The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored.

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.

Data include microbial count data (CFUs), 16S-rRNA copy number data (qPCR), and microbial community (microbiome) data from the guts of the invasive tephritid fruit flies, melon fly (Zeugodacus cucurbitae) and medfly (Ceratitis capitata). Resources in this dataset: Resource Title: R code for dada2 processing and stacked bar charts of control microbiomes File Name: Control_Processing.zip Resource Description: Data showing performance of known controls (purchased from Zymo Research) using in-house DNA extraction and PCR methods for 16S-rRNA gene amplification and sequencing. Resource Title: Data processing of 16S amplicon data File Name: 16S SSU rRNA Microbiome Data Processing and Analysis.zip Resource Description: Raw data and accompanying R scripts for analysis of figure and generation of figures and tables. Data files include both amplicon sequence variant (ASV) count data matrix and accompanying ASV sequence files and taxonomies. Analysis and figure generation are made through independent R files. Resource Title: Data and analysis of fly culturable titers File Name: CFU titers.zip Resource Description: Colony forming units (CFUs) of fruit flies at different ages and the R code for figure generation and analysis. Resource Title: qPCR of 16S rRNA of Tephritid fruit flies at different ages File Name: 16S qPCR Titers.zip Resource Description: Raw data and R code of 16S rRNA copy numbers associated with medfly and melon fly gut tissues.

An abundance matrix (BM_OTU.xlsx) contains rows as OTU, columns as samples, and entries representing the abundance of each OTU as a ratio of all sequences obtained for each individual sample. This dataset is associated with the following publication: Gomez-Alvarez, V., and R. Revetta. Monitoring of Nitrification in Chloraminated Drinking Water Distribution Systems With Microbiome Bioindicators Using Supervised Machine Learning. Frontiers in Microbiology. Frontiers, Lausanne, SWITZERLAND, 11: 2254-2267, (2020).

The fasta file (BM_OTU.fasta) contain the sequences of the bacterial 16S rRNA-encoding V4 region gene (≈250 nt) for each Operational Taxonomic Unit (OTU). This dataset is associated with the following publication: Gomez-Alvarez, V., and R. Revetta. Monitoring of Nitrification in Chloraminated Drinking Water Distribution Systems With Microbiome Bioindicators Using Supervised Machine Learning. Frontiers in Microbiology. Frontiers, Lausanne, SWITZERLAND, 11: 2254-2267, (2020).

An abundance matrix (BM_taxonomic_lineage.xlsx) contains rows as taxonomic lineage, columns as samples, and entries representing the abundance of each lineage as a ratio of all sequences obtained for each individual sample. This dataset is associated with the following publication: Gomez-Alvarez, V., and R. Revetta. Monitoring of Nitrification in Chloraminated Drinking Water Distribution Systems With Microbiome Bioindicators Using Supervised Machine Learning. Frontiers in Microbiology. Frontiers, Lausanne, SWITZERLAND, 11: 2254-2267, (2020).