Relationship between assembly behavior and molecular responses in foveal gland of unfed and fed adult Rhipicephalus appendiculatus

Abstract

Ixodid ticks respond to a limited spectrum of stimuli in their search for hospitable environments, hosts and mates hence change in their behavior. This responsiveness is mediated by pheromones which are signaled by biochemical changes that occur during different stages of feeding. Genes induced during blood feeding result in the expression of new proteins secreted into tick foveal gland. Some of these proteins may be involved in the biosynthesis of these pheromones. Assembly behavior of female and male ticks appears to be mediated by an attractant signal and an assembly pheromone blend with one well characterized component. Preliminary studies suggest that when both sexes of adults acquire blood meals from host, the female enhances the production of an attractive signal and the male suppresses it. In addition, this preliminary observation suggests that with feeding there is a pheromonal switch from mutual aggregation assembly to sex attraction i.e. fed female tick attracting feeding or fed males. The aim of this study was to examine the response of adult male and female Rhipicephalus appendiculatus ticks to assembly pheromone in a two choice bioassay technique using guanine, tick excreta and nymphal skin washings as source of assembly signal. This study also aimed at comparing protein profiles of foveal gland extracts from fed and unfed male and female R. appendiculatus. Proteins were extracted from the foveal gland of unfed, 2 days, 4 days and 6 days fed ticks. The differentially expressed proteins were determined using two-dimensional polyacrylamide gel electrophoresis followed by the computer analysis of the scanned gels. Results obtained from bioassay tests were analyzed using student t-test while the gels were analyzed using Melanie software. Response of R. appendiculatus to guanine, tick excreta and nymphal skin washings was evident during the first hour of exposure, with little change observed between 2 and 24 hours. The assembly response was strongest to excreta treated sectors followed by guanine and nymphal skin washings. Both male and female ticks assembled at the treated sectors and there were no significant difference to their attractions. Most of the expressed proteins spots were acidic and of low to high molecular weights among all the levels of feeding. Most spots in 2D were induced in day 2 and 4 fed stages in both sexes. Conversely, in the day 6 fed populations, less spots were differentially expressed with most of them in acidic range, pH 4-6 and of high molecular weight, 90-150 kDa. Most spots in day 2 fed male and female populations were also acidic, pH 4-5 and of high molecular weight 60-150 kDa. In day 4 fed populations most spots were basic pH 8-9 and molecular weight 60-120 kDa. The results provide a basis for identification of proteins associated with pheromonal switch during feeding and lays down some ground work for the development of novel control tactic for the ticks.