Aphid thermal tolerance is governed by a point mutation in bacterial symbionts

Abstract

Symbiosis is a ubiquitous phenomenon generating biological complexity, affecting adaptation, and expanding ecological capabilities. However, symbionts, which can be subject to genetic limitations such as clonality and genomic degradation, also impose constraints on hosts. A model of obligate symbiosis is that between aphids and the bacterium Buchnera aphidicola, which supplies essential nutrients. We report a mutation in Buchnera of the aphid Acyrthosiphon pisum that recurs in laboratory lines and occurs in field populations. This single nucleotide deletion affects a homopolymeric run within the heat-shock transcriptional promoter for ibpA, encoding a small heat-shock protein. This Buchnera mutation virtually eliminates the transcriptional response of ibpA to heat stress and lowers its expression even at cool or moderate temperatures. Furthermore, this symbiont mutation dramatically affects host fitness in a manner dependent on thermal environment. Following a short heat exposure as juveniles, aphids bearing short-allele symbionts produced few or no progeny and contained almost no Buchnera, in contrast to aphids bearing symbionts without the deletion. Conversely, under constant cool conditions, aphids containing symbionts with the short allele reproduced earlier and maintained higher reproductive rates. The short allele has appreciable frequencies in field populations (up to 20%), further supporting the view that lowering of ibpA expression improves host fitness under some conditions. This recurring Buchnera mutation governs thermal tolerance of aphid hosts. Other cases in which symbiont microevolution has a major effect on host ecological tolerance are likely to be widespread because of the high mutation rates of symbiotic bacteria and their crucial roles in host metabolism and development.

Figure 1. Heat-Shock Promoter Governing Expression of the Heat-Shock Gene ibpA in Buchnera

(A) Schematic of regulation of ibpA expression showing position of heat-shock promoter. (B) Sequence of E. coli consensus heat-shock promoter and of all heat-shock promoters of Buchnera of S. graminum and of A. pisum. Sequences from published complete genome data were confirmed for both species. The only variant is the ibpA promoter allele, which has a shorter spacer length, resulting in almost complete loss of the heat-shock response of ibpA.

Figure 2. Response to Heat Treatment in Buchnera-Ap lines, for Genes Typically Up-Regulated in Response to Heat

(A) Gene expression changes in response to heat treatment (35 °C) as measured from a microarray experiment, including two control genes (argG and cysG) that did not show significant responses to heat treatment. Each bar represents the mean of technical and biological replicates of each line (four arrays/aphid line) ± standard error. (B) Gene expression changes in response to heat (35 °C) as measured with RT-qPCR using several A. pisum lines containing Buchnera with either short or long ibpA promoter alleles. For the long allele, n = 4 (three lines), and for the short allele, n = 2 (two lines). Error bars indicate means ± standard errors. (C) Effects of exposure to different temperatures on ibpA expression for three aphid lines bearing Buchnera with different ibpA promoter alleles. The two 5A lines differ only in the single-base deletion affecting the length of the ibpA spacer; whereas TUC matches 5A-long for the ibpA promoter sequence, but shows other differences from 5A lines throughout the Buchnera genome. Pairwise differences between the two 5A-derived lines are significant at every temperature, with the long allele always showing higher expression relative to a control gene (cysG). TUC has other sequence differences, and these appear to have minor effects on ibpA expression.

Figure 3. Chronology of ibpA Heat-Shock Promoter Evolution in Sublines of the 5A Line

Line 5A was initiated from a single female in June 1999. In September 2000, it was transfected with a secondary symbiont to generate 5A⁺R^LONG. Subsequently the 5A line acquired the first mutation to the short promoter allele, which became fixed. An identical mutation rose to near fixation in the 5A⁺R^LONG culture in October 2005; both alleles were isolated from that colony to yield 5A⁺R^SHORT and 5A⁺R^LONG. Line 5A⁺R^SHORT lost its Candidatus Serratia symbiotica to generate line 5A⁻R^SHORT, and then in November 2005, 5A⁺R^LONG was cured of Candidatus Serratia symbiotica to generate line 5A⁻R^LONG. Thus the mutation occurred and rose to fixation or near fixation twice in sublines of the 5A laboratory line (Figure 1B).

Figure 4. Effect of Different Temperature Treatments on Day 2 on Fecundity as Adults, for Aphid Lines with Buchnera with Different ibpA Promoter Alleles

Bars show means ± standard errors.

Figure 5. Reproductive Rates under Different Temperature Conditions of Aphid Lines with Buchnera with Different ibpA Promoter Alleles

Number of nymphs per day for the first 6 d of reproduction for 5A females with Buchnera bearing the long and short alleles (5A⁻R^LONGand 5A^SHORT). Bars show means ± standard errors. Temperature conditions were constant 15 °C, constant 20 °C, and 20 °C with 4 h at 35 °C on day 2 following birth.

Figure 6. Effect of Heat Stress on Buchnera Densities in Aphids Bearing Buchnera with Different ibpA Promoter Alleles

Estimates of Buchnera and aphid genome numbers are based on qPCR assays of genomic DNA determining numbers of copies of single-copy Buchnera genes relative to a single-copy aphid gene. Heat treatment was 4 h at 35 °C on day 2 following birth, and measures were taken within 24 h of attaining adulthood. Bars show means ± standard errors.