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E COLOGY AND P OPULATION B IOLOGYEffects of Photoperiod and Light Intensity on the Genetics of Diapause in the Apple Maggot(Diptera:Tephritidae)KENNETH E.FILCHAK,1JOSEPH B.ROETHELE,AND JEFFREY L.FEDER Department of Biological Sciences,Galvin Life Science Building,University of Notre Dame,Notre Dame,IN46556Ann.Entomol.Soc.Am.94(6):902Ð908(2001)ABSTRACT Rhagoletis pomonella(Walsh)is an important pest of apples and has been at the centerof a long-standing debate concerning modes of speciation.Theßy has been proposed to speciatewithout geographic isolation(i.e.,in sympatry)in the process of shifting and adapting to new hostplants.Previous studies have shown that diapause-related traits play a key role in adapting apple-and hawthorn-infesting races of R.pomonella to a difference in the fruiting times(phenologies)oftheir respective host plants.These experiments indicated that prewinter temperature and itsduration affected the survivorship and genetics of over-wintering R.pomonella pupae.However,theearlier work did not test whether photoperiod and light intensity,two environmental factors thatalso differ between the host races,affect the genetics of diapause.Here,we report that variation inphotoperiod,but not light intensity,during the larval stage affects adult eclosion.Haw-origin larvaeexposed to longer photoperiods(18:6[L:D]h)eclosed signiÞcantly earlier that those experiencingshorter photoperiods(14:10and10:14[L:D]h).We also conÞrmed previously observed geneticrelationships between eclosion time and six allozyme loci displaying allele frequency differencesbetween the haw and apple host races.However,we did notÞnd a signiÞcant genetic response tophotoperiod for any allozyme.Our results suggest that,while photoperiod cues can regulate R.pomonella diapause,daylength is probably of secondary importance relative to temperature andseason length in genetically differentiating the host races.KEY WORDS Rhagoletis pomonella,host race,diapause,sympatric speciation,photoperiod,lightintensityD IAPAUSE IS A dynamic,hormonally mediated physio-logical state in insects characterized by low metabolic rates,limited behavioral activity,resistance to envi-ronmental extremes,and reduced morphogenesis. There are two general and interrelated reasons for insect diapause(Tauber et al.1986).First,to pass through a period unsuitable for survival(e.g.,winter), and second to coordinate an insectÕs life cycle to key resources or conditions conducive to growth and re-production(e.g.,host-plant availability).Insects use any of a number of different environmental cues, alone or in combination,as signals to initiate diapause, with photoperiod and temperature being the most common(Danilevsky1965,Morris and Fulton1970, Saunders1982,Tauber et al.1986).Diapause-related traits have been hypothesized to play a key role in sympatric speciation for phytoph-agous insects(Bush1969,1975;Smith1988;Wood and Keese1990;Abrahamson et al.1994;Feder and Filchak 1999).Speciation in sexually reproducing animals was traditionally thought to be predicated on complete geographic separation(i.e.,allopatry)of populations (Mayr1942,Futuyma and Meyer1980).But as early as the1860s,Walsh(1864)proposed that certain host-plant speciÞc phytophagous insects could speciate in the absence of geographic isolation(i.e.,in sympatry)in the process of shifting and adapting to new host pants.Subsequent studies have documented several examples of partially isolated“host races”(Feder et al. 1988,McPheron et al.1988,Wood and Keese1990, Carroll and Boyd1992,Abrahamson et al.1994)pos-sessing the hallmarks of incipient species.In several of these cases,diapause-related traits adapting the races to a seasonal difference in host availability(seasonal-ity)appears to be a primary aspect of differentiation (Smith1988,Wood and Keese1990,Abrahamson et al. 1994,Feder and Filchak1999).The apple maggotßy,Rhagoletis pomonella (Walsh),is a major economic pest of apples and a model for sympatric speciation(Bush1966,1992). Hawthorn(Crataegus spp.L.)is the native host for the ßy(Bush1966).But in the mid-1800s,a new popula-tion was reported attacking domesticated apple(Ma-lus pumila,Mill.)(Walsh1867).Subsequent studies have conferred host race status on the apple-infesting population(Feder et al.1988,McPheron et al.1988). Apple and hawthornßies differ in allele frequencies for six allozyme loci(aspartate amino transferase-2 [Aat-2],NADH-diaphorase-2[Dia-2],malic enzyme [Me],aconitase-2[Acon-2],mannose phosphate isomerase[Mpi],and hydroxyacid dehydrogenase [Had])(Feder et al.1988,McPheron et al.1988,Feder and Bush1989,Feder et al.1990a,1990b).In addition, mark-release-recapture studies have shown that adult1E-mail:Filchak.1@0013-8746/01/0902Ð0908$02.00/0᭧2001Entomological Society of Americaßies tend to return to the same species of host plant (fruit)to mate and oviposit that they fed within as larvae,a condition known as“hostÞdelity”(Feder et al.1994).Because R.pomonellaßies mate exclusively on or near the fruit of their hosts(Prokopy et al.1971, Prokopy1972),hostÞdelity translates directly into premating isolation.Although hostÞdelity is strong in R.pomonella,it is not complete(Feder et al.1994).Some intermixing still occurs between the apple and hawthorn host races at a rate ofϷ6%per generation.Given this level of geneßow and R.pomonella being univoltine,pop-ulation genetic models predict that the host races would become genetically indistinguishable within15 generations or years(Dean and Chapman1973,Boller and Prokopy1976,Feder and Filchak1999).However, long-term allozyme surveys(11yr)of naturalßy pop-ulations indicate that the apple and haw races are not fusing(Feder and Filchak1999).Therefore,some form of host-dependent selection must be occurring each generation to counteract the homogenizing ef-fects of geneßow.Several lines of evidence point to diapause-related traits associated with a difference in the fruiting times of apples and hawthorns as being the key to divergent selection between host races.Rhagoletis pomonella overwinters in a facultative pupal diapause(Prokopy 1968,Dean and Chapman1973,Boller and Prokopy 1976).Flies exposed to permissive environmental con-ditions as larvae and pupae can forgo a pronounced diapause and rapidly initiate adult development (Prokopy1968).In nature,such“nondiapause”devel-opment has disastrousÞtness consequences.Nondia-pauseßies either eclose at inappropriate times in the fall when host fruit is no longer available or commit to, but do not complete,adult development before the onset of winter and freeze/starve to death.Fruit on apple tree varieties favored by R.pomonella generally ripen from3Ð4wk earlier than haws(Feder and Fil-chak1999).As a result,the life history of appleßies is shifted earlier in the season,such that appleßy larvae and pupae are exposed to higher temperatures for a greater period of time before winter than hawthorn ßies.We hypothesized that the earlier phenology of apples selects for a more recalcitrant developmental response(deeper diapause)in the apple than the hawthorn race.Results from a series of rearing experiments have supported this“diapause”hypothesis.First,alleles at all six allozyme loci displaying frequency differences between the host races were found to correlate with the timing of adult eclosion,an event dependent on the duration of the pupal diapause(Feder et al.1993, 1997a,1997b,).Moreover,ßies possessing alleles typ-ically found in higher frequencies in the apple race eclosed later than individuals possessing“haw race”genes(Feder et al.1997a,1997b;Filchak et al.1999), as predicted by the diapause hypothesis.Second,as discussed above,elevated temperature has been shown to affect the diapause characteristics ofßies (Prokopy1968,Filchak et al.2000).Third,and most importantly,varying rearing conditions elicited ge-netic responses in the races in predicted directions. Allozyme frequencies in surviving(successfully over-wintering)adults exposed to higher temperatures for longer periods of time before winter as larvae and pupae,or to longer overwintering periods as pupae, shifted to become more“apple-like”than controls (Feder et al.1997a,1997b;Filchak et al.2000). Temperature and season length may not be the only diapause-related cues involved in the genetic differ-entiation of the host races.Photoperiod is also a good candidate because the earlier phenology of apples means that developing appleßy larvae experience longer day lengths than hawthorn larvae.Indeed, Prokopy(1968)demonstrated that variation in pho-toperiod during the larval,but not the pupal,life-stage caused signiÞcant shifts in adult eclosion times for apple-originßies.However,Prokopy(1968)did not explore the effects of photoperiod variation on survi-vorship or the genetics of host races.In addition to photoperiod,host-associated varia-tion in light intensity may be important in differenti-ating the races.Apples are physically larger than haw-thorns(mean diameter of apples at a study site(near Grant,MI)ϭ5.2cm;mean diameter hawsϭ1.6cm) (Feder1995).Consequently,light intensity near the core of fruits,where larvae prefer to feed,will likely be lower in apples than hawthorn(Prokopy1968). Fruit dissections have indicated thatßy larvae feed at much greater average depths within apples than in haws(average feeding depth in applesϭ1.38cm, hawsϭ0.13cm)(Feder1995).Moreover,competi-tion from plum curculio and codling moth larvae may force many hawthorn,but not apple-infesting,mag-gots to feed right below the surface(skin)of fruits (Feder1995),where light penetrance is greatest.Al-though Prokopy(1968)showed a photoperiod re-sponse for R.pomonella even at very low light levels (300lux),this result does not,by itself,rule out the possibility that varying light levels affect diapause and the genetics of host races.Verifying an effect of light intensity on diapause is of particular interest because it could help explain a puzzling difference in the geographic pattern of allo-zyme variation for host races.As mentioned above,six allozyme loci show consistent allele frequency differ-ences between sympatric apple and hawthorn-ßy pop-ulations across eastern North America(Feder et al. 1988,McPheron et al.1988,Feder and Bush1989; Feder et al.1990a,1990b).However,these six allo-zymes also display latitudinal frequency clines within both host races.Alleles more common to the apple than hawthorn race at the Grant,MI,site were found at higher frequencies in both host races at more south-ern locales(Feder and Bush1989,Feder et al.1990a, Berlocher and McPheron1996).Furthermore,the slopes of the clines differ between the races,being steeper for the hawthorn race(i.e.,from north to south allozyme frequencies change more dramatically among hawthorn than apple-ßy populations).One possible explanation for the pattern is that the fruiting times of hawthorns are more strongly inßuenced by latitude-related factors than apples,resulting in theNovember2001F ILCHAK ET AL.:E FFECTS OF P HOTOPERIOD AND L IGHT ON A PPLE M AGGOT903hawthorn race experiencing more variable selection pressures than appleßies.ButÞeld observations in-dicate that theϷ3Ð4wk earlier phenology of prime apple varieties is consistent across the range of overlap of apple and hawthorn trees in the Midwest(J.L.F.,un-published data).However,if increased light levels ex-perienced by hawthorn-infesting larvae exacerbate the effects of elevated temperature and a longer growing season at more southern sites,then this could account for the clinal difference between the host races.The objective of this study was to test for genetic or developmental responses to variation in photoperiod and light intensity.Our a priori hypothesis was that higher photoperiods and/or lower light intensities would select against alleles more common to the haw-thorn host race.Materials and MethodsOverview of Experiments.The experimental design consisted of exposing collections of hawthorn-origin larvae within the host fruit to varying photoperiods and light conditions in controlled environmental chambers.After a simulated winter,over-wintering survivorship and eclosion times were recorded and compared amongßies in the various environmental treatments.Surviving adults were scored for the six allozyme loci displaying frequency differences be-tween the host races to test for genetic relationships with diapause/development and for genetic responses to varying photoperiod and light-intensity conditions. Only hawthorn(not apple)ßies were used in this experiment.Ideally both apple and hawthornßies would be used in such a manipulation.However,this method was not employed herein for several reasons. One,statistical sensitivity requires large numbers of individuals to detect a response to experimental ma-nipulation.Second,genetic variation exists between individuals on different trees.If mixed samples were used,it is likely that unequal numbers of larvae would result and thus the unrepresentative proportions of this variation would bias the result.It is therefore necessary to use individuals from a single tree,which also has sufÞcient larvae numbers to detect a response to our rearing conditions.In nature infested hawthorn trees tend to support larger populations that apples. Finally,each race contains all of the variation pos-sessed by the other,although at different frequencies. Therefore,usingßies from a single race and tree is a practicalÞrst step in the majority of our investigations and is the one employed herein.Infested fruit for these experiments was collected from a hawthorn tree at aÞeld site near Grant,MI,on 28August1998(see Feder et al.1990b for a map of the Grant site).Rhagoletis pomonellaßies used herein generally eclose in early summer and have one gen-eration per year(Dean and Chapman1973,Boller and Prokopy1976).Although a small,second generation of appleßies is sometimes observed eclosing in the fall, theseßies are inevitably doomed and do not repro-duce(Dean and Chapman1973).Sexually mature adults rendezvous on or near unabscised host fruit to court and mate(Prokopy et al.1971,Prokopy1972). Females deposit one egg per oviposition bout imme-diately below the surface of host fruit(Bush1992). Eggs hatch within a few days,with subsequent larval feeding conÞned to the fruit oviposited into by the larvaÕs mother.When fruit abscise from trees in late summer or early fall,larvae leave the fruit and burrow into the soil to an average depth ofϷ2.5cm(Dean and Chapman1973,Boller and Prokopy1976).Here,they form puparia and undergo a fourth larval instar before entering a facultative pupal diapause for winter(Dean and Chapman1973,Boller and Prokopy1976). Photoperiod rval-infested fruit was transported to the laboratory and placed on0.3by 0.6m wire mesh racks that were set within plastic collecting trays(0.3m by0.6m).The fruit was divided into three equal subsamples maintained at photope-riods of18:6,14:10and10:14(L:D)h in three different constant temperature(26Ϯ1ЊC)incubators.Fruit was positionedϷ0.6m below the light source(2Ð48Љßuorescent lamps,110W,General Electric F48T12/ CW/1500,GE part#10751)in the incubators.At this distance,fruit received8500lux of light on itÕs surface, as determined with a foot candle/lux light meter(cat-alog no.L524880,Extech Instruments,Stamford,CT). As larvae completed feeding they emerged from fruit and formed puparia in plastic trays.Puparia was placed in petri-dishes containing moist vermiculite and were returned to the incubators.After10d,the petri-dishes were taken from the incubators and placed in a refrigerator(0to5ЊC cycle)to simulate winter(Data from Grant,MI,indicate that this tem-perature range is typical for pupae over-wintering in the soil there)(Feder and Filchak1999,Filchak et al. 2000).Equal samples of petri-dishes(pupae)were removed from the refrigerator after15and30wk and put into an incubator maintained at23ЊC with a pho-toperiod of14:10(L:D)h(We have estimated that temperatures are below the developmental threshold for R.pomonella for an average ofϷ26wk at the Grant site)(Filchak et al.2000).Pupal sample sizes within the15and30wk winter length treatments were nϭ529,nϭ418,and nϭ558for the photoperiods18:6, 14:10,and10:14(L:D)h,respectively.Newly eclosing adults were collected from the petri dishes on a daily basis and immediately frozen atÐ80ЊC for later genetic analysis.Light Intensity Experiment.Fruit for this experi-ment was transported to the laboratory and placed on wire racks in plastic collection trays.These trays were housed in a single incubator maintained at22.5ЊC (Ϯ1ЊC)and a photoperiod of14:10(L:D)h.Light intensity was varied by covering the fruit with no,one, or two layers of mosquito netting(0.5mm mesh size, dark gray nylon material),resulting in high(8500lux), medium(1,500lux),and low(430lux)light treat-ments.We found that fruit within and beneath host trees at the Grant site in1998typically received from 1,000Ð10,000lux of light.(These light levels were recorded using a foot candle/lux light meter.Catalog #L524880,Extech Instruments,Stamford).However, fruit outside the canopy that is exposed to full sunlight904A NNALS OF THE E NTOMOLOGICAL S OCIETY OF A MERICA Vol.94,no.6can experience as much as150,000lux of light on their surface.Consequently,the range of light intensity conditions used in our study(430Ð8500lux)was a reasonable representation of what most fruit would receive on its surface in nature.But,of course,a subset of fruit not in,or under,the canopy will be exposed to much brighter daylight.Temperature readings taken using a HOBO external temperature data logger(H08-031-08,Onset Com-puter,Pocasset,MA)indicated that the mean surface temperature of fruit in the high light treatment (23.5ЊC)averagedϷ1ЊC above that in the medium (22.5ЊC)and low treatments(22.4ЊC).Our study was therefore confounded by slight temperature differ-ences among certain light treatments,pointing to the inherent difÞculty in experimentally disentangling the two factors,as increased light intensity will almost invariably lead to increased surface heating of fruit. However,as we show in the results section,eclosion times and allozyme frequencies did not differ among light intensity treatments,allowing us to discount its importance as a diapause cue.Puparia were collected and treated in the light-intensity experiment as described above for the pho-toperiod study,except that all three light intensity samples were over-wintered for just15wk.The total number of pupae in the high,medium,and low light treatments were nϭ204,237,and238,respectively. Adults were collected on a daily basis as they eclosed in petri-dishes and immediately frozen for later ge-netic analysis.Genetic Analysis.Standard horizontal starch gel electrophoresis techniques were used to scoreßies for the six allozymes(Aat-2,Dia-2,Me,Acon-2,Mpi,and Had)displaying allele frequency differences between host races(Berlocher and Smith1983,Feder et al. 1989).Isocitrate dehydrogenase(Idh)was also scored as a genetic control because it displays no frequency differences between the host races,as well as limited geographicvariationin R.pomonella(Federetal.1990a). Flies not used for genetic analysis were saved and stored atÐ80ЊC.Theseßies are available as voucher specimens and for subsequent genetic analysis.Statistical Analysis.Eclosion time differences among photoperiod and light intensity treatments were analyzed for signiÞcance using nonparametric Kruskal-Wallis tests with tied ranks(Zar1996).Sub-sequent comparisons between pairs of treatments were conducted using the Nemenyi test(Zar1996),as modiÞed for unequal sample sizes and tied ranks by Dunn(1964).Survivorship differences among treat-ments were analyzed for signiÞcance using Fisher exact tests(Zar1996).G-heterogeneity tests were performed to test for signiÞcant genetic responses (i.e.,allozyme frequency differences)among photo-period and light intensity treatments(Zar1996).Re-lationships between eclosion time and single-locus allozyme genotypes forßies were analyzed by Spear-man rank correlation coefÞcients(r s)corrected for tied ranks(Zar1996).Flies were assigned to three different genotypic classes for each locus according to the number of Me100,Acon-295,Mpi37,Aat-2ϩ75,Dia-2100,Had100,or Idh100alleles each possessed (Note:ϩ75for Aat-2indicates the class of alleles withՆ75relative anodal mobility relative to the most common100electromorph).Correlation coefÞcients were z-transformed to test for signiÞcance(Hedges and Olkin1985).One-tailed tests were conducted for Me100,Acon-295,Mpi37,Aat-2ϩ75,Dia-2100,and Had100because of our a priori expectation from previous studies thatßies possessing these alleles should eclose earlier than others(Feder et al.1997a, 1997b,Filchak et al.1999).Two-tailed tests were done for the control locus Idh.We also conducted a meta-analysis combining correlation coefÞcients across winter length,photoperiod,and light intensity treat-ments using the methods of Hedges and Olkin(1985). These common correlation coefÞcients(known as“ef-fect magnitudes”and designated by the symbol r z)Fig.1.Mean days to eclosion versus(a)photoperiod (daylength in hours)and(b)light intensity(Lux).Treat-ments showing a letter in common within winter treatments were not statistically signiÞcant at the PϽ0.05level as determined by DunnÕs test corrected for tied ranks.All three comparisons between15-and30-wk winters in a given pho-toperiod treatment were signiÞcant at the PϽ0.05level as determined by Fisher exact tests.November2001F ILCHAK ET AL.:E FFECTS OF P HOTOPERIOD AND L IGHT ON A PPLE M AGGOT905were tested for signiÞcance by z-transformation (Hedges and Olkin1985).ResultsEclosion Time.Both photoperiod and winter length signiÞcantly affected mean time to adult eclosion (MTE)(Fig.1a).Flies exposed to longer day lengths as larvae eclosed increasingly earlier(had decreasing MTEÕs)within both the15-and30-wk overwinter treatments(Kruskal-Wallis H tied ranks for15wkϭ66.3,dfϭ2,PϽ0.0001;H for30wkϭ65.6,dfϭ2,PϽ0.0001).In addition,ßies experiencing the same pho-toperiod eclosed signiÞcantly earlier the longer they were overwintered.There was no apparent interac-tion between photoperiod and winter length.MTE decreased in a linear and parallel manner with in-creasing photoperiod between the15-and30-wk win-ter treatments(Fig.1a).In contrast to the results for photoperiod and winter length,light intensity did not affect eclosion time(Hϭ2.1,dfϭ2,Pϭ0.40);MTEs were virtually identical between the low,medium and high light treatments (Fig.1b).Survivorship.Overwintering survivorship did not vary signiÞcantly with photoperiod within either the 15-or30-wk overwinter treatments(P for15wkϭ0.152,P for30wkϭ0.069,as determined by Fisher exact tests).Althoughßy viability tended to be lower in the14:10(L:D)h(44and45%for the15and30wk, respectively)than for the other photoperiods(50%, 51%18:6[L:D]h and47%,52%10:14[L:D]h for the 15and30wk,respectively),this trend was not signif-icant.Survivorship differed signiÞcantly among light intensity treatments(Pϭ0.0003,as determined Fisher exact test).Viability was higher in the medium(76%) than in the low(64%)or high(59%)light-intensity experiments(Pϭ0.005and0.0001,respectively,as determined by Fisher exact tests).Genetic Response.No allozyme locus showed a signiÞcant allele frequency difference related to pho-toperiod within either the15-or30-wk overwinter treatments(Table1).Allozyme frequencies also did not vary signiÞcantly among light intensity treatments (Table1).Consequently,the survivorship difference seen among light intensity treatments was not accom-panied by a corresponding genetic response at any of the six allozyme loci differentiating the host races. In contrast to the muted genetic responses seen to varying photoperiod and light intensity,highly signif-icant relationships were observed between eclosion time and allozyme genotypes within every environ-mental treatment performed in this study(Table2). The signs of these relationships were negative in all cases,indicating thatßies possessing alleles(geno-types)typically found in higher frequencies in the hawthorn race at Grant,MI,eclosed earlier than in-dividuals possessing“apple race”alleles.The control locus Idh,showed no relationship with photoperiod, light intensity,or eclosion time(Table2).DiscussionOur results indicate that photoperiod is an impor-tant environmental cue affecting developmental pe-riodism in rvae exposed to longer day lengths eclosed signiÞcantly earlier as adults than those receiving shorter photoperiods.TheseÞndings are not overly surprising given that many temperate zone insects use daylength as a prime cue to regulate diapause(Saunders1982,Tauber et al.1986).More-Table1.Results for G-heterogeneity tests for significant allele frequency differences among photoperiod(10:14,14:10,and 18:6[L:D])and light intensity(430,1500,and8500lux) treatmentsTreatment/Locus Me Acon-2Mpi Aat-2Dia-2Had IdhPhotoperiod (15-wk winter)0.20.4 5.1 4.1 1.0 2.40.1Photoperiod (30-wk winter)3.9 2.7 3.10.6 1.0 2.9 1.0Light intensity(15-wk winter)1.3 1.60.22.93.4 1.5 1.1No test was statistically signiÞcant at the PՅ0.05level with2df.Table2.Spearman rank correlations between eclosion time and allozyme genotypesTreatment/Locus Me Acon-2Mpi Aat-2Dia-2Had Idh Photoperiod18:6rϪ0.36****Ϫ0.23****Ϫ0.19***Ϫ0.24****Ϫ0.19***Ϫ0.14**0.02 df273273273272271273271 14:10rϪ0.36****Ϫ0.24***Ϫ0.16*Ϫ0.07Ϫ0.93Ϫ0.13*0.03 df177177177177171177176 10:14rϪ0.43****Ϫ0.19**Ϫ0.25***Ϫ0.17*Ϫ0.14*Ϫ0.28****0.01 df180180180174180180180 Light IntensityLow rϪ0.57****Ϫ0.46****Ϫ0.14Ϫ0.04Ϫ0.03Ϫ0.21*Ϫ0.05 df90909089869090 Med rϪ0.63****Ϫ0.68***Ϫ0.15Ϫ0.25**Ϫ0.25**Ϫ0.22*Ϫ0.11 df88888888868888 High rϪ0.36***Ϫ0.22*Ϫ0.49Ϫ0.22*Ϫ0.19*Ϫ0.27**Ϫ0.07 df84848483808483Photoperiod results represent common coefÞcients(effect magnitudes)calculated across the15and30week winter treatments by meta-analysis.Correlation coefÞcients were z-transformed to test for signiÞcance(*,PϽ0.05;**,PϽ0.01;***,PϽ0.001;****,PϽ0.0001). 906A NNALS OF THE E NTOMOLOGICAL S OCIETY OF A MERICA Vol.94,no.6over,our results are consistent with the previous work of Prokopy(1968)showing that apple maggots re-spond to variation in photoperiod even in dim light (300lux).There are reasons to suspect that photoperiod does not play a dominant role in genetically differentiating the host races.Although longer photoperiods resulted in earlier mean eclosion times in our experiment,this shift was not accompanied by an increase in over-wintering mortality or a genetic response at any of the allozyme loci.The latterÞnding was true despite the observation of highly signiÞcant relationships be-tween eclosion time and allozyme genotypesÑa result reinforcing the key tenet of the diapause hypothesis that the allozymes(or linked genes)displaying host-related differences regulate the timing of develop-ment.The photoperiod experiment contrasts with ear-lier studies in which increasing the prewintering period(26ЊC)or lengthening the duration of winter not only reduced mean eclosion time,but also mark-edly decreased pupal survivorship and induced strong genetic responses favoring apple race alleles(Feder et al.1997a,1997b;Filchak et al.2000).However,past experience suggests that the10-d prewinter period used in the photoperiod experiment represents a rel-atively benign rearing condition forßies.Conse-quently,we may have pushed pupae close to,but not beyond,the point of nondiapause development to induce genotype speciÞc mortality in our study.It is, therefore,premature to completely discount a role for photoperiod in contributing to the genetic differen-tiation of the host races.Field measurements taken from our Grant,MI, study site imply that the effect of photoperiod will nevertheless be of secondary importance relative to temperature and season length in differentiating the races.In1999,the mode time for larval infestation of apples at Grant,MI,was5August and for haws1 September(Filchak et al.2000).Civic daylength,as determined from National Weather Service(USA) data,was16h,21min on1August and15h,11min on 1September a difference of70min.However,in the laboratory we failed to elicit a genetic response to photoperiod differences as great as8h(10:14com-pared with18:6[L:D]h).Therefore,while the70min longer day experienced by apple-ßy larvae could ex-acerbate the known effects of higher temperatures of apple than haws,increasing selection pressures for more recalcitrant pupal(diapause)development, photoperiod itself is unlikely to be a prime factor driving host race system.The lack of any detectable effect of light intensity on diapause,while not unexpected,was disappointing. Reports of light intensity inßuencing diapause are sparse in the insect literature(Saunders1982).In addition,Prokopy(1968)showed that R.pomonella can respond to photoperiod differences from external light sources as low as300lux,suggesting thatßies are sensitive to even very dim light.Nevertheless,light levels are likely to differ substantially for developing apple and hawthorn-ßy larvae.Moreover,as we dis-cussed above,a light intensity effect would help to explain a puzzling difference in the pattern(slope)of latitudinal allozyme clines between host races.But our results clearly reject a role for light intensity in the genetics of diapause,at least for the allozyme loci we scored.Consequently,other hypotheses(e.g.,the in-volvement of additional,as yet unidentiÞed,diapause loci partially overriding the effects of the allozymes in the apple race)must be entertained to explain the clinal differences between races.We have now accumulated information concerning the effects of a number of different environmental factors on R.pomonella diapause.Temperature,the length of the growing season,and the duration of winter all exert strong,differential selection pressures on allozymes(or linked loci)between host races (Feder et al.1997a,1997b;Filchak et al.2000).Al-though photoperiod inßuences the setting of the dia-pause clock in R.pomonella,it appears to be of only secondary importance in differentiating sympatric ap-ple and hawthorn-ßy races.Whether photoperiod plays a greater role in differentiating Rhagoletis spe-cies infesting host plants whose fruiting phenologies differ by more than apples and hawthorns is a question that remains to be investigated.In addition,light in-tensity has no detectable effect on the genetics of diapause.Given our current understanding of the rel-ative importance of various environmental cues on R. pomonella diapause,we can now concentrate on how factors such as temperature and season length interact to maintain allozyme differences between host races. Moreover,the recent development of a molecular linkage map for R.pomonella(Roethele et al.1997), and evidence for synteny between the apple maggot and Drosophila melanogaster(Meigen),will permit theÞner genetic dissection of diapause-related phe-notypes in R.pomonellaßies.AcknowledgmentsWe thank Bill Perry,Uwe Stolz,Hattie Dambroski,Xie (Frank),Nikki Wilson,and Amir Tamassebi who gave help or moral support,and Dave Prokrym and the other staff at the USDA(Niles Michigan laboratory)who allowed us to use their incubators and helped signiÞcantly with insect rearing. Additional aid was given by David Lodge,Nora Besansky,and Guy Bush.This work was supported,in part by a National Science Foundation graduate research traineeship(Grant No.9452655)to K.E.F.References Cited Abrahamson,W.G.,W.M.Brown,S.K.Roth,D.V.Sumer-ford,J.D.Horner,M.D.Hess,S.T.How,T.P.Craig,R.A.Packer,and J.K.Itami.1994.Gallmaker speciation:an assessment of the roles of host-plant characters,phenol-ogy,gallmaker competition and natural enemies,pp.208Ð122.In P.Price,W.Mattson,and Y.Baranchilov[eds.], Gall-forming DA For.Serv.N.Central Exp.Stn.Gen.Tech.Rep.NC-174.Berlocher,S.H.,and D.C.Smith.1983.Segregation and mapping of allozymes of the apple maggotßy.J.Hered.74:337Ð340.November2001F ILCHAK ET AL.:E FFECTS OF P HOTOPERIOD AND L IGHT ON A PPLE M AGGOT907。
