Copyright 0 1991 by the Genetics Society of America

Genetic Control of Pheromones in simulans. I. Ngbo, a Locus on the Second Chromosome

Jean-Franqois Ferveur

Laboratoire de Biologie et Ginitique Evolutives, Centre National de la Recherche Scientijique, 91 198 Gijhur-Yvette, France Manuscript received May 9, 1990 Accepted for publication February 4, 1991

ABSTRACT 7-Tricosene and 7-pentacosene are predominant hydrocarbons on the cuticle of both sexes in Drosophila simulans. The pheromonal role of 7-tricosene has been clearly established for conspecific males, while a synergistic effect for 7-pentacosene has been postulated. Interstrain variation for the production of both compounds is very marked, but similar for both sexes. The genetic basis of this polymorphism was investigated. A major role was found for the second chromosome,which controls the 7-tricosene:7-pentacoseneratio. The main locus involved in controlling this variation,Ngbo, was mapped to position 65.3 on the second chromosome. The production of 7-pentacosene is directly related to the Ngbo genotype, which is additively expressed with two known alleles, Seychelles and Cameroon. These alleles act codominantly and are, respectively, hypomorphic and hypermorphicwith regard to their effect on 7-pentacosene production.The production of 7-tricosene, which is partially inversely related to that of 7-pentacosene, is also affected by secondary interactionswith the second chromosome and with the autosomal background.

YDROCARBONS on the cuticle of Drosophila simulans because these compoundsare present in both H simulans females act as pheromones capable of sexes in similar amounts. inducing conspecific male courtship (JALLON 1984). BENAMARand JALLON (1983) suggested that the The hydrocarbon systems of matureflies of bothsexes level of 7-T in D. simulans was controlled by the X are virtually identical and mainly consist of long-chain chromosome.FERVEUR, COBB and JALLON (1989) hydrocarbons with one double bond, in position 7. showed that one pair of autosomes played a major The predominant cuticular hydrocarbonsshow a geo- role in controlling the ratio of 7-T:7-P, and that the graphical polymorphism: 7-tricosene(7-T; 23 car- production of 7-T was also dependent on sex-linked bons), which has been shown to induce wing display factor(s). The genetic control of male-predominant in conspecific males (JALLON1984), is the major hy- compounds (7-T and 7-P) in D. melanogaster appears drocarbon in both sexes of most strains. from to be highly complex, possibly involving all chromo- somes (SCOTTand RICHMOND1988). around theBenin Gulf in Africa have higher levels of In order to further our understanding of genetic 7-pentacosene (7-P; 25 carbons). This variation simul- controlof Drosophila pheromone production, we taneously affects both sexes (LUYTEN1982). have undertaken an exhaustive investigation testing has been studied in bioassays with 7-T D. simulans. each potential hereditary factor. In this studywe have The roleof 7-P, thepredominant hydrocarbon in chosen to concentrate on the relative and absolute some African strains, is not yet known, although 7-P production of 7-T and 7-P in D. simulans. We chose alonecannot stimulate precopulatory behavior in this species because the absence of a qualitative hydro- males from the 7-T-rich D. simulans Seychelles strain carbonsexual dimorphism allows both sexes to be (J.-M. JALLON, unpublished results). On thebasis of a analyzed and, unlike D. melanogaster, the interstrain study of inter- and intraspecific courtships, COBBand hydrocarbon polymorphism is very marked, and may JALLON (1 990)proposed that 7-P may also play a role be controlled by only a few genes. in stimulating courtship in D. simulans males, perhaps through synergistic effects. MATERIALS AND METHODS It has been suggested that 7-T or 7-P, which are Drosophila stocks and crosses: Strains of Drosophila si- predominant hydrocarbons in mulans were kept at 25 & 0.5”,under a 12: 12 dark:light males, mayplay a strain-specific “anti-aphrodisiac” cycle, on standard cornmeal food. The Cameroon strain is role in this species, following their transfer onto the derived from a small number of fertilized females collected in Yaounde in 1975. As with other D. simulans strains from femalecuticle during mating (SCOTT and JACKSON the Benin Gulf area, both sexes of Cameroon flies produce 1988). Such an effect should not be expected in D. more 7-P than 7-T. The Seychelles strain is derived from

Genetics 128: 293-301 (June, 1991) 294 J.-F. Ferveur severalinseminated females collected in the Seychelles logarithmically transformed (log,) in order to reduce intra- archipelago in 198 I. As with most D. simulans strains (all strain variations in 7-T and 7-P levels. The variation in 7- except those fromthe Benin Gulf), both sexesproduce more T:7-P levels wascompared with changes in absolute amounts 7-T than 7-P. of 7-T and 7-P in small samples of flies raised together. The inheritance of hydrocarbon phenotype variation was These results suggested that absolute amounts of the two studied by crosses between the two parental strains (Came- substances would not be a suitable parameter for studying roon and Seychelles), based on flies bred in the same con- genetic determination, assignificant intrastrain variation ditions and analyzed within a short period of time. Chro- could occur within a short space of time (see RESULTS). mosomal localization of genes involved hydrocarbonin phe- notype variationwas carried out using the following marked strains: (1) G strain, which carried the recessive markers RESULTS garnet (I,44.4), net (11,O.O) and cinnabar (II,57.5) (a gift of J. COYNE).(2) NBPSP or secondchromosome recessive Hereditarycontrol of hydrocarbon variation: multimarker strain, which carries net (11,O.O) black (I1,45.0) Flies were characterized by their hydrocarbon phe- pearly (II,74.0) spread (IIJ30.0) and plum (11,108.0) (a gift notype (S or non-S) based on two quantitative char- of J. COYNE).(3) W strain, which carries the Curly mutation acters: % and % 7-P (see Figure 1). Mean (IX,6.8) (a gift of T. WATANABE).For crosses, five pairs of 7-T 7-T 4-day-old flies were placed in vials withfood. Each cross was and 7-P percentages were alsocalculated foreach replicated at least nine timesfor each experiment. group of flies. Results of chromatographic analysis Extraction and analysis of cuticular hydrocarbons:For of males and females from all sixteenstrains ob- each experiment, flies from different strains or crosses were tained from the two parental strains, Seychelles and collected and bred simultaneously. Flies were sexed under Cameroon, their backcrosses and F2 are shown in light etherization shortly after eclosion and were kept in FI, groups offive on fresh food. Following the method of Table 1. ANTONYand JALLON(1982), 4-day-old flies were individ- When strains differedfor some of their autosomes ually washed in 50 PI hexane in a microtube and agitated they showed significant differences either for S/non- for 1 min. Washed flies were immediately removed from S characterization (SS us. strain 1 males, SS us. strain eachmicrotube. In certain samples, 20 pl of a hexane 5 females), or for their mean percentage7-T and 7-P solution containing 800 ng of hexacosane (26C) as an inter- nal standard were added to each tube. When the solvent levels within the non-S class (CC us. strain 2 females; was fully evaporated, each microtube, now containing a dry CC us. strain 6 males). These differences were com- extract, was sealed with a Teflon cap and stored at -20" posed of twoeffects. The hydrocarbonphenotype until analysis. classification differentiated flies homozygous for Sey- Immediately before analysis 20 pl hexane were added to chelles autosomes from other genotypes; variations in each microtube, and after gentle agitation 5 PI of the re- sultant solution were injected into a Girdel 300 gas chro- mean % 7-T and % 7-P within the non-S class must matograph equipped with a C.P. Si1 5 capillary column (1 = be caused by the varying autosomal genotypes present 25 m; $ = 0.23 mm) with nitrogen as the vector gas (P = in this class. 0.5 bar). During analysis the temperature increased from Otherhereditary components (X chromosomes 170 to 250 at a rate of Z"/min. Peak detection was carried " tested only between females, Y chromosome and cy- out using a Flame Ionization Detector coupled with an SP 4270 Spectraphysics integrator which yields retention times toplasmic factors) showed no significant effects. Com- and areas under each peak. Extractsof different phenotypes parisonof S/non-S distributions between males of were injected in varying order to randomize experimental strains 1 and 5 was significant (P < 0.001). This effect variability. was initially attributed to the Y chromosome but upon The majorhydrocarbon compounds of D. simulans further investigation was revealed to be a sampling Seychelleshave been chemically identified by PECHIN~ et al. (1985) and show characteristic constantretention times effect caused by delayed eclosion of certain hydrocar- (+-0.2%under constant conditions) overmany generations. bon phenotypes. For each spectrum, we calculated the total quantities Although the production of 7-T and 7-P varied in of hydrocarbon using the summed areas of 13 compounds different strains, their sum (% 7-T + % 7-P) for all which are always detected in this species. Eachhydrocarbon strains pooled remained remarkably constant both for was characterized by its percentage relative to the total hydrocarbon sum. By comparing this figure to that of the males (57.5 f 0.2; n = 719) and for females (62.3 f internal hexacosane standard, the absolute quantity in ng 0.2; n = 750). Within the sample as a whole, and for could also be calculated. all strains exceptS X S, there was a significant negative Hydrocarbonparameters: To localizegenetic factors correlation between the proportions of7-T and of 7- involved in the 7-monoene polymorphism,different hydro- P inindividual flies. The absence of asignificant carbon parameters were used. The percentages of 7-T and 7-P showed significant differences betweenparental strains, correlation in Seychelles parental flies was probably a low intrastrain variation and a marked stability over many due to the very low variability of 7-P percentage in generations. Percentages of 7-T and 7-P were used as meas- both sexes (see Figure 1). ures of hydrocarbon phenotype for studying the role of Genetic control of 7-T:7-P ratio: Strains with the various hereditary components. More than 1500 flies were same autosomes which showed no significant differ- individuafly analyzed.We chose those parameters shown to ences for their hydrocarbons parameters (see Table be the most stable over time.The ratio of 7-T:7-P produced clear interstrain differences and was used for the localization 1) were pooled. Populations of flies were character- of genetic control of 7-monoene variation. This ratio was ized by their hydrocarbon phenotype,S or non-S. The Genetic Control of Pheromones 295

females males I 80- I 60- I I I I I 0 I I 50 - 50 - I F1 Soy x Cam F1 Cam x Soy 40 - i 40 s 30-

0 F: 20 - 0 $ 20 """""""~-,~"""""""- 01 0 4 I 1 """""""""-+"9-""""""- 10 - 10 - i-3. 0 .,.,.I.I!I.I.I.I 0 .,.,.,.:-,.,.,.1 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 % 7-tricosene % 7-tricosene FIGURE1 .-Distribution of individual male and female flies from Seychelles (Sey)and Cameroon (Cam) strains and their reciprocal hybrids (FI = female X male), according to their percentage of 7-tricosene (% 7-T) and 7-pentacosene (% 7-P). % 7-T and % 7-P are expressed as a percentage of total hydrocarbons. Each fly was given a pair of x-y coordinates on the basis of its % 7-T and% 7-P. Dotted lines represent the cut-off points used to define the two hydrocarbon phenotype groups, S and non-S, and were empirically set at 40% 7-T and 20% 7-P for males, 45% 7-T and 12.5% 7-P for females. The two leading diagonal cells contain either Seychelles or Cameroon plus F1 flies and represent more than 95% of the available data (Cameroon and F1 distributions overlap and could not be discriminated by this method). When a point fell into oneof the other two diagonal cells, it was integrated into oneof the two more populated cells on the basis of immediate proximity. In this way, data were classified into two different hydrocarbon phenotypes, Seychelles (S) and non-Seychelles (non-S) with Cameroon plus F, flies. With this binary system, the misclassification probability for S and non-S cells was 0.015 for males and 0.005 for females. distributions of the Cameroon and Seychelles strains genotype being partially dominant, with flies having appeared unimodal, suggesting low intrastrain varia- Cameroon chromosomes showinga stronger effect. tion for genes controlling 7-monoene ratio (Figure 2). Further one-factor ANOVA comparisons of 7-T:7- There is no full dominance effect: pooled reciprocal P, carried out between S class flies, showed a signifi- F1 flies were normally distributed and their distribu- cant effect of autosomal background: parental Sey- tionpartially overlapped but significantly differed chelles differed from both Backcross to Seychelles and from that of Cameroon flies (t = 14.5, d.f. = 173, P Fs, for males (F = 22.4; d.f. = 2, 165; P C 0.001) and CO.001 formalesandt= 11.2,d.f.= 125,P<0.001 females (F = 11.7; d.f. = 2, 189; P C 0.001). for females).Backcrosses to Seychelles(Bc Sey) Mapping the major gene(s) involved in 7-T:7-P yielded a bimodal distribution with roughly equal variation: Chromosomal localization: The marked G peaks (49.7%:50.3% for males; 48.7%:51.3% for fe- strain, which carries garnet (X chromosome), net and males). Distributions for both sexes, tested with a x* cinnabar (second chromosome), shows the same type goodness of fit, were not significantly different from of hydrocarbon pattern as the Seychelles strain. a theoretical 1: 1 ratio. Backcrosses to the Cameroon Cameroon and G strains produce very different per- strain (Bc Cam) appeared unimodal. Their distribu- centages of 7-T and 7-P; they also showdifferences in tion overlapped those of both Cameroon and F1 flies. the absolute quantity of both substances, although the Mean values (-0.60 and -0.17 for males and females, sumsof both 7 monoenes (2 7") andthe total respectively)were roughly intermediate between hydrocarbon amount (I; Hc) are similar. The recip- those of their Cameroon (-0.76 and -0.64) and F1 rocal Fls, which showed no significant difference in (+ 0.02 and + 0.21) same sex parents. The distribu- their 7-T and 7-P absolute production (intermediate tion of F2 flies appeared bimodal with two unequal between those of their same-sex parents), were pooled peaks (81.4%:18.6% for males and 73.1%:26.9% for for furtheranalysis. Their Z7-M and ZHc values were females). The meanvalue of the majorpeak was close to those of both parental strain values. Results located between those of Bc Cam and the F1 (-0.29 are shown in Table 2. for males and -0.01 for females). Distributions for The backcross progeny was clearly dividedinto two both sexes were tested with a x2 goodness of fit test. hydrocarbon phenotype classes. Flies expressing the Males, but not females, were significantly different morphological marker cinnabar often showed a S from a theoretical 3:l ratio distribution (x2 = 4.53, hydrocarbon phenotype like that of the G strain. Flies d.f. = 1, P 0.05). This effect could be explained by which did not express the cinnabar marker often the same sampling effectdescribed previously. showed a hydrocarbon pattern similar to that of the The overall results strongly suggest that the ratio F1 (non-S) flies. of 7-T : 7-P is controlled by a Mendelian character Recombinationsbetween the S hydrocarbon and carried on a single pair of autosomes, each parental cinnabar phenotypes, only scored in Fl female X G 296 Ferveur J.-F.

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M ales Females Males out of 25) heterozygous for this segment expressed a non-S hydrocarbon phenotypelike the F1 hybrids. An 10 empirical cut-off value, discriminating both hydrocar- 5 0 bon phenotypes, was derived from these distributions 251 (Figure 3). Males were divided into thoseexpressing either black or pearly. Out of 170 “black” males, 1 16 were non-S, thus the probability for a black (non-pearly) fly being non-S was ~0.685. Outof 166 “pearly” males, 47 were non-S, thus the probability for a pearly (non- 25 i black) fly being non-S was 0.283. Note that the sum 20 ln I of these probabilities (0.97) is very close to 1, because flies from both these groups inherit complementary recombinant segments of their second chromosome from the same F1 female parents. The distance be- tween black (II,45) andpearly (11, 74) in D. simulans is 29 recombination units (J. S. BARKER,personal com- munication). The locus modifying the 7-T:7-P ratio is thus located on the second chromosome at 65.32 1.0 (the error value was calculated from the misclassifi- cation probability), while the cinnabar locus is located I ll 1 at 11, 57.5. We call this hydrocarbon locus Ngbo (the word means “forest” in the dialect of the people living in the West African equatorial forest where 7-P rich flies are found). The absolute production of 7-T and 7-P was also -2 -1 0 +1 +2 4 -2 -1 0+2 +1 4 studied. The production of7-P, which differs between loge ratio of 7-T : 7-P S flies (1 16 ng) and non-S flies (335 ng), showed no FIGURE2.-Frequency distribution of log, 7-T:7-P values (in significant differences within S or non-S classes. S flies groups of log, 0.1) for offspring of various crosses from Cameroon produced more 7-T (730 ng) than non-S flies (430 (Cam) and Seychelles (Sey) parental strains. Dotted FO values rep- ng). Within the S phenotype group, a significant dif- resent the Seychelles (S) phenotype, hatched values the Cameroon (non-S) phenotype. Dottedlines indicate the empirical cut-off point ference for 7-T production was found between pearly between S and non-S (+0.69 for males, +1.28 for females). Recip- flies varying either for black (t = 5.04, d.f. = 174, P rocal F, flies were pooled. Bc Sey and BcCam represent the < 0.001) or for spread (t = 5.36, d.f. = 175, P < offspring of male or female FI flies backcrossed to parental strain 0.001) irrespective of other markers. Thus, 7-P vari- flies (Sey or Cam). FP flies are the offspring of the four possible crosses between the two reciprocal F, hybrids. ation is mainly dependent uponNgbo, while 7-T varies with Ngbo and other genes on the second chromo- male progeny, were rare. Only 8% of cinnabar flies some. expressed the non-S hydrocarbonphenotype while Dosage effect of the second chromosome on 7-T 4.2% of non-cinnabar flies expressed the S hydrocar- and 7-P levels: Flies of the W parental strain, which bonphenotype. This suggests that the major locus bear Curly, a second chromosome dominant marker, responsible for the7-T:7-P ratio variationis relatively and express aSeychelles-like hydrocarbon phenotype, close to the cinnabar locus, on the second chromo- were reciprocally crossed with Cameroon flies (Table some. 3). Cameroon males showed a significant increase in Precise mapping on the second chromosome: The mul- the absolute amount of 7-monoenes over asix-month tiply marked second chromosome (nt b py sd pm) is period (t = 2.38, d.f. = 34, P < 0.025), while there carried by the NBPSP strain, which shows a similar was no significant change in the log, ratio of 7-T : 7- hydrocarbon pattern to the Seychelles strain. NBPSP P (t = 0.653, d.f. = 34, P = NS). F1 males with a Curly and F1 flies, which were, respectively, homozygous phenotypeand a Cameroon X chromosome back- and heterozygous forthe multiplemarker second crossed toCameroon females produceoffspring chromosome, showed clear differences in their 7-T:7- whose males share all hereditary components except P ratio distribution:all NBPSP flies (n = 13) belonged theirsecond chromosome. Comparisons between to the S phenotype, while all F1 flies (n= 35) expressed groups of wild phenotype males showed that the num- non-S phenotype. Most backcross flies (57 out of 58) ber of Cameroon second chromosomeswas responsible homozygous forthe black-pearly segmenthad a S for the main variation in the absolute level of 7-P (F hydrocarbon phenotypewhile most backcross flies (23 = 26.8; d.f. = 3, 109; P < 0.001) and 7-T (F = 21.6; 298 J.-F. Ferveur

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25 simulans (FERVEUR,COBB and JALLON 1989). In that b py homozygotes 20 I study we evaluated the effects of autosomes and sex 15 chromosomes by using the C(1)RM strain with at- 10 tached X chromosome females; however,we were not 5 able to exclude the effect of other hereditary factors. 0 In this study we have shown that the major variation 25 3 1 b py heterozygotes in the ratio of 7-monoene hydrocarbons (7-T:7-P) in simulans is controlled by Ngbo, a single locus (or a 15 D. ln i group of very tightly linked loci)on the second chro- mosome at position 65.3. The two known alleles of Ngbo (Seychelles and Cameroon) act codominantly on

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10- I production.

5- We studied both percentage measures (96 7-T and

0- . ’ . .’. % 7-P) and the ratio of 7-T:7-P, all of which show low interindividual variability and are relativelystable py homozygotes I over time, which is not the casewith absolute 7- 15 monoene levels. For example, only small nonsignifi- 10 cant changes were detected in hydrocarbon percent- 5 ages of references Seychelles and Cameroon strains, 0 as compared to the data published by JALLON (1984). -1 0 +1 +2 +3 These differences may be due to slightly different methods of analysis,and/or to genetic drift. However, logeratio of 7-T : 7-P these parameters also present certain difficulties. For FIGURE3.-Frequency distribution of log, 7-T:7-P for backcross example, we could not distinguish the distributions of male flies showing combinations of the black (b) and pearly (py) Cameroon and of the reciprocal F1 flies because of markers on their second chromosomes. Cameroon females were the overlap of their % 7-T and % 7-P values. More- crossed with NBPSP males carrying all five markers. F, females over, the variation in the percentage of either one of were backcrossed to NBPSP males. Parental NBPSP males were the two monoenes can be produced by a change in pooled with the backcross “bpy” homozygotes. FI males were pooled with the “b py” heterozygotes. Dotted lines represent the empirical the absolute production of other substances. cut-off point (+ 1.00) between the S (to the right) and non-S (to the Given that the sum of both compounds (as a pro- left) hydrocarbon phenotypes. portion of total hydrocarbons) is constant for each sex regardless of strain, weused the 7-T:7-P ratio, a d.f. = 3, 109; P 0.001). FI males,whatever the < parameter which provides a way of avoiding external cross, were significantly different from both Fo and variations and enhances phenotypic differences when Bc flies (pair-wise comparison with Fisher PLSD, P < the variations in the two compounds are negatively 0.05), which in turn were not significantly different. correlated. Such a ratio analysis of pheromone blends Comparisons of Curly males witheither oneor two W second chromosomesconfirmed that thesecond chro- has proved useful in the study of other (KLUN and MAINI 1979; COLLINSand 1985). Changes mosome was responsible for the variation of the ab- CARD^ in the ratio of 7-T:7-P were shown to be mainly due solute amount of 7-P (F = 109.7; d.f. = 3, 105; P < O.OOl), absolute amount of 7-T (F = 4.55; d.f. = 3, to a reciprocally linked variation in the absolute pro- 105; P < 0.01), and also for 27-M (F = 12.1; d.f. = duction of 7-T and 7-P. Using the NBPSP multi- 3, 105; P < 0.001) and ZHc (F = 11.2; d.f. = 3, 105; marked strain, S flies showed 300 ng more 7-T than P < 0.001). There were significant differences be- non-S FI flies, while 7-P levels were 219 ng higher in tween Fo and F1 or Bc flies (pair-wise comparisons) non-S flies. The dose-dependent increase of 7-P level but not between Bc and Fl flies. Finally, the produc- (280 ng for each Cameroon second chromosome), and tion of 7-P showed a clear dosage effect: male flies the fact that 7-P variation on the Cameroon second with none, one or two Cameroon second chromo- chromosome is exclusively linked with Ngbo, strongly some(s), respectively,produced a mean of 53, 345 or suggest that Ngbo codominantly controls 7-P synthesis. 616 ng of 7-P. The twoknown alleles of Ngbo, Seychelles and Cameroon, are respectively hypomorphic and hyper- DISCUSSION morphic with regard to 7-P production. We have previously implicated one pair of auto- Although the main variation of the ratio of 7-T:7- somes in the control of the ratio of 7-T:7-P in D. P is due to the second chromosome’s control of 7-P 300 J.-F. Ferveur

TABLE 3 Dosage effect of the second chromosomeon the absolute productionof ?-pentacosene(7-P) and 7-tricosene (7-T)

Cross Pheno- Generation (Female X Male) type n log. 7-T:7-P Q 7-T 7-M Q 74' z Hc Cam X Cam + 18 -0.83k0.09 275f27 604k44 879261 14662 78 Fo { wxw Cy 33 +2.3720.05 5452 19 53f 3 599f21 984f34 + 22 +0.72+0.06 570227 2792 15 850+34 1400f58 Cam X W { Cy 31 +0.31 2 0.06 456 2 24 3342 17 790 2 34 12962 56 FI + 30 +0.31kO.08 513232 363f21 876f 39 1405265 W X Cam i 1 Cy 18 +0.2320.10 457+31 364k26 821 242 1359f70 Cam X -0.6343 20.07342222 621 2 35 962f461518277 Backcross + (Cam Xcompressed not { Cy 27 +0.1320.06437f25 3792 16 817f341298f 58 For hydrocarbon parameters, refer to Table 2. Strains used were Cameroon (Cam) and W, a marked strain with the dominant marker Curly (Cy; 11, 6.8). Parental strains were reciprocally crossed and [Cy] males from the (Cam females X W males) cross were backcrossed to Cam females. production, variation in 7-T levels also plays a role. control of these characters in this species, based on Unlike 7-P production, 7-T levels seem to be influ- the autosomes and the X chromosome. We have ob- enced by multiple genetic factors. Independently of tained similar but not identical results for D. melano- the main effect causedby Ngbo, factors modifying the gaster; it appears thatthere are differences in the absolute production of 7-T (but not that of 7-P) were genetic control of 7-monoene polymorphism between detected on the second chromosome. The percentage D. melanogaster and D. simulans. of 7-T was also affected by factors segregating in the Hydrocarbon phenotype is only indirectly linked to autosomal background, probably located on the third gene expression. JALLON (1984) proposed a scheme chromosome. for Drosophila cuticular hydrocarbon biosynthesis in Ngbo is not the only locus involved in pheromone which it was suggested that D. melanogaster and D. synthesis: we have also isolated kete, a locus on the X simulans share mostenzymatic steps. This scheme chromosome, which mainly affects 7-tricosene levels agreed with most of the known data about (J.-F. FERVEUR,in preparation). X chromosome effects fatty acid synthesis, especially withthe biosynthesis of have previously been implicated in 7-T production in Z-9-tricosene by Musca domestica (HOWARDand D. simulans (BENAMARand JALLON 1983; FERVEUR, BLOMQUIST1982; BLOMQUIST, DILLWITHand ADAMS COBBand JALLON 1989), and in the total production 1988), and involved a small number of enzymes and of 7-monoenes in D. melanogaster (SCOTTand RICH- thus a small number of structural genes. A series of MOND 1988). Because we used the protocol of WAHLS- studies involving the injection of precursors and the TEN (1979) in this study, we could not test the hemi- use of mutants has supported this model(CHAN YONC zygous effect of this chromosome between different andJALLON 1986;JALLON et al. 1986,1988; FERVEUR, male strains.A furtherdifficulty was that most paren- COBBand JALLON 1989). tal strains, especially Cameroon and Seychelles, pro- According to this scheme,7-T and 7-P shouldresult duced equivalent amounts of total 7-monoenes (Q 7- respectively from the decarboxylation of 17-tetraco- T + Q 7-P) and no significant differences were ob- senoic acid (24 C) and 19-hexacosenoic acid (26 C), served between their reciprocal hybrids which could the 26 carbon fatty acid resulting from the 24 carbon be linked to the X chromosome. molecule by the addition of two carbons through an Nevertheless, thereare clear quantitative differ- elongation process. Given that Ngbo is involved in the ences between the sexes. Females produce more hy- 7-T:7-P ratio variation, itcould act oneither the drocarbons (Z7-M and ZHC) and have higher % 7-T elongation or decarboxylation steps in JALLON'S values and lower % 7-P values, and higher absolute 7- (1984) scheme. In either case, the result would be an T levels than homotypic males. There is an additive alteration in the relationship between the speeds of effect on 7-monoene synthesis due to an X chromo- the two reactions. somedosage effect or to the sexual determination In Seychelles-likeflies, the decarboxylation step these chromosomes control. must be faster than elongation, leading to an accu- SCOTTand RICHMOND(1988) investigated the ab- mulation of 7-T. The decrease in the ratio of 7-T:7- solute production of 7-T and 7-P in D. melanogaster, P shown by non-S flies could thus be due either to a hybridizing the 7-T-rich CS strain with the 7-P-rich decrease in the speed of decarboxylation or to an Tai strain. They concluded that therewas a polygenic increase in the speed of elongation, resulting in an Gen etic ControlGenetic of Pheromones 30 1 increase in the production of longer-chain molecules. CHANYONG, T. P., and J.-M. JALMN, 1986 Synth6se de novo The interpretation by which Ngbo modifies enzymatic d’hydrocarbures potentiellement aphrodisiaques chez les Drosophiles. C. R. Acad. Sci. Paris 303: 6 197-202. specificity is supported by the fact that 7-P-rich strains COBB,M. J., and J.-M. JALLON,1990 Pheromones, mate recogni- like Cameroon synthesize a significant amount (3-5%) tion and courtship stimulation in the Drosophila melanogaster of 7-heptacosene (27 C) which cannot be detected in species subgroup. Anim. Behav. 39: 1058-1067. 7-T-rich strains. COLLINS,R. D., and R. T. CARD^?, 1985 Variation in and herita- The behavioral and evolutionary implicationsof bility of aspects of pheromone productionin the pink bollworm moth, Pectinophora gossypiella(Lepidoptera: Gelechiidae). Ann. hydrocarbon polymorphisms in both D. simulans and Entomol. SOC.78: 229-234. D. melanogaster are still unclear. We do not know why FERVEUR,J.-F., M. J. COBBand J.-M. JALLON,1989 Complex certain geographical strains show different hydrocar- chemical messages in Drosophila, pp. 397-409 in Neurobiology bon profiles, nor what happens if and when flies from ofSensory Systems, edited by R. N. SINCHand N. J. Strausfeld. the different strains meet in the wild, although there Plenum Press, New York. HOWARD,R. W., and G.J. BMMQUIST,1982 Chemical ecology is no isolationbetween geographical strains in the and biochemistry of hydrocarbons. Annu. Rev. Entomol. laboratory (COBBand JALLON 1990). Anypossible 27: 149-172. behavioral role of the 7-monoene blend inD. simulans JALLON,J.-M., 1984 A few chemical words exchanged by Droso- will have to be studied usingbioassays combining phila during courtship. Behav. Genet. 14 441-478. different ratios and amounts of both compounds. Al- JALLON,J.-M., C. ANTONY,T. P. CHAN-YONGand S. MANIAR, 1986 Genetic factors controlling the production of aphro- though nonpheromonal cues are also important in disiac substances in Drosophila, pp. 445-452 in Advancesin mate choice in both D. simulans and D. melanogaster, Invertebrate Reproduction, Vol. 4, edited by M. PORCHET,J. C. the different hydrocarbon profiles of the two species ANDREWSand A. DHAINAUT.Elsevier, Amsterdam. probably play a role in isolation. JALLON,J.-M., G. LAUGE, L. ORSSAUDand C. ANTONY, 1988 Drosophilamelanogaster female pheromones controlled This work would not have been possible without the invaluable by the doublesex locus. Genet. Res. 51: 17-22. technical assistance of TERUYOIWATSUBO. MATTHEW COBB, KLUN,J. A., and S. MAINI, 1979 Genetic basis ofan insect chemical JEAN-MARCJALLON, MARLA B. SOKOLOWSKIand two anonymous communication system: the European cornborer. Environ. reviewers are thanked for their comments on the manuscript. Entomol. 8: 423-426. LUYTEN,I., 1982 Variations intraspirifiques etinterspkifiques des hydrocarbures cuticulaires chez Drosophila simulans. C. R. LITERATURECITED Acad. Sci. Paris 295: 723-736. ANTONY,C., and J.-M. JALLON,1982 The chemical basis for sex PECHIN~?,M., J. F. PEREZ,C. ANTONYand J.-M. JALLON,1985 A recognition in Drosophilamelanogaster. J. Insect Physiol. 28: further characterization of Drosophila cuticular monoenes 873-880. using a mass spectrometry method to localize double bonds in BAUER,S. J., and M. B. SOKOLOWSKI,1988 Autosomal and mater- complex mixtures. Anal. Biochem. 145 177-182. nal effects on pupation behavior in Drosophilamelanogaster. SCOTT, D., and L. L. JACKSON,1988 Interstrain comparison of Behav. Genet. 18: 81-97. male-predominant antiaphrodisiacs in Drosophila melanogaster. BENAMAR,O., and J.-M. JALLON,1983 The production of 7- J. Insect Physiol. 34 863-871. tricosene, an aphrodisiac of Drosophila simulans, is under the SCOTT,D., and R. C. RICHMOND,1988 A genetic analysis of male- control of one gene on the X chromosome (abstr.). Behav. predominant pheromones of Drosophila melanogaster. Genetics Genet. 13: 533. 119: 639-646. BLOMQUIST,G. J., J. W.DILLWITH and T. S. ADAMS, WALHSTEN,D., 1979 A critique of the concepts of heritability and 1987 Biosynthesis and endocrine regulation of sex phero- heredity in behavioral genetics, pp. 424-481 in Theoretical mone production in Diptera, pp. 217-243 in Pheromone Bio- Advances in Behavioral Genetics, edited by J. R. ROYCEand L. chemistry, edited by G.Prestwich and G. J. Blomquist, Academic MOS. Sijhoff and Nordhoff, Germantown, Md. Press, San Diego, Calif. Communicating editor: J. R. POWELL