Genetic drift and host-adaptive features likely underlie cladogenesis of insect-associated Lachnospiraceae.

Phylogenetic and functional group analysis of the genomes of anaerobic bacteria isolated from Periplaneta americana digestive tracts suggest that they represent novel Lachnospiraceae genera. PAL113 and PAL227 isolate genomes encoded short-chain fatty acid biosynthetic pathways and plant fiber and chitin catabolism and other carbohydrate utilization genes common in related Lachnospiraceae species, yet the presence of operons containing flagellar assembly pathways were among several distinguishing features. In general, PAL113 and PAL227 isolates encode an array of gene products that would enable them to thrive in the insect gut environment and potentially play a role in host diet processing. We hypothesize that cladogenesis of these isolates could be due to their oxygen sensitivity, reliance upon the host for dispersal and genetic drift and not necessarily as a result of an ongoing mutualism.

Microbiota Perturbation or Elimination Can Inhibit Normal Development and Elicit a Starvation-Like Response in an Omnivorous Model Invertebrate.

Omnivorous animals, including humans, harbor diverse, species-rich gut communities that impact their growth, development, and homeostasis. Model invertebrates are broadly accessible experimental platforms that enable linking specific species or species groups to host phenotypes, yet often their specialized diets and distinct gut microbiota make them less comparable to human and other mammalian and gut communities. The omnivorous cockroach Periplaneta americana harbors ∼4 × 102 bacterial genera within its digestive tract and is enriched with taxa commonly found in omnivorous mammals (i.e., Proteobacteria, Bacteroidetes, and Firmicutes). These features make P. americana a valuable platform for identifying microbe-mediated host phenotypes with potential translations to mammals. Rearing P. americana insects under germfree conditions resulted in prolonging development time by ∼30% and an up to ∼8% reduction in body size along three dimensions. Germfree rearing resulted in downregulation of gene networks involved in growth, energy homeostasis, and nutrient availability. Reintroduction of a defined microbiota comprised of a subset of P. americana commensals to germfree insects did not recover normal growth and developmental phenotypes or transcriptional profiles observed in conventionally reared insects. These results are in contrast with specialist-feeding model insects (e.g., Drosophila), where introduction of a single endemic bacterial species to germfree condition-reared specimens recovered normal host phenotypes. These data suggest that understanding microbe-mediated host outcomes in animals with species-rich communities should include models that typically maintain similarly diverse microbiomes. The dramatic transcriptional, developmental, and morphological phenotypes linked to gut microbiome status in this study illustrates how microbes are key players in animal growth and evolution. IMPORTANCE Broadly accessible model organisms are essential for illustrating how microbes are engaged in the growth, development, and evolution of animals. We report that germfree rearing of omnivorous Periplaneta americana cockroaches resulted in growth defects and severely disrupted gene networks that regulate development, which highlights the importance of gut microbiota in these host processes. Absence of gut microbiota elicited a starvation-like transcriptional response in which growth and development were inhibited while nutrient scavenging was enhanced. Additionally, reintroduction of a subset of cockroach gut bacterial commensals did not broadly recover normal expression patterns, illustrating that a particular microbiome composition may be necessary for normal host development. Invertebrate microbiota model systems that enable disentangling complex, species-rich communities are essential for linking microbial taxa to specific host phenotypes.

Microbial colonization promotes model cockroach gut tissue growth and development.

BACKGROUND: Digestive tissues are essential for diet processing and nutrient accessibility, especially in omnivores, and these functions occur despite and in collaboration with dynamic microbial communities that reside within and upon these tissues. Prolonged host development and reduced digestive tissue sizes have been observed in germ-free animals, and normal host phenotypes were recovered following the re-introduction of typical gut microbiomes via coprophagy. RESULTS: High-resolution histological analyses of Periplaneta americana cockroach digestive tissues revealed that total prevention of microbial colonization of the gut had severe impacts on the growth and development of gut tissues, especially the posterior midgut and anterior hindgut subcompartments that are expected to be colonized and inhabited by the greatest number of bacteria. Juveniles that were briefly exposed to normal gut microbiota exhibited a partial gut morphological recovery, suggesting that a single inoculation was insufficient. These data highlight gut microbiota as integral to normal growth and development of tissues they are in direct contact with and, more broadly, the organism in which they reside. CONCLUSIONS: We draw on these data, host life history traits (i.e. multigenerational cohousing, molting, and filial coprophagy and exuvia feeding), and previous studies to suggest a host developmental model in which gut tissues reflect a conflict-collaboration dynamic where 1) nutrient-absorptive anterior midgut tissues are in competition with transient and resident bacteria for easily assimilable dietary nutrients and whose growth is least-affected by the presence of gut bacteria and 2) posterior midgut, anterior hindgut, and to a lesser degree, posterior hindgut tissues are significantly impacted by gut bacterial presence because they are occupied by the greatest number of bacteria and the host is relying upon, and thus collaborating with, them to assist with complex polysaccharide catabolism processing and nutrient provisioning (i.e. short-chain fatty acids).

Nutritional symbiosis and ecology of host-gut microbe systems in the Blattodea.

Cockroaches and termites (Order: Blattodea) have been the subject of substantial research attention for over a century due, in part, to a subset of them having a strong propensity to cohabitate with humans and their structures. Recent research has led to numerous insights into their behavior, physiology, and ecology, as well as their ability to harbor taxonomically diverse microbial communities within their digestive systems, which include taxa that contribute to host growth and development. Further, recent investigations into the physiological and behavioral adaptations that enable recalcitrant polysaccharide digestion and the maintenance of microbial symbionts in cockroaches and termites suggests that symbionts contribute significantly to nutrient provisioning and processing.

Cultivable, Host-Specific Bacteroidetes Symbionts Exhibit Diverse Polysaccharolytic Strategies.

Beneficial gut microbes can facilitate insect growth on diverse diets. The omnivorous American cockroach, Periplaneta americana (Insecta: Blattodea), thrives on a diet rich in plant polysaccharides and harbors a species-rich gut microbiota responsive to host diet. Bacteroidetes are among the most abundant taxa in P. americana and other cockroaches, based on cultivation-independent gut community profiling, and these potentially polysaccharolytic bacteria may contribute to host diet processing. Eleven Bacteroidetes isolates were cultivated from P. americana digestive tracts, and phylogenomic analyses suggest that they were new Bacteroides, Dysgonomonas, Paludibacter, and Parabacteroides species distinct from those previously isolated from other insects, humans, and environmental sources. In addition, complete genomes were generated for each isolate, and polysaccharide utilization loci (PULs) and several non-PUL-associated carbohydrate-active enzyme (CAZyme)-coding genes that putatively target starch, pectin, and/or cellulose were annotated in each of the isolate genomes. Type IX secretion system (T9SS)- and CAZyme-coding genes tagged with the corresponding T9SS recognition and export C-terminal domain were observed in some isolates, suggesting that these CAZymes were deployed via non-PUL outer membrane translocons. Additionally, single-substrate growth and enzymatic assays confirmed genomic predictions that a subset of the Bacteroides and Dysgonomonas isolates could degrade starch, pectin, and/or cellulose and grow in the presence of these substrates as a single sugar source. Plant polysaccharides enrich P. americana diets, and many of these gut isolates are well equipped to exploit host dietary inputs and potentially contribute to gut community and host nutrient accessibility.IMPORTANCE Gut microbes are increasingly being recognized as critical contributors to nutrient accessibility in animals. The globally distributed omnivorous American cockroach (Periplaneta americana) harbors many bacterial phyla (e.g., Bacteroidetes) that are abundant in vertebrates. P. americana thrives on a highly diverse plant-enriched diet, making this insect a rich potential source of uncharacterized polysaccharolytic bacteria. We have cultivated, completely sequenced, and functionally characterized several novel Bacteroidetes species that are endemic to the P. americana gut, and many of these isolates can degrade simple and complex polysaccharides. Cultivation and genomic characterization of these Bacteroidetes isolates further enable deeper insight into how these taxa participate in polysaccharide metabolism and, more broadly, how they affect animal health and development.

Conspecific coprophagy stimulates normal development in a germ-free model invertebrate.

Microbial assemblages residing within and on animal gastric tissues contribute to various host beneficial processes that include diet accessibility and nutrient provisioning, and we sought to examine the degree to which intergenerational and community-acquired gut bacteria impact development in a tractable germ-free (GF) invertebrate model system. Coprophagy is a common behavior in cockroaches and termites that provides access to both nutrients and the primary means by which juveniles are inoculated with beneficial gut bacteria. This hypothesis was tested in the American cockroach (Periplaneta americana) by interfering with this means of acquiring gut bacteria, which resulted in GF insects that exhibited prolonged growth rates and gut tissue dysmorphias relative to wild-type (WT) P. americana. Conventionalization of GF P. americana via consumption of frass (feces) from conspecifics and siblings reared under non-sterile conditions resulted in colonization of P. americana gut tissues by a diverse microbial community and a significant (p < 0.05) recovery of WT level growth and hindgut tissue development phenotypes. These data suggest that coprophagy is essential for normal gut tissue and organismal development by introducing beneficial gut bacteria to P. americana, and that the GF P. americana model system is a useful system for examining how gut bacteria impact host outcomes.