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Allozyme variations between sheep or rabbit laboratory reared and goat wild populations of Trichostrongylus colubriformis
Inrernorionul Jnurndfor Prinred in Grerrr iiriruin
PurrrJirology Vol. 23. No. 8.pp. 1087-1089. 1993 0
OOZIS7519/93 $6.00 + 0.00 Pergmon Press Lid I993 Ausmdran Societyfor Purmitology
RESEARCH NOTE ALLOZYME LABORATORY
VARIATIONS BETWEEN SHEEP OR RABBIT REARED AND GOAT WILD POPULATIONS OF
TRICHOSTRONGYLUS
COLUBRIFORMIS
G. N'ZOBADILA*, J. CABARETS and N. GASNIER~ *M.N.H.N., Laboratoire de Biologie parasitaire, Protistologie, t1.N.R.A.. Station de Pathologie aviaire et de Parasitologie, (Received
Helminthologie, 61 rue Buffon, 75231 Paris Cedex 5, France Unit& d’Ecologie des Parasites, 37380 Nouzilly, France
1April 1993; accepted 11July 1993)
Abstract-N’zoaAolLA G., CABARETJ. and GASNIER N. 1993. Allozyme variations between sheep or rabbit laboratory reared and goat wild populations of Trichostrongylus colubriformis. International Journal for Porasilology, 23: 1087-1089. Allozyme variation at 3 polymorphic enzyme loci (GPI, PGM, MDH) was studied in Trichostrongylus colubriformis. By means of a multivariate analysis it was shown that populations of worms harboured by naturally infected goats were genetically different from populations laboratory reared in lambs or rabbits. A deficiency of heterozygotes was recorded in each population studied. INDEX KEY WORDS: heterozygote deficiency.
Nematode;
Trichostrongylus colubrlformis; allozymes;
Trichostrongylus colubriformis has a wide range of hosts and is found in natural infections of sheep and goats. It has been easily adapted to the rabbit in experimental infections (Ortlepp, 1939) and used as model for pathology (Hoste 8c Mallet, 1990).
THE nematode
Two major
handicaps
might
be found
in these models:
to experimental host and laboratory rearing of the nematode. The maintenance in laboratory conditions of the trichostrongylid nematode Teladorsagia circumcincta resulted in a change in allozyme frequencies (Gasnier, Cabaret & Moulia, 1992) and this may also occur in T. colubriformis maintained in the laboratory for a number of generations. The only record of strain variation in T. cofubriformis (see Sangster, 1983 unpublished PhD thesis, University of Sydney) is that of worms susceptible and resistant to thiabendazole. The aim of our work was to compare allozyme variation in individual worms from wild populations harboured by goats with strains laboratory reared in lambs or rabbits. The laboratory strain maintained in lambs originated from a Weybridge Central Veterinary Laboratory (UK) strain and has been used in our laboratory for more than 10 years. The population harboured by one 3-month-old lamb was studied. One 6-week-old rabbit was infected with the sheep strain. Both populations of worms established well (ratio of adult worm recovered in host to infective third-stage larvae administered was over 40%). Worms were also collected from goats from 10 farms (1 or 2 goats per the
switch
from
natural
enzyme
electrophoresis;
farm that harboured only T. colubriformis, based on male and female morphology of worms) located in the center-west of France. The worms were extracted from the duodenum by immersion in water at 37°C over 2-5 h. Males and females were stored in liquid nitrogen in groups of 25 in 2 ml vials without preservation liquid until required for processing. Worms were allowed to thaw, then individually crushed on 2 x 3 mm Whatman paper No. 3 and inserted into a 10% starch gel (l-l.5 mm thick). Each gel was prepared by pouring the hot starch solution onto a 160 x 140 x 2 mm glass plate; after solidification the gel was kept overnight at 15°C. A separate gel was used for each enzyme. Electrophoresis was carried out as described by Kucera (1989) for glucose-phosphate isomerase (GPI, E.C. 5.3.1.9.). This method was also adapted for malate dehydrogenase (MDH, E.C. 1.1.1.37), and phosphoglucomutase (PGM, E.C. 5.4.2.2.). Standard horizontal electrophoresis was carried out either at 16 h and 60 V (MDH) or for 4 h and 120 V (for GPI and PGM). Electrophoretic buffers and stains used for each enzyme are the same as those described in Gasnier et al. (1992). Allozyme frequencies for each population were derived from the electrophoretic results. The Biosys-1 program was used to calculate Nei genetic distance, Wright F,, (amount of differentiation among subpopulations: ratio of variance of genotypic frequencies between populations to variance of genotypic frequencies of all the populations) and eventual heterozygote deficiencies (Swofford &
1087
G. N’ZOBADILA et al.
1088
(RAPID,R AND SLOW, S) OF Trichostrongylus colubriformis
TABLE I-GENOTYPESANDALLELICFREQUENCIES
HARBOUREDBYSHEEP,RABBITSANDGOATS
Enzyme/host
GPI Sheep Rabbits Goats
PGM Sheep Rabbits Goats
MDH Sheep Rabbits Goats
Genotype
n
RR
RS
SS
obs exp obs exp obs exp
22 14 19 15 53 43
13 29 3 12 28 48
22 14 7 2 24 14
obs exp obs exp obs exp
8 4 8 8 2 1
1 8 13 13 2 5
8 4 5 5 13 11
obs exp obs exp obs exp
23 19 14 12 109 92
4 11 3 7 23 55
5 2 3
Allelic frequency
Hardy-Weinberg
R
S
equilibrium (x2. S)
51
0.50
0.50
16.5 (NS)
29
0.71
0.29
17.4 (S)
105
0.64
0.36
19.2 (S)
17
0.50
0.50
14.2 (S)
26
0.56
0.44
17
0.18
0.82
53.5 (S)
32
0.78
0.22
14.0 (S)
20
0.77
0.23
1.5 (S)
156
0.77
0.23
7.4 (S)
0.0 (NS)
1 24 8
obs, observed numbers; exp, expected numbers calculated on the basis of Hardy-Weinberg numbers of worms sampled for each host; S, significant at P
2
19%
73%
FIG. 1. Wild populations of Trichostrongylus colubriformis in goats compared to laboratory reared strains in sheep or rabbits: circle of correlations from principal component analysis of allelic frequencies for 3 enzyme loci (GPI, MDH
and PGM). Selander, 1989). Genetic distances between populations were also assessed by means of principal component as previously described (Gasnier et al., 1992). The following matrix of data was used: the rows were allelic frequencies of allozymes of rapid and slow
law; n,
migration for three enzymatic systems, and columns were the three populations tested. Three polymorphic enzymes (GPI, MDH and PGM) are interpreted in the present work. Three distinct phenotypes could be observed: single-banded (allozymes of rapid or slow migration) and double banded (PGM) or triple-banded patterns (MDH) characteristic of heterozygotes for monomeric or dimeric enzymes, respectively. The quaternary structure of GPI could not be accurately assessed, but individuals with intermediate bands located from slow to fast migrating individuals were scored as heterozygotes. Thus, the data for this enzyme should be treated with some caution since the number of heterozygous individuals may have been underscored. The allele frequencies for each putative locus in the populations harboured by the three hosts are given in Table 1; genotypic frequencies were compared to expected Hardy-Weinberg proportions. For each population a deficiency in heterozygotes (up to 46%) was recorded in all enzymes. The analysis of genetic differentiation of populations by F-statistics, and more particularly F,, (amount of differentiation between populations) showed that MDH did not differentiate the populations harboured by the three species of
1089
Research Note hosts (respective values of F,=O.OO) whereas PGM (&=0.06; J=7.2; Pt0.05) and GPI (F,,=O.O3; 2 = 11.5; P
The deficiency in goat derived populations of nematodes might more probably result from pooling samples from populations with different gene frequencies (the Wahlund effect). This is not the case in experimental nematode populations and inbreeding (mating of close relatives) could not be invoked due to the large size of breeding population in experimental populations (several thousands). The populations appeared to be structured with two genetically distinct groups present in each sample set. This population sub-structuring has been found in T. circumcincta and T. colubrijormis in both natural and laboratoryreared populations. Currently, the biological importance of such population sub-structuring is unknown. The phenomenon is evidently not a technical artifact and requires further investigations, possibly by the use of a larger array of genetic markers. A more comprehensive electrophoretic analysis at loci where heterozygous deficiencies are detected using a larger sample size for each population, together with replicate samples both from different host animals and from different times of the year may lead to an understanding as to the cause of such heterozygote deficiencies. Acknowledgements-~~
are grateful to D. Provaznik (VUBVL, Czechoslovakia) for demonstration and advice on electrophoretic procedures. H. Hoste and S. Mallet (INRA, Nouzilly) provided the experimental strains in lamb and rabbit. Financiat support in the form of grants is acknowledged fromcongo Republic (G. N’zobadila) and the French Ministry of Research (N. Gasnier); Center Region of France funded the investigation in goats. REFERENCES EVANSP. G. H. 1987. Electrophoretic variability of gene products. In: Avian Genetics-A Population and Ecological Approach (Edited by COOKEF. &BUCKLEYP. A.), pp. IOS162. Academic Press, London. GASNIERN., CABARETJ. & MOULIAC. 1992. Allozyme variation between laboratory reared and wild populations of Teladorsagia c~rcumci~c~a. ~~~ernalional Journal for Parasito~ug~ 22: 58 I-587.
HOSTE H. & MALLET S.
1990. Effects
of
size of
Trichosrrongylus colubriformis infections on pathology of
the mucosa along the whole small intestine in rabbits. Journal of Compararive Pathology 103: 457-465.
KUCERA J. 1989. Starch gel electrophoresis of lactate dehydrogenase and glucose phosphate isomerase of poultry coccidia using the LKB Multiphor. Fofia Parasitologica 36 295-299.
ORTLEPPR. I. 1939. Can hares and rabbits act as hosts of sheep and goat bankrupt (Trichostrongylus spp.) worms in South Africa? Journal of South African Veterinary Medicine Association IO: 166- 169.
SWOFFORD D. 1. % SELANDER R. B. 1989. A computer program for the analysis of allelic variation in population genetics and biochemical systematics. Release 1.7. Illinois Natural History Survey, Champaign.
PurrrJirology Vol. 23. No. 8.pp. 1087-1089. 1993 0
OOZIS7519/93 $6.00 + 0.00 Pergmon Press Lid I993 Ausmdran Societyfor Purmitology
RESEARCH NOTE ALLOZYME LABORATORY
VARIATIONS BETWEEN SHEEP OR RABBIT REARED AND GOAT WILD POPULATIONS OF
TRICHOSTRONGYLUS
COLUBRIFORMIS
G. N'ZOBADILA*, J. CABARETS and N. GASNIER~ *M.N.H.N., Laboratoire de Biologie parasitaire, Protistologie, t1.N.R.A.. Station de Pathologie aviaire et de Parasitologie, (Received
Helminthologie, 61 rue Buffon, 75231 Paris Cedex 5, France Unit& d’Ecologie des Parasites, 37380 Nouzilly, France
1April 1993; accepted 11July 1993)
Abstract-N’zoaAolLA G., CABARETJ. and GASNIER N. 1993. Allozyme variations between sheep or rabbit laboratory reared and goat wild populations of Trichostrongylus colubriformis. International Journal for Porasilology, 23: 1087-1089. Allozyme variation at 3 polymorphic enzyme loci (GPI, PGM, MDH) was studied in Trichostrongylus colubriformis. By means of a multivariate analysis it was shown that populations of worms harboured by naturally infected goats were genetically different from populations laboratory reared in lambs or rabbits. A deficiency of heterozygotes was recorded in each population studied. INDEX KEY WORDS: heterozygote deficiency.
Nematode;
Trichostrongylus colubrlformis; allozymes;
Trichostrongylus colubriformis has a wide range of hosts and is found in natural infections of sheep and goats. It has been easily adapted to the rabbit in experimental infections (Ortlepp, 1939) and used as model for pathology (Hoste 8c Mallet, 1990).
THE nematode
Two major
handicaps
might
be found
in these models:
to experimental host and laboratory rearing of the nematode. The maintenance in laboratory conditions of the trichostrongylid nematode Teladorsagia circumcincta resulted in a change in allozyme frequencies (Gasnier, Cabaret & Moulia, 1992) and this may also occur in T. colubriformis maintained in the laboratory for a number of generations. The only record of strain variation in T. cofubriformis (see Sangster, 1983 unpublished PhD thesis, University of Sydney) is that of worms susceptible and resistant to thiabendazole. The aim of our work was to compare allozyme variation in individual worms from wild populations harboured by goats with strains laboratory reared in lambs or rabbits. The laboratory strain maintained in lambs originated from a Weybridge Central Veterinary Laboratory (UK) strain and has been used in our laboratory for more than 10 years. The population harboured by one 3-month-old lamb was studied. One 6-week-old rabbit was infected with the sheep strain. Both populations of worms established well (ratio of adult worm recovered in host to infective third-stage larvae administered was over 40%). Worms were also collected from goats from 10 farms (1 or 2 goats per the
switch
from
natural
enzyme
electrophoresis;
farm that harboured only T. colubriformis, based on male and female morphology of worms) located in the center-west of France. The worms were extracted from the duodenum by immersion in water at 37°C over 2-5 h. Males and females were stored in liquid nitrogen in groups of 25 in 2 ml vials without preservation liquid until required for processing. Worms were allowed to thaw, then individually crushed on 2 x 3 mm Whatman paper No. 3 and inserted into a 10% starch gel (l-l.5 mm thick). Each gel was prepared by pouring the hot starch solution onto a 160 x 140 x 2 mm glass plate; after solidification the gel was kept overnight at 15°C. A separate gel was used for each enzyme. Electrophoresis was carried out as described by Kucera (1989) for glucose-phosphate isomerase (GPI, E.C. 5.3.1.9.). This method was also adapted for malate dehydrogenase (MDH, E.C. 1.1.1.37), and phosphoglucomutase (PGM, E.C. 5.4.2.2.). Standard horizontal electrophoresis was carried out either at 16 h and 60 V (MDH) or for 4 h and 120 V (for GPI and PGM). Electrophoretic buffers and stains used for each enzyme are the same as those described in Gasnier et al. (1992). Allozyme frequencies for each population were derived from the electrophoretic results. The Biosys-1 program was used to calculate Nei genetic distance, Wright F,, (amount of differentiation among subpopulations: ratio of variance of genotypic frequencies between populations to variance of genotypic frequencies of all the populations) and eventual heterozygote deficiencies (Swofford &
1087
G. N’ZOBADILA et al.
1088
(RAPID,R AND SLOW, S) OF Trichostrongylus colubriformis
TABLE I-GENOTYPESANDALLELICFREQUENCIES
HARBOUREDBYSHEEP,RABBITSANDGOATS
Enzyme/host
GPI Sheep Rabbits Goats
PGM Sheep Rabbits Goats
MDH Sheep Rabbits Goats
Genotype
n
RR
RS
SS
obs exp obs exp obs exp
22 14 19 15 53 43
13 29 3 12 28 48
22 14 7 2 24 14
obs exp obs exp obs exp
8 4 8 8 2 1
1 8 13 13 2 5
8 4 5 5 13 11
obs exp obs exp obs exp
23 19 14 12 109 92
4 11 3 7 23 55
5 2 3
Allelic frequency
Hardy-Weinberg
R
S
equilibrium (x2. S)
51
0.50
0.50
16.5 (NS)
29
0.71
0.29
17.4 (S)
105
0.64
0.36
19.2 (S)
17
0.50
0.50
14.2 (S)
26
0.56
0.44
17
0.18
0.82
53.5 (S)
32
0.78
0.22
14.0 (S)
20
0.77
0.23
1.5 (S)
156
0.77
0.23
7.4 (S)
0.0 (NS)
1 24 8
obs, observed numbers; exp, expected numbers calculated on the basis of Hardy-Weinberg numbers of worms sampled for each host; S, significant at P
2
19%
73%
FIG. 1. Wild populations of Trichostrongylus colubriformis in goats compared to laboratory reared strains in sheep or rabbits: circle of correlations from principal component analysis of allelic frequencies for 3 enzyme loci (GPI, MDH
and PGM). Selander, 1989). Genetic distances between populations were also assessed by means of principal component as previously described (Gasnier et al., 1992). The following matrix of data was used: the rows were allelic frequencies of allozymes of rapid and slow
law; n,
migration for three enzymatic systems, and columns were the three populations tested. Three polymorphic enzymes (GPI, MDH and PGM) are interpreted in the present work. Three distinct phenotypes could be observed: single-banded (allozymes of rapid or slow migration) and double banded (PGM) or triple-banded patterns (MDH) characteristic of heterozygotes for monomeric or dimeric enzymes, respectively. The quaternary structure of GPI could not be accurately assessed, but individuals with intermediate bands located from slow to fast migrating individuals were scored as heterozygotes. Thus, the data for this enzyme should be treated with some caution since the number of heterozygous individuals may have been underscored. The allele frequencies for each putative locus in the populations harboured by the three hosts are given in Table 1; genotypic frequencies were compared to expected Hardy-Weinberg proportions. For each population a deficiency in heterozygotes (up to 46%) was recorded in all enzymes. The analysis of genetic differentiation of populations by F-statistics, and more particularly F,, (amount of differentiation between populations) showed that MDH did not differentiate the populations harboured by the three species of
1089
Research Note hosts (respective values of F,=O.OO) whereas PGM (&=0.06; J=7.2; Pt0.05) and GPI (F,,=O.O3; 2 = 11.5; P
The deficiency in goat derived populations of nematodes might more probably result from pooling samples from populations with different gene frequencies (the Wahlund effect). This is not the case in experimental nematode populations and inbreeding (mating of close relatives) could not be invoked due to the large size of breeding population in experimental populations (several thousands). The populations appeared to be structured with two genetically distinct groups present in each sample set. This population sub-structuring has been found in T. circumcincta and T. colubrijormis in both natural and laboratoryreared populations. Currently, the biological importance of such population sub-structuring is unknown. The phenomenon is evidently not a technical artifact and requires further investigations, possibly by the use of a larger array of genetic markers. A more comprehensive electrophoretic analysis at loci where heterozygous deficiencies are detected using a larger sample size for each population, together with replicate samples both from different host animals and from different times of the year may lead to an understanding as to the cause of such heterozygote deficiencies. Acknowledgements-~~
are grateful to D. Provaznik (VUBVL, Czechoslovakia) for demonstration and advice on electrophoretic procedures. H. Hoste and S. Mallet (INRA, Nouzilly) provided the experimental strains in lamb and rabbit. Financiat support in the form of grants is acknowledged fromcongo Republic (G. N’zobadila) and the French Ministry of Research (N. Gasnier); Center Region of France funded the investigation in goats. REFERENCES EVANSP. G. H. 1987. Electrophoretic variability of gene products. In: Avian Genetics-A Population and Ecological Approach (Edited by COOKEF. &BUCKLEYP. A.), pp. IOS162. Academic Press, London. GASNIERN., CABARETJ. & MOULIAC. 1992. Allozyme variation between laboratory reared and wild populations of Teladorsagia c~rcumci~c~a. ~~~ernalional Journal for Parasito~ug~ 22: 58 I-587.
HOSTE H. & MALLET S.
1990. Effects
of
size of
Trichosrrongylus colubriformis infections on pathology of
the mucosa along the whole small intestine in rabbits. Journal of Compararive Pathology 103: 457-465.
KUCERA J. 1989. Starch gel electrophoresis of lactate dehydrogenase and glucose phosphate isomerase of poultry coccidia using the LKB Multiphor. Fofia Parasitologica 36 295-299.
ORTLEPPR. I. 1939. Can hares and rabbits act as hosts of sheep and goat bankrupt (Trichostrongylus spp.) worms in South Africa? Journal of South African Veterinary Medicine Association IO: 166- 169.
SWOFFORD D. 1. % SELANDER R. B. 1989. A computer program for the analysis of allelic variation in population genetics and biochemical systematics. Release 1.7. Illinois Natural History Survey, Champaign.