Male engorgement factor: Role in stimulating engorgement to repletion in the ixodid tick, Dermacentor variabilis.

Mating in ticks results in profound physiological changes that eventually results in egg production. In the American dog tick, Dermacentor variabilis, mating causes partially blood-fed female ticks to commence rapid engorgement to repletion and eventual detachment from the host and egg laying. The peptidic male pheromone (engorgement factor alpha/beta) transferred to the female during mating is known only from a single tick species, Amblyomma hebraeum, and was shown to consist of two peptides produced in the testis/vas deferens (TVD) and not in the male accessory gland (MAG). In the current study, we obtained 2704bp of sequence data for efalpha from D. variabilis, of 7kb as determined by Northern blot, and show that it is also present in the Southern cattle tick, Rhipicephalus microplus and the deer tick, Ixodes scapularis. Analysis of the male gonad transcriptome by pyrosequencing produced 563,093 reads of which 636 matched with efalpha; none matched with efbeta. No evidence of efbeta orthologs could be found in any publicly available database including the I. scapularis genome. Silencing efalpha in male ticks failed to significantly reduce the engorgement weight of females compared to controls. Injection of sephadex beads, replete female synganglia, fed male MAG, fed male TVD, or replete female vagina/seminal receptacle (VA/SR), separately, failed to initiate feeding to repletion like that found in normally mated females. However, a small percentage of females injected with VA/SR that fed beyond the arbitrary weight for repletion of 300mg, produced brown eggs (an indication of vitellogenin uptake by the oocytes). The greatest effect was observed in female ticks injected with a suspension of MAG and TVD combined; 50% fed to repletion and all of these dropped off from the host and laid brown eggs. The effect was abolished if the aqueous fraction of the MAG/TVD homogenate only was injected suggesting that EF in ticks is a non-secreted membrane-bound or intracellular protein. Overall, these data suggest that EFalpha in D. variabilis is not an engorgement factor.