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1.
In this study, dsRNA specific to VP28 gene of white spot syndrome virus (WSSV) of shrimp was synthesized in Escherichia coli in large scale and studied the immune response of shrimp to dsRNA‐VP28. The haematological parameters such as clotting time and total haemocytes counts, and immunological parameters such as prophenoloxidase (proPO), superoxide dismutase (SOD), superoxide anion (SOA) and malondialdehyde content, as well as the mRNA expression of ten immune‐related genes were examined to estimate the effect of dsRNA‐VP28 on the innate immunity of Litopenaeus vannamei. The activities of proPO, SOA and SOD significantly increased in haemocyte after dsRNA‐VP28 treatment, whereas MDA content did not change significantly. Among the ten immune‐related genes examined, only the mRNA expression of proPO, cMnSOD, haemocyanin, crustin, BGBP, lipopolysaccharides (LPs), lectin and lysozyme in haemocytes, gill and hepatopancreas of L. vannamei, was significantly upregulated at 12 h after dsRNA‐VP28 treatment, while no significant expression changes were observed in Toll receptor and tumour receptor genes. The increase of proPO and SOD activities, and SOA level and mRNA expression level of proPO, cMnSOD, haemocyanin, crustin, BGBP, LPs, lectin and lysozyme after dsRNA‐VP28 stimulation indicate that these immune‐related genes were involved in dsRNA‐VP28‐induced innate immunity in shrimp.  相似文献   

2.
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3.
Penaeid shrimps are among the most studied crustaceans, mainly regarding their immune system, and several proteins involved in the defense against pathogens have been described. Haemocytes are very dynamic cells responsible of recognition, phagocytosis, degranulation and nodule formation, but still little is known about their gene expression. Using differential display, we found modification in haemocyte gene expression after inoculation with an abiotic particle (DEAE‐Sephadex) or potential pathogenic bacteria (Vibrio alginolyticus). We also noticed that some of the newly expressed genes are exclusive from a specific treatment. Here we report that haemocytes from Litopenaeus vannamei are capable of recognizing and distinguishing different foreign materials, and respond specifically to each treatment, indicating some specificity in shrimp immune response.  相似文献   

4.
The capacity of reactive oxygen intermediates production upon haemocyte stimulation is one of the most important immunoparameter utilized to assess the health status in cultivated shrimps. In the present study, we compared oxidative stress potential, by measuring the superoxide anion production in three penaeid shrimps: two wild Atlantic species, the pink shrimp Farfantepenaeus paulensis and the white shrimp Litopenaeus schmitti and one cultivated Pacific species, the white shrimp, Litopenaeus vannamei, through the nitro‐blue‐tetrazolium‐reduction assay. We also proposed an optimized experimental protocol for this assay, that produces rapid and consistent results with low levels of basal superoxide anion (O2?) production by unstimulated haemocytes and high levels of this oxygen radical after cell stimulation. Among the different cell elicitors used (zymosan, laminarin, lipopolysaccharide and phorbol myristate acetate), laminarin (β‐1,3‐glucans – 2 mg mL?1) was the most potent cell activator for the haemocytes of all three penaeids and we recommend this immunostimulant to routinely evaluate shrimp respiratory burst. In general terms, the most elevated levels of O2? production, after cell stimulation with microbial components, were detected in L. schmitti. Interestingly, the stimulation profile of the haemocytes of L. vannamei was more similar to F. paulensis, than to L. schmitti, which is more phylogenetically related.  相似文献   

5.
The purpose of this study was to investigate the effects of inbreeding on the immune responses and antioxidant status of Portunus trituberculatus juveniles. Results showed that inbreeding affected the total haemocyte count, and phagocytic, pro‐phenoloxidase (propo), phenoloxidase and antibacterial activities decreased after the seventh generation. Antioxidant status showed a similar pattern: total antioxidant capacity, superoxide dismutase (SOD) activity and GSH/GSSG in the cell‐free haemolymph and hepatopancreas decreased, while catalase activity in the cell‐free haemolymph increased (P < 0.05). There were no significant differences in α2‐macroglobulin and bacteriolytic activities in the cell‐free haemolymph and glutathione peroxidase activity in the cell‐free haemolymph and hepatopancreas among nine inbreeding generations. Gene expression levels of proPO and crustin in haemocytes and SOD in haemocytes and hepatopancreas also decreased significantly as the inbreeding generations increased. The results suggest that a high level of inbreeding could severely reduce the physiological health of P. trituberculatus. Our obtained data would be particularly useful for P. trituberculatus breeding programmes.  相似文献   

6.
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7.
The crustacean haemolymph contains three main cell populations; however, it is not clear which mechanisms participate in the regulation of cells related to innate immunity. This work aimed to identify potential interleukin‐like receptors that could regulate cellular responses in Cherax quadricarinatus. By histochemical analysis with murine anti‐CD25 staining (targeting the α‐chain of the IL‐2 receptor), we identified that this antibody recognizes cytoplasmic granules in semigranular and granular haemocytes. In haemocytes stimulated with phorbol myristate acetate (PMA), increased fluorescence was observed in these cytoplasmic granules, whereas staining with a human IL‐2 antibody after stimulation with 1–10 ng/ml PMA revealed no overexpression of the receptor or oxidative burst in haemocytes. Two‐dimensional Western blot analysis of haemocyte lysates showed that anti‐CD25 identified a 27.4‐kDa protein with an isoelectric point (pI) of 7.7 and a 46‐kDa protein with a pI of 6.9. De novo sequencing of these proteins identified that they had 32% homology with a mannose‐binding lectin (MBL) from Pacifastacus leniusculus. Our results indicate that a mannose‐binding lectin‐like protein could exert a protective effect that prevents damage from other activated immune responses.  相似文献   

8.
Sixteen candidate disease‐resistant parameters were selected through which to evaluate the acute hepatopancreatic necrosis disease (AHPND)‐resistant capability of Exopalaemon carinicauda after three generations of selection for AHPND‐causing strain of Vibrio parahaemolyticus (VPAHPND) resistance in our previous study. However, these parameters required further verification. In this study, another AHPND‐resistant E. carinicauda series was obtained through a short‐term selection procedure, consisting of three virulent challenge rounds of selection (about three‐week interval for each challenge) with VPAHPND infection. After this selection, the survival rate at 144 hr post infection (hpi) increased from 23.33% to 37.78% and the observed 48‐hr LD50 of VPAHPND to shrimp increased from 105.5 cfu/ml to 106.5 cfu/ml. Then, the immune response of this AHPND‐resistant E. carinicauda was studied using the 16 candidate AHPND‐resistant parameters selected for in our previous study. The improved VPAHPND clearance rate in hpi, increased total haemocyte counts, haemocyanin concentration, alkaline phosphatase activity and expressions of six immune‐related genes (Tollip and ALF in haemocytes and hepatopancreas; lysozyme, crustin and cathepsin B in hepatopancreas; and LGBP in haemocytes) at 24 hpi after the three‐round challenge selection suggest that these immune parameters may be reliable markers for the evaluation of the physiological status and potential AHPND‐resistant phenotypes in E. carinicauda.  相似文献   

9.
At present the most common and most devastating disease of shrimp is caused by the white spot syndrome virus (WSSV), which has spread throughout the world mainly by different species of crustaceans carrying the virus. After experimental injection of Penaeus monodon with a known copy number of WSSV in the abdominal muscle, the rate of viral progression in different tissues at 12, 24, 36 and 48 hpi (hours post infection) was assessed using quantitative real‐time PCR. At 12 hpi the viral load was highest in haemocytes followed by pleopod, muscle and gills whereas at 48 hpi, the gills, the main target of WSSV, showed the highest viral load followed by pleopod, muscle and haemocytes. Viral copy number in the haemocytes was the lowest beyond 12 hpi indicating a remarkable reduction in the rate of viral replication in haemocytes compared with other tissues. The viral load in haemocytes, though increased again beyond 36 hpi, never surpassed the load in the other tissues. The real‐time PCR assay with its high sensitivity and wide dynamic range make it ideal for detecting low‐level WSSV infections that can occur in apparently healthy P. monodon.  相似文献   

10.
Rapid in vitro methods for measuring digestibility may be useful in analysing aqua feeds if the extent and limits of their application are clearly defined. The pH‐stat protein digestibility routine with shrimp hepatopancreas enzymes was previously related to apparent protein digestibility with juvenile Litopenaeus vannamei fed diets containing different protein ingredients. The potential of the method to predict culture performance of shrimp fed six commercial feeds (T3, T4, T5, T6, T7 and T8) with 350 g kg?1 declared crude‐protein content was assessed. The consistency of results obtained using hepatopancreas enzyme extracts from either pond or clear water‐raised shrimp was further verified in terms of reproducibility and possible diet history effects upon in vitro outputs. Shrimps were previously acclimated and then maintained over 56 days (initial mean weight 3.28 g) on each diet in 500‐L tanks at 114 ind m?2, clear water closed system with continuous renewal and mechanical filtering (50 μm), with four replicates per treatment. Feeds were offered four times daily (six days a week) delivered in trays at feeding rates ranging from 4.0% to 7.0% of stocked shrimp biomass. Feed was accessible to shrimp 4 h daily for 1‐h feeding period after which uneaten feed was recovered. Growth and survival were determined every 14 days from a sample of 16 individuals per tank. Water quality was monitored daily (pH, temperature and salinity) and managed by water back flushing filter cleaning every 7–10 days. Feeds were analysed for crude protein, gross energy, amino acids and pepsin digestibility. In vitro pH‐stat degree of protein hydrolysis (DH%) was determined for each feed using hepatopancreas enzyme extracts from experimental (clear water) or pond‐raised shrimp. Feeds resulted in significant differences in shrimp performance (P < 0.05) as seen by the differences in growth rates (0.56–0.98 g week?1), final weight and feed conversion ratio (FCR). Shrimp performance and in vitro DH% with pond‐raised shrimp enzymes showed significant correlation (P < 0.05) for yield (R2 = 0.72), growth rates (R2 = 0.72–0.80) and FCR (R2 = ?0.67). Other feed attributes (protein : energy ratio, amino acids, true protein, non‐protein nitrogen contents and in vitro pepsin digestibility) showed none or limited correlation with shrimp culture performance. Additional correlations were found between growth rates and methionine (R2 = 0.73), FCR and histidine (R2 = ?0.60), and DH% and methionine or methionine+cystine feed contents (R2 = 0.67–0.92). pH‐stat assays with shrimp enzymes generated reproducible DH% results with either pond (CV ≤ 6.5%) or clear water (CV ≤ 8.5%) hepatopancreas enzyme sources. Moreover, correlations between shrimp growth rates and feed DH% were significant regardless of the enzyme origin (pond or clear water‐raised shrimp) and showed consistent R2 values. Results suggest the feasibility of using standardized hepatopancreas enzyme extracts for in vitro protein digestibility.  相似文献   

11.
Diseases of shrimp have contributed to billions of dollars of economic loss in the aquaculture industry. Newly emerging strains of the bacterium Vibrio parahaemolyticus produce a condition in shrimp called early mortality syndrome or acute hepatopancreatic necrosis disease. Three different V. parahaemolyticus strains were evaluated for their respective pathogenicity on shrimp, Litopenaeus vannamei, when the bacterial strains were grown under various laboratory conditions prior to inoculating shrimp. For each trial, feed was inoculated with a known concentration of bacteria and then fed to the shrimp. The early mortality syndrome strain of V. parahaemolyticus was the most lethal resulting up to 100% mortality within 24 h after being introduced to shrimp via a single feeding. The other two strains of Vibrio, one isolated from the environment and the other from a human clinical case, resulted in 0% and 30% mortality within 96 h respectively. The concentration of the early mortality syndrome strain of V. parahaemolyticus that the shrimp were exposed to directly correlated with mortality rate, which allowed for lethal or sublethal short‐term disease challenge assays to be established. Infiltration of haemocytes was also evident in the midgut caeca of shrimp infected with the early mortality syndrome strain of V. parahaemolyticus, which has not been previously reported.  相似文献   

12.
The effects of salinity and temperature on plasma protein concentration and total haemocytic prophenoloxidase (proPO) were determined. Groups of 10 juvenile yellowleg shrimp, Penaeus californiensis Holmes, were acclimated for 20 days at different salinities (28%o, 32%o, 36%o, 40%o and 44%o at 25 °C) or temperatures (18, 22, 25, 28 and 32 °C at 36%o). While total protein levels were not affected, the quantities of proPO increased as salinity was elevated. Temperature affected both haemolymph parameters, showing a significant decrease in proPO at 32 °C, and an increase of protein at 28 and 32 °C. These results may help to explain the elevated disease susceptibility of shrimp at higher salinities and/or temperatures.  相似文献   

13.
Apoptosis is proposed to be a major cause of death in shrimp viral infections. From our previous study, an apoptosis-related gene, Pm-Alix, was identified from the black tiger shrimp. Its expression was high in defence-related tissues including haemocytes and the lymphoid organ. To clarify its possible role in shrimp, we used Pm-Alix as bait in a yeast two-hybrid analysis to search for Alix interacting proteins in shrimp. Two cDNA sequences discovered had homology to a predicted ubiquitin C of the purple sea urchin, Strongylocentrotus purpuratus, and to a guanylyl cyclase of the red swamp crayfish, Procambarus clarkii. In vitro pull-down assays confirmed positive interaction between Pm-Alix and both proteins. Tissue distribution analysis revealed that Pm-Alix and the two binding partners were widely expressed in various tissues but more highly expressed in haemocytes. However, no significant positive or negative correlation was found in the expression of these genes as shrimp approached morbidity and death after challenge with white spot syndrome virus. Thus, the results suggested that Alix and its interacting partners did not play a direct role related to shrimp death.  相似文献   

14.
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15.
The gene coding for translationally controlled tumour protein (TCTP) was polymerase chain reaction amplified from haemocyte cDNA of Indian shrimp, Penaeus indicus, and sequenced. The N‐terminal region, a conserved one among all the TCTPs, was shown to have one substitution at position 37, in the Indian isolate. Besides this, there were two substitutions in the C‐terminal region (135, 149), exclusive to the Indian isolate. Phylogenetic analysis suggested a close relatedness of TCTP from P. indicus to Fenneropenaeus chinensis compared with other isolates. Translationally controlled tumour protein gene expression was found to be elevated in the haemocytes of WSSV‐infected shrimps compared with the uninfected ones. However, tissues from the infected shrimps did not exhibit any detectable levels of TCTP expression.  相似文献   

16.
The haemocytes of the Indian edible oyster Crassostrea madrasensis were characterized using light and electron microscopy. The light microscopic study was conducted by staining a monolayer of the haemocytes with Geimsa. Cells without granules and with a large nucleus occupying much of the cytoplasmic area were grouped as hyalinocytes. Those with lesser amounts of basophilic cytoplasmic granules were characterized as semigranulocytes and those with large amounts of a mixture of acidophilic and basophilic granules were termed as granulocytes. Ultrastructural studies also revealed the presence of three types of haemocytes. Scanning electron microscopic studies were used to study the spreading behaviour of the haemocytes. Cytochemical studies revealed the presence of acidphosphatase, peroxidase and prophenol oxidase in the cells.  相似文献   

17.
栉孔扇贝血淋巴中酚氧化酶和髓过氧物酶活性↑（*）   总被引：17，自引：1，他引：16  
用电镜细胞化学和分光光度技术对栉孔扇贝血淋巴中的氧化酶（ＰＯ）和髓过氧物酶（ＭＰＯ）进行了研究。结果表明：细胞质中直径分别为３００～４３０ｎｍ和１３０～２００ｎｍ的大、小２种颗粒呈ＰＯ强阳性,次级溶酶体和小颗粒呈ＭＰＯ强阳性。生化血清中存在ＰＯ和ＭＰＯ活性,血细胞仅有ＭＰＯ活性。以ＴＭＢ为底物测得血清中不依赖卤素的ＭＰＯ活力为１０７．２Ｕ,以二乙醇胺为底物测得血清中依赖卤素的ＴＭＢ为底物测得血清中  相似文献   

18.
Phagocytosis is an important function of both invertebrate and vertebrate blood cells. In this study, the phagocytic activity of haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone), against pathogenic and non‐pathogenic particles was investigated in vitro. The haemocytes of penaeid shrimp were firstly separated by centrifugation on a continuous density gradient of iodixanol into four fractions with five subpopulations (sub), of which sub 1 (hyalinocytes) and sub 4 (semi‐granulocytes) have the main function in phagocytosis of both pathogenic and non‐pathogenic bacteria as well as fluorescent polystyrene beads. It was found that these haemocyte subpopulations engulfed virulent Vibrio campbellii and Vibrio harveyi at a higher rate than non‐virulent Escherichia coli and polystyrene beads. When these bacteria were mixed with shrimp haemocyte subpopulations and incubated for 180 min, the percentage of viable intracellular V. campbellii (25.5 ± 6.0%) recovered was significantly higher than the percentage recovered from V. harveyi (13.5 ± 1.1%). No viable intracellular E. coli was observed in this study. In contrast to V. harveyi and E. coli, V. campbellii containing endosomes did not acidify in time. Incubation of haemocyte subpopulations with the most virulent V. campbellii strain resulted in a significant drop in haemocyte viability (41.4 ± 6.3% in sub 1 and 30.2 ± 15.1% in sub 4) after 180 min post‐inoculation in comparison with the less virulent V. harveyi (84.1 ± 5.6% in sub 1 and 83.4 ± 4.1% in sub 4) and non‐virulent E. coli (92.7 ± 2.8% in sub 1 and 92.3 ± 5.6% in sub 4) and polystyrene beads (91.9 ± 1.6% in sub 1 and 84.4 ± 3.4% in sub 4). These findings may be a valuable tool for monitoring shrimp health and immunological studies.  相似文献   

19.
Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)‐killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV‐killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV‐killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV‐killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60 min post‐inoculation) and that afterwards the capsid started to become disintegrated (from 360 min post‐inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV‐killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular‐sensing molecules.  相似文献   

20.
The bioflocs technology (BFT) for shrimp production has been proposed as a sustainable practice capable of reducing environmental impacts and preventing pathogen introduction. The microbial community associated with BFT not only detoxifies nutrients, but also can improve feed utilization and animal growth. Biofloc system contains abundant number of bacteria of which cell wall consists of various components such as bacterial lipopolysaccharide, peptidoglycan and β‐1, 3‐glucans, and is known as stimulating nonspecific immune activity of shrimp. Bioflocs, therefore, are assumed to enhance shrimp immunity because they consume the bioflocs as additional food source. Although there are benefits for having an in situ microbial community in BFT systems, better understanding on these microorganisms, in particular molecular level, is needed. A fourteen‐day culture trial was conducted with postlarvae of Litopenaeus vannamei in the presence and absence of bioflocs. To determine mRNA expression levels of shrimp, we selected six genes (prophenoloxidase1, prophenoloxidase2, prophenoloxidase activation enzyme, serine proteinase1, masquerade‐like proteinase, and ras‐related nuclear protein) which are involved in a series of responses known as the prophenoloxidase (proPO) cascade, one of the major innate immune responses in crustaceans. Significant differences in shrimp survival and final body weights were found between the clear water and in the biofloc treatments. mRNA expression levels were significantly higher in the biofloc treatment than the clear water control. These results suggest that the presence of bioflocs in the culture medium gives positive effect on growth and immune‐related genes expression in L.vannamei postlarvae.  相似文献   

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