Behavioural Processes 183 (2021) 104298
Available online 18 December 2020
0376-6357/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Manipulating monoamines reduces exploration and boldness of
Mediterranean field crickets
Kristoffer A. Lundgren
a, Robin N. Abbey-Lee
a, Laura C. Garnham
a, Anastasia Kreshchenko
a,b,
Sara Ryding
a,b, Hanne Løvlie
a,*
aDepartment of Physics, Chemistry and Biology, IFM Biology, Link¨oping University, 58381 Link¨oping, Sweden bSchool of Biological Sciences, University of Manchester, M13 9PL Manchester, UK
A R T I C L E I N F O Keywords: Aggression Animal personality Boldness Dopamine Exploration Serotonin A B S T R A C T
Despite the prevalence and research interest of animal personality, its underlying mechanisms are not yet fully understood. Due to the essential role of monoamines in modulating behaviour, we manipulated the mono-aminergic systems of Mediterranean field crickets (Gryllus bimaculatus) to explore whether this altered behav-ioural responses commonly used to describe animal personality. Previous work has shown that both serotonin and dopamine manipulations can alter cricket behaviour, although results differ depending on the drug in focus. Here, we investigate the effect of Fluphenazine, a dopamine antagonist which also interacts with serotonin re-ceptors, on activity, exploration, boldness, and aggression. These results are compared with those of our earlier work that investigated the effect of drugs that more specifically target serotonin or dopamine systems (Fluoxetine and Ropinirole, respectively). Due to limited research on dose-effects of Fluphenazine, we created dose-response curves with concentrations ranging from those measured in surface waters up to human therapeutic doses. We show that compared to control animals, Fluphenazine manipulation resulted in lower levels of both exploration and boldness, but did not affect activity nor aggression. The effect on explorative behaviour contradicts our previous results of serotonin and dopamine manipulations. These results together confirm the causal role of monoamines in explaining variation in behaviour often used to describe animal personality, effects that can be both dose- and behaviour-dependent. Further, our results suggest that previous results assigned specifically to the dopaminergic system, may at least partly be explained by effects of the serotonergic system. Thus, future studies should continue to investigate the explicit underlying roles of specific monoamines in explaining behavioural variation.
1. Introduction
Animal personality (i.e. consistent among-individual variation in behaviour, Dall et al., 2004; Sih et al., 2004; R´eale et al., 2007), is widely observed (Gosling, 2001; Carere and Maestripieri, 2013), and can have ecological and evolutionary consequences (Smith and Blumstein, 2008). Although animal personality has, by now, received significant research attention, its underlying mechanisms are not yet well understood (Dall et al., 2004; Carere and Maestripieri, 2013). To further our under-standing of the evolution of personality, research should, therefore, explore underlying contributions to observed differences in animal behaviour.
Monoamines have repeatedly been shown to affect behaviour in a wide range of animals, and so variation in monoamine levels could help
explain variation in personality (e.g. Coppens et al., 2010; Rillich and Stevenson, 2014; Winberg and Th¨ornqvist, 2016; Abbey-Lee et al., 2018, 2019). For example, observed differences in the serotonergic and dopaminergic systems have been linked to variation in explorative behaviour in both birds and mammals (e.g. Schinka et al., 2002; Kem-penaers et al., 2007; Grosser et al., 2015). However, the causality and specific pathways of the relationship between monoamines and per-sonality is still mainly unknown. Examples of experimental work that has shown monoamines to causally affect behaviour include work on the confused flour beetle (Tribolium confusum), where the dopaminergic system controls activity levels and boldness (Nakayama et al., 2012), and rodents, where the serotonergic system influences aggression (e.g. Olivier et al., 1995). In a recent study, we experimentally manipulated serotonin (via manipulation by Fluoxetine, a selective serotonin * Corresponding author.
E-mail address: hanne.lovlie@liu.se (H. Løvlie).
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Behavioural Processes
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https://doi.org/10.1016/j.beproc.2020.104298
reuptake inhibitor, Invernizzi et al., 1996; Koch et al., 2002) in male Mediterranean field crickets (Gryllus bimaculatus) and showed that this causally reduced activity and aggression (Abbey-Lee et al., 2018). In that same study, we observed that manipulation of dopamine (via manipu-lation by Ropinirole, a highly selective dopamine receptor agonist, Kvernmo et al., 2008) did not significantly alter these behaviours. Generally, this result contradicts other studies that suggest a link be-tween behaviour describing personality and the dopaminergic system (e. g. Nakayama et al., 2012; Rillich and Stevenson, 2014; Korte et al., 2017). These seemingly contrasting results might be because different drugs (which may vary in selectivity for dopamine receptors and in interaction with serotonin receptors) were used in different studies. Therefore, in the current study, we repeated the methods of our previous work (Abbey-Lee et al., 2018), using different doses of a less selective dopamine receptor antagonist, Fluphenazine (Degen et al., 2000), to again experimentally manipulate the monoaminergic systems of male Mediterranean field crickets. Doing so enabled us to compare the effect on behavioural responses of a drug that is less selective for dopamine compared to a dopamine selective drug (Ropinirole). Fluphenazine is a commonly used antipsychotic medication in humans (Seeman, 2002), which affects both serotonergic, and dopaminergic, receptors, by potentiating their activity and increasing their effects, although it is uncertain how (Meltzer, 1992; Roth et al., 1992; Fink and G¨othert, 2008; Choi et al., 2017). Fluphenazine can also affect the dopaminergic system in crickets (Unoki at al., 2005; Rillich and Stevenson, 2014). Due to the predicted lower dopamine-specificity and different mode of action of Fluphenazine compared to Ropinirole (Meltzer, 2004), we predicted that monoamine manipulation using Fluphenazine would affect our measured behaviours in a more similar way to that of serotonin manipulation (via Fluoxetine) than that of manipulation via drugs that more specifically manipulate only dopamine levels (Abbey-Lee et al., 2018, 2019). We here again measured activity, boldness, exploration and aggression. Because we recently demonstrated these behavioural assays to produce repeatable among-individual behavioural responses in the species (Abbey-Lee et al., 2018), we only measured each behaviour once per cricket in the current study. Thus, in our current work we are not measuring animal personality, but test our hypothesis at the unpartitioned phenotypic level of variation. Based on the comparison to our previous work, we predicted to see reduced activity, boldness and aggression in manipulated crickets, and reduced exploration due to the observed link between the dopaminergic system and exploratory behaviour (Kempenaers et al., 2007).
2. Materials & methods
2.1. Study subjects
Mediterranean field crickets (Gryllus bimaculatus) were used because they are a model species for neuroethological studies (Horch et al., 2017; Yano et al., 2012), and display repeatable personality variation ( San-tostefano et al., 2016; Abbey-Lee et al., 2018). Further, using the same species as in our previous work on the effect of manipulation of mono-amines on behaviour describing personality (Abbey-Lee et al., 2018), enabled to us to compare results obtained across studies.
We used sexually mature males (n = 112) purchased from a local pet shop where crickets were raised as pet reptile food (i.e. we used crickets from a population with likely limited genetic and environmental vari-ation). These were housed individually in plastic containers (9cm × 16cm x 10.5 cm), which were lined with a paper towel, had a shelter in the form of a cardboard tube, and were covered by a plastic lid. All crickets were housed in 23 ± 2 ◦C, with a 12 h : 12 h light : dark cycle (7
a.m. to 7 p.m. local time). Crickets had ad libitum access to food and water, which consisted of apple slices and agar water cubes. Containers were visually isolated from one another and individuals were isolated for minimum of 12 h prior to experimental testing, to reduce effects on recent social encounters on aggression (Stevenson and Rillich, 2013).
2.2. Monoamine manipulation
The monoaminergic systems of our crickets were manipulated using dopamine receptor antagonist Fluphenazine (Sigma-Aldrich). Due to the limited availability of published dose-curve responses for this drug, and to investigate how different concentrations of this drug could influence behaviour, a control, and six biologically relevant doses of the drug were chosen, ranging from values found in surface water up to human ther-apeutic doses (Fern´andez et al., 2010). We diluted Fluphenazine with phosphate-buffered saline (PBS) to yield the concentrations 0 μM
(control dose of only PBS), 0.0002 μM, 0.002 μM, 0.2 μM, 4 μM, 20 μM
and 392 μM (highest dose based on Unoki et al., 2006). The dose (to the
total of 10 μl solution) was injected between the 4–5 th segment of the
abdominal cavity using a micro-syringe (Hamilton, Switzerland; Dyak-onova et al., 1999). Due to longer-term effects of manipulation with this drug being unknown and the relatively short adult lifespan of crickets, we did not use a within individual repeated design for our work. Instead, we used 7 randomly assigned groups of males (n = 16 / per dose). Further, prior to manipulation, we weighed each cricket to the nearest 0.01 g to enable weight-matching of males to enable weight-matching of males for equivilent pairings during aggression assay (see below). At the time of injection, each cricket was colour coded to enable later identi-fication (colour code was altered between doses). Behavioural trials were conducted 3 h post-injection, according to results from a pilot study and our previous work (Abbey-Lee et al., 2018), by treatment-blind behavioural observers.
2.3. Behavioural responses
To investigate how manipulation of monoaminergic systems altered behaviour, crickets were assayed for four behaviours commonly used to describe variation in personality: activity, exploration, boldness, and aggression (R´eale et al., 2007). Behavioural assays were performed consecutively (sensu Abbey Lee et al., 2018). Behavioural assays for activity, exploration and boldness were done for each cricket singly, using automatic tracking with Ethovision XT 10 Noldus, Wageningen, Netherlands, while aggression was scored directly by an observer when 2 males interacted. All testing took place in a lab with the same light and temperature conditions as the lab the crickets were housed in. 2.3.1. Activity
To measure activity in a familiar environment (sensu R´eale et al., 2007), each cricket was placed in the recording chamber (1 × 1 m) while in its home container. To ensure clear tracking of activity, the lid, as well as any objects within the container, were removed from the container. Crickets were given 10 min to acclimate to the new conditions, followed by 15 min during which activity was recorded as total distance moved in cm by Ethovision XT 10 software (Santostefano et al., 2016; Abbey-Lee et al., 2018).
2.3.2. Exploration and boldness
To measure variation in exploration, we measured movement in a novel arena (R´eale et al., 2007). The novel area was a plastic container (36cm × 21.5cm × 22 cm), with the floor covered with fine, white sand. Shelters from the cricket’s home container were placed in the back-left corner of the novel container and secured to the wall with sticky tape to prevent rolling. We measured exploration by using Ethovision software, recording the total distance moved (cm) within 15 min after the crickets emerged from their shelter. We measured boldness as the latency (in seconds) it took a male to move his whole head outside of his shelter, prior to the start of the exploration assay. Crickets that did not emerge within 20 min were given 20 min for boldness, and 0 for exploration. 2.3.3. Aggression
We measured the outcome of intra-sexual duels, describing aspects of aggression, by observing males in direct interaction with a conspecific
(Stevenson et al., 2005; Santostefano et al., 2016). This was carried out directly after the exploration and boldness assays. For this, the con-tainers used to measure exploration and boldness were divided in two with a cardboard divider, with one individual on each side of the divider. Contests were set up between all mixes of dose concentrations in a pre-determined random order, so that all the possible dose combina-tions were matched against each other. Four weight-matched in-dividuals (±0.05 g) were tested together in 2 pairs. Inin-dividuals were given 10 min to acclimate to the arena, prior to removal of the divider. Behaviour of the two crickets (sensu Santostefano et al., 2016; Abbey-Lee et al., 2018) was then observed for 10 min. The winner of each contest was recorded as a binomial response, with the ‘winner’ (scored as 1) being the first cricket to win 3 consecutive interactions, and the ‘loser’ (scored as 0) being the other. An interaction was defined to begin when crickets came in contact with each other, and terminated when contact ceased for over 2 s (Bertram et al., 2011). We scored a cricket to have won an interaction when it produced a victory song, while the other cricket fled (Rillich et al., 2011). If no winner was assigned, both males of that pair were scored as losers.
2.4. Statistical analyses
All statistical analyses were conducted using R software version 3.4 (R Core Team, 2017).
To explore the inter-relatedness among observed behavioural vari-ables, we did 2 pairwise comparisons of all variables (1 on all control males, n = 16, and 1 on all males injected with Fluphenazine, all doses combined, n = 96), by using Spearman’s rank correlations.
We applied linear and generalized linear mixed-effects models (GLMM; package ‘lme4’, Bates et al., 2015) to our data. We used the ‘sim’ function (package ‘arm’, Gelman and Su, 2016) to simulate the posterior distribution of the model parameters and the values extracted were based on 2000 simulations (Gelman and Hill, 2007). Statistical significance of fixed effects and interactions were assessed based on the 95 % credible intervals (CI) around the mean (β). If the 95 % CI did not overlap zero, an effect was considered to be ‘significant’ (Nakagawa and Cuthill, 2007). Sample size was 112 throughout.
We ran four models, one for each of the behaviours we studied; ac-tivity (distance moved within home container, in cm), exploration (movement in a novel arena measured, in cm), boldness (latency until a cricket moved its head outside a shelter, in s) and aggression (winner or loser, binomial). Activity was modelled following a Gaussian distribu-tion. Exploration was log transformed to meet normality assumptions and was modelled following a Gaussian distribution. Boldness was modelled following a Poisson distribution. Aggression had a binominal distribution and was modelled as such. All models included dose levels
(a factor with 7 levels) as fixed effect, and time of injection (05:00− 12:45), and time since injection (3–3.45 h) as covariates. Indi-vidual colour markings for identification (none, red, white, red and white) were included as random effect. To ensure that we modelled variation in exploration and boldness independent of activity (sensu Abbey-Lee et al., 2018), activity was included as a covariate in the models analysing variation in exploration, and boldness.
3. Results
None of the four behavioural variables measured here (activity, boldness, exploration, and aggression) correlated strongly with each other. (neither for control males, nor those injected with any dose of Fluphenazine, Rs = 0.23 – 0.40, p = 0.28− 0.36). Each behavioural variable was, therefore, analysed separately.
When comparing behavioural measures from control males with behavioural measures from those injected with Fluphenazine, the monoamine manipulation appeared to result in both lower exploratory behaviour and boldness (Table 1, Fig. 1b, c, respectively), but not affect activity nor aggression (Table 1, Fig. 1a, d, respectively). Further, when exploring how different doses affected observed behavioural responses, injections with the intermediate dose (0.2 μM) of Fluphenazine
appeared to result in less explorative crickets, while boldness was lower for all doses but, 0.2 μM, and was lowest in the 0.002, 4, and 20 μM doses
(Table 1, Fig. 1b, c, respectively). Time of injection and time since in-jection had significant positive effects on boldness (Table 1). In the model analysing variation in exploration, activity had a small, positive significant effect, whilst for boldness it had a small, negative significant effect (Table 1).
4. Discussion
We here experimentally investigated the effect of monoamine ma-nipulations on four behaviours which can describe variation in person-ality in male field crickets. We manipulated monoamine levels via injections of 6 different concentrations of the non-specific dopamine receptor antagonist Fluphenazine, and a control. We show that whether our monoamine manipulation affected the behavioural responses here measured depended on both dose given, and the behaviour in focus. In response to some doses used for our monoamine manipulation, crickets explored a novel environment less, and their latency to emerge from their shelters became longer during exploration trials (i.e. they were less bold), while our monoamine manipulation did not affect their activity in their home tank, nor their aggression during contests. Specifically, only the intermediate dose of 0.2 μM appeared to affect exploration, and
boldness was lower for all dose levels, except for 0.2 μM, and most so
Table 1
Comparison of behaviours of male crickets after manipulation of monoamine levels. Doses refer to injection with one of 6 different Fluphenazine concentrations, or control injection. Estimated effect sizes and 95 % credible intervals (CI) around the mean of predictors of: Activity (distance moved in home environment, cm); Exploration (distance moved in novel area, cm); Boldness (time to emerge from shelter, s); Aggression (winner of contest, binomial). Bold = when CI does not overlap 0 (i.e. ‘significant’ effect).
Fixed effects Activity Exploration Boldness Aggression
β (95 % CI) β (95 % CI) β (95 % CI) β (95 % CI)
Intercept (0 μM) −24.35 (− 314.42, 276.67) 5.30 (3.25, 6.05) 5.88 (5.70, 6.06) −9.63 (− 21.03, 2.23)
Activity score – 0.005 (0.001, 0.01) ¡0.001 (− 0.0013, − 0.001) –
Time of injection −8.63 (− 27.52, 10.10) 0.005 (− 0.56, 0.58) 0.02 (0.01, 0.03) −0.22 (− 0.88, 0.44) Time since injection 58.50 (− 41.28, 154.85) −0.22 (− 3.33, 2.75) 0.13 (0.10, 0.16) 2.69 (− 1.79, 7.14) Doses 0.0002 μM −69.64 (− 182.28, 44.40) −1.74 (− 3.99, 0.53) 0.34 (0.30, 0.38) −37.88(− 321*105, 319*105) 0.002 μM −16.35 (− 143.02, 105.18) −0.52 (− 2.64, 1.62) 0.12 (0.08, 0.17) 0.91 (− 1.60, 3.50) 0.2 μM −69.24 (− 186.55, 44.54) ¡1.87 (− 3.56, − 0.24) −0.02 (− 0.07, 0.02) 0.69 (− 1.79, 3.24) 4 μM 31.09 (− 104.85, 163.79) −2.48 (− 5.16, 0.28) 0.85 (0.81, 0.89) −35.81 (− 331*105, 343*105) 20 μM −36.84 (− 150.67, 69.31) −0.51 (− 2.70, 1.56) 0.37 (0.33, 0.41) 0.79 (− 1.80, 3.34) 392 μM −41.66 (− 150.74, 61.10) 0.10 (− 1.68, 1.94) 0.33 (0.29, 0.36) −30.26 (− 485*104, 449*104) Random effects σ2 (95 % CI) σ2 (95 % CI) σ2 (95% CI) σ2 (95 % CI)
when the concentration of 4 μM was used.
Similar to our results, other studies have found significant dose- dependent effects on behaviour when manipulating the mono-aminergic systems (e.g. Baarendse and Vanderschuren, 2012; Korte et al., 2017). This indicates that the dose used can have great impact on how behaviour is modified, and produce non-linear relationships be-tween drug concentration and behaviour. This is important to consider for future studies, where the use of only one or few doses may fail to detect the impact of the tested chemicals on behaviour.
In our study, monoamine manipulation appeared to have different effects on different behavioural responses. Levels of exploration and boldness were lower in male crickets injected with Fluphenazine, compared to control males. These behavioural variables did not corre-late strongly in our study (i.e. did not form a behavioural syndrome, Sih et al., 2004), hence the effects observed were independent of each other. The effect of monoamine manipulation on exploration contrasted our previous results, where we saw no effect on exploration when manipu-lating the dopaminergic or serotonergic system more individually (by using Ropinirole and Fluoxetine, respectively, Abbey-Lee et al., 2018). Studies on other species suggest exploration to be motivated through the dopamine reward pathway (Dulawa et al., 1999; Kempenaers et al., 2007). These combined results suggest that exploration in our males was potentially lowered due to Fluphenazine’s antagonistic action on the dopaminergic system. Alternatively, in the work presented here, exploration may have been lowered by Fluphenazine potentiating the activity of the serotonin receptors, thus increasing their effect, which can inhibit the dopamine reward circuit (Fink and G¨othert, 2008). Our results may, thus, suggest that exploration potentially depends on action of both the serotonergic and dopaminergic systems, both of which may be manipulated with Fluphenazine as this drug can bind with both se-rotonin and dopamine receptors (Meltzer, 1992; Roth et al., 1992). These potential explanations for the observed differences across studies of the role of monoamine manipulation on exploration thus warrant further investigation. This could, for example, be carried out by manipulating the monoaminergic system using a serotonin receptor antagonist in conjunction with Fluphenazine, aiming to provide further insight into the relationship between exploration and the serotonergic and dopaminergic systems.
Boldness, measured as latency to emerge from a shelter, is not typically recorded in insect studies exploring behavioural variation in relation to monoamine variation. However, in the confused flour beetle, how long an individual stayed in tonic-immobility (measured as tha-natosis, i.e. being immobile, which is considered an aspect of the shyness-boldness gradient, R´eale et al., 2007), was reduced after manipulation of the dopaminergic system, making individuals bolder (manipulation was carried out via caffeine, which increases the activity of dopamine receptors, Nakayama et al., 2012). These results are in line with our results, with a decrease in boldness by inhibiting dopaminergic receptors. Whether the observed effect on boldness is solely due to in-hibition of the dopaminergic system, is, however, not yet clear in male Mediterranean field crickets.
We observed limited behavioural responses in activity and aggres-sion when manipulating the potential underlying mechanisms of these behaviours via injection of Fluphenazine. This is in line with our pre-vious manipulation of the dopaminergic system with Ropinirole, where we also observed limited effect on alteration of these behaviours. On the other hand, manipulations of the serotonergic system via injection of Fluoxetine did influence these behaviour (Abbey-Lee et al., 2018). Comparing our results may, thus, suggest that variation in both activity and aggression are better explained by variation in the serotonergic system, than the dopaminergic system. This suggestion is based on the differences in binding specificity of Ropinirole and Fluoxetine versus Fluphenazine. The results of our current aggression assay are more inconclusive, as we, in the current study, had a low number of crickets engaging in fights (only 7 of 112). Our lack of agonistic interactions may be due to the reduced level of exploration and boldness from our Fig. 1. Behavioural responses of male crickets to monoamine manipulation via
Fluphenazine. Mean and standard error describing: a) Activity (distance moved in home environment, cm, n = 16 per dose); b) Exploration (distance moved in novel area, cm, n = 16 per dose); c) Boldness (time to emerge from shelter, s, n = 16 per dose) where longer latencies describe shyer males; d) Aggression (winner of fight dyad, binomial, n0.0002, 4, 392 μM =0, ncontrol =1, n0,002, 0.2, 20
μM = 2). White circles = control injection with only PBS, grey circles = Fluphenazine manipulations, one of 6 doses. Doses are visually presented at equal distances, but are of non-equal magnitude.
manipulations, reducing the likelihood of individuals coming into con-tact with each other, as crickets are less likely to fight if there is no con-tactile contact (Adamo and Hoy, 1995). Further, previous work shows that dopamine is involved in recovery behaviour after a social defeat (Rillich and Stevenson, 2014), making it possible that other aspects of contest behaviour than those recorded here are more affected by inhibition of the dopaminergic system.
Overall, our study suggests that the results of monoamine manipu-lations on behaviour depend on the drug in question. Specifically, our previous study using Ropinirole, a drug that is highly specific to dopa-mine receptors (Kvernmo et al., 2008), found no effect on behaviours used to describe personality in crickets, while by here using Fluphen-azine, a drug that interacts with both dopamine and serotonin receptors (Meltzer, 1992; Roth et al., 1992), we observe effects on measured behaviour. Altered behaviour in response to the less dopamine-specific drug, while not to a dopamine specific drug, means that analysis of only Fluphenazine alone could have led to over-estimating the impor-tance of dopamine in behavioural modification. That Fluphenazine and Ropinirole have opposite modes of action on the dopaminergic system (i. e. antagonistic and agonistic, respectively, Degen et al., 2000; Kvernmo et al., 2008), can explain our contrasting results compared to those of our previous manipulations of the dopaminergic system (Abbey-Lee et al., 2018). Our results from Fluphenazine manipulation do not exactly match results from other serotonin manipulations, which may indicate that a combined action on both the dopaminergic and serotonergic system result in different behavioural responses compared to action on the serotonergic system alone. This, in turn, suggests that research on monoamine manipulation may be highly dependent on the drug used, and thus researchers should strive to use chemicals with known speci-ficity. The potential for drugs to affect both the dopaminergic- and serotonergic systems could be explored by comparing the effect across a range of monoamine manipulating drugs with known affinity for these systems.
5. Conclusion
Taken together, our results show that pharmacological manipulation via Fluphenazine injections altered behaviour describing personality in male crickets in both a dose- and behaviour-dependent manner. Using different modes of manipulating the monoaminergic systems may impact how behaviour is altered, as we saw differences in behaviour when we used a dopamine receptor antagonist (Fluphenazine), whereas we previously saw none using a dopamine receptor agonist (Abbey-Lee et al., 2018). The generality of these results needs to be further explored as well as potential sex differences in responses. Overall, our results add support to monoamines being important underlying mechanisms in explaining variation in behaviours used to describe animal personality, and that the dose used can have a large impact on how behaviour is modified.
Author statement
KAL, RNAL, AK, SR, HL designed the study; KAL & HL coordinated the study; KAL & LCG injected crickets; KAL, SR & AK collected behavioural data; KAL carried out statistical analyses with input from RNAL & HL; KAL & HL drafted the manuscript; HL funded the study. All authors commented on the final draft and gave final approval for publication.
Data availability
Data analysed for the presented study is available as supplementary material.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
We thank Anders Hargeby for lending us cricket housing tanks. Funding was provided to HL and RNAL from Center for Systems Neurobiology.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.beproc.2020.104298.
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