Sperm capacitation of the shrimp Litopenaeus vannamei

Litopenaeus vannamei is one of the most important species of farmed shrimp. The females have an ‘open’ thelycum. Mating is accomplished by attaching the male spermatophore onto the surface of the thelycum 4–6 h before spawning. During this period, sperm may have to undergo morphological changes associated with a capacitation process that has been described for other shrimp species. The objective of this research was to extend research on sperm capacitation in L. vannamei by ultrastructural and biochemical means. The sperm of L. vannamei were divided into those freshly prepared from the spermatophore (S‐sperm), extracted from the male gonopores, and those extracted from the female thelycum (T‐sperm). Under transmission electron microscopy, ultrastructural differences were detected between the S‐ and the T‐sperm in the nuclear material, the filamentous meshwork and the cytoplasmic particles. Under scanning electron microscopy, the difference was observed in the cap and spike regions. Immunofluorescence using confocal microscopy to detect tyrosine phosphorylated proteins revealed different distribution patterns between S‐ and T‐sperm. The location of phosphorylation activity changed from the spike in S‐sperm, to the filamentous meshwork in the T‐sperm. These morphological and biochemical changes confirm that capacitation of L. vannamei sperm takes place following mating.     

温度、盐度对凡纳滨对虾精荚再生和精子质量的影响

研究了3种不同温度和盐度对凡纳滨对虾精荚再生和精子质量的影响。通过称量精荚以及采用染色或显微镜观察养殖在不同温、盐度条件下的雄虾的精子数量和正常率来评价精子质量。结果表明:随着实验养殖时间的延长和精荚再生次数的增加,26℃组的精子质量明显好于22℃组和30℃组,30℃组的活精子数由第1次再生的14.0×106个下降到4.3×106个,畸形精子百分率由6.1%增加到56.6%。温度越高,蜕皮周期越短,精荚再生时间也越短,但精子质量下降也很迅速。盐度30组的精荚重量显著高于盐度5组和15组,但盐度15的精子总数和活精子数却显著高于盐度5组和30组,随着时间的延长,3组盐度的畸形精子百分率都有所增加,其中盐度5组增加得最快。     

芽孢杆菌制剂对凡纳滨对虾Litopenaeus vannamei肠道微生物群落的影响

应用Biolog方法和传统的平板培养方法分析比较了施用芽孢杆菌制剂的虾池(B)和没有施用任何有益菌的虾池(A)在养殖后期凡纳滨对虾肠道微生物群落结构,并用Shannon指数、Simpson指数和McIntosh指数分析了2种群落的代谢功能的差异。2个虾池对虾肠道微生物群落可培养细菌优势菌属都是革兰氏阴性菌;B虾池对虾肠道可培养细菌数量比A虾池的少;但B虾池对虾肠道微生物群落Shannon指数、Simpson指数和McIntosh指数及其微生物群落代谢功能均显著高于A虾池(P<0.05)。结果表明,虾池施用了芽孢杆菌制剂,可促进养殖对虾肠道微生物群落的代谢功能。     

凡纳滨对虾G蛋白Gβ1亚基的克隆、表达与鉴定

根据G蛋白Gβ1亚基的保守序列设计简并引物,通过简并PCR和RACE技术,克隆到凡纳滨对虾(Litopenaeusvannamei)Gβ1基因的全长cDNA序列。利用Blast、DNAstar和Genedoc软件分析,发现该Gβ1编码的蛋白序列与其他物种已知的Gβ1序列具有相当高的保守性,将它命名为pvGβ1。免疫共沉淀分析发现pvGβ1能在体外适当条件下与对虾Gαs或Gαq相互结合。Westernblot-ting分析发现,pvGβ1在对虾身体各部位都有广泛分布,尤其在脑神经、眼和眼柄有大量表达。说明了Gβ1在对虾的神经系统和光信号传导等生命过程中的重要性。     

氨氮对凡纳对虾免疫指标的影响

以凡纳对虾(Litopenaeus vannamei)为研究对象,研究氨氮对其免疫指标的影响。实验氨氮质量浓度梯度设置为0.05(对照)、0.5、1.0、1.5、2.0、2.5 mg／L,各氨氮梯度用4g／L的氯化铵溶液来调节。将暂养在自然海水(对照)中的凡纳对虾分别放入各实验梯度中,对虾体长为(8.5±0.5)cm。结果表明,氨氮对凡纳对虾血细胞数量、血清中的酚氧化酶活力、溶菌和抗菌活力的影响显著(F>F0.05),且随着氨氮质量浓度的升高,血细胞数量和溶菌、抗菌活力降低,酚氧化酶活力升高;在0-24 h实验时间内,各处理组(对照组除外)对虾血细胞数量和溶菌、抗菌活力呈下降趋势,酚氧化酶活力呈上升趋势,24 h后稳定在较低水平上。实验说明,随着氨氮水平升高,凡纳对虾免疫力明显下降,对病原菌的易感性提高,因此在养殖过程中,环境氨氮变化幅度不应超过0.5 mg／L,或长时间维持在较高氨氮水平(>0.5 mg／L)。     

Morphometric relationships among spermatophore structures and their association with female fertility in the Pacific white shrimp Litopenaeus vannamei

ABSTRACT

Male reproductive performance affects production in shrimp hatcheries. To help better define the criteria required to choose male broodstock, our objective was to study associations among spermatophore morphometric traits and their relationship with fertility. The variables studied were male body weight (MBW), male body length (MBL), spermatophore weight (SW), sperm cell count (SCC), and percentage of live sperm cells (PLS). To evaluate fertility, female body weight (FBW) was considered, as well as the number of eggs in the spawn (NE), hatching percentage (HP), and the number of nauplii (NN). A random sample of 200 males and 200 females from the broodstock population were obtained. Correlations of SW with body MBW and MBL were estimated as 0.86 ± 0.02 and 0.63 ± 0.04 respectively. Correlations between the SCC with MBW and SW were estimated as 0.52 ± 0.05 and 0.61 ± 0.04 respectively. No linear associations were observed between MBW, SW, and SCC, with the NN (P > 0.05). Also, there was no linear relationship between FBW and NN (P > 0.05). On the other hand, FBW and NE were positively correlated (0.33 ± 0.07). HP showed a wide variation (2.6% to 98%) due to the multiple factors associated with artificial insemination.     

镉胁迫对凡纳滨对虾血细胞基因表达的影响

为探讨镉(Cd)胁迫下凡纳滨对虾(Litopenaeus vannamei)血细胞的分子响应,以不同浓度的Cd2+(0,0.5 mg/L和5 mg/L)进行胁迫,于不同胁迫时间取血淋巴,测定血细胞中Trx 2、Grx 2、Grx 3和MGST 3的基因表达量变化.结果显示,Trx 2、Grx 3和MGST 3表达量在胁...     

凡纳滨对虾血蓝蛋白的抗黑曲霉活性

 以凡纳滨对虾(Litopenaeus vannamei)为研究对象, 运用亲和层析、SDS-PAGE、Western-blotting、抗真菌实验等技术探索对虾血蓝蛋白的抗黑曲菌活性。结果发现, 血蓝蛋白在质量浓度为0.1～0.8 mg/mL时对黑曲霉(Aspergillus niger)生长表现出明显的抑制活性。在此基础上, 选取0.2 mg/mL血蓝蛋白作用后的黑曲霉进行显微观察分析。光学显微镜观察结果表明, 血蓝蛋白能抑制黑曲霉孢子生长。扫描电镜分析显示, 在血蓝蛋白作用下, 黑曲霉孢子囊萎缩, 分生孢子集聚成团, 菌丝裂解、扭曲、变短、无分生孢子囊。由此推测, 对虾血蓝蛋白具有抗黑曲菌活性, 其抗菌机制可能与血蓝蛋白抑制黑曲菌孢子生长和促使病态菌丝产生等有关。本研究对揭示血蓝蛋白的非特异性免疫机理和寻找新的对虾疾病免疫学防治途径等具有重要意义。

     

Silencing Pacific white shrimp Litopenaeus vannamei LvRab7 reduces mortality in brooders challenged with white spot syndrome virus

White spot syndrome virus (WSSV) is a major threat for farmed shrimp worldwide. RNA interference (RNAi) is the most recent tool against viral diseases. Rab7 silencing effectively inhibited virus infections in juvenile shrimp, but the antiviral effect in brooders remains unknown. This study found a homologue Penaeus monodon Rab7 gene in Litopenaeus vannamei brooders from Mexico. Sequence identity was >99% to a Thai LvRab7 sequence and >94% to Rab7 sequences from P. monodon or Marsupenaeus japonicus. Animals treated with a partial (494 bp) or a complete (618 bp) LvRab7 dsRNA sequences and challenged 48 h post treatment (hpt) with a high WSSV dose showed 80–88% mortality respectively. Shrimp treated with 4 or 20 μg LvRab7 dsRNA and challenged with a WSSV high dose had 80% mortality each, but it was reduced to 33% and 40%, respectively, with a low dose. Efficacy of dsRNA to reduce shrimp mortality was dependent on virus dose used regardless of dsRNA concentration. A significant reduction in LvRab7 mRNA levels was observed at 120 hpt. In conclusion, silencing LvRab7 in brooders showed a mild antiviral effect against a WSSV challenge at 48 hpt.     

凡纳滨对虾类胰岛素肽基因的结构及表达分析

类胰岛素肽(ILP)是胰岛素超家族的成员,具有进化保守性,是影响动物生命活动的重要因子之一。本研究克隆了凡纳滨对虾(Litopenaeus vannamei) ILP1 (LvILP1)全长基因,mRNA长度为812 bp,其中开放阅读框长543 bp,编码180个氨基酸。序列分析显示,LvILP1蛋白预计分子量为20.81 kDa,理论等电点为9.45,不稳定系数为96.20,具有一个信号肽,没有跨膜结构,推导其为碱性、不稳定的分泌蛋白。结构预测显示,该蛋白具有胰岛素超家族保守的IlGF结构域,由N端信号肽、B链、C肽和A链组成,同时具有6个保守的半胱氨酸位点和2个断裂位点。系统进化分析显示,LvILP1与斑节对虾(Penaeus monodon) ILP7亲缘关系最近,与甲壳动物ILP1聚为一支,然后分别与无脊椎动物ILP7、脊椎动物松弛素(Relaxin)、胰岛素(Insulin)、胰岛素样生长因子(IGF)聚在一起。无脊椎动物ILP7类与外群海葵(Actinia tenebrosa) ILP的进化关系最近,表明这类ILP可能与胰岛素超家族的祖先较为相似。转录因子预测显示,LvILP1可能的转录因子为叉头框蛋白O3 (FoxO3)、糖皮质激素受体(GR)、CAAT区/增强子结合蛋白(C/EBP)、信号传导及转录激活蛋白(STAT)等;蛋白互作分析显示,LvILP1与细胞膜上的胰岛素受体(IR)、神经信号分子(VGLUT1、SYT 1_3)、糖蛋白激素(GPHB5)、鞣化激素(Bursicon)等相互作用;对这些转录因子和互作蛋白的生物功能进行分析,进而推测LvILP1可能具有调节生长发育、激素刺激反应、神经系统稳态、碳水化合物稳态、蜕皮后组织重建以及生殖发育等作用。分析发现,LvILP1在凡纳滨对虾早期发育阶段有较高表达,在成体各个组织中均有表达,但在眼柄中表达量最高。本研究结果为深入了解凡纳滨对虾ILP的基因结构、进化、功能及表达提供了重要信息,同时为凡纳滨对虾的分子育种和健康养殖提供线索。     

pH调节法分离凡纳滨对虾壳中虾青蛋白的研究

采用pH调节法对凡纳滨对虾（Litopenaeus vannamei）虾壳中影响虾壳红变的虾青蛋白进行分离回收,以蛋白质回收率、纯度和二级结构含量为分离特性参数,以1.0为pH变化梯度,研究pH调节法从虾壳中提取虾青蛋白的规律。结果显示,pH调节法的回收率为47.5%,所得蛋白质纯度为78.23%、分子量为45 000 Da。该蛋白质在pH 3.0和11.0时有最大溶解度,分别为60.5%和55.7%;在pH 5.0时溶解度最低为15.4%;等电点为5.6。通过圆二色光谱图分析得知虾青蛋白是一种以螺旋为主要二级结构存在的物质,pH调节法所得螺旋含量为70.1%。     

凡纳滨对虾血蓝蛋白的细菌凝集活性

以凡纳滨对虾（Litopenaeaus vannarnei）为研究对象，运用亲和层析、PAGE、SDS-PAGE、Western-blotting和细菌凝集实验等方法探索凡纳滨对虾血蓝蛋白的细菌凝集活性。结果发现，血蓝蛋白对副溶血弧菌（Vibrio parahaemolyticus）、溶藻酸弧菌（Vibrio alginolyticus）、河弧菌（Vibrio fluvialis）、哈维氏弧菌（Vibrio harveyi）、嗜水气单胞菌（Agromona hydrophila）和金黄色葡萄球菌（Stphylococcus aureus）等6种虾类致病菌具有凝集活性，其凝集活性大小为0．27-1．08mg／L，其中对副溶血弧菌和溶藻酸弧菌凝集活性最大，是哈维氏弧菌的4倍。在此基础之上，选取α-D-葡萄糖、D-半乳糖、D-木糖、D-山梨糖、蔗糖、麦芽糖、α-D-乳糖、甘露醇和N-乙酰神经氨酸等9种糖考察其对血蓝蛋白细菌凝集活性的影响。结果显示，血蓝蛋白的细菌凝集活性可被α-D-葡萄糖和N-乙酰神经氨酸所部分抑制和全部抑制，其最低抑制浓度范围为50～100mmol／L。由此推测，血蓝蛋白确实具有细菌凝集活性，这对进一步研究血蓝蛋白的抗菌机理具有重要意义。     

氨氮胁迫下白斑综合征病毒对凡纳滨对虾的致病性

为了评价养殖水环境中氨氮(NH_4-N)对凡纳滨对虾(Litopenaeus vannamei)的危害性,开展了NH_4-N胁迫对凡纳滨对虾感染白斑综合征病毒(WSSV)后的死亡率、WSSV增殖速率和对虾主要免疫相关酶活性影响的实验。在NH_4-N胁迫质量浓度为15.6 mg·L-1,分别注射2×105和2×106个WSSV粒子,结果显示,NH_4-N胁迫下注射2×105个WSSV粒子的凡纳滨对虾第144小时死亡率达到53.3%,显著高于无胁迫组(40.0%)。对虾鳃组织WSSV荧光定量PCR检测结果显示,NH_4-N胁迫下凡纳滨对虾鳃组织内WSSV的增殖加快。此外,免疫相关酶活性结果显示,NH_4-N浓度突变会促使对虾血清中酚氧化酶(PO)、酸性磷酸酶(ACP)和碱性磷酸酶(AKP)活性短暂升高后持续降低。由此可见,NH_4-N胁迫会加快WSSV在患病凡纳滨对虾体内的增殖,导致更高死亡率,这可能是因为胁迫造成了对虾免疫相关酶活性降低和抗病原感染能力下降。     

Dietary protein level and natural food management in the culture of blue (Litopenaeus stylirostris) and white shrimp (Litopenaeus vannamei) in microcosms

The effect of dietary protein level and natural food management on the production parameters of blue and white shrimp, as well as on water quality, was evaluated in a microcosms system (plastic pools simulating aquaculture ponds). Two experimental trials were carried out in the facilities of DICTUS, University of Sonora, Northwest México. Treatment with low protein diet (LP) consisted of a low protein input (diet with 250 g kg^?1 crude protein) through the culture period; treatment with high protein diet (HP) consisted of a high protein input (diet with 400 g kg^?1 crude protein) through the trial, and finally treatment VP consisted of an adjustment of protein input (diets with 250, 350 or 400 g kg^?1 crude protein), depending on the abundance of biota (zooplankton and benthos) in the system. Each species responded differently to the treatments. For blue shrimp, low protein input resulted in the lowest final body weight (12.9 ± 0.6 g) and biomass (696.0 g pool^?1). Survival and feed conversion ratio were similar in the three treatments. For white shrimp, the best growth, biomass and food conversion ratio were obtained in the low protein input treatment. Water quality parameters such as nitrate, ammonia and organic matter during the two trials, were better for LP and VP treatments. White shrimp seems to have lower protein requirements than blue shrimp. For the blue shrimp culture, adjusting protein input according to natural food abundance (zooplankton and benthos) in the system, seems to be advantageous because of the possibility of getting a production similar to that obtained with a high protein input through the farming period, but at lower feed cost, and with a lower environmental impact. It is concluded that a high protein input through the whole farming period is not the best feeding strategy for any of the two species.     

Effect of dietary probiotics on apparent digestibility coefficients of nutrients of white shrimp Litopenaeus vannamei Boone

Apparent digestibility coefficients (ADCs) of dry matter (DM), crude protein (CP), lipid, phosphorus (P), essential amino acids (EAA), non‐essential amino acids (NEAA) and fatty acids were determined for white shrimp (Litopenaeus vannamei). A probiotic (Bacillus sp.) was used at 0% (diet 1), 0.5% (diet 2), 1.0% (diet 3), 3.0% (diet 4) and 5.0% (diet 5) inclusion rates in experiment diets. Apparent digestibility coefficients was determined using 0.01% Y₂O₃ as an indicator. A total of 300 shrimp (initial mean body weight 11.5±0.6 g) were randomly stocked into fifteen 500‐L outdoor concrete tanks. Faeces were collected from three replicate groups of shrimp three times a day. The ADCs of five diets were: DM, 66.1–69.6%; CP, 80.4–84.7%; lipid, 82.2–84.9%; P, 25.4–28.5%; EAA, 82.0–85.4%; NEAA, 81.5–85.1%; saturated fatty acids (SFA), 79.6–82.4%; monounsaturates (MUFA), 81.6–84.2% and polyunsaturates (PUFA)+highly unsaturates (HUFA), 83.4–86.6%. Apparent digestibility coefficients of DM, CP, lipid, P, amino acids and fatty acids in diet 1 were significantly lower than in other diets (P<0.05). Apparent digestibility coefficient of P in diets increased as probiotic increased. Apparent digestibility coefficient of lipid in diet 3 was the highest, but the difference was not significant among diets. In general, ADCs of most amino acids and fatty acids were over 80% in all diets, and PUFA+HUFA>MUFA>SFA. The results showed that ADCs of diets supplemented with 10, 30 and 50 g probiotic kg^?1 basal diet were higher than in other diets except lipid, and the difference was significant.     

凡纳滨对虾核自身抗原精子蛋白基因克隆及其表达

利用cDNA末端快速扩增技术克隆出凡纳滨对虾(Litopenaeus vannamei)核自身抗原精子蛋白(NASP)基因,NASP基因c DNA全长2258 bp,其中包括92 bp 5′端非编码区,147 bp 3′端非编码区及2019 bp开放阅读区,编码673个氨基酸,预测分子量为74.18 k D。该序列已提交至Gen Bank,序列号为KT274811。在NCBI上进行序列比对后发现,其与斑节对虾(Penaeus monodon)NASP序列相似性最高,为90%。采用荧光定量PCR方法测定了不同发育时期卵巢与肝胰腺中NASP m RNA相对表达水平,结果表明,NASP在卵巢中的表达量高于肝胰腺中的表达量,且在Ⅱ期表达量最高,Ⅲ期表达量最低。此外,通过构建系统进化树比较了凡纳滨对虾NASP与其他物种间的遗传距离。实验结果为进一步研究该基因在凡纳滨对虾卵巢发育中的作用提供了依据。     

Apparent digestibility of selected feed ingredients for white shrimp Litopenaeus vannamei,Boone

Apparent digestibility coefficients of dry matter (DM), crude protein, crude lipid, gross energy, phosphorus and amino acids in Peruvian fish meal (FM), fermented soybean meal, extruded soybean meal, soybean meal, peanut meal, wheat gluten meal, corn gluten meal, shrimp byproduct meal, meat and bone meal (MBM), poultry meat meal and plasma protein meal (PPM) were determined for white shrimp (Litopenaeus vannamei). A reference diet (RF) and test diets (consisting of 70% RF diet and 30% of the feedstuff) were used with 0.5% chromic oxide as an external indicator. A total of 1440 shrimp (initial mean body weight 1.05 ± 0.01 g) were randomly stocked into thirty‐six 500‐L fibreglass tanks with 40 shrimp per tank and three tanks per diet. Faeces were collected from triplicate groups of shrimp by a faecal collection vessel attached to the shrimp‐rearing tank. The shrimp were fed to apparent satiation four times a day and the feeding experiment lasted for 6 weeks. Statistics indicate that apparent DM digestibilities for white shrimp (L. vannamei) were the highest for FM, ranged 52.83–71.23% for other animal products and 69.98–77.10% for plant products. The protein and lipid from plant and animal sources were well digested by white shrimp. Apparent protein and lipid digestibility were in the range 87.89–93.18% and 91.57–95.28%, respectively, in plant products, and 75.00–92.34% and 83.72–92.79%, respectively, for animal products. The white shrimp demonstrated a high capacity to utilize phosphorus in the ingredients. The apparent phosphorus digestibility ranges of animal feedstuffs and plant feedstuffs were 58.90–71.61% and 75.77–82.30% respectively. Amino acid availability reflected protein digestibility, except that in MBM, for which the availability of some amino acid was lower, possibly due to protein damage during processing. Digestibility information could promote the use of ingredient substitution in least‐cost formulated diets for white shrimp.     

低盐度条件下的凡纳滨对虾幼虾亮氨酸营养需求

为了探讨低盐度(0.6～0.8 g/L)条件下初始体质量为(0.38±0.004)g的凡纳滨对虾(Litopenaeus vannamei)幼虾的亮氨酸需求量,以鱼粉、花生麸和L-晶体氨基酸为蛋白源配制含粗蛋白400 g/kg的6种等氮饲料,标记为L7～L12组,各组亮氨酸水平分别为15.95、17.95、19.95、21.95、23.95和25.95 g/kg(饲料干物质).每组3个重复,每个重复30尾虾,进行56 d生长实验.结果表明,凡纳滨对虾幼虾的增重率随饲料亮氨酸水平的升高而升高,当亮氨酸水平达到23.95 g/kg(L11组)时,增重率达到最高值(1143.11±36.40)％,显著高于L7组(P＜0.05);亮氨酸水平继续提高,增重率变化不显著(P＞0.05).幼虾的蛋白质效率、全虾体蛋白沉积率和血淋巴总蛋白随着饲料亮氨酸水平的升高呈现升高趋势,最高值出现在L11组,并显著高于L7组(P＜0.05).各实验组饵料系数、血淋巴谷丙转氨酶、谷草转氨酶活性和尿素氮含量则呈现降低的趋势,最低值出现在L11组,且显著低于L7组(P＜0.05).以增重率为指标,根据折线模型可知,低盐度条件下凡纳滨对虾幼虾的亮氨酸最适需要量为24.80 g/kg饲料,即61.99 g/kg饲料蛋白.