A method was developed for RNAi that produced reproducible results in horn flies. Functional analyses by RNAi showed the effect of some genes on horn fly mortality and oviposition. These results advanced the molecular characterization of this important ectoparasite and sug gested candidate protective antigens for the develop ment of vaccines for the control of horn fly infestations. Based on RNAi results, some of the Inhibitors,Modulators,Libraries candidate antigens to be considered for cattle vaccination experiments against horn flies include those within VTG, immune response and 5 NUC functional groups. Methods Rearing of horn flies H. irritans were reared under laboratory conditions as reported by Schmidt et al. A horn fly colony was established with flies originally collected in a cattle farm close to Ciudad Victoria, Tamaulipas, Mexico.
About 2,000 flies were collected from 2 infested animals and transported in a 20 �� 30 cm mosquito netting alumi num cage. Flies were allowed to lay eggs over a water container during 12 h. Eggs were collected and incu bated into fresh bovine feces during 5 days. Pupae were collected Inhibitors,Modulators,Libraries and placed in Petri dishes located inside mos quito netting aluminum cages Entinostat for molting into adult flies. After molting, flies were fed twice a day using pieces of cotton impregnated with fresh defibrinated bovine blood obtained from a naive cow. All the horn fly developmental phases were kept under a photoperiod of 12 h light, 12 h darkness at 28 32 C and 70 80% relative humidity. Analysis of expressed sequence tags in adult female horn flies Total RNA was isolated from whole abdominal tissues collected from 1,500 partially fed adult female horn flies using Trizol.
The cDNA library was synthesized using the SMART cDNA Library Construction Kit at Creative Biolabs. cDNAs were cloned into the pBluescript II SK vector. The library had more than 1��106 primary clones, with 90% recombinants with Inhibitors,Modulators,Libraries inserts 500 bp. A total of 2,462 ESTs were 5 sequenced. The cDNA Annotation System software, Office of Technology Information Sys tems, National Institute of Allergy and Infec tious Diseases, Bethesda, MD, USA was used for automated sequence clean up, assembly, blasting against multiple sequence specific for vector DNA sequences flanking the horn fly cDNA insert and containing T7 promoter sequences for in vitro transcription and synthesis of dsRNA.
PCR reactions were performed from individual or pooled cDNA clones using the Access RT PCR system in a 50 ul reaction mix ture. The resultant amplicons were purified using the Wizard 96 well PCR purification system. In vitro transcription and purification of dsRNA was done using the Megascript RNAi kit. Inhibitors,Modulators,Libraries The dsRNA was quantified by spectrometry. Adult partially fed female flies were injected with approximately 0. 1 ul of dsRNA in the abdominal segment.