Isolation of Enterococcus faecium with Feeding Attractant Function from Pacific White Shrimp ( Litopenaeus vannamei ) Intestine

In the present study, we isolated the lactic acid bacterium strain SC-01 from Pacific white shrimp(Litopenaeus vannamei) intestine. Using conventional and molecular methods, we identified the bacterium as Enterococcus faecium, and found it had the function of feeding attractant and could inhibit the development of Vibrio parahaemolyticus(zone of inhibition: 14 mm). The attractant effect of its fermentation broth is significantly better than that of the chemical attractant trimethylamine oxide(TMAO)(P 0.05), and is equivalent to that of dimethyl-beta-propiothetin(DMPT) based on the feeding behavior of shrimp. High performance liquid chromatography(HPLC) analysis suggested that inosine-5'-monophosphate(IMP) may be a component of the attractant. A biosecurity evaluation revealed a negative result in hemolytic assays, and no shrimp mortality was resulted from SC-01 fermentation broth challenge. Feeding trials(60 days) indicated that the SC-01 fermentation broth(viable counts: 5.7×10~9 cfu mL~(-1)) could improve feed intake, weight gain rate(WGR) and specific growth rate(SGR), and decrease the count of Vibrio sp. in the intestine of shrimp.     

Effect of bioactive peptides (BPs) on the development of Pacific white shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis> Boone, 1931)

The present study was conducted to evaluate the feasibility of replacing fish meal (FM) with bioactive peptides (BPs) in diet of white shrimp (Litopenaeus vannamei). The changes in growth performance, body composition, non-specific immunity, and water quality were examined after the shrimp were fed four diets, in which 0% (control), 33.3%, 66.7% and 100% of FM was replaced by BPs, respectively. The groups were designated as Con, 1/3BPs, 2/3BPs, and 3/3BPs. A total of 720 shrimp with an initial body weight of 1.46 ± 0.78 g were fed the experimental diets for 56 days. The results revealed that: 1) the weight gain rate (WGR) in 1/3BPs, 2/3BPs, and 3/3BPs was significantly higher than that in Con (P < 0.05), while no significant difference was found on survival rate and feed conversion ratio (FCR); 2) the whole-body crude protein (CP) and crude lipids (CL) were significantly different among groups, while there was no significant difference between crude ash and phosphorus contents; 3) the levels of acid phosphatase (ACP), lysozyme (LZM), superoxide dismutase (SOD), phenol oxidase (PO) and bactericidal activity increased significantly with the inclusion of BPs; 4) in terms of water quality, no significant difference was found in pH and dissolved oxygen among diets during the whole experimental period. Moreover, even though nitrite and ammonium levels tended to increase with time, there was no significant difference among groups. The results indicated that BPs is an applicable alternative of protein source, which can substitute FM in the diets of L. vannamei; it is able to effectively promote growth performance and improve immunity. Moreover, BPs in the diets had no negative impact on water quality.     

凡纳滨对虾肠道上皮细胞微胞子虫PCR检测方法的建立与应用

根据Gen Bank中对虾肠道上皮细胞微胞子虫(Enterocytozoon hepatopenaei,EHP)的基因保守序列,设计一对特异性引物,通过优化PCR扩增条件,建立快速检测凡纳滨对虾(Litopenaeus vannamei)EHP的PCR方法。用该方法对EHP阳性虾进行PCR扩增,得到与实验设计相符的330 bp特异性扩增条带,而对白斑综合症病毒(WSSV)、桃拉病毒(TSV)、传染性皮下及造血器官坏死病毒(IHHNV)、对虾杆状病毒(BP)、"棉花虾"微孢子虫、黏孢子虫的阳性虾,以及健康虾的扩增结果为阴性。测序比对发现,PCR产物序列与Gen Bank EHP基因序列的同源性为99.8%,表明该PCR方法检测结果准确。敏感性试验表明,该方法最低可检测出约100 fg的EHP质粒DNA。用该PCR方法检测广东、广西、江苏、山东、海南等地的755份临床样品,共检出阳性样品21份。该PCR方法可用于凡纳滨对虾EHP的快速检测。     

Effects of Hypoxia on Dopamine Concentration and the Immune Response of White Shrimp （Litopenaeus vannamei）

Effects of hypoxia on the dopamine concentration and the immune response of White Shrimp Litopenaeus vannamei were studied. The results showed that hypoxia had significant effects on the concentration of dopamine (DA) in the haemolymph, haemocyte count, phenoloxidase activity, phagocytic activity of haemocytes and bacteriolytic and antibacterial activity in the haemolvmph (P<0.05). The concentration of the dopamine in haemolymph reached its maximum in the 3.0 and 1.5mgL-1 DO groups at 12h and 6h, and then returned to normal after 24h and 12h, respectively. All immune parameters decreased with the reduction of dissolved oxygen. Total haemocyte count (THC), the hyaline cells and semi-granular cells in the 3.0mgL-1 DO group became stable after 12h,while granular cells did so after 24h, The THC and different haemocyte count (DHC) in the 1.5mgL-1 DO group became stable alter 24h. Phenoloxidase activity and bacteriolytic activity in the 3.0 and 1.5mgL-1 DO groups reached their stable levels after 24 h and 12 h respectively, while phagocytic activity and antibacterial activity became stable after 24 and 12, and 36 and 24 It, respec-tively. It was also indicated that the changes of dopamine concentrations in haemolymph, haemocyte count and phenoloxidase activ-ity were obviously related to the exposure time under hypoxic conditions.     

Effects of hypoxia on dopamine concentration and the immune response of White Shrimp (Litopenaeus vannamei)

Effects of hypoxia on the dopamine concentration and the immune response of White Shrimp Litopenaeus vannamei were studied. The results showed that hypoxia had significant effects on the concentration of dopamine (DA) in the haemolymph, haemocyte count, phenoloxidase activity, phagocytic activity of haemocytes and bacteriolytic and antibacterial activity in the haemolymph (P<0.05). The concentration of the dopamine in haemolymph reached its maximum in the 3.0 and 1.5 mg L⁻¹ DO groups at 12 h and 6 h, and then returned to normal after 24 h and 12 h, respectively. All immune parameters decreased with the reduction of dissolved oxygen. Total haemocyte count (THC), the hyaline cells and semi-granular cells in the 3.0 mg L⁻¹ DO group became stable after 12 h, while granular cells did so after 24 h. The THC and different haemocyte count (DHC) in the 1.5 mg L⁻¹ DO group became stable after 24 h. Phenoloxidase activity and bacteriolytic activity in the 3.0 and 1.5 mg L⁻¹ DO groups reached their stable levels after 24 h and 12 h respectively, while phagocytic activity and antibacterial activity became stable after 24 and 12, and 36 and 24 h, respectively. It was also indicated that the changes of dopamine concentrations in haemolymph, haemocyte count and phenoloxidase activity were obviously related to the exposure time under hypoxic conditions.     

大黄对凡纳滨对虾生长和非特异性免疫指标的影响

在凡纳滨对虾饲料中分别添加大黄0、0.5、1.0、5.0、10.0和20.0g/kg,研究大黄对凡纳滨对虾(初始体重为0.34±0.004g)生长及非特异性免疫指标的影响。结果表明,大黄对凡纳滨对虾成活率、增重率、特定生长率、饲料系数、蛋白质效率和蛋白质累积率的影响不显著(P>0.05),对全虾和尾肌肉的灰分、脂肪和粗蛋白含量影响显著,全虾的粗蛋白和粗脂肪含量以1.0g/kg组最高(P<0.05),对凡纳滨对虾血清碱性磷酸酶、酸性磷酸酶、酚氧化酶、超氧化物歧化酶、溶菌酶以及血清总蛋白量等的影响显著,溶菌酶活性以1.0g/kg组最高(P<0.05);细菌感染实验中以1.0g/kg组存活率最高(P<0.05)。以非特异性免疫反应指标及感染实验存活率为指标,凡纳滨对虾饲料中大黄的适宜添加量为1.0g/kg。     

切除眼柄对凡纳滨对虾亲虾卵巢、肝胰腺、血清生化组成的影响

为探讨眼柄切除对凡纳滨对虾性成熟的促进机理,采用镊烫法切除雌性凡纳滨对虾单侧眼柄,并测量肝胰腺指数和性腺指数,测定卵巢、肝胰腺和血清中生化组分含量的变化。结果表明,切除眼柄7 d后,1)肝胰腺指数降低,性腺指数上升,差异均有统计学意义(P0.05);2)卵巢、肝胰腺中总蛋白含量增加,差异有统计学意义(P0.05),血清中总蛋白含量无明显改变(P0.05);3)卵巢和血清中胆固醇含量增加,肝胰腺中胆固醇含量降低,差异均有统计学意义(P0.05);4)血清中钙离子和无机磷含量增加,差异有统计学意义(P0.05)。切除眼柄后,卵巢和肝胰腺中卵黄蛋白原等营养物质大量合成,肝胰腺中的总蛋白、胆固醇等营养物质先转移至血淋巴,再转移至卵巢,从而促进性腺的成熟。     

Effect of Replacing Fish Meal with Extruded Soybean Meal on Growth,Feed Utilization and Apparent Nutrient Digestibility of Juvenile White Shrimp (Litopenaeus vannamei)

Extruded soybean meal(ESBM) was evaluated as a protein source for partial replacement of fish meal(FM) in diets of juvenile Litopenaeus vannamei. In the control diet(Diet 1), FM protein was replaced with increasing dietary levels of ESBM(4.28%, 8.40%, 12.62%, 16.82%, and 25.26%) at 10%, 20%, 30%, 40%, and 60% levels(Diets 2 to 6, respectively). An eight-week feeding trial was conducted on 720 juvenile shrimp(0.67 g ± 0.01 g mean initial weight), and nutrient digestibility of the six diets was determined. ESBM could replace 20% of FM without causing a significant reduction in growth of shrimp, but other dietary treatments strongly affected whole body composition. Crude protein content of the whole body fed Diet 6 was significantly lower than that fed Diet 2(P 0.05), while crude lipid content of the whole body fed Diet 5 or 6 was significantly higher than that fed Diet 2(P 0.05). Protein digestibilities of Diets 5 and 6 were significantly lower than that of Diet 1(P 0.05). Digestibility of lipids ranged from 96.97% in Diet 6 to 98.34% in Diet 3, whereas dry matter digestibility decreased with increasing replacement level. This study indicates that 20% FM replacement with ESBM in the basic diet containing 40% protein and 30% FM is optimal for juvenile L. vannamei.     

Selection for growth performance of tank-reared Pacific white shrimp, Litopenaeus vannamei

Seven growth-related traits were measured to assess the selection response and genetic parameters of the growth of Pacific white shrimp, Litopenaeus vannamei, which had been domesticated in tanks for more than four generations. Phenotypic and genetic parameters were evaluated and fitted to an animal model. Realized response was measured from the difference between the mean growth rates of selected and control families. Realized heritability was determined from the ratio of the selection responses and selection differentials. The animal model heritability estimate over generations was 0.44±0.09 for body weight (BW), and ranged from 0.21±0.08 to 0.37±0.06 for size traits. Genetic correlations of phenotypic traits were more variable (0.51–0.97), although correlations among various traits were high (>0.83). Across generations, BW and size traits increased, while selection response and heritability gradually decreased. Selection responses were 12.28%-23.35% for harvest weight and 3.58%–13.53% for size traits. Heritability estimates ranged from 0.34±0.09 to 0.48±0.15 for harvest weight and 0.17±0.01–0.38±0.11 for size traits. All phenotypic and genetic parameters differed between various treatments. To conclude, the results demonstrated a potential for mass selection of growth traits in L. vannamei. A breeding scheme could use this information to integrate the effectiveness constituent traits into an index to achieve genetic progress.     

不同天然饵料对凡纳滨对虾亲虾性腺发育的影响

分别以双齿围沙蚕(Perinereis aibuhitensis)、杜氏枪乌贼(Loligo duvaucelii)、近江牡蛎(Ostrea riuularis)及三者的混合饵料(1∶1∶1)培育凡纳滨对虾(Litopenaeus vannamei)亲虾至性腺发育成熟,通过分析亲虾性腺发育时间、蜕壳间期、交配率、产卵量、变态率,确定培育亲虾的优质饵料。结果表明:投喂双齿围沙蚕或混合饵料的组与投喂杜氏枪乌贼、近江牡蛎组在产卵量、卵巢发育时间、蜕壳间期、交配率、孵化率、变态率方面具有显著差异(P<0.01)。投喂双齿围沙蚕或混合饵料的亲虾平均产卵量可达21.2×104粒/尾,卵巢平均发育时间为101 h,平均蜕壳间期为218 h,平均交配率可达80%,平均孵化率可达62.99%,平均变态率可达82.44%。表明在凡纳滨对虾亲虾培育的过程中,沙蚕单独或与其他天然饵料混合投喂,较乌贼、牡蛎单独投喂对亲虾性腺发育具有更好的促进效果。     

Effect of shrimp (Litopenaeus vannamei) farming waste on the growth,digestion, ammonium-nitrogen excretion of sea cucumber (Stichopus monotuberculatus)

In this study, specific growth rate (SGR), ingestion rate (IR), food conversion ratio (FCR), apparent digestion ratio (ADR) and ammonium-nitrogen excretion were determined for sea cucumber (Stichopus m...     

微囊藻毒素对凡纳滨对虾免疫相关酶活力的影响

通过毒性注射实验,探究微囊藻毒素(MC-LR)对凡纳滨对虾(Litopenaeus Vannamei)肝脏免疫相关酶活力的影响。结果表明,注射MC-LR后,凡纳滨对虾肝胰脏组织的超氧化歧化酶、谷胱甘肽过氧化酶、过氧化物酶、酸性磷酸酶、碱性磷酸酶和溶菌酶的酶活力呈先增大后减小的变化趋势。超氧化歧化酶、谷胱甘肽过氧化酶和过氧化物酶活力在对虾注射MC-LR 4 h后达到峰值,比对照组分别增加了15.62%、18.76%和32.86%,随后酶活力逐渐减小;而酸性磷酸酶和碱性磷酸酶酶活力在对虾注射MC-LR 2 h后即显著增加,比对照组分别增加了25.02%和37.99%,随后酶活力逐渐减小;溶菌酶在对虾注射MC-LR后8 h达到最大值,比对照组增加了33.57%,随后酶活力逐渐减小。MC-LR能显著影响凡纳滨对虾的免疫相关酶活力(P0.05),引起凡纳滨对虾的应激反应和抑制它们的免疫相关酶活力。     

Application of Wet Waste from Shrimp (Litopenaeus vannamei) with or Without Sea Mud to Feeding Sea Cucumber (Stichopus monotuberculatus)

In the present study, the applicability of the wet waste collected from shrimp(Litopenaeus vannamei) to the culture of sea cucumber(Stichopus monotuberculatus) was determined. The effects of dietary wet shrimp waste on the survival, specific growth rate(SGR), fecal production rate(FPR), ammonia- and nitrite-nitrogen productions of sea cucumber were studied. The total organic matter(TOM) level in the feces of sea cucumber was compared with that in corresponding feeds. Diet C(50% wet shrimp waste and 50% sea mud mash) made sea cucumber grow faster than other diets. Sea cucumber fed with either diet D(25% wet shrimp waste and 75% sea mud mash) or sole sea mud exhibited negative growth. The average lowest total FPR of sea cucumber occurred in diet A(wet shrimp waste), and there was no significant difference in total FPR between diet C and diet E(sea mud mash)(P > 0.05). The average ammonia-nitrogen production of sea cucumber in different diet treatments decreased gradually with the decrease of crude protein content in different diets. The average highest nitrite-nitrogen production occurred in diet E treatment, and there was no significant difference in nitrite-nitrogen production among diet A, diet B(75% wet shrimp waste and 25% sea mud mash) and diet C treatments(P > 0.05). In each diet treatment, the total organic matter(TOM) level in feces decreased to different extent compared with that in corresponding feeds.     

降温及生态冰温条件对凡纳滨对虾无水存活时间的影响

【目的】确定凡纳滨对虾无水存活流通的关键工艺参数。【方法】通过观察明确休眠临界温度,计时测定,确定降温程序、氧气含量及生态冰温保藏温度对存活时间的影响。【结果】凡纳滨对虾生态冰温区为-2～10℃。设定4种降温方式,其梯度降温均为≥18、18～15、15～10℃等3个区段,第Ⅰ种方式对应区段的降温速率为4、1、0.5℃/h,第Ⅱ种为4、2、0.5℃/h,第Ⅲ种为4、1、1℃/h,第Ⅳ种为4、4、4℃/h。第Ⅰ种方式下的存活时间(12.48±0.56)h,显著大于第Ⅱ种(8.75±0.77)h、第Ⅲ种(8.29±0.58)h和第Ⅳ种(6.19±0.62)h(P <0.05)。在99.9%氧气含量且维持生态冰温5℃的无水条件下可使存活时间进一步延至(23.82±0.77)h,之后常温新鲜海水中5 min可复苏至鲜活状态,成活率在98.5%以上。【结论】通过调整降温及生态冰温条件可显著延长凡纳滨对虾无水存活时间。     

酵母培养物对凡纳滨对虾肠道菌群结构的影响

在基础饲料中分别添加质量分数0(对照)和0.30%的酵母培养物,配制2种等氮等脂饲料(分别记为Y0、Y0.3组),投喂初始体质量为(1.20±0.01)g的凡纳滨对虾(Litopenaeus vannamei)56 d。提取凡纳滨对虾肠道总DNA,采用Illumina Mi Seq高通量测序比较样品菌群组成和多样性,研究饲料中添加酵母培养物对凡纳滨对虾肠道菌群结构的影响。结果表明,Y0.3组对虾肠道分类单元(OTUs)数量、丰富度指数(Chao1、ACE值)、多样性指数(Shannon、np Shannon指数)与Y0组差异无统计学意义(P0.05);从物种分类树发现,两组的主要细菌类群均为变形菌门(Proteobacteria),其中,γ-变形菌纲是对虾肠道的最主要菌群,所占比例达69.48%;从科分类水平看,Y0.3组对虾肠道菌群链球菌科(Streptococcaceae)比Y0组提高4.98%;从优势属水平来看,Y0.3组对虾肠道的乳球菌属(Lactococcus)高于Y0组,差异有统计学意义(P0.01);Y0.3组对虾肠道的发光杆菌属(Photobacterium)低于Y0组,差异有统计学意义(P0.05)。饲料中添加质量分数0.30%酵母培养物可改善凡纳滨对虾肠道菌群多样性。     

溶藻弧菌肽聚糖对凡纳滨对虾白斑综合征病毒的抑制作用

从患病凡纳滨对虾(Litopenaeus vannamei)体内提取白斑综合征病毒(WSSV),PCR证实WSSV毒性,将病毒粗提液稀释10-2、10-3、10-4和10-5倍,攻毒实验表明,最佳攻毒剂量为10-4倍稀释液。对凡纳滨对虾(Litopenaeus vannamei)分别注射0.05、0.10、0.15 mg/m L的溶藻弧菌(Vibrio alginolyticus)肽聚糖,免疫24 h后感染WSSV,测定其免疫保护率和肝胰腺的免疫学指标,探讨溶藻弧菌肽聚糖对凡纳滨对虾抗WSSV的影响。结果表明:对虾的保护率随着肽聚糖的浓度增大而增大,与对照组相比,0.05、0.10、0.15 mg/m L组的保护率分别为36.67%、46.67%、56.67%;实验组凡纳滨对虾肝胰腺中的酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)和一氧化氮合成酶(NOS)活力与对照组相比的差异有统计学意义(P0.01)。溶藻弧菌肽聚糖可有效增强凡纳滨对虾自身的非特殊性免疫能力,提高抗WSSV的能力。