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.
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:
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.
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 aver-
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.
sion.
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.
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
Nati. Acad. Sci. USA 76 (1979) ~~~~~Proc. Tompkins Genetics: al.al. etet Genetics: Tompkins
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-
885
0
0.3 C
0.2 L1_ 0.1
C 0.4-
0.30.20.1
0
1 4 2 3 Countercurrent tube
5
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
0.40
0~0.3 20. U.
0.1 0
3 1 2 4 Countercurrent tube
5
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
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Proc. Natl. Acad. Sci. USA 76 (1979)
Tompkins et al.
Table 1. Responses of Oregon R males and females and of dxsD intersexes to stimuli
Sex
QS*
Males Females Intersexes
0.90 t 0.04 0.86 + 0.05 0.90 + 0.02
Stimulus NaCl 0.84 + 0.05 0.35 + 0.08 0.72 + 0.04
None
Sucrose
0.52 + 0.03 0.50 i 0.04 0.52 i 0.02
0.89 0.85 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 Stimulus Sucrose NaCl QS* Group Mutant(s)
gusAQ2 gusAQ3 gusAQ6
0.22 4 0.04 0.20 i 0.05 0.25 + 0.03 0.26 + 0.06
0.90 4 0.02 0.86 ± 0.03 0.88 ± 0.03 0.88 ± 0.04
0.08 0.26
B
gusBQ4 gusBQ5
0.50 0.05 0.49 + 0.06
0.52 ± 0.04 0.50 ± 0.07
0.12 0.21
C
gusCNlO
0.90 ± 0.04
0.18 + 0.04
0.94
D
gusDN9
0.18 ± 0.03 0.11 ± 0.03
0.20 ± 0.03 0.15 + 0.02
0.90
gusDNl2 gusENl3
0.10 ± 0.02
0.13 ± 0.04
0.43
A
E
gusAQ1
+
0.44
0
.°0.2_
\
CI LL0.1
0
4 3 2 1 Countercurrent tube
5
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
0.78
0.92 0.50 + 0.03 0.90 ± 0.03 gusFN5 0.92 0.48 0.05 0.91 ± 0.05 gusFN32 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.
F
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, 350353. 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. 134,515532. 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, 651661. 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.
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.
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:
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.
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 aver-
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.
sion.
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.
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
Nati. Acad. Sci. USA 76 (1979) ~~~~~Proc. Tompkins Genetics: al.al. etet Genetics: Tompkins
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-
885
0
0.3 C
0.2 L1_ 0.1
C 0.4-
0.30.20.1
0
1 4 2 3 Countercurrent tube
5
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
0.40
0~0.3 20. U.
0.1 0
3 1 2 4 Countercurrent tube
5
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.
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Proc. Natl. Acad. Sci. USA 76 (1979)
Tompkins et al.
Table 1. Responses of Oregon R males and females and of dxsD intersexes to stimuli
Sex
QS*
Males Females Intersexes
0.90 t 0.04 0.86 + 0.05 0.90 + 0.02
Stimulus NaCl 0.84 + 0.05 0.35 + 0.08 0.72 + 0.04
None
Sucrose
0.52 + 0.03 0.50 i 0.04 0.52 i 0.02
0.89 0.85 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 Stimulus Sucrose NaCl QS* Group Mutant(s)
gusAQ2 gusAQ3 gusAQ6
0.22 4 0.04 0.20 i 0.05 0.25 + 0.03 0.26 + 0.06
0.90 4 0.02 0.86 ± 0.03 0.88 ± 0.03 0.88 ± 0.04
0.08 0.26
B
gusBQ4 gusBQ5
0.50 0.05 0.49 + 0.06
0.52 ± 0.04 0.50 ± 0.07
0.12 0.21
C
gusCNlO
0.90 ± 0.04
0.18 + 0.04
0.94
D
gusDN9
0.18 ± 0.03 0.11 ± 0.03
0.20 ± 0.03 0.15 + 0.02
0.90
gusDNl2 gusENl3
0.10 ± 0.02
0.13 ± 0.04
0.43
A
E
gusAQ1
+
0.44
0
.°0.2_
\
CI LL0.1
0
4 3 2 1 Countercurrent tube
5
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
0.78
0.92 0.50 + 0.03 0.90 ± 0.03 gusFN5 0.92 0.48 0.05 0.91 ± 0.05 gusFN32 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.
F
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)
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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, 350353. 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. 134,515532. 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, 651661. 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.
