- Email: [email protected]
Biochemical polymorphism in Parascaris equorum, Toxocara canis and Toxocara cati
Molecular and Biochemical Parasitology, 18 (1986) 45-54 Elsevier
45
MBP 00609
BIOCHEMICAL
POLYMORPHISM
IN PARASCARIS EQUORUM, TOXOCARA
CANIS AND TOXOCARA CATI
STEVEN A. NADLER
Department of Microbiology, Immunology, and Parasitology, Louisiana State UniversityMedical Center, New Orleans, LA 70112, U.S.A. (Received 9 July 1985; accepted 21 August 1985)
Vertical starch gel electrophoresis was used to resolve proteins encoded by 18 gene loci in ascaridoid nematodes. Estimates of genetic variability were made from population samples of the dog ascarid (Toxocara canis), cat ascarid (Toxocara cati), and the horse ascarid (Parascaris equorum). Levels of polymorphism and mean heterozygosity were high, which is not consistent with the hypothesis that the intestinal environment selects for monomorphism among endoparasites. Most observed allele frequencies conformed to Hardy-Weinberg equilibrium expectations as tested by X2goodness-of-fit, suggesting that the proteins evaluated are inherited in a Mendelian fashion and that these nematodes are mating at random. Subunit structures of the following enzymes, deduced from electrophoretic phenotypes of heterozygotes, corresponded to those of vertebrates: lactate dehydrogenase; malate dehydrogenase; 6-phosphogluconate dehydrogenase; phosphoglucomutase; esterase D; peptidase B; peptidase D; and mannose-6-phosphate isomerase. This observation substantiates the conservative nature of polypeptide subunit number across phylogeneticaUy diverse groups of organisms. Key words: Biochemical polymorphism; Ascaridoid nematodes; Heterozygosity; Subunit structure
INTRODUCTION H i g h - r e s o l u t i o n e ! e c t r o p h o r e s i s , in c o m b i n a t i o n w i t h h i s t o c h e m i c a l m e t h o d s f o r d e t e c t i n g specific p r o t e i n s c a n r e v e a l a s u b s t a n t i a l p o r t i o n o f the allelic v a r i a t i o n at a specific g e n e t i c locus. M u l t i l o c u s e l e c t r o p h o r e t i c studies h a v e m a d e p o s s i b l e the c a l c u l a t i o n o f allele f r e q u e n c i e s at m a n y loci in d i f f e r e n t p o p u l a t i o n s . S u c h studies h a v e b e e n e m p l o y e d to g a i n u n d e r s t a n d i n g o f the g e n e t i c v a r i a b i l i t y a n d p o p u l a t i o n struct u r e o f m a n y f r e e - l i v i n g o r g a n i s m s [1]. W h i l e e n z y m e e l e c t r o p h o r e s i s has b e e n a p p l i e d to m a n y p a r a s i t e species [2-5], few w o r k e r s h a v e e x p l o i t e d t h e full p o t e n t i a l o f the t e c h n i q u e f o r s t u d y i n g p a r a s i t e genetics. L e v e l s o f p o l y m o r p h i s m a n d m e a n h e t e r o z y -
Abbreviations: P, percentage of polymorphic loci; H, mean heterozygosity; A, mean number of alleles per locus. 0166-6851/86/$03.50
© 1986 Elsevier Science Publishers B.V. (Biomedical Division)
46 gosity in most parasites are largely unknown. In addition, hypotheses concerning the amount of gene flow between parasite populations have remained speculative. While several enzymes of ascaridoids have been studied electrophoretically [6-12], only two studies have addressed the question of population genetics from a multilocus approach [ 13,14]. These investigators characterized ascaridoids as having low levels of polymorphism and mean heterozygosity. Leslie et al. [14] have invoked several explanations to account for these observations in Ascaris s u u m including: (1) that the intestine of the vertebrate host is a relatively stable environment that selects for 'increased m o n o m o r p h i s m ' ; (2) A. s u u m may exist in 'small homogeneous populations with little gene flow between them'. Such a population structure has been inferred for many parasites in general [15]. In this study, levels of genetic variability in natural populations of ascaridoid nematodes were investigated using the multilocus electrophoretic approach. The results indicate that these nematodes have high levels of polymorphism and mean heterozygosity, and that phenotypes of the polymorphic proteins can be interpreted genetically. MATERIALS AND METHODS Collection. Population samples of live helminths were primarily obtained at necropsy of mammals collected in southern Louisiana. Some ascaridoids were collected following spontaneous or anti-helminthic induced expulsion. The live worms were rinsed in physiological saline, individually wrapped in aluminum foil to exclude air, sealed in plastic bags, and frozen and stored at -70°C. Worms were cataloged by sex, host, and geographic area of host collection.
Specific tissues of large ascaridoids, or whole small worms were minced, and then homogenized in two volumes of 250 mM sucrose containing 100 lag ml -~ dithiothreitol, NAD, and NADP. Homogenates were centrifuged at 5 000 X g for 10 rain at 4°C to remove cell debris. The supernatant fractions of homogenates were subjected to vertical starch gel electrophoresis [16], at 4°C. Starch gels were used in preference to polyacrylamide because of their greater solubility for the enzyme detection systems. The duration of the electrophoresis varied from 15 to 22 h. Comparisons of proteins from different individuals were done side by side on the same gel in order to avoid errors inherent in comparisons based upon relative mobilities. Each gel contained twelve samples, including one reference individual of known genotype. Approximately equal numbers of male and female worm homogenates were subjected to electrophoresis. Homogenates of host intestinal tissue were run on some gels as controls for external contamination by host a n d / o r gut flora enzymes. The following buffers were used for electrophoresis: (a) phosphate-citrate pH 6.8 (60 mM Na2HPO4, 10 mM citric acid) diluted 1:4 for gel and undiluted for electrode bath; (b) Tris-maleate pH 7.5 (100 mM Tris adjusted to p H 7.5 with 2 M maleic acid) diluted 1:3 for gel and
Electrophoresis.
47 undiluted for electrode bath; (c) Tris-EDTA borate pH 8.1 (30 mM Tris, 2 mM EDTA, adjusted to pH 8.1 with saturated boric acid) gel undiluted, borate bridge buffer pH 8.6 (see d below) used for electrode bath; (d) borate bridge buffer pH 8.6 (125 mM boric acid adjusted to pH 8.6 with 1 M NaOH) diluted 1:4 for gel, undiluted for electrode bath.
Enzyme localization.
Proteins were localized in gel slices with techniques described mainly in Harris and Hopkinson [17]. Glutamate dehydrogenase was detected by the method of Brewer [18]. Arginine kinase was localized by substituting phospho-L-arginine for phosphocreatine in the creatine kinase staining method 'B' of Harris and Hopkinson [17]. Reactions coupled to thiazoyl blue (MTT) tetrazoliums were used to detect dehydrogenases, isomerases, kinases, and phosphoglucomutase; those coupled to L-amino acid oxidase and peroxidase were used to localize peptidases. Peptidase substrates were valyl-leucine for peptidase A, leucyl-glycyl-glycine for peptidase B, and phenylalanyl-proline for peptidase D. Esterase D, which catalyzes the hydrolysis of 4-methyl-umbelliferyl esters, was visualized under ultraviolet light as bright fluorescent bands, ct-Naphthyl acetate and 13-naphthyl acetate were used as substrates for the corresponding esterases. Glutamate-oxaloacetate aminotransferase was localized by diazotization of the oxaloacetate reaction product with Fast Blue RR. Differences in phenotypes at a specific locus were used to deduce genotypes of individuals and the number of alleles segregating in the populations. When more than one presumed locus yielded products with the same activity, the locus having isozymes of faster anodal mobility was assigned the lower number; for multiple alleles, the one that yielded isozymes of fastest anodal mobility was designated 'a', those of slower mobility as 'b, c', etc., in order of decreasing electrophoretic mobility.
Analysis of genetic data.
Genotypes for single individuals at all loci were entered into the BIOSYS-1 computer program of Swofford and Selander [19]. Program outputs included values for polymorphism, allele frequencies, heterozygosity estimates, genetic similarity, and tests of conformance of observed allele frequencies in each population to Hardy-Weinberg equilibrium expectations. For small sample sizes, Levene's [20] correction for expected genotype frequencies was used. For each polymorphic protein, a Z: goodness-of-fit test was performed. However, when more than two alleles are observed at a locus the expected frequency of some genotypes can be low, and the Zz test can give misleading results [21]. In such cases, BIOSYS-I repeats the ;(2 test after pooling genotypes into three classes: (1) homozygotes for the most common allele; (2) heterozygotes for the most common allele and one of the other alleles; (3) all other genotypes. Exact significance probabilities were also calculated [22], which are analogous to Fisher's exact test for 2 × 2 contingency tables. This test avoids the difficulties encountered in using the ~2 distribution for small sample sizes.
2.6.1.1
Glutamate-oxaloacetate
Esterase D
3.1.1.1
2.7.5.1
Phosphoglucomutase
Hydrolases
2.7.3.3
A r g i n i n e kinase
transaminase
Glutamate-pyruvate
transaminase 2.6.1.2
1.15.11
Transferases
1.4.1.2
Superoxide dismutase
1.2.1.12
1.1.1.44
Glutamate dehydrogenase
dehydrogenase
phosphate
Glyceraldehyde-3-
dehydrogenase
6-Phosphogluconate
1.1.1.42
m,i,s
m,i,s
i,s
m
i,s
m
i,s
m
m,i,s
i,s
dehydrogenase
Isocitrate
m,i,s
i,s m,i,s
1.1.1.37
m,i,s
u,t
cm
cm
cm
cm
cm
cm
cm
cm
Tissue utilized
Taxa resolved
Malate dehydrogenase-2
1. I. 1.27
Lactate dehydrogenase
1.1.1.14
E.C. n u m b e r
Malate dehydrogenase- 1
dehydrogenase
Sorbitol
Oxidoreductases
Locus
P r o t e i n s resolved in a s c a r i d o i d t a x a a
TABLE 1
a
a
b
b
a
c
b
a
a
b
a
a
b
a
Buffer
m,i
i,s
none
none
none
none
none
none
s
none
none
none
s
none
Polymorphism
dimer
monomer
unknown
unknown
unknown
unknown
unknown
unknown
dimer
unknown
dimer
tetramer
unknown
Structure
i,s
3.1.1.1 3.4.13.9 3.4.1.1 3.4.13.9 3.5.4.4
4.1.2.13
5.3.1.8 5.3.1.9
a c e t a t e esterase Peptidase A
Peptidase B
Peptidase D
Adenosine deaminase
Lysases Aldolase
lsomerases Man nose-6-phosphate
isomerase Hexose isomerase
m
Hemoglobin (pseudocoelomic)
ps
cm
cm
cm
u,t
u,t
u,t
cm
cm
b
b
a
b
b
a
a
a
a
a
a
a
none
none
i
i,s
none
m
m
m,i
none
i
none
i,s
unknown
unknown
unknown
monomer
unknown
unknown
dimer
monomer
unknown
monomer
unknown
monomer
were d e d u c e d f r o m b a n d i n g p a t t e r n s o f h e t e r o z y g o t e s .
cuticle a n d muscle; u = uterus; t = testis; ps = p s e u d o c o e l o m i c fluid. Buffer c o m p o s i t i o n s a r e d e t a i l e d in M a t e r i a l s a n d M e t h o d s . S u b u n i t s t r u c t u r e s o f p r o t e i n s
equorum, i = T. cati, s = T. canis. W h o l e w o r m h o m o g e n a t e s were u s e d f o r Toxocara a n a l y s e s . A b b r e v i a t i o n s f o r tissues u s e d in s c o r i n g P. equorum loci are: c m =
a L o c u s a n d c o r r e s p o n d i n g e n z y m e c o m m i s s i o n n u m b e r s a r e listed. P o l y m o r p h i c p r o t e i n s , a n d loci s c o r e d for e a c h species a r e i n d i c a t e d b y a b b r e v i a t i o n : m = P.
m
Other proteins H e m o g l o b i n (muscle)
m,i,s
i,s
i,s
m
m
m,i,s
m,i,s
m
m,i,s
acetate esterase-2 ~Naphthyl
3.1.1.1
a c e t a t e esterase-I ct-Naphthyl
a-Naphthyl
50 RESULTS Population samples of Parascaris equorum, Toxocara canis, and Toxocara cati have been analyzed at 18 genetic loci. Buffers ranging in pH from 6.0 to 8.1 were employed in order to maximize the detection of polymorphism. The loci scored, enzyme commission numbers, tissues utilized, and the buffer yielding optimum resolution are listed in Table I. Fresh homogenates were used to assay for sorbitol dehydrogenase because its activity was greatly diminished in frozen homogenates. Although some enzymes exhibited reduced activity following freeze-thaw of homogenates, no difference was found between homogenates prepared from fresh and frozen nematode tissues. Similarly, T. canis specimens voided from hosts following organophosphate treatment had enzymatic activities comparable to those obtained from hosts at necropsy. Enzymatic activities detected in P. equorum gels, but not resolved under the conditions employed included: isocitrate dehydrogenase; glucose-6-phosphate dehydrogenase; lactate dehydrogenase; a-glycerol phosphate dehydrogenase; glutamate-oxaloacetate transaminase; arginine kinase; and aldolase. Similarly, glucose-6-phosphate dehydrogenase, peptidase D, and hexokinase activities were detected, but not resolved in experiments with Toxocara spp. No sex-specific loci were detected. The number and relative staining intensity of isozyme bands in heterozygotes was used to deduce the subunit structure of enzymes. For all proteins where heterozygous phenotypes could be clearly interpreted, the deduced subunit structures corresponded to those of vertebrate enzymes (Table I, Fig. 1). Side by side comparisons ofascaridoid homogenates and samples of their respective host's small intestine revealed no evidence of host/gut flora enzyme contamination (Fig. 1). Four enzymatic loci were polymorphic in P. equorum, five in T. canis, and seven in T. cati. Values for the percentage of polymorphic loci (P), mean heterozygosity (H), and mean number of alleles per locus (A) for each species are shown in Table II. Coefficients of Rogers' genetic similarity [23] between five T. cati populations derived from different cats in Orleans Parish ranged from 0.89 to 0.95. Observed allele frequencies of the P. equorum population conformed to H a r d y Weinberg equilibrium expectations as tested by Z 2 goodness-of-fit. This population was composed of 29 individual nematodes obtained from a single horse. Zz significance tests revealed five cases where the T. canis or T. cati populations deviated from Hardy-Weinberg equilibrium expectations at individual loci. These populations were composed of 32 individuals obtained from three dogs, and 22 individuals obtained from six cats, respectively. However, X2 results are suspect in cases with small sample size or large numbers of genotype classes. Following Z2 analyses with pooled genotype classes or an exact probability test, only two cases remained significant at the P<0.05 level (Table III). For the esterase D locus in T. cati, this result is not surprising because there are low expected frequencies in a number of genotype classes, and only 22 individuals were sampled. However, for 6-phosphogluconate dehydrogenase in T. canis there are only three genotype classes, and there appears to be a real deficiency of heterozygotes at this locus.
51
a
b
c
Fig. I. Phenotypes of some polymorphic ascaridoid enzymes. Slot numbers (in parentheses) and presumed genotypes are read left to right. The allele of fastest anodal mobility is designated 'A', and those of slower mobility sequentially as 'B, C', etc. These gel photographs do not illustrate all alleles. The anode is to the top. (a) Lactate dehydrogenase, T. canis (1,3) and T. cati (2), genotypes A/C, B/B, A/A. The heterozygote has five bands with a 1:4:6:4:1 staining intensity, indicating a tetrameric subunit structure. (b) 6-Phosphoglucohate dehydrogenase, T. canis, genotypes B/C, B/B. The heterozygote has three bands with a 1:2: I staining intensity, indicating a dimeric subunit structure. (c) Phosphoglucomutase, T. canis, genotypes B/B, A/C, A/B. Heterozygotes have two bands of equal staining intensity indicating a monomeric subunit structure. (d) Esterase D, T. cati, genotypes C/C, A/B. Heterozygote phenotype characteristic of dimeric protein. (e) Peptidase B, T. cati (1,3) and cat small intestine (2). There is no evidence of host-enzyme contamination of the ascarids. (f) a-Naphthyl acetate esterase, T. cati, genotypes B/B, A/B, A/A. Heterozygote phenotype characteristic of monomeric protein. (g) Peptidase B phenotypes, T. cati genotypes C/C, D/G, F/H. Heterozygote phenotypes characteristic of monomeric protein. (h) Peptidase D, P. equorum, genotypes B/B, B/C. Heterozygote phenotype characteristic of dimeric protein. (i) Mannose-6-phosphate isomerase, T. canis, genotypes B/B, A/B, B/B. Heterozygote phenotype characteristic of monomeric protein.
DISCUSSION T h r e e s t a n d a r d m e a s u r e s o f g e n e t i c v a r i a b i l i t y : m e a n n u m b e r o f alleles p e r l o c u s ; p e r c e n t a g e o f p o l y m o r p h i c loci; a n d m e a n n u m b e r o f h e t e r o z y g o u s loci p e r i n d i v i d u a l , r e v e a l h i g h levels o f g e n e t i c v a r i a t i o n w i t h i n p o p u l a t i o n s a m p l e s o f P. e q u o r u m , T. canis, a n d T. cati. T h i s c o n s i d e r a b l e g e n e t i c d i v e r s i t y in o r g a n i s m s i n h a b i t i n g w h a t is
52 T A B L E II
Genetic variability at 18 loci in three species of ascaridoid nematodes (mean and standard error are shown) Mean sample size per locus
Mean no. of alleles per locus
Percentage of loci polymorphic a
Mean heterozygosity Direct count
Hardy-Weinberg expected
P. equorum
27.7
1.44 + 0.22
22.2
0.085 + 0.042
0.090 + 0.046
T. canis
29.5 20.6
1.44 + 0.18 1.94 ± 0.42
33.3 38.9
0.135 + 0.061 0.137 + 0.057
0.118 + 0.051 0.139 + 0.056
T. cati
a A locus is considered polymorphic if the frequency of the most common allele does not exceed 0.99.
T A B L E III
Apparent cases of deviation from Hardy-Weinberg equilibrium in T. canis a n d 11 cati Species and locus
Genotype
Observed number
Expected number
Significance level (P) Z2
T. canis
B-B B-C
14 8
11.5 12.9
6-Phosphogluconate dehydrogenase
C-C
6
3.6
T. cati
A-A A-B
0 1
0.01 0.11
Umbelliferyl-acetate esterase
A-C
0
0.86
B-B
1
0.28
B-C
2
4.32
C-C
18
16.41
Pooled X2
0.04
0.01
Exact P
0.04
0
0.03
frequently considered a h o m o g e n o u s environment is not consistent with the hypothesis that the intestinal environment selects for m o n o m o r p h i s m a m o n g endoparasites. Levels of polymorphism and mean heterozygosity in the Baton Rouge population of P. equorum (P = 22%, H = 0.085) were similar to values reported by Leslie et al. [ 14] for an Iowa population of Ascaris suum (P = 21%, H = 0.066). These data are in marked contrast to the report of Bullini et al. [13] who found only 3.7% o f P . equorum loci polymorphic with a mean heterozygosity of 0.0088. These authors may have seriously underestimated variation due to procedural problems, such as those that may result from using horizontal gels, or in basing comparative evidence on relative mobilities. However, it is possible that these reports reflect real differences in genetic variability between P. equorum populations from Louisiana and central-eastern Europe. Animal populations established by the colonization of a small number of founding individuals, such as island populations, and those that undergo frequent bottlenecks in population
53 size often have low levels of mean heterozygosity. Although the number of T. cati individuals from each host was small, the matrix of Rogers' similarity coefficients does not reveal any marked genetic differentiation between these 'host populations'. However, studies of ascaridoid populations from a wide geographic range of hosts are required to establish the level ofgene flow between distant populations. The conformance of most observed allele frequencies to Hardy-Weinberg equilibrium expectations suggests that these proteins are inherited in a Mendelian fashion and that these nematode populations are mating at random. T. canis 6-phosphogluconate dehydrogenase genotypes did not conform to Hardy-Weinberg equilibrium expectations. A Wahlund [24] effect could account for the apparent deficiency of heterozygotes at this locus, because pooled samples may inadvertently conceal differences in allele frequencies between genetically distinct populations. This result also suggests that geographically distinct populations of ascaridoids may be genetically distinct. Among enzyme classes of the three ascaridoid species studied, oxidoreductases, transferases, hydrolases, and isomerases included at least one monomorphic and one highly polymorphic locus. Of the five major classes of enzymes, the hydrolases contributed most to overall polymorphism, the oxidoreductases the least. Of the 16 polymorphic loci distributed among the three species, nine were of the hydrolase class. Such a differential distribution of polymorphic loci among enzyme classes has been reported previously in both ascaridoids [14] and other invertebrates [25]. While no single enzymatic locus is consistently polymorphic among studied ascaridoid species, certain loci are regularly monomorphic when resolved. These loci include: sorbitol dehydrogenase, superoxide dismutase, glutamate-pyruvate transaminase, isocitrate dehydrogenase, glutamate dehydrogenase, glutamate-oxaloacetate transaminase, and aldolase. Possibly these enzymes will not tolerate many amino acid substitutions due to functional constraints. Enzymes which are monomers, dimers, or tetramers in mammals, birds, reptiles, and amphibians have identical subunit structures, where deduced, in ascaridoid nematodes. This observation further substantiates the conservative nature of polypeptide subunit number across phylogenetically diverse groups of organisms (Dessauer, H.C. and Braun, M.J. (1982) Abstr. 4th Int. Congr. Isozymes, Austin, TX). The conservation of enzyme subunit structure and substrate specificity implies that the enzymatic activities observed in diverse organisms are performed by homologous proteins. This evidence of homology further supports the inference of Mendelian inheritance of these proteins. ACKNOWLEDGEMENTS I would like to thank Dr. Herbert C. Dessauer and Dr. Joseph H. Miller for their assistance and encouragement. This work was supported in part by a Grant-in-Aid of Research from Sigma Xi, The Scientific Research Society.
54 REFERENCES 1 2 3
4
5 6 7 8 9 10
11 12 13
14 15 16 17 18 19 20 21 22 23 24 25
Nevo, E. (1978) Genetic variation in natural populations: Patterns and theory. Theor. Popul. Biol. 13, 121-177. Leriche, P.D. and Sewelt, M. (1977) Differentiation of Taenia saginata and Taenia solium by enzyme electrophoresis. Trans. R. Soc. Trop. Med. Hyg. 71, 327-328. Gibson, W.C., Marshall, T.F. and Godfrey D.G. (1980) Numerical analysis of enzyme polymorphism: A new approach to the epidemiology and taxonomy of trypanosomes of the subgenus Trypanozoon. Adv. Parasitol. 18, 175-246. Fletcher, M., LoVerde, P.T. and Woodruff, D.S. (1981) Genetic variation in S c h i s t o s o m a mansoni: Enzyme polymorphisms in populations from Africa, Southwest Asia, South America, and the West Indies. Am. J. Trop. Med. Hyg. 30, 406-421. LoVerde, P.T., DeWald, J., Minchella, D.J. Bosshardt, S.C. and Damian, R.T. (1985) Evidence for host-induced selection in Schistosoma mansoni. J. Parasitol. 71,297-301. Zam, S.G. (1973) Disc electrophoresis o f A s c a r i s suum female reproductive esterase enzymes. Comp. Biochem. Physiol. 46B, 797-803. Rhodes, M.B., Marsh, C.L. and Kelley, G.W. (1964) Studies in helminth enzymology, lII. Malic dehydrogenases of Ascaris suum. Exp. Parasitol. 15, 403-409. Zee, D.S. and Zinkham, W.H. (1968) Malate dehydrogenase in Ascaris suum: characterization, ontogeny, and genetic control. Arch. Biochem. Biophys. 126, 574-584. Zee, D.S, Isensee, H. and Zinkham, W.H. (1970) Polymorphism of malate dehydrogenase in Ascaris suum. Biochem. Genet. 4, 253-257. Langer, B.W., Jr. and Smith, W.J. (1971) The lactic acid dehydrogenase of some parasitic gastro-intestinal nematodes: Ascaris suum, O e s o p h a g o s t o m u m radiatum and H a e m o n c h u s contortus. Comp. Biochem. Physiol. 40B, 833-840. Agatsuma, T. and Suzuki, N. (1981) Electrophoretic studies on enzymes in ascarids. I. Lactate dehydrogenase isozymes in Ascaris suum. Jpn. J. Parasitol. 30, 81-83. Gockel, S.F. and Lebherz, H.G. (1981) 'Conformational' isoenzymes of ascarid enolase. J. Biol. Chem. 256, 3877-3883. Bullini, L., Nascetti, G., Ciafr~, S., Rumore, F., Biocca, E., Montalenti, S. and Rita G. (1978) Ricerche cariologiche ed elettroforetiche su Parascaris univalens e Parascaris equorum. Atti A'ccad. Naz. Lincei CI. Sci. Fis. Mat. Nat. Rend. 65, 151-156. Leslie, J.F., Cain, G.D., Meffe, G.K. and Vrijenhoek, R.C. (1982) Enzyme polymorphism in Ascaris suum (Nematoda). J. Parasitol. 68, 576-587. Price, P.W. (1977) General concepts on the evolutionary biology of parasites. Evolution 31,405-420. Smithies, O. (1959) Zone electrophoresis in starch gels and its application to studies of serum proteins. Adv. Protein Chem. 14, 65-113. Harris, H. and Hopkinson, D.A. (1976) Handbook of Enzyme Electrophoresis in Human Genetics, Elsevier/North-Holland, Amsterdam. Brewer, G.J. (1970) An Introduction to Isozyme Techniques, Academic Press, New York. Swofford, D.L. and Selander, R.B. (1981) BIOSYS-I: A FORTRAN program for the comprehensive analysis of electrophoretic data in population genetics and systematics. J. Hered. 72, 281-283. Levene, H. (1949) On a matching problem arising in genetics. Ann. Math. Stat. 20, 91-94. Sokal, R.R. and Rolfe, F.J. (1969) Biometry, W.H. Freeman and Co., San Francisco, CA. Elston, R.C. and Forthofer, R. (1977) Testing for Hardy-Weinberg equilibrium in small samples. Biometrics 33, 536-542. Rogers, J.S. (1972) Measures of genetic similarity and genetic distance. Stud. Genet. (Univ. Tex. Publ.) 7213, 145-153. Wahlund, S. (1929) Zusammensetzung von Populationen und Korrelationserscheinungenvom Standpunkt der Vererbungslehre aus betrachtet. Hereditas 11, 65-106. Johnson, G.B. (1976) Genetic polymorphism and enzyme function. In: Molecular Evolution (Ayala, F.J., ed.), pp. 46-59, Sinauer, Massachusetts.
45
MBP 00609
BIOCHEMICAL
POLYMORPHISM
IN PARASCARIS EQUORUM, TOXOCARA
CANIS AND TOXOCARA CATI
STEVEN A. NADLER
Department of Microbiology, Immunology, and Parasitology, Louisiana State UniversityMedical Center, New Orleans, LA 70112, U.S.A. (Received 9 July 1985; accepted 21 August 1985)
Vertical starch gel electrophoresis was used to resolve proteins encoded by 18 gene loci in ascaridoid nematodes. Estimates of genetic variability were made from population samples of the dog ascarid (Toxocara canis), cat ascarid (Toxocara cati), and the horse ascarid (Parascaris equorum). Levels of polymorphism and mean heterozygosity were high, which is not consistent with the hypothesis that the intestinal environment selects for monomorphism among endoparasites. Most observed allele frequencies conformed to Hardy-Weinberg equilibrium expectations as tested by X2goodness-of-fit, suggesting that the proteins evaluated are inherited in a Mendelian fashion and that these nematodes are mating at random. Subunit structures of the following enzymes, deduced from electrophoretic phenotypes of heterozygotes, corresponded to those of vertebrates: lactate dehydrogenase; malate dehydrogenase; 6-phosphogluconate dehydrogenase; phosphoglucomutase; esterase D; peptidase B; peptidase D; and mannose-6-phosphate isomerase. This observation substantiates the conservative nature of polypeptide subunit number across phylogeneticaUy diverse groups of organisms. Key words: Biochemical polymorphism; Ascaridoid nematodes; Heterozygosity; Subunit structure
INTRODUCTION H i g h - r e s o l u t i o n e ! e c t r o p h o r e s i s , in c o m b i n a t i o n w i t h h i s t o c h e m i c a l m e t h o d s f o r d e t e c t i n g specific p r o t e i n s c a n r e v e a l a s u b s t a n t i a l p o r t i o n o f the allelic v a r i a t i o n at a specific g e n e t i c locus. M u l t i l o c u s e l e c t r o p h o r e t i c studies h a v e m a d e p o s s i b l e the c a l c u l a t i o n o f allele f r e q u e n c i e s at m a n y loci in d i f f e r e n t p o p u l a t i o n s . S u c h studies h a v e b e e n e m p l o y e d to g a i n u n d e r s t a n d i n g o f the g e n e t i c v a r i a b i l i t y a n d p o p u l a t i o n struct u r e o f m a n y f r e e - l i v i n g o r g a n i s m s [1]. W h i l e e n z y m e e l e c t r o p h o r e s i s has b e e n a p p l i e d to m a n y p a r a s i t e species [2-5], few w o r k e r s h a v e e x p l o i t e d t h e full p o t e n t i a l o f the t e c h n i q u e f o r s t u d y i n g p a r a s i t e genetics. L e v e l s o f p o l y m o r p h i s m a n d m e a n h e t e r o z y -
Abbreviations: P, percentage of polymorphic loci; H, mean heterozygosity; A, mean number of alleles per locus. 0166-6851/86/$03.50
© 1986 Elsevier Science Publishers B.V. (Biomedical Division)
46 gosity in most parasites are largely unknown. In addition, hypotheses concerning the amount of gene flow between parasite populations have remained speculative. While several enzymes of ascaridoids have been studied electrophoretically [6-12], only two studies have addressed the question of population genetics from a multilocus approach [ 13,14]. These investigators characterized ascaridoids as having low levels of polymorphism and mean heterozygosity. Leslie et al. [14] have invoked several explanations to account for these observations in Ascaris s u u m including: (1) that the intestine of the vertebrate host is a relatively stable environment that selects for 'increased m o n o m o r p h i s m ' ; (2) A. s u u m may exist in 'small homogeneous populations with little gene flow between them'. Such a population structure has been inferred for many parasites in general [15]. In this study, levels of genetic variability in natural populations of ascaridoid nematodes were investigated using the multilocus electrophoretic approach. The results indicate that these nematodes have high levels of polymorphism and mean heterozygosity, and that phenotypes of the polymorphic proteins can be interpreted genetically. MATERIALS AND METHODS Collection. Population samples of live helminths were primarily obtained at necropsy of mammals collected in southern Louisiana. Some ascaridoids were collected following spontaneous or anti-helminthic induced expulsion. The live worms were rinsed in physiological saline, individually wrapped in aluminum foil to exclude air, sealed in plastic bags, and frozen and stored at -70°C. Worms were cataloged by sex, host, and geographic area of host collection.
Specific tissues of large ascaridoids, or whole small worms were minced, and then homogenized in two volumes of 250 mM sucrose containing 100 lag ml -~ dithiothreitol, NAD, and NADP. Homogenates were centrifuged at 5 000 X g for 10 rain at 4°C to remove cell debris. The supernatant fractions of homogenates were subjected to vertical starch gel electrophoresis [16], at 4°C. Starch gels were used in preference to polyacrylamide because of their greater solubility for the enzyme detection systems. The duration of the electrophoresis varied from 15 to 22 h. Comparisons of proteins from different individuals were done side by side on the same gel in order to avoid errors inherent in comparisons based upon relative mobilities. Each gel contained twelve samples, including one reference individual of known genotype. Approximately equal numbers of male and female worm homogenates were subjected to electrophoresis. Homogenates of host intestinal tissue were run on some gels as controls for external contamination by host a n d / o r gut flora enzymes. The following buffers were used for electrophoresis: (a) phosphate-citrate pH 6.8 (60 mM Na2HPO4, 10 mM citric acid) diluted 1:4 for gel and undiluted for electrode bath; (b) Tris-maleate pH 7.5 (100 mM Tris adjusted to p H 7.5 with 2 M maleic acid) diluted 1:3 for gel and
Electrophoresis.
47 undiluted for electrode bath; (c) Tris-EDTA borate pH 8.1 (30 mM Tris, 2 mM EDTA, adjusted to pH 8.1 with saturated boric acid) gel undiluted, borate bridge buffer pH 8.6 (see d below) used for electrode bath; (d) borate bridge buffer pH 8.6 (125 mM boric acid adjusted to pH 8.6 with 1 M NaOH) diluted 1:4 for gel, undiluted for electrode bath.
Enzyme localization.
Proteins were localized in gel slices with techniques described mainly in Harris and Hopkinson [17]. Glutamate dehydrogenase was detected by the method of Brewer [18]. Arginine kinase was localized by substituting phospho-L-arginine for phosphocreatine in the creatine kinase staining method 'B' of Harris and Hopkinson [17]. Reactions coupled to thiazoyl blue (MTT) tetrazoliums were used to detect dehydrogenases, isomerases, kinases, and phosphoglucomutase; those coupled to L-amino acid oxidase and peroxidase were used to localize peptidases. Peptidase substrates were valyl-leucine for peptidase A, leucyl-glycyl-glycine for peptidase B, and phenylalanyl-proline for peptidase D. Esterase D, which catalyzes the hydrolysis of 4-methyl-umbelliferyl esters, was visualized under ultraviolet light as bright fluorescent bands, ct-Naphthyl acetate and 13-naphthyl acetate were used as substrates for the corresponding esterases. Glutamate-oxaloacetate aminotransferase was localized by diazotization of the oxaloacetate reaction product with Fast Blue RR. Differences in phenotypes at a specific locus were used to deduce genotypes of individuals and the number of alleles segregating in the populations. When more than one presumed locus yielded products with the same activity, the locus having isozymes of faster anodal mobility was assigned the lower number; for multiple alleles, the one that yielded isozymes of fastest anodal mobility was designated 'a', those of slower mobility as 'b, c', etc., in order of decreasing electrophoretic mobility.
Analysis of genetic data.
Genotypes for single individuals at all loci were entered into the BIOSYS-1 computer program of Swofford and Selander [19]. Program outputs included values for polymorphism, allele frequencies, heterozygosity estimates, genetic similarity, and tests of conformance of observed allele frequencies in each population to Hardy-Weinberg equilibrium expectations. For small sample sizes, Levene's [20] correction for expected genotype frequencies was used. For each polymorphic protein, a Z: goodness-of-fit test was performed. However, when more than two alleles are observed at a locus the expected frequency of some genotypes can be low, and the Zz test can give misleading results [21]. In such cases, BIOSYS-I repeats the ;(2 test after pooling genotypes into three classes: (1) homozygotes for the most common allele; (2) heterozygotes for the most common allele and one of the other alleles; (3) all other genotypes. Exact significance probabilities were also calculated [22], which are analogous to Fisher's exact test for 2 × 2 contingency tables. This test avoids the difficulties encountered in using the ~2 distribution for small sample sizes.
2.6.1.1
Glutamate-oxaloacetate
Esterase D
3.1.1.1
2.7.5.1
Phosphoglucomutase
Hydrolases
2.7.3.3
A r g i n i n e kinase
transaminase
Glutamate-pyruvate
transaminase 2.6.1.2
1.15.11
Transferases
1.4.1.2
Superoxide dismutase
1.2.1.12
1.1.1.44
Glutamate dehydrogenase
dehydrogenase
phosphate
Glyceraldehyde-3-
dehydrogenase
6-Phosphogluconate
1.1.1.42
m,i,s
m,i,s
i,s
m
i,s
m
i,s
m
m,i,s
i,s
dehydrogenase
Isocitrate
m,i,s
i,s m,i,s
1.1.1.37
m,i,s
u,t
cm
cm
cm
cm
cm
cm
cm
cm
Tissue utilized
Taxa resolved
Malate dehydrogenase-2
1. I. 1.27
Lactate dehydrogenase
1.1.1.14
E.C. n u m b e r
Malate dehydrogenase- 1
dehydrogenase
Sorbitol
Oxidoreductases
Locus
P r o t e i n s resolved in a s c a r i d o i d t a x a a
TABLE 1
a
a
b
b
a
c
b
a
a
b
a
a
b
a
Buffer
m,i
i,s
none
none
none
none
none
none
s
none
none
none
s
none
Polymorphism
dimer
monomer
unknown
unknown
unknown
unknown
unknown
unknown
dimer
unknown
dimer
tetramer
unknown
Structure
i,s
3.1.1.1 3.4.13.9 3.4.1.1 3.4.13.9 3.5.4.4
4.1.2.13
5.3.1.8 5.3.1.9
a c e t a t e esterase Peptidase A
Peptidase B
Peptidase D
Adenosine deaminase
Lysases Aldolase
lsomerases Man nose-6-phosphate
isomerase Hexose isomerase
m
Hemoglobin (pseudocoelomic)
ps
cm
cm
cm
u,t
u,t
u,t
cm
cm
b
b
a
b
b
a
a
a
a
a
a
a
none
none
i
i,s
none
m
m
m,i
none
i
none
i,s
unknown
unknown
unknown
monomer
unknown
unknown
dimer
monomer
unknown
monomer
unknown
monomer
were d e d u c e d f r o m b a n d i n g p a t t e r n s o f h e t e r o z y g o t e s .
cuticle a n d muscle; u = uterus; t = testis; ps = p s e u d o c o e l o m i c fluid. Buffer c o m p o s i t i o n s a r e d e t a i l e d in M a t e r i a l s a n d M e t h o d s . S u b u n i t s t r u c t u r e s o f p r o t e i n s
equorum, i = T. cati, s = T. canis. W h o l e w o r m h o m o g e n a t e s were u s e d f o r Toxocara a n a l y s e s . A b b r e v i a t i o n s f o r tissues u s e d in s c o r i n g P. equorum loci are: c m =
a L o c u s a n d c o r r e s p o n d i n g e n z y m e c o m m i s s i o n n u m b e r s a r e listed. P o l y m o r p h i c p r o t e i n s , a n d loci s c o r e d for e a c h species a r e i n d i c a t e d b y a b b r e v i a t i o n : m = P.
m
Other proteins H e m o g l o b i n (muscle)
m,i,s
i,s
i,s
m
m
m,i,s
m,i,s
m
m,i,s
acetate esterase-2 ~Naphthyl
3.1.1.1
a c e t a t e esterase-I ct-Naphthyl
a-Naphthyl
50 RESULTS Population samples of Parascaris equorum, Toxocara canis, and Toxocara cati have been analyzed at 18 genetic loci. Buffers ranging in pH from 6.0 to 8.1 were employed in order to maximize the detection of polymorphism. The loci scored, enzyme commission numbers, tissues utilized, and the buffer yielding optimum resolution are listed in Table I. Fresh homogenates were used to assay for sorbitol dehydrogenase because its activity was greatly diminished in frozen homogenates. Although some enzymes exhibited reduced activity following freeze-thaw of homogenates, no difference was found between homogenates prepared from fresh and frozen nematode tissues. Similarly, T. canis specimens voided from hosts following organophosphate treatment had enzymatic activities comparable to those obtained from hosts at necropsy. Enzymatic activities detected in P. equorum gels, but not resolved under the conditions employed included: isocitrate dehydrogenase; glucose-6-phosphate dehydrogenase; lactate dehydrogenase; a-glycerol phosphate dehydrogenase; glutamate-oxaloacetate transaminase; arginine kinase; and aldolase. Similarly, glucose-6-phosphate dehydrogenase, peptidase D, and hexokinase activities were detected, but not resolved in experiments with Toxocara spp. No sex-specific loci were detected. The number and relative staining intensity of isozyme bands in heterozygotes was used to deduce the subunit structure of enzymes. For all proteins where heterozygous phenotypes could be clearly interpreted, the deduced subunit structures corresponded to those of vertebrate enzymes (Table I, Fig. 1). Side by side comparisons ofascaridoid homogenates and samples of their respective host's small intestine revealed no evidence of host/gut flora enzyme contamination (Fig. 1). Four enzymatic loci were polymorphic in P. equorum, five in T. canis, and seven in T. cati. Values for the percentage of polymorphic loci (P), mean heterozygosity (H), and mean number of alleles per locus (A) for each species are shown in Table II. Coefficients of Rogers' genetic similarity [23] between five T. cati populations derived from different cats in Orleans Parish ranged from 0.89 to 0.95. Observed allele frequencies of the P. equorum population conformed to H a r d y Weinberg equilibrium expectations as tested by Z 2 goodness-of-fit. This population was composed of 29 individual nematodes obtained from a single horse. Zz significance tests revealed five cases where the T. canis or T. cati populations deviated from Hardy-Weinberg equilibrium expectations at individual loci. These populations were composed of 32 individuals obtained from three dogs, and 22 individuals obtained from six cats, respectively. However, X2 results are suspect in cases with small sample size or large numbers of genotype classes. Following Z2 analyses with pooled genotype classes or an exact probability test, only two cases remained significant at the P<0.05 level (Table III). For the esterase D locus in T. cati, this result is not surprising because there are low expected frequencies in a number of genotype classes, and only 22 individuals were sampled. However, for 6-phosphogluconate dehydrogenase in T. canis there are only three genotype classes, and there appears to be a real deficiency of heterozygotes at this locus.
51
a
b
c
Fig. I. Phenotypes of some polymorphic ascaridoid enzymes. Slot numbers (in parentheses) and presumed genotypes are read left to right. The allele of fastest anodal mobility is designated 'A', and those of slower mobility sequentially as 'B, C', etc. These gel photographs do not illustrate all alleles. The anode is to the top. (a) Lactate dehydrogenase, T. canis (1,3) and T. cati (2), genotypes A/C, B/B, A/A. The heterozygote has five bands with a 1:4:6:4:1 staining intensity, indicating a tetrameric subunit structure. (b) 6-Phosphoglucohate dehydrogenase, T. canis, genotypes B/C, B/B. The heterozygote has three bands with a 1:2: I staining intensity, indicating a dimeric subunit structure. (c) Phosphoglucomutase, T. canis, genotypes B/B, A/C, A/B. Heterozygotes have two bands of equal staining intensity indicating a monomeric subunit structure. (d) Esterase D, T. cati, genotypes C/C, A/B. Heterozygote phenotype characteristic of dimeric protein. (e) Peptidase B, T. cati (1,3) and cat small intestine (2). There is no evidence of host-enzyme contamination of the ascarids. (f) a-Naphthyl acetate esterase, T. cati, genotypes B/B, A/B, A/A. Heterozygote phenotype characteristic of monomeric protein. (g) Peptidase B phenotypes, T. cati genotypes C/C, D/G, F/H. Heterozygote phenotypes characteristic of monomeric protein. (h) Peptidase D, P. equorum, genotypes B/B, B/C. Heterozygote phenotype characteristic of dimeric protein. (i) Mannose-6-phosphate isomerase, T. canis, genotypes B/B, A/B, B/B. Heterozygote phenotype characteristic of monomeric protein.
DISCUSSION T h r e e s t a n d a r d m e a s u r e s o f g e n e t i c v a r i a b i l i t y : m e a n n u m b e r o f alleles p e r l o c u s ; p e r c e n t a g e o f p o l y m o r p h i c loci; a n d m e a n n u m b e r o f h e t e r o z y g o u s loci p e r i n d i v i d u a l , r e v e a l h i g h levels o f g e n e t i c v a r i a t i o n w i t h i n p o p u l a t i o n s a m p l e s o f P. e q u o r u m , T. canis, a n d T. cati. T h i s c o n s i d e r a b l e g e n e t i c d i v e r s i t y in o r g a n i s m s i n h a b i t i n g w h a t is
52 T A B L E II
Genetic variability at 18 loci in three species of ascaridoid nematodes (mean and standard error are shown) Mean sample size per locus
Mean no. of alleles per locus
Percentage of loci polymorphic a
Mean heterozygosity Direct count
Hardy-Weinberg expected
P. equorum
27.7
1.44 + 0.22
22.2
0.085 + 0.042
0.090 + 0.046
T. canis
29.5 20.6
1.44 + 0.18 1.94 ± 0.42
33.3 38.9
0.135 + 0.061 0.137 + 0.057
0.118 + 0.051 0.139 + 0.056
T. cati
a A locus is considered polymorphic if the frequency of the most common allele does not exceed 0.99.
T A B L E III
Apparent cases of deviation from Hardy-Weinberg equilibrium in T. canis a n d 11 cati Species and locus
Genotype
Observed number
Expected number
Significance level (P) Z2
T. canis
B-B B-C
14 8
11.5 12.9
6-Phosphogluconate dehydrogenase
C-C
6
3.6
T. cati
A-A A-B
0 1
0.01 0.11
Umbelliferyl-acetate esterase
A-C
0
0.86
B-B
1
0.28
B-C
2
4.32
C-C
18
16.41
Pooled X2
0.04
0.01
Exact P
0.04
0
0.03
frequently considered a h o m o g e n o u s environment is not consistent with the hypothesis that the intestinal environment selects for m o n o m o r p h i s m a m o n g endoparasites. Levels of polymorphism and mean heterozygosity in the Baton Rouge population of P. equorum (P = 22%, H = 0.085) were similar to values reported by Leslie et al. [ 14] for an Iowa population of Ascaris suum (P = 21%, H = 0.066). These data are in marked contrast to the report of Bullini et al. [13] who found only 3.7% o f P . equorum loci polymorphic with a mean heterozygosity of 0.0088. These authors may have seriously underestimated variation due to procedural problems, such as those that may result from using horizontal gels, or in basing comparative evidence on relative mobilities. However, it is possible that these reports reflect real differences in genetic variability between P. equorum populations from Louisiana and central-eastern Europe. Animal populations established by the colonization of a small number of founding individuals, such as island populations, and those that undergo frequent bottlenecks in population
53 size often have low levels of mean heterozygosity. Although the number of T. cati individuals from each host was small, the matrix of Rogers' similarity coefficients does not reveal any marked genetic differentiation between these 'host populations'. However, studies of ascaridoid populations from a wide geographic range of hosts are required to establish the level ofgene flow between distant populations. The conformance of most observed allele frequencies to Hardy-Weinberg equilibrium expectations suggests that these proteins are inherited in a Mendelian fashion and that these nematode populations are mating at random. T. canis 6-phosphogluconate dehydrogenase genotypes did not conform to Hardy-Weinberg equilibrium expectations. A Wahlund [24] effect could account for the apparent deficiency of heterozygotes at this locus, because pooled samples may inadvertently conceal differences in allele frequencies between genetically distinct populations. This result also suggests that geographically distinct populations of ascaridoids may be genetically distinct. Among enzyme classes of the three ascaridoid species studied, oxidoreductases, transferases, hydrolases, and isomerases included at least one monomorphic and one highly polymorphic locus. Of the five major classes of enzymes, the hydrolases contributed most to overall polymorphism, the oxidoreductases the least. Of the 16 polymorphic loci distributed among the three species, nine were of the hydrolase class. Such a differential distribution of polymorphic loci among enzyme classes has been reported previously in both ascaridoids [14] and other invertebrates [25]. While no single enzymatic locus is consistently polymorphic among studied ascaridoid species, certain loci are regularly monomorphic when resolved. These loci include: sorbitol dehydrogenase, superoxide dismutase, glutamate-pyruvate transaminase, isocitrate dehydrogenase, glutamate dehydrogenase, glutamate-oxaloacetate transaminase, and aldolase. Possibly these enzymes will not tolerate many amino acid substitutions due to functional constraints. Enzymes which are monomers, dimers, or tetramers in mammals, birds, reptiles, and amphibians have identical subunit structures, where deduced, in ascaridoid nematodes. This observation further substantiates the conservative nature of polypeptide subunit number across phylogenetically diverse groups of organisms (Dessauer, H.C. and Braun, M.J. (1982) Abstr. 4th Int. Congr. Isozymes, Austin, TX). The conservation of enzyme subunit structure and substrate specificity implies that the enzymatic activities observed in diverse organisms are performed by homologous proteins. This evidence of homology further supports the inference of Mendelian inheritance of these proteins. ACKNOWLEDGEMENTS I would like to thank Dr. Herbert C. Dessauer and Dr. Joseph H. Miller for their assistance and encouragement. This work was supported in part by a Grant-in-Aid of Research from Sigma Xi, The Scientific Research Society.
54 REFERENCES 1 2 3
4
5 6 7 8 9 10
11 12 13
14 15 16 17 18 19 20 21 22 23 24 25
Nevo, E. (1978) Genetic variation in natural populations: Patterns and theory. Theor. Popul. Biol. 13, 121-177. Leriche, P.D. and Sewelt, M. (1977) Differentiation of Taenia saginata and Taenia solium by enzyme electrophoresis. Trans. R. Soc. Trop. Med. Hyg. 71, 327-328. Gibson, W.C., Marshall, T.F. and Godfrey D.G. (1980) Numerical analysis of enzyme polymorphism: A new approach to the epidemiology and taxonomy of trypanosomes of the subgenus Trypanozoon. Adv. Parasitol. 18, 175-246. Fletcher, M., LoVerde, P.T. and Woodruff, D.S. (1981) Genetic variation in S c h i s t o s o m a mansoni: Enzyme polymorphisms in populations from Africa, Southwest Asia, South America, and the West Indies. Am. J. Trop. Med. Hyg. 30, 406-421. LoVerde, P.T., DeWald, J., Minchella, D.J. Bosshardt, S.C. and Damian, R.T. (1985) Evidence for host-induced selection in Schistosoma mansoni. J. Parasitol. 71,297-301. Zam, S.G. (1973) Disc electrophoresis o f A s c a r i s suum female reproductive esterase enzymes. Comp. Biochem. Physiol. 46B, 797-803. Rhodes, M.B., Marsh, C.L. and Kelley, G.W. (1964) Studies in helminth enzymology, lII. Malic dehydrogenases of Ascaris suum. Exp. Parasitol. 15, 403-409. Zee, D.S. and Zinkham, W.H. (1968) Malate dehydrogenase in Ascaris suum: characterization, ontogeny, and genetic control. Arch. Biochem. Biophys. 126, 574-584. Zee, D.S, Isensee, H. and Zinkham, W.H. (1970) Polymorphism of malate dehydrogenase in Ascaris suum. Biochem. Genet. 4, 253-257. Langer, B.W., Jr. and Smith, W.J. (1971) The lactic acid dehydrogenase of some parasitic gastro-intestinal nematodes: Ascaris suum, O e s o p h a g o s t o m u m radiatum and H a e m o n c h u s contortus. Comp. Biochem. Physiol. 40B, 833-840. Agatsuma, T. and Suzuki, N. (1981) Electrophoretic studies on enzymes in ascarids. I. Lactate dehydrogenase isozymes in Ascaris suum. Jpn. J. Parasitol. 30, 81-83. Gockel, S.F. and Lebherz, H.G. (1981) 'Conformational' isoenzymes of ascarid enolase. J. Biol. Chem. 256, 3877-3883. Bullini, L., Nascetti, G., Ciafr~, S., Rumore, F., Biocca, E., Montalenti, S. and Rita G. (1978) Ricerche cariologiche ed elettroforetiche su Parascaris univalens e Parascaris equorum. Atti A'ccad. Naz. Lincei CI. Sci. Fis. Mat. Nat. Rend. 65, 151-156. Leslie, J.F., Cain, G.D., Meffe, G.K. and Vrijenhoek, R.C. (1982) Enzyme polymorphism in Ascaris suum (Nematoda). J. Parasitol. 68, 576-587. Price, P.W. (1977) General concepts on the evolutionary biology of parasites. Evolution 31,405-420. Smithies, O. (1959) Zone electrophoresis in starch gels and its application to studies of serum proteins. Adv. Protein Chem. 14, 65-113. Harris, H. and Hopkinson, D.A. (1976) Handbook of Enzyme Electrophoresis in Human Genetics, Elsevier/North-Holland, Amsterdam. Brewer, G.J. (1970) An Introduction to Isozyme Techniques, Academic Press, New York. Swofford, D.L. and Selander, R.B. (1981) BIOSYS-I: A FORTRAN program for the comprehensive analysis of electrophoretic data in population genetics and systematics. J. Hered. 72, 281-283. Levene, H. (1949) On a matching problem arising in genetics. Ann. Math. Stat. 20, 91-94. Sokal, R.R. and Rolfe, F.J. (1969) Biometry, W.H. Freeman and Co., San Francisco, CA. Elston, R.C. and Forthofer, R. (1977) Testing for Hardy-Weinberg equilibrium in small samples. Biometrics 33, 536-542. Rogers, J.S. (1972) Measures of genetic similarity and genetic distance. Stud. Genet. (Univ. Tex. Publ.) 7213, 145-153. Wahlund, S. (1929) Zusammensetzung von Populationen und Korrelationserscheinungenvom Standpunkt der Vererbungslehre aus betrachtet. Hereditas 11, 65-106. Johnson, G.B. (1976) Genetic polymorphism and enzyme function. In: Molecular Evolution (Ayala, F.J., ed.), pp. 46-59, Sinauer, Massachusetts.