- and 3′-RACE, and it was found that the mature Mee-CHH-C consisted of 72 amino acid residues containing 6 conserved cysteine residues and possessed an amidated C terminus. Mee-CHH-C had 62 and 68% identities with Mee-CHH-A and Mee-CHH-B, respectively, and was highly homologous to CHHs characterized from other penaeid shrimp species. The hyperglycemic activity of Mee-CHH-C was examined by an in vivo bioassay using the kuruma prawn Marsupenaeus japonicus. Injection of Mee-CHH-C increased hemolymph glucose levels significantly and dose-dependently. These results indicate that Mee-CHH-C is possibly one of the major molecules in M. ensis that regulate glucose levels in the hemolymph.     

Flounder metamorphosis: its regulation by various hormones

Metamorphosis in the flounder has often been compared with the transition of tadpoles into frogs. The dorsal fin rays of the Japanese flounder (Paralichthys olivaceus) elongate during prometamorphosis when thyroid hormone levels are low, and are resorbed during metamorphic climax when thyroid hormone levels are high. Using an in vitro system for the culture of the flounder fin rays, we have examined how various hormones affect the resorption process. Both thyroxine (T₄) and triiodothyronine (T₃) directly stimulated fin ray shortening, T₃ being more potent than T₄. Other hormones, such as prolactin, cortisol and sex steroids, did not directly affect the resorption process but modified the tissue's response to thyroid hormones. Similar observations were obtained from in vivo studies. We also monitored the changes in the whole body concentrations of various hormones during early development and metamorphosis, and related these with the thyroid hormone profiles in order to get a better picture of their interactions. The gaps in the present status of research on the role of thyroid hormones during metamorphosis in the Japanese flounder are also discussed.     

Purification of molt-inhibiting hormone-like peptides with hyperglycemic activity from the eyestalks of the crab Scylla serrata

ABSTRACT:   In the present study, putative molt-inhibiting hormone (MIH)-like peptides from the sinus glands of Scylla serrata ( S. serrata ) were characterized by high-performance liquid chromatography, followed by immunoassay and bioassay (inhibition of ecdysteroid synthesis by Y-organs). Results of double immunodiffusion demonstrated the cross-reactivity of Homarus americanus MIH antiserum with the S. serrata MIH-like peptides, indicating the existence of close immunological relationship between crab and lobster peptides. Of the three molt-inhibiting immuno-reactive peptides, two also possess significant hyperglycemic activity.     

Dietary supplementation with Bacillus can improve the growth and survival of the kuruma shrimp Marsupenaeus japonicus in high-temperature environments

This study examined the effects of a commercial Bacillus complex used as probiotics on the performance of Marsupenaeus japonicus under high water temperature after 60 days of rearing. Growth and survival rate of M. japonicus were significantly increased in the probiotics-treated group (P < 0.05). Vibrio count and percentage of Vibrio in the intestinal tracts of M. japonicus reared with Bacillus were significantly lower than in other treatments (P < 0.05). The expressions of immune-related gene including prophenoloxidase, lysozyme, cytosolic manganese superoxide dismutase and hemocyanin subunit L in M. japonicus were significantly increased in the Bacillus-treated group (P < 0.05). The results indicated that Bacillus can be used as a diet supplement to minimize the damages caused by free radicals generated from insufficient oxygen metabolism due to the high-temperature stress, and to enhance immunity and activate the immune response levels. Our findings can improve the growth and survival rates of M. japonicus during the high-temperature farming period.     

The influence of genetics factor on key growth traits and quantitative genetic analysis of sea cucumber <Emphasis Type="Italic">Apostichopus Japonicus</Emphasis> (Selenka) heat-resistant and fast-growing strain

Using incomplete diallels cross and North Carolina II design, this study established sea cucumber families with heat-resistant and fast-growing Apostichopus japonicus strains from different ecological areas. Then, this study analyzed the influence of paternal and maternal genetic effects on the growth of A. japonicus, evaluated the growth effect of different A. japonicus families in the early growth stage and estimated the combining ability and heritability. The study showed that, in the early growth stage of A. japonicus, the influence of paternal genetic effects on the growth was higher than that of maternal effects. The best five parents and eight offspring families in terms of genetic potential breeding and growth were selected. The 3-month weight, 9-month weight, 12-month weight, 5-month to 9-month specific growth rate, 9-month coefficient of variation (CV) and 12-month CV of A. japonicus are at moderate heritability level, in order, 28.18, 40.82, 43.56, 27.87, 44.39 and 38.59 %, respectively; these traits played a leading role in parental additive gene effect on the formation of hybrids offspring traits.     

Expression of a recombinant crustacean hyperglycemic hormone of the kuruma prawn Penaeus japonicus in methylotrophic yeast Pichia pastoris

ABSTRACT:   Until now, six crustacean hyperglycemic hormones (CHH) designated Pej-SGP-I, -II, -III, -V, -VI and -VII have been characterized in the kuruma prawn Penaeus japonicus . All CHH consist of 72 amino acid residues and have an amidated carboxyl (C)-terminus. In the present study, we expressed Pej-SGP-III in methylotrophic yeast Pichia pastoris in order to obtain a large quantity of recombinant CHH possessing biological activity. A cDNA encoding Pej-SGP-III that had been previously cloned was processed by polymerase chain reaction (PCR) and the resulting product was ligated into an expression vector. Pichia pastoris was transformed with this vector after which a recombinant Pej-SGP-III was expressed having an additional amino acid residue (glycine) at the C-terminus (rPej-SGP-III-Gly), a form considered to be a putative precursor of this hormone. rPej-SGP-III-Gly secreted into the culture medium was purified by reversed-phase HPLC, and amidated using a peptidylglycine alpha-amidating enzyme. The amidated rPej-SGP-III (rPej-SGP-III) showed hyperglycemic activity in in vivo bioassay almost comparable to that of the natural Pej-SGP-III. rPej-SGP-III thus obtained will be a useful tool not only for its physiological study but also for the determination of its 3-D structure.     

Lack of growth hormone-releasing peptide-6 action on in vivo and in vitro growth hormone secretion in sexually immature grass carp (Ctenopharyngodon idellus)

Studies in mammals have shown that synthetic Met-enkephalin derivatives, called growth hormone-releasing peptides (GHRPs), stimulate growth hormone (GH) release. In the present study, GHRP-6 action on GH secretion was examined in vivo and in vitro in sexually immature grass carp. GHRP-6 injected intraperitoneally had no influences on serum GH levels in juvenile grass carp. Following intraperitonal injection of GHRP-6 and dopamine (DA) or cysteamine hydrochloride (CSH), alone and in combination into juvenile grass carp, DA and CSH were effective in elevating serum GH levels, but GHRP-6 was not effective in this respect; in addition, the synergistic action of GHRP-6 and DA or CSH on GH secretion was not seen. In this work, we had adapted and validated a perifusion system and a culture system for GH regulation studies. In a perifusion system, GHRP-6 (1000 to 0.1 nM), GHRP-6 (0.1 to 1000 nM), GHRP-6 (1 μM), and Hexarelin (an analog of GHRP, 1 μM) had no action on GH release from juvenile grass carp pituitary fragments or cells. Under static incubation conditions, GHRP-6 was inactive on GH release from juvenile grass carp pituitary fragments after 1 h and 6 h incubation, but human growth hormone-releasing hormone (hGHRH; 1 to 100 nM) as positive control could stimulate GH release in a dose-dependent manner. Furthermore, when GHRP-6 (100 nM) in combination static incubation with neuropeptides [e.g., hGHRH (100 nM), salmon gonadotropin-releasing hormone analogue (sGnRH-A) (100 nM), or D-Ala⁶,Pro⁹-NEt-luteinizing hormone-releasing hormone (D-Ala⁶,Pro⁹-NEt-LHRH, LHRH-A) (100nM)], GHRP-6 did not strengthen GH secretion actions of neuropeptides, and at the same time neuropeptides also did not modify the effects of GHRP-6 on GH secretion. The present results obtained using in vivo and in vitro techniques adapted for GH regulation studies show that GHRP-6 does not function as a GH-releasing factor in juvenile grass carp as it does in tilapia, amphibians, chickens, and mammals.     

In vitro methyl farnesoate secretion by mandibular organs isolated from different molt and reproductive stages of the crab Oziotelphusa senex senex

ABSTRACT:   Considerable evidences indicate that mandibular organ (MO) of crustaceans secrete terpenoid hormone (methyl farnesoate [MF]), which regulates molting and reproduction. In the freshwater south Indian rice field crab Oziotelphusa senex senex , MO isolated during premolt and vitellogenesis, secreted greater amounts of MF, which indicates that the regulation of the crustacean molt and reproduction is complex and also involves MF, besides steroid (ecdysteroid) and peptide (sinus gland peptide) hormones.     

Effects of steroid and peptide hormones on in vitro growth of previtellogenic oocytes from eel, Anguilla australis

To investigate the hormonal control of previtellogenic oocyte growth in eel, Anguilla australis, ovarian explants were incubated with or without different doses of steroid (11-ketotestosterone, estradiol-17β) or peptide hormones (ovine growth hormone, human insulin-like growth factor-I, human chorionic gonadotropin, porcine insulin) for 18 days. 11-Ketotestosterone, but not estradiol-17β, significantly increased oocyte diameters at physiological doses (30–100 ng/mL). Similarly, incubation with insulin-like growth factor-I, but not any of the other peptide hormones tested, resulted in increased oocyte diameters at high physiological doses (≥30 ng/mL). These data implicate androgens and hormones from the metabolic axis in mediating growth of previtellogenic oocytes in eel.     

仿刺参体腔液的抗菌特性

为了解仿刺参体腔液的抗菌谱以及不同二价金属离子对仿刺参体腔液抗菌活性的影响,实验采用生长曲线测定法分别测定了仿刺参体腔细胞破碎液上清和体腔液上清对哈维氏弧菌、灿烂弧菌、希瓦氏菌、假交替单胞菌、金黄色葡萄球菌、溶壁微球菌、停乳链球菌、拟诺卡式菌生长的影响,并通过该法以溶壁微球菌为受试菌测定了海洋环境中常见的Fe2+、Ca2+、Cd2+、Mg2+、Mn2+、Zn2+对仿刺参体腔液上清抗菌活性的影响。结果显示,仿刺参体腔细胞破碎液上清对受试的8株细菌的生长均无抑制作用,体腔液上清对溶壁微球菌的生长有明显的抑制作用,而对其他7株受试菌的生长无明显影响;Mn2+和Zn2+可明显增强体腔液上清对溶壁微球菌的生长抑制作用,而Fe2+、Ca2+、Cd2+、Mg2+对体腔液上清的抗菌活性无明显影响。研究表明,仿刺参体腔液在体外状态下只具有窄谱抗菌活性,与抗菌相关的免疫因子主要存在于体腔液上清中;体腔液中的抗菌免疫因子对海洋环境中的常见二价金属离子有一定的适应性,而且适当浓度的Mn2+和Zn2+可能具有促进仿刺参抗菌应答能力的作用。     

GnRHa-induced spawning of wild-caught jack mackerel Trachurus japonicus

The jack mackerel Trachurus japonicus is a key commercially exploited fish species in Japan. The rearing experiment often provides information that is useful for understanding the reproductive characteristics of wild stocks; however, there has been no study on spawning in captive T. japonicus. In the study reported here, we induced spawning in T. japonicus caught in the wild by hook and line. Females with fully vitellogenic oocytes and males during spermiation were selected by gonadal biopsy and injected with gonadotropin-releasing hormone analog (GnRHa) mixed in molten coconut butter. This treatment was performed four times in different groups of four females and five to eight males, and each group was maintained in a 3-m³ concrete tank. We observed the first spawning at 1 or 2 days post-injection and collected between 41,690 and 149,450 eggs. Spawning was recorded on 18 consecutive days in one experiment and for 3 days continuously in the other experiments. In the former, spawning ended when the water temperature reached 23 °C and occurred mainly between 2100 and 2400 hours. These results indicate that GnRHa-induced spawning may be useful for evaluating the reproductive characteristics of T. japonicus and obtaining fertilized eggs to conduct larval experiments.     

Population genetic structure and gene flow in the Japanese sea cucumber Apostichopus japonicus across Toyama Bay, Japan

The Japanese sea cucumber Apostichopus japonicus has a pelagic-lecithotrophic larva. Here, we clarify larval dispersal among Japanese sea cucumber populations by describing the levels of distinctiveness and gene flow among red and green variant populations of A. japonicus across Toyama Bay using 11 polymorphic microsatellite markers. Samples of Japanese sea cucumber populations were collected from three localities (Noto, Shinminato, and Uozu) around Toyama Bay. Geographically separated populations of red and green variants were determined to be genetically distinct, supported by phylogenetic analysis and the distinctiveness range values of pairwise multilocus estimates of F _ST. Contemporary migration analyses indicated that the majority of estimated migration events occurred within A. japonicus populations comprised of only either red or green variants. In a historical gene flow analysis, two best-fit models (n-island and stepping stone models) showed circulation for the gene migration in Toyama Bay among red variant populations. Our results provide useful information on the genetic structure of Japanese sea cucumber populations and will be helpful for genetic conservation and fisheries management of sea cucumber populations in Toyama Bay.