Aphid feeding damage to wheat plants when infested with Rickettsiella, Regiella or wild type D. noxia.

Five age–matched D. noxia were placed on individual wheat plants. Wheat plants without aphids were included for comparison. (A) Aphid feeding damage to wheat plants when infested with Rickettsiella, Regiella, wild type D. noxia or when lacking aphids at Week 3.5. Each treatment had 10 replicates and measured traits were (B) tiller number, (C) leaf number, (D) total leaf area, (E) chlorotic streaking, as well as an (F) aphid infestation score and (G) overall plant damage score. Different letters represent significant pairwise differences between treatments by Bonferroni-adjusted tests. Dots represent means across all replicate wheat plants, and error bars indicate 95% confidence intervals.

Principal Component Analysis of all GC-MS components showing metabolic profiles:

(A) Schematic showing the feeding and non-feeding areas the wheat plants used in this experiment. Sixty age-mixed aphids were divided into two plastic vials. The top and bottom leaves of each plant were inserted into a vial and a sponge plug was placed into the vial openings and left for 7 days. Control plants were treated in same manner, except no aphids were added to the vials. After a 7-day infestation period, all aphids were removed and the plants remained for an additional 7 days before phytohormone analysis was undertaken. (B) Profiles of aphid feeding areas versus non-feeding areas, (C) profiles of the three aphid strains and no-aphid control on the feeding areas, and (D) profiles of the three aphid strains and no-aphid control on the non-feeding areas. Dots show data from individual replicate plants (n = 4 per aphid strain), and ovals show 95% confidence regions. For (B), data from areas with no aphids were included as non-feeding areas. For (C) and (D), the same feeding areas with no aphids were included as controls for comparison with the different aphid feeding treatments.

Rickettsiella effects on aphid population growth and alate production on whole plants.

(A) Experimental design for Rickettsiella. Five age-matched Rickettsiella or wild type aphids were placed onto wheat plants (GS14). At Day 14, 21, and 25, aphids from three replicate plants per treatment were removed and the number of alate adults, apterous adults and nymphs was recorded. (B) The number of Rickettsiella adults (alate plus apterous adults), (C) the percentage of Rickettsiella alates and (D) the number of Rickettsiella nymphs were used to characterize aphid population growth and alate production over time. “*” indicates significant differences (p < 0.05) between endosymbiont and wild type treatments at each time point, as determined by independent sample t-tests.

Regiella effects on aphid population growth and alate production on whole plants.

(A) Experimental design for Regiella. Five age-matched Regiella or wild type aphids were placed onto each of the top, middle and bottom leaves of wheat plants (GS14). A plastic vial was placed over each leave and cotton wool inserted into the vial opening to prevent aphid movement. At Day 4, 8, 12, and 16, aphids from four replicate plants per treatment were removed and the number of alate adults, apterous adults and nymphs was recorded. (B) The number of Regiella adults (alate plus apterous adults), (C) the percentage of Regiella alates and (D) the number of Regiella nymphs were used to characterize aphid population growth and alate production over time. “*” indicates significant differences (p < 0.05) between endosymbiont and wild type treatments at each time point, as determined by independent sample t-tests. Development stages of aphids on different wheat leaves are shown in Figure S9.

Rickettsiella effects on aphid dispersal and plant feeding damage to wheat plants in mesocosms.

(A) Experimental design. Populations were initiated with 5 Rickettsiella or wild type aphids on wheat plants (‘aphid-release wheat’) while adjacent plants (‘aphid-spread wheat’) were monitored. All plants were measured for (B) leaf number, (C) total leaf area, (D) chlorotic streaking, (E) aphid colonization rate, as well as (F) an aphid infestation score and (G) overall plant damage score. “*” and “**” represent significant differences at p < 0.05 and p < 0.01, respectively when comparing Rickettsiella and wild type aphids on aphid-release wheat. “Δ” and “ΔΔ” represent significant differences at p < 0.05 and p < 0.01, respectively when comparing Rickettsiella and wild type aphids on aphid-spread wheat. Aphids collected from the middle tray (“dispersal”) were counted and screened to determine (H) the percentage of alate and apterous adults in the middle that were positive for Rickettsiella, (I) the number of alate adults, and (J) the number of apterous adults. Symbols in (H) represent the percentage of 20 alate and 20 apterous adults positive for Rickettsiella at Weeks 2 and 3. “**” represents a significant difference at p < 0.01 as determined from a paired t-test.