Effects of flumethrin on the germinative cells of engorged females of Rhipicephalus annulatus.

Chemical acaricides are widely used for the effective control of ticks in India. The synthetic pyrethroids, are one of the most popular chemical acaricides with selective neurotoxic potential. Flumethrin is a type II synthetic pyrethroid used extensively in veterinary practice in India. The present study was undertaken to evaluate the cytotoxic effects of flumethrin on the engorged females of Rhipicephalus annulatus using entomological parameters, histology, electron microscopy and relative quantification of receptors of dopamine and GABAB mRNAs. Adult immersion test (AIT) using flumethrin (100 ppm), revealed twenty per cent mortality of ticks, hundred per cent inhibition of fecundity and complete blocking of hatching of the laid eggs. Microscopic analysis of the structure of the ovaries after 24 h of treatment with flumethrin (90 ppm) revealed changes, viz., reduction in size with the presence of amorphous material inside stage I oocytes, wrinkled boundary and chromatin fragmentation of nucleus of stage II oocytes, vacuoles around the germinal vesicle, thickening of the nuclear membrane and chromatin clumping of stage III oocytes and reduction in size and shape of mature stage IV and V oocytes. Also, a large number of vacuoles were observed throughout the pedicel cell region of stage II and III oocytes. Ultrastructurally, irregular nuclear membrane, swelling as well as crystolysis of mitochondria and detachment of external and internal layers of the basal lamina of oocytes were the major structural alterations confirming direct damaging effects of flumethrin on the germinative cells. The relative quantification of the expression of dopamine D1, dopamine D2 and GABAB receptors by quantitative real-time PCR (qRT PCR), revealed the upregulation of dopamine D1 receptor and downregulation of receptors of dopamine D2 and GABAB in the ovary of treated ticks.

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

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 (OCTß) 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 ±â€¯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 ß receptor (OCTß-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 OCTß-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 ß receptor mRNA induced by it in the ovary.

Ultrastructural analysis of oocytes of Rhipicephalus (Boophilus) annulatus during postengorgement period as a tool to evaluate the cytotoxic effects of amitraz and deltamethrin on the germinative cells.

The present study utilizes the ultrastructural analysis of the fully engorged female Rhipicephalus (Boophilus) annulatus ticks, as a tool to evaluate the cytotoxic potential of deltamethrin and amitraz on the germinative cells. The ultrastructural analysis of the ovary of the normal (untreated) R (B.) annulatus revealed, oocytes in different stages of development, attached to the ovary wall by pedicel cells. The attachment site of oocyte to the pedicel cell was characterized by indentations of the plasma membrane. The oocyte was bound by three cell membranes viz., plasma membrane, chorion and basal lamina. The stages of oocytes were differentiated ultrastructurally based on the features of their outer membrane and the number and size of lipid and yolk droplets. Detailed day wise analysis of ultrastructural changes in the ovary during the post-engorgement period revealed the occurrence of the degenerative changes from day five onwards. These appeared first in the oocytes followed by the germinal epithelium. The ovary of ticks treated with methanol (control), revealed similar topographies as that of a normal ovary except for the presence of very few oocytes with ring shaped nucleoli. Ultrastructurally, treatment with deltamethrin produced more prominent and extensive morphological alterations when compared to amitraz. In the case of ticks treated with amitraz, the oocytes of stage IV and V showed wavy and disrupted outer boundaries along with the loss of integrity of the yolk droplets. Uneven nuclear membranes of stage II oocytes and cristolysis of mitochondria of mature oocytes were the other changes noticed. Ticks treated with deltamethrin revealed prominent modifications such as, detachment of the basal lamina, wrinkled boundary, inconsistent nuclear membrane, ring shaped nucleoli and chromatin clumping in the case of the early stage oocytes (I and II), whereas swelling and cristolysis of mitochondria were seen in mature oocytes. The study further indicated that, in addition to the previous proven neurotoxic effects, these compounds act directly on the ovary of tick.