Boonsirm Withyachumnarnkul

Molecular characterization of gonad-inhibiting hormone of Penaeus monodon and elucidation of its inhibitory role in vitellogenin expression by RNA interference

One of the important peptide hormones that control reproduction in crustaceans is gonad‐inhibiting hormone (GIH). GIH is known to modulate gonad maturation by inhibiting synthesis of vitellogenin (Vg), the precursor of yolk proteins. In this study, a cDNA encoding a GIH (Pem‐GIH) from the eyestalk of Penaeus monodon was cloned using RT‐PCR and RACE techniques. Pem‐GIH cDNA is 861 bp in size with a single ORF of 288 bp. The deduced Pem‐GIH consists of a 17‐residue signal peptide and a mature peptide region of 79 amino acids with features typical of type II peptide hormones from the CHH family. Pem‐GIH transcript was detected in eyestalk, brain, thoracic and abdominal nerve cords of adult P. monodon. The gonad‐inhibiting activity of Pem‐GIH was investigated using the RNA interference technique. Double‐stranded RNA, corresponding to the mature Pem‐GIH sequence, can trigger a decrease in Pem‐GIH transcript levels both in eyestalk ganglia and abdominal nerve cord explant culture and in female P. monodon broodstock. The conspicuous increase in Vg transcript level in the ovary of GIH‐knockdown shrimp suggests a negative influence for Pem‐GIH on Vg gene expression, and thus implies its role as a gonad‐inhibiting hormone. This is the first report to demonstrate the use of double‐stranded RNA to elucidate the function of GIH in P. monodon.

DNA fragment of Penaeus monodon baculovirus PmNOBII gives positive in situ hybridization with white-spot viral infections in six penaeid shrimp species

Abstract PmNOBII was first described from experimentally infected shrimp, but contemporary reports showed that white-spot virus infections in several penaeid shrimp species exhibited similar gross signs and histopathology. Using laboratory infected specimens of Penaeus monodon , DNA of the non-occluded baculovirus PmNOBII was extracted and digested with BamHI and EcoRI. Resulting DNA fragments were ligated with Bluescribe vector using T4 ligase and competent cells of Escherischia coli JM 107 were transformed. Two recombinant clones that gave negative hybridization with P. monodon DNA but positive hybridization with PmNOBII DNA were selected. Inserted DNA fragments of 0.9 kbp and 4.2 kbp were obtained from these clones after plasmid digestion with BamHI and EcoRI. These fragments were subsequently labeled with digoxygenin for visualization and tested using the in situ DNA hybridization technique with tissues from PmNOBII infected and non-infected laboratory shrimp. For viral infected nuclei identified by H and E staining in parallel samples, the 4.2 kbp fragment gave a stronger DNA hybridization signal than did the 0.9 kbp fragment. The 4.2 kbp fragment was then used for in situ DNA hybridization tests with commercially or experimentally cultivated shrimp specimens showing gross signs and histopathology characteristic of white-spot virus infection. Field signs of the disease included general reddish coloration, white granules of 1–2 mm under the cuticle and rapid mortality. Normal histology (H and E) revealed Cowdry-A type nuclear inclusions that developed to produce basophilic hypertrophied nuclei typical of PmNOBII, and transmission electron microscopy revealed characteristic rod shaped virions. All these specimens gave positive hybridization results, and included cultivated shrimp specimens of Penaeus chinensis, P. indicus, P. japonicus, P. merguiensis, P. monodon and P. vannamei obtained from various countries in Asia between August 1993 and January 1995. The data indicate that PmNOBII, or closely related variants, are currently responsible for a widespread epizootic in the Asian shrimp farming industry.

Enterocytozoon hepatopenaei sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships

A new microsporidian species, Enterocytozoon hepatopenaei sp. nov., is described from the hepatopancreas of the black tiger shrimp Penaeus monodon (Crustacea: Decapoda). Different stages of the parasite are described, from early sporogonal plasmodia to mature spores in the cytoplasm of host-cells. The multinucleate sporogonal plasmodia existed in direct contact with the host-cell cytoplasm and contained numerous small blebs at the surface. Binary fission of the plasmodial nuclei occurred during early plasmodial development and numerous pre-sporoblasts were formed within the plasmodium. Electron-dense disks and precursors of the polar tubule developed in the cytoplasm of the plasmodium prior to budding of early sporoblasts from the plasmodial surface. Mature spores were oval, measuring 0.7x1.1microm and contained a single nucleus, 5-6 coils of the polar filament, a posterior vacuole, an anchoring disk attached to the polar filament, and a thick electron-dense wall. The wall was composed of a plasmalemma, an electron-lucent endospore (10nm) and an electron-dense exospore (2nm). DNA primers designed from microsporidian SSU rRNA were used to amplify an 848bp product from the parasite genome (GenBank FJ496356). The sequenced product had 84% identity to the matching region of SSU rRNA from Enterocytozoon bieneusi. Based upon ultrastructural features unique to the family Enterocytozoonidae, cytoplasmic location of the plasmodia and SSU rRNA sequence identity 16% different from E. bieneusi, the parasite was considered to be a new species, E. hepatopenaei, within the genus Enterocytozoon.

Knocking down caspase-3 by RNAi reduces mortality in Pacific white shrimp Penaeus (Litopenaeus) vannamei challenged with a low dose of white-spot syndrome virus.

Apoptosis has long been observed in viral target organs of white-spot syndrome virus (WSSV)-infected shrimp and whether the phenomenon helps the shrimp to survive the infection or is a factor leading to mortality is still controversial. If the shrimp mortality is a result of triggered apoptosis, then inactivation of caspase-3, a key protein in the induction of apoptosis, should improve shrimp survival upon challenge with WSSV. To test this prediction, we identified and characterized a caspase-3 homologue (cap-3) from the Pacific white shrimp Penaeus (Litopenaeus) vannamei and used this information to silence cap-3 expression by RNA interference prior to WSSV challenge. After confirming the efficacy of cap-3 silencing, its effects on mortality at high and low doses of WSSV were evaluated. In a high-dose WSSV challenge, cap-3 silencing had no significant effect on WSSV-induced mortality, except for a delay in mean time to death. However, at a low-dose WSSV challenge, cap-3 silencing correlated with a lower level of cumulative mortality, relative to silencing of a control gene, suggesting that apoptosis may exacerbate rather than decrease mortality in WSSV-challenged shrimp.

N-acetyltransferase and melatonin levels in the optic lobe of giant freshwater prawns, Macrobrachium rosenbergii de man

Abstract 1. 1. N-Acetyltransferase (NAT), and melatonin were determined at 3-hour intervals in the optic lobe of the giant freshwater prawn, Macrobrachium rosenbergii de Man. 2. 2. A relatively high activity of NAT, compared to that in rat pineal, was found in the tissue. The enzyme did not show a significant diurnal rhythm although a slight suppressive level was detected at night. 3. 3. Melatonin level varied considerably and showed a peak during the day (1500 hr) and a nadir during the night (2400 hr). 4. 4. The results suggest that the optic lobe has certain biochemical activities similar to those found in pineal glands of vertebrates

Solvent extracts of the red seaweed Gracilaria fisheri prevent Vibrio harveyi infections in the black tiger shrimp Penaeus monodon

Vibriosis is a common bacterial disease that can cause high mortality and morbidity in farmed shrimp. Since compounds from seaweed have been reported to have anti-bacterial and immunostimulant activity, this study was conducted to determine whether solvent extracts from the red seaweed Gracilaria fisheri might be a possible alternative for prevention and treatment of shrimp vibriosis caused by Vibrio harveyi. Seaweed extracts prepared using ethanol, methanol, chloroform and hexane were evaluated for anti-V. harveyi activity by the disc-diffusion method. The ethanol, methanol and chloroform extracts showed activity against a virulent strain of V. harveyi with potency (minimal inhibitory concentrations in the range of 90-190 μg ml(-1)) equivalent to the antibiotic norfloxacin. The ethanol extract was not toxic to the brine shrimp Artemia salina when it was fed to them for enrichment prior to their use, in turn, as feed for postlarvae of Penaeus monodon. Postlarvae fed with these enriched Artemia gave significantly lower mortality than control postlarvae after challenge with V. harveyi. In addition, P. monodon juveniles injected with the ethanol extract showed a significant increase in the total number of haemocytes and an increased proportion of semi-granulocytes and granulocytes when compared to control shrimp. The activities of phenoloxidase and superoxide dismutase were also increased, with an accompanying increase in superoxide anion production. When these juvenile shrimp were challenged with V. harveyi, mortality was markedly reduced compared to that of control shrimp. The results indicated that ethanol extracts of G. fisheri had immunostimulant and antimicrobial activity that could protect P. monodon against V. harveyi.

N-Acetyltransferase, hydroxyindole-O-methyltransferase and melatonin in the optic lobes of the giant tiger shrimp Penaeus monodon

Abstract: The activities of the enzymes N‐acetyltransferase (NAT) and hydroxyin‐dole‐O‐methyltransferase (HIOMT) and the hormone melatonin were studied in the optic lobe of the subadult giant tiger shrimp Penaeus monodon. Compared with the level in other species, a relatively high level of NAT activity that was temperature‐ and pH‐dependent were observed. The NAT enzyme had a relatively high maximum velocity (Vmax, 100 pmol/hr/μg protein) and low Michaelis constant (Km, 22 μM), when tryptamine is used as substrate. In contrast to the high level of NAT activity, HIOMT activity and melatonin levels were low in the optic lobe of the giant tiger shrimp. Sex differences in the levels of NAT activity and melatonin, which are observed in a freshwater species Macrobrachium rosenber‐gii, were not noticeable in the saltwater species P. monodon, at least not when they were in their subadult stage.

Continuous light increases N-acetyltransferase activity in the optic lobe of the giant freshwater prawn Macrobrachium rosenbergii de Man (Crustacea: Decapoda).

Giant freshwater prawns, Macrobrachium rosenbergii de Man, were reared under three different lighting conditions: continuous darkness (DD), 12 hr of light and 12 hr of darkness (LD 12:12) and continuous light (LL). After one month, the prawns were sacrificed and optic lobes isolated from the eyestalks were determined for N-acetyltransferase (NAT) activities and melatonin concentrations. Gonads were weighed and examined under light microscopy. The optic lobes from LL prawns contained significantly higher activities of NAT than those from LD 12:12 prawns. The melatonin concentrations and size and histological features of the gonads from the three groups of prawns did not differ. The results indicate that continuous light increases NAT activities in the optic lobe of M. rosenbergii but has no drastic effect on gonadal growth.

Induction of the Acrosome Reaction in Black Tiger Shrimp (Penaeus monodon) Requires Sperm Trypsin-Like Enzyme Activity

Abstract Trypsin-like enzymes in egg water (EW), a natural acrosome reaction (AR) inducer, are known for their importance in shrimp AR. In this report, we describe a unique phenomenon of the AR of black tiger shrimp (Penaeus monodon) sperm. It was completed within 45–60 sec and comprised only the acrosomal exocytosis and depolymerization of the sperm head anterior spike. We used peptidyl fluorogenic substrates to show the presence of trypsin-like enzymes in P. monodon EW and sperm, but minimal activities of chymotrypsin-like enzymes. In sperm, these trypsin-like enzymes existed both on the sperm surface and in the acrosome. The acrosomal enzyme was revealed as a 45-kDa band by fluorogenic substrate in-gel zymography. Although EW possessed high trypsin-like enzyme activities, they were not essential for the AR induction; EW pretreated with an irreversible trypsin inhibitor, or heat-inactivated EW (HI-EW), to abolish the trypsin-like activities could still induce the AR. The HI-EW-induced AR was inhibited by the presence of a membrane impermeant soybean trypsin inhibitor (SBTI) in the sperm suspension, indicating the significance of sperm-borne trypsin-like enzymes (on the surface and/or in the acrosome) in this AR process. However, pretreatment of sperm with SBTI followed by its removal from the suspension still allowed the AR to occur within 5 min of sperm exposure to HI-EW. Since trypsin-like activity of the SBTI-pretreated sperm surface at 5 min after SBTI removal was at the minimal level, our results suggest the importance of the acrosomal trypsin-like enzyme in the AR process.

Evaluation of colorimetric loop‐mediated isothermal amplification assay for visual detection of Streptococcus agalactiae and Streptococcus iniae in tilapia

Streptococcus agalactiae and Strep. iniae are bacterial pathogens that cause streptococcosis in many fish species. An accelerated colorimetric loop‐mediated isothermal amplification (LAMP) assay with pre‐addition of calcein was established, and the transmission and detection of Strep. agalactiae and Strep. iniae in tilapia under natural aquatic environment were investigated. A positive reaction was observed by a colour change from orange to green through the naked eyes after completion at 63°C for 30 min with 10 times higher sensitivity than that of nested PCR assays and without cross‐amplification with other fish bacterial pathogens. All sample types of Nile and red tilapia (broodstock, fertilized egg, fry) were Strep. agalactiae‐ and Strep. iniae positive by this new method, implying that they could be vertically transmitted. With its application for screening broodstock and fry before stocking and for monitoring fish health in grow‐out ponds, the method would become very useful in fish farming industry.