Comparative Disease Resistance in Clarias Gariepinus, Heterobranchus Longifilis, And Their Hybrid in Nigerian Aquaculture
1Oyerinde J. I. , 1Oladosu, G. A., 2Aina, O. O. and 3Ahankonye I. C
*Corresponding author: Oyerinde J. I, Department of Veterinary Medicine, Faculty of Veterinary Medicine, University of Ibadan, Nigeria, Email: oyerindejosiah@gmail.com
Citation: Oyerinde J. I, Oladosu, G. A, Aina, O. O. and Ahankonye I. C [2025] Comparative Disease Resistance in Clarias Gariepinus, Heterobranchus Longifilis, And Their Hybrid in Nigerian Aquaculture. Jr Aqua Mar Bio Eco: JAMBE-161.
DOI: 10.37722/JAMBE.2025205
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Abstract
Anecdotal evidence from fish farmers in Nigeria suggests that the hybrid of Clarias gariepinus (female) and Heterobranchus longifilis (male) are hardier to diseases compared to their parent species. There is the need to validate this assumption. Blood samples were collected from ten each of Clarias gariepinus, Heterobranchus longifilis and their hybrid for haematology. Samples of the liver, spleen, kidney, gills and posterior intestine were also collected for histology. The three catfish species were experimentally infected with 1.5×108 cfu per ml of Aeromonas carviae by immersion. Gross lesions and mortality were observed and recorded for 14 days; and these were used as indicators to measure disease resistance. Haematology and histopathology were also conducted post-infection. The pre-infection comparison of Total WBC, lymphocyte and eosinophils, across the three groups showed significant difference (p < 0.05), and the hybrids had the highest value of WBC, lymphocytes and eosinophils. However, there was no significant difference post-infection. Mortality was recorded post-infection in Clarias gariepinus only, although all the groups had clinical signs and gross lesions. Pre-infection and post-infection comparison revealed significant differences in the lymphocytes and eosinophils counts, with the hybrid having the highest increase. These results show that the hybrids have better immune response to Motile Aeromonas Septicaemia (MAS) infection as evidenced in the highest increase in lymphocyte count, and the adaptive immunity plays a major role in the line of defense against Motile Aeromonas Septicaemia (MAS) infection in the African catfish species.
Keywords: Adaptive immunity, Clarias gariepinus, Disease resistance, Heterobranchus longifilis, Hybridization, Immune response, Lymphocytes.
Introduction
The commercial culture of the African catfish has become prominent for food fish production, and has transformed into a viable industry in Nigeria, making the country the highest producer of the African catfish species globally (FAO, 2023). However, catfish farmers often experience high-level mortality events due to microbial pathogens. Previous studies revealed mortalities ranging from 1.9% to 19.7% per production cycle (Mukaila et al, 2023).
Hybridisation is a genetic technique employed to enhance economic traits in fish by breeding species that share a common evolutionary ancestry (Bondoc, 2008). Hybrid vigour or heterosis arises from the combination and interaction of genes inside gene pools, where individual populations, inbred lines, or species contribute to the various alleles (Ataguba et al., 2024). In catfish production, hybridization is used to produce progeny from a cross of Clarias gariepinus and Heterobranchus spp. It is used for stock improvement and management purposes (Owodeinde et al, 2012). A number of studies have proven that hybrids have improved survival and higher growth, when compared to their parent breeds (Madu et al, 1999). Farmers claim that the hybrid catfish are hardier than the parent species. This is the basis for the increasing demand by commercial farmers for the juveniles of the hybrids popularly referred to as Heteroclarias.
There is a need to scientifically establish the assumption that the hybrid catfish are hardier than the parent species, and also determine the factors that contribute to disease resistance observed in the cross of Clarias gariepinus and Heterobranchus spp. (Heterobranchus bidorsalis and Heterobranchus longifilis), if so established.
Materials and Methods
Study area
The study was carried out at the wet-laboratory of the Aquatic and Wildlife Medicine Unit of the Department of Veterinary Medicine, University of Ibadan.
Ethical approval
Ethical approval was obtained from Animal Care and Use Research and Ethics Committee (ACUREC), of the University of Ibadan with approval number; UI-ACUREC/058-0324/27.
Experimental design
Brood-stocks of Heterobranchus longifilis and Clarias gariepinus of known breeding records were procured locally from a reputable private catfish hatchery in Ibadan. Crossbreeding of the two fish species was conducted in the hatchery as follows: Clarias gariepinus ♀ x Clarias gariepinus ♂ (C x c) representing group A, Heterobranchus longifilis ♀ x Heterobranchus longifilis ♂) (H x h) representing group B, and Clarias gariepinus ♀ x Heterobranchus longifilis ♂ (C x h) representing group C. Therefore, Group A consists of pure line of Clarias gariepinus, group B consists of pure line of Heterobranchus longifilis while group C consists of the hybrids of the two genera popularly called Heteroclarias.
Standard spawning procedures were carried out and hatchlings were transferred to fibre-glass tanks and reared till they reached juvenile size in about 12 weeks. They were fed ad libitum with commercial fish feed (CoppensR). The feed size was changed progressively as the fishes grew from 0.2mm to 2mm. The tank water was continuously renewed every day. The fishes were sorted weekly so as to remove the shooters, and avoid cannibalism.
Sixty apparently healthy juvenile fishes were stocked in three groups in 40L tank, containing 20L of water and acclimatized for two weeks under water flow-through condition. The water parameters (temperature, pH, Dissolved Oxygen, etc) were maintained in appropriate range throughout the experiment. Group A contained pure lines of Clarias gariepinus. Group B contained pure lines of Heterobranchus longifilis. Group C contained hybrids of Clarias gariepinus (female) and Heterobranchus longifilis (male). The average weights were Clarias gariepinus: 32.14 ± 2.50g, Heterobranchus sp.: 35.78 ± 2.75g, Hybrid: 27.5 ± 0.68g. The fishes were fed to satiation, twice daily.
Haematology
Ten fishes per group were randomly selected and bled via the caudal vein, using a 2ml syringe and 23G needle. The blood samples were collected into sample bottles containing EDTA for assessment of haematological parameters. Total white blood cells (WBC) was measured using a Neubauer haemocytometer.
Blood smears were prepared and stained with Giemsa stain to determine the differential WBC (Lymphocytes, Heterophils, Monocytes, Eosinophils, and Basophils) counts. This was done to evaluate the immune status of the fishes before and after the experimental infection.
Histology and Histopathology
Ten fishes per group were sacrificed by stunning, and tissue samples of the gills, posterior segment of the intestine and other lymphoid organs (liver, spleen and kidney) were harvested and fixed in 10% formalin. Standard histological procedures were carried out using Haemoxylin and Eosin stain on 0.5µ sections of the different tissue samples, which were then observed under the microscope for the presence of immune cells. Immune cells like mucus cells, macrophages, neutrophils, lymphocytes, and dendritic cells were observed in the tissue sections and compared among the fish species. This was done before and after the experimental infection.
Bacterial culture
Samples of clinically ill fish were gotten from a commercial hatchery in Ibadan. The fish was aseptically dissected to access the internal organs. Swab samples were aseptically collected from organs showing pathological lesions. These organs include the skin, the trunk kidney and the dorsal kidneys. The swab samples were streaked on individual plates and incubated at 30°C for 24 hours. Mixed growth of different organisms was observed. Discrete colonies of individual organisms were then streaked on freshly prepared plates to get a pure culture after incubation for 24 hours at 30°C.
A bacterium was identified as Aeromonas caviae with 98% probability, using Vitek 2- Compact (BioMerieux) for characterization. A colony of the identified bacterium was inoculated in 10ml of sterile nutrient broth and incubated for 24 hours at 30°C. All bacteriological work was done in the biosafety hood with lamina flow to prevent contamination. After 24 hours, the bottles were observed for turbidity which indicated growth of bacterium, and the turbidity was adjusted using sterile normal saline, to McFarland Standard 0.5.
Experimental infection
Thirty apparently healthy catfish juveniles (ten fish per group A, B and C) were randomly selected for experimental infection and separated into 40L plastic tanks containing 20L of water and arranged in flow-through system. The groups were taken off feed for 48 hours and then infected by immersion in water containing 1.5×108 cfu per ml of the isolate at a concentration of 5ml per litre, following a method established by Madubuike et al. (2015). There was daily monitoring of the fish for 14 days, during which clinical signs, pathological lesions and mortality were observed and recorded. All water parameters were maintained within acceptable limits throughout the study.
Statistical analysis
The blood parameters obtained from the haematology were compared using Graphpad Prism version 5 for Windows software. Column statistics analysis was done to estimate the mean, standard deviation, standard error of mean etc. One-way Analysis of Variance (ANOVA) was used to calculate the level of significance between the variance of the groups of fish. Bartlett’s test for equal variance and Turkey’s Multiple Comparison tests were also done. All tests were conducted at a 95% Confidence level, with a probability value of P < 0.05 considered significant.
Results
Haematology:
The mean haematological values obtained across the three species both pre-infection and post-infection are as presented in Table 1.
| Pre infection | Post infection | ||||||
| Parameters | Heteroclarias (hybrid) | Clarias gariepinus | Heterobranchus longifilis | Heteroclarias (hybrid) | Clarias gariepinus | Heterobranchus longifilis | |
| Total WBC (103/µl) | 17000 ± 445.5 | 16678 ± 835.3 | 14075 ± 306 | 19460 ± 1643 | 16914 ± 2785 | 15550 ± 749.3 | |
| Lymphocytes (103/µl) | 10993 ± 295.4 | 10700 ± 928.7 | 8030 ± 359.1 | 12872 ± 1352 | 11740 ± 1824 | 9861 ± 470.2 | |
| Heterophils (103/µl) | 4889 ± 253.6 | 5249 ± 205.4 | 5357 ± 117.8 | 6846 ± 1662 | 3907 ± 790.1 | 4723 ± 408.4 | |
| Monocytes (103/µl) | 446.4 ± 51.71 | 258.6 ± 51.38 | 376.6 ± 78.52 | 503 ± 79.55 | 725.9 ± 306.7 | 468.1 ± 87.02 | |
| Eosinophils (103/µl) | 622 ± 57.18 | 432.6 ± 70.37 | 277.4 ± 33.51 | 802.5 ± 128.5 | 628.9 ± 102.1 | 474.9 ± 77.76 | |
| Basophils (103/µl) | 49.5 ± 25.24 | 37.67 ± 24.92 | 34.38 ± 22.5 | 76.3 ± 49.4 | 48.5 ± 31.47 | 58.69 ± 29.41 | |
| PLAT | 128400 ± 8690 | 160444 ± 11169 | 114875 ± 3632 | 227600 ± 26545 | 173857 ± 8961 | 201625 ± 9580 | |
Table 1: Mean (±SEM) of Pre-infection and Post-infection Haematological Parameters.
Generally, there were reductions in the post-infection mean PCV across all groups and these reductions were significant (p value < 0.05). There was a general increase in the post-infection mean WBC count across the groups when compared to the pre-infection groups. The highest increase in mean total WBC count was observed in the hybrids (2460), followed by the Heterobranchus longifilis group (1475), and Clarias gariepinus group(238) had the least increase in mean total WBC count. The differences observed were not significant (p value > 0.05). There was a general increase in the post-infection mean lymphocyte count across the three groups when compared to the pre-infection groups. The highest increase in mean lymphocyte count was observed in the hybrids (1879), followed by the Heterobranchus longifilis group (1831), and Clarias gariepinus group(1040) had the least increase in the mean lymphocyte count. The differences in lymphocyte count pre and post infection was significantly higher in the hybrid (p value < 0.05).
Furthermore, there was an increase in the mean heterophil count observed in the hybrids post infection (317) while there was a decrease in the values observed in Heterobranchus longifilis (-634) and Clarias gariepinus (-1342). However, these differences were not significant (p value > 0.05). Also, there was a general increase in the post-infection mean eosinophil count when compared to the pre-infection group. The highest increase was observed in Heterobranchus longifilis (197.5), followed by Clarias gariepinus (196.3) and the hybrids had the least increase (180.5). Eosinophil count was significantly higher in both H. longifilis and C. gariepinus than in Heteroclarias (p value < 0.05). There was no significant difference in the mean values of the monocytes, basophils and MCHC (p value > 0.05).
Gross lesions and mortality from experimental infection
Gross lesions such as exophthalmia, haemmorhages on the ventral and tail fin, inflammation of the foramen between the frontal bones (resembling lesions of Edwardsiella ictaluri), and ulceration dermatitis at the ventral part were observed in experimentally infected fish (Fig. 1). Mortality of one fish was observed in group A (C. gariepinus) only. The occurrence of mortality and lesions across the groups is as described in table 2 below.
Inflammation of the foramen between the frontal bones. B. Mild ulceration
Of the ventral part of the skin. C. Haemorrhage of the ventral and tail fin.
Table 2: Summary Table Showing Resistance Performance in Experimentally Infected Fish Using Severity of Gross Lesions and Mortality as Indicators.
| Clarias gariepinus | Heterobranchus longifilis | Heteroclarias (hybrid) | |||
| Lesions | Exophthalmia | + | + | + | |
| Haemorrhage on the ventral and tail fin | – | + | – | ||
| Inflammation of the foramen between the frontal bones | – | + | – | ||
| Mild skin ulceration | + | + | – | ||
| Mortality | 1 | 0 | 0 | ||
Histology and Histopathology
There were lesions observed in the liver with reduced vacuolation, degeneration and coalescing of the hepatocytes. The gills had disruption of the lamella core and loss of mucosal architecture. The kidneys had diffused tubular degeneration and ballooning. The spleen and the posterior segment of the intestine did not have prominent pathological lesions. These lesions were general to all the groups. There were no differences in the lesions observed in each group. Also, there were no significant differences in the presence of immune cells found in the tissue sections.
Discussion
Under physiological state (prior to the infection), there was a trend observed with regard to the total white blood cells across the three species. It was observed that the highest mean total WBC count was in the hybrids (17000 ± 445.5), followed by the Clarias gariepinus group (16678 ± 835.3), and Heterobranchus longifilis (14075 ± 306) had the least mean total WBC count, and these differences were significant (p value < 0.05). This implies that the hybrids will be able to elicit greater immune response against infectious agents when compared to the other two groups. A similar trend was observed with the lymphocytes, where the highest mean lymphocytes count was found in the hybrid group (10993 ± 295.4), followed by the Clarias gariepinus group (10700 ± 928.7), and Heterobranchus longifilis (8030 ± 359.1) had the least mean lymphocyte count. White blood cells and lymphocytes are a part of the major cells of defense in the body. It has been observed that WBC and lymphocyte count have implication on the immune response of the body and its ability to successfully fight infections (Douglas et al, 2010).
Other immune cells (heterophils, monocytes, basophils) did not have significant differences across the three species. This could suggest that the adaptive immunity is the main line of physiological defense in the hybrids. This negates the perspective given by Firdaus-Nawi et al. (2016) that because fishes do not have fully developed lymphoid organs, their main line of defense will be the innate immunity. As a matter of fact, the hybrids had the least mean values of pre-infection heterophils (4889 ± 253.6) when compared to the Clarias gariepinus (5249 ± 205.4) and Heterobranchus longifilis (5357 ± 117.8).
After experimental infection, similar trends were observed with the total white blood cell count and lymphocytes, where there were increments across the three species. The highest values were in the hybrids, followed by Clarias gariepinus and Heterobranchus longifilis had the least values. Although increments observed were not significant (p value > 0.05), the fact that there were increments across the three species show that there were immune responses in all species.
Comparing the values of increment in pre-infection and post-infection parameters, it was observed that the hybrids had the highest level of increase with regard to the total white blood cells (2460), followed by the Heterobranchus longifilis group (1475), and Clarias gariepinus group(238) had the least increase in mean total WBC count. Although the differences were not significant (p value > 0.05), this trend suggests that there is a much better immune response in hybrids, and moderate response in the Heterobranchus longifilis and an abysmal response in Clarias gariepinus. This supports the concept of hybrid vigour (heterosis or heterozygote advantage). In a comparative study of hybrid yellow catfish and its parental species (yellow catfish; Pelteobagrus vachelli and yellow catfish; Pelteobagrus fulvidraco) it was found that the hybrids exhibited a more prominent non-specific immune response which was interpreted as heterosis (Zhang et al, 2019).
The lymphocytes comparison also showed similar trend with the highest increment observed in the hybrids (1879), followed by the Heterobranchus longifilis (1831), and Clarias gariepinus (1040) had the least increase in mean of the lymphocyte count. These differences were significant (p value < 0.05). This trend affirms that hybrids have relatively better lymphocyte response to infections when compared to Heterobranchus longifilis and Clarias gariepinus.
The heterophils comparison showed that there was an increase in the mean heterophil count in the hybrids (317) while there were decreases in Heterobranchus longifilis (-634) and Clarias gariepinus (-1342) values. These differences were not significant (p value > 0.05). Heterophils are known to be the first line of response in innate immunity against bacterial infections. They are directed by the chemotactic factors released by the wounded tissues (Biller-Takahashi et al, 2014). However, this trend reports a reduction in the heterophil counts observed in both Clarias gariepinus and Heterobranchus longifilis. Further study should be done on this to understand what could be responsible for this deviation from the norm.
From this study, it can be inferred that the hybrids of Clarias gariepinus (female) and Heterobranchus longifilis (male) have higher disease resistance when compared to their parent species. This is indicated by the presence of consistently higher WBC and Lymphocytes both pre-infection and post-infection. It is also observed that the main line of defense in the hybrid catfish is the adaptive immune response which is depicted majorly by the lymphocytes. The lymphocytes could be B-lymphocytes which are involved in humoral immune responses; the production of plasma cells and antibodies, or T-lymphocytes which are involved in cell-mediated immune responses. It can also be deduced from the study that the disease resistance observed in the hybrids is likely linked to the Heterobranchus longifilis as the total white blood cells and lymphocyte values are not too distant to those observed in the hybrids.
Conclusion
This study reveals that the hybrids of Clarias gariepinus (female) and Heterobranchus longifilis (male) have higher disease resistance when compared to their parent species. It can also be concluded that hybrids demonstrate superior innate and adaptive immune response compared to their parent species.
Conflict Of Interest Statement: We declare that we have no conflict of interest.
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