Small genome symbiont underlies cuticle hardness in beetles

Abstract

Beetles, representing the majority of the insect species diversity, are characterized by thick and hard cuticle, which plays important roles for their environmental adaptation and underpins their inordinate diversity and prosperity. Here, we report a bacterial endosymbiont extremely specialized for sustaining beetle's cuticle formation. Many weevils are associated with a γ-proteobacterial endosymbiont lineage Nardonella, whose evolutionary origin is estimated as older than 100 million years, but its functional aspect has been elusive. Sequencing of Nardonella genomes from diverse weevils unveiled drastic size reduction to 0.2 Mb, in which minimal complete gene sets for bacterial replication, transcription, and translation were present but almost all of the other metabolic pathway genes were missing. Notably, the only metabolic pathway retained in the Nardonella genomes was the tyrosine synthesis pathway, identifying tyrosine provisioning as Nardonella's sole biological role. Weevils are armored with hard cuticle, tyrosine is the principal precursor for cuticle formation, and experimental suppression of Nardonella resulted in emergence of reddish and soft weevils with low tyrosine titer, confirming the importance of Nardonella-mediated tyrosine production for host's cuticle formation and hardening. Notably, Nardonella's tyrosine synthesis pathway was incomplete, lacking the final step transaminase gene. RNA sequencing identified host's aminotransferase genes up-regulated in the bacteriome. RNA interference targeting the aminotransferase genes induced reddish and soft weevils with low tyrosine titer, verifying host's final step regulation of the tyrosine synthesis pathway. Our finding highlights an impressively intimate and focused aspect of the host-symbiont metabolic integrity via streamlined evolution for a single biological function of ecological relevance.

Keywords: Nardonella; genome; symbiont; tyrosine; weevil.

Fig. 1.

P. infernalis and its bacterial symbiont Nardonella. (A) An adult. (B) A final instar larva. (C) A dissected larval gut with bacteriomes. Petal-like bacteriome lobes, each consisting of many bacteriocytes, are located around the foregut-midgut junction. (D) Isolated radial bacteriome lobes. (E) Detection of Nardonella in the ovarioles dissected from an adult female. Arrows highlight localization of Nardonella at the ovariole tips. (F) Visualization of Nardonella in the cytoplasm of larval bacteriocytes. (G) A transmission electron microscopic image of Nardonella cells in the larval bacteriocyte. In C–F, Nardonella 16S rRNA and host nuclear DNA are visualized in red and blue, respectively.

Fig. 2.

Weevils and their Nardonella genomes examined in this study. (A) Nardonella Rfe of the red palm weevil R. ferrugineus (Curculionidae: Dryophthorinae). (B) Nardonella Sgi of the giant weevil S. gigas (Curculionidae: Dryophthorinae). (C) Nardonella Epo of the West Indian sweet potato weevil E. postfasciatus (Curculionidae: Cryptorhynchinae). (D) Nardonella Pin of the black hard weevil P. infernalis (Curculionidae: Entiminae). Nardonella Sgi has a 2,117-bp plasmid encoding only two genes, mscS and ibpA-like. mscS, which is retained in all of the Nardonella genomes, encodes a channel for osmoregulation 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 the synteny of mscS and ibpA on the plasmid of Nardonella Sgi is conserved on the chromosome of Nardonella Epo.

Fig. 3.

Comparison of the metabolic gene repertoire between Nardonella genomes and other extremely reduced symbiont genomes. The minimal number of genes for a metabolic pathway is shown in each of the brackets. Each color indicates the ratio of retained genes to the minimal gene set for a metabolic pathway: green for 100%, orange for 75–99%, yellow for 50–74%, blue for 25–49%, and gray for 0–24%. Nardonella’s tyrosine synthesis pathway genes are highlighted in red. In the Nardonella Epo genome, aroE gene, located between def and sufE genes in the other Nardonella genomes, is lost and replaced by a 168-bp spacer sequence, which may be relevant to the fact that its host E. postfasciatus is smaller in size with thinner cuticle in comparison with the other large and hard weevil species R. ferrugineus, S. gigas, and P. infernalis.

Fig. 4.

In vitro assay of Nardonella’s tyrosine synthesis in P. infernalis. (A–C) The experimental system for hanging drop tissue culture. (A) An external view of the culture system. (B) An isolated larval bacteriome of P. infernalis in hanging drop medium. (C) A schematic view of the culture system. (D–F) Quantification of essential amino acids (D), nonessential amino acids (E), and semiessential amino acids (F) released from an isolated larval bacteriome of P. infernalis in the hanging drop medium supplemented with ¹⁵N-glutamine. Columns and bars show means and SDs, in which ¹⁵N-labeled and unlabeled amino acid fractions are depicted by dark green and light green, respectively. 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 of a generalized linear model (GLM) assuming a Gamma error distribution; ***P < 0.001]. Tukey box plots indicate the median (bold line), the 25th and 75th percentiles (box edges), the range (whiskers) and outliers, which are larger or smaller than 1.5 times the interquartile range from the box edge (dots), with sample sizes at the bottom.

Fig. 5.

Antibiotic suppression of Nardonella infection in P. infernalis. (A and B) Dissected bacteriomes. (C and D) Light microscopic images of semiultrathin sections of the larval bacteriome stained with toluidine blue. (E and F) Transmission electron microscopic images of the larval bacteriocytes. (A, C, and E) Larvae reared on the control artificial diet. (B, D, and F) Larvae reared on the artificial diet containing 0.003% rifampicin. (G–I) Nardonella titers (in terms of bacterial groEL gene copies per insect) in mature larvae (G), pupae (H), and newly emerged adults (I). (J–L) Nardonella densities (in terms of bacterial groEL gene copies per host Elongation Factor 1α [EF1α] gene copy) in mature larvae (J), pupae (K), and newly emerged adults (L). In G–L, asterisks indicate statistically significant differences (likelihood-ratio test of GLM assuming a Gamma error distribution; *P < 0.05; ***P < 0.001; ns, no significant difference). Tukey box plots are as shown in Fig. 4 G and H.

Fig. 6.

Effects of Nardonella suppression on adult color and cuticle formation in P. infernalis. (A) A control 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 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 I) The system for measuring the viscoelasticity of elytra. 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 modulus. (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 significant difference]. Tukey box plots are as shown in Fig. 4 G and H.

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 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 (E), and newly emerged adults (F). Asterisks indicate statistically significant differences (t test; *P < 0.05; ns, no significant difference). Tukey box plots are as shown in Fig. 4 G and H. (G) Color indices of newly eclosed (within 24 h) adult insects defined by the levels of cuticle pigmentation. The younger the insects, the paler their color is. (H and I) Relationships between the levels of cuticle pigmentation and the titers of hemolymph tyrosine (H) or hemolymph l-DOPA (I) in the control insects and the antibiotic-treated insects. Regression lines, Spearman’s rank correlation coefficients (ρ), and P values (P) are depicted.

Fig. 8.

Identification of GOT1, GOT2, and TAT genes of P. infernalis that are potentially involved in tyrosine synthesis in place of Nardonella’s tyrB gene. (A) Tyrosine synthesis pathway encoded by the Nardonella genome. Also see SI Appendix, Fig. S7. (B) Phylogenetic relationship of GOT1, GOT2, and TAT transcripts identified from P. infernalis to those from the fruit fly D. melanogaster, the flour beetle T. castaneum, and the bark beetle D. ponderosae. A maximum-likelihood phylogeny inferred from 515 aligned amino acid sites is shown with bootstrap values on the nodes in the order of maximum likelihood/neighbor joining. Note that three GOT1 (GOT1A, GOT1B, and GOT1C), two GOT2 (GOT2A and GOT2B) and one TAT sequences were obtained from P. infernalis, which are highlighted in red. In brackets are sequence accession numbers. (C–H) TPM values for GOT1A (C), GOT1B (D), GOT1C (E), GOT2A (F), GOT2B (G), and TAT (H) genes transcribed in bacteriomes and midguts dissected from mature larvae reared on the control diet and the antibiotic-supplemented diet. (I and J) Quantitative RT-PCR evaluating the expression levels of GOT2A (I) and GOT1A (J) in tissues dissected from mature larvae. Gene copy numbers evaluated by quantitative RT-PCR are normalized as per EF1α gene copy. Different letters (a, b, c) indicate statistically significant differences (likelihood-ratio test of GLM and post hoc multiple comparisons; P < 0.05).

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, and TAT by injecting double-stranded RNA (dsRNA) into mature larvae. Expression levels of GOT2A, GOT1A, and TAT 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. Mature larvae were injected 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 significant differences (likelihood-ratio test of GLM assuming a gamma error distribution; ***P < 0.001). (C) Images of adult insects on the day of emergence, which were 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).