Identification of abundant and informative microsatellites from shrimp (Penaeus monodon) genome

Microsatellites were isolated from P. monodon genomic libraries by direct sequencing of recombinant clones without probe screening. Forty-nine out of 83 clones sequenced contained 99 microsatellite arrays of three or more repeats. When five or more and ten or more repeats were considered, 28 and 14 microsatellites were detected, respectively. The 99 microsatellites were classified as perfect (75%), imperfect (6%), compound perfect (3%) and compound imperfect (16%). The abundance of di-, tri-, tetra- and hexanucleotide repeats were 67%, 20%, 9% and 3%, respectively. The dinucleotide repeats included 36 (CT)n, 31 (GT)n, 17(AT)n and 3 (CG)n. One octanucleotide repeat (ATTTATTC)5 was found within a large repeat sequence. Optimal annealing temperatures were determined for PCR using 11 primer sets encompassing 15 microsatellites. Ten primer sets provided successful amplifications with allele sizes generally ranging from 139 to 410 bp. All these primers amplified polymorphic loci with PIC values ranging from 0.63 to 0.96. Two primer sets amplified additional bands which can easily be distinguished from the bands of the main locus. Three out of 10 P. monodon microsatellites also amplified alleles in P. vannamei. The abundance and informative nature of P. monodon microsatellites and their potential for cross-species amplification make them useful for genetic studies.     

Development of simple sequence repeat markers from expressed sequence tags of the black tiger shrimp (Penaeus monodon)

In this study, we describe the development of expressed sequence tag-simple sequence repeat markers from expressed sequence tags of the black tiger shrimp (Penaeus monodon) deposited in public sequence databases. A total of 46 primer pairs were designed and screened on 26 individuals of P. monodon from a natural population. Of these, 16 primer pairs showed polymorphic profiles with between two and five alleles per locus. The average unbiased and direct count heterozygosities were 0.4662 and 0.3516, respectively. Cross-amplification was tested with five individuals of Penaeus vannamei and polymorphic products were detected at five loci.     

Alteration of egg activation by protease inhibitors in the black tiger shrimp,Penaeus monodon

In penaeoid shrimp, contact of spawned eggs with seawater induces egg activation. However, little is known about the factors that influence egg activation in Penaeus monodon. Therefore, the main objective of the present study was to determine whether shrimp-produced proteases that are released in seawater are essential for egg activation. Female shrimp were allowed to spawn in artificial seawater containing protease inhibitors. It was shown that 4-amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF) and soybean trypsin inhibitor (SBTI) inhibited egg activation. High doses of APMSF and SBTI induced only 1–2% complete egg activation. Moreover, when the APMSF- and SBTI- treated eggs were subsequently washed, egg activation did not resume. In contrast, other protease inhibitors, pepstatin A, E-64, and ethylene glycol tetraacetic acid, did not inhibit egg activation, as evident by approximately 98% complete activation. Our results suggest that serine proteases, which are most likely trypsin-like proteases, released in seawater may be involved in egg activation of P. monodon.     

Infectious hypodermal and hematopoietic necrosis,a newly recognized virus disease of penaeid shrimp

Populations of the Pacific blue shrimp, Penaeus stylirostris, reared at the University of Arizona's experimental shrimp culture facility on Oahu in Hawaii from late 1980 through 1981, were severely affected by a highly acute and lethal disease of viral etiology. Also found to be susceptible to the disease were P. vannamei and P. monodon. The disease was named infectious hypodermal and hematopoietic necrosis (IHHN) disease to describe the principal lesions observed. The histopathology of acute and subacute IHHN disease in these species was dominated by the presence of conspicuous eosinophilic intranuclear-inclusion bodies of the Cowdry type A variety in ectodermally (especially the cuticular hypodermis) and mesodermally (especially the hematopoietic tissues) derived tissues that were undergoing necrosis. Electron microscopy of affected tissues demonstrated the presence of two or three types of virus-like particles with cubic morphology and diameters of 17 to 27 nm that suggest IHHN virus to be either a parvo- or picornavirus.     

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     

Microsatellite and mitochondrial haplotype diversity reveals population differentiation in the tiger shrimp (Penaeus monodon) in the Indo-Pacific region

The black tiger shrimp (Penaeus monodon) is an ecologically and economically important penaeid species and is widely distributed in the Indo-Pacific region. Here we investigated the genetic diversity of P. monodon (n = 355) from eight geographical regions by genotyping at 10 microsatellite loci. The average observed heterozygosity at various loci ranged from 0.638 to 0.743, indicating a high level of genetic variability in this region. Significant departures from Hardy-Weinberg equilibrium caused by heterozygote deficiency were recorded for most loci and populations. Pairwise F(ST) and R(ST) values revealed genetic differentiation among the populations. Evidence from the assignment test showed that the populations in the West Indian Ocean were unique, whereas other populations examined were partially admixed. In addition, the non-metric multidimensional scaling analysis indicated the presence of three geographic groups in the Indo-Pacific region, i.e. the African populations, a population from western Thailand and the remaining populations as a whole. We also sequenced and analysed the mitochondrial control region (mtCR) in these shrimp stocks to determine whether the nuclear and mitochondrial genomes show a similar pattern of genetic differentiation. A total of 262 haplotypes were identified, and nucleotide divergence among haplotypes ranged from 0.2% to 16.3%. Haplotype diversity was high in all populations, with a range from 0.969 to 1. Phylogenetic analysis using the mtCR data revealed that the West Indian Ocean populations were genetically differentiated from the West Pacific populations, consistent with the microsatellite data. These results should have implications for aquaculture management and conservation of aquatic diversity.     

A parvo-like virus disease of penaeid shrimp

Cultured populations of four penaeid shrimp species (Crustacea, Decapoda) from four separate culture facilities in Asia were found to be adversely affected by a disease of presumed viral etiology. Individual shrimp with the disease displayed nonspecific signs, including poor growth rate, anorexia, reduced preening activity, increased surface fouling, and occasional opacity of tail musculature. These signs were accompanied by mortalities during the juvenile stages, after apparently normal development through the larval and postlarval stages. Accumulative mortality rates in epizootics in Penaeus merguiensis and P. semisulcatus reached as high as 50 to 100%, respectively, of the affected populations within 4 to 8 weeks of disease onset. The principal lesion, common to all four species, was necrosis and atrophy of the hepatopancreas, accompanied by the presence of large prominent basophilic, PAS-negative, Fuelgen-positive intranuclear inclusion bodies in affected hepatopancreatic tubule epithelial cells (hepatopancreatocytes). These inclusion bodies presumably developed from small, eosinophilic, intranuclear bodies that were also present in the affected tissues. Electron microscopy of affected hepatopancreatocytes revealed aggregations of 22- to 24-nm-diameter virus particles within the electron-dense granular inclusion body ground substance. The virus particle size and morphology, the close association of the nucleolus with the developing inclusion body, and the presence of intranuclear bodies within developing inclusion bodies are similar to cytopathological features reported for parvovirus infections in insects and vertebrates. It is suggested that this presumed virus disease of cultured penaeid shrimp be called HPV for Hepatopancreatic Parvo-like Virus disease.     

RpoN gene,RAPD profile,antimicrobial resistance and plasmids of Vibrio anguillarum isolates from vibriosis infected Penaeus monodon

Aim: To evaluate the diversity of Vibrio anguillarum isolates from vibriosis‐infected Penaeus monodon collected from east coast of India. Methods and Results: Thirty‐six V. anguillarum were cultured from specific V. anguillarum medium, further identified using biochemical tests and confirmed by PCR detection of rpoN gene. Randomly amplified polymorphic DNA analysis revealed that in each location, the selected V. anguillarum isolates produced a unique band pattern, indicating that the members of this species are genetically heterogeneous. Antibiotic sensitivity results showed that 85%, 72%, 70%, 58%, 45% and 34% of the isolates were resistant to erythromycin, furazolidone, chloramphenicol, oxolinic acid, ciprofloxacin and nitrofurantoin, respectively. Plasmids were found in 70% of the isolates, and nine different plasmid profiles were observed. Conclusions: Wide ranges of diversity were noted in V. anguillarum isolates collected from P. monodon at different locations of east coast of India. Significance and Impact of the Study: Molecular typing, antibiotic resistance and plasmid profiles of V. anguillarum isolates from shrimp in India enables the prediction of possible risk for diseases in shrimp culture environment and the application of alternative management plans to prevent further spread of antibiotic resistance.     

Development of polymorphic expressed sequence tag-derived microsatellites for the extension of the genetic linkage map of the black tiger shrimp (Penaeus monodon)

In this study, microsatellite markers were developed for the genetic linkage mapping and breeding program of the black tiger shrimp Penaeus monodon. A total of 997 unique microsatellite-containing expressed sequence tags (ESTs) were identified from 10 100 EST sequences in the P. monodon EST database. AT-rich microsatellite types were predominant in the EST sequences. Homology searching by the blastn and blastx programs revealed that these 997 ESTs represented 8.6% known gene products, 27.8% hypothetical proteins and 63.6% unknown gene products. Characterization of 50 markers on a panel of 35-48 unrelated shrimp indicated an average number of alleles of 12.6 and an average polymorphic information content of 0.723. These EST microsatellite markers along with 208 other markers (185 amplified fragment length polymorphisms, one exon-primed intron-crossing, six single strand conformation polymorphisms, one single nucleotide polymorphism, 13 non-EST-associated microsatellites and two EST-associated microsatellites) were analysed across the international P. monodon mapping family. A total of 144 new markers were added to the P. monodon maps, including 36 of the microsatellite-containing ESTs. The current P. monodon male and female linkage maps have 47 and 36 linkage groups respectively with coverage across half the P. monodon genome.     

Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon

AIMS: The present study evaluated the in vitro and in vivo antagonistic effect of Bacillus against the pathogenic vibrios. METHODS AND RESULTS: Cell-free extracts of Bacillus subtilis BT23 showed greater inhibitory effects against the growth of Vibrio harveyi isolated by agar antagonism assay from Penaeus monodon with black gill disease. The probiotic effect of Bacillus was tested by exposing shrimp to B. subtilis BT23 at a density of 106-108 cfu ml-1 for 6 d before a challenge with V. harveyi at 103-104 cfu ml-1 for 1 h infection. The combined results of long- and short-term probiotic treatment of B. subtilis BT23 showed a 90% reduction in accumulated mortality. CONCLUSIONS: This study reports that pathogenic vibrios were controlled by Bacillus under in vitro and in vivo conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicated that probiotic treatment offers a promising alternative to the use of antibiotics in shrimp aquaculture.     

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.     

Photobacterium damselae ssp. damselae associated with diseased black tiger shrimp Penaeus monodon Fabricius in India

AIM: To characterize and identify Photobacterium damselae ssp. damselae present in black gill diseased Penaeus monodon collected from east coast of India. METHODS AND RESULTS: Photobacterium damselae ssp. damselae was isolated from hepatopancreas, muscles and gills by using the thiosulfate citrate bile salts sucrose agar supplemented with 1.5% NaCl (TCBS-1) medium. A total of 32 Ph. damselae ssp. damselae isolates were studied together with two reference strains. The biochemical tests and analysis of ureC and 16S rRNA genes confirmed the phenotypic characterization of the isolates as Ph damselae ssp. damselae. Experimental infection studies revealed that the LD50 values of P. monodon and P. indicus ranged from 2x10(3) to 5x10(5) CFU per shrimp and from 4x10(2) to 2x10(4) CFU per shrimp, respectively. CONCLUSIONS: Photobacterium damselae ssp. damselae was found in the internal organs of P. monodon and it showed pathogenic to shrimp. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on the Ph. damselae ssp. damselae present in the black gill diseased P. monodon in India and therefore might serve as a basis for future studies and diagnosis purpose to shrimp culturists.     

Novel alternative to antibiotics in shrimp hatchery: effects of the essential oil of Cinnamosma fragrans on survival and bacterial concentration of Penaeus monodon larvae

Aims: The activity of two essential oils (EOs) of Cinnamosma fragrans, an endemic plant to Madagascar (B8: linalool‐type and B143: 1,8‐cineole‐type), against bacterial isolates from a shrimp hatchery of Penaeus monodon and their effects on the survival and bacterial concentration of larvae were determined. Methods and Results: Minimum inhibitory concentrations were determined using a broth dilution technique. The bacterial concentrations of both larvae and water tank were assessed on Marine agar and Thiosulfate Citrate Bile Sucrose agar. The assays took place in OSO Farming’s shrimp hatchery in Madagascar. EOs were directly added to the water tank. Regarding the survival, the assays in larval culture (four replicates each of B8, B143, E and control) showed that B8 oil had a similar effect (P > 0·05) as the antibiotic (Erythromycin) and was more active than B143 (P < 0·05). A negative correlation was observed between the bacterial concentration and the survival of larvae for all assays. Conclusion: Both C. fragrans essential oils, as antibiotic, exhibited significantly higher survival rates and lower bacterial concentrations of the larvae than the control (oil and antibiotic free). Significance and impacts of the study: The potential of C. fragrans essential oil to control the bacterial load in in vivo conditions, thereby enhancing survival rate of P. monodon larvae, makes it a relevant option for developing a novel alternative to antibiotics in shrimp hatchery culture.     

斑节对虾两个野生亲虾种群遗传多样性

采用RAPD和mtDNA 16S rRNA基因序列分析方法对海南和马来西亚斑节对虾(Penaeus monodon)种群进行了遗传多样性和遗传分化研究.结果表明:15个RAPD随机引物共检测到82个位点,海南和马来西亚种群的多态位点比例分别为75.90％和76.83％,杂合度分别为0.199和0.218,遗传多样性指数分别为0.276和0.288,种群间的遗传距离为0.015;16S rRNA基因检测的种内遗传变异较低,马来西亚和海南种群的核苷酸多样性分别为0.011和0,种群之间的遗传距离为0.008;马来西亚种群比海南种群的遗传变异水平要高得多,且海南种群可能起源于马来西亚种群;进行遗传选育时可考虑引进马来西亚亲虾作为奠基群体.     

Characterization of brown spot disease of gulf coast shrimp

Adult penaeid shrimp were collected from mariculture raceways, holding tanks, and near-shore waters of Galveston Island, Texas. Those with darkly pigmented lesions were chosen for study. When observed by scanning electron microscopy, diseased cuticle shows dense populations of bacteria within a lesion, whereas normal cuticle shows a relatively low number of bacteria. Some lesions exhibited a morphologically homogeneous population of bacteria while others revealed a heterogeneous or mixed infection. Bacteriological analysis of a homogenate of each lesion revealed the presence of a wide variety of organisms which produced extracellular lipases, proteases, and chitinases. Lipolytic and proteolytic organisms were consistently isolated from all 20 lesions examined, while chitinoclastic bacteria were found in all but two lesions. According to the presumptive isolation scheme used, 10 distinct phenotypes of bacteria were differentiated among the isolates from 20 lesions. These phenotypes were classified into four genera: Vibrio, Alteromonas, Spirillum, and Flavobacterium. Groups of 10 shrimp were abraded and inoculated with representative isolates from each of the 10 phenotypes. Four isolates were able to initiate lesion formation. These were two Vibrio species, one Alteromonas species, and one Spirillum species. All of these four pathogens were lipolytic, while only two were chitinoclastic. This evidence suggests that there is not one specific chitinoclastic bacterium which causes brown spot symptoms, but that a variety of bacteria acting alone or in groups may be the causal agents.     

Taura syndrome,a disease important to shrimp farms in the Americas

World Journal of Microbiology and Biotechnology -     

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.     

Abundance of potentially pathogenic micro-organisms in Penaeus monodon larvae rearing systems in India

Monodon baculovirus (MBV), external fouling organisms (EFO) and bacteria (especially Vibrio species) were monitored during 1996–1997 at nine different Penaeus monodon rearing hatcheries in India. Total cultivable heterotrophic bacteria, Vibrio-like-bacteria, presumptive Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus counts were determined from shrimp eggs, post larvae, rearing tank water, source sea water, feed (Artemia nauplii and microencapsulated feed). The MBV infected post larvae and their environment showed higher Vibrio-like-bacteria than uninfected post larvae. An over-whelming predominance of presumptive Vibrio harveyi and Vibrio anguillarum was observed in post larval rearing tank water, MBV infected and uninfected post larvae. Vibrio-like-bacteria in Artemia nauplii clearly showed the possible source of these pathogenic bacteria in the hatchery environments. Quantitative analysis of Vibrio-like-bacteria in hatcheries revealed that when the Vibrio-like-bacteria increases to 2 × 10² CFU mortality of the post larvae occurs. Abundance of these micro-organisms in hatchery samples indicated that they are opportunistic pathogens which can invade the shrimp tissue, subsequently cause disease when the post larvae were under stressful conditions.     

Early Developmental Stages of the Green Tiger Prawn, Penaeus semisulcatus de Haan (Crustacea, Decapoda, Penaeidae)

Gravid females of Penaeus semisulcatus were spawned in the laboratory by natural means. The embryos were documented and the larvae were reared from hatching to postlarval stage at 28.2–30.0 °C and 33.5–34.5 g kg⁻¹ salinity for about 10 days (223 h 55 min). Six naupliar stages, three protozoea stages, three mysis stages and the first postlarval stage were described and illustrated. The larvae were fed only with microalgae Tetraselmis tetrathele and Chaetoceros gracilis from first protozoea until the second mysis, with about 90% survival rate; from the third mysis until the first postlarva they were fed with similar microalgae coupled with rotifer Brachionus plicatilis and Artemia nauplii. The embryonic and larval stages of P. semisulcatus are generally similar to those of other closely related species in the family Penaeidae, such as Melicertus canaliculatus, Fenneropenaeus merguiensis, and Marsupenaeus japonicus, except for the size and structure of diagnostic characters, setation of appendages and duration of metamorphoses. The change in the feeding habit during ontogeny was related to morphological transformation of the feeding apparatus of larvae and postlarvae. This paper is the first comprehensive and complete account of the early developmental stages of P. semisulcatus.