Isolation and expression analysis of Bystin 1 transcript and protein during ovarian development of the giant tiger shrimp Penaeus monodon.

The full-length cDNA of bystin isoform 1 (PmBys1) of the giant tiger shrimp Penaeus monodon was characterized. It was 1553 bp in length containing an ORF of 1365 bp corresponding to a polypeptide of 454 amino acids. The level of PmBys1 mRNA in ovaries was greater than that in other tissues of females and in testes of males in both juveniles and wild broodstock (P < .05). In non-ablated wild female broodstock, PmBys1 mRNA significantly and progressively increased in ovaries from stage I of development, peaking at stage IV (P < .05). Its level in stages I-IV of eyestalk-ablated broodstock was greater than that in non-ablated broodstock (P < .05). Injection of exogenous serotonin (50 µg/g body weight) into 18-month-old shrimp resulted in a significantly increase of ovarian PmBys1 mRNA at 6-48 h post injection (hpi) (P < .05). PmBys1 protein (52 kDa) was found in ovarian stages I-V of non-ablated wild broodstock and II-IV of ablated wild broodstock, respectively. Along with the 52 kDa band, immunoreactive bands of 50 and 43 kDa were also observed in ovarian stages II-IV of both non-ablated and ablated broodstock and in ovaries of post-spawning broodstock. The 43 KDa band was not observed in ovarian stage I of wild female broodstock or in premature juveniles. PmBys1 protein was localized in the ooplasm of previtellogenic oocytes, nucleo-cytoplasmic compartments of vitellogenic oocytes and cortical rods of mature oocytes in wild broodstock. The results implied a possible role for PmBys1 during ovarian development in P. monodon.

Characterization and function of a tachylectin 5-like immune molecule in Penaeus monodon.

Tachylectin5A and its homolog, tachylectin5B both contain a fibrinogen-related domain (FReD) and have been studied in horseshoe crabs, Tachypleus tridentatus and Carcinoscorpius rotundicauda and shown to be involved in host defense. Here, we demonstrate the presence of tachylectin5-like genes in shrimp, Penaeus monodon, designated as Penlectin5-1 (PL5-1) and Penlectin5-2 (PL5-2), which both contain a signal peptide and a single FReD with an acetyl group and a calcium binding sites and they are both structurally similar to horseshoe crab tachylectin/carcinolectin5. The PL5-1and PL5-2 transcript were expressed in various shrimp tissues in normal shrimp, and their expression was upregulated in tissues such as hemocytes and hindgut following challenge with pathogenic Vibrio harveyi. The PL5-2 protein was detected in various tissues as well as in cell-free hemolymph. The biological function of the PL5-2 protein is to recognize some Gram-positive and Gram-negative bacteria regardless whether they are non-pathogenic or pathogenic. They have hemagglutination activity on human erythrocyte and bacterial agglutination activity to both Gram negative and Gram positive bacteria. Possible binding sites of PL5-2 to bacteria could be at the N-acetyl moiety of the GlcNAc-MurNAc cell wall of the peptidoglycan since the binding could be inhibited by GlcNAc or GalNAC. The presence of PL5-2 protein in both circulating hemolymph and intestine, where host and microbes are usually interacting, may suggest that the physiological function of shrimp tachylectin-like proteins is to recognize and bind to invading bacteria to immobilize and entrap these microbes and subsequently clear them from circulation and the host body, and probably to control and maintain the normal flora in the intestine.

Involvement of a tachylectin-like gene and its protein in pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in the shrimp, Penaeus monodon.

A shrimp disease, the so-called acute hepatopancreatic necrosis disease (AHPND) is caused by a specific strain of Vibrio parahaemolyticus (VP) and it has resulted in significant losses to the global shrimp farming industry. In our previous study, three of tachylectin-like genes were cloned and characterized from the intestine of Penaeus monodon, designated as Penlectin5-1 (PL5-1), Penlectin5-2 (PL5-2) and Penlectin5-3 (PL5-3). These three genes all contain fibrinogen-related domain (FReD). The expression level of PL5-1, PL5-2 and PL5-3 was elevated in the stomach after oral administration with AHPND-causing V. parahaemolyticus 3HP (VP3HP). A polyclonal antibody to PL5-2 was successfully produced in a rabbit using the purified recombinant PL5-2 as an immunogen, and this because only the predominant protein PL5-2 could be successfully purified from shrimp plasma by affinity chromatography using a N-Acetyl-d-glucosamine column allowed us to perform functional studies of this lectin. The native purified PL5-2 protein had binding and agglutination activities towards VP3HP. To further understand the functions and the involvements of this lectin in response to AHPND in shrimp, RNAi-mediated knockdown of PL5-1, PL5-2 or PL5-3 was performed prior to an oral administration of VP3HP. As a result, Penlectin5-silencing in shrimp challenged with VP3HP showed higher mortality and resulted in more severe histopathological changes in the hepatopancreas with typical signs of AHPND. These results therefore suggest a role for crustacean fibrinogen-related proteins (FRePs) in innate immune response during the development of AHPND, and maybe also during other infections.

Differentially expressed transcripts in stomach of Penaeus monodon in response to AHPND infection.

Acute Hepatopancreatic Necrosis Disease (AHPND) is an emerging disease in aquacultured shrimp caused by a pathogenic strain of Vibrio parahaemolyticus. As with several pathogenic bacteria, colonization of the stomach appeared to be the initial step of the infection for AHPND-causing Vibrio. To understand the immune responses in the stomach of black tiger shrimp (Penaeus monodon), differentially expressed transcripts (DETs) in the stomach during V. parahaemolyticus strain 3HP (VP3HP) infection was examined using Ion Torrent sequencing. From the total 42,998 contigs obtained, 1585 contigs representing 1513 unigenes were significantly differentially expressed with 1122 and 391 unigenes up- and down-regulated, respectively. Among the DETs, there were 141 immune-related unigenes in 10 functional categories: antimicrobial peptide, signal transduction pathway, proPO system, oxidative stress, proteinases/proteinase inhibitors, apoptotic tumor-related protein, pathogen recognition immune regulator, blood clotting system, adhesive protein and heat shock protein. Expression profiles of 20 of 22 genes inferred from RNA sequencing were confirmed with the results from qRT-PCR. Additionally, a novel isoform of anti-lipopolysaccharide factor, PmALF7 whose transcript was induced in the stomach after challenge with VP3HP was discovered. This study provided a fundamental information on the molecular response in the shrimp stomach during the AHPND infection that would be beneficial for future research.

Interaction of Vibrio spp. with the Inner Surface of the Digestive Tract of Penaeus monodon.

Several species of Vibrio are the causative agent of gastroenteritis in humans. In aquaculture, Vibrio harveyi (Vh) and V. parahaemolyticus (Vp) have long been considered as shrimp pathogens in freshwater, brackish and marine environments. Here we show by using scanning electron microscopy (SEM) that Penaeus monodon orally inoculated with each of these two pathogens via an Artemia diet had numerous bacteria attached randomly across the stomach surface, in single and in large biofilm-like clusters 6 h post-infection. A subsequent marked proliferation in the number of V. harveyi within the biofilm-like formations resulted in the development of infections in the stomach, the upper and middle midgut, but neither in the posterior midgut nor the hindgut. SEM also revealed the induced production of peritrichous pili-like structures by the Vp attaching to the stomach lining, whilst only a single polar fibre was seen forming an apparent physical bridge between Vh and the host's epithelium. In contrast to these observations, no such adherences or linkages were seen when trials were conducted with non-pathogenic Vibrio spp. or with Micrococcus luteus, with no obvious resultant changes to the host's gut surface. In naive shrimp, the hindgut was found to be a favorable site for bacteria notably curved, short-rod shaped bacteria which probably belong to Vibrio spp. Data from the current study suggests that pathogens of P. monodon must be able to colonize the digestive tract, particularly the stomach, where chitin is present, and then they use an array of virulent factors and enzymes to infect their host resulting in disease. Oral infection is a better way of mimicking natural routes of infection; investigating the host-bacteria interactions occurring in the digestive tract may lead to new strategies for the prevention or control of bacterial infections in penaeids.

Characterization and expression of cell division cycle 2 (Cdc2) mRNA and protein during ovarian development of the giant tiger shrimp Penaeus monodon.

The meiotic maturation of oocytes is regulated by the maturation-promoting factor (MPF), a complex of Cdc2 (Cdk1) and Cyclin B. Here, the complete open reading frame (ORF) of Cdc2 in Penaeus monodon was characterized. PmCdc2 were 900bp in length corresponding to a polypeptide of 299 amino acids with the conserved Thr14, Tyr15 and Thr161 residues. Quantitative real-time PCR indicated that the expression level of PmCdc2 in wild intact broodstock was significantly increased in stages II (vitellogenic) and III (early cortical rod) ovaries relative to stage I (previtellogenic) ovaries and peaked in stage IV (mature) ovaries (P<0.05). The expression level of PmCdc2 in stages I-IV ovaries of eyestalk-ablated broodstock was greater than that of the same ovarian developmental stages in intact broodstock (P<0.05). Expression levels of PmCdc2 in ovaries of 18-month-old P. monodon upon 5-HT injection (50µg/g body weight) were significantly increased at 1hour post injection (hpi, P<0.05). Recombinant PmCdc2 protein and its polyclonal antibody were successfully produced. Western blot analysis revealed the expected 34kDa band (PmCdc2) along with a smaller band of 23kDa (ribosomal protein S3) in ovaries of juveniles and various ovarian stages of broodstock. Using phospho-Cdc2 (Thr161) polyclonal antibody, the positive signal of 34kDa was observed in all ovarian stages but the most intense signal was found in stage IV ovaries. Results in the present study indicated that PmCdc2 gene/protein plays an important role in the development and maturation of oocytes/ovaries in P. monodon.

Expression of immune-related genes in the digestive organ of shrimp, Penaeus monodon, after an oral infection by Vibrio harveyi.

In all previous studies, to study shrimp immune response, bacteria were directly injected into the shrimp body and as a consequence the initial step of a natural interaction was omitted. In this study we have instead used an immersion technique, which is a more natural way of establishing an infection, to study immune responses in black tiger shrimp (Penaeus monodon). Normally, Vibrio harveyi (Vh) is highly pathogenic to post-larval shrimp, but not to juveniles which usually resist an infection. In post-larvae, Vh causes a massive destruction of the digestive system, especially in the hepatopancreas and in the anterior gut. We have therefore investigated changes in transcription levels of fifteen immune-related genes and morphological changes in juvenile shrimp following an immersion of shrimp in Vh suspension. We found that a pathogenic bacterium, Vh, has the capacity to induce a local expression of some immune-related genes in shrimp after such a bacterial immersion. Our results show that in the juvenile gut small changes in expression of the antimicrobial peptide (AMP) genes such as antilipopolysaccharide factor isoform 3, crustin and penaeidin were observed. However some other genes were more strongly induced in their expression compared to the AMP genes. C-type lectin, Tachylectin 5a1 and mucin-like peritrophic membrane were increased in their expression and the C-type lectin was affected most in its expression. Several other examined genes did not change their expression levels. By performing histology studies it was found that Vh infection induced a strong perturbation of the midgut epithelium in some regions. As a consequence, the epithelial cells and basement membrane of the infected site were completely damaged and necrotic and massive hemocyte infiltration occurred underneath the affected tissue to combat the infection.

Identification, characterization, and expression of the genes TektinA1 and Axonemal protein 66.0 in the tropical abalone Haliotis asinina.

The genes Tektin A1 and axonemal protein 66.0 were successfully Isolated and characterized in the tropical abalone Haliotis asinina. The full-length cDNAs of Ha-TekA1 and Ha-Axp66.0 were 2166 and 2038 bp long, with ORFs of 1350 and 1683 bp, respectively. Both Ha-TekA1 and Ha-Axp66.0 were expressed in the testes but not in the ovaries or hemocytes of H. asinina adults. In addition, HaAxp66.0 was not expressed in H. asinina juveniles (2, 3, and 5 months old). A tissue expression analysis showed Ha-Axp66.0 to be expressed specifically in the testes, whereas Ha-TekA1 was expressed abundantly in the testes but weakly in the foot, gill, digestive gland, left hypobranchial gland, and mantle. The relative expression levels of Ha-TekA1 and Ha-Axp66.0 were significantly lower in undeveloped testes (stage I) than in developed testes (stages II, III, and IV) of H. asinina (P < 0.05).

Isolation and characterization of testis-specific DMRT1 in the tropical abalone (Haliotis asinina).

The Doublesex Male abnormal-3 Related Transcription factor-1 (DMRT1) gene encodes a protein containing the DNA-binding motif called the DM domain, involved in the sexual development of various species. To gain insight into its implications for gonadal differentiation in the tropical abalone (Haliotis asinina), a DMRT1 homolog was identified and characterized. The full length cDNA of HADMRT1 (1,740 bp with an ORF of 732 bp corresponding to a putative polypeptide of 243 amino acids) and its DM domain-less variant (HADMRT1-like, 1,430 bp with an ORF of 312 bp, 103 amino acids) were successfully isolated and reported for the first time in molluscs. HADMRT1 was specifically expressed in the testes of adult H. asinina (N = 16) but not in whole juveniles (2, 3, 5 months old, N = 6 for each group) and ovaries (N = 16), and pooled hemocytes (from 50 individuals) of adults. Tissue distribution analysis further revealed testis-specific expression of HADMRT1. Semiquantitative RT-PCR illustrated that the relative expression level of HADMRT1 in developed testes (stages II, III, and IV) was significantly greater than that in undeveloped testes (stage I) of abalone broodstock (P < 0.05).

Genetic diversity of the giant tiger shrimp (Penaeus monodon) in Thailand revealed by PCR-SSCP of polymorphic EST-derived markers.

A total of 90 ESTs from normal and 157 from subtractive ovarian cDNA libraries of the giant tiger shrimp (Penaeus monodon) were sequenced. SSCP analysis of disulfide isomerase (DSl), zinc finger protein (ZFP), PMO920, and PMT1700 was carried out for population genetic studies of P. monodon in Thai waters. The number of codominant alleles per locus for overall samples was 6 for PMO920, 5 for PMT1700, and 12 for ZFP, and there were 19 dominant alleles for DSI. The observed heterozygosity of each geographic sample was 0.3043-0.5128 for PMO920, 0.3462-0.4643 for PMT1700, and 0.5000-0.8108 for ZFP. Linkage disequilibrium analysis indicated that genotypes of these loci segregate randomly (P > 0.05). Low genetic distance was found between pairs of geographic samples (0.0077-0.0178). The neighbor-joining tree constructed from the average genetic distance of overall loci allocated the Andaman samples (Satun, Trang, and Phangnga) into one cluster, and Chumphon and Trat into other clusters. Geographic differentiation between Satun-Trat and Satun-Phangnga was found only at the ZFP locus (P < 0.05), suggesting low degrees of genetic subdivision of Thai P. monodon.

Genetic heterogeneity of the tropical abalone (Haliotis asinina) revealed by RAPD and microsatellite analyses.

Genetic heterogeneity of the tropical abalone, Haliotis asinina was examined using randomly amplified polymorphic DNA (RAPD) and microsatellite analyses. One hundred and thirteen polymorphic RAPD fragments were generated. The percentage of polymorphic bands of H. asinina across overall primers was 85.20%. The average genetic distance of natural samples within the Gulf of Thailand (HACAME and HASAME) was 0.0219. Larger distance was observed when those samples were compared with HATRAW from the Andaman Sea (0.2309 and 0.2314). Geographic heterogeneity and F(ST) analyses revealed population differentiation between H. asinina from the Gulf of Thailand and the Andaman Sea (p < 0.0001). Three microsatellite loci (CUHas1, CUHas4 and CUHas5) indicated relatively high genetic diversity in H. asinina (total number of alleles = 26, 5, 23 and observed heterozygosity = 0.84, 0.42 and 0.33, respectively). Significant population differentiation was also found between samples from different coastal regions (p < 0.0001). Therefore, the gene pool of natural H. asinina in coastal Thai waters can be genetically divided to 2 different populations; the Gulf of Thailand (A) and the Andaman Sea (B).

Species identification of the tropical abalone (Haliotis asinina, Haliotis ovina, and Haliotis varia) in Thailand using RAPD and SCAR markers.

A randomly amplified polymorphic DNA (RAPD) analysis was used to identify the species- and population-specific markers of abalone; Haliotis asinina, H. ovina, and H. varia in Thai waters. Fifteen species-specific and six population-specific RAPD markers were identified. In addition, an 1650 bp band (UBC195) that was restricted to H. ovina from the Gulf of Thailand (east) was also found. All of the specific RAPD markers were cloned and sequenced. Twenty pairs of primers were designed and specificity-tested (N = 12 and 4 for target and non-target species, respectively). Seven primer pairs (CUHA1, 2, 4, 11, 12, 13, and 14) were specifically amplified by H. asinina DNA, whereas a single pair of primers showed specificity with H. ovina (CUHO3) and H. varia (CUHV1), respectively. Four primer pairs, including CUHA2, CUHA12, CUHO3, and CUHV1, were further examined against 216 individuals of abalone (N = 111, 73, and 32, respectively). Results indicated the species-specific nature of all of them, except CUHO3, with the sensitivity of detection of 100 pg and 20 pg of the target DNA template for CUHA2 and CUHA12 and CUHV1, respectively. The species-origin of the frozen, ethanol-preserved, dried, and boiled H. asinina specimens could also be successfully identified by CUHA2.