Development and application of an ethically and epidemiologically advantageous assay for the multi-locus microsatellite analysis of Schistosoma mansoni

Abstract

Non-availability of adult worms from living hosts remains a key problem in population genetic studies of schistosomes. Indirect sampling involving passage through laboratory animals presents significant ethical and practical drawbacks, and may result in sampling biases such as bottlenecking processes and/or host-induced selection pressures. The novel techniques reported here for sampling, storage and multi-locus microsatellite analysis of larval Schistosoma mansoni, allowing genotyping of up to 7 microsatellite loci from a single larva, circumvent these problems. The utility of these assays and the potential problems of laboratory passage, were evaluated using 7 S. mansoni population isolates collected from school-children in the Hoima district of Uganda, by comparing the associated field-collected miracidia with adult worms and miracidia obtained from a single generation in laboratory mice. Analyses of laboratory-passaged material erroneously indicated the presence of geographical structuring in the population, emphasizing the dangers of indirect sampling for population genetic studies. Bottlenecking and/or other sampling effects were demonstrated by reduced variability of adult worms compared to their parent field-collected larval samples. Patterns of heterozygote deficiency were apparent in the field-collected samples, which were not evident in laboratory-derived samples, potentially indicative of heterozygote advantage in establishment within laboratory hosts. Genetic distance between life-cycle stages in the majority of isolates revealed that adult worms and laboratory-passaged miracidia clustered together whilst segregating from field miracidia, thereby further highlighting the utility of this assay.

Fig. 1

Map of Uganda showing Hoima district and the sample sites for this study (1- Runga community, 2-Kibiro primary school, 3-Tonya primary school, 4-Kasenyi primary school).

Fig. 2

Comparison of mean (±standard error of the mean) (A) total number of alleles and (B) private alleles (exclusive to a particular sample type within isolates) for field miracidia (miracidia collected directly in the field), and adult worms and miracidia (laboratory miracidia) following a single generation of laboratory passage.

Fig. 3

Comparison of mean (±standard error of the mean) observed (H_o) and expected heterozygosity (H_e) (±standard error of mean) across 7 microsatellite loci for (A) field miracidia, (B) adult worms and (C) laboratory-derived miracidia.

Fig. 4

UPGMA depicting Cavalli-Sforza Edwards chord measured separately for 7 Schistosoma mansoni isolates between field-collected miracidia (FM), and adult worms (AW) and miracidia (LM) obtained following a single generation of laboratory passage.

Fig. 5

Comparison of UPGMA phenograms depicting Fst distance between 7 Schistosoma mansoni isolates from 4 geographical locations using (A) field-collected miracidia (FM), (B) adult worms (AW) and (C) laboratory-passaged miracidia (LM). Details of isolates are shown in Table 2 and P values are reported in the text.