IN SCHOOL ARTICLE
Effect of Melatonin on Trail Following Pheromone
Reception in Reticulitermes flavipes
Matthew Zipple1* and Susan Goethals2
Student1, Teacher2: Cardinal Gibbons High School, Raleigh, North Carolina
*Correspondence: menzipple@bellsouth.net
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.
Introduction
Reticulitermes 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
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.
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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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.
Materials and Methods
One 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.
Results
Termite 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.
Figure 3. Proportions of termites’ responses based on dosage
of melatonin.
Discussion
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.
The 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. Figure 4. Graph of the proportion of each level of termites’ impairment versus the dosage
Further research is needed in order to of melatonin termites were exposed to. Because “Slight Impairment” may in reality be nothing
investigate the melatonin levels of bees more than scientific optimism, the graph combines natural and slightly impaired responses..
infected with Apocephalus borealis as S1= 5mg Melatonin/Liter, S2= 10mg Melatonin/Liter, S3= 20mg Melatonin/Liter
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
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within the field of entomology.
References
1. 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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