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Endosymbiotic  Wolbachia  bacteria are known to influence the host physiology, microbiota composition, and dissemination of pathogens. We surveyed a population of  Tabanus nigrovittatus , commonly referred to as “greenheads,” from Crane Beach (Ipswich, MA, USA) for the presence of the alphaproteobacterial symbiont  Wolbachia.  We studied the COI (mitochondrial cytochrome oxidase) marker gene to evaluate the phylogenetic diversity of the studied specimens. The DNA sequences show strong similarity (between 99.9 and 98%) among the collected specimens but lower similarity to closely related entries in the NCBI database (only between 96.3 and 94.7%), suggesting a more distant relatedness. Low levels of  Wolbachia  presence necessitated a nested PCR approach, and using 5 markers ( ftsZ ,  fbpA ,  dnaA ,  coxA , and  gatB ), we determined that two recognized “supergroups” of  Wolbachia  species were represented in the studied specimens, members of clades A and B. Using next-generation sequencing, we also surveyed the insect gut microbiomes of a subset of flies, using Illumina and PacBio 16S rRNA gene sequencing with barcoded primers. The composition of  Proteobacteria  also varied from fly to fly, with components belonging to  Gammaproteobacteria  making up the largest percentage of organisms (30 to 70%) among the microbiome samples. Most of the samples showed the presence of  Spiroplasma , a member of the phylum  Mollicutes , although the frequency of its presence was variable, ranging from 2 to 57%. Another noteworthy bacterial phylum consistently identified was  Firmicutes , though the read abundances were typically below 10%. Of interest is an association between  Wolbachia  presence and higher  Alphaproteobacteria  representation in the microbiomes, suggesting that the presence of  Wolbachia  affects the host microbiome.   IMPORTANCE   Tabanus nigrovittatus  greenhead populations contain two supergroups of  Wolbachia  endosymbionts, members of supergroups A and B. Analysis of the greenhead microbiome using next-generation sequencing revealed that the majority of bacterial species detected belonged to  Gammaproteobacteria , with most of the samples also showing the presence of  Spiroplasma , a member of the  Mollicutes  phylum also known to infect insects. An association between  Wolbachia  presence and higher Alphaproteobacteria representation in the microbiomes suggests that  Wolbachia  presence affects the host microbiome composition.

Greenhead (Tabanus nigrovittatus) Wolbachia and Its Microbiome: A Preliminary Study