Experimental replacement of an obligate insect symbiont

Abstract

Symbiosis, the close association of unrelated organisms, has been pivotal in biological diversification. In the obligate symbioses found in many insect hosts, organisms that were once independent are permanently and intimately associated, resulting in expanded ecological capabilities. The primary model for this kind of symbiosis is the association between the bacterium Buchnera and the pea aphid (Acyrthosiphon pisum). A longstanding obstacle to efforts to illuminate genetic changes underlying obligate symbioses has been the inability to experimentally disrupt and reconstitute symbiont-host partnerships. Our experiments show that Buchnera can be experimentally transferred between aphid matrilines and, furthermore, that Buchnera replacement has a massive effect on host fitness. Using a recipient pea aphid matriline containing Buchnera that are heat sensitive because of an allele eliminating the heat shock response of a small chaperone, we reduced native Buchnera through heat exposure and introduced a genetically distinct Buchnera from another matriline, achieving complete replacement and stable inheritance. This transfer disrupted 100 million years (∼ 1 billion generations) of continuous maternal transmission of Buchnera in its host aphids. Furthermore, aphids with the Buchnera replacement enjoyed a dramatic increase in heat tolerance, directly demonstrating a strong effect of symbiont genotype on host ecology.

Keywords: Buchnera; aphid; maternal transmission; symbiosis; thermal tolerance.

Fig. 1.

Experimental approach for replacement of the native Buchnera symbionts within an A. pisum matriline. The recipient line (LSR1) contains a heat-sensitive Buchnera genotype, and the donor (5AY) contains a heat-tolerant Buchnera genotype. Native Buchnera are depleted by heat in the recipient line, and microinjection is used to flood the hemocoel with donor Buchnera. Most embryos are successfully colonized by the donor symbionts. In some cases, complete replacement occurs in the progeny of injected females. In other cases, progeny have a mixed Buchnera population, which can be shifted completely to the donor type through further heat exposure.

Fig. 2.

Screens for the presence of the LSR1 and 5AY Buchnera genotypes based on a PCR and diagnostic restriction digest. Each lane is from a screen of a single female aphid; lanes labeled 5AY and LSR1 are controls from stock aphids. LSR1 Buchnera contain a distinctive restriction site, yielding bands of 363 bp and 151 bp for LSR1 and a single band of 514 bp for 5AY. Results for set 1 are shown for the F1, F4, and F11 generations. Some LSR1 Buchnera remained in F4 females but were eliminated by a heat exposure in F5, and this was maintained in future generations, as shown by the assay in F11. In set 2, F1 individuals exhibited complete replacement of the original LSR1 Buchnera by the 5AY Buchnera.

Fig. 3.

Effect of symbiont replacement on aphid fitness under constant 20 °C and after heat exposure, measured as developmental time, adult weight, and fecundity (mean ± SE). The LSR1 matriline contains heat-sensitive Buchnera, whereas the LSR1 < 5AY matriline has a replacement Buchnera with normal heat tolerance. Replacement of Buchnera results in faster development, larger adult size, and increased fecundity after heat stress (****P < 0.0001, Kruskal-Wallis).