Short Communication |
Corresponding author: Charles I. Abramson ( charles.abramson@okstate.edu ) Academic editor: Ming Kai Tan
© 2021 Kiri Li N. Stauch, Riley J. Wincheski, Jonathan Albers, Timothy E. Black, Michael S. Reichert, Charles I. Abramson.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Stauch KLN, Wincheski RJ, Albers J, Black TE, Reichert MS, Abramson CI (2021) Limited evidence for learning in a shuttle box paradigm in crickets (Acheta domesticus). Journal of Orthoptera Research 30(2): 155-161. https://doi.org/10.3897/jor.30.65172
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Aversive learning has been studied in a variety of species, such as honey bees, mice, and non-human primates. Since aversive learning has been found in some invertebrates and mammals, it will be interesting to know if this ability is shared with crickets. This paper provides data on aversive learning in male and female house crickets (Acheta domesticus) using a shuttle box apparatus. Crickets are an ideal subject for these experiments due to their well-documented learning abilities in other contexts and their readily quantifiable behaviors. The shuttle box involves a two-compartment shock grid in which a ‘master’ cricket can learn to avoid the shock by moving to specific designated locations, while a paired yoked cricket is shocked regardless of its location and therefore cannot learn. Baseline control crickets were placed in the same device as the experimental crickets but did not receive a shock. Male and female master crickets demonstrated some aversive learning, as indicated by spending more time than expected by chance in the correct (no shock) location during some parts of the experiment, although there was high variability in performance. These results suggest that there is limited evidence that the house crickets in this experiment learned how to avoid the shock. Further research with additional stimuli and other cricket species should be conducted to determine if house crickets and other species of crickets exhibit aversive learning.
aversion, Avoidance behavior, comparative, invertebrate learning, Orthoptera
Aversive learning is crucial to an individual’s survival. One example of aversive learning is taste aversion, which is an important defense against potential poisoning (
Insects also demonstrate aversive learning (
Shock is commonly used as an aversive stimulus in learning experiments (
Another way that shock is administered in experiments is through the use of an apparatus called the shuttle box—one of the oldest and most widely used apparatuses for the study of learning and memory (
Crickets are ideal for studying aversive learning because this group of species exhibits a variety of learned behaviors, ranging from associative learning of olfactory cues (
Crickets also exhibit spatial learning and memory. G. bimaculatus were placed in a stadium similar to a Morris water maze in which the traditional water and the hidden platform were replaced with a hot metal surface possessing a cool area on the platform’s surface (
There is also some evidence for social learning in crickets, although this has been less explored. One social learning experiment involved naive Nemobius sylvestris (Bosc, 1792) learning anti-predator behaviors from more experienced conspecifics (
No studies of cricket learning have employed a shuttle box. The shuttle box has two major advantages that make it worth exploring as a test paradigm in crickets: it is automated, and it can be used to test a wide range of learning behaviors. The automation of the shuttle box is a major advantage since it allows for the apparatus to be used consistently and repeatedly with a variety of species and experimental designs. Furthermore, as there have been many shuttle box experiments with a wide variety of organisms, it will be interesting to compare cricket behavior in the shuttle box to that of other species to gain insight into species differences in learning. It would also open the door to the ‘psychological’ study of cricket behavior, as many interesting psychological phenomena such as social learning and spatial memory can be explored (see above).
In the present study, we tested the suitability of using the shuttle box for behavioral studies of learning using house crickets Acheta domesticus (Linnaeus, 1758) (Orthoptera: Gryllidae). Crickets have many benefits as model organisms for behavioral studies. They are usually easy to maintain and exhibit a wide range of interesting behaviors, including social behaviors and learning. In addition, crickets are short-lived and have clear developmental markers (e.g., wing development, chirping, and development of an ovipositor). These traits allow researchers to use individuals that are all at the same life stage, identify the males and females, and help to minimize differences between subjects.
Subjects.—Subjects consisted of 130 house crickets [females n = 78, males n = 52 (Acheta domesticus)] collected from colonies maintained for laboratory purposes sourced from Fluker Farms, Louisiana. Crickets were sorted into two different communal containers based on sex. In the containers, crickets had a source of cover (piece of egg carton or cardboard), ground up chicken feed in petri dishes, and distilled water in a Falcon tube closed with a cotton ball. The food and water were refilled every 48 hours. Crickets were housed in this manner until they were needed for the experiment. Only mature crickets (crickets with fully developed wings) were tested.
Apparatus.
—The present experiment made use of a modified shuttle box apparatus (see Fig.
The shuttle box apparatus with propeller controller in the upper left-hand corner. In this image, one of the plexiglass covers (A) has been removed so that the shock grid can be seen more easily. In the experiments, one cricket was placed into the compartment labeled A and one cricket was placed into the compartment labeled B. A piece of filter paper was placed under the left-hand side of the shuttle box to illustrate the midpoint for the two halves of the compartment. Once the cricket was inside the compartment, the plexiglass was placed over the compartment to ensure that the cricket maintained contact with the grid.
Each compartment was then connected to a control box containing a Propeller Experiment Controller (Parallax Inc., Rocklin, CA;
Behavioral assay.—Preliminary experiments were conducted to determine the appropriate voltage and amplitude. During the preliminary experiments, individual crickets were shocked at various voltages and amplitudes. Crickets were observed for behaviors (such as digging) that indicate the presence of a noxious stimulus (i.e., shock). Observations were made about the ability of the individuals to enact behaviors to escape the shock.
For the formal experiment, same-sex cricket pairs were captured from the container using a plastic shot glass and a notecard. Each individual was placed in a separate compartment. Once they were in the shuttle box, a three-minute habituation phase started where no shock was administered. The habituation phase provided crickets with time to recover from any stress associated with the transfer from the container to the apparatus. After the habituation phase, and once both crickets crossed the midline in either direction, the experimental trial began. During the experimental trial, shock was delivered at 12 VDC and 0.5 A.
The formal trial duration was 9 minutes. Results of previous research using this setup with honey bees (Apis mellifera Linnaeus, 1758) found that experimental fatigue occurs after 5 minutes (
Crickets were randomly assigned to the role of ‘master,’ ‘yoked,’ or ‘baseline.’ Crickets were placed in the apparatus with one cricket in each compartment. The baseline cricket pair served as an experimental control in which no cricket received a shock. The master and yoked cricket pairs were the experimental pairs where one cricket’s behavior (master) determined whether both crickets were shocked. For these pairs, the shuttle box was assigned to administer shock on one of the two sides of the grid in each compartment (i.e., either on the half away from or towards the researcher). The side that was associated with the shock was randomly chosen and did not change during the trial. If the master cricket moved to the side that was shocked, then both crickets received the shock. Once the master cricket left the side corresponding to the shock, both crickets stopped receiving the shock. The yoked cricket was unable to observe or communicate with the master cricket and therefore received the shock regardless of what side of the compartment it was on.
The reason for the pairing between the master and the yoked cricket was to set up a situation where one cricket had the opportunity to learn (the master cricket) and the other (yoked cricket) was essentially a control that experienced the same conditions, including the experience of electric shock, but could not learn because there was no consistent association between its behavior and the shock. Master crickets could learn to avoid the side associated with the shock, while yoked crickets would not be able to associate either side with a shock. As a result, the side preferences of the yoked crickets should resemble those of the baseline crickets: neither of these crickets should show a bias towards one side or the other. In contrast, if the master crickets are able to learn the association, they should spend less time on the side associated with the shock than either the baseline or the yoked cricket.
The data came from a 9-minute trial that was divided into 60-second intervals for data management purposes. The amount of time that was spent on the side that was not linked to shock (hereafter ‘correct side’) was calculated for nine 60-second intervals starting at 0 s and ending at 540 s. The proportion of time spent on the correct side was calculated in 60-second intervals by dividing the actual amount of time spent on the correct side by 60 s.
All the data were analyzed using R version 4.1.0 (
The first two sets of t-tests were used to compare the percentage of time the male and female shock-free control (e.g., baseline) crickets spent on the correct side compared to random chance (50%) (Table
One sample t-test results for male and female no shock control (baseline) crickets compared to random chance (0.5). M = proportion of time spent on the correct side; females df = 17; males df = 9; Bonferroni adjusted α value of 0.003.
Time Point (s) | Females | t | p | Males | t | p | ||
M | SD | M | SD | |||||
60 | 0.55 | 0.33 | 0.63 | 0.538 | 0.75 | 0.25 | 3.13 | 0.012 |
120 | 0.56 | 0.35 | 0.67 | 0.513 | 0.57 | 0.30 | 0.76 | 0.464 |
180 | 0.35 | 0.32 | -2.03 | 0.058 | 0.50 | 0.29 | -0.00 | 1.000 |
240 | 0.43 | 0.37 | -0.74 | 0.471 | 0.59 | 0.38 | 0.77 | 0.461 |
300 | 0.51 | 0.41 | 0.11 | 0.916 | 0.44 | 0.40 | -0.50 | 0.629 |
360 | 0.44 | 0.40 | -0.62 | 0.544 | 0.39 | 0.40 | -0.83 | 0.429 |
420 | 0.59 | 0.45 | 0.85 | 0.407 | 0.35 | 0.35 | -1.31 | 0.224 |
480 | 0.67 | 0.42 | 1.74 | 0.100 | 0.49 | 0.38 | -0.04 | 0.966 |
540 | 0.54 | 0.40 | 0.43 | 0.672 | 0.54 | 0.46 | 0.31 | 0.766 |
One sample t-test results for male and female yoked control crickets compared to random chance (0.5). M = proportion of time spent on the correct side; females df = 29; males df = 20; Bonferroni adjusted α value of 0.003.
Time Point (s) | Females | t | p | Males | t | p | ||
M | SD | M | SD | |||||
60 | 0.45 | 0.37 | -0.81 | 0.427 | 0.46 | 0.27 | -0.71 | 0.488 |
120 | 0.53 | 0.41 | 0.40 | 0.692 | 0.42 | 0.32 | -1.18 | 0.251 |
180 | 0.41 | 0.37 | -1.33 | 0.194 | 0.44 | 0.36 | -0.79 | 0.439 |
240 | 0.53 | 0.40 | 0.46 | 0.652 | 0.51 | 0.45 | 0.06 | 0.950 |
300 | 0.60 | 0.42 | 1.35 | 0.188 | 0.42 | 0.42 | -0.91 | 0.373 |
360 | 0.55 | 0.41 | 0.70 | 0.489 | 0.44 | 0.44 | -0.61 | 0.551 |
420 | 0.46 | 0.43 | -0.54 | 0.591 | 0.51 | 0.47 | 0.13 | 0.897 |
480 | 0.60 | 0.42 | 1.25 | 0.222 | 0.54 | 0.45 | 0.41 | 0.688 |
540 | 0.60 | 0.44 | 1.24 | 0.226 | 0.53 | 0.41 | 0.29 | 0.771 |
Two additional sets of t-tests were conducted on the male and female yoked crickets to see if the percentage of time they spent on the correct side differed from chance (Table
The results from the LMM showed that time point was a significant predictor of amount of time spent on the correct side, with crickets spending more time on the correct side as the experiment progressed (Table
The findings from the first LMM suggest that cricket learning occurred at different time points, as seen by the difference in male (beginning of trial) and female (end of trial) learning (Fig.
Simultaneous pairwise comparisons using Tukey’s HSD test indicated that the difference between the master group and the yoked control was statistically significant (Table
One sample t-test results for male and female behavioral (master) crickets compared to random chance (0.5). M = proportion of time spent on the correct side; females df = 29; males df = 20; Bonferroni adjusted α value of 0.003.
Time Point (s) | Females | t | p | Males | t | p | ||
M | SD | M | SD | |||||
60 | 0.63 | 0.37 | 1.94 | 0.062 | 0.73 | 0.23 | 4.620 | 0.000* |
120 | 0.57 | 0.36 | 1.10 | 0.280 | 0.66 | 0.37 | 1.972 | 0.063 |
180 | 0.58 | 0.38 | 1.17 | 0.250 | 0.51 | 0.40 | 0.089 | 0.930 |
240 | 0.59 | 0.39 | 1.24 | 0.224 | 0.64 | 0.42 | 1.555 | 0.136 |
300 | 0.60 | 0.42 | 1.29 | 0.206 | 0.60 | 0.42 | 1.108 | 0.281 |
360 | 0.59 | 0.43 | 1.10 | 0.282 | 0.60 | 0.40 | 1.142 | 0.267 |
420 | 0.67 | 0.40 | 2.37 | 0.025 | 0.65 | 0.40 | 1.690 | 0.106 |
480 | 0.72 | 0.38 | 3.22 | 0.003* | 0.64 | 0.42 | 1.596 | 0.126 |
540 | 0.65 | 0.39 | 2.13 | 0.042 | 0.65 | 0.45 | 1.474 | 0.156 |
Results of the LMM model to test the effects of time point, experimental role, and sex on time spent on the correct side. Significant predictors: * p <0 .05 and ** p < 0.001.
Independent | Predictor | Estimate | SE | t-value | 95% CI | p | |
Percent of time spent on correct side | Intercept | 29.17 | 3.73 | 7.83 | 21.87 | 36.48 | <0.001** |
Time Point | 0.01 | 0.00 | 2.04 | 0.00 | 0.01 | 0.042* | |
Exp. Role [Master] | 6.67 | 4.56 | 1.45 | -2.33 | 15.66 | 0.146 | |
Exp. Role [Yoked] | 0.72 | 4.56 | 0.16 | -8.27 | 9.71 | 0.875 | |
Sex [Male] | 1.28 | 7.25 | 0.18 | -12.93 | 15.50 | 0.860 | |
Exp. Role [Master] *Sex [Male] | -1.12 | 8.41 | -0.13 | -17.61 | 15.37 | 0.894 | |
Exp. Role [Yoked] *Sex [Male] | -5.14 | 8.4 | -0.61 | -21.63 | 11.35 | 0.541 |
Results of the master cricket LMM model to test the effects of time point, experimental role, and sex on time spent on the correct side. Significant predictors: * p < 0.05 and ** p < 0.001.
Independent | Predictor | Estimate | SE | t-value | 95% CI | p | |
Percent of time spent on correct side | Intercept | 35.02 | 3.35 | 10.45 | 28.45 | 41.59 | <0.001** |
Time Point | 0.01 | 0.01 | 1.50 | -0.00 | 0.02 | 0.134 | |
Sex [Male] | 3.36 | 5.09 | 0.66 | -6.62 | 13.33 | 0.510 | |
Time Point *Sex [Male] | -0.01 | 0.01 | -1.23 | -0.03 | 0.01 | 0.220 |
The series of experiments presented in this study had two goals. One goal was to investigate whether house crickets, A. domesticus, exhibit aversive learning. The other goal was to determine if the shuttle box is a suitable apparatus for studying aversive learning with crickets. The results show that the male master crickets’ behavior exhibited learning at the beginning of the experimental trials, while the female master crickets’ behavior exhibited learning towards the end of the experimental trials (Table
The master male crickets exhibited learning early in the experiment because they spent significantly more time than chance on the correct side. In comparison, the master female crickets exhibited learning later in the experiment, as they spent significantly more time than chance on the correct side. For both males and females, the yoked control pairs and no shock control pairs all performed similarly and were no different from the chance expectation of 50% (Fig.
Generally, honey bees hit around 60–75% on performance in this assay and maintained that performance over time, which has been taken as evidence for aversive learning (
Unlike the honey bees (
Crickets in the shuttle box responded to the shock by exhibiting digging behavior (Suppl. material 2: Cricket Digging Behavior Video). We observed the crickets digging with their front legs in response to the shock. Anecdotal observations of this behavior indicated that crickets displayed differences in digging behavior during the experimental trials when they were shocked. An experiment by
One improvement to the design that may enhance the ability of crickets to learn would be the addition of visual or olfactory stimuli. Previous research showed that individuals of other species are able to orient and can learn that if they are getting shocked, the shock will cease when they move to the other side of the arena. The addition of other cues could enhance learning but are not necessary for learning to occur (
Another possible improvement would be to replicate this experiment using a cricket species other than A. domesticus. Previous learning experiments in crickets have focused on species in the genus Gryllus, e.g., Gryllus bimaculatus (
The use of the shuttle box as described here is promising. We were able to demonstrate the predicted avoidance behavior in a majority of our animals in the master group. However, there are still some unanswered questions that must be addressed before the apparatus can gain wide applicability. These questions include appropriate spacing between the shock bars and variations in shock intensity. We believe that these are relatively minor issues and easily addressed in future studies. For example, consider a lever press situation for crabs.
This study provided important information about the learning abilities of house crickets and the suitability of using a shuttle box. Our experiment tested the house crickets’ ability to learn through aversive stimuli (i.e., shock). The behavior of both the female and male master cricket demonstrated limited aversive learning. Previous research has provided evidence of the learning abilities of crickets in other contexts. Further investigation into the learning abilities of house crickets and other cricket species though modifications of this aversive learning paradigm might provide more evidence on whether house crickets and other cricket species can learn through aversive conditioning using a shuttle box.
We would like to thank Chris Varnon for his efforts in advancing the propeller controller and for writing the program. We would also like to thank Chris Dinges for building the new shuttle box apparatuses used for this experiment. This research was funded by the NSF-PIRE 1545803 and NSF REU 1950805.