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Many insects are obligatorily associated with and dependent on specific microbial species as essential mutualistic partners. In the host insects, such microbial mutualists are usually maintained in specialized cells or organs, called bacteriocytes or symbiotic organs. Hence, potentially exponential microbial growth cannot be realized but must be strongly constrained by spatial and resource limitations within the host cells or tissues. How such endosymbiotic bacteria grow, divide, and proliferate is important for understanding the interactions and dynamics underpinning intimate host-microbe symbiotic associations. Here we report that Blattabacterium , the ancient and essential endosymbiont of cockroaches, exhibits unexpectedly high rates of cell division (20%-58%) and, in addition, the cell division is asymmetric (average asymmetry index >1.5) when isolated from the German cockroach Blattella germanica . The asymmetric division of endosymbiont cells at high frequencies was observed irrespective of host tissues (fat bodies vs ovaries) or developmental stages (adults vs nymphs vs embryos) of B. germanica , and also observed in several different cockroach species. By contrast, such asymmetric and frequent cell division was observed neither in Buchnera , the obligatory bacterial endosymbiont of aphids, nor in Pantoea , the obligatory bacterial gut symbiont of stinkbugs. Comparative genomics of cell division-related genes uncovered that the Blattabacterium genome lacks the Min system genes that determine the cell division plane, which may be relevant to asymmetric cell division. These observations combined with comparative symbiont genomics provide insight into what processes and regulations may underpin the growth, division, and proliferation of such bacterial mutualists continuously constrained under within-host conditions.IMPORTANCEDiverse insects are dependent on specific bacterial mutualists for their survival and reproduction. Due to the long-lasting coevolutionary history, such symbiotic bacteria tend to exhibit degenerative genomes and suffer uncultivability. Because of their microbiological fastidiousness, the cell division patterns of such uncultivable symbiotic bacteria have been poorly described. Here, using fine microscopic and quantitative morphometric approaches, we report that, although bacterial cell division usually proceeds through symmetric binary fission, Blattabacterium , the ancient and essential endosymbiont of cockroaches, exhibits frequent and asymmetric cell division. Such peculiar cell division patterns were not observed with other uncultivable essential symbiotic bacteria of aphids and stinkbugs. Gene repertoire analysis revealed that the molecular machinery for regulating the bacterial cell division plane are lost in the Blattabacterium genome, suggesting the possibility that the general trend toward the reductive genome evolution of symbiotic bacteria may underpin their bizarre cytological/morphological traits., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2020 American Society for Microbiology.)

German cockroaches, Blattella germanica (Blattodea: Ectobiidae), are human commensals that move freely between food and waste, disseminating bacteria, including potential pathogens, through their feces. However, the relationship between the microbial communities of the cockroach gut and feces is poorly understood. We analyzed the V4 region of the 16S rRNA gene and the V9 region of the 18S rRNA gene by next-generation sequencing (NGS) to compare the bacterial and protist diversities in guts versus feces and males versus females, as well as assess variation across cockroach populations. Cockroaches harbored a diverse array of bacteria, and 80 to 90% of the operational taxonomic units (OTUs) were shared between the feces and gut. Lab-reared and field-collected cockroaches had distinct microbiota, and whereas lab-reared cockroaches had relatively conserved communities, considerable variation was observed in the microbial community composition of cockroaches collected in different apartments. Nonetheless, cockroaches from all locations shared some core bacterial taxa. The eukaryotic community in the feces of field-collected cockroaches was found to be more diverse than that in lab-reared cockroaches. These results demonstrate that cockroaches disseminate their gut microbiome in their feces, and they underscore the important contribution of the cockroach fecal microbiome to the microbial diversity of cockroach-infested homes. IMPORTANCE The German cockroach infests diverse human-built structures, including homes and hospitals. It produces potent allergens that trigger asthma and disseminates opportunistic pathogens in its feces. A comprehensive understanding of gut and fecal microbial communities of cockroaches is essential not only to understand their contribution to the biology of the cockroach, but also for exploring their clinical relevance. In this study, we compare the diversity of bacteria and eukaryotes in the cockroach gut and feces and assess the variation in the gut microbiota across cockroach populations., (Copyright © 2018 Kakumanu et al.)

The gut microbiota of termites plays important roles in the symbiotic digestion of lignocellulose. However, the factors shaping the microbial community structure remain poorly understood. Because termites cannot be raised under axenic conditions, we established the closely related cockroach Shelfordella lateralis as a germ-free model to study microbial community assembly and host-microbe interactions. In this study, we determined the composition of the bacterial assemblages in cockroaches inoculated with the gut microbiota of termites and mice using pyrosequencing analysis of their 16S rRNA genes. Although the composition of the xenobiotic communities was influenced by the lineages present in the foreign inocula, their structure resembled that of conventional cockroaches. Bacterial taxa abundant in conventional cockroaches but rare in the foreign inocula, such as Dysgonomonas and Parabacteroides spp., were selectively enriched in the xenobiotic communities. Donor-specific taxa, such as endomicrobia or spirochete lineages restricted to the gut microbiota of termites, however, either were unable to colonize germ-free cockroaches or formed only small populations. The exposure of xenobiotic cockroaches to conventional adults restored their normal microbiota, which indicated that autochthonous lineages outcompete foreign ones. Our results provide experimental proof that the assembly of a complex gut microbiota in insects is deterministic., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Termites digest wood and other lignocellulosic substrates with the help of their intestinal microbiota. While the functions of the symbionts in the digestive process are slowly emerging, the origin of the bacteria colonizing the hindgut bioreactor is entirely unknown. Recently, our group discovered numerous representatives of bacterial lineages specific to termite guts in a closely related omnivorous cockroach, but it remains unclear whether they derive from the microbiota of a common ancestor or were independently selected by the gut environment. Here, we studied the bacterial gut microbiota in 34 species of termites and cockroaches using pyrotag analysis of the 16S rRNA genes. Although the community structures differed greatly between the major host groups, with dramatic changes in the relative abundances of particular bacterial taxa, we found that the majority of sequence reads belonged to bacterial lineages that were shared among most host species. When mapped onto the host tree, the changes in community structure coincided with major events in termite evolution, such as acquisition and loss of cellulolytic protists and the ensuing dietary diversification. UniFrac analysis of the core microbiota of termites and cockroaches and construction of phylogenetic tree of individual genus level lineages revealed a general host signal, whereas the branching order often did not match the detailed phylogeny of the host. It remains unclear whether the lineages in question have been associated with the ancestral cockroach since the early Cretaceous (cospeciation) or are diet-specific lineages that were independently acquired from the environment (host selection).

Termites and cockroaches are closely related, with molecular phylogenetic analyses even placing termites within the radiation of cockroaches. The intestinal tract of wood-feeding termites harbors a remarkably diverse microbial community that is essential for the digestion of lignocellulose. However, surprisingly little is known about the gut microbiota of their closest relatives, the omnivorous cockroaches. Here, we present a combined characterization of physiological parameters, metabolic activities, and bacterial microbiota in the gut of Shelfordella lateralis, a representative of the cockroach family Blattidae, the sister group of termites. We compared the bacterial communities within each gut compartment using terminal-restriction fragment length polymorphism (T-RFLP) analysis and made a 16S rRNA gene clone library of the microbiota in the colon-the dilated part of the hindgut with the highest density and diversity of bacteria. The colonic community was dominated by members of the Bacteroidetes, Firmicutes (mainly Clostridia), and some Deltaproteobacteria. Spirochaetes and Fibrobacteres, which are abundant members of termite gut communities, were conspicuously absent. Nevertheless, detailed phylogenetic analysis revealed that many of the clones from the cockroach colon clustered with sequences previously obtained from the termite gut, which indicated that the composition of the bacterial community reflects at least in part the phylogeny of the host.

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.

Insect endosymbiont genomes reflect massive gene loss. Two Blattabacterium genomes display colinearity and similar gene contents, despite high orthologous gene divergence, reflecting over 140 million years of independent evolution in separate cockroach lineages. We speculate that distant homologs may replace the functions of some eliminated genes through broadened substrate specificity.

In the intestinal tracts of animals, methanogenesis from CO(2) and other C(1) compounds strictly depends on the supply of electron donors by fermenting bacteria, but sources and sinks of reducing equivalents may be spatially separated. Microsensor measurements in the intestinal tract of the omnivorous cockroach Blaberus sp. showed that molecular hydrogen strongly accumulated in the midgut (H(2) partial pressures of 3 to 26 kPa), whereas it was not detectable (<0.1 kPa) in the posterior hindgut. Moreover, living cockroaches emitted large quantities of CH(4) [105 +/- 49 nmol (g of cockroach)(-1) h(-1)] but only traces of H(2). In vitro incubation of isolated gut compartments, however, revealed that the midguts produced considerable amounts of H(2), whereas hindguts emitted only CH(4) [106 +/- 58 and 71 +/- 50 nmol (g of cockroach)(-1) h(-1), respectively]. When ligated midgut and hindgut segments were incubated in the same vials, methane emission increased by 28% over that of isolated hindguts, whereas only traces of H(2) accumulated in the headspace. Radial hydrogen profiles obtained under air enriched with H(2) (20 kPa) identified the hindgut as an efficient sink for externally supplied H(2). A cross-epithelial transfer of hydrogen from the midgut to the hindgut compartment was clearly evidenced by the steep H(2) concentration gradients which developed when ligated fragments of midgut and hindgut were placed on top of each other-a configuration that simulates the situation in vivo. These findings emphasize that it is essential to analyze the compartmentalization of the gut and the spatial organization of its microbiota in order to understand the functional interactions among different microbial populations during digestion.

Proteinase 2 (Pr2) is a fungal (Metarhizium anisopliae) serine proteinase which has a tryptic specificity for basic residues and which may be involved in entomopathogenicity. Analytical and preparative isoelectric focusing methods were used to separate two trypsin components, produced during growth on cockroach cuticle, with isoelectric points of 4.4 (molecular mass, 30 kDa) and 4.9 (27 kDa). The catalytic properties of the proteases were analyzed by their kinetic constants and by a combination of two-dimensional gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme overlay membranes. Both Pr2 isoforms preferentially cleave at the carboxyl sides of positively charged amino acids, preferring arginine; the pI 4.4 Pr2 isoform also possessed significant activity against lysine. Compared with the pathogen's subtilisin-like enzyme (Pr1), the pI 4.4 Pr2 isoform shows low activity against insoluble proteins in a host (Manduca sexta) cuticle. However, it degrades most cuticle proteins when they are solubilized, with high-molecular-weight basic proteins being preferentially hydrolyzed. Polyclonal antibodies raised against each Pr2 isoform were isotype specific. This allowed us to use ultrastructural immunocytochemistry to independently visualize each isoform during penetration of the host (M. sexta) cuticle. Both isoforms were secreted by infection structures (appressoria) on the cuticle surface and by the penetrant hyphae within the cuticle. The extracellular sheath, which is commonly observed around fungal cells, often contained Pr2 molecules. Intracellular labelling was sparse.

The ciliate Nyctotherus ovalis occurs in high numbers in the hindgut of the American cockroach (Periplaneta americana) and harbors methanogenic bacteria as endosymbionts. The contribution of these hindgut microorganisms to metabolic and developmental processes of P. americana was studied by comparing cultures of cockroaches in which the composition of the hindgut microbial population was altered in various ways. Rearing the insects protozoan free resulted in increased insect generation time, decreased adult body weight, and absence of methane production. After feeding of protozoan-free adult cockroaches with a hindgut suspension containing N. ovalis and methanogens, methane increased to normal values and insect body weight was restored during the development of the second generation of insects. Feeding the protozoan-free cockroaches a hindgut suspension which was made free of N. ovalis resulted in an increase in methane production to only about 20% of the normal methane production level. This suggests that the methanogenic endosymbionts of N. ovalis are the major source of methane production in the hindgut. Inhibition of methanogens by addition of bromoethanesulfonic acid to the drinking water of a normal cockroach culture resulted in a reduction of methane production to about 2% of the normal level. No effects on insect body weight or the number of N. ovalis organisms were observed, but the fermentation pattern in the hindgut was shifted towards a relative increase in propionate levels. Similar results were obtained for in vitro cultures of hindgut microorganisms treated with bromoethanesulfonic acid. The results suggest a major role for hindgut protozoa in cockroach metabolic activities, especially during the insect growth period. The relatively large amounts of methane produced by cockroaches and by other methane-producing xylophagous insects suggest a major contribution by insects to global methane production.

The effect of high-fiber diets on microbial populations and processes in cockroach guts was investigated by feeding American cockroaches (Periplaneta americana) milled cereal leaves, milled corn cob, or commercial bran-type breakfast cereal in place of the commonly used laboratory diet of dog chow. The activities and numbers of specific gut bacteria varied significantly with the insect's diet and developmental stage. Acetate and lactate were the principal organic acids present in the gut fluid of adult cockroaches and occurred at concentrations of up to 17 and 8 mM, respectively. These acids were most abundant in the gut fluid of dog chow-fed insects, and the greatest amounts were generally found in the foregut and midgut regions. Foreguts of dog chow-fed cockroaches contained an abundant population of lactic acid bacteria that formed acetate and lactate from endogenous hexoses present in the foregut. When adult cockroaches were fed dog chow amended with antibacterial drugs, (i) the concentrations of acetate, lactate, and total hexoses in gut fluid decreased significantly, (ii) the numbers of lactic acid bacteria in the foregut also decreased significantly, and (iii) the production of acetate and lactate by foregut homogenates was suppressed. It was estimated that acetate and lactate produced by bacteria in the foregut of dog chow-fed adult P. americana could support up to 14% of the insect's respiratory requirement if taken up and used by the animal. When insects were fed high-fiber diets of bran cereal, cereal leaves, or corn cob, bacterial production of acetate and lactate in the foregut diminished.(ABSTRACT TRUNCATED AT 250 WORDS)

An immunolatex reagent was developed from antiserum to Fusobacterium varium, an obligate anaerobe isolated from the colon of the cockroach, Periplaneta americana L. A new technique that enabled the preparation of a highly efficient immunolatex conjugate was used to localize the bacterium with scanning electron microscopy, in situ, in the mixed gut contents.

A morphological study employing scanning and transmission electron microscopy was made by the alimentary tract of the American cockroach, Periplaneta americana L. A. complex microbiota of diverse morphology, which could not be readily dislodged, was observed and found to be restricted to the hindgut, particularly the colon. Numerous filamentous forms were noted, and some are described, including the morphologically distinct Methanospirillum. Flora was noted attached to the cuticular lining and cuticular filaments of the colon, and several spiral forms were observed in the luminal contents from the colon.