The bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) method was used to carry out the classification analysis of bacterial flora in adult female and male horn flies and horn fly eggs.

The bTEFAP method identified 16S rDNA sequences in our samples which allowed the identification of various prokaryotic taxa associated with the life stage examined. This is the first comprehensive report of bacterial flora associated with the horn fly using a culture-independent method. Several rumen, environmental, symbiotic and pathogenic bacteria associated with the horn fly were identified and quantified. This is the first report of the presence of Wolbachia in horn flies of USA origin and is the first report of the presence of Rikenella in an obligatory blood feeding insect.

Adult horn flies were collected on a single date from pastured cattle at the Louisiana State University Agricultural Center, St. Gabriel Research Station using aerial nets. Within 1 h after collection the flies were transferred to large sterile Erlenmeyer flasks and maintained in total darkness for 1.5 h and 30°C to allow flies to oviposit on the flask bottom [73]. Adult flies were released from the flasks into a cage and eggs were collected by rinsing with distilled water onto a filter paper. Both the eggs and adult flies were frozen at −80°C. To preserve nucleic acid integrity, adults were sexed on dry ice prior to freezing. Each sample used for DNA extraction and pyrosequencing consisted of 5 adult males, 5 adult females or 50 eggs pooled together and homogenized. Three replicates of adult male, adult female and eggs were analyzed.

The horn fly, Haematobia irritans, is one of the most economically important pests of cattle. Insecticides have been a major element of horn fly management programs. Growing concerns with insecticide resistance, insecticide residues on farm products, and non-availability of new generation insecticides, are serious issues for the livestock industry. Alternative horn fly control methods offer the promise to decrease the use of insecticides and reduce the amount of insecticide residues on livestock products and give an impetus to the organic livestock farming segment. The horn fly, an obligatory blood feeder, requires the help of microflora to supply additional nutrients and metabolize the blood meal. Recent advancements in DNA sequencing methodologies enable researchers to examine the microflora diversity independent of culture methods.