psychology of learning: a new approach to study behavior of rhodnius prolixus ...

Psychological Reports, 2005,97,721-73 1. O Psychological Reports 2005

PSYCHOLOGY OF LEARNING: A NEW APPROACH TO STUDY BEHAVIOR O F RHODNIUS PROLIXUS

STAL UNDER LABORATORY CONDITIONS

CHARLES I. ABRAMSON ENRIQUE SULBARAN ROMERO

Oklahoma State University Universidad de Los Andes Merida, Venezuela

JOSEPH FRASCA, RYAN FEHR ELIECER LIZANO

Oklahoma State University Universidad de Los Andes Merida, Venezuela

ELIS ALDANA

Universidad de Los Andes Merida, Venezuela

Summary.-Conditioning methodologies associated with the psychology of learning are suggested as a new strategy to investigate behavior of the assassin bug Rhod- nius prolixus, which is the main vector of Chagas disease in Venezuela. Chagas disease is the fourth leading cause of death in Latin America, as it causes severe chronic illness and approximately 43,000 deaths per year. To illustrate this strategy, two preliminary experiments are reported. In the first, Pavlovian conditioning was examined by pairing an olfactory conditioned stimulus with a temperature unconditioned stimulus. A temperature of 42°C elicits a complex behavioral sequence in R. prolixus consisting of proboscis extension and crawling. Over the course of 12 training trials, this behavioral sequence was not elicited by an olfactory conditioned stimulus. In the second experiment, a latent inhibition paradigm was used to pre-expose R. prolixus to an olfactory conditioned stimulus before pairing the odor with temperature. Over the course of training, an effect of pre-exposure was found. Suggestions for research are discussed and potential conditioned and unconditioned stimuli identified.

This study provides the first published data on the feasibility of extending paradigms developed for the study of invertebrate learning to the vectors of Chagas disease. Chagas disease or American trypanosomiasis, is named after the Brazilian physician Carlos Chagas (1878-1934) who first described it in 1909. Chagas disease exists only on the American Continent and is caused by the protozoan parasite Trypanosoma cruzi and is transmitted to humans and animals in Venezuela by exposure to the feces of the assassin bug Rhod- nius prolixus (G6mez-Nhez , 1969).

Chagas disease represents a major public health problem in Latin

'This research was supported by Grants CDCHT-ULA C-1199-03-01-B and CDCHT-ULA- C1341-05 from the Universidad de Los Andes, a visiting professor grant from the Oficina de Intercambio Cientifico Universidad de Los Andes Merida, Venezuela, and the National Science Fundation SES-0244 142. Address correspondence to Dr. Charles I. Abramson, Laboratory of Comparative Psychology

and Behavioral Biology, Departments of Psychology and Zoology, 215 N. Murray, Stillwater, OK 74078.

722 C. I. ABRAMSON, ET AL.

America and is associated with poverty. Chagas disease, the fourth leading cause of death in Latin America, currently infects 16 to 18 million people, causing severe chronic illness and approximately 43,000 deaths per year (WHO, 1999).

The application of learning paradigms such as habituation, sensitization, Pavlovian conditioning, and instrumental conditioning to the study of invertebrate behavior has contributed important data in many areas of research in- cluding behavioral genetics, comparative psychology, and the biochemistry of learning and memory (Abramson, 1994). The study of invertebrate learning has also contributed to our understanding of how insects are influenced by agrochemicals such as pesticides and repellents (Abramson, Squire, Sheri- dan, & Mulder, 2004) and has been suggested as a mechanism for vectors to find their hosts (Kelly & Thompson, 2000).

In surveying the behavioral literature on assassin bugs, no studies in the literature were found investigating learning. Such studies are important in the control and understanding of Chagas disease to (1) contribute to our general knowledge of assassin bug behavior, (2) assess the limits of the assassin bug's adaptive behavior, (3) shed light on how the assassin bug finds human and animal hosts, and (4) provide a class of techniques that can be used as a bioassay in the assessment of repellents and pesticides developed to control the assassin bug.

Recently, studies have appeared in the literature on learning in another blood-sucking insect, the mosquito Aedes aegypti Alonso, Wyatt, and Kelly (2003) found no evidence of learning using olfactory conditioning adapted from research with fruit flies. McCall and Eaton (2001) and Kaur, Lai, and Giger (2003)) however, did find learning in the mosquito using a pre-imagi- nal paradigm which is conceptually similar to latent inhibition experiments.

In contrast to the investigation of learned behavior in assassin bugs, attention has been given to the study of orientation responses in R. prolixus. Wigglesworth and Gillett (1934) found that this species is attracted to its host by warm air and by the smell of skin. They also reported that the olfactory and thermal senses reside chiefly in the antennae. Nunez (1982) found that, prior to molting into an adult (technically known as the fifth-instar de- velopmental stage), R. prolixus was most attracted by hamster and forearm odors and not by warmth, carbon dioxide, moisture, or lactic acid alone. Taneja and Guerin (1995) found orientation responses in R. prolixus to the odors of mouse, C 0 2 , and rabbit urine.

Two experiments are reported here. The first assessed whether the behavior of R. prolixus could be modified by simple Pavlovian conditioning by pairing an olfactory conditioned stimulus with a temperature unconditioned stimulus. A temperature of 42°C elicits a complex behavioral sequence in R. prolixus consisting of proboscis extension and movement toward the source

LAB STUDY OF RHODNIUS PROLIXUS 723

of heat. The question of interest was whether the conditioned stimulus will elicit this sequence. In the second experiment, a latent inhibition paradigm was used to pre-expose assassin bugs to a conditioned stimulus of odor before pairing the odor with temperature. The classical conditioning paradigm used in these experiments is known as the instrumental approach design (Gormezano, Kehoe, & Marshall, 1983).

EXPERIMENT 1 Experiment 1 assessed whether the behavior of R. prolixus can be mod-

ified by Pavlovian conditioning.

Method

Subjects.-Adults from the same cohort of R. prolixus were used in Exp. 1 and Exp. 2. They were reared in the laboratory at 28°C and 50% relative humidity from a colony started 30 years ago. After molting from fifth-instar, adults were starved and tested 7 to 21 days later.

Materials.-The apparatus (Aldana, Otalora, & Abramson, 2005) con- sisted of two jars separated by a latex membrane. The upper jar contained a temperature-controlled environment in which a saline solution is heated to 42°C. The lower jar contained an individual subject. The subject reached the upper jar by climbing on a filter paper strip. Once at the top of the strip, the subject was permitted to pierce the latex membrane and feed on a saline solution (NaC1 0.85%) for approximately 1 sec. before being removed. If the subject was allowed to feed for a longer period of time, it became difficult to extricate its proboscis from the membrane. The apparatus was placed near an exhaust fan to remove extraneous odors from the testing environment. In addition, experimenters used gloves to apply the odors and did not wear any perfume or cologne during the experiment. The apparatus was used in both experiments and has been used previously in this laboratory to study feeding behavior of assassin bugs under controlled conditions.

Procedure.-Insects were collected from the laboratory colony and placed individually in glass vials. Twelve insects were collected on each day of training. Insects were maintained at room temperature in the vicinity of the apparatus until tested. Thirty-six subjects were tested for each odor used as a stimulus. Six odors were used, for a total of 216 subjects. An additional 72 insects were used as a control group.

Six separate groups of insects were exposed to a conditioned stimulus (CS) of almizcle (Perfumeria Vitel S.R.L., Caracas), cinnamon (Gilbertie's, Easton, CT), citral (Sigma Chemical, St. Louis, MO), ruda (Perfumeria Vitel S.R.L.), raccoon (Hein's Quality Lures, Lahoma, OK), or buck-lure (Hein's Quality Lures). The rationale behind testing different CS odors was to provide a range of odors to estimate better the possibility of conditioning. It is known from the invertebrate learning literature that not all conditioned


stimuli condition at the same rate and for some CSs such as light, no conditioning can be detected when animals are harnessed (Abramson, 1994). Al- mizcle and ruda are common colognes used by Venezuelan men; cinnamon has been used in studies of learning by honey bees (Abramson, et al., 2004). The animal odors were selected because raccoon and buck are not found in Venezuela.

It is important to note that before testing a new CS in our series, all 36 subjects within a given CS group were run. The rationale behind running all subjects in one group before moving on to the next group was to ensure that all subjects within a group had the same physiological condition and to ensure that feeding would be elicited.

The unconditioned stimulus was exposure to 42°C temperature. Previ- ous experiments from this laboratory have shown that R. prolixus will consistently crawl up to a filter paper strip with extended proboscis when the membrane separating the two chambers of the apparatus is heated to 42°C (Spagano, 1998). Conceptually, such behavior is similar to the proboscis extension elicited in honey bees to beeswax or high molarity sucrose solutions (Abramson & Boyd, 2001). If R. prolixus would crawl up the filter paper strip to a stimulus paired with temperature, such a situation would serve as a model for host-finding behavior.

To prepare the CS odors, 1.35 pl of an odor was applied to a cotton Q-tip. The Q-tip was then applied to the latex membrane. The odors of almizcle, cinnamon, ruda, and citral were replenished every 20 min. with a new Q-tip until the termination of the experiment. The odors of buck-lure and raccoon were replenished every 40 min. because of their strong smell.

A few moments after the CS was applied, a glass vial containing an insect was connected to the upper chamber of the apparatus. Responses to the CS were visually categorized into one of two states during each trial. If the insect walked up toward the latex membrane with extended proboscis, a "1" was recorded. If the insect remained still or walked toward the membrane without extending its proboscis, a "0" was recorded. The time between the CS and US was approximately in the millisecond range. We had no way to control the stimulus intervals precisely. When the jar containing the insect was attached to the upper chamber, the insect was immediately exposed to the CS and temperature.

Each insect received 12 acquisition trials followed immediately by 12 extinction trials. During extinction, only the odor was presented, and the temperature was decreased from 42°C to room temperature 122°C to 25OC). The time between trials, the intertrial interval, was fixed at 2 min. A trial be- gan by attaching the jar containing one insect to the upper chamber of the apparatus. If the insect made a conditioned response, the jar was removed and placed on a table near the apparatus. The jar was reattached for the


next conditioning trial after the remainder of the 2-min. interval had elapsed. If no conditioned response occurred for 2 min., the jar was disconnected, placed on the desk and immediately reattached, signaling the next trial. The rationale behind using a 2-min. intertrial interval was because this was used in a previous study of feeding behavior (Aldana, Lizano, Rodriquez, & Val- derrama, 2001).

In addition to the six Pavlovian conditioning groups, two control groups were employed of 36 subjects each. One group assessed the effect of room temperature (22 to 25°C) per se on proboscis extension and movement while no odor was presented. The second control group was exposed to 42°C and assessed the ability of 42'C to evoke proboscis extension unconditionally while walking. A preliminary experiment had been conducted to examine whether assassin bugs exposed to the odor of almizcle, cinnamon, ruda, citral, and raccoon under room temperature would begin to extend the proboscis over the course of 12 trials. None of the animals tested were ever observed to extend the proboscis. The odor of buck-lure was not tested because that would waste subjects, when it was difficult to obtain Iarge Sam- ples of animals under the same physiological condition.

Results Fig. 1 shows the acquisition and extinction results of Exp. 1, in which

separate groups of assassin bugs received a pairing of odor with temperature. Also shown are the results from the two control groups. The results clearly show that the majority of assassin bugs responded to the odors of almizcle, buck, and raccoon from the outset of training. Few responses were observed in animals in which the CS was cinnamon, citral, or ruda. With re- gard to the control groups, the insects seldom responded at room temperature but responses greatly increased when they were exposed to the higher temperature. An analysis of variance comparing the two control groups indicated a significant difference (F,,, = 701.05, p < .001).

Unfortunately, assassin bugs exposed to 42'C did not walk up the filter paper strip with an extended proboscis on all trials. Of the 36 insects, 29 responded at 42°C by walking up the filter paper with extended proboscis, and on any given trial about 60% of the sample responded with this behavior. The data suggest that a combination of raccoon or buck odor in compound with a temperature of 42'C would make a potent US if used in studies. During extinction, in which only the odor was presented, the decline was rapid, with few animals responding on the first or subsequent extinction trial.

Statistical analysis indicated a difference between groups receiving almizcle, buck, cinnamon, citral, raccoon, and ruda odors across the 12 acquisition trials (F,,,, = 944.14, p < .01). Post hoc analysis (Tukey method) indi-


4- Almizcle odor

1(1 Buckodor

* Cinnamon odor

I - Citral odor

+ Raccoon odor

+ Ruda odor

+ Room environment (22-25°C)

ILC 42OC (US) temperature

Trial

FIG. 1. Acquisition and extinction performance during excitator conditioning. The performance of the mro control groups is also shown. The transition getween acquisition and extinction takes place on Trial 13.

cated that raccoon versus buck odor (q= 1.70, p = .83) and buck versus almizcle odor (q = 3.23, p = .2 1 ) were not significantly different. This confirms that the 42°C temperature combined with buck or raccoon would make a potent US.

A learning interpretation of these results should be viewed with cau- tion. A hallmark of Pavlovian conditioning is that the CS does not elicit a conditioned response prior to its first pairing with the US. Clearly, the majority of the insects responded to the odor of almizcle, buck, and raccoon on initial presentation. This would be a more definitive example of Pavlovian conditioning had the performance of insects receiving a CS of cinnamon, citral, or ruda odor improved as training progressed.

Inspection of Fig. 1 suggests that some acquisition is evident within the first five trials in animals receiving a CS of almizcle, buck, and raccoon odor. An overall analysis of variance indicated no significant differences between the groups (F,,,, = 1.89, p = .19). The power of the test is .16, well be- low recommended values (30) . This is not of concern as what is important is acquisition effects that begin with a frequency near zero. No behavioral scientist would argue that the results of Fig. 1 represent learning.

The extinction results were also of interest. A learning interpretation re- quires a gradual decrease in the frequency of a conditioned response as the number of trials progress. This certainly was not the case. As soon as the


temperature decreased from 42'C to between 22 and 25°C (room temperature), responding to the odors ceased. No statistical analysis was conducted because there was no acquisition.

EXPERIMENT 2 Latent inhibition refers to the retardation of acquisition by the unrein-

forced pre-exposure of a CS. Latent inhibition has been demonstrated in a variety of mammals (Lubow, 1973) and in honey bees (Abramson & Bitter- man, 1986)) and crayfish (Hazlett, 2003). There were three reasons to per- form this experiment. First, it provided data on what is known in the conditioning literature as conditioned inhibition. The majority of conditioning studies use methods which generate what is known as excitatory conditioning. An example of excitatory conditioning is perhaps best illustrated with vertebrates by Pavlov's experiment in which he trained a dog to salivate to a bell by pairing a bell with feeding and with invertebrates by proboscis conditioning in which an insect learned to extend its proboscis to stimuli signaling food. Exp. 2 also had a practical aspect. Although the procedure in Exp. 1 was not sensitive enough to detect excitatory conditioning, it would be er- roneous to conclude that the procedure was not sensitive enough to detect learning. The procedure might be sensitive enough to detect inhibitory conditioning. Finally, because literally nothing is known about learning in assassin bugs, the idea was to test a wide range of conditioning phenomena in these first experiments on the problem of behavior modification in assassin bugs.

Method

Subjects.-The insects were obtained from the same colony and were tested in the same apparatus as those in Exp. I .

Procedure.-The procedure was similar to the previous experiment with the exception that, for the first 12 trials only, the CS odor was presented against a background of room temperature (22 to 25OC). On Trials 13 to 24 the CS odor was then paired with a temperature of 42'C. The intertrial interval remained at 2 min. The CS-US interval remained the same as that in Exp. 1.

In the experiment were 108 insects divided into three groups of 36 each. One group received a CS of almizcle odor, the second a CS of raccoon odor, and the third a CS of buck-lure odor. These stimuli were selected because they were the most effective stimuli used in Exp. 1. Animals receiving the odors of almizcle, raccoon, and buck-lure in Exp. 1 also served as our control groups. It is customary in latent inhibition experiments to have a group in which animals receive CS-US pairings at the outset of training. The animals trained in Exp. 1 were tested under the same physiological conditions as those in the present experiment.


Results Fig. 2 shows the results of Exp. 2 in which three groups of insects re-

ceived pre-exposure to almizcle, buck, and raccoon odors for 12 trials following which the odors were paired with a temperature of 42°C. The results of the group pre-exposed to almizcle or buck odor suggested a latent inhibition effect. An overall analysis of variance indicated significant differences among the three inhibition groups (F,,,, = 13.75, p < .001). Tukey post hoc tests indicated no differences in response between almizcle and buck odors (q=2.88, p = .12). Differences were detected between raccoon and almizcle odors (q = 7.35, p < .OOl) and between raccoon and buck odors (q = 4.47, p < .01).

C Almizcle

U Buck

+ Raccoon

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Trial

FIG. 2. Latent inhibition showing the transition from pre-exposure to paired training oc- curs on Trial 13.

Also performed was an analysis comparing the performance of assassin bugs trained at the outset with almizcle, raccoon, and buck odors in Exp. 1 with the performance of insects receiving latent inhibition training in Exp. 2. It is important to note that these experiments can be properly compared because the critical factor when making the comparison is that the insects from both experiments were under the same physiological conditions. Statistical


analysis indicated a significant difference between insects responding to almizcle odor between Exp. 1 and Exp. 2 (paired t , , =2.72, p < .05) and a significant difference in responding to buck odor between Exp. I and Exp. 2 (paired t,, = 2.44, p < .03). There were no significant differences in performance in insects receiving the odor of raccoon in Exp. 1 compared with those in Exp. 2 (p = .Id). These results showed that latent inhibition was found in the insects pre-exposed to almizcle and raccoon odors but not to the odor of buck.

GENERAL DISCUSSION The goal of these experiments was to provide data on the ability of the

assassin bug R. pvolzjcus to learn. Such experiments are important because of what they tell about assassin bug behavior and because they suggest that assassin bugs can find potential victims not only by innate behavior but by learning. The experiments are also important because the techniques used can be adapted as a bioassay to test the effects of potential repellents and pesticides on assassin bugs. Moreover, this experiment might stimulate other behavioral scientists to explore learned behavior in this medically important insect.

Although no evidence was found for excitatory Pavlovian conditioning in Exp. 1, a potent US and a potential CS was identified and can be used in other studies. Moreover, there was evidence for latent inhibition in Exp. 2. The results of Exp. 1 should not be interpreted as suggesting that assassin bugs do not possess excitatory Pavlovian conditioning, as it is possible that, with a larger number of training trials, conditioning might have occurred. Also, the correct training variables may still need to be identified. Of special interest was that the US of 42°C did not consistently elicit an unconditioned response of climbing with extended proboscis by all the subjects. To obtain reliable conditioning, it is necessary to have a US elicit an unconditioned response on each trial. One would expect that such an inconsistent US would have produced curves in all experimental groups resembling those found for cinnamon, citral, and ruda odors rather than the high performance found when almizcle, buck, and raccoon odors were used. Such data suggest that certain odors could be combined with the 42°C temperature to produce a potent US for use in further conditioning studies.

The possibility that assassin bugs do not show excitatory Pavlovian conditioning when the conditioned stimulus is neutral should also be considered. It is known from the literature that associating a neutral stimulus with an unconditioned stimulus is not a general property of invertebrate behavior (Abramson, 1994). As has been pointed out, however, in the case of assassin bugs, such a conclusion is premature. The results with citral odor, in which responding was between 30% and 40% on Trial I, suggest that a form of

73 0 C. I. ABRAMSON, ET AL.

conditioning known as alpha conditioning can be used to assay assassin bug learning (Abramson, 1997). Alpha conditioning is characterized as conditioned sensitization in which the CS elicits, prior to the first pairing with the US, a small version of the conditioning response that becomes larger as training proceeds. Experiments that pair a CS of citral odor with a compound US of 42°C temperature and raccoon odor may produce traditional acquisition and extinction curves. Moreover, the fact that latent inhibition was found suggests that excitatory conditioning would be found also.

Reproducible and stable results were obtained. When the temperature was increased to 42"C, most insects responded by extending the proboscis and climbing the paper strip. When the temperature was decreased to 22 to 25"C, climbing behavior immediately decreased. Moreover, it is clear that this technique is sensitive enough to be used in the testing of potential at- tractors and repellents. This suggests that the present technique can be used for studies of habituation and sensitization. Such examples of nonassociative learning form the basis of more complex learning such as Pavlovian conditioning and instrumental conditioning. The results also suggest the technique can be used for studies of aversive conditioning in which, for example, potential repellents can be applied against a background of an attractive stimulus. Such a technique is formally knoum in the Pavlovian literature as "conditioned suppression" and has been used to demonstrate conditioning in honey bees (Abramson, 1986).

In addition to studies of Pavlovian conditioning and nonassociative learning, a form of instrumental conditioning, i.e., behavior controlled by its consequences, known as punishment can also be investigated. Electric shock or some other aversive stimulus can be applied while the animal is climbing the filter paper strip in response to a compound unconditioned stimulus consisting of raccoon, buck, or almizcle odor and a temperature of 42°C. In- dependent variables such as the schedule of punishment, pattern of punishment, and intensity of punishment can be systematically investigated. It is known from the conditioning literature that punishment mimics the effect of positive reinforcement albeit in the opposite direction (Abramson & Satter- field, 1999). The advantage of using aversive stimuli is that motivation can be easily controlled and is often the only strategy an experimenter can use in an organism which eats sparingly.

In conclusion, the study of learning in assassin bugs appears to be an area of research worthy of further study. The results illustrate the richness that studies of learning can contribute to the study of assassin bug behavior. The ability of assassin bugs to modify their behavior as a result of experi- ence may help account for their exploitation of novel environments. For example, Panstrongylus geniculatus Latreille, 1811 is usually found in the bur- rows of armadillos but recently has been found in human dwellings (Reyes-

LAB STUDY O F RHODNIUS PROLIXUS 73 1

Lugo & Rodriguez-Acosta, 2000). Perhaps P. geniculatus learned to adapt to human dwellings and, as a result, now has access to human hosts.

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Accepted October 10, 2005.

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