Isolation and partial characterization of bacteriophages infecting Vibrio harveyi from shrimp farm effluent water

Bacteriophage isolated from the semi-intensive culture of Pacific white leg shrimp Litopenaeus vannamei infects the luminous bacteria Vibrio harveyi. Lytic activity and lytic spectrum results revealed that the isolated phage had strong lytic activity in V. harveyi, V. parahaemolyticus, and V. vulnificus. Biofilm inhibition activity was performed against different pathogenic vibrios on high-density polyethylene (HDPE) template and the result revealed that the phage effectively inhibited the biofilm formation in V. harveyi. Spectrophotometric assay performed for lytic activity of the isolated phage in V. harveyi liquid culture showed that the phage significantly decreased the V. harveyi cell densities at different time intervals (P?<?0.05). To study the stability of phage at different temperature and pH revealed that the phage withstands the temperature ranged between 40 and 70 °C and the pH of 4 and 9 at a significant level (P?<?0.001). One-step growth curve depicted that the burst size gradually increased to a significant level and reached the maximum of 90% at 180 min (P?<?0.05). This study concluded that the isolated phage had specific activity against pathogenic V. harveyi infections.

     

Benthic microbial indicators of fish farm impact in a coastal area of the Tyrrhenian Sea

We studied the impact of organic loads due to the biodeposition of a fish farm in a non-impacted coastal area of the Tyrrhenian Sea (Western Mediterranean). Sediment chemistry and benthic microbial community were investigated from July 1997 to February 1998 on monthly basis at two stations: one was located under the fish farm, while the second was about 1 km away, and served as a reference site. The presence of the cage induced rapid changes in the benthic conditions: the sediments were rapidly (after 6 weeks) reduced. A significant accumulation of biopolymeric carbon was observed beneath the cage both 2 weeks after the initial cage deployment, on non-impacted sediments, and 5–7 months after, and appeared to be related to the fish farm production cycle. The density of microbial communities beneath the fish farm increased only during the first month of farming activity (July) and partially 7 months after cage disposal (i.e. in January), when there was a significant sediment organic enrichment. Additional evidence of rapid impact of the fish farm on the benthic bacterial communities is provided by the increase in the numbers of autofluorescent microbial cells. The photosynthetic eukaryotic cells displayed a highly reduced contribution to total autofluorescent microbial density, indicating that their decrease is related with biopolymeric carbon accumulation, and probably was due also to “shadow effect” induced by suspended material, coming from the fish farm, on the environment below. We propose here, to apply the ratio of culturable heterotrophic bacteria to microbial direct counts (CFU/MDC) to detect fish farm impact. In cage sediments, organic enrichment and the consequent modification of the characteristics of the benthic environment, determined an increase in aerobic heterotrophic bacteria and vibrio density indicating that they are efficient colonizers of organic-rich sediments. Densities of Escherichia coli and Enterococci were not significantly higher than in the reference site, and are likely of terrestrial origin. Thus they cannot be used as specific indicators of fish farm impact.     

Rearing the spotted seahorse Hippocampus kuda by feeding live and frozen copepods collected from shrimp ponds

This study evaluated the use of live and frozen copepods collected from shrimp ponds for rearing juveniles of the spotted seahorse Hippocampus kuda. Protein and HUFA contents in frozen copepods were all higher than in Artemia nauplii, the conventional live food for seahorse juveniles. The results of this study showed that copepods can be used as feed for rearing seahorse fry and juvenile. The spotted seahorse showed obvious preference for live copepods and rarely fed on dead copepods on the tank bottom. Furthermore, the combination of frozen copepods and live Artemia nauplii resulted in highest growth and highest survival of the experimental seahorses. Further research on possible effects of DHA:EPA ratio on survival and growth of young H. kuda is recommended.     

Discards from the artisanal shrimp fishery in a tropical coastal lagoon of Mexico: spatio-temporal patterns and fishing gear effects

Abstract  Variability in community (richness, diversity and evenness) and population (size structure) attributes of discards was evaluated in a shrimp fishery in Celestun Lagoon, Mexico, in different climatic seasons and hydrological zones. Sampling was conducted using two bottom nets with different mesh size codends: 1.3 cm (used in the fishery) and 2.5 cm. Although the shrimp:discard ratio was 1:1 in both cases, total biomass and abundance with the 1.3-cm mesh size were, respectively, 4.6 and 1.8 times higher than with the 2.5-cm mesh size. This result was consistent in time and space. Discards were dominated by fishes [60% in 1.3-cm mesh size and 96% in 2.5-cm mesh size, mainly Cichlasoma urophthalmus (Günther), Eucinostomus gula (Quoy & Gaimard) and Lucania parva (Baird & Girard)] and shellfishes (40% in 1.3-cm mesh size and 4% in 2.5-cm mesh size, mainly Palaemonetes spp. and Callinectes spp.). The highest discards were registered the nortes season with both mesh sizes. The 1.3-cm mesh size retained significantly smaller sizes than the 2.5-cm mesh size in six of the eight most important species. An increase in mesh size, together with a fishery closure during the nortes season, are suggested to mitigate the impact of the fishery on the discarded fauna.     

Transport of subarctic large copepods from the Oyashio area to the mixed water region by the coastal Oyashio intrusion

The lateral transport of organic carbon in large grazing copepods ( Neocalanus cristatus, Neocalanus flemingeri, Neocalanus plumchrus and Eucalanus bungii ) from the Oyashio area to the mixed water region (MWR) by the coastal Oyashio intrusion was estimated using the data of VMPS (vertical multiple plankton sampler) and 1500 dbar-referred geostrophic transport from the CTD (conductivity temperature depth sensor) data of five cruises during June 2001 to April 2002 on a repeat observation section OICE (Oyashio Intensive observation line off Cape Erimo), which extends southeastward from Hokkaido Island, Japan. The transport to MWR by the coastal Oyashio intrusion was estimated to be 5.3 × 10¹¹ g C for the four species. Data from profiling floats also indicated that the copepods were advected from OICE to MWR by the coastal Oyashio intrusion within about 2 months. This transport is considered to be one of the significant sources of organic carbon in MWR as it is larger than the amount of large zooplankton consumed by Pacific saury ( Cololabis saira ) in MWR, one of the dominant copepod predators in this region.     

Antioxidant effects of nanoencapsulated lipoic acid in tissues and on the immune condition in haemolymph of Pacific white shrimp Litopenaeus vannamei (Boone, 1931)

The antioxidant and oxidative damage responses and immune parameters in shrimp, Litopenaeus vannamei, reared in clear water (CW) and in a biofloc system (BFT) were evaluated. The animals were given feed supplemented with either nanocapsules containing the antioxidant lipoic acid (NCLA) or empty nanocapsules (NC). Antioxidant and oxidative stress analyses were evaluated by measuring glutathione S‐transferase activity (GST), reduced glutathione (GSH) concentration and lipid peroxidation (TBARS) in the gills, hepatopancreas and muscle. The immune parameters were then determined by the differential hemocyte count in the haemolymph. The final weight of the shrimp reared in both CW and BFT and treated with NCLA increased (p < .05). The shrimps reared in BFT showed increased GST activity in the gills and hepatopancreas after NCLA treatment (p < .05). Higher GSH levels were observed in the hepatopancreas with NCLA supplementation in CW and BFT (p < .05). Decreased levels of TBARS were observed in the gills and muscles of shrimp supplemented with NCLA and maintained in CW (p < .05). A decrease in the percentage of hyaline hemocytes and an increase in granular hemocytes were observed in shrimp treated with NCLA in CW and BFT (p < .05). The antioxidant treatment induced positive responses in terms of the zootechnical, biochemical and immunological parameters.     

The improvement of growth,digestive enzyme activity and disease resistance of white shrimp by the dietary citric acid

A 45-day feeding trial was conducted to evaluate the effects of dietary citric acid on growth, digestive enzyme and disease resistance of white shrimp, Litopenaeus vannamei. Shrimp with initial body weight of 5.57 ± 0.21 g were fed with basal diet supplemented with 0.0 g kg^?1 (control), 1.0, 2.0, 3.0, 4.0 and 5.0 g kg^?1 citric acid. Results showed that weight gain was increased by 15.9 % and feed conversion ratio was decreased by 0.17 by 2.0 g kg^?1 dietary citric acid compared with control group (P < 0.05). Intestinal protease activity of shrimp fed 2.0 g kg^?1 citric acid was significantly higher (P < 0.05) than that of control group. No significant difference was found in intestinal amylase activity among treatments (P > 0.05). The activities of serum phenoloxidase, superoxide dismutase and lysozyme in 2.0 and 3.0 g kg^?1 citric acid group were significantly higher, and accumulative mortalities of the two groups on the fourth day after injection of Vibrio alginolyticus were significantly lower than those of control group (P < 0.05). Results above demonstrated that dietary citric acid could improve growth performance, immunity and resistance against V. alginolyticus. The supplementation level of citric acid in diet was suggested to be 2.0–3.0 g kg^?1 for white shrimp.     

Laboratory and on‐farm evaluation of low‐cost salt mixtures for use during salinity acclimation and the nursery phase of Pacific white shrimp (Litopenaeus vannamei)

The current study evaluates the efficacy of a low‐cost salt mixture (LCSM) to replace expensive reconstituted sea salt (RSS) in the salinity acclimation and nursery phase of Pacific white shrimp under laboratory and farm conditions. LCSM was formulated to yield sodium, potassium, calcium and magnesium concentrations closely comparable to that of diluted seawater. Laboratory‐based nursery trials were conducted at 2, 6 and 15 g/L salinities, incrementally replacing RSS with LCSM (25%, 50%, 75% and 100%) at four replicates per treatment. Thirty postlarvae were reared for 7 days in 24‐L aquaria during the 2 and 6 g/L trials, while the nursery trial for 15 g/L salinity was conducted for 21 days with 400 postlarvae stocked in 150‐L tanks. On‐farm evaluation of LCSM was carried out in two tank‐based systems installed on levees adjacent to shrimp production ponds. RSS was incrementally replaced with LCSM (0%, 50%, 75% and 100%) and 100 postlarvae stocked into each 800‐L tank. Salinity acclimation was done from 30 g/L to 6 or 1.5 g/L within 2–3 days by pumping water from adjacent shrimp production ponds. Following salinity acclimation, the S4 system maintained flow‐through at 1.5 g/L, while N10 system was maintained static at 6 g/L salinity. At the conclusion, no significant differences were observed for either survival or growth of shrimp postlarvae between RSS and LCSM treatments at all salinities examined. Results reflect the potential use of LCSM to replace RSS, which could be an excellent solution to bring down the cost of production in inland shrimp aquaculture.     

小鼠生物法和酶联免疫法(ELISA)定量监测沿海5省养殖河豚鱼中的河豚毒素(TTX)

为进一步扩大国内养殖河豚鱼消费市场提供依据,应用小鼠生物法监测了我国辽宁、河北、山东,江苏和福建4种养殖河豚鱼(红鳍东方鲀,菊黄东方鲀,双斑东方鲀和暗纹东方鲀4种组织(皮、肉、肝脏和性腺)中河豚毒素(TTX)含量及周年变化,并用酶联免疫法(ELISA)同步检测了山东红鳍东方鲀的皮,肉和肝脏各组及个别卵巢样品。结果4种养殖河豚鱼的皮,肉和肝脏每组平均含毒量均属无毒(小鼠生物法检测结果小于0.5μg/g,ELISA检测结果小于0.8μg/g)。卵巢发现一强毒例,精巢则全部无毒。通过监测,发现养殖河豚鱼组织器官的毒力呈现一定的周年变化。ELISA法与小鼠生物试验测得的结果相符合。养殖河豚鱼皮,肉和精巢认为可以考虑科学安全的利用,肝脏须谨慎对待,由于养殖河豚鱼上市规格较小,卵巢一般无食用意义,加工过程应注意不要污染可食部分。     

凡纳滨对虾C型凝集素（LvLc1）的特征和活性分析

C型凝集素是一种依赖于Ca~(2+)而发挥功能的糖蛋白,在一线的固有免疫防御过程中发挥着重要作用。围绕对虾C型凝集素开展深入研究,不仅可以丰富无脊椎动物固有免疫学内容,还有望将其开发为具有免疫增强效果的活性饵料,应用于对虾的健康养殖。本实验根据实验室前期转录组信息提示克隆获得了凡纳滨对虾一种新的C型凝集素基因(LvLc1,Gen Bank注册号:KY937940)。生物信息学分析显示LvLc1基因的开放阅读框全长891 bp,编码296个氨基酸,该基因编码的蛋白质含有一个保守的糖识别结构域(carbohydrate recognition domain,CRD),该结构域中具有潜在的半乳糖结合位点(QPD motif),进化发生分析显示LvLc1与来自节肢动物的甘露糖结合凝集素家族成员聚类在一起。对LvLc1基因的CRD结构域进行了原核重组表达与蛋白活性分析研究,结果显示:重组目的蛋白(rLvLc1)在Ca~(2+)存在的条件下,对多种病原菌(G~+、G~–和真菌)具有凝集作用,其凝集活性可被半乳糖、甘露糖、脂多糖等多种病原相关分子模式所抑制。研究表明,LvLc1作为C-型凝集素家族一个新成员,可能通过重要的模式识别受体作用,参与机体应答病原微生物侵染的防御过程。     

山东沿海养殖和野生双齿围沙蚕营养成分比较及饵料安全性评价

为全面评估养殖和野生双齿围沙蚕(Perinereis aibuhitensis)作为亲虾饵料的营养价值和安全性,本研究分别对山东沿海养殖和野生双齿围沙蚕的基本营养成分、氨基酸和脂肪酸含量、常见重金属含量和白斑综合征病毒(WSSV)及对虾传染性皮下及造血组织坏死病毒(IHHNV)携带情况进行了检测和对比分析。结果表明,养殖沙蚕的脂肪含量显著高于野生沙蚕中含量(P0.05),粗蛋白、水分、灰分含量无显著性差异(P0.05);两种沙蚕中的对虾必需氨基酸/总氨基酸(EAA/TAA)均在40%左右,对虾必需氨基酸/非必需氨基酸(EAA/NEAA)均在60%以上,符合FAO/WHO关于高品质蛋白的标准,依据两组沙蚕的氨基酸评分(AAS)和化学评分(CS),养殖沙蚕的第一限制氨基酸含量高于野生沙蚕含量;养殖沙蚕的不饱和脂肪酸(UFA)和高度不饱和脂肪酸(HUFA)的含量分别为(66.15±3.77)%和(54.11±2.58)%,均显著高于野生沙蚕的含量[(56.13±6.60)%、(43.28±5.50)%](P0.05);对常见重金属铬(Cr)、铜(Cu)、镉(Cd)、铅(Pb)、砷(As)、汞(Hg)的检测发现,养殖沙蚕重金属的含量明显低于野生沙蚕的含量(P0.05),且野生沙蚕中As的含量严重超标;在对虾常见携带病毒WSSV和IHHNV检测中,养殖沙蚕均呈现阴性,而野生沙蚕IHHNV呈现阴性,WSSV呈现阳性,说明野生沙蚕可能携带WSSV。从营养价值、饵料安全性以及生态环境保护角度考虑,养殖双齿围沙蚕作为亲虾饵料的效果明显优于野生双齿围沙蚕。     

Effects of polysaccharides from mycelia of Cordyceps sinensis on growth performance,immunity and antioxidant indicators of the white shrimp Litopenaeus vannamei

Studies were conducted to evaluate polysaccharides from mycelia of Cordyceps sinensis (PMCS) as a feed additive for pacific white shrimp (Litopenaeus vannamei). Shrimps were randomly divided into two groups. One group received the PMCS‐supplemented diet for 52 days, and the other group received no treatment. The data showed that shrimps given PMCS gained more body weight compared with the control group (P < 0.05). Haemolymph immunity indicators, including phenoloxidase (PO), alkaline phosphatase (ALP), acid phosphatase (ACP) and lysozyme (LSZ), were remarkably greater in the PMCS group (P < 0.05). In addition, antioxidant parameters, including superoxide dismutase (SOD), glutathione reduced form (GSH), reactive oxygen species (ROS) and total antioxidant capacity (TAC), were significantly greater in the treatment group. However, there were no significant differences in malondialdehyde (MDA) levels and glutathione peroxidase (GSH‐PX) activity between the control and treatment groups. Moreover, the number of Vibrio ichthyoenteri was less in the group treated with PMCS (P < 0.05). In conclusion, PMCS can be used to improve the growth performance and health of pacific white shrimp.     

Replacement of fish meal by meat and bone meal in practical diets for the white shrimp Litopenaeus vannamai (Boone)

The use of meat and bone meal (MBM) was evaluated as a replacement for fish meal in a practical diet formulated to contain 41% protein and 8% lipid. Anchovy meal was replaced by 0%, 20%, 30%, 40%, 50%, 60% and 80% of MBM (diets 1–7) respectively. Healthy post larvae of Litopenaeus vannamei were reared in an indoor, semi‐closed recirculating system. Each dietary treatment was fed to triplicate groups of 40 shrimp per tank (260 L) arranged in a completely randomized design. The shrimp were hand‐fed to near‐satiation three times daily between 07:00 and 18:00 hours for 56 days. There were no significant differences (P>0.05) in growth performance among shrimp fed diets 1–6. However, shrimp fed diet 7 had significantly lower (P<0.05) growth than those fed diet 2 or diet 4. Survival ranged from 95% to 100% and did not significantly (P>0.05) differ. Feed conversion ratio (FCR) and carcass composition of the shrimp were not significantly (P>0.05) affected by dietary treatments. No significant differences (P>0.05) in protein efficiency ratio (PER) were found among shrimp fed diets 1‐6. However, shrimp fed diet 7 had significantly lower (P<0.05) PER than those fed diet 1 or diet 4. Results showed that up to 60% of fish meal protein can be replaced by MBM with no adverse effects on growth, survival, FCR, PER and body composition of L. vannamei.     

Cloning,characterization,and expression of themacrophage migration inhibitory factor gene from the Pacific white shrimp Litopenaeus vannamei(Penaeidae)

The macrophage migration inhibitory factor(MIF)is an important proinflammatory cytokine that mediates both innate and adaptive immune responses.In this study,we identified a homolog of MIF in the Pacific white shrimp Litopenaeus vannamei.The MIF cDNA contained a 363-bp open reading frame encoding a 120-amino acid protein with a calculated molecular mass of 13.442 kDa and a theoretical isoelectric point of 6.57.The L.vannamei MIF shared high amino acid identity with MIFs of other invertebrates.Tissue distribution analysis by quantitative real-time polymerase chain reaction(qRT-PCR)revealed that the L.vannamei MIF was abundantly expressed in the blood,heart,and hepatopancreas,was moderately expressed in the gill,and was weakly expressed in the muscle and intestine.Furthermore,in order to gain a basic understanding of the role of MIF in the shrimp immune response against viral infection,its mRNA expression was determined in the hepatopancreasofL.vannameiatdifferentstagesafter whitespotsyndromevirus(WSSV)challenge usingqRT-PCR.The result indicated that the expression of MIF was significantly upregulated after WSSV injection,suggestingthatMIFmaybeinvolved in theresponsetoviralinfection in shrimp.     

Mode of attachment and lesions associated with trypanorhynch cestodes in the gastrointestinal tracts of two species of sharks collected from coastal waters of Borneo

Lesions associated with two species of tapeworms within the digestive tract of wild-caught specimens of the bull shark, Carcharhinus leucas, and the sicklefin weasel shark, Hemigaleus microstoma, from Malaysian Borneo are described. Portions of the glandular stomach and pyloric gut with parasites were removed and fixed in 10% formalin buffered in sea water. Whole mounts, histological sections of tissues with and without worms in situ, and scanning electron microscopy images of detached worms were examined. Both species of cestodes belonged to the trypanorhynch family Tentaculariidae. Heteronybelinia estigmena was found in large numbers parasitizing the pyloric gut of C. leucas; an unidentified tentaculariid was found in relatively small numbers in both the glandular stomach and pyloric gut of H. microstoma. Both species burrowed their scoleces deeply in the mucosa and attached via hooked tentacles and unciniform microtriches of the scolex. The lesions induced by the parasites were marked in both sharks and ranged from acute necrotizing to chronic granulomatous gastroenteritis. Regenerative hyperplasia and intestinal metaplasia of gastric epithelium were also present. The severity and character of pathology was causally linked to the intensity of infection, the attachment mode of the parasites, and to the anatomophysiological relationships within the gut of the host shark.     

On condition index and percentage edibiliy of the shortneck clam Paphia malabarica (Chemintz) from estuarine regions of Ratnagiri,west coast of India

Paphia malabarica is an important clam species, which has formed vast beds in the Kalbadevi estuary (Shirgaon creek) and Kajali estuary (Bhatye creek), Ratnagiri. They are exploited here commercially throughout the year for local as well as external markets due to the heavy demand. This study was undertaken to assess seasonal variations in the quality of the meat of clams by estimating the condition index and percentage edibility. Higher values were recorded during the monsoon (June–August). A rapid decline was observed in the edibility values from November onwards. Higher condition index and percentage edibility were recorded from June to November, indicating the high quality of clam meat.     

Comparative study on the utilization of crystalline methionine and methionine hydroxy analogue calcium by Pacific white shrimp (Litopenaeus vannamei Boone)

The objective of this study was to compare the supplemental effects of crystalline DL‐methionine (DL‐Met) and methionine hydroxy analogue calcium (MHA‐Ca) on growth performance of Pacific white shrimp. Eight isoproteinic (355.3 ± 2.0 g/kg diet) and isolipidic (70.0 ± 2.2 g/kg diet) diets were prepared as positive diet (20% fish meal), negative diet (15% fish meal) and DL‐Met, MHA‐Ca‐supplemented diets with the supplementation of 0.03%, 0.06%, 0.09% DL‐Met and 0.04%, 0.07%, 0.1% MHA‐Ca in negative diet respectively. Pacific white shrimp (0.92 ± 0.03 g) were fed one of the eight diets for 49 days. The results showed that dietary DL‐Met did not affect weight gain (WG) and feed conversion ratio (FCR) (p > 0.05), but the supplementation of 0.1% MHA‐Ca significantly increased WG, protein and lipid retention, and reduced FCR (p < 0.05) when compared to the negative group, and reached the similar levels as the positive control. The total free amino acids (TFAA) in haemolymph of MHA‐Ca groups and PC, NC group peaked at the 3rd hr after feeding, but the peaking time of DL‐Met groups was advanced to the 2nd hr. Activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in haemolymph of all MHA‐Ca groups peaked at the 2nd hr, but DL‐Met groups peaked at the 1st hr after feeding. The above results indicated that the supplementation of 0.1% MHA‐Ca in a low fish meal diet could improve the growth performance and feed utilization of Pacific white shrimp, but dietary DL‐Met did not significantly affect the growth.     

Assessment of the relative contribution of dietary nitrogen from fish meal and biofloc meal to the growth of Pacific white shrimp (Litopenaeus vannamei)

The relative contribution of the dietary nitrogen supplied by fish meal and a biofloc meal to the growth of Pacific white shrimp was evaluated using stable isotope analysis. Biofloculated material was obtained from an experimental tilapia culture system. Five formulated diets were supplied. Two of them consisted in isotopic controls having only fish meal or biofloc meal as protein source. Three mixed diets were formulated with varying proportions of these ingredients on a dietary nitrogen basis (75:25, 50:50 and 25:75). At the end of the trial, survival rates were similar (92–100%) but significant differences in mean final weight were observed and a negative correlation between the inclusion of biofloc meal and weight gain was evidenced. Mean final weight in shrimp fed on diet containing only fish meal was 2.8 g, while mean final weight of animals fed on diet containing 50% biofloc was 1.9 g. Isotopic mixing models indicated that all diets contributed higher proportions of dietary nitrogen from fish meal than from biofloc meal. Dietary nitrogen available in diets containing 25%, 50% and 75% of biofloc meal was incorporated in muscle tissue as 5%, 41% and 64% respectively. Diet supplying 25% of nitrogen from biofloc was the only mixed diet eliciting growth comparable to diet containing only fish meal. Lower growth and nitrogen deposition in shrimp fed on diets containing high proportions of biofloc meal were possibly associated to the use of only two protein sources and a restriction of essential amino acids.