Journal of Chemical Ecology, VoL 18, No. 1, 1992

THE ROLE OF CHEMOSENSORY CUES IN DISCRIMINATION OF PREY ODORS BY THE

AMPHISBAENIAN Blanus cinereus

P I L A R L O P E Z * and A L F R E D O S A L V A D O R

Departamento de Ecologfa Evolutiva Museo Nacional de Ciencias Naturales

Jos~ Gutierrez Abascal, 2 28006 Madrid, Spain

(Received June 3, 1991; accepted September 30, 1991)

Abstract--Responses of amphisbaenians (Blanus cinereus) to deionized water, a control for pungency (cologne), and integumental prey odors (coleopteran larvae and adult ants) on cotton swabs were studied in experi- ments with a randomized blocks design to discover whether amphisbaenians use chemical cues to detect and identify prey. No individual bit the applica- tors. Amphisbaenians tongue-flicked at lower rates than epigean saurians, which are active foragers. Tongue-flick rate differed among treatments, but responses to prey odors were not significantly different from those to cologne. The number of directed tongue-flicks emitted during the 60-sec trials was, however, lower in response to deionized water than in response to cologne or prey odors. Response details, the low rate of tongue-flick, and absence of biting are discussed in relation to the foraging behavior and fossoriality of amphisbaenians. Evidence from this study indicates that the vomeronasal sense is used by amphisbaenians to identify odors, but our experiments failed to demonstrate that amphisbaenians discriminate between prey and nonprey odors.

Key Words--Lacertilia, Amphisbaenidae, Blanus cinereus, prey odor, tongue-flicking, fossoriality.

INTRODUCTION

Ep igean saur ians use visual and ol fac tory senses to locate prey. The p reva lence

o f one o f these senses s e e m s to be related to foraging mode . Act ive foragers

*To whom correspondence should be addressed.

87

00984)031/92/0100-0087506,50/0 �9 1992 Plenum Publishing Corporation

88 L6PEZ AND SALVADOR

appear to use chemosensory senses. They regularly tongue-flick during foraging and can detect and recognize prey by chemical cues. Sit-and-wait predators, however, do not tongue-flick at high rates and detect prey visually while for- aging (Bissinger and Simon, 1979; Huey and Pianka, 1981; Cooper, 1989, 1990). There have been few studies on senses used in prey detection by sub- terranean saurians (Hetherington, 1989).

Amphisbaenians are squamate reptiles showing many adaptations to a bur- rowing life (Gans, 1978). Their foraging behavior is unknown, specially in relation to prey detection. Vision is reduced. Hearing and olfactory capabilities, however, are enhanced (Gans and Wever, 1975; Gans, 1978), indicating an important role in predation. Nevertheless, no evidence is available of a ehemo- sensory role in any amphisbaenian species.

Blanus cinereus is an amphisbaenian endemic to the Iberian Peninsula (Busack, 1988). Its diet mainly consists of insect larvae and ants, which are the most abundant invertebrates in their habitat, but insect larvae of large size are selected (L6pez et al., 1991). The use of chemosensory cues by B. cinereus in the discrimination of prey was tested using responses to prey chemicals pre- sented on cotton-tipped applicators (Cooper and Burghardt, 1990). We tested the ability of B. cinereus to detect prey odors and to discriminate between prey odors and nonprey odors. We also address the way fossoriality influences the foraging behavior of a burrowing reptile, in comparison to epigean saurians.

METHODS AND MATERIALS

Eighteen Blanus cinereus (_six adult males, three adult females and nine immatures; snout-vent length: X + SE = 147 + 8 mm), were captured in March 1990 near Torrelodones (40~ 3~ Madrid Province, Spain). They were housed at "El Ventorrillo" Field Station laboratory (Navacerrada, Madrid Province). The photoperiod was that of the surrounding region, but ambient temperature was maintained at a constant 20~ Individuals were main- tained in 5-liter glass jars containing sand substrate from the capture area. They were fed twice weekly on coleopteran larvae, adult ants, ant eggs, and earth- worms, which they readily consumed. Humidity was raised daily with a spray. All animals were acclimatized to laboratory conditions and the experimenter's presence for at least one month before testing. Amphisbaenians were not fed for five days prior to the study.

In a first experiment, each of 18 amphisbaenians was tested with coleop- teran larvae odors, deionized water, and cologne (Eau Jeune, L'Orral). The water served as an odorless control. Cologne was used to determine response to an odorous but nonprey stimulus. Every amphisbaenian was tested with each stimulus once in a randomized block design. Order of presentation was coun-

CHEMOSENSORY DISCRIMINATION OF PREY BY AMPHISBAENIAN 89

terbalanced. One trial was conducted per day for each animal. We prepared stimuli by dipping the cotton tip (1 cm) of a wooden applicator (10 cm) in deionized water. Other stimuli were added by dipping the wetted cotton in diluted cologne or by rolling it over the body surface of coleopteran larvae. A new stimulus was used in each trial. The experiment was conducted April 23-28, 1990, between 0930 and 1900 hr. A second experiment was made using ant odor (Pheidole pallidulla), deionized water, and cologne. This experiment was conducted May 2-10, 1990, between 0930 and 1900 hr., using the pro- cedures described for experiment 1.

Because the amphisbaenians showed defensive and escape behavior on the soil surface, we simulated fossorial conditions to perform experiments by using transparent plastic tubes (one for each individual). Inside each tube, the lower half of the surface was covered with sand using adhesive. Individuals were kept in their tubes 6 hr per day during the acclimatization period, during which they showed normal behavior and were fed with larvae and ants. Individuals were kept in their tubes 10 min before trials, closing both sides with cotton. Between trials, we opened both sides of the tube for 10 min to avoid odor mixture. During experiments the laboratory was darkened and observations were made using a 50-W red light.

To begin a trial, we slowly approached a tube, removed the cotton from one side and slowly put the cotton-tipped applicator to a position 2 cm anterior to the amphisbaenian's snout. We counted total number of tongue-flicks (TFs) for 60 sec beginning with the first TF. We also noted the number of TFs directed to the swabs during this period. Latency to the first TF was computed as the period elapsed between closing the tube and the first TF. If an individual did not tongue-flick in 30 sec, we touched its snout with the swab. I f it still did not tongue-flick, the data were discarded. To demonstrate that lack of responsive- ness to stimuli was not caused by satiation, food was given to the amphis- baenians at the conclusion of trials.

To examine differences in number of TFs between trials we used nonpar- ametric Friedman two-way ANOVA (Siegel, 1956). Individual comparisons were made using Wilcoxon signed rank-matched pairs test. Spearman rank cor- relations were conducted between latency to first TF and number of TFs (Sokal and Rohlf, 1981).

R E S U L T S

Experiment 1: Using Coleopteran Larvae as Prey Odor. All of 18 individ- uals tongue-flicked. Fifteen tongue-flicked in all conditions, and three in only two conditions. Analyses were conducted only on those 15 individuals. No amphisbaenians bit an applicator. There were significant differences (X 2 = 7.58;

90 LOPEZ AND SALVADOR

df = 2,28; P = 0.001) in total number of TFs in responses to different odor conditions (X ___ SE: prey = 4.9 + 0.7; cologne = 7.7 + 1.0; water = 3.9 +__ 0.5). The tongue-flick rate in response to prey odor was significantly higher (Wilcoxon test, T --= 2.0, P < 0.05) than to deionized water but did not sig- nificantly differ (T = 1.88, P = 0.06) from those to cologne odor. Response to cologne was significantly higher (T = 3.0, P = 0.002) than to water. Num- ber o f TFs directed to the cotton-tip were also significantly different between treatments (X 2 = 7.96; df = 2,42; P = 0.019) (Figure 1).

Latency to first TF and total number of TFs in 60 sec in all trials had a negative Spearman rank correlation (r s = - 0 . 4 0 ; P = 0.008), and latency to first TF was negatively, but not significantly, correlated with the number of TFs directed to the cotton-tip (r s = - 0 . 2 1 ; P = 0.17). There was a significant treatment effect on t ime of latency to first TF (X 2 = 14.53; df = 2,28; P < 0.001). Latency to first TF in response to distil led water (~" 5- SE = 29.2 + 6.8 sec) was significantly higher than to either prey (X + SE = 13.1 + 2.5 sec; T = 2.87, P = 0.004) or cologne odor (X + SE = 10.2 ___ 1.9 sec; T = 2.73, P = 0.006). Latency in response to prey odor and cologne did not differ significantly (T = 0.68, P = 0.49).

Experiment 2: Using Ants as Prey Odor. This experiment was conducted on 16 animals. All 16 individuals tongue-flicked. Fifteen tongue-flicked in all treatments, and one in only two treatments. Analyses were conducted only on those 15 individuals. No amphisbaenians bit the applicator. The total number of TFs emitted in responses to different odor conditions (~" + SE: prey = 7.5 5- 1.1; cologne = 7.8 + 1.1; water = 3.7 + 0.5) were significantly different (X 2 = 21.83; df = 2,28; P < 0.0001). The tongue-flick rate in response to water odor was significantly lower than to either prey (T = 3.38, P = 0.0007) or cologne (T = 3.26, P = 0.001). Responses to prey and to cologne did not

FIG. 1.

LARVAE ANTS

Wale, Cologne Pr~ Water CoSine Pr~, Number of directed tongue-flicks (TFs) emitted in 60 sec in response to odor

stimuli presented on cotton-tipped applicators. Responses shown are for Blanus cinereus to larval prey (experiment 1) and to ant prey (experiment 2). Data are means (horizontal lines) +_ 1 SE.

CHEMOSENSORY DISCRIMINATION OF PREY BY AMPHISBAENIAN 91

differ significantly (T = 0.44, P = 0.66). The number of TFs directed at the cotton-tip were also significantly different between treatments (X 2 = 9.25; df = 2,42; P = 0.0098) (Figure 1).

Latency to first TF was negatively, but not significantly, correlated with the total number of TFs in 60 sec (r s = -0 .24 ; P = 0.11), and with the number of TFs directed at the cotton-tip (r s = -0 .16 ; P = 0.29). Latency to first TF in response to distilled water (X __+ SE = 15.8 + 2.6 sec) was not significantly different (X 2 = 4.28; df = 2,28; P = 0.12) from either cologne (~" ___ SE = 9.5 + 1.9 sec) or prey odor (~" _ SE = 11.8 ___ 2.7 sec).

DISCUSSION

Blanus cinereus can detect odors but did not demonstrate an ability to dis- criminate prey odors from nonprey odors as tested in the present experiment. The greater number of tongue-flicks was in response to cologne compared to water, but does not rule out recognition of food odor. However, their failure to do so does not necessarily indicate an inability to recognize food stimuli by tongue-flicking. Critical tactile or heating stimuli needed to release attack may have been absent. Lizards need not rely exclusively on one sensory cue to detect prey (Cooper, 1990). B. cinereus may use many cues to locate prey, and the ability to detect odors by chemical cues could be useful to confirm identification of potential prey (Graves and Halpern, 1990).

The tongue-flick rates of B. cinereus are close to those produced by epi- gean saurians, which are unable to detect prey odors. Nevertheless, the iguanid lizard Dipsosaurus dorsalis, although emitting a relatively low number of TFs, discriminates food odors in opposition to cologne (Cooper and Alberts, 1991). Amphisbaenians may discriminate quickly, on the basis of a few or even a single tongue-flick. Low rates of tongue-flicking could also reflect low meta- bolic requirements related to fossoriality (Withers, 1981; Kamel and Gatten, 1983).

Compacted particles of substrate that separate amphisbaenians from their potential prey probably complicate detection of invertebrates by the vomero- nasal system. Cues associated with moving prey could be much more effective, as they can be used at greater distances (Hetherington, 1989). Olfactory cues may help to identify nearby prey items whose odor can be detected through particles of substrate. The absence of attacks on applicators may indicate that amphisbaenians were not previously stimulated by hearing cues.

The construction of a permanent system of tunnels is unknown in B. ciner- eus; however, we observed permanent galleries under stones that allowed access for heating (Martfn et al., 1990). Vomerolfaction may aid detection of potential predators or prey in galleries. Both sexes of B. cinereus possess femoral pores,

92 LOPEZ AND SALVADOR

and their secret ion may impregnate the wal ls o f tunnels , in which case the vom-

eronasal sys tem may be used in individual and sex recogni t ion (Alberts , 1991;

C o o p e r and Vitt , 1986).

The exper imenta l des ign used in this paper was able to demonst ra te that

B. c inereus can detect odors using its tongue , probably via vomero l fac t ion .

Never the less , it did not demons t ra te d iscr iminat ion be tween prey odors and

nonprey odors . Al te rna t ive exper imenta l techniques , such as seal ing o f vo-

meronasa l ducts (Graves and Halpern , 1990) or teaching the animals to respond

to a par t icular chemica l s t imulus to be rewarded with food could be useful to

de te rmine the chemosenso ry abil i t ies o f amphisbaenians .

Acknowledgments--The paper has been improved by helpful comments from William E. Cooper. We thank Jos6 Martin for his help in the field and in the elaboration of the manuscript, Ross D. Johnston for critical comments and checking the English in early drafts of the manuscript, and "El Ventorrillo" MNCN Field Station for use of their facilities. Financial support was pro- vided to Pilar L6pez by a Museo Nacional de Ciencias Naturales-Comunidad de Madrid grant. This project was funded by DGICYT grant PB88-0009.

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