فصلنامه فیزیولوژی و بیوتکنولوژی آبزیان
سال دهم شماره 4 (زمستان 1401)

This study aimed to evaluate the histological effect of nonylphenol on the activity of goldfish (Carassius auratus) immune system by measuring the accumulation of melanomacrophage pigments in the kidney and spleen of goldfish. Therefore, immature goldfish were exposed to 10-6 and 10-7M nonylphenol (NP) for 25 days. Tissue processing was carried out followed by Hematoxylin-eosin (H&E), Perls Prussian blue (PPB) and periodic acid Schiff (PAS) staining to identify melanin, hemosiderin and lipofuscin, respectively. Then, image processing was performed using ImageJ software (ver. 1.53 e) to quantify the pigment content of melanomacrophage centers (MMCs). The exposure to NP led to decrease of MMC formation containing hemosiderin pigment in the spleen, and did not affect the formation of MMCs containing this pigment in the kidney. MMC formation containing lipofuscin pigment decreased and increased in the spleen and kidney, respectively, after exposure to NP (P<0.05). The exposure to NP led to the increase of MMC formation in both organs in a dose-dependent manner. Generally, melanin increased in a dose-dependent manner and seems to be a better indicator of exposure to NP.

Aeromonas hydrophila is one of the most important pathogenic bacteria in fish that causes hemorrhagic septicemia, ascites and mortality in different ages and in different fish species including carp. Immunization is one of the best ways to prevent this disease. The use of gene constructs containing bacterial-specific antigens has been developed in recent years to develop high-performance vaccines and to use them in easier methods such as oral administration in aquaculture. In this study, aopB protein of type III secretory system of A. hydrophila isolated from carp in Khuzestan province was used to design a gene construct for DNA vaccine which cloned into the expression eukaryotic plasmid pCDNA3.1. In this study, A. hydrophila isolated from carp with Aeromonas septicemia in carp farms of Khuzestan province which species confirmed by molecular and phenotypic methods. The aopB gene sequence was extracted from the NCBI gene bank and the gene encoding region was amplified using primers containing EcoRI and NheI enzyme cleavage regions. The PCR product was then inserted into the eukaryotic expression plasmid pCDNA3.1-HisA and was transfected to TOP10-F 'bacteria using heat shock. After screening for positive clones using colony PCR and sequencing, the pCDNA3.1-AopB gene construct was transferred to CHO cells using electroporation method and recombinant protein expression was assessed by Western blotting on cell lysis. PCR results and sequencing of gene constructs indicated the cloning of the aopB gene approximately 1000bp in plasmid pCDNA3.1-HisA. Western blot using anti-HisTag antibody showed the presence of a protein with a molecular weight of about 36kD. Conclusively, the pCDNA3.1-AopB gene construct containing the aopB gene was designed and validated. This gene construct can be used in clinical studies to evaluate the immunogenicity of carp in experimental pathogenicity with A. hydrophila.

Exposure of aquatic animals to silver, due to production of free radicals, causes oxidative damage and biochemical changes in organisms. Therefore, in present study, changes in the antioxidant defense system and metabolic indices of Litopenaeus vannamei were evaluated during 21 days of exposure to silver nitrate. Shrimps exposed to different concentrations of silver nitrate, including treatment 1: 10% LC50 (0.0084 mg/L), treatment 2: 25% LC50 (0.021 mg/L), treatment 3: 50% LC50 (0.042 mg/L) and treatment 4: 75% LC50 (0.063 mg/L) and one treatment as control treatment. Superoxide dismutase activity in treatments 3 and 4 and total antioxidant capacity in treatment 4 was significantly reduced (P<0.05). But malondialdehyde activity was significantly increased in treatment 4 (P<0.05). Catalase and glutathione activity in all treatments did not show a significant difference (P>0.05). Among metabolic indices, only in treatment 4 protein changes were significantly reduced, and glucose, cholesterol and triglyceride were significantly increased (P<0.05). But the amount of phosphorus and calcium did not show a statistically significant difference (P>0.05). In general, exposure to 75% LC50 of silver nitrate with a significant decrease in the activity of antioxidant enzymes and an increase in malondialdehyde caused oxidative damage and disruption of some metabolic parameters of Litopenaeus vannamei.

Bacterial L-asparaginase is used as an anti-neoplastic drug for chemotherapy of acute lymphoblastic leukemia and to process cooked or fried foods for prevention of acrylamide formation. Therefore, this enzyme is utilized for controlling and preventing cancer wildly. The aim of the present study was the isolation and identification of potential bacteria producing L-asparaginase from the intestines of Liza klunzingeri and Thunnus tonggol. Accordingly, 44 bacterial strains were initially isolated from those fishes’ intestines using nutrient agar medium. Then, L-asparaginase-producing bacteria were screened using M9 specific solid culture medium, which led to the identification of 20 potential L-asparaginase producing strains. Enzyme production by the isolated bacteria was afterwards evaluated, using as reference enzyme activity. Two strains that displayed the higher enzyme activities, 311 and 284 μmol/min, were selected and identified as Pseudomonas aeruginosa strain HR03 and Pseudomonas stutzeri strain HR04, respectively, by 16S rRNA gene sequence analysis. Overall, the results of this study showed that the intestinal bacterial flora of L. klunzingeri and T. tonggol are promising biological reservoir for the isolation of L-asparaginase producing bacteria. Additionally, marine bacteria P. aeruginosa strain HR03 and P. stutzeri strain HR04 are foreseen as potential bacterial producers of L-asparaginase with application in the pharmaceutical and food industries.

In sturgeon aquaculture, sex determination is a major challenge since culture of female sturgeon, as a valuable source of caviar production, is economically important and the differentiation of male and female sturgeons is indeed necessary to reduce costs. Indirect evidence shows that the sex determination system in sturgeon is ZW and the sex of the female determines the sex. The situation which underlies locus discovery involved in sturgeon sex determination in genomic studies. The current study, for the first time in Iran, aimed to determine the beluga sex using the above-mentioned locus specific primers. For this purpose, caudal fins samples were collected from 23 beluga broodstock (14 females and 9 males). Genomic DNA was extracted by the ammonium acetate method. After quantitative and qualitative evaluation of the extracted DNA, specific primers were used to amplify a small fragment of the female beluga locus. Based on the obtained results, the single fragment slightly larger than 100 bp in size was amplified in 14 females while no specific amplification was observed in the male. Therefore, the molecular test can be used as a reliable, efficient, relatively low-cost, and non-invasive method for early sex determination in beluga in Iran.

The present study investigated the impact of stocking density and dietary protein level on the growth, hematology, immunity and resistance against Aeromonas hydrophila in common carp reared under biofloc conditions for 60 days.  Fish (12.09±0.99g) were stocked into 15 tanks in a 2×2 factorial design consisting of two stocking densities and two protein levels in biofloc system include: moderate stocking density (10kg.m-3) with 25 and 35% protein (MD25 and MD35) and high stocking density (20kg.m-3) with 25 and 35% protein (HD25 and HD35). The control group of fish was in clean water with moderate density (10kg.m-3) and 35% protein. The growth of fish was significantly higher and the FCR was lower in MD35 group, when compared to other groups. WBC, hemoglobin and hematocrit values were reduced while red blood cell count increased in fish reared in MD35 group. IgM, ACH50 and bactericidal activity of fish serum were significantly higher in MD35 group than other groups. Based on the present results, it is recommended to culture of common carp in the biofloc system with stocking density of 10 kg.m−3. As well as, the biofloc system can compensate for up to a 10% reduction in the diet in MD group.