Journal of Chemical Ecology, Vot. 15, No. 1, 1989
BEHAVIORAL RESPONSE OF SOLITARY FATHEAD MINNOWS, Pimephales promelas, TO ALARM SUBSTANCE
B.J. LAWRENCE and R.J.F. SMITH Department of Biology, University of Saskatchewan Saskatoon, Saskatchewan, Canada, S7N OWO (Received June 29, 1987; accepted November 9,'1987)
Abstract--Single fathead minnows, Pimephales promelas, were exposed to a range of concentrations of conspecific skin extract. Their responses were observed qualitatively and quantified by a computer linked to an activity meter. The response of fathead minnows to skin extract is complex, involving at least three separate types of behavior. The minnows responded over a 1000fold range of extract concentrations with combinations of dashing, freezing, slowing, and exploring. The latency of the response increased at the lowest extract concentrations, suggesting summation of sensory, cell responses. At low stimulus concentrations, a period of exploratory behavior sometimes preceded the more typical alarm responses. The active space generated by the alarm substance in 1 cm 2 of minnow skin may exceed 58,000 liters. Key Words--Schreckstoff, alarm pheromone, fathead minnow, Pimephales
promelas, fish behavior, club cells, alarm substance cells.
INTRODUCTION
Alarm substance (Schreckstoff) is released, by mechanical damage, from specialized alarm substance cells (ASCs) in the skin of ostariophysan fishes and initiates a defensive response, or "fright reaction," in the receiver (reviews: Pfeiffer, 1977; Smith, 1986). As with most fish pheromones, the exact chemical nature of the alarm substance is not known. There is developing evidence that hypoxanthine-3(N)-oxide is an important component of the alarm signal (Pfeiffer, 1982; Pfeiffer et al., 1985), but a single compound may not account for the species specificity that has been reported in the ostariophysan alarm system (e.g., Schutz, 1956). The response of the receiving fish has been reported to 209 0098-033118910100-0209506.0010 @ 1989PlenumPublishingCoq~eation
210
LAWRENCE AND SMITH
vary among species in ways that seem generally appropriate to the habits of the species (Pfeiffer, 1977). Pfeiffer et al. (1986) examined the fright reaction of 15 species and found a generalized reaction consisting of rapid swimming (possibly controlled by Mauthner cell discharge), followed by goal-directed avoidance or shelter-seeking behavior. Research on the effects of acidic pH on the response of fathead minnows to alarm substance (Smith and Lawrence, 1988) required description of the fright reaction as measured by an automatic recording system (Lemly and Smith, 1986). This description and the experiments conducted to find appropriate stimulus levels for the pH experiment provided new information on the characteristics of the fright reaction. A combination of visual observation, using video equipment, and sensitive measurement of times and distances, using a computer-based activity meter, allowed us to distinguish several components in the response of the fish to alarm substance. The constraints of the system limited the study to lone fish confined in shallow water. In nature, the response of prey animals to predators may vary with the degree of risk or vulnerability. In general, risk and distance from the predator will be inversely related. In a chemical warning system dilution will often indicate distance. We might expect that prey will respond as if predators are close at high alarm substance concentrations, and as if predators are more distant at greater dilutions of alarm substance. Similarly there may be responses that are performed as a first response and others that are more frequently the second or third step in the fright reaction, and the appropriate first response may vary with distance and hence dilution. Active space, the volume in which concentration of the stimulus exceeds the threshold of the receiver, is a key attribute in a chemical warning system. Active space is presumably an adaptive trait based on selection of sender and receiver for a combination of stimulus concentration and sensory threshold that provides adequate warning without too many unnecessary responses. The findings of this study allow the calculation of a conservative value for the active space, per surface area of skin, of fathead minnow alarm substance.
METHODS AND MATERIALS
Experimental Animals and General Handling Procedures. The majority of the fathead minnows were collected at Zelma Reservoir, approximately 70 km southeast of Saskatoon, Saskatchewan, Canada. Other fish were from Eagle Creek, approximately 100 km southwest of Saskatoon. The fish were held in temperature-controlled outdoor pools until they were used between April and August 1986. The experimental fish were moved from the outdoor pools to glass aquaria (45-60 liters) where they were sorted by size. Fish 5-cm or more in
MINNOW RESPONSE TO ALARM SUBSTANCE
21 I
fork length were retained and held for approximately two weeks at 16°C under a 12:12 light-dark photoperiod. They were fed daily with Tetramin, a commercial fish food. After this initial adjustment period, the fish were transferred to the experimental room in groups of 10-15 and held up to a week in 45-liter glass aquaria. From this holding faci!.ity, fish were transferred in pairs to a series of Plexiglas acclimating tanks and finally to the testing tank (Lemly and Smith, 1985, 1986, 1987). The fish were acclimated and tested under incandescent lighting of 55 lux as measured at the water surface, on a 12:12 light-dark cycle. Testing occurred between 0830 and 1030 hr at a mean water temperature of 23°C. To ensure that experimental fish would display relatively "undisturbed" behavior, a series of two acclimating tanks was used prior to placing the fish in the testing tank. Acclimating and testing aquaria were identical in size, water quality, and the provision of a centrally moored floating shelter. In the first acclimating tank the water depth was 8-10 cm and two fish were present. After 24 hr both fish were transferred to a second acclimating tank with water depth of 4-5 cm, the same depth as in the testing aquarium. After 24 hr the individual fish that seemed least disturbed were selected as the experimental fish and transferred into the testing tanks for the final 24 hr. The other fish was discarded. This procedure was necessary to allow the fish to adjust to being alone in shallow water. The fish were fed after each transfer. Testing aquaria, tubing, and filters were drained, thoroughly rinsed, and refilled immediately after each trial to ensure the longest possible adjustment time to the testing tank. The washing and water exchange also minimized exposure of experimental fish to alarm substance from the previous trial. After each trial the experimental fish were measured, weighed, and sexed. For the dilution experiment the overall sex ratio was 23 females (mean wt. 2.16 g) to 47 males (mean wt. 2.69 g). Activity Tracking System. The system consisted of an Opto-Varimex-Aqua tracking meter (Columbus Instruments) that surrounds a Plexiglas aquarium. This system has been described in detail by Lemly and Smith (1986). Recessed inlet and outlet manifolds on the aquarium were attached to one another by Tygon tubing that ran through a power filter and back into the test aquarium. The activity tracking meter (ATM) was interfaced to a microcomputer that integrated the digitized signal from the grid of light beams in the meter and tabulated data on position and activity of a single fish for a designated time period. At regular intervals, the computer " s c a n s " the light beams for breaks. A minimum time between beam scans of 0.125 sec was chosen after pilot testing showed that this interval was short enough to record accurately the movement of a rapidly swimming fish. Of the eight behavior parameters measured by the meters, six proved useful in assessing fathead minnow alarm responses: total distance traveled, time spent resting, number of stereotypic movements, time
212
LAWRENCE AND SMITH
spent in stereotypic activity, time spent in ambulatory movement, and total number of beam breaks. Stereotypic activity is a term used by Columbus Instruments to describe activity in which the fish breaks light beams without moving out of a " b o x " of beams of preset dimensions, one grid square. Ambulatory movement refers to movement in which the fish progresses steadily from one region of the tank to another. The total number of beam breaks per unit time provided an index of general activity. The variables measured by the ATM are not totally independent nor are they all directly correlated. Together they allow the experimenter to characterize the response of a particular species to a stimulus. In this study each trial consisted of eight identical time periods of 2.13 rain. The alarm substance stimulus was injected into the circulation system at the end of the fourth time period. A loop of the circulation system, between the power filter and the inlet manifold, passed into an adjacent room allowing a hidden observer to inject the stimulus through the tubing. Video equipment allowed the remote observer to see and record the behavioral responses of the fish as well as to simultaneously monitor the quantitative data from the ATM. Alarm Substance Preparation. Fifteen donor fish were selected, weighed, measured (fork length), and sexed immediately after capture from an outdoor pool. Mature males that might have seasonally reduced numbers of ASCs (Smith, 1973) were not used. The sex ratio of the donor fish was nine females to six males. The mean weight was 1.5 g and the mean length was 5.1 cm. A patch of skin was removed from each side of the caudal peduncle area of each fish. A small subsample was preserved in 10% formalin for histological examination and determination of ASC abundance. These samples were embedded in paraffin and 7-/zm sections were stained with periodic acid-Schiff's reagent and counterstained with hematoxylin. ASCs were counted and measured in representative sections. The relative area of ASCs and epidermis were determined using an image analyzing computer. ASCs were present in 14 of the 15 donors. A damaged sample prevented assessment of the 15th fish. The approximate total area of minnow skin used to prepare the alarm substance was 11.9 cm 2. This skin was homogenized in 100 ml of glass-distilled water (GDW) using a Polytron homogenizer and then filtered to remove scales and tissue. A portion of the stock solution was immediately frozen ( - 1 8 ° C ) in 5-ml quantities, another portion was diluted 1 part stock to 4 parts GDW and frozen in 5-ml portions. After a period of initial testing, four different dilutions were chosen for more intensive testing: (1) stock solution, (2) a 1 : 10 dilution of the stock, (3) a 1:100 dilution, and (4) a 1:1000 dilution. With the exception of the stock solution that was used as previously prepared, the solutions were made by slowly thawing (the test tube was set in a beaker of cold water and allowed to thaw at room temperature) the original 1:4 dilution, adding appropriate amounts of GDW, and refreezing in 5-ml portions. Immediately prior to use in the exper-
MINNOW RESPONSE TO ALARM SUBSTANCE
213
imental apparatus, the prepared alarm substance was slowly thawed, as above, and 1 ml was drawn into a syringe. Fifteen fish were tested at each concentration. Solutions were tested in random order to minimize effects of seasonal changes in behavior and of possible contamination. Control injections of 1 ml of GDW were also tested on 15 fish. Statistical Analyses. Data from the three time periods immediately preceding the alarm substance injection were pooled and compared with pooled data from the three time periods immediately following the stimulus using Wilcoxon's matched-pairs, signed-ranks test (Sokol and Rohlf, 1981). Data were analyzed separately for each of the six behavioral parameters for each stimulus dilution. Differences were considered to be significant at probabilities less than 0.05.
RESULTS
Description of Alarm Behavior. Video observations indicated that the most common response to the alarm substance stimulus was to become motionless, freezing, for periods ranging from approximately 0.5 min to greater than 8 min (the entire poststimulus time). The next most frequent alarm behavior was slowing, in which the fish would slow its rate of movement, e.g., less circling and nibbling under the shelter and fewer, shorter forays away from the shelter. Nibbling and circling under a shelter are common types of fathead behavior (McMillan and Smith, 1974). Dashing, the least common response, was actually the first part of a biphasic response. Several seconds to approximately 1.5 min of very rapid, apparently disoriented swimming were followed most often by the fish becoming motionless, freezing, or by the fish showing a very low level of movement, slowing. A nonalarm behavior, exploring, typified by slow, openwater swimming towards and along the inlet channel, was observed when alarm substance stimulus levels of < 0.01 were injected. Exploratory swimming lasted from slightly under a minute to just over 2 min. In six of seven cases an obvious alarm reaction, either freezing (4/6) or slowing (2/6), was observed to follow the initial exploratory behavior. A portion of the experimental population did not respond to the alarm substance stimulus; human qualitative assessment indicated 21% nonresponders while machine assessment indicated 13% (Smith and Lawrence, 1988). Effects of Stimulus Dilution on Alarm Behavior. The majority of fathead minnows were still capable of sensing and reacting to the lowest dilution of alarm stimulus tested (Table 1 and Smith and Lawrence, 1988). Exploring was observed only at the two lowest stimulus concentrations (Figure 1) and caused an initial increase in distance traveled in the first poststimulus interval in the 0.001 treatment group (Figure 2). A comparison of the last pre-
214
LAWRENCE AND SMITH
TABLE l. THE EFFECTS OF DILUTIONS OF STOCK SOLUTION ON THE BEHAVIORAL RESPONSE OF FISH TO INTRODUCTION OF SKIN EXTRACTa
Dilutions Before/ after
Stock
0.1
0.01
0.001
Control
Distance
B A
1008 280
870 327
1463 682
1099 669
869 739 "~
Time resting
B A
185 291
142 229
187 276
196 249
214 211 "~
Stereotypic time
B A
170 84
212 141
161 90
157 11
148 161 "~
Ambulatory time
B A
30 9
29 11
35 18
Stereotypic moves
B A
298 127
336 209
287 170
251 194
235 248 "~
Beam breaks
B A
972 449
1124 602
954 564
993 595
842 806"~
Parameters
30 19"~
22 20 "~
"B = before stimulus introduction. A = after stimulus introduction. Distance was measured in
centimeters, time in seconds, and moves and beam breaks were counted. Values are the mean scores of 15 fish over the three intervals (total 6.39 min) preceding stimulus introduction and the three intervals following stimulus introduction. All differences are significant (P < 0.05), except those marked "ns,'" based on a Wilcoxon matched-pairs, signed-ranks test comparing each individual fish's cumulative score before the stimulus with that fish's score after the stimulus.
stimulus interval with the first poststimulus interval indicated that both the stock and 0.1 stimuli initiated significant decreases in distance traveled (P < 0.05, W i l c o x o n m a t c h e d - p a i r s , signed-ranks test), but the difference b e t w e e n these two intervals in the 0.01 and 0.001 stimuli was not significant. T h e f r e q u e n c y o f f r e e z i n g as the first response was positively correlated with stimulus strength (P = 0 . 0 5 , S p e a r m a n rank correlation coefficient; Siegel, 1956). H o w e v e r , freezing often f o l l o w e d e x p l o r i n g so the total f r e q u e n c y o f f r e e z i n g declined only slightly. T h e r e was an o b s e r v a b l e increase in the latency o f the response as stimulus c o n c e n t r a t i o n was reduced. With stock and 0.1 dilutions, the response was initiated almost i m m e d i a t e l y after injection, w h e r e a s at 0.001 the alarm response was delayed (Figure 2). T h i s delay could be d e m o n s t r a t e d by c o m p a r i n g stereotypic activity for the last prestimulus and first poststimulus intervals. T h e t w o intervals differed significantly in the three highest dilutions (P < 0 . 0 1 , W i l c o x o n matched-pairs, signed-ranks test) but at the 0.001 dilution the difference was not significant (P > 0.05). In the 0.001 dilution the second
215
MINNOW RESPONSE TO A L A R M S U B S T A N C E
0.7
0.6
m DASHING PP .P .P .P .P .P .P .FREEZING P .P .T~ t771 SLOWING
~ ~
NO RESPONSE EXPLORING
0.5
0 "-I0.4W 03
LL
o
0.3
>¢,_) Z W 0.2 CY W n~ b_
0.1
0.0 0.001
0.01
0.1
1.0
DILUTION FACTOR Fxc. l. Proportional frequencies, as a first response, of behaviors in the post stimulus intervals after introduction of skin extract at four dilutions of the stock solution.
poststimulus interval was significantly different from the last prestimulus interval (P < 0.05). Since we know the area of skin used in preparing the stock solution and the various dilutions performed, we can estimate the active space o f alarm substance per cm 2. The original stock solution contained 11.9 cm 2 of skin diluted in 100 ml of water. That stock solution diluted to 1 : 1000 still produced a significant reaction when 1 ml was added to an ATM containing 7 liters of water. This indicates that 1 cm 2 of fathead minnow skin contains enough alarm substance to reach threshold levels in 58,823.5 liters, equivalent to a cube approximately 3.9 m on a side. Since the I : 1000 dilution was still a very effective stimulus, the real active space may considerably exceed our estimate. The initial exploring response occurred in the 0.01 dilution but not in the 0.1 dilution, indicating that the active space at which the transition from an immediate fright reaction to an initial exploration occurs presumably lies between 588 liters/cm 2 and 5882 liters/cm 2. Our measurements o f epidermal thickness (mean = 0.052 mm), cell diameter (mean 0.025 mm), and proportion of epidermis composed of ASCs (11.7%) permit a rough calculation of the amount of ASC material in 1 cm 2 of skin in our sample (6.1 x 10 -3 mm 3) and of the active space generated per average ASC (80 liters). A conservative conclusion, considering the high
216
L A W R E N C E AND S M I T H
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1
123
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> 0
• 0.0l o E.~IT~
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-
-
-m~----~
.~f~-.~,
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7
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2
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4
5
6
7
8
9
TIME PERIOD
FIG. 2. Changes in relative levels of three behavior patterns over time, before and after introduction of skin extract at four dilutions of the stock solution. The stimulus was introduced at the end of the fourth time interval (2.13-min intervals). Average activity of each test fish over the first four intervals has been indicated on the Y axis as 1.0. Arrows indicate alarm substance introduction.
MINNOW RESPONSE TO ALARM SUBSTANCE
217
variation among samples and uncertainties such as the proportion of ASCs broken by the tissue disruption process, would be that the contents of a single ASC could generate an active space of the order of 10s of liters. DISCUSSION
Lone fathead minnows exposed to skin extracts performed four general types of behavior: dashing, reduced activity (slowing and freezing), exploring, and no response. The use of lone fish was necessitated by the limitations of the activity tracking meter. Being alone may be a relatively rare situation for minnows in nature, and the behavioral responses may have been modified by the stress of being alone. Although they are schooling fish, there are some situations in which fathead minnows are alone in nature. Male fatheads are often alone on their territories during the spawning season (Unger, 1983), and females have been observed moving singly into the territories during the spawning season (Smith, personal observation). Males retain their response to alarm substance during their territorial phase (Smith, 1976). Using lone fish had the advantage of yielding a " p u r e " response to the chemical stimulus. Schooling minnows have been reported to show a fright reaction to the sight of other fish responding (Verheijen, 1956). Any response with more than one fish present may be influenced by visual as well as chemical stimuli. The dashing component of the multiphase response to alarm substance may be an escape response controlled by the Mauthner neurons, as suggested by Pfeiffer et al. (1986). The behavior we observed was consistent with his descriptions of Mauthner responses in other fishes. Sudden unpredictable dashing may be appropriate as an initial response to imminent danger. The Mauthner response has been hypothesized to be escape behavior (Eaton and Bombardieri, 1978). It can be initiated by mechanical or visual stimuli as well as by alarm substance. Freezing and slowing may follow dashing or may occur as initial responses without a preceding dashing phase. It is not clear whether freezing and slowing are two different responses or different intensities of the same response. Both may make the minnow less conspicuous but freezing is probably more effective in this regard. Similar reductions in activity are components of the fright reaction in other cyprinids such as the pearl dace, SemotiIus margarita, where they are associated with physiological indicators of stress (Rehnberg et al., 1987). The exploring behavior that occurred at low stimulus concentrations may be a response to low alarm substance concentrations or to other chemical components of the skin extract. The behavior is generally similar to the response of fathead minnows to feeding stimuli (Lemly and Smith, 1985, 1987) and the skin extract will contain amino acids and other compounds that may elicit food searching. Bardach and Todd (1970) suggested that alarm substance may be attractive in low concentrations but provided no evidence or arguments to sup-
218
LAWRENCEAND SMITH
port this idea. Our results leave the question unresolved. The answer may have to await the availability o f chemically pure alarm substance. Increased response latency at low stimulus concentration is typical of stimulus summation (Getchell, 1986). In fathead minnows increased response latency was often associated with exploring behavior, which may bring the minnow into contact with more alarm substance, speeding the summation process. Exploring may be similar to the " p r e d a t o r inspection b e h a v i o r " described by Magurran (1986) in the European minnow, Phoxinus phoxinus, behavior thought to provide the prey with information about the predator. The active space o f a semiochemical depends on attributes o f both the sender and the receiver. The sender determines the concentration o f the semiochemical through t h e amounts produced and released. In the minnow alarm substance system this is presumably adjusted through control o f the density o f ASCs in the skin. In fathead minnows ASC density is known to be under endocrine control during the breeding season. Males lose their ASCs during the breeding season in response to increased androgen levels and regain them later as androgen declines (Smith, 1973, 1974). The factors controlling ASC numbers in females and nonbreeding fish are not known. The receiver determines the threshold o f the response, either through sensory capabilities or central controlling mechanisms. Virtually nothing is known about the sensitivity of fathead minnows to chemical stimuli. The data reported here suggest that they detect and respond to low concentrations o f skin extract. Calculations based on the work of Gandolfi et al. (1968) suggest an active space of > 10,000 liters/cm 2 o f skin in the zebra danio, Brachydanio rerio, the same order o f magnitude as our findings. Such calculations of active space provide an opportunity to compare laboratory findings with subjective impressions o f the usefulness o f the alarm substance in the wild. Active spaces with dimensions of several meters, containing 10s o f thousands o f liters seem to be within the range that should be useful to a 5-cm fish. There are a number o f uncertainties in applying these findings to natural situations, including lack o f knowledge o f the amount o f skin damage caused by natural predation or o f the spatial distribution of alarm substance plumes in natural waters.
Acknowledgments--This research was made possible through a Strategic Grant and a Strategic Equipment Grant from the Natural Sciences and Engineering Research Council of Canada. We thank Norman Cappelletto and Don Hugie for their assistance in maintaining aquaria and experimental animals and conducting experiments, and Janet Yee for assistance in analyzing behavior. We are grateful to Brad Rehnberg and Dennis Lemly for reviewing the manuscript. REFERENCES BARDACN,J.E., and TODD, J.H. I970. Chemical communication in fish, pp. 205-240, in J.W. Johnston, D.G. Moulton, and A. Turk (eds.). Advances in Chemoreception, Vol. 1, Communication by Chemical Signals. Appleton-Century-Crofts, New York. EATON, R.C., and BOMBARDIERI,R.A. 1978. Behavioral functions of the Mauthner neuron, pp.
MINNOW RESPONSE TO ALARM SUBSTANCE
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221-244, in D.S. Faber and H. Korn (eds.). Neurobiology of the Mauthner Cell. Raven Press, New York. GANDOLFI, G., 1968, Reazione di paura nel ciprinide Brachydanio rerio: variaioni della reativita indotte sperimentalmente. Arch. Zool. haL, 53:245-259. GETCHELL, T.V. 1986. Functional properties of vertebrate olfactory receptor neurons. PhysioL Rev. 66:772-818. LEMLY, A.D., and SMITH, R.J.F. 1985. Effects of acute exposure to acidified water on the behavioral response of fathead minnows, Pimephales promelas, to chemical feeding stimuli. Aquat. Toxicol. 6:25-36. LEMLY, A.D., and SMITH, R.J.F. 1986. A behavioral assay for assessing effects of pollutants on fish chemoreception. Ecotox. Environ. Safety 11:210-218. LEMLY, A.D., and SMITH, R.J.F. 1987. Effects of chronic exposure to acidified water on chemoreception of feeding stimuli by fathead minnows (Pimephales promelas): Mechanisms and ecological implications. Environ. ToxicoL Chem. 6:225-238. MAGURRAN, A.E. 1986. Predator inspection behaviour in minnow shoals: Differences between populations and individuals. Behav. Ecol. SociobioL 19:267-273. MCMILLAN, V.E., and SMITH, R.J.F. 1974. Agonistic and reproductive behaviour of the fathead minnow (Pimephales promelas Rafinesque). Z. TierpsychoL 34:25-58. PFEIFFER, W. 1977. The distribution of fright reaction and alarm substance cells in fishes. Copeia 1977:653-665. PVEIFFER, W. 1982. Chemical signals in communication, pp. 306-326, in T.J. Hara (ed.). Chemoreception in Fishes. Elsever, Amsterdam. PFEIFFER, W., RIEGELBAUER,G., MEIER, G., and SCHEIBLER, B. 1985. Effect of hypoxanthine3(N)-oxide and hypoxanthne-I (N)-oxide on central nervous excitation of the black tetra Gymnoco~mbus ternetzi (Characidae, Ostariophysi, Pisces) indicated by dorsal light response, d. ChenL Ecol. l 1:507-524. PFEIFFER, W., DENOIX, M., WEHR, R., GNASS, D., ZACHERT, I., and BREISCH, M. 1986. Videotechnische Verhaltensanalyse der Screckreaction yon Ostariophysen (Pisces) und die Bedeutung des "Mauthner Reflexes." Zool. Jb. Physiol. 90:115-165. REHNBERG, B.G., SM1TH, R.J.F., and SLOLEY, B.D. 1987. The reaction of pearl dace (Pisces, Cyprinidae) to alarm substance: time-course of behavior, brain amines, and stress physiology. Can. J. ZooL 65:2916-2921. SCHUTZ, F. 1956. Vergleichende Untersuchungen uber die Schreckreaktion bei Fischen und deren Verbreitung. Z. VergL Physiol. 38:84-135. SIEGEL, S. 1956. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. SMITH, R.J.F. 1973. Testosterone eliminates alarm substance in male fathead minnows. Can. J. Zool. 51:875-876. SMITH, R.J.F. 1974. Effects of 17 alpha-methyltestosterone on the dorsal pad and tubercles of fathead minnows (Pimephales promelas). Can. J. Zool. 52:1031-1038. SMITH, R.J.F. 1976. Male fathead minnows retain (Pimephales promelas Rafinesque) retain their fright reaction to alarm substance during the breeding season. Can. J. Zool. 54:2230-2231. SMtTH, R.J.F. 1986. The evolution of chemical alarm signals in fishes, pp. 99-115, in D. Duvall, D. Muller-Schwarze, and R.M. Silverstein (eds.). Chemical Signals in Vertebrates, Vol. 4. Plenum, New York. SMrrH, R.J.F., and LAWRENCE, B.J. 1988. Effects of acute exposure to acidified water on the behavioral response of fathead minnows, Pimephales promelas, to alarm substance (Schreckstoff). Environ. Toxicol. Chem. 7:329-335. SO~AL, R.R., and ROHLF, F.J. 1981. Biometry. W.H. Freeman, San Francisco. UNGER, L.M. 1983. Nest defense by deceit in the fathead minnow, Pimephales promelas. Behav. Ecol. Sociobiol. 13:125-130 Verheijen, F.J. 1956. Transmission of a flight reaction amongst a school of fish and the underlying sensory mechanisms. F-rperientia 12:202-204.
BEHAVIORAL RESPONSE OF SOLITARY FATHEAD MINNOWS, Pimephales promelas, TO ALARM SUBSTANCE
B.J. LAWRENCE and R.J.F. SMITH Department of Biology, University of Saskatchewan Saskatoon, Saskatchewan, Canada, S7N OWO (Received June 29, 1987; accepted November 9,'1987)
Abstract--Single fathead minnows, Pimephales promelas, were exposed to a range of concentrations of conspecific skin extract. Their responses were observed qualitatively and quantified by a computer linked to an activity meter. The response of fathead minnows to skin extract is complex, involving at least three separate types of behavior. The minnows responded over a 1000fold range of extract concentrations with combinations of dashing, freezing, slowing, and exploring. The latency of the response increased at the lowest extract concentrations, suggesting summation of sensory, cell responses. At low stimulus concentrations, a period of exploratory behavior sometimes preceded the more typical alarm responses. The active space generated by the alarm substance in 1 cm 2 of minnow skin may exceed 58,000 liters. Key Words--Schreckstoff, alarm pheromone, fathead minnow, Pimephales
promelas, fish behavior, club cells, alarm substance cells.
INTRODUCTION
Alarm substance (Schreckstoff) is released, by mechanical damage, from specialized alarm substance cells (ASCs) in the skin of ostariophysan fishes and initiates a defensive response, or "fright reaction," in the receiver (reviews: Pfeiffer, 1977; Smith, 1986). As with most fish pheromones, the exact chemical nature of the alarm substance is not known. There is developing evidence that hypoxanthine-3(N)-oxide is an important component of the alarm signal (Pfeiffer, 1982; Pfeiffer et al., 1985), but a single compound may not account for the species specificity that has been reported in the ostariophysan alarm system (e.g., Schutz, 1956). The response of the receiving fish has been reported to 209 0098-033118910100-0209506.0010 @ 1989PlenumPublishingCoq~eation
210
LAWRENCE AND SMITH
vary among species in ways that seem generally appropriate to the habits of the species (Pfeiffer, 1977). Pfeiffer et al. (1986) examined the fright reaction of 15 species and found a generalized reaction consisting of rapid swimming (possibly controlled by Mauthner cell discharge), followed by goal-directed avoidance or shelter-seeking behavior. Research on the effects of acidic pH on the response of fathead minnows to alarm substance (Smith and Lawrence, 1988) required description of the fright reaction as measured by an automatic recording system (Lemly and Smith, 1986). This description and the experiments conducted to find appropriate stimulus levels for the pH experiment provided new information on the characteristics of the fright reaction. A combination of visual observation, using video equipment, and sensitive measurement of times and distances, using a computer-based activity meter, allowed us to distinguish several components in the response of the fish to alarm substance. The constraints of the system limited the study to lone fish confined in shallow water. In nature, the response of prey animals to predators may vary with the degree of risk or vulnerability. In general, risk and distance from the predator will be inversely related. In a chemical warning system dilution will often indicate distance. We might expect that prey will respond as if predators are close at high alarm substance concentrations, and as if predators are more distant at greater dilutions of alarm substance. Similarly there may be responses that are performed as a first response and others that are more frequently the second or third step in the fright reaction, and the appropriate first response may vary with distance and hence dilution. Active space, the volume in which concentration of the stimulus exceeds the threshold of the receiver, is a key attribute in a chemical warning system. Active space is presumably an adaptive trait based on selection of sender and receiver for a combination of stimulus concentration and sensory threshold that provides adequate warning without too many unnecessary responses. The findings of this study allow the calculation of a conservative value for the active space, per surface area of skin, of fathead minnow alarm substance.
METHODS AND MATERIALS
Experimental Animals and General Handling Procedures. The majority of the fathead minnows were collected at Zelma Reservoir, approximately 70 km southeast of Saskatoon, Saskatchewan, Canada. Other fish were from Eagle Creek, approximately 100 km southwest of Saskatoon. The fish were held in temperature-controlled outdoor pools until they were used between April and August 1986. The experimental fish were moved from the outdoor pools to glass aquaria (45-60 liters) where they were sorted by size. Fish 5-cm or more in
MINNOW RESPONSE TO ALARM SUBSTANCE
21 I
fork length were retained and held for approximately two weeks at 16°C under a 12:12 light-dark photoperiod. They were fed daily with Tetramin, a commercial fish food. After this initial adjustment period, the fish were transferred to the experimental room in groups of 10-15 and held up to a week in 45-liter glass aquaria. From this holding faci!.ity, fish were transferred in pairs to a series of Plexiglas acclimating tanks and finally to the testing tank (Lemly and Smith, 1985, 1986, 1987). The fish were acclimated and tested under incandescent lighting of 55 lux as measured at the water surface, on a 12:12 light-dark cycle. Testing occurred between 0830 and 1030 hr at a mean water temperature of 23°C. To ensure that experimental fish would display relatively "undisturbed" behavior, a series of two acclimating tanks was used prior to placing the fish in the testing tank. Acclimating and testing aquaria were identical in size, water quality, and the provision of a centrally moored floating shelter. In the first acclimating tank the water depth was 8-10 cm and two fish were present. After 24 hr both fish were transferred to a second acclimating tank with water depth of 4-5 cm, the same depth as in the testing aquarium. After 24 hr the individual fish that seemed least disturbed were selected as the experimental fish and transferred into the testing tanks for the final 24 hr. The other fish was discarded. This procedure was necessary to allow the fish to adjust to being alone in shallow water. The fish were fed after each transfer. Testing aquaria, tubing, and filters were drained, thoroughly rinsed, and refilled immediately after each trial to ensure the longest possible adjustment time to the testing tank. The washing and water exchange also minimized exposure of experimental fish to alarm substance from the previous trial. After each trial the experimental fish were measured, weighed, and sexed. For the dilution experiment the overall sex ratio was 23 females (mean wt. 2.16 g) to 47 males (mean wt. 2.69 g). Activity Tracking System. The system consisted of an Opto-Varimex-Aqua tracking meter (Columbus Instruments) that surrounds a Plexiglas aquarium. This system has been described in detail by Lemly and Smith (1986). Recessed inlet and outlet manifolds on the aquarium were attached to one another by Tygon tubing that ran through a power filter and back into the test aquarium. The activity tracking meter (ATM) was interfaced to a microcomputer that integrated the digitized signal from the grid of light beams in the meter and tabulated data on position and activity of a single fish for a designated time period. At regular intervals, the computer " s c a n s " the light beams for breaks. A minimum time between beam scans of 0.125 sec was chosen after pilot testing showed that this interval was short enough to record accurately the movement of a rapidly swimming fish. Of the eight behavior parameters measured by the meters, six proved useful in assessing fathead minnow alarm responses: total distance traveled, time spent resting, number of stereotypic movements, time
212
LAWRENCE AND SMITH
spent in stereotypic activity, time spent in ambulatory movement, and total number of beam breaks. Stereotypic activity is a term used by Columbus Instruments to describe activity in which the fish breaks light beams without moving out of a " b o x " of beams of preset dimensions, one grid square. Ambulatory movement refers to movement in which the fish progresses steadily from one region of the tank to another. The total number of beam breaks per unit time provided an index of general activity. The variables measured by the ATM are not totally independent nor are they all directly correlated. Together they allow the experimenter to characterize the response of a particular species to a stimulus. In this study each trial consisted of eight identical time periods of 2.13 rain. The alarm substance stimulus was injected into the circulation system at the end of the fourth time period. A loop of the circulation system, between the power filter and the inlet manifold, passed into an adjacent room allowing a hidden observer to inject the stimulus through the tubing. Video equipment allowed the remote observer to see and record the behavioral responses of the fish as well as to simultaneously monitor the quantitative data from the ATM. Alarm Substance Preparation. Fifteen donor fish were selected, weighed, measured (fork length), and sexed immediately after capture from an outdoor pool. Mature males that might have seasonally reduced numbers of ASCs (Smith, 1973) were not used. The sex ratio of the donor fish was nine females to six males. The mean weight was 1.5 g and the mean length was 5.1 cm. A patch of skin was removed from each side of the caudal peduncle area of each fish. A small subsample was preserved in 10% formalin for histological examination and determination of ASC abundance. These samples were embedded in paraffin and 7-/zm sections were stained with periodic acid-Schiff's reagent and counterstained with hematoxylin. ASCs were counted and measured in representative sections. The relative area of ASCs and epidermis were determined using an image analyzing computer. ASCs were present in 14 of the 15 donors. A damaged sample prevented assessment of the 15th fish. The approximate total area of minnow skin used to prepare the alarm substance was 11.9 cm 2. This skin was homogenized in 100 ml of glass-distilled water (GDW) using a Polytron homogenizer and then filtered to remove scales and tissue. A portion of the stock solution was immediately frozen ( - 1 8 ° C ) in 5-ml quantities, another portion was diluted 1 part stock to 4 parts GDW and frozen in 5-ml portions. After a period of initial testing, four different dilutions were chosen for more intensive testing: (1) stock solution, (2) a 1 : 10 dilution of the stock, (3) a 1:100 dilution, and (4) a 1:1000 dilution. With the exception of the stock solution that was used as previously prepared, the solutions were made by slowly thawing (the test tube was set in a beaker of cold water and allowed to thaw at room temperature) the original 1:4 dilution, adding appropriate amounts of GDW, and refreezing in 5-ml portions. Immediately prior to use in the exper-
MINNOW RESPONSE TO ALARM SUBSTANCE
213
imental apparatus, the prepared alarm substance was slowly thawed, as above, and 1 ml was drawn into a syringe. Fifteen fish were tested at each concentration. Solutions were tested in random order to minimize effects of seasonal changes in behavior and of possible contamination. Control injections of 1 ml of GDW were also tested on 15 fish. Statistical Analyses. Data from the three time periods immediately preceding the alarm substance injection were pooled and compared with pooled data from the three time periods immediately following the stimulus using Wilcoxon's matched-pairs, signed-ranks test (Sokol and Rohlf, 1981). Data were analyzed separately for each of the six behavioral parameters for each stimulus dilution. Differences were considered to be significant at probabilities less than 0.05.
RESULTS
Description of Alarm Behavior. Video observations indicated that the most common response to the alarm substance stimulus was to become motionless, freezing, for periods ranging from approximately 0.5 min to greater than 8 min (the entire poststimulus time). The next most frequent alarm behavior was slowing, in which the fish would slow its rate of movement, e.g., less circling and nibbling under the shelter and fewer, shorter forays away from the shelter. Nibbling and circling under a shelter are common types of fathead behavior (McMillan and Smith, 1974). Dashing, the least common response, was actually the first part of a biphasic response. Several seconds to approximately 1.5 min of very rapid, apparently disoriented swimming were followed most often by the fish becoming motionless, freezing, or by the fish showing a very low level of movement, slowing. A nonalarm behavior, exploring, typified by slow, openwater swimming towards and along the inlet channel, was observed when alarm substance stimulus levels of < 0.01 were injected. Exploratory swimming lasted from slightly under a minute to just over 2 min. In six of seven cases an obvious alarm reaction, either freezing (4/6) or slowing (2/6), was observed to follow the initial exploratory behavior. A portion of the experimental population did not respond to the alarm substance stimulus; human qualitative assessment indicated 21% nonresponders while machine assessment indicated 13% (Smith and Lawrence, 1988). Effects of Stimulus Dilution on Alarm Behavior. The majority of fathead minnows were still capable of sensing and reacting to the lowest dilution of alarm stimulus tested (Table 1 and Smith and Lawrence, 1988). Exploring was observed only at the two lowest stimulus concentrations (Figure 1) and caused an initial increase in distance traveled in the first poststimulus interval in the 0.001 treatment group (Figure 2). A comparison of the last pre-
214
LAWRENCE AND SMITH
TABLE l. THE EFFECTS OF DILUTIONS OF STOCK SOLUTION ON THE BEHAVIORAL RESPONSE OF FISH TO INTRODUCTION OF SKIN EXTRACTa
Dilutions Before/ after
Stock
0.1
0.01
0.001
Control
Distance
B A
1008 280
870 327
1463 682
1099 669
869 739 "~
Time resting
B A
185 291
142 229
187 276
196 249
214 211 "~
Stereotypic time
B A
170 84
212 141
161 90
157 11
148 161 "~
Ambulatory time
B A
30 9
29 11
35 18
Stereotypic moves
B A
298 127
336 209
287 170
251 194
235 248 "~
Beam breaks
B A
972 449
1124 602
954 564
993 595
842 806"~
Parameters
30 19"~
22 20 "~
"B = before stimulus introduction. A = after stimulus introduction. Distance was measured in
centimeters, time in seconds, and moves and beam breaks were counted. Values are the mean scores of 15 fish over the three intervals (total 6.39 min) preceding stimulus introduction and the three intervals following stimulus introduction. All differences are significant (P < 0.05), except those marked "ns,'" based on a Wilcoxon matched-pairs, signed-ranks test comparing each individual fish's cumulative score before the stimulus with that fish's score after the stimulus.
stimulus interval with the first poststimulus interval indicated that both the stock and 0.1 stimuli initiated significant decreases in distance traveled (P < 0.05, W i l c o x o n m a t c h e d - p a i r s , signed-ranks test), but the difference b e t w e e n these two intervals in the 0.01 and 0.001 stimuli was not significant. T h e f r e q u e n c y o f f r e e z i n g as the first response was positively correlated with stimulus strength (P = 0 . 0 5 , S p e a r m a n rank correlation coefficient; Siegel, 1956). H o w e v e r , freezing often f o l l o w e d e x p l o r i n g so the total f r e q u e n c y o f f r e e z i n g declined only slightly. T h e r e was an o b s e r v a b l e increase in the latency o f the response as stimulus c o n c e n t r a t i o n was reduced. With stock and 0.1 dilutions, the response was initiated almost i m m e d i a t e l y after injection, w h e r e a s at 0.001 the alarm response was delayed (Figure 2). T h i s delay could be d e m o n s t r a t e d by c o m p a r i n g stereotypic activity for the last prestimulus and first poststimulus intervals. T h e t w o intervals differed significantly in the three highest dilutions (P < 0 . 0 1 , W i l c o x o n matched-pairs, signed-ranks test) but at the 0.001 dilution the difference was not significant (P > 0.05). In the 0.001 dilution the second
215
MINNOW RESPONSE TO A L A R M S U B S T A N C E
0.7
0.6
m DASHING PP .P .P .P .P .P .P .FREEZING P .P .T~ t771 SLOWING
~ ~
NO RESPONSE EXPLORING
0.5
0 "-I0.4W 03
LL
o
0.3
>¢,_) Z W 0.2 CY W n~ b_
0.1
0.0 0.001
0.01
0.1
1.0
DILUTION FACTOR Fxc. l. Proportional frequencies, as a first response, of behaviors in the post stimulus intervals after introduction of skin extract at four dilutions of the stock solution.
poststimulus interval was significantly different from the last prestimulus interval (P < 0.05). Since we know the area of skin used in preparing the stock solution and the various dilutions performed, we can estimate the active space o f alarm substance per cm 2. The original stock solution contained 11.9 cm 2 of skin diluted in 100 ml of water. That stock solution diluted to 1 : 1000 still produced a significant reaction when 1 ml was added to an ATM containing 7 liters of water. This indicates that 1 cm 2 of fathead minnow skin contains enough alarm substance to reach threshold levels in 58,823.5 liters, equivalent to a cube approximately 3.9 m on a side. Since the I : 1000 dilution was still a very effective stimulus, the real active space may considerably exceed our estimate. The initial exploring response occurred in the 0.01 dilution but not in the 0.1 dilution, indicating that the active space at which the transition from an immediate fright reaction to an initial exploration occurs presumably lies between 588 liters/cm 2 and 5882 liters/cm 2. Our measurements o f epidermal thickness (mean = 0.052 mm), cell diameter (mean 0.025 mm), and proportion of epidermis composed of ASCs (11.7%) permit a rough calculation of the amount of ASC material in 1 cm 2 of skin in our sample (6.1 x 10 -3 mm 3) and of the active space generated per average ASC (80 liters). A conservative conclusion, considering the high
216
L A W R E N C E AND S M I T H
2.5 0 Z
L.IJ '~
1.5
>
•
~
he" 0.5
/
•
..---~'
_
I
I
J
1
2
3
STOCK
a ~
t
•
0 . 0 1 Sol,~
o
~.o~,~
I
I
I
I
I
I
4
5
6
7
8
9
10
A / \ .=~
..(
W Z
.oo,~,,
\oo,
1
123
n," I.I
0
I
I
I
1
2
3
t I
t
I
I
I
I
4
5
6
7
8
9
1.2
> 0
• 0.0l o E.~IT~
• STOCK .
-
-
-m~----~
.~f~-.~,
1 0 h--
a o.lso~
7
- -
0.8
0.6 l,
12) d 0.4 Z L.I_I > 0.2 0
I
I
I
1
2
3
t I
I
I
I
I
I
4
5
6
7
8
9
TIME PERIOD
FIG. 2. Changes in relative levels of three behavior patterns over time, before and after introduction of skin extract at four dilutions of the stock solution. The stimulus was introduced at the end of the fourth time interval (2.13-min intervals). Average activity of each test fish over the first four intervals has been indicated on the Y axis as 1.0. Arrows indicate alarm substance introduction.
MINNOW RESPONSE TO ALARM SUBSTANCE
217
variation among samples and uncertainties such as the proportion of ASCs broken by the tissue disruption process, would be that the contents of a single ASC could generate an active space of the order of 10s of liters. DISCUSSION
Lone fathead minnows exposed to skin extracts performed four general types of behavior: dashing, reduced activity (slowing and freezing), exploring, and no response. The use of lone fish was necessitated by the limitations of the activity tracking meter. Being alone may be a relatively rare situation for minnows in nature, and the behavioral responses may have been modified by the stress of being alone. Although they are schooling fish, there are some situations in which fathead minnows are alone in nature. Male fatheads are often alone on their territories during the spawning season (Unger, 1983), and females have been observed moving singly into the territories during the spawning season (Smith, personal observation). Males retain their response to alarm substance during their territorial phase (Smith, 1976). Using lone fish had the advantage of yielding a " p u r e " response to the chemical stimulus. Schooling minnows have been reported to show a fright reaction to the sight of other fish responding (Verheijen, 1956). Any response with more than one fish present may be influenced by visual as well as chemical stimuli. The dashing component of the multiphase response to alarm substance may be an escape response controlled by the Mauthner neurons, as suggested by Pfeiffer et al. (1986). The behavior we observed was consistent with his descriptions of Mauthner responses in other fishes. Sudden unpredictable dashing may be appropriate as an initial response to imminent danger. The Mauthner response has been hypothesized to be escape behavior (Eaton and Bombardieri, 1978). It can be initiated by mechanical or visual stimuli as well as by alarm substance. Freezing and slowing may follow dashing or may occur as initial responses without a preceding dashing phase. It is not clear whether freezing and slowing are two different responses or different intensities of the same response. Both may make the minnow less conspicuous but freezing is probably more effective in this regard. Similar reductions in activity are components of the fright reaction in other cyprinids such as the pearl dace, SemotiIus margarita, where they are associated with physiological indicators of stress (Rehnberg et al., 1987). The exploring behavior that occurred at low stimulus concentrations may be a response to low alarm substance concentrations or to other chemical components of the skin extract. The behavior is generally similar to the response of fathead minnows to feeding stimuli (Lemly and Smith, 1985, 1987) and the skin extract will contain amino acids and other compounds that may elicit food searching. Bardach and Todd (1970) suggested that alarm substance may be attractive in low concentrations but provided no evidence or arguments to sup-
218
LAWRENCEAND SMITH
port this idea. Our results leave the question unresolved. The answer may have to await the availability o f chemically pure alarm substance. Increased response latency at low stimulus concentration is typical of stimulus summation (Getchell, 1986). In fathead minnows increased response latency was often associated with exploring behavior, which may bring the minnow into contact with more alarm substance, speeding the summation process. Exploring may be similar to the " p r e d a t o r inspection b e h a v i o r " described by Magurran (1986) in the European minnow, Phoxinus phoxinus, behavior thought to provide the prey with information about the predator. The active space o f a semiochemical depends on attributes o f both the sender and the receiver. The sender determines the concentration o f the semiochemical through t h e amounts produced and released. In the minnow alarm substance system this is presumably adjusted through control o f the density o f ASCs in the skin. In fathead minnows ASC density is known to be under endocrine control during the breeding season. Males lose their ASCs during the breeding season in response to increased androgen levels and regain them later as androgen declines (Smith, 1973, 1974). The factors controlling ASC numbers in females and nonbreeding fish are not known. The receiver determines the threshold o f the response, either through sensory capabilities or central controlling mechanisms. Virtually nothing is known about the sensitivity of fathead minnows to chemical stimuli. The data reported here suggest that they detect and respond to low concentrations o f skin extract. Calculations based on the work of Gandolfi et al. (1968) suggest an active space of > 10,000 liters/cm 2 o f skin in the zebra danio, Brachydanio rerio, the same order o f magnitude as our findings. Such calculations of active space provide an opportunity to compare laboratory findings with subjective impressions o f the usefulness o f the alarm substance in the wild. Active spaces with dimensions of several meters, containing 10s o f thousands o f liters seem to be within the range that should be useful to a 5-cm fish. There are a number o f uncertainties in applying these findings to natural situations, including lack o f knowledge o f the amount o f skin damage caused by natural predation or o f the spatial distribution of alarm substance plumes in natural waters.
Acknowledgments--This research was made possible through a Strategic Grant and a Strategic Equipment Grant from the Natural Sciences and Engineering Research Council of Canada. We thank Norman Cappelletto and Don Hugie for their assistance in maintaining aquaria and experimental animals and conducting experiments, and Janet Yee for assistance in analyzing behavior. We are grateful to Brad Rehnberg and Dennis Lemly for reviewing the manuscript. REFERENCES BARDACN,J.E., and TODD, J.H. I970. Chemical communication in fish, pp. 205-240, in J.W. Johnston, D.G. Moulton, and A. Turk (eds.). Advances in Chemoreception, Vol. 1, Communication by Chemical Signals. Appleton-Century-Crofts, New York. EATON, R.C., and BOMBARDIERI,R.A. 1978. Behavioral functions of the Mauthner neuron, pp.
MINNOW RESPONSE TO ALARM SUBSTANCE
219
221-244, in D.S. Faber and H. Korn (eds.). Neurobiology of the Mauthner Cell. Raven Press, New York. GANDOLFI, G., 1968, Reazione di paura nel ciprinide Brachydanio rerio: variaioni della reativita indotte sperimentalmente. Arch. Zool. haL, 53:245-259. GETCHELL, T.V. 1986. Functional properties of vertebrate olfactory receptor neurons. PhysioL Rev. 66:772-818. LEMLY, A.D., and SMITH, R.J.F. 1985. Effects of acute exposure to acidified water on the behavioral response of fathead minnows, Pimephales promelas, to chemical feeding stimuli. Aquat. Toxicol. 6:25-36. LEMLY, A.D., and SMITH, R.J.F. 1986. A behavioral assay for assessing effects of pollutants on fish chemoreception. Ecotox. Environ. Safety 11:210-218. LEMLY, A.D., and SMITH, R.J.F. 1987. Effects of chronic exposure to acidified water on chemoreception of feeding stimuli by fathead minnows (Pimephales promelas): Mechanisms and ecological implications. Environ. ToxicoL Chem. 6:225-238. MAGURRAN, A.E. 1986. Predator inspection behaviour in minnow shoals: Differences between populations and individuals. Behav. Ecol. SociobioL 19:267-273. MCMILLAN, V.E., and SMITH, R.J.F. 1974. Agonistic and reproductive behaviour of the fathead minnow (Pimephales promelas Rafinesque). Z. TierpsychoL 34:25-58. PFEIFFER, W. 1977. The distribution of fright reaction and alarm substance cells in fishes. Copeia 1977:653-665. PVEIFFER, W. 1982. Chemical signals in communication, pp. 306-326, in T.J. Hara (ed.). Chemoreception in Fishes. Elsever, Amsterdam. PFEIFFER, W., RIEGELBAUER,G., MEIER, G., and SCHEIBLER, B. 1985. Effect of hypoxanthine3(N)-oxide and hypoxanthne-I (N)-oxide on central nervous excitation of the black tetra Gymnoco~mbus ternetzi (Characidae, Ostariophysi, Pisces) indicated by dorsal light response, d. ChenL Ecol. l 1:507-524. PFEIFFER, W., DENOIX, M., WEHR, R., GNASS, D., ZACHERT, I., and BREISCH, M. 1986. Videotechnische Verhaltensanalyse der Screckreaction yon Ostariophysen (Pisces) und die Bedeutung des "Mauthner Reflexes." Zool. Jb. Physiol. 90:115-165. REHNBERG, B.G., SM1TH, R.J.F., and SLOLEY, B.D. 1987. The reaction of pearl dace (Pisces, Cyprinidae) to alarm substance: time-course of behavior, brain amines, and stress physiology. Can. J. ZooL 65:2916-2921. SCHUTZ, F. 1956. Vergleichende Untersuchungen uber die Schreckreaktion bei Fischen und deren Verbreitung. Z. VergL Physiol. 38:84-135. SIEGEL, S. 1956. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. SMITH, R.J.F. 1973. Testosterone eliminates alarm substance in male fathead minnows. Can. J. Zool. 51:875-876. SMITH, R.J.F. 1974. Effects of 17 alpha-methyltestosterone on the dorsal pad and tubercles of fathead minnows (Pimephales promelas). Can. J. Zool. 52:1031-1038. SMITH, R.J.F. 1976. Male fathead minnows retain (Pimephales promelas Rafinesque) retain their fright reaction to alarm substance during the breeding season. Can. J. Zool. 54:2230-2231. SMtTH, R.J.F. 1986. The evolution of chemical alarm signals in fishes, pp. 99-115, in D. Duvall, D. Muller-Schwarze, and R.M. Silverstein (eds.). Chemical Signals in Vertebrates, Vol. 4. Plenum, New York. SMrrH, R.J.F., and LAWRENCE, B.J. 1988. Effects of acute exposure to acidified water on the behavioral response of fathead minnows, Pimephales promelas, to alarm substance (Schreckstoff). Environ. Toxicol. Chem. 7:329-335. SO~AL, R.R., and ROHLF, F.J. 1981. Biometry. W.H. Freeman, San Francisco. UNGER, L.M. 1983. Nest defense by deceit in the fathead minnow, Pimephales promelas. Behav. Ecol. Sociobiol. 13:125-130 Verheijen, F.J. 1956. Transmission of a flight reaction amongst a school of fish and the underlying sensory mechanisms. F-rperientia 12:202-204.
