Molecular, histological and ultrastructural characterization of cytotoxic effects of amitraz on the ovaries of engorged females of Rhipicephalus (Boophilus) annulatus

Abstract

In the present study, the cytotoxic effects of amitraz, an octopamine receptor agonist on the reproductive system of engorged adult females of Rhipicephalus (Boophilus) annulatus were assessed using histology, electron microscopy and octopamine beta (OCTβ) receptor transcriptional expression analysis. Adult immersion test (AIT) was performed by immersing the fully engorged female ticks for 2 min in different concentrations of amitraz (200, 250, 300, 350 ppm). Amitraz at the dose of 300 ppm, caused an adult tick mortality of 16.66 ± 6.80 per cent, inhibition of fecundity of 75.80 per cent and hatching of 50 per cent of ova laid by treated ticks. Histological changes in the ovaries of ticks collected after 24 h of treatment with amitraz (300 ppm), in comparison with controls (distilled water/methanol) were identified by microscopical examination of sections (4 μm) stained using haematoxylin and eosin. These changes included reduction in size and basophilia of stage I oocytes, presence of cytoplasmic vacuoles of various sizes around germinal vesicle of stage II oocytes, wavy basement membrane of stage III oocytes and reduction in size and number of mature stage IV and V oocytes. Electron microscopy was employed for understanding the structural changes in the ultrathin sections (60 nm) of ovaries. Ticks treated with amitraz showed major ultrastructural changes such as irregular nuclear membrane, crystolysis of mitochondria and detachment of external and internal layers of basal lamina of oocytes. The cDNA synthesized from the total RNA of whole ticks and ovaries of ticks treated with amitraz along with controls were used for relative quantification of Octopamine β receptor (OCTβ-R) expression based on the 2−ΔΔCT method by quantitative real time PCR (qRT PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. Down regulation of expression of OCTβ-R mRNA in the ovaries of amitraz treated ticks was observed compared to controls. Thus, the inhibition of fecundity observed in the ticks treated with amitraz can be attributed to the major structural changes and decreased expression of OCT β receptor mRNA induced by it in the ovary.