Evolutionary origin of insect-Wolbachia nutritional mutualism

Abstract

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-vitamin-manipulated blood meals via an artificial feeding system, demonstrated that wCle certainly synthesizes biotin, and the wCle-provisioned 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.

Fig. 1.

Circular view of the wCle genome. On the GC skew circle, red and blue indicate GC rich and poor, respectively. On the CDS circle, colors indicate functional categories as shown at the bottom.

Fig. 2.

Biosynthetic pathways for B vitamins in the Wolbachia genomes. (A) Biosynthetic pathways for B vitamins in the wCle genome. Solid arrows indicate genes present in the wCle genome, whereas dotted arrows show genes absent in the wCle genome. Genes in gray color and smaller font are missing genes, whereas genes with prefix ψ are pseudogenes. Red rectangles highlight genes presumably acquired via lateral gene transfer from unrelated bacteria. (B) Presence/absence of biosynthetic pathways for B vitamins in Wolbachia strains wCle of bedbug C. lectularius, wMel of fruit fly D. melanogaster, wRi of fruit fly D. simulans, wPip of mosquito C. quinquefasciatus, wBm of filarial nematode B. malayi, and wOo of filarial nematode O. ochengi, and also obligate endosymbionts of blood-feeding insects Wigglesworthia glossinidia of tsetse fly G. brevipalpis and Riesia pediculicola of human louse Pediculus humanus. +, pathway is complete; ±, pathway is incomplete; –, pathway is absent; *, pathway contains pseudogene(s). For more details, see Table S3.

Fig. 3.

Operon structure of vitamin B synthesis genes on the genomes of wCle, allied alphaproteobacteria, and other bacteria. (A) Biotin synthesis genes. (B) Thiamine synthesis genes. Filled arrows and hatched arrows indicate intact genes and pseudogenes, respectively.

Fig. 4.

wCle-mediated provisioning of B vitamins to the host bedbug. Quantification of biotin (A), riboflavin (B), pyridoxine (C), and thiamine (D) in whole-body extract of wCle-infected (+wCle) and wCle-cured (–wCle) fourth instar nymphs. Effects of omission of biotin (E) and thiamine (F) from the B vitamin-supplemented rabbit blood meal on adult emergence rates of wCle-cured insects. Means and SDs are shown with sample sizes. Asterisks indicate statistically significant differences (likelihood ratio test: *P < 0.01; **P < 0.001; ***P < 0.0001; NS, no significant difference).

Fig. 5.

Phylogenetic relationship of Wolbachia strains on the basis of 52 ribosomal protein sequences. Unambiguously aligned 7,007 amino acid sites are concatenated and subjected to the analysis. Bootstrap probabilities of maximum likelihood analysis and posterior probabilities of Bayesian analysis are indicated at the nodes.

Fig. 6.

Location of biotin operon on the genomes of wCle and wOo. (A) Structure of biotin operon and flanking regions on the genomes of wCle and wOo. Protein-coding sequences are shown in green, transposases are in black, and hypothetical genes are in gray. (B) Structural comparison between the genomes of wCle and wOo. Locations of biotin operon are highlighted by red arrows. Corresponding genes are connected between the wCle genome and the wOo genomes by lines, whose colors are arranged in gradient on the wCle genome.