Mayfly Responses to Chemical Stimuli
Tricia Hamilton '01
Faculty sponsor: Dr. Linda Fink, Associate Professor of Biology
Honors Summer Research Program 2000
One of the most challenging problems facing conservation biologists today is
understanding the effects of introduced species on their new environments (Diamond & Chase
1986). Non-native game fishes introduced into freshwater habitats often utilize similar ecological
niches as native populations. In these cases, either the introduced species fails to successfully
inhabit the new environment or the native species is forced to shift its strategies and/or faces
serious decline. Introduced fishes also have effects on native prey organisms by increasing the
amount of predation the prey face. The prey also must shift their strategies or face decline.
The Virginia Department of Game and Inland Fisheries has programs to boost and
support many of Virginia's wildlife populations. The largest of the programs is its fish stocking
program. The VDGIF stocks many native and nonnative species of fish throughout the state,
including three species of trout. The stocking of both brown trout, Salmo trutta, and rainbow
trout, Onchorynchus mykiss, has been extremely successful from the perspective of anglers, with
little known effects on the macroinvertebrates upon which they feed. Trout are raised at large
hatcheries in various parts of the state, one of which is located near Sweet Briar in the Blue
Ridge Mountains, at Montebello. All three species are raised in a cascading trough system where
they are separated by size class and species. They are released once they have reached legal
catchable size.
The effects of the actual trout farms on the environment have been researched to some
extent. It has been shown that water that has passed through all of a trout farm's cascading
troughs, called trout farm effluent, caused a decrease in bottom dwelling organism diversity
downstream of its outlet for at least 1.5 kilometers (Loch et al 1996). The decrease in richness
downstream of the effluent outlet shows that the trout farm decreases the health of the stream,
and makes it difficult for organisms to live in it.
This summer's study sought to examine in more detail the relationship between stocked
trout and one taxon of benthic invertebrates, mayflies (order Ephemeroptera). Mayflies are
aquatic insects that coexist with trout and other fishes in Virginia's streams. Mayflies have a
unique life history, throughout which they are preyed upon by trout. They hatch out of eggs at
the bottom of streams, where they live as aquatic nymphs for six months, breathing underwater
using gills. They then emerge into the air, where they hang onto trees, rocks, or other stable
structures. During this phase they are called subimagos. After molting one more time, the
mayflies are sexually mature adults, and mating ensues. The female then rests on the surface of
the water it lived in just one to two days before, where she lays eggs that fall to the bottom of the
stream to restart the cycle. Both male and female mayflies only live a few days as adults
(McCafferty 1981). Trout of all sizes feed on mayfly nymphs, visually spotting them as the
mayflies forage for their food - algae and other plants growing on the surfaces of underwater
rocks, as well as dead leaves that have fallen to the bottom of the stream. Trout also feed on adult
mayflies while they rest on the water's surface to lay their eggs. The predator-prey relationship of
trout with adult aquatic insects is the basis of fly fishing (McCafferty 1981).
All of this makes mayflies good organisms to test for antipredator responses that would
occur only in the presence of trout. The specific objective of this study was to determine if
mayflies from the family Heptageniidae showed behavioral responses to trout odor. As mayflies
are heavily preyed upon by trout, it is reasonable to predict they have some antipredator
mechanisms to avoid them. A study by McIntosh and Peckarsky (1996) looked at the effect of
trout odor on mayflies' tendency to drift in the water as compared to their tendency to remain
stationary, hanging onto a rock or other piece of substrate. They found that in the presence of
trout odor, mayflies more often remained stationary during the day and drifted through the water
at night.
The behavioral responses that this summer's study looked at were much smaller and more
intricate than the water column position used by McIntosh and Peckarsky. I examined behavior
in units called action patterns, which are distinctive actions that are observed more than one time,
and in more than one individual. Examples include swimming, crawling, and remaining
motionless. A total of thirteen action patterns were defined: crawling, gill flap, motionless,
twitch, tail flick, back lay, wall tap, swim, wall tap/swim, abdomen press, curled sit, raise up, and
rubbing. There are no data in the scientific literature that show anyone having previously looked
at the level I did.
During the experiment I collected 5-10 millimeter nymphs from the family Heptageniidae
from the North Fork of the Tye River, and then recorded individual mayfly behavior in the
presence of one of three treatment groups. The groups were plain water, licorice extract, and
water that contained trout odor. Plain water was used to test for a response to the addition of
anything into the mayfly's environment, licorice extract was used to see if there was a response
to the addition of any chemical or smell, while the trout odor tested for a trout-specific
behavioral response. The plain water was water from the same stream as the nymphs, and was
collected at the same time as the nymphs. The licorice extract was Nature's Answer Alcohol Free
Licorice Root that was already suspended in water instead of oil to allow for dispersion during
the experiment. The water that contained trout "odor" was water that was collected from the
troughs at the Montebello Fish Hatchery that had flowed through troughs that held large numbers
of rainbow and brown trout. This water presumably contained all of the chemicals and odors that
trout give off in the form of body slime. The addition of this water into the experimental
environment simulates the presence of trout in the environment.
I studied a total of eighteen mayflies throughout the course of the experiment. The
nymphs were kept in a ten gallon aquarium filled with water from the Tye River, in an
environmental growth chamber. Environmental growth chambers keep the air temperature within
them fairly constant, which keeps the water temperature in the aquarium constant as well. I set
the temperature to be comparable to the natural stream temperature, and set lights inside the
chamber to turn on at 6:00 A.M. and off at 8:00 P.M., simulating natural photoperiod. For the
actual experiment, I transferred individual mayflies from the aquarium into a small plastic dish
with gravel substrate and one small rock. It was filled with water from the natural stream. After
an initial six minute period that allowed the mayfly to acclimate to the new environment,
reducing the chance that behavioral observations would be influenced by the change in
environment, I observed each individual's behavior for four minutes. I recorded the time that
each action pattern began, which gave me data including the duration of action patterns, as well
as how many times each mayfly performed each action pattern. Two drops of their treatment was
then added and their behavior recorded for four minutes. Then, two more drops of the treatment
were added, and behavior recorded for four more minutes, giving a total of eight minutes of
observation after treatment.
I expected to find no behavioral response to the addition of plain water, some response to
the licorice, and some distinct response from the trout water. I anticipated that the licorice and
trout odor would cause a response because they would change the environment, while the plain
water would not. I did not have any expectations for what the specific responses would be.
In actuality, I found no strong, distinct pattern at all. There seems to be a slight trend,
though not statistically significant, that mayflies who were given trout water spent less time
doing high energy, high motion action patterns compared to those given licorice or plain water.
These high energy action patterns include crawling, swimming, and tapping their legs against the
walls of their enclosure. Low energy action patterns, seen for a higher proportion of time in the
individuals given trout water, include remaining still and only flapping their gills, or remaining
still without flapping their gills. Though the trend is not strong, it makes sense. Trout are visual
predators, and high energy activities would likely make the mayflies more visible, and thus
easier to find and eat.
One other subtle trend appeared at the end of the experiment. One of the action patterns, a
tail flick, was proposed by Peckarsky (1987) to have some type of antipredator function. The
data are not clear, but it appears that individuals who were given trout water tail flicked more
often than those with the other treatment groups. This suggests that the mayflies that were given
trout water exhibited more antipredator reactions than those that were given plain water or
licorice extract.
There are a few possible reasons that I did not obtain a clear behavioral response to water
containing the trout odor. The theory behind this study was largely based on McIntosh and
Peckarsky's 1996 paper, in which they tested the drift behavior of mayflies based on the presence
or absence of trout smell in the water. The conditions in which they worked were very different
from mine. The experimental design I used did not have water flow in the dishes where I
measured the behavior. Stream flow is important to aquatic insects, and its absence in this study
may have contributed to the lack of behavioral differences. McIntosh and Peckarsky also studied
a different family of mayflies than I did, and it is possible that the family Heptageniidae simply
does not have a behavioral response to trout smell, at least at the level I studied.
Trout provide one of the major sources of recreation in much of Virginia. The stocking
programs were initiated more than a century ago without regard to the health of the waters in
which the trout are placed, or the presence or health of native organisms. It is impossible to know
what large scale effects trout stocking has had on Virginia's native streams, because data were
not collected prior to the program's start. It is imperative, however, that any further detrimental
effects are thoroughly examined and, as much as possible, anticipated, in order to prevent further
native population declines. This study provided some baseline information concerning the
relationship between trout and one type of their prey, mayflies. This type of information is
important not only for conservation of native species, but also to supply data concerning basic
predator-prey interactions. More specifically, this study gave information about the prey's
behavioral responses to their predator's chemical cues. Though the expected pattern did not
appear, and only subtle trends were found, it is important to continue basic behavioral research
similar to this, in the hopes that soon, we will not only have answers to the question of how our
recreation truly affects nature, but also have a better understanding of basic predator-prey
interactions.
Literature Cited
Diamond, Jared and Ted J. Case. (1986) Overview: Introductions, extinctions, exterminations,
and invasions. Community Ecology. (ed. by J. Diamond and T. Case), pp. 65-87. Harper
& Row Publishers, Inc., New York.
Loch D.D., West J.L., & Perlmutter D.G. (1996) The effect of trout farm effluent on the taxa
richness of benthic macroinvertebrates. Aquaculture, 147, 37-55.
McCafferty W.P. (1981) Mayflies (Order Ephemeroptera). Aquatic Entomology, pp.91-124.
Jones and Bartlett Publishers, Sudbury, MA.
McIntosh A.R. & Peckarsky B.L. (1996) Differential behavioral responses of mayflies from
streams with and without fish to trout odour. Freshwater Biology, 35, 141-148.
Peckarsky B.L. (1996) Mayfly cerci as defense against stonefly predation: deflection and
detection. Oikos, 48, 161-170.
Acknowledgements
This study could not have been done without the help of many people. Sweet Briar College's
Honors Summer Research Program provided funding and the Virginia Department of Game and
Inland Fisheries, particularly the staff at the Montebello Fish Hatchery, were very helpful. Dr.
Linda Fink was extremely supportive throughout the summer and after the program had ended.
Joe Malloy gave valuable insight into fly fishing and southwestern Virginia's trout streams. Greg
Keller and Marlena Koper’00 helped with field work and collection.