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.     

Spermatozoal ultrastructure of Penaeus kerathurus and Penaeus japonicus (Crustacea,Dendrobranchiata)

Summary An ultrastructural comparison between the unistellate spermatozoa of the shrimps Penaeus kerathurus and P. japonicus reveals a number of similarities that are common among dendrobranchiates, but also some marked differences which would confirm the validity of a potential use of sperm structure in systematic and phylogenetic studies. Typical morphological features shared by the spermatozoa of P. kerathurus and P. japonicus are: a membrane-bound acrosomal vesicle consisting of a cap and spike; non-membrane-bound filamentous chromatin; a perinuclear cytoplasmic band including degenerative membranous organelles (mostly mitochondria), small vesicles with a dense core and parallel membrane lamellae. Discordant spermatozoal characteristics between both species involve a significantly different size (ca. 5 m in length by ca. 3 m in diameter in P. kerathurus; ca. 8 m in length by ca. 4.7 m in diameter in P. japonicus), the occurrence of intranuclear lipid droplets only in P. kerathurus and the presence of a deeper subacrosomal space in this species as compared to P. japonicus. It is very likely that the most significant difference between both species is, however, the appearance of microtubule bundles in the spermatozoon of P. japonicus. So far, the occurrence of spermatozoal microtubules in decapod crustaceans appears to be restricted to reptantian species, whereby the finding of such elements in sperm of a dendrobranchiate shrimp could be of phylogenetic interest.     

Sequence and Conservation of a rRNA and tRNAVal Mitochondrial Gene Fragment from Penaeus californiensis and Comparison with Penaeus vannamei and Penaeus stylirostris

Penaeus californiensis is an important species for shrimp fisheries in the Pacific Ocean and has recently been described as a potential cultured species, mainly through the winter season in subtropical regions. A fragment of the mitochondrial 12S rRNA–tRNAVal–16S rRNA genes from P. californiensis was sequenced and compared with the corresponding regions from Penaeus vannamei and Penaeus stylirostris. Purified mitochondrial DNA was used for polymerase chain reaction amplification with primers for 12S and 16S rRNA genes. A 1379 ± 1-bp fragment was obtained, including 90% 16S rRNA, tRNAVal, and a portion of 12S rRNA, cloned, and sequenced. Genetic distances were calculated according to the Kimura 2-parameter distance model, and maximum-likelihood analysis was applied with 1000 bootstrap replications. Sequence identity of P. californiensis with both P. vannamei and P. stylirostris was 0.88, while for P. vannamei and P. stylirostris the identity was 0.92. Maximum-likelihood analysis grouped P. vannamei and P. stylirostris separately from P. californiensis.     

Penaeus vannamei isotrypsins: purification and characterization

Three isotrypsins from digestive gland of Penaeus vannamei were purified and characterized by molecular, biochemical and kinetic parameters. Purified isotrypsins A, B, and C are glycoproteins with molecular masses between 30.2 and 32.9 kDa, and, therefore similar to other trypsins. The isoelectric points are anionic and different among the three isotrypsins: pH 3.5 for isotrypsin A, pH 3.0 for isotrypsin B, and pH 4.5 for isotrypsin C. Differences in the NH(2)-terminal amino acid sequences allowed us to define three different protein entities that match isotrypsins previously deduced by cDNA. Isoform C has higher physiological efficiency and specific activity, lower K(m), and requires higher concentrations of Ca(+2) to reach the same activity as the other two isotrypsins.     

池养南美蓝对虾与南美白对虾肌肉营养品质的比较

对南美蓝对虾和南美白对虾的肌肉营养成分与营养品质进行了分析比较。结果表明:南美蓝对虾的粗蛋白含量显著高于南美白对虾(P>0.05),南美白对虾的水分、粗脂肪和粗灰分含量均显著高于南美蓝对虾(P>0.05)。两种对虾的氨基酸组成基本一致,均含有18种氨基酸,四种鲜味氨基酸总量分别为31.30%、30.73%(干样百分比),必需氨基酸指数(EAAI)分别为69.88、62.42,其构成比例符合FAO/WHO的标准。脂肪酸中EPA与DHA含量均较高,分别为20.91%、18.60%,矿物元素含量丰富。上述分析表明,南美蓝对虾和南美白对虾肌肉均为优质的动物蛋白食品,从营养学的角度,南美蓝对虾的蛋白质营养价值要高于南美白对虾,南美白对虾的脂肪酸营养价值高于南美蓝对虾。     

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.     

微生态制剂改善对虾养殖池塘底质的效果

研究了在117 d的养殖周期中微生态制剂对南美白对虾池塘底质的改良效果.结果表明,与对照组相比,施用微生态制剂可使底质中总氮、总磷和硫化物的含量显著下降;总菌数量无显著变化,而芽孢杆菌、氨化细菌以及硫氧化细菌、硫还原细菌、弧菌数量差异显著,其中弧菌数量在施用微生态制剂处理和对照条件下分别为3.65×10³ cfu·g^-1和1.16×10⁵ cfu·g^-1.表明施用微生态制剂可以减少氮、磷、硫等营养物质的积累,改善池塘底质的菌相,为南美白对虾的健康养殖提供良好的池塘底质环境.     

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.     

Effect of temperature on spawning of Penaeus merguiensis

The maturation rate and spawning performance of ablated Penaeus merguiensis were significantly (P<0.05) higher at 27°C than at 23°C. Increasing the temperature from 23°C to 27°C further improved spawning performance (P<0.05), whereas reducing the temperature from 27°C to 23°C impaired spawning. The effect of temperature during the inducing period was more critical than during the holding period. Prawn growth (% of weight gain), however, was not different between 23°C and 27°C over the 60-day experimental period (P>0.05).     

Diel activity of the pink shrimp Penaeus duorarum

Twelve juvenile pink shrimp, Penaeus duorarum Burkenroad, were tested individually for 3-day periods in electronic shuttleboxes to determine their diel patterns of locomotor activity, in relation to a natural summer photoperiod. Nocturnal activity was twice that exhibited during the daytime; however, a bimodal pattern was evident with crepuscular peaks occurring at dawn and dusk. The dusk peak was more pronounced, with activity increasing markedly before sunset (indicative of an endogenous circadian rhythm component), and continuing during the initial hours of darkness, gradually declining later during the night with a secondary peak at dawn falling off to minimal activity during daylight. Crepuscular activity (mean of dawn and dusk) was twice the nocturnal average.     

南美白对虾饲料中沙蚕的营养成分分析

以南美白对虾饲料中的沙蚕为研究对象,通过气相色谱-质谱联用仪和氨基酸自动分析仪分析检测其体内的脂肪酸和氨基酸,之后对其营养价值进行了分析评价。结果表明:沙蚕体内检测出11种脂肪酸,多不饱和脂肪酸质量分数高达46.62%,其中EPA和AA的含量达16.20%和10.41%;沙蚕体内蛋白含量丰富,必需氨基酸含量为38.39%。通过营养评价,表明沙蚕对南美白对虾来说是一种高蛋白、低脂肪的优质饲料。     

南美白对虾卵子发生的组织学

采用组织学方法研究了南美白对虾的卵子发生过程,根据卵细胞大小、核仁形态、卵黄粒的有无、皮质棒的出现以及卵母细胞与滤泡细胞的关系,将南美白对虾的卵子发生划分为卵原细胞、卵黄发生前的卵母细胞和卵黄发生的卵母细胞三个时期,并描述了各期卵细胞的形态特征。     

Biopreservation of Sardinella longiceps and Penaeus monodon Using Protective Culture Streptococcus phocae PI 80 Isolated from Marine Shrimp Penaeus indicus

The influence of Streptococcus phocae PI80 on the shelf life of Sardinella longiceps and Penaeus monodon was investigated by measurement of microbial and chemical analysis after appraising the safety of the protective probiotic culture in wistar rat’s model. The results of this safety assessment indicate that oral administration of protective culture does not demonstrate any toxicological effects. Consumption of this LAB strain had no adverse effects on animal’s general health status, hematology, blood biochemistry, histology parameters, or on the incidence of bacterial translocation. The effect of Streptococcus phocae is very evident with the reduction of Listeria monocytogenes, Vibrio parahemolyticus, and coliforms. During storage, a marked decline in total volatile base and peroxide value was observed in protective culture-treated samples than the control. This strain looks promising as a protective culture for the preservation of fish products.     

Diel activity patterns in Metapenaeus and Penaeus juveniles

Small (5–10.9 mm carapace length), medium (11–15.9 mm), and large (16–20.9 mm) juveniles of Metapenaeus anchistus, Metapenaeus sp., Penaeus monodon and P. merguiensis were stocked individually in glass tanks provided with sand substrate, sea water, artificial bamboo shelter, aeration and food. The seven activity types (recorded for each shrimp hourly for 24 h) were classified as below (burrowing) or above substrate (swimming, walking, stationary, in shelter, feeding and cleaning). Shrimp juveniles exhibited a strong diel periodicity — emergence and activity at night and burrowing in the day. The chi-square test showed that type of activity (above/below substrate) was associated with period (light/dark). Diurnal burrowing was greater among Metapenaeus than Penaeus; inversely, above substrate activities were more frequent for Penaeus species compared to Metapenaeus. Feeding was the major above substrate and nocturnal activity for M. anchistus, Metapenaeus sp. and P. monodon. Only P. Monodon used the shelter consistently. Frequency of the 7 activity types was dependent on juvenile size for Penaeus, e.g., the preference for shelters shifted to burrowing with increase in size in P. monodon. Results are discussed in relation to the importance of mangrove habitats in providing shelter to penaeids, in particular the mangrove-associated P. monodon and P. merguiensis.     

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.     

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.     

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.