Obligate insect-bacterium nutritional mutualism is among the most sophisticated forms of symbiosis, wherein the host and the symbiont are integrated into a coherent biological entity and unable to survive without the partnership. Originally, however, such obligate symbiotic bacteria must have been derived from free-living bacteria. How highly specialized obligate mutualisms have arisen from less specialized associations is of interest. Here we address this evolutionary issue by focusing on an exceptional insect-Wolbachia nutritional mutualism. Although Wolbachia endosymbionts are ubiquitously found in diverse insects and generally regarded as facultative/parasitic associates for their insect hosts, a Wolbachia strain associated with the bedbug Cimex lectularius, designated as wCle, was shown to be essential for host's growth and reproduction via provisioning of B vitamins. We determined the 1,250,060-bp genome of wCle, which was generally similar to the genomes of insect-associated facultative Wolbachia strains, except for the presence of an operon encoding the complete biotin synthetic pathway that was acquired via lateral gene transfer presumably from a coinfecting endosymbiont Cardinium or Rickettsia. Nutritional and physiological experiments, in which wCle-infected and wCle-cured bedbugs of the same genetic background were fed on B-vitaminmanipulated blood meals via an artificial feeding system, demonstrated that wCle certainly synthesizes biotin, and the wCleprovisioned biotin significantly contributes to the host fitness. These findings strongly suggest that acquisition of a single gene cluster consisting of biotin synthesis genes underlies the bedbug-Wolbachia nutritional mutualism, uncovering an evolutionary transition from facultative symbiosis to obligate mutualism facilitated by lateral gene transfer in an endosymbiont lineage.

SignificanceCicadas are dependent on the essential bacterial symbionts Sulcia and Hodgkinia. The symbiont genomes are extremely streamlined for provisioning of essential amino acids and other nutrients. In some cicada lineages, Hodgkinia genomes are fragmented into numerous minicircles, which may represent a critical stage of genomic erosion close to collapse. What would happen subsequently? Our survey of the Japanese cicada diversity revealed that while Sulcia is conserved among all species, the majority of them have lost Hodgkinia and instead harbor yeast-like fungal associates. The fungal symbionts are phylogenetically intermingled with cicada-parasitizing Ophiocordyceps fungi, indicating recurrent symbiont replacements by entomopathogens in cicadas and providing insights into the mechanisms underlying the parasitism-symbiosis evolutionary continuum, compensation of symbiont genome erosion, and diversification of host-symbiont associations.

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.

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.

Maternal investment for offspring's growth and survival is widespread among diverse organisms. Vertical symbiont transmission via maternal passage is also pivotal for offspring's growth and survival in many organisms. Hence, it is expected that vertical symbiont transmission may coevolve with various organismal traits concerning maternal investment in offspring. Here we report a novel phenotypic syndrome entailing morphological, histological, behavioral, and ecological specializations for maternal investment and vertical symbiont transmission in stinkbugs of the family Urostylididae. Adult females develop huge ovaries exaggerated for polysaccharide excretion, possess novel ovipositor-associated organs for vertical transmission of a bacterial symbiont ("Candidatus Tachikawaea gelatinosa"), and lay eggs covered with voluminous symbiont-supplemented jelly. Newborns hatch in midwinter, feed solely on the jelly, acquire the symbiont, and grow during winter. In spring, the insects start feeding on plant sap, wherein the symbiont localizes to a specialized midgut region and supplies essential amino acids deficient in the host's diet. The reduced symbiont genome and host-symbiont cospeciation indicate their obligate association over evolutionary time. Experimental deprivation of the jelly results in nymphal mortality, whereas restoration of the jelly leads to recovered nymphal growth, confirming that the jelly supports nymphal growth in winter. Chemical analyses demonstrate that the galactan-based jelly contains a sufficient quantity of amino acids to sustain nymphal growth to the third instar. The versatile biological roles of the symbiont-containing egg-covering jelly highlight intricate evolutionary interactions between maternal resource investment and vertical symbiont transmission, which are commonly important for offspring's growth, survival, and ecological adaptation.