As polyphagous, holometabolous insects, tephritid fruit flies (Diptera: Tephritidae) provide a unique habitat for endosymbiotic bacteria, especially those microbes associated with the digestive system. Here we examine the endosymbiont of the olive fly [Bactrocera oleae (Rossi) (Diptera: Tephritidae)], a tephritid of great economic importance. "Candidatus Erwinia dacicola" was found in the digestive systems of all life stages of wild olive flies from the southwestern United States. PCR and microscopy demonstrated that "Ca. Erwinia dacicola" resided intracellularly in the gastric ceca of the larval midgut but extracellularly in the lumen of the foregut and ovipositor diverticulum of adult flies. "Ca. Erwinia dacicola" is one of the few nonpathogenic endosymbionts that transitions between intracellular and extracellular lifestyles during specific stages of the host's life cycle. Another unique feature of the olive fly endosymbiont is that unlike obligate endosymbionts of monophagous insects, "Ca. Erwinia dacicola" has a G؉C nucleotide composition similar to those of closely related plantpathogenic and free-living bacteria. These two characteristics of "Ca. Erwinia dacicola," the ability to transition between intracellular and extracellular lifestyles and a G؉C nucleotide composition similar to those of free-living relatives, may facilitate survival in a changing environment during the development of a polyphagous, holometabolous host. We propose that insect-bacterial symbioses should be classified based on the environment that the host provides to the endosymbiont (the endosymbiont environment).

Insects feeding on plant sap, blood, and other nutritionally incomplete diets are typically associated with mutualistic bacteria that supplement missing nutrients. Herbivorous mammal dung contains more than 86% cellulose and lacks amino acids essential for insect development and reproduction. Yet one of the most ecologically necessary and evolutionarily successful groups of beetles, the dung beetles (Scarabaeinae) feeds primarily, or exclusively, on dung. These associations suggest that dung beetles may benefit from mutualistic bacteria that provide nutrients missing from dung. The nesting behaviors of the female parent and the feeding behaviors of the larvae suggest that a microbiome could be vertically transmitted from the parental female to her offspring through the brood ball. Using sterile rearing and a combination of molecular and culture-based techniques, we examine transmission of the microbiome in the bull-headed dung beetle, Onthophagus taurus. Beetles were reared on autoclaved dung and the microbiome was characterized across development. A ~1425 bp region of the 16S rRNA identified Pseudomonadaceae, Enterobacteriaceae, and Comamonadaceae as the most common bacterial families across all life stages and populations, including cultured isolates from the 3rd instar digestive system. Finer level phylotyping analyses based on lepA and gyrB amplicons of cultured isolates placed the isolates closest to Enterobacter cloacae, Providencia stuartii, Pusillimonas sp., Pedobacter heparinus, and Lysinibacillus sphaericus. Scanning electron micrographs of brood balls constructed from sterile dung reveals secretions and microbes only in the chamber the female prepares for the egg. The use of autoclaved dung for rearing, the presence of microbes in the brood ball and offspring, and identical 16S rRNA sequences in both parent and offspring suggests that the O. taurus female parent transmits specific microbiome members to her offspring through the brood chamber. The transmission of the dung beetle microbiome highlights the maintenance and likely importance of this newly-characterized bacterial community.

The microbiome of the olive fruit fly, Bactrocera oleae (Gmelin), a worldwide pest of olives (Olea europaea L.), has been examined for >100 yr as part of efforts to identify bacteria that are plant pathogens vectored by the fly or are beneficial endosymbionts essential for the fly's survival and thus targets for possible biological control. Because tephritid fruit flies feed on free-living bacteria in their environment, distinguishing between the transient, acquired bacteria of their diet and persistent, resident bacteria that are vertically transmitted endosymbionts is difficult. Several culture-dependent and -independent studies have identified a diversity of species in the olive fruit fly microbiome, but they have not distinguished the roles of the microbes. Candidatus Erwinia dacicola, has been proposed to be a coevolved endosymbiont of the olive fruit fly; however, this was based on limited samples from two Italian populations. Our study shows that C. Erwinia dacicola was present in all New and Old World populations and in the majority of individuals of all life stages sampled in 2 yr. Olive fruit flies reared on olives in the laboratory had frequencies of C. Erwinia dacicola similar to that of wild populations; however, flies reared on artificial diets containing antibiotics in the laboratory rarely had the endosymbiont. The relative abundance of C. Erwinia dacicola varied across development stages, being most abundant in ovipositing females and larvae. This uniform presence of C. Erwini dacicola suggests that it is a persistent, resident endosymbiont of the olive fruit fly.

The number of small proteins (SPs) encoded in the Escherichia coli genome is unknown, as current bioinformatics and biochemical techniques make short gene and small protein identification challenging. One method of small protein identification involves adding an epitope tag to the 3′ end of a short open reading frame (sORF) on the chromosome, with synthesis confirmed by immunoblot assays. In this study, this strategy was used to identify new E. coli small proteins, tagging 80 sORFs in the E. coli genome, and assayed for protein synthesis. The selected sORFs represent diverse sequence characteristics, including degrees of sORF conservation, predicted transmembrane domains, sORF direction with respect to flanking genes, ribosome binding site (RBS) prediction, and ribosome profiling results. Of 80 sORFs, 36 resulted in encoded synthesized proteins—a 45% success rate. Modeling of detected versus non‐detected small proteins analysis showed predictions based on RBS prediction, transcription data, and ribosome profiling had statistically‐significant correlation with protein synthesis; however, there was no correlation between current sORF annotation and protein synthesis. These results suggest substantial numbers of small proteins remain undiscovered in E. coli, and existing bioinformatics techniques must continue to improve to facilitate identification.