Manipulation of monoamines and effects on behavior in crickets.

Manipulation of monoamines and effects on behavior in crickets.

Louise Franzén

Student

Degree Thesis in biology 15 ECTS Bachelor’s Level

Report passed: 02 June 2017 Supervisor: Hanne Løvlie

Abstract

Animal personality is defined as behavioral differences between conspecific individuals which are relatively constant within the individual, over time or context. All species that have been studied have shown personality, which leads to the question why animals have

personality.Variation in personality is evolutionarily puzzeling and which underlying neurological and molecular explanations to why animals have personality is not fully understood. Many studies show covariances between the monoamines serotonin and dopamine, and personality. However, if the levels of monoamines affect personality or personality affects the levels of monoamines in animals is not completely known. In this study, serotonin and dopamine levels in male Mediterranean field crickets (Gryllus bimaculata) were manipulatedto investigate experimentally their effect on behaviour.

Personality traits (activity, exploration and aggressiveness) were after manipulation of monoamiones scored in standardized personality tests and compared with a control group.

Serotonin manipulation affected all behaviors scored where increased levels of serotonin led to reduced activity, exploration and aggression. However, no difference was observed in crickets that were dopamine manipulated. Further studies on this subject is needed, with comparisons among individuals and not group comparisons, to increase our understanding of the relationship between monoamines and personality and not just the relationship between monoamines and behavior.

Table of content

1. Introduction

1.1 Background

1.2 Monoamines

1.3 Aim and hypothesis

2. Materals and method

2.1 Study species

2.2 Housing of crickets

2.3 Personality assay

2.4 Injecting the monoamines

2.5 Statistical analysis

3. Result

3.1 Serotonin manipulation

3.1.1 Activity ... 4

3.1.2 Exploration and emergence ... 4

3.1.3 Aggression ... 5

3.2 Dopamine manipulation

3.2.1 Activity ... 5

3.2.2 Exploration and emergence ... 6

3.2.3 Aggression ... 7

4. Discussion

4.1 Serotonin manipulation

4.2 Dopamine manipulation

4.3 Further studies

4.4 Conclusions

Acknowledgements

References

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1. Introduction

1.1 Background

Animal personality is defined as behavioral differences between conspecific individuals which are relatively consistent within the individual, over time or context (Dall et al. 2004). Five major personality traits suggested to be used for ecological studies of personality for most animals are boldness, exploration, activity, sociability and aggressiveness (Reale et al. 2007). For example, many individuals within the same species can show aggressiveness in a situation. However, some individuals show consistent more aggressiveness than others, which then can be an example of personality. Personality has proven to affect important ecological processes (e.g. niche expansion, dispersal or social organization) and in many aspects of animal ecology, personality studies can generate important influences (Reale et al. 2007).

All species that have been studied show personality (Gosling 2001). However, personality

generates evolutionary puzzles. For example, how can less flexible traits exist stably and how can different traits coexist within the same species (Wolf et al. 2007)? This in turn generates the question why animals have personality.Variation in personality remains evolutionarily if

personality types have the same success over time and can be seen as alternative strategies (Dall et al. 2004). How variation in personality arises, i.e. which underlying neurological and molecular explanations to why animals have personality, is not fully understood. One suggestion is that variation in levels of monoamines operating in the nervous system can be an explanation for the differences in personality we can observe among different individuals (Coppens et al. 2010). This has to some extent been explored, however most studies that have focused on the relationship between monoamines and personality in animals showed only covariance (Summers et al. 2005, Murakami and Itoh 2001). Therefore, it is not fully understood if it is the behavior that affects the levels of monoamines or if the monoamines affect the behavior. To investigate this causality, experimental manipulations of monoamine levels can be done. Knowing this can help us get a better understanding of personality for both animals and humans, and perhaps make it possible to alter personality.

There are many personality studies on vertebrates (e.g. Andersson and Höglund 2012, Favati et al.

2016, Rose et al. 2017) but much fewer using invertebrates, which constitutes the majority of all animal species. Invertebrates are not often associated with personality, but previous studies show difference in personality traits among individuals in invertebrates. For example, Rose et al. (2017) tested aggression in crickets, which is an invertebrate, by studying fights. Crickets that had been isolated for 24 hours and crickets that had recently fought were put together in fights to see whether the aggression is a personality trait or a response to environment. Their results indicate that there is a difference in personality in crickets. Studying personality in invertebrates can increase our understanding of personality in general. Additionally, there are methodical advantages with invertebrates because they are easier to breed, manipulate and to obtain large amounts of (Kralj-Fišer and Schuett 2014). Insecta, which is the class of invertebrates this study will use, does not require the same ethical authorization for experiments as vertebrates do.

1.2 Monoamines

Monoamines are neurotransmitters, or hormones, that are synthesized from amino acids in the body and operate in the nervous system (Vleugels et al. 2015). It is suggested that major depressive disorders (e.g depression, anxiety, psychosis etc.) in humans are linked to the level of monoamines in the brain. (Hamon and Blier 2013). Serotonin (5-hydroxytryptamine) and dopamine are two monoamines that operate in the nervous system and affect many behaviors in humans and animals (Jensen 2009). Serotonin is often connected to fear and stress, where higher levels of serotonin have shown to reduce the fear, anxiety and stress levels in animals (Jensen 2009). Fluoxetine is a chemical that is used as a medicine for humans to relieve anxiety, depression etc. and the drug reduces energy intake which increases resting energy expenditure (Cheer and Goa 2001).

Fluoxetine increases the level of serotonin in the brain by inhibiting the reuptake of serotonin in the nerves (Cheer and Goa 2001). Dopamine is often connected to exploring and curiousness, and also with aggression and winner-loser effects (Rillich and Stevenson 2014). Dopamine has shown

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to be crucial for comeback after a loss in a fight (Rillich and Stevenson 2014). Ropinirole is a medicine that is used for humans with Parkinsons disease, where parts of the brain have a lower amount of dopamine (FASS 2017). Ropinirole is a dopamine receptor agonist, which binds to dopamine producing receptors and activate the production of dopamine. This leads to a higher level of dopamine in the brain (Matheson and Spencer 2000).

1.3 Aim and hypothesis

The aim of this study was to experimentally manipulate the levels of monoamines in

Mediterranean field crickets (Gryllus bimaculata) to investigate if the monoamines have an effect on personality. This will be done by measuring crickets personality in standardized personality tests (Santostefano 2016) and compare individuals with different treatments (fluoxetine or ropinirole and control).

The hypothesis for serotonin manipulation is that fluoxetine-injected crickets will show less aggression, as previous studies have shown (Blake and Grafman 2004, Coppens et al. 2010) and less exploration and activity due to the reduced energy levels (Cheer and Goa 2001). The

hypothesis for dopamine manipulation is that crickets injected with ropinirole will show higher activity and exploration than control crickets as previous studies have indicated (Nakayama et al.

2012) and also be more aggressive (Rillich and Stevenson 2014) than control crickets.

2. Materials and method

2.1 Study species

In this study, mature, adult Mediterranean field crickets were bought from a local pet shop (Zooariet, Linköping). The Mediterranean field cricket is found in Southern Europe along the Mediterranean, where they live in rural terrain, grasslands, pastures etc. They are common as live food for pets and often used in laboratory tests. This species was used here because it has been used before in similar laboratory studies (Dyankonova and Krushinsky 2013, Rillich and Stevenson 2014 Santostefano 2016, Rose et al. 2017,) and are easy to keep (Santostefano 2016). Only males were used in this study because they show more aggression than females.

2.2 Housing of crickets

Crickets (72 individuals for each monoamine test, total of 144 individuals) were held in containers (16 x 9.5 x 11 cm) individually with see-through plastic during april and may. All animals had ad libitum access to food in form of peeled apple slices and water in the form of gel cubes consistent of agar dissolved in water andsolidified into cubes. A cylindrical cardboard tube in each container acted as a shelter for the crickets. All containers were placed on a table with black plastic

enveloped around the containers to avoid unnecessary disturbance and cardboard between every container to avoid the individuals to see each other. Crickets were kept in a 12:12 hour day:night cycle with light available between 7 pm and 7 am. The temperature in the room was ca 23°C. All crickets were weighed and put in groups of similar weight (intervals of 0.05 grams). They were than held at least 12 hours in their home containers before personality tests were conducted, so the individuals would settle and familiarize with the environment. Behavioral test was conducted for three days for each manipulation, during the hours of light.

2.3 Personality assays

Three personality traits were used to assay various aspects of personality; activity, exploration and aggression as done previously by Santostefano (2016) (explained below). For each manipulation, crickets were divided into groups of 4 individuals with similar weight, with a total of 18 groups.

Activity

To score activity, the general level of activity in a familiar environment was recorded in the individuals home containers, so that the individuals would know their surroundings and the movement would not be considered exploration. Prior to the onset of the activity trials, each individuals within their home containers were moved to the recording setup and the lid, shelter

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and food/water dishes were removed from the home container. Individuals where then given 10 minutes to acclimatize, to minimize the stress that may occur when removing the shelter and food/water dishes. Activity was then recorded with cameras from above automatically for 15 minutes, as total amount of distance moved (in cm). The software Ethovision x 10.0 was used to analyze the total amount of movement.

Exploration

Individual exploration of a novel environment was recorded in a novel arena (36 x 21.5 x 22 cm). A novel arena was used so the individuals would not know the environment and the activity would instead be viewed as exploration (Reale et al. 2007). Prior to the onset of exploration trials, each individuals were moved (whilst within their shelters) from their home containers to individual novel arenas placed in the recording setup. Exploration distance was recorded with cameras from above and measured as the total amount of cm an individual moved during 15 minutes, starting from when they emerge from their shelters. The time it took for the crickets to emerge was also noted in seconds and if the cricket did not emerge within 20 minutes the test was aborted and the individual got a 0 in exploaration. In order to optimize for the automatic video tracking, the arenas were empty except for the shelters. The software Ethovision was used to analyze the total amount of movement in the novel arena.

Aggression

The general level of aggression was measured by placing two individuals (one individual was treated and one control) on each side of an arena (36 x 21.5 x 22 cm) divided by an opaque divider.

The crickets were given 10 minutes to acclimatize before the divider was raised and their behavior was observed for 10 minutes. During observation the number of wins was noted. A win was established when an interaction was made by the two individuals and one individual sang a

“victory” song whilst the other fled. At the end of an observation, the individual with the highest total number of wins was established as the winner. If there was no clear winner but the

individuals had interacted, a draw was set. If there was no interaction during the 10 minute trial, a no interaction was set.

2.4 Injecting monoamines

The two monoamines, serotonin and dopamine, were manipulated and compared with control one at a time with the same method on 72 different individuals for each manipulation. One group of four individuals was injected at a time in each manipulation with a micro syringe. For the serotonin manipulation, half of the individuals were injected with 10 µl fluoxetine (10 mM) and phosphate buffered saline (PBS) to block the serotonin uptake (n=36) and the other half with only PBS as control (n=36). For the dopamine manipulation, half of the individuals were injected with 10 µl ropinirole (33 mM) and PBS to block the dopamine uptake (n=36) and the other half with only PBS as control (n=36). Crickets were injected approximately 30-60 minutes prior to

personality tests were performed, because previous studies have shown that at least 30 minutes is required for the drugs to become effective (Rillich and Stevenson 2014). All individuals were exposed to an injection with a fluid of similar temperature to room temerature to minimize the effect the injection can have on the crickets. One person injected all the crickets and was the only one who knew which individuals that were injected with the fluoxetine/ropinirole and which that were injected with the control buffer. We who performed the personality tests on the crickets did not know which individuals that were injected with what, or not injected to avoid bias

observations.

2.5 Statistical analysis

To investigate variation in behavior between crickets treated with fluoxetine and control and individuals treated with ropinirole and control crickets, the total distance moved in activity and exploration were analyzed with the non-parametric statistical analysis Mann-Whitney U test (two- tailed). For both activity and exploration the mean value of total distance moved were compared between the treated groups and control group For exploration some of the individuals did not emerge from their shelter during the 20 minute trial, so if they emerged or not was noted and the likelihood of emergence was analysed in a Fisher exact test. For aggression, the total number of wins were compared between the fluoxetine treated individuals and control and the ropinirole

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treated individuals and control and Fisher exact test was used to calculate the significance. The statistical significance was defined as p<0.05.

3. Result

3.1 Serotonin manipulation

Fluoxetine reduced total distance moved for male crickets (treated individuals: 96.27 ± 6.37 cm, control individuals: 420.31 ± 16.90 cm, z=2.59, p=0.0099; fig. 1).

Figure 1. Mean of total distance moved in centimeters during the activity trial for both field crickets treated with fluoxetine (n=36) and control crickets (n=36). * p<0.05.

3.1.2 Exploration and emergence

Fluoxetine reduced total distance moved in a novel arena for male crickets (treated individulas:

36.04 ± 2.69 cm, control individuals: 207.90 ± 8.16 cm, z=2.71, p=0.0067; fig. 2). Fluoxetine also reduced emergence (p=0.015; fig. 3) for the crickets.

Figure 2. The mean values of the total distance moved in centimeters for the exploration trial for both fluoxetine treated (n=36) crickets and control crickets (n=36). * p<0.05.

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Figure 3. The fraction (%) of individuals that emerged from their shelters during the exploration trials for the control crickets (n=36) and the crickets treated with fluoxetine (n=36). * p<0.05.

3.1.3 Aggression

Fluoxetine reduced aggression in male crickets (p=0.0184; fig. 4).

Figure 4. The result from the aggression trials presented as fraction of wins (%) for male crickets, n=72. * p<0.05.

3.2 Dopamine manipulation

Ropinirole did not affect total movement in a familiar environment in male crickets (treated individuals: 212.55 ± 7.61 cm, control individuals: 165.01 ± 7.97 cm, z=1.26, p=0.2077; fig.5).

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Figure 5. The mean of the total distance moved in centimetres in a familier environment for the activity trial did not differ for ropinirole treated (n=36) crickets and control crickets (n=36).

3.2.2 Exploration and emergence

Ropinirole did not affect total movement in a novel arena for male crickets (treated individuals:

201.95 cm ± 6.85 cm, control individuals: 176.16 ± 6.08 cm, z=0.52, p=0.6031; fig. 6). Ropinirole did not affect emergence either (p=0.4736; fig. 7)

Figure 6. The mean value of the total distance moved in centimetres in a novelty arena for the exploration trial did not differ between ropinirole treated (n=36) crickets and control crickets (n=36).

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Figure 7. Fraction (%) of individuals that emerged or did not emerge from their shelters during the exploration trials for the control crickets (n=36) and for the ropinirole treated crickets (n=36).

3.2.3 Aggression

Ropinirole did not affect total number of wins for male crickets (p=0.5171; fig. 8).

Figure 8. The result of the aggression trial presented as fraction of wins (%) for male crickets, n=72.

4. Discussion

4.1 Serotonin manipulation

The result for the serotonin manipulation show a difference between the fluoxetine treated and control group of field crickets, where the control crickets are more active, more explorative and more aggressive than the treated crickets. The hypothesis, that crickets treated with fluoxetine would be less active and less explorative due to lower energy, and less aggressive as previous studies have shown (Cheer and Goa 2001, Blake and Grafman 2004, Coppens et al. 2010), is consistent with the result. Previous studies with serotonin manipulations show similar results as this study. For example, Clotfelter et al. (2007) which added fluoxetine to the water in tanks with Siamese fighting fish (Betta splendens) and tested the aggression both before and after the adding of fluoxetine. The fish with higher levels of serotonin showed less aggression than control

individuals. Even though, field crickets and fish are two different species, serotonin works relatively similar in all animals. Fluoxetine, which is a drug for humans, inhibits serotonin

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reuptake the same way in nerves of all animal species (Hird et al. 2016). Serotonin and behavior are in covariance as previous studies have shown (Summers et al. 2005, Murakami and Itoh 2001).

Furthermore, the results also indicate that behavior is affected by the levels of serotonin in the brain and not the other way around because it is the levels of monoamines that have been altered and not the environment.

4.2 Dopamine manipulation

The result for the dopamine manipulation shows no difference between the ropinirole treated crickets and the control crickets which is not consistent with previous studies. Other studies with dopamine manipulation have shown a difference in behavior between individuals with different levels of dopamine in the brain. For example, in flour beetle (Tribolium confusum), the general activity was higher for individuals with more dopamine were more active (Nakayama 2012). The explanation for this result can be that the ropinirole concentration was too low. The concentration was taken from a previous study (Waugh et al. 2014) and recalculated for crickets weight.

However, the calculations can be inaccurate and with more time for the study, more piloting could have been done to reassure a correct concentration. The time between the injection of ropinirole and the personality tests could also have been too short. If the ropinirole did not have enough time to act, there would not be a difference between the treated individuals and the control individuals.

Another explanation can be that the sample size was too small to show a significant difference.

However, the result from this study can be correct meaning that dopamine does not affect behavior in crickets.

4.3 Further studies

This study manipulated levels of serotonin and dopamine with two different medical drugs for humans, fluoxetine and ropinirole. Medical drugs can have side effects that affect the

manipulations and could perhaps contribute to the behaviors observed in this study. Therefore, tests with different medical drugs to manipulate the serotonin and dopamine levels are necessary for future studies.

Personality test for aggression can be more advanced in future studies. In this study, only the number of wins were compared which does not give the full extent of the personality trait

aggression. A prior study, by Santostefano (2016) assayed three different interactions during a 10 minute trial for aggression. “Approaching”, “singing” and “chasing” in an interaction were

considered as aggressive during the 10 minute trial, and it was noted each time an individual engaged in one of these interactions. At the end of the trial, the total number of each interaction were counted and individuals with higher numbers of interactions were considered more

aggressive. Another study (Rillich and Stevenson 2014) on aggression used a scale of 0-6, where 0=no interaction, 1=attack from one individual whilst the other retreat, 2=fencing with antennae, 3=one individual displays its spread mandibles as a threat, 4=both individuals spread its

mandibles, 5=the individuals engage in a fight with their mandibles and they interlock the mandibles and 6= an “all-out fight” with mandibles, biting and body flipping. The trial was concluded as soon as one of the individuals retreated. Both of these aggression tests give a better score for aggression and can also score the individuals that lost, was set as draw or individuals that had no interaction.

4.4 Conclusions

The result from this study show a significant difference in serotonin levels and their effect on behavior. It also indicate that it is the serotonin that effects the behavior and not the other way around. However, the result for the dopamine manipulation show no difference in behavior between individuals with different levels of dopamine. Therefore, more studies with dopamine manipulation needs to be done with perhaps a different concentration of ropinirole or a different dopamine receptor agonist.

Studies like this, with monoamines and personality, could be an important step towards

understanding personality and behavior in animals which can help improve animal welfare laws and directions by understanding the individuality in animals. It can also be applied to humans and increase our understanding of human behavior and personality and disorders connected to

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behavior (e.g. depression, anxiety etc.), and even how to alter those behaviors. Although, more studies are needed to completely understand the complexity of the different monoamines and their connection with each other and personality.

Acknowledgements

I would like to thank my supervisor Hanne Løvlie for guidance throughout the whole project. A big thanks to Robin Abbey-Lee and the rest of Hanne’s research team at Linköping University for help and guidance with the methods. I would also like to thank Elin Hessling, Kristoffer Lundgren and Simon Björklund Aksoy for working together and Laura Garnham for injecting all the crickets. I would also like to thank Rebecca Magnusson and Elisabeth Månsson for useful comments, and finally, I would like to thank Robin Forsberg for support and motivation.

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