Quantitative traits evaluation of different strains of indigenous culturable species rohu Labeorohita

Abstract

Abstract Strain evaluation and selection are crucial to address the disarray in aquaculture of any country and start a genetic improvement program. Unfortunately, Pakistan is far behind in launching any strategy for the genetic improvement of the existing fish stock, thus facing many challenges in this sector, including the availability of quality fish seed, stunted growth, higher mortality, higher FCR and overall low per acre production compared to many countries. limited literature is available on the genetic structure of indigenous culturable fish species of Pakistan. Thus, the current was designed to address this issue and determine the genetic diversity among eight different populations/strains of highly culturable species Labeorohita and then compare their growth performance, thermal tolerance, innate immunity, and disease resistance. Among eight different stains, six captive-bred strains, USs, SPs, TMs, MKs, CMs and IBD-s were collected from Fish Hatcheries, Upper Sindh Fish Hatchery, Carp Hatchery and Training Center, Peshawar, Tawakkal Fish Hatchery Muzaffargarh, Govt. Fish Hatchery, Mianchannu, Govt. Fish Hatchery, Charbanda, Islamabad Fish Hatchery and Training Centre, respectively, while two wild-strains WKs and WRs were collected from River Kabal and River Ravi, respectively. Rivers and Fish Hatcheries across the country were selected based on their geographical location and diverse fish culture practices. Genetic diversity among different strains was determined using DNA barcoding employing cytochrome oxidase subunit I (COI) and three different microsatellite markers, i.e., Lr-37, Lr-29, and Lr-28. The findings revealed an elevated level of observed heterozygosity (Ho) across all strains (Avg: 0.731), though less than the expected heterozygosity (He). Moreover, TMs and WRs exhibited the highest He, while TKs and CMs demonstrated the lowest value. Overall, FIS values observed for all strains with selected markers were positive. The DNA barcoding utilizing the CO1 gene also indicated significant genetic variation among various strains, as demonstrated by the formation of clades in the phylogenetic tree separating the strains into two distinct clusters that subsequently branch into sub-clusters. Overall, TMs showed the highest heterozygosity as compared to other strains. To evaluate quantitative traits like growth rate, thermal tolerance, disease resistance, and innate immunity, advanced fry of all stains were procured and reared at the Fisheries and Aquaculture facility at Quaid-i-Azam University. The growth performance, health status, and innate immunity of advanced fry and fingerlings of all strains were conducted under controlled conditions in an indoor facility and under semi-intensive culture conditions in the earthen communal ponds, respectively. For identification, fingerlings of all strains x were PIT tags and stocked in the earthen communal ponds. The 90-day growth performance data of all strains, both at advanced fry and fingerlings stages in controlled and semi-intensive culture systems respectively, revealed the highest final weight, weight gain %, SGR %, and final biomass of TMs followed by WRs. However, CMs, SPs and IBDs showed comparatively poor growth performance. Moreover, WKs under controlled conditions while TMs under semi-intensive culture conditions showed the highest % survival, whereas IBDs under both culture systems showed the lowest % survival. The mRNA level of growth-related genes, Myogenin, MyoD and IGF-11 in liver and muscle tissues of all stains showed a similar pattern, i.e., the highest expression in TMs and the lowest in the CMs and SPs. Moreover, significantly higher values of haematological indices (RBCs, HCT %, HGB and WBCs), innate immunity parameters (Total protein, IgM, Lysozyme activity, Respiratory burst activity and Phagocytic activity) and relative expression of Natural killer enhancing factor (NKEF), Lysozyme C, Lysozyme G, and TNF-α also indicated the comparatively improved health status and strong immunity of TMs followed by WKs. However, CMs followed by IBDs showed comparatively low values of all haemato-immunological indices. The status of antioxidant enzymes (CAT, SOD, POD, and GST of fingerlings of all strains also showed a similar trend, i.e., the highest activity in TMs followed by WRs while the lowest activity in the gills of CMs and IBDs. The proximate composition of the fingerlings of all strains after 90 days of rearing in communal ponds also showed the highest crude protein % and the lowest moisture (%) in the TMs, followed by WRs, while USs and SPs showed the lowest value of crude protein (%). For the evaluation of critical thermal tolerance (CTmax and CTmin), fingerlings of all strains were acclimatized at four acclimation temperatures (22, 26, 30 and 34°C) for 30 days before starting the trial. After that, both CTMax and CTMin tests were conducted by gradual increase or decrease in water temperature at the rate of 0.3°C per minute of each test chamber set at acclimated temperature till fish start showing loss of equilibrium (LOE), i.e., unable to maintain dorsoventral orientation for ~ 1 min. The highest and lowest temperatures at which fingerlings of each strain showed LOE were observed and considered critical thermal maxima (CTmax) and critical thermal minima (CTmin) of a particular strain, respectively. A thermal tolerance polygon over the range of 22-34°C revealed comparatively the highest CTmax with the largest total and intrinsic polygon zones as well as the upper and lower acquired thermal tolerance zones of TMs followed by WRs, however IBDs showed significantly the highest CTmin value and the smallest intrinsic and acquired thermal tolerance zones. Furthermore, this study also investigated the expression of heat shock protein genes in response to thermal shock xi by subjecting low (13°C) and high (37°C) thermal stress to 26±1oC acclimated fingerlings of all strains. Results indicated the highest mRNA levels of HPS70 and HPS90 genes in the TMs, followed by WRs groups exposed to normal (26°C) and the highest temperature (37°C) stress. However, the IBDs followed by CMs showed the highest expression of both genes after exposure to a low temperature. The current study also evaluated the comparative disease resistance of all strains by injecting the pathogen Aeromonas hydrophila (ATCC49140) at a concentration of 1.7 × 104 CFU/g body weight and monitoring the fish for any disease symptoms and mortality. The post-challenge survival rate showed a similar trend, i.e., the highest survival % of the fingerlings of TMs followed by WRs, while fingerlings of CMs and MKs showed the highest mortality rate. The post-challenge haematological indices: RBC, HB, WBC, platelets, hematocrit, and innate immunity parameters: total protein, IgM, lysozyme, respiratory burst activity, phagocytic activity, and phagocytic index also showed the highest values in the fingerlings of TMs while CMs showed the lowest values. The spatial and temporal relative expression of immunity-related genes: Complement component (C9), Transferrin and Hepcidinafter challenge to A. hydrophila were in accord to the results of immunity indices and indicated a significantly higher mRNA level of these genes in the liver and muscle tissues of the fingerlings of TMs followed by WRs and MKs while CMs showed comparatively lower expression. The overall results of this study provide the baseline data for the initiation of the genetic improvement program and indicate the genetic diversity and variation in growth performance, thermal tolerance, innate immunity, and disease resistance among different wild and captive-bred strains of L. rohita. In conclusion, TMs exhibited more vigour in terms of growth rate, stress tolerance and disease resistance, and could be suggested for improving fish production.