Changes in digestive enzyme activities during the early ontogeny of milkfish, Chanos chanos larvae.

Knowledge of the developmental ontogeny of the digestive system and nutritional requirements of marine fish larvae is a primary requisite for their successful rearing under an optimal feeding regime. In this context, we assessed the activity profile of key digestive enzymes viz., trypsin, chymotrypsin, leucine aminopeptidase, lipase, amylase, and alkaline phosphatase during the early ontogeny of milkfish, Chanos chanos (0 day, 3 days, 6 days, 9 days, 12 days, 15 days, 18 days, 21 days, 25 days, and 30 days post-hatch). Larvae for this study were obtained from the successful breeding of milkfish at ICAR-Central Institute of Brackishwater Aquaculture, India. Growth curves (length and weight) of the larvae indicated a positive morphological development under a standardized feeding regime that comprised Chlorella salina, Brachionus plicatilis, Artemia salina nauplii, and commercial weaning feed for different larval stages. With respect to protein digestion, the specific activity of pancreatic enzymes trypsin and chymotrypsin and intestinal brush border leucine aminopeptidase showed two peaks at 3 dph and 15 dph, following the introduction of rotifer and Artemia nauplii. Similar bimodal peaks were observed for alkaline phosphatase and amylase activities, with the first peak at 3 dph and the second peak at 18 dph and 21 dph, respectively. Whereas in the case of lipase, high activity levels were observed at 0 dph, 3 dph, and 18 dph, with subsequent decreases and fluctuations. Overall, as most of the enzymes were found to have peak activities at 15 to 21 dph, this period can be potentially considered as the developmental window for weaning larvae from live to formulated feeds in milkfish hatcheries.

Nervous necrosis virus titration and antigen quantitation by indirect sandwich enzyme linked immunosorbent assay.

Viral nervous necrosis (VNN) is a serious disease of marine and brackishwater fishes caused by nervous necrosis virus (NNV) resulting in up to 100% mortality in early larval and juvenile stages. Adult fish when infected are asymptomatic and spread the virus vertically to the offspring through milt and eggs. Prevention of vertical transmission of the disease is by using disease free broodstock and vaccinating the brooders. Estimation of antigen content and virus titre is essential to determine antigen/virus concentration in VNN vaccine. A monoclonal antibody based indirect sandwich ELISA was developed to quantify the NNV antigen and to estimate the virus titre by TCID50 coupled ELISA. Mouse hybridoma clones secreting monoclonal antibodies (MAb) against the capsid protein of NNV was developed and characterised. The antibodies reacted specifically with the recombinant capsid protein and purified virus in western blot. Polyclonal antibodies against NNV were used as capture antibodies and MAbs were used as detection antibodies to optimise an indirect sandwich ELISA to detect and quantify capsid protein of NNV. The developed assay had a sensitivity of 390 ng/ml and could detect the virus in clinical samples. The assay coupled with TCID50 could be used to estimate the virus titre rather than by observing the CPE which is laborious and subjective.

The effect of acclimation temperature and optimal temperature gradient for egg and larvae of silver moony (Monodactylus argenteus) during the early ontogenesis.

The early life history of a fish species is regulated by temperature, the most critical environmental cue. Thus, identifying the gradient of temperature that optimises the early development of a species is a prerequisite for standardising hatchery technology. Silver moony, Monodactylus argenteus is a tropical brackishwater ornamental fish that holds scope for the Indian and Global ornamental fish industry. This study unravels the effect of water temperature increments (26, 28, 30, 32, and 34 °C) on embryonic development, hatching, and survival rate, as well as the growth profile and survival rate of larvae at 5 days post-hatch (5 dph). Experiments were conducted to find out the optimal temperature gradient for egg incubation and early larval rearing. The experiment results revealed that the embryogenesis was accelerated at increasing temperatures, especially after the gastrula stage, and apparent differences were evident in each stage. However, the morphological development profile at each embryonic stage was similar throughout the temperature range. The incubation period differed significantly (P < 0.05) between the temperature gradients. The highest rates of hatching (90-100%) and survival after 12 hph were observed at 28 ºC and 30 ºC. Hatched-out larvae demonstrated the highest total length (1.92 ± 0.02 µm) at 34 °C, and the total length decreased at lower temperature levels. The yolk sac volume of larvae was shrunken with an increase in temperature, and a significant difference was observed between the studied temperatures. However, the oil globule diameter did not differ between the different temperatures. The total length and growth rate of 5 dph larvae were significantly different among the temperature treatments and increased with increasing temperature. In contrast, the survival rate of 5 dph larvae was highest at the range of 26 ºC and 30 ºC. The results indicated that the change in temperature from the spawning temperature (29 ± 1 °C) negatively influenced embryogenesis and the early development of M. argenteus. Based on the experimental results, the growth and survival of embryo and larvae were found to be optimum at 28 to 30 ºC. This prediction is of great importance for the effective management in the hatchery production phase, and especially the temperature could be considered the critical environmental cue.

Hypoxia-mediated inhibition of cholesterol synthesis leads to disruption of nocturnal sex steroidogenesis in the gonad of koi carp, Cyprinus carpio.

Reproductively mature koi carps (Cyprinus carpio) showed a prominent diurnal variation of sex steroids with sustained nocturnal rise. Exposure to chronic hypoxia (DO < 0.8 mg/l) disrupted nocturnal sex steroid production in koi carp gonads. Inhibition of sex steroidogenesis is linked to the down-regulation of HMG-Co A reductase (p < 0.05), which acts as a rate-limiting enzyme in the mevalonate pathway for cholesterol production. HMG-CoA reductase inhibition was obvious in the gonads and liver of both sexes during 18.00 h and 21.00 h resulting in hypocholesterolemia (p < 0.05). The levels of sex steroids, such as estradiol, testosterone, and 11-keto-testosterone in gonads were depleted below the optimum levels owing to disruption of de novo cholesterol synthesis along with attenuation of HDL-cholesterol level in serum. Inhibition of melatonin under hypoxic conditions indicates disruption of melatonin effects on the hypothalamus-pituitary-gonadal (HPG) axis of koi carp. Under severe hypoxic stress, koi carp promoted energy conservation by switching over to the triglyceride (TGA) pathway instead of the mevalonate pathway to suppress cholesterol production. Chronic hypoxia inhibited cholesterol synthesis, a prerequisite for gonadal maturation. It promoted TGA production, as an alternative energy source, suggesting a probable mitigation strategy adopted by hypoxia-tolerant fish to deal with low dissolved oxygen frequently occurring in aquatic bodies.

Broodstock development and captive maturation of hilsa (Tenualosa ilisha) in a brackishwater pond-based system.

Successful captive broodstock development of clupeid fish, hilsa, is one of the major challenges for the artificial propagation of this economically valued fish. The present study provides novel insights into the pond-reared captive broodstock development of hilsa using artificial feed. In the present study, wild collected hilsa fry (weight 1.35 ± 0.13 g, length 53.84 ± 0.95 mm) were reared in grow-out ponds for 6 months followed by maintaining them as broodstock in other ponds using formulated feed in both cases. After 6 months of pond rearing, the average body weight and length of fish were 66.76 ± 4.53 g and 184.75 ± 3.65 mm, respectively. In broodstock ponds, hilsa subadults fed with formulated feed (crude protein 41.06%) ensured significant advancement in ovarian maturity with gonado somatic index of 9.09%, enhanced secondary yolk stage oocytes of 91.66% and enlargement of oocyte diameters up to 570 µm. The broodstock reached somatic growth such as average body weight and average body length of 383.80 ± 27.38 g and 339.33 ± 9.68 mm, respectively, with a condition factor of 1.06 ± 0.15. Gut content analysis revealed the better acceptability of the artificial feed. Lipid profiling of muscle and ovary during gonadal maturation revealed mobilization of fat from muscle to ovary and selective retention of some fatty acids (i.e., C14:0, C18:0, C20:5, C22:6 and C20:4) in the ovary, which might help in gonadal maturation. STATEMENT OF SIGNIFICANCE: Successful rearing of wild-caught hilsa juvenile in captive condition using formulated feed showed its potential as aquaculture species in low saline conditions. Advance stage of maturation in low saline captive conditions showed the possibility for induced breeding of hilsa, a high priced anadromous fish. Mobilization of fat from muscle to ovary and selective retention of some fatty acids in the ovary showed the scope of dietary manipulation for broodstock development of hilsa.

Gonadal recrudescence and annual reproductive hormone pattern of captive female Spotted Scats (Scatophagus argus).

The Spotted Scat (Scatophagus argus), an important euryhaline fish inhabiting mangrove and coastal regions of Indo-Pacific waters, is both an ornamental and food fish in India. Detailed insight into maturation of Spotted Scat when maintained in aquaculture systems, therefore, needs to be elucidated. Lack of information on annual maturation dynamics of female scat collected from their natural habitat and reared in earthen ponds is the basis of this study. Oocytes were classified into five developmental stages: pre-vitellogenic, vitellogenic, late-vitellogenic, ripe, and follicular atresia. Ovarian maturity stages were subsequently categorized as immature (Stage 1), vitellogenesis (Stage 2), maturing (Stage 3), mature (Stage 4), and spent (Stage 5). In oocytes in primary, secondary and tertiary yolk stages, there are greater concentrations of E2 in vitellogenic females between March and June. Significant increases of E2, T, and 17-OHP paralleled the increase of diameter of late-vitellogenic oocytes in maturing females during July. The completion of vitellogenesis and initiation of germinal vesicle migration in the cytoplasm were evident in mature females (Stage 4) with a decreasing trend of sex steroids in and subsequent to the month of August. There were 50 % of oocytes in the final oocyte maturation stage (FOM) (490-620 µm) until completion of Stage 4 in September. The results of this study indicate there is complete ovarian maturation in female scats captured in their natural habitat and maintained in an earthen pond, which may be important information for hatchery management for induction of spawning of Spotted Scat in aquaculture systems.

Resistance of pearlspot larvae, Etroplus suratensis, to redspotted grouper nervous necrosis virus by immersion challenge.

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty-day-old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post-infection, and the infected larvae were subjected to nested RT-PCR and histology. The virus was isolated from infected larvae using SSN-1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT-PCR and the virus was isolated using SSN-1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT-PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.

Diel cyclic hypoxia alters plasma lipid dynamics and impairs reproduction in goldfish (Carassius auratus).

Diel cyclic hypoxia occurs with varying frequency and duration in freshwater habitats, yet little is known about its effects on reproduction of freshwater fishes. The present study shows that long-term exposure of goldfish (Carassius auratus) to cyclic hypoxia (0.8 ± 0.2 mg/l dissolved oxygen) for 9 h or more, per day, altered plasma lipid and sex steroid profiles, which in turn directly or indirectly suppressed ovarian growth and viable spermatozoa production. Hypoxia decreased total cholesterol and high density lipoprotein (HDL p < 0.05) and elevated triglycerides (TG; p < 0.05) in both sexes. Plasma steroid concentrations particularly of 17α-hydroxyprogesterone (17-HP), estradiol (E2), testosterone (T) in females, and T and 11-ketotestosterone (11-KT) in males were attenuated under diel hypoxic conditions. Intriguingly, both diel and continuous hypoxia elevated plasma E2 and vitellogenin levels in males. However, neither lipid nor steroid profiles recorded any variation in a dose-dependent manner in response to diel hypoxia. The reduced GSI, decreased number of tertiary oocytes, and motile spermatozoa in hypoxic fish clearly indicate suppression of gametogenesis. Thereby, prolonged diel cyclic hypoxia may affect valuable fishery resources and fish population structure by impairing reproductive performances and inducing estrogenic effects in males.

p53 dependent apoptotic cell death induces embryonic malformation in Carassius auratus under chronic hypoxia.

Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD), leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf) and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD), ultimately resulting in significant (p<0.05) embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos.