APV transmission assays: To measure maternal transmission rates and whether H. defensa impacts vertical transmission, we reared APV-infected adult aphids of the same genotype with (ND18.H3) or without (ND18) H. defensa individually in petri dishes (55mm x 15mm) containing a single V. faba leaf. Adult aphids were monitored for the production of offspring approximately every 30 min. Since APV can be transferred from infected aphids to plants during feeding we replaced fava leaves every 2-3 hours. Nymphs were collected 1-30 min after birth then surface sterilized in a 1% bleach solution and transferred to a new fava leaf containing Petri dish. Newborn aphids were individually reared to adulthood to prevent possible aphid to aphid horizontal transmission through the leaves. We allowed the first-generation cohort to produce offspring and develop into third-fourth instar nymphs before screening for APV infection using the diagnostics previously described. Fisher’s Exact Test was used to compare rates of maternal APV transmission among lines. To rule out rapid horizontal transmission in our Petri dish arenas that would potentially inflate estimates of vertical transmission, we also conducted a control assay, mimicking the conditions described above by allowing single APV+ adults to feed on a single V. faba leaf in a Petri dish. Adults were allowed to feed continuously for 1 h before removing them and any offspring they produced. We then added 8-10 second instar -APV aphids, which were allowed to feed for 30 min on the leaves previously fed upon by +APV before being separated and reared individually in petri dishes with a fresh V. faba leaf. These aphids were then allowed to develop into fourth instars and screened by PCR for the presence of APV.
Horizontal transmission of APV through plants was assessed by placing a single V. faba in cup cages with three +APV aphids (donor) and three -APV- aphids (recipient) which were distinguished by using 4 different donor and recipient lines lacking facultative symbionts that differed in color (pink or green morphs). Cup cage arenas were maintained at 20° C under 16 h light (L): 8 h dark (D) photoperiod. Eight third or fourth instar donor and recipient aphids were then collected after 1 or 3 weeks and screened by qPCR as described above to assess APV infection status. We also conducted assays to determine if oviposition by A. ervi could horizontally transfer APV from infected to uninfected aphids. A female A. ervi was allowed to oviposit into an +APV aphid and then immediately moved to a separate arena and allowed to oviposit into three -APV aphids in rapid succession. The three parasitized -APV aphids were identified by the order in which oviposition occurred and then placed into separate petri dishes with a single V. faba leaf. We allowed parasitized APV- recipient aphids to develop into fourth instars before screening them for APV infection as above.
Fitness measures: Aphid fecundity in different lines of +APV and -APV aphids was estimated by allowing cohorts of five fourth instar aphids to develop into adults on a single V. faba (equals 1 replicate). The number of offspring produced in each cup cage was carefully removed and counted every 3 or 4 days. In total, there were 9 replicates for each aphid line. Aphid mortality was also recorded and used to assess 50% survivorship. Aphid reproduction was analyzed using Analyses of Variance (ANOVA) with Tukey’s HSD to compare means among aphid lines. Aphid survival data was fit to a lognormal distribution to estimate 50% survival time.
Enemy challenge assays: Cohorts of 20 aphids that were 48-72 h old (second instars) were singly parasitized by a mated A. ervifemale and then placed onto a fresh V. faba plant in a cup cage (=1 replicate). A total of 8 replicates were conducted for each experimental aphid line (160 parasitized aphids per line). After parasitism, cup cages were maintained at 20° C under a 16 h light:8 h dark photoperiod. Ten days post-parasitism, we recorded the number of aphids that survived, mummified (a pupating wasp), or both aphid and wasp died (dual mortality) (Oliver et al. 2012). Results were then analyzed by logistic regression analyses. Since parasitoid fitness is often linked to host health, we measured hind tibia length to estimate the size of A. ervi eclosing from APV+ and APV- aphids, which served as a proxy for wasp quality (Godfray and Godfray 1994, van Lenteren 2003). One day old adult A. ervi were frozen overnight at -20°C and then dried at 60°C for 24 hours before measuring hind tibia length using an Olympus SZX16 stereomicroscope equipped with CellSens software (v. 1.4.1). Wasp tibia length was analyzed by One-way Analyses of Variance (ANOVA) to compare mean tibia length between APV+ and APV- aphid lines.
To assess whether APV infection affects fungal protection conferred byR. insecticola , we challenged aphids with P. neoaphidis as previously described (Weldon et al. 2020). Ten cohorts of ten 9-day old (early adult) aphids (total 100 aphids) from each R. insecticolaexperimental line (Table 1) were then exposed to two sporulating aphid cadavers placed in a 35 mm diameter deep Petri dish with 1.5% agar for 90 minutes. Fungal plates were inverted over aphids to mimic a natural spore shower and rotated every fifteen minutes between replicates to normalize spore exposure. Each cohort was then placed onto a freshV. fava plant and kept at 20°C with 100% humidity (via an unvented cup lid) for 24 hours under 16:8 L:D hour light cycle. After 24 hours, the unvented lid was replaced with a vented lid. Aphids were monitored every twenty-four hours for ten days post-exposure for aphid survival, dual mortality (aphid and pathogen), and fungal sporulation. The results were analyzed using logistic regression.
APV and symbiont abundance: We estimated APV and symbiont abundance by measuring genome copy number of each. Briefly, +APV and -APV adult aphids from a given experimental line were placed in separate cup cages with a fresh V. faba plant and allowed to reproduce for approximately 24 hours. Thereafter, all adults were removed and offspring were allowed to develop. Aphids were then sampled at 2, 4, 8 and 16 days old. APV genome copy number was then estimated by generating cDNA templates from 6-8 aphids at each time point (biological replicates) as described above followed duplicate qPCR (technical replication) for each sample using APV-specific primers and reaction conditions as described above. APV genome copy number per sample was then estimated by plotting the data against a standard curve generated by serial dilution of a plasmid containing the APV amplicon and normalized using a single copy aphid gene (Ef-1α ). Relative genome copy number for H. defensa was similarly determined at the same time points using previously reported primers that amplify a region of the H. defensa dnaK gene (Weldon et al. 2013, Martinez et al. 2014) while relative genome copy number for R. insecticola was determined using primers designed during this study (Reg_dnaK_Q_F: 5’-TGGTGCAGCAAAAAGTG AAG-3’ and Reg_dnaK_Q_R: 5’-CACCCATGGTTTCAATACCC-3’) that amplify a region of the R. insecticola dnaK gene. Cycle conditions for the R. insecticola primers were 95° C for 5 min; 40 cycles of 95° C for 10 s, 60° C for 10 s, 72° C for 10 s, and a final extension at 72° C for 2 min. Relative abundance of each symbiont was then determined by the 2-(ΔCT)method (Livak and Schmittgen 2001). Results were log10 transformed, and the distributions of symbiont titers in each experimental line at each time point were checked for normality using the Goodness-of-fit test. Transformed titers were then compared using ANOVA with Tukey’s post-hoc Honest Significant Difference (HSD) test. Both analyses as well as all other statistical tests performed during the study were performed using JMP Pro v. 14.0 (SAS Institute Inc., Cary, NC).