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

Five 10-day-old apterous females 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 replicate plants 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 14-day-old Rickettsiella or wild type apterous female 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. An independent sample t-test was used to compare differences between endosymbiont and wild type treatments at each time point, with “*” indicating significant differences (p < 0.05).

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

(A) Experimental design for Regiella. Five 14-day-old Regiella or wild type apterous female aphids were placed onto each of the top, middle and bottom leaves of wheat plants (GS14). A plastic vial was placed over each leaf and cotton wool inserted into the vial opening to prevent aphid movement. At Days 4, 8, 12, and 16, aphids were removed from replicate plants in each treatment, and the numbers of alate adults, apterous adults, and nymphs were recorded. Four replicate plants per treatment were sampled at Days 4 and 8, five plants infested with Regiella and wild type aphids were sampled at Day 12, and the remaining plants were sampled at Day 16. (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. The independent sample t-test was used to compare differences between endosymbiont and wild type treatments at each time point, with “*” indicating significant differences (p < 0.05). Development stages of aphids on different wheat leaves are shown in Figure 4-figure supplement 3.

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 per plants on four 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 containing eight wheat plants (“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.

Summary of effects of Rickettsiella and Regiella transinfections in Diuraphis noxia

Aphid feeding damage to barley plants when infested with Rickettsiella, Regiella or wildtype D. noxia and effects on aphid populations.

Five 10-day-old apterous females were placed on barley plants. Clean barley plants without aphids were included for comparison. (A) Aphid feeding damage to barley plants when infested with Rickettsiella, Regiella, wildtype D. noxia or when lacking aphids at Week 3. Each treatment had 9 replicates and measured traits were (B) tiller number, (C) leaf number, (D) total leaf area, (E) chlorotic streaking, as well as (F) an aphid infestation score and (G) overall plant damage score. At Week 3, aphids from each treatment were removed and the number of alate adults, apterous adults and nymphs recorded. (H) The number of adults (alate plus apterous adults), (I) the percentage of alates and (J) the number of nymphs were used to characterize the development stage of aphids. Different letters represent significant pairwise differences between treatments after Bonferroni correction. Dots represent means across all replicate barley plants, and error bars indicate 95% confidence intervals.

Comparison of jasmonic acid (JA), jasmonic acid-isoleucine (JA-Ile), and salicyclic acid (SA) levels in wheat plant areas exposed to aphid feeding (A–C) and without direct feeding (D–F).

Four replicates were set up for each treatment: Rickettsiella, Regiella, wildtype aphids, and control plants (no aphids). Dots represent data from individual wheat plants, and horizontal black lines depict medians. Error bars indicate 95% confidence intervals.

Heatmap of the top 50 GC-MS components in (A) feeding areas and (B) non-feeding areas in wheat plants.

Four replicates were set up for each treatment: Rickettsiella, Regiella, wild type, and control plants (no aphids). Components marked with ‘*’ showed significant differences compared with the wildtype aphids, with the red-marked component indicating differences in plants exposed to Rickettsiella aphids and the blue-marked component indicating differences in plants exposed to Regiella aphids.

Levels of components showing significant differences among transinfected aphids compared to wild type in wheat plants subjected to aphid feeding.

Solid dots (A, B) represent feeding areas, and open dots (C, D) represent non-feeding areas. Four replicates were set up for each treatment: Rickettsiella, Regiella, wild type, and control plants (no aphids). Different letters show significant differences in pairwise comparisons after Bonferroni correction. Error bars indicate 95% confidence intervals.

Effects of Rickettsiella on the life history, body color, body shape and Buchnera density of D. noxia.

Thirty aphids from Rickettsiella or wildtype reared at 19°C and 25°C were individually measured for life history parameters: (A) development time, (B) lifetime fecundity, and (C) longevity. Aphids reared at 19°C were measured for (D-F) body color components and (G) body shape (expressed as the ratio of body length/body width). We also quantified (H) Buchnera and (I) Rickettsiella density relative to the host actin gene, where numbers represent the average difference in Cp values between endosymbiont and actin markers, transformed by 2ΔCp. Body color was separated into three components: (D) Hue, (E) Saturation and (F) Lightness. Dots represent data from individual aphids, and horizontal black lines depict medians. Error bars indicate 95% confidence intervals.

Effects of Regiella on the life history, body color, body length and Buchnera density of D. noxia.

Thirty aphids from Regiella or wildtype reared at 19°C and 25°C were individually measured for life history parameters: (A) development time, (B) lifetime fecundity, (C) longevity, (D-F) body color components, and (G) body length. In addition, (H) Buchnera and (I) Regiella density relative to the host actin gene were measured at 19°C. Numbers represent the average difference in Cp values between endosymbiont and actin markers, transformed by 2ΔCp. Dots represent data from individual aphids, and horizontal black lines depict medians. Error bars indicate 95% confidence intervals.

Population dynamics of Rickettsiella in mixed cages at 19°C and 25°C.

(A) Experimental design. Populations were initiated with thirty Rickettsiella and thirty wildtype aphids on wheat plants placed in BugDorm cages. Five replicates were established with six wheat plants per cage and maintained at either 19°C or 25°C. Samples of 60 aphids were transferred to new plants every 3 weeks at 19°C and every 2 weeks at 25°C. The remaining aphids were stored for Rickettsiella infection dynamics at (B) 19°C and (C) 25°C. We tracked the Rickettsiella infection rate for individuals with a high density of the endosymbiont (with Cp<=30) and low density (Cp>30) of the endosymbiont at (D, F) 19°C and (E, G) 25°C. Aphids were considered negative if Cp values were absent and Tm values were not within the range of positive controls (87.5-88.3). Dots represent the proportion of individuals testing positive for Rickettsiella at 15 aphids per time point, per replicate cage. Infection density data are shown in Figure 4—figure supplement 3.

Population dynamics of Regiella in mixed cages at 19°C and 25°C.

(A) Experimental design. Populations were initiated with thirty Regiella and thirty wildtype aphids on wheat plants placed in BugDorm cages. Five replicates were established with six wheat plants per cage and maintained at either 19°C or 25°C. Samples of 60 aphids were transferred to new plants every 3 weeks at 19°C and every 2 weeks at 25°C. The remaining aphids were stored for Regiella infection dynamics at (B) 19°C and (C) 25°C. We tracked the Regiella infection rate for 20 apterous aphid individuals with a high density of the endosymbiont (with Cp<=30) and low density (Cp>30) of the endosymbiont at (D, F) 19°C and (E, G) 25°C. Dots represent the proportion of individuals testing positive for Regiella at 20 aphids per time point, per replicate cage.

Effects of Regiella on aphid development stage on the top, middle, and bottom leaves of wheat plants.

At Day 4 and Day 8, aphids from four replicate plants per treatment were removed from different plant leaves and the number of alate adults, apterous adults and nymphs recorded. At Day 12, aphids were collected from five replicate plants, while aphids from the remaining plants were collected at Day 16. “*” and “**” represent significant differences between Regiella and wildtype treatments at p < 0.05 and p < 0.01, respectively, as determined by independent sample t-tests.

Rickettsiella Cp values in (A) routine screening of laboratory Rickettsiella colonies and (B) the mixed cage experiment assessing endosymbiont frequency changes over time at 19 °C and 25 °C.

The dark red area represents overlap between the 19°C and 25°C experiments. Cp values represent the quantification cycle values obtained from qPCR, with lower Cp values indicating a higher amount of Rickettsiella target DNA. This figure shows the typical range of Cp values observed in our laboratory Rickettsiella-infected aphid colonies (A). We used this range as a reference for identifying aphids that acquired Rickettsiella through horizontal transmission, as horizontally infected aphids generally showed much higher Cp values than vertically infected aphids.