TRPM2 CHANNELS DETECTS AQUATIC POLLUTANTS IN ZEBRAFISH (Danio rerio)

Ha Nam Tran*¹, Hoa Thi Truong¹, Tran Quang Khanh Van¹, Ho Thi Tung¹, Nguyen Duy Quynh Tram¹

¹Hue University of Agriculture and Forestry, No 102 Phung Hung Street, Hue City, Thua Thien Hue Province, Viet Nam

^*Corresponding author: trannamha@huaf.edu.vn 

Pollutants such as reactive oxygen or nitrogen spices (ROS or RNS) in wastewater released into the aquatic environment cause serious impacts on fish health.  In different animal species, transient receptor potential (TRP) proteins have been well known to form sensor cation channels for detection of a variety of environmental stimulants. To elucidate sensing mechanisms that allow fish to avoid pollutants in the environment, we here characterize the zebrafish (Danio rerio) homologue of TRPM2, whose mammalian homologue has been reported to show prominent sensitivity to ROS, especially to hydrogen peroxide (H₂O₂).  Our results showed that the zebrafish TRPM2 (drTRPM2) protein forms a Ca²⁺-permeable cation channel activated by H₂O₂.  Strikingly, in contrast to mammalian TRPM2, drTRPM2 responds to nitric oxide (NO) and nitrite (NO₂^-) but not to warmth (37°C).  This distinctive RNS sensitivity of drTRPM2 channels is mediated by cooperative action of ADP ribose (ADPR) and cGMP-dependent protein kinase (PKG), and sensitivity is mediated mainly by ADPR.  Our mutation analysis suggests that the serine residues at the amino acid position 1227 and 1228 are the major phosphorylation sites for PKG, which is critical for drTRPM2 activation mechanism.  Interestingly, genetic comparison of primary structure of TRPM2 of different animal species reveals that this PKG site is conserved only among cyprinids that includes the carps and zebrafish. In addition, homozygous TRPM2 knock out zebrafish generated using the CRISPR/Cas9 system fails to show repellent behaviors against H2O2 and NO. Thus, TRPM2 channel plays an important role for fish to sense to the polluted environment. Together with this, our data suggest that a unique TRPM2-mediated mechanism enables cyprinids to adapt to their characteristic habitat rich in H₂O₂, NO, and NO₂-.

Keywords: TRPM2, sensor, fish, pollutants, envinronment

DEVELOPMENT OF ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) FOR VNN DETECTION IN Oreochromis spp.

Irfan-Hakimi, R¹, Azila Abdullah^2*, Nur-Nazifah, M¹, Firdaus-Nawi, M¹, Hazreen-Nita, M³

¹Kulliyah of Science, International Islamic University Malaysia, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia

²National Fish Health Research Centre, Batu Maung, 11960 Penang, Malaysia

³Faculty of Agro-Based Industry, University Malaysia Kelantan, Jeli Campus, 17600 Jeli, Kelantan, Malaysia

^*Corresponding author: azila@dof.gov.my

Viral Nervous Necrosis (VNN), also known as viral encephalopathy and retinopathy is a serious viral disease affecting more than 120 species including marine and freshwater farmed fishes. Adult fishes infected with VNN are usually asymptomatic and eventually become carrier that transmit the virus to the offspring through eggs. In responds to the needs of practical and validated tests for the study of Nervous Necrosis Virus (NNV) immunity, Enyzme Linked Immunosorbent Assay (ELISA) protocols were developed, improved and compared for the detection of serum antibodies against the virus in vaccinated fishes. Hence, in this study, ELISA method for VNN detection in Oreochromis spp. was developed. Indirect ELISA using non-purified NNV from cell culture supernatant as coating antigen, Tilapia anti VNN (TaVNN) as primary antibody, Rabbit anti Tilapia (RaT) as secondary antibody and Goat anti Rabbit (GaR) HRP as tertiary antibody were performed in this study. Optimum dilution for antibodies that involved for the detection process were determined. This assay was considered efficient as it does not depend on purified virus or recombinant capsid NNV protein as coating antigen. Moreover, the improved protocol was flexible as it could be applied to wider range of species.

Keywords: Viral Nervous Necrosis, Nervous Necrosis Virus, ELISA, Viral disease

REPRODUCTIVE DEVELOPMENT AND SPAWNING INDUCTION OF SPOTTED SCAT Scatophagus argus (Linnaeus, 1766) IN CAPTIVITY

Nguyen Van Huy*¹, Le Thi Thu An¹, Nguyen Khoa Huy Son¹, Nguyen Duc Thanh¹

¹Hue University of Agriculture and Forestry

102 Phung Hung Street, Hue City, Thua Thien Hue Province, Viet Nam

^*Corresponding author: huy.nguyen@hueuni.edu.vn 

The Tam Giang-Cau Hai Lagoon is the largest brackish lagoon in Southeast Asia and has a surface area of nearly 22,000 hectares. It supports a high diversity of aquatic species, especially many highly valuable fish. The spotted scat (Scatophagus argus) is considered a typical species of this ecosystem and is one of the most economically important brackish water fish species in Southeast Asia; for human consumption and as ornamental fish. The hormones of human chorionic gonadotropin (hCG) and luteinizing hormone-releasing hormone analog (LHRH-A) are commonly used in fish artificial reproduction in fish farms which has distinct differences in terms of their bioactivity and physiological function. While LHRH-A stimulates gonadotropin secretion in teleosts as a result of ovulation and spawning, hCG acts to stimulate oocyte maturation. The hormonal administration of a low dose may result in failure to induce fish spawning. This study evaluated variations in gonadosomatic index (GSI), gonadal development stages, and hormonal spawning induction of Scatophagus argus in captivity. Male and female fish were cultured separately in net-cages in Tam Giang lagoon, Central Vietnam from January to December, 2020. Five fish of each sex were randomly sampled monthly. Gonads were collected, GSI determined and subsequently prepared for histology. Gamete quality was assessed with a light microscope. Spawning was hormonally induced with different doses of human chorionic gonadotropin (hCG) and luteinizing hormone-release hormone (LHRH-A2). Gonadal development started to increase in March, peaking in July. Fish spawned from June to August, displaying the highest GSI value, sperm motility, and oocyte diameter at this time. Only female GSI fluctuated significantly month-by-month (P<0.05). Histological examination indicated that S. argus is multiple spawners. Application of LHRH-A2 (70 µg/kg) stimulated spawning and resulted in better latency periods, fertilization, and hatching rates.

Keywords: reproductive development, Scatophagus argus, spawning induction.