121 Polymorphic microsatellite markers identified in individual Plasmodium falciparum oocysts from wild-caught Anopheles mosquitoes T.G.ANTHONY ,, H. E. TRUEMAN , R. E. HARBACH  and A.P.VOGLER ,*  Department of Biology, Imperial College at Silwood Park, Ascot, Berks SL5 7PY, UK  Department of Entomology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK (Received 26 November 1999 ; revised 4 February 2000 and 18 February 2000 ; accepted 18 February 2000)  The oocysts of Plasmodium falciparum that form on the midgut wall of anopheline vectors represent the meiotic products of the sexual cycle of the parasite and are useful for genetic analyses. Recognizing the limitations of current markers for the analysis of genetic variation, we describe a protocol for the DNA isolation and PCR amplification of microsatellite loci from individual oocysts. We present a simple method for the storage and isolation of individual Plasmodium oocysts from wild-caught mosquitoes and demonstrate that it is possible to extract sufficient oocyst DNA for approximately 50 amplifications. Genotyping was successful for 10 microsatellite loci and, using Genescan technology, we found extensive microsatellite polymorphism in an analysis of a dozen oocysts from each of 2 Anopheles gambiae female specimens collected within 6 months at the same location in northern Tanzania. All of the loci exhibited at least 2 alleles, with a maximum of 5. Homozygous and heterozygous oocysts could easily be discerned, but most of the oocysts were homozygous. The procedure has great potential for shedding light on genetic mechanisms operating during meiosis in mosquito vectors and the influence this may have on the genetic structure of natural populations of P. falciparum. Key words : microsatellite variation, Plasmodium falciparum sexual stages, Tanzania, PCR amplification, heterozygote, parasite–vector interactions.  Genetic variation in P. falciparum isolated from oocysts in the mosquito vector (Ranford-Cartwright et al. 1991 ; Babiker et al. 1994; Paul et al. 1995; Babiker & Walliker, 1997) or from human blood samples has mostly been analysed using immuno- genic surface proteins such as the merozoite surface proteins 1 and 2 (MSP-1, MSP-2) (Babiker et al. 1994 ; Paul et al. 1995; Conway et al. 1999), the circumsporozoite surface protein (CSP) (Rich, Hudson & Ayala, 1997) and the glutamate-rich protein (GLURP) (Paul et al. 1995). These markers have been useful for studies of variation in popu- lations on the local (e.g. Babiker, Ranford- Cartwright & Walliker, 1999) and regional (e.g. Paul et al. 1995) scale, and to address the question of genomic recombination (Conway et al. 1999 ; Rich et al. 1997). The analysis of population polymorphisms in these markers revealed a generally high level of allelic diversity which was unexpected given that other genes, such as those involved in cell metab- olism and other cell functions, revealed virtually no variation in comparisons of P. falciparum genotypes * Corresponding author : Department of Entomology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK. Tel: 44 207 942 5613. Fax: 44 207 942 5229. E-mail : a.voglernhm.ac.uk around the world (Rich et al. 1998). This suggests that the surface proteins used in population genetic analyses either have a higher rate of evolution than other protein-coding regions surveyed or that an- cestral alleles are maintained in the populations, possibly due to selection for immunoallelic poly- morphisms. Either of these processes would affect the apparent rate of recombination (Conway et al. 1999) or estimation of levels of gene flow (Paul et al. 1995 ; Gilbert et al. 1998). For population genetic studies, it is therefore desirable to use markers presumably less affected by selection. A library of well-defined microsatellite markers (Su & Wellems, 1996; Su et al. 1999) provides a large set of novel genetic markers which potentially fulfill this re- quirement. A selection of Su & Wellems’ (1996) markers have recently been applied to the PCR- based analysis of blood samples and revealed high levels of variation in P. falciparum at all 12 loci analysed (Anderson et al. 1999). This set of markers can now be used for genotyping P. falciparum, and addressing a range of biological questions. Here we describe a procedure for PCR ampli- fication of these microsatellites from individual oocysts isolated from wild-caught Anopheles gambiae. Oocysts allow direct examination of the genetic recombination that occurs in the sexual cycle and they are a convenient stage from which to infer the parental genotypes and the shuffling of genes among Parasitology (2000), 121, 121–126. Printed in the United Kingdom  2000 Cambridge University Press