A new proteomics technology has been implemented to study the protein repertoires of developing oocytes of giant grouper (Epinephelus lanceolatus). Knowledge of the chemical composition and physiochemical properties of vitellogenin (Vtg) is necessary to interpret the functional and biological properties attributed during ovulation. Vtg, as a biomarker indicator in sex determination, has been analyzed to determine the sex and maturational status of fish in the absence of the gonad tissue. A male giant grouper was induced by 2 mg/kg of 17ß-estradiol (E2), and blood was sampled at days 0, 1, 3, 5, and 10. SDS-PAGE 1D electrophoresis was used to analyze Vtg protein, and Vtg identification was done with 4800 Plus MALDI TOF/TOF™ mass spectrophotometer (Applied Biosystems/MDS SCIEX, USA). Meanwhile, MS/MS de novo sequencing identified the proteins by matching sequences of tryptic peptides to the known sequences of other species. Vtg was confirmed by MASCOT at 95% significant level, and molecular mass was 187 kDa. Protein resolved on SDS-PAGE as a double band of approximately the same mass as determined with MALDI-TOF. The N-terminal sequences and identification of Vtg were also determined. The potential of using MS methods to understand the structure and function of Vtg is discussed.
A study was conducted to evaluate the gonad differentiation of juvenile yellow perch (YP, Perca flavencens) and determine the latest labile period related to hormone treatment. Juvenile fish were subjected to two dietary concentrations of methyltestosterone (MT; 20 and 50 mg/kg feed) for 60 days in three (3) age groups of 38-, 46-, and 67-days post-hatching (dph), where control group were fed with standard commercial feed. Following a 10-month on-growing period, sex phenotypes were determined by gross and histological gonad morphology. Results showed the juvenile YP responded to the exogenous hormone when it was applied at 38 dph for both 20 and 50 mg/kg feed resulting in 100% males. At 46 dph, only 50 mg/kg feed resulted in 100% males. Both MT-treated at 38 and 46 dph significantly differed (P
Aquatic bacterial pathogens can cause severe economic loss in aquaculture industry. An opportunistic pathogen, Aeromonas hydrophila is responsible for Motile Aeromonas Septicemia, leading to high mortality rates in fish. The present study was focused on the efficacy of Aloe barbadensis replacing fishmeal diets on hematological, serum biochemical, antioxidant, histopathological parameters, and disease resistance against A. hydrophila infection in Labeo rohita. Isonitrogenous fishmeal replaced diets (FMR) were prepared with varying levels of A. barbadensis at D1 (0%) (control), D2 (25%), D3 (50%), D4 (75%) and D5 (100%) then fed to L. rohita. After 60 days of post-feeding, the experimental fish were challenged with A. hydrophila. Blood and organs were collected and examined at 1- and 15-days post infection (dpi). The results demonstrated that on 1 dpi, white blood cells (WBC), total protein, cholesterol and low-density lipoprotein (LDL) levels were significantly increased in D3 diet fed groups. The D2 and D3 diet fed group showed decreasing trends of serum glutamic pyruvic transaminase (SGPT) and antioxidant enzymes activity on 15 dpi. The histopathological architecture results clearly illustrated that the D3 diet fed group had given a higher protective effect by reducing the pathological changes associated with A. hydrophila infection in liver, intestine and muscle. Higher percentage of survival rate was also observed in D3 diet fed group. Therefore, the present study suggested that the dietary administration of A. barbadensis up to 50% fishmeal replacement (D3 diet) can elicit earlier antioxidant activity, innate immune response and improve survival rate in L. rohita against A. hydrophila infection.
We have developed a novel single cell real-time quantitative PCR technique, which incorporates harvesting marker-identified single cells using laser-capture. Here, for the first time in a vertebrate species, using this innovative single cell gene profiling technique, we report the presence of G-protein coupled receptors in individual gonadotropin-releasing hormone (GnRH) neurons and endocrine cells of the pituitary of the tilapia Oreochromis niloticus. The differential expression of multiple combinations of three GnRH receptor types (R1, R2 and R3) in individual gonadotropic and nongonadotropic cells demonstrates cellular and functional heterogeneity. The differential use of GnRH receptors in corticotropes, melanotropes and thyrotropes during gonadal maturation and reproductive behaviors suggests new roles for these hormones. Further, we provide evidence of the structure of a novel nonmammalian G-protein coupled receptor (GPR54) for kisspeptins, encoded by Kiss-1 gene, which is highly conserved during evolution and expressed in GnRH1, GnRH2 and GnRH3 neurons. We hypothesize GPR54 stimulates GnRH secretion and is crucial for pubertal maturation. We speculate, the use of this method will allow the identification and quantification of known and unknown genes in single cells, which would greatly facilitate our understanding of the complex interactions that govern the physiology of individual cells in vertebrates species.
Increasing demand for eco-friendly botanical piscicides and pesticides as replacements for harmful synthetic chemicals has led to investigation of new sources of plant materials. Stem bark of Terminalia arjuna, which has been used as a popular folk medicine since ancient time, was examined for its piscicidal activity. This study aims to determine toxicity of ethanol extract of T. arjuna bark on fresh water stinging catfish (Heteropneustes fossilis), along with evaluation of changes in hematological parameters of the fishes exposed to a lethal concentration. The percent mortality of fishes varied significantly in response to concentrations of the extract and exposure times (between exposure time F = 36.57, p < 0.001; between concentrations F = 39.93, p < 0.001). The lethal concentrations (LC50) of ethanol extract were found to be 12.7, 8.94, 5.63 and 4.71 mg/l for 24, 48, 72 and 96 h, respectively. During acute toxicity test, blood samples of treatment fishes showed significant decreases in the red blood cells count, hematocrit content, hemoglobin concentration, mean corpuscular hemoglobin concentration and plasma protein level when compared to those of the control group, while there were significant increases in the mean corpuscular volume, mean corpuscular hemoglobin, white blood cells count and plasma glucose concentration. These results suggest that T. arjuna bark extract could be considered as a potent piscicide due to its toxic effect on fish, particularly fish hematology.
This study was conducted to investigate the growth performance, biomarkers of oxidative stress, catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST) as well as the haematological response of African catfish after being fed with fish feed containing different levels of cricket meal. The juvenile fish were assigned to three different treatments with isonitrogenous (35 %) and isoenergetic (19 kJ g(-1)) diets containing 100 % cricket meal (100 % CM), 75 % cricket meal (75 % CM), and 100 % fishmeal (100 % FM) as control groups for 7 weeks. The results indicated that a diet containing 100 % CM and 75 % CM improved growth performance in terms of body weight gain and specific growth rate, when compared to 100 % FM. The feed conversion ratio (FCR) and protein efficiency ratio (PER) did not differ significantly between all diets, but reduced FCR and increased PER were observed with a higher inclusion of cricket meal. A haematological examination of fish demonstrated no significant difference of red blood cells in all diets and white blood cells showed a significantly higher value in fishmeal-fed fish. On the other hand, haemoglobin and haematocrit significantly increased with increasing amounts of cricket meal in the diet. Antioxidant activity of CAT was higher in the 100 % CM group compared to fish fed other diets, whereas GST and SOD showed increasing trends with a higher incorporation of cricket, although insignificant differences were observed between all diets. These results suggest that cricket meal could be an alternative to fishmeal as a protein source in the African catfish diet.
The mosquitofish (Gambusia affinis) naturally inhabits freshwater (FW; 1-3‰) and seawater (SW; 28-33‰) ponds in constructed wetland. To explore the physiological status and molecular mechanisms for salinity adaptation of the mosquitofish, cytoprotective responses and osmoregulation were examined. In the field study, activation of protein quality control (PQC) mechanism through upregulation of the abundance of heat shock protein (HSP) 90 and 70 and ubiquitin-conjugated proteins was found in the mosquitofish gills from SW pond compared to the individuals of FW pond. The levels of aggregated proteins in mosquitofish gills had no significant difference between FW and SW ponds. Furthermore, the osmoregulatory responses revealed that the body fluid osmolality and muscle water contents of the mosquitofish from two ponds were maintained within a physiological range while branchial Na+/K+-ATPase (NKA) expression was higher in the individuals from SW than FW ponds. Subsequently, to further clarify whether the cellular stress responses and osmoregulation were mainly induced by hypertonicity, a laboratory salinity acclimation experiment was conducted. The results from the laboratory experiment were similar to the field study. Branchial PQC as well as NKA responses were induced by SW acclimation compared to FW-acclimated individuals. Taken together, induction of gill PQC and NKA responses implied that SW represents an osmotic stress for mosquitofish. Activation of PQC was suggested to provide an osmoprotection to prevent the accumulation of aggregated proteins. Moreover, an increase in branchial NKA responses for osmoregulatory adjustment was required for the physiological homeostasis of body fluid osmolality and muscle water content.
Tor tor is an important game and food fish of India with a distribution throughout Asia from the trans-Himalayan region to the Mekong River basin to Malaysia, Pakistan, Bangladesh and Indonesia. A new cell line named TTCF was developed from the caudal fin of T. tor for the first time. The cell line was optimally maintained at 28°C in Leibovitz-15 (L-15) medium supplemented with 20% fetal bovine serum (FBS). The propagation of TTCF cells showed a high plating efficiency of 63.00%. The cytogenetic analysis revealed a diploid count of 100 chromosomes at passage 15, 30, 45 and 60 passages. The viability of the TTCF cell line was found to be 72% after 6 months of cryopreservation in liquid nitrogen (-196°C). The origin of the cell lines was confirmed by the amplification of 578- and 655-bp sequences of 16S rRNA and cytochrome oxidase subunit I (COI) genes of mitochondrial DNA (mtDNA) respectively. TTCF cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids. Further, immunocytochemistry studies confirm its fibroblastic morphology of cells. Genotoxicity assessment of H₂O₂ in TTCF cell line revealed the utility of TTCF cell line as in vitro model for aquatic toxicological studies.
The study investigated the alleviated effects of Alpha-ketoglutaric acid (AKG) on the intestinal health of mirror carp (Cyprinus carpio Songpu) caused by soy antigenic protein. The diets were formulated from fishmeal (CON), 50% soybean meal (SBM), the mixture of glycinin and β-conglycinin (11 + 7S) and adding 1% AKG in the 11 + 7S (AKG). Carp (~ 4 g) in triplicate (30 fish per tank) was fed to apparent satiation thrice a day for six weeks. Compared with CON, SBM treatment resulted in significantly poor growth performance (P 0.05). Gene expression of tumor necrosis factor (TNF-α) and interleukin-1 β (IL-1β) in proximal intestines (PI) and distal intestines (DI) were increased (P