(a) S. symbiotica experiment
Exposure to heat shock at both the day 2 and day 6 stages had drastic effects on A. pisum performance. Compared to control aphids, heat-shocked individuals suffered from prolonged development as well as large reductions in survival and fecundity. Many aphids were sterilized by heat shock, and some gave birth to malformed offspring whose limbs were stuck to the thorax and abdomen. These aphids were visually distinguishable from healthy offspring and typically died within hours of birth. We did not include such individuals in fecundity measures.
Though all lines performed poorly when exposed to heat shock as 2 day olds, aphids infected with S. symbiotica isolates were substantially more fit than uninfected, genetically identical aphids under this treatment. S. symbiotica infection led to a higher odds of survival, with advantages over the uninfected line ranging from 19 to 30% according to logistic regression (). Similarly, S. symbiotica-infected aphids reached adulthood significantly faster than uninfected aphids, according to Wilcoxon rank-sum tests (). Uninfected aphids matured in an average time of 13.3 days. Infected lines matured, on average, at 11.0 (SsAZ), 11.6 (SsNY) and 12.4 (SsWI) days—revealing a 1–2 days acceleration conferred by the symbionts. There were also differences among the infected lines, with SsAZ reaching adulthood faster than both SsNY (p<0.0001) and SsWI (p<0.0001) and SsNY maturing faster than SsWI (p=0.0002).
Logistic regression analyses of aphid survival.
Time to adulthood for uninfected and S. symbiotica-infected A. pisum.
S. symbiotica-infected aphids heat-shocked as 2 day olds were far more fecund than aphids lacking secondary symbionts (b). Whereas uninfected aphids produced an average of 8.3 offspring per female, infected insects gave birth to averages of 19.8 (SsWI), 20.3 (SsNY) and 29.7 (SsAZ) offspring per female. This was due, in part, to higher sterility in uninfected aphids. Of aphids surviving 8 days of adulthood, 63% of uninfected individuals produced no offspring compared to 2–29% of those harbouring S. symbiotica.
Figure 1 Distributions of fecundity, for single females varying in their symbiont infection status. Offspring production of single aphid females is represented using frequency histograms. Means, medians and sample sizes are presented for each line under each of (more ...)
Lines infected with S. symbiotica also differed in their average fecundities. Specifically, aphids from the SsAZ line produced more offspring than those from either SsWI or SsNY (SsAZ versus SsWI: p=0.0047; SsAZ versus SsNY: p=0.0012), revealing variation in heat tolerance capacity among symbionts.
In aphids exposed to heat shock as 6 day olds, S. symbiotica was less beneficial. Fecundities of all aphids were reduced compared to those of aphids heat shocked on day 2. Average numbers of offspring produced by single females were 4.5 (N), 8.6 (SsWI), 2.8 (SsNY) and 10.9 (SsAZ; c). The SsWI and the SsAZ lines were both more fertile than the secondary-free line (Wilcoxon rank-sum test, p=0.0345 for SsWI versus N and p=0.0705 for SsAZ versus N). No other differences approached significance in these analyses. Overall, sterility was more common within infected lines exposed to heat shock on day 6 than those exposed on day 2, with 31–40% of S. symbiotica-bearing females yielding no offspring over 8 days of adulthood. In comparison, 59% of secondary-free aphids failed to produce any offspring during their first 8 days of maturity, indicating that sterility remained more prevalent in aphids without secondary symbionts.
Time to adulthood was shorter when aphids were heat shocked on day 6, compared to day 2, with averages ranging from 10.1 to 10.5 days (). Only SsWI and SsAZ aphids reached adulthood significantly earlier than uninfected aphids (Wilcoxon rank-sum test, p<0.0001 for SsAZ versus N and p=0.0375 for SsWI versus N). These accelerations were each less than 0.5 days in magnitude, in contrast to the 1–2 day accelerations when aphids were heat shocked as 2 day olds. Again, there were significant differences among infected lines, with SsAZ reaching adulthood faster than both SsWI (p=0.0003) and SsNY (p=0.0329).
Under the day 6 heat shock treatment the odds of survival for infected lines were reduced by 25–37% compared to uninfected aphids. Thus, symbiont infection imposed a survivorship cost when older juveniles were heat shocked (). Combining these data on survival, fecundity, and development time into an age-structured Leslie matrix model revealed, that the SsAZ and SsWI lineages had slightly higher fitness (intrinsic rates of increase) than the uninfected line, though SsNY was slightly less fit (A. Ives, unpublished data).
The highest levels of fecundity and survival, and the fastest development times, were obtained for aphids reared at a constant 18
°C throughout their lifespan (a
; and ). Under these conditions, there were no significant effects of S. symbiotica
on aphid fitness with two exceptions. First, there was a slight reduction in survival of SsWI
aphids compared to those without secondary symbionts. There was also a slight developmental acceleration within symbiont-bearing lines. Aphids harbouring S. symbiotica
reached adulthood at 9.6–9.8 days, on average, compared to 9.9 days for uninfected aphids; these developmental accelerations were statistically significant for all infected lines according to Wilcoxon rank-sum tests (). The magnitudes of these differences (2–4%) were comparable to those observed for the day 6 heat shock treatment, though substantially less than under the day 2 treatment (7–18%).
(b) H. defensa and R. insecticola experiments
To determine the generality of symbiont-mediated heat tolerance, we performed similar experiments with additional microbes, comparing the fitness of aphids infected with H. defensa or R. insecticola to that of secondary-free aphids. Again, heat shock on day 2 had drastically negative effects, causing substantial mortality, reduced fecundity and prolonged development.
The effects of the H. defensa and R. insecticola symbionts were, generally, less pronounced than those of S. symbiotica isolates. However, both had significant effects on survival under the heat shock treatment (). Uninfected aphids were 24% more likely to survive to adulthood compared to R. insecticola-bearing aphids after heat shock on day 2 (, logistic regression, p=0.0439). In contrast, H. defensa improved survival of heat-shocked aphids by 34%, relative to uninfected aphids (p=0.0111), on a par with the advantages conferred by S. symbiotica subjected to the same treatment. The significance (but not the trend) of this latter difference depended on the inclusion of one of the fourteen heat-treated H. defensa-infected cultures in which 20/20 individuals survived to adulthood.
Sterilization was also common among heat-treated aphids in this experiment, with 55% of all aphids that survived 8 days of adulthood failing to produce offspring. The average numbers of offspring produced per female were 1.4 (Ri, n=19), 3.7 (N, n=34) and 6.21 (Hd, n=29). These values were statistically indistinguishable, though our analyses were limited by small sample sizes imposed by high mortality. Neither H. defensa nor R. insecticola caused significant changes in the development time of heat-shocked aphids ().
Time to adulthood for uninfected, H. defensa- and R. insecticola-infected A. pisum.
Under the control treatment, we observed no effects of symbionts on either survival () or development time (). Average fecundities per female were 29.0 (N, n=38), 26.9 (Hd, n=34), 28.3 (Ri, n=40) under control conditions; these values were also indistinguishable according to statistical tests (data not shown).