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Female-specific regulation of cuticular hydrocarbon biosynthesis by dopamine in Drosophila melanogaster
Insect Biochemistry and Molecular Biology 34 (2004) 823–830 www.elsevier.com/locate/ibmb
Female-specific regulation of cuticular hydrocarbon biosynthesis by dopamine in Drosophila melanogaster Charlotte Marican a, Line Duportets a,, Serge Birman b, Jean Marc Jallon a a
Laboratoire NAMC UMR 8620, Universite´ Paris-Sud, Neurobiologie et Apprentissage de la Me´moire et de la Communication, rue Georges Clemenceau, 91405 Orsay, France b Laboratoire de Ge´ne´tique et Physiologie du De´veloppement, Developmental Biology Institute of Marseille, Campus de Luminy, 13288 Marseille Cedex 9, France Received 16 March 2004; received in revised form 7 May 2004; accepted 12 May 2004
Abstract The role of dopamine (DA) is investigated in cuticular hydrocarbon biosynthesis in Drosophila melanogaster with three different approaches: use of DA-deficient mutants (dopa decarboxylase temperature sensitive mutants reared at restrictive temperature, and rescued by dopamine ingestion or by pale mutants partially rescued by a tyrosine hydroxylase construction), pharmacological treatments (tyrosine hydroxylase inhibitors) and topical application on decapitated flies. We report that DA specifically regulates diene hydrocarbon biosynthesis, which is female specific. Our results suggest that DA acts in adult flies within the first hours of imaginal life and that DA production from the brain is crucial for this process. Thus, DA contributes to reproduction in D. melanogaster by acting during a critical period during development of young adults. # 2004 Elsevier Ltd. All rights reserved. Keywords: Pheromone; Drosophila; Dopamine
1. Introduction In insects, dopamine (DA) is used as a neurotransmitter in the central nervous system and as a cuticle hardening and pigmenting factor in the epidermis. Cuticular hydrocarbons (HC) can be found on the epicuticle of insects, among them Drosophila melanogaster. These molecules protect insects against desiccation (Howard and Blomquist, 1982; Rouault et al., 2004) and some of them are used for individual sex signalling (Jallon, 1984; Scott, 1994). In two D. melanogaster strains Canton S and Oregon R, mature males and females possess long chain hydrocarbon mixtures, mainly with 23–29 carbons, linear or branched. Male cuticles bear abundant linear hydrocarbons with 23 and 25 carbons and one Z7 double bond: (Z)7Tricosene (7T) and (Z)7Pentacosene (7P). Major female specific hydrocarbons have 27 and 29 carbons and two Z7 Corresponding author. Tel.: +33-1691-54963; fax: +33-169154977. E-mail address: [email protected] (L. Duportets).
0965-1748/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2004.05.002
and Z11 double bonds: (Z)7,(Z)11Heptacosadiene (7,11HD) and (Z)7,(Z)11Nonacosadiene (7,11ND) (Antony et al., 1985). However, for a short time after emergence, young fly HC are identical, irrespective of sex, with longer chains (29–37 carbons) including complex diene blends (Pe´chine´ et al., 1988). A number of genes have been shown to affect cuticular HC production (reviewed by Ferveur, 1997; Jallon and Wicker-Thomas, 2003). A number of studies have suggested that insect HC biosynthesis is under hormonal control as in Musca domestica (Tillman et al., 1999). In Drosophila, juvenile hormone has been implicated in the replacement of young fly HC by sex specific molecules (Wicker and Jallon, 1995a). Moreover, ecdysone and an unknown cephalic factor were suggested to control the female specific steps (Wicker and Jallon, 1995a; Wicker and Jallon, 1995b). Although no special gland has been shown to be involved in the biosynthesis of the pheromonal compounds as in moths (Roelofs, 1995), oenocytes are involved (Ferveur et al., 1997). Among cephalic factors, biogenic amines are putative candi-
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dates as they are regulators of the corpora allata–corpora cardiaca complexes of various insects (Thompson et al., 1990; Rachinsky et al., 1994; Kaatz et al., 1994; Woodring and Hoffman, 1994). Dopamine (DA) and some of its derivatives are essential molecules for cuticle formation and pigmentation in insects, including Drosophila (Karlson and Sekeris, 1962; Sekeris and Karlson, 1966; Wright, 1987). The pathway for DA biosynthesis in Drosophila parallels that established in vertebrates (Nagatsu et al., 1964): tyrosine is hydroxylated into l-dopa which itself is decarboxylated into DA. These steps are catalysed by the enzymes tyrosine hydroxylase (TH) (Neckameyer and Quinn, 1989; Birman et al., 1994; Vie et al., 1999) and dopamine decarboxylase (Ddc) (Hirsh and Davidson, 1981; Eveleth et al., 1986). Ddc is also involved in the serotonin biosynthesis pathway, together with a distinct hydroxylase (Neckameyer and White, 1992). Here we have tried to perturb cuticular HC biosynthesis using genetical and pharmacological tools. We show that DA mainly affects female HC production by acting on its final sex-specific steps. This control is exerted during a critical period early in ontogeny during which there is an excess of DA in females, probably in the brain pool.
2. Materials and methods
added to calculate the total quantity of fly hydrocarbons. Finally, 5 ll of individual extracts were analyzed on a gas chromatograph (GC) (Perkin Elmer Auto System) with split injection, with a flame ionisation detector and a 25QC2/BP1 0.1 column (SGE) (25 m 0:22 mm 0:1 lm) programmed to run from v v 180 to 270 C with a 3 /min gradient. 2.3. Data analysis GC chromatograms of D. melanogaster mature flies display 11 peaks for males and 16 for females which correspond to hydrocarbons which have been identified by Antony et al. (1985) (Fig. 1). Each peak may be identified by its area and associated with its percentage relatively to the sum of all areas of all peaks. All statistical analyses were performed by the software Statistica (5.1B). To assess the main factors, values of all HCs for all individuals have been used to perform a principal component analysis (PCA). With the compounds defined as determinant by PCA, an ANOVA was calculated when the distribution of data was normal. In other cases a Mann and Whitney test was performed with a Bonferroni correction. We noted if values are significantly different (p < 0:05), if p < 0:01 and if p < 0:005.
2.1. Strains
2.4. Pharmacological experiments
All flies were reared on a yeast-cornmeal-agar medium with a 12:12 light/dark photoperiod mostly at v 25 C. Flies were collected rapidly after emergence, anesthetized by CO2, sexed and placed separately in tubes by groups of 8–10. Different strains of D. melanogaster were used: wild types Oregon R and Canton S and mutants of the Ddc and TH structural genes. Ddcts1 and Ddcts2 are temperature sensitive mutants, the ts1 stock balanced by CurlyO. The two strains w:P(w+,pDdc-TH) ple2/TM6B or ple14/TM6B were crossed to produce enough viable progeny w:P ple2/ ple14 for HC analysis (see Results for P).
a-Methyl-tyrosine (a-MT) and 3iodo-tyrosine (3IY) were used to inhibit the TH activity. Solutions of 1% sucrose were prepared with or without 0.5 mg/ml of aMT or 1 mg/ml of 3IY. Aliquots (1 ml) of either solution were put on paper placed on the bottom of empty glass tubes (Neckameyer, 1996). Canton S flies were transferred every day to a new tube of either group, up to 4 days after emergence. DA was used to cure Ddcts2 mutants with 1 ml of a solution of 1% sucrose and 1 mg/ml DA.
2.2. Extraction and analysis of cuticular hydrocarbons
Oregon R females were decapitated around 1 h after emergence. Solutions of DA were prepared in water with various DA concentrations: 10, 5, 2.5 lg/ml. An aliquot of 0.15 ll was applied using a 0.5 ll Hamilton syringe, on the gap formed by the decapitation. Such flies were kept on fresh food for 3 days before HC analysis to avoid desiccation. For control, 3-day-old females were decapitated just before HC analysis. All flies had to pass a moving test (Hirsh, 1998): placed on a paper, only those moving after being touched with a brush were retained.
After emergence, wild type flies were kept on fresh v medium at 25 C for 4 days before HC extraction. v ts Mutant Ddc flies were bred at 18 C until imaginal eclosion and then either kept at the same temperature v or shifted to 29 C. Depending on the breeding temv perature, flies were studied at 7 days for 18 C and 4 v days for 25 or 29 C. For HC analysis, flies were bathed individually in 30 ll of n-hexane for 10 min. After fly removal and solvent evaporation, 20 ll of a hexane solution containing 800 ng of C26H54 were
2.5. Decapitation experiments
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Fig. 1. Variation of cuticular hydrocarbons in flies mutant for dopa decarboxylase. (A) Hydrocarbons of Ddcts1/Ddcts1 ( ) and Ddcts1/CyO ( ) v v male flies, bred at 29 C. (B) Hydrocarbons of Ddcts1/Ddcts1 ( ) and Ddcts1/CyO ( ) female flies, bred at 29 C.
2.6. Dopamine quantification Groups of 10 flies of either sex and various ages were homogenized in the mobile phase (75 mM of NaH2PO4, 1.7 mM of heptanesulfonic acid, 100 ll/l of triethylamine, 5% of methanol, pH 2.5), centrifuged for 5 min at 12500 rpm and filtered through 0.2 lm nylon membrane. The chromatographic system consisted of an ESA pump (model P582) and a Supelcosil LC-18 3 lm particle size column (Supelco). Detection and quantification were accomplished using a Coulochem detector (ESA model 5200A), an analytical cell (model 5010, channel 1 set at 50 mV and channel 2 set at 350 mV) and a guard cell (model 5020 set at 350 mV).
3. Results 3.1. Ddc mutants Ddcts1 imagos of both sexes, either homo or heterov zygous and bred at the permissive (18 C) or the v restrictive (29 C) temperature, were compared for their hydrocarbon composition, shown for the latter case in Fig. 1. PCA performed on individual male HC did not show any segregation of the 2 HC groups at either temperature; this was also the case for females
v
v
bred at 18 C. In contrast, for females reared at 29 C, the PCA showed that the second principal component (ordinate axis) represented most of the total variance, opposing two major HCs with 27 carbons, 7,11Heptacosadiene (7,11HD) (2.4) and 7 heptacosene (7H) (+1.9). This analysis strongly confirmed that the mutation mainly affected female compounds, and more specifically the balance between dienes and monoenes. v Actually at 29 C there was neither any difference in the percentage sums 7; 11HD þ 7H (29.2–29.5%) nor in the total amounts of cuticular hydrocarbons (1779– 1860 ng), between both Ddcts1 female groups. While there was more 7H in homozygotes compared to heterozygotes (MW: 21.5% vs. 11.0%, p < 0:0005), there were less 7,11 dienes in homozygotes with 27 and 29 carbons (MW: 12.7% vs. 30.3%), especially 7,11HD (MW: 7.7% vs. 18.5%, p < 0:0001). Values of the ratio 7,11HD/7H were markedly and significantly different (MW: 0.4 vs. 1.7). Another Ddc thermosensitive allele, Ddcts2, led to a low level of dienes especially of 7,11HD (12:8 1:1%) v in homozygous females bred at 29 C. However, this level was significantly increased (+28%) when these flies were fed with dopamine for 4 days. There was no significant difference in 7H percentages between mutant females fed or not with DA.
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3.2. Inhibitor studies Canton S females were fed with either one of two inhibitors of TH for different durations. When the treatment lasted 4 days, the summed levels of 7H and 7,11HD did not change much, but with 3IY the 7,11HD level decreased from 21.6% to 16.0% (p < 0:003); while the 7H level increased from 4.8% to 11.2% (p < 0:009). With a-MT, the percentages of
these compounds varied in the same direction, from 21.6% to 13.6% for 7,11HD and from 4.8% to 9.2% for 7H (in both cases p < 0:007) (Fig. 2A). The 7,11HD/7H ratio was significantly lower when flies had been treated for 4 days than for controls (3IY: p ¼ 0:005 and a-MT: p ¼ 0:003). The effect of TH inhibitors depended on the treatment duration; for both inhibitors it was most efficient during the first 2 days of imaginal life, as shown in Fig. 2B,C.
Fig. 2. Variation of female cuticular 27 carbon 7-unsaturated hydrocarbons with pharmacological or genetical manipulation of tyrosine hydroxylase (TH). Canton S wild type females were fed with TH inhibitors as described in Materials and methods and analyzed for cuticular hydrocarbons at 4 days. (A) 7,11HD and 7H percentages in flies fed during 4 days with 3IY ( ) and aMT ( ) compared to controls (sugar solution alone) ( ). (B) 7,11HD/7H ratios of females treated with aMT during 1 and 4 days. (C) 7,11HD/7H ratios of females treated with 3IY during 2, 3 or 4 days. (D) 7,11HD/7H ratios of pale homozygous (or heterozygous) females partially rescued by a TH construction (see section on Results).
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Fig. 3. Increase in the 7,11HD/7H ratio with the quantity of dopamine (DA) applied on the neck of young decapitated females. Various doses of dopamine were applied on the neck of Oregon R females decapitated within the first 40 min after emergence, and their hydrocarbon levels were measured at 3 days ( ). The controls were decapitated at 3 days and analyzed immediately for their hydrocarbons ( ).
3.3. Semi rescued pale flies Mutations in the TH structural gene pale (ple) with either ple2 or ple14 alleles are embryonic lethal but can be rescued up to adult stage by discrete insertions of one P-derived transposon containing the TH cDNA type 2 (Neckameyer and Quinn, 1989) fused downstream to the Ddc promoter (Birman and Hirsh, unpublished observations). However, the rescue is only partial and rescued homozygous ple adults present a pale cuticle and a reduced locomotor activity, suggesting a DA deficit. More adults were viable with the genotype ple2/ple14,P. Homozygous females had markedly less 7,11HD (12:6 1:0%) than either of the heterozygotes (23:9 2:2% for ple2/TM6 and 20:4 0:3% for ple14/ TM6), but percentages of 7H were closer (5.6%, 4.2% and 3.2%, respectively). Values of the 7,11HD/7H
ratio were similar in the two balanced strains, and much higher than in the ple2/ple14,P viable females (Fig. 2D). 3.4. Decapitation experiments When young Canton S females were decapitated and their HC analyzed 3 days later, there was a marked deficit of dienes especially of 7,11HD and a parallel increase in 7H (Wicker and Jallon, 1995a). A similar phenomenon was observed here when young Oregon R females were decapitated (less than 1 h of age), although of smaller amplitude, but the deficit could be cured by application of DA on the neck. The experiment was repeated four times and average values are presented in Fig. 3. The 7,11HD/7H ratio increased with the dose of applied DA and reached a plateau, close to the ratio obtained with Oregon R females
Fig. 4. Compared variations of dopamine content in wild type Oregon R males and females of different ages. The dopamine content (ng per fly) is significantly different between males ( ) and females ( ) for 3 h, 2- and 3-day-old flies. For each point n 8.
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decapitated at 3 days before HC analysis. The half effect corresponded to a DA dose around 0.5 ng. Variations of this ratio mainly reflect marked changes in 7,11HD percentages: 16.3% for females decapitated very young with no application of DA and 25.5 with 1.5 ng of DA applied (p < 0:002). The latter value has to be compared with 26.7% for control females decapitated at 3 days. 3.5. Sex specific variations of dopamine levels with age v
Oregon R male and female flies, bred at 25 C, were compared for dopamine contents (Fig. 4). During the first hour of imaginal life, the quantity of dopamine is very high but does not show any significant difference between males and females (50–75 ng/fly). The level of DA decreases sharply in both sexes at later times. However, during several consecutive hours, the DA level remains higher in females than in males: at 3 h, it is 10:9 1:6 ng=female, which is 2.3 times higher than per male (statistically significant difference). This situation is reversed from the age of 2 days. At 3 days, the DA level is 3.6 times higher in males (11:4 2:4 ng=male) than in females (significantly different). The same result was obtained considering the body mass. 4. Discussion The results reported here establish a relationship between the DA level and the production of female specific HC in D. melanogaster. The marked difference between homozygotes and heterozygotes of Ddcts1 bred at restrictive temperature was linked to a decrease in female 7,11 dienes and an increase in 7 monoenes for chains between 25 and 29 carbons, especially 7,11HD and 7H. A similar low level of dienes was also v observed in Ddcts2 homozygotes bred at 29 C from eclosion. Such an effect could be due to deficits in either DA or serotonine as the same enzyme, Ddc, is used in both biosynthetic pathways while TH is only used for DA production. Three pieces of experimental evidence support the involvement of DA: phenocopy of the hydrocarbon alterations by TH inhibitors, abnormal levels of female dienes/monoenes in ple mutants partially rescued by a TH construction, and correction of Ddc mutant effects by DA ingestion. Studies of the biosynthesis of Drosophila HC using radiolabelled precursors have suggested an elongationdecarboxylation mechanism in both sexes as in other insects (Blomquist et al., 1995) involving a small number of enzyme activities, especially desaturases and elongases (Pennanec’h et al., 1997). The first desaturation step is performed by one of two D9 desaturases with different substrate specificities (Wicker-Thomas et al., 1997; Dallerac et al., 2000). The present results show that DA modulates the production of female
compounds but does not affect that of male compounds. Thus it should act on the few additional steps in the biosynthesis which are female specific. Among them a desaturase and/or an elongase are good candidates for the target of DA action (Jallon and WickerThomas, 2003). Inhibitors of TH have an effect only if they are ingested during the early imaginal life. Actually ontogenetical studies of cuticular hydrocarbons have shown that important changes take place during that period. At eclosion, special HC which are neither sex nor species specific are present (Pe´chine´ et al., 1988). They are replaced within a day or so in D. melanogaster Canton S by sex specific compounds, monoenes in males, monoenes and dienes in females. Female monoenes appear before dienes (Jallon and Wicker-Thomas, 2003). Thus during an early critical period, the level of DA might have to be high enough to switch a few specific genes important for female HC biosynthesis. Actually Neckameyer et al. (2000) studied modifications of DA levels with age in Canton S flies. They described a male specific increase in DA around 3 days. We have confirmed this effect in Oregon R males but discovered an additional interesting effect, a female specific excess of DA a few hours after emergence. The level and duration of female DA peak might be different between strains. As DA is produced both in the nervous system to act as a neurotransmitter and in the hypoderm to play a role in the cuticle construction, the respective involvement of either organ has to be considered. Indeed, early decapitation, within 1 h of emergence of both Canton S (Wicker and Jallon, 1995a) and Oregon R blocked the feminization of their hydrocarbons, especially leading to a marked deficit in dienes of older females. Later decapitation sharply reduced this effect, which was not observed after decapitation of mature females. These results suggest the release of a cephalic feminizing factor during a critical period (Wicker and Jallon, 1995a). Here, we show that this effect can be reversed by a topical application of DA onto the open nerve cord in a dose-dependent way. It is thus suggested that brain DA might be the missing cephalic factor (or one of them). The abnormal phenotype of cuticular unsaturated HC in pale mutants being partially rescued by the insertion of a P element containing the ORF of the hypodermal form of TH triggered by the Ddc promoter supports this hypothesis. Indeed, these mutants express TH in all the DA-synthesizing cells except in a specific neural cluster in the brain (Birman, Meller and Hirsh, unpublished observations). HC biosynthesis takes place in the abdomen (Blomquist et al., 1987; Wicker-Thomas and Jallon, 2001) and partially in oenocytes (Ferveur et al., 1997). Thus, to regulate female HC biosynthesis, two modes of
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action have to be considered, either directly on the tissue producing the cuticular dienes, which should bear adequate receptors on their cell membranes (Yellman et al., 1997), or indirectly through modifications of hormone levels. A very similar sex-specific HC modification has been described in ecdysone-less mutant females (Wicker and Jallon, 1995b). Thus ecdysone could be an intermediate in this regulation. A well known source of ecdysone production in adult females is the ovary, where TH mRNA has been localized by Neckameyer (Neckameyer, 1996). Moreover, when newly eclosed females were fed with 3 YI, their ovaries had developmental problems (Neckameyer, 1996). But the absence of functional ovaries in ovo mutants did not affect the production of female cuticular dienes (Wicker and Jallon, 1995b). These results suggest that the dopamine deficit is probably the primary effect triggering ovary dysfunction and the ecdysone deficit. Finally, Neckameyer (1998) has also shown a marked decrease in female receptivity after DA depletion. However, the author did not observe any change in female attractivity. This was expected, as hydrocarbons with 27 2 carbons with at least one Z7 double bond, which includes Z7Heptacosene, are sexappeal rich, at least for Canton S males (Antony et al., 1985). Acknowledgements We thank Claude Wicker-Thomas for discussions.
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Female-specific regulation of cuticular hydrocarbon biosynthesis by dopamine in Drosophila melanogaster Charlotte Marican a, Line Duportets a,, Serge Birman b, Jean Marc Jallon a a
Laboratoire NAMC UMR 8620, Universite´ Paris-Sud, Neurobiologie et Apprentissage de la Me´moire et de la Communication, rue Georges Clemenceau, 91405 Orsay, France b Laboratoire de Ge´ne´tique et Physiologie du De´veloppement, Developmental Biology Institute of Marseille, Campus de Luminy, 13288 Marseille Cedex 9, France Received 16 March 2004; received in revised form 7 May 2004; accepted 12 May 2004
Abstract The role of dopamine (DA) is investigated in cuticular hydrocarbon biosynthesis in Drosophila melanogaster with three different approaches: use of DA-deficient mutants (dopa decarboxylase temperature sensitive mutants reared at restrictive temperature, and rescued by dopamine ingestion or by pale mutants partially rescued by a tyrosine hydroxylase construction), pharmacological treatments (tyrosine hydroxylase inhibitors) and topical application on decapitated flies. We report that DA specifically regulates diene hydrocarbon biosynthesis, which is female specific. Our results suggest that DA acts in adult flies within the first hours of imaginal life and that DA production from the brain is crucial for this process. Thus, DA contributes to reproduction in D. melanogaster by acting during a critical period during development of young adults. # 2004 Elsevier Ltd. All rights reserved. Keywords: Pheromone; Drosophila; Dopamine
1. Introduction In insects, dopamine (DA) is used as a neurotransmitter in the central nervous system and as a cuticle hardening and pigmenting factor in the epidermis. Cuticular hydrocarbons (HC) can be found on the epicuticle of insects, among them Drosophila melanogaster. These molecules protect insects against desiccation (Howard and Blomquist, 1982; Rouault et al., 2004) and some of them are used for individual sex signalling (Jallon, 1984; Scott, 1994). In two D. melanogaster strains Canton S and Oregon R, mature males and females possess long chain hydrocarbon mixtures, mainly with 23–29 carbons, linear or branched. Male cuticles bear abundant linear hydrocarbons with 23 and 25 carbons and one Z7 double bond: (Z)7Tricosene (7T) and (Z)7Pentacosene (7P). Major female specific hydrocarbons have 27 and 29 carbons and two Z7 Corresponding author. Tel.: +33-1691-54963; fax: +33-169154977. E-mail address: [email protected] (L. Duportets).
0965-1748/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2004.05.002
and Z11 double bonds: (Z)7,(Z)11Heptacosadiene (7,11HD) and (Z)7,(Z)11Nonacosadiene (7,11ND) (Antony et al., 1985). However, for a short time after emergence, young fly HC are identical, irrespective of sex, with longer chains (29–37 carbons) including complex diene blends (Pe´chine´ et al., 1988). A number of genes have been shown to affect cuticular HC production (reviewed by Ferveur, 1997; Jallon and Wicker-Thomas, 2003). A number of studies have suggested that insect HC biosynthesis is under hormonal control as in Musca domestica (Tillman et al., 1999). In Drosophila, juvenile hormone has been implicated in the replacement of young fly HC by sex specific molecules (Wicker and Jallon, 1995a). Moreover, ecdysone and an unknown cephalic factor were suggested to control the female specific steps (Wicker and Jallon, 1995a; Wicker and Jallon, 1995b). Although no special gland has been shown to be involved in the biosynthesis of the pheromonal compounds as in moths (Roelofs, 1995), oenocytes are involved (Ferveur et al., 1997). Among cephalic factors, biogenic amines are putative candi-
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dates as they are regulators of the corpora allata–corpora cardiaca complexes of various insects (Thompson et al., 1990; Rachinsky et al., 1994; Kaatz et al., 1994; Woodring and Hoffman, 1994). Dopamine (DA) and some of its derivatives are essential molecules for cuticle formation and pigmentation in insects, including Drosophila (Karlson and Sekeris, 1962; Sekeris and Karlson, 1966; Wright, 1987). The pathway for DA biosynthesis in Drosophila parallels that established in vertebrates (Nagatsu et al., 1964): tyrosine is hydroxylated into l-dopa which itself is decarboxylated into DA. These steps are catalysed by the enzymes tyrosine hydroxylase (TH) (Neckameyer and Quinn, 1989; Birman et al., 1994; Vie et al., 1999) and dopamine decarboxylase (Ddc) (Hirsh and Davidson, 1981; Eveleth et al., 1986). Ddc is also involved in the serotonin biosynthesis pathway, together with a distinct hydroxylase (Neckameyer and White, 1992). Here we have tried to perturb cuticular HC biosynthesis using genetical and pharmacological tools. We show that DA mainly affects female HC production by acting on its final sex-specific steps. This control is exerted during a critical period early in ontogeny during which there is an excess of DA in females, probably in the brain pool.
2. Materials and methods
added to calculate the total quantity of fly hydrocarbons. Finally, 5 ll of individual extracts were analyzed on a gas chromatograph (GC) (Perkin Elmer Auto System) with split injection, with a flame ionisation detector and a 25QC2/BP1 0.1 column (SGE) (25 m 0:22 mm 0:1 lm) programmed to run from v v 180 to 270 C with a 3 /min gradient. 2.3. Data analysis GC chromatograms of D. melanogaster mature flies display 11 peaks for males and 16 for females which correspond to hydrocarbons which have been identified by Antony et al. (1985) (Fig. 1). Each peak may be identified by its area and associated with its percentage relatively to the sum of all areas of all peaks. All statistical analyses were performed by the software Statistica (5.1B). To assess the main factors, values of all HCs for all individuals have been used to perform a principal component analysis (PCA). With the compounds defined as determinant by PCA, an ANOVA was calculated when the distribution of data was normal. In other cases a Mann and Whitney test was performed with a Bonferroni correction. We noted if values are significantly different (p < 0:05), if p < 0:01 and if p < 0:005.
2.1. Strains
2.4. Pharmacological experiments
All flies were reared on a yeast-cornmeal-agar medium with a 12:12 light/dark photoperiod mostly at v 25 C. Flies were collected rapidly after emergence, anesthetized by CO2, sexed and placed separately in tubes by groups of 8–10. Different strains of D. melanogaster were used: wild types Oregon R and Canton S and mutants of the Ddc and TH structural genes. Ddcts1 and Ddcts2 are temperature sensitive mutants, the ts1 stock balanced by CurlyO. The two strains w:P(w+,pDdc-TH) ple2/TM6B or ple14/TM6B were crossed to produce enough viable progeny w:P ple2/ ple14 for HC analysis (see Results for P).
a-Methyl-tyrosine (a-MT) and 3iodo-tyrosine (3IY) were used to inhibit the TH activity. Solutions of 1% sucrose were prepared with or without 0.5 mg/ml of aMT or 1 mg/ml of 3IY. Aliquots (1 ml) of either solution were put on paper placed on the bottom of empty glass tubes (Neckameyer, 1996). Canton S flies were transferred every day to a new tube of either group, up to 4 days after emergence. DA was used to cure Ddcts2 mutants with 1 ml of a solution of 1% sucrose and 1 mg/ml DA.
2.2. Extraction and analysis of cuticular hydrocarbons
Oregon R females were decapitated around 1 h after emergence. Solutions of DA were prepared in water with various DA concentrations: 10, 5, 2.5 lg/ml. An aliquot of 0.15 ll was applied using a 0.5 ll Hamilton syringe, on the gap formed by the decapitation. Such flies were kept on fresh food for 3 days before HC analysis to avoid desiccation. For control, 3-day-old females were decapitated just before HC analysis. All flies had to pass a moving test (Hirsh, 1998): placed on a paper, only those moving after being touched with a brush were retained.
After emergence, wild type flies were kept on fresh v medium at 25 C for 4 days before HC extraction. v ts Mutant Ddc flies were bred at 18 C until imaginal eclosion and then either kept at the same temperature v or shifted to 29 C. Depending on the breeding temv perature, flies were studied at 7 days for 18 C and 4 v days for 25 or 29 C. For HC analysis, flies were bathed individually in 30 ll of n-hexane for 10 min. After fly removal and solvent evaporation, 20 ll of a hexane solution containing 800 ng of C26H54 were
2.5. Decapitation experiments
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Fig. 1. Variation of cuticular hydrocarbons in flies mutant for dopa decarboxylase. (A) Hydrocarbons of Ddcts1/Ddcts1 ( ) and Ddcts1/CyO ( ) v v male flies, bred at 29 C. (B) Hydrocarbons of Ddcts1/Ddcts1 ( ) and Ddcts1/CyO ( ) female flies, bred at 29 C.
2.6. Dopamine quantification Groups of 10 flies of either sex and various ages were homogenized in the mobile phase (75 mM of NaH2PO4, 1.7 mM of heptanesulfonic acid, 100 ll/l of triethylamine, 5% of methanol, pH 2.5), centrifuged for 5 min at 12500 rpm and filtered through 0.2 lm nylon membrane. The chromatographic system consisted of an ESA pump (model P582) and a Supelcosil LC-18 3 lm particle size column (Supelco). Detection and quantification were accomplished using a Coulochem detector (ESA model 5200A), an analytical cell (model 5010, channel 1 set at 50 mV and channel 2 set at 350 mV) and a guard cell (model 5020 set at 350 mV).
3. Results 3.1. Ddc mutants Ddcts1 imagos of both sexes, either homo or heterov zygous and bred at the permissive (18 C) or the v restrictive (29 C) temperature, were compared for their hydrocarbon composition, shown for the latter case in Fig. 1. PCA performed on individual male HC did not show any segregation of the 2 HC groups at either temperature; this was also the case for females
v
v
bred at 18 C. In contrast, for females reared at 29 C, the PCA showed that the second principal component (ordinate axis) represented most of the total variance, opposing two major HCs with 27 carbons, 7,11Heptacosadiene (7,11HD) (2.4) and 7 heptacosene (7H) (+1.9). This analysis strongly confirmed that the mutation mainly affected female compounds, and more specifically the balance between dienes and monoenes. v Actually at 29 C there was neither any difference in the percentage sums 7; 11HD þ 7H (29.2–29.5%) nor in the total amounts of cuticular hydrocarbons (1779– 1860 ng), between both Ddcts1 female groups. While there was more 7H in homozygotes compared to heterozygotes (MW: 21.5% vs. 11.0%, p < 0:0005), there were less 7,11 dienes in homozygotes with 27 and 29 carbons (MW: 12.7% vs. 30.3%), especially 7,11HD (MW: 7.7% vs. 18.5%, p < 0:0001). Values of the ratio 7,11HD/7H were markedly and significantly different (MW: 0.4 vs. 1.7). Another Ddc thermosensitive allele, Ddcts2, led to a low level of dienes especially of 7,11HD (12:8 1:1%) v in homozygous females bred at 29 C. However, this level was significantly increased (+28%) when these flies were fed with dopamine for 4 days. There was no significant difference in 7H percentages between mutant females fed or not with DA.
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3.2. Inhibitor studies Canton S females were fed with either one of two inhibitors of TH for different durations. When the treatment lasted 4 days, the summed levels of 7H and 7,11HD did not change much, but with 3IY the 7,11HD level decreased from 21.6% to 16.0% (p < 0:003); while the 7H level increased from 4.8% to 11.2% (p < 0:009). With a-MT, the percentages of
these compounds varied in the same direction, from 21.6% to 13.6% for 7,11HD and from 4.8% to 9.2% for 7H (in both cases p < 0:007) (Fig. 2A). The 7,11HD/7H ratio was significantly lower when flies had been treated for 4 days than for controls (3IY: p ¼ 0:005 and a-MT: p ¼ 0:003). The effect of TH inhibitors depended on the treatment duration; for both inhibitors it was most efficient during the first 2 days of imaginal life, as shown in Fig. 2B,C.
Fig. 2. Variation of female cuticular 27 carbon 7-unsaturated hydrocarbons with pharmacological or genetical manipulation of tyrosine hydroxylase (TH). Canton S wild type females were fed with TH inhibitors as described in Materials and methods and analyzed for cuticular hydrocarbons at 4 days. (A) 7,11HD and 7H percentages in flies fed during 4 days with 3IY ( ) and aMT ( ) compared to controls (sugar solution alone) ( ). (B) 7,11HD/7H ratios of females treated with aMT during 1 and 4 days. (C) 7,11HD/7H ratios of females treated with 3IY during 2, 3 or 4 days. (D) 7,11HD/7H ratios of pale homozygous (or heterozygous) females partially rescued by a TH construction (see section on Results).
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Fig. 3. Increase in the 7,11HD/7H ratio with the quantity of dopamine (DA) applied on the neck of young decapitated females. Various doses of dopamine were applied on the neck of Oregon R females decapitated within the first 40 min after emergence, and their hydrocarbon levels were measured at 3 days ( ). The controls were decapitated at 3 days and analyzed immediately for their hydrocarbons ( ).
3.3. Semi rescued pale flies Mutations in the TH structural gene pale (ple) with either ple2 or ple14 alleles are embryonic lethal but can be rescued up to adult stage by discrete insertions of one P-derived transposon containing the TH cDNA type 2 (Neckameyer and Quinn, 1989) fused downstream to the Ddc promoter (Birman and Hirsh, unpublished observations). However, the rescue is only partial and rescued homozygous ple adults present a pale cuticle and a reduced locomotor activity, suggesting a DA deficit. More adults were viable with the genotype ple2/ple14,P. Homozygous females had markedly less 7,11HD (12:6 1:0%) than either of the heterozygotes (23:9 2:2% for ple2/TM6 and 20:4 0:3% for ple14/ TM6), but percentages of 7H were closer (5.6%, 4.2% and 3.2%, respectively). Values of the 7,11HD/7H
ratio were similar in the two balanced strains, and much higher than in the ple2/ple14,P viable females (Fig. 2D). 3.4. Decapitation experiments When young Canton S females were decapitated and their HC analyzed 3 days later, there was a marked deficit of dienes especially of 7,11HD and a parallel increase in 7H (Wicker and Jallon, 1995a). A similar phenomenon was observed here when young Oregon R females were decapitated (less than 1 h of age), although of smaller amplitude, but the deficit could be cured by application of DA on the neck. The experiment was repeated four times and average values are presented in Fig. 3. The 7,11HD/7H ratio increased with the dose of applied DA and reached a plateau, close to the ratio obtained with Oregon R females
Fig. 4. Compared variations of dopamine content in wild type Oregon R males and females of different ages. The dopamine content (ng per fly) is significantly different between males ( ) and females ( ) for 3 h, 2- and 3-day-old flies. For each point n 8.
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decapitated at 3 days before HC analysis. The half effect corresponded to a DA dose around 0.5 ng. Variations of this ratio mainly reflect marked changes in 7,11HD percentages: 16.3% for females decapitated very young with no application of DA and 25.5 with 1.5 ng of DA applied (p < 0:002). The latter value has to be compared with 26.7% for control females decapitated at 3 days. 3.5. Sex specific variations of dopamine levels with age v
Oregon R male and female flies, bred at 25 C, were compared for dopamine contents (Fig. 4). During the first hour of imaginal life, the quantity of dopamine is very high but does not show any significant difference between males and females (50–75 ng/fly). The level of DA decreases sharply in both sexes at later times. However, during several consecutive hours, the DA level remains higher in females than in males: at 3 h, it is 10:9 1:6 ng=female, which is 2.3 times higher than per male (statistically significant difference). This situation is reversed from the age of 2 days. At 3 days, the DA level is 3.6 times higher in males (11:4 2:4 ng=male) than in females (significantly different). The same result was obtained considering the body mass. 4. Discussion The results reported here establish a relationship between the DA level and the production of female specific HC in D. melanogaster. The marked difference between homozygotes and heterozygotes of Ddcts1 bred at restrictive temperature was linked to a decrease in female 7,11 dienes and an increase in 7 monoenes for chains between 25 and 29 carbons, especially 7,11HD and 7H. A similar low level of dienes was also v observed in Ddcts2 homozygotes bred at 29 C from eclosion. Such an effect could be due to deficits in either DA or serotonine as the same enzyme, Ddc, is used in both biosynthetic pathways while TH is only used for DA production. Three pieces of experimental evidence support the involvement of DA: phenocopy of the hydrocarbon alterations by TH inhibitors, abnormal levels of female dienes/monoenes in ple mutants partially rescued by a TH construction, and correction of Ddc mutant effects by DA ingestion. Studies of the biosynthesis of Drosophila HC using radiolabelled precursors have suggested an elongationdecarboxylation mechanism in both sexes as in other insects (Blomquist et al., 1995) involving a small number of enzyme activities, especially desaturases and elongases (Pennanec’h et al., 1997). The first desaturation step is performed by one of two D9 desaturases with different substrate specificities (Wicker-Thomas et al., 1997; Dallerac et al., 2000). The present results show that DA modulates the production of female
compounds but does not affect that of male compounds. Thus it should act on the few additional steps in the biosynthesis which are female specific. Among them a desaturase and/or an elongase are good candidates for the target of DA action (Jallon and WickerThomas, 2003). Inhibitors of TH have an effect only if they are ingested during the early imaginal life. Actually ontogenetical studies of cuticular hydrocarbons have shown that important changes take place during that period. At eclosion, special HC which are neither sex nor species specific are present (Pe´chine´ et al., 1988). They are replaced within a day or so in D. melanogaster Canton S by sex specific compounds, monoenes in males, monoenes and dienes in females. Female monoenes appear before dienes (Jallon and Wicker-Thomas, 2003). Thus during an early critical period, the level of DA might have to be high enough to switch a few specific genes important for female HC biosynthesis. Actually Neckameyer et al. (2000) studied modifications of DA levels with age in Canton S flies. They described a male specific increase in DA around 3 days. We have confirmed this effect in Oregon R males but discovered an additional interesting effect, a female specific excess of DA a few hours after emergence. The level and duration of female DA peak might be different between strains. As DA is produced both in the nervous system to act as a neurotransmitter and in the hypoderm to play a role in the cuticle construction, the respective involvement of either organ has to be considered. Indeed, early decapitation, within 1 h of emergence of both Canton S (Wicker and Jallon, 1995a) and Oregon R blocked the feminization of their hydrocarbons, especially leading to a marked deficit in dienes of older females. Later decapitation sharply reduced this effect, which was not observed after decapitation of mature females. These results suggest the release of a cephalic feminizing factor during a critical period (Wicker and Jallon, 1995a). Here, we show that this effect can be reversed by a topical application of DA onto the open nerve cord in a dose-dependent way. It is thus suggested that brain DA might be the missing cephalic factor (or one of them). The abnormal phenotype of cuticular unsaturated HC in pale mutants being partially rescued by the insertion of a P element containing the ORF of the hypodermal form of TH triggered by the Ddc promoter supports this hypothesis. Indeed, these mutants express TH in all the DA-synthesizing cells except in a specific neural cluster in the brain (Birman, Meller and Hirsh, unpublished observations). HC biosynthesis takes place in the abdomen (Blomquist et al., 1987; Wicker-Thomas and Jallon, 2001) and partially in oenocytes (Ferveur et al., 1997). Thus, to regulate female HC biosynthesis, two modes of
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action have to be considered, either directly on the tissue producing the cuticular dienes, which should bear adequate receptors on their cell membranes (Yellman et al., 1997), or indirectly through modifications of hormone levels. A very similar sex-specific HC modification has been described in ecdysone-less mutant females (Wicker and Jallon, 1995b). Thus ecdysone could be an intermediate in this regulation. A well known source of ecdysone production in adult females is the ovary, where TH mRNA has been localized by Neckameyer (Neckameyer, 1996). Moreover, when newly eclosed females were fed with 3 YI, their ovaries had developmental problems (Neckameyer, 1996). But the absence of functional ovaries in ovo mutants did not affect the production of female cuticular dienes (Wicker and Jallon, 1995b). These results suggest that the dopamine deficit is probably the primary effect triggering ovary dysfunction and the ecdysone deficit. Finally, Neckameyer (1998) has also shown a marked decrease in female receptivity after DA depletion. However, the author did not observe any change in female attractivity. This was expected, as hydrocarbons with 27 2 carbons with at least one Z7 double bond, which includes Z7Heptacosene, are sexappeal rich, at least for Canton S males (Antony et al., 1985). Acknowledgements We thank Claude Wicker-Thomas for discussions.
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