The female-specific W1 sequence of the Puerto Rican strain of Schistosoma mansoni occurs in both genders of a Liberian strain

Christoph G. GREVELDING

Institut für Genetik and Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität Düsseldorf, Universitätsstr.1, D-40225 Düsseldorf, Germany

Key words: Schistosoma mansoni, W chromosome, Female-specific, Genetic variability

Abbreviations: SSC, standard saline citrate; PCR, polymerase chain reaction; PDI, protein disulfide isomerase; Lib., Liberian; P.R., Puerto Rican

Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDBJ data bases under the accession numbers Z47548/ Z47549.

Among the mammalian blood flukes, the Schistosomatidae, unlike other trematodes, are dioecious. Here the female is the heterogametic sex (ZW) opposite to the male (ZZ) [1]. Adult male and female schistosomes reveal a clear sexual dimorphism in contrast to the larval stages (miracidia, sporocysts and cercariae), where sexes cannot be distinguished. To determine the sex of individuals of these larval stages, molecular markers are desirable because the applicability of cytological methods is limited [2]. Molecular discrimination of sex was achieved through the identification of a female specific DNA sequence (W1), isolated from a Puerto Rican strain of Schistosoma by subtractive hybridization [3]. This sequence is localized in the heterochromatic region of the W chromosome [4] and consists of more than 500 copies of 476-bp tandem repeats. It was shown at the Southern blot level that W1 hybridizes only to female DNA, not to male DNA [3], and that it is possible by dot blot analysis to determine the sex of cercariae, shedded by snails infected with a single miracidium [3,5]. W1 was also utilized as probe for in situ hybridization in sections of infected mouse liver to evaluate the number of female eggs in propagation studies [6]. Sexing of single larvae of S. mansoni was simplified and accelerated using the polymerase chain reaction method (PCR) with oligonucleotide primers, synthesized according to the W1 sequence [7].

In this study the PCR technique was applied to analyse sex specific differences in the Liberian strain of S. mansoni used in our laboratory. It was demonstrated that in this Liberian strain the highly repeated W1 sequence occurs in males and females. The Liberian strain of S. mansoni has been obtained from the Bayer company laboratories, where it was maintained for the last 50 years [8]. Miracidia were passaged through Biomphalaria glabrata and adult worms were recovered by perfusion [9]. DNA of worms was extracted as described elsewhere [7] except that 250 µg ml-1 proteinase K (Merck) was used instead of pronase. PCR reactions were performed with equal amounts of DNA, extracted from single or combined male and female worms, using the W1 specific primers W1a and W1b according to Gasser et al. [7] that amplify a W1 repeat reduced about 9 bp in size. As shown in Fig.1A, there is a clear banding pattern with male and female DNA of worms of our Liberian strain (lane 2,3). In contrast to this result, only the female DNA from worms of a Puerto Rican strain (kindly provided by Dr. A. Ruppel, Tropenmedizinisches Institut Heidelberg) revealed W1 specific PCR products (Fig.1A, lanes 5,7) as expected [3,6]. Due to the repetitive nature of the target DNA, bands emerged which represent 2-4 times the repeat unit. To confirm this result, a Southern blot experiment was done with equal amounts of male and female DNA that were transferred to nylon membranes (Hybond-N, Amersham) and hybridized with a W1 probe using standard protocols [10]. Beyond the appearence of a characteristic banding pattern in both sexes there is a distinct difference in the strength of the signals which is reproducible (Fig.1B, lanes 1,2). To prove that equal amounts of male and female worm DNA were present, the same blot was stripped and re-probed with part of the protein disulfide isomerase gene (PDI, Fig.1C) which has a gender independent housekeeping function [9]. The Southern blot result (Fig.1B) confirmed the previously observed difference in the amount of male and female PCR products (Fig.1A, lanes 2,3) and points to a higher W1 copy number in females. To quantify this copy number difference, a densitometric analysis was done with a Soft Laser Densitometer Scanner (Zeineh, SL-504-XL) [11] measuring the relative intensity of the hybridization signals of the monomeric repeat units (Fig. 1B, 500 bp bands). The mean value of the analysis of differently exposed autoradiographs in relation to the PDI hybridization results revealed a 2.3-fold difference in the W1 copy number between males and females of the Liberian strain (results not shown).

The relative amount of female W1 repeats in the Liberian strain compared to female W1 repeats in the Puerto Rican strain was determined by slot blot analysis [3]. This revealed no quantitative difference between both strains (result not shown). Provided that 500-1000 W1 repeats are present in the female genome [3], it is estimated that approximately 220-440 copies exist in the male genome of the Liberian strain.

Cloning and sequencing of several male and female PCR amplified W1 repeats of the Liberian strain finally confirmed that the results obtained were not due to any false positive PCR product. The comparison of these clones revealed that the homology between the female sequences of the Liberian strain (Lib.) and the published Puerto Rican (P.R.) sequence is 96-98%. The homology of the male (Lib.) W1 sequences to the female (Lib.) W1 sequences varies from 91-96%. The male sequences differ from each other by about 10% and fall into two groups. One group having a 22 bp deletion at position 329-350 and the other group without this deletion. The other differences are due to point mutations consisting of transitions and transversions. In Fig.2 the female (Lib.F) and the male (Lib.M) sequence were selected that had highest homology to the female W1 sequence of the Puerto Rican strain (P.R.F) [3].

The occurrence of W1 sequence copies in male worms of the Liberian strain of S. mansoni described in this study, represents the first gender specific strain difference in this species. In the Puerto Rican strain of S. mansoni W1 tandem repeats only exist in females but not in males, whereas both sexes of the Liberian strain possess W1 sequences, although in different copy numbers. This phenomenon could have originated from a non-homologous translocation event of parts of the heterochromatic region of the female-specific W chromosome, where W1 is located [4], to a region of the Z chromosome or an autosome.

The gender independent occurrence of W1 repeats in the Liberian strain enlarges the molecular repertoire for the discrimination of strains and will be useful for population geneticists. In genetic crosses, male W1 could serve as an inheritable marker for the characterization of hybrids and interspecific hybrids, because W1 does not cross-hybridize to DNA of other species within the same genus as S. japonicum, S. haematobium, S. mattheei and S. douthitti [3].

Acknowledgements:

The author would like to thank Dr. A. Ruppel (Tropen-Medizinisches Institut Heidelberg, Germany) for providing worms of the Puerto Rican strain. This investigation was financially supported by the Deutsche Forschungsgemeinschaft (Grant Ku 282/13-2) and by UNDP/World Bank/ WHO Special Program for Research and Training in Tropical Diseases. C.G.G. gratefully acknowledges the critical comments of Prof. Dr. W. Kunz on this manuscript and is grateful to the support of Dr. M. Finken-Eigen and Dr. K. Köhrer from the BMFZ Düsseldorf.

References:

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[6] Penschow, J.D., Gasser, R.B., Darling, P.E. and G.F. Mitchell (1993) Female-specific genomic PCR sequences from Schistosoma mansoni as probes for hybridisation histochemistry. Int. J. for Parasitol. 23(3), 407-409

[7] Gasser, R.B., Morahan, G. and Mitchel, G.F. (1991) Sexing single larval stages of Schistosoma mansoni by polymerase chain reaction. Mol. Biochem. Parasitol. 47, 255-258

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[10] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. 2nd. edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

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Figure legends:

Fig. 1

A : Polymerase chain reaction assay of total genomic DNA isolated from adult male and female worms of the Liberian (Lib.) and Puerto Rican (P.R.) strain of S. mansoni, performed with the W1a/W1b primer pair (W1a: 5`-CAACACAGTGAAATTCTTCC-3`; W1b: 5`-GAATTCACCACTCGACA-TTC-3` [7]), showing W1 sequence specific amplification products. PCR reactions were performed in a total volume of 25 µl using 0.1 ng worm DNA, 1 µM of each oligonucleotide, 200 µM of each deoxynucleotide (dATP, dTTP, dCTP, dGTP; Pharmacia) and 2.5 units Taq polymerase (Appligene). After an initial denaturation step at 95¡ C for 5 min, a temperature cycling was performed at 93¡C-45 sec, 65¡C-20 sec, 72¡C-20 sec for 30 cycles (MJ esearch MiniCyclerTM). One fourth of the reaction volume was electrophoresed in a 1.2 % TAE agarose gel and stained with ethidium bromide. Lane 1: lambda-PstI size marker, lane 2: DNA isolated from 10 male worms (Lib.), lane 3: DNA isolated from 10 female worms (Lib.), lane 4: DNA isolated from 10 male worms (P.R.), lane 5: DNA isolated from 10 female worms (P.R.), lane 6: DNA isolated from 1 male worm (P.R.), lane 7: DNA isolated from 1 female worm (P.R.) and lane 8: a control PCR with no worm DNA. B: A Southern blot of EcoRI digested male (lane 1) and female (lane 2) DNA (2 µg each) of worms of the Liberian strain hybridized with the purified 473 bp male (Lib.) specific PCR amplification product labeled with 32P-dATP. Hybridization was done overnight in the presence of formamide at 42¡C and filters were washed according to Sambrook et al. [10] followed by a final wash with 0.1 x SSC/ 0.1 % SDS at 68¡ C for 20 min. C: A Southern blot identical to B, but re-probed with a part of the protein disulfide isomerase gene (PDI, [9]) present in both males and females. Sizes shown are in kb.

Fig. 2

Sequence alignment showing the selected female (Lib.F) and male (Lib.M) W1 repeat sequence of the Liberian strain with the highest homology to the female sequence of the Puerto Rican strain (P.R.F [3]) of S. mansoni. The Liberian strain nucleic acid sequences are 98% (Lib.F) and 97% (Lib.M) identical to the P.R.F sequence according to the PC/GENE CLUSTAL program (IntelliGenetics, Inc.). Identical nucleotides are indicated by *. Sequencing wasÊperformed on an automatical sequencing device (A.L.F., Pharmacia) at the Biologisch-Medizinisches Forschungszentrum Düsseldorf (BMFZ). The cloning of PCR products was done by blunt end ligation in pUC18 plasmids according to the manufacturer«s protocol (SureCloneTM Ligation Kit, Pharmacia).

P.R.F CAACACAGTG AAATTCTTCC TTCACACATA TCTACCATCC AATGTCTTCG 50

Lib.F CAACACAGTG AAATTCTTCC TTCACACATA TCTACCATCC AATGTCTTCG 50

Lib.M CAACACAGTG AAATTCTTCC TTCACACATA TCTCTCATCG ATTGTCTTCG 50

P.R.F CAATATTTTG GAGTGAAATT TGCTTTTCTC ATTATATTGT GCATGATGAC 100

Lib.F CAATATTTTG GAGTGAAATT TGCTTTTCTC ATTATATTGT GCATGATGAC 100

Lib.M GAATATTTTG GAGTGAAATT TGCTTTTCTC ATTATATTGT GCATGATGAC 100

P.R.F TGATGTGACA GGAATGAGGA TTATGTTGAT ATCGTCTGAG TCAATGAGAA 150

Lib.F TGATGTGACA GGAATGAGGA TTATGTTGAT ATCGTCTGAG TCAATGAGAA 150

Lib.M TGATGTGACA GGAATGAGGA TTATGTGGAT ATCGTCTGAG TCAATGAGAA 150

P.R.F TTGTGAATCG GATGTGCAGA TGAGAGGTTG TGCATACTTG TTCCTTGTGA 200

Lib.F TTGTGAATCG GATGTGCAGA TGAGAGGTTG TGCATACTTG TTCCTTGTGA 200

Lib.M TTGTGAATCG GATGTGCAGA TGAGAGGTTG TGCATACTTG TTCCTTGTGA 200

P.R.F CACAAAGGAG TGGTGATGCC AGTTCGAGTG TTTGTGGATG CGATGGTGTT 250

Lib.F CACAAAGGAG TGGTGATGCC AGTTCGAGTG TTTGTGGATG CGATGGTGTT 250

Lib.M CACAAAGGAG TGGTGATGCC AGTTCGAGTG TTTGTGGATG CGATGGTGTT 250

P.R.F CACACGTGGA TTGAATAAGC GATGAACAAA TGCGATGATG CATTAGGGTG 300

Lib.F CACACGTGGA TTGAATAAGG GATGAACAAA TGCGATGATG CAAATGAGTG 300

Lib.M CACACGTGGA TTGAATAAGG GATGAACAAA TGCGATGATG CAAATGAGTG 300

P.R.F TGTGGTTGTG CTGGACCAAT GTGCATAATG GAATCGTTGC TTGTGCACAT 350

Lib.F TGTGGTTGTG CTGGACCAAT GTGCATAATG GAATCGTTGC TTGTGCACAT 350

Lib.M TATGGTTGTG CTGGACCAAT GTGCATAATG GAATCGTTGC TTGTGCACAT 350

P.R.F GGACCACCAC AAATAACACA CTCAATTCAT ACTCCGTCCA TTTAACCATG 400

Lib.F GGACCACCAC AAATAACACA CTCAATTCAT ACTCCGTCCA TTTAAACATG 400

Lib.M GGACCACCAC AAATAACACA CTCAATTCAT ACCCCGTCCA TTTAACCATG 400

P.R.F CATTGCTTTC TCATCAACAC CACAGTTTGC ATTATCATTT CGAACATTGA 450

Lib.F CATTGCGTTT TCATCACCAT CACAGTTCGC ATTATCATTT CGAACATTGA 450

Lib.M CATTGCGTTC TCATCAACAC CACAGTTTGC ATTATCATTT CGAACATTGA 450

P.R.F GTTGAATGTC GAGTGGTGAA TTC 473

Lib.F GTTGAATGTC GAGTGGTGAA TTC 473

Lib.M GTTGAATGTC GAGTGGTGAA TTC 473