Use of computer‐assisted image analysis for semi‐quantitative histology of the hepatopancreas in whiteleg shrimp Penaeus vannamei (Boone)

Despite the increasing use of novel molecular techniques in pathology, histology remains the standard method for monitoring tissue alterations and for assessing pathology. Histopathological evaluation is generally laborious and subjective with risk of discrepancies in semi‐quantitative scoring between pathologists. In contrast, computer‐assisted image analysis (CAIA) is potentially faster, more objective and thus suitable for routine screening. Limited research has been carried out on CAIA in crustacean histopathology, and the methods described were not fully automated. Therefore, the aim of this study was to develop CAIA in whiteleg shrimp (Penaeus vannamei) for the study of the hepatopancreas. Paraffin sections were immunohistochemically stained with monoclonal antibodies WSH8 against haemocytes and counterstained with Mayer′s haematoxylin for detection of haemocytes and B‐cell vacuoles, and modified toluidine blue protocol was used for detection of F‐cells; frozen sections were stained with Oil Red O for detection of lipid droplets within R‐cells. Visiopharm^® software was used to develop and validated protocols for the quantification of morphological parameters (areas of haemocyte infiltration, F‐cells, B‐cell vacuoles, lipid droplets and their ratios to total tissue area and total lumen area). These protocols enable the future use of CAIA for determination of the nutritional and pathological condition of this organ.     

Evaluation of cholesterol additions to a soyabean meal-based diet for juvenile Pacific white shrimp, Penaeus vannamei (Boone), in an outdoor growth trial

Cholesterol is considered a required nutrient for penaeid shrimps, but an optimal level has not been defined. A 68-day grow-out trial was conducted in 1300-L outdoor tanks in Hawaii, USA, to determine the cholesterol level required in a soyabean meal-based diet for juvenile Pacific white shrimp, Penaeus vannamei (Boone). Test feeds were prepared by adding six cholesterol levels (1.0-10.2 g kg^?1, as-fed basis) to a standard diet high in plant-protein meals, starch and flour. These feeds were provided to the shrimp three times daily. Harvest weights and weekly growth increments varied significantly among diets. The 2.3 g kg^?1 and 4.2 g kg^?1 cholesterol diets yielded better growth than the 1.0 g kg^?1 and 10.2 g kg^?1 diets. Survival and feed conversion ratios did not vary significantly among diets. Significant dietary cholesterol effects on growth of P. vannamei in conditions resembling commercial grow-out indicate that its inclusion in practical diets at more accurately defined levels is necessary. Optimal cholesterol levels for juvenile shrimp may be lower than those now utilized, and may vary relative to the contribution of free sterols from natural foods in shrimp ponds.     

Optimization of fixation methods for image analysis of the hepatopancreas in whiteleg shrimp,Penaeus vannamei (Boone)

Pathology in penaeid shrimps relies on histology, which is subjective, time‐consuming and difficult to grade in a reproducible manner. Automated image analysis is faster, objective and suitable for routine screening; however, it requires standardized protocols. The first critical step is proper fixation of the target tissue. Bell & Lightner's (A Handbook of Normal Penaeid Shrimp Histology, 1988, The World Aquaculture Society, Baton Rouge) fixation protocol, widely used for routine histology of paraffin sections, is not optimized for image analysis, and no protocol for frozen sections is described in the available literature. Therefore, the aim of this study was to optimize fixation of the hepatopancreas (HP) from whiteleg shrimp (Penaeus vannamei) for both paraffin and frozen sections using a semiquantitative scoring system. For paraffin sections, four injection volumes and three injection methods were compared, for frozen sections, four freezing methods and four fixation methods. For paraffin sections, optimal fixation was achieved by increasing threefold the fixative volume recommended by Bell and Lightner, from 10% to 30% of the shrimp body weight, combined with single injection into the HP. Optimal fixation for frozen sections was achieved by freezing the cephalothorax with liquid nitrogen, followed by fixation of the section with 60% isopropanol. These optimized methods enable the future use of image analysis and improve classical histology.     

Effect of starvation and refeeding on the hepatopancreas of whiteleg shrimp Penaeus vannamei (Boone) using computer‐assisted image analysis

Under normal farming conditions, shrimp can experience starvation periods attributable to disease outbreaks or adverse environmental conditions. Starvation leads to significant morphological changes in the hepatopancreas (HP), being the main organ for absorption and storage of nutrients. In the literature, limited research has described the effect on the HP of periods of starvation followed by refeeding and none in whiteleg shrimp (Penaeus vannamei) using computer‐assisted image analysis (CAIA). This study describes the effect of starvation and starvation followed by refeeding on the HP of whiteleg shrimp using CAIA. Visiopharm^® software was used to quantify the following morphological parameters, measured as ratio to the total tissue area (TLA): total lumen area (TLA:TTA), haemocytic infiltration area in the intertubular spaces (HIA:TTA), B‐cell vacuole area (VBA:TTA), lipid droplet area within R cells (LDA:TTA) and F‐cell area (FCA:TTA). Significant changes were measured for HIA:TTA and LDA:TTA during starvation (increase in HIA:TTA associated with decrease in LDA:TTA) and starvation followed by refeeding (decrease in HIA:TTA associated with increase in LDA:TTA). In the future, HIA:TTA and LDA:TTA have the potential to be used in a pre‐emptive manner to monitor the health of the HP, facilitate early diagnosis of diseases and study the pathophysiology of the organ.     

Growth and performance of the whiteleg shrimp Penaeus vannamei (Boone) cultured in low‐salinity water with different stocking densities and acclimation times

We evaluated the performance of whiteleg shrimp Penaeus vannamei (Boone, 1931) in response to different stocking densities and acclimation periods. Shrimp postlarvae were acclimated from seawater (30 g L^?1) to low‐salinity well water (<1.0 g L^?1) at a constant hourly reduction rate of 40, 60, 80 and 100 h. After acclimation to low‐salinity well water, postlarvae from each acclimation time treatment were stocked in three replicate tanks at densities of 50, 100, 150 or 200 shrimps m^?2 for 12 weeks of growth. Salinity averaged <1.0 g L^?1 for each growth study. The different treatments resulted in significant differences in both the final body weight and the survival rate (SR). Shrimp acclimated for 100 h showed substantially improved survival (83%) relative to shrimp acclimated for shorter periods. Shrimp yields for all cultured periods ranged from 0.32 kg m^?2 in tanks stocked at 50 m^?2 to 1.14 kg m^?2 in tanks stocked at 200 m^?2. We conclude that whiteleg shrimp can be successfully grown in low‐salinity well water, and that the growth, production output and SRs are significantly higher when shrimp are acclimated for longer periods.     

南美白对虾育苗及淡化试验

南美白对虾为世界养殖产量最高的三大优良虾种之一,具有繁殖季节长,营养要求低,适应性强等特点。为开发南美白对虾的淡水池塘养殖,进行了南美白对虾的育苗及虾菌淡化试验。本试验在12．6m^3水体中投入120万尾南美白对虾无节幼体(N3)撞育至仔虾P5,经逐步淡化至P13,获淡化虾菌39．39万尾。单位水体产量3．13万尾,m^3,最高产量为4．28万尾/m^3,由N3至淡化虾菌P13成活率为32．8％,经14h运输,成活率为98．5％。     

Supplementation of ascorbic acid 2-monophosphate during the early postlarval stages of the shrimp Penaeus vannamei

A culture trial was performed using five levels of ascorbic acid 2-monophosphate (AmP at 0, 10, 100, 1000 and 2000 mg kg⁻¹ diet, expressed as active ascorbic acid, AA) in a semipurified diet for early postlarval Penaeus vannamei. The experiment started 10 days after metamorphosis of the mysid larvae into postlarvae (=PL10). Each treatment was run in four replicates. P. vannamei postlarvae showed significantly better growth according to dietary AA level after 25 days of feeding, i.e. at PL36 stage. Whereas the dry weight of PL36 in the control treatment (0 mg AA kg⁻¹ diet) was only 2 mg, supplementation of 1000 mg AA kg⁻¹ increased the shrimp dry weight up to 18 mg. However, the growth in the other treatments was not significantly different from the control. Fitting a broken-line regression to the biomass yield demonstrated an optimal dietary level of 130 mg AA kg⁻¹ diet. There were no differences observed among the treatments in stress resistance of postlarval P. vannamei to a salinity shock.     

南美白对虾养殖环境及其肠道细菌多样性分析

为了探讨南美白对虾(Penaeus vannamei Boone)肠道及其养殖环境中细菌种群组成和分布,采用Illumina miseq测序平台,基于16S r RNA基因的序列测定结果,对南美白对虾肠道及其养殖池塘底泥、水体中细菌的种群结构进行分析。结果显示,在南美白对虾肠道、养殖池塘底泥、水体分别检测到206、265和270个细菌属,其中,有90个属在3个样品中均可检测到,底泥和水、底泥和虾肠道、水和虾肠道中检测到的共有细菌属分别为146、128和119个,表明养殖池塘底泥、水和肠道中细菌存在密切的相互作用。在底泥、水和虾肠道样品中丰度大于1%的优势菌属分别有20、17和10个;底泥、水体和虾肠道中丰度最高的细菌均为一种分类未明的细菌属,丰度分别为23.08%、37.13%和42.22%。除此之外,底泥中主要优势细菌属分别为假单胞菌属(Pseudomonas)(5.77%)、Ohtaekwangia(4.79%)和狭义的梭菌属(Clostridium)(3.88%);水体中主要优势细菌属分别为土壤杆菌属(Sediminibacterium)(6.64%)、Spartobacteria genera incertae sedis(3.95%)和GPIIa(3.20%),而虾肠道中主要优势细菌属分别为假单胞菌属(Pseudomonas)(14.57%)和不动杆菌属(Acinetobacter)(6.54%)。在底泥、水和肠道中分别检测到3、6和6个潜在致病细菌属,丰度最高的细菌属分别是假单胞菌属(5.77%)、黄杆菌属(Flavobacterium)(0.77%)和假单胞菌属(14.57%)。在底泥、水和肠道中检测到的益生菌可分别分配至8、6和7个细菌属,其中有5个属在3个样品中均可检测到。聚类分析显示,养殖池塘底泥、水体和南美白对虾肠道细菌种群中丰富度位于前20的分类操作单元(OUT)在聚类树的位置存在明显不同。研究结果进一步加深了对健康南美白对虾肠道细菌种群及其与养殖环境中细菌种群互作的理解,为益生菌制剂的研发提供了新的线索。     

山区淡水池塘养殖南美白对虾试验

南美白对虾 (PenaeusvannameiBoone) ,又称凡纳对虾 ,原产于南美太平洋沿岸水域 ,是当今世界上公认最有前途的三大养殖虾类之一。它具有生长速度快、适应性强、盐度适应范围广、繁殖周期长、低盐度淡化养殖中病害少等优点 ,是山区池塘调整养殖结构、优化养殖品种、提高养殖效益的优良品种。为进一步探索南美白对虾在闽西地区稳产高效养殖技术 ,笔者于 2 0 0 3年 7月进行了南美白对虾池塘养殖试验 ,取得了一定经济效益和经验。现将试验情况总结如下。1　材料和方法1 .1　池塘条件　池塘选择在临城镇西郊村 ,面积0 .2 2hm2 ,池塘平均深度 2 .…     

南美白对虾病原性豚鼠气单胞菌的分离与药敏特性

为查明引起上海青浦地区养殖南美白对虾黄腿病的病原菌,提供养殖户防控该病的有效药物,采用传统方法从患黄腿病的南美白对虾肝胰腺组织中进行了病原菌的分离,通过人工回归感染试验确定了病原菌株,采用API ID32GN生理生化鉴定和16S rRNA序列分析对病原菌株进行鉴定,通过纸片扩散法分析病原菌株的药敏特性。结果表明,从病虾的肝胰腺中分离到一株病原菌(QPX1),经生理生化特性与16S rRNA序列分析确定其为豚鼠气单胞菌(Aeromonas caviae)。此外,菌株QPX1对阿莫西林、多粘菌素B、磺胺异噁唑、复方新诺明、卡那霉素、吡哌酸、新霉素、庆大霉素、氟苯尼考、链霉素、恩诺沙星、诺氟沙星、奈替米星等抗生素高度敏感。本研究证实豚鼠气单胞菌是南美白对虾黄腿病的病原菌,生产上可选用新霉素、氟苯尼考、恩诺沙星等常规水产用抗生素作为该病的防控用药。     

南美白对虾血蓝蛋白对酚氧化酶活性的影响

以采自厦门的南美白对虾(Penaeus vannamei)为研究对象，运用病原菌人工感染、血蓝蛋白浓度和酚氧化酶(phenoloxidase，PO)活性测定等方法探索南美白对虾血蓝蛋白在体内、外对其血清酚氧化酶活性的影响。结果表明，南美白对虾人工感染副溶血弧菌后，其血淋巴中血蓝蛋白浓度和PO活性的变化趋势基本一致。在体外实验中将一定量的血蓝蛋白重组蛋白添加至血清中，其PO活性可明显升高；但同时添加血蓝蛋白重组蛋白和E．coli K12超声破碎液后，其PO活力反而降低。研究提示，血蓝蛋白在一定范围内确实可以正反馈调节PO活性，但在细菌等干扰之下，其又可表现出一定的负反馈抑制活性。     

Experimental white spot syndrome virus challenge of juvenile Litopenaeus vannamei (Boone) at different salinities

In recent years, the shrimp industry has turned to inland freshwater culture as one method to avoid problems such as the introduction of possible vectors of viral pathogens into seawater ponds. Our experiments evaluated susceptibility to white spot syndrome virus (WSSV) in Litopenaeus vannamei held under different salinity regimens. Juvenile L. vannamei that were conditioned at salinities of 35, 25, 15, 5 and 2 g L⁻¹ were challenged with WSSV. In order to assess the severity of white spot disease, histological analysis and nested polymerase chain reaction (PCR) tests were carried out on the challenged shrimp every 4 h after 48 h post challenge. The results indicated that significantly more severe infections resulted at 15‰ than at other salinities. Mortality could not be compared due to the sampling design and because severe WSSV infections occurred in all test groups such that few shrimp remained alive in each challenged group at the end of the test. Despite this, the results suggest that salinity may affect the course and outcome of WSSV infections.     

Effects of acute change in salinity and moulting on the infection of white leg shrimp (Penaeus vannamei) with white spot syndrome virus upon immersion challenge

In the field, moulting and salinity drop in the water due to excessive rainfall have been mentioned to be risk factors for WSSV outbreaks. Therefore, in this study, the effect of an acute change in environmental salinity and shedding of the old cuticle shell on the susceptibility of Penaeus vannamei to WSSV was evaluated by immersion challenge. For testing the effect of abrupt salinity stress, early premoult shrimp that were acclimated to 35 g L^?1 were subjected to salinities of 50 g L^?1, 35 g L^?1, 20 g L^?1, 10 g L^?1 and 7 g L^?1 or 5 g L^?1 and simultaneously exposed to 10^5.5 SID₅₀ mL^?1 of WSSV for 5 h, after which the salinity was brought back to 35 g L^?1. Shrimp that were transferred from 35 g L^?1 to 50 g L^?1, 35 g L^?1 and 20 g L^?1 did not become infected with WSSV. Shrimp became infected with WSSV after an acute salinity drop from 35 g L^?1 to 10 g L^?1 and lower. The mortality in shrimp, subjected to a salinity change to 10 g L^?1, 7 g L^?1 and 5 g L^?1, was 6.7%, 46.7% and 53.3%, respectively (P < 0.05)_. For testing the effect of moulting, shrimp in early premoult, moulting and post‐moult were immersed in sea water containing 10^5.5 SID₅₀ mL^?1 of WSSV. The resulting mortality due to WSSV infection in shrimp inoculated during early premoult (0%), ecdysis (53.3%) and post‐moult (26.72%) demonstrated that a significant difference exists in susceptibility of shrimp during the short moulting process (P < 0.05). The findings of this study indicate that during a drop in environmental salinity lower than 10 g L^?1 and ecdysis, shrimp are at risk for a WSSV infection. These findings have important implications for WSSV control measures.     

底泥悬浮物对海水养殖凡纳滨对虾仔虾生物毒性影响

为评价疏浚、吹填过程中产生的底泥悬浮物对南海区凡纳滨对虾仔虾的毒性影响,根据《水质-物质对淡水鱼(斑马鱼)急性致死毒性的测定》方法,对南海区凡纳滨对虾仔虾的生物毒性影响进行了分析。结果表明:疏浚区海水中底泥悬浮物浓度超过10mg/L,持续12h,对凡纳滨对虾仔虾产生一定的致死效应。底泥悬浮物浓度超高80mg/L,且持续48h以上,将对仔虾产生严重的影响,甚至导致全部仔虾的死亡。根据仔虾的死亡概率和底泥悬浮物浓度对数,分别计算出12h、24h、36h、48h的半致死量(LC50)为54.56、40.29、25.89、19.18mg/L。     

Kinetic analysis of internalization of white spot syndrome virus by haemocyte subpopulations of penaeid shrimp,Litopenaeus vannamei (Boone), and the outcome for virus and cell

Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)‐killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV‐killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV‐killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV‐killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60 min post‐inoculation) and that afterwards the capsid started to become disintegrated (from 360 min post‐inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV‐killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular‐sensing molecules.