Bugs on Drugs

Bugs on Drugs

Sidney Breth Petersen – Killarney Heights High School

This study investigates the impact of pharmaceutical drugs on the conglobation behaviour of the Armadillidium Vulgare (pill bugs). Currently, there are several studies which examine the mechanisms of this defensive behaviour, however, there are limited studies about the effects which specific drugs, such as caffeine and antihistamine, could have on this mechanism. This study compares the conglobation time of pill bugs who consumed purely water, to ones which consumed diluted solutions of pharmaceutical drugs. Results revealed that pill bugs which were exposed to the caffeine solution had significantly higher conglobation times. This proposes that caffeine induced water loss in pill bugs which caused them to conglobate. Similarly, the antihistamine group also had an increased conglobation time, but the increase was to a lesser extent. This increase may be impacting the defensive aspect of the conglobation mechanism, as central nervous system activity could have been slowed due to the sedative behaviour of antihistamines. Both drug classes impacted upon the conglobation behaviour of the pill bug which suggests that drugs polluting water sources in pill bug habitats could be placing stresses on the species and threaten their survival.

Literature Review

Worldwide, pharmaceutical compounds are traced in the aquatic environment. This exposure to pharmaceuticals has led to ecotoxicological effects on various species. Previous experiments have aimed to explore the effect of various drug classes on animals to examine the ways in which they affect the nervous system. By examining the effect of these drugs, the harmful impact of drugs in grey water can be examined and potentially limited if they pose harmful effects to species. Different drug classes affect the central nervous system (CNS) in various ways (NIDA, 2020). Typical pharmaceutical compounds which have an effect on the CNS are antihistamines and caffeine. Pill bugs are terrestrial arthropods which are sensitive to the amount of water present in their environment.

The effects of these two different drug classes have been closely examined in humans and other animals, but research is lacking on their effects on pill bug activity. In humans, antihistamines are a CNS depressant, whereas caffeine is a stimulant. Neither are harmful to humans in small doses but their effect on pill bug activity has not been examined extensively. A lack of evidence exists regarding isopod metabolism, particularly the physiological mechanisms of drug metabolism in the pill bug. Whilst a large body of literature exists regarding the cytochrome P450 enzyme in mammalian metabolism, there is a lack of knowledge on isopod metabolism.

It is crucial to understand how these drugs work in order to understand how they would affect the functionality of pill bugs. Caffeine, besides influencing cognitive performance, increases the perception of alertness, wakefulness and can induce anxiety (Cappelletti, 2015). Caffeine stimulates antagonists against adenosine receptors within the CNS (Ribeiro, J. A., & Sebastião, A. M., 2010) which prohibits cells from identifying adenosine, an indicator of fatigue. This means the brain becomes non-perceptive of fatigue. In addition, caffeine promotes diuresis, the excretion and loss of water from the body. These two effects have been thoroughly studied, yet there is a lack of literature which investigates the dangers this poses to pill bugs which are dependent on water for survival.

Histamine is a CNS neurotransmitter which is necessary in maintaining wakefulness, alertness, and reaction time (Lieberman P., 2009). This wakefulness can be impaired by antagonists, known as antihistamines. In addition to their potent activity in histaminergic pathways, first generation antihistamines also demonstrate a significant degree of pharmacologic activity in α-adrenergic pathways. Most commonly, the cognitive impairment associated with diphenhydramine is due to the α- adrenergic blockade. This means, in comparison to caffeine, the drug may cause physiological changes such as decreased movement speed and activity, if they have similar effects in pill bugs.

Pill bugs are relatively poorly adapted to land, and can only survive habitats where temperatures are moderate and damp surfaces are available (Jacob T. Smigel and Allen G. Gibbs, 2009). Evaporation from the outer surface is the most important physiological factor affecting their survival. However, pill bugs are capable of conglobation which is the process of rolling into a ball. This behavior, other than acting as protection from predators, also protects the more delicate ventral surface, allowing pill bugs to survive in harsh conditions by reducing water loss.

A journal article published in 2008, (Jacob T. Smigel and Allen G. Gibbs) ‘Conglobation in the Pill Bug, Armadillidium vulgare, as a Water Conservation Mechanism’ examined the conglobative behavior of pill bugs as a possible water conservation mechanism. Evidence found, demonstrated that pill bugs spontaneously conglobate at 40°C, with a resulting decrease in their rates of water loss. Pill bugs were found conglobated in dry soil, and unconglobated in visibly moist soil, suggestive of water conservation methods. To test this hypothesis, the water loss rate of unconglobated and conglobated isopods were measured. The data showed that conglobation significantly reduced water loss by 34.8%, suggesting that conglobation behavior may help to conserve water.

Previous studies have reported findings that certain drugs, such as caffeine, can have harmful effects on both humans and animals (P. N. Witt, 1948), such as examining the effects of psychoactive drugs on spiders. The study conducted by Witt (1948) concluded that relatively small doses of many different drugs affect the pattern of a spider web. In support of these findings, it was also examined that caffeine may function as a natural insecticide and hence may be useful in insect control (J. A. Nathanson). Subsequently, research by the National Aeronautics and Space Administration (NASA) in 1995 tested the effect of various other drugs in order to build on previous findings. The study allowed NASA to conclude that several drugs can impact spider activity by affecting the CNS.

Although we cannot link drug effects on pill bugs to drug effects on humans, we can examine through primary research, how pharmaceuticals such as caffeine and antihistamines in pill bug habitats, can cause severe impacts. This is due to the nature of caffeine and antihistamines influencing central nervous system activity.

Scientific Research Question

What are the effects of different drug classes on conglobation behaviour of the pill bug, Armadillidium vulgare?

Scientific Hypothesis

Caffeine will act as a diuretic on the pill bug, causing water loss, and will therefore increase conglobation time. Antihistamines will not have a significant effect on the conglobation period of pill bugs, but may still cause slight increase in conglobation time.

Methodology

Drugs

The antihistamine, Dexychloropheniraminemaleate (50mg/1ml, 1g of Dexchlorpheniramine in 20ml of water) and caffeine solution (50mg/1ml, 1g of Caffeine Anhydrous in 20ml of water) were purchased from Pittwater Pharmacy & Compounding Chemist and kept in small spray dispensers.

Pill bugs

100 pill bugs were collected from their natural environment and stored in containers. These containers had controlled environments, with the same type of soil, food and light availability and temperature, in each container. Hence, variables such as amount of water present, which had inevitable variations, could be controlled.

Filter paper and petri dishes

The soaked filter paper was prepared by spraying the filter paper five times each with the drug solutions. Each spray with the drug solution dispenses 0.1mL of liquid, meaning that each filter paper was soaked in 0.5mL of the selected solution. Each time a petri dish was lined with the soaked filter paper, a label was attached clearly indicating which drug was being used (Figure 1).

Drug administration

When conducting the experiment, pill bugs were placed into three groups: a petri dish environment with either water, caffeine or antihistamine. 10 specimens were placed in each group. The time the pill bugs were placed on the petri dish was clearly recorded. The pill bugs were then left in the petri dishes for 30 minutes before being removed.

Recording the effects of various drug classes on conglobation rates

As soon as the pill bugs were removed, they were kept in their groups and placed on separate dry pieces of paper. A new timer was then started to record the time taken for the pill bugs to open up and start moving around, and these results were recorded in a table which was then analysed based on the differences between the conglobation rates of each drug class. Statistical T-test was then conducted to compare the means between each group, comparing the two different drugs to water, allowing us to reject or accept hypotheses and evaluate whether or not there is statistical difference between the two selected data sets.

Figure 1) Example petri dishes with soaked filter paper

The averages for each drug class were recorded (table 2, figure 2). For the conglobation time, note that caffeine administration results significantly increased time under conglobation. That is, there was an increase of 223.22 seconds between the caffeine and water group.

Results

Table 1) Results of the first two caffeine groups

The length of the conglobation time post drug administration was recorded for each individual pill bug and the average time of the test group was taken. Table 1 indicates the length of conglobation time post drug administration for each individual pill bug in the first two study groups, as well as the average time of each test group. The mean conglobation time for study groups 1 and 2 were 220.4 and 262.6, respectively.

An increase in conglobation time was also observed in the antihistamine group (table 2) which shows an increase of 15 seconds between the respective antihistamine and water group.

Table 2) Average conglobation time of each drug class administered.

Drug class, Average conglobation time over all tests

Caffeine, 244.97

Antihistamine, 36.75

water, 21.75

Figure 2) Average conglobation time of each drug class administered

Table 3) descriptive statistics of results

A two-tailed t-test was conducted with an alpha value of 0.05 (table 6). The results showed that the water conglobation time was less than the caffeine conglobation time, and found a significant difference between the two means as the P-value was less than 0.05. This indicated that there is a statistical difference between the two means, and allows us to reject the null hypothesis that there was no statistical difference between the caffeine group and the water group.

An identical test was conducted between the mean of the antihistamine group and the water group with a P-value less than 0.05, suggesting there is significance in the results and allows us to reject the null hypothesis that there was no statistical difference between the antihistamine group and the water group.

There is a large difference between the two t-tests result, with the P value of the caffeine group being significantly smaller than the antihistamine group. This is due to the time increase of conglobation which was observed, as the antihistamine group did not have as large of an increase as that of caffeine.

Table 6) T-Test results, comparing the means between the caffeine and water results

Table 7) T-test results, comparing the means between the antihistamine and water results

Discussion

Administration of caffeine in pill bugs significantly increased the conglobation time of pill bugs (table 2). In comparison, antihistamines had very little effect on the conglobation time, however still saw an increase.

Both tests have been able to find significant differences in conglobation time after caffeine and antihistamine were administered, and suggest that the test hypothesis can be accepted.

The caffeine group results (seen in table 2), suggests that caffeine increased the conglobation time of pill bugs, which may be related to the diuretic nature of caffeine. This demonstrates the correlation between the nature of caffeine as an inducer of water loss (Stookey, J.), and the conglobation behaviour of pill bugs as a means of water conservation (Jacob T. Smigel and Allen G. Gibbs ), suggesting that caffeine acts as a diuretic in pill bugs, as it does in humans.

Water loss is important to protect pill bugs. If caffeine causes loss of H+ ions and acts as a diuretic (Maughan, R. J., & Griffin, J.), it would pose a threat to the survival of a species, which is exposed to caffeine, as they cannot survive without a sufficient amount of water. This highlights the severe effects which caffeine entering pillbug environments could have on pill bug colonies. These findings could help to examine the dangers of grey water in pill bug habitats, as pill bugs survive from the moisture in the soil, including any grey water that has entered the habitat.

The slight increase in conglobation time seen in the antihistamine group may result from the sedative effect of antihistamine. This effect could have slowed down the pill bug response times to stress and cause the increased mean conglobation time (graph 1). It is possible that the antihistamine group had a longer conglobation time than the water group, as the drug caused physiological changes such as decreased movement speed and central nervous system activity.

Each pill bug was obtained from their natural habitats but from various locations. They were all of different sizes and some even of different colour. This could be due to age or the environmental conditions, which were not taken into account when observing the effects of the drugs. If the pill bugs obtained were of the same size, this would increase the amount of controlled variables as the experiment does not observe whether or not the variance within the drug groups were due to size differences. Changes in size could increase the surface area of the bug, exposed to evaporation and hence increasing the rate of water loss. These variables were partially controlled by using a large sample size. Other variables which had inevitable variations that could have affected the rate of water loss and the effect of the drug also includes the amount of food and water they were obtaining prior to the drug administration. To minimise the effects of these factors, each pill bug habitat was sprayed with the same amount of water daily and ensured each enclosure has the same level of food availability.

There were also ethical concerns with testing on the pill bugs which need to be considered through all areas of study. Within this study, five pill bugs were tested prior to the thorough experiment conducted, to ensure that the effects observed were only temporary and did not affect survival.

The measurement of drug responses also needed to be carefully considered. A journal article (Jacob T. Smigel and Allen G. Gibbs) on conglobation measured changes in metabolic rate and water-loss rate using a flow-through respirometry chamber, which if able to be utilised in a similar investigation, could accurately measure how drug administration changes metabolic and water-loss rate. This equipment measures respiration rates of living organisms, which allows investigation into how factors such as age, or chemicals affect the rate of respiration. The major route for water loss in pill bugs is evaporation from the body surface, particularly the respiratory organs. By having a respirometry chamber, it would limit experimenter bias as to whether the drug itself is causing certain effects or other factors. Instead, the experiment conducted utilised a visual analysis and time to record changes to conglobation behaviour, which could be backed up by quantitative evidence of the changes in water loss that caffeine causes. Access to this technical equipment would be able to certify that the changes are due to the diuretic nature of caffeine and not the psychological effects that it presents to the consumer.

Further suggestions

Coffee grounds are often used in gardens to promote plant growth, which is another way that caffeine can enter habitats. Further research could include investigating the variety of organisms in soils where coffee grounds have been added, in comparison to areas where there were no coffee grounds used.

If the drug contamination levels currently present in various pill bugs habitat were obtained, these amounts of caffeine and antihistamine could be used in the experiment as this will be a more accurate indicator of the threat which soil contamination presents to pill bugs. This experiment assumes that the levels of pharmaceuticals in the soil are directly impacting the pill bugs as they are exposed to similar levels, however levels may be significantly lower or higher. In addition, if the amount present in soil is in fact higher than what was used in the experiment, this could pose an additional threat to pill bugs, as it has now been discovered that naturally occurring amounts of caffeine kill several animals, including pill bugs (Nathanson J. A.). Further studies should also investigate whether increasing moisture would mitigate the harmful effects of caffeine.

It should also be identified that we cannot assume from this paper that there is a correlation between what a drug does to the conglobation mechanisms in the pill bugs brain, and what they do to human brains. Rather, we should use it as an indication as to where similarities in drug behaviour lie and how they may affect species differently.

Conclusion

Pill bugs are sensitive to the amount of water present in their environment and have therefore adapted mechanisms such as conglobation to reduce water loss. The results of this study suggest that there was a statistically significant difference in the conglobation time of the caffeine group and antihistamine group in comparison to the pill bugs who were only exposed to water. The investigation specifically revealed that the increase in conglobation time for the caffeine group was much higher than the antihistamine group, although as hypothesised, both drugs affected the conglobation mechanism.

Further research should be conducted which examines the specific amount of water lost due to caffeine administration and consider the effects of varying caffeine concentrations. Utilising the concentration of caffeine that can be found in grey water, near pill bugs, should also be examined on its effects as this would allow for more detailed analysis of the severity of grey water effects. By conducting further research on how pill bugs metabolise drugs it could be examined whether they have a compound that may limit or enhance the efficacy of certain drug types.

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