Mitochondrial DNA Variation in Benzimidazole-Resistant and -Susceptible Populations of the Small Ruminant Parasite Teladorsagia circumcincta V. Leignel and J.-F. Humbert

The genetic diversity of the mtDNA ND4 gene in 11 Teladorsagia circumcincta populations from France and Morocco was assessed by sequencing. Some of these nematode populations were resistant to benzimidazole (BZ) anthelmintics, while others were susceptible. The nucleotide diversity in all populations studied was very high, probably due to a high mutation rate in nematodes, but there was no significant difference between them. This suggests that no strong, recurrent bottlenecks occur during the acquisition of BZ resistance by a worm population. The conservation of genetic variations during the acquisition of BZ resistance is probably due to the fact that anthelmintic treatments do not kill all the susceptible adult worms and to the presence of numerous free-living larvae that are not submitted to this anthelmintic pressure. There was no genetic subdivision between worm populations on a small geographical scale (less than 200 km), but significant FSTs were found on a larger geographical scale. This kind of subdivision cannot be explained by different genetic flows between populations because all these populations were isolated from each other. This subdivision is probably due to the breeding management practices and the large size of these worm populations, which limit genetic drift.

From INRA, Station de Pathologie Aviaire et de Parasitologie, 37380 Nouzilly, France ( Leignel) and INRA, Station d’Hydrobiologie Lacustre, BP 511, 74203 Thonon, France ( Humbert). We thank M. Peloille from the sequencing service of INRA (Station P.A.P., Tours), C. Sauve´ for technical assistance, J. Cabaret and B. Berrag for providing parasite populations, and J. Cabaret for stimulating discussions. The English text was checked by Dr. Owen Parkes. Address correspondence to J.-F. Humbert at the address above or e-mail: humbert@ thonon.inra.fr.
2001 The American Genetic Association 92:503–506

For the nematodes that parasitize small ruminants, the population dynamic is closely linked to the breeding management of their hosts. For example, anthelmintic treatment on a regular basis greatly reduces the size of the adult parasite worm population. But the population dynamic is also linked to environmental conditions, particularly for the free-living larvae. Low temperatures in winter or high temperatures in summer can kill many of the larvae living on pastures. Other breeding management practices can also influence parasite populations. Blouin et al. (1992, 1995) observed population genetic structures consistent with a high gene flow among populations of trichostrongylid nematode parasites. They suggested that the lack of differentiation between populations was due to the shipping of livestock around the United States. In contrast, studies on nematode species with small population sizes and low mobility demonstrated less genetic variation and subdivisions in the populations ( Blouin et al. 1999; Nadler 1995). Breeding management practices for small ruminants in France are very different from those of the United States. The herds are made up of animals from differ-

ent farms, but infected animals are not subsequently introduced and there is no contact between herds (Cabaret and Gasnier 1994). As trichostrongylid parasites have no intermediate hosts which can make possible the dispersion of these parasites from one herd to another, the gene flow must be dramatically limited between them. The hosts are also treated with anthelmintics two or three times a year, which reduce dramatically the worm populations. These conditions could give rise to a subdivision of these parasite populations. We looked for such subdivisions in Teladorsagia circumcincta, one of the most abundant gastrointestinal trichostrongylid species in temperate climates, examining eight populations from the center-west of France which were chosen because of our knowledge of their BZ resistance or susceptibility status, two from southwest France, and one from Morocco. These latter three populations were studied as geographically isolated outgroups. BZ resistance and BZ susceptibility were defined in terms of the proportion of BZ-resistant worms in the population. BZ resistance depends on a point mutation on the gene encoding ␤-tubulin, which is the target of

503

these drugs ( Elard et al. 1996, 1999; Elard and Humbert 1999; Kwa et al. 1993a, 1994). We also examined the link between the development of resistance in a worm population and a decrease in genetic variation. Several studies ( Beech et al. 1994; Elard and Humbert 1999; Grant and Mascord 1996; Kwa et al. 1993b) have shown that the polymorphism of the ␤-tubulin gene polymorphism is greatly decreased in BZ-resistant populations of all trichostrongylid species. This decreased polymorphism can result from the selection of the mutation involved in BZ resistance, combined with a lack of recombination in the region flanking this mutation ( hitchhiking effect). In this case, there should be no decrease in polymorphism at loci not linked to the ␤-tubulin gene (Maynard Smith and Haigh 1974). But the decrease can also reflect the existence of bottlenecks and founding events after BZ treatments, when the first resistant mutant individuals were selected. This must also result in decreased polymorphism at loci other than ␤-tubulin ( Nei et al. 1975). To evaluate the genetic diversity of the BZ-resistant or susceptible worm populations, we used the NADH dehydrogenase subunit 4 ( ND4) gene of mtDNA because of the results obtained by Blouin et al. (1992, 1995, 1998) showing that this gene is suitable for population genetics studies in nematodes.

Materials and Methods Parasite Sampling A population can be defined as the worms harbored by the entire herd of a sheep or goat farm. We studied three BZ-resistant (ReCas, ReGal, and ReECh) and five BZsusceptible (SuBou, SuLel, SuPre, SuMeu, and SuPro) worm populations in herds in the center-west of France (oceanic climate; average annual temperature ⫽ 10⬚C) ( Figure 1), two BZ-resistant (RePic and ReSer) populations from southwest France (sub-Mediterranean climate; average annual temperature ⫽ 14⬚C) ( Figure 1), and a few worms from a Morocco (Middle-Atlas; average annual temperature ⫽ 17⬚C) population (ReMor) (sub-Mediterranean climate). Only the SuBou population was isolated from a sheep herd. Adult worms used in this study were collected in two animals per farm. Gasnier (1994) demonstrated that parasites removed from two animals were sufficient to obtain a good sampling of the worms harbored by the entire herd. BZ resistance was estimated by calcu-

504 The Journal of Heredity 2001:92(6)

Figure 1. Geographic position of T. circumcincta populations studied in France.

lating the proportion of homozygous resistant worms in the populations. We used an allele-specific polymerase chain reaction (PCR) on the ␤-tubulin gene ( Elard and Humbert 1999; Humbert and Elard 1997) to determine the BZ resistance or susceptibility of single worms. Briefly, four primers were used to detect the phenylalanine ( TTC, susceptible allele) or the tyrosine ( TAC, resistant allele) at residue 200 of the isotype 1 ␤-tubulin gene. Specific electrophoretic profiles allow the identification of three genotypes: susceptible homozygote ( TTC/TTC, SS), susceptible heterozygote ( TTC/TAC, Sr), and resistant homozygote ( TAC/TAC, rr). Only adult male worms were used in order to avoid the risk of unreliable DNA amplification from the eggs of female worms. Genomic DNA was prepared from individual worms, as described in Humbert and Cabaret (1995). More than 50 worms were genotyped in each population. Sequencing the Mitochondrial ND4 Gene Genetic diversity was estimated by sequencing of the ND4 gene. Mitochondrial DNA was extracted from individual worms crushed in 25 ␮l extraction buffer (0.1 M Tris, 0.5 M EDTA, 0.45% Tween 20, 50 mg proteinase K/ml, pH 8). The extraction tubes were placed in a water-bath at 51⬚C for 7 h and then heated at 95⬚C for 20 min to inactivate the proteinase K. A 5 ␮l aliquot of the extract was added to a 20 ␮l PCR reaction mixture [1.5 mM MgCl2, 50 mM KCL, 1 U Taq polymerase (Promega), 12.5 ␮M of each primer and 0.2 mM d-

NTPs] and the LNC/ND4 mitochondrial gene amplified. The two primers used were those defined by Blouin et al. (1995) ( ND7: 5⬘-CGACAAACCACCTTGATATT-3⬘ and ND3: 5⬘CAAAGTGATTCCAAGTCATTGGC-3⬘). The thermocycling conditions were 93⬚C for 3 min, 10 cycles of 93⬚C for 1 min, 36⬚C for 1.5 min, and 72⬚C for 3 min, 25 cycles of 93⬚C for 55 s, 55⬚C for 55 s, and 72⬚C for 55 sec, followed by a final extension at 72⬚C for 3 min. The amplified fragments were sequenced on an Applied Biosystems 377 automated DNA sequencer (Perkin-Elmer) using the ND7 and ND3 primers. Data Analysis The PILEUP module of the GCG package (Genetics Computer Group, Madison, WI) and GeneDoc ( Nicholas and Nicholas 1997) were used for sequence alignment. Estimations of nucleotide diversity, analyses of molecular variance (AMOVA), and pairwise FST calculations were made using Arlequin version 1.1 software (Schneider et al. 1997).

Results Resistance or Susceptibility to BZ and Nucleotide Diversity of the ND4 Gene Five of the populations from central France were susceptible to benzimidazole ( less than 3% of homozygous resistant worms), while all the others were BZ resistant ( Table 1). The proportion of BZ resistant individuals ( homozygous worms) varied widely (13%–97%) in the BZ-resistant populations.

Table 1. Relationship between resistance or susceptibility to benzimidazole and A/T content and nucleotide diversity of the ND4 gene (mtDNA) in T. circumcincta populations from the center -west (SuBou to ReCas) and southwest (RePic and ReSer) of France, and from Morocco (ReMor)

SuBou SuPre SuLel SuMeu SuPro ReEch ReGal ReCas RePic ReSer ReMor a

Proportion (%) of BZresistant A/T (rr) conworms tent

ND4 gene nucleotide diversity

0 0 0 0 3 39 35 97 75 47 13

0.018 0.021 0.023 0.019 0.024 0.024 0.024 0.020 0.021 0.029 0.017

76.9 76.9 76.8 77.0 76.7 76.8 76.6 76.9 76.6 76.8 76.3

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.010 0.011 0.012 0.010 0.013 0.015 0.012 0.011 0.013 0.018 0.010

(12)a (12) (12) (20) (12) (6) (16) (10) (6) (5) (6)

Values are means ⫾ SD with numbers of individuals used to estimate diversity in parentheses.

The average A/T content of the ND4 gene was 77%, with no significant difference between populations ( Table 1). The average nucleotide diversity was 0.022 in all the populations ( Table 1), without any significant differences between BZ-resistant and BZ-susceptible populations or relative to geographic origin of these populations ( Table 1). No significant difference was observed in the variance of the nucleotide diversity in all these populations. Genetic Structure Almost all (99%) of the total genetic diversity in populations from the center-west of France was within, rather than among, populations (FST ⫽ 0.009). Pairwise analyses of population FST showed that all values were less than 0.055 ( Table 2). There was no genetic subdivision between BZ-resistant and BZ-susceptible populations ( Table 2). Close to 5% of the total genetic diversity in populations from the center and southwest of France was between populations (FST ⫽ 0.046; P ⬍ .05). There was no subdivision between the two populations from the southwest (FST ⫽ 0.022), but there were genetic subdivisions between the populations from the center-west and the southwest (0.105 ⬍ FST ⬍ 0.258) ( Table 2). Finally, 6% of the total genetic diversity of all the populations was between populations (FST ⫽ 0.063; P ⬍ .05). Pairwise analyses of population FST showed that the population from Morocco was distinct from all the other populations (0.130 ⬍ FST ⬍ 0.282) ( Table 2).

Table 2. Pairwise population FSTⴕs in T. circumcincta Center-west of France SuBou SuPre SuBou SuPre SuLel SuMeu SuPro ReEch ReCas ReGal RePic ReSer ReMor

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹

0.011 ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹

Southwest France

SuLel

SuMeu

SuPro

ReEch

ReCas

ReGal

RePic

ReSer

ReMor

⫺0.020 0.022

⫺0.001 0.009 0.004

⫺0.008 ⫺0.001 ⫺0.015 0.014

0.021 0.009 0.020 0.052 ⫺0.014

0.009 ⫺0.017 0.013 0.016 0.001 ⫺0.010

0.013 0.007 0.020 0.036 ⫺0.002 ⫺0.002 0.014

0.144 0.160 0.119 0.186 0.110 0.140 0.172 0.106

0.202 0.210 0.161 0.258 0.142 0.163 0.217 0.170 0.022

0.204 0.239 0.196 0.282 0.156 0.189 0.237 0.131 0.206 0.152

⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹

⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫺ ⫹ ⫹ ⫹

⫺ ⫺ ⫹ ⫹ ⫹

⫺ ⫹ ⫹ ⫹

⫹ ⫹ ⫹

⫺ ⫹

⫹

Above the diagonal gives the population pairwise FSTs; below the diagonal is the matrix of significant FST P values with a significance level of 0.01.

Discussion As previously found by Blouin et al. (1995, 1998), the trichostrongylid nematode mtDNA is very AT rich, as in other nematode species, such as Meloidogyne hapla and M. javanica ( Hugall et al. 1997) and Caenorhabditis elegans and Ascaris suum (Okimoto et al. 1992). These authors suggest that this resulted from an AT-bias mutation rate in nematodes. All the populations studied had similar genetic diversities and a similar variance for this index, which means that in partially resistant populations (ReEch, ReGal, RePic, ReSer, and ReMor), there is no difference in genetic diversity between BZ-resistant and BZ-susceptible worms. The 0.022 average value is very close to those estimated by Blouin et al. (1995) for Haemonchus contortus (0.026), H. placei (0.019), Ostertagia ostertagi (0.027), and T. circumcincta (0.024). Blouin et al. (1992, 1995) attributed this high within-population nucleotide diversity to the possibility that the mtDNA evolves faster in nematodes than in other taxa and to large longterm effective population sizes (Ne). They estimated that the populations on each farm were not large enough, but that massive gene flow between them (when livestock is shipped around the United States) makes all populations of domestic ruminants into one very large population. But there are no (or very few) exchanges of infested animals between French dairy goat farms, which must dramatically reduce the gene flow between them. On the other hand, there is new evidence that the mtDNA of nematodes mutates more rapidly than that of many metazoans ( Hoeh et al. 1996; Okimoto et al. 1994; Yokobori et al. 1999). Several explanations for differences in the rates of nucleotide substitution among taxonomic groups have been proposed. Martin and

Palumbi (1993) suggested that shorter generation times or higher metabolic rates increase the mutation rate by decreasing the ‘‘nucleotide generation time.’’ Hoeh et al. (1996) and Quesada et al. (1998) proposed for organisms with a doubly uniparental mtDNA inheritance that a relaxed selection on mtDNA constitutes an alternative proposition to explain its high evolutionary rate. But such mtDNA transmission has not been described for nematodes. The lack of differences between BZ-resistant and BZ-susceptible trichostrongylid populations shows that the decrease in polymorphism of the ␤-tubulin gene ( Beech et al. 1994; Elard and Humbert 1999; Grant and Mascord 1996; Kwa et al. 1993b) is linked only to the selection on this gene of mutations that confer BZ resistance, and not to the existence of bottlenecks when the BZ-resistant individuals are selected under anthelmintic pressure. As there is no gene flow among worm populations, the conservation of the genetic variation in BZ-resistant populations suggests that the effective size of population bottlenecks has never been small. Two factors can be proposed to explain this observation. The first of them is that anthelmintic treatment does not kill all the susceptible adult worms in the host. A recent study (Silvestre et al. 2001) demonstrated that a small number of the susceptible worms (5–10%) avoid BZ treatment. The second is that most of the parasite population is free-living larvae, which are not under anthelmintic pressure. The life span of these larvae on pastures is generally greater than that of adult worms in the ruminant. So the acquisition of resistance to BZ seems to be the result of a long process, which agrees with our field data from several goat herds ( Elard and Humbert 1999).

Leignel and Humbert • Genetic Diversity in T. circumcincta 505

There is no subdivision among the populations from the center-west of France, or among the two populations from the southwest. The same result (FST ⬍ 0.05) was obtained by isoenzyme studies on five loci (MDH, LDH, PGM, PGI, and MPI) at several farms in the center-west of France (Gasnier and Cabaret 1998). But a population subdivision was found on a larger geographical scale when comparing worm populations from the center-west of France to those from the southwest of France and from Morocco. The lack of subdivision in the two French regions studied cannot be explained by gene flow because no infected animal is exchanged between farms after the constitution of the herd (Cabaret and Gasnier 1994). But these herds include animals bought from an average of three farms geographically close together (Cabaret and Gasnier 1994). Thus each worm population results from an assemblage of different worm populations originating from the same geographical area. The effective size of each worm population seems to be large enough to maintain the polymorphism, or more probably to limit genetic drift. This was rejected by Blouin et al. (1992), but their estimations of Ne were done assuming that nematode mtDNA evolves at the same rate as vertebrate mtDNA and that there are only one or two nematode generations per year. But nematode mtDNA evolves twice as fast and there are at least three T. circumcincta generations per year in our study areas (Cabaret J, personal communication). In regard to these new parameters, as Blouin et al. (1992) estimated long-term Ne in these populations to be 4–8 million individuals, we can estimate Ne to be 1–2 million individuals. The number of animals per herd is 50–100, and the number of T. circumcincta worms per animal is at least 2000 (Cabaret and Gasnier 1994). Thus there are 0.3–0.6 million adult worms in the host population every year and a very moderate genetic drift is expected in each herd. As all the farms of this study are only 10–20 years old, the effects of such a moderate drift probably cannot be evaluated. However, the isolation between the worm populations from the center-west of France, southwest France, and Morocco is older (more than 50 years), which would explain the genetic subdivision observed between them. In addition, environmental conditions are very different between these regions, which could strongly influence the dynamic of the parasite popula-

506 The Journal of Heredity 2001:92(6)

tions (Rossanigo and Gruner 1995, 1996) and thus their evolution. This moderate genetic drift allowing the conservation of most of the genetic diversity in the T. circumcincta worm populations agrees with our observations on the diversity of the BZ resistance alleles in these populations. Silvestre and Humbert (unpublished data) occasionally found the same BZ resistance alleles in several worm populations. That means that rare BZ-resistant alleles were present in the population before the herds were formed and that these alleles were conserved until their selection when BZ drugs were used to treat the goats.

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