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Phenotypic plasticity of adult size and pigmentation in Drosophila: thermosensitive periods during development in two sibling species
Journal of Thermal Biology 27 (2002) 61–70
Phenotypic plasticity of adult size and pigmentation in Drosophila: thermosensitive periods during development in two sibling species Mohamed Chakira,c, Abdelaziz Chafikb, Patricia Gibertc, Jean R. Davidc,* Universite! Cadi Ayyad, Faculte! des Sciences et Techniques, BP 618 Marrakech, Maroc b Universite! Chouaib Doukkali, Faculte! des Sciences, El Jadida, Maroc c Laboratoire Populations, Ge!ne!tique et Evolution, CNRS, Avenue de la Terrasse Bat.13, 91198 Gif sur Yvette cedex, France a
Received 25 November 2000; accepted 3 March 2001
Abstract Variation of size-related traits and of abdomen pigmentation was investigated in D. melanogaster and D. simulans by transferring cultures from 258C to 178C at regular daily intervals. In most cases regular sigmoid curves were obtained, which were adjusted to a logistic model. For wing length, the thermosensitive period (TSP) extended during the last larval instar and most of the pupal stage, with an inflection point (IP) at 5.24 0.24 days. Pigmentation of the last abdomen segments (5–7) in females exhibited a shorter and later TSP (IP at 7.21 0.13 days) extending in the first day of adult life. In anterior segments (2–4) variations were less regular and our data suggest that TSP might extend during the first two days of adult life, at least in males. Significant differences among species were observed for size and pigmentation, but not for TSPs. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: D. melanogaster; D. simulans; Thermosensitive period; Body size; Body pigmentation; Development
1. Introduction Phenotypic plasticity, i.e. the capacity of a single genotype to produce different phenotypes in different environments, is a general property of living organisms which receives increasing attention from the scientific community (Via, 1992, 1993; Via et al., 1995; Schlichting and Pigliucci, 1993, 1995; Scheiner, 1993a, b; Van Tienderen and Koelewijn, 1994; de Jong, 1995; Gotthard and Nylin, 1995). When the trait investigated concerns an irreversible property of the adult stage (e.g. body size), plasticity provides some information on developmental mechanisms. In this respect, it is an integral part of developmental biology. A major issue is to decide whether plasticity reflects internal constraints *Corresponding author. Tel.: +33-1-69-82-37-13; fax: +331-69-07-04-21. E-mail address: [email protected] (J.R. David).
(the need to produce functionally coadapted organs) or if it may be selected directly and thus exhibits an adaptive significance of its own (see Gibert et al., 2000; David et al., 2001). In nature, a major part of phenotypic variance is due to plasticity, and relating phenotype variability to individual fitness is a major ecological problem (Lomnicki, 1988). Not surprisingly, Drosophila appears as a choice model for bridging the gap between developmental biology and ecology. In D. melanogaster, variability among individuals collected in the wild is much higher than in laboratory reared flies (David et al., 1980; Coyne and Beecham, 1987; Imasheva et al., 1997; Gibert et al., 1998b; Azevedo et al., 1998; Loeschcke et al., 2000). Such an increase in environmental variance reveals the heterogeneity of developmental conditions and two main factors, temperature and larval resources, seem to be responsible (David et al., 1998; P!etavy et al., 2000).
0306-4565/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 6 5 ( 0 1 ) 0 0 0 1 6 - X
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For unravelling temperature effects, most laboratory experiments have been done under constant thermal conditions and the phenotypic response curves (the reaction norms) of size-related traits and body pigmentation have been analyzed in several species (David et al., 1990, 1994; Gibert et al., 1996, 1998b; Morin et al., 1996, 1999; Moreteau et al., 1997; Karan et al., 1999). In nature, however, ambient temperature may be highly variable, showing not only daily cycles but also significant variations over successive days (Feder, 1997). The incidence of such variations is poorly known, although this information would be needed if we want to better understand the origin of phenotypic variability (P!etavy et al., 2000). At the level of developmental biology, changing the temperature during preadult stages is a classical means for analyzing the developmental processes which determine the adult phenotype. More precisely, adult characteristics depend on the growth of imaginal discs or embryonic cell clusters. Knowing when these cells respond to a thermal shift will help to understand the genetic and metabolic bases of plasticity, and also of cell differentiation. In this paper we analyze the temperature sensitive period (TSP) of body size and abdomen pigmentation in two Drosophila sibling species, D. melanogaster and D. simulans. We find that different characters do not react in the same way and even that different abdomen segments might react differently. Some divergences are also found between species for body pigmentation, in spite of their close phylogenetic relationship, suggesting that body pigmentation is a fast evolving trait.
2.2. Traits measured After emergence, adult flies were kept for a few days at 178C, then etherized and measured under a binocular microscope. Wing length was measured on a lateral view (left wing) from the thoracic articulation to the tip of wing. Thorax length was measured in the same way from the neck to the tip of the scutellum. Micrometer units were transformed into mm 100, and we also calculated the wing/thorax ratio (see David et al., 1994). We estimated the extension of the dark pigmentation on the abdomen tergites visually, by using 11 phenotypic classes ranging from 0 (no dark pigmentation) to 10 (completely dark) (see David et al., 1990). In females, we analyzed 6 successive segments (Abd 2–7). In males, only 6 abdomen segments are developed and the last two are almost completely black. Consequently, pigmentation variation was analyzed in segments 2–4 only. 2.3. Data analysis For all traits, we expected that the response curves would show a decreasing sigmoid shape, and we used a logistic adjustment according to the formula P ¼ Pmax þ ðPmin Pmax Þ=1 þ exp Sðt IPÞ (see Gibert et al., 1998a), where P is the phenotype, t is the time (days) at transfer to 178C, and S is a constant. Such an adjustment allows the calculation of 4 characteristic values of biological significance: *
* *
2. Material and methods
*
an upper asymptote, i.e. the phenotype at 178C (Pmax ); a lower asymptote, i.e. the phenotype at 258C (Pmin ); the position (in days) of the inflection point (IP); the slope at inflection point (b), calculated as b ¼ ½ðPmin Pmax Þ=4 S.
2.1. Strains and experimental design We used two strains collected in Marrakech in March 1999. Each strain was founded by more than 30 wild living females and was kept as a mass culture at 20–218C under day–night conditions (LD 16–8). Experiments were done in July 1999. After allowing parents (about 100 adults) to oviposit for 4 h at 208C, groups of 50 eggs were distributed on pieces of filter paper and transferred into 11 culture vials containing a high nutrient, killed yeast food (David and Clavel, 1965). One vial was immediately (time 0) put at 178C and the others were maintained at 258C. Each day, a new culture vial was transferred from 258C to 178C, until the complete development was achieved at 258C. Two successive replicate experiments were done. In each experiment, we measured 15 females from each vial. Males (15 flies per sample) were studied in the first experiment only.
Data of each experiment were adjusted separately, and a comparison was done between experiments with a two way ANOVA (not shown) for each trait. The direct effect of experiment was never significant and there was generally no significant interaction, with however three exceptions concerning the pigmentation of segments 2–4 in D. simulans females. This phenomenon is illustrated in Fig. 1 for the sum of segments 2+3 and compared to the stability of data for segments 6+7. There is no clear explanation for a discrepancy between two identical successive experiments. One might be that the anterior segments are not much pigmented so that it is difficult to assign them a precise phenotypic score. So, large measurement errors might be the explanation. But the phenomenon should also be found in D. melanogaster, which was not the case. For all traits, the mean values of both experiments should provide the best biological estimate, so we decided to pool the data for all traits.
M. Chakir et al. / Journal of Thermal Biology 27 (2002) 61–70
Fig. 1. Examples of experimental response curves obtained in the two successive experiments for female abdomen pigmentation of Drosophila simulans. Cultures were initiated at the same time at 258C and transferred to 178C at successive daily intervals. Mean developmental duration was 8 days. Durations of successive developmental stages at 258C are indicated on the graphs. (A) Pigmentation score of segments 2+3. Significant differences were found between the two experiments (significant temperature experiment interaction by ANOVA). (B) Pigmentation score of segments 6+7. The two different experiments provided similar results.
3. Results
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D. melanogaster males. D. simulans males exhibited however an almost linear decrease (see Fig. 2), with an abnormally high upper asymptote and a TIP at 2.68 days only. This may be due to large sampling errors since only 15 males were measured. Fig. 2 shows that in females of both species and in D. melanogaster males, a significant length decrease begins in the middle of the third instar larval stage (day 4, L3) and finishes in the middle of pupal stage (day 7). In other words, the TSP lasts about 96 h (day 4–7). We get the same conclusion by considering the position of the inflection point (IP=5.24 0.20 days, n ¼ 3) which indicates the middle of TSP and corresponds to early pupa. For thorax length (response curves not shown), logistic adjustments were irregular and provided an early IP, in the middle of third instar larval stage, with an average of 3.42 0.33 days (n ¼ 4) (Table 1). This value is less than that found for the wing and suggests that wing and thorax length do not have the same TSP, but such a conclusion must be taken cautiously. In D. melanogaster, the adjustments were very imprecise, as indicated by a small difference between Pmax and Pmin , and also by a huge standard error of b parameter (see Table 1). This is presumably due to the fact that, in this species, maximum thorax length occurs around 198C (Morin et al., 1996) and that a significant decrease, with respect to the maximum, is found both at 178C and 258C. A correct determination of TSP would imply the utilization of warmer temperatures, for example a transfer between 208C and 288C. In D. simulans, the maximum occurs at a lower temperature (15.98C) explaining better adjustments (Table 1). The wing/thorax ratio (Fig. 2) is much less in D. simulans than in D. melanogaster, in agreement with previous results (Morin et al., 1996). Convenient sigmoid curves were observed except for D. simulans males (Fig. 2). The average position of IP was 5.56 0.09 days (n ¼ 4) not different from that obtained for wing length, but the TSP seems to be shorter and restricted to the first half of pupal stage.
3.1. Body size traits 3.2. Pigmentation of abdomen segments 5–7 in females Data were submitted to an ANOVA (not shown), and the effect of age at transfer (from 258C to 178C) was always highly significant. We expect that a response curve comprises three parts: first a plateau, then a rapid decrease which corresponds to TSP and then a second plateau (see for example wing length variation in Fig. 2). For each character, species and sex, a logistic adjustment was used and four characteristic values were calculated (Table 1). It was sometimes difficult to adjust the response curves to a logistic equation (e.g. slope for thorax length in D. melanogaster), and in these cases, standard errors were abnormally high. For wing length (Fig. 2), convenient adjustments were obtained for the females in both species and for
Abdomen segments 5–7 in females are most reactive to temperature change (Gibert et al., 2000) and will be considered first. Results are summarized in Table 2 and illustrated in Fig. 3. All segments produced convenient logistic adjustments with one exception, segment 6 in D. simulans, which showed a regular curvilinear decrease more than a sigmoid one (see Fig. 3). The inadequacy of that adjustment is also shown by the fact that the calculated IP is found at day 9 (Table 2), i.e. after adult emergence. Significant differences between species were observed for pigmentation phenotypes, but in a complex way. For segments 5 and 6, D. melanogaster was darker in all
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Fig. 2. Determination of the temperature sensitive period of wing length and wing/thorax ratio of both sexes in D. melanogaster and D. simulans. Durations of successive developmental stages at 258C are indicated on the graphs.
cases. However, for segment 7 a lighter phenotype was found at 178C in D. melanogaster, but a darker one at 258C, indicating a higher plasticity of this segment in D. simulans. Significant differences were observed between successive segments. This is shown by different amplitudes between asymptotes and different slopes at inflection points (Table 2). More precisely, a steeper slope is correlated to a broader amplitude. In spite of differences between species and segments, data concerning TSP were similar. The positions of inflection points (excluding the abnormal value) are similar in both species, with an average of 7.21 0.13 days (n ¼ 5). Fig. 3 shows that no variations are observed up to day 5, and TSP starts between days 5 and 6, i.e. after the beginning of pupation. If we assume that TSP begins at day 5 and that IP is the middle of the TSP, then TSP ends at day 9.4, i.e. after adult emergence (8.55 and 8.18 days in D. melanogaster and D. simulans females respectively). The fact that TSP extends into early adult life is corroborated by the fact that, in all segments, pigmentation scores at day 9 are less than at day 8 (see Fig. 3).
As argued previously, sampling and measurement errors are presumably the main cause of some irregular adjustments. A means to decrease their incidence is to consider integrated traits, namely the sum of scores of the 3 segments, as already done in previous papers (David et al., 1990; Gibert et al., 1996). Fig. 3 shows the regularity of these integrative curves, in which more than 98% of the variation over time is accounted for by the logistic regression. Positions of IP are 7.13 0.09 days in D. melanogaster and 7.17 0.06 days in D. simulans, and again TSP appears to span the 5–9 day interval. 3.3. Pigmentation of abdomen segments 2–4 in both sexes In D. simulans females, as already mentioned, heterogeneous data between experiments 1 and 2 were obtained for segments 2–4. Pooled data, however, provided regular sigmoid response curves (Fig. 4) which were easily adjusted to a logistic model (Table 3). Results for segments 3 and 4 were almost identical with an IP at 7.87 days, that is close to adult emergence. For
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M. Chakir et al. / Journal of Thermal Biology 27 (2002) 61–70 Table 1 Values ( SE) of the four characteristic values calculated after a logistic adjustment for size-related traitsa Traits
Characteristic values
D. melanogaster
D. simulans
Comparison
Wing (female)
Pmax Pmin IP (days) b
287.26 0.86 260.28 0.82 5.06 0.15 8.27 1.05
269.04 1.24 240.76 1.09 5.02 0.21 7.36 1.06
11.52 13.64 0.15 0.59
*** *** NS NS
Wing (male)
Pmax Pmin IP (days) b
255.34 1.51 224.08 1.43 4.92 0.24 7.56 1.43
258.03 22.25 207.75 4.17 2.68 2.19 5.03 0.54
0.12 3.53 0.97 1.33
NS ** NS NS
Thorax (female)
Pmax Pmin IP (days) b
110.16 0.32 109.06 0.26 3.93 0.32 5.90 33.2
112.33 0.53 107.86 0.29 3.25 0.34 1.70 0.87
3.34 2.94 1.39 0.12
** ** NS NS
Thorax (male)
Pmax Pmin IP (days) b
98.70 0.40 94.30 0.33 3.92 0.12 22.79 42.58
101.21 2.86 95.18 0.49 2.56 1.42 1.46 0.76
0.83 1.42 0.91 0.48
NS NS NS NS
Ratio wing/thorax (female)
Pmax Pmin IP (days) b
2.60 0.008 2.38 0.008 5.42 0.165 0.08 0.014
2.39 0.005 2.24 0.005 5.63 0.120 0.11 0.024
21.22 14.15 0.98 1.03
*** *** NS NS
Ratio wing/thorax (male)
Pmax Pmin IP (days) b
2.57 0.007 2.39 0.008 5.79 0.146 0.12 0.038
2.46 0.106 2.15 0.088 5.41 0.965 0.03 0.009
0.99 2.59 0.37 3.66
NS * NS **
a
Pmax : upper asymptotic value (at 178C); Pmin : lower asymptotic value (at 258C); IP: position of inflection point; b=slope at inflection point. Species values are compared with a t-test. NS: non-significant, ***p50:001, **p50:01, *p50:05.
the 3 anterior segments, TSP seems to start at day 6 and end at day 9 or 10, that is from early pupa to young adults. This conclusion is enforced by considering the sum of segments 2–4 (Fig. 3) with an inflection point at day 7.63 0.29, close to adult emergence (8.18 days). In D. melanogaster females, the two experiments were homogenous but logistic adjustment provided implausible results, especially for segments 2 and 4 with inflection points after adult emergence. Pooling the 3 segments provided also a progressive smooth decrease and an inflection point at 24.9 days. Our data suggest that thermal reactivity of segments 2–4 might be different in the two species. Worth mentioning also is the fact that D. simulans females appear darker than those of D. melanogaster for segments 2–4, while the reverse was true for segments 5–7. Male data, obtained on samples of 15 individuals only, were irregular and did not reveal a sigmoid shape in any species either for single segments or for their sum (Fig. 4). Surprisingly, major pigmentation changes occurred mainly after adult emergence in D. simulans
as argued previously. Such data should be repeated on bigger samples. Interestingly, for these anterior segments, males were clearly darker than females in D. melanogaster, while both sexes were similar in D. simulans.
4. Discussion and conclusions 4.1. Experimental design and model for investigating TSP A classical determination of TSP implies the utilization of two constant developmental temperatures and a transfer of individuals from one to the other temperature at regular intervals. Such a procedure is justified, provided that the reaction norm between the two chosen temperatures is linear or at least varies monotonically. In our study, such was the case for wing length and wing/thorax ratio and also for abdomen pigmentation, but not for thorax length (see David et al., 1994). A
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Table 2 Values ( SE) of the four characteristic values calculated after a logistic adjustment on pigmentation scores of the posterior abdominal segments (Abd 5–7) and their sum in females of both speciesa Traits
Characteristic values
D. melanogaster
D. simulans
Comparison
Abd 5
Pmax Pmin IP (days) b
4.84 0.09 1.66 0.34 7.59 0.29 0.79 0.13
4.28 0.05 1.38 0.20 7.42 0.21 0.72 0.07
5.19 0.68 0.45 0.45
*** NS NS NS
Abd 6
Pmax Pmin IP (days) b
8.36 0.06 4.49 0.12 6.95 0.09 1.32 0.12
6.76 0.15 1.63 1.75 9.01 1.38 0.64 0.08
9.44 1.55 1.42 4.50
*** NS NS ***
Abd 7
Pmax Pmin IP (days) b
7.78 0.09 1.84 0.19 7.09 0.09 2.24 0.25
8.78 0.16 0.11 0.33 6.99 0.11 2.98 0.34
5.19 4.33 0.67 1.67
*** *** NS NS
Sum 5+6+7
Pmax Pmin IP (days) b
20.98 0.80 8.10 0.41 7.13 0.09 4.21 0.37
19.75 0.75 4.79 0.27 7.17 0.06 3.99 0.14
1.07 6.43 0.53 0.35
NS *** NS NS
a Pmax : upper asymptotic value (at 178C); Pmin : lower asymptotic value (at 258C); IP: position of inflection point; b=slope at inflection point. Species values are compared with a t-test. NS: non-significant, ***p50:001, **p50:01, *p50:05.
Fig. 3. Determination of the temperature sensitive period for the posterior abdomen segments (5–7) and for the sum of these segments in females. Durations of developmental stages correspond to those of D. melanogaster.
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Fig. 4. Determination of the temperature sensitive period of the 3 anterior (2–4) abdomen segments in the two species. Upper graphs: results obtained for each segment in females. Lower graphs: comparison of males and females data for the sum of the 3 segments.
precise estimate of thorax TSP should be done using higher temperatures. Experimental data may be analyzed visually or with various descriptive models. In our case, a logistic adjustment seemed appropriate since it takes into account all experimental values and describes the results by parameters with a clear biological meaning: the upper and lower phenotypic values, the position of the inflection point, which defines the middle of TSP, and the slope at IP, proportional to the duration of TSP. The logistic model has however two drawbacks. Firstly, because of the asymptotic adjustment, it is difficult to define precisely the beginning and the end of TSP. Secondly, and more seriously, the logistic curve is symmetrical around IP, while there is no biological reason for such a constraint; other sigmoid adjustments could thus be implemented. We have also found that the adjustment is quite sensitive to sampling errors. In future investigations, samples of at least 50 flies should be preferred. In spite of these limitations, our data still provided original and novel observations.
4.2. TSP and development A main conclusion of our work is that size-related traits and abdomen pigmentation do not exhibit the same TSPs. For wing length, TSP begins during larval growth and extends during the whole pupal period. TSP cannot however extend into adult life since wing and thorax length are definitely fixed during the first hour after emergence from puparium (Ashburner, 1989). Our results match what is known from anatomical description (Lawrence, 1992): wing and thorax arise from a single imaginal disk which shows a regular but slow increase during larval life and a more rapid one during the first half of pupation. The position of the IP, on average after 5.5 days at 258C, well reflects a long lasting TSP of about 6 days. For wing length at least, it is generally assumed that plasticity of overall size results mainly from a modification of cell size (el Masry and Robertson, 1978; Partridge et al., 1994). However, the fact that TSP extends during the larval life, where the imaginal disk is a massive and undifferentiated organ, suggests that cell number might also to be modified to some extend.
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Table 3 Values ( SE) of the four characteristic values calculated after a logistic adjustment on pigmentation score of anterior abdomen segments (Abd 2–4) and of their sum in femalesa Traits
Characteristic values
D. melanogaster
D. simulans
Comparison
Abd 2
Pmax Pmin IP (days) b
NC NC NC NC
2.23 0.08 1.19 0.15 6.92 0.47 0.49 0.24
NC NC NC NC
Abd 3
Pmax Pmin IP (days) b
2.71 0.16 0.66 1.47 8.38 2.23 0.35 0.10
3.52 0.07 1.99 0.17 7.94 0.25 0.72 0.28
4.42 0.86 0.19 1.22
Abd 4
Pmax Pmin IP (days) b
NC NC NC NC
3.79 0.04 1.95 0.15 7.80 0.22 0.53 0.07
NC NC NC NC
Sum 2+3+4
Pmax Pmin IP (days) b
NC NC NC NC
9.54 0.17 5.15 0.46 7.63 0.29 1.55 0.34
NC NC NC NC
*** NS NS NS
a Pmax : upper asymptotic value (at 178C); Pmin : lower asymptotic value (at 258C); IP: position of inflection point; b=slope at inflection point. Species values are compared with a t-test. NC: not calculated, NS: non-significant, ***p50:001, **p50:01, *p50:05.
For female abdomen pigmentation (last segments, Fig. 3), the TSP occurs later than for wing length and the IP is observed on average 7.2 days after egg laying. The development of adult abdomen tegument arises from clusters of embryonic cells (called histocysts) included into the larval ectoderm (Lawrence, 1992; Fristrom and Fristrom, 1993). Proliferation of these cells starts in early pupa, after the beginning of larval ectoderm histolysis, and ends during the second half of pupation, when a complete adult ectoderm is produced. The differentiation of adult segments, however, and especially the production of cuticule and microchaetae, extends up to the end of pupation. The genes, which control the spatial patterning of the tergites are progressively identified, some of which act also on the extension of the black pigment at the posterior margin of each tergite (Kopp and Duncan, 1997; Kopp et al., 1997). From all these observations it is not surprising to find that TSP begins after puparium formation. What was not expected is that TSP extends during the first day of imaginal life. This is however a plausible observation since, at emergence, Drosophila adults are not pigmented and the cuticule undergoes a progressive maturation resulting in both hardening and appearance of the pigmentation pattern. At least one day is necessary for completion of these processes. Another observation concerns the difference in plasticity between posterior segments in females although TSPs seem identical.
4.3. Sex and species comparison For size-related traits, and especially wing length, we did not find clear differences for TSPs between sexes or species (Fig. 2). The peculiar curve observed in D. simulans males more likely reflects sampling errors than a sex dimorphism. Similarities between sexes and sibling species are expected if we consider that the wing– thorax system, which is involved in the flight function, is likely to be submitted to a strong developmental canalization. For abdomen pigmentation, some differences were evidenced among females of the two species, either for the overall plasticity (Abd 7, Fig. 3) or for average pigmentation (Fig. 3). In this respect, D. melanogaster females are darker than D. simulans females for the 3 posterior segments (Fig. 3), while the reverse is true for the 3 anterior ones (Fig. 4). The shapes of the response curves and TSPs might also be different between anterior and posterior segments, but this point deserves more extensive investigations. Pigmentation differences among sexes could be analyzed only for the 3 anterior segments, since segments 5 and 6 of males are completely black. As seen in Fig. 4, D. melanogaster males were darker than females, while both sexes were almost identical in D. simulans. Moreover, our data suggest that, for the three anterior segments, TSP might extend up to day 10, that is during the first two days of adult life. It was
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recently shown (Kopp et al., 2001) that the genetic basis of pigmentation in the posterior abdominal segments was completely different between males and females. 4.4. Ecological phenotypes under changing conditions As stated in Section 1, a natural shift from one temperature (e.g. warm days) to another one (e.g. cold days) will change the phenotypes if the shift occurs mainly during pupation. So we may predict that a thermal change during embryonic development or the two first larval instars should not modify the adult phenotype. If the change occurs during TSPs, the two kinds of traits will vary in a parallel way: both size and pigmentation will decrease at a higher temperature, inducing a positive correlation in a population. The TSPs are not however identical, occurring earlier in development for size than for pigmentation. For example, a cold treatment at the time of adult emergence will not modify the size but will produce darker flies. In other words, temperature changes during development might be detected by analyzing the phenotypic correlations between different traits. Indeed, we have some preliminary observations in field collected flies (unpublished results) showing a positive correlation between body size and female abdomen pigmentation. Such a correlation does not exist in a laboratory population grown at a constant temperature.
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David, J.R., Gibert, P., Pla, E., P!etavy, G., Karan, D., Moreteau, B., 1998. Cold stress tolerance in Drosophila: analysis of chill coma recovery in D. melanogaster. J. Therm. Biol. 23, 291–299. David, J.R., Moreteau, B., Gauthier, J.R., P!etavy, G., Stockel, J., Imasheva, A., 1994. Reaction norms of size characters in relation to growth temperature in Drosophila melanogaster: an isofemale lines analysis. Genet. Sel. Evol. 26, 229–251. de Jong, G., 1995. Phenotypic plasticity as a product of selection in a variable environment. Am. Nat. 145, 493–512. el Masry, A.M., Robertson, F.W., 1978. Cell size and number in the Drosophila wing. I. The use of selection to study the inter-relations between cell size and number and body size. Egypt. J. Genet. Cytol. 7, 137–165. Feder, M.E., 1997. Necrotic fruit: a novel model system for thermal ecologists. J. Therm. Biol. 22, 1–9. Fristrom, D., Fristrom, J.W., 1993. The metamorphic development of the adult epidermis. In: Bate, M., Arias, A.M. (Eds.), The Development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, USA, pp. 843–897. Gibert, P., Moreteau, B., David, J.R., 2000. Developmental constraints on an adpative plasticity: reaction norms of pigmentation in adult segments of Drosophila melanogaster. Evol. Dev. 2, 249–260. Gibert, P., Moreteau, B., David, J.R., Scheiner, S.M., 1998a. Describing the evolution of reaction norm shape: body pigmentation in Drosophila. Evolution 52, 1501–1506. Gibert, P., Moreteau, B., Moreteau, J.C., David, J.R., 1996. Growth temperature and adult pigmentation in two Drosophila sibling species: an adaptive convergence of reaction norms in sympatric populations? Evolution 50, 2346–2353. Gibert, P., Moreteau, B., Moreteau, J.C., David, J.R., 1998b. Genetic variability of quantitative traits in Drosophila melanogaster (fruit fly) natural populations: analysis of wild-living flies and of several laboratory generations. Heredity 80, 326–335. Gotthard, K., Nylin, S., 1995. Adaptive plasticity and plasticity as an adaptation: a selective review of plasticity in animal morphology and life history. Oikos 74, 3–17. Imasheva, A.G., Loeschcke, V., Zhivotovsky, L.A., Lazebny, O.E., 1997. Effects of extreme temperatures on phenotypic variation and developmental stability in Drosophila melanogaster and Drosophila buzzatii. Biol. J. Linn. Soc. 61, 117–126. Karan, D., Morin, J.P., Gravot, E., Moreteau, B., David, J.R., 1999. Body size reaction norms in Drosophila melanogaster: temporal stability and genetic architecture in a natural population. Genet. Sel. Evol. 31, 491–508. Kopp, A., Duncan, I., 1997. Control of cell fate and polarity in the adult abdominal segments of Drosophila by optomotorblind. Development 124, 3715–3726. Kopp, A., Duncan, I., Caroll, S.B., 2001. Genetic control and evolution of sexually dimorphic characters in Drosophila. Nature 408, 553–559. Kopp, A., Muskavitch, M.A.T., Duncan, I., 1997. The roles of hedgehog and engrailed in patterning adult abdominal segments of Drosophila. Development 124, 3703–3714. Lawrence, P.A., 1992. The Making of a Fly. The Genetics of Animal Design. Blackwell Scientific Publications, Oxford. Loeschcke, V., Bundgaad, J., Barker, J.S.F., 2000. Variation in body size and life history traits in Drosophila aldrichi and
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D. buzzatii from a latitudinal cline in eastern Australia. Heredity 85, 423–433. Lomnicki, A., 1988. Population Ecology of Individuals. Princeton University Press, Princeton. Moreteau, B., Morin, J.P., Gibert, P., P!etavy, G., Pla, E., David, J.R., 1997. Evolutionary changes of nonlinear reaction norms according to thermal adaptation: a comparison of two Drosophila species. C. R. Acad. Sci. 320, 833–841. Morin, J.P., Moreteau, B., P!etavy, G., David, J.R., 1999. Divergence of reaction norms of size characters between tropical and temperate populations of Drosophila melanogaster and D. simulans. J. Evol. Biol. 12, 329–339. Morin, J.P., Moreteau, B., P!etavy, G., Imasheva, S., David, J.R., 1996. Body size and developmental temperature in Drosophila simulans: comparison of reaction norms with sympatric Drosophila melanogaster. Genet. Sel. Evol. 28, 415–436. Partridge, L., Barrie, B., Fowler, K., French, V., 1994. Evolution and development of body size and cell size in Drosophila melanogaster in response to temperature. Evolution 48, 1269–1276. P!etavy, G., David, J.R., Gibert, P., Moreteau, B., 2000. Viability and rate of development at different temperatures
in Drosophila: a comparison of constant and alternating thermal regimes. J. Therm. Biol. 26, 29–39. Scheiner, S.M., 1993a. Genetics and evolution of phenotypic plasticity. Ann. Rev. Ecol. Syst. 24, 35–68. Scheiner, S.M., 1993b. Plasticity as a selectable trait: reply to Via. Am. Nat. 142, 371–373. Schlichting, C.D., Pigliucci, M., 1993. Control of phenotypic plasticity via regulatory genes. Am. Nat. 142, 366–370. Schlichting, C.D., Pigliucci, M., 1995. Gene regulation, quantitative genetics and the evolution of reaction norms. Evol. Ecol. 9, 154–168. Van Tienderen, P.H., Koelewijn, H.P., 1994. Selection on reaction norms, genetic correlations and constraints. Genet. Res. 64, 115–125. Via, S., 1992. Models of the evolution of phenotypic plasticity. Trends Ecol. Evol. 7, 63. Via, S., 1993. Adaptive phenotypic plasticity: target or by-product of selection in a variable environment? Am. Nat. 142, 352–365. Via, S., Gomulkiewicz, R., De Jong, G., Scheiner, S.M., Schlichting, C.D., Van Tienderen, P.H., 1995. Adaptive phenotypic plasticity: consensus and controversy. Trends Ecol. Evol. 5, 212–217.
Phenotypic plasticity of adult size and pigmentation in Drosophila: thermosensitive periods during development in two sibling species Mohamed Chakira,c, Abdelaziz Chafikb, Patricia Gibertc, Jean R. Davidc,* Universite! Cadi Ayyad, Faculte! des Sciences et Techniques, BP 618 Marrakech, Maroc b Universite! Chouaib Doukkali, Faculte! des Sciences, El Jadida, Maroc c Laboratoire Populations, Ge!ne!tique et Evolution, CNRS, Avenue de la Terrasse Bat.13, 91198 Gif sur Yvette cedex, France a
Received 25 November 2000; accepted 3 March 2001
Abstract Variation of size-related traits and of abdomen pigmentation was investigated in D. melanogaster and D. simulans by transferring cultures from 258C to 178C at regular daily intervals. In most cases regular sigmoid curves were obtained, which were adjusted to a logistic model. For wing length, the thermosensitive period (TSP) extended during the last larval instar and most of the pupal stage, with an inflection point (IP) at 5.24 0.24 days. Pigmentation of the last abdomen segments (5–7) in females exhibited a shorter and later TSP (IP at 7.21 0.13 days) extending in the first day of adult life. In anterior segments (2–4) variations were less regular and our data suggest that TSP might extend during the first two days of adult life, at least in males. Significant differences among species were observed for size and pigmentation, but not for TSPs. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: D. melanogaster; D. simulans; Thermosensitive period; Body size; Body pigmentation; Development
1. Introduction Phenotypic plasticity, i.e. the capacity of a single genotype to produce different phenotypes in different environments, is a general property of living organisms which receives increasing attention from the scientific community (Via, 1992, 1993; Via et al., 1995; Schlichting and Pigliucci, 1993, 1995; Scheiner, 1993a, b; Van Tienderen and Koelewijn, 1994; de Jong, 1995; Gotthard and Nylin, 1995). When the trait investigated concerns an irreversible property of the adult stage (e.g. body size), plasticity provides some information on developmental mechanisms. In this respect, it is an integral part of developmental biology. A major issue is to decide whether plasticity reflects internal constraints *Corresponding author. Tel.: +33-1-69-82-37-13; fax: +331-69-07-04-21. E-mail address: [email protected] (J.R. David).
(the need to produce functionally coadapted organs) or if it may be selected directly and thus exhibits an adaptive significance of its own (see Gibert et al., 2000; David et al., 2001). In nature, a major part of phenotypic variance is due to plasticity, and relating phenotype variability to individual fitness is a major ecological problem (Lomnicki, 1988). Not surprisingly, Drosophila appears as a choice model for bridging the gap between developmental biology and ecology. In D. melanogaster, variability among individuals collected in the wild is much higher than in laboratory reared flies (David et al., 1980; Coyne and Beecham, 1987; Imasheva et al., 1997; Gibert et al., 1998b; Azevedo et al., 1998; Loeschcke et al., 2000). Such an increase in environmental variance reveals the heterogeneity of developmental conditions and two main factors, temperature and larval resources, seem to be responsible (David et al., 1998; P!etavy et al., 2000).
0306-4565/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 6 5 ( 0 1 ) 0 0 0 1 6 - X
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For unravelling temperature effects, most laboratory experiments have been done under constant thermal conditions and the phenotypic response curves (the reaction norms) of size-related traits and body pigmentation have been analyzed in several species (David et al., 1990, 1994; Gibert et al., 1996, 1998b; Morin et al., 1996, 1999; Moreteau et al., 1997; Karan et al., 1999). In nature, however, ambient temperature may be highly variable, showing not only daily cycles but also significant variations over successive days (Feder, 1997). The incidence of such variations is poorly known, although this information would be needed if we want to better understand the origin of phenotypic variability (P!etavy et al., 2000). At the level of developmental biology, changing the temperature during preadult stages is a classical means for analyzing the developmental processes which determine the adult phenotype. More precisely, adult characteristics depend on the growth of imaginal discs or embryonic cell clusters. Knowing when these cells respond to a thermal shift will help to understand the genetic and metabolic bases of plasticity, and also of cell differentiation. In this paper we analyze the temperature sensitive period (TSP) of body size and abdomen pigmentation in two Drosophila sibling species, D. melanogaster and D. simulans. We find that different characters do not react in the same way and even that different abdomen segments might react differently. Some divergences are also found between species for body pigmentation, in spite of their close phylogenetic relationship, suggesting that body pigmentation is a fast evolving trait.
2.2. Traits measured After emergence, adult flies were kept for a few days at 178C, then etherized and measured under a binocular microscope. Wing length was measured on a lateral view (left wing) from the thoracic articulation to the tip of wing. Thorax length was measured in the same way from the neck to the tip of the scutellum. Micrometer units were transformed into mm 100, and we also calculated the wing/thorax ratio (see David et al., 1994). We estimated the extension of the dark pigmentation on the abdomen tergites visually, by using 11 phenotypic classes ranging from 0 (no dark pigmentation) to 10 (completely dark) (see David et al., 1990). In females, we analyzed 6 successive segments (Abd 2–7). In males, only 6 abdomen segments are developed and the last two are almost completely black. Consequently, pigmentation variation was analyzed in segments 2–4 only. 2.3. Data analysis For all traits, we expected that the response curves would show a decreasing sigmoid shape, and we used a logistic adjustment according to the formula P ¼ Pmax þ ðPmin Pmax Þ=1 þ exp Sðt IPÞ (see Gibert et al., 1998a), where P is the phenotype, t is the time (days) at transfer to 178C, and S is a constant. Such an adjustment allows the calculation of 4 characteristic values of biological significance: *
* *
2. Material and methods
*
an upper asymptote, i.e. the phenotype at 178C (Pmax ); a lower asymptote, i.e. the phenotype at 258C (Pmin ); the position (in days) of the inflection point (IP); the slope at inflection point (b), calculated as b ¼ ½ðPmin Pmax Þ=4 S.
2.1. Strains and experimental design We used two strains collected in Marrakech in March 1999. Each strain was founded by more than 30 wild living females and was kept as a mass culture at 20–218C under day–night conditions (LD 16–8). Experiments were done in July 1999. After allowing parents (about 100 adults) to oviposit for 4 h at 208C, groups of 50 eggs were distributed on pieces of filter paper and transferred into 11 culture vials containing a high nutrient, killed yeast food (David and Clavel, 1965). One vial was immediately (time 0) put at 178C and the others were maintained at 258C. Each day, a new culture vial was transferred from 258C to 178C, until the complete development was achieved at 258C. Two successive replicate experiments were done. In each experiment, we measured 15 females from each vial. Males (15 flies per sample) were studied in the first experiment only.
Data of each experiment were adjusted separately, and a comparison was done between experiments with a two way ANOVA (not shown) for each trait. The direct effect of experiment was never significant and there was generally no significant interaction, with however three exceptions concerning the pigmentation of segments 2–4 in D. simulans females. This phenomenon is illustrated in Fig. 1 for the sum of segments 2+3 and compared to the stability of data for segments 6+7. There is no clear explanation for a discrepancy between two identical successive experiments. One might be that the anterior segments are not much pigmented so that it is difficult to assign them a precise phenotypic score. So, large measurement errors might be the explanation. But the phenomenon should also be found in D. melanogaster, which was not the case. For all traits, the mean values of both experiments should provide the best biological estimate, so we decided to pool the data for all traits.
M. Chakir et al. / Journal of Thermal Biology 27 (2002) 61–70
Fig. 1. Examples of experimental response curves obtained in the two successive experiments for female abdomen pigmentation of Drosophila simulans. Cultures were initiated at the same time at 258C and transferred to 178C at successive daily intervals. Mean developmental duration was 8 days. Durations of successive developmental stages at 258C are indicated on the graphs. (A) Pigmentation score of segments 2+3. Significant differences were found between the two experiments (significant temperature experiment interaction by ANOVA). (B) Pigmentation score of segments 6+7. The two different experiments provided similar results.
3. Results
63
D. melanogaster males. D. simulans males exhibited however an almost linear decrease (see Fig. 2), with an abnormally high upper asymptote and a TIP at 2.68 days only. This may be due to large sampling errors since only 15 males were measured. Fig. 2 shows that in females of both species and in D. melanogaster males, a significant length decrease begins in the middle of the third instar larval stage (day 4, L3) and finishes in the middle of pupal stage (day 7). In other words, the TSP lasts about 96 h (day 4–7). We get the same conclusion by considering the position of the inflection point (IP=5.24 0.20 days, n ¼ 3) which indicates the middle of TSP and corresponds to early pupa. For thorax length (response curves not shown), logistic adjustments were irregular and provided an early IP, in the middle of third instar larval stage, with an average of 3.42 0.33 days (n ¼ 4) (Table 1). This value is less than that found for the wing and suggests that wing and thorax length do not have the same TSP, but such a conclusion must be taken cautiously. In D. melanogaster, the adjustments were very imprecise, as indicated by a small difference between Pmax and Pmin , and also by a huge standard error of b parameter (see Table 1). This is presumably due to the fact that, in this species, maximum thorax length occurs around 198C (Morin et al., 1996) and that a significant decrease, with respect to the maximum, is found both at 178C and 258C. A correct determination of TSP would imply the utilization of warmer temperatures, for example a transfer between 208C and 288C. In D. simulans, the maximum occurs at a lower temperature (15.98C) explaining better adjustments (Table 1). The wing/thorax ratio (Fig. 2) is much less in D. simulans than in D. melanogaster, in agreement with previous results (Morin et al., 1996). Convenient sigmoid curves were observed except for D. simulans males (Fig. 2). The average position of IP was 5.56 0.09 days (n ¼ 4) not different from that obtained for wing length, but the TSP seems to be shorter and restricted to the first half of pupal stage.
3.1. Body size traits 3.2. Pigmentation of abdomen segments 5–7 in females Data were submitted to an ANOVA (not shown), and the effect of age at transfer (from 258C to 178C) was always highly significant. We expect that a response curve comprises three parts: first a plateau, then a rapid decrease which corresponds to TSP and then a second plateau (see for example wing length variation in Fig. 2). For each character, species and sex, a logistic adjustment was used and four characteristic values were calculated (Table 1). It was sometimes difficult to adjust the response curves to a logistic equation (e.g. slope for thorax length in D. melanogaster), and in these cases, standard errors were abnormally high. For wing length (Fig. 2), convenient adjustments were obtained for the females in both species and for
Abdomen segments 5–7 in females are most reactive to temperature change (Gibert et al., 2000) and will be considered first. Results are summarized in Table 2 and illustrated in Fig. 3. All segments produced convenient logistic adjustments with one exception, segment 6 in D. simulans, which showed a regular curvilinear decrease more than a sigmoid one (see Fig. 3). The inadequacy of that adjustment is also shown by the fact that the calculated IP is found at day 9 (Table 2), i.e. after adult emergence. Significant differences between species were observed for pigmentation phenotypes, but in a complex way. For segments 5 and 6, D. melanogaster was darker in all
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Fig. 2. Determination of the temperature sensitive period of wing length and wing/thorax ratio of both sexes in D. melanogaster and D. simulans. Durations of successive developmental stages at 258C are indicated on the graphs.
cases. However, for segment 7 a lighter phenotype was found at 178C in D. melanogaster, but a darker one at 258C, indicating a higher plasticity of this segment in D. simulans. Significant differences were observed between successive segments. This is shown by different amplitudes between asymptotes and different slopes at inflection points (Table 2). More precisely, a steeper slope is correlated to a broader amplitude. In spite of differences between species and segments, data concerning TSP were similar. The positions of inflection points (excluding the abnormal value) are similar in both species, with an average of 7.21 0.13 days (n ¼ 5). Fig. 3 shows that no variations are observed up to day 5, and TSP starts between days 5 and 6, i.e. after the beginning of pupation. If we assume that TSP begins at day 5 and that IP is the middle of the TSP, then TSP ends at day 9.4, i.e. after adult emergence (8.55 and 8.18 days in D. melanogaster and D. simulans females respectively). The fact that TSP extends into early adult life is corroborated by the fact that, in all segments, pigmentation scores at day 9 are less than at day 8 (see Fig. 3).
As argued previously, sampling and measurement errors are presumably the main cause of some irregular adjustments. A means to decrease their incidence is to consider integrated traits, namely the sum of scores of the 3 segments, as already done in previous papers (David et al., 1990; Gibert et al., 1996). Fig. 3 shows the regularity of these integrative curves, in which more than 98% of the variation over time is accounted for by the logistic regression. Positions of IP are 7.13 0.09 days in D. melanogaster and 7.17 0.06 days in D. simulans, and again TSP appears to span the 5–9 day interval. 3.3. Pigmentation of abdomen segments 2–4 in both sexes In D. simulans females, as already mentioned, heterogeneous data between experiments 1 and 2 were obtained for segments 2–4. Pooled data, however, provided regular sigmoid response curves (Fig. 4) which were easily adjusted to a logistic model (Table 3). Results for segments 3 and 4 were almost identical with an IP at 7.87 days, that is close to adult emergence. For
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M. Chakir et al. / Journal of Thermal Biology 27 (2002) 61–70 Table 1 Values ( SE) of the four characteristic values calculated after a logistic adjustment for size-related traitsa Traits
Characteristic values
D. melanogaster
D. simulans
Comparison
Wing (female)
Pmax Pmin IP (days) b
287.26 0.86 260.28 0.82 5.06 0.15 8.27 1.05
269.04 1.24 240.76 1.09 5.02 0.21 7.36 1.06
11.52 13.64 0.15 0.59
*** *** NS NS
Wing (male)
Pmax Pmin IP (days) b
255.34 1.51 224.08 1.43 4.92 0.24 7.56 1.43
258.03 22.25 207.75 4.17 2.68 2.19 5.03 0.54
0.12 3.53 0.97 1.33
NS ** NS NS
Thorax (female)
Pmax Pmin IP (days) b
110.16 0.32 109.06 0.26 3.93 0.32 5.90 33.2
112.33 0.53 107.86 0.29 3.25 0.34 1.70 0.87
3.34 2.94 1.39 0.12
** ** NS NS
Thorax (male)
Pmax Pmin IP (days) b
98.70 0.40 94.30 0.33 3.92 0.12 22.79 42.58
101.21 2.86 95.18 0.49 2.56 1.42 1.46 0.76
0.83 1.42 0.91 0.48
NS NS NS NS
Ratio wing/thorax (female)
Pmax Pmin IP (days) b
2.60 0.008 2.38 0.008 5.42 0.165 0.08 0.014
2.39 0.005 2.24 0.005 5.63 0.120 0.11 0.024
21.22 14.15 0.98 1.03
*** *** NS NS
Ratio wing/thorax (male)
Pmax Pmin IP (days) b
2.57 0.007 2.39 0.008 5.79 0.146 0.12 0.038
2.46 0.106 2.15 0.088 5.41 0.965 0.03 0.009
0.99 2.59 0.37 3.66
NS * NS **
a
Pmax : upper asymptotic value (at 178C); Pmin : lower asymptotic value (at 258C); IP: position of inflection point; b=slope at inflection point. Species values are compared with a t-test. NS: non-significant, ***p50:001, **p50:01, *p50:05.
the 3 anterior segments, TSP seems to start at day 6 and end at day 9 or 10, that is from early pupa to young adults. This conclusion is enforced by considering the sum of segments 2–4 (Fig. 3) with an inflection point at day 7.63 0.29, close to adult emergence (8.18 days). In D. melanogaster females, the two experiments were homogenous but logistic adjustment provided implausible results, especially for segments 2 and 4 with inflection points after adult emergence. Pooling the 3 segments provided also a progressive smooth decrease and an inflection point at 24.9 days. Our data suggest that thermal reactivity of segments 2–4 might be different in the two species. Worth mentioning also is the fact that D. simulans females appear darker than those of D. melanogaster for segments 2–4, while the reverse was true for segments 5–7. Male data, obtained on samples of 15 individuals only, were irregular and did not reveal a sigmoid shape in any species either for single segments or for their sum (Fig. 4). Surprisingly, major pigmentation changes occurred mainly after adult emergence in D. simulans
as argued previously. Such data should be repeated on bigger samples. Interestingly, for these anterior segments, males were clearly darker than females in D. melanogaster, while both sexes were similar in D. simulans.
4. Discussion and conclusions 4.1. Experimental design and model for investigating TSP A classical determination of TSP implies the utilization of two constant developmental temperatures and a transfer of individuals from one to the other temperature at regular intervals. Such a procedure is justified, provided that the reaction norm between the two chosen temperatures is linear or at least varies monotonically. In our study, such was the case for wing length and wing/thorax ratio and also for abdomen pigmentation, but not for thorax length (see David et al., 1994). A
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Table 2 Values ( SE) of the four characteristic values calculated after a logistic adjustment on pigmentation scores of the posterior abdominal segments (Abd 5–7) and their sum in females of both speciesa Traits
Characteristic values
D. melanogaster
D. simulans
Comparison
Abd 5
Pmax Pmin IP (days) b
4.84 0.09 1.66 0.34 7.59 0.29 0.79 0.13
4.28 0.05 1.38 0.20 7.42 0.21 0.72 0.07
5.19 0.68 0.45 0.45
*** NS NS NS
Abd 6
Pmax Pmin IP (days) b
8.36 0.06 4.49 0.12 6.95 0.09 1.32 0.12
6.76 0.15 1.63 1.75 9.01 1.38 0.64 0.08
9.44 1.55 1.42 4.50
*** NS NS ***
Abd 7
Pmax Pmin IP (days) b
7.78 0.09 1.84 0.19 7.09 0.09 2.24 0.25
8.78 0.16 0.11 0.33 6.99 0.11 2.98 0.34
5.19 4.33 0.67 1.67
*** *** NS NS
Sum 5+6+7
Pmax Pmin IP (days) b
20.98 0.80 8.10 0.41 7.13 0.09 4.21 0.37
19.75 0.75 4.79 0.27 7.17 0.06 3.99 0.14
1.07 6.43 0.53 0.35
NS *** NS NS
a Pmax : upper asymptotic value (at 178C); Pmin : lower asymptotic value (at 258C); IP: position of inflection point; b=slope at inflection point. Species values are compared with a t-test. NS: non-significant, ***p50:001, **p50:01, *p50:05.
Fig. 3. Determination of the temperature sensitive period for the posterior abdomen segments (5–7) and for the sum of these segments in females. Durations of developmental stages correspond to those of D. melanogaster.
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Fig. 4. Determination of the temperature sensitive period of the 3 anterior (2–4) abdomen segments in the two species. Upper graphs: results obtained for each segment in females. Lower graphs: comparison of males and females data for the sum of the 3 segments.
precise estimate of thorax TSP should be done using higher temperatures. Experimental data may be analyzed visually or with various descriptive models. In our case, a logistic adjustment seemed appropriate since it takes into account all experimental values and describes the results by parameters with a clear biological meaning: the upper and lower phenotypic values, the position of the inflection point, which defines the middle of TSP, and the slope at IP, proportional to the duration of TSP. The logistic model has however two drawbacks. Firstly, because of the asymptotic adjustment, it is difficult to define precisely the beginning and the end of TSP. Secondly, and more seriously, the logistic curve is symmetrical around IP, while there is no biological reason for such a constraint; other sigmoid adjustments could thus be implemented. We have also found that the adjustment is quite sensitive to sampling errors. In future investigations, samples of at least 50 flies should be preferred. In spite of these limitations, our data still provided original and novel observations.
4.2. TSP and development A main conclusion of our work is that size-related traits and abdomen pigmentation do not exhibit the same TSPs. For wing length, TSP begins during larval growth and extends during the whole pupal period. TSP cannot however extend into adult life since wing and thorax length are definitely fixed during the first hour after emergence from puparium (Ashburner, 1989). Our results match what is known from anatomical description (Lawrence, 1992): wing and thorax arise from a single imaginal disk which shows a regular but slow increase during larval life and a more rapid one during the first half of pupation. The position of the IP, on average after 5.5 days at 258C, well reflects a long lasting TSP of about 6 days. For wing length at least, it is generally assumed that plasticity of overall size results mainly from a modification of cell size (el Masry and Robertson, 1978; Partridge et al., 1994). However, the fact that TSP extends during the larval life, where the imaginal disk is a massive and undifferentiated organ, suggests that cell number might also to be modified to some extend.
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Table 3 Values ( SE) of the four characteristic values calculated after a logistic adjustment on pigmentation score of anterior abdomen segments (Abd 2–4) and of their sum in femalesa Traits
Characteristic values
D. melanogaster
D. simulans
Comparison
Abd 2
Pmax Pmin IP (days) b
NC NC NC NC
2.23 0.08 1.19 0.15 6.92 0.47 0.49 0.24
NC NC NC NC
Abd 3
Pmax Pmin IP (days) b
2.71 0.16 0.66 1.47 8.38 2.23 0.35 0.10
3.52 0.07 1.99 0.17 7.94 0.25 0.72 0.28
4.42 0.86 0.19 1.22
Abd 4
Pmax Pmin IP (days) b
NC NC NC NC
3.79 0.04 1.95 0.15 7.80 0.22 0.53 0.07
NC NC NC NC
Sum 2+3+4
Pmax Pmin IP (days) b
NC NC NC NC
9.54 0.17 5.15 0.46 7.63 0.29 1.55 0.34
NC NC NC NC
*** NS NS NS
a Pmax : upper asymptotic value (at 178C); Pmin : lower asymptotic value (at 258C); IP: position of inflection point; b=slope at inflection point. Species values are compared with a t-test. NC: not calculated, NS: non-significant, ***p50:001, **p50:01, *p50:05.
For female abdomen pigmentation (last segments, Fig. 3), the TSP occurs later than for wing length and the IP is observed on average 7.2 days after egg laying. The development of adult abdomen tegument arises from clusters of embryonic cells (called histocysts) included into the larval ectoderm (Lawrence, 1992; Fristrom and Fristrom, 1993). Proliferation of these cells starts in early pupa, after the beginning of larval ectoderm histolysis, and ends during the second half of pupation, when a complete adult ectoderm is produced. The differentiation of adult segments, however, and especially the production of cuticule and microchaetae, extends up to the end of pupation. The genes, which control the spatial patterning of the tergites are progressively identified, some of which act also on the extension of the black pigment at the posterior margin of each tergite (Kopp and Duncan, 1997; Kopp et al., 1997). From all these observations it is not surprising to find that TSP begins after puparium formation. What was not expected is that TSP extends during the first day of imaginal life. This is however a plausible observation since, at emergence, Drosophila adults are not pigmented and the cuticule undergoes a progressive maturation resulting in both hardening and appearance of the pigmentation pattern. At least one day is necessary for completion of these processes. Another observation concerns the difference in plasticity between posterior segments in females although TSPs seem identical.
4.3. Sex and species comparison For size-related traits, and especially wing length, we did not find clear differences for TSPs between sexes or species (Fig. 2). The peculiar curve observed in D. simulans males more likely reflects sampling errors than a sex dimorphism. Similarities between sexes and sibling species are expected if we consider that the wing– thorax system, which is involved in the flight function, is likely to be submitted to a strong developmental canalization. For abdomen pigmentation, some differences were evidenced among females of the two species, either for the overall plasticity (Abd 7, Fig. 3) or for average pigmentation (Fig. 3). In this respect, D. melanogaster females are darker than D. simulans females for the 3 posterior segments (Fig. 3), while the reverse is true for the 3 anterior ones (Fig. 4). The shapes of the response curves and TSPs might also be different between anterior and posterior segments, but this point deserves more extensive investigations. Pigmentation differences among sexes could be analyzed only for the 3 anterior segments, since segments 5 and 6 of males are completely black. As seen in Fig. 4, D. melanogaster males were darker than females, while both sexes were almost identical in D. simulans. Moreover, our data suggest that, for the three anterior segments, TSP might extend up to day 10, that is during the first two days of adult life. It was
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recently shown (Kopp et al., 2001) that the genetic basis of pigmentation in the posterior abdominal segments was completely different between males and females. 4.4. Ecological phenotypes under changing conditions As stated in Section 1, a natural shift from one temperature (e.g. warm days) to another one (e.g. cold days) will change the phenotypes if the shift occurs mainly during pupation. So we may predict that a thermal change during embryonic development or the two first larval instars should not modify the adult phenotype. If the change occurs during TSPs, the two kinds of traits will vary in a parallel way: both size and pigmentation will decrease at a higher temperature, inducing a positive correlation in a population. The TSPs are not however identical, occurring earlier in development for size than for pigmentation. For example, a cold treatment at the time of adult emergence will not modify the size but will produce darker flies. In other words, temperature changes during development might be detected by analyzing the phenotypic correlations between different traits. Indeed, we have some preliminary observations in field collected flies (unpublished results) showing a positive correlation between body size and female abdomen pigmentation. Such a correlation does not exist in a laboratory population grown at a constant temperature.
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