Copyright 0 1993 by the Genetics Society of America Genetic Control of Pheromones inDrosophila simulans. 11. kete, a Locus on the X Chromosome Jean-FranGois Ferveur and Jean-MarcJallon Laboratoire de Biologie et Ginitique Evolutives, CNRS, 91 198 Gij-sur-Yvette,France Manuscript received June 17, 1992 Accepted for publication October 28, 1992 ABSTRACT The production of Drosophila cuticular hydrocarbons, including contact pheromones, is under polygenic control. To investigate X-linked loci, EMS mutations were induced in Drosophila simulans flies. A mutant strain was discovered which in both sexes showa reduction in the biosynthesis of both 7-tricosene (7-T)the species contact pheromoneand all other linear hydrocarbons. The locus controlling this effect, kiti, is recessive and was localized to I, 18.5. Unlike a previously identified gene on the second chromosome of thisspecies, Ngbo, kiti does not affect theratio of 7-T:7- pentacosene (7-P). Other reproductive characteristics are also affected, including egg-hatching. However, courtship behaviors in both sexes appear normal. -TRICOSENE is the main hydrocarbon on the MATERIALS AND METHODS 7 cuticle of male and female flies in most strains of Drosophila stocks and crosses: All strains of D. simulans Drosophila simulans and of Drosophilamelanogaster were kept on standard cornmeal and yeast medium under a males of many strains (LUYTEN1982; PECHINEet al. 12: 12-hr 1ight:dark cycle at 25 O. Three strains were used: 1985).This substance has been shown to act as a (1) Seychelles,based on several inseminated femalescol- pheromone, able to inducewing display in D. simulans lected in the Seychelles archipelago in 1981; (2) ywmJ a strain carrying four recessive markers: yellow (I, O.O), white males UALLON 1984). The cuticle of D. simulans flies (I, 1.5), miniature (I, 36.1) and forked (I, 56.7) (a gift of J. contains at least 13 hydrocarbons which vary quanti- COYNE); and(3) C(Z)RM, a strain with attached X chromo- tatively according to sex and strain. These molecules somes carrying yellow and white (a gift of T. WATANABE). have 23, 25, 27 or 29 carbons and are linear hydro- For regular crosses, five pairs of virgin 4-day-old flies carbons or 2-methyl-branched alkanes. The predom- were placed in vials with food. Progeny from these crosses inant linear hydrocarbons are monoenes with a single were analyzed continuously throughout the course of the experiments reported here. For backcross experiments, is- double bond in position 7: 7-tricosene (7-T, 23 car- ofemale lines were set up from females randomly collected bons) and 7-pentacosene(7-P, 25 carbons) UALLON over 14 generations and inaividually mated when 4- 14 days 1984; PECHINEet al. 1985). Apart from7-T, only one old with4-day-old males. In general, crossesset up for other D.simulans hydrocarbon, 7-P, is currently sus- studying the inheritance of hydrocarbon phenotype were pected of playing a role in mate recognition or stim- studied simultaneously. Mutagenesis: Forty Seychelles males, 12-24 hr old, were ulation in this species, perhaps via a synergetic inter- fed overnight on saccharose supplemented with 0.1% EMS action (COBBand JALLON 1990). (LEWISand BACHER1968). These males were then mass- In a previous article we reported the existence of mated with 50 virgin C(Z)RM females. This procedure was Ngbo, a majorsecond chromosome locus in D. simulans replicated 10 times. Six hundred and fifty F1 males were which controls the ratio of 7-T:7-P by reciprocally produced and were individually backcrossed to four virgin C(Z)RM females, producing F2males sharing the same X changing the levels of both hydrocarbons (FERVEUR chromosome. Each line was kept inbred, except in the case 1991). We also showed that females of most D. simu- oflow fertility, where maleswere backcrossed to virgin lans strains have higher levels of 7-T than their hom- C(Z)RM females. Cuticular hydrocarbon profiles were com- otypic males. This and other data suggest that genes pared for males from 295 lines. on the X chromosome may be involved (BENAMAR and Extraction and analysis of cuticular hydrocarbons: Cu- ticular hydrocarbons were extracted from individual flies JALLON 1983;FERVEUR, COBB and JALLON 1989). with the method described in FERVEUR (1991),except for SCOTTand RICHMOND(1988) argued that theX chro- mutagenized lines, for which four males from each line were mosome could modify levels of 7-T and 7-P in D. pooled at generations FP and Fs. This “pooling” procedure melanogaster males, although it is possible that genetic enabled us to exclude intraline hydrocarbon variability control of the 7-T:7-P ratio is different in the two which was not controlled by the X chromosome. Individuals species (FERVEUR1991). To further investigate di- from lines showing X-linked hydrocarbon variation were subsequently tested separately. rectly the role of the X chromosome on pheromone Hydrocarbon parameters: Thirteen hydrocarbons were production we have used EMS mutagenesis to induce detected ingas chromatography (GC) profiles and their X-linked mutations in D. simulans. quantities were measured. For the 295 mutagenized lines Genetics 133: 561-567 (March, 1993) 562 andJ.-F. Ferveur J.-M. Jallon the percentage of each hydrocarbon was measured relative ratio and the overall production of branched com- to the sum of all hydrocarbons (CHc) for that line and pounds (CBr) were not affected in strain 430 males. compared to the percentages obtained from all lines pooled. The analysis of putative mutant lines was carried out using To localize the character(s) involved, cuticular hy- percentages and absolute amounts (Q) of hydrocarbons, in drocarbons were analyzed from crosses between strain particular of the two main alkenes (7-T and 7-P) and two 430and pmf flies, which carrya multimarked X methyl branched hydrocarbonswith 27 and 29 carbons (27 chromosome (Table 2). pmf males and reciprocal F1 Br and 29 Br). Levels of other linear and branched hydro- carbons which showed lower absolute levels were separately males sharing the ywmf X chromosome all showed pooled (Elin and ZBr). The ratios of 7-T:7-P and 7-T:27 much higher levels of 7-monoenes than strain 430 Br were also measured. males. The Q7-T value was approximately six times Statistical tests: For mutagenesis screen each hydrocar- higher in males with a ywmf X chromosome than in bon parameter was compared to the mean f 2 SD calculated strain 430 males. from all the 295 mutagenized strains(P < 0.05). When one hydrocarbon parameter was compared between two inde- However, ywmf males produced about 30% more7- pendent samples, we used Student’s t test. For comparisons T than Seychelles flies while their 7-T:27 Br ratio was between two or more samples differing for two conditions very close (6.04 and 6.6,respectively). For this reason, (genotype and/or age and/or mating status), we used a two and because males with a pmf X chromosome show a factors ANOVA. 7-T:27 Br ratiofour times higherthan strain 430 Behavioral and fertility tests: Virgin pairs of flies were observed under a watch glass for 2 hr. Courtshipand males (Table2), we mapped the locus causing the copulation latencies and durations were notedeach for pair. hydrocarbon defect with a 7-T:27 Br ratio. Mated females were individually transferred to a vial con- Recombinant males from backcrosses and F2 strains taining food colored with neutral red (0.05%; to enhance were grouped according to their morphological phe- background contrast) and were allowed to oviposit for 24 notypes and were characterized by the two predomi- hr before being discarded. At least 24 hr later the number of unhatched eggswas noted. The sex ratio of the resulting nant hydrocarbons Q7-T and Q27 Br and by their imagos was also recorded. The hydrocarbon profile of all ratio (7-T:27 Br) (Table 3).The 7-T:27 Br parameter flies was subsequently analyzed. gave the most consistent result. The factor controlling this parameter appears to segregate with white and RESULTS miniature (6.42 & 0.14 for w-m- as against 2.57 +- 0.1 1 for w+m+). On thebasis of these distributions, an Genetic control of hydrocarbon variation: Males from 295 EMS-mutagenized lines were screened for empirical cutoff point was established at 4.2, separat- their cuticular hydrocarbon profile. One strain (430), ing the two “7-T:27 Br” phenotypes with a low mis- showed a significant quantitative variation: these flies classification probability (P = 0.056) (Figure 1). With showed the same number of GC peaks as wild-type this classification criterion, the hydrocarbon profileof individuals, with similar retention times, but the pro- males showing only one of the two markers was stud- portions of 7-T and of 7-P (41.6% and 2.5%, respec- ied. Overall, males expressing either w-or m- showed tively) in males were lower than for all 295 mutagen- intermediate values; recombination between the two markersand the two 7-T:27 Br values (< 4.2 <) ized lines pooled (59.8 f 6.5%and 5.4 & 2.8%, respectively). Conversely, strain430 males showed enabled us to map a single locus or a tightly linked much higher proportions of both methyl branched group of loci on the X chromosome at 18.5 f 1.8, hydrocarbons 27 Br and 29 Br (25.8% and 9%, re- which controls the ratio of7-T:27 Br. We have called spectively) as compared tomales from all mutagenized this locus kite‘, a word meaning “small quantity” in a strains pooled (9.0 & 3.4% and 3.8 f 2.1%). Thus West African dialect, following the mutant phenotype strain 430 males have a much lower ratio of 7-T:27 which shows low 7-T:27 Br value.