1 The effect of the pesticide imidacloprid on the memory of three-spined sticklebacks (Gasterosteus aculeatus) and zebrafish (Danio rerio) Britt D.G. Sjöqvist Degree project for Bachelor of Science in Biology BIO603 Biology: Degree project 30 hec Spring 2014 Department of Biological and Environmental Sciences University of Gothenburg Examiner: Johan Höjesjö Department of Biological and Environmental Sciences University of Gothenburg Supervisors: Joacim Näslund Department of Biological & Environmental Sciences University of Gothenburg Joachim Sturve Department of Ecotoxicology University of Gothenburg Jörgen Johnsson Department of Biological & Environmental Sciences University of Gothenburg
Transcript
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The effect of the pesticide imidacloprid on the memory of three-spined sticklebacks
(Gasterosteus aculeatus) and zebrafish (Danio rerio)
Britt D.G. Sjöqvist
Degree project for Bachelor of Science inBiology
BIO603 Biology: Degree project 30 hecSpring 2014
Department of Biological and Environmental SciencesUniversity of Gothenburg
Examiner: Johan HöjesjöDepartment of Biological and Environmental Sciences
University of Gothenburg
Supervisors: Joacim NäslundDepartment of Biological & Environmental Sciences
University of Gothenburg
Joachim SturveDepartment of Ecotoxicology
University of Gothenburg
Jörgen JohnssonDepartment of Biological & Environmental Sciences
University of Gothenburg
Table of ContentsABSTRACT......................................................................................................................................... 3THEORY/INTRODUCTION...............................................................................................................4METHOD............................................................................................................................................. 6RESULTS/DISCUSSION.................................................................................................................. 10CONCLUSION ................................................................................................................................. 14REFERENCES................................................................................................................................... 15
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ABSTRACT
The neonicotinoid pesticide imidacloprid has a detrimental effect on not only insects, but other organisms as well. Terrestrial insects, aquatic crustaceans, aquatic insects, and aquatic invertebrates are all harmed by imidacloprid in surface waters; even at low concentrations. Some researches show a possible effect on aquatic vertebrates. This is a pressing issue due to high concentrations being found in, among other places, West Africa, which may be detrimental to the environment. In this study we test the effect of imidacloprid on the memory of three-spined sticklebacks (Gasterosteus aculeatus) and zebrafish (Danio rerio) since they are both model organisms that have been used in memory experiments. Both species were divided into three treatments: Control (0 µg/L), Low (10 µg/L), and High (100 µg/L). The sticklebacks were taught where to find food in a maze and the zebrafish were tested with a novel object test. The hypothesis for the stickleback experiment is that non-exposed control fish will learn to locate the food in the maze, but the fish exposed to imidacloprid will keep finding food at a rate equal to chance. The hypothesis for the zebrafish experiment is that non-exposed control fish will recognise the old object and spend more time exploring the novel object when introduced to both at the same time, but the fish exposed to imidacloprid will not spend significantly more time around the novel object. In general sticklebacks learned to find food faster in the maze, but there was no significant effect of imidacloprid. The experiment on zebrafish showed little significant evidence to indicate an effect of imidacloprid, even if there was a trend suggesting an effect of imidacloprid. However, both experiments are, due to the small scale, pilot studies and did show interesting trends suggesting an effect of imidacloprid. More research with larger sample sizes and longer exposure will be necessary to adequately evaluate the effects of imidacloprid on fish behaviour.
Den neonikotinoida pesticiden imidakloprid har negativa effekter på insekter och flera andra organismer. Terrestra insekter, akvatiska kräftdjur, akvatiska insekter, och akvatiska invertebrater blir alla skadade av imidakloprid i ytvatten, även vid låga koncentrationer. Vissa undersökningar visar också en effekt på akvatiska vertebrater. Detta är ett viktigt problem för att höga koncentrationer har hittats i, bland annat, West Afrika, vilket kan ha en skadlig effekt på miljön. Den här undersökningen testar effekten av imidakloprid på minnet hos storspigg (Gasterosteus aculeatus) och zebrafisk (Danio rerio). Båda arterna är modelorganismer som har använts i minnesexperiment. Båda arterna utsattes för tre behandlingar: Kontroll (0 µg/L), Låg (10 µg/L), och Hög (100 µg/L). Storspiggen tränades att hitta mat i en labyrint och zebrafiskarna testades i ett novel object-försök. Hypotesen för storspiggexperimentet är att de icke-utsatta kontrollfiskarna lär sig att hitta maten i labyrinten i en högre succesgrad än de för imidakloprid utsatta fiskarna. Hypotesen för zebrafiskexperimentet är att de icke-utsatta kontrollfiskarna kommer att känna igen det gamla objektet och spendera mer tid kring det nya objektet och utforska det när de blir introducerade till båda objekten samtidigt, men fiskarna utsatta för imidakloprid kommer inte spendera signifikant mer tid kring det nya objektet. Spiggarna lärde sig att hitta maten snabbare i labyrinten, men det fanns inga signifikanta resultat som visade en effekt av imidakloprid. Experimentet på zebrafiskarna visade också väldigt lite signifikanta resultat som indikerade en effekt av imidakloprid, även om det fanns en trend som suggererade en effekt av imidakloprid. Båda experimenten är småskaliga pilotstudier och visade intressanta trender som kan peka på en effekt av imidakloprid. Mer forskning med större stickprovsstorlekar och längre exponering behövs för att adekvat kunna bedöma effekterna av imidakloprid på fiskars beteende.
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THEORY/INTRODUCTION
Imidacloprid is a neonicotinoid insecticide, which means that it has an impact on the nervous system of insects through either contact or ingestion (Gervais et al, 2010). Imidacloprid blocks the post-synaptic nicotinergic receptors in the nervous system of the insect, which leads to an accumulation of the neurotransmitter acetylcholine, and this causes the insect to paralyse (extoxnet.orst.edu, 2014). Acetylcholinesterases, that metabolise acetylcholine in the synaptic cleft, are unable to break down imidacloprid, which leads to a continuous and irreversible activation of the receptors. Imidacloprid is more toxic to insects since nicotinergic pathways are more abundant in insects' nervous system than in mammals' (extoxnet.orst.edu, 2014; Matsuda and Sattelle, 2005). Imidacloprid can end up in surface water via run-off/leaching, overland flow, or via leaf fall (Kreutzweiser et al, 2007; Appelts, 2013). It is believed that imidacloprid does not affect vertebrates, including birds and aquatic vertebrates (Gervais et al, 2010), due to the blood-brain barrier in vertebrates (Gervais et al, 2010), which creates a highly selective permeable barrier between the circulating blood and the extracellular fluid of the brain. Combining this with information that imidacloprid has a short half-life in the environment suggests that imidacloprid poses a low risk in aquatic environments (Gervais et al, 2010). At least, that is what was believed until only a couple of years ago. Researchers at the university of Gothenburg found significantly high amounts (nearly 1 µg/L) of imidacloprid in the waters of Mozambique (Joachim Sturve, personal communication). Imidacloprid has also been found to be toxic towards aquatic crustaceans, aquatic insects, and aquatic invertebrates (extoxnet.orst.edu, 2014). Recent research has proven imidacloprid to have detrimental effects at even lower concentrations than previously thought (Smit, 2014). Imidacloprid is one of the neonicotinoids currently involved in the debate regarding the higher effect of lower doses compared to higher doses (Kollipara, 2014). Add all this to a shortage of research overall (Matsuda and Sattelle, 2005) and it is clear that there is more research needed on the environmental effects of imidacloprid. Research could be aimed at physiological responses to imidacloprid, the effect during different life stages of various organisms, or the effect of imidacloprid on reproductive success of various organisms.
Both three-spined sticklebacks (Gasterosteus aculeatus) and zebrafish (Danio rerio) are important model species when it comes to testing behaviour, toxicity and the effect of toxicants on behaviour (Huntingford and Ruiz-Gomez, 2009; Hill et al, 2005). Nobel prize winning Niko Tinbergen researched three-spined sticklebacks in the 1930s (Kreutzweiser et al, 2007) and George Streisinger researched zebrafish in the 1960s (Grunwald and Eisen, 2002). Research has shown that three-spined sticklebacks do use memory to locate food (Odling-Smee and Braithwaite, 2002; Odling-Smee and Braithwaite, 2003). Odling-Smee and Braithwaite (2002) provided three-spined sticklebacks with a food reward and the presence of a shoal in a T-maze (Odling-Smee and Braithwaite, 2002). The sticklebacks were trained to locate the food reward and conspecifics through either plastic landmarks, turn direction, or both (Odling-Smee and Braithwaite, 2002). Their experiments showed that the fish did not find their way to the food reward through uncontrolled visual cues or olfactory cues (Odling-Smee and Braithwaite, 2002). Therefore, the increasing success of their subjects finding the food rewards means that they have memory and learning capability which they used to locate the food.Zebrafish also have memory when trained with food as a reward (Williams et al, 2002) and can even be tested in a one-trial memory test, as proven by Lucon-Xicatta and Dadda (2014). The one-trial memory test is already known as a reliable test for investigating the memory in mammals, with the benefit of not requiring long periods of training (Lucon-Xicatto and Dadda, 2014). The zebrafish were introduced to an object, and after a resting period introduced to two objects: the old object and a novel object, different in shape and colour (Lucon-Xicatto and Dadda, 2014). Their
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testing showed that the memory of zebrafish indeed can be tested with the one-trial memory test. Sticklebacks and zebrafish are therefore appropriate test organisms to use in this research. Odling-Smee and Braithwaite (2003) review that “fish can and do use learning and memory to orientate within their natural environments”. In this project I set out to test the effect of imidacloprid on the memory of three-spined sticklebacks and zebrafish. Memory was measured by the subjects' success in locating food in a maze or recognising an earlier introduced object in a one-trial novel object test. The choice fell on testing memory because imidacloprid has been proven to have a detrimental effect on the memory and foraging behaviour of bees (Decourtye et al, 2004; Williamson and Wright, 2013). Therefore, I wanted to to investigate whether it would negatively affect the memory and learning capability of fish as well. The hypothesis is that the influence of imidacloprid lessens this capability to learn and memorise.
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METHOD
Stickleback experiment
In the first experiment three-spined sticklebacks were tested using three different treatments: control (0 µg/L), low imidacloprid dose (10 µg/L), and high imidacloprid dose (100 µg/L). 45 sticklebacks were divided over nine 10 L aquaria, with five fish per aquarium, and three aquaria per treatment.
Table 1: Aquaria distribution sticklebacks.
Tank # 1 2 3 4 5 6 7 8 9
Concen Low Control High Low Control High High Control Low
The treatments were randomly distributed among the aquaria, within the groups of 123, 456, and 789, to take away any bias due to placement in the room. See Table 1 for details. The fish were netted from a common holding tank and marked before being randomly distributed among the experimental aquaria. The fish were marked with fluorescent elastomeres (black, pink, green, blue, orange): first 9 fish with one colour and then 9 fish with the next colour until we had reached 45 fish.
As can be seen in Table 2, imidacloprid was added on Sundays, Tuesdays, and Thursdays. If the water was changed on the same day, this was always done before adding the imidacloprid. On Tuesdays, Thursdays, and a Friday the water was changed. This was done by emptying the aquarium of half its water and filling it to the level of 10 litres again. The testing (feeding in the maze) occurred on Mondays, Wednesdays, and Fridays. Thus each fish went through three trials per week, six in total.
Before the addition of imidacloprid to the aquaria the fish were fed inside the maze (Fig 1), with red mosquito larvae both to the right and left, and left to swim around and explore the maze for 30 minutes. This was to let them familiarize themselves with the maze and to not be too shy during the experiments due to a new environment. The fish were only fed inside the maze to increase the association of the maze with being fed. Imidacloprid was added on Sundays, Tuesdays, and Thursdays. When adding imidacloprid I assumed all the imidacloprid would have disappeared from the water through photolysis, microbial degradation, hydrolysis, or ingestion by the fish (Gervais et al, 2010).
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The maze consisted of a starting chamber with a wall that could be pulled up by a string to regulate when the fish would be able to swim out into the maze without disturbing the fish too much. The fish could either swim to the right or to the left of the diamond-shaped chamber in the middle. This shape was chosen to funnel the fish either left or right, decreasing the amount of “dead space” the fish could swim around in, and at the same time obscure sight of the food from the fish until they had actually swam into one of the arms.
The maze, as shown in Fig. 1, was filled with 5 cm (about 6 L) of water. Then the red mosquito larvae were added on the right side in such a location that they could not initially be spotted by the fish. The fish were then added to the starting chamber, where they rested for 10 minutes. The middle wall was pulled up and the fish swam around until they had found the food, or for a maximum of 30 minutes. The first choice (correct/wrong) was noted.
The food reward was placed on the right side, if the first choice of the fish was to swim to the right it was noted as 'correct'. If the fish swam to the left first it was noted as 'incorrect'. The choice to place the food on the right side was randomly decided by flipping a coin but the same for every fish as to not create a directional bias between the fish. The hypothesis is that the proportion of fish swimming correctly on their first choice will increase, indicating that the fish retain memory of where the food is located, except for the fish treated with imidacloprid which are hypothesised not to learn and therefore to keep on choosing the left and right side in a 50-50 ratio.
To analyse whether the proportion of sticklebacks choosing the correct side first differed among the treatment groups, I used a generalized linear model (GLM) based on the binomial data from the last trial. The first GLM analysed choice, treatment, and sex. The second GLM analysed treatment only.
Zebrafish experiment
In the second experiment I again used three treatments: Control (0 µg/L), Low (10 µg/L), and High (100 µg/L). 54 zebrafish were divided among six 12 L aquaria, with nine fish per aquarium. Table 3 shows the distribution of and across the tanks.
Table 3: Aquaria distribution zebrafish.Tank # 1 2 3
Treatment Control High Low
Tank # 4 5 6
Treatment Control High Low
These fish were divided at random, just like the sticklebacks. The placement of the different treatments was randomised as well, within the groups 123, 456, again to remove any bias due to position.
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Figure 1: Top-down view of stickleback maze. The starting chamber is at the bottom, with a wall that can be lifted up. The diamond-shaped room divides the maze in two and is essentially “dead space”.
See Table 4 for the treatment schedule. The fish were exposed to imidacloprid for four days. The water in the aquaria was refreshed on day one, by changing half of the water, and imidacloprid was added. On day three the water was again changed and more imidacloprid was added, again assuming that all the imidacloprid had disappeared from the water. The fish were fed with aquarium fish food in flake form every day. On the fifth day the fish were tested.
The zebrafish were tested by a one-trial memory test, based on the manuscript Assessing memory in zebrafish using the one-trial test by Lucon-Xiccato and Dadda (2014). The testing tank, Figure 3, contained a mirror and a plastic aquarium plant to provide comfort and cover. Two different types and colours of hardware nuts of approximately the same size were chosen as objects. One object was hexagonal and silver-coloured while the other was cylindrical with a flat head and golden-coloured, see Fig. 2. The tanks were filled with 8 cm, which is about 5 L, of water.
After the fish had been put in the test-tanks they were first given a half-hour adjustment period before the objects were hung on a bar, which was then lowered into the tank remotely via a pulley system. The type of object and the side of placement were chosen at random. Then the fish were filmed for 20 minutes with a stationary camera. After the filming the first object was lifted from the tanks, the second object added to the bar, and after two hours both objects were lowered in the tanks again whereupon the fish were filmed for another 20 minutes. After the experiment the fish were photographed in order to measure their length. The reasoning behind the test was that if the fish spent more time around the novel object they must have remembered the old one and be more curious about the new one (Lucon-Xicatto and Dadda, 2014).
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Figure 2: The silver-coloured and golden-coloured objects used in the zebrafish experiment.
Figure 3: Side view of the zebrafish test tank. 1 and 2 are the placements of the objects. 3 is a plastic aquarium plant hanging upside down for shelter. 4 is a mirror in the middle of the tank and behind the plant to give the zebrafish a feeling of safety from a shoal (it's own mirror image). 5, 6, and 7 are the different areas where the fish could be, used for measuring the interest of the fish in the object, with the hatched line being the border in between.
To analyse the zebrafish experiment the E-O ratio per fish during the second round was calculated. E stands for 'empty' in the first round and therefore it is the 'novel object' in the second round. O stands for 'object' in the first round and therefore 'known object' in the second round. In short, the E-O ratio is the time spent around the novel object compared to the time spent around the known object. The data was analysed using a Generalized Linear Model (GLM) to test the proportion of time spent in a specific place, in this case around the novel object in relation to the old object where, the sides (area 5 and area 7, see Fig. 3) were compared to each other. The decision was made to focus on minute 2-4 in the statistical testing due to two reasons: firstly, the fish were observed as swimming around frantically in a stressed manner closely after introduction of the objects; secondly, Huntingford and Ruiz-Gomez (2009) have shown that the proportion of time spent around the stimulus evens out after the first four minutes. During the first round, with one object, the E-side was empty, and the O-side contained the object. During the second round, with both objects the E-side contained the novel object and the O side the known object. The difference in time spent in the middle section per round was analysed using a Kruskal-Wallis test.
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RESULTS/DISCUSSION
As stated in the introduction the aim of this project was to test the effect of imidacloprid on the memory and learning capability of three-spined sticklebacks and zebrafish. The hypothesis was that the influence of imidacloprid lessens the fishes' capability to learn and memorise.
Stickleback experiment
Of the 45 sticklebacks when starting the experiment 12 died, leaving 33 for the final analysis. 12 out of 15 control fish survived, 11 out of 15 low-concentration fish, and 10 out of 15 high-concentration fish. All the sticklebacks that died during the two weeks were removed from the statistical analyses.
In the control group of sticklebacks there is a clear positive learning curve (Fig. 4), indicating that the fish do learn where the food is located and thus have memory. There is a dip on the fourth trial, which could possibly be due to it being after the weekend, since there would be two days in between instead of one. It would have been interesting to continue the experiment for another week. Unfortunately that wasn't an option in this case because of the high mortality rate, which would have made further statistical analysis difficult.
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Figure 4: Learning curve, control group, sticklebacks. Y-axis: percentage of sticklebacks finding the food on first try. X-axis: trial number.
Figure 5: The learning curves of sticklebacks in different treatments. The fish that died during the two-week experiment were removed from the statistical analysis. Y-axis: percentage of sticklebacks finding the food on first try. X-axis: trial number.
When comparing the different treatments (Fig. 5) we see that the control-group and high-group have very similar curves, both positive, indicating that fish in both treatment groups have undergone a learning process. This positive learning curve indicates an ability to learn and memorise new information. The low-group however does not show any sign of learning or memorisation. This hints towards a treatment with 10 µg/L imidacloprid might have a detrimental effect on sticklebacks' learning and memorising capabilities. However, the high-group does not have any heightened difficulty with finding the food. And in the last trial the amount of fish finding food on their first try are quite similar among all the groups.
The control curve and the low curve are not surprising in respect to the hypothesis, even though the control group would have been expected to have a higher success ratio, but the high curve does not support the hypothesis. The control- and high-curve are positive and the low-curve is negative. This would suggest that control and high did learn, whereas low even forgot what they already knew. Due to the low incline and decline of the curves I have chosen not to run a statistical test, as I do not expect them to show significant results. They do however show trends that hint towards an effect and considering the fact that other research has shown significant results when testing the memory of sticklebacks additional testing is required (Odling-Smee and Braithwaite, 2002; Odling-Smee and Braithwaite, 2003).
In comparing the failure rates of the different treatments in estimated marginal means there was a seemingly higher rate of failure in the treatment groups (MeanL=0,45; MeanH=0,40) than in the control group (MeanC=0,33), but this was not significant (p=0,834). Therefore the rate of failure is considered equal among the different treatments.
These differences may be insignificant due to the small sample size (33 sticklebacks), since the graphs suggest an effect; at least in the low treatment group. There could be variations due to, for example, gender, but larger groups would be necessary to determine this. It would be interesting to continue the experiment for another week, which was not worthwhile in this particular study because of the high mortality rate. A thought might be to test two treatments only. A control group (0 µg/L) and an exposed group (10 µg/L). However, a seemingly higher effect on the low group than on the high group does not necessarily mean that it is merely a coincidental indication due to small sample sizes. As mentioned before, imidacloprid is currently involved in the debate regarding the higher effect of lower doses compared to higher doses and it is therefore interesting to test low doses (Kollipara, 2014).
One speculation worth mentioning is that these sticklebacks were non-river fish. There is a difference in navigating between river sticklebacks and non-river sticklebacks (Odling-Smee and Braithwaite, 2002). Due to the flow of the water and therefore the ever changing environment in rivers these sticklebacks learn to memorise where food and places are in a different way than fish from still water (Odling-Smee and Braithwaite, 2002). Sticklebacks from still water learn to memorise their surroundings through visual cues (e.g.: rocks, sticks, human waste) (Odling-Smee and Braithwaite, 2002). The sticklebacks could have been in need of a visual cue in order for them to even be able to memorise the location of food in the maze. Even though the control group did improve over time, they did not improve as much as expected.
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Zebrafish experiment
As with sticklebacks my initial number of zebrafish was higher than the number used in the end for statistical analysis. I started out with 9 fish per aquarium, thus 18 per treatment. However, due to a flaw in the camera set-up this number was reduced to 6 fish per aquarium and therefore 12 per treatment and 36 in total.
When the E-O-ratio is positive (see Fig. 6) it means that the fish spent more time around the novel object than around the known object. These results are not significant for any treatment, as is shown with a Chi Square test (1,7, p=0,42, df=2). However, there seems to be a trend going downwards as the concentrations get higher, as seen in Fig. 6. The fish exposed to imidacloprid spend more time around the known object than the control fish. But none of this is significant, probably in part due to the small sample size of 12 fish per treatment. According to my hypothesis the control group should spend more time around the novel object, but the treatment groups should spend an equal time around the E and O objects since both should be novel to them. It is clear that the largest part of fish, over all treatments, spend their time in the middle or equal part E and O. It would be interesting to see what would happen with a larger number of fish.
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Figure 6: Zebrafish. The median and range for E-O (novel-known) ratio. The red dots are individual data points. The dotted line is the 0-line. The Y-axis is the absolute value of time in seconds with O being positive (above the 0-line) and E negative (below the 0-line). A is the first round, with only one object (O). B is the second round, with two object (E=novel).
Figure 7: Zebrafish. Amount of time spent in middle in round A (one object) and round B (two objects). Y-axis reads: M/240 and is the percentage of time spent in the middle.
Fig. 7 shows that, regardless of treatment, the zebrafish spent a significantly higher amount of time (GLM repeated measures analysis p=0,040) in the middle during round B than during round A. This figure is of all the fish, so 36 zebrafish. Perhaps the middle was too comfortable for them to leave after the 2 hour pause or perhaps the fish were too acclimatised to the tank after the wait. Each tank had a mirror and a plastic water plant. The mirror could be giving the zebrafish too little incentive to explore the objects because they have the feeling that there is another fish. Maybe they are even waiting for the other fish to make the first move. It could also simply be that they were too afraid when two objects were lowered.
As can be seen in Fig. 8 the control group spent the same amount of time in the middle during round A and round B, but the treatment groups spent a seemingly higher amount of time in the middle during the second round, even though this was not significant. Perhaps the control fish remember the first object and are therefore only faced with one new object, while the treatment groups are faced with two, which could be more intimidating.
Also, the middle section was larger and more sheltered than in the study by Lucon-Xiccato and Dadda (2014). This could also affect the outcome of the experiment. By decreasing the middle-section, and therefore increasing the outer sections, the fish are more likely to be seen as swimming in an outer section and therefore to be counted as exploring the objects. In some cases the fish were swimming close to or on the border between sections. If the border had been placed somewhere else the numbers would have been different, perhaps even significantly so. The border has to be placed in such a location that swimming across it counts as exploring and investigating behaviour. But what is considered exploring and investigating behaviour in zebrafish? Do they have to swim all the way up to the object, or will looking at the object from a safe distance suffice? Some of the fish were hovering and clearly looking at the object for periods of time, while still being inside the middle section of the tank. So while they may not show up as exploring the object in the statistics they could very well be exploring it, but from a distance. Other fish were in the area with the object, but seemingly uninterested in it and for example only swimming across the bottom.
The experiment should be repeated with a larger number of zebrafish and without the mirror in the testing tank. The plastic-plant shelter should give them enough feeling of cover and safety. I would also reduce the size of the middle part, by increasing the size of the side parts. It could even be worth to try this experiment without a mirror, which was only added here because it was used in the original experiment by Lucon-Xiccato and Dadda (2014), since it could have disturbed the fish. They may have seen the mirror image and seen it as another individual with whom they were interacting, leaving them less interested in the objects I introduced. Zebrafish aggression tests with mirrors show that they do notice the mirror image and do not realise that it is actually themselves (Decourtye et al, 2004).
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Figure 8: Time spent in the middle per treatment and round. Y-axis reads: M/240 and is the amount of time spent in the middle section.
CONCLUSION
Even though the results presented here did not prove imidacloprid to have a significant effect on the memory and learning capabilities of fish, there were still trends indicating an effect. It is therefore necessary to conduct more research. The results of this research are not sufficient to deny any and all detrimental effects of imidacloprid.
The insignificance in the stickleback results could either be due to an actual lack of effect, but may also be due to the small sample size. Continuing the experiment for another week could produce interesting, clearer, results. Unfortunately that was not worthwhile in this particular study because of the high mortality rate. An additional thought for further research could be to not test three treatments, but only two: control (0 µg/L) and low (10 µg/L).As with the sticklebacks, the small sample size of zebrafish may affect the significance. The fish do spend a higher amount of time in the middle during the second round, at least the fish with imidacloprid do, but the reason behind this is unclear. If this research were to be repeated in the future I would narrow the middle section, remove the mirror from the tank, and use a bigger number of zebrafish.
A general idea for further research on imidacloprid could be to test more concentrations, in order to determine which concentrations have an effect (if any). This could greatly benefit other research as well, as my concentrations of 10 µg/L and 100 µg/L were not based on any previous research.When assessing memory, and the effect of imidacloprid on this, there should also be some thought given to whether the short-term or long-term memory is tested. There could be a difference between the effects on these two types of memory. Another interesting thought could be to approach this problem from a different perspective. Instead of researching the effect of imidacloprid through behaviour, could it be easier to research this from a physiological angle instead? Since imidacloprid blocks the post-synaptic nicotinergic receptors in the nervous system could the effect of imidacloprid not be deduced by the amount of nicotinergic pathways in the organism? Perhaps the exact effect can not be seen through the amount of nicotinergic pathways, but it could give us an idea of whether imidacloprid even has an impact on the organism. And perhaps even the severity of the impact.
ACKNOWLEDGEMENTS
I'd like to thank my three supervisors: Joacim Näslund, Joachim Sturve, and Jörgen Johnsson.
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