Drosophila melanogaster

Proc. Natl. Acad. Sci. USA
Vol. 76, No. 2, pp. 884-887, February 1979
Genetics
Isolation and analysis of chemosensory behavior mutants in
Drosophila melanogaster
(taste/chemotaxis/countercurrent distribution/temperature-sensitive mutation)
LAURIE ToMPKINS*, M. JANE CARDOSA, FRANCES V. WHITE, AND THOMAS G. SANDERSt
Department of Biology, Princeton University, Princeton, New Jersey 08540
Communicated by Vincent G. Dethier, December 1, 1978
ABSTRACT A behavioral countercurrent paradigm has
been developed for assaying the chemotactic responses of
wild-type and mutant Drosophila melanogaster adults. Oregon
R males avoid both quinine sulfate and NaCl, whereas
Oregon R females reject the quinine salt but are attracted to
NaCl when tested in this paradigm. Wild-type behavior is sufficiently
reproducible to allow identification of mutants affecting
chemotaxis, and 12 such mutants, in six complementation
groups, have now been isolated. Three of the mutants respond
abnormally to NaCI, two in one complementation group
with atactic behavior (no chemotaxis) and the other, in a separate
group, with a mistactic response (attraction to the stimulus).
Four mutants in another group respond mistactically to quinine
sulfate. Of the remaining mutants, two in one group behave
atactically and three, in two groups, respond mistactically to
either chemical stimulus. Several of the mutants also show abnormal
behavior in a proboscis extension assay when tested
individually with sucrose solutions.
One approach to exploring the mechanisms of animal behavior
is to analyze mutations that perturb stereotyped responses in
genetically well-characterized species, such as Drosophila
melanogaster. Such a behavior, the gustatory response to
chemical stimuli in solution, has been extensively investigated
at the receptor level in closely related insects, particularly the
blowfly Phormia regina (1). In Diptera, the contact chemoreceptors
that mediate feeding behavior are located primarily
on the tarsal segments of the leg and on the labellum of the
proboscis. When its tarsal or labellar chemoreceptors are in
contact with solutions of various sugars, a hungry fly generally
extends its proboscis, whereas solutions containing both salts and
sugars usually elicit no proboscis extension. These observations
suggested the possibility of isolating mutations in Drosophila
that affected responses to chemical stimuli. Although it is possible
to select mutants by observing the behavior of individual
flies, we developed a chemosensory countercurrent distribution
(CCD) apparatus which permitted screening of many mutagen-treated
chromosomes simultaneously. We describe the use
of the CCD paradigm to analyze the chemosensory behavior
of wild-type adult flies and of 12 newly isolated mutants, in six
gustatory genes, that show abnormal CCD responses to NaCl,
to quinine sulfate, or to both compounds.
MATERIALS AND METHODS
Drosophila melanogaster stocks of the wild-type Oregon R
strain and the mutant lines were grown, on standard medium
(2) at 170, 220, or 280C as appropriate. All chemicals used were
of reagent grade, and distilled water was used for all solutions.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U. S. C. §1734 solely to indicate
this fact.
884
Chemosensory Countercurrent Distribution. The apparatus
has been described (3). It consists of two opposing rows of five
glass vials, the inner surfaces of which are coated 0.2 mm thick
with a solution of 2% (wt/vol) Bacto-Agar (Difco) and 15-30
mM sucrose, provided for nutrition. The solution applied to one
of the two rows of vials also contains either 0.01 M quinine
sulfate (Sigma) or 1 M NaCl. To begin an experiment, the apparatus
was placed at 280C in the dark or in red light (Kodak
#2 Safelight), to which Drosophila do not respond (4). Males
or females (150-200 flies, 2-4 days posteclosion) were introduced
into the first vial in the row that contained the chemical
stimulus and were allowed to distribute themselves between
the two opposing stimulus-containing and non-stimulus-containing
vials. At the end of the trial (6 hr with NaCl, 8 hr with
quinine sulfate), the apparatus was manipulated as described
by Benzer (5) to bring each vial containing flies opposite a fresh
vial, thus beginning a new trial. Each experiment consisted of
five such trials, after which flies were distributed in six fractions
according to their tendency to choose a stimulus-containing vial.
If the vial positions are consecutively numbered, with the vial
in which flies were initially placed being designated zero, the
fraction in which an individual is found corresponds to the
number of times that the fly has appeared in the non-stimulus-containing
vial at the end of a trial. Assuming that each
individual acts independently and with a constant partition
coefficient P, the distribution of flies recovered is expressed
according to the binomial expansion (5, 6) as:
N (n - r)!r! ( )
where N is the number of flies in the total population, NM the
number in fraction r, n the number of trials, and P the probability
that a fly chooses a non-stimulus-containing vial. In our
experiments, with n = 5, N and N, were determined by
counting the flies at the end of the experiment, and, thus, P
could be estimated by fitting the data to theoretical curves of
known P values. Flies that were unresponsive to the stimulus
(i.e., atactic or "taste-blind") should be distributed with P =
0.5; a P value less than 0.5 indicates attraction to the stimulus
(a mistactic response in comparison to that of wild-type males)
and, conversely, a P value greater than 0.5 indicates aversion.
Assay of Proboscis Extension. To test the responses of individual
flies to sucrose, adults (2-4 days posteclosion) were
starved for 24 hr in a humid atmosphere and then lightly
anesthetized with carbon dioxide and restrained by embedding
Abbreviation: CCD, chemosensory countercurrent distribution.
* Present address: Department of Biology, Brandeis University,
Waltham, MA 02154.
t Present address: Department of Biology, Lake Forest College, Lake
Forest, IL 60045.

Genetics: Tompkins et al.~~~~~Proc.
Nati. Acad. Sci. USA 76 (1979) 885
Genetics: Tompkins et al.
the wings in Takiwax (Cenco, Chicago). After recovery, each
fly was stimulated with distilled water until it no longer extended
its proboscis and then stimulated tarsally with a'0.06 M
sucrose solution while the position of the proboscis was observed.
In no case was the proboscis permitted to contact the sugar solution.
Mutant Isolation. To generate populations that were predominantly
male, virgin females bearing an attached X chromosome
homozygous for the temperature-sensitive lethal
mutation 1(1 )E6's were mass-mated to Oregon R males at 280C
(7). This mutation causes females, which inherit attached X
chromosomes from their Mothers, to die as larvae or pupae (8).
Male progeny of this cross were treated with 25 mM ethyl
methanesulfonate (9) and then mass-mated to virgin attached-X
females. The male progeny of this cross, which inherit mutagen-treated
X chromosomes from their fathers, were tested by
CCD. Each fly remaining in the starting vial at the end of the
experiment, which had thus chosen the stimulus-containing vial
five times in five trials, was mated individually to virgin attached-X
females to generate a putative gus mutant stock.
Males from each stock were subsequently tested twice by CCD,
and stocks that displayed abnormal phenotypes in both experiments
were considered confirmed gus mutants and retained
for additional analysis.
Genetic Analysis. Hemizygous males were maintained in
stock with attached-X females. To generate females to be tested
with quinine sulfate, the following crosses were done. Hemizygous
gus males were mated to virgin females homozygous
for the FM7a X chromosome balancer, Ino() FM7a, y3ld sc8
Wa v B (10), to generate heterozygous gus/FM7a females.
These were then mated as virgins to males bearing the same gus
mutation to generate parents for homozygous gus stocks. For
complementation analysis, virgin females homozygous for one
gus allele were crossed to males hemizygous for another, and
their female offspring were tested by CCD.
Unlike Oregon R males, Oregon R females are attracted to
NaCl when tested by CCD (see below). Thus it was not possible
to observe the effect of gus mutations in normal females by
using NaCl as stimulus, because mutant and wild-type phenotypes
could not readily be distinguished. For analyzing such
mutants, this problem was circumvented by using diplo-X flies
bearing the dsXD mutation on the third chromosome (1 1, 12),
which transforms genetic females into morphological intersexes
(13). We observed that dsxD flies avoid NaCl. Accordingly, we
tested populations of transformed flies that were heterozygous
and homozygous for gus mutations to determine dominance
and complementation of gus mutants with NaCI as stimulus.
The gus genes were roughly mapped with respect to the
visible markers yellow (y; 1-0.0), crossveinless (cv; 1-13.7),
vermilion (v; 1-33.0) and forked (f; 1-56.7) (11). We observed
that these markers had no effect on contact chemoreception.
Ten to 20 F2 males representing each of the six possible single
recombinant classes, as well as an equal number from each of
the two parental classes, were mated to virgin attached-X females,
and males from each of the resulting eight progeny
populations were tested separately by CCD (14).
RESULTS
Before using the countercurrent device for mutant selection,
it was necessary to ascertain that flies were distributed solely
on the basis of their chemotactic behavior. This assumption
predicts that the distribution of flies tested by CCD without a
stimulusrt be%, cha-rn-acterized by, a P valuea equval to.5,-%Ar as is0 true
for the Oregon R wild-type strain from which all mutant lines
were derived (Fig. 1). In the next experiments, the concentra-
0
C
0.3
0.2
L1_ 0.1
C
0.4-
0.3-
0.2-
0.1
0 1 2 3 4 5
Countercurrent tube
FIG. 1. Countercurrent distributions of Oregon R males in the
absence of a stimulus (A) and with NaCi (B) and quinine sulfate (C)
as stimuli. Data from one representative experiment are shown.
tions of NaCl, quinine sulfate, and the background sucrose, as
well as the duration of the trials, were adjusted to maximize
negative chemotaxis by wild-type males. For the optimal
variables, both Oregon R males and females exhibited pronounced
avoidance of quinine sulfate. In contrast, whereas
Oregon R males avoided NaCl (Fig. 1), Oregon R females were
attracted to the stimulus (Fig. 2). Intersexes produced with the
dsxD mutation were repelled by NaCl, although their avoidance
was not as pronounced as that of wild-type males. These data
are presented in Table 1.
After mutagenesis, approximately 17,500 chromosomes were
screened, yielding 69 putative gustatory mutants of which 12
were confirmed. All were sex-linked and fully recessive, the
0.4-
0
0~0.3
20.
U. 0.1
0 1 2 3 4 5
Countercurrent tube
FIG. 2. Countercurrent distributions of Oregon R females (0)
and genetically transformed (dsxD) intersexes (0) with NaCl as
stimulus. Data from one representative experiment are shown.

886 Genetics: Tompkins et al.
Table 1. Responses of Oregon R males and females and of dxsD
intersexes to stimuli
Stimulus
Sex QS* NaCl None Sucrose
Males 0.90 t 0.04 0.84 + 0.05 0.52 + 0.03 0.89
Females 0.86 + 0.05 0.35 + 0.08 0.50 i 0.04 0.85
Intersexes 0.90 + 0.02 0.72 + 0.04 0.52 i 0.02 0.90
Responses to quinine sulfate, NaCl, and no stimulus were assayed
by CCD; P values are +SEM. Responses to sucrose were assayed by
proboscis extension; the fraction of flies responding to 0.06 M sucrose
is shown.
* Quinine sulfate.
phenotype in each case being wild-type in gus/+ females tested
with quinine sulfate or transformed gus/+ intersexes tested
with NaCl. In addition, all populations of gus males responded
abnormally whether the experiment was begun in a stimuluscontaining
or non-stimulus-containing vial, demonstrating that
the genetic lesion in each case affects contact chemoreception
rather than simply reducing mobility.
The gus mutants were distributed among six complementation
groups, designated A through F. All except C, which is
located between the markers crossveinless and vermilion, map
between vermilion and forked, which are 23.7 map units apart.
The mutants may be classified by their patterns of CCD behavior:
those in complementation groups A, C, D, and E exhibited
mistactic responses to one or both stimuli (P values less
than 0.5), whereas those in the B and F groups behaved atactically
(P = 0.5), as shown in Table 2. No mutant exhibited
atactic behavior with one stimulus and a mistactic response to
the other.
In the mistactic class, all four gusA mutants responded abnormally
to quinine sulfate (Fig. 3) and normally to NaCl. In
contrast, gusCNlO behaved normally when tested with quinine
Table 2. Responses of the gustatory mutants to stimuli
Group Mutant(s) QS*
Stimulus
NaCl Sucrose
A gusAQ1 0.22 4 0.04 0.90 4 0.02 0.08
gusAQ2 0.20 i 0.05 0.86 ± 0.03 0.26
gusAQ3 0.25 + 0.03 0.88 ± 0.03 +
gusAQ6 0.26 + 0.06 0.88 ± 0.04 0.44
B gusBQ4 0.50 0.05 0.52 ± 0.04 0.12
gusBQ5 0.49 + 0.06 0.50 ± 0.07 0.21
C gusCNlO 0.90 ± 0.04 0.18 + 0.04 0.94
D gusDN9 0.18 ± 0.03 0.20 ± 0.03 0.90
gusDNl2 0.11 ± 0.03 0.15 + 0.02 0.78
E gusENl3 0.10 ± 0.02 0.13 ± 0.04 0.43
F gusFN5 0.90 ± 0.03 0.50 + 0.03 0.92
gusFN32 0.91 ± 0.05 0.48 0.05 0.92
Responses to quinine sulfate and NaCl were assayed in males by
CCD; P values are +SEM. Responses to sucrose were assayed by
proboscis extension; the fraction of flies responding to 0.06 M sucrose
is shown. Letters A through F designate the different gus complementation
groups. Superscripts Q and N refer to the stimulus, quinine
sulfate or NaCl, with which each mutant was originally isolated. For
conditional mutants, the responses shown are those observed when
flies were raised at nonpermissive temperatures.
* Quinine sulfate.
Proc. Natl. Acad. Sci. USA 76 (1979)
0
.°0.2_ \
LL0.1 CI
0 1 2 3 4 5
Countercurrent tube
FIG. 3. Countercurrent distributions of gusA Ql males, raised at
22°C, with quinine sulfate as stimulus. Data from one representative
experiment are shown. All other mistactic distributions are similar,
the distance of the peak from the center being proportional to the
degree of attraction to the stimulus.
sulfate but exhibited a mistactic response to NaCl. The four
gusA mutants and the single gusC mutant are all cold sensitive:
the mistactic phenotype was expressed in flies grown at 220 C,
whereas populations grown at 28°C responded as Oregon R
males did.
The remaining mistactic mutants exhibited abnormal phenotypes
with both quinine sulfate and NaCl. Of the gusD
mutants, gusDN9 is expressed unconditionally, while gusDNl2
is heat sensitive, males raised at 17°, 220, and 280C being distributed
with P values of 0.90 + 0.02, 0.60 + 0.04, and 0.11 ±
0.03, respectively, when tested with NaCl. The gusE mutant
is cold sensitive, showing mistactic behavior when raised at
220C and normal behavior when raised at 280C.
Of the atactic mutants, the two gusB mutants responded
abnormally to either quinine sulfate or NaCl when tested by
CCD (Fig. 4). The two gusF mutants were atactic when tested
with NaCl but normal with quinine sulfate. All four atactic
mutants were unconditional, the abnormal phenotype in each
case being expressed in flies grown at either 220 or 280C.
In addition to their CCD phenotypes, several of the gus
mutants displayed abnormal behaviors when tested by the
proboscis extension assay with 0.06 M sucrose (Table 2). For all
of the mutants in complementation groups A, B, and E, fewer
than half of the flies tested gave positive responses to the sugar.
In contrast, almost all Oregon R and gusC, gusD, and gusF flies
responded to the sucrose solution. We asked whether the abnormal
response to sugar of gusE N13, the most extreme coldsensitive
mistactic mutant, was similarly temperature sensitive.
Accordingly, males raised at 220and 280C were tested by the
proboscis extension assay. The response of mutant flies grown
at 28°C was indistinguishable from that of Oregon R males
Countercurrent tube
FIG. 4. Countercurrent distributions of gusBQ4 males with quinine
sulfate as stimulus. Data from one representative experiment
are shown. All other atactic distributions are similar.

Genetics: Tompkins et al.
raised at that temperature, which is true of the mistactic CCD
phenotype also.
DISCUSSION
Populations of wild-type (Oregon R) D. melanogaster adults
respond with highly reproducible distributions to quinine sulfate
and NaCl when tested by CCD. The two chemical stimuli
used in the CCD paradigm were chosen because they had been
shown to be repellent to flies in other assays (15-17); thus, the
responses of Oregon R males to both compounds and of Oregon
R females to the quinine salt were not unexpected. However,
the positive chemotaxis observed with Oregon R females tested
with NaCl was not expected from previous observations. One
finding that may be relevant to this point is that dsxD intersexes,
whose ovipositors are rudimentary and heavily sclerotized (18),
avoid NaCl. In Phormia there are chemoreceptors on the ovipositor
that respond electrically to NaCl (19, 20); if analogous
receptors mediate the behavior of normal Drosophila females
to NaCl, then dsxD flies would be expected to behave more like
males, because their ovipositors do not appear to bear functional
chemoreceptors. This interpretation is also supported by the
observation that female Oregon R larvae avoid NaCI (L.
Tompkins, unpublished results).
Analysis of gus mutant behavior shows that the chemosensory
countercurrent device can be used to select various mutants
with aberrant chemotaxis. Some mutants respond abnormally
to quinine sulfate or to NaCl but not to both compounds. This
result would be expected for mutations that act at the receptor
level, because quinine salts hyperpolarize blowfly chemoreceptors,
antagonizing generator potentials induced by sugars
and salts (21), whereas 1 M NaCI usually stimulates electrical
activity in the blowfly cation receptor (22). On the other hand,
mutations perturbing functions in the central nervous system
could affect responses to NaCI, to quinine sulfate, or to both
compounds. In this regard, the existence of gus mutations which
alter the proboscis extension response is of practical significance,
because mutations that are detectable in individual flies can be
analyzed with genetic mosaics to locate anatomical foci for
mutant behavior (23). It is also possible to record electrical activity
from chemoreceptors in Drosophila (17, 24), which will
allow direct observation of the effect of gus mutations on receptor
function. Finally, the heat- and cold-sensitive mutants
are particularly useful, because they can be exploited for
temperature-shift analysis to determine the time in development
during which the gus gene products act (25). By identifying
when and where the gustatory genes function, it should
thus be possible to dissect parts of the nervous system mediating
contact chemoreception in Drosophila.
Proc. Natl. Acad. Sci. USA 76 (1979) 887
Note Added in Proof. Rodrigues and Siddiqi (28) have isolated six gus
mutants, in four genes, with abnormal responses to NaCl, quinine
sulfate, sucrose, or all three compounds.
We thank the reviewers for their comments on the manuscript. This
work was supported by U.S. Public Health Service Grant GM 20770
and was presented in part at the annual meetings of the Genetics Society
of America in 1974 and 1977 (refs. 26 and 27).
1. Dethier, V. G. (1969) Adv. Study Behav. 2, 111-266.
2. Lewis, E. B. (1960) Drosophila Inform. Serv. 34, 117-118.
3. Tompkins, L., Fleischman, J. A. & Sanders, T. G. (1978) Drosophila
Inform. Serv. 53, 211.
4. Hamilton, W. F. (1922) Proc. Natl. Acad. Sci. USA 8, 350-
353.
5. Benzer, S. (1967) Proc. Natl. Acad. Sci. USA 58, 1112-1119.
6. Craig, L. C. (1944) J. Biol. Chem. 155,519-534.
7. Grigliatti, T. A., Hall, L., Rosenbluth, R. & Suzuki, D. T. (1973)
Mol. Gen. Genet. 120, 107-114.
8. Grigliatti, T. & Suzuki, D. T. (1970) Proc. Natl. Acad. Sci. USA
67, 1101-1108.
9. Lewis, E. B. & Bacher, F. (1968) Drosophila Inform. Serv. 43,
193.
10. Merriam, J. R. (1969) Drosophila Inform. Serv. 44, 101.
11. Lindsley, D. L. & Grell, E. H. (1968) Genetic Variations of
Drosophila melanogaster, Carnegie Institute Publication No.
627 (Carnegie Institute, Washington, D.C.).
12. Denell, R. E. & Jackson, R. (1972) Drosophila Inform. Serv. 48,
44-45.
13. Gowen, J. W. (1942) Anat. Rec. 84, 458 (abstr.).
14. Konopka, R. J. & Benzer, S. (1971) Proc. Natl. Acad. Sci. USA
68,2112-2116.
15. Dethier, V. G. (1951) J. Gen. Physiol. 35,55-65.
16. Quinn, W. G., Harris, W. & Benzer, S. (1974) Proc. Natl. Acad.
Sci. USA 71, 708-712.
17. Falk, R. & Atidia, J. (1975) Nature (London) 254,325-326.
18. Fung, S.-T. C. & Gowen, J. W. (1957) J. Exp. Zool. 134,515-
532.
19. Wolbarsht, M. L. & Dethier, V. G. (1958) J. Gen. Physiol. 42,
393-412.
20. Wallis, D. I. (1962) J. Insect Physiol. 162, 453-467.
21. Morita, H. & Yamashita, S. (1959) Science 130, 922.
22. Evans, D. R. & Mellon, D. (1962) J. Gen. Physiol. 45, 651-
661.
23. Hotta, Y. & Benzer, S. (1970) Proc. Natl. Acad. Sci. USA 67,
1156-1163.
24. Isono, K. & Kikuchi, T. (1974) Jpn. J. Genet. 49, 113-124.
25. Suzuki, D. T. (1970) Science 170, 695-706.
26. Cardosa, M. J., Fleischman, J. A., & Sanders, T. G. (1974) Genetics
77, s9 (abstr.).
27. Tompkins, L. & Sanders, T. G. (1977) Genetics 86, s64
(abstr.).
28. Rodrigues, V. & Siddiqi, 0. (1978) Proc. Indian Natl. Acad. Sci.
Sect. B 87, 147-160.