Journal of Insect Physiology 55 (2009) 943–951

The effect of environmental temperature on olfactory perception inDrosophila melanogaster

Jacob Riveron, Tamara Boto, Esther Alcorta *

Department of Functional Biology, Facultad de Medicina, Universidad de Oviedo, Calle Julian Claveria s/n, 33006 Oviedo, Spain

A R T I C L E I N F O

Article history:

Received 28 April 2009

Received in revised form 15 June 2009

Accepted 16 June 2009

Keywords:

Drosophila melanogaster

Environmental temperature

Insects

Olfaction

Olfactory acclimatization

A B S T R A C T

Olfaction provides chemical information to an animal about its environment. When environmental

conditions change, individuals should be able to adequately maintain function. Temperature may

influence olfaction in a double manner, as it modifies the concentrations of gaseous compounds and

affects biological processes. Here, we address acclimatization to environmental temperature in the

olfactory system of Drosophila melanogaster using heat and cold treatments. Because the consequences of

temperature shifts persist for some time after the treatment’s end, comparison of olfactory behaviors at

the same temperature in treated and untreated flies allows us to infer the biological effects of

temperature in olfaction.

At intermediate odorant concentrations heat always generates a reduction of olfactory sensitivity, as

they would be expected to compensate for the increase of volatiles in the air. Cold produces the opposite

effect. These changes are observed in both sexes and in natural populations as well as in standard

laboratory stocks.

Short applications suffice to cause detectable olfactory perception changes, but even prolonged

temperature treatments have only a transitory effect. Together, these results suggest that olfaction in

Drosophila underlies acclimatization to environmental temperature. However, sensitivity changes are

not immediate and may cause imperfect adjustment of olfactory function for short time periods.

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1. Introduction

In many species, olfaction is the primary sense that providesenvironmental information. In insects, for example, it is used tofind food, mates and oviposition sites. Given the importance of thefunctions it performs, the system should be able to retain its abilityto provide adequate information in the face of environmentalchanges.

Two main mechanisms serve to overcome temperaturevariations in nature; one involves thermosensation and mobility,which allows an organism to choose a habitat with theappropriate temperature range (McKemy, 2007), and the secondmechanism is related to the ability of the animal to adapt to newconditions.

Numerous biological phenomena have been associated withfluctuations in temperature. In poikilothermic animals, bodytemperature shifts with changes in environmental temperature.In insects, body size and the length of the developmental period areshaped by temperature (Ashburner, 1989). It has also been shownthat Drosophila populations undergo thermal adaptation in the

* Corresponding author.

E-mail address: ealcorta@uniovi.es (E. Alcorta).

0022-1910/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jinsphys.2009.06.009

laboratory (Laayouni et al., 2007) and in nature (Balanya et al.,2006; Overgaard and Sørensen, 2008; Kristensen et al., 2008).

Various effects of temperature shifts have been reported. At thebehavioral level, it has been shown for poikilothermic vertebratesthat nervous system activity can partially compensate fortemperature effects to preserve adequate function within a certainrange of temperatures. At the cellular level, however, temperaturefluctuations have a major impact on the functions of the nervoussystem and its components, inducing changes in conduction delayand synaptic gain (Montgomery and Macdonald, 1990). At themolecular level, several genes have been described that directlyrespond to heat and cold stresses, including the heat-shock-protein (Hsp) gene (Feder and Hofmann, 1999) and the cold-shock-protein (Csp) gene (Al-Fageeh and Smales, 2006).

Temperature may strongly influence olfaction, both by affectingbiological processes (especially in small poikilothermic insects, inwhich the large surface-to-volume ratio results in a rapid thermalequilibrium with the ambient temperature (Zeiner and Tichy,2000) and by modifying the gaseous odorant concentrationssurrounding the animal.

Several reports have addressed the ability of the olfactorysystem to adapt to high odorant concentrations in the environment(Dalton, 2000). In Drosophila, adaptation to high concentrations ofodorant has been described in larvae (Wuttke and Tompkins, 2000)

J. Riveron et al. / Journal of Insect Physiology 55 (2009) 943–951944

and adults. In adults, prolonged exposure may induce structuralchanges in the central nervous system (Devaud et al., 2001, 2003).Short exposures to undiluted odorants diminish subsequentbehavioral responses to the odorant. Electrophysiological signalsfrom the antenna in response to short pulses of odorant alsodiminished; this is related to the function of TRP Ca2+ channels(Stortkuhl et al., 1999; Deshpande et al., 2000).

The global effects of environmental temperature on olfactoryperception in the short term are not yet well understood.Temperature variations in nature constantly occur at differenttime scales, at different times of the day, and also when movingfrom one place to another (from sun to shade, for example). Ifenvironmental temperature variation is followed by olfactorysystem adjustment, do these changes compensate for the alteredconcentrations of odorants in the air? To answer this question, weinvestigated the effects of small temperature shifts at intermediatetime scales (3–48 h). We either increased or decreased tempera-ture (21 + 9 8C or 24 � 9 8C) in the range between 30 8C and 15 8Cand measured the olfactory sensitivity of Drosophila melanogaster.These conditions are quite common in temperate climates. Sincethe consequences of heat or cold treatments persist for some timeafter the treatment’s end, comparison of the olfactory behaviors oftreated and untreated flies at the same temperature allowed us toinfer the biological effects of temperature on olfactory sensitivity.

2. Materials and methods

2.1. Fly stocks

The wild-type Canton-S stock from the Bloomington StockCenter (Bloomington, Indiana, USA) was the only material used,except in one experiment. In the experiment performed toestablish the general characteristics of the effect, we utilized thewild-type stock Lausanne-S (Bloomington, ID, USA) and two othernatural populations captured in different locations in Asturias,Spain. P-1 and P-2 were each founded from more than 30 isofemalelines in 2004 and 2006, respectively.

2.2. Temperature treatments

In order to perform enough behavioral tests to completelycharacterize the possible effects of temperature, a maximum of 60behavioral tests (Y-mazes) with 40 flies in each were carried outsimultaneously. This set-up allowed us to apply temperaturetreatments during very precise time periods, but also prevented usfrom using behavioral paradigms like the T-maze, which requirescontinuous manipulation. T-mazes were therefore used onlyoccasionally. Since the Y-maze measures olfactory preferenceover the course of 30 min, large temperature treatments (usually of48 h) were initially chosen in order to maintain the effectthroughout the test duration. The presence of the same tempera-ture effect for shorter treatments was directly addressed in asubsequent experiment.

Fig. 1 depicts the different temperature treatments applied tothe flies before the behavioral tests. The basic protocol for heat orcold treatments used in most experiments is shown in Fig. 1A. Forthe heat treatment experiments, the control (CH) and experi-mental (EH) groups differed only in the heat period (H), where fliescultured at 21 8C were subjected to 30 8C conditions for a certaintime period. Afterward, flies were maintained at the testtemperature (24 8C) for 90 min prior to the behavioral tests (foracclimation purposes). The duration of the acclimation period wasdetermined by the results of the preliminary experimentsdescribed in supplemental material (S1 and S2). The durationwas dependent upon the time necessary for our experimentalconditions stabilize the flies’ biology and to reach the environ-

mental temperature inside the tubes containing the flies. After thistime, we could assume that body temperature would remainconstant during the 30-min Y-maze test. Thus, flies completing themaze in the first few minutes could be considered similar to thoseresponding more slowly. The same principles were applied to thecold treatment protocol, in which the experimental group (EC)cultured at 24 8C was subjected to a 15 8C cold period (C).

In order to evaluate the durability of the effect produced by thetemperature treatment, a second protocol was used (as depicted inFig. 1B). The new IH and IC groups (for heat or cold treatments,respectively) incorporated an intermediate extinction period aftertemperature treatment.

Fig. 1C depicts the heat treatment applied in the T-mazeexperiments. In this case, no acclimation time was incorporateddue to the short duration of the behavioral tests (1 min) and to thefact that flies were moved to a space other than the tube wherethey received temperature treatments prior the test. Thus, thisprotocol was developed to show the short-term effects of thetemperature treatment.

Control and experimental flies were maintained in a specialculture medium containing agarose gel (5 g/l) and sucrose (50 g/l)during the temperature treatment protocols. The medium is nearlyodorless, and therefore did not differentially affect the two groups.

2.3. Behavioral tests

Two different behavioral paradigms have been used to testolfactory preference: the Y-maze and the T-maze. For mostexperiments, a double choice, horizontally placed Y-maze wasselected (Alcorta and Rubio, 1989; Martin et al., 2002). During the30-min experiment, 40 females (except where otherwise indi-cated) that had been starved for 24 h chose between two tubes: astimulus tube containing filter paper soaked with 0.5 ml of acertain concentration of odorant, and a control tube with 0.5 ml ofsolvent.

The second assay, the T-maze (Helfand and Carlson, 1989), wasalso a double-choice olfactory preference test. In this case, 30 flieswere introduced into a central chamber in the sliding vertical platefrom the start compartment. Once the plate was slid into thebottom position, flies could choose between the left and right sidesduring the 1-min experiment. One side was connected to a tubecontaining a filter paper soaked with the odorant at a certainconcentration, and the other side was connected to a tubecontaining the solvent. This assay was performed in completedarkness.

An olfactory index (OI) was calculated by the number of flies inthe stimulus tube divided by the total number of flies reaching theend of the maze at either end. This index measures olfactorypreference from the total number of flies that chose one of the armsin the Y-maze, and disregards those flies that do not move from theinitial tube. The choice of such an index, which is dependent onlyon the number of moving flies, is especially important in this reportsince temperature could directly affect the mobility of the flies.

OI values ranged from 0 (maximum repulsion) to 1 (maximumattraction), with the threshold of indifference at 0.5. Twentyreplicate tests were performed for each line and stimuli. Thenumber of replicate tests was increased in cases where differenceswere at the limit of statistical significance.

2.4. Odorants

Responses to different concentrations of ethanol and acetone inwater and ethyl acetate and benzaldehyde in paraffin oil wereexamined. All chemicals were obtained from Merck (Darmstadt,Germany). All of these compounds are known to be odorousstimuli for D. melanogaster.

Fig. 1. Temperature treatment protocols. For heat treatment, flies cultured at 21 8C were transferred to 30 8C. In the cold treatment protocol, flies were changed from 24 8C to

15 8C. Acclimation period and tests were always carried out at 24 8C. (A) BT = breeding temperature, H = heat period, C = cold period, A = acclimation (90 min), Y = olfactory

test in the Y-maze (30 min). CH = control group and EH = experimental group for the heat treatment. CC = control group and EC = experimental group for the cold treatment.

(B) E = extinction time. IH = intermediate group for heat treatment and IC = intermediate group for cold treatment. Both I groups include heat or cold treatments and

additional extinction times before acclimation and behavioral test in the Y-maze. (C) T = olfactory test (1 min) in the T-maze.

J. Riveron et al. / Journal of Insect Physiology 55 (2009) 943–951 945

2.5. Statistical analysis

Direct comparisons between the experimental and controlgroups for each odorant concentration and experimental conditionwere performed using Student’s t-test for the Olfactory Index (OI)after applying the arcsine transformation to assure normality(Martin et al., 2002). For the last experiment, which includedseveral controls, an analysis of variance (ANOVA) followed by amean comparison a posteriori test was applied for each condition.

3. Results

3.1. Olfactory perception changes depend on environmental

temperature

Flies exposed to 48 h of heat or cold treatment (Fig. 1A) weretested for behavioral response to odorants. Because the effectsinduced by the temperature change persisted for some time afterthe treatment’s end, we can compare the olfactory responsesdisplayed by this experimental group and untreated controls at thesame temperature for direct evaluation of the biological effects oftemperature change on perception.

A complete dose–response curve in the Y-maze was performedin response to two odorants: ethanol and acetone (both producedin natural conditions of fruit fermentation) in wild-type Canton-SD. melanogaster females (Fig. 2). The square at the right side of the

figure summarizes the temperature protocol. Twenty to 25 Y-mazereplicated tests (each testing 40 female flies) were carried out foreach group (EH or CH) and each concentration. Statisticalsignificance for each concentration was estimated by Student’st-test comparing the EH and CH group values at that concentration.Data including Olfactory Index values and standard error of themean (SEM) are shown in Fig. 2.

For concentrations of ethanol evoking an attractive response(above the dashed line), the previous heat treatment experiencedby the experimental group (EH) tended to induce more extremeattractive responses than those displayed by the control group(CH). The differences became statistically significant at the 10�3

concentration (t = 2.043, nEH = 21, nCH = 20, p = 0.0479). For con-centrations evoking a repellent response (under the dashed line),no differences in olfactory behavior were observed between thegroups for extremely high concentrations of odorant. However,responses to the intermediate concentration of 10�1 appearedsignificantly less repellent in the heat-treatment group (t = 2.335,nEH = 22, nCH = 21, p = 0.0245).

In response to acetone, no differences were observed betweenthe control and experimental groups at the concentrations thatevoked attractive responses. However, in the region of repellentresponses, the heat treatment induced a diminished sensitivity atthe intermediate concentration 10�2 (t = 4.429, nEH = 20, nCH = 23,p = 0.0001) and 10�1.5 (t = 2.061, nEH = 23, nCH = 23, p = 0.0453),which disappeared at extremely high concentrations of odorant. In

Fig. 2. Dose–response curves to ethanol and acetone after applying the treatment

protocol summarized in the square at the right of the figure. H = heat period,

A = acclimation time and Y = Y-maze test. EH = experimental group (30 8C),

CH = control group (21 8C).

Fig. 3. Effects of the heat treatment protocol presented at the top of the figure in the

response to four different odorants at concentrations that trigger intermediate

repellent responses. H = heat period, A = acclimation time and Y = Y-maze test.

Odorant concentrations (v/v): ethanol 10�1, acetone 10�2, ethyl acetate 10�1.625

and benzaldehyde 10�3. EH = experimental group (30 8C), CH = control group

(21 8C).

Fig. 4. Dose–response curve to ethanol after the cold treatment protocol included in

the square. C = cold period, A = acclimation time and Y = Y-maze test.

EC = experimental group (15 8C), CC = control group (24 8C).

J. Riveron et al. / Journal of Insect Physiology 55 (2009) 943–951946

this report, the concept of ‘‘olfactory sensitivity’’ refers broadly tothe odorant concentration required to evoke the same behavioralresponse in different conditions. Thus, a diminished sensitivityindicates that a higher odorant concentration is needed to obtainthe same OI.

The effect that induced intermediate repellent responses foracetone resembles that described for ethanol.

The same effect was observed at intermediate repellentconcentrations (Fig. 3) in response to ethyl acetate 10�1.625

(t = 2.386, nEH = 19, nCH = 19, p = 0.0224) and benzaldehyde 10�3

(t = 2.121, nEH = 29, nCH = 32, p = 0.0381), although we observeddiminished attraction to ethyl acetate and no difference forbenzaldehyde at low concentrations (data not shown).

A dose–response curve was also constructed to compare flies inthe control group to those subjected to 48 h of cold treatment priorto the test in response to ethanol (Fig. 4). In this case, 8–14replicate tests were carried out for each odorant concentration andgroup (EH or CH).

The effect of the cold treatment was exactly the opposite of thatof the heat treatment for both low and intermediate concentra-tions of ethanol. Olfactory index differences became significant atthe 10�3.5 (t = 2.323, nEC = nCC = 8, p = 0.0358) and 10�3 concentra-tions (t = 3.522, nEC = nCC = 9, p = 0.0028). At the 10�1.5 concentra-tion, which induced intermediate repellent responses, weobserved an increased sensitivity after the cold treatment(t = 2.186, nEC = 14, nCC = 13, p = 0.0384).

All the results obtained at the intermediate odorant concentra-tions are compatible with the hypothesis that olfactory sensitivitychanges compensate for the changing concentration of odorant inthe airborne phase induced by temperature shifts in naturalconditions. Thus, heat that induces an increase of odorantconcentration in the air produced a decrease in olfactorysensitivity. The opposite is observed after cold treatments.

In light of this finding and the constant effect observed at theintermediate repellent region, we plan to further analyze thetemperature-drive perception changes in this region. Moreover,the repellent region is the only part of the dose–response curvewhere we can establish a linear relationship between olfactoryresponse and odorant concentration and therefore can estimatesensitivity changes at the behavioral level (Devaud, 2003).

J. Riveron et al. / Journal of Insect Physiology 55 (2009) 943–951 947

3.2. Olfactory sensitivity changes due to temperature are

general in D. melanogaster

Because the experiments described above involved onlyfemales of the Canton-S stock, the question of possible sexualdimorphism was addressed. The responses of males of the Canton-S line to ethanol and acetone after heat or cold treatments arepresented in Fig. 5. In all cases, a single concentration of odorant inthe intermediate repellent response region was tested. Althoughthere is some variability in OI values among experiments with fliessubjected to different conditions, control and experimental groupsfor each comparison were always tested together at the sameodorant concentration, which was chosen because it evokedintermediate repellent responses.

Heat treatment induced a significant decrease of sensitivity inresponse to ethanol 10�1.75 (t = 3.671, nEH = nCH = 20, p = 0.0007) aswell as acetone 10�1 (t = 4.368, nEH = 40, nCH = 37, p = 0.0001), andresponses appeared less repellent in the experimental group (EH)than in the control group (CH).

After the cold treatment, olfactory sensitivity increasedsignificantly, producing more repellent responses in experimentalflies (EC) than in control flies (CC) in response to ethanol 10�1.75

(t = 2.280, nEC = nCC = 33, p = 0.0259) and acetone 10�2 (t = 2.898,nEC = nCC = 26, p = 0.0056).

The observed differences in males are completely in line withthe results obtained in females. Again, heat and cold treatmentshad opposite effects on olfactory perception.

To extend our conclusions on the effects of temperature onolfactory function to Drosophila stocks other than the Canton-Sline, we tested olfactory responses in the intermediate repellentregion to ethanol and acetone in four other stocks: Canton-S andLausanne-S, as wild-type laboratory stocks, and P-1 and P-2, asnatural populations captured 2 years and 2 months before theexperiment, respectively.

Fig. 5. Effects of heat and cold treatment (see the square at the right side of the

figure) on the olfactory response of males of Drosophila melanogaster to

intermediate repellent concentrations of ethanol and acetone. EH = experimental

group (30 8C), CH = control group (21 8C). EC = experimental group (15 8C),

CC = control group (24 8C).

Fig. 6A depicts the OI values obtained in response tointermediate repellent concentrations of ethanol for the control(CH) and the experimental (EH) groups submitted to heattreatment. Significant differences were found for the Lausanne-Sline (t = 2.294, nEH = 40, nCH = 39, p = 0.0245) in the same directionas the Canton-S flies (t = 2.335, nEH = 22, nCH = 21, p = 0.0245).However, no significant differences were found for the two naturalpopulations, P-1 (t = 1.025, nEH = nCH = 21, p = 0.3121) and P-2(t = 1.108, nEH = 28, nCH = 29, p = 0.2725). The lack of a pattern inboth natural populations in response to ethanol in the Y-maze hastwo potential explanations: either the effect is not present in theselines, or it disappeared more quickly than in the wild-typelaboratory stocks. To discriminate between the two hypotheses,olfactory perception after heat treatment was measured in adifferent behavioral paradigm: the T-maze. This apparatusmeasures olfactory preference in only one minute and thereforedoes not require flies to undergo the 90-min acclimation periodafter heat treatment and before the test (as is required for the Y-maze). When flies of the natural populations P-1 and P-2 weretested in the T-maze for olfactory preference after heat treatment,EH flies displayed decreased repellent responses compared to thecorresponding control flies (CH). Differences were near statisticallysignificant for population P-1 (t = 1.704, nEH = 21, nCH = 22,p = 0.096) and were significant for population P-2 (t = 2.295,nEH = 33, nCH = 32, p = 0.0251) in the same direction as both theCanton-S and Lausanne-S wild-type stocks. These data suggest thatthe ethanol perception changes induced by temperature also existfor natural populations.

When acetone was used as the olfactory stimulus (Fig. 6C), heatinduced a diminished sensitivity (less repellence) in all the testedstocks in the same direction as was observed for ethanol in the Y-maze. Thus, responses in the Canton-S stock were significantlydifferent after heat treatment than those in untreated Canton-Sflies (t = 4.429, nEH = 20, nCH = 23, p = 0.0001). The same was truefor Lausanne-S flies (t = 3.701, nEH = nCH = 22, p = 0.0006), P-1(t = 4.011, nEH = nCH = 19, p = 0.0003) and P-2 (t = 3.028, nEH = 19,nCH = 17, p = 0.0047) stocks.

3.3. How much time must the temperature treatment last in

order to affect olfactory perception?

These experiments were intended to test whether the treat-ments produced rapid acclimation to normal daily temperaturefluctuations or more lasting temperature changes. Since the Y-maze allowed us to observe only those perception differences thatwere maintained through the 90-min acclimation period and 30-min test, our results do not precisely reflect natural conditions,though they may help to discriminate between ephemeral andlong-lasting temperature effects.

Previous experiments were performed using a 48-h period oftemperature treatment to magnify the effect on perception. In thepresent experiments (Fig. 7), the time of treatment wasprogressively reduced to 3 h for heat and cold treatments, buttreatments lasting less time were not used. In all cases, we mustconsider that the duration of the temperature treatment extendsfrom the moment that flies are introduced in the heat or coldchamber, though approximately 90 min are required to achieve thecorresponding temperature inside the tube containing the flies(see the supplemental figure S1). Statistically significant differ-ences in ethanol perception were observed in the heat treatmentafter as little as 3 h of treatment (t = 2.840, nEH = 38, nCH = 40,p = 0.0058). These results clearly suggest a rapid effect oftemperature on fly olfactory perception.

In response to the cold treatments, a minimum of 6 h wasrequired to produce a significant perception effect in ourexperimental conditions (t = 2.204, nEC = nCC = 13, p = 0.0374).

Fig. 6. EH = experimental group (30 8C), CH = control group (21 8C). (A and C) Effects of the heat treatment protocol (right side of the figure) on the olfactory response to

intermediate repellent concentrations of ethanol and acetone of four different strains. C-S = Canton-S and L-S = Lausanne-S wild-type laboratory stocks, and P-1 and P-2

natural populations captured in 2004 and 2006, respectively. (B) Effects of the corresponding heat treatment protocol (see box at the right side) on the olfactory response to

intermediate repellent concentrations of ethanol measured for the P-1 and P-2 populations in the T-maze.

J. Riveron et al. / Journal of Insect Physiology 55 (2009) 943–951948

Although biological changes in response to heat and cold protocolsshowed slightly different kinetics, we can assume in both casesthat we are describing a rapidly arising effect that may reflectnormal adjustments to short-term temperature fluctuations.

3.4. Changes in olfactory perception due to temperature

shifts are transitory

If the effects we are describing do in fact reflect a normal responseto environmental conditions, we would expect rapid extinction aftercessation of the temperature treatment. Although extinction timewill probably depend on the duration of the previous heat or coldtreatment, we tested it only for our longest treatment (48 h). In theseexperiments, new periods at control temperature (E) were addedafter treatment and before the 90-min acclimation time in thetemperature protocol. A square at the right side of each graphsummarizes the temperature protocol that was applied. To detectstatistically significant differences, an ANOVA compared the threegroups and an a posteriori test (Fisher PLSD) compared pairs of thethree means when differences were found by ANOVA.

We studied responses to ethanol in the intermediate repellentresponse region after heat treatment (Fig. 8A) for two differentconditions, testing extinction times of 3 h (F = 16.165, nCH = 20,nIH3 = nEH = 19, p = 0.0001) and 6 h (F = 6.338, nCH = nEH = 20,nIH6 = 19, p = 0.033). When flies were subjected to three additionalhours at the control temperature before the test (IH3), the originaldifferences in olfactory sensitivity seen among treated (EH) andcontrol groups (CH) persisted, and no differences were observedbetween the IH3 and EH groups. However, when the extinctionperiod in the treatment protocol was extended to 6 h (IH6), OIdifferences with the control group of untreated flies (CH)disappeared. This suggests that the olfactory perception differ-ences we observed in response to the 48-h heat treatmentpersisted for at least three additional hours (during which wefound intermediate values), but ceased before 6 h. Therefore, evenfor the most extreme conditions (48 h of treatment), the effect wastransitory.

Parallel experiments were carried out for flies subjected to 48 hof cold treatment (Fig. 8B) and 3 h (F = 6.680, nCC = 17, nIC3 = 16,nEC = 14, p = 0.0029) or 6 h (F = 4.754, nCC = 19, nIC6 = 18, nEC = 14,

Fig. 7. Olfactory responses to intermediate ethanol concentrations as a function of temperature treatment duration of flies subjected to different heat or cold periods in the

temperature treatment protocol (see the square boxes at the right for details). EH = experimental group (30 8C), CH = control group (21 8C). EC = experimental group (15 8C),

CC = control group (24 8C).

J. Riveron et al. / Journal of Insect Physiology 55 (2009) 943–951 949

p = 0.0131) of extinction time. In these cases, treated flies did notdiffer significantly from untreated flies (CC) after three additionalhours at control temperature following cold treatment (IC3). Asexpected, the result was confirmed in the experimental groupsubjected to 6 h at control temperature before the experiment(IC6). Therefore, changes in olfactory perception induced by 48 h ofcold treatment were even more transient than those due to heattreatments in our experimental conditions. Moreover, persistenceof olfactory perception changes due to temperature seem to beindependent of the age of the tested animals, since young fliesrecently emerged from pupae did not display behavior thatdiffered significantly from that of older flies (data not shown). Suchpermanent effects involving developmental processes have beendescribed for adaptation to the presence of high concentrations ofodorant in the environment (Devaud et al., 2003).

4. Discussion

These results suggest that olfactory preference is affected by thethermal history of the fly, and that preference changes when theanimal has previously been subjected to a different environmentaltemperature. Moreover, the type of change we found depends onthe direction of the previous environmental temperature shift. Ifwe compare the odorant concentration needed to evoke abehavioral response after the temperature treatment betweenexperimental flies and control flies, we can deduce how olfactorysensitivity changes. Thus, we found that sensitivity eitherincreased or decreased when flies were subjected to cold or heat,respectively. We would expect to observe this pattern if the fliesadjust their perception to the odorous background level. The

described effect reflects olfactory changes in response to previoustemperature conditions and, as such, may represent flies’responses to temperature changes over the course of a day orseveral days in natural conditions, although such adjustment is notimmediate. However, some degree of variation in the time-courseof adaptation to a certain temperature has been found, dependingon the strain and the odorant. In fact, the two natural populationsanalyzed for response to ethanol showed a faster sensitivityadjustment after heat treatment compared to the other tested linesand to their own acetone response. Genetic variation for somemetabolic processes and changes in membrane fluidity bysequestering ethanol in cell membranes may account for thisdifference (Montooth et al., 2006). The need for a habituationperiod has been previously described for other sensory modalities,such as the visual system (Kohn, 2007) and the olfactory system(Dalton, 2000), though previous studies focus on the adaptation todifferent levels of sensory stimulation.

Environmental temperature affects two processes: the con-centration of volatiles in the air, and also the internal physiologyof the animal, especially body temperature in poikilothermicanimals, membrane fluidity (Wodtke, 1981; Kashiwayanagiet al., 1997a; Ohtsu et al., 1998; Overgaard et al., 2005) andmetabolic rate (Gillooly et al., 2001; Clarke, 2006). Our finding ofa persistent component of previous acclimation allows us todisentangle both components, since olfactory behavior wastested after the treatments’ end in experimental and control fliesat the same temperature. Moreover, to prevent side effectsduring temperature treatments, the culture medium was nearlyodorless (agarose gel + sucrose) for both experimental andcontrol groups.

Fig. 8. Analysis of extinction times for heat and cold effects following the corresponding treatment protocols (see the square boxes at the right for details) in response to

ethanol. EH = experimental group (30 8C), CH = control group (21 8C). IHN = N hours intermediate period after heat treatment and before the test. EC = experimental group

(15 8C), CC = control group (24 8C). ICN = N hours intermediate period after cold treatment and before the test.

J. Riveron et al. / Journal of Insect Physiology 55 (2009) 943–951950

One may ask whether the direction of the observed changes inolfactory sensitivity due to temperature corresponded to the shiftin concentration of volatiles. Our results suggest that appropriateacclimation occurred in the olfactory system, though this was notthe case for all the odorant concentrations range. At intermediateconcentrations, changes in olfactory perception do adjust to theexpected concentration if the system compensates for the changesin odorant concentration in the air due to temperature fluctuation.Olfactory sensitivity diminished when environmental temperature– and therefore odorant concentration in the air – increased and,conversely, olfactory sensitivity increased when colder tempera-tures were applied.

Although some changes in olfactory behavior were found at lowodorant concentrations, they depended on the odorant type andcould not be easily related to the olfactory function adjustmentsdue to the temperature. However, we must point out that olfactoryresponses in adult flies follow an attractive–repellent patterndepending on odorant concentration, which is only linear in therepellent region. Therefore, for attractive responses, no clearcorrelation could be established between olfactory index andodorant concentration. Finally, at extremely high concentrations(probably out of the natural range), odorant becomes extremelyrepellent to both experimental as well as control flies.

From a biological point of view, several processes can explainthe concentration dependency of the effects. It has beendemonstrated in turtles that heat-induced changes in cellmembrane fluidity of the olfactory receptor neurons (ORNs)differentially affect both main olfactory transduction cascades

(mediated by cAMP and IP3, respectively), making them morepronounced for the IP3 cascade (Kashiwayanagi et al., 1997b). Ifthe response to different concentrations of the same odorantinvolves a different relative contribution of both cascades, thismay explain the diverse effects of temperature on olfactoryperception across a range of odorant concentration. Moreover,olfactory discrimination among related compounds diminishesat high temperatures (Linn et al., 1988; Hanada et al., 1994) andhigh odorant concentrations (Kashiwayanagi and Nagasawa,1995).

From the ecological point of view, accurate estimation ofolfactory information could be important not only for finding foodor mates but also for avoiding predators. Thus, predation riskassessment by scent has been studied in birds (Roth et al., 2008). Ingeneral, correct olfactory information may be more critical atintermediate than at low concentrations of odorant. For example,volatility changes cause the number of ethanol molecules in thegas phase to increase by 63.45% when the temperature shifts from21 8C to 30 8C in a closed system at the environmental conditions ofhumidity and pressure of the city of Oviedo (Reid and Sherwood,1966; Prausnitz et al., 1998; Poling et al., 2000). Such an increasemay become critical for finding the odorant source over a shortdistance (corresponding to intermediate concentrations), but maynot be important at low concentrations, especially considering thatmany flying insects follow odor plumes (Carde, 1996; Vickers,2000; Budick and Dickinson, 2006). However, the relatively slowtime-course of adaptation deduced from our experiments impliesthat, when such temperature conditions appear in nature, there

J. Riveron et al. / Journal of Insect Physiology 55 (2009) 943–951 951

may be time periods of imperfect adjustment of the olfactorysystem to environmental conditions. These effects may also beimportant for especially variable climates.

Additional work at the cellular and molecular levels may help tofurther elucidate the basis of olfactory sensitivity changes andbiological constraints that lead to better adaptation to environ-mental conditions.

Acknowledgments

We thank Fernando Martin and Carolina Gomez-Diaz for helpwith the experimental work and Jose Maria Berrueta for hiscontribution to the theoretical calculations of odorant concentra-tion in the gas phase across temperatures. This work wassupported by the Spanish Ministry of Education and Science andthe PCTI Program of the Principado de Asturias. J. Riveron is a FICYTpredoctoral fellow and T. Boto is a FPU predoctoral fellow of theSpanish Ministry of Education and Science.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jinsphys.2009.06.009.

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