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.     

Yolk Synthesis in the Marine Shrimp, Penaeus vannamei

Eyestalk neuroendocrine factors control specific yolk proteinsynthesis in the ovaries of the shrimp, Penaeus vannamei. Abioassay was developed to measure specific yolk protein synthesisin vitro. The eyestalk neuroendocrine complex may also producea peptide capable of stimulation of yolk synthesis.     

Histopathology of aflatoxicosis in the marine shrimp Penaeus stylirostris and P. vannamei

The acute and subacute toxicity of aflatoxin B₁ to the marine shrimp Penaeus stylirostris and P. vannamei (Order: Decapoda, Class: Crustacea) was investigated. Experimental shrimp were exposed to a range of concentrations of the toxin directly by intramascular injection (from 2 to 160 μg aflatoxin B₁/g body weight), or by multiple per os dosing with the feed (from 53 to 300 μg aflatoxin B₁/g feed) for up to 25 days. The histopathogenesis of aflatoxicosis in the aflatoxin-exposed animals was followed and found to be time and dose dependent in the hepatopancreas, mandibular organ, and in the hematopoietic organs. Less significant and/or inconsistent lesions were also observed in other organs and tissues, but a time-dose dependency was not noted. The principal lesions of aflatoxicosis in penaeid shrimp occur in the hepatopancreas and the mandibular organ. In the former organ, subacute and acute aflatoxicosis is expressed as necrosis of the hepatopancreatic tubule epithelium that proceeds from the proximal (older) portion of the tubules, in the center of the organ, to the peripheral (younger) tubule tips. A marked intertubular hemocytic inflammation followed by encapsulation and fibrosis of affected tubules follows in subacute aflatoxicosis, but is not as developed as in acute aflatoxicosis. The mandibular organ in aflatoxicosis displays a necrosis of the peripheral epithelial cells of the cords within the gland that progresses proximally to the central vein. Only a slight hemocytic inflammation accompanies the degenerative changes in this latter organ.     

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。     

Possible toxic effects of the marine blue-green alga, Spirulina subsalsa, on the blue shrimp, Penaeus stylirostris.

Blooms of a marine species of blue-green algae identified as Spirulina subsalsa (Cyanophyta, Oscillatoriacae) were found to be related to a particular disease syndrome in raceway-reared blue shrimp, Penaeus stylirostris. The disease was characterized by necrosis of the lining epithelium of the midgut, dorsal cecum, and hindgut gland, and a consequent hemocytic enteritis. Bacterial infections due predominately to Vibrio alginolyticus were common in affected shrimp and presumed to be a secondary condition resulting from necrosis of the gut epithelium. These bacterial infections were expressed as local abscesses near or on the gut or as fulminating septicemias.     

Microsatellite Polymorphism and the Population Structure of the Black Tiger Shrimp (Penaeus monodon) in Thailand

: Genetic variation and differentiation of Thai Penaeus monodon from five geographic locations (Chumphon, Trad, Phangnga, Satun, and Trang) were investigated using five microsatellite loci (CUPmo18, Di25, Di27, CSCUPmo1, and CSCUPmo2). The number of alleles across the five loci ranged from 19 to 30, and heterozygosities ranged from 0.49 to 0.95. The mean number of alleles and effective number of alleles per locus were 21.0 to 26.6 and 13.1 to 20.4, respectively. The average heterozygosity across all investigated samples was 0.78, indicating high genetic diversity in this species. Geographic heterogeneity analysis of the results from two of the loci, CUPmo18 and Di25, showed significant differences among the Gulf of Thailand (Trad and Chumphon) but not the Andaman samples. Comparison between regions revealed significant heterogeneity of the Andaman and Trad P. monodon (P < .001), whereas those from Chumphon and the Andaman were genetically similar (P > .05). Significant genetic differentiation was consistently observed between the Andaman-Trad samples (FST = 0.0101, P < .0001) and the Chumphon-Trad samples (FST = 0.0101, P < .0001). On the basis of our analyses, the investigated samples from five geographic locations were allocated to three distinct populations composed of the Andaman Sea (A), Chumphon (B), and Trad (C).     

A comparison of the capabilities of juvenile and adult Penaeus setiferus and Penaeus stylirostris to regulate the osmotic,sodium, and chloride concentrations in the hemolymph

• 1.1. The capabilities of juvenile and mature adult Penaeus setiferus and P. stylirostris to regulate the osmotic, sodium, and chloride concentrations in the hemolymph are compared.
• 2.2. In P. setiferus and P. stylirostris acclimated to salinities of 9.8 and 10.8%. respectively, juvenile shrimp are stronger hyperosmotic and hyperionic regulators than adults. However, the reduced regulatory capabilities of adult shrimp are not sufficient to require migration to offshore waters for survival.
• 3.3. At 40.4%. juvenile P. setiferus are more effective hypoosmotic and hypoionic regulators than adults. However, there is no difference between the regulatory capabilities of juvenile and adult P. stylirostris at 36.2%.
• 4.4. Differences in hemolymph concentration between juvenile and adult P. setiferus at 23.5%. indicate that the isosmotic and isoionic crossover concentrations are elevated with maturation.

     

Microbial Flora of Pond-Reared Brown Shrimp (Penaeus aztecus)

Agar plate counts and microbial types are reported for brown shrimp reared in 2-acre natural marshland and in 0.5-acre artificial ponds during June to October 1970. Bacterial counts of pond-reared shrimp ranged from 5 × 10⁴ to 5.5 × 10⁶ per g. At final harvest in October, bacterial counts ranged from 2 × 10⁵ to 5.5 × 10⁶ per g. In marsh ponds, bacterial counts of shrimp and pond water were lowest in August when both water temperature and salinity were high. Coryneform bacteria and to a lesser extent Vibrio were the predominant isolates from fresh pond shrimp. Shrimp stored at 3 to 5 C for 7 days were acceptable as judged by appearance and odor. Between 7 and 14 days of refrigerated storage, bacterial counts increased sharply and about 50% of the samples became unacceptable. Refrigerated storage of pond shrimp caused increases in coryneform bacteria and micrococci and decreases in Vibrio, Flavobacterium, Moraxella, and Bacillus species. Pseudomonas species were not significant in fresh or stored pond shrimp. The microbial flora of pond water usually was dominated by coryneform bacteria, Flavobacterium, Moraxella, and Bacillus species.     

Protection of Shrimp Penaeus monodon from WSSV Infection Using Antisense Constructs

White spot syndrome caused by white spot syndrome virus (WSSV) is one of the most threatening diseases of shrimp culture industry. Previous studies have successfully demonstrated the use of DNA- and RNA-based vaccines to protect WSSV infection in shrimp. In the present study, we have explored the protective efficacy of antisense constructs directed against WSSV proteins, VP24, and VP28, thymidylate synthase (TS), and ribonucleotide reductase-2 (RR2) under the control of endogenous shrimp histone-3 (H3) or penaedin (Pn) promoter. Several antisense constructs were generated by inserting VP24 (pH3–VP24, pPn–VP24), VP28 (pH3–VP28, pPn–VP28), TS (pH3–TS, pPn–TS), and RR2 (pH3–RR2) in antisense orientation. These constructs were tested for their protective potential in WSSV infected cell cultures, and their effect on reduction of the viral load was assessed. A robust reduction in WSSV copy number was observed upon transfection of antisense constructs in hemocyte cultures derived from Penaeus monodon and Scylla serrata. When tested in vivo, antisense constructs offered a strong protection in WSSV challenged P. monodon. Constructs expressing antisense VP24 and VP28 provided the best protection (up to 90 % survivability) with a corresponding decrease in the viral load. Our work demonstrates that shrimp treated with antisense constructs present an efficient control strategy for combating WSSV infection in shrimp aquaculture.     

Hemolymph analysis and evaluation of newly formulated media for culture of shrimp cells (Penaeus stylirostris)

Summary Creation of a shirmp cell line has been an elusive goal. This failure may be due to the composition of the cell culture medium, which may be inadequate to support primary cultured cells. Shrimp hemolymph should contain the nutritional components needed to support cell growth and division. We report here the comprehensive biochemical analysis of hemolymph from the blue shrimp,Penaeus stylirostris (Litopenaeus stylirostris) (see Holthuis, L. B. Shrimps, and prawns of the world, in: FAO species catalog. Vol. 1. Rome: Food and Agriculture Organization of the United Nations; 1980), for free amino acids (FAAs), carbohydrates, electrolytes, metals, pH, and osmolality. Levels of hemolymph components were compared to 2×L-15 with 20% fetal bovine serum, a commonly used culture medium for crustacean cells. The FAAs, taurine and proline, and the metals, strontium and zinc, were significantly higher in hemolymph than in the 2×L-15 medium. In contrast, other FAAs were up to 50 times higher in the 2×L-15 medium than in the hemolymph. To mimic more closely the hemolymph composition, we created two new media based on either the 0.2×L-15 or the M199 medium. We compared the microscopic appearance of cells cultured in these media and evaluated deoxyribonucleic acid (DNA) and protein synthesis by³H-thymidine uptake and³⁵S-methionine uptake assays. The ovary cells ofP. stylirostris cultured in either of the new media formed monolayers, while the cells cultured in 2×L-15 medium did not. Despite these differences, there was no evidence of sustained DNA or protein synthesis with any of the media. Future studies to establish a shrimp cell line should focus on analysis of the cell cycle and on overcoming the molecular blocks to cell division.     

Development of EST-SSR Markers by Data Mining in Three Species of Shrimp: Litopenaeus vannamei, Litopenaeus stylirostris, and Trachypenaeus birdy

We report on the data mining of publicly available Litopenaeus vannamei expressed sequence tags (ESTs) to generate simple sequence repeat (SSRs) markers and on their transferability between related Penaeid shrimp species. Repeat motifs were found in 3.8% of the evaluated ESTs at a frequency of one repeat every 7.8 kb of sequence data. A total of 206 primer pairs were designed, and 112 loci were amplified with the highest success in L. vannamei. A high percentage (69%) of EST-SSRs were transferable within the genus Litopenaeus. More than half of the amplified products were polymorphic in a small testing panel of L. vannamei. Evaluation of those primers in a larger testing panel showed that 72% of the markers fit Hardy-Weinberg equilibrium, which shows their utility for population genetic analysis. Additionally, a set of 26 of the EST-SSRs were evaluated for Mendelian segregation. A high percentage of monomorphic markers (46%) proved to be polymorphic by singles-stranded conformational polymorphism analysis. Because of the high number of ESTs available in public databases, a data mining approach similar to the one outlined here might yield high numbers of SSR markers in many animal taxa.     

Size-dependent haemagglutinating activity in the haemolymph from sub-adult blue shrimp (Penaeus stylirostris stimpson)

• 1.1. The blue shrimp (Penaeus stylirostris) haemolymph is capable of agglutinating the red blood cells of several vertebrates to different titres. However, the haemagglutinin is considered non-specific because it is incapable of differentiating erythrocytes of human blood types A, B and O.
• 2.2. Haemagglutinating activity and serum protein content were determined for male and female blue shrimp ranging in size from 8.5 to 16 cm. Haemagglutinating activity decreased significantly with animal size, while protein content was unaffected.
• 3.3. The above finding is probably related to maturation of the immune system and could explain the higher susceptibility of young shrimp to parasitic and viral diseases.

     

Tissue-Specific Expressed Sequence Tags from the Black Tiger Shrimp Penaeus monodon

Expressed sequence tag data were generated from complementary DNA libraries created from cephalothorax, eyestalk, and pleopod tissue of the black tiger shrimp (Penaeus monodon). Significant database matches were found for 48 of 83 nuclear genes sequenced from the cephalothorax library, 22 of 55 nuclear genes from the eyestalk library, and 6 of 13 nuclear genes from the pleopod library. The putative identities of these genes reflected the expected tissue specificity. For example, genes for digestive enzymes were identified from the cephalothorax library and genes involved in the visual and neuroendocrine system from the eyestalk library. A few sequences matched anonymous EST or genomic sequences, and others contained mini-satellite or microsatellite repeat sequences. The remainder, 31 from the cephalothorax library, 25 from the eyestalk library, and 5 from the pleopod library, were sequences of high nucleotide complexity with no matches in any database searched and thus may represent novel genes. Received February 12, 1999; accepted April 13, 1999     

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.     

Lack of Genetic Differentiation in the Shrimp Penaeus Chinensis in the Northwestern Pacific

Genetic differentiation of the shrimp Penaeus chinensis in the Yellow Sea and Bohai Sea was investigated using the mitochondrial control region (CR). RFLP of a partial CR segment (613 bp) shows that 106 out of 122 (86.9%) individuals from six sampling localities along the coast of northern China and the west coast of the Korean Peninsula share the same haplotype, and the haplotype frequencies among localities are not significantly different. The findings are further confirmed by sequencing the complete CR. Divergence of the complete CR (992 bp) is less than 1.6% in 14 individuals from the six localities. F-statistics based on RFLP data and the TCS network of sequencing data suggest little genetic differentiation of P. chinensis in the Yellow Sea and Bohai Sea. Mismatch analysis suggests a rapid expansion of P. chinensis population to the Yellow Sea and the Bohai Sea, which probably occurred with the rapid rise in sea level after the last glacial maximum. Despite the lack of genetic heterogeneity, we propose that P. chinensis populations in this region should be treated as separate management units, as fishery management programs have to be applied on a local basis by different governments.