Molecular cloning of a cDNA that encodes a serine protease with chymotryptic and collagenolytic activities in the hepatopancreas of the shrimp Penaeus vanameii (Crustacea, Decapoda).

Two clones were isolated by screening a shrimp hepatopancreas cDNA library with a DNA fragment obtained by PCR amplification using two oligonucleotides based on the partial protein sequence of Penaeus vanameii chymotrypsin purified earlier. One of these clones, PVC 7 contains a complete cDNA coding for a serine protease. The deduced amino acid sequence shows the existence of a 270 residue-long preproenzyme containing a highly hydrophobic signal peptide of 14 amino acids. This suggests the existence of a putative zymogen form of the enzyme containing a 30 amino acid-long peptide which is cleaved to give a mature protein of 226 residues. A highly preferred codon usage is observed for this protein. The other obtained cDNA was found to encode the less predominant variant of the protein. Sequence alignments show that shrimp chymotrypsin is highly homologous with crab collagenase (77% homology taking into account the same amino acid at the same position, and 83% homology taking into account amino acids with conserved function) and that it is more similar to mouse trypsin (41% homology of strictly conserved amino acids) than to hornet chymotrypsin (35% homology).     

A lymphoma-like neoplasm arising from hematopoietic tissue in the white shrimp, Penaeus vannamei Boone (Crustacea: Decapoda)

A pond-reared adult female Pacific white shrimp (Penaeus vannamei Boone, Crustacea: Decapoda) from an experimental shrimp culture facility in Hawaii was found to possess a lymphatic neoplasm. Present in this animal were bilateral hypertrophied hematopoietic nodules present ventral and lateral to the ventral nerve cord, and smaller multiple ectopic foci of similar appearing lymphoid cells in the gills, the subcutis, and other tissues. The lesions contained numerous anaplastic and hypertrophied lymphoid cells, many of which displayed bizarre mitotic figures, including polypolar metaphase figures. Numerous multinuclear giant cells present in the lesion were presumed to have originated from cells with polypolar mitotic figures. The characteristics of the cells composing these lesions, the expansive and invasive nature of the lesions, and the presence of ectopic foci of neoplastic cells support classification of this lesion as a hematopoietic sarcoma. Focal lesions of the type that are diagnostic of infections by the penaeid shrimp virus IHHN were present in the neoplastic hematopoietic tissue and other tissues of this shrimp, suggesting the possible role of viral infection in the development of neoplastic lesions in this animal.     

Pleonal muscle development in the shrimp Penaeus (Litopenaeus) vannamei (Crustacea: Malacostraca: Decapoda: Dendrobranchiata)

Penaeoidean shrimp pleonal muscle is a valuable economic resource worldwide, but little is known of its development during larval stages. The development of pleonal muscle in Penaeus (Litopenaeus) vannamei was studied by rhodamine-phalloidin staining and laser-scanning confocal microscopy. Dorsal pleonal muscle was first evident at the protozoea I stage while ventral pleonal muscle was present by the protozoea II stage. Identifiable ventral pleonal muscles were evident by the protozoea III stage and all ventral muscle types were present in the mysis I. The tail flex response began at the mysis stage and growth of existing pleonal muscles continued. The pleopods formed during the mysis stages, with coxal and basis muscles developed by mysis III. The pleopods became functional beginning with the first post-larval stage. We conclude that the pleonal muscle pattern of P. vannamei larvae is similar to that of adult Penaeus setiferus, and that homologous muscles are present. The major formation of dorsal pleonal muscles occurs during the protozoea II stage, while significant development of ventral pleonal muscles occurs during the protozoea III stage.     

Cleavage and gastrulation of the dendrobranchiate shrimp Penaeus monodon (Crustacea,Malacostraca, Decapoda)

The cleavage pattern of the black tiger shrimp Penaeus monodon was analyzed from the first division until gastrulation. Observations were based on microscopy combined with the use of fluorescent dyes, histological techniques, and computer based three-dimensional reconstructions. Early cleavage is holoblastic and follows a stereotypic pattern, which largely corresponds to what is known from other dendrobranchiate decapods. However, for the first time in this group, we report the presence of an intracellular structure throughout early development. This intracellular body (icb) marks the lineage of one of the two enlarged and division-delayed mesendoderm cells that initiate gastrulation. The identity of the icb as a natural marker and putative determinant of the germ line and its implications on the establishment of the body axes are discussed. The icb as a landmark reveals that the same stereotypic cell division pattern can lead to different fates of individual cells. Hence, the results of this study permit an additional approach to study the relation between cell lineage pattern and the identity of cell lineages.     

Lecithin essay in the diet of young Penaeus vannamei (Crustacea: Decapoda)

The effect of different lecithin sources and presentations on growth, food conversion ratio and survival of P. vannamei (290 mg +/- 0.02) was studied. The bioassay was designed in order to compare different dietary levels and different quality of lecithin. Squid lecithin, crude soybean (7%), deoiled soybean lecithin (3.48%) in combination with fish oil or squid neutral lipids, in a partially dilapidate formula. The isoenergetic diets were fed ad libitum to four replicate groups (tanks) of 15 shrimps each (5 x 4 x 15), during 28 days. The result of the bioassay with the partially dilapidate formulas was; the best growth rate (191%) and FCR (1.69 +/- 0.041) were obtained with the diet containing 7% of soybean crude lecithin as the unique lipid source. Followed by the diet countering 3.94% deoiled lecithin and 2.42% Menhaden oil (172% and 2.03 +/- 0.054 respectively). As expected, the worst results were obtained without the dietary lecithin 121% and 2.42 +/- 0.129). Crude soybean lecithin alone covered the phospholipid and neutral lipids requirements as well as the combination of deoiled soybean lecithin with fish or squid oil.     

Purification and characterization of alpha 2-macroglobulin from the white shrimp (Penaeus vannamei)

alpha(2)-Macroglobulin (alpha(2)M) is a broad-spectrum protease-binding protein abundant in plasma from vertebrates and several invertebrate phyla. This protein was purified from cell-free hemolymph of the white shrimp, Penaeus vannamei, using Blue-Sepharose and Phenyl-Sepharose chromatography. The shrimp alpha(2)M is a 380 kDa protein, a homodimer of two apparently identical subunits of approximately 180 kDa linked by disulphide bridges. The amino acid sequence of the N-terminus is similar to the Limulus alpha(2)M counterpart. The shrimp alpha(2)M has a wide inhibition spectrum against different proteinase types including trypsin, leucine amino peptidase, chymotrypsin, elastase and papain. The secondary structure of shrimp alpha(2)M is mainly beta-sheet (36%), with a characteristic minimum elipticity at 217 nm. Evidence for a thiolester-mediated inhibition mechanism of proteases by alpha(2)M was provided by inactivation with methylamine.     

The basic isoelectric form of alpha-L-fucosidase from the hepatopancreas of the shrimp Penaeus monodon (Crustacea: Decapoda).

1. alpha-L-Fucosidase was purified ca 10,889-fold to homogeneity from Penaeus monodon, with a final spec. act. of 31,250 U/mg of protein. 2. By using SDS-polyacrylamide gel electrophoresis, the monomers of shrimp alpha-L-fucosidase were discovered to have mol. wts of 63,000 and those of human placental enzyme, 46,000 and 20,000. Since the active shrimp alpha-L-fucosidase was found to have a mol. wt of 233,000 by Superose 12 FPLC, it was concluded that the purified shrimp enzyme was tetrameric. 3. In contrast to the discovery of thermolability with human placental alpha-L-fucosidase, the shrimp enzyme was found to be stable to heating at 65 degrees C for 10 min. 4. The shrimp alpha-L-fucosidase has an isoelectric point (pI) of 8.5, but the human placental enzyme has a pI of 4.0. The shrimp enzyme was sialyated. 5. The shrimp alpha-L-fucosidase has a pH optimum at 5.5 and its Km was 22.2 microM with 4-methyl-umbelliferyl-alpha-L-fucopyranoside as substrate. The human enzyme has a broad pH optimum between 5.0 and 6.5.     

Isolation and characterization of microsatellites in Pacific white shrimp Penaeus (Litopenaeus) vannamei

Five polymorphic microsatellite loci were characterized for Penaeus (Litopenaeus) vannamei. Loci were isolated using a partial Sau3A1 genomic library by the sequencing of randomly selected clones and by a biotinylated (CT)₁₀ and (GT)₁₀ probes screening procedure. The last strategy resulted in the most useful data. About 40% of the clones showed a previously reported satellite/microsatellite (PVS1), reducing the chance of finding new microsatellite regions. Whereas two of the microsatellite loci with more than 10 alleles will be useful for mating analysis in a breeding program, the others might prove useful for population genetic studies.     

Purification and characterization of hatching enzyme from shrimp Penaeus chinensis

By using Penaeus chorion as a specific substrate, the hatching enzyme (HE) from Penaeus chinensis was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties in this study. It was found that the molecular weight of Penaeus HE is about 43.0 kDa in SDS-PAGE. The Penaeus HE had obvious choriolytic activity, which was optimal at pH 6.0 and temperature of 40 degrees C, respectively. The Km value of the HE for casein was 7.47 mg ml(-1). The HE activity was almost completely inhibited by SBTI, p-APMSF, bestatin, and NEM, greatly inhibited by ovomucoid, TLCK, IAM, chymostatin, and PMSF, and slightly inhibited by pepstatin A, TPCK, LBTI, and leupeptin. These results indicate that the HE is most probably a trypsin-type serine protease. Besides of these, the HE was extremely sensitive to EDTA, Zn2+, Ca2+, Mg2+, and Cu2+. Combined with the results that the EDTA-pretreated HE activity could be perfectly recovered by Zn2+, it is indicated that shrimp HE is most probably a kind of Zn-metalloprotease.     

Ultrastructural and sequence characterization of Penaeus vannamei nodavirus (PvNV) from Belize

The Penaeus vannamei nodavirus (PvNV), which causes muscle necrosis in Penaeus vannamei from Belize, was identified in 2005. Infected shrimp show clinical signs of white, opaque lesions in the tail muscle. Under transmission electron microscopy, the infected cells exhibit increases in various organelles, including mitochondria, Golgi stacks, and rough endoplasmic reticulum. Cytoplasmic inclusions containing para-crystalline arrays of virions were visualized. The viral particle is spherical in shape and 19 to 27 nm in diameter. A cDNA library was constructed from total RNA extracted from infected shrimp. Through nucleotide sequencing from the cDNA clones and northern blot hybridization, the PvNV genome was shown to consist of 2 segments: RNA1 (3111 bp) and RNA2 (1183 bp). RNA1 contains 2 overlapped open reading frames (ORF A and B), which may encode a RNA-dependent RNA polymerase (RdRp) and a B2 protein, respectively. RNA2 contains a single ORF that may encode the viral capsid protein. Sequence analyses showed the presence of 4 RdRp characteristic motifs and 2 conserved domains (RNA-binding B2 protein and viral coat protein) in the PvNV genome. Phylogenetic analysis based on the translated amino acid sequence of the RdRp reveals that PvNV is a member of the genus Alphanodavirus and closely related to Macrobrachium rosenbergii nodavirus (MrNV). In a study investigating potential PvNV vectors, we monitored the presence of PvNV by RT-PCR in seabird feces and various aquatic organisms collected around a shrimp farm in Belize. PvNV was detected in mosquitofish, seabird feces, barnacles, and zooplankton, suggesting that PvNV can be spread via these carriers.     

A sialidase from the hepatopancreas of the shrimp Penaeus japonicus (Crustacea: Decapoda): reversible binding with the acidic beta-galactosidase

1. The sialidase purified from the hepatopancreas of Penaeus japonicus is able to bind the acidic beta-galactosidase in vitro. No protective protein, Mr 32,000, was detected in either purified enzyme preparation. 2. The specific activity of the isolated sialidase is 55.0 mU/mg of protein. After polyacrylamide gel electrophoresis under denaturing conditions, the purified shrimp enzyme was found to consist of monomers of Mr 32,000. 3. The sialidase from shrimp has an isoelectric point (pI) of 4.6 +/- 0.1. 4. The shrimp enzyme has the pH optimum at 5.0 and its Km was 5.5 microM with 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid as substrate. The enzyme activity was inhibited by either Hg2+ or Cu2+ ions.     

Ontogeny of gut morphology in the white shrimp Penaeus setiferus (Decapoda,Penaeidae)

Ontogeny of the gut in Penaeus setiferus was investigated by reconstruction of serial sections examined by light microscopy. Development of the gut into the adult form is protracted over several weeks beyond metamorphosis in steps that may be directly related to the unique postlarval life history of Penaeus. The gastric mill is lacking in larval stages of P. setiferus. In protozoeal stages Z₁-Z₃, the pyloric ampullae are blind sacs that do not communicate with the midgut. The gland filter first appears in mysis stage M₂. The gastric mill in early postlarval (PL) stages consists of poorly chitinized lobes with flexible setae. By PL₂₁ the ossicles of the gastric mill are rigid and setae are replaced by spine-like denticles, but even by PL₃₅ the gastric mill is neither as massive nor heavily chitinized as in adults. During the mysis stages and early PL stages, the hepatopancreas communicates freely with both the foregut and the midgut trunk. By PL₃₅ the hepatopancreatic ducts are essentially isolated from the remainder of the midgut by foregut ossicles. The midgut in Z₁ consists of two pairs of simple caeca and the midgut trunk. During larval growth, each of the lateral midgut caeca develops into a number of lobes. After metamorphosis these lobes begin to ramify into small-diameter tubules, and by PL₃₅ have completely ramified into the hepatopancreas of adults. From M₁ to PL₄, the anterior midgut caeca decrease in absolute size and become a single anterior diverticulum. The posterior midgut diverticulum first appears in PL₂₁ as a simple sac and thereafter increases in size and complexity.     

Molecular characterization of vitellin from the ovaries of the white shrimp Penaeus (Litopenaeus) vannamei

Vitellin (Vt) was purified from ovary extracts of mature females of the white shrimp Penaeus vannamei using Sepharose CL-4B and Q-Sepharose columns. Native Vt had an apparent molecular weight of 388 kDa as detected in Native-PAGE, bound the lipophilic dye Oil Red O and had a total lipid content of approximately 43.8%. Under reducing and denaturing conditions (SDS-PAGE), Vt is composed of three major subunits of 87, 78 and 46 kDa, although minor bands of 65, 61 and 31 kDa are also detected. The 87- and 78-kDa polypeptides were strongly recognized by Penaeus semisulcatus anti-Vt polyclonal and Penaeus monodon anti-Vt monoclonal antibodies. Furthermore, the N-terminal amino acid sequence of the 78-kDa polypeptide is very similar to Penaeus japonicus vitellogenin (Vg) and P. semisulcatus Vt, with an identity of 76%. Circular dichroism indicates that the beta-helix content of Vt is 25% while beta-sheets correspond to 37 and 14% of unordered secondary structure. These values are similar to insect microvitellogenin. Vt has an emission fluorescence maximum at 329 nm, comparable to the shrimp high-density lipoprotein/beta-glucan binding protein (HDL/BGBP).     

Purification and characterization of a lectin from the white shrimp Litopenaeus setiferus (Crustacea decapoda) hemolymph

A 291-kDa lectin (LsL) was purified from the hemolymph of the white shrimp Litopenaeus setiferus by affinity chromatography on glutaraldehyde-fixed stroma from rabbit erythrocytes. LsL is a heterotetramer of two 80-kDa and two 52-kDa subunits, with no covalently-liked carbohydrate, and mainly composed by aspartic and glutamic acids, glycine and alanine, with relatively lower methionine and cysteine contents. Edman degradation indicated that the NH2-terminal of the 80-kDa subunit is composed DASNAQKQHDVNFLL, whereas the NH2-terminal of the 52-kDa subunit is blocked. The peptide mass fingerprint of LsL was predicted from tryptic peptides from each subunit by MALDI-TOF, and revealed that each subunit showed 23 and 22%, respectively, homology with the hemocyanin precursor from Litopenaeus vannamei. Circular dichroism analysis revealed beta sheet and alpha helix contents of 52.7 and 6.1%, respectively. LsL agglutinate at higher titers guinea pig, murine, and rabbit erythrocytes its activity is divalent cation-dependent. N-acetylated sugars, such as GlcNAc, GalNAc, and NeuAc, were the most effective inhibitors of the LsL hemagglutinating activity. Sialylated O-glycosylated proteins, such as bovine submaxillary gland mucin, human IgA, and fetuin, showed stronger inhibitory activity than sialylated N-glycosylated proteins, such as human orosomucoid, IgG, transferrin, and lactoferrin. Desialylation of erythrocytes or inhibitory glycoproteins abolished their capacity to bind LsL, confirming the relevance of sialic acid in LsL-ligand interactions.     

Isolation and characterization of 14 polymorphic microsatellite markers for the blue and red shrimp, Aristeus antennatus (Crustacea, Decapoda)

Eighteen microsatellite loci were isolated and characterized from the blue and red shrimp, Aristeus antennatus Risso 1816, a commercially exploited marine crustacean widely distributed throughout the Mediterranean Sea and in the eastern Atlantic. Polymorphism was assessed in a population (n = 20) from the southwestern Sardinian seas; 14 loci resulted polymorphic and showed from three to 13 alleles. The observed heterozygosity ranged from 0.2 to 0.85. These microsatellites will be potentially useful for the study of A. antennatus population genetic structure.     

Burrowing shrimp of the infraorders Gebiidea and Axiidea (Crustacea: Decapoda)

This review is devoted to the different aspects of biology of burrowing shrimp of the infraorders Gebiidea and Axiidea (Crustacea: Decapoda). Information on their taxonomy, morphology and physiology, distribution, burrow architecture, and trophic mode is reviewed. The peculiarities of breeding and development, life history, and the role in the ecosystem of these infaunal species are analyzed. Special attention is given to the species that inhabit Peter the Great Bay of the Sea of Japan.     

Diversity and characterization of oxytetracycline-resistant bacteria associated with non-native species, white-leg shrimp (Litopenaeus vannamei), and native species, black tiger shrimp (Penaeus monodon), intensively cultured in Thailand

Aims: This study aimed at surveying prevalence of oxytetracycline (OTC)‐resistant bacteria in the white‐leg shrimp Litopenaeus vannamei, and the black tiger shrimp Penaeus monodon, intensively cultured in Thailand. We investigated the phylogenetic diversity of the bacterial isolates, as well as the minimum inhibitory concentration (MIC) of OTC, the occurrence of major OTC‐resistant genes and multiple‐antibiotic resistance in the isolates. Methods and Results: Shrimps were collected from culture ponds, and the homogenates of whole bodies were plated on tryptic soy agar supplemented with or without OTC. Percentages of OTC‐resistant bacteria were 0·3–52·1% in white‐leg samples and 0·008–22·3% in black tiger samples. Analyses of 16S rDNA sequences indicated that most OTC‐resistant isolates were closely related to Aeromonas spp. and Lactococcus garvieae. MICs of OTC were 4–128 μg ml^?1 in the OTC‐resistant aeromonads and 128–256 μg ml^?1 in OTC‐resistant L. garvieae. OTC resistance was found to be conferred by the genes tet(A), tet(C), tet(D), tet(E), tet(M) and tet(S), detected either singly or in pairs. No resistance to ceftazidime, imipenem or chloramphenicol was observed in any isolate. Conclusions: Both species of shrimp are associated with OTC‐resistant bacteria, occasionally at high densities exceeding 10⁶ cfu g^?1. The associated bacteria, predominantly Lactococcus and Aeromonas genera, are potential pathogens and are reservoirs of a variety of OTC‐resistant genes. Significance and Impact of the Study: Cultured shrimps can be vehicle to carry OTC‐resistant bacteria to domestic and foreign consumers via the food chain. Very low populations of OTC‐resistant bacteria observed in the several ponds suggest that levels of the resistant bacteria are artificially high and should be reduced in farmed shrimps.     

Na+-K+ ATPase and carbonic anhydrase activities in larvae,postlarvae and adults of the shrimp Penaeus japonicus (Decapoda,Penaeidea)

• 1.1. Activities of Na⁺-K⁺ ATPase and carbonic anhydrase were measured through the early post-embryonic development of Penaeusjaponicus. In adults, only the Na⁺-K⁺ ATPase activity was measured.
• 2.2. ATPase activity was variable in the successive development stages. From zero in nauplii, the activity slightly increased in zoeae, and rose sharply in mysis stages 2 and 3.
• 3.3. A further significant increase in activity was noted at the transition from late mysis to early postlarvae, concomitant with a change from the larval osmoconforming pattern of osmoregulation to the postlarval and adult hyper-hyporegulating pattern.
• 4.4. The activity of Na⁺-K⁺ ATPase, measured in isolated cephalothorax, increased from PL3 to PL4 to its maximum value in PL5; at this stage, osmoregulatory capacity was fully efficient.
• 5.5. In young stages of P. japonicus, the variations in Na⁺-K⁺ ATPase activity appear correlated with the development of osmoregulatory ultrastructures, and with osmoregulation and salinity tolerance.
• 6.6. These results are discussed with regard to their ecological and physiological implications.
• 7.7. In adults, the activity of Na⁺-K⁺ ATPase was high in gills and epipodites and no activity was detected in branchiostegites. These results are related to the ultrastructure of these organs.
• 8.8. The activity of carbonic anhydrase did not change significantly in larval and postlarval stages.
• 9.9. From these results, it is proposed that the effector sites of osmoregulation are located in branchiostegites, pleurae and epipodites in postlarvae, and in epipodites and mainly in gills in adults.