Identification of a putative egg-laying hormone in neural and ovarian tissues of the black tiger shrimp, Penaeus monodon, using immunocytochemistry

The existence of an egg-laying hormone (ELH) was identified for the first time in the black tiger shrimp, Penaeus monodon, by means of immunoenzyme and immunofluorescence techniques. This was achieved using a polyclonal antibody produced against expressed recombinant ELH of the female Australian blacklip abalone, Haliotis rubra. The shrimp ELH reactive material was found to be localised within female neurosecretory tissues and the secretory tissue of the antennal gland, but was not identified in the X-organ sinus gland within the eyestalk. It was also present in the ovary, where the amount of ELH present was observed to be greatest in the period prior to spawning. These findings implied that the induction of P. monodon spawning might be involved with humoral regulation relating to ELH expression.     

Up-regulation of ribophorin I after yellow head virus (YHV) challenge in black tiger shrimp Penaeus monodon

This work constitutes the second report from a continuing investigation of shrimp genes that may be involved in apoptosis associated death resulting from yellow head virus (YHV) infection. Here, we describe from the black tiger shrimp Penaeus monodon, a ribophorin I-like gene that is probably a subunit of the oligosaccharyltransferase complex (OST), a key enzyme in N-linked glycosylation that occurs in the endoplasmic reticulum. The OST complex also contains DAD1 (defender against apoptotic death 1) that has been reported to control apoptosis and that we have previously reported from P. monodon. The full length ribophorin I of P. monodon comprised 2157 bp with the ORF of 1806 bp corresponding to 601 deduced amino acids and three putative N-linked glycosylation sites. Analysis revealed hydrophobic properties implying that it could be a membrane protein. Tissue distribution analysis using real-time RT-PCR with SYBR Green revealed that ribophorin I was endogenously expressed in all examined tissues of normal shrimp. However, unlike DAD1 that was down-regulated after YHV challenge, ribophorin I expression was up-regulated and remained high until the moribund stage.     

Hepatopancreatic nuclease of black tiger shrimp Penaeus monodon unlikely to be involved in viral triggered apoptosis

Nucleases are phosphodiesterases that hydrolyze DNA and/or RNA. In a search for shrimp nucleases involved in apoptosis, we discovered a nuclease from hepatopancreatic cDNA of the black tiger shrimp Penaeus monodon. The full-length nuclease gene was amplified and revealed to contain 1668bp corresponding to 381 deduced amino acid residues in the mature enzyme. Sequence analysis indicated 83% nucleic acid identity and 89% amino acid identity to a nuclease from the Kuruma shrimp Penaeus japonicus (also called Marsupenaeus japonicus). Comparative analysis of sequences, conserved motifs and phylogenetic trees indicated that P. monodon nuclease (PMN) belonged to the family of DNA/RNA non-specific endonucleases (DRNSN). RT-PCR analysis using primers specific for PMN mRNA with seven different shrimp tissues revealed that expression in normal shrimp was restricted to the hepatopancreas. Semiquantitative RT-PCR analysis of PMN using hepatopancreatic mRNA from normal shrimp and from shrimp challenged with white spot syndrome virus (WSSV) indicated significant up-regulation of PMN in the hepatopancreas (P<0.05) at the early stage of viral infection but a return to baseline levels as gross signs of disease developed. At the same time, expression was always confined to the hepatopancreas and never seen in other tissues, including those reported to be prime targets for WSSV and subject to increased levels of apoptosis after infection. The results suggested that PMN is probably a digestive enzyme that is unlikely to be involved in hallmark DNA digestion associated with apoptosis.     

YHV-protease dsRNA inhibits YHV replication in Penaeus monodon and prevents mortality

Yellow head virus infects cultured shrimps and causes severe mortality resulting in a great economic loss. Haemolymph injection of dsRNA(pro) corresponding to the protease motif of YHV genome resulted in a complete inhibition of YHV replication. The effect of dsRNA lasted for at least 5 days. Injecting sequence-unrelated dsRNA(gfp) or dsRNA(TSV-pol) also resulted in an inhibition of YHV replication but at a comparatively much less extent. Shrimp mortality was monitored for 10 days when more than 90% shrimps receiving no dsRNA died within 8 dpi. However, those receiving dsRNA(pro) showed no mortality. A partial mortality was observed among the shrimps receiving dsRNA(gfp) or dsRNA(TSV-pol). Thus, Penaeus monodon possesses the sequence-specific protection to YHV infection, most likely through the RNAi pathway, in addition to sequence-independent protection. It gives a new notion that dsRNA induction of antiviral immunity in shrimp goes through two pathways, sequence-independent and sequence-dependent.     

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.     

Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides previously identified in various crustaceans. Out of five isoforms identified in Penaeus monodon, ALFPm3 is the best characterized, exhibits antibacterial and antifungal activities and can protect the shrimp from viral infections. Herein, the most recent identified ALFPm, called ALFPm6, is characterized for its potential role in the shrimp’s immunity. RNA interference-mediated gene silencing was used to study the function of ALFPm6 in comparison to ALFPm3. Knockdown of ALFPm3 gene led to rapid death with a cumulative shrimp mortality of 86% within 7 days, accompanied by a 12- and 50-fold higher bacterial count after 2 days in the haemolymph and hepatopancreas, respectively, compared to the control shrimp injected with GFP dsRNA. In contrast, gene silencing of ALFPm6 alone had no effect on the shrimp mortality, but led to a significant increase in the cumulative mortality and a faster mortality rate following Vibrio harveyi and white spot syndrome virus (WSSV) infections, respectively. These results support the roles of ALFPm6 and ALFPm3 in the protection of shrimp against microbial infections.     

Protection of Penaeus monodon against white spot syndrome virus using a WSSV subunit vaccine

Although invertebrates lack a true adaptive immune response, the potential to vaccinate Penaeus monodon shrimp against white spot syndrome virus (WSSV) using the WSSV envelope proteins VP19 and VP28 was evaluated. Both structural WSSV proteins were N-terminally fused to the maltose binding protein (MBP) and purified after expression in bacteria. Shrimp were vaccinated by intramuscular injection of the purified WSSV proteins and challenged 2 and 25 days after vaccination to assess the onset and duration of protection. As controls, purified MBP- and mock-vaccinated shrimp were included. VP19-vaccinated shrimp showed a significantly better survival (p<0.05) as compared to the MBP-vaccinated control shrimp with a relative percent survival (RPS) of 33% and 57% at 2 and 25 days after vaccination, respectively. Also, the groups vaccinated with VP28 and a mixture of VP19 and VP28 showed a significantly better survival when challenged two days after vaccination (RPS of 44% and 33%, respectively), but not after 25 days. These results show that protection can be generated in shrimp against WSSV using its structural proteins as a subunit vaccine. This suggests that the shrimp immune system is able to specifically recognize and react to proteins. This study further shows that vaccination of shrimp may be possible despite the absence of a true adaptive immune system, opening the way to new strategies to control viral diseases in shrimp and other crustaceans.     

In vivo therapeutic potentiality of red seaweed,Asparagopsis (Bonnemaisoniales,Rhodophyta) in the treatment of Vibriosis in Penaeus monodon Fabricius

The crude extract of the red seaweed, Asparagopsis sp. was evaluated for in vivo antibacterial activity against the shrimp vibrio pathogens. The algal extract was rationalized with commercial shrimp feed and orally administered for different duration of time followed by the artificial bacterial challenge experiment. In dose titration experiments, the oral administration of Asparagopsis sp. at a dosage of 850 mg kg^–1 of biomass was highly efficacious in the treatment of natural infestations of Vibriosis in Penaeus monodon. The results of the confirmatory dose experiment revealed that the prophylactic treatment with moderate dose of 850 mg kg^–1 of biomass day^–1 for four weeks followed by 14 days of post infection therapy was highly effective in controlling Vibrio infection in shrimps. Moreover, results of the percent survival index and microbiological analysis clearly show that Asparagopsis extract incorporated medicated feed had broad therapeutic potential for managing shrimp Vibriosis. In addition, in vivo trials and results obtained in this work are based on the crude organic extract sourced from an unidentified Asparagopsis cryptic lineage, therefore further molecular analysis to identify the species will be required.     

Molecular cloning of the black tiger shrimp (Penaeus monodon) elongation factor 2 (EF-2): sequence analysis and its expression on the ovarian maturation stage

The techniques of homology cloning and anchored PCR were used to clone the elongation factor 2 (EF-2) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp EF-2 (btsEF-2) contained a 5' untranslated region (UTR) of 73 bp, an ORF of 2541 bp encoding a polypeptide of 846 amino acids with an estimated molecular mass of 95 kDa, and a 3( UTR of 112 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btsEF-2 was homological to the EF-2 of other species and even the mammalians. The conserved signature sequence of EF-2 gene family, GTPase effector domain and ADP-ribosylation domain were found in the btsEF-2 deduced amino acid sequence. The temporal expressions of gene in the different ovarian stages were measured by real time PCR. The mRNA expressions of the gene were constitutively expressed in ovary and different during the maturation stages. The result indicated that EF-2 gene was constitutively expressed and could play a critical role in the ovarian maturation stage.     

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.     

广西防城港东湾红树林湿地春季小型底栖动物丰度与生物量

于2019年春季在广西防城港东湾红树林湿地,设置4个断面共13个站位采集沉积物样品,对小型底栖动物的类群组成、丰度和生物量进行研究。共鉴定出11个以上的小型底栖动物类群,分别为自由生活海洋线虫、桡足类、多毛类、寡毛类、介形类、等足类、双壳类、涟虫、海螨类、腹足类、枝角类和其他未鉴定类群;小型底栖动物总平均丰度为(10364±8012) ind·10 cm-2;线虫是绝对优势类群,占小型底栖动物总丰度的95.38%,平均丰度为(9886±7746) ind·10 cm^-2,其次为桡足类,占比为2.14%,平均丰度为(221±358) ind·10cm^-2;小型底栖动物的总平均生物量为(10502±7894)μg·10 cm^-2,各断面的生物量趋势依次为:断面3>断面2>断面1>断面4。     

A novel medium for the development of in vitro cell culture system from Penaeus monodon

Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 ‰ salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8–7.2. Osmolality was adjusted to 720 ± 10 mOsm kg⁻¹ and temperature of incubation was 25 ºC. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (2×), modified L-15 and Grace’s insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation “cocktail”.     

Low salinities adversely affect photosynthetic performance of the mangrove,Avicennia marina

Photosynthetic performance of the highly salt tolerant mangrove, Avicennia marina, was compared at two sites differing insubstrate soil salinities. Carbon dioxide exchange and chlorophyll fluorescence weremonitored at a high salinity site in Durban Bay (35) and at a low salinitysite in Beachwood (< 12). Mean CO₂ exchange, conductanceand transpiration were consistently higher at the high salinity site. Carbondioxide response curves indicated that carboxylation efficiency was higherand stomatal limitation lower at the Durban Bay site. PSII quantum yield,electron transport rates (ETR) and intrinsic PSII efficiency(F_v/F_m) were significantly higher at the high salinity site.Quenching analysis indicated a higher degree ofphotoinhibition/photoprotection in leaves at the low salinity site. Predawnand midday leaf water potentials were –1.6 and –3.1 MPa at Beachwood,compared to –2.6 and –3.8 MPa, respectively, at Durban Bay. Leafconcentrations of Na⁺, K⁺, Ca²⁺, Mg²⁺,Cl^- and N were significantly higher at Durban Bay. Photosyntheticperformance is apparently impaired at the low salinity site in Beachwood asa result of K⁺ and N deficiencies in the leaves.     

Hydrographic Status of Sundarbans Mangrove, Bangladesh with Special Reference to Post-larvae and Juveniles Fish and Shrimp Abundance

Environmental factors, including water parameters were correlated with assemblages of shell and finfish post-larvae and juveniles in the 5 rivers of Sundarbans mangrove for 2 years, located in the south-west of Bangladesh. Fifteen species of shellfish belonging to 6 families and 37 species of finfish belonging to 27 families were recorded. Four species of shrimp (Metapenaeus monoceros, M. brevicornis, Macrobrachium villosimanus and Acetes spp.) and megalopa of crabs (79.3–96.83%), and 4 species of finfish (Panchax melastigma, Liza parsia, L. tade and Gobidae) (40–76%) were the most abundant species. Significant (p<0.05) inter-month variations in temperature, transparency and chlorophyll-a; inter-month and inter-river variations in salinity, conductivity and TDS were observed in the Sundarbans water. Ordination on correlation coefficients between temperature and salinity with the abundance of dominant species showed that Acetes spp. and Gobidae were abundant in high salinity and high temperature areas; Stolephorus tri in low salinity and high temperature areas; Macrobrachiun villosimanus, Leiognathus spp. and Liza tade in low salinity and low temperature areas, however, other species showed variations among 2 years of study. Out of 7 dominant shellfishes, 4 species showed positive correlation to temperature, pH and TDS; 3 species to salinity, transparency, conductivity and chlorophyll-a in the river water. Among 8 dominant fish species, 4 species were found to positively correlate with temperature and pH; 3 with salinity, conductivity and chlorophyll-a; 6 with transparency and 5 species showed positive correlation to TDS in Sundarbans water. Abundance of Penaeus monodon also showed positive correlation to observed water parameters except chlorophyll-a.     

Vegetation dynamics in the Bangladesh Sundarbans mangroves: a review of forest inventories

The Sundarbans, the largest contiguous mangrove forest in the world, covers 6,017 km² of the coastal zone of Bangladesh. Heritiera fomes Buch.-Ham., Excoecaria agallocha L. and Ceriops decandra (Griff.) Ding Hou jointly cover 95% of the forest area. In this study, the results of four forest inventories have been analyzed to understand observed vegetation dynamics of the Sundarbans from 1926 to 1997. The diversity of forest types has been gradually reduced over time, but H. fomes and E. agallocha have maintained their dominance over large portions of the forest. H. fomes is spread over 67% of the vegetated area of the forest, concentrated mostly in the northeastern area, and constitutes 75% of the density of trees with >15 cm dbh. The distribution and stem density of H. fomes show negative relationships with that of E. agallocha and C. decandra. In terms of coverage, E. agallocha is the most common species, spread over 74% of the vegetated area of the forest, and constitutes 39% of the density of trees with >2.5 cm dbh. On a longer timeframe (1926–1997), the dominance (coverage and density of larger diameter trees) of H. fomes as well as that of E. agallocha is declining. Even on parameters such as density of trees with >15 cm dbh, the dominance of E. agallocha is declining at a much greater rate than H. fomes. This observation contradicts the successional schemes proposed by different authors. This might indicate that theorizing successional schemes based on short-term observations on vegetation dynamics is not sufficient. The effect of human interference, changes in hydroedaphic condition and species interaction should be taken into consideration during explaining observed vegetation dynamics. Moreover, the need to understand vegetation trajectories at the micro-scale should be emphasized.