Hepatopancreas is the extraovarian site of vitellogenin synthesis in black tiger shrimp, Penaeus monodon.

The site of yolk protein synthesis in crustaceans has long been a subject of controversy. The vitellogenin gene structure was partially reported only very recently in Macrobrachium rosenbergii, after which the hepatopancreas was confirmed as the extraovarian site of vitellogenin synthesis in that species. Ovaries are the most frequently reported as the site of yolk protein synthesis in penaeid shrimp. Using cDNA reversed-transcribed from mRNA isolated from the hepatopancreas of vitellogenic female shrimp, Penaeus monodon, we found that its deduced amino acid sequence had high identity of 48% with that from M. rosenbergii vitellogenin. A similar location of the intron in the sequenced region of genomic DNA was also found between these two species. We therefore concluded that the hepatopancreas the extraovarian site of vitellogenin synthesis in P. monodon in vivo. The partial structure of vitellogenin gene is presented in this study.     

News & Notes: Virulence of Vibrio alginolyticus Isolated from Diseased Tiger Prawn,Penaeus monodon

Outbreaks of mass mortality among cultured tiger prawns (Penaeus monodon) with white spotted syndrome (WSS) in the carapace occurred in the summer of 1994 in I-Lan, Taiwan. A swarming strain Val was isolated from hemolymph of the moribund prawns with tryptic soy agar (TSA, supplemented with 1% NaCl, Oxoid) and/or thiosulfate citrate bile salt sucrose (TCBS, Difco) agar. This strain was characterized and identified to be Vibrio alginolyticus. The strain was susceptible to antibiotics such as chloramphenicol, ciprofloxacin, doxycycline hydrochloride, nalidixic acid, oxolinic acid, and oxytetracycline while resistant to ampicillin, novobiocin, penicillin G, sulfisoxazole, and sulfonamide. The bacteria and their extracellular products (ECP) were lethal to both tiger prawns (P. monodon) and kuruma prawns (P. japonicus) with LD₅₀ values of 1.13 × 10⁵, 2.46 × 10⁵ CFU/g, and 0.23, 0.63 μg protein/g prawn body weight, respectively.     

Folate Transport by Prawn Hepatopancreas Brush-Border Membrane Vesicles

The transport system of folic acid (Pte-Glu) by brush-border membrane vesicles (BBMV) isolated from prawn (Penaeus japonicm) hepatopancreas, was studied by measuring the uptake of Pte-Glu. This uptake was found to have two components, intravesicular transport and membrane binding. Membrane binding was not affected by the presence of a transmembrane pH-gradient at a short incubation period. However, a transmembrane pH-gradient increased membrane binding at 60 min. The transport of Pte-Glu appeared to be carrier-mediated, was stimulated by an inwardly proton gradient (pH 5.5 outside, 7.4 inside) and was unaffected by a sodium-gradient. The relationship between pH gradient-driven Pte-Glu uptake and medium Pte-Glu concentration followed saturating Michaelis–Menten kinetics. Eadie–Hofstee representation of the pH gradient-driven Pte-Glu uptake indicated a single transport system with a Km of 0.37 M and Vmax of 1.06 pmol/mg protein/15 s. These findings indicate that BBMV isolated from prawn hepatopancreas possesses a Pte-Glu transport system similar to that described in mammalian intestine.     

克毒丹防治斑节对虾Penaeus monodon Fabricius苗种病毒的试验

全球气候异常,“埃厄尔诺”现象的频频发生,在一定程度上造成生态不平衡,至使１９９３年世界范围虾病的大流行,而我国尤为严重,产量大幅度下降。在斑节对虾ＰｅｎａｅｕｓｍｏｎｏｄｏｎＦａｂｒｉｃｉｕｓ育苗期间,发生虾病毒而引起苗种大量死亡的现象,使用抗生素仍无法控制其死亡。使用克毒丹０２×１０－６～０６×１０－６浸洒苗池,以便改变其微生态环境,又相间使用虾丹特号药物。结果苗种成活率比对照组平均提高１１％以上,从而达到控制病毒的试验效果。     

Molecular Characterization of a cDNA Encoding Vitellogenin and Its Expression in the Hepatopancreas and Ovary during Vitellogenesis in the Kuruma Prawn, Penaeus japonicus

In Crustacea, reproductive function and mechanisms regulating vitellogenesis have not been fully elucidated. This is due in great part to a lack of information concerning the biochemical nature of the vitellogenin molecule, the hemolymph precursor of yolk protein, vitellin, as well as the functional expression of the vitellogenin-encoding gene. We have therefore cloned a cDNA encoding vitellogenin in the kuruma prawn, Penaeus japonicus based on the N-terminal amino acid sequence of the 91 kDa subunit of vitellin. The open reading frame of this cDNA encoded 2,587 amino acid residues. This is the first investigation reporting a full-length cDNA and its corresponding amino acid sequence for vitellogenin in any crustacean species.Northern blot analysis and in situ hybridization have revealed that mRNA encoding vitellogenin was expressed in both the follicle cells in the ovary and the parenchymal cells in the hepatopancreas. In nonvitellogenic females, vitellogenin mRNA levels were negligible in both the ovary and hepatopancreas, but in vitellogenic females, levels were dramatically increased in both tissues. In the ovary, highest levels were observed during the early exogenous vitellogenic stage, and thereafter rapidly decreased, whereas in the hepatopancreas, high levels were maintained until the onset of the late vitellogenic stage. Differing profiles of vitellogenin mRNA levels in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenin synthesis harbor separate and complementary roles during vitellogenesis.     

Dopamine depresses immunity in the tiger shrimp Penaeus monodon

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, phagocytic activity and clearance efficiency to the pathogen Photobacterium damsela were measured when tiger shrimp Penaeus monodon (13.5+/-1.5 g) were individually injected with saline or dopamine at 10(-8), 10(-7), or 10(-6)mol shrimp(-1). Results showed that a transient period of immunosuppression occurred between 2 and 8h after injection of dopamine for all immune parameters except circulating haemocytes, and all immune parameters had returned to control values within 8-16 h after receiving dopamine. The injection of dopamine also significantly increased the mortality of P. monodon challenged with the pathogen Pho. damsela. These results suggest that stress-inducing dopamine suppresses the immune system, which in turn promotes the susceptibility of P. monodon to Pho. damsela.     

Ontogenic change of digestive enzymes in Penaeus monodon

• 1.1. Ontogenic changes of both proteases and carbohydrases of Penaeus monodon from different larva stages to adult were investigated.
• 2.2. Total protease activity was low during nauplius and zoea but peaked up in mysis. This was due to the activity increase of both trypsin and chymotrypsin.
• 3.3. The change of isozyme pattern of these two enzymes from different life stages of the shrimp was further determined by functional staining on an electrophoregram.
• 4.4. Activity of α-amylase increased after the post-larva stage, while that of chitinase and maltase showed a peak in zoea then gradually decreased to adult.
• 5.5. The ratio of α-amylase activity to protease coincided with the dietary change of the shrimp in different life stage.

     

Diseases of the eye of farmed shrimp Penaeus monodon

Lesions were found in the eyes of cultured shrimp Penaeus monodon that displayed non-specific signs of disease, including lethargy, dark pigmentation, brown gills, empty midgut, anorexia, white tail muscle, necrosis of uropods and fouled cuticle. Eye lesions were associated with sexual development in moribund shrimp in at least 1 disease event. Suppurative inflammation, granuloma and malacia were observed in histological examination of the eye and the causative agents of lesions appear to be Vibrio spp. and a rod-shaped virus (similar to Lymphoid Organ Virus, Gill-Associated Virus [GAV] and Yellow-Head Virus). Suppurative inflammation was characterised by edema, infiltration of haemocytes and local sites of abscesses. Eyes with granuloma usually appeared white in pond-side examinations, and histology showed that fibrous tissue replaced ommatidia, ganglia and internal structures of the eye. Malacia of the eye was characterised by necrosis of nervous tissue, vacuolation and vascular proliferation in the medulla ganglia. Levels of presumptive Vibrionaceace were high in moribund specimens and Gram-negative rods were observed in some specimens as free particles in the interstitial fluid and haemolymph in the eye. Transmission electron microscopy showed that nerve cells in the fasciculated zone (near the basement membrane) contained cytoplasmic vesicles (1 to 3 microm in diameter) with particles (15 to 26 nm in diameter) and rod-shaped nucleocapsids. The rods were similar to those of GAV and were 130 to 260 nm long, 10 to 16 nm in diameter and had helical symmetry with a screw-like thread (2.4 to 3.5 nm pitch). Also, unidentified enveloped virions, averaging 74 nm in diameter, were observed in cytoplasmic vesicles in the fasciculated zone. In conclusion, it is suggested that bacterial and viral infections of the eye could result in impaired neuroendocrine functions, which may cause a range of clinical signs of disease.     

Haemocyte reactions in WSSV immersion infected Penaeus monodon

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture worldwide in the past decades. In this study, WSSV infection (by immersion) and behaviour recruitment of haemocytes is investigated in gills and midgut, using an antiserum against the viral protein VP28 and a monoclonal antibody recognising haemocytes (WSH8) in a double immunohistochemical staining and in addition transmission electron microscopy was applied. More WSH 8(+) haemocytes were detected at 48 and 72 h post-infection in the gills of infected shrimp compared to uninfected animals. Haemocytes in the gills and midgut were not associated with VP28-immunoreactivity. In the gills many other cells showed virus replication in their nuclei, while infected nuclei in the gut cells were rare. Nevertheless, the epithelial cells in the midgut showed a clear uptake of VP28 and accumulation in supranuclear vacuoles (SNV) at 8h post-infection. However, epithelial nuclei were never VP28-immunoreactive and electron microscopy study suggests degradation of viral-like particles in the SNV. In contrast to the gills, the midgut connective tissue shows a clear increase in degranulation of haemocytes, resulting in the appearance of WSH8-immunoreactive thread-like material at 48 and 72 h post-infection. These results indicate recruitment of haemocytes upon immersion infection in the gills and degranulation of haemocytes in less infected organs, like the midgut.     

Anti-CHH antibody causes impaired hyperglycemia in Penaeus monodon

Crustacean hyperglycemic hormone (CHH) plays a major role in controlling glucose level in the haemolymph and also triggers important events during molting and reproductive cycles. In Penaeus monodon, three types of CHH, namely Pem-CHH1, Pem-CHH2 and Pem-CHH3, have been previously characterized. In this study, mouse polyclonal antibody was raised against recombinant Pem-CHH1 that was expressed in Escherichia coli. The anti-Pem-CHH1 antibody recognized all three types of Pem-CHHs but did not cross-react with either related hormone, molt-inhibiting hormone of P. monodon, or unrelated human growth hormone. The hyperglycemic activity in the extract from the eyestalk neural tissues was significantly depleted after incubating with anti-Pem-CHH antibody. Direct injection of the antibody into shrimp caused about 30-50% reduction in the haemolymph glucose level. The result demonstrates the ability of anti-Pem-CHH1 antibody to deplete the activity of CHH in vivo, and thus provides a possibility of using anti-Pem-CHH1 antibody to inhibit the hormone activity as a strategy to modulate growth and reproduction in this species.     

The midgut chymotrypsins of shrimps (Penaeus monodon, Penaeus japonicus and Penaeus penicillatus).

The midgut chymotrypsins (EC 3.4.4.5) of three species of shrimps, Penaeus monodon, Penaeus japonicus and Penaeus penicillatus were purified and studied in detail to clarify previous ambiguity in their identification. In each of the species there are two major forms of chymotrypsin, both single-chained with three disulfide bonds. One has a pI of 3.2 and Mr 27,000 or 28,000, while the other has a pI of 3.0 and Mr 25,000 or 26,000. The N-terminal amino acid sequences of the P. monodon enzymes are homologous to those of the crab (Uca pugilator) collagenase and to the other chymotrypsins. However, the active sites of the shrimp chymotrypsins are different from that of the well studied bovine alpha-chymotrypsin in some respects: (1) in spite of showing the typical specificity of chymotrypsin, the shrimp enzymes are more stringently selective for substrates with extended polypeptide chain; (2) some titration agents of alpha-chymotrypsin, including t-cinnamoylimidazole, 4-nitrophenyl guanidinobenzoate and its fluorescent derivative, do not react with the shrimp enzymes, neither do some of the alpha-chymotrypsin inhibitors: Tosyl-PheCH2Cl, methyl-4-nitrobenzenesulfonate and benzeneboronic acid; (3) the shrimp chymotrypsins are more reactive than the bovine enzyme toward native protein substrates including collagen; (4) the kinetic-salt-effects of the shrimp enzyme toward N-succinyl- and acetyl-Ala-Ala-Pro-Phe-4-nitroanilide mainly reflect electrostatic rather than hydrophobic interactions between the substrates and the enzyme. The shrimp enzymes are acid-labile but resistent to autolysis. Our results suggest that most Crustacea decapods contain chymotrypsins as one of the major digestive endopeptidases.     

斑节对虾杆状病毒包涵体研究

利用显微图像分析技术,对斑节对虾肝胰腺正常细胞胞核和斑节对虾杆状病毒(MBV)感染细胞胞核横切面的直径、周长和面积进行了测量分析.结果表明:正常细胞胞核的直径为3.47±0.30μm、周长为13.03±1.36μm、面积为10.87±1.78μm²;MBV感染细胞胞核的直径为3.81±0.79μm、周长为14.00±2.87μm、面积为13.52±5.37μm²;两者的直径、周长和面积均存在极显著的差异(P<0.01).对3种类型的包涵体进行了测量,其中1类包涵体最大,2类包涵体次之,3类包涵体最小,并以1类包涵体占大多数.     

Genetic Diversity and Geographic Differentiation of the Giant Tiger Shrimp (Penaeus monodon) in Thailand Analyzed by Mitochondrial COI Sequences

Genetic diversity and geographic differentiation of the giant tiger shrimp, Penaeus monodon, in Thai waters (Satun, Trang, Phangnga, and Ranong in the Andaman Sea and Chumphon and Trat in the Gulf of Thailand) were examined by COI polymorphism (N = 128). We observed 28 COI mitotypes across all investigated individuals. The sequence divergence between pairs of mitotypes was 0.00–20.76%. A neighbor-joining tree clearly indicated lineage separation of Thai P. monodon and large nucleotide divergence between interlineage mitotypes but limited divergence between intralineage mitotypes. High genetic diversity was found (mean sequence divergence = 6.604%, haplotype diversity = 0.716–0.927, π = 2.936–8.532%). F-statistics (F _ST) and an analysis of molecular variance (AMOVA) indicated that the gene pool of Thai P. monodon was not homogeneous but genetically differentiated intraspecifically (P < 0.05). Six samples of P. monodon could be allocated into three different genetic populations: Trat (A), Chumphon (B), and the Andaman samples Satun, Trang, Phangnga, and Ranong (C). Contradictory results regarding patterns of geographic differentiation previously reported by various molecular approaches were clarified by this study.     

Quaternary structure and functional properties of Penaeus monodon hemocyanin

The hemocyanin of the tiger shrimp, Penaeus monodon, was investigated with respect to stability and oxygen binding. While hexamers occur as a major component, dodecamers and traces of higher aggregates are also found. Both the hexamers and dodecamers were found to be extremely stable against dissociation at high pH, independently of the presence of calcium ions, in contrast to the known crustacean hemocyanins. This could be caused by only a few additional noncovalent interactions between amino acids located at the subunit-subunit interfaces. Based on X-ray structures and sequence alignments of related hemocyanins, the particular amino acids are identified. At all pH values, the p50 and Bohr coefficients of the hexamers are twice as high as those of dodecamers. While the oxygen binding of hexamers from crustaceans can normally be described by a simple two-state model, an additional conformational state is needed to describe the oxygen-binding behaviour of Penaeus monodon hemocyanin within the pH range of 7.0 to 8.5. The dodecamers bind oxygen according to the nested Monod-Whyman-Changeaux (MWC) model, as observed for the same aggregation states of other hemocyanins. The oxygen-binding properties of both the hexameric and dodecameric hemocyanins guarantee an efficient supply of the animal with oxygen, with respect to the ratio between their concentrations. It seems that under normoxic conditions, hexamers play the major role. Under hypoxic conditions, the hexamers are expected not to be completely loaded with oxygen. Here, the dodecamers are supposed to be responsible for the oxygen supply.     

Mitochondrial DNA Diversity in Three Populations of the Giant Tiger Shrimp Penaeus monodon

Mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) was utilized for determination of genetic variation and population structure in Penaeus monodon collected from Satun (the Andaman Sea) and Surat and Trat (the Gulf of Thailand). Twenty-eight composite haplotypes were generated from 52 restriction profiles of P. monodon mtDNA digested with 11 restriction endonucleases. The size of the entire P. monodon mitochondrial genome was estimated to be 15.913 ± 0.177 kb. The average haplotype diversity in P. monodon was 0.864, whereas the mean nucleotide diversity within populations was 2.51%, 2.22%, and 1.91% for Satun, Trat, and Surat, respectively. Geographic heterogeneity analysis indicated population differentiation between P. monodon from the Andaman Sea and P. monodon from the Gulf of Thailand (p < .0001). On the basis of the high genetic diversity level of P. monodon in Thailand, the Satun and Trat P. monodon populations from the west and east of the pennisula were selected to be founder stocks in our selective breeding program. Received February 23, 1998; accepted September 30, 1998.