Plant and aphid growth conditions
Two lines of cowpea (Vigna unguiculata) ), susceptible California blackeye (CB46) and its resistant NIL (CB77), were grown in UC Mix 3 soil (agops.ucr.edu/soil/) in 24 oz plastifoam cups and fertilized weekly with MiracleGro (18 − 18 − 21; Stern’s MiracleGro Products). Plants were grown in a pesticide free plant growth room at 28 ± 2°C and 16:8 light:dark photoperiod.
The cowpea aphids were collected from a field in Riverside, California, in the summer of 2016. Since then, the aphids were reared on the susceptible cowpea line CB46 in a pesticide free greenhouse or a plant growth chamber and maintained at 28 ± 2°C with a 16:8 light:dark photoperiod. Aphis gossypii (cotton melon aphid) was used as a conspecific non-specialist in damage experiments. The colony was established from aphids collected from squash about a decade ago near Reedley, CA, USA. Melons (Cucumis melo cv. “Iroquois”) served as the host in all experiments and were used to maintain the aphid colony under the same conditions as for A. craccivora.
Aphid damage assay
One-week-old susceptible cowpea plants were infested with 15 mixed stages of 3rd and 4th instars, and adults of cowpea aphids or cotton melon aphids for 15 days. Plants were encased in plastic pollination bags with minute holes (Seedburo, SKU: S27) to restrict aphids to the plant and were maintained at 26 ± 2°C with a 16:8 light:dark photoperiod for 15 days. Plants were exposed to two different aphid densities, with one density of aphids maintained at a constant 15 aphids on the entire plant (constant cowpea aphid density), and a second density where aphids were allowed to grow and reproduce without any numeric restriction (variable cowpea aphid density). To keep the cowpea aphids at a constant density, newborn nymphs were removed daily, and the population was maintained at 15 aphids. Newborn nymphs were kept when the number of the original aphids dropped below 15. The cotton melon aphids were allowed to grow without any numeric restriction like the variable cowpea aphid density (variable cotton melon aphid density). The presence or absence of damage symptoms induced by the aphids were monitored daily throughout the experiment (chlorosis, pseudogalling, and/or stunted growth). For each aphid density or species, 8–10 plants were used per experiment and the experiment was performed three times.
Local vs systemic damage assay
A single unifoliate leaf of one-week-old susceptible cowpea plants was infested with 15 mixed stages of 3rd and 4th instars and adults of cowpea aphids using a mesh sleeve bag. Plants were maintained at 22–28°C with a 16:8 light:dark photoperiod for 15 days. On the uninfested areas of the plants, damage symptoms including chlorosis, pseudogalling, and/or stunted growth were monitored daily throughout the 15-day period. After 15 days, the mesh sleeves were removed and the symptoms on the infested leaves as well as on the entire plants were documented. Three plants were infested per experiment and the experiment was performed three times.
Aphid no-choice assay
Two-week-old susceptible and resistant cowpea plants were infested with 20 adult apterous aphids on a single unifoliate leaf and enclosed in a mesh sleeve bag. The plants were left undisturbed for six days after which the total number of aphids was counted on the leaf. A total of 15 plants were used for each cowpea line.
Aphid fecundity assay
Age synchronized cohorts of one-day-old adult aphids were developed by clip-caging adult apterous aphids to the adaxial side of a susceptible cowpea plant for 24 h to lay progeny. After this period, the adult aphids were removed with a fine tip paint brush and the first instars they produced were allowed to develop to maturity. A single, age synchronized, one-day-old adult cowpea aphid was transferred to a naive two-week-old unifoliate leaf of either a susceptible or a resistant cowpea plant and clip-caged on the adaxial side. The cages were monitored daily, for one week, and the survival of the adult aphid and the number of nymphs produced were recorded. After counting, the newly produced laid nymphs were removed. A single leaf per plant was infested and a total of 16 plants of each cowpea line were used.
Aphid growth rate assay
Ten cowpea aphid adults were clip-caged onto the adaxial side of a susceptible or resistant cowpea leaf. After 24 h, the adult aphids were removed, and ten newborn nymphs were left on the leaf in the clip cage. After six days, the surviving aphids were counted and weighed with a microbalance (CN HG 01.121, Sartorius).
The mean relative growth rate (MRGR) was calculated as the difference of logarithms of the mean weight of day-old aphid nymphs and the mean weight of the surviving aphid nymphs divided by the number of days (MRGR = (logWsurviving – logWday−old) / Number of days). Twelve cages were used per cowpea line.
Electrical penetration graph
A DC-electrical penetration graph (DC-EPG system) was used for the EPG analysis (Tjallingii 1988). The EPG technique has been developed to monitor the probing activities of arthropods with piercing mouthparts when probing inside plants. The EPG waveforms are determined depending on the stylet tip positions in leaf tissue and the insect’s behavior (Tjallingii 1988). An EPG is performed by securing an electrode onto an aphid and placing a second electrode in the soil next to plant roots. Cowpea aphids were tethered to a 12.5 µm gold wire on their dorsal side of the abdomen using a water-based, conductive silver glue (Cervantes and Backus 2018). After an hour-long starvation period, tethered aphids were placed on the abaxial side of a two-week-old unifoliate leaf and a second electrode was placed in the soil of the potted plant. Simultaneous recordings for eight aphids were performed on a Giga-8 DC-EPG amplifier for 8 h. The aphid-plant systems were housed in a Faraday cage in a climate-controlled room at 24 ± 1°C. Each recording session had half of the aphids on susceptible plants and half on resistant plants. At the end, a total of 25 susceptible and 30 resistant EPGs were obtained and analyzed. The analysis of the EPG variables and waveforms were performed with PROBE 3.5 software (EPG systems, www.epgsystems.eu) naming convention based (Ebert et al. 2015). These EPG parameters were based on six different waveforms corresponding to stylet pathways in plant tissues other than phloem and xylem (C), to potential drops (i.e., intracellular stylet punctures) (pd), to salivation in phloem elements (E1), to passive phloem sap ingestion (E2), to active xylem sap ingestion (G), and to derailed stylet mechanics (F). The calculations were performed with EPG-Calc 6.1 software (Giordanengo 2014).
Aphid choice assay
A large, modified petri dish arena was placed above two 2-week-old cowpea plants, one of each resistant and susceptible line, to evaluate aphid preference. The arena had two holes of 2 cm diameter cut out of the bottom of the plate, directly across from each other. When positioned over paired leaves from each treatment, the aphids were able to choose between equal tissue amounts of each leaf, spaced equidistant within the plate. Aphids were introduced to the arena through a third 2 cm hole directly in the center area between the two choice options. This hole was fitted on the underside (exterior) of the arena with a screw cap to which a modified (shortened) 50 mL conical tube containing the aphids could be affixed (Online Resource 1). A group of twenty aphids (starved for 1 h prior to use) was introduced to the arena through this release mechanism. The number of aphids feeding on each leaf was recorded at 2, 3, 6, and 24 h after release. Four to five plant pairs were used per experiment and the experiment was performed four times.
Aphid dispersal assay
The rate of cowpea aphid dispersal in response to resistant cowpea was used to monitor aphid deterrence. Aphid dispersal was measured using a behavior dispersal assay similar to that described in Mauck et al. (2010). Bioassay arenas were constructed from 100 x 15 mm petri dishes, with two conjoining holes (17 mm2) cut in the center. A unifoliate leaf from one of the two cowpea plant types being compared was exposed on one side of the conjoining holes, and the other choice option was immediately adjacent occupying the second conjoined hole (Online Resource 2). After a 15-min chill at 4°C, twenty 4th stage instars and adult aphids were placed on a piece of filter paper. The filter paper with the chilled aphids was then placed directly on one of the exposed leaves. The placement of the filter paper on the leaf ensured that the aphids contacted the initial leaf they were placed on before dispersal. The filter paper was removed after an h when all aphids had dispersed and the location of the aphids were documented at 1, 2, 6, and 24 h. The cowpea plants screened against each other included, susceptible with resistant, susceptible with susceptible and resistant with resistant. Five plant pairs were used per experiment and the experiment was performed twice.
We used one-way analysis of variance (ANOVA) followed by Tukey HSD tests to assess aphid population damage assay using GraphPad Prism version 8 (GraphPad Software, San Diego, California USA). The aphid no-choice experiment was analyzed using a two-tailed t-test using GraphPad Prism version 8 (GraphPad Software, San Diego, California USA). We used generalized linear models (GLM) with a likelihood ratio and chi-square test to analyze adult aphid fecundity and survival rate using R software (version 3.6.0) (R Core Team, 2019). Two-tailed t-test using GraphPad Prism version 8 (GraphPad Software, San Diego, California USA) was used to analyze the nymph survival and MRGR. Aphid feeding behavior was analyzed using generalized linear models (GLM) with a likelihood ratio and Chi-square test (χ²). We had three types of data: (a) event duration, (b) frequency of penetration events and (c) time delay until the first occurrence of an event. Event durations were modeled using GLM with a gamma (link = ”inverse”) distribution and frequency of penetration were modeled using GLM with poisson (link = “identity”) distribution. Time delay data were modeled using the Cox proportional hazards (CPH) model and we treated cases where the given event did not occur as censored. The assumption of validity of proportional hazards was checked using the functions “coxph” and “cox.zph”, respectively (package R: “survival”). The fit of all generalized linear models was controlled by inspecting residuals and QQ plots. Aphid choice assays were analyzed using Friedman tests for the overall model and Kruskal-Wallis tests for each individual time point (Minitab v. 14). Aphid dispersal assays were analyzed using GLMs for the overall repeated measures model, followed by univariate tests to explore treatment effects within each time point (Minitab v. 14).