Role of the tongue and senses in feeding of naive and experienced garter snakes

Physiology and Behavior, Vol. 14, pp. 185-194. Brain Research Publications Inc., 1975. Printed in the U.S.A.

Role of the Tongue and Senses in Feeding of Naive and Experienced Garter Snakes GORDON M. BURGHARDT AND CHERYL H. PRUITT 2

Department o f Psychology, University o f Tennessee Knoxville, Tennessee 37916

(Received 3 April 1974)

BURGHARDT, G. M. AND C. H. PRUITT. Role of the tongue and senses in feeding of naive and experienced garter snakes. PHYSIOL. BEHAV. 14(2) 185-194, 1975. - Prey attack behavior was studied in two species of garter snakes (Thamnophis sirtalis and T. radix). Newborn, ingestively naive, and experienced snakes had their tongues severed surgically, while control groups retained their tongues. Attack latency, tongue flick frequency and an orientationinterest measure were recorded for each subject on responses to extracts prepared from species-characteristic prey. Feeding, as well as responses to prey extracts, were found to be suppressed almost totally in the tongueless naive snakes. A detongued adult, however, readily ate although its behavior was abnormal. Temporary blind and anosmic conditions did not have a significant effect on response rates of the tongueless or control groups. While importance of the tongue-Jacobson's organ system is demonstrated, the length of tongue removed and presurgery experience are important factors. Chemical senses

Tongue

Olfaction

Feeding

Garter snake

Jacobson's organ

would not eat. Noble and Clausen [7] report that their garter snakes with cauterized Jacobson's organs ate normally. In each case the authors did not report the previous feeding histories of their subjects. Wilde fed his snakes individually with forceps; the conditions in Noble and Clausen are unspecified but probably involved grouped animals. It has been also reported that if the tongue is severed trailing ability in a number of species is impaired [3] and the chemically mediated defense posture of rattlesnakes to king snakes is abolished [2]. The experiments reported here direct themselves to the question of the role of the tongue in the perception of prey. Does the absence of the tongue disrupt the animal's response to prey or chemical cues from prey? Does tongue removal further back than the fork have more detrimental effects than lesser amputation? Does removal of the tongue have greater, lesser, or equivalent effects on young experienced and inexperienced snakes? The elimination of visual and olfactory senses appears to have no significant effect on the frequency of prey attacks in naive newborn plains garter snakes (Thamnophis r. radix} [5]. These experiments strongly implicated the tongue-Jacobson's organ system via elimination of alternative sensory channels. But, by not interrupting the system itself, direct involvement was not demonstrated, regardless of its plausibility. In order to answer these questions, we studied the

THE chemical senses are of great importance in the behavior of reptiles, especially snakes (see review in [3 ] ). In addition to gustation and olfaction, snakes possess another chemical receptor, Jacobson's organ, a pair of sacs which open into the anterior roof of the mouth and contain typical olfactory cells in the epithelial lining of the sacs [10]. Tongue flicking delivers chemical information to Jacobson's organ while passing over the paired openings [ 1,12]. Taste seems unimportant in prey attack. Wilde [12] discovered that Eastern garter snakes (Thamnophis s. sirtalis} would emit open-mouthed attacks to cotton wads dipped in aqueous extracts of earthworm substances. If the snakes' olfactory nerves were severed, the attack on earthworm extracts persisted; however, cutting the vomeronasal nerves leading from the accessory olfactory bulb to Jacobson's organ abolished the response, as did severing the olfactory tract. He concluded that prey attack was more dependent on Jacobson's organ than on olfaction. To test the view that the forked tongue might actually be inserted into the sac openings of Jacobson's organ, Wilde progressively removed sections of the tongue up to the fork. Response to prey was impaired but not abolished. Wilde concluded that the tongue certainly aided in transporting chemical information to Jacobson's organ, but was not critical to the process. Additionally, he reported that snakes with severed vomeronasal nerves

1This study was supported in part by research grant MH 15707 from the National Institute of Mental Health to G.M.B. We thank Doris Carey and Lori Burghardt for typing and editing, respectively.

Now at Northern Kentucky State College, Highland Heights, Kentucky. 185

186

BURGHARDT AND PRU1TT

behavior of young newborn garter snakes. Newborn garter snakes typically respond to chemical releasing stimuli from normally eaten prey animals [3]. The use of ingestively naive snakes eliminates possible conditioning effects on the food preferences prior to testing and allows us to assess their role in later behavior. In an attempt to delineate the perceptual basis of prey attack, newborn snakes in these experiments were presented with extracts of normally ingested prey. The experimental groups were surgically rendered tongueless, while control groups retained their tongues. In some tests, both experimental and control snakes were also subjected to reversible blind and/or anosmic conditions to assess the role of sensory channels other than Jacobson's organ. Different concentrations of prey extracts were also employed to detect threshold differences. GENERAL METHOD

Animals Newborn snakes were from litters borne by gravid, wild-caught females. Except for the first experiment using Thamnophis r. radix, all the newborn young were born to gravid female T. s. sirtalis obtained from Midwest Reptile Co., Ft. Wayne, Ind. Shortly after birth the young were weighed, measured, and isolated in glass aquaria measuring 23 × 14 × 17 cm. Each tank was placed on white shelf paper and the four outside walls were covered with white partitions. A small plastic water dish was present within each tank. Plate glass tops covered each tank except during testing periods. Temperatures in the air conditioned room were maintained at 2 4 - 2 7 ° C ( 2 4 - 2 5 ° C during testing). The room was lighted by sunlight and fluorescent fixtures.

Tongue and Sense Elimination Procedures Members of each litter were randomly assigned to sham and surgical groups. To cut the tongue, the snake was held slightly back of its neck area under a dissecting scope. The investigator's fingers were placed such that a slight pressure on the underside of the throat caused the tongue tips to protrude. Viewed through a dissecting scope, the tongue tips were gently pulled away from the animal with tissue forceps. While the tongue was extended another person carefully cut the tongue with a scalpel (Experiment I only) or surgical tissue scissors. The guideline for the incisions was a small vein which crossed the tongue at the junction of the white and red areas (see Fig. 1). After cutting, each tongue was measured and placed in a 10% Formalin solution. Sham controls received essentially identical treatment, but without an incision. The tongue was held out from the mouth for 10 sec and then allowed to retract intact. In the first group the amount of tongue removed was considerably less than for the later groups (Table 1). Additionally, the behavioral decrements observed were not as great as expected and more complete tongue removal was called for. The weight and length of the newborn snakes was not significantly correlated with the total length of tongue removed (Table 2). However, the length of the removed tongue did correlate with both the length of the tongue tips (i.e., length to fork) and, to a lesser extent, to the length into the white base of the tongue. Blind and anosmic conditions were produced by covering the eyes and/or nostrils with a mixture of collodion and

JoR rE!.

LACK

RED

/HITE

FIG. 1. The tongue in garter snakes showing the color divisions and the vein used as a guideline during surgery. carbon black powder following an earlier developed procedure [5]. A fine artist's brush was used to paint a double coating of the collodion mixture on the eyes and nostrils approximately 25 and 20 rain before testing. The covering was removed easily with forceps after testing and feeding, thus making these conditions reversible. In the animals not made blind and/or anosmic, a patch of the collodion mixture was placed on each lateral side of the snout between the eye and nostril.

Extract Preparation Except for Experiment 1, nightcrawter (NC), Lumbricus terrestris, was the only prey species used. Chemical surface substance extracts were prepared by first rinsing a small quantity of the prey with water which was then carefully dried and weighed to the nearest tenth of a gram. Distilled water in the proportion of 20 cc of distilled water to 6 gm of prey animal was heated to 64°C and the prey was placed in the warm water and stirred for one minute (holding the water temperature at 60°C). After prey removal the resulting liquid was centrifuged at 3000 rpm for I0 m and the supernatant poured into small glass scintillation bottles, labeled, and placed in a freezer for storage. Dilutions of these extracts to 10, 1,0.1 and 0.01 percent were prepared with distilled water and used in Experiments 2, 3, and 4.

Extract Tests Frozen extracts were thawed and brought to room temperature each day shortly before testing. Control bottles contained distilled water. The plate glass cage tops were removed 10 rain before testing and left off the tanks until the end of each day's test session. Commercial 15 cm cotton swabs were used to present the stimuli to the" snakes. A swab was dipped into the extract or control, the excess flicked off, and slowly introduced into the tank to within 1 - 2 cm of the snake's snout. The snake was typically in a resting position, with little body or tongue movement. With the introduction of a worm swab, however, a normal animal's tongue flicks usually sharply increased and it sometimes lunged toward the swab in attack. As the snake lunged, the experimenter withdrew the swab. The tongue flicks emitted d u r i ~ a test were recorded, both total flicks and those directed at the

187

T O N G U E A N D S E N S E S IN F E E D I N G O F G A R T E R S N A K E S TABLE 1 MEASUREMENTS (ram) OF TONGUES REMOVED FROM SNAKES IN ALL EXPERIMENTS Experiment

No.

Total Length

Length to Fork

Length into White*

1

5

'X range S.D.

7.5 7.2 - 8.0 0.45

3.4 2.5 - 4.0 0.58

1.8 0.5 - 2.5 0.74

2

12

.X range S.D.

12.0 11.0 - 14.8 0.93

7.0 6.2 - 9.8 0.94

2.4 1.5 - 5.0 0.89

3

14

.X range S.D.

12.4 11.0 - 13.8 0.71

7.5 6.2 - 8.5 0.59

2.5 1.5 - 3.2 0.54

4

11

"X range S.D.

11.6 11.0 - 12.2 0.33

6.7 6.0 - 7.2 0.44

2.1 1.5 - 2.8 0.28

*Length into red area in Experiment 1. TABLE 2 SPEARMAN

RANK CORRELATIONS OF TONGUE MEASUREMENTS T O LENGTHS AND WEIGHTS OF SNAKES IN ALL EXPERIMENTS

SNOUT-VENT

Total Tongue Length and Experiment

Snake Weight

1

0.18

2 3 4

0.22

Snout-Vent Length

Tongue Length to Fork

Distance Severed into White

-0.10

1.00+

N.A.

0.02

-0.30

0.93+

0.65"

0.10

-0.27

0.94+

0.95+

0.24

0.82+

0.55 *

*p<0.05 +p<0.01

swab; o n l y t h e l a t t e r were used in t h e analyses p r e s e n t e d here. W h e n a snake a t t a c k e d t h e test t e r m i n a t e d a n d t h e l a t e n c y was r e c o r d e d . . If n o a t t a c k o c c u r r e d t h e swab was r e m o v e d at t h e e n d o f 30 sec. Tests w i t h w o r m and c o n t r o l swabs i n t e r m i x e d were r u n d o u b l e blind ( E x p e r i m e n t 1). A c o n s t a n t intertrial interval was used in each r o u n d o f testing ( E x p e r i m e n t 1: 15 r a i n ; E x p e r i m e n t 2 : 2 0 m i n ; Experim e n t s 3 a n d 4 : 1 0 rain). A n a d d i t i o n t o t h e testing p r o c e d u r e was i n c o r p o r a t e d a f t e r E x p e r i m e n t 1 to p r o v i d e a n a d d i t i o n a l m e a n s o f c o m p a r i n g tongueless a n d c o n t r o l animals, since t o n g u e flicking is n o t available to tongueless subjects. In earlier e x p e r i m e n t s , p r e p a r a t o r y m o v e m e n t s b e f o r e swab a t t a c k s were o b s e r v e d in n o r m a l snakes. Most o b v i o u s was a h e a d o r i e n t a t i o n t o t h e swab. If t h e s n a k e m o v e d a w a y f r o m t h e swab, it generally e x h i b i t e d a lesser n u m b e r o f t o n g u e flicks; t h e vast m a j o r i t y o f t o n g u e flicks were d i r e c t e d t o w a r d s t h e swab w i t h t h e head d i r e c t l y facing t h e swab.

A n I n t e r e s t Score was m e a s u r e d b y recording the time e a c h snake, tongueless o r c o n t r o l , s p e n t w i t h his h e a d o r i e n t e d t o w a r d t h e swab. If t h e a n i m a l m o v e d or t u r n e d away f r o m the swab, t i m i n g s t o p p e d and r e s u m e d o n l y w h e n its head was r e o r i e n t e d t o w a r d the swab. In E x p e r i m e n t s 1 and 2 t h e swab was slowly m o v e d closer t o t h e s u b j e c t ' s s n o u t and a c t u a l l y t o u c h e d it o n c e if n o tongue-flicks or interest was s h o w n w i t h i n t h e first 15 sec. This was an a t t e m p t t o s t i m u l a t e o r a r o u s e a possibly resting or sleeping snake (snakes have n o eyelids and sleep is difficult, if n o t impossible, t o d e t e r m i n e f r o m overt behavior). S u c h p r o c e d u r e s h a d little, if any, effect a n d were d i s c o n t i n u e d in Experim e n t s 3 a n d 4. A c o r r e c t i o n o f t h e i n t e r e s t score was m a d e for all animals w h i c h a t t a c k e d t h e swab. If a snake a t t a c k e d b e f o r e t h e end o f t h e 3 0 sec test, its i n t e r e s t score m i g h t b e s h o r t e r t h a n an aroused snake t h a t did n o t a t t a c k . In o r d e r to c o m p e n s a t e for this bias, all i n t e r e s t scores for snakes w h i c h

1~8

BURGHARDT AND PRUITT TABLE 3 SUMMARY OF AGE OF SNAKES 1N DAYS 1N RELATION TO FEEDING (F) TESTING (T) AND SURGERY (S) SCHEDULES IN ALL FOUR EXPERIMENTS

Experiment

1

Age 10(T) 11 (F) 14(F) 17(F) 20 (TF) 24 (F) 29(S) 34 (TF) 38 (F) 43 (F) 48 (F) 53 (TF) 58 (TF) 63 (TF) 68 (TF) - 312 (T) 4(S) 8(T) 9(T) 10(T) !l (TF) 2 (S) 8 (Y) 9 (TF) 6iS) 10(T) 12(TF) 17(TF) 22(TF)

attacked were corrected by the formula: 30 sec - latency of attack + interest time. This procedure also introduces other, hopefully smaller, biases.

Feeding Tests Unless otherwise noted, each animal was fed small (2 cm) sections of dead (hot water killed) nightcrawler in the center of each tank immediately following all extract testing sessions. During such feedings, any nostril and eye patches remained in place. The worm pieces were placed in the center of the tank after a cardboard partition was introduced between the snake and the tank center, thereby preventing observation of the food placement. Each snake was given 30 min to find and eat the worm. If the worm was eaten within this period, another piece of worm was presented in the same manner 5 min after the previous worm was eaten. If the worm was not eaten within the 30 min period, it was presented 1 - 2 cm in front of the snake's jaws with forceps. If no attack occurred in 60 sec, the worm was moved in closer until it actually touched the jaws. If still no attack occurred, the worm was returned to the center of the tank and remained there overnight at which time uneaten worms were removed. A summary of the four experiments is shown in Table 3. EXPERIMENT 1

This experiment evaluated the effect of tongue removal, blinding, and anosmia on the prey attack and feeding behavior of young snakes with limited prior feeding and extract experience.

Method The animals were 10 newborn eastern plains garter snakes, Thamnophis r. radix, born in captivity to a gravid female caught in Cook County, Illinois. Preoperative testing. All animals were fed 6 times and tested twice with extracts before surgery. At the time of first testing, the animals were 10 days old and had no previous feeding experience. At 10 days each snake was tested 3 times with swabs dipped in NC extract and 3 times with distilled H20 utilizing an alternating order with H20 first. The following day each snake was fed a piece of NC presented with a forceps. This feeding was repeated twice at 3 day intervals. After another 3 days, the snakes were tested again on NC extract, H20, and also with a fish

extract prepared in the same manner (Carrasius auratus). The 3 stimuli were ordered randomly and each snake was tested on 3 such series. After testing each animal was fed 2 pieces of NC with forceps. The feedings were repeated twice at 4 day intervals. At the first repetition each snake was fed 4 pieces of NC, the following feeding each received 3 pieces of NC. The snakes were matched on the basis of scores from the two initial testing sessions, giving experimental and control groups of 5 snakes each. Tongue surgery was performed on the 5 experimental animals at age 29 days, and sham procedures were performed on the controls at the same time. Postoperative testing. Each group of 5 animals was subjected to 5 conditions, in the same order and on identical days for each group. Each sham (N) and tongueless (T) snake received control (C), blind (B), anosmic (A), anosmic and blind tAB), and control (C) test conditions in that order, on 5 separate test days. The first test session was 5 days after surgery and included the fish extract as well as NC and H20. In other words, this was an exact replication of the second preoperative testing. After testing, all snakes were offered 3 pieces of NC presented with forceps and all ate at least 2. Four days later the snakes were fed again using the method for postoperative feeding described under General Methods. This feeding procedure was repeated twice at 5 day intervals. The second test session under condition B occurred 5 days later. The standard NC extract and distilled water were randomly presented to each animal on swabs 3 times per test day, for a total of 6 trials per session per snake. The A, AB, and C conditions were similarly run at subsequent 5 day intervals. After each session the animals were fed in the standard manner. About 8 months later, at 3 1 2 d a y s of age, the 5 normal and 4 surviving tongueless snakes were retested on the NC and H20 extracts 3 times each in an identical fashion as the last session of the postsurgery period.

Results and Discussion Only 3 attacks were given to the goldfish extract by the sham snakes before and after surgery, while 3 attacks were given by the other group before surgery and none after surgery. A larger number of attacks were given to nightcrawlers extract by each group throughout the experiment (Table 4). Although the tongueless group attacked the swab

189

TONGUE AND SENSES IN FEEDING OF GARTER SNAKES TABLE 4 ATTACKS (PERCENT) GIVEN TO HIGHTCRAWLEREXTRACT UNDER VARIOUS CONDITIONS BY SNAKES IN EXPERIMENT 1 Prior to Surgery

Post Surgery Tests

8 Months Later

Group

Control

Control

Blind

Anosmie

Blind and Anosmic

Control

Control

Sham

80

80

47

53

47

47

93

Tongueless

87

7

27

33

27

53

100

TABLE 5 NUMBER OF SNAKES EATING OR NOT EATING IN ALL EXPERIMENTS Experiment (No. of Feeding Sessions)

Animals Eating During Sessions

Animals Not Eating During Any Session

Experiment t (5) Sham Tongueless

5 5

0 0

Experiment 2 (3) Sham Tongueless

8 1

4 11

Experiment 3 (3) Sham Tongueless

7 2

7 12

Experiment 4 (3) Sham Tongueless

12 1

0 11

hardly at all on the first postsurgery session, they did begin to approach sham attack rates on the second session. By the end of testing, the tongueless animals' attack rate was equal to that of the controls. There is a highly significant difference between the postsurgery sessions. A Friedman two-way analysis of variance [9] on the number of attacks to nightcrawler extract showed a significant difference over the five postsurgery days for both tongueless (Xr 2 = 78, d f = 4, p<0.001) and normal (Xr 2 = 85, df = 4, p<0.001) groups regardless of condition. There was no significant difference between groups in total attacks except on the first day of testing (p<0.01, binomial test). These results seem due to the suppressed responding of tongueless snakes on the first day of testing and the enhanced responsivity of normals on the same date. The results for normal snakes closely parallel those for rat snake neonates (Elaphe vulpina) tested repeatedly [4]. The recovery of the tongueless animals may be partially due to their ability to adapt to the use of a shorter, but still protrusible organ. During the first two test days, the tongueless animals frothed at the mouth area during presentation of the swab and some showed a stub. By the

third day all tongueless animals extended a minute stub of tongue along with a bit of froth. This observation, teamed with the high attack rate of tongueless snakes under the final control condition, indicates that the blind and anosmic conditions may have depressed the increasing effect of visible tongue stubs. Average lengths of tongue cut were shorter for this litter (Table I) than in the subsequent experiments. Feeding habits of each group were nearly identical (Table 5). After surgery, both tongueless and control groups ate over 90 percent of the time. B, A, and AB conditions may have depressed the tongueless snakes' attack scores, but did not affect their feeding behavior. The attack scores about 40 weeks after surgery were over 90 percent for both groups indicating complete recovery as far as this test procedure is concerned (Table 4). EXPERIMENT 2 The results of Experiment 1 left several unanswered questions. The presence of tongue stubs that could extend outside the mouths of the experimental group prevented an

190 evaluation of their necessity. If part of the tongue had not been present during testing, would those animals still have exhibited prey attacks to the swab? Although he presented no data, Wilde [12] claimed that a completely tongueless adult snake would attack if the prey chemicals came in contact with its lips. On the other hand, the prey attacks of tongueless snakes may be at least partially attributed to prior feeding experiences and resultant sensory conditioning. This experiment investigated tongueless and sham animals prior to any feeding experience under control, anosmic, blind, and blind plus anosmic conditions. Method The animals were 24 eastern garter snakes, T. s. sirtalis, randomly selected from a litter of 29. Tongue surgery or sham treatment was performed on 12 snakes each at age four days. All tongues were cut as far behind the small red vein crossing the white area of the tongue as possible (Fig. 1). No tongue stubs were seen in any animals in subsequent testing and feeding in this and subsequent experiments. Nostril and eye patches were placed and removed using the standard collodion and lampblack method. The standard neightcrawler extract and 4 dilutions were used giving 5 solutions with strengths of 100, 10, 1.0, 0.1 and 0.01 percent extract. Each animal was tested 5 times every test day on each of the 5 extract dilutions. The concentrations were presented in descending order. Latency of attacks, tongue flicks, and interest scores were recorded. Each snake was tested under each of the 4 conditions on 4 consecutive days, beginning four days after surgery. The order of the conditions was different for each tongueless and sham snake, with one in each group receiving the same sequence of conditions. First feeding occurred upon completion of the fourth day of testing. Further testing was not carried out as a skin infection afflicted several members of the litter in both groups.

BURGHARDT AND PRUITT TABLE6 MEAN INTEREST SCORES IN SECONDS TO VARIOUS CONCENTRATIONS OF NIGHTCRAWLER EXTRACT AS A FUNCTION OF AVAILABLE SENSE ORGANS

Group C B A AB T TA TB TAB

100% 8.5 8.3 7.0 5.8 2.9 6.2 5.5 3.3

Concentration 10% 1%

O.1%

0.01%

10.2 10.3 6.6 9.1 0.6 0.8 1.9 2.1

2.9 4.9 4.0 3.2 0.0 0.2 0.3 0.0

3.5 5.0 2.8 2.1 0.0 0.0 0.0 0.4

4.2 8.3 7.7 4.5 0.4 2.0 0.0 0.0

C = Control Shams, A = Anosmic, B = Blind, T = Tongueless. TABLE 7 PRODUCT-MOMENT CORRELATIONS OF INTEREST AND TONGUE-FLICK SCORES FOR SHAM SNAKES IN EXPERIMENT 2 Extract Concentration 1.0% 0,1% 0.01%

Condition

100%

10%

X r

C

0.62*

0.81+

0.86+

0.80+

0.89+

0.80

B

0.76+

0.85+

0.89+

0.82+

0.77+

0.82

A

0.92+

0.62*

0.90+

0.97+

0.96+

0.87

AB

0.94+

0.73+

0.98+

0.94+

0.94+

0.90

,X r

0.77

0.75

0.91

0.88

0.89

Results and Discussion Tongueless snakes in this experiment did not attack the swab at any time. Shams did attack but at a relatively low rate, which may be partially attributed to the skin infection. However, the difference in attack rates between controls and tongueless groups to 100% NC extract is striking. There were 16 attacks by the controls to the 100% NC extract. Blinding and/or anosmia apparently had little or no effect on attacks (C: 3; A: 4; B: 4, AB: 5). There were 8 attacks to the 10% extract, one to the 1% and none to the lesser dilutions. As seen in Table 6, mean interest scores for each group also reflect the effect of the tongue on prey attack. A split plot analysis of variance on interest scores to 100% NC showed a significant difference between tongueless and sham animals across conditions, F(1,22) = 13.85, p<0.01. No differences were found, however, between the A, B, AB, or C conditions for either sham or tongueless animals, F(3,66) = 0.164. With all the dilutions the interest scores of the tongueless snakes were greatly reduced. Since tongue-flicks are the usual recorded measure of interest short of an attack and since the orientation measure is a postulated measure of interest in tongueless animals, it is necessary to ask how these are related in snakes with tongues. Table 7 shows the product-moment

*p<0.05 +p<0.01 C = Control, B = Blind, A = Anosmic.

correlations of orientation and tongue-flicks at swab for all conditions and concentrations in the sham animals. Since all the correlations were substantial (>0.60) and statistically significant, it appears that the one is indeed a good substitute for the other. During the feeding sessions after testing, only one of the 12 tongueless animals ate (Table 5). In contrast, 8 of the 12 control animals ate during 3 feeding periods. This experiment demonstrated that tongue removal in naive snakes can seriously suppress feeding as well as responses to prey extracts. EXPERIMENT 3 Although there was a significant difference between interest scores of control ~nd experimental groups in Experiment 2, illness forced a premature end to the postfeeding testing. These tongueless snakes did show an absence of prey attack responses which, coupled with their refusal to eat, corroborate Wilde's findings. It was the

TONGUE AND SENSES IN FEEDING OF GARTER SNAKES

191

purpose of Experiment 3 to further replicate these findings.

0~ extract,

However, the blind conditions (B and AB) were eliminated as having little effect.

)NO--~100*/,extract

)Die ~ends test

Method The animals were 28 newborn T. s. sirtalis, from a litter of 29. Surgery was performed at age 2 days, employing the surgical methods of Experiment 2, half being detongued and half being sham controls. First testing to extracts began at age 8 days, with no previous feeding or swab exposure. Randomly assigned anosmic and normal conditions were administered to the animals using the standard collodion and lampblack method; 7 tongueless and 7 normals were assigned to each condition on each of two test days in counter balanced fashion. The same 5 concentrations were used as in Experiment 2. They were, however, presented in an u p - d o w n sequencing to avoid problems of overtesting and accompanying habituation effects (see Fig. 2). In effect this was a stair step version of the method of limits. The snake was always given the 100% NC extract first. No attack to the initial 100% extract meant a second presentation of that concentration to the snake. An absence of an attack to the second presentation of 100% extract ended that snake's testing for the day. If he attacked that concentration, he was next presented with the 10% dilution. An attack to 10% extract, forwarded the snake on to 1.0% extract, while no attack to 10% meant another test with 100%. End of testing for each snake was determined when it attacked an extract more concentrated than one it did not attack or when it did not attack a given extract twice in succession (regardless of response to interpolated extracts). First feeding occurred immediately following the second test day.

1 ° % .... $ 0,1%

~L

Yes

extract ~No . . . . . . . -~ends test ~ _

0~01*/.extract ~No FIG. 2. Scheme of the up-down presentation of extract concentration used in Experiments 3 and 4. No attacks were made to 0.01% extract. Dashed arrows reflect pathways following a second no attack response. TABLE8 INTEREST SCORES AND TONGUE FLICK-INTEREST SCORE CORRELATIONS OF ANIMALS IN EXPERIMENT 3 TO 100% NC EXTRACT (NUMBER OF PAIRS IN PARENTHESES) Mean Condition

Interest Time (sec)

Correlation

C

10.8

0.88 (28)*

A

10.6

0.82 (29)*

T

2.1

TA

1.3

Results and Discussion The tongueless snakes did not attack the extract swabs whereas a total of 11 attacks were given by shams under control conditions and 4 attacks under anosmic conditions. Attack rates were low, however, and were made only on the first day of testing indicating rapid habituation in the 4 snakes that did attack. All but one of the attackers responded to dilutions; one each responding down to 10, 1, and 0.1 percent. The interest scores reflect a significant difference between the sham and tongueless animals (Table 8). In the control condition the shams responded significantly more than the tongueless animals (t = 3.00, d f = 26, p<0.01); the same result is obtained for the anosmic condition (t = 3.80, d r = 26, p<0.001). There was no significant difference between anosmic and control conditions for either the sham (dep. t = ].00, d f = 13) or tongueless conditions (dep. t = 0.095, d f = 13) although in both groups the scores in the anosmic condition averaged slightly lower. This again indicates that the tongue has a greater influence on interest in prey than functioning nostrils. Table 8 also shows that, as in Experiment 2, there is a high correlation between orientation time and tongue flicks at swab.

Further substantiating Wilde's earlier research, tongueless animals characteristically did not eat during feeding sessions (Table 5). The 2 animals which took food did so only when the nishtcrawler was presented to them with forceps and touched their snout. The remainder of the

*p<0.01

tongueless animals, which did not eat, were eventually force fed to maintain their health. In the sham group only 7 (50%) ate during the feeding sessions. Based on previous research in our laboratory, the shams' scores on feeding and extract testing reflect below normal responsivity, although differences between litters in general extract responsivity as well as in the effectiveness of specific prey extracts have been shown [3]. However, even in this situation the absence of tongues proved to significantly reduce prey finding abilities in those animals. EXPERIMENT 4 Because of the low level of attack response by the snakes in Experiment 3, another litter was studied in an identical fashion. In addition, the effects of feeding experience on

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response to prey extracts in initially naive snakes were evaluated in an a t t e m p t to compare the results with the more experienced snakes in E x p e r i m e n t 1. Me th od

Animals in this experiment were an entire litter of 22 garter snakes. N o n e of the snakes were fed or received test experience before tongue surgery, which was p e r f o r m e d at age 6 days. Surgery e m p l o y e d the m e t h o d of E x p e r i m e n t 2. First testing to extract occurred at age 10 days, with no exposure to food or extract in the four day interim between surgery and initial testing. All snakes were r a n d o m l y assigned anosmic or normal conditions in balanced groups. T w o days later each snake was tested in the other condition. A f t e r the second testing, each animal was fed. All animals were tested again under either anosmic and normal conditions five days after feeding. They were then fed and all snakes were tested under the other c o n d i t i o n (A or C) after another five days except for one control subject that inexplicably died prior to the final test. There was no use of force feeding until c o m p l e t i o n of the fourth testing or after the second feeding. Nightcrawler extract and dilutions were presented as in E x p e r i m e n t 3.

F

tal ¢w 0 ot5 o3 I---

T

TONGUELESS

kl

kl~O&J

At

1

la a:~O Iz m

z < w

5

0

N A T T-A BEFORE FEEDING

N A T T-A AFTER FEEDING

FIG. 3. Interest scores before and after food exposure by snakes in Experiment 4. TABLE 9 TOTAL NUMBER OF ATTACKS TO EXTRACT CONCENTRATIONS IN EXPERIMENT 4

Results and Discussion

The tongueless group did not give attack responses to the swab, either before or after feeding; however the sham group did attack the swab, under b o t h normal and anosmic conditions (Table 9). Attacks were given to all but the least concentrated extract. If anything, the anosmic snakes responded more o f t e n to the lower concentrations, indicating no detrimental effects due to anosmia. Interest scores to the extract (Fig. 3) reflect similar results: the tongueless group exhibited little interest ('X range = 0 sec - 1.1 sec), while the control group maintained a fairly consistent interest in the swab (X range = 12.5 - 17.8 sec). U n d e r anosmic conditions sham interest is lowered slightly, but not significantly so, F ( 1 , 2 0 ) = 1.24. Split plot analyses of variance for b o t h before and after feeding interest scores show a significant difference between tongueless and control scores in b o t h cases (before: F(1,20) = 24.84, p < 0 . 0 1 ; after: F(1,20) = 16.42, p < 0 . 0 1 ) . The interest scores of tongueless animals actually decreased after exposure to food. Habituation was conceivably responsible. In the sham animals there were again substantial correlations b e t w e e n orientation and tongue flicks (Table 10). During the feeding sessions only one tongueless snake ate (Table 5) yielding a 3 percent feeding rate for the experimental group. The 11 controls however, ate 91 percent of the possible times. No controls required force feeding, as did the tongueless animals. The one tongueless snake who ate encountered the nightcrawler in mid tank and in doing so, its lips brushed across the worm. In further feeding tests it subsequently refused food. GENERAL DISCUSSION Taken together the present experiments establish several facts about the sensory control of appetitive, prey attack, and feeding behavior in garter snakes. (1) Blinding has little effect on any of the behaviors

Concentration 1% 0.1%

Condition

100%

10%

0.01%

C

20

14

3

0

0

A

14

17

6

1

0

T

0

0

0

0

0

TA

0

0

0

0

0

T A B L E 10 CORRELATION BETWEEN INTEREST (ORIENTATION) TIME SCORE AND TONGUE-FLICKS TO SWAB TO THE MOST EFFECTIVE EXTRACTS IN EXPERIMENT 4. ALL WERE S I G N I F I C A N T AT p<0.01 (NUMBER OF PAIRS IN PARENTHESES) Concentration 10%

Condition

100%

1%

C

0.80 (41)

0.90 (22)

0.88 (11)

A

0.85 (40)

0.86 (19)

0.78 (14)

studied here in b o t h T h a m n o p h i s radix and T. sirtalis as measured under the conditions that prevailed during testing ( E x p e r i m e n t s 1 and 2). The latter proviso is crucially important. Visual cues, especially prey m o v e m e n t , can be decisive in simultaneous choice tests (unpublished observations), as well as elicit orientation in the absence o f chemical cues [ 3 ] . In neonate snakes of this genus,

TONGUE AND SENSES IN FEEDING OF GARTER SNAKES however, prey attacks do not apparently occur without at least one tongue-flick directed at the object that, in fact, touches the object [8]. The present experimental arrangement precluded both the role of distance cues as well as prey movement since dead prey and relatively motionless swabs were used. The attacks that occurred did not seem well directed, however, and the animals appeared functionally blind as in an earlier study [7]. (2) Anosmia, though perhaps not complete since the internal nares were not blocked, also seemed relatively unimportant in that no significant differences occurred with normals in all 4 experiments. On the other hand, in almost every case the anosmic animals scored slightly lower with the swab tests. Nasal olfaction may, in fact, be more important for distance reception. Unpublished experiments from this laboratory using an olfactometer tend to support this view. (3) Blinding and anosmia together had little additive effect (Experiments 1 and 2). (4) Highly significant correlations between tongue flicks at swab and the interest (orientation) time score were the rule at all concentrations of prey extract. (5) Virtual complete removal of the tongue in ingestively naive neonate snakes did have a marked effect. Interest scores, prey attacks, and food ingesion all fell to near nonexistence (Experiments 2, 3, and 4). This gives direct proof of the involvement of the tongue-Jacobson's organ system in the mediation of prey attack in newborn snakes and thereby extends the findings on adult snakes [ 12]. (6) Removal of the tongue in young experienced snakes had only a temporary effect on prey attack and virtually none on feeding under the experimental conditions ( E x p e r i m e n t 1). This study was confounded by the incomplete removal of the tongue and the compensatory movements involving the remaining stub that soon developed. In Experiment 4 it was shown that tongue removal before initial feeding was not followed by compensation of any type following exposure to food. It remains to be shown whether experienced adult snakes undergo similar decrements with virtual complete tongue removal. While not the goal of this study, the results of a pilot experiment seem pertinent and will be briefly presented. An adult male T. s. sirtalis was chosen for tongue surgery to test the hypothesis that the tongue is less critical to food preference if the animal has had extensive previous prey experience. The animal had been captured in the field and was maintained in good health in our laboratory for over a year prior to tongue surgery. During that year the snake had eaten readily both earthworms and fish. At the time of surgery, he weighed 77 g and was 53 cm in s n o u t - v e n t length. The severed tongue measured 29 mm in length, 8 mm into the white area, and 19 mm from the fork. Bleeding around the mouth was profuse; however, after 20 rain the bleeding subsided and the snake showed no more adverse effects. Two days after surgery forceps were used to offer 2 cm nightcrawler pieces to the animal. It immediately displayed an aggressive attack to the food, then gave a normal prey attack and ate each of the four pieces. Prior to the prey attack there was no visible evidence that it touched his lips or nostrils to the nightcrawlers, clearly in opposition to the studies with newborn snakes of the same species [8]. After having determined that the tongueless adult would eat, further tests commenced, utilizing a realistic brown plastic nightcrawler, in addition to dead nightcrawlers. In

193

these tests, the plastic or real nightcrawler was placed on a 15 X 10 cm piece of plate glass in the snake's home tank while the snake was restricted to a corner of the 30 X 60 cm aquarium with an opaque partition. The partition was then removed, allowing the subject full view of, and access to, the glass plate. The standard order of presentation, repeated 6 times at several day intervals, was as follows: plastic nightcrawler, plastic nightcrawler with nightcrawler scent, dead worm, plastic worm with scent, and dead worm. The unscented plastic worm had to be presented prior to the introduction of any worm odors to the cage. In applying the worm scent to the plastic nightcrawler, four worms were rinsed with hot water, thereby killing them and causing a mucous substance to be released from their skin. These dead worms were then placed atop the plastic worm in a small bowl for 30 min. The plastic worm without scent was stored in a tightly sealed plastic container when not in use in the test. Latency to prey attack was measured for each trial. If the snake did not attack the worm after 20 min, the worm was held by forceps and dangled 12 cm from the snake's head. The dead nightcrawler was most effective in eliciting a prey attack in the plate glass test. The snake exhibited a prey attack in 92 percent of the presentations of the dead worm, as compared with only 30 percent to the plastic worm with scent, and 17 percent (1 attack) to the plastic worm without scent. Normal snakes offered the unscented plastic worms under these conditions never attacked. But it is interesting to note that in the closely related water snakes (Natrix) prey contrast with background was a major factor in prey selection [6]. Attacks to the scented plastic worm were followed by attempts to swallow it, which necessitated forceful dislodging of the plastic worm from the snake's mouth by the experimenter. The attack latencies to the dead worm, following these attempts to eat, were considerably longer than the other latencies to the dead worm. When the plastic worms were dangled in front of the snake, four additional attacks were made to both the scented and unscented plastic worms. Dangling was unnecessary for the nightcrawlers. These attacks did seem aggressive in nature, and, to a lesser extent, were also elicited from normal snakes on the temperamental side. Behavioral observation showed that there was little rubbing of the nostrils Or lips on the plate glass or the test objects prior to an attack on the motionless stimuli, often seen in normal adults along with tongue flicking. Aggressive attacks to the forceps and human hands were quite frequent and were not limited to test situations. One particular sequence of actions, however was noted throughout the testing. The snake's head first oriented towards the stimulus and the entire body moved to the edge of the glass. The actual attack followed a brief period during which the head was raised and poised toward the prey object. That this adult tongueless snake ate readily, although somewhat abnormally (and continues to do so after 3 years), while the newborn tongueless snakes would not (Experiments 2, 3, and 4), leads us to speculate that the role of previous experience with prey objects can not be underestimated. Through past experience the adult has enlarged upon auxilliary means of recognition of prey. In addition to cues from Jacobson's organ, it may also rely upon visual and olfactory cues, as well as cues of movement in feeding behavior. Evidence from Experiment 1 supports this hypothesis. However, a lowered threshold for agonistic

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responses to visual stimuli t h e snake c a n n o t evaluate chemically via t h e t o n g u e m a y be partially r e s p o n s i b l e for the difference. N e w b o r n g a r t e r s n a k e s f r o m these species are n o t n o r m a l l y very aggressive at b i r t h and a m a t u r a t i o n a l factor m a y be involved. Regardless of t h e m e c h a n i s m , we m a y c o n c l u d e t h a t J a c o b s o n ' s organ and the t o n g u e working in c o n j u n c t i o n are necessary and sufficient to elicit prey a t t a c k s in naive, n e w b o r n snakes, b u t t h a t t h e t o n g u e is n o t a b s o l u t e l y essential in adults.

M a n y m o r e studies are n e e d e d o n the f u n c t i o n of the t o n g u e a n d J a c o b s o n ' s o r g a n using diverse species, behaviors, and m e t h o d s o f analyiss. T h e m o v e m e n t s of t h e t o n g u e itself need to b e carefully studied [ 11 ], as well as gross b o d y m o v e m e n t s . T h e i m p o r t a n c e o f studies o n this specialized c h e m o s e n s o r y s y s t e m goes b e y o n d an interest in snakes per se. It is clear t h a t s u c h studies also need to consider o n t o g e n e t i c variables.

REFERENCES 1. Bellairs, A. The Life of Reptiles. Vol. 2. New York: Universe, 1970. 2. Bogert, G.M. Sensory cues used by rattlesnakes in their recognition of ophidian enemies. Ann. N.Y. Acad. ScL 41: 329-344, 1941. 3. Burghardt, G. M. Chemical perception in reptiles. In: Communication by Chemical Signals, edited by J.W. Johnston Jr., D. G. Moulton and A. Turk. New York: Appleton-CenturyCrofts, 1970, pp. 241-308. 4. Burghardt, G. M. and J. P. Abeshaheen. Responses to chemical stimuli of prey in newly hatched snakes of the genus Elaphe. Anita. Behav. 19: 4 8 6 - 4 8 9 , 1971. 5. Burghardt, G.M. and E.H. Hess. Factors influencing the chemical release of prey attack in newborn snakes. J. comp. physiol. Psychol. 66: 2 8 9 - 2 9 5 , 1968. 6. Czaplicki, J.A. and R.H. Porter. Visual cues mediating the selection of goldfish (Carassius auratusJ by two species of Natrix. J. of Herpetol. 8: 129-134, 1974.

7. Noble, G.K. and H. J. Clausen. The aggregation behavior of Storeria dekayi and other snakes with especial reference to the sense organs involved. Ecol. Monogr. 6 : 2 7 1 - 3 1 6 , 1936. 8. Sheffield, L. P., J. H. Law and G. M. Burghardt. On the nature of chemical food sign stimuli for newborn snakes. Communs. Behav. Biol. 2: 7 - 1 2 , 1968. 9. Siegel, S. Nonparametric Statistics for the Behavioral Sciences. New York: McGraw-Hill, 1956. 10. Tucker, D. Nonolfactory responses from the nasal cavity: Jacobson's organ and the trigeminal system. In: Handbook of

Sensory Physiology. Vol IV. Chemical Senses, Part 10lfaction, edited by L. M. Beidler. New York: Springer-Verlag, 1971, pp. 151-181. 11. Ulinski, P.S. Tongue movements in the common boa (Constrictor constrictor). Anim. Behav. 20: 3 7 3 - 3 8 3 , 1972. 12. Wilde, W.S. The role of Jacobson's organ in the feeding reaction of the common garter snake, Thamnophis sirtalis sirtalis (Linn.). J. exp. Zool. 77: 4 4 5 - 4 6 5 , 1938.