Background: Serine proteinase inhibitors (Serpins) are a large superfamily of structurally related, but functionally diverse proteins that control essential proteolytic pathways in most branches of life. Given their importance in the biology of many organisms, the concept that ticks might utilize serpins to evade host defenses and immunizing against or disrupting their functions as targets for tick control is an appealing option.

SUMMARYIn order to successfully feed and transmit disease agents, ticks are thought to inject serine protease inhibitors (serpins) into the host to modulate host defense responses to tick feeding, such as inflammation, the complement activation pathway and blood coagulation. In this study, we show that Amblyomma americanum (Aam) serpin (S) 6 is putatively injected into the host during tick feeding, in that the antibody to recombinant (r) AamS6 specifically reacted with the expected ~43/45kDa AamS6 protein band on western blots of pilocarpine-induced tick saliva. Additionally, antibodies to tick saliva proteins that were generated by repeated 48h infestations of rabbits with adult A. americanum specifically reacted with rAamS6. We speculate that AamS6 is associated with regulating events at the start of the tick feeding process, as temporal and spatial RT-PCR and western blot analyses revealed that both AamS6 mRNA and protein are strongly expressed during the first 24-72h of feeding time before starting to fade from 96h. The AamS6 protein has an apparently slow turnover rate in that, although the injection of AamS6 dsRNA into unfed ticks triggered complete disruption of the AamS6 mRNA by the 48h feeding time point, western blot analysis of protein extracts of the same animals showed that the AamS6 protein that may have been expressed prior to disruption of the AamS6 mRNA was not depleted. We speculate that the presence of the AamS6 protein in ticks despite the complete disruption of the AamS6 mRNA explains the observation that RNAi-mediated silencing of the AamS6 mRNA did not affect the ability of A. americanum ticks to attach onto host skin, successfully feed and lay eggs. These findings are discussed in regards to advances in the molecular biology of ticks.

The induction of the naturally occurring phenomenon of RNA interference (RNAi) to study gene function in insects is now common practice. With appropriately chosen targets, the RNAi pathway has also been exploited for insect control, typically through oral delivery of dsRNA. Adapting current methods to deliver foreign compounds, such as amino acids and pesticides, to mosquitoes through sucrose solutions, we tested whether such an approach could be used in the yellow fever mosquito, Aedes aegypti. Using a non‐specific dsRNA construct, we found that adult Ae. aegypti ingested dsRNA through this method and that the ingested dsRNA can be recovered from the mosquitoes post‐feeding. Through the feeding of a species‐specific dsRNA construct against vacuolar ATPase, subunit A, we found that significant gene knockdown could be achieved at 12, 24 and 48 h post‐feeding.

SUMMARYThe organic anion transporting polypeptides (Oatps in rodents and other organism; OATPs in human) are Na + -independent transporters that shuttle a wide range of endogenous and xenobotic amphipathic compounds across plasma membranes. We previously discovered an Amblyomma americanum tick (Aam) Oatp cDNA among genes that were upregulated or induced in ticks that were stimulated to start feeding. In this study, we have characterized a 2860 bp full-length cDNA that encode a 724 amino acid putative protein. Bioinformatics and hydropathy analyses revealed that, in addition to the kazal-type serine proteinase inhibitor motif, AamOatp possess typical features that characterize the Oatp/OATP protein family, including 12 transmembrane (TM) domains, the consensus amino acid motif D-X-RW-(I,V)-GAWW-X-G-(F,L)-L and 11 consensus cysteine residues in the large extracellular domain between TM9 and TM10. AamOatp is constitutively and ubiquitously expressed, as determined by RT-PCR amplification of the transcript, in all organs of ticks that fed for 1-7 days. Analysis of the normalized transcript abundance revealed that from days 1 to 5 of feeding, AamOatp mRNA expression in the midgut (MG) was 60-80-fold higher than levels found in the salivary gland (SG), ovary (OV) and carcass (CA). By contrast, by day 7 of feeding, the AamOatp mRNA was 60-80-fold more strongly expressed in the OV than in the SG, MG and CA. These data strongly indicate that changing physiological needs during the tick feeding process influences transcriptional regulation of AamOatp. Our data also show that RNAi-mediated suppression of the AamOatp caused ticks to obtain smaller blood meals, which consequently resulted in ticks laying fewer eggs. The results are discussed in the context of AamOatp as a potential pharmacological or anti-tick vaccine target.

This study was conducted to determine the efficacy of truck-mounted ultra-low volume applications of pyriproxyfen against Aedes aegypti larvae in artificial water containers and wild adult Ae. albopictus populations in an urban setting. The study was conducted over a 3.5-month period (June-October 2012), during which 3 pyriproxyfen applications were conducted. Beginning 6 wk prior to the 1st pyriproxyfen spray, 10 Biogents-Sentinel® traps were used each week to survey the adult Ae. albopictus population at each experimental plot through the end of the study. The treatment and control plots contained specimen cups, each containing 10 laboratory-reared Ae. aegypti larvae, placed at 8, 15, and 23 m from the spray line. Emergence inhibition (EI) of 82% or greater was observed among Ae. aegypti larvae exposed to the 3 pyriproxyfen sprays. The EI of these same Ae. aegypti larvae at the 3 distances from the spray ranged from 84% to 92% and were not significantly different. Laboratory analysis of water samples taken from the larval cups independently confirmed the presence of pyriproxyfen. Similar levels of EI were achieved in Ae. aegypti and Ae. albopictus larvae when the measured field concentrations of pyriproxyfen were recreated in laboratory assays. Trap captures of wild adult Ae. albopictus were not markedly reduced following the 1st pyriproxyfen spray, perhaps due to heavy rainfall at the time and the lower rate of pyriproxyfen applied. Within 2 wk following Spray 2, however, Ae. albopictus collections from the treatment plot averaged approximately 50% of those from the control plot, and the reduction trend continued following Spray 3.