Rapid, asexual reproduction results in clonal lines of aphids that can be kept indefinitely under long-day conditions. This allows the manipulation of facultative symbiont presence through antibiotic curing or artificial infections while maintaining an essentially identical aphid genotype. Two pea aphid genotypes were used for this study, both collected from the UK (Supplementary Table S1). Genotype 218 was collected naturally infected with Fukatsuia and Hamiltonella, and was cured more than a year before use. This was achieved by feeding young aphids with broad bean leaves suspended in a tube of antibiotic solution (0.5% Gentomicin, 1% Ampicillin, 0.5% Cefotaxime in distilled water) over 4 days (McLean et al., 2011). Genotype 200 was collected naturally uninfected, harboring no known facultative symbionts. All aphid lines were screened for Hamiltonella, Regiella, Serratia symbiotica, Fukatsuia, Spiroplasma sp., Rickettsia sp., and Rickettsiella viridis following protocols in Tsuchida et al. (2010) and Ferrari et al. (2012) to ensure that they had the appropriate symbiont infection and were not infected with any other known facultative symbionts. The symbiont infections were regularly checked to detect possible contamination. The symbiont-specific PCR primers can be found in Supplementary Table S2. The PCR mix comprised 6.25 μl BioMix (Bioline), 0.1 μl (20 μM) of forward and 0.1 μl (20 μM) reverse primer, 5.55 μl distilled water and 1.0 μl sample DNA. The PCR reaction was performed at 94°C for 2 min, followed by 35 cycles of: 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. It concluded with 6 min at 72°C and then cooled the sample to 4°C indefinitely. PCR products were run on a 1% agarose gel and the presence of a band confirmed the presence of the symbiont.

Five aphid lines were used in the experiment, two uninfected with facultative symbionts (200 and 218) and the remainder infected singly with one of three facultative symbionts, Regiella, Fukatsuia, and Hamiltonella (Supplementary Table S1). Regiella was injected into aphid genotype 200, while the other two symbionts were injected singly into genotype 218. These five aphid lines comprise three pairwise comparisons between uninfected and infected aphids, with the uninfected line of 218 used in two comparisons. This design aimed to compare host fitness and symbiont densities within each pair across the same aphid genetic background and thus we conducted no analyses across multiple pairs. These specific isolates of symbiont were chosen because preliminary results indicated that they were likely to provide heat shock protection.

To produce these infections of Hamiltonella, Regiella, and Fukatsuia we used hemolymph injections from infected donor aphids (Supplementary Table S1). Hemolymph was extracted from donor aphids under a microscope using glass needles and re-injected into the appropriate aphid line and surviving aphids raised to adulthood. Glass needles were pulled from Kwik-Fil™ borosilicate glass capillaries (1B100-4, World Precision Instruments, 1 mm diameter) using a P-97 Flaming/Brown micropipette Puller (Sutter Instrument Co.). The offspring of the surviving aphids were tested for the successful establishment of the new infection when they were adults, and these aphid lines were retested regularly to ensure the maintenance of the new aphid-symbiont combinations. All injected lines had been maintained in the laboratory for at least a year before being used for experimental assays.

This assay was designed to understand symbiont dynamics after aphids have been exposed to heat shock. Aphids were exposed to either a single peak of high temperatures or were maintained at a steady control temperature. The “heat shock” temperature chosen was 38.5°C, which was based on a series of pilot studies (data not shown). Our aim was to find a temperature that had a strong negative effect on fitness, but at which approximately half the individuals in an aphid population still survived. The aim was to explore the phenotypes of the symbionts and not to model natural situations directly. The temperature experienced by aphids near the ground is often considerably higher than meteorological records and depends, for example, on aspect and slope of the site, but it is likely that aphids are exposed to similarly high temperatures in Northern England on hot summer days (Bennie et al., 2008; Suggitt et al., 2011).

To produce age-controlled populations of each of the five lines, groups of young adults were placed into petri dishes (9 cm diameter) that contained a single broad bean (Vicia faba var. Sutton Dwarf) leaf, placed in 2% agar. V. faba is a host plant that almost all pea aphids perform on (Ferrari et al., 2008). The adults were left to reproduce for 24 h at 20°C, and the offspring were subsequently put onto 2 week old V. faba plants in groups of 50 and enclosed in a vented, transparent cage. On the following day (aphid age 24–48 h) the populations were moved into cabinets where they were either exposed to heat stress or 20°C as a control. Temperature cabinets were rigorously checked before and during the experiments to ensure even distribution of heat and the same relative humidity (50%) in both cabinets. Plants containing aphids were also placed in a randomized block pattern within the cabinet to remove any potential effects of uneven heat distribution.

While the control treatment was left at 20°C, the temperature in the heat treatment was increased from 20 to 38.5°C steadily over the course of 2 h, held at 38.5°C for 4 h, and then decreased back to 20°C over a further 2 h. Surviving aphids from both treatments were moved onto fresh 2-week-old plants on the day after heat shock to mitigate any temperature effects on the plant itself. These plants were moved into a different controlled-temperature room (20°C), where aphids from both heat treatments were kept together until being collected for analysis.

Aphids were removed to measure symbiont density at two time points. The first was 24–26 h after the start of the peak heat shock period (when the aphids were 3 days old), and the second 11 days post-heat shock (when the aphids were 14 days old, ~6 days after an aphid would usually begin reproducing). These two time points were chosen to investigate symbiont densities immediately after stress, and to test if recovery by the onset of reproduction was possible. Buchnera densities are known to decrease as aphids age (Simonet et al., 2016) and so this second time point was chosen to be the potential highest density of Buchnera during an aphid's development.

The aphids were flash frozen using dry ice and kept at −80°C until DNA extraction. In addition, one surviving apterous individual from each group was placed on a petri dish with a V. faba leaf (as above) to measure the number of offspring produced. These dishes were refreshed every 3–4 days to ensure healthy V. faba leaves. Offspring counts continued until all aphids had died, measuring total lifetime fecundity. There were 5–6 replicates for fecundity counts and symbiont density for each of the five aphid lines in each treatment (i.e., 10–12 replicates in total for each line), these were performed in two temporal blocks with approximately half the replicates of each treatment in each block.

DNA was extracted from the aphids after samples were defrosted at room temperature. Aphids were homogenized in a 200 μl 5% Chelex solution made in distilled water. Ten microliters of proteinase K (10 mg/ml) was added per sample, and samples were incubated for 6 h at 56°C to facilitate digestion. They were then “boiled” at 100°C for 10 min before being centrifuged at 13,000 rpm for 3 min and the supernatant containing the DNA pipetted into a clean 1.5 ml Eppendorf tube which was stored at −20°C until use. Five aphids per replicate were pooled to generate a sample for the first time point (24–26 h after heat shock) and one aphid for the second time point (11 days later).

Samples were run in duplicate using SYBR^® Green reagent on a StepOnePlus™ Real Time PCR machine (Applied Biosystems). Each reaction consisted of 10 μl FAST SYBR 2 × mastermix (Applied Biosystems), 1 μl forward primer (7 μM), 1 μl reverse primer (7 μM), 6 μl nuclease-free water, and 2 μl DNA sample. qPCR primers for Regiella, Fukatsuia, Hamiltonella, and the aphid housekeeping gene elongation factor-1 alpha (EF-1α) (Supplementary Table S2) were tested to ensure high efficiency and similarity between primer sets. Melt curves were performed on each plate to ensure the primers were specific to each target and only bound once. Cycling conditions were 95°C for 20 s, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. The melt curve involved a further 95°C for 15 s, 60°C for 1 min and then a gradual increase to 95°C over 15 min. Each 96-well qPCR plate was analyzed using StepOne Software v2.2.2 (Applied Biosystems) and Cycle threshold (Ct) values were obtained by comparing each primer sample to a single standard curve of known concentration and using identical threshold and baseline levels for each primer target across plates. Standard curves were created by amplifying positive control samples using PCR, calculating DNA concentrations using a High Sensitivity DNA Assay on a 2100 Bioanalyzer system (Agilent), and then serially diluting the sample 1:10 with distilled water to create a 5-sample curve comprising known concentrations decreasing from 10 pmol/ml. Samples with Ct values over 30 were classed as negative, confirmed by our negative controls. This corresponds to a copy number of <52 per 2 μl sample for all primers, and is below the threshold of detection. Where the difference in Ct values between technical replicates was >1.5, the sample was either rerun or not used in the analysis.

The standard curves were used to calculate the DNA concentration of each sample, and this was converted into copy numbers per 2 μl of DNA extract. To control for aphid size and extraction quality, copy numbers for each sample were presented relative to those of a housekeeping aphid gene as a control, giving a ratio of symbiont copy numbers to aphid copy numbers.

Data were analyzed using the R software v. 3.4.1 (R Core Team, 2018). Since our core question was to test whether the three symbiont species can provide heat shock protection, but not to compare the extent of this protection, the data were analyzed separately for each symbiont species. Thus, in each analysis the infected line was compared with the same uninfected aphid genotype, within and across heat treatments. The data for the uninfected line 218 was therefore used twice, paired with line 218 infected with Hamiltonella or Fukatsuia. Similarly, we analyzed the two time points separately because symbiont densities change during aphid development, which would complicate the interpretation of the analysis.

Lifetime fecundity of the set of Regiella lines was analyzed using a general linear model assuming a normal error distribution. The number of offspring was the response variable, and temporal block, facultative symbiont presence, heat treatment and the interaction between symbiont presence and heat treatment were the explanatory variables. The sets of Fukatsuia and Hamiltonella lines were analyzed with a non-parametric Kruskal-Wallis test, because the model assumptions of parametric models were not met. This was followed by Wilcoxon tests to identify differences between specific treatments.

The densities of the symbionts were also analyzed with a general linear model assuming a normal error distribution. This was split into six analyses, separate for the lines relating to each symbiont species at each time point, to simplify the interpretation. For Buchnera densities, the explanatory variables were again temporal block, facultative symbiont presence and heat treatment as well as the interaction between the latter factors. For the densities of the facultative symbionts, only block and heat treatment were explanatory variables. In most cases model assumptions were met without transforming the data, only the Buchnera densities at the first time point in the Regiella lines were log-transformed. For Regiella densities at the first time point and Buchnera densities in the set of Regiella lines at the second time point, Kruskal Wallis and Wilcoxon tests were used as described for the fecundity data.

For all general linear models, post-hoc tests were only performed when the factor or interaction was significant in the main analysis. This was conducted using the R package “phia” (De Rosario-Martinez, 2015), with Holm's correction for multiple comparisons. All data are available as Supplementary Material (Data Sheets 2–4).

We exposed aphids to a short spike of high temperature and measured facultative and obligate symbiont densities and aphid fitness after 1 and 11 days. As expected, heat shock decreased the number of offspring produced in an aphid's lifetime in all three sets of lines [Regiella F_{(1, 19)} = 103.23, P < 0.001; Fukatsuia: W = 109, P = 0.03, Hamiltonella: W = 136.5, P = 0.001; Figure 1]. However, the extent of this decrease was modified by the presence of Regiella and Fukatsuia [Regiella, symbiont × heat treatment: F_{(1, 19)} = 5.78, P = 0.03; Fukatsuia: heat treatment in uninfected lines W = 36, P = 0.004 and in infected lines W = 13, P = 0.48]. Fukatsuia provided the greatest protection from heat as there was no difference in the fecundity of the infected lines in the control and heat shock treatment, whereas there was a greater reduction in fecundity in the uninfected aphids than in the infected aphids for the Regiella lines. In contrast, there was a similar decrease in fecundity for both uninfected and infected Hamiltonella lines following heat shock (uninfected: W = 36, P = 0.004; infected: W = 34.5, P = 0.008; Figure 1). At benign temperatures, two of the symbionts also affected lifetime fecundity: the presence of Hamiltonella increased lifetime fecundity (W = 4, P = 0.03), whereas Fukatsuia decreased it (W = 34, P = 0.013), and there was no difference for Regiella (Figure 1).

We measured the densities of the three facultative symbionts at two time points after exposure to heat, 24–26 h and 11 days post-heat shock (Figure 2). Compared to non-heat shocked controls the densities of two of the symbionts, Fukatsuia and Hamiltonella, were lower on the day after heat shock [Fukatsuia: F_{(1, 8)} = 65.05, P < 0.001, Hamiltonella: F_{(1, 8)} = 6.64, P = 0.03], whereas densities of Regiella are unaffected (W = 28, p = 0.13; but note that this is significant in a less conservative parametric test). By the second time point, taken when the aphids were young adults, there was no difference between population densities in heat stressed or control aphids for any of the three facultative symbionts [Fukatsuia: F_{(1, 8)} = 0.35, P = 0.57, Hamiltonella: F_{(1, 7)} = 0.95, P = 0.36, Regiella: F_{(1, 6)} = 0.01, P = 0.91; Figure 2], suggesting that heat did not have long-term effects on facultative symbiont populations.

Compared to the control treatment densities of Buchnera were decreased on the day after heat shock in each of the three pairs of lines, regardless of facultative symbiont infection [Regiella lines: F_{(1, 19)} = 80.36, P < 0.001, Fukatsuia lines: F_{(1, 18)} = 70.71, P < 0.001, Hamiltonella lines: F_{(1, 18)} = 56.07, P < 0.001; Figure 3]. Regardless of treatment, Buchnera densities were higher in the lines harboring Fukatsuia [F_{(1, 18)} = 33.86, P < 0.001] or Hamiltonella [F_{(1, 18)} = 24.52, P < 0.001] compared to uninfected lines, an effect that was not seen in the Regiella lines [F_{(1, 19)} = 0.15, P = 0.71]. For the Fukatsuia and Hamiltonella lines there was also a significant interaction between symbiont presence and heat treatment [Fukatsuia: F_{(1, 18)} = 9.57, P = 0.006; Hamiltonella: F_{(1, 18)} = 4.80, P = 0.04]: in both cases, Buchnera densities in the control treatment were higher in lines with facultative symbionts compared to uninfected aphids but there was no difference between these lines after heat shock. The interaction was not significant for the Regiella lines [F_{(1, 19)} = 2.58, P = 0.13] where the extent of the loss of Buchnera did not differ between infected and uninfected lines.

At the later time point, when aphids were young adults, heat shock again reduced Buchnera densities on average [Regiella lines: W = 105, P = 0.004; Fukatsuia lines: F_{(1, 19)} = 6.85, P = 0.02; Hamiltonella lines: F_{(1, 17)} = 24.84, P < 0.001]. Fukatsuia presence on average also significantly increased the density of Buchnera regardless of treatment which was due to high Buchnera densities in the heat shocked aphids [F_{(1, 19)} = 7.89, P = 0.01]; a difference that was not found for Regiella (W = 46, P = 0.37) or Hamiltonella presence [F_{(1, 17)} = 0.17, P = 0.69]. Importantly, there was a significant interaction between symbiont infection and temperature in the Fukatsuia lines [F_{(1, 19)} = 7.55, P = 0.01] and an equivalent effect in the Regiella lines (heat treatment in uninfected lines: W = 36, P = 0.004 and in infected lines: W = 17, P = 0.42) that was not seen for the Hamiltonella lines [F_{(1, 17)} = 1.95, P = 0.18]: Buchnera densities after heat shock were significantly reduced in uninfected aphids but not when Fukatsuia or Regiella were present.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.