.4,.. J. Trop. Med. Hyg., 3c@2),1981, pp. 406—421Copyright © 1981 by The American Society of Tropical Medicine and Hygiene

GENETIC VARIATION IN SCHISTOSOMA MANSONI:

ENZYME POLYMORPHISMS IN POPULATIONS

FROM AFRICA, SOUTHWEST ASIA, SOUTH

AMERICA, AND THE WEST INDIES*

MADELEINE FLETCHER,t PHILIP T. LoVERDE, AND DAVID S. WOODRUFFI

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

Abstract. To assess the genetic differences underlying geographic variation in Schistosomamansoni, individual adult worms from 22 populations, from East and South Africa, Southwest Asia, South America, and the West Indies, were processed for enzyme electrophoresison starch gels. Fourteen enzyme systems were analyzed. An estimated seven of 18 loci werepolymorphic, and the most variable population was polymorphic at six of the loci (P = 0.33),with a heterozygosity H of 0.07. These results suggest that S. mansoni is as variable genetically as most other organisms. Most S. mansoni strains showed relatively low variability,however (P = 0. 13 ±0.02, H = 0.04 ±0.005). This may be attributed to small foundingpopulations and passage in the laboratory through low numbers of infected snails and throughabnormal laboratory hosts, resulting in random fixation of alleles by the action of genetic driftand possibly in selection against particular alleles. This finding implies that geographic cornparisons of any traits should be based on several isolates from each region compared, so asto adequately sample the total variation occurring within each region. Genetic distancesbetween all strains were low (mean 0.052 , range 0—0.275), suggesting that little intraspecificdifferentiation has occurred in S. mansoni, even between Old and New World populations.These results contrast with published electrophoretic evidence of significant divergence between geographic strains of S. japonicum. Most polymorphisms were consistent with a simpleMendelian interpretation, although formal genetic crosses were not performed. For thoseenzymes, the banding patterns of heterozygotes indicated that subunit structure is the samein S. mansoni as in many other organisms. Sexual differences in mobility and in number ofbands were found in a few enzymes. The polymorphisms uncovered can eventually be usedas genetic markers to map chromosomes and to study various traits, such as infectivity tosnails and drug resistance.

There is substantial experimental evidence toshow that geographic strains of Schistosoma mansoni differ in many respects. Divergent traits in

dude minor morphological characters,' infectivityfor snails,2 response to chemotherapy,3 infectivityfor laboratory hosts, prepatency periods, egg production, and pathogenicity.@6 Yet these characters, while of epidemiological relevance, fail toprovide a basis for understanding the genetic differences underlying geographic variation. Changesin electrophoretic mobility of enzymes, on the other hand, are usually related to changes in amino

Accepted 14 October 1980.

* Address reprint requests to: Dr. Philip T. LoVerde,

DepartmentofBiologicalSciences,PurdueUniversity,WestLafayette,Indiana47907.

t Present address: Yale University School of Medicine, New Haven, Connecticut 06510.

@ Present address: Department of Biology, Universityof California at San Diego, Ja Jolla, California 92037.

acid sequence. Thus, electrophoresis of an adequate sample of enzymes provides a more directmethod of evaluating genetic differentiation within and between populations. The systematic valueof electrophoretic data has recently been the subject of extensive reviews.7'o Although the existence of considerable genetic variation has beendocumented for many organisms, information ongenetic variability of parasitic species is minimal.

The primary objective of this study was to establish baseline data on the occurrence and dis

tribution of enzyme polymorphisms in S. mansoniby means of electrophoresis of individual schistosomesfromdifferentgeographicpopulations.Inthismanner we canbegintodocument thegeneticdiversity that exists in S. mansoni and to identifypatterns of geographic variation.

This report marks the first time that specificenzyme polymorphismshave been identifiedinaSchistosoma species. Previous electrophoretic

studies―'7were based on pooled samples of

406

407GENETIC VARIATION IN SCHISTOSOMA MANSONI

FIGURE 1. World distribution of Schistosoma mansoni (reproduced from Wright@'), with the range indicatedby the shaded areas. The origins of the S. mansoni strains studied are indicated by solid circles, with the numberreferring to the number of isolates from each area.

worms. This method, however, does not allow thedetection of differences between individuals within a population, thus precluding a genetic interpretation of the data. Wright et al.'8 did use individual S. intercalatum in an isoelectric focusingstudy of seven enzyme systems but found only oneworm expressing a variant phosphoglucomutasepattern (the total number of worms analyzed wasnot given).

Information on enzyme polymorphisms willalso extend our knowledge of schistosome genetics, which at present goes little beyond thenumber of chromosomes (2n = 16) and standardkaryotype. 19.20The polymorphisms identified mayeventually provide genetic markers, which wouldbe of use in mapping chromosomes and in studying the inheritance of various traits, such as infectivity to snails and drug resistance.

Strains

studied cover most of the distribution range of S.mansoni, except for Central and West Africa, andMadagascar (Fig. 1). The samples obtained represent most S. mansoni strains currently maintamed in U.S. laboratories and, in addition, indude six fresh isolates derived from human feces.

Information on the mode of isolation and of laboratory maintenance of the different strains, together with our strain designations, are shown inTables 1 and 2. Most of the strains had been isolated from one infected human patient or from asmall number of naturally-infected snails.

Sample preparation

Infected mice and hamsters were killed by injection of a sodium pentobarbitol-heparin solution, at least 45 days postinfection. Sexually mature schistosomes were recovered by perfusionand dissection. Schistosomes were rinsed clear ofhost blood in Eagle's Minimal Essential Mediumwith Earle's salts (Grand Island Biological Co.,New York, USA). Individual motile male and female worms were then crushed with a groundglass stopper on the frosted end of a glass slide in10 pi of distilled water at room temperature (re

MATERIALS AND METHODS

Mice, hamsters, snails, or human feces infectedwith S. mansoni were obtained from differentlaboratories in the U.S. and abroad. The strains

DateRemarksonStrainsisolated LocalityMode of isolation laboratory maintenance Nt

408 FLETCHER, LOVERDE, AND WOODRUFF

TABLE 1

Origin and laboratory maintenance of Old World Schistosoma mansoni strains *

Southwest AsiaSAR 1977 Saudi Arabia Feces from 3—5 Worm recovery 40—45%; 31

light to passaged through 10—20B.moderate glabrata (Puerto Rico,infections susceptibility over 80%) for

10—12 generations

East AfricaEGY1 1977 Qualyub, Egypt About 30 snails Passaged through 10—50B. 10—50

infected from alexandrina (Qualyub,human source susceptibility 50-80%)

EGY2f 1978 Qualyub, Egypt About 30 snails Passaged through 10—50B. 53infected from alexandrina (Qualyub,human source susceptibility 50—80%)(differs fromEGY1)

KEM 1968 Nairobi, Kenya 20—30snails Worm recovery 30—35%; 25infected from passaged through 10—20B.human source sudanica (Ethiopia,

susceptibility 50—80%)for30—40 generations

KEB 1@ 1968 Nairobi, Kenya Same origin as Passaged through baboons, overKEM worm recovery 20—65%; 10

usually passaged throughover 20 B. pfeiffer-i(Tanzania, susceptibility50—80%; sometimes lower)for three generations

KEB2* 1968 Nairobi, Kenya Same origin as Same as KEB1, but different NA―KEM pool of shedding snails

TANF@ 1979 Mwanza, 5 snails infected Passaged in S B. sudanica 5Tanzania with feces from (Tanzania, susceptibility

about S light below 50%), then onceinfections through albino mice

TANL 1966 Mwanza, About 50 snails Usually passaged through aboutTanzania infected with over 20 B. glabrata (Puerto 20

feces from 1 Rico, susceptibility overinfected 80%) for 45 generationsindividual

South Africa

SA11@ 1979 Near Neispruit, 19 snails infected Passaged in 15 B. pfezfferi 19Transvaal, with feces from (South Africa) and 4 B.South Africa 1 10-year-old glabrata (Puerto Rico), then

once through albino mice

SA2t 1979 Same as SA11 8 snails infected Passaged in 8 B. pfeifferi 8with feces from (South Africa), then once1 10-year-old throughalbinomice

* All strains were passaged through albino mice unless otherwise stated.t Number of infected snails from which the worms processed were derived.* Freshisolates.* Passaged through baboons since 1968, but derived from the same original isolate as KEM.‘Information unavailable.

409GENETIC VARIATION IN SCHISTOSOMA MANSONI

sults did not differ when samples were preparedat 4°C).The resulting homogenate was immediately absorbed on a 9.5 x 5 mm tab of chromatography paper (Whatman No. 1), which was then

inserted along a cut made across the width of acold starch gel. Up to 23 individuals could thusbe processed at the same time.

Electrophoresis

Horizontal electrophoresis was performed on

12.5% starch gels (Lot 307 , Electrostarch Co.,Madison, Wisconsin, USA) at 4°C, following the

techniques described by Ayala et al.21 Starch wasused in the present study, because it has been themedium of choice in most surveys of genetic variation and comparisons with the existing literature

would thus be facilitated. Following electrophoresis, each gel was sliced horizontally into five

slices, 2 mm thick. The top slice was discarded,and each remaining slice was stained for a different enzyme system, thus allowing four enzymeassays to be obtained from each individual schistosome. The gel slices were stained at 37°Cin thedark, until the enzyme patterns developed. Thestained gel slices were then rinsed and fixed in a5:5: 1 solution of water, methanol, and glacial ace

tic acid. Enzyme activity patterns were recordedand photographed immediately.

The electrophoretic buffer and stain recipes arethose given in Fletcher et al.22 The following 14enzyme systems were analyzed, with the abbreviation and enzyme commission number23 givenin parentheses: glucose-6-phosphate dehydrogenase (G6PD, E. C. 1. 1. 1.49), glutamate dehydrogenase (GDH, E.C. 1.4. 1.3), lactate dehydroge

nase (LDH, E.C. 1. 1. 1.27), malate dehydrogenase(MDH, E.C. 1. 1. 1.37), acid phosphatase (ACP,E.C. 3.1.3.2), alkaline phosphatase (AKP, E.C.3.1.3.1), mannose phosphate isomerase (MPI,E.C. 5.3.1.8), phosphoglucose isomerase (PGI,E.C. 5.3.1.9), adenylate kinase (AK, E.C.2.7.4.3), aldolase (ALD, E.C. 4.1.2.13), leucyl

glycylglycine aminopeptidases (LGG, E. C.3.4.1.3), hexokinase (HK, E.C. 2.7.1.1), NADH

diaphorase (DIA, E.C. 1.6.2.2) and phosphoglucomutases (PGM, E.C. 2.7.5.1). The only differ

ence from the methods given in Fletcher et al.22was that both buffer system 1 (amine citrate pH7.0), and buffer system 2 (amine citrate pH 6.4),

were used to differentiate the five malate dehydrogenase alleles detected.

Designation of electromorphs

The term “electromorph―is used to refer to aband of enzyme activity.24 To designate a locuscoding for a specific enzyme, the same abbreviation used to represent the enzyme is set in lowercase and italicized. When several forms of thesame enzyme exist, each controlled by a differentlocus, the electromorphs or sets of electromorphsrepresenting separate loci are numbered sequentinily, from the most anodal to the most cathodal.Allelic variants of enzymes encoded by the samelocus are designated a, b, c, . . . in order of decreasing electrophoretic mobility. Thus Mdh-larepresents the most anodal allelic variant of themost anodal enzyme encoded by one of the locicontrolling the malate dehydrogenases. To represent a given genotype, the two alleles presentare separated by a slash, as in Pgm-2a/b. In thecase of ALD and LDH, a simple genetic interpretation could not be made, and so the differentphenotypes are designated by capital letters, e.g.,

Ldh-N.

To prevent misidentification of electrophoreticvariants, schistosornes sampled from unfamiliarstrains were processed on the same gel with schis

tosomes from strains whose electromorphs hadalready been identified. Occasionally, blood samples from mice or hamsters or samples of Biom

phalaria head-foot tissue were also included as acheck against host contamination of the schistosome homogenates.

RESULTS

Assays for 42 enzymes were tested using 15 dcc

trophoretic buffer systems. Good resolution wasachieved with 14 enzyme systems. The differentbanding patterns observed for each of these enzymes, obtained from individual worms from thevarious S. mansoni strains, are illustrated in Fig

ure 2 . Five other enzymes, carbonic anhydrase,esterase, glyceraldehyde-3-phosphodehydrogenase, 6-phosphogluconate dehydrogenase, andtetrazoliurn oxidase, showed some activity instained gel slices, but, because resolution was

poor, they were not included in the electrophoreticsurvey.

Progeny studies to verify the genetic basis of

electrophoretic differences between individualschistosomes (Fig. 2) were not performed. However, the following observations support the as

DateRemarksonStrainsisolated LocalityMode of isolation laboratory maintenance Nt

410 FLETCHER, LOVERDE, AND WOODRUFF

TABLE 2

Origin and laboratory maintenance of New World Schistosoma mansoni strains*

West IndiesNMRI early Puerto Rico NAt Worm recovery 30—40%; 400—500

40's snail susceptibility over80%; before 197 1 strainwas passaged through10—20snails

NPR1* 1950 Arecibo, NW. About 170 Snail susceptibility over NAPuerto Rico naturally- 80%

infectedB.glabrata

PRl@ 1950 Arecibo, NW. Same origins as Snail susceptibility over 10—30Puerto Rico NPR1 80%; passaged through

10—20snails

JHPR early Puerto Rico NA Snail susceptibility over 740's 80%; after 1975 strain

was passaged throughover 20 snails

PR2 1975 Luquillo, N.E. Feces from 1 NA 28

Puerto Rico acute patient

PR3 1976 Boquerón,SE. Feces from 1 4 substrains differing in NAPuerto Rico chronic patient infectivity to snails

were studied

PR9 1977 Boquerón,SE. 6 B. glabrata 7 substrains differing in NAPuerto Rico infected with infectivity to snails

feces from 1 were studiedchronic patient

PR13 1977 Boquerón,SE. Feces from 1 5 substrains differing in NAPuerto Rico chronic patient infectivity to snails

were studied

PRFA― 1979 Boquerón, SE. 91 B. glabrata Passaged once through 91Puerto Rico infected with albino mice

feces from 1chronic patient

PRFD' 1979 Boquerón,SE. 6 B. glabrata Passaged once through 6Puerto Rico infected with albino mice

fecesfrom1chronic patient

STLU 1967 St. Lucia NA 3 substrains differing in NAinfectivity to snailswere studied

South AmericaVENZ 1948 Venezuela About 60 Snail susceptibility over 7

naturally- 80%; usually passagedinfectedsnails throughover20snails

(Venezuela)

BRAZ 1974 Catolandia, 12—15naturally- Worm recovery30—40%; NABahia, Brazil infected snails snail susceptibility 50—

80%; passaged through10—20snails (Bahia) for18—20generations

* All strains were passaged through albino mice and B. glo.broJo from Puerto Rico, unless otherwise stated.t Number of infected snails from which the worms processed were derived.* Information unavailable.* Derived from the same original isolate but maintained in different laboratories.‘Freshisolates.

a bca/ba,tb/ca ba/baaba/b+(d)Mdh-1(e)Mdh-@(f)@j@malefemale

GENETIC VARIATION IN SCHISTOSOMAMANSON! 411

sumption that the variation observed is genetic:1) the number of bands in presumed heterozygotes was usually consistent with the subunitstructure typical of the corresponding enzyme in

other organisms. For example, malate dehydrogenases are usually dimeric (i.e. , composed of twosubunits), thus heterozygotes show three bands ofactivity.25 Presumed heterozygotes for two S.mansoni malate dehydrogenases, Mdh-1 andMdh-2, did show three bands of activity (Figs. 2,4), suggesting genetic variation affecting dimeric

molecules; 2) for all presumed polymorphic loci,electrophoretic variants were not distributed randomly among the strains. Rather, each variantwas found at specific frequencies in some of thestrains and was absent altogether in others, andthese strain-specific differences were consistentfrom gel to gel. This was true even of two polymorphic enzymes, AId and Ldh, for which heterozygotes could not readily be identified. Thisagain suggests that the variation is genetic ratherthan the result of differences in experimental conditions; 3) for those schistosome populations derived from a large number of infected snails, theobserved phenotypic class proportions were usually in close agreement with those expected on thebasis of the Hardy-Weinberg model for a randomly mating population, with correction for bias insmall 26

None of the schistosome enzymes coincided inmobility with analogous enzymes in mouse orhamster blood or in Biomphalaria head-foot tissue. There were no differences in the mobility ofschistosome electromorphs that could be relatedto differences in the age of the adult worms processed. Schistosome electromorph mobility wasalso not affected by passage through a differentspecies of final or intermediate host (this does notexclude the possibility of host-related differencesin the frequency of specific schistosome electromorphs). For example, four of the schistosomestrains in the study, EGY1 and 2 and PR! and 2,

could be passaged through mice or hamsters without affecting electromorph mobility. Likewise, enzymes derived from worms of the SAil strainpas

saged through B. glabrata did not differ inelectromorph mobility from those from worms of

the same strain passaged through B. pfe@fferi. Thepossibility that differences in band patterns between individual schistosomes reflected geneticvariation in the snails through which they cycledrather than in S. mansoni could likewise be discounted; for instance, two strains in the study,

+ (a) PGM (b) LGG (c).@gj.male female

—_— 1811

L.oo-3

= = = = = =@— — — — = j@es-I

0

0

a ba/b a baYb'a ba/bc b/c A BC

+ (g) i=@ti (h) monomorphic loci

pooledmole females

@:::;@1@N L H T B F

FIGURE 2 . Electrophoretic patterns for 14 enzymesystems in individual male and female Schistosomamansoni. The direction of migration is indicated by anarrow with + = anode, —= cathode, 0 = point ofsample insertion. Loci, allele, and phenotype abbreviations are explained in text.

PR! and 2, were passaged many times throughthe same snail strain, but PR2 showed variationin Mdh-1, Ldh and Lgg-1, while PR! was invariant at all loci examined.

Genetic interpretations

In the absence of formal genetic crosses, certainassumptions were made to facilitate genetic interpretations and analysis of the data. These geneticinterpretations remain to be verified by progeny

studies.When for a given enzyme system two zones of

activity varied independently of each other, or ifone showed variation while another did not, twodifferent loci were assumed to be involved. Whenan electromorph or set of electromorphs showedno variation, it was assumed to be controlled bya single locus without allelic variation. Each ofthe variable zones of activity was assumed to becontrolledby a singlepolymorphiclocus,evenwhen the variation was not readily interpretable,asinLDH and ALD. Thus,estimatesofthenum

0

412 FLETCHER, LOVERDE, AND WOODRUFF

quencies were in accordance with those expectedon the basis of the Hardy-Weinberg equilibrium.

Leucylglycylglycine aminopeptidases. Theaminopeptidases detected may be relatively nonspecific, since they hydrolyzed at least one other

tripeptide (L-phenylalanyl-L-leucine) besides L-leucylglycylglycine. Three loci are probably involved,

of which only one, Lgg-1, was polymorphic, withtwo alleles segregating in most S. mansoni populations. The heterozygote was usually detectedas a broad band of activity encompassing the positions of the slow and fast bands representing thetwo alleles (Fig. 2); under optimal conditions,however, the heterozygote appeared to be doublebanded, suggesting that Lgg-1 is monomeric, asare aminopeptidases in many other organisms.25In half of 12 cases in which the chi-square testcould be applied, the observed allele frequencieswere not in accordance with those expected on thebasis of the Hardy-Weinberg equilibrium. In eachof these six cases, the discrepancy could be attributed to a deficiency in the number of heterozygotes. Since the two electromorphs stained heavilyand differed little in their migration, it was oftendifficult to determine whether an individual was

homozygous or heterozygous. It is possible thatsome individuals classified as homozygous werein fact heterozygous.

Phosphoglucose isomerase. This enzyme waspolymorphic in S. mansoni from Kenya, with two

alleles detected. The heterozygote was triplebanded (Fig. 2), suggesting that S. mansoni PGIis a dimeric enzyme, as is typically the case in

other organisms.25 Because of the low frequencyof the Pgi-b allele, it was not possible to apply thechi-square test for goodness of fit with the HardyWeinberg model.

Malate dehydrogenases. Two loci were involved, both polymorphic, with three alleles detected for Mdh-1 and two alleles for Mdh-2. Forboth loci, the heterozygotes were triple-banded(Fig. 4), suggesting that malate dehydrogenasesare dimeric in S. mansoni. In the three cases (PR2and two STLU substrains) in which the chi-squaretest could be applied, Mdh-1 allele frequencieswere in accordance with those expected on thebasis of the Hardy-Weinberg equilibrium. Therewere no cases suitable for analysis for Mdh-2,because the frequency of the Mdh-2a allele wastoolow.

Aldolase. Three single-banded variants differing in mobility were found (Fig. 2). Genetic interpretation is difficult, because heterozygotes,

..... ••ff

3

II@

@ m@

4

sIEie@:ea5FIGURES 3—5. Enzyme polymorphisms in Schisto

soma mansoni. 3. Individual male worms stained forphosphoglucomutases; topmost band is Pgm-1 ; genotypes for Pgm-2 are, from left to right, a/a, a/b, a/a, andthe six following individuals are b/b. 4. Individual maleworms stained for malate dehydrogenases; genotypes forthe anodal Mdh-1 are, from left to right, first four a/b,then a/a, b/b, a/b, b/b, a/b, b/b; genotypes for the cathoctal Mdh-2 are all b/b. 5. Individual male wormsstained for lactate dehydrogenase; phenotypes are, fromleft to right, N, L, N, L, N, N, N, L, N, N. See textfor enzyme, allele, and phenotype designations.

ber of loci involved in the study and of the proportion of polymorphic loci are conservative.

Polymorphic enzymes

Phosphoglucomutases. Two loci are involved.Pgm-1, a doublet, was monomorphic in all populations, and Pgm-2 was polymorphic, with threealleles detected in the populations examined. Theheterozygotes were double-banded (Fig. 3), suggesting that S. mansoni Pgm-2 is monomeric, as

is typical of phosphoglucomutases.25 In the twocases (EGY2 and SAl!) in which expected phenotypic classes were large enough for the chisquare test (not less than one),27 Pgm-2 allele fre

GENETIC VARIATION IN SCHISTOSOMAMANSONI 413

usually identified by multiple banding, were notdetected. In the absence of progeny studies, it wasassumed that the three different electromorphsrepresent three alleles at the same locus.

Lactate dehydrogenase. Five electromorphs

were present for individual male worms, as ischaracteristic of most vertebrates.25 In other organisms, the five bands are accounted for by therandom association of two different polypeptidesubunits to form five tetramers. No activity wasdetected for individual female worms. Two typesof variation were encountered in male worms.The most common form of variation was in therelative activity of the five bands (Figs. 2 , 5).Numbering the electromorphs from the anode tothe point of sample insertion, bands showinggreatest activity were 1, 3 , and 4 for the mostcommon phenotype, Ldh-N; 1, 4, and 5 for LdhL; and 1 and 3 for Ldh-H. This suggests variationat a locus regulating the relative amounts or activity of the five LDH enzymes rather than at alocus directly coding for LDH subunits. Tissuespecific variation in the relative amounts of thetwo types of subunits, leading to a gradual increase in intensity of the five LDH bands towardsthe anode or towards the cathode, is known.25Variation between individuals of the type seen inS. mansoni, on the other hand, has apparentlynot been reported. Cases in which polymorphismsin regulatory genes cause quantitative variationin enzyme activity between individuals are knownfor other enzymes.25

Another form of variation, seen in some PuertoRican strains, affects the mobility of all five bandsof activity. Two variant phenotypes were present,Ldh-T and Ldh-B, Ldh-B electromorphs beingblurred, and intermediate in mobility betweenLdh-T and Ldh-N electromorphs. This could represent variation in a locus coding for LDH subunits. Again this form of variation is atypical, because a mutation in one of the two types ofsubunits would result in the formation of 15-banded heterozygotes. The variation in the mobility ofS. mansoni LDH enzymes must be due to achange affecting both subunits, since the mobilityof all five bands is modified.

The two types of variation described could represent variation at two different loci, but this hypothesis remains wholly conjectural in the absenceof progeny studies. Manwell and Baker revieweda number of cases in which polymorphisms inboth structural and regulatory genes result incomplex patterns of enzyme variation.25

In calculating heterozygosity, H, a measure ofgenetic variation, and genetic distance, D, a measure of genetic differentiation, the five Ldh phenotypes were assumed to be five “alleles―at onelocus, as were also the three Aid phenotypes.Equating phenotypic classes to alleles would tendto bias the estimate for H slightly downward andthat for D slightly upward, as there are usuallymore phenotypic classes than alleles.

Monomorphic enzymes

Lgg-2, Lgg-3, Gopd, Gdh, Hk, Akp, Acp, Mpi,and Ak were represented by one band of activityunder the electrophoretic conditions used, whilePgm-i and Dia appeared as doublets (Fig. 2).

One of the enzymes classified as monomorphic,G6PD, showed very slight variation in electromorph mobility between individuals, but with noconsistent pattern and no occurrence of multiplebanding indicative of heterozygotes. Such slightindividual variation in G6PD mobility was notedfor wild-caught butterflies by Johnson,28 who suggested that genetic factors may be involved. Morerefined techniques may clarify the situation in thecase of S. mansoni GÔPD, and may uncover ad

ditional genetic variation, even in apparentlymonomorphic enzymes.293o

Sexual differences

Differences in activity, mobility, and numberof bands were apparent between individual female and male worms for some enzymes (see Fig.2). Extracts from single female worms usually

stained less intensely than those from single maleworms, and for LDH and AK there was usuallyno detectable activity for individual females.LDH and AK activity was present, however,when pooled samples of female worms were analyzed, and in this case LDH electromorphs fromfemales migrated faster than those from males(Fig. 2). Conde-del Pino et al.@ and Coles'3 alsofound sexual differences in LDH mobility. Md/i2a and b electromorphs from female worms were

distinctly slower in mobility than those from males(Fig. 2). Neither Conde-del Pino et al.1' nor Coles'3found sexual differences in MDH mobility. Ingels stained for LGG, a band of activity was de

tected in extracts from female worms that wasabsent in those from males (Fig. 2), although theband could be present in males but too close inmobility to Lgg-3 to be detected.

414 FLETCHER, LOVERDE, AND WOODRUFF

061

056

050

0.44

039

0.33

028

022

0.17

0.11

0.06

0

P

H

0

0.18

0 16

0 14

012

010

008

0.06

004

002

093 OTHER

INVERTEBRATE

SPECIES32

FIGURE 7 . Average heterozygosity per locus H inthe various Schistosoma mansoni strains studied. SeeFigure 6 for an explanation of the symbols used.

Iflwns@

saii @.eas

+‘@f6fresI,solmes

th=9?@e 93OTHER1j@9@ja4Oyears -J IIWERTEBRATE

@ MANSONI SPECIES32

FIGURE 6. Proportion of polymorphic loci P in thevarious Schistosoma mansoni strains studied. The solidcircles represent the means, the bars the standard errorsof the mean, and the vertical lines the ranges. The numbers by the means are the number of P values averaged.

Differences in activity could be attributed to thesmaller size of female schistosomes, as well as todifferences in specific activity.3' The extra LGGband could be a sex-limited enzyme, as is spermspecific LDH in certain birds and mammals.25 The

mobility differences in the Mdh-2 and Ldh enzyrnes, on the other hand, were unexpected andmay well be the first reported cases of sexual differences in enzyme mobility. This could be because most other organisms analyzed are largeenough so that homogenates from specific organsare used rather than whole body extracts. In S.mansoni, the reproductive system takes up muchof the body volume and could create enough of achemical difference between male and femaleworms to affect the electrophoretic mobility ofsome enzymes, for example by slight differencesin pH or the binding of sex-specific molecules tothe enzymes. Alternatively, the sexual differencesin enzyme mobility could be due to more subtledifferences in metabolism, such as NAD availability. It should be determined whether thesesexual differences hold for cercariae as well, sincethis would be of practical importance in determining the sex of cercariae in monomiracidialsnail infections.

2

f6T possoged

t flwo@t Pseosm10! 3 qese!o!oos

15lest! sololes

possogedth'ot.tghmce

totlto4Oyeo,s _________________S MANSONI

There were occasionally sexual differences inallele frequencies at polymorphic loci, but theseare most likely due to the small number of schistosome genomes represented in the samples analyzed.

Genetic variation

One quantitative index of genetic variation, P,is simply the proportion of loci that are polymor

phic in a given population. The range of P valuesfound for different groups of organisms is verylarge, but usually P tends to fall between 25 and50% . ‘°For natural populations of 93 inverte

brates, P = 0.397 ±0.20!.32

An estimated seven of 18 loci, or 39% , werepolymorphic in at least one S. mansoni population. In individual strains, P varied from 0—0.33(see Tables 3 and 4, Fig. 6). P averaged over allS. mansoni strains was a comparatively low

0.13 ±0.02. The average P for six recent isolateswas slightly higher (Student's t-test, 0.05 level ofsignificance) than that for 15 mouse-adaptedstrains (Fig. 6). The highest P values, 0.28 to0.33, were for two samples from an S. mansonistrain from Kenya that had been maintained in

GENETIC VARIATION IN SCHISTOSOMA MANSONI 415

TABLE 3

Allele and phenotype frequencies at polymorphic loci for male worms of Old World Schistosoma mansoni strains;11 other loci were monomorphic

Strains

Loci* SAR EGY1 EGY2 KEM KEB1 KEB2 TANF TANL SAil 5A2

Pgm-2 a 0.63 0.44 0.08 0.06 0.61 0.29 0.20b 0.99 0.37 0.56 1.00 0.89 0.94 0.33 1.00 0.54 0.70c 0.01 0.03 0.06 0.17 0.10

d 69 153 78 58 47 18 9 31 26 159 29 30 38 36 13 21 0 10 11 6

Lgg-1 a 1.00 1.00 1.00 1.00 0.67 1.00 1.00 0.72 1.00 1.00b 0.33 0.28

cf 41 9 45 66 26 17 6 152 50 45

9 11 4 4 31 8 2 0 17 1 20

Ldh N 1.00 1.00 1.00 0.75 0.22 1.00 0.75 0.92 0.81L 1.00 0.04 0.04 0.25 0.08 0.19H 0.21 0.74TB

97 236 45 78 48 23 11 52 40 26

Mdh-1 ab 1.00 1.00 1.00 1.00 0.98 0.98 0.95 1.00 1.00 1.00c 0.02 0.02 0.05

d 169 281 142 132 66 53 21 121 43 359 36 189 39 59 15 10 0 7 1 15

Mdh-2 a 0.78 0.89 0.88 0.91 0.03 0.06b 1.00 1.00 1.00 0.22 0.11 0.12 0.09 1.00 0.97 0.94

d 169 281 142 149 66 53 22 111 43 359 36 189 39 64 7 7 0 3 1 11

Aid A 0.10B 0.25 1.00 0.50 0.30C 0.75 1.00 1.00 1.00 1.00 1.00 1.00 0.40 0.70

d 88 99 87 93 43 34 2 31 8 10@ 26 46 25 27 16 2 0 12 19 6

Pgi a 1.00 1.00 1.00 1.00 0.96 0.96 1.00 1.00 1.00 1.00b 0.04 0.04

d 133 150 35 118 57 48 16 43 23 20

? 37 32 20 50 16 16 0 4 0 5

P1: 0.11 0.06 0.06 0.06 0.33 0.28 0.17 0.11 0.22 0.22

H* 0.02 0.03 0.03 0.02 0.07 0.05 0.04 0.04 0.08 0.07

a Abbreviations of enzyme loci, alleles, and phenotypes are explained in text;@ = number of male worms processed; 9 = number of female wormsprocessed.

t OfllYmalewormsshowedactivityfor 14k.* Proportionof polymorphicloci.* Averageheterozygosityper locus.

baboons since 1968. For the same 1968 isolate the two strains can be attributed to differences inmaintained in a different laboratory in mice, a laboratory maintenance. The most striking differmuch lower P of 0.06 was obtained. Of the five ence was in the number of generations of laboloci that were polymorphic in the baboon-adapted ratory passage: three for the baboon-adaptedstrain but not in the mouse-adapted strain, two strain versus 30—40 for the mouse-adapted strain.

showed variants that were absent in any of the There were also differences in the number of inmouse-adapted strains examined (Pgi-b and Ldh- fected snails used to cycle the two strains (10—20

H). The possibility that these variants are selected snails for the mouse-adapted strain versus over 20out in mice should be explored further. Nonethe- snails for the baboon-adapted strain), in the numless, at least part of the discrepancy in P between ber of cercariae used to infect the animals, and in

416 FLETCHER, LOVERDE, AND WOODRUFF

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GENETIC VARIATION IN SCHISTOSOMAMANSONI 417

the snail species used as intermediate host (although snail susceptibility was moderate in bothlaboratories). Another pair of strains originatingfrom the same 1950 isolate but maintained in diiferent laboratories, PR1 and NPR1, also differedin P, with P = 0 and P = 0. 11, respectively.

A second measure of genetic variation is provided by the average proportion of heterozygotesper locus in a population, H, estimated from allelefrequencies at each locus. Usually H tends to fallbetween 5 and 15% .‘°For natural populations of93 invertebrates, H = 0.1123 ±0.0720.32 In theS. mansoni strains examined, H ranged from 0—0.08, with a mean of 0.04 ±0.005 (see Tables 3,

4, Fig. 7). The average H for six recent isolateswas significantly higher at the 0.005 level thanthat for 15 mouse-adapted strains. Again H forthe baboon-adapted strain was high (0.05 to 0.07),while the highest H (0.08) was for a recent isolate(SAl 1).

Geographic variation

For each of the strains examined, allele frequencies at polymorphic loci for male worms areshown in Tables 3 and 4, together with the number of female and male worms processed. Becausegreater activity was obtained with male worms,more males were processed than females.

The inferences on geographic variation thatmay be derived from such data are limited for thefollowing reasons: 1) the strains are probably notrepresentative of natural populations, because ofsmall founding populations and prolonged laboratory maintenance (see Tables 1, 2); 2) the actualnumber of genomes sampled from a given straincan only be estimated (from the number of infected snails used to infect the animals from whichthe samples were derived, when known), sinceasexual reproduction occurs in the snail; 3) in mostcases, laboratory animals were infected with cercariae obtained from a group of shedding snails;

because some snails release many more cercariaethan others, some schistosome genomes may havebeen disproportionately represented relative toothers. These limitations must be kept in mind inattempting to analyze geographic variation in S.mansoni by means of the data in Tables 3 and 4.

In all strains examined, monomorphic loci werefixed for the same allele. This is characteristic ofconspecific natural populations.7 At each polymorphic locus there was at least one electrophoretic variant common to all geographic regions.

Only two strains were fixed for less common variants, KEM (for Ldh-L and Md/i-ic and JHPR(for LDH-L). Because both strains had beenmaintained in the laboratory for over 10 years,these cases can probably be accounted for by theaction of genetic drift, causing random fixation ofalleles in small populations. Seven relatively uncommon variants at five loci were found exclusively in African strains (Mdh-ic, Mdh-2a, Pgm2a, Ldh-H, Ald-A, and B, and Pgi-b), while threevariants were restricted to American strains(Mdh-ia, and Ldh-T and B).

A quantitative measure of the amount of geneticdifferentiation between strains was obtained bycalculating Nei's index of genetic distance D33 foreach pairwise comparison of populations, usingthe data on allele frequencies. D is defined as aminimum estimate of the average number of codon differences per locus between two populations. The formula for Nei's D is given by:

D = —lnI,

where I is an index of genetic identity varyingfrom 0 to 1, defined as the normalized identity ofgenes, with the equation for I given in Nei.33 Byusing the allele frequency data in the present studyto calculate I and D, it is assumed that the lociinvestigated are a representative sample of structural loci in the S. mansoni genome as a whole.It must also be stressed that the estimates of I andD calculated relate only to the strains studied, not

to actual geographic populations of S. mansoni.The index of genetic identity I averaged over all666 pairwise comparisons of S. mansoni populations was a comparatively high 0.950, well withinthe usual range for conspecific populations.a@ Twostrains, TANF and KEM, diverged significantly

from the other strains studied, and, to a muchlesser extent, so did KEB2 and JHPR. As discussed above, their genetic distinctiveness can be

attributed to genetic drift. (For TANF, samplingerror may be implicated, because the number ofschistosomes examined was very small.) Whendivergent populations are eliminated from the calculations so that the average genetic distance between African strains (D = 0.045 ±0.007) is thesame as that between American strains (0.044 ±0.005), the average genetic distance between African and American populations (0.062 ±0.006)is not significantly greater at the 0.025 level than

the average genetic distances within each continent. Thus the apparent existence of variants restricted to one or the other continent does not aS

418 FLETCHER, LOVERDE, AND WOODRUFF

fect the genetic distance between African andAmerican strains, probably because these particular variants are mostly present at low frequencies.

DISCUSSION

Although electrophoresis of pooled schistosomeshas provided indirect evidence of individual vari

@7this is the first report on enzyme poly

morphisms in a Schistosoma species, based on

electrophoresis of single worms. An estimated seven of 18 loci were found to be polymorphic invarious populations of S. mansoni, with P ranging up to 0.33 and H up to 0.08. This suggeststhat S. mansoni is as variable genetically as most

other organisms (see Figs. 6, 7). This is significantin view of the fact that the reduced morphologicalcomplexity of parasites and physiological dependence on their hosts might lead one to assume aparallel reduction in genetic complexity. On thecontrary, high levels of genetic variation were alsoencountered in a nematode parasitic in fishes,35while a DNA renaturation study showed highergenetic complexity in several helminth parasitesthan in their free-living relatives, based on thenumber of nonrepetitive DNA sequences.36

However, estimates of P and H were low formost S. mansoni populations studied, with the

average values for recent isolates only slightlyhigher than those for laboratory strains (Figs. 6,7). This may be attributed to several factors: 1)

typically, the strains examined were isolated fromone human patient or from a small number ofnaturally-infected snails; these isolates probably

lacked the full array of alleles present in the natural population of origin (the founder effect);37 2)laboratory strains were probably subjected to atleast one population bottleneck (i.e. , a severe reduction in population size), by being occasionallypassaged through a low number of infected snails,

for instance; this would lead to further restrictionsto the gene pool through genetic drift; 3) the cycling of schistosomes through laboratory hostsmight have subjected the parasite to selectionagainst particular alleles. One example of this,which remains to be confirmed, could be the absence in any of the mouse-adapted strains exammed of two phenotypes, present in the strainmaintained in baboons. Coles noted the loss of

two bands of MDH activity in pooled samples ofS. mansoni after two passages through@ A

similar loss in two bands of MDH activity in

pooled samples of S. bovis and S. leiperi, occurring after four passages through mice, has alsobeen reported. 16.17 However, these data are inconclusive in the absence of information on allelefrequencies and on the number of snails used topassage the strains; the loss of two bands could bedue, for instance, to genetic drift causing fixationof an allele at a locus coding for a dimeric enzyme.In the present study, all five MDH alleles detectedwere segregating in mouse-adapted strains, evenin laboratory strains of long standing. Geneticchanges in S. mansoni strains passaged in the laboratory have been documented, however. Examples include alterations in infectivity to intermediate and final hosts,―38 and changes in thearrangement of sensory receptors in cercariae and

miracidia from human strains passaged in micesuch that the mouse-adapted strains came to resemble strains cycling through rats in the wild.'

The possibility that laboratory strains of S.mansoni may lack much of the genetic variabilityfound in natural populations and may be otherwise atypical because of founder effects, randomgenetic drift, and inbreeding should be an issueof major concern to workers involved in medicaland epidemiological research. Such research areasinclude drug trials, immunology, and geographiccomparisons of pathogenicity to laboratory mammals and of infectivity to snails. In particular, thecommon practice of considering one strain as representative of an entire geographic area is erro

neous. The significance of differences betweengeographic strains cannot be evaluated withouttaking into account differences between isolatesfrom the same locality. Thus, geographic comparisons of any trait should be based on a numberof isolates from each region compared. This alsoapplies to comparisons of electrophoretic patternsbetween different species (discussed by Fletcheret al.3@).

Careful records should be kept of the localityof origin of any given strain and the manner inwhich it was isolated (i.e. , the number of naturally-infected snails or of human patients used assource), as well as of the mode of laboratory maintenance. When it is important that experimentalstrains be as representative as possible of natural

populations, care should be taken in the originalisolation of the strain and in subsequent laboratory maintenance to retain as much genetic variability as possible (e.g. , by using large numbersof naturally-infected snails or several human patients as source and by using at least 50—100shed

GENETIC VARIATION IN SCHISTOSOMA MANSON! 419

ding snails to maintain the cycle). The choice oflaboratory host may also be important. In someinstances, on the other hand, it may be preferableto use strains of limited genetic variability. Forexample, to study the genetics of inheritance of aparticular trait, such as infectivity to snails ordrug resistance, it is necessary to cross individualsfixed for different alleles regulating the trait, withthe overall genetic background remaining as constant as possible.

The present study shows that genetic divergence between geographic strains of S. mansoniis slight. The is a significant finding, because it

contrasts with evidence of marked intraspecificdifferentiation in two other human schistosomes,S. haematobium and S. japonicum. For these

species, intraspecific divergence, as evidenced inparticular by intermediate host-parasite relation

ships, may have proceeded to the stage where

species or subspecies status may by warranted forthe different strains. 40.41At least for S . japonicumthis is supported by electrophoretic evidence.39

The lack of large genetic differences betweenAfrican and American strains of S. mansoni, asevidenced by electrophoretic data, lends supportto the theory that S. mansoni was introduced relatively recently into the New World, via the extensive slave trade of the 15th to 19th centuries.42 Seven variants were apparently restrictedto African populations, while three variants wererestricted to American populations. The reduced

number of variants in American populations, ifreal, may reflect founder effects. However, thepresent study is limited as an assessment of geographic differentiation in S. mansoni, becauseadequate samples of natural populations could notbe obtained. Looking at more isolates will doubtless uncover more genetic variation, because P is

strongly dependent upon sample size.43 Thus someof the alleles that seem at present to be restrictedto definite geographic areas may in reality be morewidespread.

Further studies of genetic differentiation inSchistosoma species through enzyme electrophoresis should be based on a number of field isolates

from each geographic area. To be able to estimateallele frequencies in natural populations, it wouldbe preferable to analyze directly cercariae derivedfrom individual snails, each infected with onemiracidium obtained from human stools, ratherthan adult schistosomes.

This study paves the way for more in-depthwork on S. mansoni genetics. The polymorphic

loci uncovered could be used as genetic markers,once the mode of inheritance of each polymorphism is determined through genetic crosses, andlinkage relationships established. A correlationbetween degree of infectivity to snails and the frequency of certain LDH variants, suggestive ofchromosomal linkage, has already been shown onthe basis of electrophoretic data.22

ACKNOWLEDGMENTS

We thank Ms. W. Knight and Drs. R. Damian,R. Kuntz, Y. S. Liang, E. Loker, R. J. Pitchford,

C. S. Richards, M. Stirewalt, and P. 5. Visser forproviding us with strains of Schistosoma mansoni; Dr. R. K. Selander for help in finding enzyme systens suitable for analysis in S. mansoni;Drs. V. Ferris and M. Levy, and Mr. M. Goldman for critical evaluation of the manuscript;John DeWald for technical assistance and BettyGick for typing services.

This work has been supported, in part, by

grants from the Edna McConnell Clark Founda

tion and NSF, RIAS SER7706731.

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