Molecular characterization of Aeromonas hydrophila detected in Channa marulius and Sperata sarwari sampled from rivers of Punjab in Pakistan.

Aeromonas hydrophila is one of the major pathogenic bacteria responsible for causing severe outbreaks at fish farms and is also a major global public health concern. This bacterium harbors many virulence genes. The current study was designed to evaluate the antidrug and virulence potential of A. hydrophila by amplifying its antimicrobial resistance and virulence genes using PCR and examining their effects on fish tissues and organs. A total of 960 fish samples of Channa marulius and Sperata sarwari were collected from four sites of the rivers of the Punjab, Pakistan. A. hydrophila isolates were subjected to biochemical identification and detection of virulence and antimicrobial resistance (AMR) genes by PCR. We retrieved 181 (6.46%) A. hydrophila isolates from C. marulius and 177 (6.25%) isolates from S. sarwari. Amplification through PCR revealed the incidence of virulence genes in 95.7% of isolates in C. marulius and 94.4% in S. sarwari. Similarly, amplification through PCR also revealed occurrence of AMR genes in 87.1% of isolates in C. marulius and 83.9% in S. sarwari. Histopathological examination revealed congestion (5.2%) and hepatocyte necrosis (4.6%) in liver, lamellar fusion (3.3%) and the presence of bacterial colonies (3.7%) in gills, fin erosion (6%), and the presence of biofilms (3.5%) in tail fins of infected fish. Phylogenetic tree analysis of 16S rRNA and gyrB gene of A. hydrophila revealed 100% and 97% similarity, respectively, with 16S rRNA gene and gyrB of A. hydrophila isolated in previous studies. The results of antimicrobial susceptibility testing showed that all isolates demonstrated resistance to sulfamethoxazole, ampicillin, neomycin, and norfloxacin, while susceptibility to gentamicin, chloramphenicol, and tetracycline, and intermediate resistance was observed against cefotaxime. The results concluded that examined fish samples were markedly contaminated with virulent and multidrug strains of A. hydrophila which may be of a potential health risk. The study emphasizes the responsible antimicrobial use in aquaculture and the urgent need for effective strategies to control the spread of virulence and antimicrobial resistance genes in A. hydrophila.

Effects of Different Aquafeed Sources on Growth Performance, Oxidative Capacity, and Fatty Acid Profile of Three Carps Reared in the Semi-Intensive Composite Culture System.

The current experiment is designed to evaluate the effect of different aquafeeds (farm-made versus commercial) on growth, body composition, oxidative capacity, and fatty acid profile in the semi-intensive composite culture system. For this, 1,100 fingerlings/acre having initial body weight and length, Labeo rohita (61.34 g, 171 mm), Catla catla (71.45 g, 181 mm), and Cyprinus carpio (30.80 g, 91 mm) were randomly distributed to 16 ponds and randomly fed on eight different diets (n = 2 pond/diet) in a completely randomized research design. Aquafeed were farm-based diets (D1-D2) and commercial aquafeed (D3-D8). The farm-made diets contained various crude protein levels of maize gluten (24.9%) and rice polish (7.3%), whereas commercial diets were procured from commercial feed plants (AMG, Supreme, Aqua, Star Floating, Hi-Pro, and Punjab feed). The growth performance of carps (L. rohita and C. catla) was significantly improved (p < 0.05) by feeding D3 as compared to other diets. Similarly, white blood cell concentration was greater (p < 0.05) in all species fed by D3 than in those fed on D7, D8, D5, D6, D1, and D2 fed groups, respectively. Alanine transaminase, aspartate transaminase, and alanine phosphatase activities were significantly lower (p < 0.05) in the D3-fed L. rohita, C. catla, and C. carpio compared with those fed on the rest of the treatments. The activities of glutathione peroxidase and superoxide dismutase were also higher (p < 0.05) for the D3 fed L. rohita, C. catla, and C. carpio than those fed on the rest diets. The groups fed on D3 and D4 had greater (p < 0.05) concentrations of myristic (14), palmitic acid (16), and stearic (18) acids than those fed on the rest of the commercial diets. However, meat chemical composition was similar (p > 0.05) across the treatments. These results also prove that the increase in the dietary protein level and lipid content can improve the fish's body's crude protein and fat levels. Feeding D3 improved the production performance, oxidative status, and fatty acid profile in composite major carps culture systems. Thus, based on growth, survival, and body composition, it is concluded that D3 and D4 may be recommended for a commercial culture of major carps. Dietary treatments had no significant impact (p > 0.05) on water's physical-chemical properties. Calcium content and alkalinity varied (p < 0.05), with D5 showing the lowest calcium and the highest alkalinity.

Optimizing rice-fish co-culture: Investigating the impact of rice spacing density on biochemical profiles and production of genetically modified tilapia (Oreochromis spp.) and Cyprinus carpio.

Rice fish co-culture synergistically boosts food production, resulting in numerous advantages across the environmental, social, and financial domains. A study was conducted to investigate the effects of three different rice spacing densities, rice high density (RHD) 9inch×12inch, rice medium density (RMD) 12inch × 12inch, rice low density (RLD) 15inch×12inchon both rice and fish. Various parameters were assessed to evaluate the performance of the co-culture system, including water quality, growth parameters, muscle quality, soil characteristics, rice stem characteristics, and rice yield parameters. When comparing the species, it was observed that GMT (Genetically Male Tilapia) demonstrated superior weight gain (303.13g vs 296.41g) and specific growth rate (1.16 vs 1.15). Regarding the proximate composition, results showed that RMD had the highest crude protein and fat content compared to RLD and RHD. GMT also exhibited greater crude protein and fat content than Cyprinus carpio, with RMD showing the highest values. Treatment groups significantly influenced the amino acid profile of experimental species, with RMD exhibiting the highest values. GMT showed significantly higher levels of essential, non-essential, half-essential, and umami amino acids compared to Cyprinus carpio. The interaction between RMD and GMT further demonstrated significant differences in various amino acid categories with RMD. A non-significant difference was observed among the treatments regarding soil biochemical characteristics. Regarding the rice stem characters, the height of the plant, panicle length, and stem length of rice were found to be comparable in the RMD and RLD groups however, significantly higher in RHD. Regarding rice yield parameters, no significant differences were observed among the other treatment groups, except for yield per hectare (yield/ha), which was significantly higher in the RHD group compared to RMD and RLD. Additionally, 1000-grain weight and panicle number (ears per hill) were significantly higher in the RLD treatment than in the other treatments. In conclusion, our findings indicate that the RMD treatment consistently yielded superior results compared to RLD and RHD. Furthermore, within the rice-fish co-culture system, GMT proved to be a more competent species compared to Cyprinus carpio. The study provides data to understand the interactions between rice spacing density, fish growth and overall productivity can guide the development of sustainable and profitable rice-fish co-culture systems.

Impact of duck stocking density on growth performance, digestive enzymes, blood biochemistry, and antioxidant capacity of the Labeo rohita reared in an integrated ponds system.

This study investigated the effects of duck stocking density (SD) on growth performance, meat quality, hematology, serum metabolites, and antioxidant status of L. rohita reared in an integrated pond system. A total of 9000 fingerlings of L. rohita average weighing 150.5±1 grams were reared in nine ponds (n = 1000/pond) and randomly allotted one of three SDs under a completely randomized design. The stocking densities were 100 (SD-100), 150 (SD-150), and 200 (SD-200) ducks/pond and each group had three replicates. The final body weight, weight gain, and specific growth rates were greater (p<0.05) in SD-200 than in the rest of the treatments. The activities of proteases and amylase were also greater (p<0.05) for SD-200 than for the rest of the SD. However, hematological parameters like red blood cells, white blood cells, hematocrit, met hematocrit, and hemoglobin contents were not influenced (p>0.05) by SD in ponds. The serum total protein, albumin, globulin, creatinine, and glucose contents were also similar (p>0.05) across the ponds. However, blood urea nitrogen was higher (p<0.05) in SD-200 than in the rest of the SD. Moreover, alanine transaminase, aspartate transaminase, and alkaline phosphatase activities were linearly decreased (p<0.05) with increasing SD across the ponds. The changes in catalases and superoxide dismutase were non-significant (p>0.05) among the groups. However, glutathione peroxidase was greater for SD-200 than for the rest of the SD. The carcass compositional characteristics such as dry matter, protein, fat, and ash contents were not changed (p>0.05) by varying SD. In conclusion, SD over 200 birds/pond improved growth performance, digestive enzyme functions, and oxidative capacity without any negative impact on the hematology and carcass composition of L. rohita.

Parasite Diversity in a Freshwater Ecosystem.

Parasites are a significant component of biodiversity. They negatively affect fish appearance, growth, and reproduction. In this study, the prevalence of infection, diversity, and mean intensity of parasites were examined in 9 freshwater fish species (45 samples per fish species). Ecto-parasites were examined on the skin, gills, and fins with a hand lens. Wet mounts were prepared using mucosal scrapings from all the external and internal organs of the sampled fish. Microscopy, muscle compression, and the pepsin-HCL artificial digestion technique were also performed. In this study, 26 species of parasites were identified including three taxa belonging to 9 species of protozoan parasites, 11 treamtodes, and 6 monogenean parasites. The identified protozoan parasites were Entamoeba histolitica, Chilodonella sp., Coccidia sp., Costia sp., Cryptobia sp., Ichthyopthiris-multifilis, Microsporidia, Piscinoodinium sp., and Ichthyobodo necator. The identified trematode parasites were Fasciola gigantica, Echinostoma revolutum, Fasciola hepatica, Haplorchis pumilio, Brachylaima cribbi, Echinostoma cinetorchis, Neascus sp., Deropegus sp., Trematode Soldier, Centrocestus formosanus, and Clinostomum marginatum. The identified monogenean parasites were Dactylogyrus limipopoensis, Dactylogyrus anchoratus, Dactylogyrus myersi, Dactylogyrus vastator, Gyrodactylus salaris, and Ancyrocephalus. The diversity of parasites was maximum at the Okara site. The host's organs that were targeted for parasitic infection included the intestine, liver, gills, fins, skin, and kidneys. The majority of the parasites were identified in Labeo rohita followed by Hypophthalmichthys molitrix, Ctenopharyngodon idella, Oreochromis niloticus, Cyprinus carpio, and Wallagu attu. Two species appeared to be resistant species because none of the parasites were observed in Notopterus notopterus or Sperata seenghala. This study also concluded that the prevalence of parasites increased with increasing length, size, and age of fish.

First record of Euphlyctis kalasgramensis (Anura: Dicroglossidae) from Punjab, Pakistan.

The present study documented the first record of Euphlyctis kalasgramensis from Punjab, Pakistan. The specimens were collected during field visits from June through August 2018. Various morphometric measurements of E. kalasgramensis were taken and compared with Euphlyctis cyanophlyctis. Snout-vent length (SVL) was 38.11 ± 0.87 mm (n = 5), snout length was 3% of SVL, foot length was 55% of SVL, head length was 32% of SVL and weight was 8.01 ± 0.12 g (n = 5). A few specimens (n = 2) were euthanized and preserved for molecular analysis through mitochondrial 16S rRNA gene sequences. The newly obtained DNA sequences of E. kalasgramensis were submitted to GenBank and accession numbers were obtained (MK881165.1 and MK920114.1). The Maximum likelihood and Neighbor-joining trees based on Kimura 2-parameter distance resulted in similar phylogenetic trees. Euphlyctis kalasgramensis was out group in both phylogenetic trees. The interspecific divergence of E. kalasgramensis and E. cyanophlyctis was high ranging from 4% to 6% as compared to low intraspecific divergence 0% and 1%. The diversity and distribution ranges of many amphibians species are not well known in Pakistan due to lack of taxonomic information. In our recommendation, a large scale DNA barcoding is required to report more cryptic or new species from Pakistan.

Isolation, Characterization, and Application of a Bacteriophage Infecting the Fish Pathogen Aeromonas hydrophila.

Bacteriophages are increasingly being used as biological control agents against pathogenic bacteria. In the present study, we isolate and characterize bacteriophage Akh-2 from Geoje Island, South Korea, to evaluate its utility in controlling motile Aeromonas septicemia. Akh-2 lysed four of the seven Aeromonas hydrophila strains tested. Transmission electron microscopy analysis showed that Akh-2 belongs to the Siphoviridae family, with head and tail sizes of 50 ± 5 and 170 ± 5 nm, respectively. One-step growth curve analysis revealed that the phage has a latent period of 50 ± 5 min and a burst size of 139 ± 5 plaque-forming units per infected cell. The phage appeared stable in a pH range of 6-8 and a temperature range of -80 to 46 °C. Based on next-generation sequencing analysis, its genome is 114,901 bp in size, with a 44.22% G + C content and 254 open reading frames. During an artificial induction of the disease, loach (Misgurnus anguillicaudatus) treated with Akh-2 showed an increased survival rate and time compared with the non-treated control. Our results suggest that Akh-2 is a potential biological agent for the treatment of Aeromonas infections in fish.