Production of intergeneric hybrid of Catla catla × Labeo rohita and evaluation of genetic and phenotypic variations

Abstract

Hybridization is a simple and well-known approach to genetic improvement via heterosis. However, heterosis in any hybrid comparative to parents is trait-dependent and may not be achieved in every case. Rohu (R) and Catla (C) are the highly culturable freshwater fish species in Pakistan, India, and Bangladesh. They are variable in qualitative and quantitative traits like C.catla is fast-growing than L.rohita but carcass value, nutritive value, and immunity of L. rohita is better than C. catla. Here, a comprehensive study was designed to combine the valuable traits of these closely and phylogenetically related species and produce F1 hybrid of superior quality. Induced breeding of these species was conducted and homospecific (R♀×R♂ and C♀× C♂) and reciprocal heterologous (C♀ × R♂ and R♀ × C♂) crosses were made for the production of parent species (RR and CC, rohu and catla respectively) and reciprocal hybrids (RC, L. rohita♀, and CR, C. catla♀). To evaluate the impact of hybridization, fertilization, and hatching rate, growth performance, blood profile, immunity, disease resistance, thermal tolerance, nutritive value, and morphological traits of reciprocal hybrids (RC &CR) to parental species (RR & CC) were compared. Results indicated statistically comparable and significantly higher fertilization and hatching rate of both purebred cross-types (RR and CC) as compared to hybrid cross-types (CR and RC). Furthermore, both hybrid cross-types also showed statistically similar fertilization and hatching rate. The growth performance data of all cross-types at different developmental stages, both in controlled and semi-intensive culture systems showed the highest final weight, weight gain % and SGR %, of CC followed by CR hybrids while RR showed the lowest values. Moreover, pairwise comparison among hybrids indicated considerably (p<0.05) higher weight gain and SGR % of CR hybrid. The relative expression of growth-related genes MyoD, Myogenin, and IGF-1 in all cross-types showed tissue-specific expression, i.e., higher expression of MyoD and Myogenin genes in the muscle compared to IGF-1 that showed higher expression in the liver. The expressions are in accordance with growth performance data, i.e., significantly (P<0.001) higher expression of Myogenin, MyoD and IGF-1 genes in the CC (C. catla) followed by F1 CR hybrid while the lowest expressions in RR (L.rohita). Based on growth performance, all cross-types followed the following rank CC > CR >RC>RR. Both F1 hybrids showed low parent heterosis, with the CR hybrid favorable mid parent heterosis. Phenotypically, both hybrids (RC and CR hybrids) were intermediate in most of the traits, while some traits resemble their mother species. In morphometric features, xi the CR hybrid resembled C. catla in body appearance and L. rohita in mouth profile. The body of CR hybrid was compressed, relatively deep with a far more convex dorsal profile compared to ventral profile, and like CC, pectoral, ventral, and anal fins were black. However, the body depth of the RC hybrid was wider than RR but not to CC, while the head size was smaller compared to CC and somewhat larger than RR. The mouth of both hybrids was terminal with a circular opening. For accurate identification and phylogenetic relationship of all cross-types (RR, CC, CR, and RC), DNA barcoding by mitochondria COX 1 was also conducted. The constructed Neighbour Joining (NJ) tree (K2P) based on COI gene sequence analysis categorized all four crosses into two genetically distant groups. Each hybrid showed an identical phylogenetic relationship with the mother species, i.e., CR hybrids showed the minimum distance with CC, and RC hybrid showed the minimum distance with RR. Moreover, the genetic distance between CC and RC hybrid was higher than between RR and RC hybrid. For the evaluation of health status, immunity, and disease resistance against pathogens, a randomly designed experiment under controlled conditions was also designed, and 200 fingerlings of each cross-type after about two weeks of acclimation were evenly distributed in their respective 8 tanks at a stocking density of 2g/L (25 fingerlings/tank). The first two tanks of each cross-type were marked as control without exposure to pathogen while the other six tanks were exposed to the pathogen. Among pathogen exposed tanks, 2 of each were used for evaluation of cumulative mortality (%) during 20 days exposure, the other 2 for the evaluation of immunological indices, and the last two for the temporal and spatial expression of immune-related genes. All fingerlings except in two tanks of each cross-type were exposed to pathogen Aeromonas hydrophila, at a concentration of 2.24×107 CFU/g. The control fish were injected with 100µL sterile PBS. For evaluating disease resistance, fish in each group were observed closely for disease symptoms and mortality was recorded. For hematological indices, at random 18 fingerlings of each cross-type were collected from their respective control group while for evaluation of immunological indices, after 24 hrs post infection, the blood of 18 fingerlings (9/tank), both from control and pathogen exposed groups of each cross-type were collected. Moreover, 6 fingerlings from the last two tanks of each cross-type (3 fish/tank) were captured after 0, 6, 12, 24, 48, and 168 hrs post-challenge, and their liver and muscle were stored in RNA Later™ for gene expression study. Results of this part of the study indicated that RC hybrids had the highest values of RBCs, WBC, MCH, MCHC, HB, and Hct% followed by CR hybrid as compared to parental species. The post- xii challenge survival rate showed a similar trend i.e., the highest survival % of the fingerlings of RC followed by CR hybrid while CC fingerlings showed the highest mortality rate. The pre and post-challenge immunity parameters: serum total protein, IgM, lysozyme level, phagocytic activity, phagocytic index and respiratory burst activity, also showed the highest values in the RC hybrids than CR hybrids while CC showed the lowest values. The spatial and temporal relative expression of immunity-related genes Lysozyme C, Lysozyme G, and TNF-α after challenge to A. hydrophila were in accordance to the results of immunity indices and indicated a significantly higher mRNA level of lysozyme C, lysozyme G, and TNF-α genes in the liver and muscle of the fingerlings of RC hybrid followed by CR and RR while fingerlings of CC showed comparatively lower expression. Overall, both hybrids showed higher immunity, and heterosis (LPH, MPH, and HPH) in all studied immunity parameters before and after challenge to A. hydrophila. Based on immunity, F1 fingerlings of all cross-types followed the following rank RC > CR >RR>CC before and after pathogen challenge, while based on heterosis, RC hybrid followed the CR hybrid. The critical thermal tolerance (CTmax and CTmin) of all cross-types: RR, CC, and their reciprocal hybrids RC and CR being acclimatized at four acclimation temperatures (22, 26, 30, and 34°C) indicated, comparatively the highest CTmax with the largest total and intrinsic polygon zones as well as the upper and lower acquired thermal tolerance zones of RR followed by RC and CR hybrids. However, CC showed the highest CTmin value and the smallest intrinsic and acquired thermal tolerance zones. Both RC and CR hybrids showed intermediate thermal tolerance with low parent heterosis. The nutritive value of all cross-types indicated a significantly higher percentage of crude protein and fat contents, monosaturated fatty acids (MSFA, 27.45%) and polyunsaturated PUFA (>33%) in the muscle of RR followed by both hybrids, while muscle of CC contained a significantly higher concentration of moisture, ash content, non-essential amino acids (>92%) and the lowest values of MSFA and PUFA. Moreover, both hybrids (RC and CR) showed intermediate values of crude protein and fat contents, MSFA and PUFA with a lower ratio of ω3 to ω6 PUFA as compared to parental species (RR and CC). Additionally, the RC hybrid showed significantly higher concentration of essential amino acids (EAA) especially alanine, yet the CR hybrid showed a significantly higher value of PUFA (p<0.05) as compared to both RR and CC. Overall results indicated intermediate growth performance, thermal tolerance, nutritive value, and morphological features while improved immunity and disease resistance of both hybrids compared to both parental species. These results also indicated the higher disease resistance, immunity, thermal xiii tolerance, and nutritive value of RC hybrid compared to CR hybrids which showed a higher growth rate.