Genetic Diversity and Population Structure of Haemonchus contortus

CHAPTER TWO

Genetic Diversity and Population Structure of Haemonchus contortus J.S. Gilleard1, E. Redman University of Calgary, Calgary, AB, Canada 1 Corresponding author: E-mail: [email protected]

Contents 1. Introduction 2. Background Information on Reproduction and Genetics 3. Genetic Diversity and Population Structure of Haemonchus contortus in the Field 3.1 Many factors influence genetic diversity and population structure of Haemonchus contortus 3.2 Extremely high levels of genetic diversity are seen within Haemonchus contortus populations 3.3 Large population size is a major determinant of the high genetic diversity within Haemonchus contortus populations 3.4 Haemonchus contortus has substantial global population structure 3.5 Haemonchus contortus has a low but discernable regional population structure within countries 3.6 Current evidence regarding genetic differentiation between Haemonchus contortus populations from different host species 3.7 Effect of anthelmintic selection on the overall genetic diversity of Haemonchus contortus populations in the field 4. Consequences of Haemonchus contortus Population Structure for the Emergence and Spread of Anthelmintic Resistance in the Field 4.1 Consequence of high genetic diversity 4.2 Consequence of low regional population structure within a country 4.3 Consequence of substantial global population structure 4.4 Consequence of low population structure between hosts 5. Genetic and Phenotypic Variation in Laboratory Strains 5.1 Genetic variation within and between laboratory strains 5.2 Phenotypic variation within and between laboratory strains 5.2.1 Variation in gene expression and function 5.2.2 Variation in morphological traits 5.2.3 Variation in life history traits and pathogenicity

Advances in Parasitology, Volume 93 ISSN 0065-308X http://dx.doi.org/10.1016/bs.apar.2016.02.009

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Abstract Haemonchus contortus is one of the most successful and problematic livestock parasites worldwide. From its apparent evolutionary origins in sub-Saharan Africa, it is now found in small ruminants in almost all regions of the globe, and can infect a range of different domestic and wildlife artiodactyl hosts. It has a remarkably high propensity to develop resistance to anthelmintic drugs, making control increasingly difficult. The success of this parasite is, at least in part, due to its extremely high levels of genetic diversity that, in turn, provide a high adaptive capacity. Understanding this genetic diversity is important for many areas of research including anthelmintic resistance, epidemiology, control, drug/vaccine development and molecular diagnostics. In this article, we review the current knowledge of H. contortus genetic diversity and population structure for both field isolates and laboratory strains. We highlight the practical relevance of this knowledge with a particular emphasis on anthelmintic resistance research.

1. INTRODUCTION Current knowledge indicates that Haemonchus contortus evolved in wild ungulates in sub-Saharan Africa before being translocated around the globe by anthropogenic livestock movement (Hoberg et al., 2004). Over this time, it has adapted to a wide range of different host species and climatic zones, and is now essentially ubiquitous in grazing small ruminants worldwide. This parasite has a remarkably high propensity to develop anthelmintic drug resistance, even within a few years of drug use (Gilleard, 2013; Prichard, 2001). This adaptive capacity is largely due to the very high level of genetic variation in parasite populations upon which selection can act (Gilleard and Beech, 2007; Prichard, 2001). Understanding this genetic variation, and how it is partitioned within and among populations, is central to understanding how parasite populations respond to selective pressures, such as drug treatments, host genetics, immune responses, climate change and other environmental factors (Gilleard and Beech, 2007). It is also important for interpreting apparent associations of particular genetic markers with a drug resistance phenotype and for applying genome-wide approaches to identify novel drug resistance loci (Gilleard, 2013; Gilleard and Beech, 2007). In this article, we review the current understanding of genetic variation and population structure of H. contortus. We first consider field populations and then laboratory strains.

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2. BACKGROUND INFORMATION ON REPRODUCTION AND GENETICS Haemonchus contortus is sexually dioecious and undergoes obligate sexual reproduction. The karyotype of H. contortus has been defined for a number of different isolates, and comprises of five pairs of autosomes and one sex chromosome pair (Le Jambre and Royal, 1980; Redman et al., 2008a). The sex chromosomes have been identified by sperm karyotyping and confirmed by genotyping single female broods using sex-linked microsatellite markers (Le Jambre and Royal, 1980; Redman et al., 2008a). The male sex karyotype is XO, and the female sex karyotype is XX. As in the case for Caenorhabditis elegans, all five autosomes and the X-chromosome are of a similar size, whilst in the closely related species Haemonchus placei, the X-chromosomes are considerably larger than the autosomes (Bremner, 1954; Le Jambre, 1979). Inheritance studies using both autosomal and X-linked microsatellite markers have demonstrated that mating is polyandrous with each female mating with multiple males (Redman et al., 2008a). Genotypes derived from up to four different parental males have been identified in broods from single females derived from experimental infections (Redman et al., 2008a). A similar level of polyandry has also been shown for Teladorsagia circumcincta, suggesting that it may be a common feature of the trichostrongyloid group (V. Grillo and J.S. Gilleard, unpublished data). The relative costs and benefits of polyandry are an ongoing subject of debate for a variety of organisms (Jennions and Petrie, 2000; Tregenza and Wedell, 2000). In the case of H. contortus, the potential costs of polyandry include the expenditure of energy in finding and copulating with multiple mates, together with the associated disruption of mucosal attachment and feeding. However, a major benefit might be greater genetic variation associated with an increased opportunity for recombination between different parental haplotypes at each generation. Polyandry is also expected to reduce the impact of population bottlenecks on genetic diversity within populations, and so reduce genetic drift between populations. The extent to which polyandry occurs in natural H. contortus infection has not been investigated and may vary with infection intensity. It is possible to undertake genetic crosses of H. contortus by experimental transplantation of male and female adult worms from different strains into the sheep abomasum (Le Jambre et al., 1979; Redman et al., 2015; Sangster et al., 1998). This approach has allowed the inheritance of anthelmintic resistance to be studied and now offers the potential for forward genetic mapping

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of anthelmintic resistance loci utilizing the rapidly improving H. contortus genomic resources and sequencing technologies. This aspect will not be discussed here as it has been recently reviewed elsewhere (Gilleard, 2013).

3. GENETIC DIVERSITY AND POPULATION STRUCTURE OF HAEMONCHUS CONTORTUS IN THE FIELD 3.1 Many factors influence genetic diversity and population structure of Haemonchus contortus Population genetic structure essentially describes the total genetic diversity and its distribution within and among a set of populations. It is shaped by many factors, including life history, population size, geographical or environmental barriers, gene flow, selection and population crashes or bottlenecks (Charlesworth, 2009; Slatkin, 1987; Wright, 1931). These factors are more complex for parasites than for free-living organisms, since the interactions between parasite and host have additional impacts. The population dynamics of parasites is intimately associated with that of their hosts, since changes in host numbers and/or geographic range can drive associated changes in parasite populations (Blouin et al., 1995; Donnelly et al., 2001; Morrison and Hoglund, 2005). This aspect is particularly relevant to livestock parasites, since their hosts are commonly subject to major changes in their number and distribution due to human activity. For example, parasites of human-associated hosts, including H. contortus, show evidence of recent population expansions in their mitochondrial (mt)DNA sequences more often than do hosts not directly subject to human intervention (Mes, 2003; Morrison and Hoglund, 2005). Haemonchus contortus has both parasitic and free-living stages of its life cycle, and these stages are subject to very different environmental influences (Gilleard, 2013). The free-living stages can be exposed to dramatic fluctuations in temperature and humidity that will affect population size and will differ depending on geographical location and season. In contrast, the host provides a much more stable environment, allowing a proportion of the parasite population to avoid adverse external environmental conditions, and this has been an essential element in the successful expansion of H. contortus around the world. The parasite originates from sub-Saharan Africa and is consequently best adapted to warm and humid conditions (Gilleard, 2013; Hoberg et al., 2004). Hence, its ability to survive inside the host during periods when the external environment is inhospitable has allowed it to establish, and even thrive, following its introduction into much colder and more

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arid regions (chapter: The Pathophysiology, Ecology and Epidemiology of Haemonchus contortus Infection in Small Ruminants by Besier et al., 2016). However, the host environment is not completely benign from the parasite’s perspective. The parasitic stages are subject to host immune responses, and drug treatments and these responses will also influence population size and apply strong selective pressures to the parasite. Hence, a large number of factors, both inside and outside of the host, affect parasite populations and apply selection pressure. Another major factor that contributes to the population genetic structure of parasites is their dispersal by host movement that can potentially lead to high rates of gene flow, even across large distances. This factor disrupts the pattern of isolation by distance that is often seen for free-living organisms as a result of their dispersal capacity being less than their geographical distribution (Koop et al., 2014). In the case of H. contortus, anthropogenic movement of its livestock hosts can be extensive, long range and complex. In summary, there are many variables that shape the population genetic structure of H. contortus and these will differ between regions, seasons and production systems.

3.2 Extremely high levels of genetic diversity are seen within Haemonchus contortus populations The high genetic diversity of nematodes in the superfamily Trichostrongyloidea was first suggested by a restriction fragment polymorphism analysis of mtDNA of Ostertagia ostertagi in US cattle (Dame et al., 1993; Tarrant et al., 1992). This was followed by a second, more detailed, study of sequence diversity in the nicotinamide adenine dinucleotide dehydrogenase subunit 4 (nad4) gene in five species of trichostrongyloid nematodes from four or five different locations in the United States; H. contortus and T. circumcincta from sheep, O. ostertagi and H. placei from cattle, and Mazamastrongylus odocoilei from the white-tailed deer (Blouin et al., 1995). Extremely high levels of within-population genetic diversity were found in all of these species (Blouin et al., 1995). In the case of H. contortus, the within-population diversity was 0.026 substitutions per site of the nad4 sequence, which was much higher than that observed for other taxa (Lynch and Crease, 1990). Numerous other studies have subsequently confirmed these findings using a variety of different approaches or nuclear DNA markers, including amplified fragment length polymorphism (AFLP) analysis, transposons, single nucleotide polymorphisms (SNPs), indels and microsatellites. (Hoekstra et al., 1997, 2000a,b; Otsen et al., 2000a,b; Redman et al., 2008b; Silvestre et al., 2009; Troell et al., 2006a). For

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example, a study of laboratory strains of H. contortus reported the diversity of AFLP patterns between individual H. contortus worms to be similar to the level of variation found between closely related mammalian species, such as cattle and bison (Otsen et al., 2001). Similar levels of within-population diversity of AFLP markers have also been reported for field populations (Troell et al., 2006a). Microsatellite markers also show high levels of genetic diversity with a high proportion being polymorphic and having imperfect repeat structures (Hoekstra et al., 1997; Otsen et al., 2000b; Redman et al., 2008b). The high genetic diversity within H. contortus populations is also reflected by the frequent presence of null alleles for microsatellite markers (Hunt et al., 2008; Otsen et al., 2000b; Redman et al., 2008b, 2015; Silvestre et al., 2009). This observation has also been made for other trichostrongyloid nematodes, including T. circumcincta and Trichostrongylus tenuis (Grillo et al., 2007, 2006; Johnson et al., 2006). For several loci, these null alleles have been directly shown to be due to sequence polymorphisms within the flanking primer sites (Redman et al., 2015). In spite of extensive screening, even the best available H. contortus microsatellite markers contain null alleles in at least some populations, resulting in heterozygote deficiencies in population genetic data (Otsen, 2000b; Redman et al., 2015). Although methods are available to detect and partially compensate for null alleles in population genetic data, the presence of null alleles still results in some limitations in the analyses that can be performed and the interpretation of data produced (Chapuis and Estoup, 2007). Nevertheless, microsatellite markers have proved to be useful tools for studying the genetic diversity and population structure of H. contortus, and this work is reviewed in further detail below (Chaudhry et al., 2015a; Hunt et al., 2008; Redman et al., 2015; Silvestre et al., 2009). As yet, there are no published studies of genetic diversity of H. contortus in natural field populations using genome-wide data. However, whole genome sequencing of laboratory strains provides some insight into the overall levels of genome-wide variation present in this parasite. Both of the H. contortus genome sequencing consortia have found a very high level of sequence polymorphism in the raw sequence reads used to produce the consensus reference genome sequences (Laing et al., 2013; Schwarz et al., 2013). Indeed, these high levels of sequence polymorphism have been a major challenge for genome assembly (chapter: Haemonchus contortus: Genome Structure, Organization and Comparative Genomics by Laing et al., 2016; chapter: Understanding Haemonchus contortus Better Through Genomics and Ranscriptomics by Gasser et al., 2016 e in this volume). To provide

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some indication of the level of sequence polymorphism between laboratory strains, genome-wide short (100 bp) Illumina sequence reads generated from 20 to 30 adult worms from each of a number of strains were aligned to the 338,499,134 bp MHco3(ISE) reference genome assembly (14.11.2014 version). The number of SNPs identified across the genome assembly, after filtering for read depth and quality with the vcf_annotate script (http:// vcftools.sourceforge.net), was 921,246 (1/367 bp) for Hco3(ISE), 1,650,368 (1/205 bp) for Hco4(WRS), 1,671,886 (1/202 bp) for Hco10 (CAVR), 1,194,992 (1/283 bp) for MHco18(UGA2004) and 1,373,491 (1/246 bp) for MHco16 (A. Martinelli, J. Cotton and J. Gilleard, unpublished data). This information illustrates the high density of SNPs across the genome, relative to MHco3(ISE) reference genome assembly for laboratory strains derived from field populations from different countries. In addition, there is likely to also be a large number of indels across the genomes as indicated by studies of specific genes (Otsen et al., 2000a; Rufener et al., 2009).

3.3 Large population size is a major determinant of the high genetic diversity within Haemonchus contortus populations Genetic diversity in a population is a function of mutation rate (m) and effective population size (Ne) and, in an idealized diploid population, the pairwise nucleotide diversity is equal to 4 mNe (Charlesworth, 2009; Wright, 1931). Consequently, high levels of genetic diversity within H. contortus populations could be due to high mutation rates and/or due to large effective population sizes. An accurate determination of mutation rates is difficult for parasitic species, and we do not have meaningful values for H. contortus. However, mutation rates have been directly measured for the free-living nematode C. elegans, and were originally estimated to be 2.1  108 and 1.6  107 mutations per site per generation for the nuclear and mitochondrial genomes, respectively (Denver et al., 2004, 2000). Whilst these estimates are significantly higher than those of many other organisms, more recent estimates suggest the nuclear genome mutation rate in C. elegans is actually lower. Genome-wide analysis, and conversion of the data to a per-germ-line cell division mutation rate, yielded an estimate of 3.2  1010 mutations per site per-cell division. This value is very similar to the per-cell division mutation rate estimate for Saccharomyces cerevisiae (3.3  1010) and only approximately threefold higher than those for Drosophila melanogaster (1.5  1010) and humans (1.0  1010) (Denver

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et al., 2009). Consequently although it is possible that the mutation rate in H. contortus is higher than that of C. elegans, the current evidence suggests that it is unlikely that mutation rate alone accounts for the high genetic diversity of trichostrongyloid nematode populations. Instead, population size is likely to be a key factor. The effective population size (Ne) is the size of an idealized, sexually reproducing population that would provide the same outcome of a random sampling of alleles as that observed in the real population under study (Charlesworth, 2009; Wright, 1931). A number of different outcomes can be used to calculate Ne for a population including levels of heterozygosity, genetic drift and inbreeding. Ne is generally much smaller than the actual number of individuals in a population e referred to as the census population size (N) e due to factors including nonrandom mating, breeding sex ratios, overlapping generations and nonuniform spatial dispersion (Charlesworth, 2009). However, Ne is a useful concept, since it, rather than the census population size, determines how a population is likely to respond to selection. Values of Ne will generally be large for H. contortus populations by virtue of the high levels within-population genetic diversity that are observed and this has a number of important consequences. For example, genetic drift is likely to be low in populations with high Ne and this, along with migration, helps explain why genetic differentiation is generally low among different H. contortus populations within a region (Charlesworth, 2009). In addition, positive selection has more impact when effective population sizes are large, which helps explain why anthelmintic resistance alleles commonly arise in H. contortus populations (Charlesworth, 2009; Gilleard and Beech, 2007). One important question is whether the remarkably high levels of genetic diversity, and consequently large Ne, observed within each H. contortus population is dependant on migration of genotypes between populations. Blouin et al. (1995) was the first to note that, although sequence diversity in nad4 mtDNA within the four populations of H. contortus examined in the United States was extremely high (0.026 substitutions per site), the diversity among populations was very low (0.0004 substitutions per site) (Blouin et al., 1995). Hence, more than 96% of the genetic diversity was within, and not among, separate H. contortus populations. This lack of population structure was suggested to be a consequence of gene flow between populations as a result of anthropogenic animal movement. It was further suggested that this gene flow might result in a single ‘meta-population’ across the United States with a huge effective population size. However, in the same study, even higher levels of genetic diversity were seen within

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populations of M. odocoilei, in spite of there being substantial partitioning of this variation among populations, suggesting lower levels of gene flow (Blouin et al., 1995). Hence, it seems unlikely that gene flow was solely responsible for the high diversity observed in the trichostonglyoid nematode populations examined in that study. Furthermore, two subsequent studies, using microsatellite markers, have shown that sheep and goat farms, in France and Pakistan, which have been closed to animal movement for more than 30 years have similarly high levels of diversity as farms open to animal movement (Chaudhry et al., 2015b; Redman et al., 2015; Silvestre et al., 2009) and (U. Chaudhry, E. Redman, K. Ashraf, M. Shabbir, M. Rashid, S. Ashraf and J. Gilleard, unpublished data). It is also noteworthy that laboratory isolates passaged for many years typically retain very high levels of genetic diversity, in spite of being effectively closed to gene flow in this wildlife parasites (Hunt et al., 2008; Otsen et al., 2001; Redman et al., 2008b). Hence, a high level of contemporary gene flow between populations does not seem necessary to maintain high levels of genetic diversity within H. contortus populations. One other factor that could potentially increase observed levels of genetic diversity within H. contortus populations is admixture (ie, when individuals derived from previously allopatric and genetically differentiated populations are mixed in a single population) (Dlugosch et al., 2015). The large amount of long-distance livestock movement that has historically occurred in many parts of the world might be expected to result in such admixture being commonly seen. However, there are no obvious discontinuities in phylogenetic relationships of nad4 haplotypes reported for H. contortus populations and little evidence of admixture from the various studies that have been performed in different countries using microsatellite markers (Chaudhry et al., 2015b; Redman et al., 2015; Silvestre et al., 2009). However, one caveat to this evidence is that the presence of null alleles for microsatellites used in these studies makes definitive testing of admixture difficult. Nevertheless there is little evidence to suggest that admixture of diverse populations is a major feature of most H. contortus populations. The balance of evidence overall suggests that the high genetic diversity observed within H. contortus populations is largely due to their large census population sizes. This information is consistent with our knowledge of the life history of the parasite. A single small ruminant host can contain thousands, or tens of thousands, of adult female H. contortus worms, each of which can produce up to 4000 eggs per day (Fleming, 1988). Consequently, a single pasture grazed by a flock of several hundred sheep will

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be seeded with billions of new progeny every few days. Although only some of these progeny are then ingested by a host and contribute to the next generation, the census population size of H. contortus is generally very large, even at a single location. If census population size is the most important determinant of the high genetic diversity of H. controtus populations, one would predict that parasite species with lower infection intensities (lower numbers of adult worms per host) would have lower levels of genetic diversity. There are few studies to date that have directly addressed this question, but there is some evidence that mtDNA diversity in sexually reproducing nematode species with direct life cycles is positively correlated with mean infection intensities (Criscione et al., 2005). For example, the ascaridoid nematodes Ascaris suum and Ascaris lumbricoides, which have much lower infection intensities than trichostrongyloid nematodes, also have much lower levels of mtDNA diversity. In addition, an estimate of the effective population size (Ne) of A. lumbricoides in a village in Nepal, using microsatellite data, was just w1300 compared with estimates of several million on a single farm for trichostrongyloid nematodes such as O. ostertagi (Blouin et al., 1992; Criscione, 2013).

3.4 Haemonchus contortus has substantial global population structure Haemonchus contortus has substantial population structure on a global scale. Troell et al. (2006a,b) examined AFLP profiles and nad4 mtDNA sequences from 8 to 10 worms from each of 19 isolates distributed across 14 different countries (Troell et al., 2006a). Of a total of the 150 individual worms analysed, there were no identical AFLP profiles and 94 different nad4 haplotypes, reflecting the high overall genetic diversity. For the AFLP data, genetic differentiation between continental areas was significant at P < 0.001 for all pairwise comparisons. For the nad4 sequence data, 38.0% of the genetic variation was among individuals within populations, 27.1% among populations within continents and 34.8% among continents. When the AFLP data were used to construct phylogenetic trees, almost all individuals from the same isolate clustered together and, in most cases, isolates from the same continent were also clustered. The mitochondrial nad4 marker showed less phylogenetic resolution overall, but broadly supported the phylogenetic relationships determined using AFLP data. In summary, this study not only found very high levels of overall genetic diversity, but also demonstrated significant genetic differentiation between H. contortus populations from different countries, suggesting strong barriers to gene flow

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at this scale. A few findings did not fit the expected pattern based on geographical location. Most notably, the Greek isolate clustered with the Australian rather than the other European isolates, suggesting a possible introduction of H. contortus to Greece from Australia. However, a limitation of the study was that only a single isolate was examined from each country, and so further work is needed to test this hypothesis. We have genotyped H. contortus populations from the UK, southern India and Pakistan with panels of microsatellite markers in separate population genetic studies (Chaudhry et al., 2015b; Redman et al., 2015) (U. Chaudhry, E. Redman, K. Ashraf, M. Shabbir, M. Rashid, S. Ashraf and J. Gilleard, unpublished findings). Although different microsatellite marker panels were used in each of these studies, five markers were common to all three panels. To assess the genetic differentiation of H. contortus populations between countries, we have analysed the data from these five markers for six H. contortus populations from each country. The populations clearly cluster by country on principal coordinate analysis (PCoA), even using as few as five microsatellite markers (Fig. 1A). The populations from southern India and Pakistan appear more closely related to each other than they are to the UK population, as expected based on their geographical relationships. Eight microsatellite markers were shared among the panels used in the Pakistan and southern India studies, and the populations clustered clearly by country when these eight markers were applied to all populations from the two studies (Chaudhry et al., 2015b; Redman et al., 2015) (Fig. 1B). This latter point illustrates that the detection of genetic differentiation increases in sensitivity as a greater number of discriminatory markers are used. Hence, the future application of genome-wide approaches is expected to reveal finer scale population structure that can be detected using the microsatellite marker panels employed to date. The characterization of H. contortus laboratory strains is also suggestive of significant population structure among countries since there is substantial genetic differentiation between laboratory strains derived from different countries (Redman et al., 2008b). Consequently, laboratory strains may not be representative of field populations if originally isolated from a different geographical region. This aspect is discussed in more detail in Section 5.1.

3.5 Haemonchus contortus has a low but discernable regional population structure within countries Although, on a regional level, most genetic variation in H. contortus is within and not between populations, some population structure is still

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Figure 1 Principal coordinate analysis based on Fst values calculated from microsatellite genotype data from UK, southern India and Pakistan Haemonchus contortus populations using Arlequin 3.11. Panel (A) Six loci e Hcms36, Hcms25, Hcms33, Hcms3086, Hcms53265, Hcms8a20 e were used to genotype 25e30 worms from six populations from three different countries, UK, southern India and Pakistan. Each data point represents a different population with the country of origin coded by its colour. Panel (B) Eight loci e Hcms36, Hcms25, Hcms33, Hcms3086, Hcms53265, Hcms22193, Hcms2561 and Hc8a20 e were used to genotype 25e30 worms from 13 H. contortus populations from southern India and 11 H. contortus populations from Pakistan. Each data point represents a different population with the country of origin coded by its colour and the colour of the text label indicating the host species of origin.

evident. Although the study of Troell et al. (2006a,b) only examined a single isolate from most countries, four different isolates were examined from Sweden. There was low but significant genetic structure among these populations, with an overall Fst of 0.13 based on the AFLP data and an Nst of

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0.16 based on the mtDNA nad4 data. In addition, in the minimumevolution tree constructed using AFLP data, all eight worms from each Swedish isolate clustered together but separately from those of the other Swedish isolates (Troell et al., 2006a). A study in southern and central France also revealed detectable population structure at the regional scale. Pairwise Fst values, based on a panel of seven microsatellite markers, ranged from 0.045 to 0.183, with 11 of 15 being significantly different from zero (p ¼ 0.08 with Bonferroni correction) (Silvestre et al., 2009). Although this is a clear example of genetic differentiation between H. contortus populations within a region of a country, the herds of goats studied had been closed to animal movement for more than 30 years. Consequently the lack of gene flow may mean that genetic drift of these H. contortus populations may be higher than is typical for most farms that are open to animal movement. However, in a separate study of seven H. contortus populations on UK sheep farms, genetic differentiation could also be detected on the regional scale using a panel of 10 microsatellite markers (Redman et al., 2015). In this case, pairwise Fst values ranged from 0.0198 to 0.0757, with 10 of 21 being significantly different from 0 (p ¼ 0.01). In contrast to the French study, these sheep flocks were not closed, and so even with the considerable movement of sheep that occurs in the UK, low but detectable population substructure of H. contortus occurs at a regional level. One hypothesis to potentially explain regional population structure of H. contortus is suggested by comparison with the closely related trichostrongyloid nematode T. circumcincta. In both the French and UK studies described above, T. circumcincta was also examined on the same farms. In contrast to H. contortus, this nematode species showed no significant genetic differentiation between farms in either of the studies. In the French study, pairwise Fst values between T. circumcincta populations ranged from 0.001 to 0.057, with none being significantly different from 0 (p ¼ 0.08). In the UK study, pairwise Fst values between T. circumcincta populations ranged from 0.0269 to 0.0340, with only 2 of 21 being significantly different from zero (p ¼ 0.08). In this latter study, H. contortus and T. circumcincta were collected from the same individual hosts, suggesting that their contrasting population structures must be directly related to differences in the life histories of the two parasites. Haemonchus contortus is primarily adapted to warmer climates (being originally native to sub-Saharan Africa), and so in temperate and colder regions, very few infective larvae survive on pastures over the winter (Falzon et al., 2014; Sargison et al., 2007; Thomas and

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Waller, 1979; Waller et al., 2004). Instead, the parasite primarily overwinters inside the host, which is likely to represent a population bottleneck, particularly if hosts are treated with anthelmintic drugs when larval counts on pasture are low. In contrast, T. circumcincta is native to temperate regions, and so a larger numbers of infective larvae usually survive on pastures over the winter, making population bottlenecks less likely. The relative prevalence and infection intensities of these two parasite species in UK sheep are consistent with this model. In a survey of 118 UK sheep farms, T. circumcincta was found to be present in all flocks and, in most cases, at high frequencies (Burgess et al., 2012; Redman et al., 2015). In contrast, H. contortus was only detected in w50% of flocks and was present at a very low frequency (<5%) in most cases. If the population structure of H. contortus observed in temperate regions (UK, France and Sweden) is predominantly due to population bottlenecks caused by the death of larvae on winter pastures, one would predict less population structure in countries with year-round warm humid climates. Preliminary evidence suggests that this might be the case. Haemonchus contortus populations show little population structure in southern India; our recent study reported pairwise Fst values, based on microsatellite data, to be very low, ranging from 0.0244 to 0.0351, with only 5 of 66 pairwise comparisons being significantly different from 0 (p ¼ 0.01) (Chaudhry et al., 2015b). We also see a similar lack of genetic structure for H. contortus populations in Pakistan using a similar panel of microsatellite markers (U. Chaudhry, E. Redman, K. Ashraf, M. Shabbir, M. Rashid, S. Ashraf and J. Gilleard, unpublished data). In that case, pairwise Fst values were not significantly different from zero, even among three government farms closed to animal movement for more than 30 years In summary, most of the genetic variation is within, and not among, H. contortus populations from an individual country. However, a low level of regional genetic differentiation is sometimes discernable, even with relatively small microsatellite marker panels. It is hypothesized that population genetic structure is more marked in temperate than in tropical regions due to an increased population bottleneck occurring in regions with colder climates. However, this suggestion is based on relatively few studies, and more work comparing H. contortus, and other trichostrongyloid nematodes, in different climatic zones is needed to test this hypothesis. The use of larger sets of genetic markers, such as genome-wide SNPs, should also provide more discriminatory power for such studies.

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3.6 Current evidence regarding genetic differentiation between Haemonchus contortus populations from different host species Although its ‘preferred’ hosts are sheep and goats, H. contortus has been reported to infect a range of wild and domestic ungulate species (Hoberg et al., 2004). In the few reports published to date, there is little evidence to suggest the presence of cryptic species or of discernable genetic differentiation between H. contortus populations in different host species. For example, there was no clustering by host species of nad4 mtDNA sequences derived from 78 H. contortus specimens from alpine chamois (Rupicapra r. rupicapra), roe deer (Capreolus capreolus), alpine ibex (Capra ibex ibex), domestic goat (Capra hircus) and sheep (Ovis aries) (Cerutti et al., 2010). To date, most comparisons have been between populations of H. contortus from sheep and goats. Troell et al. (2003) investigated the species identity of Haemonchus specimens isolated from a sheep and goat from Sweden and a sheep from Kenya using pyrosequencing of the ITS-1, ITS-2 and the 5.8S rRNA regions of the rDNA cistron (Troell et al., 2003). The worms from the Swedish sheep and goat were more closely related to each other than to the worms from the Kenyan sheep. Similarly, in the study of 19 H. contortus isolates from 14 different countries, the only 2 isolates from goats (from Cambodia and Guadaloupe) clustered by geographical region and not by host species for both the AFLP and mtDNA sequence data (Troell et al., 2006a). This finding is further supported by our study of H. contortus in southern India in which six populations were from sheep and six from goats (Chaudhry et al., 2015b). No differences were found between H. contortus populations in the two host species based on a panel of eight polymorphic microsatellite markers. Indeed, the Fst values were lower for many of the pairwise comparisons between the H. contortus populations from sheep and goats than between those from the same host species (Chaudhry et al., 2015b). The same result was found for our recent study of H. contortus in Pakistan, with no Fst values for pairwise comparisons between the two host species being significantly different from zero (U. Chaudhry, E. Redman, K. Ashraf, M. Shabbir, M. Rashid, S. Ashraf and J. Gilleard, unpublished data). This lack of clustering by host species in both of these studies is illustrated by PCoA (Fig. 1B). There is only one study that has suggested genetic differences can occur between H. contortus from sheep and goats. This is a phylogenetic analysis of H. contortus mtDNA nad4 sequences from sheep and goats in Malaysia and

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Yemen (Gharamah et al., 2012). In this study, marked clustering of sequences between the two countries revealed significant geographical population substructuring. Although all of the nad4 sequences from H. contortus from Malaysia and most from Yemen did not show any evidence of clustering by host species, there was one separate clade of nad4 sequences that was derived exclusively from H. contortus from goats from Yemen (Gharamah et al., 2012). This information suggested the possibility of a subpopulation of genetically distinct parasites present in Yemen goats that was not present in sheep in the same region. The authors speculated that this might be evidence of goat-specific cryptic species of H. contortus in Yemen, analogous to that which has been described for Teladorsagia cricumcincta in France (Grillo et al., 2007). However, these authors also noted that there is significant importation of small ruminants into Yemen from Africa, and so the results could also be explained by admixture of allopatric parasites with the indigenous H. contortus populations. A subsequent, detailed morphometric analysis of the same populations, based on eight morphological characters, revealed significant grouping based on country but not by host species in either country (Gharamah et al., 2014). In summary, most of the evidence suggests that H. contortus is generally freely shared between sheep and goats, with little or no host species barrier or population substructuring.

3.7 Effect of anthelmintic selection on the overall genetic diversity of Haemonchus contortus populations in the field In general, field populations of parasitic nematodes that are resistant to anthelmintic drugs appear have a similar level of overall genetic diversity as susceptible populations. For example, H. contortus populations on two farms with a low frequency of benzimidazole resistance mutations showed no significant difference in allelic richness or expected heterozygosity compared with five farms with much higher frequencies of resistance mutations assessed using a panel of 10 microsatellite markers (Redman et al., 2015). Similarly 2 H. contortus populations from southern India, from which benzimidazole resistance mutations were absent, showed no significant difference in allelic richness or expected heterozygosity for a panel of 8 microsatellite markers compared with 10 populations in which benzimidazole resistance mutations were present (in some cases at high frequency) (Chaudhry et al., 2015b). In these two examples, there was significant animal movement between farms that might have reduced the impact of anthelmintic selection on overall genetic diversity. However, we have recently compared H. contortus populations on three government (small

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ruminant) farms in Pakistan with a history of intense benzimidazole drug selection pressure and that have been closed to animal movement for more 20 years with those from four pastoral locations with little or no drug treatment. Although the frequency of benzimidazole resistance mutations in H. contortus was very high on the government farms and very low for the pastoral locations, consistent with their respective drug treatment histories, there was again no significant difference in allelic richness, expected heterozygosity or inbreeding coefficient for a panel of eight microsatellite markers (U. Chaudhry, E. Redman, K. Ashraf, M. Shabbir, M. Rashid, S. Ashraf and J. Gilleard, unpublished data). In contrast to these studies, we are not aware of any evidence to date showing an overall loss of genetic diversity of H. contortus populations in response to drug selection in the field.

4. CONSEQUENCES OF HAEMONCHUS CONTORTUS POPULATION STRUCTURE FOR THE EMERGENCE AND SPREAD OF ANTHELMINTIC RESISTANCE IN THE FIELD 4.1 Consequence of high genetic diversity The high level of genetic diversity within H. contortus populations underlies the remarkable adaptive capacity of this parasite. Assuming a mutation rate of w2.1 mutations per genome per generation (based on data for C. elegans), mutations for each position in the >300 Mb genome of H. contortus will occur many times within the billions of progeny seeded on to a typical pasture grazed by small ruminants every few days. This process results in both a large amount of standing genetic variation and a constant supply of new mutations on which selection can act. It also provides the parasite with an extraordinary capacity to respond not only to drug selection, but also to other changes, such as climate, geographical location and host species. The consequence of this high genetic diversity of H. contortus for anthelmintic resistance is best illustrated by studies of the population genetics of benzimidazole resistance. There are three mutations that occur in the H. contortus isotype-1 b-tubulin gene: F200Y (TAC), E198A (GCA) and F167Y (TAC) (Ghisi et al., 2007; Prichard, 2001; Silvestre and Cabaret, 2002). The F200Y (TAC) mutation is commonest and present in most geographic locations studied to date, the F167Y (TAC) is less common but can be at high frequency in some regions and the E198A (GCA) is the rarest based on current studies (chapter: Anthelmintic Resistance in Haemonchus contortus: History, Mechanisms and Diagnosis by Kotze and Prichard, 2016 e in this

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volume). For the common F200Y (TAC) mutation, several studies (Chaudhry et al., 2015b; Redman et al., 2015; Silvestre and Humbert, 2002; Silvestre et al., 2009) have reported a high level of haplotype diversity for resistance alleles within H. contortus populations. Phylogenetic network analysis of these haplotypes suggests that the F200Y (TAC) mutation has originated multiple independent times within a region and is derived from both recurrent mutation and from the standing genetic variation (Chaudhry et al., 2015b; Redman et al., 2015; Silvestre et al., 2009). The same results have been found even for H. contortus populations that have been closed to animal movement for many years and, hence, in the absence of contemporary gene flow (Silvestre et al., 2009). This hypothesis, and the evidence for it, is discussed in more detail in Redman et al. (2015). This repeated appearance of an anthelmintic drug resistance mutation is a direct consequence of the high genetic diversity of H. contortus. This information provides a persuasive argument that the emergence of anthelmintic resistance is inevitable when intensive drug selection is applied to a parasite that has such high levels of genetic diversity.

4.2 Consequence of low regional population structure within a country As discussed in Section 3.5, the low levels of population structure of H. contortus within a region, at least in part, reflects high levels gene flow between populations. This observation is consistent with the high levels of anthropogenic host movement for sheep for most farms studied. If gene flow is high, then even rare resistance mutations have the potential to spread widely in regions under the influence of drug selection. This observation has been made recently in a study (Chaudhry et al., 2015b) showing that the E198A (GCA) mutation is relatively widespread in southern India, in spite of being rare or absent from most countries studied to date (Barrere et al., 2013; Prichard, 2001; Redman et al., 2015; Silvestre and Humbert, 2002). Phylogenetic analysis of the resistant and susceptible isotype-1 btubulin haplotypes showed that this E198A (GCA) mutation was represented by a single haplotype in the region, despite high levels of susceptible haplotype diversity. This finding strongly suggests that this mutation has arisen once and has subsequently spread throughout populations of H. contortus in this region of India (Chaudhry et al., 2015b). The spread of a relatively rare mutation, such as E198A (GCA), can be clearly demonstrated by phylogenetic analysis of resistant and susceptible haplotypes. However, it is more difficult to demonstrate for a common mutation

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with multiple origins, such as the F200Y (TAC), because of the diversity of resistance haplotypes. Further, if resistance is well established, the lack of susceptible haplotypes makes the interpretation of the phylogenetic analysis difficult. Nevertheless the population genetic data, overall, is also consistent with the spread of the F200Y (TAC) between farms in the UK (Redman et al., 2015). This conclusion emphasizes the role of animal movement in spreading anthelmintic resistance, and the need for stringent biosecurity and quarantine dosing procedures in minimizing the spread of resistance between farms.

4.3 Consequence of substantial global population structure The high level of population genetic structure of H. contortus among different countries has a number of consequences for anthelmintic resistance. It suggests that there is more limited gene flow between parasite populations in different countries. Consequently, the spread of resistance mutations between countries is likely to be much less than that which occurs at the regional level. In addition, the genetic background on which selection acts is different among countries, such that one might expect different mutations to be important in different regions. In the case of benzimidazole resistance, we know this to be true; although the F200Y (TAC) mutation appears to occur in all countries examined to date, the rarer E198A (GCA) and F167Y (TAC) mutations differ markedly among regions. For example, in the UK, the F167Y (TAC) mutation is almost as frequent as F200Y (TAC), but has not yet been detected in southern India (Redman et al., 2015). Conversely, the E198A (GCA) mutation is widespread in southern India, but the F167Y (TAC) has not yet been found (Chaudhry et al., 2015b). This information has important implications for the use of molecular diagnostic tools and the surveillance of resistance. It also emphasizes the importance of biosecurity measures for imported livestock, such as anthelmintic dosing in quarantine to avoid the importation of new resistance mutations to a particular geographical region. The global population structure of H. contortus also has a number of consequences for anthelmintic resistance research. One cannot assume that a mutation implicated as an important cause of resistance in one region is necessarily important in another region or is of general global importance. Specific regional studies will always be needed. In addition, care must be taken when comparing the diversity or association of candidate gene haplotypes between resistant and susceptible parasite isolates from different geographical regions. It is likely that differences will be found between

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such populations, even at neutral genetic loci, and, therefore, it is critical to take into account the genetic background of parasite populations in such studies. This will also be important for future genome-wide population genomic and association studies.

4.4 Consequence of low population structure between hosts The low population structure found between H. contortus populations in different host species in the same region suggests that the parasite is freely shared with little or no host species barrier. Hence, it is likely that the same resistance mutations will be found in the different host species within the same geographical region. There have been few studies directly testing this aspect, but it is supported by the observation that the same benzimidazole resistance mutations were found in H. contortus from sheep and goats, both southern India and Pakistan (Chaudhry et al., 2015b) (U. Chaudhry, E. Redman, K. Ashraf, M. Shabbir, M. Rashid, S. Ashraf and J. Gilleard, unpublished data).

5. GENETIC AND PHENOTYPIC VARIATION IN LABORATORY STRAINS Understanding and monitoring the genetic and phenotypic variation between laboratory strains is an important and neglected aspect of H. contortus research. The substantial levels of genetic diversity present within H. contortus field populations will be inevitably reflected in laboratory strains, since they are derived from field populations. The mode of obligate sexual reproduction of this parasite means that clonal lines cannot be established. Instead, laboratory strains are typically maintained as populations of large numbers of interbreeding parasites, which are serially passaged through experimentally infected hosts. Consequently, there is often considerable genetic and phenotypic variation both within and among laboratory strains, and there is potential for this variability to change over time. There is no generally accepted definition of what constitutes an ‘isolate’ or a ‘strain’ for a sexually reproducing organism, such as H. contortus. In this chapter, we use the term ‘isolate’ for a population of parasites recovered directly from the field and the term ‘strain’ for an isolate that has been subsequently serially passaged by experimental infection, and then studied and archived in the laboratory. In the case of H. contortus, isolates are generally recovered from an infected animal from the field by harvesting infective third-stage larvae (L3s) from faecal cultures. Such field isolates are sometimes

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contaminated with other species. Although these can be removed by transplantation of morphologically identified adult worms into the abomasum of a recipient sheep, they are often ignored if only present in trace amounts. Harvested L3s can be stored for several months in water or exsheathed L3s can be cryopreserved in liquid nitrogen where they remain viable and infective for many years (Van Wyk et al., 1977). Laboratory strains are usually passaged every few months by oral or rumenal infection of sheep or goats, typically using between 2000 and 5000 L3s (Wood et al., 1995). Faeces from such animals are then cultured to obtain the next generation of infective L3s. There are a number of aspects of these processes that may have important impacts on experimental work. First, strain contamination can occur in a variety of ways, including donor animals that are not parasite-free, by contamination of feed or bedding with infective L3s or by human error during strain handling or archiving. If contamination occurs with a different nematode species, it should be readily detectable. However, if contamination occurs with a different H. contortus population, then there is a significant risk that it will go undetected, and could lead to erroneous experimental results. Second, there is an ongoing risk of population bottlenecks due to variability in infective dose or rates of establishment in the host animal. For example, larvae that have been stored incorrectly, or for too long a period, can lose infectivity, leading to experimental infections with low parasite numbers. This reduction in population size could result in a loss of overall genetic diversity or to genetic drift of the population. Third, the number of passages of a strain is not always recorded and captured in the parasite strain nomenclature and, hence, experiments performed on the same strain over time may not be equivalent. Fourth, strains are often exchanged between laboratories without any monitoring of genetic integrity, and so contamination events or errors may only be detected if clear differences exist in phenotype or if specific molecular markers are used. Finally, there is no standardized nomenclature system as there is for model organisms, such as C. elegans and D. melanogaster (Attrill et al., 2015; Harris et al., 2004). As a result, strains, such as the ‘ISE’ or ‘McMaster’, which are commonly used in experimental studies around the world, may differ genetically and phenotypically among laboratories. We use a nomenclature system for the reference strains that are maintained at the Moredun Research Institute, Scotland, to help minimize some of these problems (Gilleard, 2013). For example, the version of the CAVR strain that is passaged at this institute is named MHco10(CAVR), to distinguish it from other versions of this isolate passaged in other

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laboratories (Redman et al., 2008b). There have been relatively few publications specifically addressing genetic and phenotypic variation of H. contortus laboratory strains. However, some information is available in a variety of papers that are reviewed here.

5.1 Genetic variation within and between laboratory strains In spite of some of the limitations discussed in Section 5, H. contortus is still one of the best-characterized parasitic nematode species, in terms of genetic variation within and between laboratory strains. As for natural field populations, there is a substantial amount of genetic variation within H. contortus laboratory strains. The earliest work examining genetic diversity of H. contortus laboratory strains focused on sequence polymorphisms in candidate anthelmintic resistance genes (Beech et al., 1994; Blackhall et al., 1998a,b; Kwa et al., 1993; Prichard, 2001; Sangster et al., 1999). Typically, these studies compared the frequency of particular haplotypes of candidate genes between resistant and susceptible strains or between populations of the same strain before and after drug selection. In all cases, high levels of haplotypic diversity have been reported both within and between strains. A number of these studies have reported increased frequencies of particular haplotypes in resistant relative to susceptible strains or following drug selection (chapter: Anthelmintic Resistance in Haemonchus contortus: History, Mechanisms and Diagnosis by Kotze and Prichard, 2016 e in this volume). However, to interpret such studies, it is important to consider differences and changes that occur throughout the genome as well as at the locus or loci under investigation. A variety of marker systems have been developed for H. contortus that can be used for this purpose, including random amplified polymorphic DNA, restriction fragment length polymorphism, AFLP, transposon-associated markers, SNPs, indels and microsatellites (Hoekstra et al., 1997, 2000a,b; Hunt et al., 2008; Otsen et al., 2000a, 2001; Redman et al., 2008a; Roos et al., 1998). Panels of well-characterized microsatellites are available to assess, compare and monitor the genetic diversity within and among laboratory isolates (Hoekstra et al., 1997; Otsen et al., 2000b; Redman et al., 2008b, 2015). For instance, Redman et al. (2008a,b) used a panel of eight microsatellite markers to characterize five laboratory isolates that had been passaged by serial experimental infection for many years, following their original field isolation from different countries: MHco1(MOSI) and MHoc3(ISE) of unknown field origin (possibly UK); MHoc4(WRS) from South Africa; MHco10(CAVR) from Australia and HcSwe(VAST) from Sweden.

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All pairwise Fst values were very high (0.1385e0.333), except for MHco1(MOSI) and MHoc3(ISE) (0.1008), which was lower. This observation is consistent with our understanding of global population structure of the parasite in the field (Troell et al., 2006a), as discussed in Section 3.4. MHoc3(ISE) is derived from the MHco1(MOSI) strain, and so the closer relationship of these two strains is consistent with their known history (Roos et al., 2004). Amplification of the five microsatellite markers from pools of worms generates repeatable genetic ‘fingerprints’ for individual strains, and provides a convenient and rapid system with which to monitor strain integrity during passage and exchange between laboratories (Redman et al., 2008b). Hunt et al. (2008) used a number of different microsatellite markers to characterize six commonly used laboratory strains (called McMaster1931, Wallangra2003, Gold Coast2004, Arding2005 and Cannawigara2005), originally isolated from the field in south eastern Australia. Depending on the markers used, pairwise Fst values varied from 0.00007 to 0.04532 (Hunt et al., 2008). Although these values are lower than those reported by Redman et al. (2008a,b), many pairwise comparisons were statistically significant. This information demonstrates that there can be significant genetic differentiation between laboratory strains, even when isolated from different regions of the same country (Hunt et al., 2008). These results suggest that a laboratory strain is likely to be more representative of field populations located in the same region from it was originally isolated. This hypothesis has not been rigorously tested, but is supported by comparison of a Swedish laboratory isolate with global field populations (Troell et al., 2006a). Also our recent results show that field populations of H. contortus, isolated from the south-east United States, are genetically closer to the UGA2004 laboratory strain than to the MHoc3(ISE), MHoc4(WRS) and MHco10(CAVR) laboratory strains (M. Miller, E. Redman, R. Kaplan and J. Gilleard, unpublished data). Although there are no published studies specifically comparing laboratory strains and field populations, the overall evidence suggests that laboratory strains are generally as genetically diverse as field populations. Microsatellite markers typically show similar levels of allelic richness, expected heterozygosity and inbreeding coefficients in studies of passaged H. contortus laboratory strains as they do for field populations (Hunt et al., 2008; Redman et al., 2008b, 2015; Silvestre et al., 2009). As discussed in Section 4.6, from the limited data available, it appears that anthelmintic selection does not lead to an overall reduction of genetic diversity in H. contortus populations in the field. Similarly from the limited analyses conducted to date, drug selection

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does not seem to substantially reduce the overall genetic diversity of laboratory strains. To date the most direct analysis to address this question used AFLP analysis of individual worms, to monitor changes in genetic diversity within and between strains during consecutive stages of selection for increased benzimidazole or levamisole resistance (Otsen et al., 2001). In the case of benzimidazole selection, eggs from a susceptible laboratory strain were incubated at a drug concentration (ED80) such that w20% of the eggs survived and were used to infect a donor sheep following culture to L3. Five rounds of such in vitro selection resulted in a significantly increased ED50, but no reduction in the overall genetic diversity was detected by AFLP analysis. In the same study, six rounds of levamisole selection were applied to a susceptible laboratory strain by in vivo drug treatments of experimentally infected animals. Although a small reduction in overall diversity was detected by AFLP analysis after the first round of selection, there was no further loss of diversity detected even by the sixth generation (Otsen et al., 2001). In addition, as discussed in Section 3.2, genome-wide SNP analysis has revealed that several anthelmintic laboratory strains, namely Hco4(WRS), Hco10(CAVR), MHco18(UGA2004) and MHco16, retain very high levels of sequence polymorphism across the genome. Although microsatellite markers have been useful for the genetic characterization of H. contortus strains, more extensive genome-wide marker analyses using various methods, such as SNParrays, restriction site-associated DNA markers and whole genome sequencing, should provide much greater resolution in the future (Davey et al., 2011; Salgotra et al., 2014). Recent progress in the assembly of the H. contortus reference genome (Laing et al., 2013; Schwarz et al., 2013), together with the rapidly diminishing costs of next-generation sequencing, is now making such approaches increasingly feasible.

5.2 Phenotypic variation within and between laboratory strains Although much of the genetic variation within and between H. contortus laboratory strains consists of sequence polymorphisms in noncoding regions, there is also a substantial number of nonsynonymous mutations in coding regions. For example, in an analysis of 927 gene models, in a 11.2-Mb region of the H. contortus draft genome sequence, nonsynonymous SNPs resulted in 2104 and 1666 amino acid substitution mutations in the MHco10(CAVR) and MHco4(WRS) strains compared with the MHco3(ISE) reference genome, respectively (A. Martinelli, A. Rezansoff, J. Cotton and J. Gilleard,

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unpublished data). Hence, there is clear potential for phenotypic variation between laboratory strains. Here, we review the information currently available on phenotypic variation among laboratory strains. 5.2.1 Variation in gene expression and function Most of the work investigating differences in gene expression and function between H. contortus laboratory isolates has been aimed at understanding the mechanisms of anthelmintic resistance (chapter: Anthelmintic Resistance in Haemonchus contortus: History, Mechanisms and Diagnosis by Kotze and Prichard, 2016 e in this volume). Anthelmintic resistance research has provided a number of examples of genes that are differentially expressed or transcribed between different H. contortus isolates and strains. For example, differences in gene expression between levamisole resistant and susceptible H. contortus populations has been described for the nicotinic acetylcholine receptors, Hco-unc-29.3 and Hco-unc-63 and acr-8 as well as ancillary proteins Hco-unc-74, -unc-50, -ric-3.1 and -ric-3 (Sarai et al., 2013; Williamson et al., 2011). Similarly, expression differences have been described between ivermectin resistant and susceptible populations for dyf7 e a gene that encodes a protein involved in amphid sensory neuron development e and several members of the P-glycoprotein efflux pump family (Urdaneta-Marquez et al., 2014; Williamson et al., 2011; Xu et al., 1998). This topic is not discussed in detail here, as it is reviewed by Kotze and Prichard (2016) (chapter: Anthelmintic Resistance in Haemonchus contortus: History, Mechanisms and Diagnosis e in this volume) as well as in other articles (Gilleard, 2006; Gilleard and Beech, 2007; Kotze et al., 2014; Prichard, 2001). Other than anthelmintic resistance research, there has been relatively little work investigating differences in gene expression between H. contortus laboratory strains. One study compared the soluble proteome of adult female worms of the MHco3(ISE) and MHco10(CAVR) strains (Hart et al., 2012). The data from three replicate two-dimensional (2-D) gels for each strain identified 23 protein spots appearing to differ in abundance between the two strains. Four of these had a greater than twofold difference and were statistically significant; a cysteine protease, a glutathione-S-transferase, an actin and a heat shock protein 60. This paper also reported some differences in the antigens detected by immune sera taken from experimentally infected sheep (Hart et al., 2012). One caveat to these experiments is that the data appear to be derived from a single aqueous worm homogenate extract for each strain examined on 2-D gels (run three times) rather than from three independent

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bio-replicates for each strain. We have compared the transcriptomes of three independent bio-replicates for each the MHco3(ISE), MHco4(WRS) and the MHco10(CAVR) laboratory strains using DESeq2 analysis of RNAseq data (Love et al., 2014). A total of 1239, 1803 and 718 transcripts were greater than twofold differentially expressed (significance: p ¼ 0.01) between MHco3(ISE)/MHco4(WRS), MHco3(ISE)/MHco10(CAVR) and MHco4(WRS/MHco10(CAVR), respectively (A. Martinelli, A. Rezansoff, J. Cotton and J. Gilleard, unpublished data). Other than anthelmintic resistance candidates, the molecules most thoroughly investigated for strain differences in expression and function are the secreted and intestinal microvilli proteases. This is primarily because of the interest in these molecules as vaccine candidates and concerns about potential antigenic variation among different geographical regions. Initial evidence of geographical variation came from differences in the protease profiles of excretoryesecretory (ES) products between strains derived from the United States and the UK (Karanu et al., 1993). Subsequent analysis of the ES proteases from one US and two Kenyan strains revealed differences in the mobility of the major enzyme species detected on substrate gels and in the effect of inhibitors (Karanu et al., 1997). The cysteine protease inhibitors E64 and iodoacetic acid abolished substrate gel protease activity of ES products from the US strain but had little effect on either of the Kenyan strains. Conversely, protease activity from the Kenya strains, but not the US strain, was completely inhibited by the metallo- and serine protease inhibitors ethylenediaminetetraacetic acid, 1,10-phenanthroline and phenylmethylsulfonyl fluoride (PMSF). Redmond and Wyndham (2005) characterized the protease activity profiles of integral membrane protein extracts isolated from the gut of three different H. contortus strains (MOSI, ISE and WRS) (Redmond and Windham, 2005). At pH 5, there were three clear zones of proteolysis on gelatin substrate gels in all three strains, with only minor differences in mobility. However, at pH 7 and pH 9, although there was a high level of activity at >200 kDa in MOSI and WRS, it was completely absent from ISE. Hemoglobinase activity was detected in the MOSI and WRS strains but not in the ISE strain, and fibrinogen b-degradation was observed at much higher levels for the MOSI than for the WRS and ISE strains. Overall the results suggested a more limited enzymatic profile for the ISE strain, that the authors speculated might be related to its inbred nature (Redmond and Windham, 2005). The genetic basis for differences in protease activity is presently unknown. The proteases and amino-peptidase gene families are much larger in

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H. contortus than in most other organisms including other nematode species (Jasmer et al., 2004; Laing et al., 2013). For example, the cathepsin D aspartic protease and cathepsin B cysteine protease gene families are predicted to consist of 83 genes and 63 genes, respectively (Laing et al., 2013). These numbers appear to relate to a recent evolutionary expansion, since the gene families predominantly consist of large monophyletic clades with many of the genes organized in large tandem arrays in the genome (Laing et al., 2013). Differences in these gene families, either in copy number, sequence polymorphism or expression have not yet been examined in detail. Indeed, analyses are a difficult proposition for such large and complex gene families encoded in a draft reference genome. Some differences in expression are suggested by the observation that only 60% of the 194 cathepsin B-like ESTs (50 clusters) sequenced from a UK isolate were present in a data set of 686 ESTs (123 clusters) from a US isolate (Jasmer et al., 2004). However, our RNAseq comparisons of these strains reveal just 5, 2 and 1 of 74 annotated cathepsin B genes that are greater than fourfold differentially expressed (significance: p ¼ 0.01) between MHco3(ISE)/MHco10(CAVR), MHco3(ISE)/MHco4(WRS), and MHco4(WRS)/MHco10(CAVR), respectively (A. Martinelli, A. Rezansoff, J. Cotton and J. Gilleard, unpublished data). In summary, we still have a poor understanding of differences in gene expression and transcription between H. contortus laboratory strains, but recent progress in the H. contortus reference genome assembly should make systematic analyses more feasible. 5.2.2 Variation in morphological traits The simple body plan of nematodes limits the amount of visible morphological variation apparent within and among species. However, detailed morphological and morphometric analyses reveal that significant variation occurs. For example, 25 distinct morphological characters were used to study the phylogenetic relationships between 12 different species of Haemonchus (Hoberg et al., 2004). Although there is within-species variation for some of these traits, care must be taken when interpreting some of the earlier studies due to the lack of a definitive specific identification. For example, Gibbons (1979) considered H. contortus and H. placei to be the same species, and a number of studies before that time classified both these species as H. contortus (Gibbons, 1979). Nevertheless subsequent studies have shown significant variation for a number of morphological characters within and among H. contortus isolates. The traits most commonly examined are the series of ridges on the anterior region of the cuticle called the synlophe,

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the male bursa and the female vulva. Although these traits are generally used to distinguish between different Haemonchus species, there is significant within-species variation. For example, the number of ridges comprising the synlophe varies between 22 and 30 among H. contortus individuals, and there is significant variance in the morphometrics of the spicules among individual H. contortus worms (Jacquiet et al., 1997; Lichtenfels et al., 1994). One clear example of variance of morphometric traits in different geographical isolates of H. contortus comes from a study in Yemen and Malaysia (Gharamah et al., 2014). In that case, the majority of 200 male H. contortus worms taken from sheep and goats were separated into two distinctive groups by PCoV analysis using morphometric data of body length, length of cervical papillae and spicule length. Most worms clustered by country of origin, with only a slight overlap between countries. This differentiation was supported by molecular analysis, where mtDNA sequences also clustered by country (Gharamah et al., 2012). Similarly we have found a number of statistically significant morphometric differences between the MHco3(ISE), MHco4(WRS) and MHco10(CAVR) strains, including oesophagus length and spicule length in males as well as the extent of the synlophe cuticular ridges in females (E. Hoberg, E. Redman and J. Gilleard, unpublished data). The clearest example of a morphological trait that varies between isolates is vulval morphology. At least 14 morphological types have been described for the H. contortus female vulval, which have been grouped into three major types; smooth, knobbed and linguiform, the proportions of which differ among different isolates (Das and Whitlock, 1960; Hunt et al., 2008; Le Jambre, 1977). Although some of the minor variations are suggested to be environmentally determined e since they vary with parasite population density e there is evidence that the major morphotypes are genetically determined (Le Jambre, 1977; Le Jambre and Ractliffe, 1976). Experiments with US isolates have shown that several generations of selection for offspring of one vulva type increases the frequency of that phenotype in the population. In addition, test crosses between female worms of one vulva type with male worms derived from an isolate with a different predominant vulva type have suggested a genetic basis and an order of dominance of linguiform over knobbed over smooth morphotypes (Le Jambre, 1977). Similar genetic crosses using Bulgarian isolates also supported a genetic basis, but suggested a different dominance hierarchy, with the linguiform type being recessive to both knobbed and smooth morphotypes (Daskalov, 1975). These apparent geographical differences in the respective dominance of these traits were suggested to be due to differences in the genetic

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backgrounds between different regions (Le Jambre, 1977). In conclusion, there are clear differences in morphology and morphometrics between H. contortus isolates and strains derived from different geographical regions, consistent with the pronounced global population genetic structure. 5.2.3 Variation in life history traits and pathogenicity There have been relatively few detailed studies investigating differences in life history traits or pathogenicity of H. contortus isolates. Aumont et al. (2003) investigated whether there was evidence of H. contortus being better adapted to host breeds derived from the same geographical region. These authors compared H. contortus populations from two different geographical regions, one isolate from France and another derived by pooling five different isolates from Guadeloupe. Groups of 10 lambs of two different sheep breeds were infected with each parasite isolate: the ‘Martinik’ Black Belly breed, derived from the Barbados (BB) and the INRA 401 breed from France (Aumont et al., 2003). In both breeds, the resultant faecal egg counts (FECs) were significantly higher in lambs infected with the H. contortus population from Guadaloupe than those infected with the French parasite population (p ¼ 0.008). The establishment rate was same for both H. contortus populations in INRA 401 lambs (w50%); however, in the more resistant BB lambs, it was higher for the ‘sympatric’ Guadaloupe population (15.2%) than for the ‘allopatric’ French population (7.4%). There was no significant difference in haematocrit or eosinophil count between the two H. contortus populations in either breed, indicating that the two strains had a similar pathogenicity. In another study, Troell et al. (2006b) investigated potential differences between isolates adapted to temperate and tropical climates. Groups of six sheep were infected with either a Swedish or a Kenyan isolate using larvae freshly developed from eggs or following storage at 5 C for 9 months. For the fresh larvae only, there were significant differences in both the prepatent period (p ¼ 0.025) and the proportion of larvae undergoing hypobiosis; 70% of the Swedish larvae underwent inhibition compared with 36% for Kenyan isolate (p ¼ 0.0104). This result is consistent with the high propensity of H. contortus to arrest development inside the host in Sweden and the suggestion that this arrest may be the parasite’s ‘genetic default’ in that region as an adaptation to survive cold winters (Waller et al., 2004). No other traits, including worm length, establishment rate, sex ratio or any of the haematological parameters reflective of pathogenicity, were significantly different between the two isolates (Troell et al., 2006b).

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A number of studies have suggested differences in pathogenicity occur between different H. contortus isolates. For example, Poeschel and Todd (1972a,b) undertook a series of experimental infections using 18 different H. contortus isolates obtained from different regions of the United States. These authors reported that three isolates had statistically significant reduced pathogenicity, and two isolates had increased pathogenicity with respect to a control isolate (based on a reduction in blood haemoglobin concentrations, corrected for adult worm number). Although these experiments were replicated several times for the main isolates of interest, the host group size was small (three animals per group), and minimal detail of statistical analyses was reported. More recently, Hunt et al. (2008) compared five laboratory strains (McMaster1931, Wallangra2003, Gold Coast2004, Arding2005 and Cannawigara2005) that were originally derived from south eastern Australia and showed significant genetic differentiation based on microsatellite markers (Hunt et al., 2008). An experiment, in which 10 sheep were infected with each strain, suggested a number of differences in life history traits and pathogenicity. There were significant differences, at least between some of the isolates, in the establishment rate, the rate of increase in FEC and in worm fecundity (FECs divided by the number of adult worms) (p < 0.001). In addition, significant differences in erythrocyte and neutrophil counts as well as wool growth between isolates were reported. ANOVA analysis suggested that these differences were only partially due to the intensity of infection, suggesting differences in pathogenicity among the isolates. Angullo-Cubilan et al. (2010) compared experimental infection of groups of six Spanish Manchego breed lambs with a ‘sympatric’ Spanish H. contortus isolate, Aran 99, with infections with two ‘allopatric’ non-Spanish isolates MRI (Moredun Research Institute, Edinburgh UK) and MSD (Merck Sharp and Dohme) (Angulo-Cubillan et al., 2010). The prepatent period of the Aran 99 isolate was significantly longer (mean 28.1 days) than those of the MSD and MRI isolates (means of 21.3 and 21.7 days, respectively) (p < 0.05). Although there were no differences in the intensity of infection between the isolates, the MRI infected group had significantly lower packed cell volume values than those infected with the other two isolates, again suggesting differences in pathogenicity. We have compared the genetically divergent MHco3(ISE), MHco4(WRS) and MHco10(CAVR) laboratory strains for differences in basic life history traits in an experiment, in which groups of 15 sheep were infected with each isolate (Redman et al., 2012). Only a small difference in the prepatent period was detected between the MHco3(ISE) and the other

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two strains (significantly more animals positive for eggs at 18 days after infection). Day 18 was the only day on which there was a statistically significant difference in FEC between the groups up to day 36 after infection (D. Bartley, N. Sargison, E. Redman and J. Gilleard, unpublished data). We have also recently investigated whether there are competitive differences between these strains during coinfection. We coinfected sheep with 4000 L3s of each of two different strains, to test for differences in overall fitness or fecundity by genotyping F1 progeny with microsatellite markers to determine their parental strain identity. Two sheep were coinfected with strains MHco3(ISE) and MHco4(WRS) and two sheep with strains MHco3(ISE) and MHco10(CAVR). In both cases, MHco3 (ISE) homozygous progeny were significantly overrepresented compared with progeny homozygous or heterozygous for the second strain, suggesting a competitive advantage to the MHco3(ISE) strain during experimental coinfection (N. Sargison, E. Redman, D. Bartley and J. Gilleard, unpublished data). In summary, a number of studies have suggested phenotypic differences in life history traits (including establishment rate, prepatent period and worm fecundity) between different field isolates and laboratory strains. Several studies (Angulo-Cubillan et al., 2010; Hunt et al., 2008) have also suggested that observed differences in the extent of anaemia induced by different strains was not completely accounted for by differences in infection intensity. However, other studies (Aumont et al., 2003; Newton et al., 1995) have found no difference in pathogenicity between isolates. Hence, only a very limited number of studies to date have suggested phenotypic differences in life history traits between isolates and strains of H. contortus.

6. CONCLUDING REMARKS Genetic diversity and population structure are poorly understood for most parasitic nematode species. However, a substantial amount of research has been undertaken of H. contortus, and this parasite serves as a useful model for the trichostrongyloid nematode group. Studies have consistently shown that H. contortus field isolates have remarkably high levels of genetic diversity, which is predominantly due to extremely large population sizes. There is also usually substantial anthropogenic gene flow among populations within a geographical region. Although most of the genetic diversity occurs within populations, there is low, but discernable population structure within a region and substantial genetic differentiation among populations from different

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countries. This population genetic structure underlies the propensity of the parasite to develop resistance to anthelmintic drugs and predisposes to the repeated emergence and potentially rapid spread of drug resistance mutations. There is also substantial genetic diversity within and among H. contortus laboratory strains. It is important that this diversity is considered during experimental studies, particularly when investigating apparent associations between candidate genes and drug resistance or other phenotypes. There has been much less research on phenotypic variation between field isolates and laboratory strains, other than for drug resistance and certain morphological traits. There is some suggestion in the literature of potential variation in life history traits and pathogenicity, but these aspects are still poorly defined and more research is needed. Most of the information on genetic diversity and population structure of H. contortus to date is based on the study of specific genes or the application of panels of relatively low-coverage, neutral genetic markers. Whilst such studies have been very informative, these marker systems are of limited resolution. However, in recent years, there have been substantial improvements in H. contortus genomic resources, together with major advances in sequencing technologies. Consequently larger scale genome-wide approaches, using much larger panels of genetic markers, are becoming both practically and economically feasible. This context should not only enable a more detailed view of genetic variation and population structure of the parasite, but also allow the application of more powerful population genomic approaches to identify drug resistance loci and to study the emergence and spread of drug resistance.

ACKNOWLEDGEMENTS We are grateful to Charles Criscione, James Cotton, Axel Martinelli, Andrew Kotze, Ray Kaplan, Melissa Miller and Andrew Rezansoff for discussion and for sharing unpublished data and information. We are also grateful to Robin Gasser for his valuable comments.

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