One trial learning in the American cockroach, Periplaneta americana

J. Insect Physiol., Vol. 26. pp. 499 to 504. 0 Peryamon Press Ltd. 1980. Printed in Great Britain.

ONE TRIAL COCKROACH,

0022-1910/80/0801-0499E02.0010

LEARNING

fN THE AMERICAN

PERZPLANETA

AMERICANA

NORBERTOBALDERRAMA*

Laboratorio Fisiolog’a de! Comportamiento, Institute

Venezolano

(Receiletl

Centro Biofisica y Bioquimica,

Investigaciones Cientificas (IVIC), Apartado Caracas 101, Venezuela

No. 1827,

24 April 1979: rerlised 4 Fehruur!~ 1980)

Abstract--A new and simple method of training intact cockroaches was designed. (1) Cockroaches were maintained before and during the experiment with a high motivation to look for sugar. They were kept

in a chamber with water and yeast extract ud lib&urn but without any sugar. (2) Cockroaches were individually trained to associate two artificial scent signals, i.e. menthol and vanilla, with sugar and salt solutions. Discriminatory learning performance was measured by testing the scent preference before and after training. Care was taken to exclude other than olfactory cues in the discriminatory paradigm. (3) Cockroaches exhibited a spontaneous initial preference for vanilla over menthol. This preference could be modified by training. The insect showed fast learning and long retention. One training trial was enough to reverse the initial preference with a significant retention after 7 days. Reversal of trained preference was accomplished by retraining. (4) The method provides an opportunity to study sensory performance and memory consolidation in cockroaches.

INTRODUCTION THE PROCESSESof memory consolidation in an intact insect have been studied in mantids (MALDONADO and TABLANTE,1975; JAFF~:and MALDONADO, 1979; MALDONADOet al.. 1979). bees (BECKMANN,1974; MENZEL et ul., 1974, 1975, ERBER, 1975a, b). Drosophila (DUDAI, 1977) and cockroaches (FRECKLETONand WAHLS~IN, 1968; LOVELL and EISENSTEIN. 1973). Cockroaches offer special advantages due to easy mass rearing and their independence from a colonial organization. Experiments on learning with free cockroaches have been done primarily with dark preference and searching behaviour. Dark avoidance training (SZYMANSKY, 1912; TURNER, 1912; MINAMI and DALLENBACH, 1946; LOVELL and EISENSTEIN, 1973; CHENEY et al.. 1974) required a strong punishment (electric shock) which might result in undesirable effects on the cockroach (BEAMENT,1958; EWING and EWING, 1973; COOK and HOLT, 1974). On the other hand, training to search required no strong punishment (TURNER, 1913; ELDERING, 1919: GATES and ALLEE, 1933; LONGO, 1964). However, maze experiments with the cockroach have other drawbacks. (a) Insufficiently rigorous definition of the discriminatory paradigm can lead to inconclusive evidence concerning the sensory cues (ALLOWAY, 1972). (b) A great number of trials is required to obtain a long retention. This entails frequent handling of the animals and regular cleaning of the maze between trials. This may not eliminate odour marks reliably. (c) A long training process renders an investigation of the memory consolidation process by means of interference with amnesic agents unduly laborious (JAFFB and MALDONAW. 1979; MALDONADO et al., 1979). (d) Finally, * Present address: Instituto Zoologia Agricola, Facultad de Agronomia (UCV), Apartado Postal 4579, Maracay 2101. Edo. Aragua, Venezuela. I P

26

x-

A

both types of learning experiments with cockroaches have often underestimated the influence of circadian rhythms which are well known in these insects (SAUNDERS.1976). The purpose of the present work is to test a new method of training cockroaches in an odour discriminatory task (i.e. a modification of the searching behaviour) free of the shortcomings mentioned above. The experimental design provides fast and reliable learning with a device not requiring a maze and with a precise identification of the sensory cues involved.

MATERIALS

AND

METHODS

Animals

The animals were adult female Periplaneta Llmerireared in covered plastic cages (50 x 25 x 25cm) at constant temperature (25’C) with reversed light-dark cycle of 12:12 hr (photophase 8 pm-8 am). They were fed with Purina Laboratory Chow. Water was provided ad lihitum.

cana

Chemicals

Sugar solution 50% (w-w), used as the positive reinforcement during training, was prepared with D( +) sucrose (E. Merck A. G. Darmstadt). The saline solution 20:, (w-v) used as the negative reinforcement, was prepared with sodium chloride (E. Merck A. G. Darmstadt). Yeast extract of autolysed cells (Becton. Dickinson and Co.) was used as the only amino acid source. Vanilla and menthol artificial essences (C. A.. Especieras Indian, Venezuela) were employed as odour sources. Experimental

arrangement

(Fig. 1)

The experimental arrangement consisted of two chambers, the ‘resident chamber’ and ‘training chamber’. 499

NORBERTO BALDERRAMA

500

Fig. 1. Experimental container. RCH: resident camber. R: refuge. DJ: jar with water. Y: container with yeast extract. TCH: training chamber. SD: sliding door. NS: nylon string. SH: source holder. RA: rotating axle. SS: odour sources. N: gauze net. FP: filter paper. H: holes connecting with two SS. T:

supporting table. (i) The resident chamber was a transparent lucite cage (30 x 50 x 8 cm) with a removable cover pierced with many holes to ensure ventilation. A black cardboard sheet covered the floor. This provided a reasonably normal substratum for locomotion. eliminated light reflection, and absorbed urine excretion and humidity. The chamber also included (a) a refuge made of a black cardboard cage ( 15 x IO x 6 cm), (b) an inverted water-filled jar over a plastic plate with grooves on its surface, and (c) a small container of yeast extract. (ii) The training chamber was a transparent lucite cage (15 x 30 x 7 cm), again with a removable cover. Between the chambers there was a sliding door that could be opened and closed manually with a nylon string. On the floor there were two round holes (3 cm dia) that could connect the chamber with two of the six odour sources. Every source consisted of a vertical plastic tube (3.5 cm dia. x 4 cm high) with its upper part covered by a fine gauze net. The six containers were mounted on the source holder (consisting of a 20 cm dia. x 2 mm. thick plastic disc) which had a rotating axle in its centre. fixed to the front wall of the training chamber. The axle supported the source holder horizontally and immediately below the training chamber. Two scent sources could be located simultaneously just below the holes at the ‘offer position’ by rotation of the source holder. (iii) The resident and training chambers were mounted over a supporting table. in an experimental room with a reversed light-dark cycle (scotophase from 8 am to 8 pm) and at constant temperature (25 C). During the scotophase the experimental chamber was illuminated with a red light, to allow observation of the animals. Procedure

(I) Previous treatment of the experimental animals. One week before the beginning of the experiment, cockroaches were marked individually with a paper label (on the back of the right elytron) and were left in a container with water and yeast extract ad libitum. Two days before the first trial, a group of 20 cockroaches was placed in the resident chamber with yeast

extract, water ud libitum, and no sugar. The sliding door was closed. (2) Odour source arrangement in the training chamber. Pilot experiments showed that cockroaches are more attracted by vanilla than by menthol. Therefore, except for the reversal experiments, menthol was used as the ‘positive’ cue (associated with the sugar solution) and vanilla as the ‘negative’ cue (associated with the saline solution). Before each experimental session, rectangular filter papers FP (5 x 7 cm) were soaked with either menthol or vanilla essence, rolled and put inside the scent sources (Fig. 1). The menthol (+ ) and vanilla ( - ) sources were distributed in two groups: the ‘training’ sources and the ‘preference’ sources, placed in different halves of the source holder (see Fig. 2). The positive (menthol) training sources had a 5 ~1 drip of sucrose solution on their top nets. The net of every negative (vanilla) training source was soaked with saline solution. All the preference sources had no solution on their top nets. Rotation of the source holder allowed the observer to alternate at the ‘offer position’ a chosen combination of the positive and negative sources (Fig. 2a, b). (3) The preference test (Fig. 2a, b). Before every preference test, positive and negative preference sources with their respective odours (i.e., menthol and vanilla) were located at the ‘offer position’ and the sliding door was opened. When a cockroach entered the training chamber, the door was closed and the preference test started. Every minute the source holder was rotated in order to change the relative positions of the positive and negative preference sources (Fig. 2a, b). Thus, three changes took place during every preference test. A source was considered to be visited when the cockroach explored the top net with its mouth parts. Time spent during a visit to one scent source was registered cumulatively with an electric counter. Every preference test lasted 4 min and was immedi,ately followed by a training trial. (4) The training trial (Fig. 2c, d). Immediately after the end of a preference test the source holder was rotated locating positive and negative training sources at the ‘offer position’. When the cockroach explored a

One trial learning in the American cockroach

T OFFER

501

l--?

’ PREFERENCE TEST

a

b

4d minute

I

I

d

I

I

C

Fig. 2. Experimental design. Views from above of the source holder. (+) stands for a positive odour source. (-) stands for a negative source. The stippled half of the source holder includes three “training” sources. The white half three “preference” sources. a. b: positions of the holder source during the preference test. c. d: positions of the holder source during the training trial.

negative training source it tested the net soaked with saline solution and retreated almost immediately from the source. When the cockroach explored a positive training source it drank the sugar solution drop (5 ~1) and continued licking the net of the source. A rewarded visit, i.e. a visit to a positive training source, was limited by the observer to 20sec and after this time the training source was alternated (Fig. 2c, d). Tht training trial was concluded after two rewarded visits or after 4 min from the beginning. Thus, the preference test and training trial lasted a maximum of 8 min. Depending on the experimental program, cockroaches could receive one or two such sets during a day. If the preference and training trial set was the second one or the only one the animal was given during the day, the roof of the training chamber was removed at the end of the training trial and the cockroach was placed in a container. On the other hand, if the animal had to be given a second preference and training trial the sliding door was opened at the end of training and the cockroach encouraged to move into the resident chamber. Daily preference and training sessions began at 1 p.m. and had a maximum duration of 5 hr. (5) The retention test. After the last preference and training trial, cockroaches underwent a retention test that was identical to the preference test but not fol-

lowed by a training trial. The interval between the last preference and training trial and the retention test is called the retention interval. Experimental

program

(a) Nomenclature. PI and TI stand respectively for the preference test and the training trial corresponding to a first preference test and training trial, Pz and T2 for those corresponding to a second such test. R stands for retention test and RI for retention interval. (b) Experimental groups. (1) Naive cockroaches trained to menthol as positive scent. (M groups) (a) M-l groups: Animals received (Pl + Tl) and a test R. Depending on the duration of the RI, four subgroups were distinguished: M-l-O (RI = Smin-5 hr), M-l-l (RI = 1 day), M-l-2 (RI = 2 days) and M-l-7 (RI = 7 days).

retention

(b) M-2 groups: Animals (P2 + T2) and a retention

received

(Pl + Tl) and

test R. Two subgroups were similarly distinguished: M-2-2 (RI = 2 days) and M-2-7 (RI = 7 days). (2) Reversal groups (V groups): Animals of the M-groups with RI = 7 days were retrained to vanilla as the positive scent after having been trained to menthol as positive scent, i.e. the retention test R corresponding to the M-animals was

followed by a new training trial with vanilla as the

65.6 &-20.7

72.3 4 20.8

65.5 t 20.7

29.4 + 25.7

35.1 + 18.8 12.7 + 18.4 69.9 + 23.2 81.5 2 15.8

38.3 &-22.7

43.8 + 25.4

35.1 & 22.9

70.6 t_ 19.1

+36.2 + 31.6(P < 0.001)

52.7 k 25.2

34.6 f. 18.6

60.1 f 22.1

+18.1 + 29.4(P < 0.025)

61.1 + 24.4

28.9 + 200.5

+46.4 F 30.5 (P < 0.001)

+21.9 + 20.8(P < 0.001)

f34.3 + 25.O(P < 0.001)

+24.8 + 23.4(P < 0.001)

+32.2 5 31.4(P < 0.001)

+26.4 & 26.9 (P < 0.001)

63.3 f 24.8

t41.4 &-28.O(P < 0.001)

R-P,

36.9 f. 19.6

R

72.6 * 20.8

p2

31.2 + 23.2

PI

+ 10.8 + 21.0 (NS)

-5.4 +_ Zl.S(NS) +30.4 + 25.6(P < O.til,

+35.5 & 26.7 (P < 0.001)

- 13.2 + 22.4 (NS)

R-P2

+ 34.0 + 33.9 (P < 0.001)

Pz-PI

to preference and retention test in the different groups. (Means + SD)

PI = First preference test; P2 = second preference test; R = retention test, N = Number of animals in each group. NS = Not significant (t-test for paired data, a = 0.25 one tailed). M = Menthol-trained cockroaches. V = Cockroaches trained to vanilla after menthol training.

M-1-0 (N = 34) M-l-l (N = 31) M-l-2 (N = 22) M-l-7 (N = 16) M-2-2 (N = 23) M-1-1 --. (N = 26) ’ V-I-O (N = 15) V-l-l (N = 12) V-2-f (N = 13)

Groups

Table 1. Comparisons between percentages of preference corresponding

$ n

B d

z 8

One trial learning in the American cockroach positive scent. Thus, the R of the M-animals the Pl of the V-animals.

became

(a) V-l groups: Animals received (Pl + Tl) and a retention test R. Two subgroups: V-1-O (RI = 5 min-5 hr) and V-l-l (RI = 1 day). (b) V-2-l group: Animals received (Pl + Tl) and (P2 + T2) and a retention test R (RI =G1 day). Statistics and comparisons. For each experimental group results of Pl, P2 and R are given as the mean + SD of the individual relative preferences to the positive scent. These relative preferences were estimated for each preference test according to the following relationship: total time of visits to the positive sources over total time of visit to both (positive and negative) sources, multiplied by 100. For all comparisons, the t-test was used.

RESULTS The usual behaviour of cockroaches in the training chamber was as follows. During the first preference test (PI) cockroaches visited the odour sources intensively, at least for the first minute of the test. During the training trial the animals retreated immediately after testing the saline solution associated with the negative scent. Mouth part-probing was necessary for the retreat response to appear. On the other hand, when the animals found the drop of sugar solution at a positive source and drank it, intense licking of the net followed. Alternation of the sources at this moment produced a rapid orientation toward the new position of the positive source and intense movements of the antennae. As a rule, the animals completed two rewarded visits during every training trial but rarely visited a negative source twice. It is interesting to note that when an animal was withdrawn from the training chamber to resident chamber, at the and of a (PI + T,), it first ran away to the refuge but soon returned to a position near to the sliding door. The initial preference test Pl for menthol in the M-groups proved to be significantly lower than that for vanilla (mean equal to 34.3%, SD + 22.3%, significantly less than SO%, t-test, P < 0.001, N = 159 animals) and not significantly different from the initial preference P, for vanilla in the three ‘reversal’ groups (38.9%, SD + 23.3, N = 40, t-test, a = 0.05). Table 1 summarises the results. Intragroup comparisons, i.e. R-PI, P2-PI and R-P2, show that (a) preference for positive odour during R became significantly greater than that for P, in all the groups; (b) preference for positive odour during P2 proved to be significantly greater than that for PI in those groups with two (P + T), i.e. M-2-2, M-2-7 and M-2-2; (c) no significant difference was found between R and P2 preferences (t-test for paired data, a = 0.025, one tailed). In order to assess the influence of the number of training trials and the span of retention intervals on values of preference during R, multiple intergroups were done (independent comparisons of R-P, t-test, one tailed). When the criterion samples, z = 0.25 was used, significant differences were only found comparing M-l-O vs M-l-7 i.e. only that comparison involving the most extreme span of retention intervals and the minor number of trials. All other

503

intergroup comparisons failed to show a significant decay of retention with increasing retention intervals or an improvement of retention with an additional training trial.

DISCUSSION It is interesting to attempt a comparison between behaviour of cockroaches during their search for food and that of bees during their search for nectar. Foraging bees show a very specific and predictable searching behaviour. i.e. they are highly motivated to search for one type of food source and quickly learn source signals (VON FRISCH, 1915, 1919. and 1967). On the other hand, cockroaches seem to have non-specific and unpredictable searching behaviour, since they may be looking for a mate, refuge and/or a great variety of food sources with changing motivation. In order to overcome this ambiguity of the cockroach exploratory behaviour the following precautions were taken during these experiments: (1) only female insects were used, (2) they were provided with a refuge, (3) they were submitted to sugar deficit in such a way that their motivation for sugar was increased, and (4) training was accomplished during the natural period of maximum activity. Results of the present paper lead us to the following conclusions: (a) Cockroaches can learn to associate one odour with food and another with no food or punishment. The experimental design excluded the possibility that this discrimination could be accomplished using spatial, tactile or visual cues. or their own odour marks. i.e. cockroaches seem able to learn on the basis of external olfactory cues alone. (b) Learning to choose a scent against an initial preference seems fo take place very fast. One trial is enough to reverse the preference. (c) Learning is characterized by a long lasting memory. (d) No significant decay was observable with increasing retention interval from 1 to 7 days. (e) An additional training trail does not improve retention at least for those intervals which were tested here (2 and 7 days). Conclusions (b) and (c) indicate that cockroaches show fast learning and long retention, features that are comparable only with those reported for bees in colour learning experiments (MENZEL et ul.. 1974). Mass conditioning of Drosophila to avoid an odorant associated with an electric shock (DUDAI. 19771 is also accomplished in one trial but with a fast memory decay. Mantids can modify their attack or defensive behaviour by a type of learning with long retention (MALDONAOO and TABLANTE, 1975: MALD~NADCI er al., 1979). but they require many trials to achieve learning. Conclusions (d) and (e) suggest that neither the number of training trials nor the span of the retention interval has any influence on retention. However. it is possible that real differences between experimental groups were obscured by the great data dispersion. This dispersion might result from lack of resolution of the methods used to test odour preferences. since

NORBERTOBALDERRAMA

504

some overlapping of odour gradients in the training chamber during trials could not be completely discounted. The present results indicate that high plasticity of behaviour patterns among insects is not only found among social insects (MENZEL ef al., 1975). Cockroaches also show a comparably high learning performance. Thus, the process of memory consolidation and sensory performance can be investigated using one of the commonest laboratory insects.

behaviour in the cockroach, Nauphoeta cinerea. Anim. Behav. 21, 579-584. FRECKLETONW. C. and WAHLSTEIN D. (1968) Carbon dioxide induced amnesia in the cockroach. Psychom. Sci. 12, 179-180. FRIXH K. VON (1915) Der Farbensinn und Formsinn der Bienen. Zool. Jnhrb. Abr. Zoo/. Physiol. 35, 1-182. FRI~CH K. VON (1919) Uber der Geruchssin der Bienen und seine bliitenbiologische Bedeutung. Zoo/. Jahrb. Abf. Zoo!. Physiol. 37, l-225. FRISCH K. VON (1967) The dance, language, and orientation of bees. Transl. L. Chadwick, Harvard Univ. Press, Camsubordinate

bridge. Acknowledgements-The author thanks Dr. H. MALD@ NADO and Dr. G. WHITTEMBURYfor advice and critical review of the manuscript and Dr. J. N~REz for valuable suggestions and discussions. He is also indebted to Mrs. MARIA CENTENOfor drawing the figures, to Mr. JOAQU~N SALCED~ for further correcting them, and to Mrs. R. EASTAWAY for her secretarial work.

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