凡纳滨对虾感染白斑综合症病毒后的新症状

2007年6-7月,辽宁营口地区凡纳滨对虾养殖场陆续出现死虾现象,通过组织病理观察和PcR检测,确定为白斑综合症.患病初期,对虾并未表现出典型白斑、甲壳易剥离等症状,而在鳃区头胸甲内侧滋生一种囊状胶状物,导致头胸甲内侧肿胀,病理观察发现该滋生物为对虾感染白斑综合症病毒后,皮下组织"胶质化"的结果,同时胃和皮下组织的结缔组织中有大量的典型性病变核;细菌分离结果显示,伴随着弧菌和假单胞菌的混合感染.     

A Profound Effect of Hyperthermia on Survival of Litopenaeus vannamei Juveniles Infected with White Spot Syndrome Virus

This study was conducted to examine the effect of increasing seawater temperature on White Spot Syndrome Virus (WSSV) infection in juvenile Pacific White shrimp ( Litopenaeus vannamei ). Infection by WSSV was achieved using two methods, intramuscular injection and per os (oral) administration. Forty injected and 20 per os infected animals were kept in heated tanks at 32.3 ± 0.8 C, and the same number of WSSV infected animals were maintained in tanks at ambient temperature (25.8 ± 0.7 C). Despite the route of exposure, there were no survivors among the animals kept at ambient temperature; whereas, in heated tanks the survival of the WSSV infected juvenile shrimp was always above 80%, suggesting the existence of a beneficial effect from hyperthermia that mitigated the progression of WSSV disease. Moreover, this beneficial effect was not attributable to viral inactivation. Infected animals kept at 32 C had histologically detectable lymphoid organ spheroids suggestive of a chronic viral infection but were PCR negative (hemolymph) for WSSV. These findings might be related to low viral replication in WSSV-infected shrimp held at the higher environmental temperature. When the WSSV-infected shrimp were transferred from 32 C to ambient temperature, the mortality from WSSV ensued and was always 100%. Although the mechanism related to the beneficial effect of heating was not determined, our results indicate that increasing the water temperature modifies dramatically the natural history of the WSSV disease and the survival curves of WSSV-infected juvenile Pacific White shrimp.     

凡纳滨对虾白便综合征的组织病理学观察

本文比较观察了不同规格的健康和患白便综合征凡纳滨对虾Litopenaeus vannamei肝胰腺、中肠、后肠、后盲囊等的组织结构。组织病理显示:白便综合征凡纳滨对虾肠道严重病变,肠腔空,环肌两侧增生大量成纤维细胞,这些增生细胞取代了肠上皮,而不见正常柱状上皮细胞;肝胰腺有不同程度和性质的病变,如腺管萎缩、崩解、坏死等。凡纳滨对虾患白便综合征的组织病变演变过程为:最初肠上皮基膜下出现一圈增生细胞,肠上皮柱状细胞脱离基膜;增生细胞持续增生增多,肠上皮崩解脱落至肠腔,增生细胞完全取代肠上皮;增生细胞不断增生,增生细胞层逐渐变厚,随着时间的推移,增生细胞层近腔端的一圈增生细胞坏死,出现一圈棕黄色物质,最终坏死细胞连同棕黄色物质脱落至肠腔。脱落至肠腔的腺管细胞、上皮细胞、增生细胞排出体外即为肉眼可见的白便。     

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.     

氨氮促使携带白斑综合症病毒凡纳滨对虾发病及其血细胞、酚氧化酶和过氧化氢酶变化

通过白斑综合症病毒(WSSV)感染得到携带WSSV病毒的凡纳滨对虾,它们由0.05 mg/L氨氮水体进入不同氨氮浓度水体,在7 d内,1.75 mg/L,2.25 mg/L,3.00 mg/L氨氮水体内个体发生病毒病,并产生死亡。在14 d内,0.75 mg/L氨氮以及大于这一浓度水体内个体发生病毒病,并产生死亡,其中3.00 mg/L氨氮水体内死亡100%,2.25 mg/L氨氮水体内死亡80%,1.75 mg/L水体内死亡60%。在0.35 mg/L氨氮水体,7-14 d内对虾开始发病,没产生死亡。发病个体血细胞数量下降迅速,血清酚氧化酶先增高再迅速下降,其中3 mg/L氨氮水体中的携带病毒对虾只有下降。同时氨氮浓度越大,携带病毒凡纳滨对虾血清过氧化氢酶活力下降越快,越多。     

A Laboratory Challenge Method for Estimating Taura Syndrome Virus Resistance in Selected Lines of Pacific White Shrimp Litopenaeus vannamei

Among the strategies being developed to improve survival and harvest yields in the farming of Pacific white shrimp Litopenneus vannumei is breeding domesticated family lines and the selection for further development of those lines that demonstrate resistance to Taura syndrome virus (TSV) challenge in the laboratory. A standardized laboratory challenge method for measuring TSV resistance by per os exposure to the virus, relative to a reference stock of L. vannamei , was developed and used to screen a total of 176 family lines provided by five different companies over a period of several months. All challenged shrimp were exposed to TSV per os by feeding minced TSV-positive shrimp carcasses at ∼10% of the shrimp biomass once per day for three consecutive days. Studies were carried out for a minimum of 14 d from the first day (day 0) of exposure to TSV. The survival rates obtained following TSV challenge of the selected L. vannamei families ranged from 0% to 100%, with a mean of 31%. The reference line of L. vannamei ("Kona line") gave survival rates of 0% to 37% with a mean of 13%. The results of the present study demonstrate that the use of a relatively simple laboratory challenge procedure provides a mechanism to evaluate and compare resistance to TSV among selected L. vannamei families and to predict the performance of selected stocks in farm environments where TSV is enzootic.     

不同温度下凡纳滨对虾和中国明对虾对白斑综合征病毒(WSSV)耐受性比较

本研究分别对凡纳滨对虾"壬海1号"(Litopenaeus vannamei"Renhai No.1")和中国明对虾"黄海2号"(Fenneropenaeus chinensis"Huanghai No.2")在3种温度条件下(24℃、28℃和32℃),采用单尾定量口饲感染白斑综合征病毒(White spot syndrome virus,WSSV)的方法进行感染实验,比较2种对虾在不同温度情况下对WSSV的耐受性差异(L代表凡纳滨对虾,F代表中国明对虾)。结果显示,L-24℃和F-24℃组的平均存活时间分为(184.05±69.56)h和(101.68±38.45)h;L-28℃和F-28℃组的平均存活时间分别为(100.25±26.79)h和(73.38±22.22)h,相同温度组内均存在显著性差异(P0.05);截至第15天,L-32℃和F-32℃组的存活率分别为45.74%和23.47%。3个温度组对虾在50%的死亡率时的存活时间分别为178 h和98 h、98 h和74 h、292 h和78 h;死亡高峰时间分别为第5天和第4天、第5天和第4天、第10天和第4天。另外,分别在感染后的12 h、1 d、2 d、3 d、4 d、5 d、6 d、7 d、15 d共9个时间点对每组对虾进行活体取样,利用实时荧光定量RT-PCR技术对其进行病毒载量检测,从对虾体内肌肉组织病毒载量的角度探寻不同对虾抗病性能的差异。6 d时,L-24℃和F-24℃组对虾肌肉内病毒载量分别达到(2.97×10~6±7.44×10~6)和(8.08×10~6±3.22×10~6)copies/ng DNA,差异极显著(P0.01),L-28℃和F-28℃组分别达到(6.73×10~6±1.49×10~6)和(1.20×10~7±6.15×10~5)copies/ng DNA,差异极显著(P0.01);15 d,L-32℃和F-32℃组分别达到(5.18×10~4±4.32×10~4)和(3.78×10~4±8.97×10~4)copies/ng DNA,差异显著(P0.05)。研究表明,2种对虾在3种温度环境下感染WSSV后,凡纳滨对虾耐受WSSV能力要高于中国明对虾;不同温度下同种对虾肌肉体内WSSV的增殖能力从强到弱依次为28℃组、24℃组和32℃组。     

Use of Porcine Meal in Plant‐based Practical Diets for Pacific White Shrimp,Litopenaeus vannamei

Two growth trials were conducted to evaluate and confirm the efficacy of a porcine meal (PM) with high protein content (>90%) as an alternative feed ingredient in commercial‐type feed formulation for Pacific white shrimp, Litopenaeus vannamei. Six experimental diets were formulated for the two growth trials. The first five diets contained increasing levels (0, 1, 2, 4, and 6%) of PM as a replacement for soybean meal in a plant‐based diet with low inclusion level (6%) of fish meal (FM). The last experimental diet was produced utilizing 4.2% PM to completely replace FM. In Trial 1, shrimp (1.5 g initial mean weight, 20 shrimp/tank, n = 4) were offered test diets for 6 wk in a semirecirculation system. At the end of Trial 1, shrimp fed with the diet containing 6% PM exhibited significantly enhanced weight gain (WG), feed conversion ratio (FCR), and survival compared to those fed with the diet devoid of FM. As survival was poor across all treatments and different densities could mask growth results the trial was repeated. In Trial 2, shrimp (0.85 g, 15 shrimp/tank, n = 4) were offered diets for 6 wk. Dietary supplementation of PM at 6% significantly improved WG, FCR, and apparent net protein retention in contrast with the treatment devoid of FM, confirming the same trends in Trial 1. No significant difference was detected in protein, lipid, moisture, and mineral profiles of whole‐body shrimp as well as survival across all the treatments. Results of this study indicate that PM is a good high protein source in shrimp feeds, which can be included up to 6% in the low FM‐based diet without compromising the growth of shrimp.     

Pigmentation of Pacific White Shrimp,Litopenaeus vannamei,by Dietary Astaxanthin Extracted from Haematococcus pluvialis

This study investigated the effectiveness of dietary supplementation of astaxanthin (Ax) from the Haematococcus pluvialis on growth, survival, and pigmentation in Pacific white shrimp. Ten test diets were processed to contain five levels of Ax (25, 50, 75, 100, and 150 mg/kg as fed basis) by adding the natural or synthetic Ax to a basal diet containing no Ax. Each diet and a commercial shrimp feed were fed to four replicate tanks of shrimp (12 shrimp/tank) for 8 wk. Neither the natural nor synthetic Ax affected shrimp growth or survival. After cooking, shrimp fed the diets containing the natural Ax exhibited a strong red color, compared to the light pink color of shrimp fed the remaining diets. Colorimetric readings and Ax content in cooked shrimp demonstrated that the natural esterified Ax had greater pigmentation efficiency than synthetic free Ax (P < 0.05). The Ax contents in shrimp tail muscle demonstrated significant correlation with the levels of dietary Ax. The supplementation level of the natural Ax for optimum pigmentation efficiency is in a range of 75–100 mg/kg diet. The Ax product used in this study contained only a small amount (ca. 5.0%) of other carotenoids, indicating that the high pigmentation efficiency was mainly due to algal esterified Ax.     

Digestibility of Glandless Cottonseed Protein in Diets for Pacific White Shrimp,Litopenaeus vannamei

Accurate digestibility coefficients for protein, energy, and lipid are needed by feed formulators to optimize diets to meet nutritional requirements and to substitute ingredients cost‐effectively. Of particular interest is protein, which accounts for the majority of shrimp feed content and expense. The current study evaluated seven cottonseed meal and protein products. Most of the samples were derived from a glandless cotton variety that lacks significant levels of the antinutritive compound, gossypol. The various protein fractions were evaluated for apparent crude protein, crude lipid, and energy digestibility when fed to juvenile Pacific White Shrimp, Litopenaeus vannamei. Apparent energy digestibility for the protein fractions derived from the glandless seed ranged from 76.7% for ground kernels to 94.6% for protein isolate, and these values were greater than the value obtained for the commercial meal that contained gossypol (64%). Apparent protein digestibility for the six glandless‐cotton‐based samples varied from 72.3% for the ground full‐fat kernels to 94.1% for the protein isolate, and these values were mostly higher than the value obtained for the commercial meal (82.3%). The high apparent digestibility values demonstrate that low‐gossypol cottonseed protein products may be useful as a cost‐effective replacement for more expensive protein sources in L. vannamei diets.     

不同盐度对生物絮团、对虾生长以及酶活性的影响

在不同盐度条件下进行凡纳滨对虾的生物絮团养殖试验,研究盐度对生物絮团养殖水质和对虾生长及其酶活性的影响。试验设5个盐度梯度(10、15、20、25、30),生物絮团初始量为20 mL/L,对虾密度为500尾/m^3,试验周期30 d。试验结果显示,15盐度组与20盐度组的对虾体质量增长率最大,达70.73%,10盐度组的体质量增长率最小,达50.24%。盐度越高生物絮团生长越快,30盐度组17 d生物絮团沉降量达200 mL/L,之后逐渐降至43 mL/L,其他组呈相同变化趋势。试验过程中水体总碱度与pH持续降低,但不同组间差异不显著(P>0.05)。盐度越高氨氮累积越快,30盐度组在第6 d达到最大质量浓度8.62 mg/L,之后降至0 mg/L,其他组呈相同趋势变化。盐度越低亚硝态氮累积越快,10盐度组在第6 d达到最大质量浓度9.18 mg/L,之后降至0 mg/L,其他组呈相同趋势变化。硝态氮在不同盐度中呈前期上升的趋势,第16 d之后开始缓慢下降。15盐度组的淀粉酶活性显著高于其他组(P<0.05),其他各组之间无显著差异(P>0.05)。脂肪酶在25盐度组活性最高,盐度升高或者降低酶活性均降低。在10、15、20盐度组中,超氧化物歧化酶、碱性磷酸酶、酸性磷酸酶活性均维持在较高水平,在相同盐度下,肌肉酶活性低于肝胰脏。     

Efficacy of Three Methionine Sources in Diets for Pacific White Shrimp, Litopenaeus vannamei

Abstract.— To examine the need for supplemental dietary methionine and to determine the ability of different methionine sources to meet this need for Pacific white shrimp, Litopenaeus vannamei, a growth trial was conducted. A control diet (30% crude protein) was formulated to be deficient in methionine by inclusion of high levels of dehulled, solvent‐extracted soybean meal and with no fish meal (methionine level was 0.45% of diet as fed, 1.5% of total amino acids). Three test diets were manufactured based on the control diet but supplemented with l ‐methionine, dl ‐methionine, or an analog of methionine (2‐hydroxy‐4‐methylthio butanoic acid [HMTBA], calcium salt) at the level of 0.5% as fed (equivalent to 1.5% of total amino acids). Cystine was constant among these diets at 0.5% of diet as fed. A commercial feed (30% crude protein) was included as a reference. The culture vessels were flat‐bottomed, cylindrical, black fiberglass tanks (1.5‐m diameter) initially filled with 1000 L seawater and supplied with aeration and seawater from a well at a rate of 2 L/min. At the end of the trial, all surviving shrimp were counted and weighed. The trial was run outdoors, with a mean water temperature of 25.9 C. The mean survival, final weight, growth rate, and feed efficiency of shrimp in the control group were 95.9%, 10.8 g, 0.93 g/wk, and 0.44 g/g, respectively. The mean values of all the test treatments were 96.9%, 11.6 g, 1.00 g/wk, and 0.47 g/g, respectively. The final weight and growth rate of shrimp fed the control diet were significantly lower than those of shrimp fed the methionine‐supplemented diets, indicating that this species has a requirement for dietary methionine. There were no significant differences (P > 0.05) in any of the parameters among the test treatments, indicating that l ‐methionine, dl ‐methionine, and HMTBA can be used to meet the methionine requirement of this species under these experimental conditions. It is concluded that the three sources of methionine examined in this trial, l ‐methionine, dl ‐methionine, and HMTBA, are similar in their ability to meet the methionine requirement of this species.     

凡纳滨对虾虾头蛋白的内源酶水解研究

试验结果表明,凡纳滨对虾虾头蛋白白内源的最优水解条件为:40℃,pH 6,固液比1∶3,7 h,此时得氨基态氮达6.90 mg/g。从水解液检出16种氨基酸,其中含7种人体必需氨基酸。     

Survival of White Spot Syndrome Virus–Infected Litopenaeus vannamei Fed with Ethanol Extract of Uncaria Tomentosa

With the present project, the phytochemical composition of Uncaria tomentosa cultivated in Chapecó (Brazil) was analyzed and then the potential of using this local plant extract to protect by oral feeding virus‐infected Litopenaeus vannamei shrimp was also investigated. For this study, five treatments were tested: uninfected shrimp fed with pelleted feed (T_C), WSSV‐infected shrimp fed with pelleted feed (T₁), infected shrimp fed with pelleted feed with 1% ethanol extract of U. tomentosa (EEUT) (T₂), infected shrimp fed with pelleted feed with 2% EEUT (T₃), and infected shrimp fed pelleted feed with 4% EEUT (T₄). The chemical analysis of EEUT showed the presence of alkaloids, flavonoids, sterols, terpenes, coumarins, and tannins and also a high in vitro free radical‐scavenging activity as well as high total phenolic content. Shrimp fed with 2 and 4% EEUT (T₃ and T₄) showed a survival probability significantly higher than T₁ and T₂ treatments and no clinical symptoms of WSSV infection. Immunological assay also showed a positive phenoloxidase activity effect on shrimp fed with EEUT.