2.1 Plants and insects
Two strains of the pea aphid (Acyrthosiphon pisum), characterized by red and green body colors, and exhibiting similar locomotion and fecundity traits, were employed in the subsequent experiments (refer to Supplementary Information). These strains were sourced from Lanzhou, Gansu (36°07′7″N, 103°42′20″E), and Golmud, Qinghai (36°23’55.2"N 94°51’03.4"E) in China, respectively. Aphids were cultivated on broad beans (Vicia faba) under long-day conditions (16L:8D; 20 ± 1ºC) for more than five years.
This study exclusively utilized wingless aphids. Aphids were acquired by rearing at a low density of no more than 50 individuals per plant for at least three generations before each treatment.
The ladybirds, Harmonia axyridis, were collected from wheat fields and apple orchards in Qingdao, Shandong, China. Subsequently, the beetles were maintained under laboratory conditions at 25 ± 1°C, 50% relative humidity, and a 14 L:10D photoperiod in preparation for further experiments.
Unmated female ladybirds (approximately 7 days post-eclosion) subjected to continuous excessive feeding were selected. After several attempts, feeding-disabled ladybirds were achieved by removing the maxillary and mandibular setae from the mouthparts. Subsequently, they were placed for 24 hours to allow for wound recovery, simultaneously serving as a 24-hour starvation treatment before subsequent experiments.
In the experiments described below, individual red aphids and varying numbers of green aphids were mixed and placed into the observation chamber. Subsequent analysis relied on video recordings (by Microscope Camera HY-2037, Hayear Electronics Co. Ltd. Shenzhen, China) and behavioral analysis software (EthoVision XT, V16.0.1536; Noldus Information Technology, Wageningen, The Netherlands). The distinctive coloration of the red aphids, contrasting sharply with both the leaf color and the green aphids, served as a prominent feature for software identification (Fig. 1D-1).
2.2 Experimental design for behavioral analysis
In order to verify whether the presence of ladybirds affects the activity level of aphids, we designed an experiment to track the movement of the target aphids.
2.2.1 Device setup: We designed and flattened an experimental observation chamber for locomotion behavior and fecundity studies. The chamber was constructed from a petri dish, with the lower third filled with 1% moist agar. In the center of the petri dish on agar, a leaf of V. faba, trimmed to a diameter of 30 mm, was placed. The remaining space was covered with a waterproof circular paper to facilitate the movement of aphids (Fig. 1D-1).
2.2.2 Video record and analysis: Red and green aphids were arranged in three different combinations: 1:0, 1:4, and 1:9 ratios, and placed into the observation chamber. After waiting for approximately half an hour for the majority of aphids (especially the red ones) to settle, the treated ladybirds were introduced, and the entire process was recorded for 6 hours. The treatment without the introduction of ladybirds was designated as the control group. Each treatment group was repeated 10 times.
After the video files underwent EthoVision XT analysis, data regarding the movement visualization, total distance, speed during movement, and the duration of aphid stay on the leaf were exported and analyzed (Spink et al., 2001).
2.3 Feeding and fecundity
Experiments were also designed and conducted to investigate changes in the feeding capacity and reproductive capability of aphids.
2.3.1 Feeding: We assessed the feeding situation by quantifying the quantity of honeydew secreted by aphids. The experiment involved selecting bean plants of approximately 20 days in size, potted with the removal of the bottommost pair of leaves. A clean A4-sized white paper was placed at the base of the plant, with a hole in the middle to accommodate the plant stem. Fifty aphids were then inoculated, and after the aphids had settled, treated ladybirds were introduced. The white paper was collected and taken to visualize honeydew drops after 12 hours (Fig. 1E-1). This experiment was repeated 5 times, with a control group that did not have ladybirds introduced.
2.3.2 Visualization of honeydew drops: The visualization of honeydew drops was achieved using a chromogenic agent spray (based on Anderson et al., 2000 and modified). The agent was prepared using the following recipe: diphenylamine: aniline: phosphoric acid solution (85%): acetone at a ratio of 1:1:5:50. The solution was thoroughly mixed and then sprayed using a glass atomizer (3 ml per repeat). After undergoing a baking process in an oven for 10 minutes at 110°C, the drops became distinctly visible, appearing in a clear, dark brown color (Fig. E).
2.3.3 Fecundity: Ten red aphids were placed into the observation chamber, and after waiting for the majority of aphids to settle, treated ladybirds were introduced. After 24 hours, the ladybirds and mature aphids were removed, and the number of nymphs were counted. The treatment group without the introduction of ladybirds served as the control, and the experiment was repeated 15 times.
2.4 Chemical mechanism
At the physiological, biochemical, and molecular levels, our research delves into various aspects related to the feedback mechanisms of aphids. This investigation encompasses the examination of the impact on aphid behavior in response to aphid alarm pheromones and residues from ladybird crawling. Additionally, we explore alterations in the synthesis levels of monoamine neurotransmitters within aphids themselves under diverse stimuli.
2.4.1 Locomotion responses to Ladybird footprints marks: The experiment involved selecting the same observation chamber. One ladybird was placed into the setup and allowed to walk freely for 12 hours. After removing the ladybird, one red aphid was introduced for behavioral tracking experiments. Recording time and analysis methods were as previously described, with the behavioral experiment lasting for 6 hours and the reproductive experiment lasting for 24 hours. The experiment was repeated 10 times.
2.4.2 Locomotion responses to Aphid alarm pheromone:
E-β-farnesene (EBF) detection: The gas chromatograph system utilized in this study was the GC-2030 (Shimadzu, Japan) equipped with a Flame Ionization Detector (FID). A hand-held Solid Phase Microextraction (SPME) device with a 100 μm Polydimethylsiloxane (PDMS) coating was initially introduced into the system for a 12-hour adsorption period. Subsequently, it was employed for manual sample injection (Fig. 3E-1). Separation was achieved using the SH-Rtx-5 capillary column (length = 30 m, inner diameter = 0.25 mm, stationary phase thickness = 0.25 μm, Shimadzu, Japan). The LabSolutions workstation, also from Shimadzu, Japan, was utilized to control the GC system and manipulate the chromatograms.
Locomotion responses: We designed an experiment to verify the physiological and behavioral impact of alarm pheromones on aphids. The experiment involved selecting the same observation chamber and methods. One red aphid was introduced for behavior monitoring experiments, with EBF introduced. The moving track was lasted for 6 hours and the fecundity experiment lasting for 24 hours. The experiment was repeated 10 times.
2.5 Gene expressions of Neurotransmitters synthesis
2.5.1 Experimental Treatment and Brain RNA Extraction: Red aphids were placed into the observation chamber, and treated ladybirds were introduced. The aphids were quickly transferred into liquid nitrogen after 6 hours. Adult aphids were then carefully selected, and the thorax and abdomen were removed. The heads were rapidly placed into RNA extraction reagent for RNA extraction.
The treatment group without the introduction of ladybirds served as the control, and the experiment was repeated a total of 5 times.
2.5.2 Measurement of Gene Expression Related to Neurotransmitter Synthesis: Q-RT-PCR was conducted to assess the genes expression levels of key enzymes (AP-TH, AP-TβH, and AP-TPH) involved in the synthesis of three monoamine neurotransmitters (dopamine, octopamine, and serotonin). APRPL7 was chosen as the reference gene (Table S1; Zhang et al., 2016; 2020).