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Small Genome Symbiont Underlies Cuticle Hardness in Beetles

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Small Genome Symbiont Underlies Cuticle Hardness in Beetles Small genome symbiont underlies cuticle hardness PNAS PLUS in beetles Hisashi Anbutsua,b,1,2, Minoru Moriyamaa,1, Naruo Nikohc,1, Takahiro Hosokawaa,d, Ryo Futahashia, Masahiko Tanahashia, Xian-Ying Menga, Takashi Kuriwadae,f, Naoki Morig, Kenshiro Oshimah, Masahira Hattorih,i, Manabu Fujiej, Noriyuki Satohk, Taro Maedal, Shuji Shigenobul, Ryuichi Kogaa, and Takema Fukatsua,m,n,2 aBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan; bComputational Bio Big-Data Open Innovation Laboratory, National Institute of Advanced Industrial Science and Technology, Tokyo 169-8555, Japan; cDepartment of Liberal Arts, The Open University of Japan, Chiba 261-8586, Japan; dFaculty of Science, Kyushu University, Fukuoka 819-0395, Japan; eNational Agriculture and Food Research Organization, Kyushu Okinawa Agricultural Research Center, Okinawa 901-0336, Japan; fFaculty of Education, Kagoshima University, Kagoshima 890-0065, Japan; gDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan; hGraduate School of Frontier Sciences, University of Tokyo, Chiba 277-8561, Japan; iGraduate School of Advanced Science and Engineering, Waseda University, Tokyo 169-8555, Japan; jDNA Sequencing Section, Okinawa Institute of Science and Technology Graduate University, Okinawa 904-0495, Japan; kMarine Genomics Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa 904-0495, Japan; lNIBB Core Research Facilities, National Institute for Basic Biology, Okazaki 444-8585, Japan; mDepartment of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan; and nGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan Edited by Nancy A. Moran, University of Texas at Austin, Austin, TX, and approved August 28, 2017 (received for review July 19, 2017) Beetles, representing the majority of the insect species diversity, are symbiont transmission over evolutionary time (4, 6, 7). Some bac- characterized by thick and hard cuticle, which plays important roles terial symbionts of plant-sucking insects like cicadas, leafhoppers, for their environmental adaptation and underpins their inordinate spittlebugs, psyllids, and mealybugs belonging to the order Hemi- diversity and prosperity. Here, we report a bacterial endosymbiont ptera, which are associated with multiple endocellular bacterial extremely specialized for sustaining beetle’s cuticle formation. cosymbionts within the symbiotic organ called the bacteriome, have γ Many weevils are associated with a -proteobacterial endosymbiont experienced extreme genome reduction down to 0.2 Mb or smaller Nardonella lineage , whose evolutionary origin is estimated as older with 200 or less protein-coding genes (8–13), suggesting that met- EVOLUTION than 100 million years, but its functional aspect has been elusive. abolic complementation between the cosymbionts may have fur- Sequencing of Nardonella genomes from diverse weevils unveiled ther facilitated the reductive genome evolution entailing losses of drastic size reduction to 0.2 Mb, in which minimal complete gene – sets for bacterial replication, transcription, and translation were pre- otherwise essential genes in either of the cosymbionts (7, 14 17). In this study, we report another case of extremely reduced sent but almost all of the other metabolic pathway genes were missing. Notably, the only metabolic pathway retained in the symbiont genome in a different insect group through a different Nardonella genomes was the tyrosine synthesis pathway, identifying evolutionary trajectory. Beetles, comprising the insect order tyrosine provisioning as Nardonella’s sole biological role. Weevils are Coleoptera, represent the majority of the biodiversity described armored with hard cuticle, tyrosine is the principal precursor for cu- ticle formation, and experimental suppression of Nardonella resulted Significance in emergence of reddish and soft weevils with low tyrosine titer, confirming the importance of Nardonella-mediated tyrosine produc- Beetles are successful in the terrestrial ecosystem, which is tion for host’s cuticle formation and hardening. Notably, Nardonella’s attributable to, at least partly, their highly sclerotized exo- tyrosine synthesis pathway was incomplete, lacking the final step skeleton. Here, we report a bacterial symbiont extremely spe- transaminase gene. RNA sequencing identified host’s aminotransfer- cialized for underpinning the beetle’s hardness. The ancient ase genes up-regulated in the bacteriome. RNA interference target- endosymbiont Nardonella associated with weevils has an ex- ing the aminotransferase genes induced reddish and soft weevils tremely small genome devoted to a single biological function, with low tyrosine titer, verifying host’s final step regulation of the tyrosine provisioning, which is needed for insect’s cuticle for- tyrosine synthesis pathway. Our finding highlights an impressively mation and hardening. Notably, only the final step reaction of intimate and focused aspect of the host–symbiont metabolic integ- the tyrosine synthesis pathway is complemented by host- rity via streamlined evolution for a single biological function of encoded aminotransferases up-regulated in the bacteriome, ecological relevance. highlighting a highly focused aspect of the host–symbiont metabolic integrity. Both symbiont suppression by an antibiotic weevil | Nardonella | symbiont | genome | tyrosine and RNA interference of the host aminotransferases induce reddish and soft weevils, verifying the pivotal role of the ymbiotic associations with microorganisms are ubiquitously symbiosis for the beetle’s hardness. Sfound in a variety of insects, which are rated among the im- portant factors underpinning their adaptation, diversity, and pros- Author contributions: H.A., M.M., N.N., T.H., and T.F. designed research; H.A., N.N., T.H., – M.T., X.-Y.M., T.K., N.M., K.O., M.F., T.M., and R.K. performed research; M.H., N.S., and S.S. perity (1 3). Many bacterial symbionts are indispensable for contributed new reagents/analytic tools; H.A., M.M., N.N., T.H., R.F., M.T., K.O., M.F., and growth, survival, and reproduction of their insect hosts via, for T.M. analyzed data; and H.A., M.M., N.N., R.F., and T.F. wrote the paper. example, provisioning of essential nutrients like amino acids and The authors declare no conflict of interest. vitamins, where the host and the symbiont are integrated into an This article is a PNAS Direct Submission. almost inseparable biological entity (4, 5). In such obligate symbi- Freely available online through the PNAS open access option. otic associations, the symbiont genomes tend to exhibit conspicu- Data deposition: The sequences reported in this paper have been deposited in the DNA ous structural degeneration, massive gene losses, and drastic size Data Bank Japan Read Archive, www.ddbj.nig.ac.jp (accession nos. AP018159–AP018162, reduction, which are attributable to relaxed natural selection acting LC260175–LC260180, LC260491, and DRR095964–DRR095975). on many symbiont genes no longer necessary for the permanent 1H.A., M.M. and N.N. contributed equally to this work. intrahost lifestyle, and also to accumulation of deleterious muta- 2To whom correspondence may be addressed. Email: [email protected] or t-fukatsu@ tions driven by attenuated natural selection acting on the symbiont aist.go.jp. genomes due to strong population bottlenecks and restricted This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. horizontal gene acquisitions associated with continuous vertical 1073/pnas.1712857114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1712857114 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 – ’ A C lasting host symbiont coevolution, Nardonella s biological role Bacteriome has been poorly understood (26). Previous studies have identi- fied a number of weevil lineages in which Nardonella infections have been lost or replaced by different bacterial lineages, uncovering a strikingly dynamic aspect of the endosymbiotic evolution in the insect group (23, 24, 32–35). Here, we report genomic, transcriptomic, and functional 1 cm analyses of the Nardonella symbionts associated with diverse 5 mm weevils, which unveiled their extremely reduced genomes down B to as small as 0.2 Mb in the absence of any cosymbionts. The tiny Midgut genomes encode minimal but complete gene sets for bacterial 500 m Foregut replication, transcription, and translation, while lacking almost E all of the other metabolic pathway genes, which indicate Nar- donella’s near-complete dependence on host-derived metabolites D toward a minimal cellular entity through the ancient coevolu- tionary history. Notably, a set of metabolic genes is conspicu- ously retained in the Nardonella genomes, namely synthesis pathway genes for a specific amino acid, tyrosine. Weevils are Oocyte armored with hard cuticle, tyrosine is the principal precursor 1 mm needed for cuticle formation, and the Nardonella genome has G 1 m been streamlined for a single biological function, tyrosine pro- 200 µm visioning, for sustaining the weevil’s highly sclerotized exoskeleton, which elucidates the general importance of endosymbiont- F provisioned tyrosine for cuticle formation in weevils (36, 37), and potentially also in other beetles. Furthermore, we demon- strate that, reflecting the absence of transcriptional regulators in the tiny symbiont
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