克氏原螯虾大颚器合成甲基法尼酯的研究

利用放射化学方法测定了克氏原螯虾大颚器中甲基法尼酯合成速率。结果发现，同一尾克氏原螯虾的左、右侧大颚器中甲基法尼酯的合成速率无明显差异(P＞0.05)；雄螯虾甲基法尼酯的合成速率比雌螯虾大(P＜0.05)；处于精巢发育期的雄螯虾甲基法尼酯合成速率明显大于发育前期的雄螯虾(P＜0.05)；在雌螯虾卵巢发育周期中，甲基法尼酯的合成速率依次为次级卵黄发生期＞初级卵黄发生期＞成熟期＞卵黄发生前期＞恢复期，表明大颚器合成甲基法尼酯与促进卵黄发生密切相关。     

孕酮对克氏原螯虾大颚器激素生物合成的体外调控

运用体外放射化学方法研究孕酮对克氏原螯虾（Procambarus clarkii）大颚器官（MO）生物合成甲基法尼酯（MF）的调控作用.结果表明,低浓度孕酮（10 pmol/L,100 pmol/L）对MO生物合成MF的抑制作用不明显;高浓度孕酮（1 nmol/L,5 nmol/L,10 nmol/L）对MF生物合成具有极显著的抑制作用（P＜0.01）.表明较高的血淋巴孕酮水平对克氏原螯虾MO合成MF产生反馈抑制作用.研究中对涉及MF合成的眼柄-大颚器官-卵巢轴的调节机理作了探讨.[中国水产科学,2006,13（3）：471-474]     

脊尾白虾(Exopalaemon carinicauda)FAMeT基因在卵巢发育周期中的表达分析

为了研究法尼酸甲基转移酶(Farnesoic acid O-methyltransferase,FAMeT)在脊尾白虾卵巢发育中的作用,采用实时荧光定量PCR方法分析连续3次繁殖过程中卵巢不同发育时期(Ⅰ-Ⅴ期)大颚器、卵巢和肝胰腺中FAMeT基因的表达规律.结果显示,FAMeT基因在鳃、心脏、肌肉、肝胰腺、卵巢、大颚器等组织中均有稳定的表达,其中,在鳃和大颚器的表达量显著高于其他组织;在卵巢发育周期内,FAMeT基因在大颚器、卵巢、肝胰腺中的表达量变化趋势基本一致,从Ⅰ-Ⅳ期逐渐下降,到Ⅴ期时表达量上调;在每个卵巢发育时期,FAMeT基因在大颚器中表达量最高,约为卵巢中表达量的20-30倍.研究结果表明,大颚器是合成FAMeT的主要组织,脊尾白虾繁殖过程中FAMeT基因主要在卵巢发育前期表达,可以合成大量甲基法尼酯(Methylfarnesoate,MF),促进卵巢发育.     

三疣梭子蟹FAMeT基因克隆及其在蜕皮周期中的表达水平

法尼酸甲基转移酶(farnesoic acid O-methyl transferase, FAMeT)是甲基法尼酯(methyl farnesoate, MF)生物合成途径中最后一步的关键酶,MF是一种类似昆虫保幼激素(juvenile hormone, JH)的倍半萜,在甲壳动物的生长发育及繁殖过程中起到了重要作用。本文克隆得到了三疣梭子蟹FAMeT的全长cDNA序列（GenBank登录号：KC192659）,它包括一个201bp 的5非编码区、一个318bp的 3非编码区,和一个825bp的开放阅读框（ORF）,编码274个氨基酸;推导的氨基酸序列与已公布的其他甲壳动物FAMeT进行比对,发现一致性达75%-97%,其中与远海梭子蟹FAMeT的一致性最高;而且该氨基酸序列由两个CF（CPAMD8/FAMeT）区域组成,这两个CF区域是FAMeT的标志,在所有甲壳动物的FAMeT里均有发现,因此推导的氨基酸序列是三疣梭子蟹FAMeT基因。我们运用实时荧光定量PCR(qRT-PCR)的方法分析了其在不同组织中、不同蜕皮周期中的表达量变化,发现FAMeT在三疣梭子蟹的各个组织里均有表达且在胸神经节(Taoracic ganglia)里表达最强;在三疣梭子蟹蜕皮过程中,大颚器FAMeT在D1期表达最强,然后逐渐下降至D4最低。该结果表明FAMeT在三疣梭子蟹蜕皮调控中起着重要的作用。     

中华绒螯蟹大颚器激素生物合成与性早熟的关系

运用放射化学方法跟踪测定中华绒螯蟹早熟个体和正常发育个体的大颚器合成和分泌的激素-甲基法尼酯(methyl farnesoate，MF)，发现早熟雌、雄蟹当年9月的MF合成速率约为正常蟹的2倍，早熟蟹的MF在当年秋季(10月或11月)均达到峰值，之后：MF合成速率显著降低，而成熟系数继续升高。正常发育的雌、雄幼蟹，当年至翌年4月的MF合成速率始终维持较低水平，翌年6月开始，MF合成速率迅速增大，至翌年10月的MF合成速率已超过早熟蟹的峰值，11月的MF合成速率明显降低，性腺发育至Ⅳ期末。研究结果表明：早熟蟹大颚器的提早发育，大量合成和分泌促性腺激素-MF，刺激卵巢或精巢发育成熟是导致性早熟的内分泌因素。     

去眼柄后中华绒螯蟹法呢酸甲基转移酶表达和卵巢发育

为了解甲基法呢酯(methyl farnesoate,MF)在中华绒螯蟹(Eriocheir sinensis)卵巢发育中的调控作用,采用离体方法研究了中华绒螯蟹大颚器(mandibular organ,MO)以及X-器官窦腺复合体(X-organ-sinus gland,XO-SG)对卵巢发育的作用。将MO与离体卵母细胞共培养作为实验组1,将MO和XO-SG与离体卵母细胞共培养作为实验组2,仅添加培养液的卵母细胞作为对照组。实验发现,蟹去眼柄(eyestalk ablation,ESA)后的第1、3、6、14天,同对照组相比,MO对其卵母细胞的发育均有极显著促进作用。ESA处理第6天,MO对卵母细胞发育的促进作用最强。XO-SG能逆转ESA处理后MO对卵巢发育的促进作用(P<0.01)。荧光定量PCR技术检测结果显示,ESA处理后,MO中法呢酸甲基转移酶(farnesoic acid O-methyltransferase,FAMeT)mRNA相对表达量上升。同未去眼柄的对照组相比,第1、3天FAMeT mRNA表达丰度无明显变化(P>0.05),第6、14天表达丰度提高至265%左右(P<0.01)。结果表明,中华绒螯蟹MO功能性物质能够促进卵母细胞发育,且ESA处理后第6天MO生物效应最强。MO中FAMeT mRNA的合成受XO-SG的抑制调节。在离体条件下,中华绒螯蟹XO-SG能够抑制卵母细胞的发育。     

Methyl farnesoate inhibition of late stage ovarian development and fecundity reduction in the black tiger prawn, Penaeus monodon

There is mounting evidence that the terpenoid hormone methyl farnesoate (MF) plays important roles in regulating reproductive processes in crustaceans. To gain further information on its roles and possible modes of action, MF was orally administered to ablated Penaeus monodon at a concentration of 5.5 μg per gram of diet, and a range of reproductive performance criteria measured. Results confirmed that MF plays a role in the reproductive process of this species. Specifically, under the conditions of this study, MF inhibited late stage ovary development and reduced fecundity in ablated prawns. The impact of the artificial diet (without additional MF) relative to a squid–mussel diet, was also assessed in this study and although it increased the quality of larvae produced, it also increased inhibition of late stage ovary development. Thus while the current study has increased our knowledge of MF by isolating an ovary developmental stage at which MF regulates reproduction in P. monodon, factors that determine the extent of its effect and whether it has a stimulatory or inhibitory effect, remain unknown. Until these factors are identified, the application of MF as a means of predictably manipulating egg production in captive prawns remains problematic.     

Validation of diagnostic assays to screen broodstock for Flavobacterium psychrophilum infections

It is hypothesized that the frequency of bacterial coldwater disease outbreaks can be reduced through the detection of the aetiologic agent, Flavobacterium psychrophilum, in broodstock followed by culling of eggs from heavily infected broodstock. Before a culling programme can be instituted, however, it is necessary to determine the sensitivity and specificity of existing assays for the detection of F. psychrophilum. In this study, tissue and ovarian fluid samples were collected from 224 fish at five hatcheries and screened using an enzyme‐linked immunosorbent assay (ELISA), a membrane‐filtration fluorescent antibody test (MF‐FAT), bacteriological culture and nested PCR. Latent class analysis was used to estimate sensitivity and specificity of kidney culture, kidney ELISA, nested PCR and MF‐FAT. Analytical sensitivity of the ELISA varied but was greatest when bacteria were cultured under iron‐limiting conditions. Diagnostic sensitivity estimates ranged from 0.02 (kidney culture) to 0.97 (kidney ELISA). Specificity estimates ranged from 0.02 (MF‐FAT) to 0.98 (kidney ELISA). In a separate challenge experiment, the ELISA confirmed the presence of F. psychrophilum in sub‐clinically infected fish. Results from this study demonstrate that the ELISA is an appropriate tool to screen broodstock and provides an indication of infection severity, which is crucial for implementation of a screening/culling programme.     

Identification of angiotensin I-converting enzyme inhibitory peptides derived from salmon muscle and their antihypertensive effect

ABSTRACT:   The salmon peptide digested from salmon muscle showed a strong inhibitory activity against the angiotensin I-converting enzyme (ACE). The antihypertensive effect of the salmon peptide on spontaneously hypertensive rats (SHR) was examined. After the single intravenous administration of the salmon peptide at a dose of 30 mg/kg body weight, the systolic blood pressure (SBP) was significantly reduced against the control. Further, a double-blind, placebo-controlled, parallel-group study determined the efficacy of the salmon peptide in mild hypertensive subjects. The SBP, after a 1.0 g of salmon peptide intake, was significantly reduced at 4 weeks after the intake, and 2 weeks after the intake finished, compared to the value before ingestion. Bioassay-guided separation of the salmon peptide, using a combination of column chromatographic techniques, led to the identification of 20 active di- and tri-peptides, including Ile-Val-Phe and Phe-Ile-Ala as two new ACE inhibitory tripeptides. Ile-Trp had the strongest ACE inhibitory activity (IC₅₀ = 1.2 μM) in vitro , and contributed 5.2% to the total ACE inhibitory activity. The salmon peptide and Ile-Trp showed a digestive resistance by in vitro assay, which mimicked the digestive organ, and had no affinity for factors related to blood pressure regulation, except for the ACE inhibitory activity.     

Molecular characterization,tissue distribution of Follicle‐Stimulating Hormone (FSH) beta subunit and effect of kisspeptin‐10 on reproductive hormonal profile of Catla catla (Hamilton, 1822)

Gonadotropin (GTH) hormones are glycoprotein which stimulates gonadal maturation in vertebrates. Follicle stimulating hormone is involved in initiation of gametogenesis and regulation of gonadal growth. FSHβ has been cloned and characterized from the brain of Catla catla. The FSHβ full‐length of cDNA sequence of 523 bp comprised 3, 394 and 128 bp of 5′‐UTR, open reading frame (ORF) 3′‐UTR respectively. The coding region of C. catla FSHβ encoded a peptide of 130 amino acids. Phylogenetic analysis of C. catla FSHβ deduced amino acid sequence showed high similarity with Gobiocypris rarus followed by goldfish, Carassius auratus. The qPCR result shows that FSHβ mRNA is mainly expressed in pituitary while moderate and low expression was observed in testis and ovary respectively. Chitosan‐nanoconjugated kisspeptin‐10 (CK‐10) of particle size 125 nm, polydispersity index of 0.335 to 0.65 and zeta potential of ?34.95 mV were synthesized and evaluated at against naked kisspeptin‐10 for their reproductive hormonal profile. Treatment of fish with CK‐10 showed controlled and sustained surge of the reproductive hormones (FSH & LH) with peak at 12 h. The hormone levels of naked kisspeptin‐10 treated fish decline after 6 h. The sustained release of this CK‐10 will help in reducing maturation age, synchronization of ovulation and spawning in fish. This is the first report on use of chitosan‐nanoconjugated kisspeptin‐10 (CK‐10) for reproduction in fish.     

Energy budget adjustment of sea cucumber Apostichopus japonicus during breeding period

Reproductive success is unpredictable for many cultured echinoderms. And cost of reproduction plays an important role in animal life‐history. Therefore, understanding cost of reproduction contributes to improving breeding techniques during broodstock rearing in aquaculture. In this study, energetic costs during breeding were explored in the sea cucumber, Apostichopus japonicus, an important aquaculture species in China, Japan and South Korea. The food intake, digestive functions and energy budget patterns were estimated at different breeding stages. Unexpectedly, in the growing gonad phase of the sea cucumber lifecycle, animals showed a decline in feed consumption (from 4.49 to 2.6 g ind^?1 day^?1), ingestion rate (from 0.4 to 0.13 g g^?1 day^?1) and apparent digestive rate (from 14.73% to 10.92%), resulting in a reduced energy input. In addition, the increased energy investment in reproduction (from 1.76% to 15.61%) resulted in a decrease in energy allocated to soma growth (from 17.1% to 5.64%) and self‐maintenance (from 47.82% to 34.67%). These results suggest that reproduction impairs energy acquisition ability in breeding A. japonicus. And broodstock can adapt energy shortage by internal adjustment of energy allocation strategy.     

Multigenerational effects of 17β-estradiol and nonylphenol on euryhaline cladoceran <Emphasis Type="Italic">Diaphanosoma celebensis</Emphasis>

ABSTRACT:     The effects of parental exposure to 17β-estradiol (E2) and 4-nonylphenol (4-NP) on life history parameters of three successive generations of the euryhaline cladoceran Diaphanosoma celebensis were assessed under laboratory conditions. Less than 24-h-old neonates (P) were exposed to five sublethal concentrations of E2 (0.1, 1, 10, 100 and 1000 μg/L) and 4-NP (0.01, 0.1, 1, 10 and 50 μg/L) at 25°C. Age at first reproduction, reproductive period, fecundity and lifespan were investigated. Successive generations (F₁–F₃) were monitored further in the absence of toxicants. Results showed that cladocerans exposed to 10–1000 μg/L E2 produced more neonates at a younger age compared to the control. The same effects were also observed in the F₁ and F₂ but ceased in F₃. Cladocerans exposed to 1 μg/L 4-NP also produced significantly more neonates compared to the control, but this effect was not found in successive generations. The results suggest that vertebrate hormone can modify the reproduction of D. celebensis , and the effects are multigenerational.     

Growth hormone-like substance in the rotifer Brachionus plicatilis

ABSTRACT:   A growth hormone (GH)-like substance was extracted from the rotifer Brachionus plicatilis and subsequently purified by gel filtration and ion exchange chromatography. The GH-like substance had a molecular weight of approximately 28 kDa and had cross-reactivity with salmon GH antibody. In vivo bioassay showed a higher intrinsic rate of increase and net reproduction rate of B. plicatilis treated with the GH-like substance.     

The effect of Ibuprofen on female and male reproduction of the open thelyca marine shrimp,Litopenaeus

A research was designed to explore the effect of ibuprofen (IBU) as an intervening agent for prostaglandin biosynthesis on male and female reproduction of three species of Litopenaeus. Ovarian maturation was studied in L. stylirostris and L. vannamei injected periodically with IBU at 0.01 and 0.1 μg g^?1 body weight (b.w.) and treated for unilateral eyestalk ablation. IBU at 0.1 μg induced a statistically (P < 0.05) higher rate of L. vannamei females with developing ovaries (45 ± 19%) as compared with controls (12 ± 8%) during 5 weeks; L. stylirostris did not activate vitellogenesis under this experimental protocol. IBU was also evaluated for male sexuality in L. occidentalis, L. stylirostris and L. vannamei. Periodic injections of IBU had no effect on sperm counts and spermatophore weights; however, sperm abnormalities in IBU 0.1 μg treated L. occidentalis and L. stylirostris were very low. In L. stylirostris, the renovation of normal spermatophores was remarkably improved in IBU treated males as compared with controls, but in L. vannamei spermatophore deterioration was not affected. The findings of this research support the hypothesis that prostaglandins are involved in penaeid reproduction playing a negative physiological role; however, altered steroidogenic pathways could also be involved.     

The effects of salinity on reproductive performance and plasma levels of sex steroids in Caspian kutum Rutilus frisii kutum

Sexually mature kutum, Rutilus frisii kutum, captured from its natural habits, the Caspian Sea and the Khoushkrood River, reared at 0.5 g L^?1 and 8–13 g L^?1 for approximately 1 year in experimental condition, for assessing the effect of salinity on reproduction. Plasma concentrations of sex steroid hormones (17β‐estradiol, testosterone and 17α‐hydroxyprogestrone) were measured in the three stages of gametogenesis. Female kutum held at <0.5 g L^?1 or 8–13 g L^?1 had no ovulated oocytes in their ovaries. In contrast, males held in captivity were spermiated, similar to their wild counterparts. The average sperm volume of males held at <0.5 g L^?1 (2.36 ± 0.46 mL) was lower than males held at 8–13 g L^?1 (3.65 ± 0.73 mL) at the end of the experimental period. The highest concentration of testosterone was observed in mid‐gametogenesis in wild fish that was significantly higher than the concentration seen in fish held in either <0.5 g L^?1 or 8–13 g L^?1. Female kutum showed suppressed steroid hormones in captivity, resulting in failure in the gonad development. However, male kutum adapt well to captivity and showed synchrony in steroid hormone variations with the wild fish, resulting in the testicular development. Results of this study also indicate that salinity plays a minor, but vital, role in reproduction of kutum, a factor that needs to be considered for keeping broodstock of brackish water fish species like kutum.     

Dietary reduction of fish meal,supplementation of vitamin C and fish oil may promote ovary maturation by improving digestibility and immunity in Procambarus clarkii (Crustacea: Decapoda)

Fish meal, vitamin C and fish oil are known to play essential roles in reproduction and immunity in animals. To investigate how the diet affect reproduction under the condition of reducing costs, we designed an experimental diet that decreased the ratio of fish meal with soybean meal from 1:1 to 1:1.5, and supplemented with 0.06% vitamin C and 3.8% fish oil for the crayfish, Procambarus clarkii. After a 100‐day feeding experiment, female crayfish fed the test diet had significantly greater gonadosomatic index (GSI) compared with those fed the basal diet (P < 0.05). At the middle stage of ovarian development, the test diet gave significantly higher trypsin‐specific activity (340.94 ± 57.32 U mg^?1 protein) in hepatopancreas of the crayfish than the basal diet (89.48 ± 10.01 U mg^?1 protein) (P < 0.05). However, hepatosomatic index (HSI) was remarkably decreased for those females fed test diet (P < 0.05). In addition, the experimental diet resulted in markedly lower superoxide dismutase (SOD) activity and malonaldehyde (MDA) levels, especially in the ovaries (P < 0.01). These results suggest that dietary reduction of fish meal, supplementation of appropriate amounts of vitamin C and fish oil may promote ovary development probably by increasing digestibility, as well as by promoting transferring of nutrients from hepatopancreas to ovary and raising immunity of P. clarkii.     

Cloning of kuruma prawn Marsupenaeus japonicus crustin-like peptide cDNA and analysis of its expression

ABSTRACT:   Antimicrobial peptides serve as an important component of the innate immune system of all species by functioning to provide a rapid first line defense against infection. Arthropod antimicrobial peptides have been well described in insects, whereas only a few molecules have been identified in crustaceans. Five variants (types 1–5) of Marsupenaeus japonicus crustin-like peptide cDNA that were obtained from a hemocyte cDNA library and polymerase chain reaction (PCR) amplification are reported here. Marsupenaeus japonicus crustin-like peptide type 1, the predominant type, has a cDNA consisting of 679 nucleotides and an open reading frame consisting of 573 base pairs coding for 191 amino acid residues. Other types contain varying glycine-rich repeats at the N-terminal amino acid sequences. The deduced amino acid sequences of these variants are highly similar to those of Litopenaeus setiferus (80% identity), Litopenaeus vannamei (80% identity) and Carcinus maenas crustins (44% identity). Expression of Marsupenaeus japonicus crustin-like peptide mRNA was detected in hemocytes, but not in the heart, hepatopancreas, gill, fore-gut, mid-gut, muscle, subcuticular epithelium or ovary. The expression level of crustin-like peptide mRNA increased significantly 1, 3 and 7 days post-peptidoglycan feeding as determined by quantitative real-time PCR. These results suggest that crustin-like peptide could have an important role in shrimp defense mechanisms.