Lipid-based transfection as a method for gene delivery in zebrafish (Danio rerio) embryos

A major challenge to the widespread production of transgenic, knockout and knockdown zebrafish has been the absence of a simple and effective procedure for introducing macromolecules into the fertilized egg. None of the existing techniques for gene transfer in fish embryos has proven to be a major advance over cytoplasm microinjection, which is a technically demanding and time‐consuming procedure. This report addresses this need, considering that the development of protocols for lipid‐based transfection with fish embryos would considerably simplify gene transfer in this complex biological model. In this study, lipid‐based transfection with two different reporter vectors was carried out in zebrafish embryos at different developmental stages. The parameters tested included different plasmid/transfection reagent ratios as well as the influence of an added transfection enhancer reagent. When embryos were transfected in the blastula stage with a pEGFP‐N1 vector, more than 35% successfully incorporated the plasmid and expressed the fluorescent protein 24 h after transfection. The transfection enhancer did not show any significant effect in our experiments. This work presents an approach to implement this technique as a faster, cheaper and more practical alternative than microinjection.     

显微注射生长激素基因导入中国对虾（Penaeus chinensis）受精卵的研究

本实验采用显微微量注射方法，交羊生长激素基因导入中轻地虾受精卵，受精卵发育到蚤状幼体第三期后采样检测。ＰＣＲ检测结果表明：７个样品共９３尾幼体，有３个样品呈出阳性信号，基因转移比率至少在３％以上。同时斑点杂交结果也证明有两个明显阳性斑点。     

The molecular detection of Taura syndrome virus emerging with White spot syndrome virus in penaeid shrimps of Thailand

This study is to investigate the outbreak of the Taura syndrome (TS) in penaeid shrimps of Thailand. Following the outbreak in 2003, the Taura syndrome virus (TSV) was detected in two closely related penaeids; the Pacific white shrimp, Penaeus vannamei, and the black tiger prawn, Penaeus monodon. Multiple viral infections were detected, we found that the TSV-infected P. vannamei also presented evidence of the White spot syndrome virus (WSSV) as evidenced by histological examination and molecular diagnostic methods. Infections of TSV and WSSV were investigated over a period of ten months between October 2003 and July 2004. TSV infection in shrimps was diagnosed by RT-PCR using the OIE recommended 9195/9992 primers. Positive shrimp gave the expected amplicon of 231 bp. We also amplified and sequenced a fragment of the vp1 gene (354 bp). The pairwise comparisons of vp1 revealed that Thai TSV was phylogenetically related to the Taiwanese strains (98.3% identity).     

江苏地区对虾3种病毒病的流行病学调查及5株IHHNV的编码区基因序列分析

对虾白斑综合征病毒(WSSV)、桃拉综合征病毒(TSV)、传染性皮下及造血组织坏死病毒(IHHNV)是威胁对虾养殖的重要病毒。为了调查这3种病在江苏境内的流行情况,根据世界动物卫生组织(OIE)推荐的《水生动物疾病诊断手册》的PCR检测法对2015年5月—2016年5月采集的1436尾对虾样品进行3种疾病的流行病学调查。结果显示WSSV阳性率为17.20%,TSV阳性率为0%,IHHNV阳性率为39.48%。对江苏不同地区分离出的5株IHHNV进行基因序列分析,数据显示这5个地区的毒株均属于Ⅰ型感染株,与韩国株的进化关系较为接近。本研究通过调查以上3种病毒病在江苏境内的流行情况,并进行序列分析,发现江苏不同地区的凡纳滨对虾IHHNV感染均为Ⅰ型,研究结果对养殖虾的疾病防控有重要参考价值。     

Growth and digestibility by three sizes of Penaeus vannamei Boone: Effects of dietary protein level and protein source

Growth, survival and digestibility of Penaeus vannamei were studied in three experiments. P. vannamei averaging 4.0, 9.8 and 20.8 g were monocultured in circular fiberglass tanks (2650 l) for 30 days. Six diets having protein contents of 22, 29 and 36% and an animal: plant protein source ratio of 2:1 or 1:1 were fed ad libitum. Survival ranges for small, medium and large shrimp were 81.7–93.3, 65.6–90.0 and 65.0–80.0%, respectively, and were unrelated to diet.Growth (g/day) decreased significantly with increased size of shrimp, whereas digestibility varied independently of size. Growth by all three sizes of shrimp fed the 2:1 diet series was not significantly (α = 0.05) higher than that of the 1:1 diet series.Digestibility ranges for protein, lipid and total diet were 78.6–85.8, 45.1–64.8 and 43.9–53.8%, respectively, for small shrimp; 80.7–84.5, 52.8–63.8 and 46.9–53.0%, respectively, for medium shrimp; and 78.7–85.4, 49.2–63.2 and 41.8–58.4%, respectively, for large shrimp. Protein digestibility by small shrimp was strongly correlated with dietary protein level. Only small shrimp showed a strong correlation between protein digestibility and growth. No size exhibited a relationship between growth and either lipid or total diet digestibility.Growth of small P. vannamei appeared to be more influenced by protein level where-as growth of medium and large P. vannamei appeared to be more influenced by protein source.     

基于营养通道模型的海州湾中国明对虾生态容纳量

通过增殖放流,增加优质渔业资源、改善种群结构是渔业资源养护的重要手段,而增殖生态容量的研究是科学实施增殖放流的前提。为确定海州湾中国明对虾的生态容纳量,根据2013年连云港海州湾渔业生态修复水域的调查资料,应用Ecopath with Ecosim(EwE)软件中的Ecopath模块,构建了该区域的生态系统能量流动简易模型,计算了放流种类中国明对虾的增殖生态容纳量。结果表明:系统各功能组营养级范围在1~4.42。系统总流量9335.191 t·km~(–2)·a~(–1),系统总初级生产力3892.630 t·km~(–2)·a~(–1),系统初级生产力与总呼吸量的比值为1.331,连接指数为0.415,杂食指数为0.174,Finn循环指数为11.4%,平均能流路径为2.8系统尚处于由衰竭状态向恢复状态转变,还未恢复到成熟态。中国明对虾不是本海域的关键种,当前中国明对虾的生物量为0.04 t·km–2·a–1,中国明对虾的生态容纳量为0.846 t·km~(–2)·a~(–1)。     

Retention efficiency and release of nutrients in the digestive tract of larval shrimp (Penaeus japonicus Bate) using different microencapsulated diets

Retention efficiency and release of the nutrients in the digestive tract of larval shrimp (Penaeus japonicus) using the diets microencapsulated with gelatin and ethyl cellulose respectively were evaluated. The microencapsulated diets were produced using the fluidized bed coating process. 7.8 % gelatin and 4.2 % ethyl cellulose to the whole microencapsulated diet were respectively adopted as coating material. After immersion in 35 ‰ NaCl solution for 1 h, the nitrogen retention efficiency was significantly lower in the diet microencapsulated with gelatin compared with the diet microencapsulated with ethyl cellulose. Free amino acid retention efficiency of the diet microencapsulated with gelatin and ethyl cellulose was 12.9, 17.2 %, respectively. The mysis II larval shrimp (P. japonicus) 10 days after hatch were fed two different microencapsulated diets for 20 days and grew significantly more than the control larval shrimp fed with Artemia and shrimp flake. The nutrient components in intermediate intestine of larval shrimp were increased gradually in the order of the control (50 % shrimp flake + 50 % Artemia), Group I (50 % diet microencapsulated with gelatin + 25 % shrimp flake +25 % Artemia), Group II (100 % diet microencapsulated with gelatin), and Group III (100 % diet microencapsulated with ethyl cellulose). This study confirmed that the microencapsulated diets with slow and controlled release characteristic in the digestive tract of the larval shrimp (P. japonicus).     

Water quality,phytoplankton composition and growth of Litopenaeus vannamei (Boone) in an integrated biofloc system with Gracilaria birdiae (Greville) and Gracilaria domingensis (Kützing)

A 28-day indoor trial was conducted to evaluate the water quality, phytoplankton composition and growth of Litopenaeus vannamei in an integrated biofloc system with Gracilaria birdiae and Gracilaria domingensis. The experimental design was completely randomized with three treatments: control (shrimp monoculture); SB (shrimp and G. birdiae) and SD (shrimp and G. domingensis), all with three replicates. Random sampling was done (6 % of total population per experimental unit) to confirm white spot syndrome Virus (WSSV) infection using nested-PCR analysis due to suspicion of presence of the virus in the experiment (treatment and control groups). Shrimp L. vannamei (2.63 ± 0.10 g) were stocked in experimental tanks at a density of 425 shrimp m^?3, and the Gracilaria was stocked at a biomass of 2.0 kg m^?3. Shrimp mortality began in both the experimental and control groups at 10 days of culture. The integrated biofloc system (shrimp and seaweed) increased settleable solids (by 26–52 %); final weight (by 6–21 %); weekly growth (by 17–43 %); weight gain (by 17–43 %); specific growth rate (by 16–36 %); and yield (by 5–7 %) and decreased feed conversion ratio (by 21–28 %) and Cyanobacteria density about 16 % as compared to the control (shrimp monoculture). The use of red seaweed Gracilaria in an integrated biofloc system can enhance shrimp growth and reduce Cyanobacteria density in the presence of WSSV.     

New type of heat shock protein 70 homologue gene abounds in the genomic sequence of kuruma shrimp Marsupenaeus japonicus

Heat shock proteins (HSPs) are a group of highly conserved molecular chaperones. Shrimp HSPs have recently been a topic of increasing interest because of their roles in shrimp immunity and homeostasis. In penaeid shrimp, HSP70s and the cognate forms, heat shock cognate (HSC) 70s, have been reported, but their responses towards various stimulations are different. We found a novel type HSP70 (MjHSP70-2) from the hyperexpansion of the large segmental duplication that is present in kuruma shrimp Marsupenaeus japonicus, which shows about 60 % identity with reported shrimp HSP70s. In a phylogenetic tree, MjHSP70-2 formed a sister clade with eukaryote HSP70 family while MjHSP70 was located close to the shrimp HSP70 and HSC70 group. MjHSP70-2 gene expression was not significantly increased by heat shock or pathogen challenge by Vibrio penaeicida, but it was significantly increased by infection with white spot syndrome virus. In contrast, MjHSP70 gene expression was increased by heat shock but decreased by infection with V. penaeicida. The kuruma shrimp genome was found to have 400-fold more copies of the MjHSP70-2 gene than the putative single-copy gene transglutaminase. In conclusion, our results reveal the presence of a novel type HSP70 gene, HSP70-2, from kuruma shrimp. There are multiple forms of HSP70 in crustaceans, and these HSP70s behave differently under various stressors.     

Water quality and growth of Pacific white shrimp Litopenaeus vannamei (Boone) in co-culture with green seaweed Ulva lactuca (Linaeus) in intensive system

An indoor trial was conducted for 28 days to evaluate the effects and interactions of biofloc and seaweed Ulva lactuca in water quality and growth of Pacific white shrimp Litopenaeus vannamei in intensive system. L. vannamei (4.54 ± 0.09 g) were stocked in experimental tanks at a density 132 shrimp m^?2 (566 shrimp m^?3) and the U. lactuca was stocked at a density 0.46 kg m^?2 (2.0 kg m^?3). Biofloc with seaweed (BF-S) significantly reduced (P < 0.05) total ammonia nitrogen (TAN) by 25.9 %, nitrite–nitrogen (NO₂–N) by 72.8 %, phosphate (PO ₄ ³ -P) by 24.6 %, and total suspended solids by 12.9 % in the water and significantly increased (P < 0.05) settleable solids by 34.2 % and final weight of shrimp by 6.9 % as compared to biofloc without seaweed. The BF-S can contribute by reducing nitrogen compounds (TAN and NO₂–N), phosphate (PO ₄ ³ -P), and total suspended solids in water and increased final weight of shrimp.     

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.     

Chemical composition and physical properties of salted shrimp paste (Kapi) produced in Thailand

Chemical composition and physical properties of 11 salted shrimp pastes (Kapi) obtained from various places of Thailand were determined. Based on proximate composition, protein constituted the major component (29.44–53.27 %, dry wt. basis). All samples contained 22.77–35.47 % NaCl with A _w of 0.695–0.774. Various formal nitrogen contents (11.96–22.87 mg N/g sample) were in agreement with different degrees of hydrolysis (12.68–20.76 %), suggesting the varying cleavage of peptides among the samples. From electrophoretic study, salted shrimp paste contained a large amount of small molecular weight proteins and peptides. Different samples had different colors with \( \Delta E^{*} \) of 47.10–60.43 and \( \Delta C^{*} \) of 9.46–20.76. The samples had total carotenoid content of 0.54–1.97 mg/g sample. Free astaxanthin, astaxanthin diester and canthaxanthin were the major carotenoids in salted shrimp paste. Thus, salted shrimp paste is a good source of protein and serves as the nutritious condiment.     

Substitution of fishmeal with soybean meal in practical diets for juvenile white shrimp Litopenaeus schmitti (Pérez‐Farfante & Kensley 1997)

The feasibility of substituting soybean meal for fishmeal diets for juvenile white shrimp Litopenaeus schmitti (0.35±0.01 g) was evaluated, and an adequate substitution level was determined. Five diets were evaluated using 46%, 59%, 75%, 88% and 100% substitution levels. Pellet water stability was significantly affected by dietary soybean content (P<0.05). Increased soybean content produced lower pellet stability, ranging from a dry matter loss of 14–22% after a 2‐h immersion, and 20–33% after an 8‐h immersion. After 52 days, significant differences (P<0.05) were found in shrimp weight, feed conversion ratio and protein efficiency ratio. The values were 0.64–1.06 g, 2.8–7.9 and 0.45–1.21, respectively, for the three measurements. Overall, better results were obtained with diets where soybean meal was substituted for fishmeal up to 75%. The 100% soybean meal diet resulted in poor growth performance of shrimp. Survival rates were acceptable for all treatments (90% or higher) and no significant differences were found in survival between treatments. Regression analysis using the broken‐line methodology indicated that 76.5±2% is an optimum soybean substitution level in diets that contained fishmeal and soybean as the major protein sources for grow‐out of juvenile white shrimp.     

Dietary β-Glucan and Nucleotide Effects on Growth,Survival and Immune Responses of Pacific White Shrimp,Litopenaeus vannamei

Effects of dietary supplementation of β-glucans and nucleotides on growth, survival and immune responses of the Pacific white shrimp (Litopenaeus vannamei) at a low salinity (5 ppt) were evaluated during a 30 d feeding trial. Final mean weight of shrimp fed nucleotides at 0.5%?was highest and significantly different from those fed the basal diet or diets supplemented with β-glucans. Survival was significantly higher for shrimp fed the diet with 0.2%?β-glucans compared to all other diets and was lowest for shrimp fed the basal diet. Shrimp fed diets containing β-glucans and nucleotides had generally better immune responses than shrimp fed the basal diet with higher total hemocyte count (THC) recorded for shrimp fed 0.2%?β-glucans followed by those fed 0.2%?nucleotides. Shrimp fed diets with 0.2%?and 0.5%?nucleotides and 0.2%?β-glucans had significantly higher respiratory burst values than shrimp fed the basal diet. These results indicated that dietary supplementation of either nucleotides or β-glucans has beneficial effects in improving shrimp performance when cultured at low salinity.     

Organization and promoter analysis of a tiger shrimp Penaeus monodon single WAP domain-containing protein gene

The main function of the single whey acidic protein domain (SWD)-containing protein in shrimp is unknown. To elucidate the function of the SWD-containing protein in vivo, the SWD-containing protein gene was isolated and characterized. A 9.3-kb shrimp SWD-containing protein gene, and a 3.9-kb 3′-flanking region. The shrimp SWD-containing protein gene contained three exons and two introns. Different fragments of the shrimp SWD-containing protein 5′-flanking region were transfected into HeLa cells. The promoter activities were assayed by basal human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone (LRH), and gonadotropin-releasing hormone (GnRH) treatments. The in vitro actions of the SWD-containing protein promoter expression pattern were studied by transfection of an SWD-containing protein promoter (1 kb)-driven green fluorescent protein (GFP) encoding the GFP cDNA transgene into the HeLa cell line, which was then microinjected into zebrafish Danio rerio embryos. These results indicate that the shrimp SWD-containing protein promoter might play an important role in gene regulation of sex hormones in mammalian cell lines and in gene regulation of developmental stages in zebrafish.     

Some effects of oral doses of oxytetracycline on growth,survival and disease in Penaeus aztecus

Two size groups of brown shrimp (Penaeus aztecus) were fed a formulated feed containing 0, 100, 1 000 or 5 000 mg of oxytetracycline/kg of feed. Growth and survival were measured after a 3-week drug consumption period. The shrimp were then inoculated with Vibrio alginolyticus, and survival was monitored during the following 24 h.At all three concentrations of oxytetracycline, small shrimp (mean initial wet weight 143.4 mg) consumed approximately one-third the amount of feed consumed by those fed the control diet with no oxytetracycline, yet growth was more rapid with diets containing 100 and 1 000 mg of oxytetracycline/kg of food than with the control diet. Larger shrimp (mean initial wet weight 458.1 mg) receiving oxytetracycline consumed about one-fourth the feed consumed by those on the oxytetracycline-free diet. Some growth inhibition was apparent in these shrimp at all oxytetracycline concentrations. Maximum drug consumption rate, based on actual feed intake, was approximately 1 300 mg oxytetracycline per kg body weight per day for small shrimp, but only 370 mg per kg body weight per day for larger shrimp.All shrimp fed 0, 100 or 1 000 mg of oxytetracycline/kg of feed died within 24 h following inoculation with a standard dose (70% light transmission at 520–540 nm) of Vibrio alginolyticus. All the small shrimp and 70% of the large shrimp fed at the 5 000-mg drug level died, but death generally took place later in the 24-h period than with those fed at the lower drug concentrations. All shrimp fed 5 000 mg oxytetracycline/kg of feed and inoculated with a 1 : 100 dilution of the standard dose of Vibrio alginolyticus survived. All small shrimp and 90% of the large shrimp survived injection of sterile saline.     

Replacement of fish meal in practical diets for the Pacific white shrimp (Litopenaeus vannamei) reared under pond conditions

Increasing economical and ecological concerns regarding the use of fish meal in diets for marine shrimp have led to the development of replacement strategies where soybean meal has received ample attention. Most studies evaluating these strategies have been carried out under laboratory conditions which greatly differ from production conditions in ponds. This study evaluated a fish meal replacement strategy using vegetable protein sources in practical feeds for marine shrimp reared in ponds. Juvenile Pacific white shrimp (Litopenaeus vannamei) (0.03 g) were stocked into 16 0.1-ha low-water exchange ponds and reared over an 18-week period. Four commercially extruded diets formulated to contain 35% crude protein and 8% lipids were evaluated. These diets included varying levels of fish meal (9, 6, 3, and 0%) which was replaced by a combination of increasing levels of soybean meal (32.5, 34.9, 37.2 and 39.6% respectively) and corn gluten meal (0.0, 1.7, 3.2, and 4.8% respectively) to replace the protein originating from fish meal. At the conclusion of the experimental period, there were no significant differences (P ≥ 0.05) in shrimp production among the test diets. Mean final yield, final weight, feed conversion ratio and survival values ranged from 5363–6548 kg ha^− 1, 18.4–20.7 g, 1.38–1.12 and 84.0–94.0%, respectively. Although not significant, as higher levels of plant protein sources were included in the diets, the economic analysis showed a general increase in the partial gross returns of shrimp production. Results from this study demonstrate that fish meal can be completely replaced using alternative vegetable protein sources in practical shrimp feeds without compromising production and economic performance of L. vannamei reared in ponds.