Takema Fukatsu (Prime Senior Researcher and Leader of Symbiotic Evolution and Biological Functions Research Group), Takahiro Hosokawa (Co-researcher, post doctoral research fellow of the University of the Ryukyus) and Minoru Moriyama (Collaborative Researcher, JSPS fellow), Bioproduction Research Institute (BRI; Director: Tomohiro Tamura), the National Institute of Advanced Industrial Science and Technology (AIST; President: Ryoji Chubachi), in collaboration with the Open University of Japan and the University of Tokyo, determined the complete genome sequence of the symbiotic bacterium Wolbachia, which is essential for survival of the hygienic pest bedbug, and demonstrated that a cluster of vitamin B7 (biotin) synthesis genes on the Wolbachia genome, which had been acquired via lateral gene transfer from a distinct bacterium, supports survival of the host bedbug.

Wolbachia is known as a parasitic symbiotic bacterium ubiquitously associated with various insects. However, this study unveiled the evolutionary mechanism by which Wolbachia-bedbug nutritional mutualism has been established exceptionally. This finding exemplifies an evolutionary transition from parasitism to mutualism facilitated by lateral gene transfer, which provides insights into physiological and molecular bases underpinning survival of the hygienic pest. These results are also expected to contribute to the prevention and control of the bedbug.

Details of the results will be published online in an American scientific journal, “Proceedings of the National Academy of Sciences USA,” on June 30, 2014 (US Eastern Time).

Young nymphs of the bedbug immediately after blood-feeding The black coloration within the abdomen is feces of the previous blood-feeding in the intestine.

Blood-sucking insects, such as mosquitoes, fleas, lice, bedbugs, and tsetse flies, cause serious problems as hygienic pests, because they vector various infectious diseases as well as cause itching and discomfort due to blood-sucking. In developing countries, they are still a serious social burden that threatens the health and lives of many people. In advanced countries, on the other hand, many of the serious insect-borne infectious diseases have been almost eradicated, and classical blood-sucking hygienic pests, such as lice and bedbugs, were rarely observed up until approximately the 1960s. Recently, however, with the emergence of pesticide-resistant insects and the globalization of human and material transportations, these hygienic pests have again become a serious problem. In advanced countries such as North America, Australia and Japan, the recent outbreaks of the bedbug elicit strong discomfort and disgust for those who are bitten, which can be serious problems in lodging facilities, public facilities and residential complexes. Therefore, the bedbug is a risk factor that cannot be ignored by the tourism industry, the housing industry and public institutions.

The prevention and control measures for blood-sucking insects, which cause various problems as hygienic and/or nuisance pests, must be of low impact on human health and the environment. Therefore, in addition to conventional control methods like pesticide spraying, development of new pest control technologies is anticipated.

AIST previously elucidated important biological functions of symbiotic microorganisms associated with insects, such as plant adaptation, pest evolution and pesticide resistance. (AIST press releases on March 26, 2004, June 13, 2007, and April 24, 2012). AIST has also been working for understanding of the sophisticated biological interactions between host insects and symbiotic microorganisms, including reproductive manipulation, vertical transmission and maintenance of symbiosis (AIST research results on July 2, 2007 and May 28, 2012, and AIST press release on June 11, 2013). Research results highlighting the evolutionary importance of lateral gene transfers between symbiotic microorganisms and host insects deserve special mention (AIST press releases on October 29, 2002 and June 21, 2013). In addition, research on the relationship between blood-sucking insects and their symbiotic bacteria revealed that the parasitic symbiotic bacterium Wolbachia, which is ubiquitously associated with various insects, is a nutritional mutualist of the bedbug that supplies B vitamins deficient in the blood diet of the host (AIST press release on December 22, 2009).

In this study, based on the previous research and using genomic, experimental and physiological approaches, the researchers investigated why Wolbachia evolved from being parasitic to being mutualistic, as well as which B vitamin was especially important for the mutualistic relationship between them.

This research was supported by NARO Bio-oriented Technology Research Advancement Institution and Grants-in-Aid for Scientific Research by the Ministry of Education, Culture, Sports, Science and Technology.

DNA samples were prepared from the Wolbachia-harboring bacteriomes dissected from bedbugs, and the complete genome sequence of Wolbachia, a circular DNA of 1,250,060 base pairs, was determined (Fig. 1). The bedbug’s Wolbachia genome was, in general, similar in gene number and composition to the parasitic Wolbachia genomes associated with other insects like Drosophila melanogaster, Drosophila simulans, or Culex quinquefasciatus, unveiling paucity of unique characteristics in the genome of the Wolbachia strain associated with the bedbug (wCle). However, comparison of vitamin B synthesis genes showed that the wCle genome possessed all six genes for vitamin B7 (biotin) synthesis, whereas the other Wolbachia genomes were devoid of these genes (Fig. 2).

Figure 1 : Genome structure of the Wolbachia strain wCle associated with the bedbug

Figure 2 : Presence of synthetic pathways of B vitamins in various Wolbachia genomes “Complete”, “Incomplete” and “Absent” respectively indicate that all of, a part of, and none of the genes for the synthetic pathway is present. Only wCle possesses the complete synthetic pathway of vitamin B7 (marked with a red circle).

In the wCle genome, all six genes for vitamin B7 synthesis formed a compact operon, which was not observed in the genome of a related bacterium Anaplasma phagocytophila. On the other hand, this operon structure was shared with the genomes of various bacteria that are not closely related, such as Cardinium hertigii, a parasitic bacterial symbiont found in various insects; a plasmid of Rickettsia sp., a parasitic bacterial symbiont of ticks and insects; and Lawsonia intracellularis, a bacterial pathogen that causes swine enteritis (Fig. 3). Molecular phylogenetic analysis showed that all the six genes for vitamin B7 synthesis in the wCle genome were closely related to those in the bacteria described above. In particular, they were the closest to those in the genome of Cardinium (Fig. 4).

Figure 3 : Structure of vitamin B7 synthesis genes in the genomes of the Wolbachia strain wCle and other bacteria

Figure 4 : Molecular phylogeny inferred from the amino acid sequence of the vitamin B7 synthesis gene product BioH encoded by the wCle genome The bacterial group is described in each bracket and the statistical support (%) of each clade is indicated on the node. Similar results were obtained from the analysis of other vitamin B7 synthesis genes.

Together, this research shows that: (1) most Wolbachia genomes are deficient in genes for vitamin B7 synthesis; (2) only wCle possesses a complete set of these genes; (3) on the wCle genome, the genes for vitamin B7 synthesis form a compact operon; (4) a similar operon structure is found in the genome of Cardinium hertigii; (5) molecular phylogenetic analysis revealed that the vitamin B7 synthesis genes of wCle are most closely related to those of Cardinium; and (6) both Wolbachia and Cardinium hertigii are symbiotic bacteria widely associated with insects, and often the same host insect host both bacteria simultaneously. From these facts, it seems likely that the gene cluster for vitamin B7 synthesis on the wCle genome was acquired as a whole operon via lateral gene transfer from a strain of Cardinium, which was perhaps present together with wCle in an ancestor of the bedbug.

The researchers also examined the biological significance of the Wolbachia‘s vitamin B7 synthesis genes for the host bedbug.

Fourth-instar bedbug nymphs were fed with either normal rabbit blood or antibiotic-supplemented rabbit blood to eliminate Wolbachia infection. The vitamin B7 concentrations in the Wolbachia-cured insects were lower than those in the Wolbachia-infected insects (Fig. 5A), which confirmed that the vitamin B7 synthesis genes on the wCle genome function and supply vitamin B7 to the host bedbug.

When the Wolbachia-cured bedbug nymphs were fed with normal rabbit blood, the adult emergence rate was very low (Fig. 5B, the right bar), whereas addition of all B vitamins to the rabbit blood restored the adult emergence rate up to over 90 % (Fig. 5B, the left bar). In addition, when the Wolbachia-cured bedbug nymphs were fed with rabbit blood supplemented with all B vitamins except for B7, the emergence rate was approximately 60 % (Fig. 5B, the middle bar), which was lower than the emergence rate when all B vitamins were added. This nutritional physiological analysis showed that the ability of wCle to supply vitamin B7 plays an important role in supporting the growth and survival of the host bedbug.

Figure 5 : Synthesis and provisioning of vitamin B7 by the Wolbachia strain wCle in the host bedbug (A) Concentrations of vitamin B7 in Wolbachia-infected and Wolbachia-cured fourth-instar nymphs; (B) Adult emergence rates of Wolbachia-cured bedbug nymphs after feeding with rabbit blood supplemented with all B vitamins (the left bar), all B vitamins except for B7 (the middle bar), or no B vitamins (the right bar); The asterisks indicate statistically significant differences. The numbers in the bars are the numbers of insects analyzed.

The researchers plan to analyze the function of bedbug genes expressed in the Wolbachia-harboring bacteriomes, and investigate the molecular mechanisms of the host side involved in establishment and maintenance of symbiosis. Development of new drugs, such as inhibitors of enzymes that are produced by the vitamin B7 synthesis genes of wCle, or drugs that prevent the functions of the genes for maintenance of symbiosis, which are expected to be discovered in the future, may provide novel approaches to prevention and control of the bedbug, a recently re-emerging hygienic pest. The researchers are planning to promote researches in these directions.