The microbiome – the community of microorganisms that is associated with an individual animal – has been an important driver of insect biodiversity globally, enabling insects to specialize on narrow, nutrient deficient diets. The importance of maternally inherited, obligate bacterial endosymbionts to provisioning nutrients missing from these narrow dietary niches has been well studied in insects. However, we know comparatively little about the processes that dictate the composition of non-maternally inherited bacteria in insect microbiomes, despite the importance of these bacteria in insect health, fitness, and vector competence. Here, we used two species of obligate insect ectoparasites of bats, the bat flies (Streblidae) Trichobius sphaeronotus and Nycterophilia coxata, to examine whether the microbiome, beyond obligate bacterial endosymbionts, is conserved or variable across geographic space, between ectoparasite species, or covaries with the external microbiome of their bat hosts or the cave environment. Our results indicate that ectoparasite microbiomes are highly conserved and specific to ectoparasite species, despite these species feeding on the blood of the same bat individuals in some cases. In contrast, we found high geographic variation in the fur microbiome of host bats and that the bat fur microbiome mimics the cave microbiomes. This research suggests that there is constraint on blood-feeding insect ectoparasites to maintain a specific microbiome distinct from their host and the environment, potentially to meet their nutritional needs. Given many of these bacteria are not known to be maternally inherited, this research lays the foundation for future examinations of how blood-feeding arthropods acquire and maintain bacteria in their microbiomes.

As microbiome research moves away from model organisms to wildlife, new challenges for microbiome high throughput sequencing arise caused by the variety of wildlife diets. High levels of contamination are commonly observed emanating from the host (mitochondria) or diet (chloroplast). Such high contamination levels affect the overall sequencing depth of wildlife samples thus decreasing statistical power and leading to poor performance in downstream analysis. We developed an amplification protocol utilizing PNA-DNA clamps to maximize the use of resources and to increase the sampling depth of true microbiome sequences in samples with high levels of plastid contamination. We chose two study organisms, a bat (Leptonyteris yerbabuenae) and a bird (Mimus parvulus), both relying on heavy plant-based diets that sometimes lead to traces of plant-based faecal material producing high contamination signals from chloroplasts and mitochondria. On average, our protocol yielded a 13-fold increase in bacterial sequence amplification compared with the standard protocol (Earth Microbiome Protocol) used in wildlife research. For both focal species, we were able significantly to increase the percentage of sequences available for downstream analyses after the filtering of plastids and mitochondria. Our study presents the first results obtained by using PNA-DNA clamps to block the PCR amplification of chloroplast and mitochondrial DNA from the diet in the gut microbiome of wildlife. The method involves a cost-effective molecular technique instead of the filtering out of unwanted sequencing reads. As 33% and 26% of birds and bats, respectively, have a plant-based diet, the tool that we present here will optimize the sequencing and analysis of wild microbiomes.