Small symbiont underlies cuticle hardness in

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 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 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 -sucking 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 ptera, which are associated with multiple endocellular bacterial extremely specialized for sustaining ’s cuticle formation. cosymbionts within the symbiotic organ called the bacteriome, have γ Many 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 (8–13), suggesting that met- than 100 million years, but its functional aspect has been elusive. abolic complementation between the cosymbionts may have fur- Sequencing of Nardonella from diverse weevils unveiled ther facilitated the reductive genome evolution entailing losses of drastic size reduction to 0.2 Mb, in which minimal complete – sets for bacterial replication, , and 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 | 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.

E8382–E8391 | PNAS | Published online September 18, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1712857114 Downloaded by guest on October 2, 2021 – ’ PNAS PLUS 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 , 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 genome, Nardonella’s tyrosine provisioning is controlled by host’s final step regulation of the synthesis 20 µm pathway.

Fig. 1. P. infernalis and its bacterial symbiont Nardonella.(A)Anadult.(B)A Results and Discussion finalinstarlarva.(C) A dissected larval gut with bacteriomes. Petal-like bac- Weevil-Nardonella Endosymbiotic System. In the black hard weevil teriome lobes, each consisting of many , are located around the Pachyrhynchus infernalis (Fig. 1 A and B), Nardonella exhibited foregut-midgut junction. (D) Isolated radial bacteriome lobes. (E)Detectionof characteristic and specific endocellular localizations. In larvae, Nardonella in the ovarioles dissected from an adult female. Arrows highlight localization of Nardonella at the ovariole tips. (F) Visualization of Nardonella the symbiotic were localized within a conspicuous sym- in the cytoplasm of larval bacteriocytes. (G) A transmission electron micro- biotic organ, called the bacteriome, consisting of petal-like lobes scopic image of Nardonella cells in the larval . In C–F, Nardonella surrounding the foregut-midgut junction (Fig. 1 C and D). In 16S rRNA and host nuclear DNA are visualized in red and blue, respectively. adult females, the larval bacteriome was not found, and the symbiotic bacteria were concentrated at a tip region of the ovarioles and also in developing oocytes (Fig. 1E). In the bac- (18–20), of which weevils comprise the most species-rich group, teriomes, the symbiotic bacteria were located within the cyto- the superfamily Curculionoidea, with some 70,000 described plasm of numerous bacteriocytes (Fig. 1F) as slender bacterial species in the world (19, 21, 22). Many, if not all, weevils are cells (Fig. 1G). Similar localization patterns of Nardonella were associated with an ancient γ-proteobacterial endosymbiont observed in the red palm weevil Rhynchopholus ferrugineus, the lineage, Nardonella, in the bacteriome, whose evolutionary origin giant weevil gigas, the West Indian sweet poptato is estimated as older than 100 My (23–31). Despite the long- weevil postfasciatus, and other weevil species, although

Rhynchophorus ferrugineus Pachyrhynchus infernalis ABCD Fig. 2. Weevils and their Nardonella genomes ex- amined in this study. (A) Nardonella Rfe of the red palm weevil R. ferrugineus (: Dryoph- thorinae). (B) Nardonella Sgi of the giant weevil S. gigas (Curculionidae: ). (C) Nardo- nella Epo of the West Indian sweet potato weevil 1 cm 1 cm 2 mm 5 mm E. postfasciatus (Curculionidae: ). (D) Nardonella Pin of the black hard weevil P. infer- nalis (Curculionidae: ). Nardonella Sgi has a EVOLUTION 2,117-bp encoding only two genes, mscS and Nardonella Nardonella Nardonella Nardonella ibpA-like. mscS, which is retained in all of the Nar- Rfe Sgi Epo Pin donella genomes, encodes a channel for osmoregu- 200,316 bp 230,546 bp 219,841 bp 226,299 bp lation that exports water and ions upon hypotonic conditions (77). ibpA, which is found in Nardonella Epo but absent in Nardonella Rfe and Nardonella Pin, encodes a small chaperone Hsp20 (78). Note that pNSgi1 2,117bp the synteny of mscS and ibpA on the plasmid of Translation Transcription Replication, recombination and repair RNA processing wall/membrane/envelope Intracellular trafficking, secretion Signal transduction Amino acid Coenzyme Nucleotide Carbohydrate Lipid Energy production Inorganic ion Cell cycle control, Posttranslational modification Defense Nardonella Sgi is conserved on the of General function prediction only Function unknown Unassigned rRNA,tRNA Nardonella Epo.

Anbutsu et al. PNAS | Published online September 18, 2017 | E8383 Downloaded by guest on October 2, 2021 size and shape of the bacteriomes as well as those of the bacterial dissected bacteriomes was also drastically suppressed in the larvae cells were considerably different between the species (25, 29) (SI reared at 30 °C (about 1/150 in terms of 15N-labeled tyrosine titers) Appendix, Fig. S1). in comparison with the control larvae reared at 25 °C (Fig. 4H). These results indicate that the Nardonella symbiont is certainly Extremely Reduced Nardonella Genomes. From these four weevil involved in tyrosine synthesis and that the Nardonella-harboring species, the larval bacteriomes were dissected, extracted for DNA preparation, and subjected to library construction, se- bacteriome may function as a tyrosine-producing organ. quencing, and assembly, by which we obtained the complete Nardonella genome sequences (Fig. 2 and SI Appendix, Table S1). The main circular genomes of the Nardonella symbionts Escherichia coli K12 Tremblaya PCIT Hodgkinia Nardonella Carsonella DC Buchnera APS Nardonella Nardonella Nardonella

were strikingly small, ranging from 0.20 Mb to 0.23 Mb in size, Sulcia GWSS with only 196–226 putative protein-coding ORFs. In the Nar- donella Sgi genome, a 2.1-kb plasmid encoding only two genes Dsem Pin Rfe Sgi was identified. Although extremely reduced, the Nardonella ge- Epo nomes retained a minimal but complete set of genes needed for replication, transcription, and translation: three or more ribo- somal RNAs, 27 or more transfer RNAs, and all 20 aminoacyl tRNA synthetases (SI Appendix, Tables S1–S3). Besides some minor bacterial reads and contigs derived from contaminants, no other predominant bacterial contigs were identified, confirming the histological observations that these weevils harbor no major bacterial symbionts other than Nardonella in the bacteriome (25, 29) (Fig. 1 and SI Appendix, Fig. S1).

Extreme Metabolic Capacity of Nardonella Specialized for Tyrosine Synthesis. Reflecting their extreme genome reduction, the gen- eral metabolic capacity of the Nardonella symbionts was strin- gently limited: no synthesis genes for amino acids (aside from the exception involving tyrosine described later), no synthesis genes for B vitamins and cofactors (but a few genes for Fe-S cluster assembly), no functional nucleotide synthesis pathways, no TCA cycle genes, almost no pentose-phosphate pathway genes, no functional glycolysis pathway, and substantial absence of genes for energy metabolism, sugar transport, lipid synthesis, and fla- gellar apparatus (Fig. 3 and SI Appendix, Table S2). The only pathways that seemed to be complete were the synthesis pathway for tyrosine and the synthesis pathway for peptidoglycan (Fig. 3). Considering that the peptidoglycan synthesis is needed for cell wall construction to ensure the bacterial cell integrity, the Nar- donella genome seems to be specialized toward production of a single amino acid, tyrosine.

In Vitro Assay of Nardonella’s Tyrosine Synthesis. Hence, we con- structed an in vitro assay system for testing whether Nardonella is capable of synthesizing tyrosine and other amino acids (Fig. 4 A–C). When bacteriomes dissected from mature larvae of P. infernalis were individually incubated with a culture medium containing 15N-labeled glutamine as a source of the amino group for newly synthesized amino acids, we observed that (i) essential amino acids were scarcely 15N-labeled, reflecting the absence of the synthesis pathways in both the host and the symbiont (Fig. 4D); (ii) some nonessential amino acids were 15N-labeled, likely owing to the synthesis pathways operating in the host bacteriocytes (Fig. 4E); and (iii) among semiessential amino acids, tyrosine exhibited a remarkably high proportion (about 50%) of 15N-labeling (Fig. 4F). These results suggest that the Nardonella symbiont in the bacteriome is preferentially synthesizing a substantial quantity of tyrosine. Fig. 3. Comparison of the metabolic gene repertoire between Nardonella genomes and other extremely reduced symbiont genomes. The minimal High Temperature Eradication of Nardonella Infection in P. infernalis. As number of genes for a metabolic pathway is shown in each of the brackets. previously described in a variety of insect-microbe symbiotic sys- Each color indicates the ratio of retained genes to the minimal gene set for a tems (38, 39), rearing of P. infernalis larvae at an elevated tem- metabolic pathway: green for 100%, orange for 75–99%, yellow for 50–74%, – – ’ perature, 30 °C, resulted in drastically suppressed symbiont titers in blue for 25 49%, and gray for 0 24%. Nardonella s tyrosine synthesis pathway the bacteriomes (about 1/530 in terms of symbiont groEL gene genes are highlighted in red. In the Nardonella Epo genome, aroE gene, lo- cated between def and sufE genes in the other Nardonella genomes, is lost copies) in comparison with control larvae reared at 25 °C (Fig. 4G). and replaced by a 168-bp spacer sequence, which may be relevant to the fact Considering that quantitative PCR also detects DNA molecules that its host E. postfasciatus is smaller in size with thinner cuticle in comparison derived from dead bacterial cells, the level of symbiont suppression with the other large and hard weevil species R. ferrugineus, S. gigas,and may actually be more severe. Accordingly, tyrosine synthesis by the P. infernalis.

E8384 | www.pnas.org/cgi/doi/10.1073/pnas.1712857114 Anbutsu et al. Downloaded by guest on October 2, 2021 Antibiotic Suppression of Nardonella Infection in P. infernalis. While PNAS PLUS A B the larvae of P. infernalis reared at 25 °C normally became adults, none of the larvae reared at 30 °C attained adulthood, suggesting that Nardonella is essential for pupal/adult survival or the high temperature condition is detrimental to pupal/adult survival. Hence, we attempted to develop a rearing condition for 76 mm P. infernalis under which the Nardonella infection is not eradi- cated but suppressed significantly without high temperature. Finally, in addition to the standard rearing system on sweet 1 mm potatoes (SI Appendix, Fig. S2 A–C), we established an agar- based artificial diet rearing system for P. infernalis, to which Chambered glass slide Medium drop Bacteriome antibiotics can be conveniently supplemented, to generate and C attached on the lid + 15N-Gln investigate normal and symbiont-suppressed insects (SI Appen- dix, Fig. S2 D–F). On the control artificial diet, the larvae grew normally with their bacteriomes full of Nardonella cells (Fig. 5 A, C,andE and SI Appendix,TableS4). On the antibiotic- supplemented artificial diet containing 0.003% rifampicin, the larvae also grew, but their bacteriomes were depleted of Nar- donella cells (Fig. 5 B, D, and F and SI Appendix, Table S4). In Petri dish Wet filter paper 3 mature larvae, the Nardonella titers in the antibiotic-treated in- D sects were estimated by quantitative PCR to be around 10 times Labeled lower than those in the control insects (Fig. 5 G and J). In pupae Unlabeled 2 and adults, notably, the Nardonella titers in the antibiotic-treated n = 17 each insects were still lower but recovering toward the levels of the pmol 2 control insects (Fig. 5 H, I, K, and L). Plausibly, intake of the 1 antibiotic stopped upon the cessation of larval feeding before x 10 pupation, and remaining Nardonella cells proliferated and re- stored the infection density during the prepupal and pupal pe- 0 * riods. The life tables of P. infernalis reared on the control diet Lys His Thr Val Leu Ile Phe Trp Met and the antibiotic-supplemented diet revealed that the antibiotic treatment resulted in (i) lower mortality during the larval period E 20 F 6 (P < 0.05), (ii) higher mortality during the prepupal period (P < 16 5 0.05), (iii) higher mortality of pupae and newly emerged adults 4 (although statistically not significant), and (iv) higher overall 12 lifetime mortality (SI Appendix, Tables S4 and S5). These pat- pmol pmol 2 2 3 terns were generally concordant with the results of fitness mea- 8 surements: The antibiotic treatment resulted in greater larval

x 10 x 10 2 < < 4 body weight (P 0.05) and prolonged pupal period (P 0.05) 1 (SI Appendix, Fig. S3). The superior performance of the 0 0 * antibiotic-treated larvae was likely relevant to the condition of Pro Ala Glu Asn Asp Gly Ser Arg Tyr Cys the artificial diets. While the antibiotic-supplemented diet plates were usually clean throughout the rearing period (SI Appendix, GH*** Fig. S2D), the control diet plates suffered microbial contami- 10 3 *** nations, in particular bacterial ones, more frequently than the 9 antibiotic-supplemented diet plates, which entailed change of 2 color and smell of the diet (SI Appendix, Fig. S2E), and larvae on 8 such contaminated diet plates tended to exhibit retarded growth 1 or death. Meanwhile, the inferior performance of the antibiotic- 7 treated prepupae, pupae, and adults seemed to be due to the groEL copies) ( 0 antibiotic-induced symbiont depletion. It should be noted that 10 Symbiont titer 6 [pmol]) Tyr (labeled almost all adult insects that successfully emerged were Nardo- 10 Tyrosine synthesis Tyrosine

Log nella-infected (Fig. 5 I and L) and a few Nardonella-deficient 5 17 9 -1 17 9 o o Log o o adult insects were frail and died early as described later, favoring 25 C30C 25 C30C the notion that Nardonella is essential for pupal/adult survival. ’ – Fig. 4. In vitro assay of Nardonella s tyrosine synthesis in P. infernalis.(A C) Nardonella The experimental system for hanging drop tissue culture. (A)Anexternalview Effects of Suppression on Adult Color and Cuticle Formation P. infernalis of the culture system. (B) An isolated larval bacteriome of P. infernalis in in . The majority of the adult insects that emerged hanging drop medium. (C) A schematic view of the culture system. (D–F) from the control diet were morphologically normal with black Quantification of essential amino acids (D), nonessential amino acids (E), and and rigid elytra (Fig. 6A), whereas the Nardonella-suppressed EVOLUTION semiessential amino acids (F) released from an isolated larval bacteriome of P. adult insects that emerged from the antibiotic-supplemented diet infernalis in the hanging drop medium supplemented with 15N-glutamine. frequently suffered morphological abnormalities with reddish, 15 Columns and bars show means and SDs, in which N-labeled and unlabeled crumpled, and/or deformable elytra (Fig. 6 B–D and SI Appendix, amino acid fractions are depicted by dark green and light green, respectively. Fig. S4). Elytra color analysis (Fig. 6 E–G and SI Appendix, Fig. Asterisks indicate undetected amino acids, methionine, and cysteine. (G and H) Nardonella titers (G) and tyrosine synthesis activities (H) in the bacteriomes dissected from control larvae reared at 25 °C and heat-treated larvae reared at 30 °C. Asterisks indicate statistically significant differences [likelihood-ratio test 75th percentiles (box edges), the range (whiskers) and outliers, which are of a generalized linear model (GLM) assuming a Gamma error distribution; larger or smaller than 1.5 times the interquartile range from the box edge ***P < 0.001]. Tukey box plots indicate the median (bold line), the 25th and (dots), with sample sizes at the bottom.

Anbutsu et al. PNAS | Published online September 18, 2017 | E8385 Downloaded by guest on October 2, 2021 Control larvae Antibiotic-treated larvae Physical property analysis of the elytra using a viscoelastometer A B (Fig. 6 H and I) revealed that, unexpectedly, the elastic modulus, a qualitative index of physical hardness, was not significantly different between the control insects and the antibiotic-treated insects irrespective of adult aging (Fig. 6K). However, elytra thickness increased as the insect age proceeded, and the control adult insects developed thicker elytra than the antibiotic-treated adult insects (Fig. 6L). These results indicate that the Nardo- 5 mm 5 mm nella-suppressed adult insects exhibit reddish and thinner CD5 m elytra in contrast to black and thicker elytra of the control adult insects.

Effects on Nardonella Suppression on Levels of Tyrosine and L-DOPA During Pupal and Adult Development of P. infernalis. In the process of cuticular pigmentation and hardening in beetles and other insects, 5 m tyrosine and its derivative L-dopa (L-DOPA) play essential roles as principal substrates for initiating a series of chemical reactions EF5 m 5 m toward cuticle tanning, polymerization, and melanization (40, 41) (SI Appendix,Fig.S6). Quantification of the levels of tyrosine and L-DOPA in hemolymph of mature larvae, pupae, and newly emerged adults of P. infernalis revealed that (i) the levels of ty- rosine were consistently 10–100 times higher than the levels of L-DOPA irrespective of the developmental stages and the symbiotic conditions (Fig. 7 A–F), probably reflecting the fact that L-DOPA Mature larvae Pupae New adults is a transient intermediate in the cuticle formation process (SI Appendix,Fig.S6); (ii) both the levels of tyrosine and L-DOPA GH9 ns I *** * were the highest at the pupal stage (Fig. 7 A–F), suggesting active 8 8 mobilization of these metabolites at the developmental stage of 8

titer active adult body formation; (iii) at the mature larval stage, the 6 levels of tyrosine were significantly higher in the control insects 7 6 than in the antibiotic-treated insects (Fig. 7A), suggesting that the

( groEL copies) normal symbiotic larvae are able to synthesize and accumulate 10 6 Nardonella 4 4 more tyrosine than the symbiosis-deficient larvae; and (iv)atthe Log pupal stage, the levels of L-DOPA were significantly higher in the 5 10 11 20 20 8 13 control insects than in the antibiotic-treated insects (Fig. 7E), suggesting the possibility that the normal symbiotic pupae are JL3 K *** ns ns recruiting L-DOPA and constructing adult cuticles more actively

copy) 2 than the symbiosis-deficient pupae. Moreover, by carefully moni- 2 2 toring plastic sandboxes in which pupae were observable in pupal chambers adjacent to a transparent wall (SI Appendix,Fig.S2G–I),

density 1 0 0 we collected teneral adult insects newly eclosed within 24 h (Fig. 7G) and measured the levels of tyrosine and L-DOPA in their 0 -2 hemolymph (Fig. 7 H and I). As the cuticle pigmentation in the -2 teneral adult insects proceeded, the tyrosine levels tended to de- Nardonella

( groEL copies/ EF1 -1 10 11 -4 20 20 8 13 crease in both the control adult insects and the antibiotic-treated 10 Control Antibiotic Control Antibiotic Control Antibiotic adult insects (Fig. 7H). Log Nardonella Fig. 5. Antibiotic suppression of Nardonella infection in P. infernalis.(A and as an Ancient Symbiont Specialized for Provisioning B) Dissected bacteriomes. (C and D) Light microscopic images of semi- Tyrosine That Underpins Hard Cuticle of Weevils. All these results ultrathin sections of the larval bacteriome stained with toluidine blue. (E and taken together, we conclude that the ancient endosymbiont F) Transmission electron microscopic images of the larval bacteriocytes. (A, C, Nardonella has experienced an extremely streamlined genome and E) Larvae reared on the control artificial diet. (B, D, and F) Larvae reared evolution toward a specific biological function, namely tyrosine on the artificial diet containing 0.003% rifampicin. (G–I) Nardonella titers (in provisioning, which underpins the formation of the hard cuticle of terms of bacterial groEL gene copies per insect) in mature larvae (G), pupae weevils. Weevils constitute the most species-rich group – (H), and newly emerged adults (I). (J L) Nardonella densities (in terms of with some 70,000 described species, including many highly bacterial groEL gene copies per host Elongation Factor 1α [EF1α] gene copy) – sclerotized lineages such as Pachyrhynchus spp., Eupholus spp., in mature larvae (J), pupae (K), and newly emerged adults (L). In G L,as- – terisks indicate statistically significant differences (likelihood-ratio test of Rhynchophorus spp., and spp. (19, 21, 22, 42 44). GLM assuming a Gamma error distribution; *P < 0.05; ***P < 0.001; ns, no The hard cuticle of these and other weevils has been shown to significant difference). Tukey box plots are as shown in Fig. 4 G and H. confer mechanical strength, antipredator effects, tolerance to desiccation, and other beneficial consequences, which facilitate their survival and adaptation (45–48). In this context, the Nardo- S5) revealed that the antibiotic-treated adult insects exhibited nella-mediated tyrosine production and cuticle sclerotization are significantly more reddish elytra in comparison with the control likely to play pivotal roles in the biology of weevils, which po- adult insects, while the reddish color turned into black as the tentially contribute to their diversity and prosperity. Whether such insect age proceeded (Fig. 6J). The red-black color transition was symbiont-assisted cuticle hardening is also found in other insect partly because reddish frail adult insects, which were presumably groups deserves future survey of diverse beetle–microbe associa- suffering severe symbiosis deficiency, tended to die early, and tions. Recent detailed structural studies unveiled that, among di- also because surviving adult insects became darker in color. verse beetles, weevils are prominent in that their cuticle exhibits a

E8386 | www.pnas.org/cgi/doi/10.1073/pnas.1712857114 Anbutsu et al. Downloaded by guest on October 2, 2021 PNAS PLUS ABCD

Fig. 6. Effects of Nardonella suppression on adult EFGH color and cuticle formation in P. infernalis.(A)Acon- trol adult insect with black and hard elytra. (B–D) Antibiotic-treated adult insects with reddish elytra (B), crumpled fragile elytra (C), and soft and deformable elytra (D). (E–G) The process of quantifying the redness of elytra. From each of dorsal images of adult insects (E), a square area of maximal size (dotted square) was I extracted. On the square image (F), the pixels whose brightness was either over top 10% or below bottom 10% were masked in blue (G) and excluded from the analysis to minimize the effects of highlights and shadows. Then, red-green-blue (RGB) values for all (=n) pixels were measured and averaged to obtain the redness index by Σ (R − mean [R, G, B])/n. (H and J KL I) The system for measuring the viscoelasticity of el- ytra. On the stage of a viscoelastometer (H), each sample elytron was set by gluing onto two plastic plates with epoxy resin (I) to measure elastic modu- lus. (J–L) Elytra redness (J), elytra elastic modulus at 10 Hz (K), and elytra thickness (L) of the control and antibiotic-treated adult insects 0, 7, and 35 d after emergence. Asterisks indicate statistically significant differences [Wilcoxon rank sum test for (J), and t test for (K) and (L): ***P < 0.001; **P < 0.01; ns, no sig- nificant difference]. Tukey box plots are as shown in Fig. 4 G and H.

peculiar microstructure of densely interlocked exocuticle and than the Nardonella genome and retaining many metabolic endocuticle, which may be relevant to the mechanical strength of pathways intact; however, the Sodalis genome was full of am- their exoskeleton (48, 49). Whether the symbiont-provisioned ty- plified IS elements and pseudogenes (50, 51), representing an rosine contributes to formation of the cuticular structure, and if so, early stage of the degenerative genome evolution after replacing how, is of interest and open to future investigation. the original Nardonella symbiont. A number of classic and recent studies have documented a variety of biological roles of the Insights into Symbiont Replacements and Diversification in Evolution Sodalis symbiont for the grain weevils: at phenotypic levels, en- of Weevils. While the Nardonella symbionts have been conserved hanced growth, survival, and fecundity (37, 52, 53), improved among diverse weevils and cospeciated with the weevil hosts for flight activity (52, 54) and facilitated cuticular tanning and over 100 My (23–31), previous studies have identified a number hardening (36, 37, 52); and at biochemical and metabolic levels, of weevil lineages in which the Nardonella symbiont had been provisioning of B vitamins such as pantothenic acid, biotin, and either lost or replaced by novel bacterial symbionts (23, 24, 32– riboflavin (55), supply of aromatic amino acids phenylalanine 35). The extremely reduced Nardonella genome down to 0.2 Mb and tyrosine (36, 37), metabolism of methionine and sarcosine (Fig. 2 and SI Appendix, Table S1) must be relevant to the re- (56, 57), and involvement in mitochondrial energy metabolism current symbiont losses and replacements as a consequence of (58, 59). Our results strongly suggest that the tyrosine pro- genomic decay theoretically expected and empirically observed visioning, which underpins the cuticle hardening and the fitness in ancient symbiont lineages subjected to strict vertical trans- improvement, is the original essential role of the weevil- mission and strong population bottleneck (4, 6, 7, 14–17). The bacterium endosymbiosis, and the other biological functions highly specific Nardonella’s function, tyrosine provisioning only, are likely acquired following the symbiont replacement from is also likely to have facilitated the symbiont losses and re- Nardonella to Sodalis in an ancestor of the grain weevils. placements. For example, in weevil lineages that had evolved feeding habits on tyrosine-rich food sources, it is expected that Incomplete Tyrosine Synthesis Pathway of Nardonella. It should be

evolutionary losses of Nardonella may proceed easily. Consid- noted that, despite the importance of tyrosine provisioning for EVOLUTION ering that many bacteria are capable of synthesizing amino acids the host weevil, Nardonella’s tyrosine synthesis pathway is in- including tyrosine, any bacterial infections with secondary fac- complete, lacking the final step gene, tyrB, that encodes a tyro- ultative symbionts or gut microbial associates may potentially sine transaminase (Fig. 8A and SI Appendix, Fig. S7). We result in complementation of Nardonella’s biological function. expected that the final step reaction of converting 4-hydrox- Such a symbiont replacement has been presumed and best yphenylpyruvate (HPP) to tyrosine may be catalyzed by some documented for grain weevils of the genus Sitophilus, in which gene(s) expressed in the host bacteriome. In the genome of Nardonella was replaced by a γ-proteobacterial lineage Sodalis Drosophila melanogaster (60), there are three aminotransferase pierantonius (23, 24, 32, 50). The weevil-associated Sodalis ge- genes, namely glutamate oxaloacetate transaminase 1 (GOT1), nome was determined as 4.5 Mb in size, which was much larger glutamate oxaloacetate transaminase 2 (GOT2), and tyrosine

Anbutsu et al. PNAS | Published online September 18, 2017 | E8387 Downloaded by guest on October 2, 2021 Mature larvae Pupae New adults GOT1A, GOT1B,andGOT1C), two GOT2 (GOT2A and GOT2B) A BC – 12.5 ns ns and one TAT gene sequences (accession numbers LC260175 4 * LC260180), which were orthologous to those of D. melanogaster 10.0 6 and other insects based on molecular phylogenetic analyses 3 (Fig. 8B). TPM (transcripts per million reads) values indicated 7.5 that, among them, GOT1A and GOT2A represented the over- 4 whelmingly predominant transcripts in the bacteriomes of 2 5.0 Nardonella-infected control insects (Fig. 8 C–H). Quantitative 2 RT-PCR analysis of dissected tissues confirmed preferential 1 2.5 expression of GOT2A and GOT1A in the larval bacteriomes

Tyrosine in hemolymph (mM) Tyrosine 10 11 20 20 813 (Fig. 8 I and J). D E 0.4F ns ns ’ Nardonella 0.6 * Host s Aminotransferase Genes Involved in -Mediated 0.15 Tyrosine Production and Cuticle Formation. Based on these re- 0.3 sults, we attempted to suppress the expression levels of GOT2A 0.10 0.4 and GOT1A (and additionally TAT) by RNAi. Injection of 0.2 dsRNA into mature larvae resulted in significant suppression of GOT2A and GOT1A, while we could not detect RNAi effects on 0.05 0.2 0.1 TAT because of the originally very low expression level (Fig. 9A). In vitro culturing of bacteriomes dissected from mature larvae,

L-DOPA in hemolymph (mM) L-DOPA which had been injected with dsRNAs targeting GOT2A, 0.00 10 11 0.0 20 20 0.0 813 Control Antibiotic ControlCR Antibiotic ControlCR Antibiotic GOT1A, and TAT, revealed significantly reduced tyrosine pro- G 12 3 4 5duction in association with drastically reduced expression levels of GOT2A and GOT1A (Fig. 9B). To observe phenotypic con- sequences of the mixed dsRNA injection, some of the injected larvae were reared to adulthood but in vain: Most died as pupae and a few died as teneral adults in pupal chambers without cu-

Color index ticle sclerotization. Hence, we performed another experiment in which only GOT2A was suppressed by RNAi. The treated larvae 0.4 HI = -0.33 = -0.28 injected with dsRNA targeting GOT2A attained 40% (10/25) P = 0.42 P = 0.36 adult emergence rate, which was similar to the 42% (10/24) adult emergence rate of the control larvae injected with dsRNA 6 0.3 = -0.47 targeting GFP (Fig. 9C). Notably, the GOT2A-suppressed adult P = 0.10 insects tended to exhibit morphological abnormalities, repre- = -0.12 sented by reddish, crumpled, and/or deformed elytra, in compar- 4 0.2 P = 0.77 ison with the control adult insects that were mostly with normally Treatments sclerotized black elytra (Fig. 9C). Elytra color analysis confirmed 0.1 Control that the GOT2A-suppressed adult insects were more reddish in 2 (n = 8) comparison with the control adult insects (although the differ- Antibiotic L-DOPA in hemolymph (mM) L-DOPA

Tyrosine in hemolymph (mM) Tyrosine ences were statistically not significant) and the reddish color be- (n = 13) 0.0 came darker as the insect age proceeded (Fig. 9D). These results 12345 12345 strongly suggest that the host’s aminotransferase genes up- Color index Color index regulated in the bacteriome are needed for Nardonella-mediated tyrosine production in the bacteriome and involved in formation Fig. 7. Effects of Nardonella suppression on levels of tyrosine and L-DOPA during pupal and adult development of P. infernalis.(A–C) Tyrosine levels in and pigmentation of adult cuticle. the hemolymph of mature larvae (A), pupae (B), and newly emerged adults ’ ’ (C). (D–F) L-DOPA levels in the hemolymph of mature larvae (D), pupae Host s Final Step Control over Symbiont s Metabolic Pathway. All these (E), and newly emerged adults (F). Asterisks indicate statistically significant results taken together, we conclude that the Nardonella’s sole and differences (t test; *P < 0.05; ns, no significant difference). Tukey box plots most important role, tyrosine synthesis, is subjected to the final are as shown in Fig. 4 G and H.(G) Color indices of newly eclosed (within step regulation by host’s enzymes up-regulated in the symbiotic 24 h) adult insects defined by the levels of cuticle pigmentation. The younger organ, which highlights an impressively intimate and focused as- the insects, the paler their color is. (H and I) Relationships between the levels pect of the host–symbiont metabolic integrity. We suggest that the of cuticle pigmentation and the titers of hemolymph tyrosine (H) or hemo- regulation by the host side may have been evolutionarily facilitated lymph L-DOPA (I) in the control insects and the antibiotic-treated insects. Regression lines, Spearman’s rank correlation coefficients (ρ), and P values by, on one hand, the extremely reduced Nardonella genome that (P) are depicted. has purged out transcriptional and translational regulator genes, and on the other hand, the drastic change in demand for tyrosine, much more for highly sclerotized adults than for nonsclerotized aminotransferase (TAT), which are potentially capable of convert- larvae, in the developmental course of the weevil host. In the ing HPP to tyrosine by transamination. Hence, we surveyed the aphid-Buchnera endosymbiosis, similar patterns are observed in transcriptomes of P. infernalis for these aminotransferase genes. metabolic pathway genes for synthesis of essential amino acids isoleucine, leucine, valine, and phenylalanine, and also tyrosine. Genes of P. infernalis Up-Regulated in the Bacteriome and Complementing Specifically, the Buchnera genome encodes almost all of the syn- Nardonella’s Tyrosine Synthesis Pathway. We performed RNA ex- thesis pathway enzymes for these amino acids except for the final traction of bacteriomes and midguts dissected from mature larvae step enzymes, which are complemented by host-encoded enzymes, of P. infernalis reared on the control diet and the antibiotic- namely, branched-chain aminotransferase for isoleucine, leucine, supplemented diet, and the RNA samples were subjected to and valine; aspartate aminotransferase for phenylalanine; and RNA sequencing analysis (SI Appendix, Table S6). From the as- phenylalanine 4-monooxigenase for tyrosine (61–68). Notably, in sembled contigs, we identified three GOT1 (designated as contrast to the synthesis pathway for tyrosine via phenylalanine in

E8388 | www.pnas.org/cgi/doi/10.1073/pnas.1712857114 Anbutsu et al. Downloaded by guest on October 2, 2021 PNAS PLUS A Erythrose 4- 4-hydroxy phosphate phenyl + pyruvate Tyrosine Phosphoenol (HPP) pyruvate

[ ] B 75/60 [ ] 75/ 66 [ ] 65/95 98/ -/90 100

99/100 [ ] 84/81 [ ] 97/99 [ ] GOT/AST 74/86 99/100 [ ] 71/50 [ ] Fig. 8. Identification of GOT1, GOT2, and TAT genes 100/100 [ ] of P. infernalis that are potentially involved in tyro- 94/60 [ ] 91/84 TAT sine synthesis in place of Nardonella’s tyrB gene. C D E (A) Tyrosine synthesis pathway encoded by the Nar- 400 40 20 donella genome. Also see SI Appendix, Fig. S7. 350 GOT1A 35 GOT1B 18 GOT1C 16 300 30 (B) Phylogenetic relationship of GOT1, GOT2,and 14 250 25 12 TAT transcripts identified from P. infernalis to those 200 20 10

TPM 150 15 8 from the fruit fly D. melanogaster, the flour beetle 6 100 10 4 T. castaneum, and the bark beetle D. ponderosae. 50 5 2 0 0 0 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1#1 #2 #2 #3#3 #1#1 #2 #2 #3#3 #1#1 #2 #2 #3#3 #1#1 #2 #2 #3#3 A maximum-likelihood phylogeny inferred from Bacteriome Midgut Bacteriome Midgut Bacteriome Midgut Bacteriome Midgut Bacteriome Midgut Bacteriome Midgut 515 aligned amino acid sites is shown with bootstrap F Control Antibiotic G Control Antibiotic H Control Antibiotic 900 20 20 values on the nodes in the order of maximum like- 800 GOT2A 18 GOT2B 18 TAT 700 16 16 lihood/neighbor joining. Note that three GOT1 600 14 14 12 (GOT1A, GOT1B,andGOT1C), two GOT2 (GOT2A 500 12 10 10 TPM 400 8 8 and GOT2B) and one TAT sequences were obtained 300 6 6 200 4 4 from P. infernalis, which are highlighted in red. In 100 2 2 0 0 0 brackets are sequence accession numbers. (C–H)TPM #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 Bacteriome Midgut Bacteriome Midgut Bacteriome Midgut Bacteriome Midgut Bacteriome Midgut Bacteriome Midgut values for GOT1A (C), GOT1B (D), GOT1C (E), GOT2A Control Antibiotic Control Antibiotic Control Antibiotic (F), GOT2B (G), and TAT (H) genes transcribed in IJ−1-1 a −1-1 ) GOT2A expression in GOT1A expression in bacteriomes and midguts dissected from mature ) different tissues a different tissues larvae reared on the control diet and the antibiotic- ef1α ef1α −2/ -2 b −2-2 bc ab supplemented diet. (I and J) Quantitative RT-PCR b bc bc b b c evaluating the expression levels of GOT2A (I)and GOT2A

b GOT1A /

( -3 GOT1A (J) in tissues dissected from mature larvae. −3-3 −3( 10 10 Gene copy numbers evaluated by quantitative RT- Expression level Expression level Log

Log α −4-4 6 6 6 6 6 6 −4-4 6 6 6 6 6 6 PCR are normalized as per EF1 gene copy. Differ- ent letters (a, b, c) indicate statistically significant differences (likelihood-ratio test of GLM and post hoc multiple comparisons; P < 0.05).

the aphid-Buchnera system, the synthesis pathway for tyrosine in ancient endosymbiotic evolution. The only functional metabolism the weevil-Nardonella system circumvents phenylalanine (SI Ap- retained in the Nardonella genome is the tyrosine synthesis path- pendix,Fig.S7), which is probably relevant to the facts that (i) way, uncovering the sole biological role of Nardonella as a tyrosine distinct from the Buchnera of aphids, Nardonella of weevils is in- supplier. Experimental suppression of Nardonella infection results capable of synthesizing phenylalanine; (ii)weevilsrequirein- in emergence of reddish and soft weevils, verifying the importance comparably more tyrosine for building their hard cuticle than of Nardonella for host’s cuticle formation and hardening. In ac- aphids; (iii) thus, if Nardonella could synthesize tyrosine from cordance with the stringently limited gene repertoire of Nardonella phenylalanine, it would readily end up with depletion of the es- lacking transcriptional and translational regulators, the Nardo- sential amino acid phenylalanine in weevils; and (iv)thisisprobably nella-mediated tyrosine synthesis is regulated by host amino- the reason why Nardonella has evolved the phenylalanine- transferases up-regulated in the bacteriome at the final step of the – independent synthesis pathway for tyrosine. In the aphid-Buchnera synthesis pathway, highlighting an intricate host symbiont meta- system, amino acid transporter proteins located at the host sym- bolic integration that underpins the bacteriome function as a biosomal membrane in the bacteriocyte play pivotal roles for me- tyrosine-producing organ. In an evolutionary perspective, the ex- tremely reduced Nardonella genome specialized for a single met- tabolite exchange and host–symbiont metabolic integration (69, 70). abolic function may have facilitated the recurrent symbiont losses In the weevil-Nardonella system, it is of interest but unknown how and replacements observed in weevils. In an ecological perspec- the precursor of tyrosine, 4-HPP, produced by the symbiont is tive, the symbiont-assisted formation of hard cuticle may have transported to the host cytoplasm within the bacteriome (SI Ap- contributed to, at least to some extent, the adaptation, diversity, pendix,Fig.S7),whichisopentofutureinvestigation. and prosperity of weevils. The weevil-Nardonella endosymbiotic EVOLUTION Conclusion and Perspective system presents an impressive example as to how far such an in- timate and ancient host–symbiont association can go over evolu- The weevil-Nardonella system represents an unprecedented form tionary time and why such a highly sophisticated symbiotic system of nutritional symbiosis. The extremely reduced symbiont genome, inherently entails evolutionary consequences of instability, losses, around 0.2 Mb in size, encodes minimal but complete gene sets for and replacements. bacterial cell replication, transcription, and translation, while lacking almost all of the other metabolic pathway genes, which is Materials and Methods indicative of Nardonella’s near-complete dependence on host- Weevil samples used in this study are as listed (SI Appendix, Table S7). derived metabolites toward a minimal cellular entity through the P. infernalis was reared in the laboratory for experiments (SI Appendix, Fig.

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C D

Fig. 9. RNA interference (RNAi) suppression of host’s tyrosine synthesis genes up-regulated in the bacteriome of P. infernalis, and its effects on cuticle formation and pigmentation. (A) RNAi targeting GOT2, GOT1,andTAT by injecting double-stranded RNA (dsRNA) into mature larvae. Expression levels of GOT2A, GOT1A,andTAT were measured by quantitative RT-PCR 3 d after the injection. Different letters (a, b, c) indicate statistically significant differences (likelihood-ratio test of GLM and post hoc multiple comparisons; P < 0.05). (B) Tyrosine synthesis activity of dissected larval bacteriomes suppressed by RNAi of GOT2 and GOT1.Maturelarvaewereinjected with dsRNAs, their bacteriomes were dissected 7 d after the injection and cultured in a medium containing 15N-labeled glutamine for 2 h, and the bacteriomes were subjected to quantitative RT-PCR, whereas the culture media were analyzed by LC-MS for quantification of 15N-labeled tyrosine. Asterisks indicate statistically sig- nificant differences (likelihood-ratio test of GLM assuming a gamma error distribution; ***P < 0.001). (C)Imagesofadultinsectsonthedayofemergence,whichwere subjected to larval injection with either GFP dsRNA or GOT2A dsRNA. Numbers on the top of the images indicate the values of elytra redness. (D) Comparison of elytra redness between the adult insects 0, 7, and 35 d after emergence. “ns” indicates no statistically significant difference (Wilcoxon rank sum test; P > 0.05). Tukey box plots are as shown in Fig. 4 G and H. Note that the primers for quantitative RT-PCR are highly specific, whereas dsRNAs for RNAi may potentially cause some cross- suppressions: dsRNA for GOT2A may also recognize GOT2B, and dsRNA for GOT1A may also target GOT1B and GOT1C (SI Appendix,Fig.S8).

S2). Histological analyses were conducted as described (28, 71). Symbiont dynamic viscoelastometer (Rheovibron DDV-25FP; A&D) was used to mea- genome sequencing and annotation were performed as described (72–75). sure mechanical properties of elytra. Transcriptomic data of the host bacteriomes were obtained by RNA se- See SI Appendix, SI Materials and Methods for complete details on the quencing using TruSeq RNA Sample Preparation Kit v2 (Illumina) and materials and methods. HiSeq2000 sequencer (Illumina), and analyzed using the program Trinity (76) implemented in the MASER pipeline (cell-innovation.nig.ac.jp/). ACKNOWLEDGMENTS. We thank Takuya Aikawa, Katsunori Nakamura, Symbiont visualization by fluorescence in situ hybridization, symbiont Takuma Takanashi, Wataru Toki, and Yosuke Usui for insect samples; Norikuni Kumano, Munetoshi Maruyama, Katsunori Nakamura, and Katsushi Yamaguchi quantification by quantitative PCR, and evaluation of host gene ex- for insect photos; Akira Oyafuso for artificial diet ingredients; Katsushi Yamaguchi pression by quantitative RT-PCR were conducted using the probes and Tomoko F. Shibata for supporting genomic and transcriptomic data and primers listed (SI Appendix,TableS8). Tracer experiments with [2-15N] processing; Hiroshi Shimizu and Hideyuki Tsukada for supporting measurement of glutamine (Cambridge Isotope Laboratories) using tissue culture physical properties of insect cuticle; and Wakana Kikuchi, Junko Makino, and and amino acid analyses were performed using a high-performance liq- Kaoru Nikoh for technical and secretarial assistance. This study was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research uid chromatography system (Prominence, Shimadzu) with a Shim-pack Grants JP25221107 (to T.F., N.N., T.H., and R.K.), JP22128001, and JP22128007 (to × FC-ODS column (150 mm 2 mm i.d., Shimadzu) and an electrospray T.F., N.N. and S.S.); the Program for Promotion of Basic and Applied Researches for ionization mass spectrometry system (LCQduo; Thermo). Elytra color analysis Innovations in Bio-Oriented Industry (to T.F.); and National Institute for Basic was conducted using the software Natsumushi version 0.99. An automatic Biology Collaborative Research Projects for Integrative Genomics (to T.F. and S.S.).

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