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ABSTRACT Diverse insects are intimately associated with specific symbiotic bacteria, where host and symbiont are integrated into an almost inseparable biological entity. These symbiotic bacteria usually exhibit host specificity, uncultivability, reduced genome size, and other peculiar traits relevant to their symbiotic lifestyle. How host-symbiont specificity is established at the very beginning of symbiosis is of interest but poorly understood. To gain insight into the evolutionary issue, we adopted an experimental approach using the recently developed evolutionary model of symbiosis between the stinkbug Plautia stali and Escherichia coli. Based on the laboratory evolution of P. stali-E. coli mutualism, we selected ΔcyaA mutant of E. coli as an artificial symbiont of P. stali that has established mutualism by a single mutation. In addition, we selected a natural cultivable symbiont of P. stali of relatively recent evolutionary origin. These artificial and natural symbiotic bacteria of P. stali were experimentally inoculated to symbiont-deprived newborn nymphs of diverse stinkbug species. Strikingly, the mutualistic E. coli was unable to establish infection and support growth and survival of all the stinkbug species except for P. stali, uncovering that host specificity can be established at a very early stage of symbiotic evolution. Meanwhile, the natural symbiont was able to establish infection and support growth and survival of several stinkbug species in addition to P. stali, unveiling that a broader host range of the symbiont has evolved in nature. Based on these findings, we discuss what factors are relevant to the establishment of host specificity in the evolution of symbiosis.IMPORTANCEHow does host-symbiont specificity emerge at the very beginning of symbiosis? This question is difficult to address because it is generally difficult to directly observe the onset of symbiosis. However, recent development of experimental evolutionary approaches to symbiosis has brought about a breakthrough. Here we tackled this evolutionary issue using a symbiotic Escherichia coli created in laboratory and a natural Pantoea symbiont, which are both mutualistic to the stinkbug Plautia stali. We experimentally replaced essential symbiotic bacteria of diverse stinkbugs with the artificial and natural symbionts of P. stali and evaluated whether the symbiotic bacteria, which evolved for a specific host, can establish infection and support the growth and survival of heterospecific hosts. Strikingly, the artificial symbiont showed strict host specificity to P. stali, whereas the natural symbiont was capable of symbiosis with diverse stinkbugs, which provide insight into how host-symbiont specificity can be established at early evolutionary stages of symbiosis.

ABSTRACT Microbial mutualists are pivotal for insect adaptation, which often entails the evolution of elaborate organs for symbiosis. Addressing what mechanisms underpin the development of such organs is of evolutionary interest. Here, we investigated the stinkbug Plautia stali, whose posterior midgut is transformed into a specialized symbiotic organ. Despite being a simple tube in newborns, it developed numerous crypts in four rows, whose inner cavity hosts a specific bacterial symbiont, during the 1st to 2nd nymphal instar stages. Visualization of dividing cells revealed that active cell proliferation was coincident with the crypt formation, although spatial patterns of the proliferating cells did not reflect the crypt arrangement. Visualization of visceral muscles in the midgut, consisting of circular muscles and longitudinal muscles, uncovered that, strikingly, circular muscles exhibited a characteristic arrangement running between the crypts specifically in the symbiotic organ. Even in the early 1st instar stage, when no crypts were seen, two rows of epithelial areas delineated by bifurcated circular muscles were identified. In the 2nd instar stage, crossing muscle fibers appeared and connected the adjacent circular muscles, whereby the midgut epithelium was divided into four rows of crypt-to-be areas. The crypt formation proceeded even in aposymbiotic nymphs, revealing the autonomous nature of the crypt development. We propose a mechanistic model of crypt formation wherein the spatial arrangement of muscle fibers and the proliferation of epithelial cells underpin the formation of crypts as midgut evaginations. IMPORTANCE Diverse organisms are associated with microbial mutualists, in which specialized host organs often develop for retaining the microbial partners. In light of the origin of evolutionary novelties, it is important to understand what mechanisms underpin the elaborate morphogenesis of such symbiotic organs, which must have been shaped through interactions with the microbial symbionts. Using the stinkbug Plautia stali as a model, we demonstrated that visceral muscular patterning and proliferation of intestinal epithelial cells during the early nymphal stages are involved in the formation of numerous symbiont-harboring crypts arranged in four rows in the posterior midgut to constitute the symbiotic organ. Strikingly, the crypt formation occurred normally even in symbiont-free nymphs, revealing that the crypt development proceeds autonomously. These findings suggest that the crypt formation is deeply implemented into the normal development of P. stali, which must reflect the considerably ancient evolutionary origin of the midgut symbiotic organ in stinkbugs.

ABSTRACT 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.

Many insects are intimately associated with microbial symbionts, which are passed to developing oocytes in the maternal body for ensuring vertical transmission to the next generation. Previous studies uncovered that some symbionts utilize preexisting host’s molecular and cellular machineries for targeting oocytes.

ABSTRACT 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.

ABSTRACT 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.

ABSTRACT Global warming impacts diverse organisms not only directly but also indirectly via other organisms with which they interact. Recently, the possibility that elevated temperatures resulting from global warming may substantially affect biodiversity through disrupting mutualistic/parasitic associations has been highlighted. Here we report an experimental demonstration that global warming can affect a pest insect via suppression of its obligate bacterial symbiont. The southern green stinkbug Nezara viridula depends on a specific gut bacterium for its normal growth and survival. When the insects were reared inside or outside a simulated warming incubator wherein temperature was controlled at 2.5°C higher than outside, the insects reared in the incubator exhibited severe fitness defects (i.e., retarded growth, reduced size, yellowish body color, etc.) and significant reduction of symbiont population, particularly in the midsummer season, whereas the insects reared outside did not. Rearing at 30°C or 32.5°C resulted in similar defective phenotypes of the insects, whereas no adult insects emerged at 35°C. Notably, experimental symbiont suppression by an antibiotic treatment also induced similar defective phenotypes of the insects, indicating that the host’s defective phenotypes are attributable not to the heat stress itself but to the suppression of the symbiont population induced by elevated temperature. These results strongly suggest that high temperature in the midsummer season negatively affects the insects not directly but indirectly via the heat-vulnerable obligate bacterial symbiont, which highlights the practical relevance of mutualism collapse in this warming world. IMPORTANCE Climate change is among the biggest environmental issues in the contemporary world, and its impact on the biodiversity and ecosystem is not only of scientific interest but also of practical concern for the general public. On the basis of our laboratory data obtained under strictly controlled environmental conditions and our simulated warming data obtained in seminatural settings (elevated 2.5°C above the normal temperature), we demonstrate here that Nezara viridula, the notorious stinkbug pest, suffers serious fitness defects in the summer season under the simulated warming conditions, wherein high temperature acts on the insect not directly but indirectly via suppression of its obligate gut bacterium. Our finding highlights that heat-susceptible symbionts can be the “Achilles’ heel” of symbiont-dependent organisms under climate change conditions.

ABSTRACT Endosymbiotic bacteria of the genus Wolbachia represent the most successful symbiotic bacteria in the terrestrial ecosystem. The success of Wolbachia has been ascribed to its remarkable phenotypic effects on host reproduction, such as cytoplasmic incompatibility, whereby maternally inherited bacteria can spread in their host populations at the expense of their host's fitness. Meanwhile, recent theoretical as well as empirical studies have unveiled that weak and/or conditional positive fitness effects may significantly facilitate invasion and spread of Wolbachia infections in host populations. Here, we report a previously unrecognized nutritional aspect, the provision of riboflavin (vitamin B2), that potentially underpins the Wolbachia-mediated fitness benefit to insect hosts. A comparative genomic survey for synthetic capability of B vitamins revealed that only the synthesis pathway for riboflavin is highly conserved among diverse insect-associated Wolbachia strains, while the synthesis pathways for other B vitamins were either incomplete or absent. Molecular phylogenetic and genomic analyses of riboflavin synthesis genes from diverse Wolbachia strains revealed that, in general, their phylogenetic relationships are concordant with Wolbachia's genomic phylogeny, suggesting that the riboflavin synthesis genes have been stably maintained in the course of Wolbachia evolution. In rearing experiments with bedbugs (Cimex lectularius) on blood meals in which B vitamin contents were manipulated, we demonstrated that Wolbachia's riboflavin provisioning significantly contributes to growth, survival, and reproduction of the insect host. These results provide a physiological basis upon which Wolbachia-mediated positive fitness consequences are manifested and shed new light on the ecological and evolutionary relevance of Wolbachia infections. IMPORTANCE Conventionally, Wolbachia has been regarded as a parasitic bacterial endosymbiont that manipulates the host insect's reproduction in a selfish manner, which tends to affect a host's fitness negatively. Meanwhile, some theories predict that, at the same time, Wolbachia can directly affect the host's fitness positively, which may potentially reconcile the negative effect and facilitate spread and stability of the symbiotic association. Here we demonstrate, by using comparative genomic and experimental approaches, that among synthetic pathways for B vitamins, the synthetic pathway for riboflavin (vitamin B2) is exceptionally conserved among diverse insect-associated Wolbachia strains, and Wolbachia's riboflavin provisioning certainly contributes to growth, survival, and reproduction in an insect. These findings uncover a nutritional mechanism of a Wolbachia-mediated fitness benefit, which provides empirical evidence highlighting a “Jekyll and Hyde” aspect of Wolbachia infection.