Many insects are dependent on bacterial symbionts that provide essential nutrients (ex. aphid-Buchnera and tsetse-Wiglesworthia associations), wherein the symbionts are harbored in specific cells called bacteriocytes that constitute a symbiotic organ bacteriome. Facultative and parasitic bacterial symbionts like Wolbachia have been regarded as evolutionarily distinct from such obligate nutritional mutualists. However, we discovered that, in the bedbug Cimex lectularius, Wolbachia resides in a bacteriome and appears to be an obligate nutritional mutualist. Two bacterial symbionts, a Wolbachia strain and an unnamed γ-proteobacterium, were identified from different strains of the bedbug. The Wolbachia symbiont was detected from all of the insects examined whereas the γ-proteobacterium was found in a part of them. The Wolbachia symbiont was specifically localized in the bacteriomes and vertically transmitted via the somatic stem cell niche of germalia to oocytes, infecting the incipient symbiotic organ at an early stage of the embryogenesis. Elimination of the Wolbachia symbiont resulted in retarded growth and sterility of the host insect. These deficiencies were rescued by oral supplementation of B vitamins, confirming the essential nutritional role of the symbiont for the host. The estimated genome size of the Wolbachia symbiont was around 1.3 Mb, which was almost equivalent to the genome sizes of parasitic Wolbachia strains of other insects. These results indicate that bacteriocyte-associated nutritional mutualism can evolve from facultative and prevalent microbial associates like Wolbachia, highlighting a previously unknown aspect of the parasitism-mutualism evolutionary continuum.B vitamins | bacteriome | Cimex lectularius | nutritional mutualism

Background: Host-symbiont co-speciation and reductive genome evolution have been commonly observed among obligate endocellular insect symbionts, while such examples have rarely been identified among extracellular ones, the only case reported being from gut symbiotic bacteria of stinkbugs of the family Plataspidae. Considering that gut symbiotic communities are vulnerable to invasion of foreign microbes, gut symbiotic associations have been thought to be evolutionarily not stable. Stinkbugs of the family Acanthosomatidae harbor a bacterial symbiont in the midgut crypts, the lumen of which is completely sealed off from the midgut main tract, thereby retaining the symbiont in the isolated cryptic cavities. We investigated histological, ecological, phylogenetic, and genomic aspects of the unique gut symbiosis of the acanthosomatid stinkbugs.

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.

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.