Effects of early feeding experience on the responses of garter snakes to food chemicals

.EAHNING

Effects

.iNIl

MOTIVATION

of Early

2,

Feeding

of Garter JANNON

(1971)

L.

271-279

Experience

Snakes FUCHS’

AND

University

to Food GORDON

on the

Responses

Chemicals’ M.

BURGHARDT:’

of Chicago

A litter of newborn garter snakes (Thamnophis sirtaks) was divided into two groups. In the first eight feedings, one group was fed redworms and the other group was fed guppies. For the next eight feedings the diets were reversed with each group receiving the food it had not been fed previously. Prior to any feeding the snakes were tested with water extracts of these foods and two related prey (nightcrawler and minnow) which initially elicited the same response strengths as redworm and guppy. The tests were repeated after each diet. In addition, all snakes were tested on a short test series of redworm and guppy extracts after every two feedings. Each group showed an increasing responsivity to chemical extracts from the food fed during the first period although responses to these extracts tended to return to their original levels during the second period. Changes in responsivity to nightcrawler and minnow extracts did not parallel those to redworm or guppy extracts in that they steadily decreased in effectiveness throughout the experiment. The snakes were, therefore, capable of discrininating chemical cues of related prey organisms that initially have similar releasing valnes.

When a previously unfed neonatal garter snake (Thamnophis sirt&s) is presented with a cotton swab dipped in the water extract of a prey animal, the snake responds initially by orienting toward the swab and flicking its tongue. Some animal extracts lead to a prey-attack response which is characterized by the snake’s opening its jaws and striking at the swab (Burghardt, 1966). This response is mediated by the Jacobson’s organ (Wilde, 1938; Burghardt & Hess, 1968). The tongue serves to transport food chemicals to the paired Jacobson’s organ, which opens into the anterior portion of the roof of the oral cavity. ’ This research was supported by an NSF Undergraduate Research Participation Program Grant CY-3095 to J. L. Fuchs, and NIMH Grants MH-13375 and MH15707 to C. M. Burghardt. Requests for reprints should be sent to Cordon hl. Rurghardt. Department of Psychology, University of Tennessee, Knoxville, Ten,>. 37916. 1%‘~ thank Dr. Robert Tsutakawa for his help in the statistical analysis 01 the data and Edward Lace and the other naturalists of the Cook County Forest Preserve System (Palos Division) for the gravid snakes. ’ Now at Massachusetts Institute of Technology. J No\\, at the University of Tennessee, Knoxville. 271

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In comparative studies with previously unfed snakes, Burghardt (1967a, 1969) s h owed that different species may respond differentially to the same series of prey extracts. Such differences correspond to the normal food preferences shown by the species. Since naive snakes were involved in the comparative studies, it would appear that these chemical preferences are innate. However, unmodifiable food preferences could be potentially disastrous to a predatory species. The question remains as to what experiences, if any, can alter the prey extract preferences of newborn garter snakes. Burghardt and Hess (1968) tested part of a litter of newborn Butler’s garter snakes (T. butleri) to obtain response profiles to a series of prey extracts. They then force-fed the remaining naive snakes with strained liver, a substance which the species will not eat. At the end of 2 or 6 months, the response profiles for these subjects were essentially unchanged from snakes tested at birth. Therefore, in the absence of normal feeding experience, prey chemical preferences in these snakes are quite stable. Snedigar (1963) reports that garter snakes can be trained to accept raw beef by mixing it with earthworms and gradually reducing the proportion of worm to zero over a number of meals. The junior author has replicated this procedure using worm extract rather than pieces of whole worm. Adult and newborn garter snakes then readily attack beef extract presented on cotton swabs. Some adult snakes and a few newborn snakes ( T. sirtalis) will, however, eat the chopped beef without any prior exposure to beef mixed with prey extract. Thus an increased responsivity has been observed toward a substance initially low in effectiveness after association with a prey extract which readily elicits prey attacks in neonatal snakes. The present experiment was designed to systematically investigate how feeding experience can alter responses to extracts of foods which garter snakes have eaten. We were also interested in whether such changes are accompanied by similar changes in responses to extracts of related prey which are excluded from their diets. Our interest in this question stems from the fact that worm or fish extracts frequently cluster together in their effectiveness in newborn snakes of this and other species, indicating that a common chemical cue may be involved. If this is true, then changes induced toward redworm, for example, should be accompanied by parallel changes in responsivity toward nightcrawler, even if excluded from the diet. The reliance of snakes on the chemical senses, however, would lead US to predict that they would discriminate related prey if it was made meaningful for them to do so. In addition, three concentrations of the extracts were used to increase the chance of detecting poss:ble changes in response thresholds to extracts. “Food imprinting,” as measured by the greater effect on food prefer-

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“3

ences of early over later feeding, has been reported in turtles (Burghardt & Hess, 1966; Burghardt, 1967b). Using the extract technique, the present experiment might reveal whether such a phenomenon would generalize to another reptilian group.

A litter of 22 garter snakes (T. .sirtuIis se??Gf&sci&r) was born in captivity to a female captured in Cook County, Ill. Shortly after birth they were weighed, measured, and housed individually in r&s tanks (23 x 14 x 17 cm) covered with plate glass. The tanks were placed on a white surface and were surrounded by white partitions SO that each snake was visually isolated. Plastic water dishes were provided. The temperature of the room ranged from 22” to 26”.

Testing Procedure The procedure for making extracts and testing the snakes was similar to that used by Burghardt ( 1969). T we 1ve extracts were prepared from the skin substances of four prey animals: (a) two species of wormredworm ( Eisenia foetidu) and nightcrawler ( Lumbricus terretiris) ; ( b) two species of fish-guppy (Poecilfa reticzhta) and minnow (Notropis

atherinoidees acutus). Before preparing extracts, a small number of each prey species wer( rinsed in tap water and blotted dry between sheets of absorbent paper. The animals were then weighed to the nearest 0.1 g. The worm extracts were prepared by immersing the worm in 50” distilled water for 1% min, stirring occasionally. A ratio of 3 g of worms/20 ml of II,0 was used. In preparing fish extracts, 6 g of prey/20 ml of water and 3-min stirring time were employed so that the initial effectiveness of all extracts would be nearly equal. After the elapsed period, the animals were removed and the resulting extracts were centrifuged at 2500 rpm for 10 min and the supernatants decanted. Portions of each extract were diluted to make solutions of one-tenth and one-hundredth of the original strengths. Several milliliters of each extract were stored in glass vials and kept frozen when not in use. Fresh extracts were prepared every 2 weeks. Before being used in testing, extracts were allowed to warm to room temperature. In testing a subject a cotton swab was dipped in the extract, rubbed on the side of the glass vial to remove excess liquid, and then brought to a position about 2 cm in front of the snake’s head. If the snake did not attack the swab within 30 set, the swab was touched gently to its snout. If there was 110 attack within the remainder of the 66set test period.

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the number of tongue flicks during this period was recorded. If the snake opened its jaws to attack the swab within the 60-set period, the swab was quickly removed before the attack was completed, and the attack latency was recorded to the nearest 0.1 sec. Extracts were tested according to random sequences assigned to each snake. Every snake was tested on the first extract in its sequence before the experimenter proceeded to the next round of testing. For any individual approximately 35 min elapsed between successive tests given on any single day. Feeding

and Testing

Schedules

During a 4-day period beginning on the 6th day after birth (and prior to any feeding experience), each snake was tested twice with three concentrations each of redworm, guppy, nightcrawler, and minnow extracts and a distilled H,O control. The 13 stimuli were each tested once on the 6th and 7th days and again on days 8 and 9. Either six or seven stimuli were tested on a given day. The snakes were then separated into two groups of 11 subjects each such that average response scores for the two groups were nearly equal. The division was made in this way so that later differences between the groups would be primarily a function of differential treatment with minimal influences from differences in initial chemical responsivities. The snakes were fed once every 4 days beginning with the 10th day after birth. For the first eight feedings, group I received redworms and group II received guppies. On each feeding day every snake received two pieces of food weighing a total of .30 t .02 g. The prey animals were killed by immersion in 55” water before being placed in the tanks. After the first eight feedings, the foods given each group were switched for an additional eight feedings. Group I received guppies and group II received redworms. After the first and second sets of eight feedings, all subjects were again tested twice on H,O and the three concentrations of the four prey extracts (long test series ) . These extracts were coded so that the experimenter could not identify them during testing. On the day prior to alternate feeding days, beginning with the Ist, each snake was tested once on distilled H?O, once on undiluted redworm extract, and once on undiluted guppy extract. This short test series was included so that the course of any chemical responsivity changes could be followed. RESULTS The method used to score extract effectiveness was developed by Burghardt (1967a). Since Jacobson’s organ is stimulated via action

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was considered a measure of of the tongue, tongue flick frequency arousal by, or interest in, a given test swab in the absence of an actual attack. For a test in which there was no attack, the score was simply the number of tongue flicks for that 60-set test period. If the snake attacked, its score for that test was 60 (the maximum number of seconds) minus the attack latency, plus the “base unit” which was the highest number of tongue flicks without an attack given by any snake on any test in a given series, The formula for attacking snakes can be represented by: score = base unit + (60 - response latency). Thus an attack is considered a stronger response than any number of tongue flicks alone, and a short attack latency indicates a stronger response than an attack with a long latency. In computing average attack scores for the worm and fish extracts for group I and group II, the water control scores were subtracted. Since fluctuations in the magnitude of average control and extract scores from one test day to another were roughly parallel, subtracting control scores should reduce some of the variability caused by factors unrelated to specific food preferences. Mean scores, which include 8 subjects from group I and 10 subjects from group II, do not include two snakes which escaped during the experiment and two snakes from

60 50 40 30 i

I *

1

012341234 Y Food

v A fed TEST

Food

B lad

PERIOD

FIG I. Comparison of tongue flick-attack scc~es of group I and group redworm and guppy extracts. Test period O-prior to feeding. Subsequent periods occurred after every second meal. Group I-Food A -~ redwornl, I3 -= guppy. Group II-Food A = guppy, Food R := redworm.

I1 to test I~ootl

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group I which refused the new food. One snake refused fish for 20 days after the diets were switched, and the other refused the new food for 53 days and then apparently died of starvation. Figure 1 shows a comparison of group I and group II responses to guppy and redworm extracts. These averages were computed for each test day in the short test series. Throughout the first half of the experiment, each group showed an increasingly stronger response to the extract of its own food as compared to the other group’s response to the same extract. Responsivities tended to return to their original levels during feeding on the new foods. During the first diet, redworm scores of group I became significantly higher than those of group II (p < .025 after 4 meals, decreasing to p < 601 after eight meals, MannWhitney U test, one-tailed), while guppy scores of group II became significantly higher than those of group I (p < .OW after six meals). Differences were no longer statistically significant after the first two meals of the second diet. GROUP

M

5

0

i y

60-

3 g

so-

? Y’

40-

* 5

JO-

z

20-

5: Y

Redwarm Nightcrarlrr

(, GROUP

IO 0

I

[I

y

Initial (no feeding)

Mid (food A) TEST

Final (foods A b 6)

PERIODS

FIG. 2. Tongue flick-attack scores of both groups to undiluted redworm nightcrawler extracts as related to diet. Group IDiet A = redworm, Diet guppy. Group II-Diet A = guppy, Diet B = redworm.

and B =

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3;;

E:XI’I’l-IIESCl~

results for the long test scrics comparing the rcdworm and guppy extracts arc shown in extracts with the nightcrawlrr and minnow Figs, 2 and 3. For each feeding period there was an incrcasc in responsivity to extracts of prey included in a group’s diet, and a decrcasc in responsivity to extracts of prey which had never bcc~ included in the group’s diet. This result is in agreement with the findings from thrk short test series. Thus changes in responses to undiluted rtdworm and guppy extracts did not parallel those of undiluted nightcrawler and minIlo\% extracts, respectively. The Wilcoxon matched-pair signed-rank test (onctailed) was used to test for divergence between responses to both pairs of extracts shown in Figs. 2 and :3. For each subject the difl’(rences between the average scores of both worm and fish extract pairs were compared with differences between these scores before and after the relevant prey had been fed. In Fig. 2 the overall worm scor(‘s are given. Group I scores diverged in favor of redworm at the em1 of the redworm feeding period (p < 425). Group II worm scores also The

GROUP

I /

H

GUPPY Mimow

GROUP

I

I

Initial (no feeding)

Mid (food A) TEST

FIG. extracts. guppy,

II

1

Final (foodsA &B)

PERIODS

3. Tongue flick-attack scores of each group to undiluted guppy and Group I-Diet A = redworm, Diet B = puppy. C:roup II-Divt Diet B = redworm.

u~innow .4 :-_

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after the snakes were significantly diverged in favor of redworm switched from guppy to redworm ( p < .025). In the fish scores given in Fig. 3, group I scores diverged in favor of guppy after the animals were switched from redworm to guppy, while the minnow extract effectiveness continued to decline (‘p < ,025). The difference scores for group II fish extracts did not quite reach significance when the initial scores were compared with the midpoint scores, but were significant when compared with the final scores (p < .005). However, the guppy scores were significantly higher than the minnow scores after the first period (p = .O5, Wilcoxon signed-rank matched-pair tests, one-tailed). Because of the scarcity of attacks to weaker extracts, these scores were based almost solely on tongue flick frequencies, which are more variable than attack data, and therefore not as amenable to analysis with the number of subjects utilized in this experiment. DISCUSSION

It may be concluded that the responses of newborn snakes to food chemicals were influenced by feeding experience. Although the reversibility of the extract scores obtained does not support a food imprinting hypothesis, it should be noted that two snakes refused the new food and hence were not used in the above analysis (Fig. 1). These two subjects may have been imprinted or “fixated” on the food first experienced. Indeed, this experiment may not provide an adequate test for food imprinting since the snakeswere exposed to more than one food chemical during the first feeding period. The observed increase in responsivity to the extract of a food present in a subject’s diet suggests the possibility that snakesmay develop chemically based “search images” dependent upon prior encounters with prey. This is compatible with the suggestion that animals such as birds may become more efficient in searching for prey through the development of visual search images associated with certain foods (see Hinde, 1970). The differences which developed between redworm and guppy scores within eight feedings show that the snakes were able to distinguish between redworm and guppy extracts. The significant divergences found in the extract pairs in Figs. 2 and 3 indicate that the snakes were also discriminating between the two worm extracts and between the two fish extracts. Since we did not have comparison groups fed on nightcrawler and minnow, the validity of this conclusion depends on the assumption that extracts made throughout the experiment did not vary significantly in composition. Precise control of extract concentration can not yet be attained. Only preliminary studies of the effective chemical cues have been reported (Sheffield, Law, & Burghardt, 196S).

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\\Tc suggest that a similar paradigm could be used to further study the chemical discriminations of snakes. Known chemicals could even be added to a food (i.e., redworm), and the snake’s response to a similarly adulterated and normal extracts compared to determine discrimination of known chemicals without elaborate odor discrimination procedures. Little research has been done on chemical discrimination in reptiles despite the fact that much of the behavior of many reptiles is guided by chemical cues. Electra-physiological recording studies from the olfactory apparatus of a few species of amphibians and reptiles and have resulted in differential responses to various reagent chemicals (e.g., Gesteland, Lettvin. Pitts, & Rojas, 1963; Tucker, 1963). However, behavioral correlates of these observations are lacking, and the relevance of such findings to discrimination of food substances remains to be determined. REFERENCES BUWHARDT, C. M. Stimulus control of the prey attack response in naive gal trl snakes. Psychonomic Science, 1966, 4, 3738. BURGHARDT, G. M. Chemical-cue preferences of inexperienced snakes; comparative aspects. Science, 1967, 157, 718-721. (a) BUHGHARDT, G. M. The primacy effect of the first feeding experience in the snapping turtle. Psychonomic Science, 1967 7, 383384. (b) BUIEHAKDT, G. M. Comparative prey-attack studies in newborn snakes of the grnus Thamnophis. Behaviour, 1969, 33, 77-114. BUHGHARDT, G. M., & HESS, E. H. Food imprinting in the snapping turtle, Chelydm serpentinu. Science, 1966, 151, 108-109. BURGHARDT, G. M., & HESS, E. H. Factors influencing the chemical release of prey attack in newborn snakes. Journal of Comparative and Physiological Psychology, 1968, 66, 289-295. GESTELAND, R. C., LETTVIN, J. Y., PITTS, W. H., & ROJAS, A. Odor specificities of the frog’s olfactory receptors. In Y. Zotterman (Ed. ), Olfaction and taste: First internutional symposium On olfaction unrl taste. New York: Macmillan. 1963. Pp. 19-34. HINDE, R. A. Animal behaviour. (2nd ed.), New York: McGraw-Hill, 1970. SHEFFIELD, L. P., LAW, J. M., & BURGHARDT, G. hl. On the nature of chemical food sign stimuli for newborn snakes. Communications in Behavioral. Biology, 1968, Z(A), 7-12. SNEDICAR, R. Our small native anzmu~: their habits and care. New York: Dover, 1963. TUCKER, D. Physical variables in the olfactory stimulation process. Jo~ml of General Physiology, 1963, 46, 453489. WILDE, W. S. The rob of Jacobson’s organ in the feeding reaction of the common garter snake, Thamnophis sirtulis sirtalis ( Linn. ), Jollrnal of Experimental Zoology, 1938, 77, 445-465. (Received

March

23,

1970)