Genetica 114: 195–205, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

195

Male sterility thermal thresholds in Drosophila: D. simulans appears morecold-adapted than its sibling D. melanogaster

Mohamed Chakir1!3, Abdelaziz Chafik2, Brigitte Moreteau3, Patricia Gibert3 & Jean R. David3,"1Universite Cadi Ayyad, Faculte des Sciences et Techniques, BP 618 Marrakech, Maroc; 2Universite ChouaibDoukkali, Faculte des Sciences, El Jadida, Maroc; 3Lab. Populations, Genetique, Evolution, CNRS, Avenue de laTerrasse, 91198 Gif sur Yvette cedex, France; "Author for correspondence (Phone: 33 1 69 82 37 13; Fax: 33 1 6907 04 21; E-mail: david@pge.cnrs-gif.fr)

Received and accepted 8 January 2002

Key words: cold stress, heat stress, male fertility, spermatogenesis, thermal adaptation

Abstract

Numerous different criteria may be used for analysing species thermal adaptation. We compared male sterilitythresholds in the two most investigated cosmopolitan siblings, D. melanogaster and D. simulans. A survey ofvarious populations from Europe and North Africa evidenced consistent differences between the two species, anda detailed analysis was made on flies from Marrakech. Sharp sterility thresholds were observed in both species butat different temperatures: D. simulans appeared more tolerant to cold than its sibling (difference 1#C) but moresensitive to heat (difference 1.5#C). When transferred to an optimum temperature of 21#C, D. simulans males,sterilized by a low temperature, recovered more rapidly than males of D. melanogaster; the reverse was true onthe high temperature side. The analysis of progeny number also revealed the better tolerance of D. simulans malesto cold but a lesser tolerance to heat. From these observations, we might expect that D. simulans should be moresuccessful in cold temperate countries than its sibling, while ecological observations point to the contrary. Our dataclearly show the difficulty of comparing ecophysiological data to field observations, and also the need of extensivecomparative life history studies in closely related species.

Introduction

The two cosmopolitan siblings Drosophila melano-gaster and D. simulans are often considered as aparadigm both for speciation and ecological investi-gations. The two species, included in the D. melano-gaster subgroup, are native to tropical Africa but theyhave been able to spread over the world thanks to ar-tificial, man-linked transportations (David & Tsacas,1981; Parsons & Stanley, 1981; Parsons, 1983;Lachaise et al., 1986; Lemeunier et al., 1986; David& Capy, 1988). A specially interesting feature of thetwo species is that they are able to proliferate notonly in tropical places but also in temperate countrieswhere overwintering is a major ecological challenge(Izquierdo, 1991; David et al., 1983a; Parsons, 1983).The role of ambient temperature as a strong selectivefactor is also suggested by the fact that both species

exhibit latitudinal clines (Parsons, 1983; Lemeunieret al., 1986; Capy, Pla & David, 1993). The magnitudeof latitudinal genetic variation seems, however, lesspronounced in D. simulans, either for genetic mark-ers (Hyytia et al., 1985; Singh, Choudhary & David,1987) or for several morphometrical traits (Capy, Pla& David, 1993). This might suggest that D. simulansis less adapted to cold temperate climates and has keptmore strongly its native physiological characteristicsof a tropical species.

Ecological observations confirm this hypothesis.At the level of Europe, it is well known (Louis, 1983)that D. simulans is a dominant species around theMediterranean sea, and that its relative abundance,compared to D. melanogaster, regularly decreasestoward northern countries. In France, D. simulans ap-pears every year in the North, but quite later in theseason (David et al., 1984) as if such populations

196

were founded by migrants coming in summer fromthe south. A similar situation, that is prevalence of D.simulans in warm temperate and subtropical places, isknown in other continents, such as Australia, Northand South America (McKenzie & Parsons, 1974;Parsons, 1975, 1983; David et al., 1983b, 1984).

Numerous physiological traits, often geneticallyindependent, can be compared among species in or-der to define their thermal ecological niches (Prechtet al., 1973 ; Leather, Walters & Bale, 1993). Not sur-prisingly, many comparative investigations have beenmade on D. melanogaster and D. simulans, and theprevalent overall conclusion (Parsons, 1975; Parsons& Stanley, 1981; Parsons, 1983; Hoffmann & Parsons,1991) is that D. melanogaster is more tolerant to ex-treme stressful conditions, either heat or cold, whileD. simulans does better under more stable, milderenvironments.

In D. melanogaster, a complete development fromegg to adult is possible at constant temperatures rang-ing between 11 and 32#C. Successive generations,however, are obtained only between 13 and 29#C, be-cause males are sterile when grown either at 30#C(David, Arens & Cohet, 1971) or at 12#C (Cohet,1973). We hypothesised that these thermal sterilitythresholds could be useful criteria for comparing thethermal adaptation of different species. In the presentwork, we compared the fertility of the two siblingspecies grown at extreme, low or high, temperatures.We found that D. simulans males were sterile at 28#C,but still fertile at 12#C. In other words, the sterilitythresholds are always less in D. simulans than in D.melanogaster. Such characteristics are expected froma cold adapted species, and this unexpected conclusionis discussed and compared with extant knowledge onthe two species.

Material and methods

Drosophila strains

Wild living flies were collected in 1999 in Marrakecharea (Morocco). Females were assigned either to D.melanogaster or D. simulans by phenotypic examina-tion (see Moreteau et al. 1995); then isofemale lineswere established and the specific status confirmed byinspection of male progeny genitalia. Thirty isofemalelines of each species were pooled to make a mass pop-ulation, which was then kept in bottles at 20#C undera LD 16-8 photoperiod. Adults of these strains wereused as parents of the experimental flies. A detailed

analysis was made on these Moroccan strains and ispresented below. Other sympatric populations of thetwo species were also compared in order to confirm thesterility thresholds evidenced for the Moroccan flies.These populations, kept as mass laboratory cultureswere collected in France (Bordeaux, Lorient and Vil-leurbanne), in Italy (Capri) and in Spain (Cordoba).Sterility tests were made after less than a year oflaboratory culture, except for Cordoba (5 years).

Experimental flies

Groups of 10 pairs were established after light ether-isation and used as parents. Egg laying occurred invials on a killed yeast, high nutrient food whichprevents crowding effects (see Karan et al., 1999).Oviposition was limited to a few hours and popula-tion density in each vial was about 100 larvae. Afterremoval of the parents, these vials were immediatelytransferred to incubators at various experimental tem-peratures. The precision of the temperature controlwas ± 0.1#C. Such a high precision is achieved byusing small size incubators (about 225 litres) witha permanent ventilation. These incubators are them-selves kept in controlled rooms and thermal regulationof each incubator is implemented only by a heat-ing system. Low temperature incubators were keptin a cold room (6#C), while high temperature in-cubators were kept in a room at 20#C. Finally, aprecision thermometer (0.1#C) was put in each incub-ator close to the rearing vials, and the temperaturechecked regularly. After emergence, the adults weretransferred to fresh food and either kept at the sametemperature or transferred to another one, as explainedbelow.

Male fertility assays: presence of mobile sperm

Samples of males were kept at the same temperatureused for their development, and then dissected forchecking the presence of mobile sperm in the seminalvesicles. For males grown at high temperatures, theage at dissection was generally 4–6 days. Such a delayfavours the accumulation of sperm in seminal vesiclesand facilitates observation. Dissections were made inDrosophila saline using minutia pins under a binocularmicroscope. Seminal vesicles were opened with thepins and the presence of sperm observed directly asa qualitative observation. For critical growth temper-atures (e.g., 29.5#C for D. melanogaster), the amountof mobile sperm among males classified as fertile wasclearly variable. We did not try, however, to quantify

197

this variability. A similar procedure was used with thelow temperature grown males. Since maturation timeis much lower at such temperatures, the males wereaged for at least 2 weeks before dissection. Again,quantitative differences were noticed among fertilemales. For each growth temperature, we always usedmales grown from at least three different culture vials.

Progeny production after transfer at middletemperature

Young males were transferred after emergence at thepermissive temperature of 21#C, and mated each withthree virgin females of their own species. These fe-males had been grown at 21#C, which is close tothe physiological optimum of the two species (Pétavyet al., 2001) and were aged 3–4 days at the beginningof the experiment. Each group of one male and threefemales was transferred daily to a new vial (killedyeast food). A male was considered as fertile whenat least one larva was observed in the vial. In theseexperiments, the number of progeny was also countedand provided an information on fertility level of eachmale.

Results

Fertility as a function of growth temperature

For the two species we verified that males, grown atmiddle temperatures, 21 and 25#C, were all fertile,

Figure 1. Variation of the percentage of fertile males according to growth temperature in the two sibling species. (A) Effects of low tempera-tures; (B) Effects of high temperatures. Male age at dissection was 5–6 days for high temperatures and at least 2 weeks for low temperatures.Sample size was 50 in all cases.

as indicated by the presence of mobile sperm intheir seminal vesicles (sample size 50 males for eachspecies).

Fertility at low temperatures was analysed bygrowing the two species between 11 and 14#C, witha thermal interval of 0.5#C (Figure 1(A)). Sterilitywas complete at 11#C in D. simulans and at 12#C inD. melanogaster. Then a progressive increase in theproportion of fertile males was observed at higher tem-peratures, with a proportion close to 100% at 13.5#Cin D. simulans and 14#C in D. melanogaster. As seenFigure 1(A), the two species produced regularly in-creasing sigmoid curves, analogous to dose-responsecurves in toxicity studies. We thus used the classicalmethod of log-probit transformation for estimatingthe temperature producing 50% of sterile male (malesterility temperature 50 or MST50) (Table 1): valueswere 12.25 and 13.21#C for D. simulans and D.melanogaster, respectively. We also used anothermethod, adjusting the data to a logistic function(Gibert et al., 1998) as shown Figure 2. With thismethod the MST50 is given by the position of theinflection point (Table 1). The goodness of fit canbe appreciated by the fact that the upper asymptotesare close to 100% and the lower asymptotes close to0%. More interestingly, the method allows to calculatethe slope at the inflection point. We see that MST50sat low temperatures are almost identical to the val-ues calculated with the log-probit transformation. Theslopes are similar in the two species (on average 75),

198

Table 1. Calculation of male sterility temperature 50 (MST50)

Low temperature High temperaturesimulans melanogaster simulans melanogaster

Log probit adjustmentMST50 (#C) 12.25 13.21 27.62 29.13CI 12.14–12.36 13.10–13.32 27.54–27.70 29.06–29.21

Logistic adjustmentUA 99.18 ± 2.46 102.81 ± 3.63 97.05 ± 0.17 99.72 ± 0.32LA !1.72 ± 4.61 !1.10 ± 2.43 !0.07 ± 0.07 !0.27 ± 0.71TIP (#C) 12.21 ± 0.06 13.25 ± 0.05 27.65 ± 0.00 29.14 ± 0.01SIP 78.38 ± 13.73 72.70 ± 8.82 !168.03 ± 10.78 !186.97 ± 7.78

Comparison of data obtained with a log probit adjustment and a logistic adjustment.CI: confidence interval; UA: upper asymptote; LA: lower asymptote; TIP: temperature at inflection point; SIP: slopeat inflection point.

Figure 2. Example of logistic adjustments of male fertility data at low temperatures in the two sibling species. The 50% line shows the positionof the inflection point.

meaning that, at the MST50 level, an increase of0.1#C would increase the proportion of fertile malesby 7.5%.

Similar investigations were done on the high tem-peratures side, between 27 and 31#C, with also a pro-gression of 0.5#C (Figure 1(B)). Data (Table 1) werealso very similar with the two adjustment techniques.MST50s were 27.6#C in D. simulans and 29.1#C in D.melanogaster, that is a difference of 1.5#C. The slopesat inflection points were still much steeper than at lowtemperatures (average 177). The sterility threshold athigh temperatures is, in both species, narrower than atlow temperatures.

Recovery of male fertility after transfer to middletemperature

As indicated in Methods section, young males (1-dayold) were mated with three virgin females, put at 21#C

and transferred daily to fresh medium. Males wereconsidered as fertile when larvae were observed invials.

On the low temperature side, four developmentaltemperatures were used for each species, namely 11,12, 13 and 14#C. Recovery curves are shown Fig-ure 3. Most males grown at 14#C were fertile fromthe beginning of the experiment in the two species. Aslight increase in the proportion of fertile individualswas observed over time in D. melanogaster. A similarobservation was made after a development at 13#C,and the initial proportion of fertile males was about50% in the two species. After a development at 12 or11#C, all D. melanogaster males were sterile at thebeginning. About 70% of the 12#C grown flies re-covered, but only 30% of those grown at 11#C, after15 days at 21#C. In D. simulans, higher proportionsrecovered: 90% after a development at 12#C and 80%after a development at 11#C.

199

Figure 3. Increase in the proportion of fertile males after a return at the optimum temperature of 21#C in the two sibling species. Upper graphs:low developmental temperatures; lower graphs: high developmental temperatures; growth temperature is shown for each curve; sample sizesare given in Table 2.

The difference between the two species can beanalysed not only by the proportion of males re-covering after 2 weeks, but also by the kineticsof recovery. For this we calculated a mean reco-very time, using for the calculation only the indi-viduals which effectively produced progeny duringthe experiment. Results given in Table 2 reveal twomain features. First, and in both species, the timeneeded for recovering fertility increased at extremetemperatures, either low or high, and sometimesoverpassed 10 days. Second, significant differenceswere observed between species, but in opposite di-rections. At low growth temperatures, D. simulansrecovered faster than D. melanogaster, a confirma-tion of its higher cold hardiness. At high temper-atures, the reverse was true: D. melanogaster didbetter.

Progeny number in males transferred at 21#C

Males recovering fertility produced different offspringnumbers, and we hypothesised that this number mightbe proportional to the number of viable sperm. Re-sults (Figure 4) revealed a general tendency for anincrease of progeny number over time, but even inthe control flies grown and kept at 21#C. Since, toour knowledge, this kind of experiment is originalfor analysing male reproductive capacity, we will firstconsider, with some details, the results of controlflies.

Individual curves are shown Figure 5. In the twospecies, a regular linear increasing trend is observedfor each male, and the progeny number is less in D.simulans than in D. melanogaster. Data, submitted toANOVA (not shown) revealed a highly significant age

200

Table 2. Mean recovery time to fertility of males grown at various temperatures and transferred after emergence at 21#C withunexposed females

Growth D. simulans D. melanogaster Differencetemperature N n M ± s.e. N n M ± s.e. sim-mel(#C)

11 30 24 7.46 ± 0.75 32 10 13.30 ± 0.37 !5.84 ± 1.20"""

12 30 27 4.74 ± 0.73 30 24 9.13 ± 0.51 !4.39 ± 0.91"""

13 15 15 2.87 ± 0.90 14 14 4.11 ± 1.07 !1.24 ± 1.39 ns14 15 15 1.33 ± 0.33 15 15 2.20 ± 0.78 !0.87 ± 0.85 ns21 15 15 1.13 ± 0.13 10 10 1 ± 00 +0.13 ± 0.16 ns28 15 13 7.00 ± 0.52 – – – –29 15 12 11.25 ± 0.37 14 13 2.77 ± 0.61 8.48 ± 0.73"""

30 15 6 11.00 ± 0.26 10 8 8.75 ± 0.41 2.25 ± 0.53"""

31 – – – 10 7 9.14 ± 0.34 –

N : total number investigated; n: number of males which recovered fertility and which were used for calculating the recovery time.M: mean in days; s.e.: standard error.Significance of differences (t-test): "p < 0.5; ""p < 0.01; """p < 0.001, ns: non significant.

Figure 4. Variation of progeny number from males grown at various experimental temperatures (indicated for each curve) and transferredto 21#C with unexposed females. Upper graphs: results for low developmental temperatures; lower graphs: results for high developmentaltemperatures; vertical bars indicate the standard errors.

201

Figure 5. Number of daily progeny produced by individual males, grown and kept at 21#C. (A) Results for 10 males of D. melanogaster.(B) Results for 15 males of D. simulans.

effect in both cases. We tried to compare more pre-cisely the two species by assuming a linear increaseof progeny number over time. A linear regressionwas calculated for each male and the coefficients av-eraged (Table 3). Intercept values, corresponding tothe fertility at the beginning of life, are very differentbetween the two species, with a clear superiority ofD. melanogaster (60.3 ± 3.1 vs. 40.4 ± 3.6). A secondinteresting observation concerns a major difference inslopes, with again a superiority in D. melanogaster.More precisely the number of progeny increased by 5per day in D. simulans and more than 10 in D. melano-gaster. The durations of experiments were not thesame in the two species, that is, 15 days in D. simulansbut only 10 in D. melanogaster. As seen Figure 5,there was no tendency for a stabilisation of the pro-geny number in ageing males. Especially there was

still a significant increase in D. simulans between day10 (mean daily progeny number 88.7 ± 4.7) and day15 (mean number 124.5 ± 7.0).

Males grown at low temperatures (11, 12, 13 and14#C) also showed an increasing progeny number overtime when transferred to 21#C (Figure 4 and Table 3).Negative intercepts indicate that several days were ne-cessary before the start of fertility. The slopes, whichmay be proportional to the speed of recovery, weresuperior in D. simulans at 11#C, identical in the twospecies at 12 and 13#C, superior in D. melanogaster at14#C (but also at 21#C).

On the high temperatures side, negative interceptswere also observed when a delay was necessary forrecovery. Slopes were higher in D. melanogaster thanin D. simulans, and decreased in each species withincreasing growth temperatures.

202

Table 3. Quantitative analysis of progeny number at 21#C for various developmental temperatures of males (data ofFigure 4)

Growth Coefficients D. simulans D. melanogaster Comparisontemperature n m ± s.e. CV n m ± s.e. CV (t)(#C)

21 a 15 40.43 ± 3.56 34.1 10 60.26 ± 3.11 16.3 3.92"""

b 5.19 ± 0.29 22.0 10.50 ± 0.65 19.5 8.35"""

11 a 24 !68.63 ± 31.02 230 10 !122.13 ± 64.15 157 0.82 nsb 8.93 ± 2.07 118 10.31 ± 4.29 125 0.32 ns

12 a 27 !2.23 ± 5.87 1368 24 !44.35 ± 8.07 91 4.20"""

b 4.14 ± 0.44 56 6.58 ± 0.89 68 2.49""

13 a 15 3.79 ± 5.99 611 14 !8.75 ± 1.31 310 1.91"

b 5.35 ± 0.56 41 1.77 ± 0.28 27 5.40"""

14 a 15 32.18 ± 3.17 38 15 10.79 ± 15.67 562 1.29 nsb 4.24 ± 0.18 16 6.94 ± 0.82 46 3.11""

28 a 13 !59.96 ± 10.92 66 – –b 12.09 ± 1.01 30

29 a 12 !59.17 ± 5.50 32 13 14.26 ± 7.01 177 7.82"""

b 7.60 ± 0.57 26 8.83 ± 0.92 38 1.07 ns30 a 6 !82.93 ± 10.37 31 8 !151.96 ± 22.78 40 2.30 ns

b 8.65 ± 1.00 28 20.67 ± 2.47 32 3.73""

31 a – – – 7 !102.37 ± 11.79 28 –b 13.12 ± 1.14 26

A linear regression was calculated for each recovering male. a: intercept; b slope; n: number of males; m ± s.e.:mean ± standard error; CV: coefficient of variation.The two species are compared with a student’s t test.Significance: "p < 0.5.; ""p < 0.01; """p < 0.001. ns: non significant.

Comparative data with European strains

The five European populations of each species, indi-cated in the Methods section, were grown at variouscritical temperatures and investigated for their ca-pacity to produce F1 progeny. A complete lack ofprogeny from a mass culture was considered as evi-dence of a complete male sterility. We verified that thefemales always produced viable eggs when mated tonormal males.

On the high temperature side, all the D. melano-gaster strains failed to produce any progeny whengrown and kept at 30#C. Successive generations wereeasily obtained at 29#C, and progeny, although some-times in small number, were also obtained at 29.5#C.For D. simulans, the complete sterility thresholdwas 28#C. Permanent cultures could be easily es-tablished at 27#C and, with more difficulty, at27.5#C.

On the low temperature side, D. melanogaster cul-tures were completely sterile when grown at 12#C,while permanent cultures of D. simulans were easily

established at that temperature. Permanent cultures ofD. melanogaster could be kept at 13#C. All these ob-servations, made on five European strains, also applyto the strains from Marrakech.

Discussion and conclusions

A comparison of various populations of D. melano-gaster and D. simulans from Europe and North Africahas revealed consistent differences between the twospecies: D. melanogaster cultures do not produce pro-geny when grown at 30#C, in agreement with previousdata (David et al., 1983a). D. simulans also exhibits asterility threshold, but at a lower temperature of 28#C.At low temperatures, a reverse situation is observed:D. melanogaster is completely sterile at 12#C, whileD. simulans can be grown permanently at that temper-ature. The detailed functional analysis, which has beendone on the Marrakech populations, is certainly validfor other temperate populations of the two species, atleast in the European region.

203

On the Marrakech populations, we used three dif-ferent criteria for a deeper analysis of male sterilityat high and low temperature: the percentage of malesshowing mobile sperm when reared and kept at an ex-treme temperature; the proportion of males recoveringfertility when transferred to a permissive middle tem-perature and mated to unexposed females; the numberof progeny produced by such males under permissiveconditions.

Slight differences could be observed between dif-ferent kinds of experiments. For example, D. melano-gaster males grown at 29#C were considered as 75%fertile by dissection (Figure 1(B)) while the fertilitypercent was only 50% when progeny production wasanalysed at 21#C (Figure 3). The most likely interpre-tation is that the difference is due to male age: youngmales may be sterile after emergence but become fer-tile after a few days even when kept at the stressfultemperature used for their development. We thus maysuggest that our three criteria have provided a coherentinformation: compared to its sibling, D. simulans ismore tolerant to cold (1#C difference) but more sensi-tive to heat (about 1.5#C difference). Interestingly, thesterility thresholds are extremely narrow in the twospecies, and their breadth can be appreciated by theslopes at the inflection points (Table 1). At low tem-peratures, an increase of 2#C is sufficient to pass froma complete sterility to an almost complete fertility.At high temperatures, the threshold is still narrower,since one degree increase is sufficient for inducing acomplete sterility.

D. simulans males recovered more quickly from acold sterilizing stress than D. melanogaster, but moreslowly from heat sterility. From a practical point ofview, we have found that it was easily possible to keepstrains of D. simulans at 12#C for successive gener-ations, while the same was possible for D. melano-gaster at 13#C only. On the high temperature side, D.melanogaster is easily kept at 29#C but not at 30#C,while D. simulans can be reared over successive gen-erations at 27#C but not at 28#C. This observation hasalso been made on several European populations of thetwo species and reflects a stable divergence betweenspecies. We have some evidence that the male steril-ity thermal thresholds are stable characteristics whichdo not change over time in laboratory culture and arealso difficult to change by selection. For example, wetried to increase, in D. melanogaster (Bordeaux strain)the sterility threshold by directional selection. Cul-tures were grown at the high sterilizing temperatureof 31#C. The emerged adults were transferred at 20#C

for recovery of male fertility and then used to producethe next generation at 31#C. After 20 generations ofsuch selection the threshold of complete male sterilitywas still 30#C.

The recovery time presumably corresponds to thenovel production of viable sperm (David, Arens &Cohet, 1971). The long delay, which may exceed10 days (Table 2) suggests that, at least for extremetemperatures, spermatogenesis has to start again frommale germ cells. Of course, cytological investigationsof this process would be needed. Males recoveringfertility have regularly shown a progressive increasein their progeny number over time, and a likely inter-pretation is that it reflects an increase in the numberof viable sperm. We consider this interpretation to bebasically valid, that is, that progeny number is a meansfor estimating the number of sperm. Surprisingly,however, we found a similar phenomenon for controlmales, and this observation deserves discussion. Weused, in all cases, three virgin females in excellentphysiological condition and aged 3–4 days. It has beenknown for a long time that, at least in D. melano-gaster, egg laying curves have a triangular shape, witha maximum on day 4 and then a slow linear decreaseup to the end of the fertile period (David, Biémont &Fouillet, 1974). In other words, females aged between4 and 15 days have a maximum egg production, whichis close to two eggs per ovariole and per day (David,1970).

In Moroccan populations of D. melanogaster,ovariole number lies between 40–45 (Capy, Pla &David, 1993), so that three females at their maximumegg production should produce about 240 eggs a day.It is also known that, when experimental sex-ratio is1, egg hatching always overpasses 90% and larvalviability is also above 90% (David & Croissant, 1956).In other words, three females could produce a max-imum of 200 offspring a day. As seen Figure 5(A),our data were much less, especially with young males.Only after 10 days did the progeny number (160 perday) approached the maximum expectation. Also, anunexpected observation was the linear increase (10per day) in progeny number over time. This mayresult from sperm storage in female and regular re-mating, and presumably reflects the total sperm num-ber produced by the male. Further investigations are,however, needed for a better understanding of such aphenomenon.

The technique revealed major difference with D.simulans (Figure 5(B)). The overall lesser progenynumber in that species may be due to its lower ovariole

204

number (around 37) (Capy, Pla & David, 1993). Butthe lesser rate of increase in progeny number over time(5 against 10 in D. melanogaster) is something differ-ent: a possible interpretation is that sperm productionis much less in D. simulans.

The fact that, with respect to male sterility thermalthreshold, D. simulans may be described as more coldtolerant (and heat sensitive) than its sibling, comes asa surprise especially if we consider the ecology andabundance of the two species (see Introduction). Fromour physiological observations, we might expect thatD. simulans would overwinter more easily than its sib-ling in temperate countries (Izquierdo, 1991). In factthat is true but only in places with a mild winter, suchas Mediterranean countries. D. melanogaster is, how-ever, more abundant at higher latitudes, with a colderwinter. The likely interpretation of this paradox is thatD. melanogaster overwinters in human constructions,thanks to its domestic status. D. simulans, on the otherhand, exhibits a specific behaviour, avoids to enterbuildings (Rouault & David, 1982), remains outdoorand is more directly submitted to ambient temperature.

If we consider other physiological traits in the twospecies, some contradictory observations are worthmentioning. For several traits, D. simulans appears asa cold adapted species: viability is less at high temper-atures (Parsons, 1983; Pétavy et al., 2001) maximumbody size, due to phenotypic plasticity is observed ata lower temperature than in D. melanogaster (Morinet al., 1996). There are, however, other traits whichpresent D. simulans as more adapted to a warm envir-onment. One is wing loading. Wing loading is a plastictrait, related to wing beat frequency and flight capa-city, and which decreases regularly with increasinggrowth temperature (Pétavy et al., 1997). Also, likebody size, wing loading seems to increase with lat-itude in D. melanogaster (Azevedo et al., 1998). Thesame trend is observed in D. simulans (Morin et al.,1999) and D. kikkawai (Karan et al., 1998). Such in-direct evidences suggests that wing loading should behigher in warm adapted species. But, in this respect,D. simulans is remarkable by a higher wing loading(Pétavy et al., 1997; Morin et al., 1999) than in D.melanogaster. Another difference concerns the effectsof cold treatment, which can be estimated either byconsidering survival time or chill coma recovery. Sur-vival time at !1#C is significantly less in D. simulansthan in D. melanogaster (Parsons, 1983). It was re-cently shown that, after a treatment at 0#C, adult fliesrecover progressively from chill coma (David et al.,1998). Convincing evidence exists that the speed of

recovery is a measure of cold adaptation, with a fastrecovery in temperate species and a slow recovery intropical species (Gibert et al., 2001). With that cri-terion, however, D. simulans appears slightly moresensitive to cold than D. melanogaster (Gibert et al.,2001).

In conclusion, the comparison of the two cos-mopolitan sibling species is progressively unravellingnumerous functional differences, which presumablyinvolve a divergence in the responsible genetic sys-tems. For the moment, empirical observations on theirlife histories are difficult to integrate into a simple andgeneral evolutionary interpretation and more extensivecomparative investigations are still needed. We wouldalso like to point out the interest of investigating malesterility thresholds in more numerous species and re-lating the data to geographic distribution. Finally, intemperate countries, sterile male phenotypes are likelyto be found in natural populations either in summerdue to heat, or in winter due to cold. Looking forsuch phenotypes should help understand the speciesabundance and their population dynamics.

Acknowledgements

This work benefited from a cooperative agreementbetween France (CNRS) and Morocco (CNCPRST).

References

Azevedo, R.B.R., A.C.C. James, J. McCabe & L. Partridge,1998. Latitudinal variation of wing: thorax size ratio and wing-aspect ratio in Drosophila melanogaster. Evolution 52: 1353–1362.

Capy, P., E. Pla & J.R. David, 1993. Phenotypic and genetic variabil-ity of morphometrical traits in natural populations of Drosophilamelanogaster and D. simulans. I. Geographic variations. Genet.Sel. Evol. 25: 517–536.

Cohet, Y., 1973. Stérilité mâle provoquée par une basse températurede développement chez Drosophila melanogaster. C. R. Acad.Sci. Paris 276: 3343–3345.

David, J. & J. Croissant, 1956. Recherche sur la fécondité et la fer-tilité chez Drosophila melanogaster Meigen. Annls. Univ. Lyon9: 73–128.

David, J., 1970. Le nombre d’ovarioles chez Drosophila melano-gaster: relation avec la fécondité et valeur adaptative. Arch. Zool.Exp. Gen. 111: 357–370.

David, J., M.F. Arens & Y. Cohet, 1971. Stérilité mâle à haute tem-pérature chez Drosophila melanogaster: nature, progressivité,reversibilité des effets de la chaleur. C. R. Acad. Sci. Paris 272:1007–1010.

David, J., C. Biémont & P. Fouillet, 1974. Sur la forme des courbesde ponte chez Drosophila melanogaster et leur ajustementà des modèles mathématiques. Arch. Zool. Exp. Gen. 115:263–277.

205

David, J.R. & L. Tsacas, 1981. Cosmopolitan subcosmopolitan andwidespread species: different strategies within the Drosophilidfamily (Diptera). C. R. Soc. Biogeog. 57: 11–26.

David, J.R., R. Allemand, J. Van Herrewege & Y. Cohet, 1983a.Ecophysiology: abiotic factors, pp. 105–170 in The Genetics andBiology of Drosophila, Vol. d, edited by M. Ashburner, H.L.Carson & J.N. Thompson. Academic Press, London.

David, J.R., Y. Carton, Y. Cohet, D. Lachaise, J. Louis, J. Rouault,L. Tsacas & J. Voudibio, 1983b. Influence des activités humainessur la structure de certains peuplements de drosophilides. ActaOecol., Oecol. Gener. 4: 185–200.

David, J.R., J. Louis, J.A., McKenzie, M.T. Rocha-Pite & J. Voudi-bio, 1984. Comparative demography of the cosmopolitan siblingspecies, Drosophila melanogaster and D. simulans, under tem-perate and tropical climate. Ann. Soc. Entomol. Fr. (N.S.) 20:135–142.

David, J.R. & P. Capy, 1988. Genetic variation of Drosophilamelanogaster natural populations. Trends Genet. 4: 106–111.

David, J.R., P. Gibert, E. Pla, G. Pétavy, D. Karan & B. Moreteau,1998. Cold stress tolerance in Drosophila: analysis of chill comarecovery in D. melanogaster. J. Therm. Biol. 23: 291–299.

Gibert, P., B. Moreteau, J.R. David & S.M. Scheiner, 1998. Describ-ing the evolution of reaction norm shape: body pigmentation inDrosophila. Evolution 52: 1501–1506.

Gibert, P., B. Moreteau, E. Pla, G. Pétavy, D. Karan & J.R. David,2001. Chill coma recovery: a major adaptive difference betweentemperate and tropical Drosophila species. Evolution 55: 1063–1068.

Hoffman, A.A. & P.A. Parsons, 1991. Evolutionary Genetics andEnvironmental Stress. Oxford University Press, Oxford.

Hyytia, P., P. Capy, J.R. David & R. Singh, 1985. Enzymatic andquantitative variation in European and African populations ofDrosophila simulans. Heredity 54: 209–217.

Izquierdo, J.I., 1991. How does Drosophila melanogaster over-winter? Entomol. Exp. Appl. 59: 51–58.

Karan, D., A.K. Munjal, P. Gibert, B. Moreteau, R. Parkash & J.R.David, 1998. Latitudinal clines for morphometrical traits in Dro-sophila kikkawai: a study of natural populations from the Indiansubcontinent. Genet. Res. 69: 31–38.

Karan, D., J.P. Morin, E. Gravot, B. Moreteau & J.R. David, 1999.Body size reaction norms in Drosophila melanogaster: temporalstability and genetic architecture in a natural population. Genet.Sel. Evol. 31: 491–508.

Lachaise, D., J.R. David, F. Lemeunier, L. Tsacas & M. Ashburner,1986. The reproductive relationships of Drosophila sechelliawith D. mauritiana, D. simulans and D. melanogaster from theAfrotropical region. Evolution 40: 262–271.

Leather, S.R., K.F.A. Walters & J.S. Bale, 1993. The Ecology ofInsect Overwintering. Cambridge University Press.

Lemeunier, F., J.R. David, L. Tsacas & M. Ashburner, 1986. The D.melanogaster species group, pp. 147–256 in The Genetics andBiology of Drosophila, Vol. e, edited by M. Ashburner, H.L.Carson & J.N. Thompson. Academic Press, London.

Louis, J., 1983. Les espèces dominantes de Drosophiles dans lespeuplements de l’Europe atlantique et méditerranéenne. Ann.Soc. Entomol. Fr. (N.S.) 19: 167–173.

McKenzie, J.A. & P.A. Parsons, 1974. Numerical changes andenvironmental utilization in natural populations of Drosophila.Aust. J. Zool. 22: 175–187.

Moreteau, B., G. Pétavy, P. Gibert, J.P. Morin, A. Munoz &J.R. David, 1995. New discriminating traits between femalesof two sibling species: Drosophila melanogaster and D. simu-lans (Diptera: Drosophilidae). Ann. Soc. Entomol. Fr. (N.S.) 31:249–257.

Morin, J.P., B. Moreteau, G. Pétavy, A. Imasheva & J.R. David,1996. Body size and developmental temperature in Drosophilasimulans: comparison of reaction norms with sympatric Droso-phila melanogaster. Genet. Sel. Evol. 28: 415–436.

Morin, J.P., B. Moreteau, G. Pétavy & J.R. David, 1999. Divergenceof reaction norms of size characters between tropical and temper-ate populations of Drosophila melanogaster and D. simulans. J.Evol. Biol. 12: 329–339.

Parsons, P.A., 1975. The comparative evolutionary biology ofthe sibling species, Drosophila melanogaster and Drosophilasimulans. Quart. Rev. Biol. 50: 151–169.

Parsons, P.A. & S.M. Stanley, 1981. Domesticated and widespreadspecies, pp. 349–393 in The Genetics and Biology of Drosophila,Vol. a, edited by M. Ashburner, H.L. Carson & J.N. Thompson.Academic Press, London.

Parsons, P.A., 1983. The Evolutionary Biology of ColonizingSpecies. Cambridge University Press, Cambridge.

Pétavy, G., J.P. Morin, B. Moreteau, B. & J.R. David, 1997. Growthtemperature and phenotypic plasticity in two Drosophila siblingspecies: probable adaptive changes in flight capacities. J. Evol.Biol. 10: 875–887.

Pétavy, G., J.R. David, P. Gibert & B. Moreteau, 2001. Viabilityand rate of development at different temperatures in Drosophila:a comparison of constant and alternating thermal regimes. J.Therm. Biol. 26: 29–39.

Precht, H., J. Christophersen, H. Hensel & W. Larcher, 1973. Tem-perature and Life. Springer Verlag, Berlin.

Rouault, J. & J.R. David, 1982. Evolutionary biology of Droso-phila melanogaster and D. simulans: a behavioral divergence inmicrohabitat selection. Acta Oecol., Oecol. Gener. 3: 333–338.

Singh, R.S., M. Choudhary & J.R. David, 1987. Contrasting patternof geographic variation in cosmopolitan sibling species Dro-sophila melanogaster and D. simulans. Biochem. Genet. 25:27–40.