Modulatory role of dietary Thymus vulgaris essential oil and Bacillus subtilis against thiamethoxam-induced hepatorenal damage, oxidative stress, and immunotoxicity in African catfish (Clarias garipenus)

Thiamethoxam (TMX) is a widely used neonicotinoid insecticide for its effective potential for controlling insects from the agricultural field, which might induce toxicity to the aquatic biota. In this study, the role of the probiotic Bacillus subtilis (BS) and a phytogenic oil extract of Thymus vulgaris essential oil (TVEO) in the modulation of thiamethoxam (TMX)-induced hepatorenal damage, oxidative stress, and immunotoxicity in African catfish (Clarias garipenus) has been evaluated. Fish were subjected to TMX (5 mg L⁻¹) and fed with a diet either supplemented with BS (1000 ppm) or TVEO (500 ppm). The experiment lasted for 1 month. By the end of the experiment, blood was sampled for biochemical analysis and fish organs and tissues were collected for histopathological and immunohistochemical examinations. Results showed a substantial increase of serum markers of hepatorenal damage such as the activities of aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) and levels of blood urea nitrogen (BUN) and creatinine with an obvious decrease of serum protein levels in the TMX-intoxicated group. Also, there was a considerable increase in malondialdehyde (MDA) levels and glutathione-S-transferase (GST) activity. TMX remarkably suppressed serum lysozyme activity, respiratory burst activity, and phagocytosis with a conspicuous elevation of the levels of interleukins (interleukin-1 beta (IL-1β) and interleukin-6 IL-6). The histopathological findings showed that TMX induced degenerative changes and necrosis in the gills, liver, head kidneys, and spleen of the intoxicated fish. Significant alterations of frequency, size, and area percentage of melanomacrophage centers (MMCs), decreased splenocyte proliferation, and increased number of caspase-3 immunopositive cells were also observed. Contrariwise, the concurrent supplementation of either BS or TVEO in the diets of catfish partially mitigated both the histopathological and histomorphometric lesions of the examined tissues. Correspondingly, they improved the counts of proliferating cell nuclear antigen (PCNA) and caspase-3 immunopositive splenocytes. In conclusion, the co-administration of either BS or TVEO in catfish diets partially diminished the toxic impacts of TMX. Nonetheless, the inclusion of TVEO in the diets of catfish elicited better protection than BS against TMX-induced toxicity in response to its potential anti-inflammatory, antioxidant, anti-apoptotic, and immune-stimulant effects.