Proc. Nati. Acad. Sci. USAVol. 86, pp. 8118-8122, October 1989Neurobiology

A simple chemosensory response in Drosophila and the isolation ofacj mutants in which it is affected

(olfaction/behavior genetics/sensory system/brain/antennae)

MICHAEL MCKENNA, PAULA MONTE, STEPHEN L. HELFAND, CRAIG WOODARD, AND JOHN CARLSONDepartment of Biology, Yale University, Box 6666, New Haven, CT 06511

Communicated by Alan Garen, July 10, 1989

ABSTRACT Although the Drosophila visual system hasbeen described extensively, little is known about its olfactorysystem. A major reason for this discrepancy has been the lackof simple, reliable means of measuring response to airbornechemicals. This paper describes a jump response elicited byexposing Drosophila to chemical vapors. This behavior pro-vides the basis for a single-fly chemosensory assay. The be-havior exhibits dose dependence and chemical specificity: it isstimulated by exposure to ethyl acetate, benzaldehyde, andpropionic acid but not ethanol. Animals can respond repeatedlyat short intervals to ethyl acetate and propionic acid. Theresponse relies on the third antennal segments. To illustrate theuse of this behavior in genetic analysis of chemosensory re-sponse, nine acj mutants defective in response are isolated (acj= abnormal chemosensory jump), and their responses to twochemicals are characterized. All of the acj mutants are normalin giant fiber system physiology, and two exhibit defects invisual system physiology.

Although the visual system of Drosophila has received agreat deal of attention, very little is known about its othersensory systems. A substantial portion of the animal's ner-vous system subserves the perception of chemical stimuli,which play an important role in the location of food andoviposition sites and which mediate various forms of sexualbehavior (1, 2).

Genetic analysis has proven a valuable means of address-ing the function and development of the Drosophila visualsystem. A wide variety of molecules essential for vision havebeen defined by mutation and investigated by genetic ma-nipulation (3). The power of the genetic approach, however,is critically dependent on the means available for isolatingand analyzing mutants. The success of genetic analysis ininvestigating the visual system is in large measure due to theavailability of a variety of approaches for measuring thebehavioral, physiological, and morphological consequencesof genetic perturbation.The example set by analysis of the visual system suggests

the potential of genetic analysis in addressing a number ofcentral problems in chemosensory function and develop-ment, including the mechanism of transduction and theprinciples of chemosensory coding. Precedent for the use ofsingle-gene mutations in analysis of the Drosophila olfactorysystem has already been established (4). The potential ofgenetic analysis, however, will not be fully realized until thefunction of this system can be measured effectively in avariety of ways.

In this article we describe a jump response elicited byexposure of Drosophila to high concentrations of certainvolatile chemicals, a behavior that provides a means ofmeasuring response to airborne chemicals. We discuss the

advantages this behavior offers for genetic analysis ofchemosensory function, and its use as a means of geneticanalysis is illustrated by the isolation and characterization ofa set of mutants defective in this behavior.The chemosensory jump assay was designed to be per-

formed on a single fly. For purposes of mutant isolation,single-fly assays allow testing of the F1 generation. Previousassays used in isolating olfactory mutants have been based onthe testing of populations, which requires testing of the F2generation and thus places severe constraints on the numberof chromosomes that can be tested. This limitation is partic-ularly critical in the isolation of alleles marked by physicalrearrangements useful in molecular cloning, since the muta-gens commonly used to generate such alleles-such as x-raysand transposable elements-generally induce mutations atlow frequency. The availability of a single-fly assay alsoallows certain forms of genetic analysis, such as mosaicanalysis, that are useful in investigating the function ofgenesdefined by mutation.

MATERIALS AND METHODSDrosophila melanogaster. Canton-S-5 (CS-5) is homozygous

for an X chromosome derived from a Canton-S strain ob-tained from 0. Siddiqi (Tata Institute, Bombay). It wasconstructed from this Canton-S strain and from an FM7balancer stock whose autosomes were of Canton-S origin,from S. Benzer (California Institute of Technology). TheNasobemia (Ns) allele, isolated in W. Gehring's laboratory(University of Basel), was obtained on a Ns st cu chromo-some, and AntennapediaB (AntpB) was obtained in anIn(3R)AntpB/TMI stock, both from E. Frei (Yale Universi-ty). Both Ns and AntpB chromec.omes were placed andmaintained in a Canton-S genetic background. The Ns stockwas made homozygous for aNs chromosome, and AntpB wasbalanced over TM3 Ser. Drosophila were cultured as de-scribed elsewhere (5).The CS-5 stock was mutagenized by means of ethyl meth-

anesulfonate (6), x-irradiation (4300 rads; 1 rad = 0.01 Gy),or hybrid dysgenesis. In the case of ethyl methanesulfonateor x-rays, mutagenized males were allowed to mate withattached-X females obtained from a C(1)A y stock in aCanton-S background from D. Kankel (Yale University). Asingle X chromosome and autosomes from a defined back-ground were used in these screens in order to reduce varia-tion in olfactory response due to genetic background effects(7). Hybrid dysgenesis was performed as in ref. 5. Mutant acjchromosomes are maintained both over the attached-X chro-mosome C(1)A y and, separately, over the balancer chromo-some FM6 or FM7c.The Jump Apparatus. The apparatus, depicted in Fig. 1,

has as its base a block of Teflon, 25 mm high, into which twoholes =17.5 mm in diameter have been drilled. A nylon mesh

Abbreviations: CS-5, Canton-S-5; ERG, electroretinogram.

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is placed across the opening of the uppermost hole. A plasticculture tube (Falcon, 17 mm x 100 mm) containing a 4.76-mmdiameter hole in its rounded bottom is fitted tightly, upsidedown, into the uppermost hole, so that the lip of the tube isimmediately above the mesh. A section of glass tubing fitsinto the other hole in the block and is connected to a vacuumsource (Cole-Parmer, KNF 7056-25; flow rate of 1 liter/min)by means of a Teflon adapter and Tygon tubing. As a sourceof chemical vapor, 10 ml of odorant is placed in a sidearmvessel (25 mm X 200 mm). Glass tubing (3-mm internaldiameter) extends through a silicone stopper and terminates=2 mm above the surface of the odorant. The glass tubing isconnected tightly to Teflon tubing (3.18 mm) by means oftwotruncated pipette tips; pipette tips are also firmly attached tothe other end of the tubing and are inserted through the holein the culture tube to connect the odorant to the assay tube.Thus air is drawn over the surface of the odorant, through thetube containing the fly, and out of the system through a longstretch of tubing by the pump, which is located in an adjacentroom. Chemicals were from Fluka and were of the highestpurity available. Ethyl acetate, propionic acid, and ethanolwere used undiluted, except that ethyl acetate was diluted inwater for the experiment shown in Fig. 3. Benzaldehyde wasused either undiluted or following dilution in paraffin oil(Fluka, no. 76235).Measuring the Jump Response. Flies were tested individ-

ually as described in the legend to Fig. 1, with no priorexposure to anesthesia. After each trial, the tubing from theodorant source was removed from the tube containing the fly.The fly was then removed by raising the jump apparatus,inverting it, and agitating it against the inner surface of afunnel inserted into a fly morgue or collecting vial. The flyfalls out the hole at the top of the tube, and the apparatus isthen righted and used again. Unless indicated otherwise, flieswere discarded after being tested once. A fresh tube is usedfor the next trial; tubes are allowed exposure to fresh air forat least 20 min between uses as a precaution against accu-mulation ofodor on the plastic surface. Using these methods,on the order of 100 flies can be tested in a period of 1 hr.

Typically, a group of 30 or 50 flies was tested individually,and the group score was defined as the percentage offlies thatjumped. Control flies were tested in parallel with experimen-tal flies in all experiments. Male flies were used exclusivelyexcept in the complementation analysis or where indicated

otherwise. All indicated experimental errors are SEM. Testsof statistical significance were performed with Student's t test(for comparison of two means) or analysis of variance (forcomparison of more than two means) following arcsinetransformation of the jumping percentages.

Physiology. Electroretinograms (ERGs) (8) were recordedas described elsewhere (5). Giant fiber pathway physiologywas recorded from three individuals of each genotype, es-sentially as described by Tanouye and Wyman (9). Methodsfor measuring antennal physiology will be described in detailelsewhere. Briefly, chemical vapor was injected into a streamof air directed at the antenna of an immobilized fly. Glassrecording and reference electrodes contained Ringer's solu-tion and were placed below the surface of the third antennalsegment and in the head capsule, respectively. Electricalresponses were measured on an oscilloscope.

RESULTSThe Jump Response. We have found that flies jump when

exposed to certain chemical vapors, and we have used thisbehavior as the basis for a chemosensory assay, the jumpassay. Fig. 1 shows an apparatus constructed to measure thisresponse. A fly is inserted into the tube and is allowed tocrawl up the wall. When it reaches an intermediate position,a chemical vapor is caused to pass through the tube. Ajumping event is scored if the fly lands on a nylon screenbelow within 3 s after the onset of the pulse of chemicalvapor.

Fig. 2a shows that thejump response is strong and that thesignal-to-noise ratio of this assay is very high. When pulsesof benzaldehyde vapor were delivered to flies, 96% ± 2%(SEM; n = 4 sets of 50 trials each) responded by jumping;when control pulses of air were delivered, only 5% ± 1% (n= 6)jumped, yielding a signal-to-noise ratio of 19. Fig. 2a also

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FIG. 1. Apparatus for testing jump response. A single fly isintroduced into the tube with a mouth aspirator and is allowed to walkapproximately one-third to one-half of the way up the side. Air isdrawn through the system continuously by means of a pump. Whenthe Teflon tubing from the sidearm vessel is inserted into the hole atthe top of the tube, air is drawn over the surface of the chemicalplaced in the sidearm vessel and through the tube. The tubing isinserted into the tube for 3 s, giving the animal a 3-s exposure tochemical vapors. Ajump event is scored if the fly lands on the screenat the base of the tube during this period.

FIG. 2. (a) Jump response to chemicals of different classes. Allchemicals were undiluted. n indicates the number of experiments,each experiment consisting of 50 trials, one CS-5 fly per trial. Errorbars indicate SEM. (b) Jump response to sequential stimuli. CS-5flies were assayed with vapor of the indicated chemical, transferredto a vial, allowed to rest 1 min, and then assayed again. Thisprocedure was repeated on each of 30 flies per experiment; fiveexperiments were performed in the case ofeach chemical. Error barsindicate SEM. EA, ethyl acetate; PA, propionic acid; Bz, benzal-dehyde, undiluted.

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shows that this response is not limited to aldehydes but canbe elicited by representatives of at least two other chemicalclasses: organic acids (propionic acid) and acetate esters(ethyl acetate). Interestingly, there appears to be chemicalspecificity to this response: ethanol provoked a responseamong only 8% ± 2% (n = 6) of the tested flies, although itsvapor pressure is intermediate between that of ethyl acetateand propionic acid.The jump is not a manifestation of trauma. After the jump,

the fly resumes apparently normal behavior within seconds.In fact, a fly thatjumps in response to a pulse of ethyl acetateis fully capable ofjumping in response to a second pulse ofthe same chemical 1 min later. Fig. 2b shows a series ofexperiments in which flies scored 70o ± 4% (n = 5) inresponse to an initial pulse and 70% + 3% (n = 5) in responseto a second pulse 1 min later. As a more sensitive test foreffects of this treatment, in one experiment flies were givensix consecutive pulses of ethyl acetate at 1-min intervals, andthey continued to demonstrate a comparably strong response(not shown). Fig. 2b also shows that animals exhibit a robustresponse to repeated stimulation with propionic acid. Re-sponse to the first and second stimuli did not differ signifi-cantly (P > 0.1), and delivery of six consecutive pulses to onegroup of flies did not lead to reduced response (not shown).

In the case of benzaldehyde, response declined signifi-cantly, from 92% ± 1% on the first trial to 73% ± 3% on thesecond (P < 0.001). Further experimentation will be requiredto determine whether benzaldehyde causes injury or, amongother possibilities, whether the chemosensoryjump behaviormight be a useful paradigm for studies of the mechanism ofhabituation.The response exhibits dose dependence (Fig. 3). Jump

response to both ethyl acetate and benzaldehyde increased asthe concentration of the stimulus increased from a 10-2dilution to a 10-1 dilution. Above this level, response ap-peared to reach saturation.

a

b

Dependence of the Response on the Antenna. The physio-logical basis of this behavior is unknown. Flies are known tojump in response to a visual stimulus, a response that hasbeen interpreted as an escape response and that has been wellcharacterized physiologically (9, 11). As a first step in char-acterizing the physiology of the chemosensory jump behav-ior, we sought to determine whether the response wasmediated through the antenna, of which the third segment isthe principal organ of olfaction (12). This question has beenaddressed through genetic and surgical manipulations. Fig.4a shows that the homeotic mutants Nasobemia and Anten-napediaB, both of which suffer partial transformation ofantennae into legs, exhibit diminished chemosensory jumpresponse to ethyl acetate. Fig. 4b shows that surgical removalof the third antennal segments severely diminished the jumpresponse to ethyl acetate, propionic acid, and benzaldehyde.As a control for effects of the surgery on motor activity, theexperiment shown in Fig. 4c shows that the same surgicaltreatment did not reduce the jump response to a visualstimulus.

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FIG. 3. Dose dependence of jump response. CS-5 flies weretested with each concentration of the indicated chemicals on each of5 days. For ethyl acetate, 30 flies, males and females, were used ineach test. For benzaldehyde, 60 flies, 30 of each sex, were used ineach test. The responses to benzaldehyde were calculated separatelyfor each sex and showed no significant differences (P > 0.1 at allconcentrations); thus, combined data for the two sexes are shown.These data were collected in a study of a chemosensory mutant (10).Error bars represent SEM. (a) Ethyl acetate. (b) Benzaldehyde.

* Control: Chem.M Operated: Chean.J Operated: Air

* 1 TrialM 10 Trials

uperated

FIG. 4. Dependence of jump response on antennae. (a) Jumpresponse of homeotic mutants with partially transformed antennae.Each experiment (n = 8 for CS-5; n = 9 for Ns; n = 10 for AntpB)represents trials of 30 flies each, using ethyl acetate as the stimulus.A number of AntpB flies were unable to climb the walls of the tubeand were not scored. Perhaps for related reasons, AntpB flies werefound to give relatively high scores (up to 20%) in response to controlair pulses; thus for all three genotypes the background levels ofjumping in response to a control pulse of air were subtracted. Errorbars indicate SEM. (b) Jump response of CS-5 following removal ofthird antennal segments with fine forceps as described elsewhere (5).Flies were tested 1-3 hr after surgery. Error bars indicate SEM. EA,ethyl acetate; PA, propionic acid; Bz 10-2, benzaldehyde diluted10-2; Bz 100, undiluted benzaldehyde. n = four or five sets of 30 flieseach. Operated animals exhibited stronger responses to chemicalvapors than to control pulses of air: P < 0.001 for both concentrationsof benzaldehyde. (c) Jump response of CS-5;bw;st stimulated indi-vidually with a "lights-off" visual stimulus (13); the bw and stmutations reduce the levels of screening pigment and are used toincrease the frequency of the visually induced jump response. Thefraction offlies thatjumped in response to an initial stimulus is shownfor operated and control populations. Each fly was subsequentlygiven nine additional stimuli, at intervals of 1 min, and the totalnumber of responses was recorded. The jump response averagedover these 10 trials is also shown. Ninety flies of each type weretested in a total of three experiments, 30 flies per experiment.

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These experiments together indicate that the chemosen-sory jump response is mediated primarily through the anten-nae. The residual response following surgical removal of theantennae, however, is greater than that observed followingstimulation with control pulses of air, indicating thatchemosensory information is also received from organs otherthan the third antennal segments. Chemosensory hairs aredistributed on several other parts of the adult cuticle. Thedorsal surfaces of the maxillary palps, for example, have ahigh density of sensilla basiconica (14), and projections fromthese sensilla to the antennal lobes have in fact been docu-mented by dye-filling (15). Experimentation with flies surgi-cally deprived of both the third antennal segments and themaxillary palps, however, suggests that these two pairs oforgans are not the only ones through which chemosensoryinformation can produce a jump response, since removal ofboth pairs did not abolish all response (not shown).

Isolation of acj Mutants. We have screened 2000 linescarrying mutagenized X chromosomes for defects in thejumpresponse to ethyl acetate. Three males were tested initiallyfrom each line, and those lines from which at least one malefailed to jump were tested further. From 1000 lines mutagen-ized with ethyl methanesulfonate, nine acj mutants wererecovered (Fig. 5) (acj = abnormal chemosensory jump;numerals following the three-letter designation indicate mu-tants and are not intended to indicate loci). Mean jumpresponses to ethyl acetate among these mutants ranged from20% to 45%, compared to a control value of83%. No mutantswere obtained from 1000 lines established after mutagenesisby means of x-irradiation or hybrid dysgenesis and enrich-ment for olfactory mutants by a procedure described else-where (5).

Characterization of acj Mutants. Abnormal response in thechemosensory jump assay may be caused by lesions at any ofseveral levels, including the transduction, processing, ormotor levels. By using an analogous behavioral assay, basedon a visually drivenjump response, Thomas and Wyman (13)isolated and characterized a number of mutants with defectsin the neurons driving the motor output. In these mutants,electrical stimulation of the giant fiber does not lead to thenormal electrical responses in the tergotrochanteral muscles(leg extensor muscles) and the dorsal longitudinal muscles(wing depressor muscles). By using the same physiologicaltest that reveals defects in these mutants, and using thebendless mutant of Thomas and Wyman (16) as a negativecontrol, all of the acj mutants tested normally.We note that the physiology of the chemosensory jump

response has not been characterized, and could act througha different motor circuit than that of the visually drivenjump

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response. In any case, it is clear from this testing that all ofthe acj defects are sufficently specific as not to disrupt axonalpropagation through several defined neurons, synaptic trans-mission across several synapses-both electrical and chem-ical-or electrical response of multiple muscle fibers.

If the acj defects do not affect giant fiber physiology, doany of them affect sensory physiology? We first examinedvisual system physiology by the convenient method of re-cording ERGs; initial testing of antennal physiology, which ismore difficult, is described below. Two of the acj mutantsshow obvious ERG abnormalities (Fig. 6). acj2 and acjS areaffected in the on and off transients that arise in wild typefrom excitation of the laminar neurons following synaptictransmission from the retinal photoreceptor neurons (17).Neither on nor off transients were observed in any of 19 acj2individuals or 12 acjS individuals examined. Normal on andoff transients were observed in 15 of 15 CS-S control flies. Inaddition to the effect on the transients, retinal depolarizationalso appeared to be reduced in acj2, although the penetranceof this phenotype was incomplete.One strategy for determining whether a mutation affects

olfactory transduction or processing, as opposed to actingexclusively at other levels, is to test response to multiplechemicals. Many types of lesions, such as various types ofmotor defects, would be expected to affectjump response toall chemicals. On the other hand, genes whose mutationsaffect response only to a specific subset of chemicals wouldbe good candidates for genes playing direct roles in olfactorytransduction or processing, such as receptor genes. For thisreason, the nine mutants were also tested for response to asecond chemical, benzaldehyde.

Fig. 5 shows the ratio of response to ethyl acetate andbenzaldehyde among the different mutants. In several mu-tants the response to both chemicals is reduced equally. Inacj3 for example, response is approximately one-third that ofwild type for both chemicals. By contrast, in some mutants,of which acj2 is the most notable example, response to ethylacetate is reduced much more than is response to benzalde-hyde. A thorough phenotypic characterization of the acj

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FIG. 5. Jump responses of acj mutants. EA, ethyl acetate; Bz,benzaldehyde at a 3 x 10-2 dilution. n = five experiments of 30 flieseach. All genotypes, including controls, were scored blind. Errorbars indicate SEM.

FIG. 6. ERG analysis of acj mutants. The light stimulus is of0.5-sec duration. Stimulus artifacts indicate the beginning and end ofthe flash directly on the trace. (a) CS-5. A normal "on" transient,retinal depolarization (typically 5-15 mV), "off" transient, andreturn to resting potential are visible. (b) acjS. (c) acj2.

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mutants will require measurement of response to a series ofconcentrations of each chemical; however, we note thatmutants exhibiting chemically specific phenotypes have beenreported previously (4), and interestingly, certain mutantsisolated by other means show a reciprocal phenotype. Amongthese is the mutant ptg3D18, which responds defectively tobenzaldehyde but normally to ethyl acetate across a broadrange of stimulus concentrations in the chemosensory jumpassay (10).

Genetic analysis has been performed on two mutants thathave lower responses to ethyl acetate than to benzaldehyde,acj2 and acjS, and two mutants that have severely reducedresponses to both chemicals, acjl and acj3. A complemen-tation matrix was established from these four mutants andfrom the CS-5 wild type; all pairwise combinations of thesegenotypes were tested five times each, and the results forCS-5/CS-5 were compared to those for the other genotypes,using P < 0.05 as a criterion for significance.The defective response to ethyl acetate was found to be

recessive in all cases. The four mutants define three com-plementation groups. One complementation group is repre-sented by a single mutant, acjl, which complements the otherthree mutants. A second group is represented by acj3, whichalso complements the other three mutants. The third group,however, contains both acj2 and acjS, which fail to comple-ment each other either with respect to ethyl acetate responseor with respect to their ERG phenotypes, which are alsorecessive. The fact that neither phenotype complementssuggests strongly that the chemosensory and visual pheno-types arise in each case from a single mutation. It is unlikelythat two distinct genes would be independently mutated ineach of two independent mutants. acjS will be referred tohenceforth as acj22.

Electrophysiological recording from the antennae of thenine acj mutants has so far revealed a defect in one, acj6:extracellular recordings following odorant stimulation haveshown that the change in electrical potential exhibited by acj6is significantly less than for wild type (P < 0.05) in responseto all tested concentrations-spanning five orders of magni-tude-of either benzaldehyde or ethyl acetate (R. Ayer andJ.C., unpublished results). A detailed investigation of thegenetic and physiological basis of the acj6 defect is now ofinterest.

DISCUSSIONThe function, organization, and development of the Drosoph-ila olfactory system remain largely unexplored. A majorimpediment to study of this system has been the lack ofsimple, reliable means of measuring olfactory function. Inthis paper we describe a chemosensory behavior that can bemeasured quickly and precisely, which enjoys a high signal-to-noise ratio and which can be measured using a single fly.The behavior has further been used to isolate a set of acjmutants in which it is defective.The ability to test a single fly provides several important

advantages. First, it offers the possibility of using an F1screen to isolate new mutants, a possibility that has substan-tial logistical advantages in that it obviates the need toestablish lines for screening. The feasibility of an F1 screenhas not yet been tested-the acj mutants described here wereisolated from a set of lines already available-but is sup-ported by the high signal-to-noise ratios obtained using theassay. Moreover, data shown here indicate that for at leasttwo chemicals a fly can be tested multiple times in quicksuccession, allowing rapid retesting of putative mutants.With regard to the utility of this response as a means of

mutant isolation, it may be possible to enlarge the physicalparameters ofthe assay so as to allow its use as an enrichmentprocedure. The extant acj mutants could be used in recon-struction experiments so as to optimize and quantitate the

efficiency ofenrichment. A large-scale enrichment procedurehas been used by Thomas and Wyman (16) in the successfulisolation of mutants defective in the visually induced jumpresponse.Another advantage accorded by the availability ofa single-

fly assay is the possibility of analyzing mutants by mosaicanalysis. In fact, determination of the foci of some of the acjmutants described here might aid in clarifying the basis oftheir phenotypes. Again, the high signal-to-noise ratio andability to retest mosaic animals should facilitate this analysis.Does the chemosensory jump response have adaptive

value? We do not know how often flies come into contactwith high concentrations of chemical vapors in the wild norare we aware ofDrosophila alarm pheromones like those thatcause aphids, for example, to undergo jerking movementsand fall from plants (18). Equally little is known about theresponse at the cellular level. Experiments described hereindicate that the response is mediated primarily, although notexclusively, by the antennae, suggesting that the chemosen-sory jump response may depend on some of the samemolecular mechanisms used in generating an olfactory at-traction response. However, whether overlap between thetwo responses extends to include olfactory receptor mole-cules is a question that will require further analysis. A role forspecific receptor molecules in the jump response might besought, for example, by determining whether response to anindividual chemical is diminished by prior or concurrentstimulation with structurally related chemicals but not withunrelated chemicals.The genetic analysis of the chemosensory jump response

that has been begun here may be a useful means of investi-gating the mechanisms by which chemical information issensed and processed. Further genetic and phenotypic anal-ysis will be necessary to determine the basis of the acjphenotypes. The finding that two acj mutants have ERGdefects could reflect an interaction between visual andchemosensory pathways at a central level. Alternatively, itsuggests the possibility that some molecules required invisual transduction or processing are also required forchemosensory function, a possibility that can be explored bytesting of defined visual system mutants.

We thank Haig Keshishian and Henry Sun for ideas and discus-sion. This work was supported by National Institutes of Health GrantGM-36862 (to J.C.). J.C. is an Alfred P. Sloan Research Fellow.

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