Host range of naturally and artificially evolved symbiotic bacteria for a specific host insect

Abstract

Diverse insects are intimately associated with specific symbiotic bacteria, where host and symbiont are integrated into an almost inseparable biological entity. These symbiotic bacteria usually exhibit host specificity, uncultivability, reduced genome size, and other peculiar traits relevant to their symbiotic lifestyle. How host-symbiont specificity is established at the very beginning of symbiosis is of interest but poorly understood. To gain insight into the evolutionary issue, we adopted an experimental approach using the recently developed evolutionary model of symbiosis between the stinkbug Plautia stali and Escherichia coli. Based on the laboratory evolution of P. stali-E. coli mutualism, we selected ΔcyaA mutant of E. coli as an artificial symbiont of P. stali that has established mutualism by a single mutation. In addition, we selected a natural cultivable symbiont of P. stali of relatively recent evolutionary origin. These artificial and natural symbiotic bacteria of P. stali were experimentally inoculated to symbiont-deprived newborn nymphs of diverse stinkbug species. Strikingly, the mutualistic E. coli was unable to establish infection and support growth and survival of all the stinkbug species except for P. stali, uncovering that host specificity can be established at a very early stage of symbiotic evolution. Meanwhile, the natural symbiont was able to establish infection and support growth and survival of several stinkbug species in addition to P. stali, unveiling that a broader host range of the symbiont has evolved in nature. Based on these findings, we discuss what factors are relevant to the establishment of host specificity in the evolution of symbiosis.IMPORTANCEHow does host-symbiont specificity emerge at the very beginning of symbiosis? This question is difficult to address because it is generally difficult to directly observe the onset of symbiosis. However, recent development of experimental evolutionary approaches to symbiosis has brought about a breakthrough. Here we tackled this evolutionary issue using a symbiotic Escherichia coli created in laboratory and a natural Pantoea symbiont, which are both mutualistic to the stinkbug Plautia stali. We experimentally replaced essential symbiotic bacteria of diverse stinkbugs with the artificial and natural symbionts of P. stali and evaluated whether the symbiotic bacteria, which evolved for a specific host, can establish infection and support the growth and survival of heterospecific hosts. Strikingly, the artificial symbiont showed strict host specificity to P. stali, whereas the natural symbiont was capable of symbiosis with diverse stinkbugs, which provide insight into how host-symbiont specificity can be established at early evolutionary stages of symbiosis.

Keywords: Escherichia coli; Pantoea; Plautia stali; gut symbiosis; host specificity; stinkbug.

Fig 1

Stinkbugs and their symbiotic bacteria. (A) Systematic relationship of the stinkbugs used in this study. The relationship of the stinkbug families is after Wu et al. (31). Stinkbug superfamilies are displayed on the right side. (B) Phylogenetic relationship of the symbiotic bacteria. A maximum-likelihood phylogeny inferred from bacterial 16S rRNA gene sequences (1,462 aligned nucleotide sites) is shown. On each node, statistical support values are indicated as posterior probability of Bayesian inference/bootstrap probability of maximum-likelihood analysis. Gut symbiotic (GS) bacteria of the stinkbugs are shown in green; E. coli is highlighted in red; and free-living proteobacterial 16S rRNA gene sequences retrieved from the DNA databases are displayed in black. Bacterial families and classes are shown on the right side. Corresponding host stinkbugs are indicated by line connections. Note that the symbiont phylogeny does not agree with the host systematics, reflecting recurrent symbiont acquisitions and replacements in the evolutionary course of the stinkbugs (29).

Fig 2

Infection, survival, and growth of P. stali experimentally deprived of the original mutualistic symbiont and inoculated with either a wild-type E. coli strain (ΔintS), a mutant E. coli strain mutualistic to P. stali (ΔcyaA), or a natural cultivable symbiont strain mutualistic to P. stali (Sym C). (A) Survival curve. (B) Adult emergence curve. (C) Survival rate on the 37th day after hatching. (D) Adult emergence rate on the 37th day after hatching. (E) Adult female body size. (F) Adult male body size. (G) PCR check of bacterial infection in second instar nymphs (left) and adult insects (right). Statistical tests were conducted by a likelihood ratio test of a generalized linear model in panels C and D (a–d, P < 0.05) and by pair-wise t-test with Bonferroni correction in panels E and F (a and b, P < 0.05). NA, statistical analysis not applicable.

Fig 3

Infection, survival, and growth of G. subpunctatus experimentally deprived of the original mutualistic symbiont and inoculated with either a wild-type E. coli strain (ΔintS), a mutant E. coli strain mutualistic to P. stali (ΔcyaA), or a natural cultivable symbiont strain mutualistic to P. stali (Sym C). (A) Survival curve. (B) Adult emergence curve. (C) Survival rate on the 38th day after hatching. (D) Adult emergence rate on the 38th day after hatching. (E) Adult female body size. (F) Adult male body size. (G) PCR check of bacterial infection in second instar nymphs (left) and adult insects (right). Statistical tests were conducted by likelihood ratio test of a generalized linear model in panels C and D (a–c, P < 0.05) and by pair-wise t-test with Bonferroni correction in panels E and F (a and b, P < 0.05. In panels C and D, for example, “n = 5 (112 eggs)” indicates “five experimental groups were prepared, which consisted of 112 eggs in total.” The same applies to panels C and D in Fig. 2 and 4–9. NA, statistical analysis not applicable; NS, no significant difference.

Fig 4

Infection, survival, and growth of N. viridula experimentally deprived of the original mutualistic symbiont and inoculated with either a wild-type E. coli strain (ΔintS), a mutant E. coli strain mutualistic to P. stali (ΔcyaA), or a natural cultivable symbiont strain mutualistic to P. stali (Sym C). (A) Survival curve. (B) Adult emergence curve. (C) Survival rate on the 38th day after hatching. (D) Adult emergence rate on the 38th day after hatching. (E) Adult female body size. (F) Adult male body size. (G) PCR check of bacterial infection in second instar nymphs (left) and adult insects (right). Statistical tests were conducted by likelihood ratio test of a generalized linear model in panels C and D. In panels E and F. NA, statistical analysis not applicable; NS, no significant difference.

Fig 5

Infection, survival, and growth of H. halys experimentally deprived of the original mutualistic symbiont and inoculated with either a wild-type E. coli strain (ΔintS), a mutant E. coli strain mutualistic to P. stali (ΔcyaA), or a natural cultivable symbiont strain mutualistic to P. stali (Sym C). (A) Survival curve. (B) Adult emergence curve. (C) Survival rate on the 49th day after hatching. (D) Adult emergence rate on the 49th day after hatching. (E) Adult female body size. (F) Adult male body size. (G) PCR check of bacterial infection in second instar nymphs (left) and adult insects (right). Statistical tests were conducted by likelihood ratio test of a generalized linear model in panels C and D (a and b, P < 0.05). and by pair-wise t-test with Bonferroni correction in panels E and F. NA, statistical analysis not applicable; NS, no significant difference.

Fig 6

Infection, survival, and growth of L. miyakonus experimentally deprived of the original mutualistic symbiont and inoculated with either a wild-type E. coli strain (ΔintS), a mutant E. coli strain mutualistic to P. stali (ΔcyaA), or a natural cultivable symbiont strain mutualistic to P. stali (Sym C). (A) Survival curve. (B) Adult emergence curve. (C) Survival rate on the 45th day after hatching. (D) Adult emergence rate on the 45th day after hatching. (E) Adult female body size. (F) Adult male body size. (G) PCR check of bacterial infection in second instar nymphs (left) and adult insects (right). Statistical tests were conducted by likelihood ratio test of a generalized linear model in panels C and D (a and b, P < 0.05) and by pair-wise t-test with Bonferroni correction in panels E and F. NA, statistical analysis not applicable; NS, no significant difference.

Fig 7

Infection, survival, and growth of Po. lewisi experimentally deprived of the original mutualistic symbiont and inoculated with either a wild-type E. coli strain (ΔintS), a mutant E. coli strain mutualistic to P. stali (ΔcyaA), or a natural cultivable symbiont strain mutualistic to P. stali (Sym C). (A) Survival curve. (B) Adult emergence curve. (C) Survival rate on the 50th day after hatching. (D) Adult emergence rate on the 50th day after hatching. (E) Adult female body size. (F) Adult male body size. (G) PCR check of bacterial infection in second instar nymphs (left) and adult insects (right). Statistical tests were conducted by likelihood ratio test of a generalized linear model in panels C and D (a and b, P < 0.05) and by pair-wise t-test with Bonferroni correction in panels E and F. NA, statistical analysis not applicable; NS, no significant difference.

Fig 8

Infection, survival, and growth of E. grandis experimentally deprived of the original mutualistic symbiont and inoculated with either a wild-type E. coli strain (ΔintS), a mutant E. coli strain mutualistic to P. stali (ΔcyaA), or a natural cultivable symbiont strain mutualistic to P. stali (Sym C). (A) Survival curve. (B) Adult emergence curve. (C) Survival rate on the 57th day after hatching. (D) Adult emergence rate on the 57th day after hatching. (E) Adult female body size. (F) Adult male body size. (G) PCR check of bacterial infection in second instar nymphs (left) and adult insects (right). Statistical tests were conducted by likelihood ratio test of a generalized linear model in panels C and D (a and b, P < 0.05). In panels E and F, NA indicates statistical analysis not applicable.

Fig 9

Infection, survival, and growth of E. grandis experimentally deprived of the original mutualistic symbiont and inoculated with either a wild-type E. coli strain (ΔintS), a mutant E. coli strain mutualistic to P. stali (ΔcyaA), or a natural cultivable symbiont strain mutualistic to P. stali (Sym C). (A) Survival curve. (B) Adult emergence curve. (C) Survival rate on the 37th day after hatching. (D) Adult emergence rate on the 37th day after hatching. (E) Adult female body size. (F) Adult male body size. (G) PCR check of bacterial infection in second instar nymphs (left) and adult insects (right). Statistical tests were conducted by likelihood ratio test of a generalized linear model in panels C and D and by pair-wise t-test with Bonferroni correction in panels E and F. NA, statistical analysis not applicable; NS, no significant difference.