Inlernario~lfournalfor Parasitology Vol. 22, No. 5, pp. 581-587, 1992 Printed in Greor Britain 0

ALLOZYME VARIATION WILD POPULATIONS

ooze-7519/92 $5.00 + 0.00 Pergamon Press Lfd I992 Awrralian Socieryfor Parasirology

BETWEEN LABORATORY REARED AND OF TELADORSAGIA CIRCUMCINCTA

N. GASNIER,*

J. CABARET*~

and C. MOULIA$

* INRA, Station de Pathologie aviaire et de Parasitologie, Unite d’Ecologie des Parasites, 37380 Nouzilly, France $ Laboratoire de Parasitologie cornpar& USTL, Place E. Bataillon, 34060 Montpellier CCdex, France (Received 20 May 199 1; accepted 16February 1992) Abstract- GASNIERN., CABARETJ. and MOULIA C. 1992. Allozyme variation between laboratory reared and wild populations of Teladorsagia circumcincta. International Journal for Parasitology 22: 581-587 The technique of allozyme electrophoresis was applied to two laboratory strains (isolated from the center or south-east of France) and wild populations of Teludorsugiu circumcincta from the center of France. Five systems out of 13 (GPI, MPI, MDH, LDH, PGM) could be interpreted. By means of a multivariate analysis, it was shown that the laboratory strains were very similar with each other and genetically different from the wild populations. A deficiency of heterozygotes was recorded for each enzyme locus (except for MDH) in all populations studied. INDEX

KEY WORDS:

Nematode;

Teludorsagiu circumcincta; isoenzyme;

INTRODUCTION

MOST of our basic knowledge on the biology of digestive-tract strongyles has been established using laboratory strains. -These are maintained in standard conditions: infection of young and naive hosts, cultivation from eggs to larvae at optimum temperature and humidity, and storage of infective larvae at 4°C over several months. Artificial breeding conditions induce a reduction of genetic variability (see Maynard Smith, 1989) and the literature is replete with arguments concerning the effect of genetic drift and selection (McDonald, 1983). A very demonstrative example in recently domesticated animals (mimicking the situation of nematode laboratory strains) is found in French brown trout (Sulmo trutta L.): large geographical differentiation of wild populations vs high similarity in hatchery strains (Krieg & Guyomard, 1985). Nadler (1990) reported a substantial decrease in genetic variability in laboratory maintained ‘colonies’ of schistosomes. A similar reduction of variability might occur in digestive-tract strongyles reared for several generations in the laboratory. Allozyme electrophoresis is a particularly powerful technique for assessing the genetic variability within and between populations. Very few studies are available for digestive-tract strongyles (Baker & Fisk, 1976; Andrews & Beveridge, 1990) and electrophoresis was

t To whom all correspondence

should be addressed.

enzyme electrophoresis;

strains.

conducted on pools of worms. The purpose of the present work was to assess allozyme variation in individuals of laboratory reared strains or wild populations of the digestive-tract strongyle Teludorsugia circumcincta. MATERIALS AND METHODS Nematodepopulationsstudied. Two laboratory strains were investigated. The maintenance of these strains was standard: (i) infection of naive lambs with 500&7000 infective thirdstage larvae (L3), (ii) collection of feces (5 and 6 weeks postinfection), culture of nematode eggs to L3 at 23’C and 80% relative humidity over 10 days, and baermannization to extract L3, (iii) storage at 4’C of L3 concentrated at a maximum of 2500 L3 mll’. The first strain (Provence region, coded as P) originated from an isolate of 300 females obtained on several naturally infected sheep in a flock grazed on irrigated pastures in the south-east of France (Salon de Provence). The strain P has been bred for 3 years in laboratory conditions (fourth generation). The second strain (Touraine region: coded as T) originated from an isolate of 100 worms collected in 1983 at the abattoir of Tours (centerwest of France) on several sheep and goats of various flocks. The worms from the 10th generation were used. These two strains were monomorphic on morphological grounds: only T. circumcincta morph circumcincta was recovered. The natural populations of T. circumcincta were collected on sheep slaughtered at the abattoir of Tours in May and June 1990. The recruited sheep were of center-west origin. On every sampling occasion, the abomasa of five sheep from one flock were collected. The worms were extracted from the abomasum by immersion in water at 37’C over 2-5 h. A total

N. GASNIER, J. CABARET and C. MOULIA

582

of eight flocks was then investigated and the electrophoretic data were pooled due to the small size of the worm burdens (referenced as pooled natural populations: PNP). One flock that yielded larger numbers of nematodes was studied separately (single natural population: SNP). Males of 7’. circumcincta morph circumcincta were stored in liquid nitrogen in groups of 25 in 2 ml vials without preservation liquid until required for processing; males were utilized as freshly collected on four occasions. Electrophoresis. The worms in vials were allowed to thaw and then washed with deionized water. Each worm was crushed on 2 x 3 mm Whatman paper No. 3 and inserted in to a 10% starch gel tray. Standard horizontal electrophoresis was carried out at 100-160 V (two migrating lines of 20 worms each) for 4-5 hat 4’C. Gels were then sliced in two and each part stained for a specific enzyme. The following enzymes were studied: alcohol dehydrogenase (ADH, E.C. 1.1.1.1). fumarate hydratase (FUM, E.C. 4.2.1.2), guanine deaminase (GDA, E.C. 1.4.1.3),glucose-6-phosphate dehydrogenase (G6PD. E.C. 1.1.1.49), glucose-phosphate isomerase (GPI, E.C. 5.3.1.9), lactate dehydrogenase (LDH, E.C. I. 1.1.27), malate dehydrogenase (MDH, E.C. 1. I .I .37), malic enzyme (ME, E.C. 1.1.1.40), mannose-phosphate isomerase (MPI, E.C. 5.3.1.8), purine nucleoside phosphorylase (NP, E.C. 2.4.2.1), phosphoglucomutase (PGM, E.C. 5.4.2.2), sorbitol dehydrogenase (SDH, E.C. 1.1.1.14), and superoxide dismutase (SOD, E.C. 1.15.1.1). Electrophoretic buffers (Table 1) and stains were modified either from Richardson, Baverstock & Adams (1986) or Pasteur, Pasteur, Bonhomme & Britton-Davidian (1987) (Table 2). Detailed recipes are available upon request in the form of a hypertext running under the MS-DOS operating system on IBM-compatible computers. Dafa analysis. Allozyme frequencies for each population were derived from the electrophoretic results. The departure from the Hardy-Weinberg equilibrium was tested according

TABLE I-COMPOSITIONOFBUPFERSYSTEMS

Buffer system

Electrodes

Gel

TE 9.4

12.11 gTris 200 mg EDTA H,O 1 liter pH 9.4

20 ml TC 9.4 180 ml H,O 20 g starch

TEB 7.8

3.64 g Tris 3.72 g Na,EDTA I .90 g M&l, H,O I liter pH 7.8 with boric acid

100 ml TEB 7.8 100 ml H,O 20 g starch

to Elston & Forthofer (1977) because of the small size of samples. F,, of Wright, 1965 (ratio of variance of genotypic frequencies between populations to variance of genotypic frequencies of all the populations) was used to assess the reality of subdivisions into different populations of T. circumcincta from the strains or wild populations. The statistical significance was derived from Chi-square values for each isozyme. Genetic distances between populations were assessed by means of principal component analysis (PCA) using a computer statistical package (Stat-Itcf, 1988). The following matrix of data was used: the rows were genotypic frequencies of rapid electromorph and heterozygotes for five enzymatic systems, and columns were the four populations tested. The frequencies (a and 1 -a for each system) were estimated on the variable number of worms (n) for each system; minimum and maximum values (frequency n ) were estimated at P < 0.05 for each * 2 Ja(l-a)lJ frequency. PCA was performed successively on mean, maximum and minimum values of these frequencies.

TABLET--ELECTROPHORETICPROCEDURES

Enzymes

LDH

Buffer systems

Volts

Time (h)

TEB 7.8 160

4

Staining buffers

Coenzymes enzymes

0.1 M-Tris-HCI pH 8 20 ml

NAD

Linking methods

1ml

MDH

TEB7.8

160

4

0.1 t+Tris-HCI pH820ml

NAD I ml NADP 1 ml

GPI

TE9.4

100

5

0.2 M-Tris-HC1 pH8 10ml

NAD 1 ml G6PDH NADP 0.5 ml

MPI

TE9.4

100

5

0.2 M-Tris-HCI pH8 10ml

NAD I ml G6PDH 6 ~1 NADP 0.5 ml PGI 15 ~1

PGM

TE9.4

100

5

0.2 t+Tris+HCl pH8 10ml

NAD 1 ml G6PDH NADP 0.5 ml

MTT, Methyl thiazolyl blue; PMS, phenazine

methosulfate;

Substrates

Activators

L-Lactic acid 50mgml-‘pH8 I ml

MTT

1ml

L-Malic acid 50mgml ‘pH8 1 ml 20 ~1 Fructose-6phosphate IO mg Mannose-6phosphate 20 mg Pyruvate 20 mg

20 ~1 Glucose- lphosphate 300 mg

NBT, nitroblue

tetrazolium.

Visualization

MgCI, 0. I I ml

PMS

I ml

MTT PMS

I ml 1ml

M NBT 1 ml PMS 0.5 ml MTT I ml PMS I drop

MgCl, 0.1 M NBT 1 ml PMS 0.5 ml 1 ml

Allozyme

variation

of T. circumcincta

of Teludorsugia circumcincta isoenzyme pattern (R, rapid FIG. LDH, MDH and MPI photographs S isozyme is not shown for MDH. Quaternary structure of LDH heterozygote elect! ronnorphs, H, heterozygote). MDH has a three-banded heterozygote and MPI a two-banded heterozygote.

583

and

S,

: not asse

N. GASNIER,J. CABARETand C. MOULIA

584 RESULTS

OPI

+

+

MPI

+

MD&l

+

LDH

+

PQM-1

Genetic structure of populations at enzyme loci

Thirteen enzymes were screened, from which a total of five enzymes (GPI, MDH, MPI, LDH, PGM) could be interpreted. Photographs of the gels for the enzymes LDH, MDH and MPI are shown in Fig. 1. The electrophoretic patterns are given in Fig. 2, with the exception of the anodal migration of MDH (not interpreted) and of the invariant locus PGM-2. Three distinct phenotypes could be observed: single-banded phenotypes (rapid, R and slow, S migration) and two(MPI, PGM) or three-banded (MDH) phenotypes characteristic of heterozygotes for monomeric/dimeric enzymes, respectively. The quaternary structure of GPI and LDH could not be assessed, but individuaIs with inte~ediate bands located from the S to R position were recorded as heterozygotes. These intermediate band individuals were found in all populations and were not apparently related to storage in nitrogen and its duration. The allele frequencies for each putative locus (five loci) in each studied population are given in Table 3.

Deflclenoy

in hetwozygotee

.*t

FIG. 2. Diagrams of Teladorsagiacircumcinctafiveisoenzyme patterns (arranged in the following order: rapid, slow and heterozygote). Quaternary structure of GPI and LDH not assessed.

Genotypic frequencies were compared to expected Hardy-Weinberg proportions (see Table 3). Significant differences were recorded between observed genotypic frequencies and those expected under the Hardy-Weinberg equilibrium. A significant deficiency of heterozygotes (up to 41%) was observed for each enzyme locus except for MDH (Fig. 3).

(W)

10 0

I

B&l

P

PNP

8ilP

Poputatkns II

QPI

MPI

aPOM-1

m

MDH-1

BLDH

FIG. 3. Deficiency in heterozygotes (observed - expected frequencies) for five enzyme loci. Wild populations: single, SNP and pooled, PNP; strains: Touraine, T and Provence, P.

Allozyme

variation

of T. circumcincta

585

TABLE~~ENOTYPESANDALLELICFREQUENCIES(RAPID, Rand SLOW,S)INDIFFERENTPOPULATIONSOF Teladorsagia circumcincta

Enzyme RR GPI Strain T

obs

Strain P

exp obs

SNP

cxp obs

PNP

exp obs exp

MPI Strain T

obs cxp

Strain P

obs

SNP

cxp obs

PNP

exp obs cxp

PGM-1 Strain T

obs

Strain P

exp obs

SNP

exp obs

PNP

exp obs exp

MDH-1 Strain T

obs

Strain P

exp obs

SNP

exp obs

PNP

exp obs exp

LDH Strain T

obs

Strain P

cxp obs

SNP

exp obs

PNP

exp obs exp

Genotype RS

n ss

Allelic frequency R S

35 35

8 9

1 0

44

0.89

0.11

60 48

3 28

17 4

80

0.77

0.23

18 13

9 18

10 6

37

0.60

0.40

15 12

5 11

6 3

26

0.67

0.33

18 9

4 22

22 13

44

0.45

0.55

6 3

5 10

10 8

21

0.40

0.60

12 8

13 21

18 14

43

0.43

0.57

10 5

15 25

37 32

62

0.28

0.72

11 3

6 23

55 46

72

0.20

0.80

7 2

4 14

32 27

43

0.21

0.79

23 18

9 18

9 5

41

0.67

0.33

28 28

16 17

3 2

47

0.77

0.23

34 37

25 20

1

60

0.78

0.22

3

78 76

27 31

5 3

110

0.83

0.17

39 40

15 13

0 I

54

0.86

0.14

47 47

6 6

0 0

53

0.94

0.06

14 4

5 24

44 35

63

0.26

0.74

16 7

20 38

59 50

95

0.27

0.73

6 2

10 17

32 29

48

0.23

0.77

20 13

6 21

17 9

43

0.54

0.46

obs, Observed numbers; exp, expected numbers calculated on the basis of the Hardy-Weinberg law; n, numbers of male worms sampled for each population.

N.

586

GASNIER,

J. CABARET and C. MOULIA

Genetic dtyerentiation betweenpopuiations From F,,, the significant (PcO.01 except for MPI) Chi-squares were obtained for 3 degrees of freedom: 31.8 (GPI), 8.5 (MPI), 149.8 (PGM), 19.4 (MDH) and 48.8 (LDH). The circle of correlations (axes 1 and 2, respectively 75 and 22% of inertia) of principal component analysis is given in Fig. 4. The analyses performed on minimum and maximum values are not presented as they did not differ significantly from that performed on mean values. The angle between vectors representing strains on the one hand and natural populations on the . . other is approximately 90”, which means that they are different on the basis of allozyme frequencies. The two laboratory strains are very similar according to this analysis.

FIG. 4. Wild populations (single, SNP and pooled, PNP) and laboratory strains (Touraine, T and Provence, P): principal component

analysis

of electromorph isoenzymes.

frequencies

for five

DISCUSSION

Andrews & Beveridge (1990) considered GPI, LDH and PGM as invariant among pools of individuaIs of T. circumcincta whereas these enzyme loci were polymorphic when individual worms were examined. FUM was interpreted as polymorphic by the previous authors but we were only able to detect activity and no genetic interpretation was proposed. MPI was found to be monomeric and polymorphic in both studies. Deficiency in the number of heterozygotes was recorded. Inbreeding may be invoked to explain the deficiency of heterozygotes within strains. A deficiency of heterozygotes is to be expected when a total sample is made up by pooling samples from populations with different gene frequencies (the Wahlund effect); it may partly account for the relative lack of heterozygotes in pooled sample sets of wild populations. The Wahlund effect should not interfere in the single wild population set. The deficiency is grossly of the same order of magnitude in strains and natural populations and it does not affect all loci: population substructuring (two

or more genetically distinct groups or cryptic species present in the sample set) is thus a possible explanation. The maintenance of variation is achieved by combination of three major causes according to Maynard Smith (1989): (i) variation is selectively neutral, (ii) selection is balanced by mutation, or (iii) equilibrium of selective forces. No information is readily available on points (i) and (ii) whereas partial knowledge exists on the latter, in particular on fitnesses that vary in space and time. It is well documented that development and survival of free-living stages of T. circumcincfa depend on the environment and that selective pressure of climatic factors (temperature and humidity) is important (Kates, 19.50). Our laboratory breeding conditions for nematodes were constant and the strains (Touraine and Provence) did resemble each other more than the original natural populations (pooled or single). This gives indirect support to the selectionist viewpoint, that reduced genetic variation among populations is correlated with ecological homogeneity. Further investigations are thus required to assess the representativeness of laboratory strains of digestive-tract strongyles in ecological studies. Acknon$edgements-We are grateful to F. Renaud (Universitt des Sciences et Techniques du Languedoc, Laboratoire de Parasitologie comparte) for critical comments on the manuscript. Financial support from the French Ministery of Research was obtained in the form of a grant to N.G. REFERENCES ANDREWS R.H. & BEVERIDCEI. 1990. Apparent absence of genetic differences among species of Teladorsagia (Nematoda: Trichostrongylidae). Journal of Helminfhology 64:

290-294. BAKER N.F. & FISK R.A. 1976. Phenotypic differences in female ~sfert~~~u CircMrncjn~fa as demonstrated by isoenzymes hydrolyzing alpha-naphthyl acetate. American

Jourrtai of Veferinary Research 37: 325-327.

ELSTON R.C. & FORTHOFER R. 1977. Testing for HardyWeinberg equilibrium in small samples. Biometrics 33: 536-542. KATES K.C. 1950. Survival on pastures of free-living stages of some common nematodes of sheep. Proceedjngs of the Hefm~n fhologi~al Society of Wa.~hi~gfan17: 39-58. KRIEG F. & GUYOMARD R. 1985. Population genetics of French brown trout (Salmo frufta L.): large geographical differentiation of wild populations and high similarity of domesticated stocks. G&htique S6lection Evolution 17:

225-242. MAYNARD S~rrt-1 J. 1989. Evolufi(~~ary Genetics. Oxford University Press, Oxford. MCDONALD J.F. 1983. The molecular basis of adaptation: a critical review of relevant ideas and observations. Annual

Review of Ecology and Systematics 14: 77-102. NADLER S.A.

1990.

Molecular

approaches

to

studying

helminth population genetics and phylogeny. International

Allozyme variation of Z’. circumcincta

587

atics and Population Studies. Academic Press, San Diego. Journalfor Parasitology 20: 11-29. PASTEUR N., PASTEURG., BONHOMME F. & BRITTON-DAVIDIAN STAT-ITCF1988. Manuel d’l3tilisation. Institut des C&ales et J. 1987. Manuel technique de gh&ique par Plectropkor&e des Fourrages, Paris. WRIGHTS. 1965. The interpretation of population structure desprot&nes. Lavoisier, Paris. by F-statistics with special regard to systems of mating. RICHARDSON B.J., BAYERSTOCK P.R. & ADAMS M. 1986. Evolution 19: 395420. Allozyme Electropkoresis. A Handbook for Animal System-