It is well established that genetic exchange occurs between Trypanosoma brucei parasites when two stocks are used to infect tsetse flies under laboratory conditions and a number of such crosses have been undertaken. Both cross and self-fertilisation can take place and, with the products of mating being the equivalent of F1 progeny in a Mendelian system and. Recently, analysis of a large collection of independent progeny using a series of polymorphic micro and minisatellite markers, has formally demonstrated that the allelic segregation at loci on each of the 11-megabase chromosomes conforms to ratios predicted for a classical diploid genetic system involving meiosis as well as independent assortment of markers on different chromosomes. Further extensive analysis of these F1 progeny, using a large panel of micro and minisatellite markers, has led to the construction of a genetic map of one parasite stock A. MacLeod, A. Tweedie and S. McLellan et al., The genetic map of Trypanosoma brucei, Nucleic Acids Res 33 (2005), pp. 6688–6693. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (10)

Alison Tweedie

Andy Tait

Annette MacLeod

Bingle

C. Michael R. Turner

Dan Masiga

Gibson

MacLeod

Masiga

Schweizer

Sternberg

Tait

Tetley

Turner

Van den Abeele

Vickerman

English

PubMed

Tait, A.

MacLeod, A.

Tweedie, A.

Masiga, D.

Turner, C.M.R.

Enlighten

      Tait, A. and MacLeod, A. and Tweedie, A. and Masiga, D. and Turner, C.M.R. (2005) Genetic exchange in Trypanosoma brucei: evidence for mating prior to metacyclic stage development. Molecular and Biochemical Parasitology 151(1):pp. 133-136.       http://eprints.gla.ac.uk/4606/  10th September 2008   Glasgow ePrints Service https://eprints.gla.ac.uk 1Genetic exchange in Trypanosoma brucei: evidence for mating prior to metacyclic stage development.Andy Tait1*, Annette MacLeod1, Alison Tweedie1, Dan Masiga 2,3 and C. Michael R. Turner1,21. Wellcome Centre for Molecular Parasitology,University of Glasgow,Glasgow Biomedical Research Centre,120, University Place,Glasgow G12 8TA, UK2. Division of Infection and Immunity, IBLSUniversity of GlasgowGlasgow Biomedical Research Centre,120, University Place,   Glasgow G12 8TA, UK3. Current Address: Department of Biochemistry and Biotechnology,Kenyatta University,P.O. Box 4384400100 NairobiKenya *  To whom correspondence should be addressed: Tel 0141-330-8472; Fax 0141-330-5422; E-mail: gvwa01@udcf.gla.ac.ukAbbreviations: AFLP, Amplified Fragment Length Polymorphism; TREU, Trypanosomiasis Research, Edinburgh University; STIB, Swiss Tropical Institute, Basel; PCR, Polymerase Chain Reaction Key words: Trypanosoma brucei; genetic exchange; life cycle stageIt is well established that genetic exchange occurs between Trypanosoma bruceiparasites when two stocks are used to infect tsetse flies under laboratory conditions and a number of such crosses have been undertaken [1,2]. Both cross and self-fertilisation can take place [3,4], with the products of mating being the equivalent of F1 progeny in a Mendelian system [2,3]. Recently, analysis of a large collection of independent progeny using a series of polymorphic micro and minisatellite markers, has formally demonstrated that the allelic segregation at loci on each of the 11-megabase chromosomes conforms to ratios predicted for a classical diploid genetic system involving meiosis as well as independent assortment of markers on different chromosomes [5]. Further extensive analysis of these F1 progeny, using a large panel of micro and minisatellite markers, has led to the construction of a genetic map of one parasite stock [6].One of the remaining unknowns about this system of genetic exchange is the life cycle stage at which mating takes place.  Mating clearly takes place in the tsetse fly [1,2] 2and in principal could take place at any point during cyclical development. In the tsetse, procyclic form trypanosomes migrate anteriorly from the midgut into the foregut and proboscis of a tsetse fly and thence to the salivary glands, from where free-swimming mature metacyclic forms are expelled with saliva during tsetse feeding. The development from procyclic to metacyclic is via a succession of morphologically distinct stages, the most prominent of which are the mesocyclic and epimastogote stages in the proventiculus and salivary glands respectively [7,8]. Gibson et al. [9], using parental stocks tagged with selectable markers, provided evidence that mating does not occur between procyclic stage trypanosomes in the mid-gut of the tsetse fly, in contrast to a previous report [10]. Using an innovative approach involving GFP tagging, the Gibson group showed that the products of mating only occur in the salivary glands although the precise stage in trypanosome development was not specified [11]. The genotypes of single metacyclic trypanosomes (amplified vegetatively in mice) obtained from crosses, clearly show that they are the products of mating [1,3]. Taken together, the available evidence is consistent with mating taking place at one of three possible life cycle stages in the tsetse fly: (1) between metacyclic forms, (2) between epimastigotes, or (3) at a pre-epimastigote stage post the procyclic stage in the mid-gut.To test the possibility that mating occurs between metacyclic stage trypanosomes, we genotyped a series of progeny clones derived from single metacyclic stage trypanosomes taken from tsetse flies infected with mating mixtures of two pairs of parental stocks (TREU 927 x STIB 247 or STIB 386 x STIB 247). Single metacyclic trypanosomes were isolated from either dissected salivary glands or guinea pig serum into which infected tsetse had been allowed to probe, each expanded by injection into a single mouse and the resulting infected blood cryopreserved. The metacyclic stage trypanosomes were isolated from two flies (F532 and F974) in the TREU 927/STIB 247 cross and two flies in the STIB 386/STIB 247 cross (F9 and F492)  Theseprogeny have been described previously and been shown to be hybrid for a series of RFLP and iso-enzyme markers [3,12], indicating that they are the products of genetic exchange between the parental stocks. The cryopreserved  metacyclic clones were expanded in immuno-suppressed mice and DNA prepared from purified trypanosomes as previously described [3]. A total of eleven progeny clones from the first cross and nine from the second were generated (listed in Table 1).  Each DNA preparation of the metacyclic derived progeny clones from the cross STIB 247 x TREU 927 was genotyped with three polymorphic minisatellite markers (TB1/1, TB3/13 and TB10/14) located on chromosomes 1, 3 and 10, respectively- [5]) and ten microsatellite markers (listed in Table 2) located on chromosomes 1, 2, 3, 4 and 5 [6] using the PCR conditions described previously [5]. Similarly, the preparations from the metacyclic derived progeny of the cross STIB 247 x STIB 386 were genotyped with the same three minisatellite markers and nine microsatellite markers located on chromosomes 1, 2, 3, 4, 5 and 10. The markers on different chromosomes will segregate independently [5] while those on the same chromosomes have been chosen on the basis of being separated by genetic distances (Table 2) to give a high probability of cross-over between them. The genetic distances correspond to physical distances of 185-525 kb in the sequence of TREU 927 but the physical distances have not been determined for STIB 386. The genotyping allowed the construction of a multilocus genotype (MLG 1-17) for each progeny clone and comparative analysis showed that the multilocus genotypes of the clones from the 247 x 927 cross were unique, except for clones F532/72 mcl 1 and 4 (MLG 2), while the same analysis with the       Tait, A. and MacLeod, A. and Tweedie, A. and Masiga, D. and Turner, C.M.R. (2005) Genetic exchange in Trypanosoma brucei: evidence for mating prior to metacyclic stage development. Molecular and Biochemical Parasitology 151(1):pp. 133-136.       http://eprints.gla.ac.uk/4606/  10th September 2008   Glasgow ePrints Service https://eprints.gla.ac.uk 3progeny from the 247 x 386 cross identified two pairs of clones (F9/45 mcl 7 and F9/45 mcl 11 – MLG 13; F9/45 mcl 9 and F9/45 mcl 10 – MLG 14) that were identical to each other for all micro- and minisatellite markers (Table 1), although the remaining clones were unique. To test the identity of these pairs of clones further, genome wide markers were used. Previous work, using the technique of amplified fragment length polymorphism (AFLP [13,14]), has been undertaken to construct linkage maps of TREU 927 and STIB 386 using some of the metacyclic derived progeny and provides genome wide analyses of polymorphic segregating markers [2]. DNA was prepared for AFLP analysis  from the parental and progeny clones after growth in immunosuppressed mice. (independent preparations from those used for the microsatellite analysis).  A section of one gel is shown in Fig 1 to illustrate the differences observed between the parental stocks and the inheritance of these differences in the resulting progeny. The parental clones (247 and 386) show a series of identical bands as well as differences in which a band is present in one parent but not the other and some of these bands segregate in the F1 progeny clones as a prescence/absence (Fig 1 arrows). This can be interpreted as the homozygous absence of a polymorphic site in one parent (therefore no amplification product) with the second parent being heterozygous for that polymorphism. These polymorphisms arise from the presence/absence of a restriction site, insertion/deletion or single base change in the sequence matching the 3’ extension of one primer. This polymorphism will then segregate into the progeny, which will inherit one ‘absent’ allele from one parent and either the amplified band or ‘absent’ allele from the second parent [2,13]. Applying this technique to the detection of differences between the three parental clones (247, 386 and 927), using the restriction enzyme pairs EcoR1/Mse1 and HindIII/Taq1, and screening the F1 progeny clones to detect markers that segregate, 157 segregating polymorphic fragments were detected in the 247 x 386 cross and 185 in the 247 x 927 cross. Figure 1 illustrates the different patterns of segregation of the parental bands in progeny that differ in genotype (lanes 3-7), as well as the identity of clones F9/45 mcl 9 and 10 (lanes 7 and 8).  These data allow a detailed genetic fingerprint of each progeny clone to be generated and a comparison between the clones in terms of their similarity. The AFLP data confirmed the findings with the using min- and microsatellite markers. In the cross 927 x 247, seven of the metacyclic derived clones were confirmed to be of distinct, independent genotypes and two clones were identical to each other (AFLP genotype A, Table1). These data indicate that most of the metacyclic stage clones are genotypically distinct and that the metacyclic stage is of hybrid genotype and therefore a product of mating (Table 1). AFLP analysis of six of the metacyclic derived clones from the 247 x 386 cross, showed that five were of unique genotype and two were identical for all AFLP markers (AFLP genotype J, Table1). When these data are combined with the multilocus genotypes generated from the micro- and minisatellite data, there is clear evidence that one pair of clones from the 927 x 247 cross and two pairs from the 386 x 247 cross are identical in terms of genotype. While a proportion of these markers will be genetically linked, there is a sufficiently large number of independent markers to make the conclusion of identity a robust one. If mating occurred between the metacyclic stage trypanosomes  the probability that two F1 progeny, in the absence of division, would be identical in the sample sizes from each cross is extremely low as the products of meiosis will all be different. The evidence that metacyclics do not divide is based on extensive electron microscopy studies of infected 4salivary glands, where there is ample evidence for dividing epimastigotes but no evidence for the division of either the mature or nascent metacylics that have aqcuired a VSG coat [15]. Additionally, immuno-electron microscopy with monoclonal antibodies specific a particular VSG, shows that the metacyclics expressing a particular VSG are randomly distributed throughout the gland and occur in isolation. If the metacyclic divided you would predict clusters of such cells expressing the same VSG-this is not observed [16]. On this basis, mating cannot be occurring between the mature metacyclic stage trypanosomes and the identical clones must have arisen by vegetative division of a single original product of mating. This result suggests that mating takes place either amongst epimastigote or pre-metacyclic forms in the salivary glands or at a stage in the foregut/proboscis. However, in both crosses most metacyclic stage clones are unique, therefore suggesting there is very limited vegetative growth between mating and the development of the metacyclic otherwise many identical clones would be observed.  In order to explain the results from the two crosses, taken together, mating cannot take place between the metacyclic stages but is most likely to occur between the epimastigotes with limited subsequent mitotic division between mating and the development of the metacyclic stage. If mating occurred prior to the epimastigote stage between some of the morphologically distinct stages identified in the foregut and proboscis [7], the subsequent multiple rounds of division of the epimastigotes [7,8] would lead to many of the metacyclic stage trypanosomes being of identical genotype. This is clearly not the case. Our data strongly support mating and meiosis occurring between attached epimastigotes or dividing pre-metacyclic stages in the salivary glands. Two indirect arguments suggest that the epimastigote is the most likely stage at which mating takes place. Firstly, pre-metacyclics are relatively rare and this is thought to be a very transient phase in development [8, 15]. Secondly, in other flagellates, the process of gamete fusion that leads to syngamy is first mediated via flagella attachment [17] and it is only in the attached epimastigote phase of the life cycle that T. brucei has the required flagellum-substratum and flagellum-flagellum interactions [18]. AcknowledgementsThis work was supported by grants from The Wellcome Trust and World Health Organisation (TDR) to AT and CMRT and by Fellowships from The Leverhulme Trust (CMRT) and The Royal Society of Edinburgh (AMacL). We would like to thank Andrew Parker of IAEA, Vienna for the supply of tsetse pupae.Figure LegendFigure 1 Autoradiograph of labelled AFLP gel with DNA samples digested with HindIII and Taq1. Each primer had a two-nucleotide extension (H-CA and T-AA) for selective amplification and fragments were separated on a 6% acrylamide gel. Tracks: 1-STIB 247; 2-STIB 386; 3-F9/45 mcl 2; 4- F9/45 mcl 11; 5- F492/50 mcl 12; 6- F492/50 mcl 13; 7- F9/45 mcl 9; 8-F9/45 mcl 10. Arrows indicate fragments that are heterozygous for polymorphisms in one of the two parental stocks and segregate in the progeny.5References[1] Gibson WC, Stevens J. Genetic exchange in the Trypanosomatidae. Adv Parasitol 1999;43:1-45.[2] Tait A, Masiga D, Ouma J, MacLeod A., Sasse J, Melville M. et al. The genetic analysis of complex phenotypes in T.brucei. Phil. Trans. Roy. Soc. B. 2002;367:89-99.[3] Turner CMR, Sternberg J, Buchanan N, Smith E, Hide G, Tait A. Evidence that the mechanism of gene exchange in Trypanosoma brucei involves meiosis and syngamy Parasitology 1990;101:377-86.[4] Tait A, Buchanan N, Hide G, Turner CMR, Self-fertilisation in Trypanosoma brucei.  Mol Biochem Parasitol 1996;76:31-42.[5] MacLeod A, Tweedie A, McLellan S, Hope M, Taylor S, Cooper A, et al. Allelic segregation and independent assortment in T.brucei crosses: proof that the genetic system is Mendelian and involves meiosis. Mol Biochem Parasitol 2005;143:131-3.  [6] MacLeod A,  Tweedie A,  McLellan S, Taylor S, Hall N, Berriman M, El-Sayed, N  Hope M, Turner C.M.R. and Tait A. The genetic map of Trypanosoma brucei.   Nucleic. Acids. Res. 2005;33:6688-93. [7] Van den Abeele J, Claes Y, Van Bockstaele D, Le Ray D, Coosemans M. Trypanosoma brucei spp. Development in the tsetse fly: characterization of the post-mesocyclic stages in the foregut and proboscis. Parasitology 1999;118:469-78.[8] Vickerman K, Developmental cycles and biology of pathogenic trypanosomes. Brit Med Bull 1985;41:105-14.[9] Gibson W, Bailey M. Genetic exchange in T.brucei: evidence for meiosis from analysis of a cross between drug-resistant transformants. Mol Biochem Parasitol 1994;64:241-52.[10] Schweizer J, Jenni L. Hybrid formation in the life cycle of Trypanosoma (T.) brucei: detection of hybrid trypanosomes in a midgut-derived isolate. Acta Tropica 1991;48:1319-21.[11] Bingle LEH, Eastlake JL, Bailey M, Gibson WC. A novel GFP approach for the analysis of genetic exchange in trypanosomes allowing the in situ detection of mating events. Microbiology 2001;147:3231-40[12] Sternberg J, Turner CMR, Wells JM, Ranford-Cartwright LC, Le Page RFW, Tait A. Gene exchange in African trypanosomes: frequency and allelic segregation.  Mol Biochem Parasitol 1989;34:269-80.[13] Masiga DK, Tait A, Turner CMR. The use of the amplified fragment length polymorphism (AFLP) technique in parasite genetics.  Parasitol. Today. 2000;16:350-3.[14] Masiga DK, Turner CMR. Amplified (restriction) fragment length polymorphism (AFLP) analysis. In: SE Melville editor. Parasite Genomics Protocols  Humana Press New Jersey. 2004;p.173-86.[15] Tetley L, Vickerman K. Differentiation in Trypanosoma brucei: host-parasite cell junctions and their persistence during acquisition of the variable antigen coat. J. Cell Sci. 1985;74:1-19.[16] Tetley L, Turner CMR, Barry J D, Crowe J S, Vickerman K. Onset of expression of the variant surface glycoproteins of Trypanosoma.brucei in the tsetse fly studied using immunoelectron microscopy. J.Cell Sci. 1987; 87:363-372.6 [17] Pan J, Snell WJ. Signal transduction during fertilization in the unicellular green alga, Chlamydomonas. Curr. Opinion Microbiol. 2000;3:596-602.[18] Vickerman K, Tetley L. Flagellar surfaces of parasitic protozoa and their role in attachment. In: RA Bloodgood editor. Ciliary and flagellar membranes.  Plenum Press. 1990;p.267-304.Table 1.The genotypes of the metacyclic stage F1 progeny clones from two crosses, determined by micro and minisatellite markers as well as AFLP genotyping. Each multilocus genotype (MLG) was based on the analysis of 12-13 micro- and minisatellite markers and each MLG that was different has been given a different number. The genotypes determined by AFLP used 157 segregating markers (247 x 386) and 185 markers (247 x 927). Each AFLP that was different has been given a different letter. ND-not determined.Cross Clone MLG AFLP247 x 927 F532/53 mcl 1 1 NDF532/72 mcl 1 2 AF532/72 mcl 2 3 BF532/72 mcl 3 4 CF532/72 mcl 4 2 AF532/72 mcl 5 5 DF532/72 mcl 6 6 EF532/72 mcl 7 7 FF532/72 mcl 9 8 GF532/72 mcl 8 9 NDF974/70 mcl 4 10 ND247 x 386 F9/34 mcl 1 11 NDF9/45 mcl 2 12 HF9/45 mcl 7 13 NDF9/45 mcl 11 13 IF9/45 mcl 9 14 JF9/45 mcl 10 14 JF9/45 mcl 12 15 NDF492/50 mcl 12 16 KF492/50 mcl 13 17 L7Table 2Micro- and minisatellite markers used for genotyping. All markers are ‘TB’ markers except where shown. Markers TB 3/13, TB 5/4 and TB 10/14 on chromosomes 3, 5 and 10 were also used.Cross Chromosome Marker Genetic distance (cM)927 x 247 1 TB 1/1    17.5TB 1/7    18.3TB 1/132 TB 2/3    42.7TB 2/10    35.6TB 2/204 TB 4/1    32.2TB 4/4    25.9TB 4/10    31.2TB 4/16386 x 247 1 TB 1/1    23.1TB 1/4    35.8TG 1/42 TB 2/3    32.6TB 2/10    20.7TB 2/204 TB 4/1    44.7TB 4/8    31.8TB 4/1481    2    3    4    5    6    7    8Figure 1

A novel GFP approach for the analysis of genetic exchange in trypanosomes allowing the in situ detection of mating events.

Allelic segregation and independent assortment in T.brucei crosses: proof that the genetic system is Mendelian and involves meiosis.

Amplified (restriction) fragment length polymorphism (AFLP) analysis. In:

Developmental cycles and biology of pathogenic trypanosomes.

Differentiation in Trypanosoma brucei: host-parasite cell junctions and their persistence during acquisition of the variable antigen coat.

Evidence that the mechanism of gene exchange in Trypanosoma brucei involves meiosis and syngamy Parasitology

Flagellar surfaces of parasitic protozoa and their role in attachment.

Gene exchange in African trypanosomes: frequency and allelic segregation. Mol Biochem Parasitol

Genetic exchange in T.brucei: evidence for meiosis from analysis of a cross between drug-resistant transformants. Mol Biochem Parasitol

Genetic exchange in the Trypanosomatidae. Adv Parasitol

Hybrid formation in the life cycle of Trypanosoma (T.) brucei: detection of hybrid trypanosomes in a midgut-derived isolate.

Onset of expression of the variant surface glycoproteins of Trypanosoma.brucei in the tsetse fly studied using immunoelectron microscopy. J.Cell Sci.

Self-fertilisation in Trypanosoma brucei.

Signal transduction during fertilization in the unicellular green alga,

The genetic analysis of complex phenotypes in T.brucei.

The genetic map of Trypanosoma brucei.

The use of the amplified fragment length polymorphism (AFLP) technique in parasite genetics.

Trypanosoma brucei spp. Development in the tsetse fly: characterization of the post-mesocyclic stages in the foregut and proboscis. Parasitology

Genetic exchange in <i>Trypanosoma brucei</i>: evidence for mating prior to metacyclic stage development

Enlighten: Publications

 
 
 
 
 
 
Tait, A. and MacLeod, A. and Tweedie, A. and Masiga, D. and Turner, 
C.M.R. (2005) Genetic exchange in Trypanosoma brucei: evidence for 
mating prior to metacyclic stage development. Molecular and 
Biochemical Parasitology 151(1):pp. 133-136.
 
 
 
 
 
 
 
http://eprints.gla.ac.uk/4606/ 
 
10th September 2008 
 
 
Glasgow ePrints Service 
https://eprints.gla.ac.uk 
1Genetic exchange in Trypanosoma brucei: evidence for mating prior to metacyclic 
stage development.
Andy Tait1*, Annette MacLeod1, Alison Tweedie1, Dan Masiga 2,3 and C. Michael R. 
Turner1,2
1. Wellcome Centre for Molecular Parasitology,
University of Glasgow,
Glasgow Biomedical Research Centre,
120, University Place,
Glasgow G12 8TA, UK
2. Division of Infection and Immunity, IBLS
University of Glasgow
Glasgow Biomedical Research Centre,
120, University Place,
   Glasgow G12 8TA, UK
3. Current Address: Department of Biochemistry and Biotechnology,
Kenyatta University,
P.O. Box 43844
00100 Nairobi
Kenya 
*  To whom correspondence should be addressed: Tel 0141-330-8472; Fax 0141-330-5422; 
E-mail: gvwa01@udcf.gla.ac.uk
Abbreviations: AFLP, Amplified Fragment Length Polymorphism; TREU, 
Trypanosomiasis Research, Edinburgh University; STIB, Swiss Tropical Institute, Basel; 
PCR, Polymerase Chain Reaction 
Key words: Trypanosoma brucei; genetic exchange; life cycle stage
It is well established that genetic exchange occurs between Trypanosoma brucei
parasites when two stocks are used to infect tsetse flies under laboratory conditions and a 
number of such crosses have been undertaken [1,2]. Both cross and self-fertilisation can 
take place [3,4], with the products of mating being the equivalent of F1 progeny in a 
Mendelian system [2,3]. Recently, analysis of a large collection of independent progeny 
using a series of polymorphic micro and minisatellite markers, has formally demonstrated 
that the allelic segregation at loci on each of the 11-megabase chromosomes conforms to 
ratios predicted for a classical diploid genetic system involving meiosis as well as 
independent assortment of markers on different chromosomes [5]. Further extensive 
analysis of these F1 progeny, using a large panel of micro and minisatellite markers, has 
led to the construction of a genetic map of one parasite stock [6].
One of the remaining unknowns about this system of genetic exchange is the life 
cycle stage at which mating takes place.  Mating clearly takes place in the tsetse fly [1,2] 
2and in principal could take place at any point during cyclical development. In the tsetse, 
procyclic form trypanosomes migrate anteriorly from the midgut into the foregut and 
proboscis of a tsetse fly and thence to the salivary glands, from where free-swimming 
mature metacyclic forms are expelled with saliva during tsetse feeding. The development 
from procyclic to metacyclic is via a succession of morphologically distinct stages, the 
most prominent of which are the mesocyclic and epimastogote stages in the proventiculus 
and salivary glands respectively [7,8]. Gibson et al. [9], using parental stocks tagged with 
selectable markers, provided evidence that mating does not occur between procyclic stage 
trypanosomes in the mid-gut of the tsetse fly, in contrast to a previous report [10]. Using an 
innovative approach involving GFP tagging, the Gibson group showed that the products of 
mating only occur in the salivary glands although the precise stage in trypanosome 
development was not specified [11]. The genotypes of single metacyclic trypanosomes 
(amplified vegetatively in mice) obtained from crosses, clearly show that they are the 
products of mating [1,3]. Taken together, the available evidence is consistent with mating 
taking place at one of three possible life cycle stages in the tsetse fly: (1) between 
metacyclic forms, (2) between epimastigotes, or (3) at a pre-epimastigote stage post the 
procyclic stage in the mid-gut.
To test the possibility that mating occurs between metacyclic stage trypanosomes, we 
genotyped a series of progeny clones derived from single metacyclic stage trypanosomes taken 
from tsetse flies infected with mating mixtures of two pairs of parental stocks (TREU 927 x 
STIB 247 or STIB 386 x STIB 247). Single metacyclic trypanosomes were isolated from either 
dissected salivary glands or guinea pig serum into which infected tsetse had been allowed to 
probe, each expanded by injection into a single mouse and the resulting infected blood 
cryopreserved. The metacyclic stage trypanosomes were isolated from two flies (F532 and 
F974) in the TREU 927/STIB 247 cross and two flies in the STIB 386/STIB 247 cross (F9 and 
F492)  Theseprogeny have been described previously and been shown to be hybrid for a series 
of RFLP and iso-enzyme markers [3,12], indicating that they are the products of genetic 
exchange between the parental stocks. The cryopreserved  metacyclic clones were expanded in 
immuno-suppressed mice and DNA prepared from purified trypanosomes as previously 
described [3]. A total of eleven progeny clones from the first cross and nine from the second 
were generated (listed in Table 1).  Each DNA preparation of the metacyclic derived progeny 
clones from the cross STIB 247 x TREU 927 was genotyped with three polymorphic 
minisatellite markers (TB1/1, TB3/13 and TB10/14) located on chromosomes 1, 3 and 10, 
respectively- [5]) and ten microsatellite markers (listed in Table 2) located on chromosomes 1, 
2, 3, 4 and 5 [6] using the PCR conditions described previously [5]. Similarly, the preparations 
from the metacyclic derived progeny of the cross STIB 247 x STIB 386 were genotyped with 
the same three minisatellite markers and nine microsatellite markers located on chromosomes 
1, 2, 3, 4, 5 and 10. The markers on different chromosomes will segregate independently [5] 
while those on the same chromosomes have been chosen on the basis of being separated by 
genetic distances (Table 2) to give a high probability of cross-over between them. The genetic 
distances correspond to physical distances of 185-525 kb in the sequence of TREU 927 but the 
physical distances have not been determined for STIB 386. The genotyping allowed the 
construction of a multilocus genotype (MLG 1-17) for each progeny clone and comparative 
analysis showed that the multilocus genotypes of the clones from the 247 x 927 cross were 
unique, except for clones F532/72 mcl 1 and 4 (MLG 2), while the same analysis with the 
 
 
 
 
 
 
Tait, A. and MacLeod, A. and Tweedie, A. and Masiga, D. and Turner, 
C.M.R. (2005) Genetic exchange in Trypanosoma brucei: evidence for 
mating prior to metacyclic stage development. Molecular and 
Biochemical Parasitology 151(1):pp. 133-136.
 
 
 
 
 
 
 
http://eprints.gla.ac.uk/4606/ 
 
10th September 2008 
 
 
Glasgow ePrints Service 
https://eprints.gla.ac.uk 
3progeny from the 247 x 386 cross identified two pairs of clones (F9/45 mcl 7 and F9/45 mcl 11 
– MLG 13; F9/45 mcl 9 and F9/45 mcl 10 – MLG 14) that were identical to each other for all 
micro- and minisatellite markers (Table 1), although the remaining clones were unique. 
To test the identity of these pairs of clones further, genome wide markers were used. Previous 
work, using the technique of amplified fragment length polymorphism (AFLP [13,14]), has 
been undertaken to construct linkage maps of TREU 927 and STIB 386 using some of the 
metacyclic derived progeny and provides genome wide analyses of polymorphic segregating 
markers [2]. DNA was prepared for AFLP analysis  from the parental and progeny clones after 
growth in immunosuppressed mice. (independent preparations from those used for the 
microsatellite analysis).  A section of one gel is shown in Fig 1 to illustrate the differences 
observed between the parental stocks and the inheritance of these differences in the resulting 
progeny. The parental clones (247 and 386) show a series of identical bands as well as 
differences in which a band is present in one parent but not the other and some of these bands 
segregate in the F1 progeny clones as a prescence/absence (Fig 1 arrows). This can be 
interpreted as the homozygous absence of a polymorphic site in one parent (therefore no 
amplification product) with the second parent being heterozygous for that polymorphism. 
These polymorphisms arise from the presence/absence of a restriction site, insertion/deletion or 
single base change in the sequence matching the 3’ extension of one primer. This 
polymorphism will then segregate into the progeny, which will inherit one ‘absent’ allele from 
one parent and either the amplified band or ‘absent’ allele from the second parent [2,13]. 
Applying this technique to the detection of differences between the three parental clones (247, 
386 and 927), using the restriction enzyme pairs EcoR1/Mse1 and HindIII/Taq1, and screening 
the F1 progeny clones to detect markers that segregate, 157 segregating polymorphic fragments 
were detected in the 247 x 386 cross and 185 in the 247 x 927 cross. Figure 1 illustrates the 
different patterns of segregation of the parental bands in progeny that differ in genotype (lanes 
3-7), as well as the identity of clones F9/45 mcl 9 and 10 (lanes 7 and 8).  These data allow a 
detailed genetic fingerprint of each progeny clone to be generated and a comparison between 
the clones in terms of their similarity. The AFLP data confirmed the findings with the using 
min- and microsatellite markers. In the cross 927 x 247, seven of the metacyclic derived clones 
were confirmed to be of distinct, independent genotypes and two clones were identical to each 
other (AFLP genotype A, Table1). These data indicate that most of the metacyclic stage clones 
are genotypically distinct and that the metacyclic stage is of hybrid genotype and therefore a 
product of mating (Table 1). AFLP analysis of six of the metacyclic derived clones from the 
247 x 386 cross, showed that five were of unique genotype and two were identical for all 
AFLP markers (AFLP genotype J, Table1). When these data are combined with the multilocus 
genotypes generated from the micro- and minisatellite data, there is clear evidence that one pair 
of clones from the 927 x 247 cross and two pairs from the 386 x 247 cross are identical in 
terms of genotype. While a proportion of these markers will be genetically linked, there is a 
sufficiently large number of independent markers to make the conclusion of identity a robust 
one. 
If mating occurred between the metacyclic stage trypanosomes  the probability that 
two F1 progeny, in the absence of division, would be identical in the sample sizes from 
each cross is extremely low as the products of meiosis will all be different. The evidence 
that metacyclics do not divide is based on extensive electron microscopy studies of infected 
4salivary glands, where there is ample evidence for dividing epimastigotes but no evidence 
for the division of either the mature or nascent metacylics that have aqcuired a VSG coat 
[15]. Additionally, immuno-electron microscopy with monoclonal antibodies specific a 
particular VSG, shows that the metacyclics expressing a particular VSG are randomly 
distributed throughout the gland and occur in isolation. If the metacyclic divided you would 
predict clusters of such cells expressing the same VSG-this is not observed [16]. On this 
basis, mating cannot be occurring between the mature metacyclic stage trypanosomes and 
the identical clones must have arisen by vegetative division of a single original product of 
mating. This result suggests that mating takes place either amongst epimastigote or pre-
metacyclic forms in the salivary glands or at a stage in the foregut/proboscis. However, in 
both crosses most metacyclic stage clones are unique, therefore suggesting there is very 
limited vegetative growth between mating and the development of the metacyclic otherwise 
many identical clones would be observed.  In order to explain the results from the two 
crosses, taken together, mating cannot take place between the metacyclic stages but is most 
likely to occur between the epimastigotes with limited subsequent mitotic division between 
mating and the development of the metacyclic stage. If mating occurred prior to the 
epimastigote stage between some of the morphologically distinct stages identified in the 
foregut and proboscis [7], the subsequent multiple rounds of division of the epimastigotes 
[7,8] would lead to many of the metacyclic stage trypanosomes being of identical genotype. 
This is clearly not the case. Our data strongly support mating and meiosis occurring 
between attached epimastigotes or dividing pre-metacyclic stages in the salivary glands. 
Two indirect arguments suggest that the epimastigote is the most likely stage at which 
mating takes place. Firstly, pre-metacyclics are relatively rare and this is thought to be a 
very transient phase in development [8, 15]. Secondly, in other flagellates, the process of 
gamete fusion that leads to syngamy is first mediated via flagella attachment [17] and it is 
only in the attached epimastigote phase of the life cycle that T. brucei has the required 
flagellum-substratum and flagellum-flagellum interactions [18]. 
Acknowledgements
This work was supported by grants from The Wellcome Trust and World Health 
Organisation (TDR) to AT and CMRT and by Fellowships from The Leverhulme Trust 
(CMRT) and The Royal Society of Edinburgh (AMacL). We would like to thank Andrew 
Parker of IAEA, Vienna for the supply of tsetse pupae.
Figure Legend
Figure 1 
Autoradiograph of labelled AFLP gel with DNA samples digested with HindIII and Taq1. 
Each primer had a two-nucleotide extension (H-CA and T-AA) for selective amplification 
and fragments were separated on a 6% acrylamide gel. Tracks: 1-STIB 247; 2-STIB 386; 3-
F9/45 mcl 2; 4- F9/45 mcl 11; 5- F492/50 mcl 12; 6- F492/50 mcl 13; 7- F9/45 mcl 9; 8-
F9/45 mcl 10. Arrows indicate fragments that are heterozygous for polymorphisms in one 
of the two parental stocks and segregate in the progeny.
5References
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Table 1.
The genotypes of the metacyclic stage F1 progeny clones from two crosses, determined by 
micro and minisatellite markers as well as AFLP genotyping. Each multilocus genotype 
(MLG) was based on the analysis of 12-13 micro- and minisatellite markers and each MLG 
that was different has been given a different number. The genotypes determined by AFLP 
used 157 segregating markers (247 x 386) and 185 markers (247 x 927). Each AFLP that 
was different has been given a different letter. ND-not determined.
Cross Clone MLG AFLP
247 x 927 F532/53 mcl 1 1 ND
F532/72 mcl 1 2 A
F532/72 mcl 2 3 B
F532/72 mcl 3 4 C
F532/72 mcl 4 2 A
F532/72 mcl 5 5 D
F532/72 mcl 6 6 E
F532/72 mcl 7 7 F
F532/72 mcl 9 8 G
F532/72 mcl 8 9 ND
F974/70 mcl 4 10 ND
247 x 386 F9/34 mcl 1 11 ND
F9/45 mcl 2 12 H
F9/45 mcl 7 13 ND
F9/45 mcl 11 13 I
F9/45 mcl 9 14 J
F9/45 mcl 10 14 J
F9/45 mcl 12 15 ND
F492/50 mcl 12 16 K
F492/50 mcl 13 17 L
7Table 2
Micro- and minisatellite markers used for genotyping. All markers are ‘TB’ markers except 
where shown. Markers TB 3/13, TB 5/4 and TB 10/14 on chromosomes 3, 5 and 10 were 
also used.
Cross Chromosome Marker Genetic distance (cM)
927 x 247 1 TB 1/1
    17.5
TB 1/7
    18.3
TB 1/13
2 TB 2/3
    42.7
TB 2/10
    35.6
TB 2/20
4 TB 4/1
    32.2
TB 4/4
    25.9
TB 4/10
    31.2
TB 4/16
386 x 247 1 TB 1/1
    23.1
TB 1/4
    35.8
TG 1/4
2 TB 2/3
    32.6
TB 2/10
    20.7
TB 2/20
4 TB 4/1
    44.7
TB 4/8
    31.8
TB 4/14
81    2    3    4    5    6    7    8
Figure 1


Genetic exchange in Trypanosoma brucei: Evidence for mating prior to metacyclic stage development

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Genetic exchange in <i>Trypanosoma brucei</i>: evidence for mating prior to metacyclic stage development

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