Abstract When exposed to a fresh trail of ink, termites of the species Reticulitermes flavipes, the Eastern Subterranean Termite, will follow the trail as they confuse it for a trail pheromone released by other termites that would lead them to a food source. To date only two trail pheromones have been identified with certainty: dodecatrienol and neocembrene. Melatonin is a hormone present in both vertebrates and invertebrates that manages circadian rhythms and effects release of other hormones. This study examines interference of pheromone reception as a result of altering termites’ natural response to pheromones by artificially increasing levels of melatonin in the termites. Termites were run through a commonly performed experiment where they followed a trail of ink which contains ingredients that mimic trail pheromones. It is known that melatonin has an effect on the hormonal balance within insects’ brains, as demonstrated in a study of the effects of melatonin on the release of prothoracicotropic hormone from the brain of the cockroach Periplaneta Americana. Furthermore, the recently observed light-seeking behavior of some honeybees could suggest a hormonal imbalance. If this is the case the hormone is question is likely that which regulates light responses: melatonin. Thus, the question of the effect of melatonin imbalances on the pheromonal responses of insects was examined. Termites not exposed to abnormal melatonin levels in their environment followed the ink trail correctly for an extended period of time. Severely impaired termites represent a significant alteration of termites’ natural response and are indicative of a disruption in the pheromone reception process or possibly a different neurological disruption caused by unnaturally high melatonin levels. If the hypothesis that increased melatonin levels in termites effects pheromone reception is confirmed, then it is possible that hormone imbalances are somewhat responsible for phenomena observed in other insects such as the errant behavior seen in honeybees. This possible connection should be further explored in higher level research of melatonin levels in affected honey bees.

IntroductionReticulitermes flavipes, the Eastern Subterranean termite, is a social insect that lives in colonies in the ground. Termites are broken up into three classes: soldiers, workers, and reproductives. Workers are the only termites that gather food for the whole colony and they do so by eating and transferring rotten wood. Their role as providers of food for the colony is vital to the success of the colony1. When gathering food, worker termites

Effect of Melatonin on Trail Following Pheromone Reception in Reticulitermes flavipesMatthew Zipple1* and Susan Goethals2

Student1, Teacher2: Cardinal Gibbons High School, Raleigh, North Carolina *Correspondence: menzipple@bellsouth.net

IN SCHOOL ARTICLE

release a pheromone from their sternal glands that leads other termites to the food source. These pheromones, known as trail pheromones, are received by other workers through the use of their antennae and then are followed to the food source.

Termites’ natural response to trail pheromones is to follow them until they find a food source. Trail pheromones optimize the foraging process, and are a vital component of the health of a colony. If termites are unable to accurately perceive and make use of trail pheromones, it is unlikely that the colony would be able to survive2. Termites are very sensitive to the presence of trail pheromones, which can be detected in amounts as small as 0.01 pg/cm of trail3. Two trail pheromones have been identified to date in termites. These two pheromones are dodecatrienol and neocembrene4. The chemical structures of these two trail pheromones are shown in Figure 1.

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Figure 1. The two known trail following pheromones in Reticulitermes flavipes, dodecatrienol (bottom) and neocembrene (top).

There is an ingredient within the ink of ballpoint pens that closely resembles termite trail pheromones and, if exposed to it, termites will follow a trail of ink as if it is a trail of pheromones. This ingredient has been described as a glycol derivative, but the exact chemical name is unknown3. There appears to be no significant difference in termites’ response to a trail of ballpoint pen ink and a trail of pheromones.

Melatonin is a hormone that is present in both vertebrates and invertebrates. In vertebrates, melatonin is released in order to signal environmental darkness and acts as a neurological mediator between environmental conditions and an organism’s

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ResultsTermite performance is sorted into the following four categories: natural, slightly impaired, moderately impaired, and severely impaired. Natural termites acted in the same manner as the control termites. They followed the ink line almost flawlessly and correct any errors within 1 to 2 seconds. Slightly impaired termites had some trouble following the trail. They occasionally lose the trail, but they are able to find it again without human assistance. Moderately impaired termites occasionally lose the trail and are unable to find it again without human assistance in the form of a paint brush positioned to direct them back to the trail. They moved away from the brush; they did not investigate it. Severely impaired termites performed two or more of the following actions: 1. Total inability to follow the trail for

more than five seconds, 2. Movement in small circles, suggesting disorientation, and/or 3. Investigating the paint brush with their legs and antennae instead of avoiding it, suggesting a disruption in the termites’ analysis of environmental factors. Severe impairment represents a marked deviation from the natural response. The responses of termites in each trial are shown in Figure 2. Figure 3 display the proportions of termite responses for a given dosage of melatonin. Figure 4 displays the proportions graphically.

Materials and MethodsOne liter of water was placed in three separate fishbowls. The three fishbowls then had a varying number of crushed 5 mg melatonin tablets stirred into solution. Melatonin is a polar molecule, and therefore dissolved into solution. The cellulose and dicalcium phosphate filler that made up the majority of the tablets did not dissolve. The numbers of crushed tablets in each solution are as follows: Solution 1: 1 tablet (5mg), Solution 2: 2 tablets (10mg), Solution 3: 4 tablets (20mg). Approximately equal amounts of wood were then added to the solutions such that the wood filled the fishbowls to approximately the water line. The wood was allowed to soak for 48 hours in the solution.Groups of 10 termites were placed in clear plastic containers with approximately equal masses of rotting wood treated with varying solutions of melatonin. Four termites at a time were then placed in a cardboard box on whose floor was drawn an oval with a red Pilot pen. The termites were then observed and recorded in their attempts to follow the trail of ink. It was expected that some termites would die during the first 24 hours that they spent in the plastic container as a result of the stress of transfer. To account for this expectation, more than the number of trial organisms (four) were placed in each container. Ten termites were placed in each plastic container and left to live and eat the treated wood for 24 hours. Because termites are subterranean and therefore have little natural exposure to light, the containers were placed inside of a dark cardboard box. Four termites from each group were then placed in a cardboard box with a red Pilot ink trail drawn in the shape of an oval. The termites’ response and movements were then recorded with a video camera.

physical activity. In insects, the release of melatonin acts to keep a regular circadian rhythm, governing an insect’s response to light/dark cycles. Furthermore, melatonin levels have been demonstrated to influence the release of other hormones which govern insects’ activities. For example, melatonin levels were shown to be an influencing factor in the release of prothoracicotropic hormone from the brain of the cockroach Periplaneta Americana5. The necessity of certain levels of melatonin in insect brains suggests that an unnatural level of melatonin would result in unnatural behavior or hormonal or pheromonal activity. This leads to the hypothesis that artificially increasing the levels of melatonin in termites would interfere with termites’ reception of and response to trail pheromones. This study artificially elevated termites’ melatonin levels in order to observe their response to the presence of trail pheromones.

Figure 2. This table displays the number of termites that display each level of impairment in a given trial. Melatonin doses are 5mg/L, 10 mg/L, and 20mg/L.

Figure 3. Proportions of termites’ responses based on dosage of melatonin.

DiscussionThe results yield significant deviations from the expected natural response of termites exposed to a trail pheromone, but do not demonstrate a correlation between dosage of melatonin and termites’ response . It is important to note that it is possible that the observed slight impairment is likely simply the result of variation between different individuals or groups of termites. For this reason, slight impairment was treated as equivalent to a natural response in the statistical analysis of the data. It is also possible that the stress levels that termites encountered were different enough to explain the slightly and possibly the moderately impaired termites. Stress levels could not be completely controlled and it is possible that one or many undetected differences existed

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between holding containers. However, efforts were made to make the different living environments as equivalent as possible.

Melatonin levels’ effect on the life of insects may have implications for other entomological questions. For example, honey bees’ recently described behavior may be due in part to chemical imbalances of melatonin or other hormones. When infested with the parasitic flea Apocephalus borealis, honey bees appear to abandon their hives in favor of street lamps6. This seeking of light may be the result of an imbalance in melatonin levels that alter bees’ natural response to pheromones that would otherwise keep them at the hive. The draw to street lamps suggests an imbalance in bees’ circadian rhythms which could perhaps be explained by a melatonin imbalance. Further research is needed in order to investigate the melatonin levels of bees infected with Apocephalus borealis as compared to healthy bees.

It is important to note that for analysis purposes “slight impairment” and “natural” responses were combined. Though slight impairment was initially thought to be potentially significant, the difficulty in distinguishing between slightly impaired termites and natural termites, combined with slight variations in termites’ individual performance over time led to the conclusion that slight impairment did not represent a significant deviation from natural behavior and should therefore be grouped with natural responses in analysis.

A simple glance at the data suggests that there is not enough evidence to show correlation between melatonin dosage and impairment. Furthermore, a chi squared test yields a X2 value of 4.217 which falls far below the required P value for a 90% confidence interval of 10.64, which supports this conclusion. The lack of demonstrable correlation between dosage and response may demonstrate a saturation effect. A repeated experiment with lower doses of melatonin could confirm this hypothesis. The data does show that termites that were exposed to artificially high melatonin levels had significantly different responses from those termites that were not exposed. Because all the control termites that were tested displayed natural responses, any moderate or severe impairment in treated termites represent definite (1.0) statistical differences. However, it is likely that termites’ ability to follow a line of trail pheromones varies between termites at least slightly. A T-test is therefore rendered mostly useless for this data, for confirmation purposes. Further, larger-scale research is needed in order to confirm the natural response of control termites and to better understand the relationship between melatonin dosage and termites’ response.

Despite the lack of demonstrable correlation between dosage and response, the hypothesis that termites’ perception of and response to the presence of pheromones would be interfered with by increased levels of melatonin was. Severely impaired termites represent a significant deviation from the natural response to the presence of trail pheromones. Severely impaired termites were either unable to perceive the pheromone trail or else were totally disinterested in following it. The former explanation is more attractive for a variety of reasons. If behavior is being modified by the increased melatonin levels, then that would suggest that some other stimulus is overriding the termites’ natural response to trail pheromones. The draw of the trail pheromones is very strong. As part of the background research, termites were exposed to a container that had areas of both light and dark. Being subterranean, the termites tended to avoid the light in favor of the dark. The termites showed no hesitancy, however, in following a pheromone trail through areas of both light and dark. Thus, the attraction to the trail pheromone is stronger than the termites’ negative response to light. No uncontrolled variable can be accounted for that might be able to produce a response stronger than the termites’ response to light. Therefore, it seems highly unlikely that the termites’ were ignoring the pheromone in favor of another stimulus. Furthermore, the other observable symptoms of walking in circles and being aggressive or at least investigative of a foreign object suggest some kind of chemical imbalance within the termites that is responsible for their being unable to perceive the trail pheromone. Moving in small circles was observed only in severely impaired termites and was unlike anything observed in control termites not only while following the trail of ink, but also in their regular movements outside of the experiment. The circular motions represent major disorientation and inability to react to termites’ environment

Further experimentation is needed to better understand the role of melatonin in governing insects’ hormonal and pheromonal interactions with other termites and with their environment. This study demonstrates the complexity of the insect brain and its neurological activities. The impact of melatonin on insects’ behavior and interaction with other insects could be significant in a variety of different ways

Figure 4. Graph of the proportion of each level of termites’ impairment versus the dosage of melatonin termites were exposed to. Because “Slight Impairment” may in reality be nothing more than scientific optimism, the graph combines natural and slightly impaired responses.. S1= 5mg Melatonin/Liter, S2= 10mg Melatonin/Liter, S3= 20mg Melatonin/Liter

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within the field of entomology.

References1. Klass, Carolyn and Kowalsick, Thomas. “Eastern Subterranean Termites.” Cornell University Cooperative Extension Chemung County. Web.

2. Vander Meer, Robert K., Breed, Michael D., Espelie, Karl E., Winston, Mark L. Pheromone Communication in Social Insects: Ants, Wasps, Bees, and Termites, Westview Press. 4, 15-16.

3. Schulz, Stephen. “Trail Following of Termites.” University of Texas Field Biology 208L. University of Texas. Web.

4. Sillam-Dussès, D., Sémon, E., Robert, A., Cancello, E., Lenz, M., Valterová, I. And bordereau, c. (2010), Identification of Multi-Component Trail Pheromones in the Most Evolutionarily Derived Termites, The Nasutitermitinae (Termitidae). Biological Journal of the Linnean Society, 99: 20–27.

5. Richter, Klaus, Peshke Elmar, and Peschke Dorothee. (1999). Effect of Melatonin on the Release of Prothoracicotropic Hormone From the Brain of Periplaneta americana. European Journal of Entomology 96 341-345.

6. Core A, Runckel C, Ivers J, Quock C, Siapno T, et al. (2012) A New Threat to Honey Bees, the Parasitic Phorid Fly Apocephalus borealis. PLoS ONE 7(1).

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