Breaking Barriers with Bread: Using the Sourdough Starter Microbiome to Teach High-Throughput Sequencing Techniques.

Widespread usage of high-throughput sequencing (HTS) in the LIFE SCIENCES has produced a demand for undergraduate and graduate institutions to offer classes exposing students to all aspects of HTS (sample acquisition, laboratory work, sequencing technologies, bioinformatics, and statistical analyses). Despite the increase in demand, many challenges exist for these types of classes. We advocate for the usage of the sourdough starter microbiome for implementing meta-amplicon sequencing. The relatively small community, dominated by a few taxa, enables potential contaminants to be easily identified, while between-sample differences can be quickly statistically assessed. Finally, bioinformatic pipelines and statistical analyses can be carried out on personal student laptops or in a teaching computer lab. In two semesters adopting this system, 12 of 14 students were able to effectively capture the sourdough starter microbiome, using the instructor's paired sample as reference.

Feasting, not fasting: winter diets of cave hibernating bats in the United States.

Temperate bat species use extended torpor to conserve energy when ambient temperatures are low and food resources are scarce. Previous research suggests that migratory bat species and species known to roost in thermally unstable locations, such as those that roost in trees, are more likely to remain active during winter. However, hibernating colonies of cave roosting bats in the southeastern United States may also be active and emerge from caves throughout the hibernation period. We report what bats are eating during these bouts of winter activity. We captured 2,044 bats of 10 species that emerged from six hibernacula over the course of 5 winters (October-April 2012/2013, 2013/2014, 2015/2016, 2016/2017, and 2017/2018). Using Next Generation sequencing of DNA from 284 fecal samples, we determined bats consumed at least 14 Orders of insect prey while active. Dietary composition did not vary among bat species; however, we did record variation in the dominant prey items represented in species' diets. We recorded Lepidoptera in the diet of 72.2% of individual Corynorhinus rafinesquii and 67.4% of individual Lasiurus borealis. Diptera were recorded in 32.4% of Myotis leibii, 37.4% of M. lucifugus, 35.5% of M. sodalis and 68.8% of Perimyotis subflavus. Our study is the first to use molecular genetic techniques to identify the winter diet of North American hibernating bats. The information from this study is integral to managing the landscape around bat hibernacula for insect prey, particularly in areas where hibernating bat populations are threatened by white-nose syndrome.

Microbiome Variation Across Two Hemlock Species With Hemlock Woolly Adelgid Infestation.

The hemlock woolly adelgid (Adelges tsugae, HWA), an invasive insect, is devastating native hemlock populations in eastern North America, and management outcomes have so far had limited success. While many plant microbiomes influence and even support plant immune responses to insect herbivory, relatively little is known about the hemlock microbiome and its interactions with pathogens or herbivores such as HWA. Using 16S rRNA and ITS gene amplicon sequencing, we characterized the needle, branch, root, and rhizosphere microbiome of two hemlock species, Tsuga canadensis and T. sieboldii, that displayed low and high levels of HWA populations. We found that both archaeal/bacterial and fungal needle communities, as well as the archaeal/bacterial branch and root communities, varied in composition in both hemlock species relative to HWA population levels. While host species and plant-associated habitats explained a greater proportion of the variance in the microbiome than did HWA population level, high HWA populations were associated with enrichment of 100 likely fungal pathogen sequence variants across the four plant-associated habitats (e.g., needle, branch, root, rhizosphere) compared to trees with lower HWA populations. This work contributes to a growing body of literature linking plant pathogens and pests with the changes in the associated plant microbiome and host health. Furthermore, this work demonstrates the need to further investigate plant microbiome effects across multiple plant tissues to understand their influences on host health.

Disparities in second-generation DNA metabarcoding results exposed with accessible and repeatable workflows.

Different second-generation sequencing technologies may have taxon-specific biases when DNA metabarcoding prey in predator faeces. Our major objective was to examine differences in prey recovery from bat guano across two different sequencing workflows using the same faecal DNA extracts. We compared results between the Ion Torrent PGM and the Illumina MiSeq with similar library preparations and the same analysis pipeline. We focus on repeatability and provide an R Notebook in an effort towards transparency for future methodological improvements. Full documentation of each step enhances the accessibility of our analysis pipeline. We tagged DNA from insectivorous bat faecal samples, targeted the arthropod cytochrome c oxidase I minibarcode region and sequenced the product on both second-generation sequencing platforms. We developed an analysis pipeline with a high operational taxonomic unit (OTU) clustering threshold (i.e., ≥98.5%) followed by copy number filtering to avoid merging rare but genetically similar prey into the same OTUs. With this workflow, we detected 297 unique prey taxa, of which 74% were identified at the species level. Of these, 104 (35%) prey OTUs were detected by both platforms, 176 (59%) OTUs were detected by the Illumina MiSeq system only, and 17 (6%) OTUs were detected using the Ion Torrent system only. Costs were similar between platforms but the Illumina MiSeq recovered six times more reads and four additional insect orders than did Ion Torrent. The considerations we outline are particularly important for long-term ecological monitoring; a more standardized approach will facilitate comparisons between studies and allow faster recognition of changes within ecological communities.

Predator-prey interaction reveals local effects of high-altitude insect migration.

High-altitude nocturnal insect migrations are ubiquitous and represent significant pulses of biomass, which impact large areas and multiple trophic levels, yet are difficult to study and poorly understood. Predation on migratory insects by high-flying bats provides potential for investigating flows of migratory insects across a landscape. Brazilian free-tailed bats, Tadarida brasiliensis, provide valuable ecosystem services by consuming migratory pests, and research suggests migratory insects are an important resource to bats in autumn. We sequenced insect DNA from bat feces collected during the 2010-2012 autumn migrations of insects over southern Texas, and tested the utility of predator-prey interactions for monitoring migratory insect populations by asking: 1) how extensively do bats consume migratory insects during autumn? (2) does the prey community reflect known drivers of insect migrations, e.g. cold fronts? and (3) are migratory insects increasingly important to bats when local food resources decline in autumn? Bats consumed at least 21 species of migratory insects and 44 species of agricultural pests. Prey community richness increased with cold front passage. Bats consumed migratory moths over the entire autumn season, and the proportion of migratory moths in the bat diet increased over the course of the autumn season in all 3 years. This study confirms extensive consumption of migratory insects by bats, links patterns in prey communities to mechanisms driving insect migration, and documents a novel approach to tracking patterns of migratory insect movement. As an important resource for T. brasiliensis in autumn, migratory insects provide stabilizing effects to the local animal community.

Bats track and exploit changes in insect pest populations.

The role of bats or any generalist predator in suppressing prey populations depends on the predator's ability to track and exploit available prey. Using a qPCR fecal DNA assay, we document significant association between numbers of Brazilian free-tailed bats (Tadarida brasiliensis) consuming corn earworm (CEW) moths (Helicoverpa zea) and seasonal fluctuations in CEW populations. This result is consistent with earlier research linking the bats' diet to patterns of migration, abundance, and crop infestation by important insect pests. Here we confirm opportunistic feeding on one of the world's most destructive insects and support model estimates of the bats' ecosystem services. Regression analysis of CEW consumption versus the moth's abundance at four insect trapping sites further indicates that bats track local abundance of CEW within the regional landscape. Estimates of CEW gene copies in the feces of bats are not associated with seasonal or local patterns of CEW abundance, and results of captive feeding experiments indicate that our qPCR assay does not provide a direct measure of numbers or biomass of prey consumed. Our results support growing evidence for the role of generalist predators, and bats specifically, as agents for biological control and speak to the value of conserving indigenous generalist predators.

Population growth of Mexican free-tailed bats (Tadarida brasiliensis mexicana) predates human agricultural activity.

BACKGROUND: Human activities, such as agriculture, hunting, and habitat modification, exert a significant effect on native species. Although many species have suffered population declines, increased population fragmentation, or even extinction in connection with these human impacts, others seem to have benefitted from human modification of their habitat. Here we examine whether population growth in an insectivorous bat (Tadarida brasiliensis mexicana) can be attributed to the widespread expansion of agriculture in North America following European settlement. Colonies of T. b. mexicana are extremely large (~10(6) individuals) and, in the modern era, major agricultural insect pests form an important component of their food resource. It is thus hypothesized that the growth of these insectivorous bat populations was coupled to the expansion of agricultural land use in North America over the last few centuries. RESULTS: We sequenced one haploid and one autosomal locus to determine the rate and time of onset of population growth in T. b. mexicana. Using an approximate Maximum Likelihood method, we have determined that T. b. mexicana populations began to grow ~220 kya from a relatively small ancestral effective population size before reaching the large effective population size observed today. CONCLUSIONS: Our analyses reject the hypothesis that T. b. mexicana populations grew in connection with the expansion of human agriculture in North America, and instead suggest that this growth commenced long before the arrival of humans. As T. brasiliensis is a subtropical species, we hypothesize that the observed signals of population growth may instead reflect range expansions of ancestral bat populations from southern glacial refugia during the tail end of the Pleistocene.