Under the canopy: disentangling the role of stemflow in shaping spatial patterns of soil microbial community structure underneath trees.

Stemflow is a spatially concentrated input of rainwater at the base of trees, resulting from precipitation draining down tree branches to the stem. Depending on tree shape, stemflow can represent a significant fraction of the total rainfall that contacts the tree's canopy area, and can become chemically enriched along its drainage path. As a result, stemflow has been hypothesized to influence microbial communities in the receiving soil proximal to the stem. However, previous studies have (i) yielded conflicting results on the significance of stemflow as a driver in bacterial community composition, and (ii) not directly compared communities in soils with and without stemflow receipt. In this study, a stemflow diversion system was employed on Quercus virginiana trees in Skidaway Island (Georgia, USA) to directly compare soil bacterial communities receiving no stemflow to those beneath trees with no diversion system in place. In both treatments, sample distance from the stem significantly influenced bacterial community structure. However, the absence of stemflow resulted in increased bacterial community diversity across all samples. Stemflow diversion also significantly altered longitudinal patterns in the abundance of multiple taxonomic groups. These results support the hypothesis that Q. virginiana stemflow has a significant impact on bacterial soil inhabitants and is a key factor in taxon selection in stem-proximal communities.

Analysis of Salmonella enterica Isolated from a Mixed-Use Watershed in Georgia, USA: Antimicrobial Resistance, Serotype Diversity, and Genetic Relatedness to Human Isolates.

As the cases of Salmonella enterica infections associated with contaminated water are increasing, this study was conducted to address the role of surface water as a reservoir of S. enterica serotypes. We sampled rivers and streams (n = 688) over a 3-year period (2015 to 2017) in a mixed-use watershed in Georgia, USA, and 70.2% of the total stream samples tested positive for Salmonella. A total of 1,190 isolates were recovered and characterized by serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). A wide range of serotypes was identified, including those commonly associated with humans and animals, with S. enterica serotype Muenchen being predominant (22.7%) and each serotype exhibiting a high degree of strain diversity by PFGE. About half (46.1%) of the isolates had PFGE patterns indistinguishable from those of human clinical isolates in the CDC PulseNet database. A total of 52 isolates (4.4%) were resistant to antimicrobials, out of which 43 isolates were multidrug resistant (MDR; resistance to two or more classes of antimicrobials). These 52 resistant Salmonella isolates were screened for the presence of antimicrobial resistance genes, plasmid replicons, and class 1 integrons, out of which four representative MDR isolates were selected for whole-genome sequencing analysis. The results showed that 28 MDR isolates resistant to 10 antimicrobials had blacmy-2 on an A/C plasmid. Persistent contamination of surface water with a high diversity of Salmonella strains, some of which are drug resistant and genetically indistinguishable from human isolates, supports a role of environmental surface water as a reservoir for and transmission route of this pathogen. IMPORTANCE Salmonella has been traditionally considered a foodborne pathogen, as it is one of the most common etiologies of foodborne illnesses worldwide; however, recent Salmonella outbreaks attributed to fresh produce and water suggest a potential environmental source of Salmonella that causes some human illnesses. Here, we investigated the prevalence, diversity, and antimicrobial resistance of Salmonella isolated from a mixed-use watershed in Georgia, USA, in order to enhance the overall understanding of waterborne Salmonella. The persistence and widespread distribution of Salmonella in surface water confirm environmental sources of the pathogen. A high proportion of waterborne Salmonella with clinically significant serotypes and genetic similarity to strains of human origin supports the role of environmental water as a significant reservoir of Salmonella and indicates a potential waterborne transmission of Salmonella to humans. The presence of antimicrobial-resistant and MDR Salmonella demonstrates additional risks associated with exposure to contaminated environmental water.

Phylosymbiosis across Deeply Diverging Lineages of Omnivorous Cockroaches (Order Blattodea).

The gut microbiome is shaped by both host diet and host phylogeny. However, separating the relative influence of these two factors over long periods of evolutionary time is often difficult. We conducted a 16S rRNA gene amplicon-based survey of the gut microbiome from 237 individuals and 19 species of omnivorous cockroaches from the order Blattodea. The order Blattodea represents an ancient lineage of insects that emerged over 300 million years ago, have a diverse gut microbiota, and have a typically gregarious lifestyle. All cockroaches shared a broadly similar gut microbiota, with 66 microbial families present across all species and 13 present in every individual examined. Although our network analysis of the cockroach gut microbiome showed a large amount of connectivity, we demonstrated that gut microbiota cluster strongly by host species. We conducted follow-up tests to determine if cockroaches exhibit phylosymbiosis, or the tendency of host-associated microbial communities to parallel the phylogeny of related host species. Across the full data set, gut microbial community similarity was not found to correlate with host phylogenetic distance. However, a weak but significant phylosymbiotic signature was observed using the matching cluster metric, which allows for localized changes within a phylogenetic tree that are more likely to occur over long evolutionary distances. This finding suggests that host phylogeny plays a large role in structuring the cockroach gut microbiome over shorter evolutionary distances and a weak but significant role in shaping the gut microbiome over extended periods of evolutionary time.IMPORTANCE The gut microbiome plays a key role in host health. Therefore, it is important to understand the evolution of the gut microbiota and how it impacts, and is impacted by, host evolution. In this study, we explore the relationship between host phylogeny and gut microbiome composition in omnivorous, gregarious cockroaches within the Blattodea order, an ancient lineage that spans 300 million years of evolutionary divergence. We demonstrate a strong relationship between host species identity and gut microbiome composition and found a weaker but significant role for host phylogeny in determining microbiome similarity over extended periods of evolutionary time. This study advances our understanding of the role of host phylogeny in shaping the gut microbiome over different evolutionary distances.

Rapid and Stable Microbial Community Assembly in the Headwaters of a Third-Order Stream.

Small streams and their headwaters are key sources of microbial diversity in fluvial systems and serve as an entry point for bacteria from surrounding environments. Community assembly processes occurring in these streams shape downstream population structure and nutrient cycles. To elucidate the development and stability of microbial communities along the length of a first- through third-order stream, fine-scale temporal and spatial sampling regimes were employed along McNutt Creek in Athens, GA, USA. 16S rRNA amplicon libraries were constructed from samples collected on a single day from 19 sites spanning the first 16.76 km of the stream. To provide context for this spatial study and evaluate temporal variability, selected sites at the stream's upper, mid, and lower reaches were sampled daily for 5 days preceding and following the spatial study. In a second study, three sites at and near the creek's headwaters were sampled daily for 11 days to understand initial bacterioplankton community assembly. Both studies revealed decreasing alpha and beta diversity with increasing downstream distance. These trends were accompanied by the enrichment of a small fraction of taxa found at low abundance in headwater-proximal sites. Similar sets of taxa consistently increased in relative abundance in downstream samples over time scales ranging from 1 day to 1 year, many of which belong to clades known to be abundant in freshwater environments. These results underpin the importance of headwaters as the site of rapid in-stream selection that results in the reproducible establishment of a highly stable community of freshwater riverine bacteria.IMPORTANCE Headwater streams are critical introduction points of microbial diversity for larger connecting rivers and play key roles in the establishment of taxa that partake in in-stream nutrient cycling. We examined the microbial community composition of a first- through third-order stream using fine-scale temporal and spatial regimes. Our results show that the bacterioplankton community develops rapidly and predictably from the headwater population with increasing total stream length. Along the length of the stream, the microbial community exhibits substantial diversity loss and enriches repeatedly for select taxa across days and years, although the relative abundances of individual taxa vary over time and space. This repeated enrichment of a stable stream community likely contributes to the stability and flexibility of downstream communities.

Temporal and spatial dynamics in microbial community composition within a temperate stream network.

The water column of streams hosts a unique microbial community that is distinct from the microbial communities of the stream benthos and surrounding soil. This community is shaped by complex interacting forces, including microbial dispersal from surrounding environments and in-stream selection. However, how the processes structuring stream communities change over space and time remains poorly understood. In this study, we characterize spatial and temporal trends in microbial community composition throughout a stream network spanning first through fifth order streams. We found that the microbial communities of headwater streams are compositionally diverse, with low representation of freshwater microbial taxa and high representation of soil and sediment-associated taxa. In three out of five seasonal samplings, a successional pattern was identified in which phylotype richness and compositional heterogeneity decreased while the proportion of known freshwater taxa increased with increasing cumulative upstream dendritic distance. However, in two samplings, streams instead exhibited uniformly high microbial diversity across the watershed, and the fraction of freshwater taxa showed no relationship with dendritic distance. Overall, our data suggest that the successional processes that drive microbial diversity in streams are highly dynamic and can be disrupted at landscape scales, potentially in response to variation in temperature and precipitation.

The Core Gut Microbiome of the American Cockroach, Periplaneta americana, Is Stable and Resilient to Dietary Shifts.

The omnivorous cockroach Periplaneta americana hosts a diverse hindgut microbiota encompassing hundreds of microbial species. In this study, we used 16S rRNA gene sequencing to examine the effect of diet on the composition of the P. americana hindgut microbial community. Results show that the hindgut microbiota of P. americana exhibit a highly stable core microbial community with low variance in compositions between individuals and minimal community change in response to dietary shifts. This core hindgut microbiome is shared between laboratory-hosted and wild-caught individuals, although wild-caught specimens exhibited a higher diversity of low-abundance microbes that were lost following extended cultivation under laboratory conditions. This taxonomic stability strongly contrasts with observations of the gut microbiota of mammals, which have been shown to be highly responsive to dietary change. A comparison of P. americana hindgut samples with human fecal samples indicated that the cockroach hindgut community exhibited higher alpha diversity but a substantially lower beta diversity than the human gut microbiome. This suggests that cockroaches have evolved unique mechanisms for establishing and maintaining a diverse and stable core microbiome. IMPORTANCE: The gut microbiome plays an important role in the overall health of its host. A healthy gut microbiota typically assists with defense against pathogens and the digestion and absorption of nutrients from food, while dysbiosis of the gut microbiota has been associated with reduced health. In this study, we examined the composition and stability of the gut microbiota from the omnivorous cockroach Periplaneta americana. We found that P. americana hosts a diverse core gut microbiome that remains stable after drastic long-term changes in diet. While other insects, notably ant and bee species, have evolved mechanisms for maintaining a stable association with specific gut microbiota, these insects typically host low-diversity gut microbiomes and consume specialized diets. In contrast, P. americana hosts a gut microbiota that is highly species rich and consumes a diverse solid diet, suggesting that cockroaches have evolved unique mechanisms for developing and maintaining a stable gut microbiota.

Localizing transcripts to single cells suggests an important role of uncultured deltaproteobacteria in the termite gut hydrogen economy.

Identifying microbes responsible for particular environmental functions is challenging, given that most environments contain an uncultivated microbial diversity. Here we combined approaches to identify bacteria expressing genes relevant to catabolite flow and to locate these genes within their environment, in this case the gut of a "lower," wood-feeding termite. First, environmental transcriptomics revealed that 2 of the 23 formate dehydrogenase (FDH) genes known in the system accounted for slightly more than one-half of environmental transcripts. FDH is an essential enzyme of H2 metabolism that is ultimately important for the assimilation of lignocellulose-derived energy by the insect. Second, single-cell PCR analysis revealed that two different bacterial types expressed these two transcripts. The most commonly transcribed FDH in situ is encoded by a previously unappreciated deltaproteobacterium, whereas the other FDH is spirochetal. Third, PCR analysis of fractionated gut contents demonstrated that these bacteria reside in different spatial niches; the spirochete is free-swimming, whereas the deltaproteobacterium associates with particulates. Fourth, the deltaproteobacteria expressing FDH were localized to protozoa via hybridization chain reaction-FISH, an approach for multiplexed, spatial mapping of mRNA and rRNA targets. These results underscore the importance of making direct vs. inference-based gene-species associations, and have implications in higher termites, the most successful termite lineage, in which protozoa have been lost from the gut community. Contrary to expectations, in higher termites, FDH genes related to those from the protozoan symbiont dominate, whereas most others were absent, suggesting that a successful gene variant can persist and flourish after a gut perturbation alters a major environmental niche.

Pattern and synchrony of gene expression among sympatric marine microbial populations.

Planktonic marine microbes live in dynamic habitats that demand rapid sensing and response to periodic as well as stochastic environmental change. The kinetics, regularity, and specificity of microbial responses in situ, however, are not well-described. We report here simultaneous multitaxon genome-wide transcriptome profiling in a naturally occurring picoplankton community. An in situ robotic sampler using a Lagrangian sampling strategy enabled continuous tracking and repeated sampling of coherent microbial populations over 2 d. Subsequent RNA sequencing analyses yielded genome-wide transcriptome profiles of eukaryotic (Ostreococcus) and bacterial (Synechococcus) photosynthetic picoplankton as well as proteorhodopsin-containing heterotrophs, including Pelagibacter, SAR86-cluster Gammaproteobacteria, and marine Euryarchaea. The photosynthetic picoplankton exhibited strong diel rhythms over thousands of gene transcripts that were remarkably consistent with diel cycling observed in laboratory pure cultures. In contrast, the heterotrophs did not cycle diurnally. Instead, heterotrophic picoplankton populations exhibited cross-species synchronous, tightly regulated, temporally variable patterns of gene expression for many genes, particularly those genes associated with growth and nutrient acquisition. This multitaxon, population-wide gene regulation seemed to reflect sporadic, short-term, reversible responses to high-frequency environmental variability. Although the timing of the environmental responses among different heterotrophic species seemed synchronous, the specific metabolic genes that were expressed varied from taxon to taxon. In aggregate, these results provide insights into the kinetics, diversity, and functional patterns of microbial community response to environmental change. Our results also suggest a means by which complex multispecies metabolic processes could be coordinated, facilitating the regulation of matter and energy processing in a dynamically changing environment.

Distinct dissolved organic matter sources induce rapid transcriptional responses in coexisting populations of Prochlorococcus, Pelagibacter and the OM60 clade.

A considerable fraction of the Earth's organic carbon exists in dissolved form in seawater. To investigate the roles of planktonic marine microbes in the biogeochemical cycling of this dissolved organic matter (DOM), we performed controlled seawater incubation experiments and followed the responses of an oligotrophic surface water microbial assemblage to perturbations with DOM derived from an axenic culture of Prochlorococcus, or high-molecular weight DOM concentrated from nearby surface waters. The rapid transcriptional responses of both Prochlorococcus and Pelagibacter populations suggested the utilization of organic nitrogen compounds common to both DOM treatments. Along with these responses, both populations demonstrated decreases in gene transcripts associated with nitrogen stress, including those involved in ammonium acquisition. In contrast, responses from low abundance organisms of the NOR5/OM60 gammaproteobacteria were observed later in the experiment, and included elevated levels of gene transcripts associated with polysaccharide uptake and oxidation. In total, these results suggest that numerically dominant oligotrophic microbes rapidly acquire nitrogen from commonly available organic sources, and also point to an important role for carbohydrates found within the DOM pool for sustaining the less abundant microorganisms in these oligotrophic systems.

Purified fibers in chemically defined synthetic diets destabilize the gut microbiome of an omnivorous insect model.

The macronutrient composition of a host's diet shapes its gut microbial community, with dietary fiber in particular escaping host digestion to serve as a potent carbon source for gut microbiota. Despite widespread recognition of fiber's importance to microbiome health, nutritional research often fails to differentiate hyper-processed fibers from cell-matrix derived intrinsic fibers, limiting our understanding of how individual polysaccharides influence the gut community. We use the American cockroach (Periplaneta americana) as a model system to dissect the response of complex gut microbial communities to diet modifications that are impossible to test in traditional host models. Here, we designed synthetic diets from lab-grade, purified ingredients to identify how the cockroach microbiome responds to six different carbohydrates (chitin, methylcellulose, microcrystalline cellulose, pectin, starch, xylan) in otherwise balanced diets. We show via 16S rRNA gene profiling that these synthetic diets reduce bacterial diversity and alter the phylogenetic composition of cockroach gut microbiota in a fiber-dependent manner, regardless of the vitamin and protein content of the diet. Comparisons with cockroaches fed whole-food diets reveal that synthetic diets induce blooms in common cockroach-associated taxa and subsequently fragment previously stable microbial correlation networks. Our research leverages an unconventional microbiome model system and customizable lab-grade artificial diets to shed light on how purified polysaccharides, as opposed to nutritionally complex intrinsic fibers, exert substantial influence over a normally stable gut community.

Long-term gut colonization with ESBL-producing Escherichia coli in participants without known risk factors from the southeastern United States.

We evaluated gut carriage of extended spectrum beta lactamase producing Enterobacteriaceae (ESBL-E) in southeastern U.S. residents without recent in-patient healthcare exposure. Study enrollment was January 2021-February 2022 in Athens, Georgia, U.S. and included a diverse population of 505 adults plus 50 child participants (age 0-5). Based on culture-based screening of stool samples, 4.5% of 555 participants carried ESBL-Es. This is slightly higher than reported in studies conducted 2012-2015, which found carriage rates of 2.5-3.9% in healthy U.S. residents. All ESBL-E confirmed isolates (n=25) were identified as Escherichia coli. Isolates belonged to 11 sequence types, with 48% classified as ST131. Ninety six percent of ESBL-E isolates carried a blaCTX-M gene. Isolated ESBL-Es frequently carried virulence genes as well as multiple classes of antibiotic resistance genes. Long-term colonization was common, with 64% of ESBL-E positive participants testing positive when rescreened three months later. One participant yielded isolates belonging to two different E. coli sequence types that carried blaCTX-M-1 genes on near-identical plasmids, suggesting intra-gut plasmid transfer. Isolation of E. coli on media without antibiotics revealed that ESBL-E. coli typically made up a minor fraction of the overall gut E. coli population, although in some cases they were the dominant strain. ESBL-E carriage was not associated with a significantly different stool microbiome composition. However, some microbial taxa were differentially abundant in ESBL-E carriers. Together, these results suggest that a small subpopulation of US residents are long-term, asymptomatic carriers of ESBL-Es, and may serve as an important reservoir for community spread of these ESBL genes.

Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite.

From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding 'higher' Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H2 metabolism, CO2-reductive acetogenesis and N2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-microl environment can be.

Disentangling hindgut metabolism in the American cockroach through single-cell genomics and metatranscriptomics.

Omnivorous cockroaches host a complex hindgut microbiota comprised of insect-specific lineages related to those found in mammalian omnivores. Many of these organisms have few cultured representatives, thereby limiting our ability to infer the functional capabilities of these microbes. Here we present a unique reference set of 96 high-quality single cell-amplified genomes (SAGs) from bacterial and archaeal cockroach gut symbionts. We additionally generated cockroach hindgut metagenomic and metatranscriptomic sequence libraries and mapped them to our SAGs. By combining these datasets, we are able to perform an in-depth phylogenetic and functional analysis to evaluate the abundance and activities of the taxa in vivo. Recovered lineages include key genera within Bacteroidota, including polysaccharide-degrading taxa from the genera Bacteroides, Dysgonomonas, and Parabacteroides, as well as a group of unclassified insect-associated Bacteroidales. We also recovered a phylogenetically diverse set of Firmicutes exhibiting a wide range of metabolic capabilities, including-but not limited to-polysaccharide and polypeptide degradation. Other functional groups exhibiting high relative activity in the metatranscriptomic dataset include multiple putative sulfate reducers belonging to families in the Desulfobacterota phylum and two groups of methanogenic archaea. Together, this work provides a valuable reference set with new insights into the functional specializations of insect gut symbionts and frames future studies of cockroach hindgut metabolism.

Distribution of Antibiotic Resistance in a Mixed-Use Watershed and the Impact of Wastewater Treatment Plants on Antibiotic Resistance in Surface Water.

The aquatic environment has been recognized as a source of antibiotic resistance (AR) that factors into the One Health approach to combat AR. To provide much needed data on AR in the environment, a comprehensive survey of antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs), and antibiotic residues was conducted in a mixed-use watershed and wastewater treatment plants (WWTPs) within the watershed to evaluate these contaminants in surface water. A culture-based approach was used to determine prevalence and diversity of ARB in surface water. Low levels of AR Salmonella (9.6%) and Escherichia coli (6.5%) were detected, while all Enterococcus were resistant to at least one tested antibiotic. Fewer than 20% of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae (17.3%) and carbapenem-resistant Enterobacteriaceae (CRE) (7.7%) were recovered. Six ARGs were detected using qPCR, primarily the erythromycin-resistance gene, ermB. Of the 26 antibiotics measured, almost all water samples (98.7%) had detectable levels of antibiotics. Analysis of wastewater samples from three WWTPs showed that WWTPs did not completely remove AR contaminants. ARGs and antibiotics were detected in all the WWTP effluent discharges, indicating that WWTPs are the source of AR contaminants in receiving water. However, no significant difference in ARGs and antibiotics between the upstream and downstream water suggests that there are other sources of AR contamination. The widespread occurrence and abundance of medically important antibiotics, bacteria resistant to antibiotics used for human and veterinary purposes, and the genes associated with resistance to these antibiotics, may potentially pose risks to the local populations exposed to these water sources.

Draft Genome Sequence of Cupriavidus basilensis SRS, a Bacterium Isolated from Stream Sediments.

Cupriavidus basilensis SRS was isolated from stream sediments from the Savannah River Site in South Carolina. Here, we report the draft genome sequence and annotation of Cupriavidus basilensis SRS. The genome contains 8,918,236 bp and 7,916 predicted protein-coding genes, with a total G+C content of 65.2%.

Non-point source fecal contamination from aging wastewater infrastructure is a primary driver of antibiotic resistance in surface waters.

Antibiotic resistance is a global threat to human health. Many surface water resources are environmental hotspots of antibiotic resistant gene (ARG) transfer, with agricultural runoff and human waste highlighted as common sources of ARGs to aquatic systems. Here we quantified fecal marker genes and ARGs in 992 stream water samples collected seasonally during a 5-year period from 115 sites across the Upper Oconee watershed (Georgia, USA), an area characterized by gradients of agricultural and urban development. Widespread fecal contamination was found from humans (48% of samples), ruminants (55%), and poultry (19%), and 73% of samples tested positive for at least one of the six targeted ARGs (ermB, tet(B), blaCTX-M-1, blaKPC, blaSHV, and qnrS). While ARGs were strongly correlated with human fecal markers, many highly contaminated samples were not associated with sewage outfalls, an expected source of fecal and ARG pollution. To determine sources of contamination, we synthesized ARG and fecal marker data with geospatial data on land use/land cover and wastewater infrastructure across the watershed. This novel analysis found strong correlations between ARGs and measures of sewer density, sewer length, and septic system age within sample watersheds, indicating non-point sources of fecal contamination from aging wastewater infrastructure can be critical disseminators of anthropogenic ARGs in the environment.

Formyltetrahydrofolate synthetase gene diversity in the guts of higher termites with different diets and lifestyles.

In this study, we examine gene diversity for formyl-tetrahydrofolate synthetase (FTHFS), a key enzyme in homoacetogenesis, recovered from the gut microbiota of six species of higher termites. The "higher" termites (family Termitidae), which represent the majority of extant termite species and genera, engage in a broader diversity of feeding and nesting styles than the "lower" termites. Previous studies of termite gut homoacetogenesis have focused on wood-feeding lower termites, from which the preponderance of FTHFS sequences recovered were related to those from acetogenic treponemes. While sequences belonging to this group were present in the guts of all six higher termites examined, treponeme-like FTHFS sequences represented the majority of recovered sequences in only two species (a wood-feeding Nasutitermes sp. and a palm-feeding Microcerotermes sp.). The remaining four termite species analyzed (a Gnathamitermes sp. and two Amitermes spp. that were recovered from subterranean nests with indeterminate feeding strategies and a litter-feeding Rhynchotermes sp.) yielded novel FTHFS clades not observed in lower termites. These termites yielded two distinct clusters of probable purinolytic Firmicutes and a large group of potential homoacetogens related to sequences previously recovered from the guts of omnivorous cockroaches. These findings suggest that the gut environments of different higher termite species may select for different groups of homoacetogens, with some species hosting treponeme-dominated homoacetogen populations similar to those of wood-feeding, lower termites while others host Firmicutes-dominated communities more similar to those of omnivorous cockroaches.

Differences in Gut Microbiome Composition Between Sympatric Wild and Allopatric Laboratory Populations of Omnivorous Cockroaches.

Gut microbiome composition is determined by a complex interplay of host genetics, founder's effects, and host environment. We are using omnivorous cockroaches as a model to disentangle the relative contribution of these factors. Cockroaches are a useful model for host-gut microbiome interactions due to their rich hindgut microbial community, omnivorous diet, and gregarious lifestyle. In this study, we used 16S rRNA sequencing to compare the gut microbial community of allopatric laboratory populations of Periplaneta americana as well as sympatric, wild-caught populations of P. americana and Periplaneta fuliginosa, before and after a 14 day period of acclimatization to a common laboratory environment. Our results showed that the gut microbiome of cockroaches differed by both species and rearing environment. The gut microbiome from the sympatric population of wild-captured cockroaches showed strong separation based on host species. Laboratory-reared and wild-captured cockroaches from the same species also exhibited distinct gut microbiome profiles. Each group of cockroaches had a unique signature of differentially abundant uncharacterized taxa still present after laboratory cultivation. Transition to the laboratory environment resulted in decreased microbiome diversity for both species of wild-caught insects. Interestingly, although laboratory cultivation resulted in similar losses of microbial diversity for both species, it did not cause the gut microbiome of those species to become substantially more similar. These results demonstrate how competing factors impact the gut microbiome and highlight the need for a greater understanding of host-microbiome interactions.

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana).

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 °C for approximately 4-6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Diversity of formyltetrahydrofolate synthetases in the guts of the wood-feeding cockroach Cryptocercus punctulatus and the omnivorous cockroach Periplaneta americana.

We examined the diversity of a marker gene for homoacetogens in two cockroach gut microbial communities. Formyltetrahydrofolate synthetase (FTHFS or fhs) libraries prepared from a wood-feeding cockroach, Cryptocercus punctulatus, were dominated by sequences that affiliated with termite gut treponemes. No spirochete-like sequences were recovered from the omnivorous roach Periplaneta americana, which was dominated by Firmicutes-like sequences.