Corresponding author: Sean McCann (
Academic editor: J. Neff
Social wasps commonly exhibit impressive, pheromone-mediated nest defenses with stinging attacks on potential vertebrate nest predators. Studying this type of nest defense and comparing results across studies is challenging because there is no standardized method for quantifying defense intensities. For that reason, we developed a simple, paired-target apparatus coupled with easy and inexpensive data recording and analysis technologies. Each target is formed by two conjoined black plastic weigh boats that generate distinct percussive sounds when struck by attacking wasps. A battery-powered microphone inside each target converts the sounds into electrical signals that are transferred to a digital audio recorder. These audio files are then split into left- and right-channel files, saved as 16-bit WAV files, and the strikes to each target are counted using the open-source software SoundRuler. Using this apparatus, we show that workers of
McCann S, Moeri O, Jimenez SI, Scott C, Gries G (2015) Developing a paired-target apparatus for quantitative testing of nest defense behavior by vespine wasps in response to con- or heterospecific nest defense pheromones. Journal of Hymenoptera Research 46: 151–163. doi:
Nest defense is an integral life history trait of social insects. Many social wasps and bees are capable of coordinated, massed stinging attacks against potential vertebrate nest predators. During nest defense, large numbers of workers are mobilized and engage in stinging, biting or venom spraying to dissuade potential nest predators (
Nest defense is often coordinated by alarm and marker pheromones released by worker wasps. The alarm pheromone recruits nest mates out of the nest, and the marker pheromone that is deposited on potential predators directs the attacking workers toward them (
Pheromone-mediated nest defense seems to be widespread in vespines (
Studying nest defense of vespines is destined to reveal complex and intricate communication systems, but these studies are challenging in that nest defense is difficult to quantify. Moreover, results of previous studies are difficult to compare because they were obtained using rather different recording technologies and experimental designs. Our objectives were (1) to design an apparatus for quantifying nest defense behavior, and (2) to test experimentally whether
We worked with western yellowjackets,
The experimental nests we studied in behavioral tests were located near Vancouver in the municipalities of Langley, Burnaby, and Richmond. We sourced workers for pheromone extraction at separate nests in these municipalities.
We captured worker wasps emerging from their nests by placing a 4-litre glass jar with a steel-mesh cone over the nest entrance. We immediately killed and froze captured wasps by crumbling powdered dry ice into the capture jar, emptied the frozen wasps into polyethylene bags in an icebox, and transported them back to the laboratory for dissection. We excised their venom sacs, placed them in acetonitrile, macerated the tissue with a clean metal rod, and filtered the extract through glass wool to remove tissue fragments. We kept extracts frozen at a concentration of one venom sac per 10 µl, and transported extracts in an ice chest to the field for testing.
The design of our paired-target apparatus and its recording technology was inspired by
The paired-target, tripod-mounted apparatus consists of a crossbar supporting two targets separated by 1 m (Fig.
Graphical illustration (
We studied nest defense behaviour with nests of
To assess the suitability of our paired-target apparatus and its recording technology for quantifying nest defense responses by vespines, we repeated the experiment by
We ran nest defense pheromone experiments the same way as the color experiment, except that (
In experiments 2, 5, 8 and 9, we tested the response of nest mates to
The effect of color of two paired targets (Fig. venom sac extract
Color stimulus | Olfactory stimulus | P < 0.05 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Exp. | Date | n | S1 | S2 | S1 | S2 | Species tested | Strikes on S1 | Strikes on S2 | t-test | WSR |
1 | 21Aug10 | 10 | Black | White | None | None |
|
16.2 ± 12.75 | 0.1 ± 0.31 | Y | Y |
2 | 22Aug10 | 9 | Black | Black | CH3CN |
|
70.7 ± 33.9 | 51.3 ± 46.4 | N | N | |
3 | 7Sep11 | 10 | Black | Black | CH3CN |
|
12.0 ± 11.1 | 3.5 ± 5.5 | Y | Y | |
4 | 13Sep11 | 12 | Black | Black | CH3CN |
|
28.8 ± 20.5 | 10.2 ± 10.6 | Y | Y | |
5 | 20Sep11 | 12 | Black | Black | CH3CN |
|
122.1 ± 133.3 | 43.3 ± 120.8 | Y | Y | |
6 | 12Sep11 | 10 | Black | Black | CH3CN |
|
143.6 ± 152.6 | 18.0 ± 18.1 | Y | Y | |
7 | 12Sep11 | 10 | Black | Black | CH3CN |
|
46.1 ± 48.2 | 6.6 ± 9.9 | Y | Y | |
8 | 12Sep11 | 12 | Black | Black | CH3CN |
|
24.8 ± 26.1 | 8.0 ± 11.2 | Y | N | |
9 | 13Sep12 | 10 | Black | Black | CH3CN |
|
6.1 ± 3.2 | 1.4 ± 1.1 | Y | Y | |
10 | 9Sep11 | 12 | Black | Black | CH3CN |
|
31.2 ± 23.0 | 13.8 ± 9.5 | Y | Y |
Venom sac extract (
50 µl of acetonitrile (CH3CN)
Wilcoxon Signed Rank test
For each replicate, we used Audacity (Audacity Team) to split the audio file into a left and right channel, and saved them as mono 16-bit WAV files under appropriate filenames. We then opened each file in SoundRuler (
Representative example of paired oscillograms (obtained during replicate 6 of Exp. 2), depicting strikes caused by wasps hitting the control and treatment target of the bioassay apparatus (Fig.
Because our protocol produced paired data, we compared proportions of strikes on treatment and control targets in each replicate. In initial tests, we found a high variation in the total number of strikes on treatment and control targets between replicates, but the proportions of strikes on treatment targets was almost invariably higher, so a treatment effect became evident as a higher proportion of strikes on the treatment target than on the control target. We used a Wilcoxon Signed Rank test to determine whether the proportion of strikes on the treatment target differed from 0.5, and we also report the results of parametric paired T-tests for the same data. The more conservative Wilcoxon test has lower power because it uses ranks and discards ties, however, in all but one experiment the results agreed with those of the parametric tests.
The audio data underpinning the analyses reported in this paper, as well as supplemental videos, figures and a SoundRuler settings file, are deposited at
Worker wasps of
Proportion of strikes by various
Worker wasps of
There was also recognition of nest defense pheromones from heterospecifics, as evident by nest mates striking targets treated with
Our experimental data coupled with personal observations in field experiments indicate that the paired-target apparatus meets all the criteria to effectively quantify nest defense behavior by vespine wasps in response to nest defense pheromones.
The apparatus is assembled from inexpensive parts, its light weight facilitates transport to and from test sites, and the tripod-mount with height adjustment of the paired targets allows easy placement in uneven terrain. The conjoined plastic weigh boats serving as paired targets have surprisingly good resonant properties, thus facilitating recordings of the percussive sounds when they are struck by attacking wasps, with each strike becoming a quantifiable data point. The weigh boat targets are easily treated with test stimuli and can be readily replaced between replicates, thus avoiding the need to repeatedly clean the apparatus in a series of trials. The microphone and the digital audio recorder were sufficiently sensitive to record the wasps’ strikes on targets, and “bandpass filtering” further improved the signal-to-noise ratio of these strikes. As a result, the number of strikes could be accurately counted by a software program (Sound Ruler), provided that the strike recognition parameters (amplitude, duration and inter-strike intervals) were finely tuned. Because the microphones also picked up sounds from a nearby construction site, it is advisable though to seek nests in quiet settings for data recording.
Automated counting of strikes has the advantage of expedient data processing, which is helpful when quantitative data are needed to decide on the composition of test stimuli in follow-up experiments. The ability to run multiple sets of experiments in rapid succession is particularly critical in nest-defense pheromone research, where often many experiments are required to unravel the composition of complex pheromone blends (
Exposing nests to paired rather than single targets provided the option to compare and analyze proportions, instead of absolute numbers, of strikes on treatment and control targets. This option proved valuable because a nest’s propensity to defend in response to a test stimulus varied between days or replicates, a fact that renders the absolute number of strikes as an assessment criterion for the potency of a test stimulus more difficult to interpret.
Our data support evidence for the presence of nest defense pheromones in
Intriguingly, our data also provide evidence that vespines respond not only to their own nest defense pheromones but also to those of heterospecifics. Workers of
Each species will likely respond most vigorously to its own nest defense pheromone, because the alarm message is released by nest mates when they sense an immediate threat to the nest and when concerted defense by nest-mates is needed to protect the nest’s offspring. Nonetheless, the recognition of nest defense pheromones from
In conclusion, we have described a paired-target apparatus that facilitates the quantification of pheromone-mediated nest defense behavior by vespine wasps, and provide evidence that some
We thank various homeowners for kindly allowing permission to conduct wasp pheromone research on their properties. The study was supported by a Natural Sciences and Engineering Research Council of Canada
Call recognition parameters used in SoundRuler software for detecting and counting wasp strikes against plastic targets. In addition to these settings, we also set a bandpass filter of 900 Hz.