Synergistic role of pipronyl butoxide in improving the Spodoptera littoralis nucleopolyhedrosis virus effectiveness against Spodoptera littoralis and studying its histopathological biosafety

Spodoptera littoralis nucleopolyhedrosis virus (SlNPV) has been considered as a vital and safe alternative to chemical insecticides. It is effectively used in biological control of Spodoptera littoralis which is considered the most destructive and detrimental economic pest in Egypt. However, its slow speed of action limits its application among other bio-control agents. So, great attention was directed to solve this problem by using insecticide synergists. In this study the toxicity of both SlNPV and the insecticide synergist pipronyl butoxide (PBO) was investigated against 2nd and 4th instar S. littoralis larvae. Different concentrations of the tested viral isolate used (106, 107, 108, 109 and 1010 POB/ml) showed high mortality percentage ranging from 60 to 93% and 56 to 83% for the two tested instar larvae respectively 10 days post treatment. The three PBO concentrations used (0.1, 0.2 and 0.3%) revealed weak toxic effect as they produced 13, 20 and 26% mortality rate for the three tested concentrations respectively 4 days post treatment. To examine the biosafety of the three PBO concentrations (0.1, 0.2 and 0.3%), alone or in combination with SlNPV; in vivo animal studies, and in vitro cell line studies were applied. In vivo evaluations included histomorphometric and immunohistochemical alterations in Caspase-3, Proliferating cell nuclear antigen (PCNA), tumor necrosis factor – alpha (TNF-α) in hepatic and renal tissues, plus serum assessments of malondialdehyde (MDA) and total antioxidant capacity (TAC). The sulforhodamine B (SRB) cytotoxicity assay has been applied on OEC (Oral epithelial cell) and Vero (Green monkey kidney) cell lines. Results revealed that 0.1% PBO concentration was safe and didn’t induce any pathogenicity, as a dose concentration – dependent decreased pathological changes by comparing 0.3%, 0.2% PBO treated rats . Furthermore, SlNPV was safe to rat tissues and cells and PBO didn’t alter the safe nature of SlNPV on mammalian cells. According to the results of histopthological bio-safety, 0.1% of PBO was applied to 4th instar larvae for estimation its effect on larval enzyme activity and the results proved significant differences in the metabolic tested enzymes between the control and treated larvae with inhibition in both of Actylcholin esterase (AchE), Glutathione S-transferase (GST), Alpha esterases (α-esterases), Beta esterases (B- esterases), and Glutamic oxaloacetic transaminase (GOT) and stimulation in Lactate dehydrogenase (LDH), Glutamic pyruvic transaminase (GPT) and Multi-function oxidases (MFO). Moreover, 0.1% of PBO has been mixed with viral LC50 for 2nd and 4th instar larvae and the results showed highly synergistic effect of PBO as 100 and 96 % mortality rate respectively fourth day post treatment.