Field trials
Field trials were conducted in three raised beds (1 × 2 × 0.6 m) on the Penn State University campus from July to August 2020. The raised beds were separated by at least 8 m to avoid treatment cross-contamination. Faba bean (Vicia faba L.) seeds were planted at a density of 20 seeds/ m2 (50 plants per bed), and each bed was caged using a metal-framed tent. “Noseeum” nylon mesh (Outdoor Wilderness Fabric s, Inc., Caldwell, ID) was draped over the frame and the edges buried in the soil of the bed. The sides of the cages were fastened closed with zippers to allow access.
Insects
Aphid and predator beetle colonies were raised separately on faba bean plants in cages (BugDorm 20 cm × 40 cm × 20 cm, BioQuip Products, Inc., Rancho Dominguez, CA) in the field. Larvae and adults of predator beetles were fed with a combination of A. pisum and Rhopalosiphum padi every other day (Supplementary information Fig. S1). Trials involving plants, insects, and entomopathogenic fungi were conducted according to institutional, national, and international guidelines and legislation.
Fungal inoculations (Beavueria bassiana)
We released first instar aphid nymphs on each faba bean plant on the raised beds (~ 1100 aphids) by gently shaking plastic containers with groups of 20 nymphs and placing them on the plants using a paintbrush. They were allowed to grow and reproduce for fifteen days. During the night, we sprayed spore suspension of the Beauveria strain GHA (BotaniGard ®, MT, USA) at 1.4 × 106 and 1.4 × 1012 spore ha−1, low and high load respectively. Two days after inoculation, we collected adult aphids (~ 4–5 days old) from the experimental plots and measured physiological parameters (see details below). Next, we released 300 adult beetles inside each aphid–fungal inoculated cage, allowed them to feed for 2–3 days in our experimental cages, and then collected beetles for physiological measurement.
Identification of critical thermal limits (CTMax and CTMin) of healthy and infected insects
To determine critical thermal maximum for locomotion (CTMax) of healthy and infected individuals of each species, we employed a protocol modified from Ribeiro et al.25, using a hotplate with a programmable heating rate controlled by a computer interface (Sable Systems, LV, USA). The temperature was monitored by independent thermocouple channels connected to a Hobo 4-channel data logger. One thermocouple was attached to the surface of the hotplate, and the other sensor was attached inside the glass tube plugged by a cotton ball in which we placed an individual insect. This equipment was located inside an automated thermal chamber (interior dimensions: width 40.5 cm × 35 cm length × 40 cm height). We transferred an adult aphid (4-day-old) into the glass tube and exposed it to increasing temperatures at a rate of 0.3 °C min−1 until its locomotion stopped. CTMax was recorded when the insect turned upside down and could no longer return to the upright position within 5 s. The insect was returned to a faba bean leaf for recovery (n = 10 individuals per treatment).
To measure the critical thermal minimum for locomotion (CTMin) of healthy and infected individuals of each species (n = 10 individuals per treatment), we used an insulated incubator where the temperature was monitored by independent thermocouple channels connected to a Hobo 4-channel data logger. The sensors were attached inside three glass tubes, each tube with an adult (3 to 4-day-old), and plugged by a cotton ball. The glass tube was exposed to decreasing temperature at a rate of 0.3 °C min−1 until its locomotion stopped. CTMin was recorded when no movement was recorded within 5 s. The insect was returned to an aphid-infested faba bean leaf for recovery. Data were only considered valid if the insect displayed normal activity 2 h after a CTMax or CTMin test.
Impacts of infection on voluntary exposure aphids and predator beetles to extreme thermal zones
To examine how voluntary exposure to ETZ was affected by fungal infection, we collected aphids and predator beetles (3 to 5 day-old) from our field plots and transferred them to a dark plastic bottle. Next, a bottle containing the insects was attached to a choice test arena following a modified protocol from Navas et al.24. This experimental arena allows insects to freely move across extreme temperatures to access food in containers located at each end of the device. To reach food, individuals had to cross an ETZ, either warm or cold. The location of each insect was recorded after 60 min, and it was classified as: exploration for individuals that left the initial black bottle, warm or cold ETZ crossings. The experiment was replicated ten times for each species and treatment condition [aphid: healthy, infected (low and high spore load); predator beetle: healthy, infected (low and high spore load)].
Effects of fungal infection and thermal conditions (critical thermal limits and voluntary exposure to ETZs) on longevity of aphids and predator beetles
To examine whether fungal infection and thermal conditions alter longevity in aphids and beetles, we isolated three individuals from each factor combination (low, high fungal load, CTMin, CTMax, behavior: crosses to ETZ cold, warm, and no cross) from previous experiments, and counted the number of days the adults survived after the exposure to the thermal condition (n = 3 factor combination).
Energetic cost associated with fungal infection of aphid and predator beetles under critical thermal limits and voluntary exposure to ET
Intracellular ATP content was determined in neutralized perchloric acid extracts and by a spectrophotometric coupled enzyme assay, based on modified protocol from Churchill and Storey26 content (n = 3 per treatment condition). An insect was ground to powder using a mortar and pestle cooled in liquid nitrogen, and then weighed into 1.5 mL microcentrifuge tubes (Eppendorf). Powder was dissolved with 0.1 mL ice-cold TE buffer (50 mM Tris–HCl, pH 7.5 plus 1 mM EGTA) and homogenized by sonication (15 s, three times), using a Q500 Sonicator system (QSonica, Newtown, CT, USA). An aliquot (10 µL) of the well-mixed homogenate was removed for protein determination. Cells were lysed by adding 6% (v/v) ice-cold perchloric acid, strongly vortexed for 2 min and incubated at 4 °C for 10 min. Next, the cell homogenate was centrifuged at 14,462 rpm and 4 °C for 5 min. The resulting supernatant was neutralized by adding KOH/Tris (3 M/0.1 M) and centrifuged again to discard the perchlorate salts. Extracts were kept at 4 °C for their immediate utilization. ATP content was determined spectrophotometrically by following the production of NADPH at 340 nm (ε = 6.22 mM−1 cm−1) and using CARY WinUV-Vis Spectrophotometer (Agilent, Santa Clara, CA, USA). The following reagents were used for the spectrophotometric coupled enzyme assay: 5 U Hexokinase, 10 U Glucose 6-phosphate dehydrogenase, 1 mM NADP + , 5 mM MgCl2 and 10 mM Glucose in HE buffer (100 mM Hepes-HCl plus 1 mM EGTA, pH 7.0) at 25 °C. Chemicals were purchased from Roche (Manheim, Germany) and Sigma (St Louis, MO, USA).
Infection status
We used two different protocols to confirm fungal infection: (1) placing each individual in wet towel paper inside a Ziploc bag to observe hyphal growth27. (2) For insects used in ATP measurements, we followed a modified protocol from Wraight and Ramos28 and Castrillo et al.29. Insect were washed using a serial dilution technique, vortexed for 10 s, and mounted in a drop of lactophenol blue, diluted with distilled water. We then preserved insect body parts (i.e., legs and abdomen terga) at − 80 °C for 12 months and placed in Petri dishes containing potato dextrose agar (PDA HiMedia-GM096) medium (pH 6.8), and incubated for ten days. To confirm infection by B. bassiana, we observed plates every 3 days, identified fungal growth (dense white mycelia), then randomly chose three samples, collected mycelia, and DNA was extracted using PureLink Genomic DNA Kit (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer’s protocol. Next, we used PCR essays (25 µL) contained 1 × Q5 Hot Start High-Fidelity Master Mix (New England BioLabs), following a protocol modified from Castrillo et al.29 using primers GHTqF1 (5′-TTTTCATCGAAAGGTTGTTTCTCG) and GHTq R1 (5′-CTGTGCTGGGTACTGACGTG) amplified a 96-bp region of the SCAR fragment. The PCR protocol was initial denaturation at 98 °C, followed by 30 cycles at 98 °C for 1 min, annealing at 58 °C for 1 min; and extension at 72 °C for 1 min. PCR products were visualized in a 1.0% (wt/vol) agarose gel stained with ethidium bromide.
Data analysis
All data were tested for statistical test assumptions using a qqplot, Levene’s homogeneity test and the Shapiro–Wilk normality test at alpha = 0.05 significance level. For critical thermal limits (CTMax and CTMin) experiments, the data sets were non-normal and transformation did not normalize the residuals, so we used nonparametric ANOVAs (Kruskal–Wallis) followed by post-hoc nonparametric multiple comparisons. For voluntary exposure to ETZs, we used a generalized linear model with treatment (healthy, low and high spore load) with Poisson distribution, followed by comparisons within each treatment group. For healthy insects, we used a t-test to compare crosses between warm or cold ETZs; for infected insects, we conducted ANOVAS for comparisons among 23 °C, warm or cold ETZs.
ATP data: Data for CTMax of A. pisum were non-normal, and transformation did not normalize the residuals, nonparametric ANOVAs (Kruskal–Wallis) were then used and followed by post-hoc nonparametric pairwise comparisons with Wilcoxon tests. ATP data sets from voluntary exposure to ETZs were analyzed following the same protocol as described previously for in crosses analysis of ETZ experiment. Longevity was analyzed using a two-way ANOVA with fungal load and thermal condition (critical temperature and behavior) as factors. Analyses were performed in the R programming environment (v. 3.4.3., CRAN project)30 and JMP-Pro version 15 (SAS Institute 2020).
Source: Ecology - nature.com
