Anti-oomycetes and immunostimulatory activity of natural plant extract compounds against Saprolegnia spp.: Molecular docking and in-vitro studies.

This study aimed to investigate the effectiveness of five natural plant extract compounds Curcumin (CUR); Eugenol (EUG), Cinnamaldehyde (CIN), Stigmasterol (ST) and Morin (MOR), on two species of Saprolegnia; Saprolegnia parasitica and S. australis. Selective compounds were screened for the minimum inhibitory concentration, first for anti-oomycetes activity and then mycelium growth inhibition, spore germination inhibition and colonisation test. Nitric oxide production and myeloperoxidase activity of the compounds were tested in head kidney leukocytes of rainbow trout, Oncorhynchus mykiss to assess the immunostimulatory potential. Molecular docking of effective compounds was carried out with effector proteins of S. parasitica to investigate the target binding sites. Among all, CUR could completely inhibit zoospore production and significantly (p ≤ .05) inhibit hyphal growth at 16 mg l-1 against S. parasitica and S. australis. CIN at the concentration of 50 mg l-1 completely inhibited hyphal growth of both Saprolegnia spp., although the zoospore production of S. parasitica and S. australis was reduced at 25 mg l-1 and 10 mg l-1. In the case of EUG, significant inhibition of the hyphal growth and germination of S. parasitica zoospores was observed at 50 mg l-1. ST and MOR did not show antioomycetes activity. The molecular docking results were consistent with in vitro studies, possibly due to the binding with the vital proteins (Plasma membrane ATPase, V-type proton ATPase, TKL protein kinase, Host targeting protein 1) of S. parasitica and ultimately inhibiting their activity. CUR and CIN showed increased nitric oxide production at the highest concentration of 250 and 256 mg l-1 but the value was not significant (p ≤ .05) with control. CUR showed significantly higher peroxidase activity (p ≤ .05) at a concentration of 256 mg l-1 though values were significantly similar with concentration from 16 to 128 mg l-1. The nitric oxide and total peroxidase activity of rainbow trout leukocytes in the case of CIN showed a significant difference only at 250 mg l-1 against the control. The results conclude that CUR, CIN showed the better anti-Saprolegnia activity and could be used as phyto-additives in aquaculture. Among all, the inclusion of CUR as phyto-additives will provide additional immunostimulatory activity.

Effects of rearing temperature on egg incubation, growth, standard metabolic rate, and thermal tolerance of chocolate mahseer, Neolissochilus hexagonolepis.

The present study was aimed to assess the effect of temperatures on egg incubation, growth, standard metabolic rate (SMR), and thermal tolerance of a near threatened Himalayan hill stream chocolate mahseer (Neolissochilus hexagonolepis). For the hatching study, eggs were incubated in four temperatures (17, 20, 23, and 26 °C). The total hatching and free-swimming larvae percentage were higher at 23 °C (p < 0.05). Experiment I (for validation of the CTmax method) was carried out by incubating eggs at 17 °C and 23 °C. The CTmax was estimated in response to different warming rates (1-18°C h-1), acclimation temperatures (17 and 23°C), and the age of fishes (8, 15, 35 dph). The results suggested that a warming rate of 18°C h-1 could be used for the thermal tolerance study of yolk-sac larvae (8 dph) and 35 dph larvae, but for free-swimming larvae (15 dph) up to 3°C h-1 is suitable. Experiment II (for growth, SMR and thermal tolerance) was carried by acclimatizing 15 dph larvae in five temperatures (15, 19, 23, 27, and 31 °C) for 60 days. The mean growth rate increased with the increase in temperature from 15°C to 27°C (1.30-3.58% day-1) and decreased at 31°C. The mean SMR of the chocolate mahseer in the above acclimation temperatures was ranged from 1.14 ± 0.36 to 2.81 ± 0.15 µgO2h-1mg-1 and were significantly different (p < 0.01). The Q10 with the SMR of the fish suggested the preferred temperature ranged between 23 and 27 °C, and the optimum temperature for growth (ToptG) was estimated to be 25 °C. Chocolate mahseer is an eurythermal species which is advantageous for aquaculture practices due to its wide thermal tolerance zone (411.68°C2 in 15 to 31 °C acclimation temperature range) and high ARR values (0.49 - 0.54).

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.

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.

Tenets in Microbial Endocrinology: A New Vista in Teleost Reproduction.

Climate vulnerability and induced changes in physico-chemical properties of aquatic environment can bring impairment in metabolism, physiology and reproduction in teleost. Variation in environmental stimuli mainly acts on reproduction by interfering with steroidogenesis, gametogenesis and embryogenesis. The control on reproductive function in captivity is essential for the sustainability of aquaculture production. There are more than 3,000 teleost species across the globe having commercial importance; however, adequate quality and quantity of seed production have been the biggest bottleneck. Probiotics are widely used in aquaculture as a growth promoter, stress tolerance, pathogen inhibition, nutrient digestibility and metabolism, reproductive performance and gamete quality. As the gut microbiota exerts various effects on the intestinal milieu which influences distant organs and pathways, therefore it is considered to be a full-fledged endocrine organ. Researches on Gut-Brain-Gonad axis (GBG axis) and its importance on physiology and reproduction have already been highlighted for higher mammals; however, the study on fish physiology and reproduction is limited. While looking into the paucity of information, we have attempted to review the present status of microbiome and its interaction between the brain and gut. This review will address a process of the microbiome physiological mechanism involved in fish reproduction. The gut microbiota influences the BPG axis through a wide variety of compounds, including neuropeptides, neurotransmitter homologs and transmitters. Currently, research is being conducted to determine the precise process by which gut microbial composition influences brain function in fish. The gut-brain bidirectional interaction can influence brain biochemistry such as GABA, serotonin and tryptophan metabolites which play significant roles in CNS regulation. This review summarizes the fact, how microbes from gut, skin and other parts of the body influence fish reproduction through the Gut-Brain-Gonad axis.

Spawning substrate preference and spawning behavior of chocolate mahseer, Neolissochilus hexagonolepis.

Captive breeding programs for Neolissochilus hexagonolepis are essential for population restoration. To develop an efficacious method for enhancing N. hexagonolepis spawning in captivity, there was examination of: (1) different types of spawning substrate and (2) area of spawning in the substrate. The study was conducted to describe spawning behavior of males and females. There was a choice of three substrates in which to spawn: gravel, small cobble, and coarse sand. There was preferential choosing of gravel followed by cobble with there being no use of sand for spawning. Behavior of N. hexagonolepis included preparation of a spawning pit by females, a behavior that has not been previously ascertained for cyprinids. Males expressed courting behaviors, including chasing, nudging, and quivering. Courting males expressed aggressive behaviors towards other males. Results from the present study are the first on the volitional spawning of N. hexagonolepis in captivity using spawning substrate. It was further revealed that using a gravel substrate tray would also be a feasible approach for egg production. Mean total eggs per female and mean fertilized eggs collected were less when there was siphoning used for egg collections in the preference study. Hence, stripping was implemented to increase the egg collection when spawning behaviors were observed. Total eggs collected were 40,540 with 3685 eggs per female, 90.3% fertilization rate, 82.8% hatching rate, and 97.4% free-swimming larvae survival rate. The implications of this study could be beneficial for enhancing the natural population through environmental management and developing a viable egg production technique in captivity.

Duration of stunting impacts compensatory growth and carcass quality of farmed milkfish, Chanos chanos (Forsskal, 1775) under field conditions.

An 18-months field trial was performed to explore the effect of duration of stunting on growth, digestive enzymes and carcass quality in Chanos chanos. Milkfish fry (weight of 1.25 ± 0.03 g and length of 5.53 ± 0.03 cm) were stocked in earthen ponds of 0.02 ha, in triplicate, for different duration of stunting, viz., 4 months (Treatment-1; T4), 8 months (Treatment-2; T8) and 12 months (Treatment-3; T12) and a normal seed (Control; C) separately. In the stunting phase, fish were stocked at higher stocking density (0.2 million/ha) and fed de-oiled rice bran at sub-optimal level. Post-stunting or re-feeding phase commenced immediately after completion of respective stunting duration and fish were reared for the rest of the period to complete the total rearing period of 18 months. In post-stunting, fish stocking density was adjusted to (5000 pieces/ha) and fed at an optimum level (3%). At the end of stunting phase, the study found a significant reduction in growth, survival, digestive enzymes activity, except protease in the T4 group, and carcass nutrients composition of stunted fish. However, in the initial phase of post-stunting, T8 group exhibited an elevated specific growth rate (5.00 ± 0.092%/day), body weight gain (80.82 ± 1.28 g), amylase (0.585 ± 0.021 U/mg protein), protease (5.48 ± 0.13 U/mg protein), and lipase activity (7.92 ± 0.32 U/mg protein). All stunted fish groups displayed a compensatory growth response in post-stunting, but a complete growth compensation was observed in T8 group, which resulted in better feed conversion ratio (3.03 ± 0.04) feed efficiency ratio (0.33 ± 0.01), protein efficiency ratio (1.91 ± 0.03), survival (91.38 ± 0.07%) and digestive enzyme activities. Similarly, at the end of post-stunting, carcass analysis revealed a complete restoration of nutrients in stunted fish and significantly higher protein content in T8 group. Further, the study found lower meat and higher bone contents in normally reared fish than the post-stunted fish which revealed the carcass quality improvement in post-stunted fish thus indicates superiority of the stunting process over normal rearing. Overall, the study suggests that stunting of milkfish, for 8 months (T8), positively affects its growth, survival, digestive enzyme activities and carcass quality which in turn, shall help to overcome the contemporary challenges in milkfish culture.

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.