425
Epidemiology and genetic variability of two species of nematodes (Heligmosomoides polygyrus and Syphacia stroma) of Apodemus spp. C. D. M. MULLER-GRAF 1 ,2*, P. DURAND 2 ,3, c. FELIU\ J.-P. HUGOT 5 , C. J. O'CALLAGHANl, F. RENAUD 3 , F. SANTALLA4 and S. MORAND 2 Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK Centre de Biologie et d' Ecologie Tropicale et Mediterraneenne, Laboratoire de Biologie Animale, CNRS UMR 5555, Universite de Perpignan, Avenue de Villeneuve, 66860 Perpignan Cedex, France 3 Laboratoire de Parasitologie Comparee, CNRS UMR 555 Universite Montpellier 2, Place E. Bataillon, 34095 Montpellier Cedex OS, France 4 Laboratori de Parasitologia, Facultat de Farmacia, Universitat de Barcelona, Av. Diagonal, sin. 08028 Barcelona, Spain 5 Museum National d'Histoire Naturelle, Mammiferes et Oiseaux, (FR CNRS 1541) 55, rue Buffon, 75231 Paris Cedex OS, France 1 2
-I
SUMMARY
The epidemiology and genetic variability of 2 parasitic nematodes Heligmosomoides polygyrus and Syphacia stroma of Apodemus spp. were investigated. Both are parasites of the same host, exhibit a direct life-cycle and are dioecious. However, H. polygyrus has a diploid and S. stroma a haplodiploid mode of reproduction. Haplodiploidy may lead to a more female biased sex ratio and reduced genetic variability. Levels of genetic diversity were analysed using both isoenzyme electrophoresis and RAPDs (random amplified polymorphic DNA). Both parasites showed a female biased sex ratio with a stronger bias for the haplodiploid parasite. Results showed significantly fewer genetic polymorphisms as measured by RAPDs for the haplodiploid parasite S. stroma in comparison with H. polygyrus. Despite the observed female biased sex ratio this could not be explained by a significant amount of inbreeding. Heterozygote deficiency for individual allozyme loci - which could indicate inbreeding - was not found in either parasite species. Other features of the particular lifehistory of these species are likely to have an impact on the sex ratio and genetic variability too. Key words: trichostrongyloids, oxyuroid, haplodiploidy, genetic diversity, sex ratio.
INTRODUCTION
Over recent years studies have begun to investigate parasite life-histories in a comparative way Skorping, Read & Keymer, 1991 ; Poulin, 1995; Morand, 1996; Morand & Sorci, 1998). Parasite life-histories are likely to be reflected in the epidemiological and genetic structure of parasite species. However, the picture is not clear and empirical research is needed to study the interactions. Factors like demography, life-cycles, mechanisms of dispersal, host specificity, mating patterns and effective population size may have an impact (Nadler, 1995). We were interested to study whether a life-history trait, such as the mode of reproduction, influences the epidemiology, for instance, the sex ratio of the parasite, and levels of genetic diversity. It is difficult and challenging to assess the impact of life-history patterns. In order to control for the confounding effect caused by host parameters, it seems appropriate to use a comparative approach in which 2 different parasite
*
Corresponding author: Laboratoire d'Ecologie, CNR5UMR 7625, Pierre et Marie Curie Universite, Case 237,7 quai 5t Bernard, 75252 Paris Cedex OS, France. Tel: 0033 (0) 144 273 809. Fax: 0033 (0) 144 273 516. E-mail:
[email protected] Parasitology (1999), 118, 425-432.
Printed in the United Kingdom
species of the same host are investigated simultaneously. We have compared 2 nematodes of the same host with a similar life-cycle, but with different modes of reproduction. We studied Heligmosomoides polygyrus (Dujardin, 1845) (== Nematospiroides dubius) , a trichostrongyloid, a nematode with a diploid mode of reproduction, and Syphacia stroma (von Linstow, 1884), an oxyuroid parasite with a haplodiploid mode of reproduction. Both parasites exhibit a direct lifecycle, are dioecious and parasitize the same host (Apodemus spp.). The characteristics of a haplodiploid mode of reproduction (males are haploid, from unfertilized eggs; females are diploid, from fertilized eggs) may have an impact on the epidemiology (i.e. sex ratio) and the levels of genetic diversity of this parasite species as compared to a diploid species. A persistent component of haplodiploidy is considered to be an elevated level of inbreeding (Adamson, 1989). Haplodiploids are often colonizing species with small semi-isolated subpopulations. New habitats are colonized by females which can then produce males parthenogenetically. Since not many males are necessary and local mate competition may lead to unnecessary cost, only a small number of males is needed (Hamilton, © 1999 Cambridge University Press
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Miiller-Graf and others
1967). Hence, a slightly more skewed sex ratiobiased towards the females - is expected in comparison with a diploid parasite population which may be more outbreeding. MATERIALS AND METHODS
The parasites Two species of murine nematodes were studied: H. polygyrus, a trichostrongyloid, which is diploid, and S. stroma an oxyuroid parasite with a haplodiploid mode of reproduction. Both species are compared in Table 1. Both have a direct life-cycle. H. polygyrus eggs are excreted with the host faeces, the larvae hatch and develop, migrate to the surface of the substratum and are then ingested by the next potential host. S. stroma eggs are deposited via faeces in the external environment or the gravid females leave the host (at night) and deposit their eggs around the anus. The eggs are ingested by the next host or reinfect the same host (auto-infection) when ingested during grooming. Eggs of oxyurids are not very resistant to environmental conditions (Adamson, 1989).
Sampling Apodemus spp. (n = 65) were trapped between 16 and 26 September 1996 in the Spanish Pyrenees (Province of Navarra) in the area around Eugi (coordinates: 43° 00 1 04 11 N, 1° 31 1 1011 W). The mice were analysed for their parasites at a field laboratory and the species and number of male and female parasites recorded according to morphological criteria (Genov, Yanchev & Jancev, 1981; Hugot, 1988). The mice were weighed and their age classified according to external criteria into 3 categories: adult, subadult and juvenile as detailed by Niethammer & Krapp (1978) and Corbet & Harris (1991). Morphological measurements and isoenzyme electrophoresis of the host allowed the identification of the host species. The majority of the hosts belonged to the species Apodemus sylvaticus (Linnaeus, 1758). The only other host was the closely related species A. fiavicollis (Melchior, 1834). Previous electrophoretic analyses had shown that S. stroma and H. polygyrus in A. sylvaticus and A. fiavicollis belong to the same species (F. Santalla, unpublished data). Specimens of the parasites H. polygyrus and S. stroma were stored immediately after dissection in liquid nitrogen and stored upon arrival in the laboratory at - 80°C. Parasites used for RAPD (random amplified polymorphic DNA) analyses were obtained from hosts caught in the same area at the same date (i.e. Eugi). Parasites used for isoenzyme analysis were obtained from hosts caught in Eugi in 1996 and, in order to augment the sample size for the isoenzyme electrophoresis, samples taken from a mouse population in the same area in the
Spanish Pyrenees from 1995 and from a mouse population trapped near Barcelona (coordinates 41 ° 25 N; 2° 10 E) in 1996 were added. Details of parasite sample sizes used for isoenzyme and RAPD analyses are given in Table 3 and Table 6. Parasites within an individual host may be more genetically similar than between hosts. However, we concentrated on the analysis of overall genetic variability across hosts in this study. To control for the impact of individual hosts on the genetic variability, especially for the RAPDs, we tried to use a similar number of samples from a similar number of hosts for both parasite species when this was possible. For the RAPDs parasite DNA was extracted from specimens from 12 hosts for H. polygyrus and from 10 hosts for S. stroma. For the analysis only females of S. stroma were used, since males are haploid. Isoenzyme electrophoreses were carried out to study heterozygosity and genetic polymorphism, whereas RAPDs were used to estimate the genetic polymorphism only. Using these 2 complementary methods together seemed an appropriate approach for testing our hypothesis. 1
1
Electrophoresis Starch isoenzyme electrophoresis was carried out according to the technique of Pasteur et ale (1987) optimized for the 2 parasite species. For each electrophoresis for each well a single worm was crushed on to a piece of filter paper. Horizontal electrophoresis was performed at 4 °c at 70 m Volt, 70 Ampere for approx. 5 h. Altogether 17 enzymatic systems were tested. The following enzymes were used for the final analysis: malate-dehydrogenase (MDH, EC 1.1.1.37), malic enzyme (ME, -EC 1 .1 .1 .40), mannose-phosphate-isomerase (MPI, EC 5.3.1.8), peptidase A, C (PEP, EC 3.3.11; A = peptide: Val-Leu; C = peptide: Lys-Leu), glucosephosphate-isomerase (PG I, EC 5.3.1 .9) and phospho-glucomutase (PGM, EC 2.7.5.1). Also tested were: peptidase B, D (PEP, EC 3 .4. 11 ; B = peptide: Leu-Gly-Gly; D = peptide: Phe-Ala-Pro), isocitrate dehydrogenase (IDH, EC 1 .1 . 1 .42), adenylate kinase (AK, EC 2.7 .4.3) creatine kinase (CK, EC 2.7.3.2), nucleoside phosphorylase (NP, EC 2.4.2.1), aspartate aminotransferase (AAT, EC 2.6. 1 .1), esterase alpha (EC 3 . 1 .1 .1), but these did not work or only worked for 1 speCIes.
RAPDs The DNA was extracted according to the slightly modified method of Barral et ale (1993). Each individual worm was squashed with a pestle in an Eppendorf tube with 50 pJ of extraction buffer (NaCI 0·15 M; Tris 10 mM, pH 8; EDTA 1 mM; sucrose 0·88 M) on ice. Following another 50 pI of extraction buffer, 15 pI of proteinase K (10 mgjml) and 7 pI of
Epidemiology and genetic variability of 2 nematodes
427
Table 1. Comparison of life-history characteristics of Heligmosomoides polygyrus and Syphacia stroma
Mode of reproduction Average length (fem. in mm) Adult life-expectancy (days) Pre-patent period (days) Eggs/ day/ female
H. polygyrus
s.
Diploid
Haplodiploid
12 84
10 600
stroma
4.1
60t 10* 1500* (Total reproductive output)
Life-expectancy of free-living stages (days) Life-cycle
510 Monoxenous
35t Monoxenous
* S. obvelata. t Enterobius vermicularis. Estimates are for related oxyurids as no data are available for S. stroma (for references see Morand, 1996). 20 % SDS were added. The solution was incubated for 2 h at 57°C. After the incubation 60 #1 of phenol and 60 #1 of chloroform-isoamyl alcohol were added, tubes were gently shaken for 10 min and then centrifuged for 15 min at 12000 rpm at room temperature. The supernatant containing the DNA was transferred into a clean tube and 1 j 10 volume of 3 M NaAc and 2 vols of cold absolute ethanol were added. The tubes were left approx. 12 h at - 20°C and subsequently centrifuged at 14000 rpm for 20 min at 4°C. The remaining pellet was washed with 5 vols of 70 % ethanol, centrifuged for 10 min at room temperature and then dried with a speed vac. The pellet was dissolved in 25 #1 of TE (Tris 10 mM, EDTA 1 mM). A total of 22 decamer primers were tested (from kits A, B and G) from Operon Technologies Inc., USA for RAPDs. Out of these, 10 primers were used for the final analyses (A02: 5/_TGCCGAGTCG-7 / ; / A03: 5/-AGTCAGCCAC-7 ; All: 5/-CAATCGC/ / CGT-7 ; A13: 5 CAGCACCCAC-7 / ; A18: 5/-AGGTGACCGT-7 / ; B04: 5/-GGACTGGAGT-7 / ; / G08: 5 /-TCACGTCCAC-7 ; G09: 5/-CTGACG/ / TCAC-7 ; G14: 5 -GGATGAGACC-7 / ; G18: 5/GGCTCATGTG-7 /). Primers were always tested for both species and selected according to the quality of revealed bands for both species. The volume for each amplification reaction was 25 #1 with 3 #1 of the DNA solution of S. stroma or 2 #1 for H. polygyrus, 100 #M of each dNTP, 3 mM MgCI 2 , 0·2 #M primer, 1·0 unit of Taq DNA polymerase (Promega Biotech, USA), 2·5 #1 of buffer (10 mM Tris-HCl pH 9 at 25°C, 50 mM KCl, 0·1 % Triton X-l00) and sterilized water. A control tube with no template DNA was run with each primer. The amplification was performed in a MJ -Researchers PTC100 thermal cycler and the PCR cycle was carried out for 3·5 min at 92°C, followed by 40 cycles at 92 °c for 1 min, 35°C for 2 min and 72 °C for 2 min for denaturing, annealing and primer extension respectively and a terminal extension for 5 min at
72°C. Amplification products were analysed on 1 % agarose gels stained with ethidium bromide (0·5 #gjml). Polymorphic bands (absence or presence of a band) were detected in each RAPD primer selected. A comparison of RAPD profiles was carried out within samples to score the number of different phenotypes detected. Bands were scored between 300 and 2000 bp. A table of the number of bands scored per individual per primer is available on request.
Statistical analyses Prevalence is defined as the percentage of individuals of a host species infected with a particular parasite species. Abundance describes the number of parasites in an individual host and includes infected and uninfected hosts (Margolis et ale 1982). Epidemiological data were analysed with non--parametric techniques and the G-test using Statview 4 (Abacus Concepts, Berkeley, USA). The significance level was set at 0·05. For the purpose of analysis, juveniles and subadults were considered as one category. Heterozygote deficiency at each putative enzymatic locus was tested according to Rousset & Raymond (1995) using the software Genepop 3 (1997) (Raymond & Rousset, 1995). The level of significance was adjusted to the number of tests carried out and set at 0·007. Levels of heterozygosity between the 2 species were compared using a MannWhitney U test. Data from both isoenzyme electrophoresis and RAPD profile analysis were expressed as the proportion of polymorphic samples observed by loci and the proportion of unique phenotypes observed by primer. Differences between the number of phenotypes for each particular species using RAPDs were tested with a Fisher-Exact test and corrected for the number of tests carried out. Additionally, differences in phenotype proportions between species across all
C. D. M. Miiller-Graf and others
428
Table 2. Epidemiology of Heligmosomoides polygyrus and Syphacia stroma in the wild wood mouse (Apodemus) (n = 65)
Variable
H. polygyrus Females Total
Mean worm 20·02 burden 2·44 S.E. Maximum 73 Skewness 0·83 Prevalence 84·62 (55) 0/0 (no.)
11·88
Males
Larvae
7·63
0·53
S. stroma Total
10·86
Females 6·63
Males 0·26
1·48 1·03 0·13 37·52 0·12 2'78 42 32 4 5 > 188* > 160* 0·96 1·13 1·87 4·82 5·65 4·01 78·46 (51) 78·46 (51) 26.15 (17) 30·77 (21) 32·31 (21) 10·77 (7)
Larvae 4·37 2·58
> 103* 6·03 15·39 (10)
* Per intestine segment the number of worms was only counted up to 100. 30
~
:.....
25
~
~ c ~
2
~
c.o.....
0 \-<
0
15
£J
8
=' c: c:
10
c..s
0
8 5
o juveniles
adults
Fig. 1. Differences in Heligmosomoides polygyrus abundance between young and old Apodemus spp. Error bars indicate S.E.
RAPD primers were analysed by logistic regression using MLN software (Institute of Education, London). Initial tests demonstrated that the assumption of a binomial error distribution was valid for all models. Owing to the small sample size, a further analysis incorporating host effects in a multilevel approach was not possible. The measure of diversity was estimated by the index of Nei & Li (1979): S = (2 X NAB)/(N A + N B) where NAB is the number of bands shared in common between individuals A and B, and N A and N B are the total number of bands observed in A and B respectively. The distances (D = 1 - S) were performed with the RAPDistance Program, vl·04 (Armstrong et ale 1994). Distances between the 2 species were compared with a non-parametric test (Mann-Whitney U test).
RESULTS
The epidemiological analyses showed that there was a higher prevalence of infection with H. polygyrus than with S. stroma (Table 2). However, the maximum number of worms found was higher for S. stroma.
The sex ratio for both parasites was biased towards females. The sex ratio was more skewed for S. stroma (sex ratio m/f = 0'04; m/total = 0'02) than for H. polygyrus (sex ratio m/f = 0'64; m/total = 0'38). It was noted that almost each infected host contained H. polygyrus males as well as females. In 4 hosts only H. polygyrus females were found and in 4 hosts only males. For this parasite species, in 70·9 % of the cases more females than males could be observed in one host, while in 23·6 % of the cases more males than females were detected. For the remainder an equal number was reported. For S. stroma, more females were found in every infected host. Out of 21 infected hosts only 7 were also infected with males. The worm burden for both species and both sexes was aggregated (H. polygyrus overall variance/mean = 19'1, females vim = 11'97, males vim = 9'04; S. stroma overall vim = 129'65, females vim = 75'56, males vim = 3'38). Female and male abundance was correlated in both cases (Spearman-Rank P = < 0·0001 ; n = 65; H. polygyrus: rs corrected for ties = 0'886, S. stroma: rs corrected for ties = 0'561). Abundance of S. stroma and H. polygyrus were not correlated (Spearman-Rank P = 0'1643, rs corrected for ties = 0'174, n = 65). No significant difference in prevalence could be found between adult and non-adult hosts for either parasite species (H. polygyrus G-test X2 = 0'58, P = 0'45, S. stroma G-test X2 = 1'90, P = 0'17). The number of mice caught for each category was 57 adults and 8 younger individuals. However, older animals had a higher abundance of H. polygyrus than younger ones (Mann-Whitney U test, Z-value = -2'714, P = 0'0067; Fig. 1). The same trend was shown when correlating weight with H. polygyrus burden (Spearman-Rank P = 0'018, rs corrected for ties = 0'30, n = 65). No difference between ageclasses in abundance was apparent for S. stroma (Mann-Whitney U test, Z-value = -1'455, P = 0'146) or a correlation with weight (Spearman-Rank P = 0'36, rs corrected for ties 0'12, n = 65). Host sex had a significant impact on prevalence and abundance of S. stroma (G-test X2 = 5'80, P = 0·016 and Mann-Whitney U test, Z-value = -2'38, P =
Epidemiology and genetic variability of 2 nematodes
429
Table 3. Results of the isoenzyme electrophoresis (Numbers and origin of worms analysed at each locus are given in parentheses (El : Eugi 1995; E2: Eugi 1996; B: Barcelona 1996, see text). H. polygyrus individuals were obtained from 1El, 15E2 and 11 B hosts respectively. S. stroma individuals were obtained from 6El, 14E2 and 8B hosts respectively.)
Locus
Heligmosomoides polygyrus Number heterozygotes
MDH ME MPI PEPA PEPC PGI PGM
4 2 8 1 4 17 5
Sample size
Syphacia stroma Number heterozygotes
Sample size
39(IEI; 22E2; 16B) 23(IEI; IOE2; 12B) 36(OEI; 26E2; lOB) 15 (OEI;7E2;8B) 27(OEI; 21E2; 6B) 44(OEI; 30E2; 14B) 44(OEI; 30E2; I4B)
3 0 0 0 4 5 3
47(7EI;33E2;7B) 11(2EI;7E2;2B) 21(6EI;8E2;7B) 6(OEI;6E2;OB) 30(4EI; 20E2; 6B) 33 (5EI; 25E2; 3B) 24(OEI; 24E2;OBO)
Table 4. Genotypes and allelic frequencies (in parentheses) for 7 enzyme loci Enzymes
H eligmosomoides polygyrus
Syphacia stroma
MDH ME MPI PEP A PEP C PGI PGM
35aa 4ab (a = 0'95, b = 0'05) 21aa 2ab (a = 0'96, b = 0·04) 28bb 7ab 1bc (a = 0·10, b = 0·89, c 14aa lab (a = 0'97, b = 0·03) lcc 4cd 22dd (c = 0·11, d = 0·89) 21cc 17cd 6dd (c = 0·67, d = 0'33) 2aa 5ab 37bb (a = 0'1, b = 0·9)
44dd lcd 2de (d = 0'97, c = 0·01, e llcc (c = 1) 21dd (d = 1) 6cc (c = 1) 26bb 4ab (a = 0·07, b = 0·93) 28bb 5ab (a = 0·08, b = 0'92) lcc 3cd 20dd (c = 0·1, d = 0·9)
= 0·01)
= 0·02)
Table 5. Observed and calculated heterozygosities of Heligmosomoides polygyrus and Syphacia stroma (assuming a Hardy-Weinberg distribution) Locus
Parasite
Observed H
Expected H
MDH
H. polygyrus S. stroma H. polygyrus S. stroma H. polygyrus S. stroma H. polygyrus S. stroma H. polygyrus S. stroma H. polygyrus S. stroma H. polygyrus S. stroma
0·10 0·06 0·09 Monomorphic 0·22 Monomorphic 0·07 Monomorphic 0·15 0·13 0·39 0·15 0·11 0·13
0·10 0·06 0·08
ME MPI PEP A PEP C GPI PGM
P-value
0·20 0·06 0·20 0·12 0·44 0·14 0·18 0·19
0·27 1 0·28 1 0·05 0·21
Level of significance was adjusted to the number of tests carried out (P = 0·007).
0'017). More males than females were shown to be infected (m = 72%, f= 160/0)' Overall 22 female and 43 male mice were caught. No significant influence of host sex on prevalence and number of worms was observed for H. polygyrus (G-test X2 = 3·56, P = 0·059 ; Mann-Whitney U test, Z-value = -0'563, P = 0'57). Table 3 shows the number of heterozygotes observed and the overall sample size for the iso-
enzyme electrophoresis. The number of genotypes and allele frequencies for the isoenzymes are presented in Table 4. The observed heterozygosity across loci was for H. polygyrus H = 0·161 and for S. stroma H = 0·068 and did not differ significantly across loci (Mann-Whitney U test, Z = -1' 54, P = 0'12) between the 2 nematode species. Table 5 shows the observed and expected heterozygosity (assuming Hardy-Weinberg) and the test for heterozygote
430
C. D. M. Muller-Gra! and others
Table 6. Number of bands, phenotypes and mean genetic distances of Nei & Li (1979) for each primer (All worms were obtained from hosts trapped in Eugi 1996.) A2
A3
All
A13
A18
B4
G8
G9
G14
G18
lleligor.nosor.noides polygyrus n No. of bands No. of phenotypes Mean D
24 15 18 0·336
24 15 18 0·347
21 15 16 0·211
24 17 19 0·226
24 21 21 0·326
22 23 19 0·395
22 19 21 0·353
23 10 19 0·307
24 15 21 0·440
24 23 21 0·269
Syphacia stror.na n No. of bands No. of phenotypes Mean D
24 18 7 0·131
24 21 6 0·138
24 13 7 0·068
18 17 8 0·222
20 14 6 0·114
23 18 18 0·328
24 12 9 0·160
21 8 3 0·045
29 22 27 0·522
21 16 13 0·228
n, Number of individual worms used for each primer.
deficiency per locus. We did not observe any significant deficit of heterozygotes at any locus of either nematode species. Out of 7 loci tested only 4 were polymorphic for S. stroma whereas all were polymorphic for H. polygyrus. RAPD distances are presented in Table 6. With 1 exception, G 14, the distances were greater in the diploid nematode than in the haplodiploid nematode. For most of the primers, with the exception of A13, B04, G14 and G18, a significantly higher proportion of phenotypes, that is more variation in the band pattern, was found for H. polygyrus when using Fisher Exact tests (Table 7). RAPD phenotypes had also significantly more variation in the band pattern of H. polygyrus than S. stroma (xi = 64·96, P < 0·001) when using a logistic regression summarizing across all primers; however, the 3 RAPD primers B04, G14 and G18 showed up as quite variable as well, as demonstrated by significant interaction terms (xi = 38·71, P < 0'001) between these primers and the covariate for comparing the differences between species.
DISCUSSION
This study investigates the epidemiological patterns and genetic variability of 2 parasite species of the same host with different modes of reproduction. The genetic analysis was carried out intra-locus for the analysis of isoenzyme heterozygosity and inter-loci for the analyses of isoenzyme and RAPD variability. We observed less polymorphism as measured by the majority of RAPDs and isoenzymes for S. stroma, the haplodiploid parasite, than H. polygyrus, the diploid parasite, as predicted. In particular, the results for the RAPDs show that for the majority of markers the haplodiploid parasite exhibits less genetic variability than the diploid parasite. The RAPD band patterns for H. polygyrus are very mixed
and show a high variability whereas for most of the primers the bands for S. stroma are fairly similar and do not show many differences. Exceptions are especially 3 primers (B04, G 14, G 18) which demonstrate a considerable amount of variability for S. stroma as well, indicating that for certain nucleotide sequences, S. stroma is more variable. The RAPD distances were for most primers greater in H. polygyrus as well. When analysing the isoenzymes, 3 out of 7 loci were found to be monomorphic in the case of S. stroma. Even though the level of heterozygosity (i.e. H) is lower for S. stroma than H. polygyrus, there was no significant difference in the overall levels of heterozygosity between the 2 species. Furthermore, the isoenzyme data of each individual locus do not show a significant difference from the expected values. No heterozygote deficiency can be observed for either parasite at any locus. Inbreeding leads to a reduction in heterozygosity, hence heterozygote deficiency would be expected. The observed reduced polymorphism for S. stroma is therefore not the result of elevated levels of inbreeding in the haplodiploid parasite. There are other explanations. For instance haplodiploidy, a skewed sex ratio and an isolated local population can lead to a smaller effective population size and hence lead to a faster fixation of certain genotypes in the population (Crozier, 1985; Nadler, 1995). Not only the particular characteristics of the haplodiploid system, but also the demographic history and other lifehistory patterns of the parasite may be important for the levels of genetic diversity. For example, the lifeexpectancy of the free-living stages of H. polygyrus, the diploid' nematode, is much longer than the lifeexpectancy of S. stroma eggs and therefore certain genotypes may be preserved for a considerable time. The epidemiological results show that the sex ratio is far more female biased for S. stroma, the haploid parasite, than for H. polygyrus, the diploid parasite
431
Epidemiology and genetic variability of 2 nematodes
Table 7. Difference between the phenotypes and RAPDistances of Heligmosomoides polygyrus and Syphacia stroma (Signs indicate whether the RAPDistance was larger for H. polygyrus than for S. stroma. P-value adjusted for the number of tests carried out: P == 0·005. Differences between the number of phenotypes of the species were tested using a FisherExact Test. Differences between the RAP Distances of the species were tested using a Mann-Whitney U test.) Primer
A02
A03
All
A13
A18
B04
0·0269 0·0034 0·0007 0·0027 0·6995 P-value Fisher0·0001 Exact -4,66 -12,83 -13,94 -5,02 -13,25 Z (corrected for -12,11 ties) 0·0001 0·0001 0·0001 0·0001 P-value Mann0·0001 0·0001 Whitney U H.p. > S.s. + + + + + +
as observed in a previous study (Lewis, 1968). However, we also reported more females than males for H. polygyrus. This finding is supported by other studies (Lewis, 1968; Keymer 1985; Slater & Keymer, 1986; Quinnell, 1992; for H. mixtum Haukisalmi, Henttonen & Vikman, 1996), but stands in contradiction to observations made by Gregory, Keymer & Clarke (1990). Gregory et ale (1990) reported that the female worm burden decreased over time and the initially female biased sex ratio became increasingly more male biased. Therefore, we may assume that most of our animals were newly infected and/or constantly acquiring new infections. This explanation seems plausible since it may be quite likely that wood mice are being constantly infected with H. polygyrus. Even though the very female-biased sex ratio found for the haplodiploid parasite supports the hypothesis that there may be a considerable amount of parthenogenesis (Adamson & Ludwig, 1993) and consequently sister and brother or mother and son matings (Hamilton, 1967), this was not borne out by the genetic data. They did not show evidence for inbreeding. Hence, there may be other reasons for the biased sex ratio. It has been mentioned that lifeexpectancies of males and females are different for some oxyurid species (Adamson, 1989). A difference in life-expectancy between the 2 sexes could result in a biased sex ratio. Furthermore, theoretical calculations have shown that the difference in sex ratio between haplodiploid and diploid species may not be big, depending on the number of foundresses (Bulmer, 1994). Therefore, the noticeably greater sex bias in the haplodiploid parasite may indicate the influence of other mechanisms. We also studied the impact of other factors, such as host sex and age on parasite burden. We did not find any impact of host sex on H. polygyrus in contrast to Gregory et ale (1990) who observed a significantly higher burden of H. polygyrus in male than in female mice (see also Lewis, 1968). However, this observation was made under a regime of constant
G08
< 0·0001 -14,47
G09
< 0·0001 -16,52
0·0001
+
0·0001
+
G14
G18 0·0808
0·6468 -5,01
-3,78 0·0002
0·0001
+
repeated infections. Hence, the parasite burden in the wild mice may not have been high enough to notice a similar effect. We did observe, on the other hand, a higher S. stroma burden in male Apodemus, as did Lewis (1968). Differences in infection between the 2 sexes have been frequently observed (Bundy, 1988; Brabin & Brabin, 1992; Roberts, Satoskar & Alexander, 1996). These may be, for instance, due to different hormonal states in the 2 genders (Folstad & Karter, 1992). The observation that older mice have a higher parasite burden of H. polygyrus than younger mice confirms previous findings (Elton et ale 1931; Lewis 1968). Elton et ale (1931) also reported an increase of infections with weight/age for Syphacia but noted that it was less marked than for H. polygyrus. Further studies are needed to look at epidemiological and genetic patterns such as sex ratio, genetic variability and population structure in different parasite populations of the same species. It has been suggested that the amount of skew of the sex ratio is also a function of prevalence and intensity of infection and, therefore, may vary among localities (J. Lewis, personal communication; for theoretical discussion see Adamson & Ludwig, 1993). We require more information about the stability of the systems to be able to take the effective population size into account. Also the importance of the individual host for the genetic variability of the parasite will have to be investigated. This is a first study to compare 2 species of parasite with different modes of reproduction in the same species of host. We will need further information from different haplodiploid and diploid species to answer the question whether there is a systematic difference in epidemiology and genetic variability between these 2 types of parasites. C. D. M.-G. was supported by a travel grant of the European Science Foundation. The project is part of the PICASSO initiative (HF 1996-0186). We would like to thank especially the other members of the fieldwork team in Eugi. We also thank C. Dowson for support to undertake this work, J. Lewis, T. Anderson, P. Wiener, C. Gabrion,
C. D. M. Muller-Graf and others D. Couvet and the scientists in Perpignan and Montpellier for discussions and help and two anonymous referees for constructive comments.
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