Shrimp Intestine Research Articles

Dual oxidases participate in the regulation of intestinal microbiotic homeostasis in the kuruma shrimp Marsupenaeus japonicus

The metazoan gut lumen harbors numerous microbial communities. Tolerance for high bacterial counts and maintenance of microbiota homeostasis remain insufficiently studied. In this study, we identified a novel dual oxidase (MjDUOX2) involved in reactive oxygen species (ROS) production in the kuruma shrimp Marsupenaeus japonicus. MjDUOX2 is a transmembrane protein with an N-signal peptide region (19 aa) and a peroxidase homology domain (PHD, 554 aa) in the extracellular region; seven transmembrane regions; and three EF (calcium-binding region) domains (110 aa), a FAD-binding domain (104 aa), and a NAD-binding domain (156 aa) in the intracellular region. The novel MjDUOX2 exhibits a relatively low similarity (26.84% identity) to a previously reported DUOX in the shrimp (designated as MjDUOX1). The mRNA of MjDUOXs was widely distributed in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine. Oral infection of the shrimp with pathogenic bacteria upregulated the mRNA expression of MjDUOXs and increased the ROS level in the intestine. However, High ROS level could inhibit the expression of MjDUOXs in shrimp after Vibrio anguillarum infection. Knockdown of MjDUOXs by RNA interference (RNAi) decreased the ROS level, increased the bacterial count in the intestine, and decreased the survival rate of the MjDUOX-RNAi shrimp infected with V. anguillarum. These results suggest that MjDUOXs play an important role for microbiota homeostasis in intestine of shrimp.

Effect of an Indigenous Probiotic (<i>Shewanella algae</i>) Isolated from Healthy Shrimp (<i>Penaeus monodon</i>) Intestine on <i>Clarias gariepinus</i>

The efficacy of Shewanella algae , isolated from healthy shrimp ( Penaeus monodon ) intestine, as a biocontrol agent against Aeromonas hydrophila infection in Clarias gariepinus was evaluated. Spread plated technique was employed for bacterial isolation. The bacterial isolates were screened for in vitro antimicrobial activity against pathogenic Aeromonas hydrophila using agar well diffusion assay. The most active isolate (B2) was identified using phenotypic and molecular methods. The safety of the indigenous probiotic (B2) and the pathogenicity of the Aeromonas hydrophila on Clarias gariepinus were investigated. Feeding experiment was designed to evaluate the in vivo antibacterial efficiency of the most active strain using three groups of fish ( Shewanella algae - supplemented feed group, control infected and control non-infected groups). At the end of the experiment, five fish from each group were randomly selected for haematological and serum biochemical response investigation. The parameters analysed were haemoglobin, packed cell volume (PCV), red blood cell (RBC), white blood cell (WBC), lymphocytes, monocytes, total protein and albumin. A total of four genera belonging to Pseudomonas, Shewanella, Vibrio and Proteus which were isolated from shrimp intestine had antagonistic ability against Aeromonas hydrophila with zone of inhibition of 8.0 ± 0.0 mm, 10.0 ± 0.0 mm, 8.0 ± 0.0 mm and 8.0 ± 0.0 mm respectively. Molecular analysis conducted on the most active isolate (B2) revealed that it is closely related to Shewanella algae strain KJ-W32 gi: 385880930. The virulence test carried out on Clarias gariepinus using Aeromonas hydrophila which was injected intraperitoneally, showed 50% lethal dose value at 6.4 x 104 cfu ml-1. The result from the probiotic trial showed that fish fed with incorporated Shewanella algae strain KJ-W32 exhibited better resistance to pathogenic Aeromonas hydrophila than the control infected fish. The mortality rate during the in vivo challenge experiments observed for Shewanella algae - supplemented feed group, control infected and control non -infected fish groups were 0%, 50% and 5% respectively. Fish fed with supplemented diet showed better haematological and serum biochemical performance than those fed with normal fish feed. The study showed that Shewanella algae strain KJ-W32 is beneficial to Clarias gariepinus . Therefore, this indigenous bacterium could be used as effective biocontrol agent for management of aeromonasis in aquaculture.

Studies on an Indigenous Probiotic (<i>Shewanella algae</i>) Isolated from Healthy Shrimp Intestine

An indigenous probiotic ( Shewanella algae ) previously isolated from healthy shrimp ( Penaeus monodon ) intestine and found to protect Clarias gariepinus from Aeromonas hydrophila infection was screened for antibiotic susceptibility, enzymatic activities and abiotic stress tolerance. Optimization of the cultivation conditions and identification of its bioactive compounds were also carried out. The antibiotic susceptibility was performed by disk diffusion method. A total of fifteen antibiotic discs were employed. Qualitative screening for extracellular enzyme-producing ability was done using skim milk agar, starch agar, egg yolk agar, gelatin agar and cellulose agar for protease, amylase, lipase, gelatinase and cellulase activities respectively. The optimal conditions for growth of the isolate were assessed by growing on nutrient broth at various pH, temperature and salinity levels. Cell growth was estimated by standard plate count. The probiotic was screened for antimicrobial activity by agar well diffusion assay against the pathogen ( Aeromonas hydrophila ) using cell-free supernatant (crude extract) and methanolic extract. The methanolic extract was analyzed using gas chromatography- mass spectrometry (GC-MS). The isolate showed a wide range of environmental tolerance with pH (4-11), temperature (28-40℃) and salinity (0-100 ppt). The optimum conditions for cell growth were pH 6-8, temperature 37℃ and salinity 5-15 ppt. The isolate was susceptible to all tested antibiotics which supported the ideal probiotic characteristic. The isolate was capable of producing extracellular enzymes such as protease, amylase, lipase and gelatinase which could improve feed digestibility and feed utilization. The crude and methanolic extracts were active against the pathogen with inhibition zones of 13.0 ± 0.02 mm and 18.0 ± 0.03 mm respectively. Two bioactive compounds (Tromethamine and Pyrrolo [1, 2-a] pyrazine-1, 4-dione, hexahydro-) were identified. These results suggest that the Shewanella algae could probably be used as a potential probiotic in aquafeed formulation and a good source of novel drug development.

颗石藻Pleurochrysis carterae抗捕食特征

PDF HTML阅读 XML下载 导出引用 引用提醒 颗石藻Pleurochrysis carterae抗捕食特征 DOI: 10.5846/stxb201401170133 作者: 作者单位: 宁波大学海洋学院,宁波大学海洋学院,宁波大学海洋学院,宁波大学海洋学院 作者简介: 通讯作者: 中图分类号: 基金项目: 浙江省自然科学基金(LY12D06001);教育部博士点优先发展领域课题(20133305130001);浙江省重大科技创新团队(2011R10029);浙江省重点基金(Z3100565) Characterization of anti-predator abilities of Pleurochrysis carterae Author: Affiliation: Marine Biology Institute of Ningbo University,,Marine Biology Institute of Ningbo University, Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:颗石藻Pleurochrysis carterae是沿海水域中常见钙化微藻,易形成高密度水华,也是养殖环境致害种之一。抗捕食防御能力可能是其种群增殖优势的一个重要原因。以卤虫作为捕食者,分析了颗石藻P.carterae抗捕食现象,以及在捕食压力下的重要生理生化响应特征,以期为颗石藻P. carterea抗捕食机制研究及其高密度增殖机理提供参考。研究结果显示:(1)当颗石藻P.carterae比例增加时,卤虫对微藻的摄食率显著降低,且存活率显著下降,显示该藻具抗捕食能力。(2)以卤虫饵料微藻球等鞭金藻(Isochrysis galbana)为对照,比较研究发现,相同的捕食压力下,饵料金藻的叶绿素荧光参数(电子传递速率ETR和最大量子产率Fv/Fm)显著降低,但颗石藻P.carterae的ETR和Fv/Fm没有显著变化,显示颗石藻P.carterae对卤虫抗捕食作用。(3)相对于没有捕食压力的对照组,捕食压力下,饵料金藻I.galbana的脂类组成没有显著差异。但是,颗石藻P.carterae的脂类组成则发生了显著变化,主要表现在对细胞叶绿体有重要作用的单半乳糖甘油二酯(MGDG),双半乳糖甘油二酯(DGDG),磷脂酰甘油二酯(PG)含量上升,与促细胞分裂相关的二酰甘油(DAG)和磷脂酰肌醇(PI)也上升。这些脂类代谢物的变化可能在其种群水平上抵抗捕食并实现种群增殖中发挥作用。(4)培养介质中磷的状态对颗石藻P.carterae细胞二甲基巯基丙酸(Dimethyl sulfonio propionate, DMSP)含量有显著影响,且影响颗石藻P.carterae对卤虫的致害效应:缺磷条件下生长的颗石藻P. carterae首先使卤虫受害。当培养液中仅以ATP为磷源时,颗石藻P. carterae的卤虫致害效应则降低。研究证明,颗石藻P.carterae具有抗捕食能力,细胞的脂类代谢物质以及DMSP可能在抗捕食防御中发挥作用。 Abstract:The coccolithophorid species Pleurochrysis carterae is a common coastal calcifying phytoplankton, which is also able to form dense blooms that harm marine aquaculture. The anti-predator ability of P. carterae is supposed to be one reason why it is a dominant species. In this study, this hypothesis is tested by using brine shrimp Artemia salina as the model predator. Another Prymnesiophyceae species, Isochrysis galbana, which is considered as the benign live food source for A. salina, was used as prey. Changes to the grazing rate or survival rate of A. salina were analyzed when P. carterae was present in the prey mixture. Algal chlorophyll fluorescence efficiency and lipid changes in these two algal species under predation pressure were also studied. Some very interesting P. carterae features were observed, which may provide insights into its anti-predator abilities. These were as follows:(1) When the P. carterae cell numbers increases in the microalgal prey mixture, the brine shrimp A. salina has a lower predation capacity, which is shown by the gradient decrease of chlorophyll content in the brine shrimp intestines. The survival rate is also lower as the number of P. carterae cells increase. Therefore, these results clearly indicated the anti-predator ability of P. carterae.(2) In comparison, the chlorophyll fluorescence(ETR and Fv/Fm) for the two microalgae facing the same predation pressure decreased significantly from 0.75 to 0.60 for Fv/Fm, and from 88% to 65% for ETR, respectively, for I. galbana, but were almost unchanged for P. carterae. This means that photosynthesis of P.carterae is virtually unaffected by the presence of the predator. Such unconstrained ability against the predator was very prominent.(3) Lipidomic analyses, with without the predator, between the two microalgal species indicated that I. galbana did not show any significant changes, whereas P. carterae did significant increase its monogalactosyl diacylglycerol(MGDG), digalactosyldiacylglycerol(DGDG), phosphatidylglycerol(PG), diacylglycerol(DAG), and phosphatidylinositol(PI) contents. Since MGDG, DGDG, and PG are the major components of the chloroplast membrane, the increase in these compounds can have protective effects on the microalgal cell, and both DAG and PI are considered important signals for cell division, so their increase can stimulate the population growth. Hence, the lipid changes clearly provide the biochemical evidence for the physiologically positive changes to P. carterae, in the presence of the predator.(4) Dimethyl sulfoniopro pionate(DMSP) is supposed to contribute in the anti-predator abilities of microalgae. We further studied whether the mortality of A. salina was affected by the algal cellular DMSP content. Cultures containing different cellular DMSP contents were set up by adjusting the phosphorus chemical states in the f/2 culture medium, namely phosphate deficiency, normal f/2, and f/2 medium with ATP as the phosphate supplier. The results showed that, the ambient phosphorus chemical states significantly affect the intracellular DMSP level in P. carterae. After three days of exponential growth, the phosphate deficiency group increased DMSP from 2.35 to 2.42 mg/g lyophilized cells, whereas the normal f/2 medium group reduced DMSP from 2.35 to 2.26 mg/g lyophilized cells, and the ATP enriched group showed the largest decrease in DMSP from 2.35 to 1.90 mg/g lyophilized cells. This indicated that the phosphorus level and chemical state had negative effects on the accumulation of DMSP. Furthermore,, the phosphorus state can affect the preying behavior and mortality of A.salina. Phosphorus deficiency in the culture medium can increase the algal cell's fatal effects on the predator, while ATP, as the sole phosphorus source, may decrease the fatal effects. We conclude that P. carterae contains positive biochemical mechanisms that underlie its anti-predator abilities, possibly through cell membrane component protective reconstruction and DMSP metabolism. Its active anti-preying abilities mean that P. carterae can reach a sustainable population size at the ecological level. 参考文献 相似文献 引证文献

Bacterial dynamics in intestines of the black tiger shrimp and the Pacific white shrimp during Vibrio harveyi exposure

The intestinal microbiota play important roles in health of their host, contributing to maintaining the balance and resilience against pathogen. To investigate effects of pathogen to intestinal microbiota, the bacterial dynamics upon a shrimp pathogen, Vibrio harveyi, exposures were determined in two economically important shrimp species; the black tiger shrimp (BT) and the Pacific white shrimp (PW). Both shrimp species were reared under the same diet and environmental conditions. Shrimp survival rates after the V. harveyi exposure revealed that the PW shrimp had a higher resistance to the pathogen than the BT shrimp. The intestinal bacterial profiles were determined by denaturing gradient gel electrophoresis (DGGE) and barcoded pyrosequencing of the 16S rRNA sequences under no pathogen challenge control and under pathogenic V. harveyi challenge. The DGGE profiles showed that the presence of V. harveyi altered the intestinal bacterial patterns in comparison to the control in BT and PW intestines. This implies that bacterial balance in shrimp intestines was disrupted in the presence of V. harveyi. The barcoded pyrosequencing analysis showed the similar bacterial community structures in intestines of BT and PW shrimp under a normal condition. However, during the time course exposure to V. harveyi, the relative abundance of bacteria belong to Vibrio genus was higher in the BT intestines at 12h after the exposure, whereas relative abundance of vibrios was more stable in PW intestines. The principle coordinates analysis based on weighted-UniFrac analysis showed that intestinal bacterial population in the BT shrimp lost their ability to restore their bacterial balance during the 72-h period of exposure to the pathogen, while the PW shrimp were able to reestablish their bacterial population to resemble those seen in the unexposed control group. This observation of bacterial disruption might correlate to different mortality rates observed between the two shrimp species. Our findings provide evidence of intestinal bacterial population altered by a presence of the pathogen in shrimp intestines and intestinal bacterial stability might provide colonization resistance against the invading pathogen in the host shrimp. Hence, intestinal microbial ecology management may potentially contribute to disease prevention in aquaculture.

Crustacean cardioactive peptide (CCAP) of the Pacific white shrimp (Litopenaeus vannamei): Molecular characterization and its potential roles in osmoregulation and freshwater tolerance

Osmoregulation is one of the most fundamental and delicately controlled processes in euryhaline marine species like penaeid shrimp. Here we first report a cDNA encoding for the crustacean cardioactive peptide (CCAP) precursor cloned from the intestine of Pacific white shrimp, Litopenaeus vannamei, a prominent commercial culture species. The CCAP mature peptide (PFCNAFTGC-NH2) is generated by post-translational processing and found to be highly comparable with its counterparts in insects and other crustaceans. Semi-quantitative RT-PCR reveals that L. vannamei CCAP (Lv-CCAP) mRNA is predominantly expressed in the brain and intestine. However, only the expression level of intestine-derived Lv-CCAP mRNA showed responsiveness to salinity changes in culture. The inducible expressed Lv-CCAP mRNA by transfer to high-salinity condition was continuous, while mRNA expression stimulated by transfer to low-salinity condition was transient. Functionally, the stimulatory effects of CCAP on heart rates in the shrimp cultured in low- and normal-salinity conditions were confirmed. Furthermore, injection of CCAP could up-regulate the freshwater tolerance of shrimp by increasing their survival rates. Our study, as a whole, provides new insights into hormone-regulated crustacean osmostasis and may prove instructive to the inland freshwater penaeid shrimp aquaculture. State of relevanceIt proves instructive to freshwater shrimp aquaculture.

Antibacterial activity of the methanolic extracts of Eclipta alba against bacteria in intestinal tract of black tiger shrimp (Penaeus monodon)

Antibacterial activity of the methanolic extracts of Eclipta alba were carried out by the disc diffusion method on 12 strains of bacteria isolated from 30 samples of tiger shrimp (intestines) collected from shrimp farms in Tan Thanh hamlet, An Loi commune, Tran Van Thoi district, Ca Mau province. All strains isolated were grown for 24h on TCBS agar plates at 30°C in yellow colony or green colony. Antibacterial activity assay of the extract was carried out in the concentration of 8 µg/mL, 16 µg/mL, 32 µg/mL, 64 µg/mL and 128 µg/mL. The extract of Eclipta alba were tested against 10 bacterial strains were isolated. The highest antibacterial potentiality was exhibited by the methanolic extract of E. alba with G5 strain, means of zones of bacterial growth inhibition are 30,3mm. Three of twelve isolates (Y1, G2, G5) were chosen to examine the biochemical characteristics with the API 20E Kit and identified by two method: Maldi toff mass and sequencing 16S rDNA using primers pair 27F (5¢-AGAGTTTGATCCTGGCTCAG-3¢) and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The results showed that Y1 isolate was similarity of 97% with E. Cloacae; G2 isolate was similarity of 98% with V. brasiliensis and G5 isolate was similartty of 99% with V. Parahaemolyticus.

Effects of dietary supplementation with β-glucan and synbiotics on immune gene expression and immune parameters under ammonia stress in Pacific white shrimp

Abstract The goal of this study was to investigate the effects of dietary supplementation with β-glucan and synbiotics (i.e., microencapsulated Bacillus subtilis and Pediococcus acidilactici ) on immune gene expression and immune parameters of Pacific white shrimp ( Litopenaeus vannamei ) as well as modulation of immune parameters when shrimp were exposed to ammonia stress. Shrimp were fed one of four treatment diets [basal diet (C), β-glucan-containing diet (β-glu), β-glucan plus B. subtilis -containing diet (β-glu + Bs ), and β-glucan plus P. acidilactici -containing diet (β-glu + Pa )] for 90 days. The microencapsulated bacteria colonized in the proximal and central parts of the shrimp intestine. The β-glu diet increased expression of lipopolysaccharide and β-1,3-glucan-binding protein ( LGBP ), and the β-glu + Bs diet induced expression of the serine protease and prophenoloxidase genes. In addition, β-glu + Pa increased the LGBP transcription. However, the mRNA level of peroxinectin did not differ significantly among the experimental groups. Shrimp fed the β-glu and β-glu + Pa diets had higher superoxide dismutase activity (SOD) compared to shrimp fed the basal diet. In addition, the β-glu + Bs and β-glu + Pa diets increased phenoloxidase activity (PO). However, there were no significant differences in total hemocyte count (THC) and lysozyme activity among the treatment diets. Ammonia stress led to increased THC and decreased SOD activity, but it seemed to have no adverse effects on PO and lysozyme activity. Thus, both β-glucan and synbiotic supplementation had beneficial effects on several immune parameters, and the effect of dietary synbiotics was superior to that of β-glucan only for PO activity.

Bdellovibrio and like organisms enhanced growth and survival of Penaeus monodon and altered bacterial community structures in its rearing water.

In this study, a 96-h laboratory reduction test was conducted with strain BDHSH06 (GenBank accession no. EF011103) as the test strain for Bdellovibrio and like organisms (BALOs) and 20 susceptible marine bacterial strains forming microcosms as the targets. The results showed that BDHSH06 reduced the levels of approximately 50% of prey bacterial strains within 96 h in the seawater microcosms. An 85-day black tiger shrimp (Penaeus monodon) rearing experiment was performed. The shrimp survival rate, body length, and weight in the test tanks were 48.1% ± 1.2%, 99.8 ± 10.0 mm, and 6.36 ± 1.50 g, respectively, which were values significantly (P < 0.05) higher than those for the control, viz., 31.0% ± 2.1%, 86.0 ± 11.1 mm, and 4.21 ± 1.56 g, respectively. With the addition of BDHSH06, total bacterial and Vibrio numbers were significantly reduced (P < 0.05) by 1.3 to 4.5 log CFU · ml(-1) and CFU · g(-1) in both water and shrimp intestines, respectively, compared to those in the control. The effect of BDHSH06 on bacterial community structures in the rearing water was also examined using PCR amplification of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE). The DGGE profiles of rearing water samples from the control and test tanks revealed that the amounts of 44% of the bacterial species were reduced when BDHSH06 was added to the rearing water over the 85-day rearing period, and among these, approximately 57.1% were nonculturable. The results of this study demonstrated that BDHSH06 can be used as a biocontrol/probiotic agent in P. monodon culture.

Quorum-quenching activity of the AHL-lactonase from Bacillus licheniformis DAHB1 inhibits Vibrio biofilm formation in vitro and reduces shrimp intestinal colonisation and mortality.

Vibrio parahaemolyticus is a significant cause of gastroenteritis resulting from the consumption of undercooked sea foods and often cause significant infections in shrimp aquaculture. Vibrio virulence is associated with biofilm formation and is regulated by N-acylated homoserine lactone (AHL)-mediated quorum sensing. In an attempt to reduce vibrio colonisation of shrimps and mortality, we screened native intestinal bacilli from Indian white shrimps (Fenneropenaeus indicus) for an isolate which showed biofilm-inhibitory activity (quorum quenching) against the pathogen V. parahaemolyticus DAHP1. The AHL-lactonase (AiiA) expressed by one of these, Bacillus licheniformis DAHB1, was characterised as having a broad-spectrum AHL substrate specificity and intrinsic resistance to the acid conditions of the shrimp intestine. Purified recombinant AiiA inhibited vibrio biofilm development in a cover slip assay and significantly attenuated infection and mortality in shrimps reared in a recirculation aquaculture system. Investigation of intestinal samples also showed that AiiA treatment also reduced vibrio viable counts and biofilm development as determined by confocal laser scanning microscopy (CLSM) imaging. These findings suggest that the B. licheniformis DAHB1 quorum-quenching AiiA might be developed for use as a prophylactic treatment to inhibit or reduce vibrio colonisation and mortality of shrimps in aquaculture.

The cellular community in the intestine of the shrimp Penaeus penicillatus and its culture environments

In the present study, bacterial community diversities were investigated in the shrimp intestines, outside seawater and culture-associated environments using clone library analysis. Comparative analyses revealed that the bacterial community in the intestinal contents was most similar to that of the sediment. Bacteria in the phyla Proteobacteria and Firmicutes constituted the dominant components of the intestinal bacteria in the shrimp. The potential pathogens and probiotics were dominant members of the intestine in healthy or diseased shrimp. Intestinal bacterial communities in the healthy shrimp were composed of Bacillales, Rhodobacterales, Vibrionales, and Alteromonadales, whereas fewer bacterial groups were observed in the intestines of diseased shrimp, which were dominated by Vibrionales and Bacillales. Phylogenetic analysis revealed that nearly half of the sequences in the intestines of diseased shrimp were closely related to the potential pathogen Photobacterium damselae and Vibrio harveyi. Obvious differences in microbial diversity were also observed between outside seawater and culture-associated environments. Eukaryotes in the outside seawater were dominated by Skeletonema, Cyclotella, Thalassiosira, Guinardia, and Chaetoceros. The pond water showed less diversity in the eukaryotic community and was dominated by Skeletonema, Thalassiosira, Chaetoceros, and Oithona. The implications of the present study may be significant in the management of shrimp cultivation, as well as the detection of potential pathogens and the application of probiotics.

Effect of calcium on amino acid and dipeptide transport by intestines of American lobster (Homarus americanus) and Atlantic white shrimp (Litopenaeus setiferus) (908.6)

Previous work using L‐histidine and zinc in American lobster intestine has shown these solutes can be transported as bis‐complexes (Histidine‐Zinc‐Histidine) across the intestine using a peptide transporter. Furthermore, transport of L‐leucine has been shown to be inhibited by high calcium concentrations. Dipeptide and bis‐complex transport and the role of calcium were investigated in the perfused intestine of lobster and Atlantic white shrimp. Samples of serosal medium were analyzed for amino acid composition by gas chromatography. In lobster, the transport of glycylsarcosine (Gly‐Sar) from mucosal to serosal medium was measured for a range of pH values and either 0.5mM or 25mM calcium chloride. The rate of Gly‐Sar transport was stimulated two‐fold with pH 8.5 in the lumen. Reduced calcium chloride concentration stimulated Gly‐Sar transport two‐fold. In shrimp, the transport of L‐histidine from mucosal to serosal medium was measured. The addition of 50µM zinc chloride increased the rate of histidine transport, while Gly‐Sar inhibited transport. The rate of histidine transport was significantly higher with 1mM calcium chloride than with 25mM. These results indicate that shrimp transport bis‐complexes in a manner similar to lobster, which is inhibited by the presence of a competing dipeptide. High calcium concentration has an inhibitory effect on both amino acid and dipeptide transport.Grant Funding Source: Supported by USDA Agricultural and Food Research Initiative Competitive Grant no. 2010‐65206‐20617

Characterization of Intestinal Bacteria in Wild and Domesticated Adult Black Tiger Shrimp (Penaeus monodon)

The black tiger shrimp (Penaeus monodon) is a marine crustacean of economic importance in the world market. To ensure sustainability of the shrimp industry, production capacity and disease outbreak prevention must be improved. Understanding healthy microbial balance inside the shrimp intestine can provide an initial step toward better farming practice and probiotic applications. In this study, we employed a barcode pyrosequencing analysis of V3-4 regions of 16S rRNA genes to examine intestinal bacteria communities in wild-caught and domesticated P. monodon broodstock. Shrimp faeces were removed from intestines prior to further analysis in attempt to identify mucosal bacterial population. Five phyla, Actinobacteria, Fusobacteria, Proteobacteria, Firmicutes and Bacteroidetes, were found in all shrimp from both wild and domesticated environments. The operational taxonomic unit (OTU) was assigned at 97% sequence identity, and our pyrosequencing results identified 18 OTUs commonly found in both groups. Sequences of the shared OTUs were similar to bacteria in three phyla, namely i) Proteobacteria (Vibrio, Photobacterium, Novosphingobium, Pseudomonas, Sphingomonas and Undibacterium), ii) Firmicutes (Fusibacter), and iii) Bacteroidetes (Cloacibacterium). The shared bacterial members in P. monodon from two different habitats provide evidence that the internal environments within the host shrimp also exerts selective pressure on bacterial members. Intestinal bacterial profiles were compared using denaturing gradient gel electrophoresis (DGGE). The sequences from DGGE bands were similar to those of Vibrio and Photobacterium in all shrimp, consistent with pyrosequencing results. This work provides the first comprehensive report on bacterial populations in the intestine of adult black tiger shrimp and reveals some similar bacterial members between the intestine of wild-caught and domesticated shrimp.

Isolation of Lactic Acid Bacteria from Kuruma Shrimp (Marsupenaeus japonicus) Intestine and Assessment of Immunomodulatory Role of a Selected Strain as Probiotic

Fifty-one lactic acid bacteria (LAB) strains were isolated and identified based on 16S ribosomal DNA sequence from the intestinal tracts of 142 kuruma shrimps (Marsupenaeus japonicus) collected from Kanmon Strait, Fukuoka and Tachibana Bay, Nagasaki, Japan. Cellular immunomodulatory function of 51 isolated LAB strains was assessed by measuring the level of interferon (IFN)-γ induction in mouse spleen cell culture. The strain Lactococcus lactis D1813 exhibited the highest amount of IFN-γ production and also bactericidal activity and was selected for testing its immunomodulatory role as a probiotic in kuruma shrimp. We also assessed the effect of dietary incorporation of this probiotic on resistance to Vibrio penaeicida infection in the kuruma shrimp. Our results demonstrate that probiotic L. lactis D1813-containing diet-fed (10⁵ cfu g⁻¹) shrimps displayed a significant up-regulation of lysozyme gene expressions in the intestine and hepatopancreas. However, insignificantly higher expression of anti-lipopolysaccharide factor, super oxide dismutase, prophenoloxidase, and toll-like receptor 1 was recorded in the intestine of shrimps fed the probiotic diet. Moreover, significantly increased (P < 0.01) resistance to the bacterial pathogen in term of better post-infection survival (61.7 %) was observed in the shrimps fed with the probiotic-incorporated diet compared with the control diet-fed group (28.3 %). The present study indicates the immunomodulatory role of the LAB L. lactis D1813 on the kuruma shrimp immune system and supports its potential use as an effective probiotic in shrimp aquaculture.

Effects of probiotics from the shrimp intestine on the non-specific immunity and antiviral capacity of Litopenaeus vannamei

We evaluated the effects of probiotics on the non-specific immunity and antiviral capacity of Lito-penaeus vannamei.We fed groups of shrimp(body weight 3.2 g±0.26 g) a diet supplemented with either 1.0×108 CFU/g Bacillus firmus(live) plus 1% Photobacterium damsela(inactivated),1.0×108 CFU/g Bacillus firmus(live),or 1.0×108 CFU/g Bacillus firmus(live) plus 1% Vibrio alginolyticus(inactivated).The control groups were fed a non-supplemented diet.Each diet was fed to triplicate groups of 60 shrimp that were reared in aquaria.We ran-domly selected 10 shrimp per group every 5 d and measured a range of humoral immune parameters(superoxide dismutase SOD,acidic phosphatase ACP,alkaline phosphatase AKP,nitric oxide synthase NOS and lysozyme UL) in each individual.The shrimp were challenged with white spot syndrome virus(WSSV) after two weeks.We then calculated the cumulative mortality of each experimental group.Supplementation with bacteria significantly in-creased serum SOD,ACP,AKP,and NOS compared with the controls.There was significantly lower cumulative mortality in shrimp that were fed the probiotics diet(10.7%,39.3% and 26.2%,respectively) compared to the control(64.28%).Our results suggest that supplementing the diet with shrimp intestine probiotics improves im-munity and the antiviral capacity of Litopenaeus vannamei.

Bacterial Population in Intestines of the Black Tiger Shrimp (Penaeus monodon) under Different Growth Stages

Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A total of 25,121 pyrosequencing reads (reading length = 442±24 bases) were obtained, which were categorized by barcode for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20 bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15 and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles. This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth development stages in rearing facilities.

F0ATP synthase b-chain of Litopenaeus vannamei involved in White Spot Syndrome Virus infection

White Spot Syndrome Virus (WSSV) is one of the most common and distrous diseases for shrimp. In this study, we show that the protein VP292 that is a envelop protein of WSVV interacts with F0ATP synthase b-chain from Litopenaeus vannamei using far-western blot, ELISA, and indirect immunofluorescence analysis. Tissue distribution analysis of F0ATP synthase b-chain showed that it's transcription can be detected in muscle, hepatopancreas, intestine, hemocytes, lymphoid, and gills. Cellular localization of F0ATP synthase b-chain in shrimp hemocytes showed that F0ATP synthase b-chain was primarily located in the cytoplasm of hemocytes. The transcription levels of F0ATP synthase b-chain were significantly upregulated in intestine, hepatopancreas, hemocytes, and gills of WSSV-infected shrimp at 12 h after infection. Far-western, ELISA, and indirect immunefluorescence assay indicated that F0ATP synthase b-chain interacted with VP292. In the in vivo neutralization experiment, F0ATP synthase b-chain attained 18% protection rate of the shrimp challenged by WSSV. To the best of our knowledge, this is the first report to show that F0ATP synthase b-chain is involved in WSSV infection.

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).

Isolasi Dan Karakterisasi Bakteri Asam Laktat Dari Usus Udang Penghasil Bakteriosin Sebagai Agen Antibakteria Pada Produk-Produk Hasil Perikanan

L actid Acid bacteria are capable of fermenting sugars or carbohydrates to produce lactic acid in large numbers. The characteristics of lactic acid bacteria in general are cells reacted positively to the Gram stain, catalase react negatively and do not form spores. And fermentation of glucose would result Iaktat acid. Isolation of lactic acid bacteria carried in the intestines of three species of shrimp that Tiger Shrimp (Penaeus monodon), shrimp Seker (Metapenaopsis sp.), Guts White shrimp (Penaeus merguensis). Selection of bacteriocin -producing lactic acid bacteria were in a qualitative and semi-quantitative (pitting) in MRS medium. Selected isolates were identified by morphological characteristics, biochemical, physiological, A number of 209 isolates were isolated from three species of shrimp. The results of the qualitative selection there were 54 isolates produced bacteriocins. The results of the quantitative selection of 54 isolates produced 24 fine isolates. From the 24 isolates there are 2 isolates seed SFE-7 (33) and P12A (25). Characteristics of seed isolates had morphological characteristics of spherical shape, cell arrangement tetrad, gram positive, negative motility. While the results of the biochemical approach to gram-negative, catalase negative, homofermentative (no gas in the fermentation of glucose), can ferment D-Galactose, D-Glucose, D-mannose, D-lactose. Physiological approaches to isolate growth SFE-7 (33) and P12A (25) can be generated that both isolates able to live in the range of 4 ° C to 50 ° C, in the range of pH between 4-10, and able to grow at levels of 5-10% NaCl . Based on these characteristics can be inferred featured isolates was Pediococcus acidilactici Key words : Lactid Acid Bacteria, intestine of shrimp, Pediococcus acidilactici

Effects of a yeast-based additive on growth and immune responses of white shrimp,Litopenaeus vannamei(Boone, 1931), and aquaculture environment

A 60 days feeding experiment was conducted to determine the effects of YK-6 (a yeast culture feed supplement) on growth and immune responses of white shrimp, Litopenaeus vannamei and aquaculture environment in commercial scale farms composed of nine ponds. Three treatments (control, Diet 1 and Diet 2) were designed to contain YK-6 levels of 0.0, 1.0 and 1.5 g kg−1 respectively. At the end of the test, the mean production of Diet 1 (0.649 ± 0.030 kg m−2) and Diet 2 (0.648 ± 0.033 kg m−2) were significantly higher than that of the control by 29.5% and 29.3% respectively. The feed conversion ratio decreased significantly by 12.3% (Diet 1) and 8.5% (Diet 2) compared with the control group respectively. The environmental indices indicate that the quality of water and pond sediment was improved. The endotoxin concentrations, the number of Vibrios and heterotrophic bacteria in the shrimp intestine of the treatment groups were significantly lower than that of the control, particularly in the later stage of the farming period. The YK-6 extended positive effects on growth, improved sediment quality, reduced endotoxin in shrimp intestine, and enhanced activities of lysozyme and phenoloxidase.