A 56

The Mekong Schistosome, 1980, pp. 113-122MDUzcologiCDlReview, supple 2

GENETIC DIFFERENTIATION BETWEEN 'SCHISTOSOMA MEKONG!AND S. JAPONICUM: AN ELECTROPHORETIC STUDY

, Madeleine Fletcher, David S. Woodruffl and Philip T. Loverde

Department of Biological SciencesPurdue University

West Lafayette, Indiana 47907, U.S.A.

and

Harold L. Asch

Center for Tropical DiseasesUniversity of Lowell

Lowell, Massachusetts 01854, U.S.A.

INTRODUCTION

The detection of enzyme variation by electrophoresis provides a convenient basis for thegenetic characteriz.ation of populations and species, and for the assessment of the degree ofdifferentiation between such groups. By revealing the products of structural genes, the elec-trophoretic method provides an estimate of phylogenetic affinity that is unattainable by moreconventional taxonomic methods. Using this technique, we- are.able, 10 add an importantdimension to the characterization of Schistosoma mekongi, a newly-described species whichis morphologically very similar to S. japonicum (Davis et al., 1976; Voge et al., 1978). Spe-cifically, we demonstrate that S. mekongi is genetically distinct from S. japonicum and thatthe divergence found between the two species is greater than that between various geographicstrains of S. japonicum,

The application of the techniques of gel electrophoresis of enzymes to systematic and evo-lutionary problems is now well established. The reader unfamiliar with the development ofthis field is referred to extensive reviews by Avise (1974, 1976), Lewontin (1974), Ayala(1975), Nei (1975), and Selander (1976). Surprisingly, these powerful techniques have notbeen applied to the genus Schistosoma; previous electrophoretic studies have involved pooledsamples of worms and genetic interpretations have thus been precluded (e.g., Conde-del-Pineet al., 1966, 1968; Yoshimura,1968; Oya et al., 1970; Coles, 1970, 1971a,b; Ruffet al.,1971, 1973; Ross, 1976). In contrast, the present study is based on samples derived fromindividual schistosomes. The separate allelic variants segregating at a given locus can thus bedetected on the basis of differences in mobility in the gel, and intra- and inter- populationvariation can be assessed.

Previous work on Schistosoma japonicum (Ruff et al., 1973) demonstrated differences be-tween the Japanese, Philippine and Formosan strains based on polyacrylamide disc gel elec-trophoresis of total soluble proteins. The authors concluded that the Japanese and Formosanstrains were more closely related to each other than to the Philippine strain. The present studyinvolves a horizontal starch gel technique, which presents definite advantages over acrylarnidefor surveys of genetic variation. For instance, up to 20 samples may be electrophoresed on a

IPresent address: Department of Biology C-OI6, University of California-San Diego, LaIolla, California92093, U.S.A.

(113)

I ;;.:l~

114 ELECTROPHORESIS OF SCHISTOSOMA

single gel, thus greatly facilitating comparisons of electrophoretic mobility between samples.By cutting the starch slabs in up to four slices and staining each slice for a different enzymesystem, considerably greater infonnation can be derived from a single electrophoretic runthan with acrylamide. Furthermore, although greater resolution may be achieved for someenzymes electrophoresed on polyacrylamide disc gels, starch as a support medium providessatisfactory separation of a wider range of enzyme systems (Brewer, 1970). The use of stainsfor specific enzyme systems rather than for total soluble proteins enables a more accurate esti-mate of the number of loci involved in the comparisons. The sample of loci surveyed in thepresent study was moderate, but representative of a wide range of enzymatic functions.

MATERIALS AND METHODS

Schistosoma mekongi was characterized on the basis of a strain isolated from Khong Island, Laos, in1971_ It W3Scompared with four geographic strains of S. Iaponicum, The four strains were isolated, re-spectively, from OIin3 in 1928, from OIang-HII3, Formosa, in 1962, from Kofu province,Japan, in 1968,and from Palo, Leyte, Philippines, in 1969. A strain of S. mansoni isolated from Puerto Rico in 1969(NIH-Sm-PR-l) served as a reference. The strains of S. mekongi and S. laponieum were obtained from theCenter for Tropical Diseases, University of Lowell, Massachusetts, U.S.A. The S. manson; strain was origi-nally obtained from Dr. C.S. Richards, U.S. National Institutes of Health, and is maintained at the Depart-ment of Biological Sciences, Purdue University, Indiana, U.S.A., where the present study was conducted.S. mtkongi and S. Iaponicum samples were derived by infecting female mice with 25-50 cercariae from apool of 5-54 shedding snails. S. mansoni samples were derived by infecting female mice with 150-200 eer-earlae from a pool of 10-25 shedding snails.

Infected mice were killed by injection 'Il-ith sodium pentobarbitol and heparin, 34-77 days postinfec-tion, and sexually mature schistosomes were recovered by perfusion and dissection. Schistosomes weretinsed dear of blood in Eagle's Minimal Essential Medium with Eaf~'Bahs (Grand Island Biological Co.,New York, U.S.A.). Individual motile worms were then crushed with a pound glass stopper on a glassslide in approximately 10 }J of distilled water at 4ce. The resulting homogenate was immediately absorbedon a 9.5 x 3.5 mm tab of filter paper (\\'hatman No.3), which was then inserted along a transverse cutmade in a cold starch gel.

Horizontal electrophoresis was performed on 12.5% starch ~e1s (Electrostarch Co., Madison, Wiscon-sin, U.s.A.) at 4cC, following the techniques of Selander et ale (1971). The follOwing buffer systems wereemployed: 1) continuous tris-citrate I, pH 6.7 (Selander et al., 1971); 2) histidine. pH 7.0 (Brewer .1970);3) uis-glydne, pH 8.3, consisting ofO.o09M ms, O.o2SM~ydne for the gel. and O.3Mborate, pH 8.0 forthe electrode buffer (Dr. Morris Levy, Purd ue University, pcrs. comm.). Following electrophoresis, eachgel was cut into four horizontal slices, 2 mm thick, and stained for various enzyme systems. Enzyme stain-ing techniques followed Brewer (l970), Shaw &. Prasad (I970) and Selander et al. (I 971). The gel sliceswere incubated at 37cC until the enzyme patterns developed, then were rinsed and fixed in a 5:5:1 solu-tion of water, methanol and glacial acetic add. Enzyme activity patterns were recorded and photographedimmediately. ,

For greater accuracy. comparisons between strains and species were only made from worms electro-phoresed on the same gel. Occasionally. samples of whole mouse blood were also electrophoresed to checkfor the possible presence of host proteins in the schistosome homogenates. Bands of enzyme activity, orelectromorphs (King &. Ohta. 1975), were characterized by the distance they migrated on the gel, relativeto the migration distance of the corresponding electromorph of Schistosoma mansonl controls run on thesame gel. For example, under the specified conditions, the aldolase electrornorph in S. mekongi migrated1.9 times as far as the S. mansonl electromorph, which was given a mobility of 100; it was accordinglydesignated as Ald190• The relative mobility values thus obtained are approximations, and only served todesignate different electrophoretic variants of one enzyme. The exact determination of a protein's elec-trophoretic mobility requires, the use of more elaborate methods (Johnson,1977) and is not directly rele-vant to the purposes of this study. No attempt was made to correlate distance between electromorphs oftwo strains with the degree of genetic differentiation between them, since there is no simple relationshipbetween a genetic change, such as a single amino acid substitution, and a change in relative mobility. Infact, many amino acid substitutions are not even detectable electrophoretically. We used NIH-Sm-PR-l asa control.because we have found this strain to be invariant at 18 loci (Fletcher et al., in prep.), and becauseit is readily accessible to workers in the U.S. For most enzymes studied, we were able to examine the fol-lowing number of adult male and female schistosomes: S. mekongi (males, 19; females,7); S. IaponicumPhilippines (73; 6). Japan (51; 8), Formosa (32; 8), Olina (19; 3). Except where noted, aU comparativestatements refer to male worms.

FLETCHER, WOODRUFF, LoVERDE AND ASCH 115

Comparisonsbetween strains were based on the proportion ofloci that diffend in electrophoretic mo-bility rather than direetly on the p:oportion of diffenn, electromorp.tls,since a particular locus may ex-press more \han one band of acthity. Because none of \he strains included in \he p:esent study showedany inuutnin electrophoretic \'ariuion, \he lenetle interpretations we made are tentative, and are u,"trap-olated from patterns of \'I1iation found in other sUainsof Schitlos017l6 mfmsoni (Fletcher et al., in prep.).For example, in some suains of S. mDnsoni, \he anodal band pattern for malate dehydrogenase s.bowsvari-ation while \he cathodal band panern does not. Wemay thuslSSume t!lat two 'enzyme loci are involved,~ mipatin, anodaDyand ~ ~ntin, c:athodalJy.

RESULTS

Consistent and interpretable patterns were obtained with ten enzyme systems, representinga conservative estimate of 13 loci. All electromorphs were distinct from those of host pro-teins.' Each strain surveyed was invariant at the loci examined. Schistosoms mekong; clearlydiffered in electrophoretic mobility from S. japonicum in at least 82% of the loci (Table 1)_The actual difference varied between 82% (nine of 11 loci) for the Chinese and Formosanstrains, 83% (10 of 12 loci) for the Japanese strain, and 91% (lO of 11 loci) for the Philip.pine strain. No significance is attached to these differences in the estimate of interspecificdifferentiation in view of the small number of genes sampled. In contrast, differences be-tween the four geographic strains of S. laponicum affected only 17 to 36% of the loci (twoof 12 10 four of 11 loci). Both species of Asian schistosomes diverged from S. mansoni at92 to lOO%of\he loci (12 of 13 to 12 of 12 loci).

There were consistent differences in enzyme acthity (as detected by stain intensity) andin mobility between male and female schistosomes of a given species. The speCific differ.ences found are descnoed below, but not commented on, as they are of peripheral interest tothe present study. The adult worms compared were not aiways of equal age (time after infec-tion of the mouse host), but no age-related variation in enzyme mobility was detected. Thefollov.ing are descriptions of the electrophoretic phenotypes and presumed genotypes of thevarious schistosome strains examined.

Phosphoglucomutase (E.C. 2.7.5.1)

Two loci were detected on tris·glycine gels. ~ appeared as a two-banded pattern ofidentical mobility in male and female worms (fijS:'T," 3). The mobility of this doublet wasspecies-specific (Table 1). The Chinese strain of Schistosoma japonicum differed slightlyfrom the other three strains in the mobility of the doublet. A faint slower band found inS. 17U1IIS0l7i was considered to represent a second locus,~, because it showed alleHc vari-ation in a recently-isolated strain of S. manson; from Egypt (Fletcher et al., in prep.). In thatstrain, indh'idual ""onns exhibit either a slow band, a fast band, or a double-banded hetero-zygote. In N1H.Sm.PR.l,.!l!!2:1is flXed for the slow band. In S. mekong; and S. japonicum,this locus, if expressed, has not yet been identified.

Aldolase (E.C. 4.1.2.13)

On ais·glycine gels, a single well-defined band of enzyme activlty was resolved for maleand female worms. TIle three species were characterized by electromorphs of different mo.bility (Figs. l , 4; Table 1). There was variation between the Schistosoma laponicum strains,wiih the electromorph in the Philippine and Japanese strains migrating slightly further thanthat in the Chinese and Fonnosan strains_ Female scbistosomes stained less intensely thandid the males, presumably due to their smaller siz.e, although intrinsic differences betweenthe sexes have been noted for other enzymes (Coles, 1973). In female S. mekong; and S.japonicum, the electromorphs migrated at slightly different rates than those in the males.No differences in mobility were found between male and female S. mansont,

'".....: ...

116 ELECTROPHORESIS OF SCHISTOSOMA

+

0I

jp p f-M j e m M j c m M p f, m

f . c

PGM ALD G6PD

- -- -----__

_-- _-_- - -_--

-c m M J m

PGI GDHFIG. 1. Schematic diagram of v:l!iation in electromorph patterns of indhidual adult male schistosomes.

Patterns shown are those resolved on uis-glydne gels after 2.s hrs at 250 V. Various grades of shading in-dicate differences in stain intensity. See Table 1 for relative mobilities.

Enzyme system abbreviations: PGM" phosphoglucomutase; ALD. aldolase; G6PD. glucose-6.phosphate dehydrogenase; PGI" phosphoglucoisomerase; GDH. glutamate dehydrogenase.

Schistosome strain abbreviations: M· SchiSTOSOma mekongi; J. S. jeponicum • all strains; p,j, r,c"S. japonicum • Philippines, Japan, Formosa and China, respectively; m" S. mansoni.

Glucose-e-phosphate dehydrogenase (E.C. 1.1.1.49)

A single band of activity was resolved in male worms on tris-glycine gels (Fig. 1). The mo.bility of the Schistosoma mekong; electromorph was considered identical to that of the elec-tromorphs of S. iaponicum from Japan, Formosa and China (Table 1). The electrornorphs ofs. japonicum from the Philippines and S. mansoni from Puerto Rico each differed distinctlyin mobility. Female worms of all three species showed a band identical in mobility to thatfound in the males, but of lesser stain intensity. In individual female S. mekongi and S. japon-icum and in pooled samples of the smaller S. monson; females. an additional band migratingat a faster rate was also present. This faster band was similar in mobility to an electromorphpresent in mouse blood samples, so it may be of host origin.

Phosphoglucoisomerase (E.C. 5.3.1.9)

On tris-glycine gels, a single band of activity was resolved for male and female worms.Schistosoma mekongi showed a band of slower mobility than S. japonlcum, while differenceswere also apparent between S. japonicum strains (Fig. 1, Table 1). An additional cathodalelectromorph in the Japanese strain was considered to represent a second locus. After moreprolonged staining, the first set of bands faded and another system of bands, independentfrom the first, appeared. This second system, possibly representing another enzyme, is notincluded in the present survey due to insufficient data.

Glutamate dehydrogenase (E.C. 1.4.1.3)

A faint but well-defined band was resolved after staining tris-glyeine gels overnight. Schis-tosoma mekong; and S. japonicum migrated the same distance and S. manson; migrated slight-ly further (Fig. 1, Table 1). Females of the two Asian species migrated a little faster andstained more intensely than did the male worms.

Hexokinase (E.C. 2.7.1.1)

Both on tris-citrate and histidine gels, Schistosoma mekong; and S. japonicum males were

FLETCHER, WOODRUFF, loVERDE AND ASCH 117

+I

~I-.m - -•.. --• - -01 - -~ ~ :! -M J m M J rn M J m M J mHK MDH ACP LDH

~ -~.J

~,._ -M J m

GA3PDFIG.2. Schematic diagram of \"Uiation in electromorph patterns ofindhidual adult male schistosomes.

Pallerns shown are those resolved on trls-citrate ~els after 3 hrs at 120 Y, and on histidine gels after 3 hrsat 80 Y_ See Table 1 for relative mobilities.

Enzyme system abbreviations: HK" hexokinase; MOH .. malate dehydrogenase; ACP" acid phos-phatase; 10H" lactate dehydrogenase; GA3PO .. glyceraldehyde-3-phosphate dehydrogenase.

Schistosome strain abbre\iations are as in Fig. I.

characterized by a single band of different width and relative mobility, while S. mansoni malesshowed a two-banded pattern (Fig. 2, Table I). No variation was detected between S. [apon-icum strains. Female worms of all three species were characterized by a single band, identicalin mobility to that of male worms (in S. mansoni, it was identical to the slower male electro.morph). In S. mekong; and S. Iaponicum, a few individuals, male and female, showed an ad:ditional slow band. More study is needed to determine whether these variants are due to arti-facts in sample preparation or reflect actual genetic heterogeneity. Proteolytic degradationhas been incriminated as a source of heterogeneity in yeast hexokinase (Gazith et al., 1968).Malate dehydrogenase (E.C. 1.1,] .37)

Evidence for two loci was detected both on tris-citrate and histidine gels (Fig. 2, Table 1).Mdh·1 migrated as a single band toward the anode, with different mobilities observed for thethree species. No variation was detected between Schistosoma japonicum strains. The S.mekongi electrornorph showed signs of degradation, usually appearing as a streak migratinganodally rather than a well-defined band. Mdh·2 was poorly resolved under all conditionstested; it migrated cathodally as a smear close to the origin for S. [aponicum and S. mansoni,with no distinct interspecific differences, and was usually absent for S. mekongi. Male andfemale worms showed identical electrophoretic mobility at both loci.Acid phosphatase (E.C. 3.13.2)

Interspecific variation was detected both on tris-citrate and histidine gels (Figs. 2, S; Table1)- For both male and female worms, a single well-defined band migrated cathodally in Schis-tosoma mekongi and anodally in S. japonicum. Again, no variation was detected betweenS_ joponicum strains. S. mansoni worms showed a streak remaining at the origin rather thana well-defined band. Extensive smearing extending below the band of activity to the originoccurred under the specified conditions, especially for S. japonlcum, and suggests the pres-ence of other electromorphs, or partial degradation of the enzyme.Lactate dehydrogenase (E:~. 1,]. I .27)

Both on iris-citrate and histidine gels, enzyme activity was detected only in male schisto-somes, presumably because enzyme activity in females is too low to be detected by the methods

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118 ELECTROPHORESIS OF SCHISTOSOMA

TABLE 1_ Mobility of various enzyme electromorphs in male Schistosoma mekongi and S. japoni.cum, expressed relative to that of the corresponding electromorph in S. mansoni. Dee-tromorph mobility was measured from the lower margin of the band of activity. Catho-dally-migrating electrornorphs are designated by a negative sign preceding the relativemobility value. Enzymes are abbreviated as in text.

Enzyme S. japonicumlocus S. mekongi S. manson;

Philippine Japanese Formosan Chinese

pgm-l 108 89 89 89 92 11195 78 78 78 81 100

Pgm·2 - - - - - 100

AId 190 270 270 260 260 100

G6pd 85 90 85 85 85 100

Pgi-l 200 333 333 333 700 -100• • • • • 0

Pgi·2 - - -400 - - -Gdh 97 97 97 97 97 100

Hk 200 225 225 225 225 167• • • • • 100

Mdh-l 110 500 500 500 500 100

Mdh-2 - ·100 ·100 -100 -100 ·100

Ac:p -250 1700 1700 1700 1700 100

Ldh ·50 1300 1300 1300 1300 500• 700 700 700 700 300• 0 0 0 0 100

Ga3pd 150 133 133 133 133 10042 25 25 25 25 •

- • absence of any activity at a locus identified in congeners; ... absence of second or third bandof activity. .

used. Patterns for Schistosoma [aponicum were not consistently well resolved, possibly dueto lower stability of the enzyme in that species. Optimal resolution of S. japonicum and S.manson; gave three-banded anodal patterns of different mobilities for each species. For S.mekongi, the enzyme migrated cathodally as an intensely-staining band close to the origin(Fig. 2, Table I).

Glyceraldehyde-Svphosphate dehydrogenase (E.C- 1.2.1.12)

This enzyme was partially degraded under all conditions tested, and zones of activity were

.....«t,

FLETCHER, WOODRUFF, loVERDE AND ASCH 119

FIG. 3. Phosphoglucomutase patterns for adult male schistosomes as resolved on ttis-glYcine gels after2.5 hrs at 250 V_ FIrst position: Schlstosoma mlJnsoni; second and third positions: S. mekongi; fourthand fifth positions: S. japonicum ; Philippine strain. FIG.4. Aldolase patterns for adult male schlstoscmesas resolved on tris-glycine gels after 2.5 hrs at 250 V. Positions as in Fig. 3. FIG_ 5. Acid phosphatasepatterns for adult male schistosomes as resolved on histidine sels after 3 hrs at SOV. Positions as in Fig. 3.

smeared. Schistosoma mekongi, however, did show a pattern distinct from that seen in S.japonicum and S. mansoni. Again, no variation was detected within S. japonicum (Fig. 2,Table 1).

DISCUSSION

The results presented here provide strong evidence for the species status of Schistosomamekongi. S. mekongi is genetically distinct from S. japonicum at between 82 and 91% ofthe 11·12 loci involved in the comparison, while differences between S. japonicum strainsaffect only 17 to 36% of the same loci. Using these data, Nei's (1972) indices of genetic iden-tity illand distance (0) were calculated for all pairwise comparisons between strains of S.mekongi, S. japonicum and S. mansoni (Table 2). Nei's lis defined as the normalized iden-tity of genes between two populations and .Q as a minimum estimate of the average numberof codon differences per locus between the two populations. Because no genetic variationwas found for any of the strains examined, the formulae for! and .Q between any two strainsare reduced to: ! = 1 • c

Q = -log1e being the proportion of loci that differ in electrophoretic mobility. The genetic distancesbetween S. mekongi and each strain of S. japonicum, 1.71-2.40, fall in the upper range ofthose reported for congeneric animal species (the range being 0.05-2.80 (Nei, 1975)).

The taxonomic status of the geographic strains of Schistosoma japonicum, as well as thephylogenetic affmities between them, have been the subject of much debate (Hstl & Hsil,1962; Davis, 1970). Although the genetic distances among the four laboratory strains aretypical of those found among subspecies in other taxa, it would be premature to employour data for formal phylogenetic purposes. The genetic distances given in Table 2 are basedon a limited number of loci and so must be considered as preliminary estimates. For example,the 12 value of ~ between S. mekongi and S. mansoni is surely due to sampling error. Thusthe sample of loci examined should be increased, but, even more important, additional field-collected populations of S. [aponicum from each geographic area should be surveyed.

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120 ELECTROPHORESIS OF SCHISTOSOMA

TABLE 2. Nei's (1972) genetic identity ill,above diagon3l. and genetic distance (0), below diago-nal, between various strains of Schistosoma, based on available electrophoretic data(Table 1) for 11-13 loci. Strains abbreviated as in Fig. I.

Schistosome M p j f c m

S. mekongi (M) - 0.091 0.167 0.182 0.182 0.000,

S. jl1ponicum Philippines (p) 2.398 - 0.833 0.818 0.636 0.083Japan (j) l.792 0.182 - 0.833 0.667 0.077Formosa (f) 1.705 0.201 0.182 - 0.818 0.083China (c) 1.705 0.452 0.405 0.201 - 0.083

S. mansoni (m) coo 2.485 2.565 2.485 2.485 --

A rough estimate of divergence time between Schistosoma mekongi and S. japonicum maybe obtained from the electrophoretic data generated in this study using Nei's (1975) formula:1= 5 x 106 Q, where 1is time in years. Based on this relationship. the various strains of S.japonicum have been evolving separately from one another for 1·2 million years, S. japanl-cum and S. mekongi diverged from one another 8.5 -12 million years ago, and the S. japonl-cum stock separated from the S. mansoni stock 12.5 -13 million years ago. This relationshipis based on certain assumptions about the constancy of mutation rates across different loci,and Nei (1975) should be consulted for a discussion of the validity of this method. The fig.ure of approximately 10 million years ago- for the divergence-time of the S. iaponicum andS. mekongi stocks is of particular interest in that it is remarkably close to the estimate arrivedat by Davis (1980) on the basis of paleogeographical considerations.

In the present studies. intrastrain genetic variation within each of the schistosome strainsexamined was not detected. It is possible that Individual variation will be detected whenmore enzyme systems are surveyed and when more refined techniques of enzymology areemployed. For example, separation at different pH and gel concentrations and thermostabll-Ity studies have uncovered a surprising amount of cryptic variation (Johnson. 1977; Throck-morton. 1977). However, in comparison to similar studies on other animal species (reviewedby Powell, 1975; Selander, 1976; Nevo, 1978). the lack of genetic variation in the schisto-some strains examined is remarkable. For instance, in 93 other invertebrates, the mean pro.portion of polymorphic loci per population (f) was 0.397 ± 0.201, and the average propor-tion of heterozygotes per locus per Individual (H) was 0.1123 ± 0.0720 (Nevo, 1978). Usingcomparable techniques, the present study obtained.f = 0.000 and H = 0.000 for Schistosomamekongi, S. japonicum (four strains), and S. manson; (NIH-Sm-PR.l). It should be notedthat all these strains have been maintained in the laboratory for periods ranging from eightto 51 years. The results obtained thus probably reflect inbreeding and selection due to labo-ratory conditions, as well as small original founding populations (the founder principle (Mayr,1970»). Some support for this conclusion is emerging from ongoing studies on recent schis-tosome isolates (Fletcher et al., in prep.). For example, a freshly-collected sample of S. man-son; from Qalyub, Egypt, shows allelic variation at Pgm-2, and variation was found at Mdh-l,Ldh, G6pd, and a peptidase, in newly-isolated S. manson; strains from Puerto Rico. This in-dicates that most of the laboratory strains of Schistosoma presently available may be atypi-cal of natural pcpulations. This finding has important consequences for some areas of schis-tosome research, such as chemotherapy or systematics. Future taxonomic work on the vari-ous geographic strains of S. [aponicum should be based on recent isolates, of sufficient sizeand geographic diversity to adequately represent each strain. '

FLETCHER, WOODRUFF, LoVERDE AND ASCH

SUMMARY

The taxonomic status of Schistosoma mekongi was investigated by comparing it to fourstrains of S. iapantcum, originally collected in China, Formosa, Japan and the Philippines,using starch gel electrophoresis of 10 enzyme systems, representing an estimated 13 loci. Theenzymes were lactate, malate, glucose-6-phosphate, glyceraldehyde-3-phosphate and gluta-mate dehydrogenases, hexokinase, phosphoglucomutase, acid phosphatase, aldolase and phos-phoglucoisomerase. Electromorphs were characterized by their electrophoretic mobility rela-tive to that of an invariant strain of S. mansoni (NIH·Sm-PR·1). Each of the schistosomestrains examined was invariant at the loci surveyed. S. mekongi and S. japonicum divergedin electrophoretic mobility at 82 to 91% of the loci examined, while the S. japonicum strainsdiffered from each other at 17 to 36% of the same loci. These data provide strong evidencefor the species status of S. mekongi, Nei's genetic distance between S. mekongi and S. iapon-icum is estimated at 1.71-2.40, suggesting that these specieshave been diverging for 8.S-12million years. The absence of individual variation was attributed to the founder effect andprolonged laboratory maintenance of the strains examined. Future taxonomic work on schis-tosomes should be based on recently-isolated samples that more adequately represent naturalpopulations.

ACKNOWLEDGEMENTS

We are indebted to Dr. A.I. Grossm3JI for having suggested this study. We are grateful to Dr. C.S.Richards, who kindly supplied us with the NIH-Sm·PR-l strain; to Dr. Y.-S. liang and Ms. V. Knowltonfor infecting animals with Schistosoma mekongi; and to the U.S.-Japan Cooperative Program. GeographicMedicine Branch, National Institutes of Health, Bethesda, Maryland, U.S.A., for making animals infectedwith S. japonicum available. We thank Mr. F. C. Debs for preparing the figures and Mr. M.A. Goldman forcomments on the manuscript.

This investigation was supported by a grant from The Edna McConnell Clark Foundation.

LITERA TURE CITED

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Sinauer Associates,lnc., Sunderland, Massachusetts. 277 p. Ip.l06-122].AYALA, F.J. 1975. Genetic differentiation during the speciation process. In: Dobzhansky, T., Hecht,

M.K. It Steere, W.C. (Eds.), EI'OlutionDry Biology, vol. 8. Plenum Press, New York. 396 p. [p, 1-78].

BREWER, G.J. 1970. An Introduction to Isozyme Techniques. Academic Press, New York. 186 p. ,COLES, G.C. 1970. A comparison of some isoenzymes of Schistosoma mansoni and Schistosoma haema-

tobium. Compo Biochem, Phystol., 33: 549-558.COLES, G.C. 1971a. Variations in malate dehydrogenase isoenzymes of Schistosoma mansoni. Compo

Biochem. Physiol .• 39B: 35-42.COLES, G.C. 1971b. Alteration of Schlstosoma mansonl malate dehydrogenase Isoenzyrnes on passage

in the laboratory. Compo Biochem. Physiol..• 40B: 1079-1083.COLES, G.C. 1973. The metabolism of schistosomes: a review. Int. J. Biochem .• 4: 319-337.CONDE-DEL-PINO, E., ANNEXY-MARTINEZ, A.M., PEREZ-VILAR, M. It CINTRbN·RIVERA, A.A.

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