Introduction
Many organisms have the ability to assess predation risk and react in order to escape a dangerous situation (1). Mayflies exhibit anti-predator behaviors, including retreat into interstitial crevices and downstream drift, in response to predator cues (2, 3). These behaviors enhance mayfly fitness by reducing encounters with predators (1). Mayfly drift, a low-energy cost mechanism of leaving a risky environment, exhibits a diel periodicity, with drift peaking nocturnally, due to an evolved behavior for reducing daytime encounters with visually foraging drift feeding fish or reducing nocturnal encounters with benthivorous fish (4, 5).
Because predators forage in different ways and pose different risks to various prey, it may be expected that prey can distinguish between chemical cues of different predators. The direction and magnitude of changes in mayfly drift due to predator chemical cues should depend on the type of risk the predator presents. For instance, it may be safer for a mayfly to suppress drift and stay hidden in interstitial crevices in the presence of a drift feeder, but may be more advantageous to drift in the presence of a benthic feeder. In 2004, McIntosh and Peckarsky demonstrated that nocturnal mayfly drift due to fish chemical cues was negatively correlated with predation risk posed by two different drift feeding predators (6). In contrast in 1991 Culp, et al. found that benthivorous fish predators tended to increase nocturnal drift of mayflies, presumably as an escape mechanism (3). However, the limitation of these studies, and most empirical studies on mayfly anti-predator drift, is that they examine only one predator at a time, but in natural systems prey face multiple predators (7). As such, the reasoning behind our study is that it would be advantageous for mayflies living in a stream where both benthic and drift feeders pose a predation risk to be able to distinguish between different predator chemical cues and respond to them appropriately according to the risk they present.
Interestingly, other studies have found contrasting responses in the direction of change in nocturnal mayfly drift. Benthivorous fish have been found to decrease mayfly drift (8), and drift feeding salmon have been found to increase mayfly drift (9). However, the magnitude of increase in drift caused by the salmon in this study was smaller than the magnitude of increase in drift caused by benthic sculpin (9).
Mayflies, aquatic insects with very short adult lifespans, are though to decrease their drift in response to cues from their fish predators. them pest-resistant.
We tested the mass-specific risk of predation posed by drift-feeding brook trout (Salvelinus fontinalis) and benthic-feeding slimy sculpin (Cottus cognatus) on mayflies (Oligoneuriidae) in a tank. Our in-stream experiment sought to measure drift of all mayflies in a stream in response to elevated chemical cues from these two fish. We hypothesized that change in mayfly drift (from prior to amplification of predator cues compared to during amplification of predator cues) would differ in direction (increase vs. decrease in drift) and magnitude depending on predation risk posed by the species of the predator cue released.
In pre-trial observations, mayflies in tanks appeared to spend most of their time clinging to rocks or the tank bottom, not actively swimming into open water. Consequently, we predicted that the benthivorous sculpin would more effectively predate the mayflies. We also predicted that, in the stream, mayfly drift should be suppressed with an amplification of chemical cues from the trout and increased in the amplification of chemical cues from the sculpin, compared to drift in ambient fish stream water.
Materials and Methods
Study Site
Blood Brook in VT, USA. is a 3rd order north temperate stream. We conducted our experiment in a riffle/run habitat about 10 m wide, with discharge approximately 36 m3/min. Substrate was mostly small cobbles and larger rocks (5-30 cm diameter) on top of a sandy bottom. We compared mayfly drift before and after our treatment to account for background response to cues. Additionally, it was important to our study that the mayfly populations in our stream had adapted to predation by both types of predators. Mayflies from fishless streams may not have been able to respond to novel predator cues as appropriately (found in mussels, 10; 11).
Feeding Trials
We captured Brook Trout and Slimy Sculpin from Blood Brook, downstream of our experimental site, by electro-shocking in early November 2009. They were fed both stream invertebrates and bloodworms on a sporadic basis, dictated by how often we could obtain the food. Low densities of moderately sized macroinvertebrates in local streams in autumn posed a significant challenge to maintaining fish and conducting feeding trials.
Each species of fish was sorted into pairs, with one pair of fish placed into each of three 10 gallon tanks. Tanks each contained two large rocks (about 10-20cm diameter) similar in color and roughness. Combined mean mass in trout tanks was 6.07+0.145 g, and in sculpin tanks was 7.45+0.267 g. Prior to the feeding trial, fish were starved for at least 24 hours.
We added 40 Oligoneuriidae of approximately equivalent size distribution to each tank prior to adding fish so that they would have a chance to colonize the rocks. We allowed fish to feed for six hours, three light and three dark, before removing the fish and counting remaining mayflies, both dead and alive. We did not run the trial in control tanks with no fish because mayflies could not have escaped the aquaria, and there should not have been an effect of the tank on number of mayflies remaining after 6 hours.
Measuring Drift in the Stream
We ran the experiment in a riffle/run area of Blood Brook in which we had confirmed mayfly presence by electrofishing for macroinvertebrates one month prior. Because mayfly drift peaks at night in trout streams (4, 12), we conducted the experiments at night.
On the bank, about 2 m above the stream bottom, we placed three 121 L trash cans that had been modified with 50 foot garden hoses attached to a spigot at the bottom of the can that could gravity-feed water from the can into the stream. In each of two cans, we placed 200 g of fish (trout in the first, sculpin in the second), and no fish in the third can, which acted as the control. We offered the fish a mixture of stream invertebrates to feed on and let them sit in the cans for 90 minutes.
In the stream, we placed three drift nets evenly across the width of the stream so that they were far enough apart that plumes of drift into each net did not cross, tested simply with a leaf drop. We positioned the mouth of each hose about 3 m upstream of each drift net. We collected 15 minute drift samples in these nets to assess ambient mayfly drift. We then reset our drift nets and turned on the spigots of the trash cans to release the water and fish chemical cues for 15 minutes. At the end of this second 15 minute period, spigots were shut off and we collected our drift samples to assess the change in mayfly drift in the presence of amplified fish chemical cues compared to drift during the initial samples with ambient stream water.
We decided to use Oligoneurids in the tank feeding trial because they are good swimmers, based on our own observations. We intended to measure the drift of “good swimmers” as our response variable in the stream experiment, but due to the small number of mayflies collected, decided to focus on total mayflies.
Statistical Analysis
For the feeding trials, we converted the count of remaining mayflies in each tank into Predation risk, mass-specific consumption by fish: (mayflies introduced – mayflies remaining) / (total fish mass in tank) for the six hour feeding trial period. We used a two-sided, pooled t-test to test for any difference between species in mass-specific consumption. Levene’s test (F1,4=0.0097, p=0.9261) supported the assumption of equal variances.
For the in-stream drift experiment, we converted counts of mayflies caught in drift nets in the 15-minute periods prior to and during release of fish cues to the proportional change in mayfly drift: (# of mayflies drifting during release of fish cues / # of mayflies drifting prior to release of fish cues). We tested for differences between treatments (trout, sculpin, control) with a one-way ANOVA. An arcsin-square root transformation was considered, but strong non-normality was not evident. All following statistics were calculated in JMP 7.2, and data organization and processing was done in Microsoft Excel 2004 (v.11.5.4). All means are reported ± 1 std. error.
Results
Laboratory feeding trials
During the six hours of feeding trials, overall mean consumption of mayflies by pairs of fish was 15.3 ±2.9 individuals, resulting in average consumption of 2.4 ±0.5 individual mayflies per gram of fish mass (N=6 tanks). We made no formal assessment of mayfly space use in the tanks, however, observations made immediately after removing fish from the tanks found mayflies to be highly concentrated on the surfaces of the rocks and to some degree in the corners of the tanks, where plastic edging provided a dark background. While trout did tend to consume more mayflies than sculpin on an absolute and a mass-specific basis, the difference was not significant (Figure 1; t4=-1.6249, p=0.1795, N=6). No control tanks were run, but potential disappearance of mayflies from tanks was assumed to be zero.
In-stream nighttime drift of mayflies
There were no significant differences in Proportional change in mayfly drift among the trout, sculpin, and control chemical cue treatments (one-way ANOVA, F2,6=1.7632, p=0.2498, N=6). In all replicates, the Proportional change in mayfly drift was either negative or zero, and overall mean Proportional change was -0.36 ±0.11 (N=9).
Figure 1. While no differences between fish treatments were significant, data suggest that trout may pose a greater predation risk to mayflies (in laboratory feeding trials), and amplified trout cues result in a greater decrease in drift relative to the control and sculpin treatments (i.e. a stronger negative change in mayfly drift in-stream: # of mayflies drifting during release of fish chemical cues / # drifting beforehand). Error bars represent ±1 standard error of the mean. Low sample sizes, due to time and resource constraints, reduced statistical power.
Discussion
Contrary to our expectations, brook trout tended (non-significantly) to consume more Oligoneuriidae in tanks. However, since our tanks were not representative of natural conditions, results may not be applicable in the stream environment. Since the rocks we used were relatively tall compared to the fish, and did not provide a true substrate (leaving more than half of the tank bottom exposed as glass), the ability of sculpin to forage may have been sharply diminished. Instead of being able to rest on top of the substrate, as they normally would in streams, we observed the sculpin in our tanks spending most of their time on the bottom of the tank, next to the bottom of the rocks. They may have avoided foraging at the top of the rocks, which may have afforded the mayflies a refuge. In contrast, the brook trout spent most of their time swimming in the open water. It is likely that they had better access to a larger number of mayflies than the sculpin did as an artifact of our tank habitat design. Our results tentatively suggest that since brook trout consume more mayflies on a mass specific basis (non-significantly), and since there is greater brook trout biomass in Blood Brook, that brook trout pose a higher predation risk to mayflies than sculpin do.
Number of replicates for the predation risk trial was constrained by our ability to catch enough mayflies and fish of appropriate masses. We had originally intended to run the feeding trials with a wide range of masses within each fish taxa. However, even without these data, we could still determine mass specific consumption of mayflies for each fish taxa. There is also the possibility that trout and sculpin adapt differentially to stressful capture situations and that this affected their foraging ability in tanks. In the future, it would be better to assess predation risk posed by different predators by examining gut contents of stream fish.
Mayfly drift in the stream experiment did not significantly change compared to the control due to exposure to either fish chemical cue. However, the low number of replicates sharply reduced statistical power. The trend for stronger suppression of mayfly drift due to trout cues relative to sculpin cues is in concord with our hypothesis that even nocturnal mayfly drift may be dangerous in the presence of drift feeding fish. Our findings, that both fish tended to suppress mayfly drift (non-significantly) agree with the broad conclusion of Winkelman et al. in 2008 that high predation risk generally reduces invertebrate drift (8). However, we cannot conclude that either fish induced a behavioral change in mayfly drift.
The inconclusiveness of our stream trial may also have been due to background fish cues in the stream. If the cues released from our trash cans were not potent enough to overcome background noise, we would not expect either treatment to be different from the control (13). Another problem with our study design was determining the distance between upstream release of cue and downstream placement of the drift net. We aimed to sample a short enough distance from the cue release that mayflies responding to the chemical cues by drifting would all drift into the net and not re-adhere to a rock before reaching the net. We also wanted the distance to be long enough that the cue released would affect a large number of mayflies. We are concerned that, if the distance we chose was too short, mayflies drifting from upstream of the cue release site may have drowned out any response that we could have observed for the treatment where we expected an increase in drift. This may be why the sculpin treatment shows such little difference from the control treatment. We speculate that drift from upstream poses less of a problem in the trout treatment, where we expected drift suppression, because mayflies drifting from upstream would suppress their drift as they encountered the trout cue.
Our results, albeit non-significant, place this study in the midst of an ongoing debate in the literature about the drift response of mayflies to fish chemical cues. Laboratory microcosm and in-stream studies by McIntosh and Peckarsky (2004), and McIntosh et al. (1999) for large mayflies only, found the chemical cues of drift-feeding salmonids to cause suppression of mayfly drift (6, 14). In contrast, Miyasaka and Nakano (2001) and McIntosh et al. (1999) for small mayflies only found that chemical cues of drift-feeding salmonids to cause an increase in mayfly drift (9, 14).
Mayfly size could be a confounding factor that explains some discrepancies in the literature. Few studies, to our knowledge, classified drifting mayflies by size, but McIntosh et al. (1999) found that large Baetidae mayflies decreased drift, while small Baetidae increased drift, in response to increasing trout odor concentration.
We propose that some discrepancies in the literature may also be explained by light pollution in streams in more developed areas. Salmonids are primarily visual predators and pose a significant threat to mayflies drifting during the daytime, but can also consume prey at night (15). If light levels are sufficient at night (e.g., during a full moon) for salmonids to remain effective predators (or at least pose a greater risk of mortality than benthic predation), mayfly drift suppression would be expected, given that risk of mortality is high enough and selective pressures have had time to act on the mayfly population in question. Alternatively, if nighttime light intensity is too low for effective hunting by salmonids (i.e. risk posed to mayflies by drifting is less than that posed by staying on the stream bottom), mayflies might be expected to utilize drift in order to depart areas where trout are present to search for high-quality food patches, as Kohler found in 1985 (16). At our study site, light pollution from nearby residential houses could have produced enough light for visually foraging drift feeders to be able to forage at night. This pressure would negate the benefits of nocturnal drift as an escape mechanism from drift feeders. We speculate that, if this is true, it may also be generalized to many streams in urbanized areas that experience light pollution at night. Light pollution, or lack thereof, may explain some discrepancies in results of various studies of mayfly drift in response to drift feeding fish chemical cues.
There has also been controversy over whether benthivorous fish should cause an increase or decrease in nocturnal mayfly drift. A large-scale field experiment found that drift was suppressed (8), and a laboratory stream experiment found that drift increased (9). Winkelman, et al. (2008) attribute the discrepancy to differential vulnerability of different prey species to predator species used in different studies that were conducted at varying spatial and temporal scales. Additionally, they posit that mayflies may employ other anti-predator tactics that do not involve drift in response to benthivorous predators. In our study, since we observed much less mayfly drift due to amplified sculpin cues, the utilization of alternative anti-predator behaviors may explain the unexpectedly low response.
We also postulate that mayfly size might also play a role in response to benthic predators, as it has been shown in response to drift feeding fishes, as discussed above. Additionally, most literature that we encountered addressed drift feeder effects on mayfly drift, but there are fewer studies that address benthivorous fish effects on mayfly drift.
In conclusion, the findings of our combined laboratory and field experiments cannot relate differential mayfly drift responses to fish chemical cues from fish that pose different predation risks. However, the unresolved debate in the literature is a compelling reason to tease out what factors influence the direction and magnitude of change in mayfly drift. In the future, it may be interesting to examine how light pollution near urban streams may modify mayfly response to more effective nocturnal drift feeding, and the variation in size-specific mayfly drift response to benthivorous predation cues.
Acknowledgements
We would like to acknowledge Brad Taylor, Ramsa Chaves, and Craig Layne for assisting in the design and implementation of the project. The Biology Department at Dartmouth College provided funding.
References
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