Evolutionary origin of insect–<em>Wolbachia</em> nutritional mutualism

Naruo Nikoh

Takahiro Hosokawa

Minoru Moriyama

Kenshiro Oshima

Masahira Hattori

Takema Fukatsu

Supplementary Material

Supporting Information:

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^{Department of Liberal Arts, The Open University of Japan, Chiba 261-8586, Japan;}

^{Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan; and}

^{Center for Omics and Bioinformatics, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa 277-8561, Japan}

^{To whom correspondence should be addressed. E-mail:}pj.og.tsia@ustakuf-t.

Edited by Nancy A. Moran, University of Texas at Austin, Austin, TX, and approved June 3, 2014 (received for review May 20, 2014)

Author contributions: N.N., T.H., M.M., and T.F. designed research; N.N., T.H., M.M., and K.O. performed research; M.H. and T.F. contributed new reagents/analytic tools; N.N., T.H., M.M., K.O., and M.H. analyzed data; and T.F. wrote the paper.

^{N.N., T.H., and M.M. contributed equally to this work.}

Edited by Nancy A. Moran, University of Texas at Austin, Austin, TX, and approved June 3, 2014 (received for review May 20, 2014)

Freely available online through the PNAS open access option.

Significance

How sophisticated mutualism has arisen from less-intimate associations is of general interest. Here we address this evolutionary issue by looking into the bedbug. Wolbachia endosymbionts are generally regarded as facultative/parasitic bacterial associates for their insect hosts, but in the bedbug, exceptionally, Wolbachia supports the host’s growth and survival via provisioning of vitamins. In the bedbug’s Wolbachia genome, we identified a gene cluster encoding the complete synthetic pathway for biotin (vitamin B7), which is not present in other Wolbachia genomes and is presumably acquired via lateral transfer from a coinfecting endosymbiont. The Wolbachia-provisioned biotin contributes to the bedbug’s fitness significantly, uncovering an evolutionary transition from facultative symbiosis to obligate mutualism facilitated by lateral gene transfer in the endosymbiont lineage.

Significance

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.

Abstract

Symbiotic associations are ubiquitous in the biological world, in which obligate insect–bacterium endosymbiotic associations are among the most sophisticated forms wherein the host and the symbiont are integrated into a coherent biological entity and cannot survive without the partnership (1, 2). For example, in the aphid–Buchnera nutritional mutualism, the host depends on the symbiont for supply of essential amino acids that are needed for host’s protein synthesis but are scarce in the host’s plant sap diet (3). In the tsetse–Wigglesworthia nutritional mutualism, the symbiont provides B vitamins that are deficient in vertebrate blood the host exclusively feeds on (4). Through the intimate relationship over evolutionary time, these and other endosymbiont genomes have been reduced drastically, losing many genes needed for independent life and streamlined for specific biological roles to support their hosts (5, 6). Novel biological properties acquired through endosymbiosis have played substantial roles in adaptation, evolution, and diversification of insects and other organisms (1, 2). Although currently comprising elaborate symbiotic systems, such endosymbionts must have originally been derived from free-living ancestors. How highly specialized obligate endosymbionts have arisen from less specialized bacterial associates is of evolutionary interest.

Members of the genus Wolbachia are well known as facultative bacterial endosymbionts ubiquitously associated with diverse insects, generally conferring negative fitness consequences to their hosts and often causing hosts’ reproductive aberrations to enhance their own transmission in a selfish manner (7, 8). Recently, however, a Wolbachia strain associated with the bedbug Cimex lectularius, designated as wCle, was shown to be essential for normal growth and reproduction of the blood-sucking insect host via provisioning of B vitamins (9). Hence, it is expected that a transition from facultative association to obligate mutualism may have occurred in an ancestor of wCle. What evolutionary processes and mechanisms are involved in the emergence of the insect–Wolbachia nutritional mutualism?

In this study, we determined the complete genome of wCle, which was similar in size and composition to the genomes of facultative Wolbachia endosymbionts associated with other insects, except for the presence of an operon encoding biotin synthesis pathway that was presumably acquired via lateral gene transfer from an unrelated bacterium. Using wCle-infected and wCle-cured bedbug strains under the same genetic background, we experimentally demonstrated that wCle is capable of synthesizing biotin and wCle-provisioned biotin significantly contributes to the host fitness, thereby uncovering a genomic basis of the insect–Wolbachia nutritional mutualism. Through comprehensive survey of Wolbachia genomic data, we discuss evolutionary hypotheses as to how and when the biotin operon was acquired by wCle in the course of insect–Wolbachia coevolution.

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Acknowledgments

This study was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-Oriented Industry and by Grants-in-Aid for Scientific Research on Innovative Areas (Grants 22128001 and 22128007) from Japan Society of the Promotion of Science.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences reported in this paper have been deposited in the DNA Data Bank of Japan database, www.ddbj.nig.ac.jp/index-e.html (accession nos. {"type":"entrez-nucleotide","attrs":{"text":"AP013028","term_id":"651086863"}}AP013028 and {"type":"entrez-nucleotide","attrs":{"text":"AB934986","term_id":"656322931"}}AB934986–{"type":"entrez-nucleotide","attrs":{"text":"AB934989","term_id":"656322963"}}AB934989).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1409284111/-/DCSupplemental.

Footnotes

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