Wolbachia as a bacteriocyte-associated nutritional mutualist

Abstract

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 gamma-proteobacterium, were identified from different strains of the bedbug. The Wolbachia symbiont was detected from all of the insects examined whereas the gamma-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.

Fig. 1.

The common bedbug Cimex lectularius and localization of its symbiotic bacteria. (A) A mating pair of adult bedbugs. (B and C) Gonad-associated bacteriomes in adult male (B) and female (C) highlighted by orange arrows, from Davis (10). (D and E) FISH detection of Wolbachia in adult male (D) and female (E); orange arrows and arrowheads indicate bacteriome infections and ovarial infections, respectively. (F and G) FISH detection of Wolbachia and γ-proteobacteria in bacteriomes of monosymbiotic bedbug strain JESC (F) and disymbiotic bedbug strain TUA (G). (H and I) Transmission electron microscopy of rod-shaped Wolbachia cells in a bacteriocyte of JESC insect (H) and a tubular γ-proteobacterial cell in addition to Wolbachia cells in a bacteriocyte of TUA insect (I). (J and K) Infection patterns of Wolbachia and γ-proteobacteria in germalia of JESC female (J) and TUA female (K); white arrowheads and arrows indicate Wolbachia infections in somatic stem cell niche and nutritive cord, respectively. (L and M) Localization of Wolbachia and γ-proteobacteria at posterior pole of developing oocytes in JESC female (L) and TUA female (M). (N) Localization of Wolbachia in primordial bacteriome (orange arrow) in developing embryo. Abbreviations: Mn, mandible; Mx, maxilla; Lb, labium; T1–3, first-third thoracic appendages. In D–G and J–N, red, green, and blue signals indicate Wolbachia, γ-proteobacteria and insect nuclei, respectively.

Fig. 2.

Phylogenetic placement of symbiotic bacteria from C. lectularius and allied bugs. (A) A 16S rRNA gene phylogeny of Wolbachia from the bedbugs (1,441 aligned nucleotide sites). (B) A 16S rRNA gene phylogeny of γ-proteobacteria from the bedbugs (1,089 sites). Bayesian (BA) phylogenies are shown. Host insect names are in italic, accession numbers in brackets, and Wolbachia supergroups A–F on the right side are shown in A. Posterior probabilities for BA analyses and bootstrap probabilities for maximum parsimony (MP) and maximum likelihood (ML) analyses greater than 50% are indicated at the nodes in the order of BA/MP/ML.

Fig. 3.

Quantification of Wolbachia density in different organs of C. lectularius. (A and B) Infection densities in male insects; and (C and D) infection densities in female insects in terms of Wolbachia wsp gene copies per insect elongation factor 1α gene copy. Filled and open dots indicate insects of the monosymbiotic strain JESC and the disymbiotic strain TUA, respectively.

Fig. 4.

Biological role of Wolbachia for C. lectularius demonstrated by antibiotic treatment and nutritional supplementation using an artificial feeding system. (A and B) An illustration (A) and a photo (B) of the artificial feeding system for bedbug. (C) FISH of an antibiotic-treated adult insect, wherein Wolbachia signal in the bacteriome disappeared (dotted circle). (D and E) Effects of antibiotic treatment and vitamin B supplementation on adult insects: (D) number of deposited eggs; and (E) percentage of developing eggs. (F) Eggs 5 days after oviposition: (Upper) eggs laid by control insects, wherein arrows indicate red eyespots; (Lower) eggs laid by antibiotic-treated insects, which are darkened, deformed, and dead. (G and H) Effects of antibiotic treatment and vitamin B supplementation on nymphal growth and development: (G) percentage of adult emergence; and (H) nymphal period.

Fig. 5.

Genome size of Wolbachia from the bacteriome of C. lectularius estimated by pulsed field gel electrophoresis. (A) I-CeuI digestion yielded a single band of 1.3 Mb. (B) AscI digestion produced four bands, which were summed up to 1.3 Mb. Lanes S and M indicate samples and DNA size markers, respectively.