Evidence for the presence of white spot syndrome virus (WSSV) and monodon baculovirus (MBV) in wild Penaeus monodon (Fabricius) broodstock,in the southeast coast of India

A survey on the presence of the viruses of two economically significant diseases, white spot syndrome virus (WSSV) and monodon baculovirus (MBV) in wild‐collected Penaeus monodon broodstock, was conducted during different seasons of the year in two major coastal areas of southeast India. The broodstock were collected along the coast of Tamil Nadu and Andhra Pradesh during summer, premonsoon, monsoon and post‐monsoon seasons for three consecutive years. A total of 7905 samples were collected and subjected to MBV screening, and 6709 samples that were screened as MBV negative were diagnosed for WSSV. MBV was detected using rapid malachite green staining and WSSV by nested polymerase chain reaction. Prevalence data of the viruses were analysed using the EpiCalc 2000 program at 95% confidence interval. Samples collected from the Andhra Pradesh coast displayed a slightly higher prevalence of WSSV and MBV infection than those collected from Tamil Nadu, although this difference was not statistically significant (P > 005). In addition, it was found that the prevalence of both WSSV and MBV infections fluctuated according to season. Data on prevalence of these viruses in broodstock would be useful to develop strategies for shrimp health management along the southeast coast of India.     

Development of a Penaeus monodon-type baculovirus (MBV) DNA probe by polymerase chain reaction and sequence analysis

Abstract. Penaeus monodon -type baculovirus (MBV) was isolated and purified from the hepatopancreases of MBV-infected Penaeus monodon Fabricius. MBV DNA was extracted and used as a template in a polymerase chain reaction (PCR). The primers were chosen from conserved regions of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV). One DNA fragment (674 base pairs) was amplified after PCR. There was a 65% homology between the predicted amino acid sequence of this PCR product with that of the polyhedrin polypeptide of AcNPV. Nucleotide sequence analysis indicated that the amplified DNA is the open reading frame of the MBV polyhedrin gene. This 674 bp DNA fragment was subsequently used as a probe in a dot blot analysis. The probe was able to hybridize with the DNA extracted from the purified MBV and from the MBV-infected P. monodon , but not from the MBV uninfected P. monodon.     

Detection of Penaeus monodon-type baculovirus (MBV) infection in Penaeus monodon Fabricius by in situ hybridization

Abstract. A digoxigenin-labelled DNA probe was used for in situ detection of the Penaeus monodon -type baculovirus (MBV) derived from cloned MBV polyhedrin genome in cultured Penaeus monodon Fabricius. First, the specificity of the probe against MBV DNA with dot blot hybridization analysis was verified. This probe indicated that cloned MBV polyhedrin fragment can be used as an MBV-specific probe. This was then used to microscopically examine sections of MBV-infected tissues for a blue-purple precipitate indicative of a positive reaction for MBV. MBV-positive cells were located only in the epithelium of the hepatopancreatic tubules and of the midgut. Furthermore, comparison of the susceptibility to MBV infection among several life-stages of the shrimp showed that the MBV genome was found in the zoea, mysis, post-larva, and adult stages, whereas MBV DNA was not detected in either eggs or nauplii. The results were quantified from in situ hybridization with an image analyser to compare the degree of cell infection among groups of cultured P. monodon collected from various farms in Taiwan.     

斑节对虾杆状病毒在感染对虾中肠腺中的分布

利用组织切片法研究了斑节对虾杆状病毒（MBV）在养殖早期班节对虾及养成期斑节对虾中肠腺各部位的分布特点。斑节对虾的中肠腺可分为头部、亚头部、中部、亚尾部和尾部等5个部分，其上皮细胞包括E细胞、F细胞、B细胞和R细胞等4种类型。头部和尾部均以E细胞为主、亚头部、中部和亚尾部均以R细胞为主。在感染度较轻的样本中，MBV一般只感染F细胞、R细胞和B细胞。感染度较重时，E细胞也同时受累。在养殖早期虾和养成期虾中，MBV相对感染度在中肠腺各部分的分布均以中部最高，而且以中部的中间最高，离中部越远，相对感染度越低。     

Detection of monodon baculovirus and white spot syndrome virus in apparently healthy Penaeus monodon postlarvae from India by polymerase chain reaction

The simultaneous presence of monodon baculovirus (MBV) and white spot syndrome virus (WSSV) in apparently healthy postlarvae of Penaeus monodon from different hatcheries in India was studied by nested polymerase chain reaction (PCR). MBV could be detected in 54% of the samples. However, only 15% of samples were positive by non-nested reaction. WSSV could be detected in 75% of samples, 19% being positive by non-nested reaction. The results show simultaneous presence of WSSV and MBV in many samples at various degrees of infection. Only 14% of the samples analysed were negative for both viruses.     

Effect of Penaeus monodon-type baculovirus (MBV) on survival and growth of larval Penaeus monodon Fabricius

Abstract. The present study attempts to investigate the effect of Penaeus monodon -type baculovirus (MBV) infection on growth and survival of the larval stages of Penaeus monodon Fabricius. The results showed that MBV may significantly retard larval growth and cause mortalities during the larval stage. The MBV-infected larvae of P. monodon varied more in body size than uninfected control shrimp, and were discoloured. To detect the presence of MBV in larval prawn, histopathological techniques and an enzyme-linked immunosorbent assay ( elisa ) were used. Positive diagnoses were routinely detected as early as the zoea 1 stage when ELISA was used.     

The relationship between paralarval feeding and morphological changes in the proboscis and beaks of the neon flying squid <Emphasis Type="Italic">Ommastrephes bartramii</Emphasis>

We compared the diet of Ommastrephes bartramii paralarvae with morphological changes in their beaks and proboscis (fused tentacles). The paralarvae were collected north of the Hawaiian Islands during 2001 and 2002 and ranged in mantle length (ML) from 1.1 to 13.2 mm. They fed on crustaceans, including copepods (copepodite stage) and amphipods. The rostral tips of upper and lower beaks began to protrude anteriorly at around 3–4 mm ML, and the smallest paralarva with identifiable prey in its digestive tract was 4.2 mm ML, which suggests that the paralarvae can masticate prey soon after the beaks protrude. The proboscis separated into two tentacles at 9.3–13.2 mm ML, but the newly formed tentacles were weakly developed even in the largest specimen, suggesting that tentacles do not operate functionally and that the arms are used to capture prey.     

Competition of infectious hypodermal and haematopoietic necrosis virus (IHHNV) with white spot syndrome virus (WSSV) for binding to shrimp cellular membrane

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) are two widespread shrimp viruses. The interference of IHHNV on WSSV was the first reported case of viral interference that involved crustacean viruses and has been subsequently confirmed. However, the mechanisms underlying the induction of WSSV resistance through IHHNV infection are practically unknown. In this study, the interference mechanisms between IHHNV and WSSV were studied using a competitive ELISA. The binding of WSSV and IHHNV to cellular membrane of Litopenaeus vannamei was examined. The results suggested that there existed a mutual competition between IHHNV and WSSV for binding to receptors present on cellular membrane of L. vannamei and that the inhibitory effects of WSSV towards IHHNV were more distinct than those of IHHNV towards WSSV.     

Daily rhythms of cortisol and glucose and the influence of the light/dark cycle on anaesthesia in Nile tilapia (Oreochromis niloticus): Does the timing of anaesthetic administration affect the stress response?

Although daily variations in drug pharmacokinetics have been reported for a variety of teleost species, the influence of this daily variation on the cortisol response following anaesthesia remains poorly understood. To address this, two experiments were performed. The first experiment described the daily patterns of cortisol and glucose secretion in tilapia (Oreochromis niloticus). The second experiment investigated how the timing of anaesthetic administration (specifically at mid‐light [ML] or at mid‐dark [MD]) affects the induction and recovery times and plasma cortisol and glucose levels of juvenile Nile tilapia exposed to benzocaine, clove oil or tricaine methanesulphonate (MS‐222). The results revealed that the effect on the stress response associated with the moment when anaesthesia took place (ML or MD) varied according to the treatment (p < 0.05). Cortisol levels were significantly higher at ML for MS‐222 (ML = 116.23 ± 25.55; MD = 48.25 ± 22.33 ng/dl) (p < 0.05) and clove oil (ML 59.73 ± 14.27; MD 38.26 ± 12.07 ng/dl) (p < 0.05), whereas no significant differences were found between ML and MD cortisol levels for the control treatment (ML = 72.91 ± 18.42; MD = 64.80 ± 10.68 ng/dl) (p > 0.05) or in the benzocaine‐treated group (ML = 38.7 ± 4.90; MD = 38.60 ± 3.69 ng/dl) (p > 0.05). The highest plasma cortisol level in ML was found in the MS‐222‐treated group. All the tested anaesthetics had similar cortisol levels at MD (p > 0.05).     

肝胰腺细小病毒和斑节对虾杆状病毒的PCR复合检测

肝胰腺细小病毒和斑节对虾杆状病毒是常见的2种DNA对虾病毒,在虾类养殖业中经常会出现混合感染。因此,建立这2种病毒的复合检测方法显得尤为重要。根据基因库中肝胰腺细小病毒和斑节对虾杆状病毒的保守序列,设计了肝胰腺细小病毒和斑节对虾杆状病毒的特异性引物,对这2种病毒PCR复合检测的反应条件和试剂浓度进行优化,退火温度55℃,主要试剂Mg2+终浓度为3mmol/L,dNTP终浓度为0.4 mmol/L,引物终浓度为0.8μmol/L。进一步比较了在优化后的PCR反应体系下,检测单种病毒和同时检测这2种病毒的灵敏度差异。     

Spatial distribution of the Japanese common squid, Todarodes pacificus, during its northward migration in the western North Pacific Ocean

The spatial distribution of Todarodes pacificus in and near the Kuroshio/Oyashio Transition Zone during its northward migration was examined by comparative surveys using two types of mid‐water trawl net and supplementary squid jigging from June to July 2000. The vertical and horizontal distribution patterns varied for different body sizes in relation to the oceanographic structure. Todarodes pacificus of 1–20 cm dorsal mantle length (ML) were widely distributed from the coastal waters of Japan to near 162°E longitude, probably due to transport by the Kuroshio Extension (KE). Todarodes pacificus smaller than 10 cm ML were mainly distributed in temperate surface layers at sea surface temperatures (SSTs) >15°C near the KE meander probably because of their poor tolerance to lower temperatures and limited swimming ability. Squid of 10–15 cm ML were distributed in the offshore waters of 10–15°C SST and in the coastal waters of northern Honshu, and underwent diel vertical migrations between the sea surface at night and deeper layers during the daytime. Squid larger than 15 cm ML were distributed in the coastal feeding grounds of northern Honshu and Hokkaido until they began their southward spawning migration. They also underwent diel vertical migrations, but remained deeper at night than the squid of 10–15 cm ML; this migration pattern closely matched that of their main prey such as euphausiids. We concluded that as T. pacificus grow, they shift their distribution range from the temperate surface layer around the KE toward the colder deeper layers, above 5°C, in the Oyashio and coastal areas.     

Isolation,identification and pathogenicity of Vibrio harveyi,the causal agent of skin ulcer disease in juvenile hybrid groupers Epinephelus fuscoguttatus × Epinephelus lanceolatus

The hybrid grouper, Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂), is a newly bred cultivated marine fish species of high economic value. However, a skin ulcer disease with high mortality has occurred, and the responsible pathogen remains unknown. In this study, we summarized the epidemic status and external signs of this disease. We screened potential pathogens and finally isolated one bacterial strain ML01 from affected fish. We subjected healthy juvenile hybrid groupers to bacterial challenge tests with the isolate by immersion, immersion after dermal abrasion and intraperitoneal injection, respectively. Within 14 days post‐infection, the isolate ML01 caused mass mortality of juveniles infected via immersion after dermal abrasion or intraperitoneal injection. Diseased juveniles displayed obvious signs of skin ulcers. The median lethal dose of ML01 by intraperitoneal injection was 1.10 × 10⁵ colony‐forming units. ML01 was identified as Vibrio harveyi by bacterial morphology, analytical profile index identification, 16S rDNA sequencing and multilocus sequence analysis. Antibiotic susceptibility tests showed that ML01 was sensitive to ceftriaxone, doxycycline and minocycline. The results of this study suggest that V. harveyi is the causal agent of skin ulcer disease in juvenile hybrid groupers, thus providing a basis for effective control and prevention of this disease.     

Ontogeny of schooling behavior in the oval squid <Emphasis Type="Italic">Sepioteuthis lessoniana</Emphasis>

In contrast to schooling behavior in fish, which is well documented, the schooling behavior of an evolutionary counterpart, squid, has not been adequately described in the literature. In the current study, we investigated the ontogeny of schooling behavior in the oval squid Sepioteuthis lessoniana reared from hatching to about two months of age. During the two months of development, the distance and angle between nearest-neighbor individuals decreased from 5.3 mantle lengths (ML) to 1.8 ML and from 42.7° to 24.2°, respectively. In addition, the swimming distance of individuals in experimental duration decreased from 530.4 to 79.9 ML, and the relative swimming speed also decreased from 17.1 to 4.6 ML/s during the same period, with increasing synchronized hovering of individuals. These observations clearly suggest that oval squid swim randomly until 20 days after hatching, then form a school 30–60 days after hatching with an inter-individual distance of 2.0 ML. At two months after hatching, oval squid individuals swam in a parallel orientation and formed a belt-shaped arrangement, similar to the adult schooling behavior observed in natural populations.     

Presence of multiple viruses in non-diseased, cultivated shrimp at harvest

Histological examinations were carried out with 400 cultivated black tiger shrimp (Penaeus monodon) from 12 commercial rearing ponds from three different areas in Thailand over a period of 3 years. The shrimp were collected at or near harvest time as two arbitrary size groups of 10–20 each from each pond. Aside from size difference, they showed no gross signs of disease and were normally active. Pathognomonic histopathological lesions were found only for hepatopancreatic parvovirus (HPV) or monodon baculovirus (MBV). Although these were relatively frequent, no unusual shrimp mortality had occurred in any of the ponds examined. Severity of these infections was negatively correlated with shrimp size. When grouped together, HPV-infected shrimp gave mean lengths of approximately 6.5 cm that were significantly different from uninfected shrimp at 9 cm length, early in the cultivation cycle while MBV-infected groups of approximately 9 cm length were not readily distinguishable until uninfected shrimp were 10 cm or more, later in cultivation. Thus, HPV infection was correlated with more severe stunting than MBV. In addition to histopathological examination, polymerase chain reaction (PCR) assays for HPV, white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) were carried out on one large sample of 240 shrimp from 6 ponds where visible lesions were apparent for MBV only. Surprisingly, 94% of the specimens gave a positive test for at least one of the four viruses. HPV and IHHNV alone or in combination were detected at high prevalence (approximately 60%) despite the absence of visible histological lesions and were confirmed by southern blot hybridization. Although the prevalence of the four viral pathogens was very high, it would normally have gone unnoticed, since normal shrimp are rarely examined for viruses.     

基于人工神经网络的日本海舍氏贝乌贼角质颚色素沉积特性分析

根据中国鱿钓船于2018年12月在日本海采集的303个舍氏贝乌贼样本,进行基础生物学分析,对其角质颚色素沉积等级进行划分和判定,利用人工神经网络模型分析色素沉积等级与胴长、体质量、性腺成熟度、角质颚形态参数和胃级的关系,并对各生长因子的中位数进行线性拟合。结果显示,胴长对角质颚色素沉积的贡献率最大,为22.90%,其次分别为下头盖长、性腺成熟度、体质量、下喙长和下翼长,贡献率分别为16.50%、14.40%、11.90%、11.70%和11.60%,下侧壁长和胃级对角质颚色素沉积的贡献率较小,分别为6.30%和4.70%。舍氏贝乌贼角质颚的色素沉积与胴长、体质量、性腺成熟度和胃级这4项生长因子的关系均不存在性别间显著性差异。角质颚色素沉积等级与胴长、体质量和角质颚外部形态参数均呈正相关,并随着性腺成熟度的增加而增加,但与胃级的线性关系不明显。研究表明,日本海舍氏贝乌贼角质颚色素沉积等级与胴长、性腺成熟度、体质量和角质颚形态参数均呈正相关关系,可以利用胴长、性腺成熟度、体质量和角质颚形态参数预估色素沉积等级。     

Influence of one selected Tisochrysis lutea strain rich in lipids on Crassostrea gigas larval development and biochemical composition

Effects of a remarkably high overall lipid Tisochrysis lutea strain (T+) upon gross biochemical composition, fatty acid (FA), sterol and lipid class composition of Crassostrea gigas larvae were evaluated and compared with a normal strain of Tisochrysis lutea (T) and the diatom Chaetoceros neogracile (Cg). In a first experiment, the influence of different single diets (T, T+ and Cg) and a bispecific diet (TCg) was studied, whereas, effects of monospecific diets (T and T+) and bispecific diets (TCg and T+Cg) were evaluated in a second experiment. The strain T+ was very rich in triglycerides (TAG: 93–95% of total neutral lipids), saturated FA (45%), monounsaturated FA (31–33%) and total fatty acids (4.0–4.7 pg cell^?1). Larval oyster survival and growth rate were positively correlated with 18:1n‐7 and 20:1n‐7, in storage lipids (SL), and negatively related to 14:0, 18:1n‐9, 20:1n‐9, 20:4n‐6 and trans‐22‐dehydrocholesterol in membrane lipids (ML). Surprisingly, only the essential fatty acid 20:5n‐3 in SL was correlated positively with larval survival. Correlations suggest that physiological disruption by overabundance of TAG, FFA and certain fatty acids in larvae fed T+ was largely responsible for the poor performance of these larvae. ‘High‐lipid’ strains of microalgae, without regard to qualitative lipid composition, do not always improve bivalve larval performance.     

Effects of light intensity on molting,growth, precocity,digestive enzyme activity,and chemical composition of juvenile Chinese mitten crab <Emphasis Type="Italic">Eriocheir sinensis</Emphasis>

A 74-day growth trial was carried out to investigate the effects of light intensity on the juvenile Chinese mitten crab, Eriocheir sinensis, under semi-natural conditions. The experiment had three light intensity treatment groups, natural light (NL), middle light (ML), and low light (LL), as light intensity became weaker. The results indicated that light intensity had no significant effect on molting interval and molting frequency but did have a significant effect on the molting weight gain of the crab. Molting weight gain in group NL was significantly higher than that in group LL (P < 0.05). Specific growth rate in weight (SGRw) and in carapace width (SGRcw), weight gain, and final body weight were significantly affected by light intensity (P < 0.05). No significant difference was detected in survival between groups (P > 0.05). The precocious rate in groups NL, ML, and LL was 26.14, 15.48, and 17.14%, respectively. The precocious rate in group NL was significantly higher than that in groups ML and LL (P < 0.05). Chemical composition of the crab body was significantly affected by light intensity, but the activity of alkaline phosphatase, trypsin, and pancreatic lipase was not significantly affected by light intensity. The results indicated that the submitted light intensity was useful in reducing the precocious rate without affecting the normal growth of juvenile E. sinensis.     

Effects of complete fish meal replacement by rice protein concentrate with or without lysine supplement on growth performance,muscle development and flesh quality of blunt snout bream (Megalobrama amblycephala)

An 8‐week feeding trial was conducted to evaluate the effects of fish meal (FM) replacement by rice protein concentrate (RPC) with supplementation of microcapsule lysine (ML) or crystalline lysine (CL) on growth performance, muscle development and flesh quality of blunt snout bream. Four isonitrogenous and isoenergetic diets were formulated, including FM diet (containing 50 g/kg FM), RPC diet (FM replaced by RPC), MRPC diet (FM replaced by RPC with ML supplementation) and CRPC diet (FM replaced by RPC with CL supplementation). Fish fed FM diet had significantly higher weight gain, feed efficiency, protein efficiency ratio and nitrogen and energy utilization than that of RPC group, but showed no statistical difference with other treatments. In addition, fish fed RPC diet showed higher muscle fibre frequency in the 20‐ to 50‐μm class but lower >50‐μm class and higher cooking loss than that of the other groups. Furthermore, no significant difference was found in whole‐body proximate compositions, frequency distribution of <20‐μm‐diameter fibres, texture, muscle content, collagen, pH 24 hr post‐mortem and sensory quality. The results showed that RPC supplemented with ML or CL could replace fishmeal without any adverse effects on growth performance and flesh quality for blunt snout bream.     

哈维氏弧菌(Vibrio harveyi)ML01株胞外产物及分泌性蛋白的分离与特性分析

本研究以引起珍珠龙胆石斑鱼[Epinephelus fuscoguttatus(♀)×Epinephelus lanceolatus(♂)]幼鱼“皮肤溃疡病”的哈维氏弧菌(Vibrio harveyi) ML01株为研究对象,采用平板膜覆盖技术和柱层析技术,分离、纯化了ML01株的胞外产物及分泌性蛋白.应用毒性试验、质谱分析与分子克隆技术,对纯化的胞外产物和3种主要的分泌性蛋白进行了特性分析与鉴定.结果显示,哈维氏弧菌ML01株的胞外产物(Extracellular products,ECPs)具有酯酶、明胶酶、淀粉酶、酪蛋白酶活性,无脲酶活性.ECPs对羊红细胞无溶血性,对斑马鱼(Danio rerio)的半数致死剂量(LD50)为19.55μg/g鱼体重.从ML01株中分离到3种主要的分泌蛋白P42、P36、P31,其分子量分别为42、36、31 kDa.经质谱鉴定和分析,这3种蛋白分别为哈维氏弧菌的外膜蛋白OmpU和OmpN,以及一种功能未知的蛋白.利用同源克隆,成功地从ML01株基因组中扩增到了P42、P36、P31的基因.序列测定和比对结果显示,ML01株的这3个基因与哈维氏弧菌ATCC 33843(GenBank CP009467)的相应基因相比,其开放阅读框(Open reading frame,ORF)序列的相似性分别为97.08％、100％、99.67％,其编码的多肽序列的相似性分别为99.71％、100％和99.93％.本研究对进一步分析哈维氏弧菌ML01株的致病机理、研发该菌的亚单位疫苗具有重要的参考价值.     

湖北省潜江地区克氏原螯虾(procambarus clarkii)白斑综合征(White Spot Syndrome)PCR诊断及组织病理学观察

近年来在湖北省范围内人工养殖的克氏原螯虾暴发了严重的疾病,其中白斑综合征病毒（WSSV）已成为危害克氏原螯虾健康养殖的重要病原。2016年5月湖北省潜江市养殖区暴发了一种传染性疾病,为探究此次疾病病因和流行规律,将染病虾进行临床症状观察、对病料进行PCR检测、系统发育树分析、人工感染和组织病理学观察。结果显示,发病克氏原螯虾临床症状主要表现为摄食减少,活力下降,反应迟钝;组织病理学观察结果显示,克氏原螯虾的肝胰腺、肠、肌肉、鳃组织均出现不同程度变性和坏死以及炎性细胞浸润等典型病理学变化,与WSSV感染克氏原螯虾出现的病变相似;PCR检测患病克氏原螯虾样品,结果显示WSSV呈阳性,阳性检出率为55.56%（15/27）,未检测到斑节对虾杆状病毒（MBV）和传染性皮下及造血组织坏死病毒（IHHNV）;检测产物测序并进行系统发育树分析,结果显示,该基因序列与WSSV的EG3株（KR083866.1）核苷酸序列同源性为100%。将病虾的肝胰腺、肠和肌肉组织投喂健康克氏原螯虾,投喂组均表现为急性死亡（累积死亡率为100%）,并出现与自然发病虾相同的症状。WSSV的巢式PCR检测结果显示,人工感染病虾为WSSV阳性。根据以上显示,本次养殖克氏原螯虾大规模死亡的病原是WSSV。