Sex-specific gene expression in distinct tissues of the shrimp Penaeus vannamei

• 1.1. Soluble proteins extracted from male and female Penaeus vannamei tissues such as eyes, eyestalks, brain, nerve cord, hemolymph, heart, muscle, hepatopancreas, hepatopancreas membrane and cuticular epidermis were analyzed and compared by high-resolution mini-two-dimensional polyacrylamide gel electrophoresis (mini-2D-PAGE).
• 2.2. In each shrimp tissue a large number of discrete polypeptides was observed.
• 3.3. The polypeptide patterns from the same tissue of female and male shrimp were mostly similar but both qualitative and quantitative differences were noted, suggesting the presence of sex-specific gene products in various shrimp tissues.
• 4.4. Future applications of these results are discussed.

     

脊尾白虾血蓝蛋白大亚基基因的克隆及表达分析

应用RACE技术克隆脊尾白虾血蓝蛋白大亚基基因, 并通过攻毒实验揭示脊尾白虾血蓝蛋白基因的先天免疫防御作用, 为脊尾白虾(Exopalaemon carinicauda)的免疫防治研究提供依据和思路。研究成功克隆了脊尾白虾血蓝蛋白大亚基基因全长cDNA序列, 该大亚基cDNA全长 2192 bp, 开放式阅读框长 2034 bp, 5′非编码区长 21 bp, 3′非编码区长 137 bp, 将该基因命名为 EcHcL。EcHcL编码 667 个氨基酸, 前 21 个氨基酸组成信号肽, 推测成熟肽的分子量为 78.5 kD。Blast比对结果显示, 由脊尾白虾血蓝蛋白EcHcL序列推导的氨基酸序列与日本沼虾、凡纳滨对虾血蓝蛋白氨基酸序列的同源性分别达到 87%、73%, 其M结构域氨基酸序列与斑节对虾、日本对虾等物种同源性性高达 90% 左右, 由此推断该cDNA序列属于血蓝蛋白家族。组织表达分析结果显示, EcHcL基因在脊尾白虾鳃、卵巢、肝胰腺、心脏、肠、肌肉、胃、腹神经节、眼柄、血细胞中均有表达, 肝胰腺中相对表达量最高。Real-time PCR分析发现EcHcL基因在金黄色葡萄球菌、副溶血弧菌和对虾白斑综合征病毒(WSSV)感染后脊尾白虾肝胰腺和血细胞中的表达量显著增加, 并具有不同的时空表达模式, 推测脊尾白虾EcHcL基因在免疫防御中具有重要作用。     

Molecular characterization of a Ran isoform gene up-regulated in shrimp immunity

Diseases caused by viruses are the greatest challenge to worldwide shrimp aquaculture. Ran gene was an important antiviral gene identified from shrimp and its mRNA level was up-regulated in response to viral infection. In this investigation, a Ran isoform gene (named Ran-iso) cDNA was cloned from shrimp, Marsupenaeus japonicus. The full-length cDNA of Ran-iso was 1286 bp, including a 5′-terminal untranslated region (UTR) of 272 bp, 3′-terminal UTR of 366 bp and an open reading frame (ORF) of 648 bp encoding a polypeptide of 215 amino acids. The deduced protein was highly homologous, it shared 90.64%, 84.19%, 81.48% and 67.58% identities with Ran protein of shrimp, honey bee, human and tobacco respectively. Ran-iso gene was constitutively expressed in 6 tissues examined, including gill, hepatopancreas, hemolymph, heart, intestine and muscle. However, Ran-iso was highest expressed in hepatopancreas (p < 0.01), whereas the expressions of other five tissues were equal and relatively low. Time course analysis showed that the expression level of Ran-iso was obviously up-regulated 2.8 times (at 6 h) as much as that in the control in the hepatopancreas challenged by WSSV. This investigation might provide a clue to elucidate the shrimp innate immunity and would be helpful to shrimp disease control.     

Superoxide radical production in response to environmental hypoxia in cultured shrimp

Markers of oxidative stress in response to hypoxia and reoxygenation were assessed in Pacific white shrimp (Litopenaeus vannamei). Adult shrimp were either exposed to hypoxia (1 mg O(2)/L) for 6, 12, or 24 h followed by 1-h reoxygenation, or exposed to hypoxia for 24 h followed by 1- to 6-h reoxygenation. In all cases, shrimp maintained at constant normoxia were used as controls. Spectrophotometric techniques were applied to analyze lactate concentration, superoxide radical (O(2)(*-)) production, lipid peroxidation (TBARS), and antioxidant capacity status in muscle, hepatopancreas, and gill samples. Results indicate differences among tissues, even under control conditions. O(2)(*-) production and TBARS levels were higher in hepatopancreas than in gill or muscle. No effect of exposure to hypoxia was found. However, reoxygenation following exposure to hypoxia was found to affect the oxidative metabolism of muscle and hepatopancreas from cultured shrimp. Lactate concentration and O(2)(*-) production increased while antioxidant capacity decreased in hepatopancreas and muscle in the first hours of reoxygenation. This could translate into tissue damage, which may significantly jeopardize the commercial aquaculture product.     

Clearing mechanisms of Vibrio vulnificus biotype I in the black tiger shrimp Penaeus monodon

Vibrio species' infections are a common sequelae to environmental stress or other disease processes in shrimp, but the mechanism by which the shrimp eliminate the bacteria is poorly understood. In this study, the penetration, fate and the clearing of V. vulnificus were investigated in Penaeus monodon. A bacterial disease isolate from a shrimp farm was identified as V. vulnificus biotype I. Polyclonal antiserum was raised in rabbits against the bacterium and the specificity was verified by ELISA and immunoblot against a range of Vibrio spp. and other gram-negative bacteria. The bacteria were then administered to P. monodon juveniles by injection, immersion and oral intubation. An indirect immunoperoxidase technique was employed in a time course study to follow the bacteria and bacterial antigens in the tissue of the shrimp. Bacteria were cleared by a common route, regardless of the method of administration. Observations in immersion challenge were similar to a combination of those for oral and injection challenges. With immersion, bacteria entered the shrimp through damaged cuticle or via insertion points of cuticular setae. Shortly after entry, whole bacterial cells were observed in the haemolymph and connective tissue. They were either phagocytosed by haemocytes, or broken down outside host cells. Haemocytes containing bacterial cells or antigens (HCB) were observed in the connective tissue and haemolymph. HCB accumulated around the hepatopancreas, midgut, midgut-caecum, gills, heart and lymphoid organ. Free bacterial antigens also accumulated in the heart and lymphoid organ. Bacteria entering through the mouth by oral intubation or immersion were broken down so that only soluble or very fine particles entered the hepatopancreas. Bacterial antigens passed through the hepatopancreas into the haemolymph. Antigens were initially observed in the haemolymph sinuses and subsequently accumulated in the heart and lymphoid organ. Bacterial antigens were released from the shrimp, initially through the gills and subsequently through hepatopancreatic B-cells, branchial podocytes and sub-cuticular podocytes.     

Histopathological and ultrastructural changes in the antennal gland,midgut, hepatopancreas,and gill of grass shrimp following exposure to hexavalent chromium

Grass shrimp, Palaemonetes pugio, were exposed for 1 month to subacute concentrations of hexavalent chromium (0.5, 1.0, 2.0, 4.0 ppm) after which the gills, midgut, hepatopancreas, and antennal glands were examined for histopathological and ultrastructural changes. Pathological changes were greatest in the antennal glands, followed by hepatopancreas, gills, and midgut. Severe changes occurred in some shrimp, even at 0.5 ppm chromium. Cells of all tissues frequently had both swollen mitochondria and rough endoplasmic reticulum. Small, spherical or ring-like intranuclear inclusions, possibly indicative of cellular hyperactivity or manifestions of chromium and/or protein complexes, were most prevalent in the hepatopancreas and antennal glands but also occurred in the midgut and gills. Other major degenerative changes in the antennal glands were restricted to the labyrinth and included diminution of basal plasmalemmal infoldings and cytoplasmic density, nuclear hypertrophy followed by widespread nuclear pyknosis and epithelial desquamation. In severely altered hepatopancreas hypertrophy was indicated for the basal laminae, nuclei, and possibly for the nucleoli. There was an apparent reduction in mitotic events and many observed mitotic nuclei were abnormal. Abnormal midgut hypertrophy was present in only 8 of 20 examined shrimp, exposed to 0.5 and 1 ppm chromium. Further, the gills of only 10 of the 40 examined chromium-exposed shrimp possessed abnormal features detectable with ligh microscopy. Ultrastructural analysis of the latter indicated an increase in lysosomes and a decrease in cytoplasmic density. In addition, there was a pronounced diminution in the degree of lamellar, subcuticular plasmalemmal infolding. This latter feature is postulated to be a mechanism for the regulation of chromium influx. Possible explanations for most observed alterations in the above tissues are proposed.     

The effect of dietary Panax ginseng polysaccharide extract on the immune responses in white shrimp, Litopenaeus vannamei

The immunostimulatory effects of orally administered Panax ginseng root or its polysaccharides (GSP) in white shrimp, Litopenaeus vannamei, were investigated in this study. Shrimp were fed a diet containing 0.4 g kg^?1 GSP over a period of 84 days, during which the activities of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), acid phosphatase (ACP), and alkaline phosphatase (AKP), as well as malondialdehyde (MDA) content, and expressions of cytosolic superoxide dismutase (cyt-SOD), CAT, GSH-Px, and peroxiredoxin (Prx) genes were determined in various tissues of the shrimp. Results showed that the shrimp fed the GSP diet had significantly increased ACP and AKP activities in the gills. The GSP-fed shrimp also displayed significantly increased T-SOD and GSH-Px activities in the gills and hepatopancreas of the shrimp; meanwhile there was enhanced CAT activity in the gills, but decreased MDA content in the gills, hepatopancreas and muscle. The mRNA expressions of cyt-SOD, CAT, GSH-Px and Prx were significantly elevated in the gills and hepatopancreas of the shrimp fed the GSP diet for 84 days, compared with that of the control. Therefore, GSP can be used as an immunostimulant for shrimp through dietary administration to increase immune enzyme activity and modify expression of immune genes in shrimp.     

Time course of necrotizing hepatopancreatitis (NHP) in experimentally infected Litopenaeus vannamei and quantification of NHP-bacterium using real-time PCR

Necrotizing hepatopancreatitis (NHP), a severe bacterial disease affecting penaeid shrimp aquaculture, is caused by a gram-negative, pleomorphic, intracellular alpha-proteobacterium referred to as the NHP-bacterium (NHPB). The time course of NHP was investigated in experimentally infected juveniles of Kona stock Litopenaeus vannamei. Susceptible animals were individually isolated in 41 of aerated artificial seawater at salinity 30 +/- 1 ppt and maintained in a water bath at 30 +/- 1 degree C for 60 d. A total of 120 individuals were exposed per os to a 0.05 g piece of NHPB-infected hepatopancreas and 100 controls were exposed to uninfected tissue. At intervals of 3, 6, 9, 16, 23, 30, 37, 44, and 53 d post-exposure, 6 shrimp exposed to NHPB-infected tissue and 4 controls were randomly removed from the experiment; hepatopancreas samples were processed for histological and molecular analysis, and feces were processed for molecular diagnosis of NHPB infection. NHPB was first detected in the hepatopancreas through histology at 6 d post-exposure. All control shrimp were diagnosed as NHPB negative. NHPB infections classified as stage I (scattering of hepatopancreatic tubules with adjacent epithelial cells containing NHPB) and stage II (numerous infected tubules with occasional hemocyte infiltration) were observed from 6 to 37 d post-exposure. All animals that experienced NHPB-induced mortality from 16 to 51 d post-exposure were at stage III (numerous necrotic tubules, dense hemocyte infiltration, and presence of granulomas). NHPB is capable of infecting all hepatopancreatic cell types including embryonic, resorptive, fibrillar and blister-like cells. The percent of hepatopancreatic tubules containing NHPB in epithelial cells increased over time, representing bacteria multiplication and spread. Real-time PCR allowed for quantification of NHPB in hepatopancreas and feces. Over the course of infection, NHPB was present at 10(3) to 10(7) copies mg(-1) of hepatopancreas and 10(1) to 10(5) copies mg(-1) of feces. Lethal infections contained 10(6) to 10(7) copies mg(-1) of hepatopancreas and 10(3) to 10(6) copies mg(-1) of feces.     

Loose shell syndrome of farmed Penaeus monodon in India is caused by a filterable agent

Loose shell syndrome (LSS) of farmed black tiger shrimp Penaeus monodon has been reported from Indian shrimp farms since 1998 and is recognized as a major disease problem causing significant economic loss to the shrimp aquaculture sector. Unlike the rapid mortalities associated with viral pathogens such as white spot syndrome virus and yellow head virus, progression of LSS is gradual, leading to low-level progressive mortalities. The signs of LSS include a flaccid spongy abdomen due to muscular dystrophy, space between the exoskeleton and muscle, and a shrunken hepatopancreas. The feed conversion efficiency is reduced, and shrimp have poor meat quality, caused by impairment of the hepatopancreatic functions such as digestion and absorption as evidenced by the atrophy of the hepatopancreas. Histopathological investigations on LSS-affected shrimp showed shrinkage of extensor and flexor muscles with occasional hemocytic infiltration. The hepatopancreas showed inflammation of hepatopancreatic tubules with enlargement of intertubular spaces, hemocytic infiltration, and low levels of lipid reserves in the R cells. In advanced stages of LSS, many tubules were in highly necrotic condition with a sloughed epithelium, reflecting the dysfunction of the digestive gland. LSS could be induced in healthy tiger shrimp by challenge studies using membrane-filtered LSS-affected shrimp tissues, suggesting involvement of a filterable infectious agent.     

The effects of arginine administration on the levels of arginine,other amino acids and related amino compounds in the plasma,heart, aorta,vena cava,bronchi and pancreas of the rat

Arginine (0.8g/kg, ip) or a vehicle was administered to rats and the levels of arginine and a large number of related amino compounds were measured in plasma, heart, aorta, vena cava, pancreas and bronchi at specified time intervals. Arginine levels (nmol/ml) increased in the plasma from 237 to 3172 at 15 min, 1236 at 30 min and 509 at 120 min. Peak concentrations (nmol/g) of arginine are reached in the tissues at 15 or 30 minutes with control and postinjection values of 500 and 1769 in the heart, 314 and 1563 in the aorta, 575 and 2976 in the vena cava, 760 and 1943 in the bronchi, and 234 and 3638 in the pancreas. Arginine injection also affects a number of amino acids and related compounds in the plasma and tissues most notably ornithine, isoleucine, phosphoserine, leucine and ethanolamine. However, plasma level changes do not predict tissue level changes which are highly specific for an individual compound and tissue. There was no general indication that arginine injection stimulates nitric oxide (NO) formation in any tissue. Thus, arginine is rapidly absorbed from the abdominal cavity into the blood stream, is quickly taken up by the tissues studied and disappears after about 2 to 3 hours. The effects seen after arginine administration could be caused by arginine per se and/or changes in one or more of the related amino compounds but not by NO.     

Ultrastructural and histological study of degenerative changes in the antennal glands, hepatopancreas, and midgut of grass shrimp exposed to two dithiocarbamate biocides

The histological and ultrastructural alterations observed in the antennal glands, hepatopancreas, and midgut of grass shrimp exposed to either a 50% potassium dimethyldithiocarbamate biocide (Busan-85; 5–60 ppb) for 14 days, or to a different biocide, composed of 15% sodium dimethyldithiocarbamate and 15% disodium ethylene bisdithiocarbamate (Aquatreat DNM-30), for 3–4 days (60–140 ppb) and 28–35 days (40–120 ppb), were compared and contrasted with the normal morphological features in control shrimp. Only those experimental shrimp that exhibited various degrees of branchial abnormality were examined. Although the alterations in Busan-exposed shrimp were generally more pronounced, the antennal glands of 32 out of 36 experimental shrimp exhibited abnormalities that were manifested primarily as increased secretory activity by the labyrinth cells. In dithiocarbamate-exposed shrimp with “black gills”, the labyrinth epithelium exhibited moderate nuclear hypertrophy, apparent cell sloughing, intense secretory activity, and occasional melanized lesions; alterations in the antennal gland coelomosac included nuclear pyknosis, a general deterioration of podocyte organization, and an unusual increase in hemolymph density adjacent to affected tissues. Although there was an apparent increase in mitotic activity in the hepatopancreatic tubules of shrimp exposed to Aquatreat for 28–35 days, degenerative changes were most frequent and extensive in the hepatopancreas and midgut of dithiocarbamate-exposed shrimp with “black gills”. These observed changes included the diminution of the basal midgut and hepatopancreatic tubular system, moderate midgut hypertrophy, pronounced activity by the hepatopancreatic fixed phagocytes, development of mitochondrial inclusions and megamitochondria, loss of cytoplasmic density, hepatopancreatic nuclear pyknosis, and irreversible degeneration of hepatopancreatic tubule apices. This study suggests that some of the observed abnormal/pathological changes are the indirect consequence of branchial degeneration. A number of possible defensive reactions to dithiocarbamate poisoning, including heterostasis, phagocytosis, encapsulation, and the possible participation of reserve inclusion cells are proposed.     

Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein

The gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (MrNV) was cloned into pGEX-6P-1 expression vector and then transformed into the Escherichia coli strain BL21. After induction, capsid protein-glutathione-S-transferase (GST-MrNV; 64 kDa) was produced. The recombinant protein was separated using SDS-PAGE, excised from the gel, electro-eluted and then used for immunization for monoclonal antibody (MAb) production. Four MAbs specific to the capsid protein were selected and could be used to detect natural MrNV infections in M. rosenbergii by dot blotting, Western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was 10 fmol μl-1 of the GST-MrNV, as determined using dot blotting. However, the sensitivity of the MAb on dot blotting with homogenate from naturally infected M. rosenbergii was approximately 200-fold lower than that of 1-step RT-PCR. Immunohistochemical analysis using these MAbs with infected shrimp tissues demonstrated staining in the muscles, nerve cord, gill, heart, loose connective tissue and inter-tubular tissue of the hepatopancreas. Although the positive reactions occurred in small focal areas, the immunoreactivity was clearly demonstrated. The MAbs targeted different epitopes of the capsid protein and will be used to develop a simple immunoassay strip test for rapid detection of MrNV.     

Metabolic responses of the white shrimp, Penaeus vannamei, to ambient ammonia

Juvenile shrimp were individually exposed during 24 h to 0.007 (control), 0.36, 1.07, and 2.14 mmol/l total ammonia-N at 28°C and 39 ppt salinity. After 22 h of ammonia-N exposure, oxygen consumption was measured for 2 h, and then hemolymph, hepatopancreas, and muscle tissues were sampled. Oxygen consumption, and levels of lactate and glycogen in the hepatopancreas increased significantly at the highest ammonia-N concentration. Concentration of oxyhemocyanin, acylglycerol, and cholesterol in hemolymph, and lactate in muscle decreased significantly in the group exposed to the highest ammonia levels. The changes observed in hemolymph and tissue metabolic fuels suggest a reduced use of carbohydrate through anaerobic metabolism and an increase in the use of lipids to satisfy the metabolic demand.