Much of the focus on selection in population genetics is on events that occur over an evolutionary time frame, but when selection pressure is exerted by use of antimicrobial agents, the effect of a selection event can be observed after a very short period of time. In prokaryotes, antimicrobial selection often results in overgrowth of a clone (Soares et al., 1993
; Davis et al., 1999
; Manges et al., 2001
; Martin et al., 2002
) and is thus easy to identify using common molecular tools. Sexual reproduction never produces clones, but rather families in which the genotypes among the members are more closely correlated than unrelated or more distantly related members of a population. Since useful mutations often develop on one or a few genotypes, in the short-term, the average degree of relationship among resistant organisms is likely to be higher than the average degree of relationship among susceptible organisms. In the absence of isolation and over many generations, polymorphisms associated with resistance may be distributed to other genotypes, and familial relationships may be less apparent. At the moment of a selective sweep and prior to reproduction, however, the distribution of surviving genotypes is likely not to be random. They are likely to represent an extended family of individuals more closely related to each other at many loci than the susceptible population.
Measurement of accuracy and error are complex for the population genetics of schistosomes since there are two scales to be considered. At one scale there is the error of individual genotyping compared with genotyping pools of individuals. This error ranges from 2–11% based on our own studies (Silva et al., 2006
; Blank et al., 2009
; Hanelt et al., 2009
) and those of others (Redman et al., 2008
). However, when we compared the FST
based on genotyping individuals or pools, we found that the differences had little effect on the FST
and is unlikely to significantly affect other measures of differentiation. The second source of error exemplified in results from the compartmentalization of genotypes in infrapopulations. The almost complete sampling of all infrapopulations here avoids error due to undersampling, which we show could potentially be substantial given the degree of differentiation between infrapopulations.
Given a model where resistance arises in a limited number of organisms, we would predict that if schistosomes persist following treatment with PZQ due to a shared selective advantage, the persistent infrapopulations would be more related to each other than to the susceptible populations. What we observed was that the schistosome populations that persist are essentially randomly drawn from the overall population and not a selected population. This is consistent with the recent introduction of PZQ for widespread treatment of S. mansoni
infections in Brazil and its limited use to date in these communities. The lack of differentiation from the overall population and lack of similarity within the persistent populations supports the common presumption that persistent populations represent recent transmission (Cioli, 2000
; Gryseels et al., 2001
; Cioli and Pica-Mattoccia, 2003
). Since for the first 4 weeks following infection S. mansoni
is relatively insensitive to PZQ, any recently acquired infections will not be affected by the administration of these drugs. These will appear as persistent infections, although the parasites will be drawn from the overall susceptible population. It is also possible worms may be in a location protected from the drug or that host factors governing drug metabolism randomly affect worm survival.
There are other models for the development of resistance under which these assumptions would not hold. Resistance to an antimicrobial agent may develop on multiple genotypes or by means of mobile elements in which case the genomes of resistant organisms will not show an overall correlation. Another caveat for any interpretation is that the marker panel used is not large and does not provide dense coverage of the genome. We are, however, able to show infrapopulation variation within these small communities, genetic drift within a laboratory strain (Blank et al., 2010
), and demonstrate variation between subsamples of an S. mansoni
laboratory population using a marker panel of similar size (Blank et al., 2011
). Finally, there is a precedent for selection producing detectable differentiation across neutral unlinked markers (Merilä and Crnokrak; Freeland et al., 2010
; Johansson et al., 2010
). Since in this study we see little pre- and post-treatment differentiation for the component populations of the communities studied, this is suggestive evidence of a lack of selection.
One other study has examined S. mansoni
population structure in the context of a treatment program. Norton et al. (2010)
conducted a study in Tanzania analyzing individual miracidia from 80 children prior to PZQ treatment and from 47 children at follow-up 1 year post-treatment. The individuals included in the pre- and post-treatment groups did not completely overlap. The study found a marked reduction in parasite allelic richness, which was attributed primarily to a population bottleneck resulting from mass community-wide administration of PZQ. Pre- and post-treatment populations a year later were not similar, however this study primarily examined re-infection rather than persistent infections. The potential for drug resistance was considered as an explanation for some observations, but thought unlikely given the modest prior exposure of the population to PZQ in these communities. It is difficult to make comparisons with the present study since the location and sampling strategies were so different and Norton et al, (2010)
did not analyze infrapopulation differentiation. Two-year follow-up studies in Jenipapo and Volta do Rio are planned to determine the longer-term impact on the parasite populations.
In addition to investigating selection, the approach taken in this study has allowed us to sample whole communities widely and begin to understand important aspects of baseline genetic diversity within and among subpopulations. We find that at least over the period of 1 week, the excretion of parasite genotypes is consistent day-to-day. In theory, a single stool would suffice to represent the individual host’s infrapopulation. We also found that in these communities, there is a moderate degree of differentiation of the infrapopulations among hosts. Parasites are probably acquired a few at a time by each host at different locations and under different circumstances. When the component population is very heterogeneous, any individual host will carry a very incomplete sample of the component population. For this reason the component population differentiation between villages is lower than the infrapopulation differentiation within each of the communities. This is consistent with each infrapopulation representing a limited draw from the overall pool. Sampling of infrapopulations needs to be extensive to capture the variability in the component population and under sampling is likely to overestimate differentiation at this level. We will continue to monitor the response to therapy and the population structure in these communities following repeated rounds of PZQ treatment using the techniques we have developed.
> Schistosoma mansoni egg genotypes stable for at least 1 week.
> Moderate infrapopulation differentiation within villages.
> Moderate geographic differentiation between villages.
> Little differentiation between susceptible and persistent populations after praziquantel.
> Evidence for lack of resistance in populations that persist after praziquantel.