Our good neighbors: Understanding ecosystem services provided by insectivorous bats in Rwanda.

Bats are prodigious consumers of agricultural and forest pests, and are, therefore, a natural asset for agricultural productivity, suppressing populations of such pests. This study provides baseline information of diet of 143 bats belonging to eight insectivorous bat species from agricultural areas of Rwanda while evaluating the effectiveness of bats as pest suppressors. Using DNA metabarcoding to analyze bat fecal pellets, 85 different insect species were detected, with 60% (n = 65), 64% (n = 11) and 78% (n = 9) found to be agricultural pests from eastern, northern and western regions, respectively. Given the high percentages of agricultural pests detected, we submit that Rwandan insectivorous bats have the capacity for biocontrol of agricultural pests. Rwandan bat populations should be protected and promoted since they may foster higher crop yields and sustainable livelihoods.

Once upon a time in Mexico: Holocene biogeography of the spotted bat (Euderma maculatum).

Holocene-era range expansions are relevant to understanding how a species might respond to the warming and drying climates of today. The harsh conditions of North American deserts have phylogenetically structured desert bat communities but differences in flight capabilities are expected to affect their ability to compete, locate, and use habitat in the face of modern climate change. A highly vagile but data-deficient bat species, the spotted bat (Euderma maculatum), is thought to have expanded its range from central Mexico to western Canada during the Holocene. With specimens spanning this latitudinal extent, we examined historical demography, and used ecological niche modeling (ENM) and phylogeography (mitochondrial DNA), to investigate historic biogeography from the rear to leading edges of the species' range. The ENM supported the notion that Mexico was largely the Pleistocene-era range, whereas haplotype pattern and Skyline plots indicated that populations expanded from the southwestern US throughout the Holocene. This era provided substantial gains in suitable climate space and likely facilitated access to roosting habitat throughout the US Intermountain West. Incongruent phylogenies among different methods prevented a precise understanding of colonization history. However, isolation at the southern-most margin of the range suggests a population was left behind in Mexico as climate space contracted and are currently of unknown status. The species appears historically suited to follow shifts in climate space but differences in flight behaviors between leading edge and core-range haplogroups suggest range expansions could be influenced by differences in habitat quality or climate (e.g., drought). Although its vagility could facilitate response to environmental change and thereby avoid extinction, anthropogenic pressures at the core range could still threaten the ability for beneficial alleles to expand into the leading edge.

Bat Use of Hollows in California's Old-Growth Redwood Forests: From DNA to Ecology.

The loss of roosting resources, either through disturbance or removal, negatively affects bats. Identifying sensitive species and determining roost requirements are critical components in conserving their habitat. Cavity-roosting bats on the North Coast of California are known to use hollows in large redwood trees. In this study, we examined the factors determining the use of basal tree hollows by different bat species at eight redwood forest sites in Del Norte, Humboldt, and Mendocino Counties, California. Bat guano was collected from 179 basal hollow roosts from 2017 to 2018, and guano mass was used as an index of roosting activity. Nine bat species and one species group were identified by analysis of DNA in guano. We made a total of 253 identifications from 83 hollows into the 10 species categories. The most prevalent species were Myotis californicus (California myotis; 28.5% of all identifications), the Myotis evotis-Myotis thysanodes group (17.4%), Corynorhinus townsendii (17.0%), and Myotis volans (15.0%). We evaluated the extent to which habitat variables at the scales of the hollow, vicinity, and site influenced the level of roost use. The correlations between guano mass and habitat variables were examined using generalized additive mixed models. At the hollow scale, guano mass increased with ceiling height above the opening. At the vicinity scale, guano mass increased with less cover of small trees. At the site scale, there was no association between guano mass and distance to foraging areas, elevation, or the number of nearby hollows. These tree hollow roost preferences can inform land managers when planning the management and conservation of redwood forests.

RELATEDNESS AND GENETIC STRUCTURE OF BIG BROWN BAT (EPTESICUS FUSCUS) MATERNITY COLONIES IN AN URBAN-WILDLAND INTERFACE WITH PERIODIC RABIES VIRUS OUTBREAKS.

Big brown bats (Eptesicus fuscus) are the bat species in North America most frequently found to be rabid because of their high rate of human contact and thus submissions for rabies testing, of which, 4-5% are positive. The social behavior of big brown bats during the summer months may drive space use and potential viral exposure to conspecifics and mesocarnivores. We collected 88 unique genetic samples via buccal swabs from big brown bats captured at four maternity roosts surrounding a golf course during the summer of 2013. We used seven microsatellite loci to estimate genetic relatedness among individuals and genetic structure within and among colonies to infer whether females selected roosts based on kinship and used genetics and radio telemetry to determine the frequency of roost switching. We found roost switching through genetics and telemetry, and no evidence of elevated genetic relatedness within colonies or genetic structure among colonies. Social cohesion based on relatedness may not act to constrain the pathogen to a particular roost area, and thus, geographic mobility may increase viral exposure of bats in neighboring areas.

A fecal sequel: Testing the limits of a genetic assay for bat species identification.

DNA metabarcoding assays are powerful tools for delving into the DNA in wildlife feces, giving unprecedented ability to detect species, understand natural history, and identify pathogens for a range of applications in management, conservation, and research. Next-generation sequencing technology is developing rapidly, which makes it especially important that predictability and reproducibility of DNA metabarcoding assays are explored together with the post-depositional ecology of the target taxon's fecal DNA. Here, we defined the constraints of an assay called 'Species from Feces' used by government agencies, research groups, and non-governmental organizations to identify bat species from guano. We tested assay sensitivity by examining how time and humidity affect the ability to recover and successfully sequence DNA in guano, assessing whether a fecal pellet from a rare bat species could be detected in a background of feces from other bat species, and evaluating the efficacy of Species from Feces as a survey tool for bat roosts in temperate and tropical areas. We found that the assay performs well with feces over two years old in dry, cool environments, and fails by 12 months at 100% relative humidity. We also found that it reliably identifies rare DNA, has great utility for surveying roosts in temperate and tropical regions, and detects more bat species than do visual surveys. We attribute the success of Species from Feces to characteristics of the assay paired with application in taxa that are particularly well-suited for fecal DNA survival. In a time of rapid evolution of DNA metabarcoding approaches and their use with feces, this study illustrates the strengths and limitations of applied assays.

Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay.

Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha+] or orfX+). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters.IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type (ha+ or orfX+) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest.

Species From Feces: Order-Wide Identification of Chiroptera From Guano and Other Non-Invasive Genetic Samples.

Bat guano is a relatively untapped reservoir of information, having great utility as a DNA source because it is often available at roosts even when bats are not and is an easy type of sample to collect from a difficult-to-study mammalian order. Recent advances from microbial community studies in primer design, sequencing, and analysis enable fast, accurate, and cost-effective species identification. Here, we borrow from this discipline to develop an order-wide DNA mini-barcode assay (Species from Feces) based on a segment of the mitochondrial gene cytochrome c oxidase I (COI). The assay works effectively with fecal DNA and is conveniently transferable to low-cost, high-throughput Illumina MiSeq technology that also allows simultaneous pairing with other markers. Our PCR primers target a region of COI that is highly discriminatory among Chiroptera (92% species-level identification of barcoded species), and are sufficiently degenerate to allow hybridization across diverse bat taxa. We successfully validated our system with 54 bat species across both suborders. Despite abundant arthropod prey DNA in guano, our primers were highly specific to bats; no arthropod DNA was detected in thousands of feces run on Sanger and Illumina platforms. The assay is extendable to fecal pellets of unknown age as well as individual and pooled guano, to allow for individual (using singular fecal pellets) and community (using combined pellets collected from across long-term roost sites) analyses. We developed a searchable database (http://nau.edu/CEFNS/Forestry/Research/Bats/Search-Tool/) that allows users to determine the discriminatory capability of our markers for bat species of interest. Our assay has applications worldwide for examining disease impacts on vulnerable species, determining species assemblages within roosts, and assessing the presence of bat species that are vulnerable or facing extinction. The development and analytical pathways are rapid, reliable, and inexpensive, and can be applied to ecology and conservation studies of other taxa.