Plants and insects
A pea aphids colony, originating from a parthenogenetic female collected in Kiryat Tiv’on, northern Israel, was reared in the laboratory on fava bean (Vicia faba) plants potted in commercial planting soil, at 22-24 °C, 65 ± 5% RH and a photoperiod of 16:8 L:D. Fava bean plants were used for experimentation 2 weeks after sowing. Aphid colonies that were used for experimentation were established on fava bean plants individually grown in plastic cups. For the ease of handling, the experimental plants were stripped of all leaves, leaving only a bare stem and an apical bud. Only wingless aphids were used in this study. The evening before each experiment, approximately 10 adult aphids (8–12) were placed on each fava bean plant. On the next morning, each plant was ready for experimentation with an aphid “colony” composed of adults and first instar nymphs (henceforth nymphs).
Lentil plants (Lens culinaris, a highly attractive host for pea aphids), used as destination host plants for surface-moving aphids, were planted in pots in commercial planting soil and after approximately 2 weeks, transplanted into the plant-surrounded arena (described below). Every morning the lentil plants in the arena were trimmed to a height of 10 cm. We chose lentil plants because they can be planted very densely (forming a highly uniform target) and they perform well after being trimmed repeatedly. Every 5 days, the lentil plants were replaced with new ones.
Setup and working procedures
The experiments were performed in a fluorescent-light lit and air-conditioned laboratory at 22-24 °C. All experiments were performed under a 300 W halogen flood light that was situated 75 cm above the center of the working table, illuminating and moderately warming it (surface temperature of the experimental arenas was ~ 28 °C; surface temperature was measured using Extech© 42545 infrared thermometer). The different experiments in this study were performed using several types of arenas, as described below. In all experiments, two observers sitting on opposite sides of each arena tracked the aphids’ movement and behavior.
Aphids were induced to drop by tilting the fava bean plant so that the stem was 10 cm above the center of each arena, and then slowly exhaling on the stem (see Gish et al. 2010) while gently tapping on its base with a finger. In response to this stimulus, the average ratio of the nymphs:adults that dropped from our created colonies to the surface at the start of the experiments was 2.8 ± 0.2 (6.5 ± 0.3 adults vs. 18.3 ± 1.7 nymphs). Adults that were in the middle of giving birth were quickly removed from the arena using gentle forceps and were not included in the collected data. In experiments where only nymphs were needed, 2–6 h before the beginning of the experiment all adult aphids were removed from the plant using forceps. When only adults were needed, they were placed on the stem 2–6 h before the experiment. This minimized the number of nymphs that dropped to the arena and any nymphs that did drop were quickly removed.
Description and analysis of aphid behavior
The description and analysis of the riding behavior was done in three experiments, according to our three main questions:
Experiment 1: Does riding behavior differ from the natural tendency of surface-walking aphids to climb on objects they encounter?
In this experiment, we compared the time nymphs spent on three types of objects: plastic beads (as schematic aphid models), dead adults and live adults. The experiment was necessary for determining whether the riding behavior is a new behavior or simply a manifestation of the tendency to climb on objects.
The arena was constructed by arranging 64 thumbtacks with green, spherical, plastic bead heads (head diameter: 4 mm) that were tacked on a sheet of brown cardboard, in an 8X8 matrix (distance between adjacent insertion points was 1 cm). Only nymphs were dropped onto this arena. The arena was video filmed with a handheld Sanyo Xacti HD1010 high-definition video camera for 8 min in each trial. We used a video camera in this study since it provided detailed information on aphid movement and precise timing of events. We extracted data from the films on the movements of 35 individual nymphs (nymphs that did not contact other nymphs). We timed only the first climbing event of each nymph, from the moment it started climbing a bead until the moment it got off.
We constructed another arena, similar to the one described above, using dead adult aphids instead of thumbtacks. Sixty-four adult aphids were killed by freezing in -20 °C. The aphids were then arranged in an 8X8 matrix using fine forceps. The dead aphids were laid down facing forward and their limbs and antennae were arranged so that they were in a natural standing posture. Whenever an aphid’s color started turning dark (this happened with some of the aphids after several trials), it was replaced with a freshly killed aphid. The experiment was conducted and videotaped as described in the plastic bead experiment above (n = 35).
This experiment was performed on a brown Bristol paper- covered table (table dimensions 2 X 1.5 m). Thirty created aphid colonies containing both adults and nymphs were separately used in this experiment (n = 30). Aphids were dislodged in the center of the arena and two observers manually timed riding events and clasified each adult as “static” or “mobile”. A static adult was defined as an adult that did not change its location while the nymph being tracked was on its body, as opposed to a mobile adult, which was defined as one that did change its location during that time. The observers timed each riding event from initial contact until the nymph was completely off the adult. Riding events of multiple nymphs on one adult were rare, and were not included in the collected data. In addition, the observers recorded whether and how the adults tried to remove the riding nymphs.
Experiment 2: What are the costs and benefits of riding behavior?
To answer this question, we measured: a) the difference in movement speed between nymphs and adults. This data was a baseline for the following experiments; b) the effect of riding on return time to the plant. Shorter return times would indicate a benefit while longer return times would indicate a cost; c) the effect of riding on the chances of finding a plant. Better chances would indicate a benefit while worse chances would indicate a cost.
Difference in movement speed between nymphs and adults
A doughnut shaped trench (outer diameter: 28 cm; inner diameter: 5 cm; depth: 3 cm) was made in a Styrofoam board. The central circle that was left intact was covered with brown Bristol paper. This circle served as the starting point for the aphids that were dropped using a plastic funnel (described below). The trench was filled with dry commercial planting soil, which contained small pebbles and some small dry twigs. The surface of the soil was flat and flush with the middle circle and the trench’s edge. Thirty colonies were used in this experiment (n = 30). After the aphids were dropped onto the central circle, a stopwatch was started and the two observers recorded the time it took each aphid to reach the edge of the arena (the outer edge of the trench).
The effect of riding on return time and chances of finding a plant
This experiment was performed on a circular arena, surrounded by groups of lentil plants (details in Additional file 3). The high-definition video camera was positioned above the arena. The observers added information to the recorded video by providing verbal descriptions of events and using skewers to point out and refer to specific aphids.
At the beginning of each experiment, a plastic funnel (bottom/top opening diameters: 4/10 cm, respectively) was placed on the center of the arena and the aphids were dropped (n = 30 colonies) through the funnel, which was then removed. The funnel reduced the bouncing and scattering of aphids, thus raising the frequency of riding events and lowering the necessary number of trials. Although this caused some aphids to become temporarily entangled with each other, the aphids quickly disentangled. Tracking of aphid behavior always started after aphids disentangled.
To measure the probability of an aphid reaching a plant, we noted whether each aphid exited the arena in front of a plant or a gap. “Plant” was defined as a segment of the circumference of the arena that was planted with lentil plants and “gap” was defined as a segment that contained no plants. Only riding events that lasted until the adult exited the arena were included in the data, thus excluding partial and short riding events. In addition, during the experiments in the plant-surrounded arena we recorded the riding positions of nymphs on adults’ bodies.
Experiment 3: do adults prefer carrying nymphs of their own kin?
In order to test whether adults prefer carrying nymphs of their own kin (an indication for parental care), we compared the riding frequency among members of our laboratory line of aphids (the one used in all other experiments in this study) with that of aphids from a mixed- line colony, where the chances of aphids encountering their kin are low. The mixed-line colony was established by 10 adult wingless pea aphids that were collected from 10 different locations near Haifa and Kiryat Tiv’on, northern Israel. The arena used in this experiment was a 20 cm diameter brown Bristol paper circle, pasted on a Styrofoam board. We performed two identical experiments, one with our laboratory line and one with the mixed-line colony. In each experiment we used 20 colonies (n = 20). The time it took each aphid to reach the edge of the arena was recorded. Only riding events that lasted until the adult exited the arena were recorded.
It was not possible to record data blindly because our study involved close observation of the behavior of animals in the laboratory. All data were tested for normality using the Shapiro-Wilk test. Data that did not meet the assumptions of parametric tests were tested with equivalent nonparametric tests. All tests used were two-tailed. Specifications of the tests used are given in the results. Statistical analyses were performed using IBM SPSS software v.20. Means are presented with standard error in parentheses.