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Predator-prey interactions among fish and larval amphibians: use of chemical cues to detect predatory fish
Anita. Behav., 1987, 35, 420~425
Predator-prey interactions among fish and larval amphibians: use of chemical cues to detect predatory fish J A M E S W. P E T R A N K A , * LEE B. K A T S & A N D R E W SIH
Behavioral and Evolutionary Ecology Research Group, T. H. Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky 40506~)225, U.S.A.
Abstract. Gravitational flow-through systems were used to determine whether larvae of two-lined
salamanders, Euryeea bislineata, and Cope's grey treefrog, Hyla chrysoscelis, use chemical cues to detect predatory fish. Eurycea larvae were attracted to water conditioned with food, showed no response to water conditioned with green frog, Rana elamitans, tadpoles, and avoided water conditioned with a dangerous predator, the green sunfish, Lepomis cyanellus. Hyla tadpoles exposed to water conditioned with green sunfish spent significantly more time in refuges than did controls exposed only to water. In a comparable experiment performed at a natural breeding site, Hyla tadpoles significantly increased refuge use when exposed to water conditioned with fish. These data suggest that amphibian larvae use chemical cues in their natural habitats to minimize predation risk from fish.
Aquatic predators play a crucial role in determining the composition and structure of larval amphibian communities (Walters 1975; Morin 1983; Smith 1983; Wilbur et al. 1983; Woodward 1983). Of the numerous organisms that prey upon amphibian larvae, fish are possibly the most destructive. Heyer et al. (1975) considered fish to be the only aquatic predators capable of eliminating entire populations of tadpoles from ponds. Burger (1950) reported the widescale elimination of tiger salamander, Ambystoma tigrinum, larvae from ponds in Colorado following stocking with trout. Macan (1966) noted a dramatic decrease in numbers of toad, Bufo sp. and frog, Rana sp. tadpoles following the introduction of brown trout into a British tarn, and Petranka (1983) documented decimation of small-mouthed salamander, A. texanum larvae in local pools in streams following colonization by green sunfish. Despite such well-documented cases of predation, the larvae of many amphibians characteristically coexist with predatory fish. These include such familiar North American species as the bullfrog, Rana catesbeiana, the green frog, R. clam# tans, and the mudpuppy, Necturus rnaculosus. These and other amphibians have a battery of defences, which minimize the risk offish predation, * Present address: Department of Biology, 0104 Coker Hall. University of North Carolina, Chapel Hill, North Carolina 27514, U.S.A.
including chemical repellents (Voris & Bacon 1966; Wassersug 1971; Kruse & Francis 1977), cryptic coloration (Wassersug 1971), rapid growth rates coupled with large body size (Heyer et al. 1975), and antipredator behaviours. Behaviours that are thought to reduce predation risk include schooling (e.g. Waldman 1982; Kruse & Stone 1984), reduced movement (Woodward 1983) and protean flight (Taylor 1983). A growing body of data suggests that larval behaviour is a more important defence against predators than was previously suspected. Taylor (1983, 1984), for example, reported that larvae of the northwestern salamander, Ambystoma gracile, are active day and night in lakes lacking predatory fish, but are strictly nocturnal in those with trout. Differences in f~ight behaviour of A. graeile also correlate with fish presence and may reflect differential predation from fish versus terrestrial predators. Tiger salamander larvae show strong shifts in diel patterns of habitat use which correlate with the presence or absence of predaceous beetles (Holomuzki 1986), and spring peeper, Hyla crucifer, tadpoles are more secretive in the presence of predaceous newts (Morin, in press). These observations indicate that amphibian larvae regularly sample their environments for predators and respond with appropriate behaviour to minimize risk. A key missing link in understanding this phenomenon, however, involves the proximate
420
Petranka et al.: Chemical detection of predators
EXPERIMENT
I
B
A
cues used to monitor predators. Here we show that one factor, the use of chemical cues, is important in detecting predatory fish.
421
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Materials and Methods
We used a completely randomized design to determine whether larvae of two-lined salamanders, Eurycea bistineata, chemically detect and discriminate between: (1) green sunfish, Lepomis cyanellus, a dangerous predator that commonly coexists with Eurycea, (2) tadpoles of the green frog, Rana clamitans, an innocuous herbivore that occasionally coexists with Eurycea, and (3) San Francisco Bay brine shrimp, Artemia spp., a commercial fish food that larvae readily consume in the laboratory. We tested larvae in a gravitational flow-through system consisting of plastic tubs, 48 • 33 cm, arranged in linear sequences at different heights on laboratory benches (Fig. 1A). When operative, flow occurred sequentially from the uppermost to the lowermost tubs via 4-mm (inside diameter) plastic aquarium tubing at rates of 0.5 0.6 litres/min. We filled all of the tubs, except the lowermost which served for catchment, with 10 litres of Mount's 100, a synthetic lakewater solution consisting of distilled water and mineral salts (Birge et al. 1979). Tub 5 received effluent from two higher tubs (3, 4), one served as a control and held only lakewater, the other held one of three experimental treatments: (1) lakewater and one green sunfish; mean wet weight of three animals used = 14.2 g, (2) lakewater and 41 green frog tadpoles; total wet weight= 14.2 g, and (3) lakewater and 14-2 g of brine shrimp. Thirty min prior to beginning each trial, we added one of the three test items to either tub 3 or 4, and placed screening over the outflow drain to prevent all but extremely fine particles from passing into tub 5. We used a random number table to determine which tub (3 or 4) received the test item, and used the remaining tub as the control. A solid Plexiglas partition in tub 5 prevented effluents from tubs 3 and 4 from mixing until they passed through a perforated Plexiglas plate into an experimental chamber, 8 x 33 cm, at the far end of the tub (Fig. 1A). A stepped chemical gradient was generated as effluents passed through the plate into drains located at either corner of the tub.
7-
Figure 1. Gravitational flow-through systems used in the
experiments. System A was used for twoqined salamanders; system B for grey treefrogs.
In preliminary trials using food colouring, we found that coloured water began entering the experimental chamber 1.5 3 rain after flow was initiated between tubs. Spectrophotometric analyses of samples taken at intervals of 1 min for 20 rain from the side of the chamber receiving coloured water showed that optical density increased sharply during the first 10-12 min of the experiment, then levelled off. We subsequently elected to run trials for 20 min, but to use only the last 10 rain of the trials to judge larval responses to treatments. This allowed time for the conditioned water to concentrate in the experimental chamber, and provided 10 min for larvae to adjust to the experimental conditions. We initiated flow between tubs immediately after adding eight larvae (mean total length = 27 ram) to the experimental chamber in tub 5. To minimize human disturbance, we filmed larvae with a video camera and observed behaviour on a monitor located in an adjoining room. We scored the number of larvae in the half of the experimental chamber receiving conditioned water at intervals of 1 min, then used the mean of these data to estimate the average proportion of time that larvae spent in the end of the chamber receiving conditioned water. The tubs were washed repeatedly in distilled water after each trial, and the experiment was replicated 10 times. We used a random number
422
Animal Behaviour, 35, 2
table to randomize all aspects of the experiment including the treatment sequence, the tubs holding control and conditioned water, and the side of the chamber receiving conditioned water. We collected animals for this and other experiments from natural habitats, maintained them in the laboratory for 1-2 weeks prior to testing, and fed them trout pellets (tadpoles) or brine shrimp (fish and salamander larvae) every 1 2 days. To minimize confusion with odours emitted from faeces, we deprived all animals of food 12-18 h prior to running the experiments.
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Data Analysis The assumption of independence of error terms is violated if test organisms are re-used in subsequent trials, if repeated measures taken on the same organism are used in statistical tests, or if individual readings of animals housed in a common chamber or environment are used in statistical tests. To assure independence of data points, we did not re-use larvae in subsequent trials, i.e. we drew new groups of larvae from a population of several hundred maintained in the laboratory. In addition, we used the mean (expressed as a proportion) of counts made at l-min intervals during the last 10 min of the trials in our statistical tests. We elected to use groups of larvae, rather than test larvae individually, because social interactions which might override responses to chemical cues occur under natural conditions. Again, by using group means in our statistical tests we avoided violating the assumption of independence. Angular transformations were made of all proportions before we ran parametric tests with SAS (Ray 1982a, b). Assumptions of normality (Shapiro-Wilk test) and homogeneity of variances (folded F-statistic or Bartlett's test) were tested and satisfied for all sets of transformed data.
Results We first used a two-tailed t-test to test the null hypothesis that the proportion of time spent by Eurycea larvae on the side of the chamber receiving treated water was 0.50. The analyses indicated a significant positive response (attraction) to brine shrimp (t = 2.5, df= 9, P = 0-03), a neutral response (t = 1.1, df= 9, P = 0.30) to green frog tadpoles, and a negative response (avoidance) to green sunfish (t=2"5, df=9, P=0'03; Fig. 2). Larvae exposed to
Figure 2. Responses of two-lined salamander larvae to water conditioned with brine shrimp (bs), green frog (gf) tadpoles and green sunfish (gs). Symbols reflect the percentage of time (mean _ 1 SE) that larvae remained on the side of the experimental chamber receiving conditioned water during sequential 5-min time intervals.
effluents from tubs holding green sunfish showed a consistent decline in the proportion of time spent in chemical-laden water. Responses to the two other treatments were relatively constant throughout the duration of the experiment. ANOVA showed significant treatment effects for the last 10 min of the trials ( F = 5.52, df= 2,27, P < 0.001), and pairwise comparison using Tukey's procedure indicated that larval responses to green sunfish were significantly different from those to green frog tadpoles and brine shrimp. The ability of Eurycea to detect brine shrimp chemically is probably greater than the data in Fig. 2 imply. As effluent began entering the experimental chamber at the 2-3 min mark of the experiment, larvae quickly moved to the side receiving conditioned water. During the 4-6 min interval, larvae spent about 69% of their time on the side receiving conditioned water ( P < 0-0001). Soon thereafter, they began to explore all areas of the chamber in a manner similar to that of laboratory stock when fed daily rations of brine shrimp. Larvae would periodically return to the side of the chamber receiving conditioned water, often orient towards the current, then continue exploring the entire chamber for food. In nine of 10 replicates, larvae spent greater than 50% of their time on the side of the chamber receiving conditioned water. Thus, it was clear that larvae responded to brine shrimp even though the magnitude of the response was not great.
Petranka et al.: Chemical detection of predators EXPERIMENTS
II A N D III
The results of experiment I indicated that larvae can detect fish chemically under simplified laboratory conditions. But can they do so in chemicallycomplex, natural habitats? We designed two experiments using tadpoles of Cope's grey treefrog, Hyla chrysoscelis, to address this question. We used Hyla tadpoles rather than Eurycea larvae because Hyla natural habitats are more amenable to experimental manipulations.
Methods
We first used a modified flow-through system in the laboratory to test the ability of tadpoles to detect fish chemically (Fig. 1B). The system and experimental protocol were similar to that of experiment I, except that the solutions flowed from tubs 3 and 4 into separate tubs (5, 6) that each contained eight tadpoles at Gosner stages 3440. Tubs 5 and 6 held a 23 x 31-cm translucent, grey Plexiglas plate supported 1.5 cm above the bottom by wood screws fastened to the corners. The plate served as a refuge and covered about 50% of the bottom surface of the tubs. We added tadpoles to the open (non-refuge) portion of the tubs immediately before initiating flow between the tubs, then recorded the number of larvae outside of refuges at intervals of 1 min for 20 min. We estimated the proportion of time that larvae spent outside of refuges from these data. As in experiment I, we used only data for the last 10 min of the trials in statistical comparisons of the control and experimental groups. We alternated the positions of the plates and tubs between replicates ( N = 5). We examined tadpole responses to chemical cues in a field experiment conducted 22-23 August 1985 in two temporary pools adjoining the Red River in Powell County, Kentucky. The pools were 2 m apart, and were apparently remnants of a larger pool that subdivided as seasonal water levels dropped. Both pools lacked fish, had silt-mud bottoms, and were relatively small (surface area less than or equal to 6 m2), and shallow (maximum depth less than or equal to 10 cm). Odonate larvae and dytiscid beetles were conspicuous invertebrate predators in both pools. We gently sank water-tight, open-bottomed plywood boxes into the bottom mud and silt, then packed mud around the outside bottom edges to minimize leakage of water from the boxes. The
423
dimensions of the boxes were the same as those of the tubs used in the laboratory experiment. We paired control and experimental boxes (N = 5) with respect to microhabitat and pool. We collected tadpoles from the pools 1 week before running the experiment, maintained them in the laboratory on trout pellets, then returned them to the pools for testing. We placed Plexiglas refuges identical to those used in the laboratory experiment into the boxes and added eight tadpoles (Gosner stages 34-40) to each of the non-refuge halves at the start of the experiment. We initially attempted to mimic the laboratory experiment by flowing Mount's 100 solutions from elevated tubs into the boxes below, but abandoned this approach because the flow caused increased turbidity that made it impossible to count larvae. Instead, we poured 0.5 litres of Mount's solution (control) or 0.5 litres of Mount's solution in which a green sunfish was placed for 10 rain (experimental) into the non-refuge half of the box at the 0 and 10 min mark of the experiment. We scored the number of larvae outside of refuges at intervals of 1 min for 20 min, but used only the last 10 rain of data in our statistical test.
Results
In laboratory trials, ttyla showed a strong negative response to effluent from tubs with fish. Experimental tadpoles spent 68% less time outside of refuges than controls during the last 10 min of the trials (paired, two-tailed t-test: t=9-3, df=3, P < 0.001; Fig. 3). Hyla reacted very quickly to fish effluent. During the first 5 min of the experiment, differences in the percentage of time spent outside of refuges nearly equalled that which occurred during the final 10 min. Experimental animals in the field showed responses that were similar in direction, but weaker in magnitude. Experimental tadpoles spent 30% less time outside of refuges than controls during the last 10 rain of the trials (paired, one-tailed t-test: t = 3 3 , dr=3, P=0.02). Variation among replicates for both controls and experimentals was nearly twice as high in the field as in the laboratory.
DISCUSSION Chemically-mediated predator avoidance occurs in a variety of vertebrate and invertebrate groups
Animal Behaviour, 35, 2
424
Hyla showed an equally strong response to Lepomis in the laboratory and quickly moved
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(Pfeiffer 1966, 1977; Dayton et al. 1977; Stoddart 1980), but, to our knowledge, a direct mechanism has not been previously demonstrated in amphibians. Pfeiffer (1966) demonstrated an indirect mechanism in which skin extracts of Bufo tadpoles elicit a fright reaction in conspecifics. Chemicals released from the skin following attacks by predators indirectly signal potential danger. Anosmic tadpoles do not respond to skin extracts, which indicates chemical detection via olfaction. Our data show that Eurycea larvae can discriminate between water conditioned with food, a dangerous predator, and a benign herbivore. Larvae showed no response to Rana tadpoles, but strongly avoided water conditioned with a single Lepomis. Eurycea larvae are palatable to Lepomis in the laboratory (Petranka 1983), yet the two species often coexist in small streams in central Kentucky. Thus, chemical detection of Lepomis may be an important mechanism allowing Eurycea to exploit habitats occupied by predatory fish.
beneath cover. Although our field and laboratory experiments with Hyla are not entirely comparable because of minor differences in protocol, the data suggest that responses to Lepomis are weaker in nature. This may be because larvae receive conflicting chemical stimuli from food, fish and invertebrate predators in breeding pools. We find the strong response of Hyla to Lepomis somewhat surprising because Hyla usually breeds in seasonally ephemeral pools that lack fish. Nonetheless, we have occasionally collected larvae from farm ponds that contain predatory fish. The ability to detect predators chemically is particularly advantageous for organisms whose activity patterns or environments preclude the effective use of other sensory systems (Madison 1977; Stoddart 1980). Amphibian larvae fulfil this criterion reasonably well: many species are nocturnally active, while others live in turbid habitats. An additional advantage of using chemical cues is that organisms can detect predators while remaining in refuges, that is, they can gauge predation risk without exposing themselves to danger. A major disadvantage is that chemical cues can provide erroneous information about predator presence because of information time lags. We find that small-mouthed salamander, Ambystoma texanum, larvae, for example, respond to water conditioned with green sunfish by significantlyincreasing refuge use for as long as 3 days after the fish are removed from the water (Sih, Petranka & Kats, unpublished data). Remaining in refuges long after predators vacate an area reduces opportunities for feeding and growth which, in turn, may lessen the probability of surviving to metamorphosis (Wilbur 1980). Our data help to explain how palatable amphibian larvae successfully coexist with aquatic predators (Taylor 1983; Morin, in press). We have conducted a general survey of amphibians and find that the larvae of many species avoid water conditioned with green sunfish. We do not know if larvae respond similarly to invertebrate predators, but hope to address this and other questions relating to chemically-mediated predator avoidance in the future.
ACKNOWLEDGMENTS We thank John Just and Kevin Strohmeier for help
Petranka et al.: Chemical detection o/predators with the s p e c t r o p h o t o m e t r i c analysis. T h e w o r k was s u p p o r t e d by N a t i o n a l Science F o u n d a t i o n G r a n t BSR-8500329.
REFERENCES Birge, W. J., Black, J. A., Westerman, A. G. & Hudson, J. E. 1979. The effects of mercury on reproduction offish and amphibians. In: Biogeochemistry of Mercury in the Environment (Ed. by J. O. Nriagu), pp. 629 655. Amsterdam: Elsevier/North Holland Biomedical Press. Burger, W. L. 1950. Novel aspects of the life history of two Ambystomas. J. Tenn. Acad. Sci., 25, 252 257. Dayton, P. K., Rosenthal, R. J., Mabem L. C. & Autezana, T. 1977. Population structure and foraging biology of the predaceous Chilean asteroid Meyenaster gelatinosus and the escape biology of its prey. Mar. Biol., 39, 361-370. Heyer, W. R., McDiarmid, R. W. & Weigmann, D. L. 1975. Tadpoles, predation and pond habitats in the tropics. Biotropica, 7, 100-I 11. Holomuzki, J. R. 1986. Predator avoidance and diel patterns of use of larval tiger salamanders. Ecology, 67, 737 748. Kruse, K. C. & Francis, M. G. I977. A predation deterrent in larvae of the bullfrog, Rana eatesbeiana. Trans. A m Fish. Soc., 106, 248-252. Kruse, K. C. & Stone, B. M. 1984. Largemouth bass (Micropterus salmoides) learn to avoid feeding on toad (Bufo) tadpoles. Anim. Behav., 32, 1035-1039. Macan, T. T. 1966. The influence of predation on the fauna of a moorland fishpond. Arch. Hydrobiol., 61, 432~452. Madison, D. M. 1977. Chemical communication in amphibians and reptiles. In: Chemical Signals in Vertebrates (Ed. by D. Muller-Schwarze & M. M. Mozell), pp. 135 168. New York: Plenum. Morin, P. J. 1983. Predation, competition, and the composition of larval anuran guilds. Ecol. Monogr., 53, 119-138. Morin, P. J. In press. Interactions between intraspecific competition and predation in an amphibian predator prey system. Ecology.
425
Petranka, J. W. 1983. Fish predation: a factor affecting the spatial distribution of a stream-breeding salamander. Cope&, 1983, 624~628. Pfeiffer, W, 1966. Die Verbreitung der Schreckreaktion bet Kaulquappen und die Herkunft des Schreckstoffes. Z. ver. Physiol., 52, 79 98. Pfeiffer, W. 1977. The distribution of fright reaction and alarm substance in cells of fishes. Copeia, 1977, 653665. Ray, A. A. 1982a. SAS User's Guide." Basics. Ca W, North Carolina: SAS Institute. Ray, A. A. 1982b. SAS User's Guide: Statistics. Cary, North Carolina: SAS Institute. Smith, D. 1983. Factors controlling tadpole populations of the chorus frog (Pseudacris triseriata) on Isle Royale, Michigan. Ecology, 64, 501 5 I0. Stoddart, D. M. 1980. The Ecology qf Vertebrate Ol/~wtion. New York: Chapman & Hall. Taylor, J. T. 1983. Orientation and flight behavior of a neotenic salamander (Ambystoma gracile) in Oregon. Am. Midl. Nat., 109, 40 49. Taylor, J. T. 1984. Comparative evidence for competition between the salamanders Ambystoma gracih" and 7~wieha granulosa. Copeia, 1984, 672-683. Voris, H. K. & Bacon, J. P., Jr. 1966. Differential predation on tadpoles. Copeia, 1966, 594 598. Waldman, B. 1982. Sibling association among schooling toad tadpoles: field evidence and implications. AnDs7. Behav., 30, 700-713. Waiters, B. 1975. Studies ofinterspecific predation within an amphibian community. J. Herpetol., 9, 267 279. Wassersug, R. J. 1971. On the comparative palatability of some dry-season tadpoles from Costa Rica. Am. Midl. Nat., 86, 101-109. Wilbur, H., M. 1980. Complex life cycles. A. Rer. Ecr Syst., 11, 67-93. Wilbur, H M., Morin, P. J. & Harris R. N. I983. Salamander predation and the structure of experimental communities: anuran responses. Ecology, 64, 1423-1429. Woodward, B. D. 1983. Predator-prey interactions and breeding-pond use of temporary-pond species in a desert anuran community. Ecology. 64, 1549 1555.
(Receized 21 February 1986; rerised 1 April 1986," MS. number: A4670)
Predator-prey interactions among fish and larval amphibians: use of chemical cues to detect predatory fish J A M E S W. P E T R A N K A , * LEE B. K A T S & A N D R E W SIH
Behavioral and Evolutionary Ecology Research Group, T. H. Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky 40506~)225, U.S.A.
Abstract. Gravitational flow-through systems were used to determine whether larvae of two-lined
salamanders, Euryeea bislineata, and Cope's grey treefrog, Hyla chrysoscelis, use chemical cues to detect predatory fish. Eurycea larvae were attracted to water conditioned with food, showed no response to water conditioned with green frog, Rana elamitans, tadpoles, and avoided water conditioned with a dangerous predator, the green sunfish, Lepomis cyanellus. Hyla tadpoles exposed to water conditioned with green sunfish spent significantly more time in refuges than did controls exposed only to water. In a comparable experiment performed at a natural breeding site, Hyla tadpoles significantly increased refuge use when exposed to water conditioned with fish. These data suggest that amphibian larvae use chemical cues in their natural habitats to minimize predation risk from fish.
Aquatic predators play a crucial role in determining the composition and structure of larval amphibian communities (Walters 1975; Morin 1983; Smith 1983; Wilbur et al. 1983; Woodward 1983). Of the numerous organisms that prey upon amphibian larvae, fish are possibly the most destructive. Heyer et al. (1975) considered fish to be the only aquatic predators capable of eliminating entire populations of tadpoles from ponds. Burger (1950) reported the widescale elimination of tiger salamander, Ambystoma tigrinum, larvae from ponds in Colorado following stocking with trout. Macan (1966) noted a dramatic decrease in numbers of toad, Bufo sp. and frog, Rana sp. tadpoles following the introduction of brown trout into a British tarn, and Petranka (1983) documented decimation of small-mouthed salamander, A. texanum larvae in local pools in streams following colonization by green sunfish. Despite such well-documented cases of predation, the larvae of many amphibians characteristically coexist with predatory fish. These include such familiar North American species as the bullfrog, Rana catesbeiana, the green frog, R. clam# tans, and the mudpuppy, Necturus rnaculosus. These and other amphibians have a battery of defences, which minimize the risk offish predation, * Present address: Department of Biology, 0104 Coker Hall. University of North Carolina, Chapel Hill, North Carolina 27514, U.S.A.
including chemical repellents (Voris & Bacon 1966; Wassersug 1971; Kruse & Francis 1977), cryptic coloration (Wassersug 1971), rapid growth rates coupled with large body size (Heyer et al. 1975), and antipredator behaviours. Behaviours that are thought to reduce predation risk include schooling (e.g. Waldman 1982; Kruse & Stone 1984), reduced movement (Woodward 1983) and protean flight (Taylor 1983). A growing body of data suggests that larval behaviour is a more important defence against predators than was previously suspected. Taylor (1983, 1984), for example, reported that larvae of the northwestern salamander, Ambystoma gracile, are active day and night in lakes lacking predatory fish, but are strictly nocturnal in those with trout. Differences in f~ight behaviour of A. graeile also correlate with fish presence and may reflect differential predation from fish versus terrestrial predators. Tiger salamander larvae show strong shifts in diel patterns of habitat use which correlate with the presence or absence of predaceous beetles (Holomuzki 1986), and spring peeper, Hyla crucifer, tadpoles are more secretive in the presence of predaceous newts (Morin, in press). These observations indicate that amphibian larvae regularly sample their environments for predators and respond with appropriate behaviour to minimize risk. A key missing link in understanding this phenomenon, however, involves the proximate
420
Petranka et al.: Chemical detection of predators
EXPERIMENT
I
B
A
cues used to monitor predators. Here we show that one factor, the use of chemical cues, is important in detecting predatory fish.
421
2
[~
~q~2'm
Materials and Methods
We used a completely randomized design to determine whether larvae of two-lined salamanders, Eurycea bistineata, chemically detect and discriminate between: (1) green sunfish, Lepomis cyanellus, a dangerous predator that commonly coexists with Eurycea, (2) tadpoles of the green frog, Rana clamitans, an innocuous herbivore that occasionally coexists with Eurycea, and (3) San Francisco Bay brine shrimp, Artemia spp., a commercial fish food that larvae readily consume in the laboratory. We tested larvae in a gravitational flow-through system consisting of plastic tubs, 48 • 33 cm, arranged in linear sequences at different heights on laboratory benches (Fig. 1A). When operative, flow occurred sequentially from the uppermost to the lowermost tubs via 4-mm (inside diameter) plastic aquarium tubing at rates of 0.5 0.6 litres/min. We filled all of the tubs, except the lowermost which served for catchment, with 10 litres of Mount's 100, a synthetic lakewater solution consisting of distilled water and mineral salts (Birge et al. 1979). Tub 5 received effluent from two higher tubs (3, 4), one served as a control and held only lakewater, the other held one of three experimental treatments: (1) lakewater and one green sunfish; mean wet weight of three animals used = 14.2 g, (2) lakewater and 41 green frog tadpoles; total wet weight= 14.2 g, and (3) lakewater and 14-2 g of brine shrimp. Thirty min prior to beginning each trial, we added one of the three test items to either tub 3 or 4, and placed screening over the outflow drain to prevent all but extremely fine particles from passing into tub 5. We used a random number table to determine which tub (3 or 4) received the test item, and used the remaining tub as the control. A solid Plexiglas partition in tub 5 prevented effluents from tubs 3 and 4 from mixing until they passed through a perforated Plexiglas plate into an experimental chamber, 8 x 33 cm, at the far end of the tub (Fig. 1A). A stepped chemical gradient was generated as effluents passed through the plate into drains located at either corner of the tub.
7-
Figure 1. Gravitational flow-through systems used in the
experiments. System A was used for twoqined salamanders; system B for grey treefrogs.
In preliminary trials using food colouring, we found that coloured water began entering the experimental chamber 1.5 3 rain after flow was initiated between tubs. Spectrophotometric analyses of samples taken at intervals of 1 min for 20 rain from the side of the chamber receiving coloured water showed that optical density increased sharply during the first 10-12 min of the experiment, then levelled off. We subsequently elected to run trials for 20 min, but to use only the last 10 rain of the trials to judge larval responses to treatments. This allowed time for the conditioned water to concentrate in the experimental chamber, and provided 10 min for larvae to adjust to the experimental conditions. We initiated flow between tubs immediately after adding eight larvae (mean total length = 27 ram) to the experimental chamber in tub 5. To minimize human disturbance, we filmed larvae with a video camera and observed behaviour on a monitor located in an adjoining room. We scored the number of larvae in the half of the experimental chamber receiving conditioned water at intervals of 1 min, then used the mean of these data to estimate the average proportion of time that larvae spent in the end of the chamber receiving conditioned water. The tubs were washed repeatedly in distilled water after each trial, and the experiment was replicated 10 times. We used a random number
422
Animal Behaviour, 35, 2
table to randomize all aspects of the experiment including the treatment sequence, the tubs holding control and conditioned water, and the side of the chamber receiving conditioned water. We collected animals for this and other experiments from natural habitats, maintained them in the laboratory for 1-2 weeks prior to testing, and fed them trout pellets (tadpoles) or brine shrimp (fish and salamander larvae) every 1 2 days. To minimize confusion with odours emitted from faeces, we deprived all animals of food 12-18 h prior to running the experiments.
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TIME INTERVAL (min)
Data Analysis The assumption of independence of error terms is violated if test organisms are re-used in subsequent trials, if repeated measures taken on the same organism are used in statistical tests, or if individual readings of animals housed in a common chamber or environment are used in statistical tests. To assure independence of data points, we did not re-use larvae in subsequent trials, i.e. we drew new groups of larvae from a population of several hundred maintained in the laboratory. In addition, we used the mean (expressed as a proportion) of counts made at l-min intervals during the last 10 min of the trials in our statistical tests. We elected to use groups of larvae, rather than test larvae individually, because social interactions which might override responses to chemical cues occur under natural conditions. Again, by using group means in our statistical tests we avoided violating the assumption of independence. Angular transformations were made of all proportions before we ran parametric tests with SAS (Ray 1982a, b). Assumptions of normality (Shapiro-Wilk test) and homogeneity of variances (folded F-statistic or Bartlett's test) were tested and satisfied for all sets of transformed data.
Results We first used a two-tailed t-test to test the null hypothesis that the proportion of time spent by Eurycea larvae on the side of the chamber receiving treated water was 0.50. The analyses indicated a significant positive response (attraction) to brine shrimp (t = 2.5, df= 9, P = 0-03), a neutral response (t = 1.1, df= 9, P = 0.30) to green frog tadpoles, and a negative response (avoidance) to green sunfish (t=2"5, df=9, P=0'03; Fig. 2). Larvae exposed to
Figure 2. Responses of two-lined salamander larvae to water conditioned with brine shrimp (bs), green frog (gf) tadpoles and green sunfish (gs). Symbols reflect the percentage of time (mean _ 1 SE) that larvae remained on the side of the experimental chamber receiving conditioned water during sequential 5-min time intervals.
effluents from tubs holding green sunfish showed a consistent decline in the proportion of time spent in chemical-laden water. Responses to the two other treatments were relatively constant throughout the duration of the experiment. ANOVA showed significant treatment effects for the last 10 min of the trials ( F = 5.52, df= 2,27, P < 0.001), and pairwise comparison using Tukey's procedure indicated that larval responses to green sunfish were significantly different from those to green frog tadpoles and brine shrimp. The ability of Eurycea to detect brine shrimp chemically is probably greater than the data in Fig. 2 imply. As effluent began entering the experimental chamber at the 2-3 min mark of the experiment, larvae quickly moved to the side receiving conditioned water. During the 4-6 min interval, larvae spent about 69% of their time on the side receiving conditioned water ( P < 0-0001). Soon thereafter, they began to explore all areas of the chamber in a manner similar to that of laboratory stock when fed daily rations of brine shrimp. Larvae would periodically return to the side of the chamber receiving conditioned water, often orient towards the current, then continue exploring the entire chamber for food. In nine of 10 replicates, larvae spent greater than 50% of their time on the side of the chamber receiving conditioned water. Thus, it was clear that larvae responded to brine shrimp even though the magnitude of the response was not great.
Petranka et al.: Chemical detection of predators EXPERIMENTS
II A N D III
The results of experiment I indicated that larvae can detect fish chemically under simplified laboratory conditions. But can they do so in chemicallycomplex, natural habitats? We designed two experiments using tadpoles of Cope's grey treefrog, Hyla chrysoscelis, to address this question. We used Hyla tadpoles rather than Eurycea larvae because Hyla natural habitats are more amenable to experimental manipulations.
Methods
We first used a modified flow-through system in the laboratory to test the ability of tadpoles to detect fish chemically (Fig. 1B). The system and experimental protocol were similar to that of experiment I, except that the solutions flowed from tubs 3 and 4 into separate tubs (5, 6) that each contained eight tadpoles at Gosner stages 3440. Tubs 5 and 6 held a 23 x 31-cm translucent, grey Plexiglas plate supported 1.5 cm above the bottom by wood screws fastened to the corners. The plate served as a refuge and covered about 50% of the bottom surface of the tubs. We added tadpoles to the open (non-refuge) portion of the tubs immediately before initiating flow between the tubs, then recorded the number of larvae outside of refuges at intervals of 1 min for 20 min. We estimated the proportion of time that larvae spent outside of refuges from these data. As in experiment I, we used only data for the last 10 min of the trials in statistical comparisons of the control and experimental groups. We alternated the positions of the plates and tubs between replicates ( N = 5). We examined tadpole responses to chemical cues in a field experiment conducted 22-23 August 1985 in two temporary pools adjoining the Red River in Powell County, Kentucky. The pools were 2 m apart, and were apparently remnants of a larger pool that subdivided as seasonal water levels dropped. Both pools lacked fish, had silt-mud bottoms, and were relatively small (surface area less than or equal to 6 m2), and shallow (maximum depth less than or equal to 10 cm). Odonate larvae and dytiscid beetles were conspicuous invertebrate predators in both pools. We gently sank water-tight, open-bottomed plywood boxes into the bottom mud and silt, then packed mud around the outside bottom edges to minimize leakage of water from the boxes. The
423
dimensions of the boxes were the same as those of the tubs used in the laboratory experiment. We paired control and experimental boxes (N = 5) with respect to microhabitat and pool. We collected tadpoles from the pools 1 week before running the experiment, maintained them in the laboratory on trout pellets, then returned them to the pools for testing. We placed Plexiglas refuges identical to those used in the laboratory experiment into the boxes and added eight tadpoles (Gosner stages 34-40) to each of the non-refuge halves at the start of the experiment. We initially attempted to mimic the laboratory experiment by flowing Mount's 100 solutions from elevated tubs into the boxes below, but abandoned this approach because the flow caused increased turbidity that made it impossible to count larvae. Instead, we poured 0.5 litres of Mount's solution (control) or 0.5 litres of Mount's solution in which a green sunfish was placed for 10 rain (experimental) into the non-refuge half of the box at the 0 and 10 min mark of the experiment. We scored the number of larvae outside of refuges at intervals of 1 min for 20 min, but used only the last 10 rain of data in our statistical test.
Results
In laboratory trials, ttyla showed a strong negative response to effluent from tubs with fish. Experimental tadpoles spent 68% less time outside of refuges than controls during the last 10 min of the trials (paired, two-tailed t-test: t=9-3, df=3, P < 0.001; Fig. 3). Hyla reacted very quickly to fish effluent. During the first 5 min of the experiment, differences in the percentage of time spent outside of refuges nearly equalled that which occurred during the final 10 min. Experimental animals in the field showed responses that were similar in direction, but weaker in magnitude. Experimental tadpoles spent 30% less time outside of refuges than controls during the last 10 rain of the trials (paired, one-tailed t-test: t = 3 3 , dr=3, P=0.02). Variation among replicates for both controls and experimentals was nearly twice as high in the field as in the laboratory.
DISCUSSION Chemically-mediated predator avoidance occurs in a variety of vertebrate and invertebrate groups
Animal Behaviour, 35, 2
424
Hyla showed an equally strong response to Lepomis in the laboratory and quickly moved
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TIME INTERVAL (min) Figure 3. Response of grey treefrog tadpoles in the laboratory (A) and field (B) to water (c) versus water conditioned with green sunfish (gs). Symbols are the percentage of time larvae spent outside of refuge plates during sequential 5-min intervals. Probability values are for comparisons during the last 10 min of the experiment (N = 5). Note the different scales on the y-axis.
(Pfeiffer 1966, 1977; Dayton et al. 1977; Stoddart 1980), but, to our knowledge, a direct mechanism has not been previously demonstrated in amphibians. Pfeiffer (1966) demonstrated an indirect mechanism in which skin extracts of Bufo tadpoles elicit a fright reaction in conspecifics. Chemicals released from the skin following attacks by predators indirectly signal potential danger. Anosmic tadpoles do not respond to skin extracts, which indicates chemical detection via olfaction. Our data show that Eurycea larvae can discriminate between water conditioned with food, a dangerous predator, and a benign herbivore. Larvae showed no response to Rana tadpoles, but strongly avoided water conditioned with a single Lepomis. Eurycea larvae are palatable to Lepomis in the laboratory (Petranka 1983), yet the two species often coexist in small streams in central Kentucky. Thus, chemical detection of Lepomis may be an important mechanism allowing Eurycea to exploit habitats occupied by predatory fish.
beneath cover. Although our field and laboratory experiments with Hyla are not entirely comparable because of minor differences in protocol, the data suggest that responses to Lepomis are weaker in nature. This may be because larvae receive conflicting chemical stimuli from food, fish and invertebrate predators in breeding pools. We find the strong response of Hyla to Lepomis somewhat surprising because Hyla usually breeds in seasonally ephemeral pools that lack fish. Nonetheless, we have occasionally collected larvae from farm ponds that contain predatory fish. The ability to detect predators chemically is particularly advantageous for organisms whose activity patterns or environments preclude the effective use of other sensory systems (Madison 1977; Stoddart 1980). Amphibian larvae fulfil this criterion reasonably well: many species are nocturnally active, while others live in turbid habitats. An additional advantage of using chemical cues is that organisms can detect predators while remaining in refuges, that is, they can gauge predation risk without exposing themselves to danger. A major disadvantage is that chemical cues can provide erroneous information about predator presence because of information time lags. We find that small-mouthed salamander, Ambystoma texanum, larvae, for example, respond to water conditioned with green sunfish by significantlyincreasing refuge use for as long as 3 days after the fish are removed from the water (Sih, Petranka & Kats, unpublished data). Remaining in refuges long after predators vacate an area reduces opportunities for feeding and growth which, in turn, may lessen the probability of surviving to metamorphosis (Wilbur 1980). Our data help to explain how palatable amphibian larvae successfully coexist with aquatic predators (Taylor 1983; Morin, in press). We have conducted a general survey of amphibians and find that the larvae of many species avoid water conditioned with green sunfish. We do not know if larvae respond similarly to invertebrate predators, but hope to address this and other questions relating to chemically-mediated predator avoidance in the future.
ACKNOWLEDGMENTS We thank John Just and Kevin Strohmeier for help
Petranka et al.: Chemical detection o/predators with the s p e c t r o p h o t o m e t r i c analysis. T h e w o r k was s u p p o r t e d by N a t i o n a l Science F o u n d a t i o n G r a n t BSR-8500329.
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(Receized 21 February 1986; rerised 1 April 1986," MS. number: A4670)