Targeting TtVgR via siRNA Knockdown Elicits Ovarian Cell Death in the Tri-spine Horseshoe Crab.

The vitellogenin present in the bloodstream undergoes internalization into developing oocytes through the vitellogenin receptor (VgR), a process mediated by receptor-mediated endocytosis. VgR plays a crucial role in facilitating the accumulation of vitellogenin and the maturation of oocytes. In this study, we characterized a Tachypleus tridentatus vitellogenin receptor (TtVgR) gene from the tri-spine horseshoe crab, revealing a length of 1956 bp and encoding 652 amino acid residues with 12 exons. TtVgR has a molecular weight of 64.26 kDa and an isoelectric point of 5.95. Predictions indicate 85 phosphorylation sites and 7 glycosylation sites within TtVgR. Transcriptional analysis demonstrated specific expression of TtVgR in the ovary and yellow connective tissue. TtVgR was identified and distributed in the plasma membrane of oocytes. The siRNA-mediated TtVgR knockdown significantly reduced the transcriptional activity of TtVgR. This depletion induced excessive ROS production, resulting in DNA damage in ovarian primary cells. TUNEL and flow cytometry analyses confirmed ovarian cell apoptosis following TtVgR knockdown, indicating DNA damage in ovarian primary cells. These findings underscore the importance of TtVgR in ovarian cell development, suggesting its potential involvement in vitellogenesis and oocyte maturation. This knowledge may inform innovative breeding strategies and contribute to the sustainable management and conservation of the tri-spine horseshoe crab.

Hoven's carp Leptobarbus hoevenii strategized metabolism needs to cope with changing environment.

Current water warming and freshwater acidification undoubtedly affect the life of aquatic animals especially ammonotelic teleost by altering their physiological responses. The effect of temperature (28 °C vs 32 °C) and pH (7 vs. 5) on the metabolic compromising strategies of Hoven's carp (Leptobarbus hoevenii) was investigated in this study. Fishes were conditioned to (i) 28 °C + pH 7 (N28°C); (ii) 32 °C + pH 7 (N32°C); (iii) 28 °C + pH 5 (L28°C) and (iv) 32 °C + pH 5 (L32°C) for 20 days followed by osmorespiration assay. Results showed that feeding performance of Hoven's carp was significantly depressed when exposed to low pH conditions (L28°C and L32°C). However, by exposed Hoven's carp to L32°C induced high metabolic oxygen intake and ammonia excretion to about 2x-folds higher compared to the control group. As for energy mobilization, Hoven's carp mobilized liver and muscle protein under L28°C condition. Whereas under high temperature in both pH, Hoven's carp had the tendency to reserve energy in both of liver and muscle. The findings of this study revealed that Hoven's carp is sensitive to lower water pH and high temperature, thereby they remodeled their physiological needs to cope with the environmental changes condition.

Female tilapia, Oreochromis sp. mobilised energy differently for growth and reproduction according to living environment.

This study was conducted to investigate the energy mobilisation preference and ionoregulation pattern of female tilapia, Oreochromis sp. living in different environments. Three different treatments of tilapia as physiology compromising model were compared; tilapia cultured in recirculating aquaculture system (RAS as Treatment I-RAS), tilapia cultured in open water cage (Treatment II-Cage) and tilapia transferred from cage and cultured in RAS (Treatment III-Compensation). Results revealed that tilapia from Treatment I and III mobilised lipid to support gonadogenesis, whilst Treatment II tilapia mobilised glycogen as primary energy for daily exercise activity and reserved protein for growth. The gills and kidney Na+/K+ ATPase (NKA) activities remained relatively stable to maintain homeostasis with a stable Na+ and K+ levels. As a remark, this study revealed that tilapia strategized their energy mobilisation preference in accessing glycogen as an easy energy to support exercise metabolism and protein somatogenesis in cage culture condition, while tilapia cultured in RAS mobilised lipid for gonadagenesis purposes.

siRNA Silencing of FpVtg Induces Ovarian Cell Apoptosis in Redtail Prawn, Fenneropenaeus penicillatus.

Inadequate gonadal maturation and poor spawning performance increasingly threaten the sustainability of shrimp aquaculture. Unraveling the mechanisms regulating ovarian development and maturation hence is critical to address industry challenges. Vitellogenin (Vtg), a precursor of yolk protein found in the hepatopancreas and ovary of shrimp, plays a key role in facilitating shrimp's oocyte maturation and embryonic development after oviposition. This study found that FpVtg was specifically expressed in F. penicillatus hepatopancreas and ovary. FpVtg was localized predominantly in the oocyte cytoplasm and distributed uniformly in the hepatopancreas tissue. Silencing FpVtg led to apoptosis in both hepatopancreas and ovary tissues. Furthermore, FpVtg depletion upregulated the expression of ovarian peritrophin 1, ovarian peritrophin 2, serine proteinase inhibitor 6, and juvenile hormone esterase-like carboxylesterase 1, while downregulated that of vitellogenin, delta-9 desaturase, and insulin-like receptor. KEGG pathway analysis implicated such as PI3K-AKT signaling, RNA transport, ECM-receptor interaction, hippo signaling, oocyte meiosis, and apoptosis were enriched and involved in ovarian development. These findings have provided insights into the FpVtg's reproductive role and the associated regulatory genes and pathways in F. penicillatus. This knowledge can contribute to establishing strategies to improve the breeding and aquaculture production of F. penicillatus by elucidating its vitellogenesis regulation in redtail prawn and other penaeid species. Further characterization of the implicated pathways and genes will clarify the intricacies underlying ovarian maturation.

Sensory-mediated feeding behaviour in the larvae of marble goby (Oxyeleotris marmorata).

This study was conducted to determine the senses that facilitate prey detection in the marble goby (Oxyeleotris marmorata) larvae. The ingestion ratios of live (generate chemical and mechanical stimuli) or frozen Artemia nauplii (generate chemical but no mechanical stimuli) by the intact or free neuromast (mechanoreceptor)-ablated O. marmorata larvae (11 mg/L streptomycin treatment before feeding) under the light or dark (fish vision was obstructed) condition were examined. Vision, mechano-, and chemoreceptions were all found to be essential in prey detection of the O. marmorata larvae. Prey movement has a significant influence as a visual stimuli on the O. marmorata larval feeding as the Artemia nauplii ingestion ratio was approximately 40% higher with significant (p = 0.001, d = 3.0), when the intact larvae were fed with the live (78.1 ± 1.5%), rather than the frozen (40.9 ± 2.8%) Artemia nauplii, under the light condition. This result was assured when no significant difference (p = 0.572, d = 0.2) was found between the ingestion ratios of frozen Artemia nauplii by the intact O. marmorata larvae under light and dark conditions. These findings demonstrate that prey detection in the O. marmorata larvae was facilitated by multi-modal senses, allowing O. marmorata larvae to survive in their natural habitats.

Evaluation of crystalline amino acids as potent stimulatory chemoattractants for the slipper lobster Thenus orientalis.

Intensive research on the effectiveness of chemoattractants has been widely explored to improve the feed qualities in expanding crustacean farming. Taste preferences in slipper lobster remained unknown despite their significant contribution to the lobster fisheries. Chemoattractants allow better performance in aquaculture species by increasing food attractiveness and palatability. Amino acids (AA) have been leading in previous research on crustacean feeding behavior. Given that slipper lobster possesses chemoreceptors to detect and orient towards food, this study investigated an approach to identify the AA with the most potent chemoattractant in eliciting a response from slipper lobster. Behavioral assays were performed to evaluate the responses of slipper lobster Thenus orientalis (carapace length, 52.34 ± 1.52 mm) on 15 crystalline AA and three derivatives of AA (DAA) at three concentrations between 10-1 and 10-3 M as test substances (TS). Meretrix sp. extract was used as a positive control and clean filtered seawater as a negative control. The behavioral responses of 14 T. orientalis were evaluated based on their antennular flicking rate, third maxillipeds activity, and substrate probing by the pereiopods. T. orientalis responded to the solutions of single AA down to a concentration of 10-3 M, excluding histidine and serine. The behavioral activity displayed by T. orientalis increased with the TS concentrations. L-glutamic acid monosodium salt monohydrate, betaine, and glycine solutions elicited the most behavioral responses, whereas histidine exhibited the lowest behavioral responses. Conclusively, L-glutamic acid monosodium salt monohydrate, betaine, and glycine can be potential chemoattractants for T. orientalis.

Silencing of novel TtVtg6-like induced ovarian cell apoptosis in ancient chelicerate Tachypleus tridentatus.

Vitellogenin (Vtg) serves as the precursor of yolk protein and exhibits widespread distribution in tissues, including in the ovary of both vertebrates and invertebrates. Vtg plays a critical role in facilitating oocyte maturation and embryonic development following oviposition. In this study, we have successfully elucidated the complete transcript sequence of TtVtg6-like from an ancient chelicerate Tachypleus tridentatus. The TtVtg6-like transcript encompassed a length of 4887 bp and encoded 1629 amino acids residues. Notably, TtVtg6-like was found to contain 25 exons. Furthermore, the molecular weight and isoelectric point of TtVtg6-like were determined to be 191.6 KDa and 6.73, respectively. Subsequent mRNA expression analysis demonstrated the specific expression of TtVtg6-like in ovary and yellow connective tissue. In addition, TtVtg6-like was located and distributed in both ovary and yellow connective tissue. Intriguingly, employing an siRNA approach to silence TtVtg6-like resulted in a decrease in TtVtg6-like transcription levels. Concomitantly, TtVtg6-like silencing led to increase production of ROS, ultimately resulting in DNA damage and cell apoptosis within the ovarian primary cell. The induction of apoptosis ovarian primary cells due to TtVtg6-like silencing was further corroborated through TUNEL assay and flow cytometry analysis. Overall, our findings underscore the significance of TtVtg6-like in ovarian cell development, revealing its potential association with ovarian cell apoptosis. Consequently, the insights gained from this study contribute to the future exploration of vitellogenesis and ovarian development in T. tridentatus.

Impact of face mask microplastics pollution on the aquatic environment and aquaculture organisms.

Microplastic pollution in our environment, especially water bodies is an emerging threat to food security and human health. Inevitably, the outbreak of Covid-19 has necessitated the constant use of face masks made from polymers such as polypropylene, polyurethane, polyacrylonitrile, polystyrene, polycarbonate, polyethylene, or polyester which eventually will disintegrate into microplastic particles. They can be broken down into microplastics by the weathering action of UV radiation from the sun, heat, or ocean wave-current and precipitate in natural environments. The global adoption of face masks as a preventive measure to curb the spread of Covid-19 has made the safe management of wastes from it cumbersome. Microplastics gain access into aquaculture facilities through water sources and food including planktons. The negative impacts of microplastics on aquaculture cannot be overemphasized. The impacts includes low growth rates of animals, hindered reproductive functions, neurotoxicity, low feeding habit, oxidative stress, reduced metabolic rate, and increased mortality rate among aquatic organisms. With these, there is every tendency of microplastic pollution to negatively impact fish production through aquaculture if the menace is not curbed. It is therefore recommended that biodegradable materials rather than plastics to be considered in the production of face mask while recycle of already produced ones should be encouraged to reduce waste.

Low water pH depressed growth and early development of giant freshwater prawn Macrobrachium rosenbergii larvae.

Macrobrachium rosenbergii is one of the shellfish species with high aquaculture value due to its increasing market demand. However, the comparatively low production volume compared to demand coupled with the rapid decline of the natural environment, consequently, drives the potential depletion of the wild population. The decrease in water pH related to anthropogenic pollution is one of the most critical factors affecting the early life performances of M. rosenbergii. Therefore, this study was designed to examine the effect of low water pH on feeding, growth and development of M. rosenbergii early life stages. Experimental water pH was set as neutral (7.7 ± 0.4); mild-acidic (6.4 ± 0.5) and acidic (5.4 ± 0.2) with triplication at a stocking density of 2 larvae/L for 30 days. As expected, M. rosenbergii larvae were highly sensitive to acidic pH with no larvae survived beyond 48 h of exposure. Feeding, survival and growth of larvae were adversely affected by mild-acidic pH exposure as compared to neutral pH. Larvae exposed to mild-acidic water pH experienced a prolonged larval period and only metamorphosed to the post-larval stage at day-30. Whilst under neutral water pH, larval that metamorphosed to post-larval was first observed on day-23. The negative impact of decreased pH, even in mild-acidic pH exposure, on the feeding, survival, growth and development of M. rosenbergii larvae highlights the urgency of periodic pH monitoring during M. rosenbergii larviculture.

Aquaculture wastewater-raised Azolla as partial alternative dietary protein for Pangasius catfish.

Azolla is a freshwater floating aquatic fern found in the tropical, subtropical and temperate regions with a high nitrogen-fixing rate from the result of symbiotic relationship with the blue-green cyanobacterium, Anabaena azollae. Azolla can effectively remediate aquaculture wastewater owing to its high production capacity and the ability to absorb nutrients and toxic compounds. The Azolla biomass generated as a by-product is currently underutilized and could potentially benefit the aquafeed industry in replacing the unfeasible and expensive fishmeal protein at a certain level. This study evaluates the incorporation of red tilapia wastewater-raised Azolla as a dietary protein for the growth performance, feed efficiency, survival, body indices, body composition and nutrient utilization of Pangasius catfish Pangasianodon hypophthalmus during a 90-days feeding experiment. Dried Azolla was incorporated into four isonitrogenous (30 g kg-1) and isolipidic (12 g kg-1) practical diets containing 0 g kg-1 (Control), 10 g kg-1 (A10), 20 g kg-1 (A20) and 30 g kg-1 (A30) fishmeal protein replacement. One hundred and twenty juveniles with an initial mean weight of 45 ± 15 g were distributed into 12 tanks representing four dietary treatments in triplicates. Results showed significant (p < 0.05) improvement in weight gain (WG), specific growth rate (SGR), protein efficiency ratio (PER) and feed conversion ratio (FCR) in Pangasius catfish fed 10 g kg-1 Azolla protein. Beyond that, WG, SGR, PER and FCR decreased to the lowest value when fed with 30 g kg-1 Azolla protein. No significant (p > 0.05) effects were recorded for feed intake, survival, body indices and nutrient utilization amongst all dietary treatments. In conclusion, Azolla raised from red tilapia aquaculture wastewater can replace fishmeal protein up to 10 g kg-1 in the diet of Pangasius catfish juveniles having better growth, feed efficiency and nutrient utilization without affecting its survival, body indices and body composition.

Chemosensitivity and role of swimming legs of mud crab, Scylla paramamosain, in feeding activity as determined by electrocardiographic and behavioural observations.

Swimming crabs have a characteristic fifth pair of legs that are flattened into paddles for swimming purposes. The dactyl of these legs bears a thick seta along its edge. The chemoreceptive and feeding properties of the seta are supported with scientific evidence; however, there is no available data on the sensitivity of the setae in portunid crabs. The underlying mechanisms of the chemo- and mechano-sensitivity of appendages and their involvement in feeding activities of the mud crab (Scylla paramamosain) were investigated using electrocardiography and behavioural assay, which focused on the responses of the mud crab to chemical and touch stimulus. Electrocardiography revealed the sensory properties of the appendages. The dactyls of swimming legs and the antennules were chemosensitive, but not mechanosensitive and vice versa for the antennae. However, the mouthparts, claws, and walking legs were chemo- and mechanosensitive. Only the chemosensitive appendages, including the swimming legs, were directly involved in feeding. The flattened dactyls of the swimming legs were more efficient than the pointed dactyls of the walking legs in detecting the food organism crawling on the substrate. The structural features enhanced the capacity of the crab in coming into contact with scattered food items. This study revealed that the swimming legs are important appendages for feeding in the mud crab.

High environmental temperature and low pH stress alter the gill phenotypic plasticity of Hoven's carp Leptobarbus hoevenii.

Climate warming and low pH environment are known to negatively impact all levels of aquatic organism from cellular to organism and population levels. For ammonotelic freshwater species, any abiotic factor fluctuation will cause disturbance to the fish, specifically at the gills which act as a multifunctional organ to support all biological processes. Therefore, this study was designed to investigate the effect of temperature (28 vs. 32°C) and pH (7.0 vs. 5.0) stress on the gill plasticity of Hoven's carp after 20 days of continuous exposure. The results demonstrated that high temperature and low pH caused severe changes on the primary and secondary lamellae as well as the cells within lamellae. An increasing trend of the proportion available for gas exchange was noticed at high temperature in both pH exposures, which resulted from a reduction of the primary lamellae width with elongated and thinner secondary lamellae compared to fishes at ambient temperature. Following exposure to high temperature and acidic pH, Hoven's carp experienced gill modifications including aneurysm, oedema, hypertrophy, curling of secondary lamellae, epithelial lifting, hyperplasia and lamellae fusion. These modifications are indicators of the coping mechanism of Hoven's carp to the changing environment in order to survive.

Valorization of Bokashi leachate as feed additive in tilapia farming.

The Bokashi leachate (BL) is a by-product from the anaerobic digestion of food waste, following the Bokashi composting method. Bokashi leachate is acidic and it contains effective microorganisms hence it has potential to be a functional feed additive to the plant proteins based diets for fish farming. This study evaluated the growth performance and feed utilization of the red tilapia (Oreochromis sp.) fingerlings fed with the BL supplemented soybean meal (SBM) based diets. After an 8-week feeding trial, fish fed with the 5% BL supplemented SBM diet attained the highest weight gain. This result was significantly higher (p < 0.05) than those fed with the 0% BL supplemented SBM diet, and comparable (p > 0.05) to those fed with the control full fish meal (FM) diet. Generally, dietary inclusion of BL enhanced the fish feed intake on the SBM diet but it did not show clear sign of improvement in their feed utilization. In addition, no significant difference was found across the hepatosomatic index and viscerosomatic index from all dietary treatments. These outcomes concluded that dietary inclusion of BL can enhance the feed intake and growth performance of the red tilapia fingerlings fed with the SBM based diet without compromising their health, and the optimum BL inclusion level was 5%. Nevertheless, further study on the properties and substances content of the BL produced from different types and ratios of food waste is strongly recommended. In this study, BL was also discovered to be capable of reducing the crude fiber content in the SBM diets. Such observation deserves a further exploitation on the application of BL to manipulate the crude fiber content in the plant proteins based diets in fish farming.

Diel osmorespiration rhythms of juvenile marble goby (Oxyeleotris marmorata).

Oxyeleotris marmorata is an ambush predator. It is known for slow growth rate and high market demand. Farming of O. marmorata still remains a challenge. In order to establish a proper feeding practice to stimulate growth, knowledge of its metabolic processes and cost should be examined. Therefore, this study was designed to investigate the diel osmorespiration rhythms of O. marmorata in response to feeding challenge by using an osmorespirometry assay. The results have shown that oxygen consumption rate of the fed fish was approximately 3 times higher than that of the unfed fish in early evening to support specific dynamic action. Digestion and ingestion processes were likely to be completed within 18-20 h in parallel with the ammonia excretion noticeable in early morning. Under resting metabolism, metabolic oxygen consumption was influenced by diel phase, but no effect was noted in ammonia excretion. As a nocturnal species, O. marmorata exhibited standard aerobic metabolic mode under dark phase followed by light phase, with high oxygen consumption rate found in either fed or unfed fish. It can be confirmed that both the diel phase and feeding have a significant interactive impact on oxygen consumption rate, whereas ammonia metabolism is impacted by feeding state. High metabolic rate of O. marmorata supports the nocturnal foraging activity in this fish. This finding suggested that feeding of O. marmorata should be performed during nighttime and water renewal should be conducted during daytime.

Roles of Carotenoids in Invertebrate Immunology.

Carotenoids are biologically active pigments that are well-known to enhance the defense and immunity of the vertebrate system. However, in invertebrates, the role of carotenoids in immunity is not clear. Therefore, this study aims to review the scientific evidence for the role of carotenoids in invertebrate immunization. From the analysis of published literatures and recent studies from our laboratory, it is obvious that carotenoids are involved in invertebrate immunity in two ways. On the one hand, carotenoids can act as antioxidant enzymes to remove singlet oxygen, superoxide anion radicals, and hydroxyl radicals, thereby reducing SOD activity and reducing the cost of immunity. In some organisms, carotenoids have been shown to promote SOD activity by up-regulating the expression of the ZnCuSOD gene. Carotenoids, on the other hand, play a role in the expression and regulation of many genes involved in invertebrate immunity, including thioredoxins (TRX), peptidoglycan recognition receptor proteins (PGRPs), ferritins, prophenoloxidase (ProPO), vitellogenin (Vg), toll-like receptor (TLRs), heat shock proteins (HSPs), and CuZnSOD gene. The information in this review is very useful for updating our understanding of the progress of carotenoid research in invertebrate immunology and to help identify topics for future topics.

Low pH Water Impairs the Tactile Sense of the Postlarvae of the Giant Freshwater Prawn Macrobrachium rosenbergii.

The effect of low pH on the tactile sense of Macrobrachium rosenbergii postlarvae was determined in the laboratory by means of two behavioural assays: shelter (netting) occupancy and jumping response to touch stimuli (taps) by a glass micropipette. The postlarvae were acclimated to pH 4, pH 5, pH 6 and pH 7.5 (control) in 45 L aquaria 5-7 d before the experiments. Shelter occupancy decreased with pH and was significantly lower at pH 4 and pH 5 than at pH 6 and in the control. The jumping response instantly followed a tap 93-98% of the time in the control, pH 6 and pH 5 treatments. However, the postlarvae showed significantly lower jumping response (65%) at pH 4, indicating an impaired tactile sense. Low pH 4-5 probably degrades the chitin of the sensory setae and inhibits the surface mechanoreceptors of the prawn postlarvae.