Temperature affects the ontogeny of sexually dimorphic cuticular hydrocarbons in Drosophila melanogaster
1 LEEC, CNRS- FRE 2413, Université Paris 13, 93430 Villetaneuse,
France
2 CNRS-UMR 5548, Faculté des Sciences, Université de
Bourgogne, 6 Boulevard Gabriel, 21000 Dijon, France
* Author for correspondence (e-mail: jean-francois.ferveur{at}u-bourgogne.fr)
Accepted 5 August 2002
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Summary |
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Key words: mate recognition, temperature, cuticle, cuticular hydrocarbon, Drosophila melanogaster, ontogeny, heat shock, biosynthesis, sex difference
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Introduction |
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Predominant CHs of mature Drosophila melanogaster flies are
sexually dimorphic, both in their occurrence and in their effect on male
courtship behaviour (Antony and Jallon,
1982; Ferveur,
1997
). Male flies synthesize monoenes (containing one double bond)
with 23 and 25 carbons [cis7-tricosene (7-T) and
cis7-pentacosene (7-P)]. 7-T tends to inhibit intraspecific male
excitation (Ferveur and Sureau,
1996
). Female flies produce dienes (containing two double bonds)
with 27 and 29 carbons [cis, cis7, 11-heptacosadiene (7, 11HD) and
cis, cis7, 11-nonacosadiene (7, 11ND)]. Both dienes reduce
interspecific male excitation but only slightly reinforce intraspecific male
excitation, the main stimulation/inhibition signal being provided by 7-T and,
eventually, by 7-P (Savarit et al.,
1999
; Sureau and Ferveur,
1999
). Other CHs are found on the cuticle of immature imagoes but
these are not sexually dimorphic; these substances generally have longer
carbon chains and more double bonds than mature CHs
(Pechiné et al., 1988
).
In D. melanogaster, these immature CHs strongly stimulate the
courtship of mature homospecific males
(Antony and Jallon, 1981
;
Cobb and Jallon, 1990
).
Several experiments suggest that mature CHs are synthesised during early
imaginal life. Immature CHs are almost completely replaced by shorter chain
CHs after 20 h (Antony and Jallon,
1981). Misexpression of the sex determination gene
transformer suggests that the ontogeny of mature Drosophila
CHs occurs in at least two distinct steps: (i) the elongation of fatty acids
into long or very long alkane chains, which occurs 6 h after adult eclosion
(AE) (Savarit et al., 1999
),
in contrast to (ii) the sexual differentiation of elongated CHs, which occurs
slightly later (12-48 h after AE) (Ferveur
et al., 1997
).
We measured the influence of temperature on the production of predominant and sexually dimorphic CHs in D. melanogaster. Two laboratory strains, Canton-S (Cs) and Tai, with different CH morphs, were either raised at a constant temperature (20 or 25°C), or shifted between the two temperatures immediately after adult eclosion. The influence of temperature on CH maturation was studied during the first 3 days of imaginal life. During this period, males and females of control and transgenic strains carrying the heat-inducible hsp70-GAL4 transgene were subjected to a single 1 h heat-shock pulse. The production of immature long chain CHs was also measured in heat-shocked flies.
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Materials and methods |
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Two wild-type strains were used: (i) the Canton-S (Cs) strain, originating
from North America; Cs males produce mainly 7-T
(Antony and Jallon, 1982), and
(ii) the Tai strain, originating from Ivory Coast; Tai males produce mainly
7-P (Jallon, 1984
). Females of
both strains show different principal hydrocarbons: 7, 11 HD and 7, 11 ND in
Cs females; cis,cis5,9 heptacosadiene (5,9HD) and cis,cis5,9
nonacosadiene (5,9ND) in Tai females
(Jallon and Péchiné,
1989
).
In the transgenic hsp70-GAL4 strain
(Brand et al., 1994), the Gal4
sequence is under the control of the hsp70 gene promoter. The
production of GAL4 protein can be induced ubiquitously subsequently to a heat
shock at 37°C. In turn, GAL4 can activate a second transgene carrying a
UAS-reporter sequence linked to the gene of interest. Therefore, the heat
shock ubiquitously activates the transgene shortly (less than 1 h) after
application (Greig and Akam,
1993
). Here we used the UAS-transformer
(UAS-tra) strain, which dominantly expresses the female form of the
TRA protein (Ferveur et al.,
1995
), and the UAS-lacZ strain, which expresses the
ß-galactosidase (Brand and Perrimon,
1993
). The transgenic strain combining hsp70-GAL4 with
UAS-tra was chosen because we noted that the overall amount of CHs
was very reduced after heat shock in young flies of this strain
(Savarit et al., 1999
;
Ferveur and Savarit, 2000
). In
order to study the effect on CHs of both reporter transgenes, the effect of
the UAS-lacZ transgene (also driven by hsp70-GAL4) was
compared to that of UAS-tra. The heat-shock effect was also measured
on Cs flies carrying one or no copies of the hsp70-GAL4 transgene.
The comparison of `Cs' and `hsp70-GAL4; Cs' genotypes allowed us to
distinguish between the general effect of the heat shock and that of the
activation of the hsp70-GAL4 transgene.
Manipulation of temperature and heat-shock procedure
In order to test the influence of temperature on CH production, flies were
raised at a constant temperature of either 20°C or 25°C throughout
their life (these flies being the offspring of flies kept at that same
temperature), or shifted between the two temperatures (2025°C or
25
20°C) less than 1 h after imaginal eclosion. Given that
developmental time is a function of temperature, the age of flies was
standardized for CH extraction: 4 days old for flies raised at 25°C, and 6
days old for flies raised at 20°C.
Flies were lightly anaesthetised with CO2 soon after eclosion,
sexed and placed in groups of ten in small polypropylene vials (11
cm3) containing food. Heat-shock pulses were applied by submerging
the vials in a water bath at 37°C for 1 h during the period between adult
eclosion (AE) and 72 h later. We have shown elsewhere that variations in the
tube volume and food alter heat shock efficiency
(Ferveur and Savarit, 2000).
Immediately after heat shock, flies were returned to 25°C, in standard
food vials to await CH extraction.
Hydrocarbon extraction
CH extraction was performed on individual flies following the standard
procedure (Ferveur, 1991). We
give the absolute amount of each CH because (1) all experiments were
simultaneously performed and (2) absolute amounts provide more complete
information than relative quantities, the variation of which can indirectly
depend upon other compounds (for a complete discussion, see
Savarit and Ferveur,
2002
).
Extraction was performed on mature flies (4 or 6 days old, see above), and
also on immature flies (6-48 h after AE); at this age flies still produce
longer carbon chain, immature CHs. For the latter experiment, 3 h imagoes were
heat shocked and immature CHs were extracted at least 3 h later (i.e. at 6 h
after AE), thus allowing the heat shock to affect CH biosynthesis. Individual
flies were soaked in a microtube containing 50 µl hexane for 10 min. This
short time was sufficient to extract most of the external but not the internal
CHs. After removal of the flies, 20 µl of hexane containing 800 ng of
hexacosane (C26; used as an internal standard) were added to each microtube. 5
µl of each sample were then injected into a Perkin-Elmer Autosystem
gas-phase chromatograph (GPC) equipped with a 25 m capillary column (25
QC2/BP1 0,1), using hydrogen as the carrier gas. During chromatography the
temperature was programmed to increase from 180°C to 270°C at a rate
of 3°C min-1. Peak detection was carried out using a Flame
Ionization Detector coupled with a Chromjet integrator (Thermo
Separation Product Inc.) that yielded retention times and areas under each
peak. All the predominant CHs of D. melanogaster have already been
identified and characterized (Antony and
Jallon, 1982; Pechiné et al.,
1985
,
1988
;
Jallon and Pechiné,
1989
).
Statistical analysis
The CHs of strains raised at constant or shifted temperatures were compared
using non-parametric statistical tests. First, the level of each major CH was
compared between flies raised at the four temperature conditions using a
KruskalWallis test. When a significant difference was detected,
temperature conditions were compared two-by-two using a MannWhitney
U-test. We used the sequential protocol of Bonferroni
(Holm, 1979;
Rice, 1989
) to correct the
significance levels, depending on the number of comparisons. Briefly, the
levels of significance for each comparison between conditions were ranked from
lowest to highest value. The lowest value was then compared with the threshold
divided by the total number of comparisons (n). Only where the first
value was significant did we compare the second with the threshold divided by
(n-1), and so on. This procedure was performed until a value showed a
non-significant difference.
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Results |
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Cs males showed significant differences in their production of 7-P (but not 7-T) when raised at a constant 20°C or 25°C (Table 1). Indeed, when raised at 25°C, Cs males produced 45% more 7-P (+48 ng) and 16% less 7-T (-165 ng) than sibling males raised at 20°C. When males were shifted from 20°C to 25°C, the levels of both 7-monoenes were significantly different from those measured in Cs males raised at constant 20°C. Furthermore, in shifted males, production of 7-P (but not 7-T) was significantly different from that in males raised at constant 25°C. Conversely, the levels of both 7-monoenes were the same in Cs males shifted from 25°C to 20°C and males raised at constant 20°C. There was no significant difference in the total amount of CHs (Sum CHs) in the four different conditions.
|
At 25°C, Cs females produced significantly less 7-T (-65%; -103 ng),
less 7-P (-47%; -80 ng) and more 7,11ND (+41%; + 89 ng) than females raised at
20°C. At 25°C, the level of 7,11HD also decreased, although this was
not significant (-22%; -117 ng) (Table
1). After a 2025°C shift, the levels of the four
unsaturated CHs were not significantly different from those measured in
females raised at constant 25°C, but were all different from the levels in
females raised at constant 20°C. With the reciprocal temperature shift
(25
20°C), CH levels were similar to those in females raised at
constant 20°C, but levels of 7-monoenes were different from those of
females raised at constant 25°C or shifted from 20°C to 25°C.
Although the SumCHs showed no difference between females raised at either
constant temperature, slight differences were detected between flies raised at
20
25°C and 20°C, and between flies raised at 25
20°C
and 25°C. In conclusion, in Cs flies of both sexes, the levels of the
principal CHs seem to be related to the temperature experienced by the flies
during early imaginal life: a temperature increase resulted in decreased
amounts of shorter CH chains and more longer CH chains.
In Tai males, the effect of temperature was different to that observed in
Cs males (Table 1). Tai males
raised at constant 25°C produced less 7-T than those raised at constant
20°C (-61%; -162 ng). However, we noted no significant effect on 7-P
production, although it had a tendency to decrease as temperature increased
(-15%; -154 ng). Tai males shifted from 2520°C significantly
increased their levels of 7-T in comparison to homotypic flies raised at a
constant 25°C. Comparison of the sumCHs over the four conditions suggests
that Tai males have a tendency to produce fewer CHs when they experience a
higher temperature during imaginal development.
In Tai females kept at 25°C after adult eclosion, the levels of 7,11HD decreased compared with those in adults held at 20°C (-31%; -42 ng for constant temperature; -58%; -105 ng, for shifted temperature). However, no changes in the levels of other CHs (including 5,9HD and 7,11ND) or in the SumCHs were detected (Table 1).
For the Tai strain, temperature variation thus only had a marked effect on 7-T in males and on 7,11HD in females. The effect of the temperature increase was different from that observed in Cs flies: the level of all predominant CHs seems to decrease in Tai flies with increased developmental temperature.
Precise characterization of the critical period for the maturation of
male-predominant CHs
The previous experiment indicates that the temperature after adult eclosion
largely determines the CH profile in male and female flies of two wild-type
strains. In order to characterize the critical period for the processing of
mature CHs precisely, we subjected flies to a single 1 h heat-shock pulse (at
37°) during the period between adult eclosion (AE) and 72h after AE, by
which time control flies show a mature CH profile. CH levels were measured in
4-day-old flies that had or had not been heat shocked. Four genotypes were
compared: Cs flies, Cs flies carrying a single copy of the hsp70-GAL4
transgene (hsp-Cs), and the two double transgenic strains hsp70-GAL4
x UAS-lacZ (hsp-lacZ) and hsp70-GAL4 x
UAS-tra flies (hsp-tra; see Materials and methods). These
four genotypes allowed us to measure the heat-shock effect (in Cs flies), the
effect of the hsp70-GAL4 transgene, and that of two UAS-reporter
transgenes driven by hsp70-GAL4, after heat shock.
In males (Fig. 1), there was a marked effect on predominant CHs when the heat shock was induced at different times between 0 and 72 h after AE, with further differences between the genotypes.
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Heat-shocked hsp-Cs males also showed a substantial and lasting decrease in 7-T levels (with a short refractory period approximately 6 h after AE). 7-P showed a smaller decrease when heat shock was induced between 9 and 24 h after AE, but not between eclosion and 6 h after AE.
Heat-shocked hsp-lacZ males showed a substantial decrease in both 7-monoenes. As in heat-shocked hsp-Cs females, 7-T levels were less affected when the heat shock was induced between eclosion and 6 h after AE. The level of 7-P decreased at all times.
The situation was more complex in heat-shocked hsp-tra males because, as expected, they produced a mixture of male- and female-predominant CHs. Although the levels of 7-monoenes were relatively low in control flies, the heat-shock effect was very strong in this genotype and induced two distinct phenotypes: (i) the feminization of CHs (between AE and 6 h after AE, and between 24 and 48 h AE, and (ii) a drastic reduction of male- and female-predominant CHs between 6 and 12 h after AE. The effect of heat shock on 7-T production lasted longer (up to 72 h after AE), than on 7-P production (up to 24 h after AE).
Precise characterization of the critical period for the maturation of
female predominant CHs
The heat-shock effect in transgenic females was similar to that observed in
males of the corresponding genotypes. Females of the three strains carrying
the hsp70 sequence showed a marked reduction in the production of
7,11 HD and 7,11 ND when the heat shock was induced between 0 and 72 h after
AE (Fig. 2). When the heat
shock was induced between 3 and 18 h after AE, the production of 7,11 dienes
and of 7-monoenes was drastically reduced, or was absent. Between 18 and 72 h
AE, the heat-shock effect was undetectable for 7-monoenes and a milder, but
still substantial, decrease in the production of 7,11 dienes was induced.
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Cs females that were subjected to a single heat-shock pulse at 9 h after AE showed a slight but significant decrease of 7,11 HD when compared to `no HS' flies (626±25 ng and 469±51 ng, respectively; d.f.=28; z=2.64; P=0.008). 7,11 ND was similarly affected (343±18 ng and 212±31 ng, respectively; z=3.12; P=0.0017).
Heat-shock effect on all detected CHs
We also measured the levels of all CHs in flies of the four genotypes
following heat shock at 9 h after AE (Fig.
3). We selected 9 h after AE because it was generally at this time
that the heat-shock effect on the predominant CHs was the strongest (Figs
1,
2). In Cs males, heat induction
mainly caused a decrease in 7-T and 7-P levels, with a smaller effect on 23C
and 25C saturated linear alkanes (23LIN and 25LIN). The activation of the
hsp70-GAL4 transgene (in both hsp-Cs and in hsp-lacZ
genotypes) decreased levels of most linear CHs (with the exception of 9-P),
but not those of the two branched CHs with 27C and 29C (27BR and 29BR). In
hsp-tra males, heat shock also affected the level of the predominant
branched compound 27BR. In Cs females, heat shock decreased only 7,11HD and
7,11ND levels, whereas the three other genotypes (with the hsp70-GAL4
transgene) all showed a drastic reduction in their CHs levels. In these
females, 29BR was always the least affected compound.
The levels of immature and mature CHs are independent
Two generations of CHs are present on the cuticle of D.
melanogaster flies. The long-chain CHs (29C-33C; non-sexually dimorphic)
are normally present on the cuticle of young imagoes (until 36-48 h after AE,
at 25°C). During this period, they are gradually replaced by shorter chain
molecules (23C-29C), which form the definitive sex-specific profile of mature
flies. To investigate the relationship between the two types of CHs, we
induced a single heat shock at 3 h after AE and measured the levels of both
immature and mature CHs in 6-48 h-old hsp-tra flies.
In hsp-tra males, heat shock had no detectable effect on the production of immature CHs (27C-31C; Fig. 4A). Strikingly, heat shock altered the levels of mature CHs by increasing the quantity of 27C CHs at the expense of shorter-chain CHs (those with 23C). In females, the heat shock induced at 3 h after AE mainly decreased levels of 25C and 27C CHs, with a small effect on 23C CHs (Fig. 4B). Although the level of 29C CHs was not affected by heat shock, the levels of longer chain compounds (31C-33C) remained slightly higher after heat shock.
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We cannot exclude the possibility that we missed slight quantitative differences in CHs because we were frequently unable to detect position isomers using gas chromatography.
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Discussion |
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We found intraspecific variation in the response to temperature that can be
compared with the variations in physiological mechanisms observed in D.
melanogaster strains selected for desiccation resistance
(Gibbs et al., 1997). Mature
Tai and Cs flies were affected differently by the temperature increase: in Cs
flies, levels of shorter carbon chains decreased and those of longer chains
increased, whereas in Tai flies the levels of all CHs decreased. While both
strains responded differently to temperature variation, we do not know whether
or not this difference is adaptative. Originally, Tai flies were collected in
a humid, warm habitat (Ivory Coast), whereas the Cs strain comes from a drier
and colder environment (northern USA). Strikingly, both strains, which have
been maintained under constant laboratory conditions for at least several
hundred generations, still retain different genetic potentials to react to
temperature variation. However, it should be noted that the plastic response
within species is not comparable to the differences in reaction to temperature
noted between species. For example, we have found that D.
melanogaster strains maintain their original CH profile over many
generations, whereas other Drosophila species (D. mojavensis, D.
pseudoobscura) can rapidly change as a response to laboratory conditions
(Toolson and Kuper-Simbron,
1989
; Markow and Toolson,
1990
). We do not know whether the differential plasticity observed
between Drosophila species reflects a greater capacity of some
species to adapt their CH profile in response to changing environmental
conditions. An alternative argument is that some species keep their CH profile
more constant than others because of the role played by some CHs in sexual
communication (Cobb and Jallon,
1990
; Savarit et al.,
1999
). Also, in D. melanogaster and in other insects,
internal hydrocarbons occur in the same composition and proportion as
cuticular hydrocarbons (for a review, see
Tillman et al., 1999
).
Therefore, it is possible that the high stability of the CH profile in D.
melanogaster reflects the involvement of internal hydrocarbons in key
physiological processes (such as reproduction). The balance between internal
and cuticular hydrocarbons can change, as in Blattella germanica
nymphs, where food intake affects the allocation of both hydrocarbon types
(Young et al., 1999
). In
Musca domestica, quantitative variations of CHs depend upon age and
sex. For example, in 6-day-old females, increased Z-9-tricosene is compensated
by a decrease of Z-9-heptacosene (Mpuru et
al., 2001
).
No relationship was found between immature and mature CHs in heat-shocked
flies. However, it seems probable that both generations of CHs share
biosynthetic mechanisms. In this case, the biosynthesis of immature CHs would
occur some time before imaginal life and would not be sensitive to the
misexpression of the sex-determination gene transformer
(Ferveur et al., 1997). This
observation is consistent with the fact that immature CHs are not sexually
dimorphic.
The present data confirm that mature CHs are processed in at least two
steps that occur during early imaginal life: (i) elongation of relatively long
saturated carbon chain alkanes and (ii) sexual differentiation after
desaturation (Ferveur et al.,
1997; Savarit et al.,
1999
), and that biosynthesis is still active in 3-day-old flies
(Chan Yong and Jallon, 1986
).
They also provide two new insights into CH biosynthesis. Firstly, there is a
short period of time, between adult eclosion (AE) and 6 h after AE in most
genotypes, during which the heat-shock effect on CH production seems to be
less dramatic. This refractory period could correspond to a heat-shock-induced
protective mechanism equivalent to the neuroprotection at synapses observed
with elevated HSP70 levels in Drosophila
(Karunanithi et al., 1999
).
Selection at different temperatures has been shown to change the transcription
of the HSP factor (Lerman and Feder,
2001
). Secondly, a sex difference was found for sensitivity to
heat shock: D. melanogaster females carrying at least one copy of the
hsp70-GAL4 transgene showed a much higher CH decrease than homotypic
males, specially when the heat shock was induced 6-12 h after AE. CHs in
females are probably much more sensitive to stress than CHs in males. To
explain the difference between transgenic strains, we propose that the heat
induction of hsp70-GAL4 induces a toxic effect in those tissues, such
as the oenocytes, that are involved in CH processing. The sexual difference in
reaction to heat shock could indicate sex differences in tissues involved in
the sexual maturation of CHs (oenocytes in males; oenocytes and fat body in
females) (Savarit and Ferveur,
2002
), or sex differences in the sensitivity of these tissues to
heat shock. On the other hand, it is possible that CHs in D.
melanogaster female are more sensitive to heat shock because, being
characterized by dienes, they depend upon a more complex biosynthetic pathway
than male monoenes.
In summary, the production of CHs in mature flies depends upon a series of
biosynthetic mechanisms that occur during the first day of imaginal life. We
propose that a non-sex-specific enzyme, acting like a fatty acid synthetase
(FAS), controls the amount of most linear CHs between 6 and 12 h after AE. At
12 h after AE, the sexual differentiation of CHs precursor (including 16C
palmitate) occurs in the oenocytes and/or in the fat body
(Wicker-Thomas et al., 1997;
Ferveur et al., 1997
;
Savarit and Ferveur, 2002
).
This sex-specific process could involve the coupling of one or several
elongase and desaturase enzymes. The process of sexual differentiation would
be under the control of a brain factor released before 24 h after AE
(Wicker and Jallon, 1995
) and
would remain sensitive to the action of the transformer gene until 48
h after AE. The biosynthesis of CHs would be less active in 3 day-old flies,
although it would still be present. Tests of these hypotheses will involve
interspecific studies, in particular of D. simulans, which has no
qualitative sexual dimorphism, but has a similar ecology to D.
melanogaster. Studies of other insect genera involving temperature shifts
even without the advantage of the genetic technology available for
D. melanogaster could provide valuable insights into the
generality of our findings, and help deepen our understanding of the evolution
of insect cuticular hydrocarbons and their multiple roles in ecology,
physiology and communication.
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Acknowledgments |
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