by Lauren Dunavant and Treva Hickey
Armadillidium vulgare is a common isopod found throughout the United States that has an important role in breaking down plant litter. In order to test the stress responses of A. vulgare to acute environmental stressors, these isopods (commonly identified as “pill bugs”) were exposed first to a control environment (which was created to mimic the conditions of their naturally preferred dark, damp terrestrial habitats) and then to one of five stress stimuli: heat, nitrogen fertilizer-containing soil, flooding, carbaryl insecticide, and permethrin insecticide. Observational data gave evidence that A. vulgare were indeed stressed by all of these stressors, as evidenced by behavioral signals such as conglobation (“balling-up”), clustering, attempted escapes, and mortality, to name a few. The pill bugs showed the highest levels of stress when exposed to flooded and nitrogen-rich environments, and oddly, the carbaryl insecticide did not cause mortality in any of the isopods. Furthermore, previous research suggested that isopods such as A. vulgare might have an ability to detect and avoid permethrin toxins, but in this environment, the pill bugs showed the lowest level of stress during observation, although they died soon after being removed from the contaminated environment. The experiment showed that in many stressful situations the pill bugs do show signs of stress but are only harmed in trials with insecticide. Although experimental conditions were extreme, as this was an acute exposure study, stress responses may indicate that changing environmental conditions could have an effect on terrestrial ecosystems.
The current and prospective states of the environment have become a hot topic in global news in recent years, as modern technologies now allow scientists to predict the impending environmental effects of certain phenomena, such as global warming, possible species extinction, and pollution. Furthermore, environmentalists are calling into question the safety of certain chemicals and practices that were formerly thought of as harmless, such as the use of fertilizers and pesticides on commercial farmlands. Many biological systems have also been deeply affected by climatic changes linked to global warming, including flooding and deforestation. This experiment will show how some of these environmental changes may specifically affect the common pill bug species, Armadillidium vulgare.
During the past several decades, environments in which these organisms live have been heavily impacted by anthropogenic activity, and the widespread effects of pesticides and other potentially toxic chemicals on insect and arthropod populations have been the subjects of many experimental studies. A substantial percentage of commercial farmers use herbicides, insecticides, fungicides, and other varieties of chemicals to protect their crops from parasites and pests, and while most of these chemicals are effective in keeping crops safe, they may jeopardize the vitality of agricultural ecosystems across the globe by creating unintended cascading effects that may interfere with the current balance of the environment’s biotic and abiotic components.
Pyrethrin-based pesticides are one example of these commercially used insecticides. Derived from extracts of the Chrysanthemum family, they are some of the most common types of pesticides used by farmers to eradicate insects that are harmful to crops. Pyrethrin-based insecticides work by keeping the sodium channels of nerve cells open, inducing an ion imbalance that leads to paralysis and, ultimately, death (1). Because the isopods A. vulgare generally inhabit moist terrestrial environments (common agricultural conditions), they may be at risk of exposure to pyrethrin-based insecticides. In a study done on the effects of pyrethrins in closely related isopods, the terrestrial crustaceans showed not only a susceptibility to pyrethrin poisoning, but also the potential ability to detect and avoid pyrethrin-contaminated foods and soils (2). In this experiment, A. vulgare will be exposed to a pyrethrin-contaminated environment as well as an environment contaminated with another commercial fertilizer (GardenTech Sevin®- 5 Ready-To-Use 5% Dust), and their response to these substances will be observed (2).
The effects of commercial fertilizers on the stress levels of A. vulgare will also be measured during this experiment. Because all terrestrial plants require fixed nitrogen (in the bio-accessible forms of nitrate or ammonia) in order to survive, many farmers add nitrogen-containing fertilizers to their crops. While this practice has noticeably improved the abundance of harvest, it has become clear that runoff of these fertilizers into other environments (such as lakes and streams) can cause imbalance in other ecosystems, resulting in remarkable changes, such as out-of-control algal blooms and changes in population densities. Even more alarming, perhaps, is the fact that the effects of these fertilizers on numerous other environments with which they come into contact are largely unknown. Therefore, the imbalances caused by commercial fertilizers may be even more widespread than is yet understood. During the experimental procedure, it will be determined whether or not high levels of ammonia-based fertilizers are harmful to pill bugs.
Finally, global warming is another recent environmental change that could potentially affect the vitality of many terrestrial organisms like A. vulgare. This climatic phenomenon introduces two new possible stressors: increased rainfall/flooding in certain environments and increased global temperature. Although pill bugs breathe through gill-like organs (which is why they generally inhabit moist environments), these organisms are not aquatic, and it is possible that too much moisture could lead to drowning (3). During the experimental procedure, the stress levels of A. vulgare will be measured when they are exposed to flooded environments. In addition to humid environments, pill bugs also seem to favor habitats that are dark and shaded, yet temperature may or may not play a role in this preference. Due to global temperature increases, it is possible that warmer environments may become a reality for pill bugs, and like any other abrupt changes in the environment, increased temperature will likely have an effect on their stress levels. A recent study done on the environmental impact of fluctuations in temperature and rainfall suggests that climate is directly correlated to the quality of food available to terrestrial isopods, such as A. vulgare. This finding provides further evidence to justify our hypothesis that the survival of A. vulgare can be affected by weather, food quality, and density (4). While this experiment is merely a measurement of the stress responses of A. vulgare to acute changes in a controlled environment, the findings address knowledge gaps regarding the ecology of this organism and will provide insight to the possible effects of global warming on species similar to A. vulgare.
In the first part of this experiment, the responses of the isopod A. vulgare to four environmental stressors (heat, flooding, ammonia-based fertilizer contamination, and insecticide contamination) were observed and recorded. Activity levels and stress indicators, such as frequent turns, conglobation (“balling up”), and clustering were recorded throughout the observation periods. Two replications using five experimental chambers each were completed using the experimental design shown in Figure 1.
The first chamber was the control chamber, and it contained uncontaminated damp organic soil at a temperature of about 25°C (or room temperature) with natural lighting conditions. Natural activity and behavior were observed (i.e., average speed of movement, approximate frequency of turns, etc.). In the second chamber, A. vulgare were exposed to the same conditions as in the control chamber, but the temperature was kept higher (40°C). In the third chamber, A. vulgare were exposed to the same conditions as in the control chamber, but the soil was contaminated with 0.148 g GardenTech Sevin®- 5 Ready-To-Use 5% Dust, whose active ingredient is carbaryl(1-napthyl-N-methylcarbamate) 5.0%), also known as Carbaryl. In the fourth chamber, A. vulgare were exposed to the same conditions as in the control chamber, but there were excess amounts of water in the soil (20 mL added to 0.5 in soil in a 100 mm petri dish). The soil was completely water-saturated and pooling slightly. In the fifth chamber, A. vulgare were exposed to the same conditions as in the control chamber, but they were exposed to soil containing 0.21% nitrogen (0.113% ammonaical, 0.097% nitrate).
Fig. 1. A prototype of the experimental design. Chamber 1 was held at room temperature and soil was damp. Chamber 2 was held at 40°C, Chamber 3 contained 0.148 g GardenTech Sevin®- 5 Ready-To-Use 5% Dust, Chamber 4 contained 20 mL of water/0.5 in. soil, Chamber 5 had soil containing 0.21% nitrogen, and the follow-up experiment chambers contained 5 mL and 0.5 mL 1% permethrin.
There were two shifts of 6 for each chamber. At the beginning of the experiment, all pill bugs were placed into the same control chamber, where their behavior was monitored for 13 total minutes (1 min observing, 5 min off, 1 min observing, 5 min off, 1 min observing) in order to most effectively monitor activity quantitatively at various points of exposure. After the pill bugs had been observed for 13 minutes in the control chamber, 6 were placed in each of the four experimental chambers.
Each experimenter monitored one of the chambers closely, loosely following the same 1-5-1-5-1 minute pattern of observation as used to observe the control (making note of any remarkable behaviors during the off time as well). After they had been observed for 13 minutes, the pill bugs in each experimental chamber were placed into a “used bug” control environment that was identical to but separate from the control chamber into which all the “unused” pill bugs were initially deposited. Any notable changes in activity were recorded once the pill bugs were placed back into this control environment. The exact same experiment was executed a second time with fresh pill bugs from the first control environment. Their turn patterns, speeds, conglobation, and color were again observed closely and quantified in the same fashion as the first replication for a total of 13 minutes. The pill bugs were again deposited into the used-bug control environment and monitored for a short time.
In a subsequent experiment, A. vulgare were exposed to the same conditions as in the control chamber (with a slight difference in soil— compost was used instead of organic soil as a result of availability), but the soil was contaminated with 5.0 mL (diluted with 10 mL water) of a pyrethrin-based insecticide, Nix® Lice Killing Creme Rinse (1% permethrin).
This experimental concentration is somewhat higher than what would be found in the soil of a field treated with a standard pyrethrin insecticide, as it was the experimental goal to measure the effects of acute exposure (9). At the beginning of the experiment, 12 pill bugs were placed into the same control chamber, where their behavior was monitored for 13 total minutes.
The first shift of 6 pill bugs was placed in an environment containing 5 mL of 1% permethrin insecticide diluted to 15 mL, but based on the mortality rate of the first shift of pill bugs, the second shift was only exposed to 0.5 mL 1% permethrin insecticide diluted to 15 mL. Turn patterns, speeds, conglobation, and color were observed closely, loosely following the 1-5-1-5-1 minute observation pattern used in the main experiment. The pill bugs were deposited into a new control environment and monitored for a short time after their 13-minute exposure to the permethrin insecticide.
Photo 1. A pill bug with slightly purple coloring at the end of the nitrogen fertilizer observation period for trial 2.
Fig. 2. (Control) The movement of 6 A. vulgare was monitored for a 13-minute period in an experimental chamber containing damp soil held at about 25°C (control conditions).
Fig. 3. (Heat) Movement was monitored for two 13-minute trials of 6 (each) A. vulgare in an experimental chamber held at 40°C.
Fig. 4. (Carbaryl Insecticide) Movement was monitored for two 13-minute trials of 6 (each) A. vulgare in an experimental chamber containing 0.148 g GardenTech Sevin®- 5 Ready-To-Use 5% Dust, whose active ingredients is carbaryl(1-napthyl-N-methyl carbamate) 5.0%).
Fig. 5. (Flooding) Movement was monitored for two 13-minute trials of 6 (each) A. vulgare in an experimental chamber containing 20 mL water in 0.5 in. of soil (100 mm petri dish).
Fig. 6. (Nitrogen Fertilizer) Movement was monitored for two 13-minute trials of 6 (each) A. vulgare in an experimental chamber with soil containing 0.21% nitrogen (0.113% ammoniacal, 0.097% nitrate).
Fig. 7. (Permethrin) Movement was monitored for two 13-minute trials of 6 (each) A. vulgare in an experimental chamber with soil containing 5.0 mL (diluted with 10 mL water) of a pyrethrin-based insecticide, Nix® Lice Killing Creme Rinse (1% permethrin).
Table 1. Rate of activity over time, burrowing, conglobation, attempted escapes, clustering patterns, color change, and other behaviors were observed for A. vulgare exposed for 13 min. to a chamber containing damp soil held at about 25°C (control conditions).
Table 2. Rate of activity over time, burrowing, conglobation, attempted escapes, clustering patterns, color change, and other behaviors were observed for the two trials of 6 A. vulgare exposed for 13 min. to the experimental chamber held at 40°C.
Table 3. Rate of activity over time, burrowing, conglobation, attempted escapes, clustering patterns, color change, and other behaviors were observed for the two trials of 6 A. vulgare exposed for 13 min. to the experimental chamber containing 0.148 g GardenTech Sevin®- 5 Ready-To-Use 5% Dust, whose active ingredients is carbaryl(1-napthyl-N-methyl carbamate) 5.0%).
Table 4. Rate of activity over time, burrowing, conglobation, attempted escapes, clustering patterns, color change, and other behaviors were observed for the two trials of 6 A. vulgare exposed for 13 min. to the experimental chamber containing 20 mL water in 0.5 in. of soil (100 mm petri dish).
Table 5. Rate of activity over time, burrowing, conglobation, attempted escapes, clustering patterns, color change, and other behaviors were observed for the two trials of 6 A. vulgare exposed for 13 min. to the experimental chamber with soil containing 0.21% nitrogen (0.113% ammonaical, 0.097% nitrate).
Table 6. Rate of activity over time, burrowing, conglobation, attempted escapes, clustering patterns, color change, and other behaviors were observed for the two trials of 6 A. vulgare exposed for 13 min. to the experimental chambers with soil containing 5.0 mL and 0.5 mL 1% permethrin insecticide.
During the experimental procedure, all A. vulgare placed in experimental chambers showed various observable signs of stress. Overall, A. vulgare in Trial 1 generally showed much more fluctuation in activity, which could be related to the fact that these pill bugs spent only 13 minutes in the control chamber while the Trial 2 pill bugs spent upwards of 26 minutes in the control environment (perhaps allowing them to adjust better to a new environment). In both trials, the pill bugs generally showed declining movement with time, which is consistent with their movement patterns in the control environment. Movement in the control chamber consisted of very infrequent sharp turns, relatively quick walking in the first 5 minutes, little clustering, almost no climbing on top of one another, burrowing near the chamber edge, and highly reduced movement after 5-10 minutes. See Table 1, Fig. 2.
In the heat environment, movement declined steadily. The pill bugs were somewhat sluggish in this environment, and they tended to move a bit slower in the ~40°C environment than they did in the room temperature environment. However, aside from a few incidents of clustering, the pill bugs exposed to increased temperature exhibited nearly normal behavior (See Table 2, Fig. 3).
In the carbaryl insecticide environment, movement decreased steadily in both Trial 1 and Trial 2, and although this may seem comparable to the movement of the pill bugs in the control environment, those in the insecticide-contaminated chamber exhibited some conglobation and clustering while those in the control chamber did not. Pill bugs exposed to the Sevin®- 5 insecticide also tried to escape multiple times, and just as in the nitrogen fertilizer chamber, one of the pill bugs in the insecticide chamber looked slightly purple in color. However, mortality was rare. See Table 3, Fig. 4.
In the flooding environment, A. vulgare were generally more active than they were in the other experimental chambers. There was one instance of conglobation, and the rate of movement did not decrease as rapidly in the flooded environment as it did in the control environment. Furthermore, the pill bugs attempted to climb over the edge of the chamber to escape numerous times. Their only other deviation from normal behavior was the repeated incidence of pill bugs climbing on top of one another (perhaps to escape the pooling water). See Table 4, Fig. 5.
Finally, A. vulgare showed many signs of stress in the environment containing nitrogen fertilizer. First, movement fluctuated quite a bit during Trial 1, indicating that the pill bugs were having difficulty adjusting to the new environment. They also showed multiple instances of clustering and conglobation, and they became almost restless, which was shown in their burrowing and then un-burrowing throughout the 13-minute observation period. However, probably the most diagnostic signal of their stress was that one pill bug looked slightly purple in color after exposure to the nitrogen fertilizer (Photo 1). See Table 5, Fig. 6.
In the follow-up experiment, the susceptibility of pill bugs to permethrin was tested. The pill bugs showed minimal signs of stress (1-2 instances of conglobation, sparse clustering), but they did not exhibit strange behavior until they were removed from the permethrin environment and placed back into a control environment. Almost immediately, the pill bugs rolled onto their backs (following the 5 mL and .5 mL exposure), began twitching, and then ceased to move almost completely. On rare occasions, the pill bugs would make small leg movements, but overall movement was strained and they seemed fully incapable of behaving normally. Eventually, all twelve of the pill bugs died. See Table 6, Fig. 7.
Patterns of movement were used to quantify the stress levels of A. vulgare in the experimental chambers. Several studies indicate that pill bugs tend to move more quickly when they are stressed by environmental changes such as dry, bright conditions or when predators are in close proximity (5). The effects of environmental stressors can also be measured by the number of turns a pill bug makes when exposed to a stress stimulus compared to the number of turns it takes on average in a given amount of time under normal conditions. When exposed to stressors, the amount and directions of their turns changes (more sporadic turns, different directions). They also express stress by rolling up into a tight ball and, in some extreme cases (such as iridovirus infection), turning a bright blue or purple color (6, 7).
There are two major types of toxicological studies: acute-exposure and chronic-exposure studies. Acute data is usually generated after a single, short exposure to a chemical in a high dosage (a few hours to a few days). Mortality is the endpoint of interest for most acute studies, but in this experiment, stress responses such as increased turns, quicker speed, conglobation, or color change will be sufficient indicators of stress (5). This experiment was focused on acute exposure, but it is important to note that if ecological trends continue, many of these stressors could become chronic obstacles for the A. vulgare species.
Overall, there were noticeable signs of stress for all A. vulgare placed in the experimental stressor chambers, and stress was most commonly manifested as clustering events, conglobation, and attempted escapes. None of these behaviors were observed in the control environment, meaning that they are most likely stress responses. Research suggests that conglobation allows A. vulgare to protect themselves from predators and to conserve water (8). Furthermore, clustering was most noticeable in the flooded environment, which may have been an attempt to avoid pooling and reach higher ground (as pill bugs were seen climbing on top of one another). Clearly, attempted escapes indicate the pill bug’s instinct to abandon the stressful experimental environment and seek more normal conditions to which it is better adapted. The Sevin®- 5 insecticide-treated environment also seemed to be a significant stressor for pill bugs, as there were several conglobation events and attempted escapes; however, mortality was very rare following exposure to this environment, and the effects of the carbaryl contamination were much less devastating compared to those of the permethrin contamination.
Perhaps the most unexpected results were the data from the flooding environment. It was hypothesized that although pill bugs breathe through gills, their preference for terrestrial environments may indicate that these gills could be overloaded, so to speak, and flooding might cause them to drown (3). However, none of the pill bugs exposed to the flooding environment died, and they all exhibited relatively normal activity in the used-bug control chamber after being removed from the flooded environment.
The permethrin-contaminated environment was the only stressor after which significant mortality ensued, but A. vulgare oddly showed virtually no signs of stress while in the experimental chamber. As stated in the introduction, it was hypothesized (based on previous research) that A. vulgare might have an ability to detect the presence of pyrethrins in the soil and try to avoid these toxins (1). However, this hypothesis was not supported by the data gathered during this experimental procedure, as there were no attempts by the pill bugs to escape the environment. Surprisingly, they exhibited very normal behavior until placed in the “used bug” control environment after the 13-minute observation period, where they almost immediately ceased to move and died soon after. It took much longer for the permethrin to cause paralysis than was expected – about 15 minutes total, but because of the 100% mortality rate, permethrin was concluded to be lethal to pill bugs, even in low doses.
Interestingly, though, in almost all of the experimental trials, activity rates declined over time, just as in the control environment. There were also very few instances of color variation, and former research indicates that this type of color-change response may occur exclusively in pill bugs with iridovirus infections (6). The unique slightly purple coloring of the A. vulgare might be the result of a genetically controlled exoskeleton coloring or even the fluorescent lighting in the laboratory (pill bugs tend to inhabit very dark environments where they are almost never exposed to direct light, so their exoskeletons may look different in this setting).
If further experiments were to be conducted to study the effects of these variables on the stress behaviors of A. vulgare, researchers would be strongly encouraged to allow ample space for natural movement, allowing pill bugs the opportunity to give stronger data in regards to their environmental preferences. In this experiment, it is concluded that the pill bugs’ actions demonstrated that they are able to detect stressful situations and subsequently seek more comfortable environments. If this is the case, A. vulgare may not be as greatly affected by environmental stressors, such as temperature, flooding, and heat. However, as far as insecticides, permethrin-based chemicals will likely always have an effect on the nervous systems of A. vulgare, and when exposed to this insecticide, pill bugs will almost always die (2). However, carbaryl insecticide does not seem to have the same effects as permethrin-based insecticides. Only one of the twelve pill bugs exposed to the carbaryl insecticide displayed mortality. Therefore, carbaryl based insecticides do not seem to be as fatal to A. vulgare as permethrin insecticides. Also, in future experiments, it would be beneficial to measure more quantitative variables (such as the amount of plant litter consumed) rather than collecting primarily observational data.
In conclusion, this study gave strong evidence that the experimental environments caused stress to A. vulgare. Although these organisms may not face the exact same conditions in nature (as the experimental environments were specifically formulated to provide data about acute exposure), it is possible that A. vulgare may begin to be exposed to higher temperatures, increased rainfall and flooding (in some places), and higher concentrations of insecticides and fertilizers as a result of global warming and a steady demand for high-yield crops. It is important to take into account the effects of these conditions on A. vulgare, as they, and many other similar, coexisting organisms, are vital to terrestrial ecosystems.
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Acknowledgments: This article was written by co-authors Lauren Dunavant and Treva Hickey based on research done in collaboration with Callee Manna and Mi’Kayla Scott.
Citation style: PNAS