Defensive fungal symbiosis on insect hindlegs.

Tympanal organs as "insect ears" have evolved repeatedly. Dinidorid stinkbugs were reported to possess a conspicuous tympanal organ on female's hindlegs. Here we report an unexpected discovery that the stinkbug's "tympanal organ" is actually a novel symbiotic organ. The stinkbug's "tympanum" is not membranous but a porous cuticle, where each pore connects to glandular secretory cells. In reproductive females, the hindleg organ is covered with fungal hyphae growing out of the pores. Upon oviposition, the females skillfully transfer the fungi from the organ to the eggs. The eggs are quickly covered with hyphae and physically protected against wasp parasitism. The fungi are mostly benign Cordycipitaceae entomopathogens and show considerable diversity among insect individuals and populations, indicating environmental acquisition of specific fungal associates. These results uncover a novel external fungal symbiosis in which host's elaborate morphological, physiological and behavioral specializations underpin the selective recruitment of benign entomopathogens for a defensive purpose.

Genome analysis of "Candidatus Aschnera chinzeii," the bacterial endosymbiont of the blood-sucking bat fly Penicillidia jenynsii (Insecta: Diptera: Nycteribiidae).

Insect-microbe endosymbiotic associations are omnipresent in nature, wherein the symbiotic microbes often play pivotal biological roles for their host insects. In particular, insects utilizing nutritionally imbalanced food sources are dependent on specific microbial symbionts to compensate for the nutritional deficiency via provisioning of B vitamins in blood-feeding insects, such as tsetse flies, lice, and bedbugs. Bat flies of the family Nycteribiidae (Diptera) are blood-sucking ectoparasites of bats and shown to be associated with co-speciating bacterial endosymbiont "Candidatus Aschnera chinzeii," although functional aspects of the microbial symbiosis have been totally unknown. In this study, we report the first complete genome sequence of Aschnera from the bristled bat fly Penicillidia jenynsii. The Aschnera genome consisted of a 748,020 bp circular chromosome and a 18,747 bp circular plasmid. The chromosome encoded 603 protein coding genes (including 3 pseudogenes), 33 transfer RNAs, and 1 copy of 16S/23S/5S ribosomal RNA operon. The plasmid contained 10 protein coding genes, whose biological function was elusive. The genome size, 0.77 Mbp, was drastically reduced in comparison with 4-6 Mbp genomes of free-living γ-proteobacteria. Accordingly, the Aschnera genome was devoid of many important functional genes, such as synthetic pathway genes for purines, pyrimidines, and essential amino acids. On the other hand, the Aschnera genome retained complete or near-complete synthetic pathway genes for biotin (vitamin B7), tetrahydrofolate (vitamin B9), riboflavin (vitamin B2), and pyridoxal 5'-phosphate (vitamin B6), suggesting that Aschnera provides these vitamins and cofactors that are deficient in the blood meal of the host bat fly. Similar retention patterns of the synthetic pathway genes for vitamins and cofactors were also observed in the endosymbiont genomes of other blood-sucking insects, such as Riesia of human lice, Arsenophonus of louse flies, and Wigglesworthia of tsetse flies, which may be either due to convergent evolution in the blood-sucking host insects or reflecting the genomic architecture of Arsenophonus-allied bacteria.

Single mutation makes Escherichia coli an insect mutualist.

Microorganisms often live in symbiosis with their hosts, and some are considered mutualists, where all species involved benefit from the interaction. How free-living microorganisms have evolved to become mutualists is unclear. Here we report an experimental system in which non-symbiotic Escherichia coli evolves into an insect mutualist. The stinkbug Plautia stali is typically associated with its essential gut symbiont, Pantoea sp., which colonizes a specialized symbiotic organ. When sterilized newborn nymphs were infected with E. coli rather than Pantoea sp., only a few insects survived, in which E. coli exhibited specific localization to the symbiotic organ and vertical transmission to the offspring. Through transgenerational maintenance with P. stali, several hypermutating E. coli lines independently evolved to support the host's high adult emergence and improved body colour; these were called 'mutualistic' E. coli. These mutants exhibited slower bacterial growth, smaller size, loss of flagellar motility and lack of an extracellular matrix. Transcriptomic and genomic analyses of 'mutualistic' E. coli lines revealed independent mutations that disrupted the carbon catabolite repression global transcriptional regulator system. Each mutation reproduced the mutualistic phenotypes when introduced into wild-type E. coli, confirming that single carbon catabolite repression mutations can make E. coli an insect mutualist. These findings provide an experimental system for future work on host-microbe symbioses and may explain why microbial mutualisms are omnipresent in nature.

Endosymbiotic bacteria of the boar louse Haematopinus apri (Insecta: Phthiraptera: Anoplura).

Insects exclusively feeding on vertebrate blood are usually dependent on symbiotic bacteria for provisioning of B vitamins. Among them, sucking lice are prominent in that their symbiotic bacteria as well as their symbiotic organs exhibit striking diversity. Here we investigated the bacterial diversity associated with the boar louse Haematopinus apri in comparison with the hog louse Haematopinus suis. Amplicon sequencing analysis identified the primary endosymbiont predominantly detected from all populations of H. apri with some minor secondary bacterial associates. Sequencing and phylogenetic analysis of bacterial 16S rRNA gene confirmed that the endosymbionts of the boar louse H. apri, the hog louse H. suis and the cattle louse Haematopinus eurysternus form a distinct clade in the Gammaproteobacteria. The endosymbiont clade of Haematopinus spp. was phylogenetically distinct from the primary endosymbionts of other louse lineages. Fluorescence in situ hybridization visualized the endosymbiont localization within midgut epithelium, ovarial ampulla and posterior oocyte of H. apri, which were substantially the same as the endosymbiont localization previously described in H. suis and H. eurysternus. Mitochondrial haplotype analysis revealed that, although the domestic pig was derived from the wild boar over the past 8,000 years of human history, the populations of H. apri constituted a distinct sister clade to the populations of H. suis. Based on these results, we discussed possible evolutionary trajectories of the boar louse, the hog louse and their endosymbionts in the context of swine domestication. We proposed 'Candidatus Haematopinicola symbiotica' for the distinct clade of the endosymbionts of Haematopinus spp.

Evolutionary Dynamics of Host Organs for Microbial Symbiosis in Tortoise Leaf Beetles (Coleoptera: Chrysomelidae: Cassidinae).

Diverse insects host specific microbial symbionts that play important roles for their growth, survival, and reproduction. They often develop specialized symbiotic organs for harboring the microbial partners. While such intimate associations tend to be stably maintained over evolutionary time, the microbial symbionts may have been lost or replaced occasionally. How symbiont acquisitions, replacements, and losses are linked to the development of the host's symbiotic organs is an important but poorly understood aspect of microbial symbioses. Cassidine leaf beetles are associated with a specific gammaproteobacterial lineage, Stammera, whose reduced genome is streamlined for producing pectin-degrading enzymes to assist the host's digestion of food plants. We investigated the symbiotic system of 24 Japanese cassidine species and found that (i) most species harbored Stammera within paired symbiotic organs located at the foregut-midgut junction, (ii) the host phylogeny was largely congruent with the symbiont phylogeny, indicating stable host-symbiont association over evolutionary time, (iii) meanwhile, the symbiont was not detected in three distinct host lineages, uncovering recurrent losses of the ancient microbial mutualist, (iv) the symbiotic organs were vestigial but present in the symbiont-free lineages, indicating evolutionary persistence of the symbiotic organs even in the absence of the symbiont, and (v) the number of the symbiotic organs was polymorphic among the cassidine species, either two or four, unveiling a dynamic evolution of the host organs for symbiosis. These findings are discussed as to what molecular mechanisms and evolutionary trajectories underpin the recurrent symbiont losses and the morphogenesis of the symbiotic organs in the herbivorous insect group. IMPORTANCE Insects represent the biodiversity of the terrestrial ecosystem, and their prosperity is attributable to their association with symbiotic microorganisms. By sequestering microbial functionality into their bodies, organs, tissues, or cells, diverse insects have successfully exploited otherwise inaccessible ecological niches and resources, including herbivory enabled by utilization of indigestible plant cell wall components. In leaf beetles of the subfamily Cassininae, an ancient symbiont lineage, Stammera, whose genome is extremely reduced and specialized for encoding pectin-degrading enzymes, is hosted in gut-associated symbiotic organs and contributes to the host's food plant digestion. Here, we demonstrate that multiple symbiont losses and recurrent structural switching of the symbiotic organs have occurred in the evolutionary course of cassidine leaf beetles, which sheds light on the evolutionary and developmental dynamics of the insect's symbiotic organs and provides a model system to investigate how microbial symbionts affect the host's development and morphogenesis and vice versa.

Host's guardian protein counters degenerative symbiont evolution.

Microbial symbioses significantly contribute to diverse organisms, where long-lasting associations tend to result in symbiont genome erosion, uncultivability, extinction, and replacement. How such inherently deteriorating symbiosis can be harnessed to stable partnership is of general evolutionary interest. Here, we report the discovery of a host protein essential for sustaining symbiosis. Plataspid stinkbugs obligatorily host an uncultivable and genome-reduced gut symbiont, Ishikawaella Upon oviposition, females deposit "capsules" for symbiont delivery to offspring. Within the capsules, the fragile symbiotic bacteria survive the harsh conditions outside the host until acquired by newborn nymphs to establish vertical transmission. We identified a single protein dominating the capsule content, which is massively secreted by female-specific intestinal organs, embedding the symbiont cells, and packaged into the capsules. Knockdown of the protein resulted in symbiont degeneration, arrested capsule production, symbiont transmission failure, and retarded nymphal growth, unveiling its essential function for ensuring symbiont survival and vertical transmission. The protein originated from a lineage of odorant-binding protein-like multigene family, shedding light on the origin of evolutionary novelty regarding symbiosis. Experimental suppression of capsule production extended the female's lifespan, uncovering a substantial cost for maintaining symbiosis. In addition to the host's guardian protein, the symbiont's molecular chaperone, GroEL, was overproduced in the capsules, highlighting that the symbiont's eroding functionality is compensated for by stabilizer molecules of host and symbiont origins. Our finding provides insight into how intimate host-symbiont associations can be maintained over evolutionary time despite the symbiont's potential vulnerability to degeneration and malfunctioning.

Environmental Acquisition of Gut Symbiotic Bacteria in the Saw-Toothed Stinkbug, Megymenum gracilicorne (Hemiptera: Pentatomoidea: Dinidoridae).

Many plant-sucking stinkbugs possess a specialized symbiotic organ with numerous crypts in a posterior region of the midgut. In stinkbugs of the superfamily Pentatomoidea, specific γ-proteobacteria are hosted in the crypt cavities, which are vertically transmitted through host generations and essential for normal growth and survival of the host insects. Here we report the discovery of an exceptional gut symbiotic association in the saw-toothed stinkbug, Megymenum gracilicorne (Hemiptera: Pentatomoidea: Dinidoridae), in which specific γ-proteobacterial symbionts are not transmitted vertically but acquired environmentally. Histological inspection identified a very thin and long midgut symbiotic organ with two rows of tiny crypts whose cavities harbor rod-shaped bacterial cells. Molecular phylogenetic analyses of bacterial 16S rRNA gene sequences from the symbiotic organs of field-collected insects revealed that (i) M. gracilicorne is stably associated with Pantoea-allied γ-proteobacteria within the midgut crypts, (ii) the symbiotic bacteria exhibit a considerable level of diversity across host individuals and populations, (iii) the major symbiotic bacteria represent an environmental bacterial lineage that was reported to be capable of symbiosis with the stinkbug Plautia stali, and (iv) the minor symbiotic bacteria also represent several bacterial lineages that were reported as cultivable symbionts of P. stali and other stinkbugs. The symbiotic bacteria were shown to be generally cultivable. Microbial inspection of ovipositing adult females and their eggs and nymphs uncovered the absence of stable vertical transmission of the symbiotic bacteria. Rearing experiments showed that symbiont-supplemented newborn nymphs exhibit improved survival, suggesting the beneficial nature of the symbiotic association.

Genome Analysis of "Candidatus Regiella insecticola" Strain TUt, Facultative Bacterial Symbiont of the Pea Aphid Acyrthosiphon pisum.

The genome of "Candidatus Regiella insecticola" strain TUt, a facultative bacterial symbiont of the pea aphid Acyrthosiphon pisum, was analyzed. We determined a 2.5-Mb draft genome consisting of 14 contigs; this will contribute to the understanding of the symbiont, which underpins various ecologically adaptive traits of the host insect.

Bacteriocytes and Blattabacterium Endosymbionts of the German Cockroach Blattella germanica, the Forest Cockroach Blattella nipponica, and Other Cockroach Species.

Cockroaches are commonly found in human residences and notorious as hygienic and nuisance pests. Notably, however, no more than 30 cockroach species are regarded as pests, while the majority of 4,500 cockroaches in the world are living in forest environments with little relevance to human life. Why some cockroaches have exceptionally adapted to anthropic environments and established pest status is of interest. Here we investigated the German cockroach Blattella germanica, which is a cosmopolitan pest species, and the forest cockroach Blattella nipponica, which is a wild species closely related to B. germanica. In contrast to easy rearing of B. germanica, laboratory rearing of B. nipponica was challenging-several trials enabled us to keep the insects for up to three months. We particularly focused on the distribution patterns of specialized cells, bacteriocytes, for harboring endosymbiotic Blattabacterium, which has been suggested to contribute to host's nitrogen metabolism and recycling, during the postembryonic development of the insects. The bacteriocytes were consistently localized to visceral fat bodies filling the abdominal body cavity, where a number of single bacteriocytes were scattered among the adipocytes, throughout the developmental stages in both females and males. The distribution patterns of the bacteriocytes were quite similar between B. germanica and B. nipponica, and also among other diverse cockroach species, plausibly reflecting the highly conserved cockroach-Blattabacterium symbiotic association over evolutionary time. Our study lays a foundation to experimentally investigate the origin and the processes of urban pest evolution, on account of possible involvement of microbial associates.

Reduced Genome of the Gut Symbiotic Bacterium "Candidatus Benitsuchiphilus tojoi" Provides Insight Into Its Possible Roles in Ecology and Adaptation of the Host Insect.

Diverse animals, including insects, harbor microbial symbionts within their gut, body cavity, or cells. The subsocial parastrachiid stinkbug Parastrachia japonensis is well-known for its peculiar ecological and behavioral traits, including its prolonged non-feeding diapause period and maternal care of eggs/nymphs in an underground nest. P. japonensis harbors a specific bacterial symbiont within the gut cavity extracellularly, which is vertically inherited through maternal excretion of symbiont-containing white mucus. Thus far, biological roles of the symbiont in the host lifecycle has been little understood. Here we sequenced the genome of the uncultivable gut symbiont "Candidatus Benitsuchiphilus tojoi." The symbiont has an 804 kb circular chromosome encoding 606 proteins and a 14.5 kb plasmid encoding 13 proteins. Phylogenetic analysis indicated that the bacterium is closely related to other obligate insect symbionts belonging to the Gammaproteobacteria, including Buchnera of aphids and Blochmannia of ants, and the most closely related to Ishikawaella, an extracellular gut symbiont of plataspid stinkbugs. These data suggested that the symbiont genome has evolved like highly reduced gamma-proteobacterial symbiont genomes reported from a variety of insects. The presence of genes involved in biosynthesis pathways for amino acids, vitamins, and cofactors in the genome implicated the symbiont as a nutritional mutualist, supplementing essential nutrients to the host. Interestingly, the symbiont's plasmid encoded genes for thiamine and carotenoid synthesis pathways, suggesting the possibility of additional functions of the symbiont for protecting the host against oxidative stress and DNA damage. Finally, possible involvement of the symbiont in uric acid metabolism during diapause is discussed.

Diversity and function of multicopper oxidase genes in the stinkbug Plautia stali.

Multicopper oxidase (MCO) genes comprise multigene families in bacteria, fungi, plants and animals. Two families of MCO genes, MCO1 (laccase1) and MCO2 (laccase2), are conserved among diverse insects and relatively well-characterized, whereas additional MCO genes, whose biological functions have been poorly understood, are also found in some insects. Previous studies reported that MCO1 participates in gut immunity and MCO2 plays important roles in cuticle sclerotization and pigmentation of insects. In mosquitoes, MCO2 was reported to be involved in eggshell sclerotization and pigmentation, on the ground that knockdown of MCO2 caused deformity and fragility of the eggshell. Here we identified a total of 7 MCO genes, including PsMCO1 and PsMCO2, and investigated their expression and function in the brown-winged green stinkbug Plautia stali. RNA interference (RNAi) knockdown of MCO genes by injecting double-stranded RNA (dsRNA) into nymphs revealed that MCO2, but not the other 6 MCOs, is required for cuticle sclerotization and pigmentation, and also for survival of P. stali. Trans-generational knockdown of MCO2 by injecting dsRNA into adult females (maternal RNAi) resulted in the production of unhatched eggs despite the absence of deformity or fragility of the eggshell. These results suggested that MCO2 plays an important role in sclerotization and pigmentation of the cuticle but not in eggshell integrity in P. stali. Maternal RNAi of any of the other 6 MCO genes and 3 tyrosinase genes affected neither survival nor eggshell integrity of P. stali. Contrary to the observations in the red flour beetle and the brown rice planthopper, RNAi knockdown of MCO6 (MCORP; Multicopper oxidase related protein) exhibited no lethal effects on P. stali. Taken together, our findings provide insight into the functional diversity and commonality of MCOs across hemipteran and other insect groups.

Morphogenesis and development of midgut symbiotic organ of the stinkbug Plautia stali (Hemiptera: Pentatomidae).

Diverse insects are intimately associated with microbial symbionts, which play a variety of biological roles in their adaptation to and survival in the natural environment. Such insects often possess specialized organs for hosting the microbial symbionts. What developmental processes and mechanisms underlie the formation of the host organs for microbial symbiosis is of fundamental biological interest but poorly understood. Here we investigate the morphogenesis of the midgut symbiotic organ and the process of symbiont colonization therein during the developmental course of the stinkbug Plautia stali. Upon hatching, the midgut is a simple and smooth tube. Subsequently, symbiont colonization to the posterior midgut occurs, and thickening and folding of the midgut epithelium proceed during the first instar period. By the second instar, rudimentary crypts have formed, and their inner cavities are colonized by the symbiotic bacteria. From the second instar to the fourth instar, while the alimentary tract grows and the posterior midgut is established as the symbiotic organ with numerous crypts, the anterior midgut and the posterior midgut are structurally and functionally isolated by a strong constriction in the middle. By the early fifth instar, the midgut symbiotic organ attains the maximal length, but toward the mid fifth instar, the basal region of each crypt starts to constrict and narrow, which deforms the midgut symbiotic organ as a whole into a shorter, thicker and twisted shape. By the late fifth instar to adulthood, the crypts are constricted off, by which the symbiotic bacteria are confined in the crypt cavities and isolated from the midgut main tract, and concurrently, the strong midgut constriction in the middle becomes loose and open, by which the food flow from the anterior midgut to the posterior midgut recovers. This study provides the most detailed and comprehensive descriptions ever reported on the morphogenesis of the symbiotic organ and the process of symbiont colonization in an obligatory insect-bacterium gut symbiotic system. Considering that P. stali is recently emerging as a useful model system for experimentally studying the intimate insect-microbe gut symbiosis, the knowledge obtained in this study establishes the foundation for the further development of this research field.

Genome Sequence of "Candidatus Serratia symbiotica" Strain IS, a Facultative Bacterial Symbiont of the Pea Aphid Acyrthosiphon pisum.

"Candidatus Serratia symbiotica" is a facultative bacterial symbiont of aphids that affects various ecological traits of the host insects. Here, we report the complete genome sequence of "Candidatus Serratia symbiotica" strain IS, consisting of a 2,736,352-bp chromosome and an 82,605-bp plasmid, from the pea aphid Acyrthosiphon pisum.

Genome Evolution of Bartonellaceae Symbionts of Ants at the Opposite Ends of the Trophic Scale.

Many insects rely on bacterial symbionts to supply essential amino acids and vitamins that are deficient in their diets, but metabolic comparisons of closely related gut bacteria in insects with different dietary preferences have not been performed. Here, we demonstrate that herbivorous ants of the genus Dolichoderus from the Peruvian Amazon host bacteria of the family Bartonellaceae, known for establishing chronic or pathogenic infections in mammals. We detected these bacteria in all studied Dolichoderus species, and found that they reside in the midgut wall, that is, the same location as many previously described nutritional endosymbionts of insects. The genomic analysis of four divergent strains infecting different Dolichoderus species revealed genes encoding pathways for nitrogen recycling and biosynthesis of several vitamins and all essential amino acids. In contrast, several biosynthetic pathways have been lost, whereas genes for the import and conversion of histidine and arginine to glutamine have been retained in the genome of a closely related gut bacterium of the carnivorous ant Harpegnathos saltator. The broad biosynthetic repertoire in Bartonellaceae of herbivorous ants resembled that of gut bacteria of honeybees that likewise feed on carbohydrate-rich diets. Taken together, the broad distribution of Bartonellaceae across Dolichoderus ants, their small genome sizes, the specific location within hosts, and the broad biosynthetic capability suggest that these bacteria are nutritional symbionts in herbivorous ants. The results highlight the important role of the host nutritional biology for the genomic evolution of the gut microbiota-and conversely, the importance of the microbiota for the nutrition of hosts.

Genomic Insight into Symbiosis-Induced Insect Color Change by a Facultative Bacterial Endosymbiont, "Candidatus Rickettsiella viridis".

Members of the genus Rickettsiella are bacterial pathogens of insects and other arthropods. Recently, a novel facultative endosymbiont, "Candidatus Rickettsiella viridis," was described in the pea aphid Acyrthosiphon pisum, whose infection causes a striking host phenotype: red and green genetic color morphs exist in aphid populations, and upon infection with the symbiont, red aphids become green due to increased production of green polycyclic quinone pigments. Here we determined the complete genome sequence of the symbiont. The 1.6-Mb circular genome, harboring some 1,400 protein-coding genes, was similar to the genome of entomopathogenic Rickettsiella grylli (1.6 Mb) but was smaller than the genomes of phylogenetically allied human pathogens Coxiella burnetii (2.0 Mb) and Legionella pneumophila (3.4 Mb). The symbiont's metabolic pathways exhibited little complementarity to those of the coexisting primary symbiont Buchnera aphidicola, reflecting the facultative nature of the symbiont. The symbiont genome harbored neither polyketide synthase genes nor the evolutionarily allied fatty acid synthase genes that are suspected to catalyze the polycyclic quinone synthesis, indicating that the green pigments are produced not by the symbiont but by the host aphid. The symbiont genome retained many type IV secretion system genes and presumable effector protein genes, whose homologues in L. pneumophila were reported to modulate a variety of the host's cellular processes for facilitating infection and virulence. These results suggest the possibility that the symbiont is involved in the green pigment production by affecting the host's metabolism using the secretion machineries for delivering the effector molecules into the host cells.IMPORTANCE Insect body color is relevant to a variety of biological aspects such as species recognition, sexual selection, mimicry, aposematism, and crypsis. Hence, the bacterial endosymbiont "Candidatus Rickettsiella viridis," which alters aphid body color from red to green, is of ecological interest, given that different predators preferentially exploit either red- or green-colored aphids. Here we determined the complete 1.6-Mb genome of the symbiont and uncovered that, although the red-green color transition was ascribed to upregulated production of green polycyclic quinone pigments, the symbiont genome harbored few genes involved in the polycyclic quinone biosynthesis. Meanwhile, the symbiont genome contained type IV secretion system genes and presumable effector protein genes, whose homologues modulate eukaryotic cellular processes for facilitating infection and virulence in the pathogen Legionella pneumophila We propose the hypothesis that the symbiont may upregulate the host's production of polycyclic quinone pigments via cooption of secretion machineries and effector molecules for pathogenicity.

Drastic Genome Reduction in an Herbivore's Pectinolytic Symbiont.

Pectin, an integral component of the plant cell wall, is a recalcitrant substrate against enzymatic challenges by most animals. In characterizing the source of a leaf beetle's (Cassida rubiginosa) pectin-degrading phenotype, we demonstrate its dependency on an extracellular bacterium housed in specialized organs connected to the foregut. Despite possessing the smallest genome (0.27 Mb) of any organism not subsisting within a host cell, the symbiont nonetheless retained a functional pectinolytic metabolism targeting the polysaccharide's two most abundant classes: homogalacturonan and rhamnogalacturonan I. Comparative transcriptomics revealed pectinase expression to be enriched in the symbiotic organs, consistent with enzymatic buildup in these structures following immunostaining with pectinase-targeting antibodies. Symbiont elimination results in a drastically reduced host survivorship and a diminished capacity to degrade pectin. Collectively, our findings highlight symbiosis as a strategy for an herbivore to metabolize one of nature's most complex polysaccharides and a universal component of plant tissues.

Dramatic Differences in Gut Bacterial Densities Correlate with Diet and Habitat in Rainforest Ants.

Abundance is a key parameter in microbial ecology, and important to estimates of potential metabolite flux, impacts of dispersal, and sensitivity of samples to technical biases such as laboratory contamination. However, modern amplicon-based sequencing techniques by themselves typically provide no information about the absolute abundance of microbes. Here, we use fluorescence microscopy and quantitative polymerase chain reaction as independent estimates of microbial abundance to test the hypothesis that microbial symbionts have enabled ants to dominate tropical rainforest canopies by facilitating herbivorous diets, and compare these methods to microbial diversity profiles from 16S rRNA amplicon sequencing. Through a systematic survey of ants from a lowland tropical forest, we show that the density of gut microbiota varies across several orders of magnitude among ant lineages, with median individuals from many genera only marginally above detection limits. Supporting the hypothesis that microbial symbiosis is important to dominance in the canopy, we find that the abundance of gut bacteria is positively correlated with stable isotope proxies of herbivory among canopy-dwelling ants, but not among ground-dwelling ants. Notably, these broad findings are much more evident in the quantitative data than in the 16S rRNA sequencing data. Our results provide quantitative context to the potential role of bacteria in facilitating the ants' dominance of the tropical rainforest canopy, and have broad implications for the interpretation of sequence-based surveys of microbial diversity.

Small genome symbiont underlies cuticle hardness in beetles.

Beetles, representing the majority of the insect species diversity, are characterized by thick and hard cuticle, which plays important roles for their environmental adaptation and underpins their inordinate diversity and prosperity. Here, we report a bacterial endosymbiont extremely specialized for sustaining beetle's cuticle formation. Many weevils are associated with a γ-proteobacterial endosymbiont lineage Nardonella, whose evolutionary origin is estimated as older than 100 million years, but its functional aspect has been elusive. Sequencing of Nardonella genomes from diverse weevils unveiled drastic size reduction to 0.2 Mb, in which minimal complete gene sets for bacterial replication, transcription, and translation were present but almost all of the other metabolic pathway genes were missing. Notably, the only metabolic pathway retained in the Nardonella genomes was the tyrosine synthesis pathway, identifying tyrosine provisioning as Nardonella's sole biological role. Weevils are armored with hard cuticle, tyrosine is the principal precursor for cuticle formation, and experimental suppression of Nardonella resulted in emergence of reddish and soft weevils with low tyrosine titer, confirming the importance of Nardonella-mediated tyrosine production for host's cuticle formation and hardening. Notably, Nardonella's tyrosine synthesis pathway was incomplete, lacking the final step transaminase gene. RNA sequencing identified host's aminotransferase genes up-regulated in the bacteriome. RNA interference targeting the aminotransferase genes induced reddish and soft weevils with low tyrosine titer, verifying host's final step regulation of the tyrosine synthesis pathway. Our finding highlights an impressively intimate and focused aspect of the host-symbiont metabolic integrity via streamlined evolution for a single biological function of ecological relevance.

A Novel, Extremely Elongated, and Endocellular Bacterial Symbiont Supports Cuticle Formation of a Grain Pest Beetle.

The saw-toothed grain beetle, Oryzaephilus surinamensis (Silvanidae), is a cosmopolitan stored-product pest. Early studies on O. surinamensis in the 1930s described the presence of peculiar bacteriomes harboring endosymbiotic bacteria in the abdomen. Since then, however, the microbiological nature of the symbiont has been elusive. Here we investigated the endosymbiotic system of O. surinamensis in detail. In the abdomen of adults, pupae, and larvae, four oval bacteriomes were consistently identified, whose cytoplasm was full of extremely elongated tubular bacterial cells several micrometers wide and several hundred micrometers long. Molecular phylogenetic analysis identified the symbiont as a member of the Bacteroidetes, in which the symbiont was the most closely related to the endosymbiont of a grain pest beetle, Rhyzopertha dominica (Bostrichidae). The symbiont was detected in developing embryos, corroborating vertical symbiont transmission through host generations. The symbiont gene showed AT-biased nucleotide composition and accelerated molecular evolution, plausibly reflecting degenerative evolution of the symbiont genome. When the symbiont infection was experimentally removed, the aposymbiotic insects grew and reproduced normally, but exhibited a slightly but significantly more reddish cuticle and lighter body mass. These results indicate that the symbiont of O. surinamensis is not essential for the host's growth and reproduction but contributes to the host's cuticle formation. Symbiont genome sequencing and detailed comparison of fitness parameters between symbiotic and aposymbiotic insects under various environmental conditions will provide further insights into the symbiont's biological roles for the stored-product pest.IMPORTANCE Some beetles notorious as stored-product pests possess well-developed symbiotic organs called bacteriomes for harboring specific symbiotic bacteria, although their biological roles have been poorly understood. Here we report a peculiar endosymbiotic system of a grain pest beetle, Oryzaephilus surinamensis, in which four oval bacteriomes in the abdomen are full of extremely elongated tubular bacterial cells. Experimental symbiont elimination did not hinder the host's growth and reproduction, but resulted in emergence of reddish beetles, uncovering the symbiont's involvement in host's cuticle formation. We speculate that the extremely elongated symbiont cell morphology might be due to the degenerative symbiont genome deficient in bacterial cell division and/or cell wall formation, which highlights an evolutionary consequence of intimate host-symbiont coevolution.

Novel bacteriocyte-associated pleomorphic symbiont of the grain pest beetle Rhyzopertha dominica (Coleoptera: Bostrichidae).

BACKGROUND: The lesser grain borer Rhyzopertha dominica (Coleoptera: Bostrichidae) is a stored-product pest beetle. Early histological studies dating back to 1930s have reported that R. dominica and other bostrichid species possess a pair of oval symbiotic organs, called the bacteriomes, in which the cytoplasm is densely populated by pleomorphic symbiotic bacteria of peculiar rosette-like shape. However, the microbiological nature of the symbiont has remained elusive. RESULTS: Here we investigated the bacterial symbiont of R. dominica using modern molecular, histological, and microscopic techniques. Whole-mount fluorescence in situ hybridization specifically targeting symbiotic bacteria consistently detected paired bacteriomes, in which the cytoplasm was full of pleomorphic bacterial cells, in the abdomen of adults, pupae and larvae, confirming previous histological descriptions. Molecular phylogenetic analysis identified the symbiont as a member of the Bacteroidetes, in which the symbiont constituted a distinct bacterial lineage allied to a variety of insect-associated endosymbiont clades, including Uzinura of diaspidid scales, Walczuchella of giant scales, Brownia of root mealybugs, Sulcia of diverse hemipterans, and Blattabacterium of roaches. The symbiont gene exhibited markedly AT-biased nucleotide composition and significantly accelerated molecular evolution, suggesting degenerative evolution of the symbiont genome. The symbiotic bacteria were detected in oocytes and embryos, confirming continuous host-symbiont association and vertical symbiont transmission in the host life cycle. CONCLUSIONS: We demonstrate that the symbiont of R. dominica constitutes a novel bacterial lineage in the Bacteroidetes. We propose that reductive evolution of the symbiont genome may be relevant to the amorphous morphology of the bacterial cells via disruption of genes involved in cell wall synthesis and cell division. Genomic and functional aspects of the host-symbiont relationship deserve future studies.