فصلنامه فیزیولوژی و بیوتکنولوژی آبزیان
سال سیزدهم شماره 2 (تابستان 1404)

This study aimed to extract, characterize and evaluate the antibacterial activity of the polysaccharide derived from the halophilic microalga Cyanothece sp. The strain was cultivated under laboratory conditions, and the resulting biomass was harvested. The polysaccharide extraction process was optimized by investigating the effects of extraction temperature (40, 60 and 80°C) and time (60, 120 and 180 minutes). Following initial extraction, the crude polysaccharide was purified using chromatographic techniques, specifically gel filtration chromatography. Physicochemical characteristics, including homogeneity, molecular weight and monosaccharide composition, were determined. Antibacterial efficacy was assessed in vitro using the disk diffusion method. The results indicated that the optimal extraction yield was achieved at 80°C over a duration of 180 minutes. The purified polysaccharide exhibited a molecular weight of 103kD. The monosaccharide profiling revealed a complex structure comprising rhamnose, fucose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid, present in the following molar ratios: 1.7, 0.8, 1.0, 3.2, 6.1, 4.5, 5.0 and 6.0%, respectively. Also, the extracted polysaccharide significantly inhibited the growth of Escherichia coli and Staphylococcus aureus bacteria (P<0.05). Based on these findings, the polysaccharide derived from Cyanothece sp. possesses promising antimicrobial properties and warrants further investigation as a potential candidate for pharmaceutical and nutraceutical applications.

Proteases, as one of the most widely used industrial enzymes, play a role in breaking protein molecules and converting them into peptides and amino acids. Serine proteases, which are considered as endopeptidases, are divided into four subgroups, among which alkaline serine proteases are considered the most important group from an industrial point of view. In this study, serine alkaline metalloprotease gene from Salinivibrio proteolyticus from Bakhtegan hyper saline lake was cloned and recombinantly expressed. The obtained results confirmed the production of a monomeric protein with a molecular weight of about 66kDa. The results of this research indicate that chaperone plasmids do not have a significant effect on the soluble expression of the studied protease, and even if used, it disrupts the protein purification process due to the presence of high amounts of chaperone proteins in the soluble phase. Enzyme activity measurement by zymography method and also with Folin-Ciocalteu reagent showed its protease activity on casein substrate. By observing the inhibitory effect of EDTA with a concentration of 1mM as an inhibitor of metalloproteases and the effect of reducing the enzyme activity in the presence of PMSF with a concentration of 1mM as an inhibitor of serine proteases, it is concluded that the investigated protease belongs to the category of serine metalloproteases. Also, the proteolytic activity of the enzyme on the casein substrate with a concentration of 0.65% was calculated as 1.65±0.08U/mg based on the spectrophotometer results and using the L-tyrosine standard curve. Finally, in order to achieve the optimal specific activity of the enzyme, the evaluation of different reaction conditions such as optimal temperature and pH and its desired metal ions should be considered.

In this study, the effect of replacing fish meal with hydrolyzed fish protein was investigated on the levels of selected oxidative stress indicators and the activity of digestive enzymes in juvenile beluga (Huso huso). For this purpose, 750 juvenile belugas with an initial weight of 3±0.5g were reared in five experimental groups with three replicates, for 48 days. The groups included a control group (without hydrolyzed protein), three treatments with different replacement levels of fish meal with hydrolyzed fish protein (treatments 1, 2 and 3 containing 2.75, 5.5 and 8.25%, respectively), and treatment 4 (positive control) with commercial diet. The results for oxidative stress indicators showed no significant differences in the activities of catalase and superoxide dismutase and in the malondialdehyde levels among the experimental groups (P>0.05). For digestive enzymes trypsin, pepsin, amylase and lipase, no significant statistical differences were observed in any of the four stages (5, 10, 20 and 35 days after stocking) of enzyme activity assessment or in any of the types of studied enzymes (P>0.05). Overall, the results of this study showed that replacing fishmeal with hydrolyzed fish protein up to 8.25% in the diet of beluga juveniles has no significant effect on the activities of the antioxidant and digestive systems of these fish.

The pdx1 gene, which is also known as insulin factor 1, is the main key to the growth and development of the pancreas, as well as the maturation of beta cells and their survival. By targeting this gene, various transgenic models have been produced in stem cells as well as animal models that are used in the field of diabetes research. In this research, the aim is to make gene plasmids based on Tol2 using conventional cloning method, which can be used to create Tg(pdx1:GFP) zebrafish transgenic model. In order to label this gene, a part of its promoter sequence needs to be placed upstream of the reporter gene. Since the regulatory region of the pdx1 gene has not been defined, two pairs of primers were designed to amplify the 5' sequence of 2500 and 6600 base pairs upstream of the ATG start codon in the zebrafish pdx1 gene, and to the 5' and 3' ends of each of these primers, the sequence of restriction enzymes (SalI, Sph1) was placed. For this purpose, after designing suitable primers for two different lengths of the promoter sequence of the pdx1 gene expressed in beta cells, these fragments were amplified and inserted in the Tol2 plasmid containing the green fluorescent reporter gene using the conventional cloning method including enzymatic digestion and the ligation reaction. Confirmation of the correctness of the cloning process was done using colony PCR, enzymatic digestion and sequencing. The results showed that the conventional cloning process, in addition to the low cost, has good efficiency for cloning of fragments, with different sizes.

Microplastic particles smaller than 5mm are referred to as microplastics (MPs). Microplastic pollution is a widespread global threat to aquatic ecosystems. Considering that various sources of pollutants, including microplastics, enter the Siyahdarvishan River, in present study, the level of microplastic pollution in the bivalve Anodonta cygnea, water and sediments of the Siyahdarvishan River (Guilan) was studied. Samples of bivalves, sediments, and water were collected from three stations from June to September 2023. A total of 65 bivalve specimens were sampled and analyzed. The mean length and total weight of the sampled bivalves were 113.90±3.97mm and 143.78±14.96g, respectively. Microplastics were observed and identified in all samples. The average abundance of microplastics in bivalve tissue was 34.13±11.88 particles per individual and 0.38±0.15 particles per gram of soft tissue, 853.33±296.31 particles per kilogram of dry weight in sediment and 0.77±0.15 particles per liter in water. There was no significant correlation in terms of the amount of microplastics and shape, between water and bivalves, sediment and bivalves, and sediment and water (P<0.05). But a significant positive correlation was observed in terms of particle size between water and bivalves, sediment and bivalves, and sediment and water (P<0.05). Additionally, there was no correlation between microplastic concentration and the total weight, soft tissue weight or shell length (P<0.05). The predominant shapes of microplastics were fibers and fragments, and mainly black in color. Among the plastic polymers identified by the ATR-FTIR device, polypropylene (PP) and polyethylene (PE) were the most common polymers. The average size of particles in bivalve soft tissue and sediment across all stations was 0.5-1mm and 1-2mm in water.

This study was conducted to evaluate the effect of the aqueous extract combination of brown macroalgae and the bacterium Bacillus subtilis IS0 on the hemolymph indices and non-specific immunity of the white shrimp (Litopenaeus vannamei). A total of 2400 post-larvae with an average weight of 29±0.87mg were stocked at a density of 100 individuals in 8 groups (with three replicates). The control group received a diet without the combined macroalgae extract (MPE) and single cell probiotic (P), while the other groups were fed diets containing 15g/kg MPE, 1% (P1), 2% (P2) and 3% (P3) probiotics and combinations of the macroalgae extract with the probiotic (P1+MPE, P2+MPE and P3+MPE) for a period of 60 days. The results showed that the highest total hemocyte count was observed in the groups fed with P2+MPE and P3+MPE, at 15.36±0.35 and 15.10±0.36 ×106/mL), respectively, which were significantly different from the other experimental groups and control (P<0.05). The simultaneous use of an aqueous extract of macroalgae with different concentrations of single-cell probiotics significantly increased the number of granular, semi-granular and hyaline cells compared to the control group and other experimental groups. The highest counts of these cells were found in the groups fed with P2+MPE and P3+MPE, showing a significant difference from the other experimental groups (P<0.05). The highest protein level (111.47±9.21mg/mL) and the lowest glucose level (9.0±0.91mg/dL) were observed in the group fed P2+MPE, showing a significant difference compared to the control and other experimental groups (P<0.05). The results of this study demonstrated that the simultaneous use of the combined extract of brown macroalgae and 2% single-cell probiotic improved hemolymph indices and innate immune system in white shrimp.