's. J. Le Magnen, Behav. Brain Sac. 4, 596 (I98I).I6. J. C. Newman and D. A. Booth, Physiol. Bchav. 27,

929 (1981).17. A rat's food was withheld for the last 4 hours of the

dark phase. A chow pellet (diet 422, Heygate andSons) of known dry weight (-i.8 g) was presentediS to 60 minutes after the lights came on in the I2: I2light-dark cyde and was consumed within about iominutes. The rat (Sprague-Dawley hooded) wasweighed and anesthetized I20 minutes after presen-tation of the pellet, and the stomach was clamped atthe pylorus and esophageal junction. Rats that hadbrain operations were perfused for neurohistology.Water washings ofgastnc contents were centrifugedtwice, and the pellet and supematants were driedseparately to constant weight. Abnormalities inautonomic nervous system control of the stomachcan have opposite effects on the emptying of liquidsand solids [J. D. Davis and D. A. Booth, Physiol.Behav. 12, 685 (i974)]; furthermore, gastric "dump-ing" of a load differs from the later main phase ofcontrolled emptying [J. N. Hunt and D. F. Stubbs,J. Physiol. (London) 245, 209 (I975)]. Thus ourfindings are compatible with the slower emptying ofmilk in the first 60 seconds after loading m VMHrats [T. Ralph and P. R. Sawchenko, Brain Res.BuJl. 3, I (I975)]-

I8. C. M. Brooks and E. F. Lambert, Am. J. Physiol.147, 695 (i946).

I9. N. E. Miller, C. J. Bailey, J. A. F. Stevenson, Scienc112, 256 (I950); H. Graff and E. Stellar, J. Comp.Physiol. Psychol. 55, 4I8 (I962).

2o. S. C. Wang, G. Clarke, F. L. Dey, S. W. Ranson,Am. J. Physiol. 130, 81 (1940).

2I. F. Rohner et al., Diabetologia 13, 239 (I977).22. H. R. Berthoud and B. Jeanrenaud, Endocinology

IO5, I46 (I979); J. Louis-Sylvestre and J. Le Mag-nen, Neurosci. Biobehav. Rev. 4 (suppl. I), I3 (1980).

23. H. R. Berthoud et al., Diabetologia 20, 393 (I98I).24. H. S. Sitren and N. R. Stevenson, J. Nutr. I10, 558

(I980).25. T. Powley [Psychol. Rev. 84, 89 (i977)] and L. A.

Campfield, F. J. Smith, and K. F. Fung [in TheNeural Basis ofFeeding and Reward, B. G. Hoebeland D. Novin, Eds. (Haer Institute, Brunswick,ME, I982), p. 203] reported signs of cholinergicreceptor "down-regulation" and adrenergic "up-regulation" in chronic VMH obesity.

26. W. J. Malaisse et al.,J. Lab. Clin. Med. 71, 56 (I968);D. A. Booth and S. P. Jarman, Behav. Bio. IS, I59(I975)-

27. Campfield et al. (2S) have made the glucose and fattyacid components ofBooth's (I4) models explicit andadded autonomic nervous system control of insulin

secretion to the net lipid synthesis-mobilizationcomponents. Their calculations confirm the earlierconclusion (I4) that insulin hypersecretion is neces-sary in addition to rapid gastric emptying, but theydo not establish that the hyperinsulinism is auto-nomic and primary rather than secondary to rapidabsorption and even perhaps to purely metabolichypertrophy.

28. Rapid gastric emptying has been reported in someclinically obese populations [J. N. Hunt, R. Cash,P. Newland, Lancet g97s-IH, 905 (I97s); C. Johnsonand K. Ekeland, Gut 17,456 (I976); R. D. Wright atal., Gastrenterolgy 84, 747 (1983)]. Slow gastricemptying has been reported in the psychopatholog-icak self-starvation condition ofanorexia nervosa [A.Dubois et al., Gastmenteroloy 77, 319 (I979)]. Theappetite suppressant, antiobesity agent fenfluraminereduces food intake in freely feedmg rats primarilyby slowing gastric emptying and so extending thesatiating effects of a meal [D. A. Booth and D.Stribling, Proc. Nutr. Soc. (London) 37, i8I (1978); R.F. Davies et a., Physiol. Behav. 30, 73 (1983); N.Rowland and P. Carlton, Life Sci. 3.4, 2495 (1984);D. A. Booth, E. L. Gibson, B. Baker, Appetite, inpress].

2a May I985; accepted 22 November 1985

Postindustrial Melanism in the Peppered Moth

L. M. CooK, G. S. MANI, M. E. VARLEY

New data show the geographical pattern offrequency ofthe melanic morph carbonariaof the peppered moth, Biston betularia, in 1983-84. These frequencies are comparedwith data from 1952 to 1970. After 20 years ofsmoke control, the area ofhigh melanicfrequency has contracted to the northeast. The change indicates a disadvantage tocarbonaria of about 12 percent compared with 20 years ago. Computer simulations,which do not include the assumption ofheterozygote advantage, provide a good matchto the surface for the period 1952 to 1970, and also the 1983-84 surface. Experimentson visual predation have been criticized as giving unrepresentative estimates ofselection but they permit satisfactory simulations to be made.

T HE RESPONSE OF THE PEPPEREDmoth, Biston betulaia, to environ-mental changes brought about by

industrialization remains one of the mostfully documented cases of microevolution-ary change. The first survey backed up byquantitative data was published byKettlewell; it was based on data obtainedbetween 1952 and 1956 and on the use of avariety of moth traps and capture methods.The results were presented in the familiar"pie diagrams" showing the frequencies ofthe two melanic types, carbonaria and insu-lana, and of the typicals. Similar surveyswere carried out in 1957 to 1964 and in1965 to 1970. All three sets of results werelisted together by Kettlewell (1), and themap of morph frequencies was updated bySheppard (2). A detailed survey was carriedout by Clarke and Sheppard (3), Whittle etal. (4), and Bishop (5) ofthe transition froma high melanic frequency in Liverpool to alow frequency in rural northern Wales, anarea where insularia is rare so that the clinemeasures the ratio of carbonaria to typical.

7 FEBRUARY I986

By 1972, smoke control legislation and re-placement ofold coal-burning housing stockhad reduced atmospheric pollution. Therewas also a drop in carbonania frequency (6).In order to provide a baseline for futurecomparison, a survey was carried out thatextended the northern Wales-Liverpooltransect to the Pennines east of Manchester(7). Data from individual trap sites weremapped with a computer program that in-terpolates values between points, to providecontours or three-dimensional maps. Thesurface produced may be altered by varyinga smoothing factor, which was chosen togive a good fit to the data available for thenorthern Wales cline. Over 90 percent car-bonaria in Cheshire dropped to less than 5percent in Wales to the southwest of anarrow transition region (7).One of the purposes of the study was to

investigate the possibility that heterozygotesfor the melanic gene, indistinguishable inappearance from the melanic homozygote,have a greater net fimess than either homo-zygote, as had been suggested (1, 8, 9). It

was concluded that the evidence could aswell be explained by a migration-selectionhypothesis, with the same fitness being as-cribed to the two melanic genotypes. Theimplications of the model were investigatedtheoretically (10-12), and additional evi-dence was available from breeding experi-ments (13). Using all the available evidence,Mani (12) obtained a good fit to the UnitedKingdom data on melanic morph frequencywithout the assumption ofheterozygote ad-vantage.

Since the early 1970's study ofB. betulariahas been part of the Foundation Year biolo-gy program in the Open University. Stu-dents are asked to collect moths in standardtraps over 5-day periods. Records are credit-ed to the Open University Study Centernearest to their collecting site. Since 1983specimens of each morph collected havebeen sent to L. M. Cook for checking. Thesurvey covers most of England and Walesand part of southem Scotland. We presenthere results for carbonaria frequency, basedon a total of 1825 moths recorded at 190locations.To illustrate morph frequencies, we have

used the same computer program and thesame smoothing factor that were used in theearlier survey. A spurious sense ofcontinuityis given to point records, but the position ofcontours is objectively determined once pro-gram parameters have been chosen. Fre-quency surfaces for Kettlewell's survey are

L. M. Cook, Department of Zoology, University ofManchester, Manchester MI3 9PL, Umited Kingdom.G. S. Mani, Department of Physic, University of Man-chester, Manchester MI3 9PL, United Kingdom.M. E. Varley, Department of Biology, Open University,Walton Hal, Mton Keynes MK7 6AA, United King-dom.

REPORTS 611

on

Dec

embe

r 18

, 201

4w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

o

n D

ecem

ber

18, 2

014

ww

w.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

on

Dec

embe

r 18

, 201

4w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

(P,/Q/) on n is linear with a slope of log w.Contour maps were produced separately forthe three series of data presented byKettlewell (1). Sixty-one points at 50-kmintervals over England, Wales, and southernScotland were then taken from the resultingsurfaces, and their values compared between

b

Fig. 1. Computer-generated surfaces for the frequency of carboaria in the peppered moth (a) in 1952

to 1970 and (b) in 1983-84 (14). The vertical scale ranges from 0 percent in the southwesternpeninsula to 99 percent at the highest point to the northeast. The cline running diagonally acrossEngland and Wales shifts to the northeast. Data are incomplete for northern England and southernScotland, so change there is unconfirmed. Simulations of the early survey (c) and of the later one (d)show the patterns generated by the use of a set of parameters derived from experiments by severalworkers.

3

2

aIL

c

%d

Fig. 2. Change in carbonaria frequency with time at Caldy, Wirral,based on data of Sir Cyril Clarke. The straight line represents thechange expected if carbonaria has been at a constant 12 percentdisadvantage since 1969. The continuous curve represents changeresulting from a disadvantage increasing at a rate linearly related tochange in sulfur dioxide concentrations in the area. This assumptionwas used in producing the simulation in Fig. ld.

1960 1970

Date

1980

the different surveys. For the transition from1952 to 1957 (time elapsed, 5 generations)the geometric mean of w was 0.98 ± 0.04.For 1952 to 1970 (time elapsed, 18 genera-tions) the geometric mean was 0.99 ± 0.02.These changes cannot be tested for signifi-cance since they are not based on indepen-dent records. Standard errors measuringvariation between estimates from the 61locations are given, however, to indicate theamount ofvariation observed. The estimatesare sufficiently near to unity to suggest thatthere is no detectable selection operatingover the period elapsed. We have thereforepooled the data to indicate the pattern at theend ofthe period ofhigh atmospheric pollu-tion (Fig. la). The 1983-84 data provide afrequency surface in the same way (Fig. lb).Considering 15 generations to have elapsedbetween the two data sets, we calculate w tobe 0.88 ± 0.02 from the 50-km point data.On average, since the introduction ofsmokecontrol, there has been 12 percent selectionagaist melamcs.The area of high melanic frequency in

northern England is still present but hascontracted in range. The result is that somelocalities have experienced very substantialdeclines in melanic frequency, while in oth-ers there is little change. One site at whichthe morph frequency cine has contracted isat Caldy, Wirral, where collections havebeen made by Sir Cyril Clarke from 1959 tothe present (15). The change observed ispresented in Fig. 2 as the logarithm of theratio ofcarbonaria to noncarbonaria. For thelast 16 years the change is more or lessconstant, and, if a regression line is fitted tothese points, provides an estimate of w of0.88 ± 0.01, as does the comparison ofKettlewell's and the Open University data.Selection against carbonaria may have beenvery low from 1959 to 1968 and thereafterreached a more or less steady level of 12percent, as the general survey also suggests.

It is possible that w, and w2 are not equal.Clarke and Sheppard (3) inferred a higherfitness for the heterozygote than the melanichomozygote from examination of changesin morph frequency. Bishop et al. disagreedwith this interpretation (7) and consider theexisting data to be compatible with theassumption that w1 and w2 are the same. If itis assumed that the gene frequency q is thesquare root ofQ (which is an approxima-tion), so thatp = 1 - q, then for any pair ofgenerations 0, 1 the quantity PtQo/Qlpo =2w2 - po(2w2 - w1). Considering the Caldydata, we find w1 = 1.09 and w2 = 0.76. Thedifference is in the reverse direction from theone proposed, so that there is no evidence ofthe existence of heterozygote advantage.The two surveys have been compared,

with large-scale computer simulations de-

SCIENCE, VOL. 231

compared with those for 1983-84 (Fig. 1).In discussing the changes observed, the no-tation in (14) is used.

Ifthe fitness ofthe two melanic genotypesis the same, so that w, = w2, then w may beestimated from the change over n genera-tions, since P,/Q/ = wnPo/Qo, and log

a

I

I1

612

signed to test the effectiveness with whichmorph frequency pattern can be predicted.A model was developed (10, 12) in whichEngland and Wales were represented by agrid of 360 square cells of linear dimension22.5 km. Values for the mninimum set ofparameters required were obtained from theliterature and from modeling the northernWales cline (11). Mani (12) presented com-parisons with the Kettlewell data for threetransects. Figure lc shows the completesurface, for comparison with Fig. la. Theagreement is sufficiently close as to indicatethat a set of assumptions derived from asmall part of the area (the cline in the north-west) may be used to explain the morphfrequency distribution over the whole ofEngland and Wales, and that these assump-tions need not include heterozygote advan-tage.

For the period from the 1970's to thepresent, fitness of carbonaria and isularia isassumed to have decreased linearly by anamount estimated from the change in re-corded atmospheric sulfur dioxide on theWirral (15). This produced a good match tothe change in morph frequency at Caldy,and consequently the same change is as-sumed throughout the country. The resultof the simulation projected to 1984 isshown in Fig. Id. In this case also there is asatisfactory match to the observed data, withthe plateau of high carbonania frequencyretreating to the northeast, although thepredicted frequency in the London area ishigher than the frequency observed. Withthe selective intensities involved in the mod-el, such discrepancies are not surprising. Theselection is associated with industrial pollu-tion and is assumed to act through theagency ofvisual predation. Mikkola (16, 17)has pointed out, rightly, that more observa-tions are needed before we can have a truepicture of the role of visual selection, andthat experiments to measure visual selectionhave design deficiencies. He questionswhether the evidence even gives useful in-formation on selective predation. The selec-tive pressures assumed in these simulationsare based on the visual selection experi-ments, however, and for the most part havea good predictive value. In our opinion, thissuggests that the experiments do give acorrect indication of the order and directionof the selective pressure involved.

REFERENCES AND NOTES

i. B. Kettlewell, The Evolution ofMelanism (Claren-don, Oxford, 1973).

2. P. M. Sheppard, Natural Selection and Hercdity(Hutchinson, London, i975).

3. C. A. Clarke and P. M. Sheppard, Proc. R. Soc.London Ser. B I65, 424 (I966).

4. P. D. J. Whittle, C. A. Clarke, P. M. Sheppard, J. A.Bishop, ibid. 194, 467 (I976).

s. J. A. Bishop, J. Anim. Ecol. 4I, 2o9 (1972).6. D. R. Lecs, in Genetic Consequences ofMan Made

Change, J. A. Bishop and L. M. Cook, Eds. (Aca-demic Press, London, I98I), p. 129.

7. J. A. Bishop, L. M. Cook, J. Muggleton, Philos.Trans. R. Soc. London Ser. B 281, 4.89 (I978).

8. E. B. Ford, Biol. Rev. (Cambridge) I2, 461 (I937).9. D. R. Lees and E. R. Creed, J. Anim. Ecol. 4, 67

('975).io. L. M. Cook and G. S. Mani, Bio. J. Linn. Soc. 13,179

(I980).iI. G. S. Mani, Proc. R. Soc. London Ser. B. 210, 299

(1980).I2. , Biol. J. Linn. Soc. 17, 259 (1982).I3. E. R. Creed, D. R. Lees, M. G. Bulmer, ibid. I3, 251

(1980).I4.. The following notation was used in preparing the

contour maps for melanic frequency in the pe redmoth. The genotype before selection for carbonarais represented byp- or 2pq, and the typical genotypebefore selection is represented by q2; the pnenotypeof carbonaria by P and the typical phenotype byQ;the fitness for carbonaria by w, or w2 and the typicalfitness by i.

is. C. A. Clarke, G. S. Mani, G. Wynne, ibid. 26, I89(1985).

I6. K. Mikkola, Ann. Entomol. Fenn. 45, 8i (I979).17. , Biol. J. Linn. Soc. 21, 409 (I984).I8. This survey could not have been made without the

participation of Open University students. We aregrateful to A. C. Arnold for help with the graphics.

23 July i985; accepted 26 November 1985

Expression of an Epidermal Antigen Used to StudyTissue Induction in the Early Xenpus laevis Embryo

REBECCA M. AKERS, CAREY R. PHILLIPS, NoRMAN K. WESSELLS*

A monoclonal antibody (Epi 1) has been produced that recognizes an antigenexpressed in epidermal cells ofXenpus laevs embryos. The Epi 1 antigen appears inembryonic epidermis at the end of gastrulation and is not expressed in nonepidermalstructures derived from ectoderm (for example, neural tube or cement gland). Thecapacity to express the Epi 1 antigen is restricted to cells of the animal hemisphereprior to the midblastula stage ofdevelopment (stage 8), and tissue interactions duringgastulation inhibit the expression of the Epi 1 antigen in neural ectoderm. Thisepidermal antigen will be a valuable marker for studies of ectodermal commitment.

T HE FATES OF MANY EMBRYONICcells are determined during the timebetween fertilization and gastrula-

tion. In some tissues, such as the nervoussystem, cell fate is thought to be influencedby control signals arising in other parts ofthe embryo; these interactions are referredto as "induction" (1-3). Little is knownabout the molecular events underlying in-duction. This is due in part to the fact thatthe experimental analysis of this processrelies on the appearance of morphologicalfeatures appropriate to particular tissuetypes. Tissue differentiation may be far re-moved in time from the actual inductionevents and may involve complex patterns ofcellular behavior that are not amenable tostudy by molecular techniques. One strategyis to identify molecules that are expressed ina tissue-specific fashion in the early embryoand to study the factors controlling theappearance of these molecules during devel-opment. We have produced a monoclonalantibody that distinguishes between epider-mal ectoderm and neural ectoderm at theonset of neurulation. The antigen recog-nized by this antibody is restricted to epider-mal ectoderm, and its expression in neuralectoderm appears to be inhibited by tissueinteractions. This antibody may therefore beuseful in molecular studies of neural induc-tion.

A membrane preparation obtained fromlate neurula (stages 19 to 22) Xenpus laeirembryos (4) was used to immunize mice forthe production of monoclonal antibodies(5). Several antibodies demonstrating tis-sue-specific binding were isolated and char-acterized. One of these, referred to as Epi 1,recognizes an antigen expressed in epider-mal tissue. Immunocytochemical prepara-tions of embryos processed with the Epi 1antibody exhibited intense staining in theventral and lateral surface ectoderm, whereasthe dorsal ectoderm overlying the archenter-on lacked detectable staining (Fig. 1, A andD). The boundaries of the unstained regioncorrespond to the lateral borders of thedeveloping neural plate (Fig. 1B). Immuno-cytochemical and Western blot analysis re-vealed that this antigen appears in the em-bryo at the time of blastopore closure (stage13) and persists in the epidermis throughthe feeding larva stage. At no time duringembryonic or larval development does theEpi 1 antigen appear in the nervous systemor other nonepidermal structures derived

R. M. Akers and N. K. Wessels, Department of Biologi-cal Sciences, Stanford University, Stanford, CA 94305.C. R. Phillips, Department of Zoology, University ofCalifornia, Berkeley 94720.

*To whom correspondence should be addressed.

7 FEBRUARY I986 REPORTS 613