J. Asia-Pacific Entomol. 8(4): 345-351 (2005)www.entomology.or.kr

PHYSIOLOGY AND BIOCHEMISTRY

Spatial Conditioning in the Flesh Fly, Sarcophaga crassipalpiS: Disruptionof Learning by Cold Shock and Protection by Rapid Cold HardeningYoung-Soo Kim*, D. L. Denlinger! and B. Smith!

Agricultural Biology Department, National Institute of Agricultural Science and Technology, Suwon 441-100, Korea'Department of Entomology, Ohio State University, Enarson Hall 154 W 12th Avenue, Columbus, Ohio 43210, USA

Abstract We have developed a new paradigm forexamining classical conditioning in the flesh fly,Sarcophaga crassipalpis, and specifically apply it asa sensitive measurement for sublethal effects of coldshock. When water was applied as a conditionedstimulus to the right tarsus and reinforced with abrief opportunity to feed on a sucrose-water solution,flies quickly leam to discriminate stimulation of theright from the left tarsus. Further analyses revealedthat the discrimination has a strong spatial com­ponent. Flies also retain this spatial discriminationover four days as indicated by the fact that thenumber of correct responses increases with continuedtraining over that time. Cold shock (-10°C for 3 min)applied 30 and 60 min after first training interferedwith learning. But, cold shock applied 120 minbefore or 90 min after conditioning did not signi­ficantly affect learning. The spatial memory we haveidentified therefore has a sensitive period duringwhich it can be disrupted with cold shock. Fur­thermore, we demonstrate that the disruptive effectof cold shock on learning formation can be preventedby rapid cold hardening, a brief pre-exposure to aless severe low temperature.

Key words classical conditioning, cold shock, fleshfly, rapid cold hardening, Sarcophaga crassipalpis,spatial discrimination

Introduction

Injury inflicted by cold shock can be subtle. Whilesevere forms of cold shock injury, the form of injuryelicited by a brief exposure to low temperaturesabove the insect supercooling point (Lee, 1991), cancause death, more mild forms of injury are manifestedin such activities as slight phase shifts of the insect's

*Corresponding author.E-mail: kbugguy@rda.go.krTel: +82-31-290-8528; Fax: +82-31-290-8503

(Received September 2, 2005; Accepted December 7, 2005)

circadian rhythm (Yocum et al., 1994). Much of theavailable evidence is derived from the nervous systemamong the systems most vulnerable to cold injury(Denlinger and Lee, 1998). In these experiments, wefurther investigate the possibility of nervous systeminjury by evaluating the capacity of cold shock todisrupt the processes of associative learning.

Learning enables animals to infer a correlationamong environmental stimuli and to use this in­formation to predict future rewards or threats. Evenstimuli that are not originally correlated in an animal'snatural environment can be experimentally correlatedto provide biologically meaningful information con­cerning such factors as food availability. This typeof associative learning, pioneered by Thorndike(1911) and Pavlov (1927), temporally links a condi­tioned stimulus (CS) with an unconditioned stimulus(US), which results in a change of behavior to theinitially neutral CS.

The capacity for associative learning has beendemonstrated in a number of insects including flies(Nelson, 1971), honey bees (Bitterman et al., 1983),an aphid parasitoid (Poppy et al., 1997), a cockroach(Li and Strausfeld, 1997), and a butterfly (Hem,1996). In flies and honey bees, the proboscis extensionreflex (PER) has been used effectively to demonstratea learned association for food expectation betweenCS and US (Dethier et al., 1965; Nelson, 1971;Menzel and Mueller, 1996). In this paradigm, appli­cation of a feeding stimulus (US) to taste receptorselicits the extension of proboscis among the otherappetitive feeding movements. Association of a neu­tral CS, eliciting strong mouth part movement, withthe US typically increases the probability of proboscisextension to the CS.

In this study, we demonstrate using the PERprocedure that adults of the flesh fly, Sarcophagacrassipalpis, have the capacity for associative lear­ning, and our detailed analysis revealed that spatialcues provide the basis for discrimination in thisparadigm. By administering cold shock to the fliesboth before and after training, we also demonstratethe impairment of memory in flies that have beencold shocked immediately after a training episode.

346 J. Asia-Pacific Entomol. Vol. 8 (2005)

We provide evidence that the cold shock impairmentofmemory can be ameliorated by rapid cold hardeningachieved within a short period of time at a moderatelylow temperature (Chen et al., 1991).

Materials and Methods

Insect rearing

The colony of S. crassipalpis was reared as described(Denlinger, 1972). To avoid pupal diapause inductionand associated cold hardening, insects were main­tained at 25°e under a long-day photoperiod (l5L:9D h). One or two days after adult eclosion, flieswere transferred from the main rearing cage to a'motivation cage' that contained only water. Flieswere given access to water alone for 48-60 h, thusmotivating them to respond to sucrose.

Conditioning protocol

Flies from the motivation cage were restrained withinthe tip of 1,000 ,ul micropipette. Approximately 5 mmof the micropipette tip was cut off so that the fly'shead and front legs could protrude through theopening. The fly's abdomen was held in place with200 ,ul micropipette embedded in plastic clay. Flieswere restrained for at least 30 min to adapt the restraintbefore the experiment.

The left tarsus was first touched with either distilledwater or 3 M salt solution for 4-5 s (CS 1), and thenthe right tarsus was touched with the rewardingsolution for 4-5 s (CS2). CS2 was associated with "1 M sucrose reward. The alternation between tarsiwas necessary to avoid mixing CS residues. Therewas approximately one second between the two tarsalcontacts because we had to switch from the watersyringe to the salt. When the right tarsus receivedCS2, flies were permitted to feed on the sucrose for4-5 sec. The inter trial interval (ITl) was always 7-10min. In the initial experiment, we always reinforcedCS2 when it was applied to the right tarsus. To resolvethis issue we performed two replicates of the originalexperiment; 1) randomized solutions: we consistentlyreinforced one tarsus but randomized which solutionwas associated with reinforcement; 2) randomizedtarsi: we consistently reinforced either the water orsalt component of the CS, but we randomized thetarsus that was stimulated with each solution. Sixteentrials were completed, with an alternative CS2 (A orB). The two options were presented in the followingsequence: ABBABAABABBABAAB (Bhagavan et

al., 1994).After determining that the solution quality (salt vs.

water) did not affect the association process, waterwas consistently used as the conditioned stimulus forall cold shock experiments. Tarsal side was used asthe CS. In trial A, the left tarsus was touched withwater, followed by sucrose. In trial B, the right tarsuswas touched with water, followed by 3 M salt.Sucrose was used as a reward in trial A and salt wasused as a punishment in trial B.

The sequence of learning trials was carried out atthe same time each day for four consecutive days.The response was measured on whether the fliesextended their proboscis to one or both CSs.

The error ratio expresses the mean number of errorsfor each fly. Because the A trials were a reward, thefly should learn to extend its proboscis in responseto trial A. If the fly did not respond to trial A, itwas assumed to have: made an error. Trial B, however,was a punishment, thus the fly should not have ex­tended its probosciis, and any extension was con­sidered an error. If a fly responded seven times outof eight to trial A and two times out of eight to trialB, we interpret this to mean that it made one mistakeon A and two on B. Thus, 3 errors out of 16 totaltrials could be calculated as an error ratio of 3/16(0.19).

Cold shock

For the administration of cold shock, groups of 3 flieswere enclosed in a small, cotton-plugged test tube(10 X 1.5 em) and placed for 3 min in a -10 oe ofLauda RM 20 (Brinkmann, OR, USA) bath filled withwater and ethylene glycol (I: I).

Flies were cold shocked at various times, eitherbefore conditioning or after completion of the trainingprocedure. The experimental procedure was schemati­cally summarized to investigate the effect of coldshock on learning formation in the adult stage, coldshock was administered 2 h before the first trainingsection, and a control group was trained without acold shock. To investigate the effect of cold shockon flies that had already been trained, cold shock wasadministered 1 h after the first training, and after 2h passed, the second training session began. A controlgroup was not cold shocked between the first andsecond training sessions. A 3 h interval separated theend of the first training session and the beginningof the second session (Fig. 1). In another set ofexperiments with adult flies, the period between thefirst training session and cold shock was varied. Theseexperiments were designed to define the time windowin which cold shock affects memory formation. Threedifferent intervals were tested: 30, 60, and 90 min.

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Fig. 1. Time schedule for administering cold shock after orbefore conditioning in 4-5 days-old adults. Cold shockconsisted of 3 min exposure to -10 'C. Cold shock wastreated 1 h after first training or 2 h before the first training.Usually the training time lasted 2 h. In another experiment,we varied the time interval between the end of first trainingand cold shock treatment: 30, 60, and 90 min, to look fora time window which the formed memory could be easilyaffected by cold shock.

Rapid cold hardening

To test whether rapid cold hardening could preventthe injury caused by cold shock, adult flies were firstexposed to O°c for 30 min and then exposed to coldshock (-I O'C for 3 min). Cold shock was consistentlyadministered 2 h before the first or second trainingsession.

Statistical analyses

Two-sample t-tests were used for most of the data.We normalized the distribution by log- or squareroot-transformation of the data. To compare the firsttraining responses with the second training responsesin the cold shock experiment, paired t-test was usedbecause the same individuals had two different res­ponses in this experiment set. Trial effect was ex­cluded in these analyses because it was obvious thatthe responses were always higher than 50 % and noother changes were noted after 3-4 trials. All datawas analyzed using the statistic package, MINITAB.

Results

Acquisition curve based on side-specifictarsal contact

This experiment was designed to test the learning

Spatial Conditioning in Sarcophaga Crassipalpis 347

capacity of the flesh fly using a traditional acquisitioncurve to evaluate a conditioned response. In the firstsequence, water was CS 1 and salt was CS2 and viceversa in the second sequence. The result revealed amuch more rapid increase in the rate of respondingto CS2, which was the stimulus most directly relatedto reinforcement. The relative difference between theresponse to CS1 or CS2 is typical for discriminationconditioning, reflecting by the positive slope of thedifference curve (Fig. 2A,B). In each sequence, theresponses to CS 1 and CS2 were significantly different(paired t-test, T == 9.71 for the first sequence; T ==14.06 for the second sequence; both df== 29, P <0.001).

The central component of CS2 was the side onwhich tarsal contact was made, but not the chemicalmakeup (salt/water) of the stimulus. We varied thechemical makeup, but always associated reinforce­ment with stimulation of the right tarsus. Subjectsrevealed the same pattern of discrimination as before,an increased frequency of response to stimulation ofthe right tarsus relative of the left. Their responseswere not significantly different from the nonrando­mized ones (two sample t-test, T == 0.70, df == 58, P== 0.49). When we randomized the side that wasstimulated, but always associated reinforcement witheither salt or water, the discrimination performancewas dramatically reduced (Fig. 2D: two sample t-test,T == 3.81, df== 42, P == 0.0014).

Gradual improvement in responses to bothreward and punishment

The memory of side-specific tarsal contact improvesover a four-day period (Fig. 3). The error ratio droppedfrom 6.13 in the first day to 3.07 on the fourth dayalthough overall responses to punishment as well asreward were increased. Interestingly, subjects appearto retain little from one day to the next as indicatedby comparing the responses to the first trial. But, fasteracquisition on subsequent days indicates some reten­tion of memory that is initially below a response­eliciting threshold.

Responses to cold shock administered atdifferent times

Fig. 4A shows the effect of 3 min cold shock at -10'Cadministered before and after conditioning training.As before, flies that were not cold shocked showeda classical acquisition curve over a period of 15 trialswhen the right tarsus was associated with sucrose.The response to cold shock administered before trai­ning was not significantly different from the response

348 J. Asia-Pacific Entomo!. Vo!. 8 (2005)

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Fig. 3. Discrimination learning over four days. A: sucrosewas applied to right tarsi and proboscis (reward). B: salt wasapplied to left tarsi and proboscis (punishment). These twodifferent trials, A and B, were pseudo-randomized as AB­BABAABABBABAAB (Bhagavan et al., 1994). Althoughthere was an overall response increase to reward as well aspunishment, the error ratio (ER) decreased from 6.13 to 3.07during the four days of testing.

in the control. In contrast, cold shock administered1 h after training significantly impaired learning(paired t-test, T = 4.97, df = 29, P < 0.001). Theresponses of proboscis extension after 4 trials in thecontrol group were above 80 %, while the responsesin flies' cold shocked after training dropped to 45% after 4 trials. Response variations also differed:the standard deviation in the responses of the controlflies (9.73) was much larger than in flies cold shockedafter training (5.70), indicating that the responsefollowing cold shock was quite consistent.

When flies were cold shocked at -10 °C within 30or 60 min after their first training session, the acqui­sition curve was low, with responses usually lowerthan 50 % (Fig. 4B). However, 90 min delay inadministering the cold shock did not adversely affectthe second training (not significantly different fromcontrol, two-sample t-test: T = 2.30, df = 56, P =

0.074).

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Spatial Conditioning in Sarcophaga Crassipalpis 349

A: Effect of cold shock on two trainingperiods Discussion

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This paper reports a new paradigm for studying spatialconditioning in flies. A major finding is that the fleshflies used in this experiment exhibit spatial learning.The flies were able to discriminate between differen­tially reinforced stimuli for up to four days. Normally,subjects would be expected to discriminate based onthe chemical composition of the conditioned stimuli(CS). However, when we randomized the side thateach CS stimulated, the discrimination was markedlyreduced. Further analysis revealed that subjects atten­ded to the spatial component of the CS. A secondmajor finding is the cold shock's amnesiac effect onmemory or its retrieval. The experiments revealed atime window most vulnerable to cold shock injuryand a cold-hardening mechanism against the injuryof cold shock.

Spatial learning permits animals to discriminate aplace by reference to surrounding landmarks (Bitter­man, 1996), and the animal' s body position relativeto these landmarks is important (Collett et al., 1996,1997). It is likely that the spatial learning we haveidentified relates to this kind of learning. Tactilestimuli influence a number of behaviors includingegg-laying in a generalist herbivore (Foster et al.,1997), posture adjustment of locusts (Newland andBurrows, 1997), communication between aphid para­sitoids and trophobiotic ants (Voelkl et al., 1996),and grooming or escape responses of cockroaches(Ritzmann and Pollack, 1998). Erber et al., (1997)showed that honeybees exhibit tactile motor learningin the antennal system. After honeybees scan an objectwithin the reach of the antennae with frequent, short­lasting antennal contacts, they continue to scan thearea where the object was previously located forseveral minutes. This suggests that bees acquireinformation about the position of an object via an­tennal scanning, and this acquisition of informationis proposed to represent a simple form of associativelearning.

In our experiments, two conditioned stimuli (CS)consisted of touching the left or right tarsi inassociation with sucrose reward. In his experiment,Dethier (1965) used the first conditioned stimulus,CS 1, as a pre-test which discharged the centralexcitatory state. However, CS 1 was offered as apre-stimulus that was not associated with sucrose (US)in this experiment and used as a signal for thefollowing associative process. CS2 was more closelylinked to US in time, thus it would be expected toelicit a greater response than CS 1. The reason thatthe flesh fly showed a stronger response to thephysical contact direction than to differences incomposition of solution could be explained several

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be prevented by exposing the flies to O°C for 30 minbefore subjecting them to a -10 DC cold shock for 3min (Fig. 4C). As noted, learning was impaired inflies that were directly exposed to -10 'C, but rapidcold hardening prevented this form of injury(Two-sample t-test: T = 0.31, df= 57, P = 0.75).

350 J. Asia-Pacific Entomo!' Vo!. 8 (2005)

ways. First, the physical contact ofCS2 was so strongthat the fly had no opportunity to distinguish whichsolutions were salt or water. Second, perhaps theextent ofhunger was too high to discriminate the smalldifferences in solutions. Because the flies wereseparated from the raising cage and moved to a'motivation cage' , in which they were offered onlywater for 48-60 h, they may have had a much strongerdrive to feed on sucrose. Our results suggest that bothphysical contact and the sense of taste can be usedto induce the PER, but the physical stimulation wasthe more powerful stimulus in this experiment design.

The second major finding involved the use of thisconditioning paradigm to develop a sensitive assayto detect sublethal effects of cold shock. Once theconditioning response was defined, we were able toevaluate perturbations of learning by exploiting coldshock as a tool to disrupt the response. Cold shockinjury can be manifested in diverse forms (Denlingeret al., 1998), and suggest that the conditioningparadigm can indeed be a powerful tool for evaluatingsubtle forms of this type of injury.

In these experiments, the cold shock (-10°C for 3min) decreased the fly's responses as revealed bothby a lower rate of acquisition and by a lower plateauof the acquisition curve. Although approximately 50% of flies survived all of the trials, they failed toextend their proboscis in response to sucrose. Thisimplies that they either lost their ability to associatethe CS and US, or alternatively they were unable toexpress the response, i.e. cold shock may havedamaged the central nervous system pathways thatare involved in memory formation or it may havedamaged the motor system that controls proboscismovement. Kelty et al. (1996) demonstrated impairedneuromuscular function in cold-shocked flesh flies asevidenced by a reduction in evoked end platepotentials.

The most likely scenario, however, is that coldshock exerted a direct effect on the memory processes.We suspect this is the case because the timing ofthe cold shock greatly influenced the fly's responses.The impact of cold shock was largely dependent onthe timing of the shock in relation to the first training.If the muscular system were injured by the cold shock,we would predict that the timing of the shock wouldnot influence the response. Cold shock within 60 minof the first training severely impaired learningformation, but a shock administered 90 min aftertraining had only a weak effect on the response. Thisimplies that more than an hour is needed to consolidatethe memory.

Similar time-dependent effect of memory formationhas been reported for the honey bee (Menzel et al.,1988), fruit fly (Xia et al., 1999), and a nematode(Morrison and van der Kooy, ]997). The memory

immediately after training exists as a short-lived,disruptable form, which then is consolidated withina few hours into a longer-lasting, stable form (Tullyet al., 1994). The transition from the labile form tostable form of memory can be reached by a differenttraining protocol such as spaced training (repeatedtraining sessions with a rest interval between each)and the labile memory can easily be disrupted by coldshock and cooling treatment. Tully performed amnesiaexperiments on fruit flies using cold shock andseparated the memory retention period into anes­thesia-sensitive memory (ASM) and anesthesia­resistant memory (ARM). The cold treatment orcooling was employed to disrupt memory and tolocalize the phase of memory consolidation. From ourexperiments with flesh flies, we conclude that thememory formed within 60 min is anesthesia-sensitiveand short-term because the formed memory wasdisrupted by cold shock. Previous studies with theflesh fly have demonstrated that other forms of coldshock injury can be eliminated or greatly reduced byfirst exposing the fly briefly to a less severe coldtemperature before subjecting it to the cold shock.For example, if S. crssipalpis is transferred directlyfrom 25°C to -10°C for I h, the injury is lethal, butif there flies are placed at O°C as briefly as 10 minbefore being placed at -10°C, they survive quite well(Chen et al., 1987). One ofthe events that occur duringthis period of rapid cold hardening is an accumulationof glycerol, a polyol that acts as a classic antifreezer(Lee and Denlinger, 1985), but it is likely that otherbiochemical responses also contribute to this form ofcold hardening. In this study, we tested whether aperiod of rapid cold hardening could also prevent thecold shock injury we Observed in association withmemory formation.

Our results clearly demonstrated that rapid coldhardening protected against the injury inflicted by coldshock on memory formation. Brief exposure to O°Cbefore the -10°C exposure prevented the memoryimpairment associated with a direct exposure to -10°C.Although the precise manner in which this protectionis achieved remains unknown, these results indicatethat the rapid cold hardening operating in these fliesto protect against development malformation (Chenet al., 1987), disturbance in circadian rhythmicity(Yocum et al., 1994), and reproductive malfunctionalso prevents injury to memory formation.

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