Abstract
BackgroundAntimalarial interventions designed to impact on the transmissible sexual stages of Plasmodium falciparum are evaluated by measurement of peripheral gametocyte carriage in vivo and infectivity to mosquitoes. Drug or vaccine-elicited effects may differentially affect the relative abundance of mature male and female sexual forms, and this can be measured by estimation of sex ratios before and after intervention in vivo and in vitro. Measuring the impact of anti-gametocyte drugs on sexual commitment of immature gametocyte stages in vitro is not currently possible as male and female parasites cannot be distinguished by morphology alone prior to stage IV.Methodology/Principal FindingsWe have modified an existing immunofluorescence-based approach for distinguishing male and female gametocytes during development in vitro, by using highly synchronised magnetically-enriched gametocyte preparations at different stages of maturity. Antibodies recognising α-tubulin II (males) and Pfg377 (females) were used to attempt to discriminate the sexes. Transcription of these two proteins was not coordinated during in vitro development, with pfg377 transcripts accumulating only late in development, immediately prior to immunofluorescent signals from the PfG377 protein appearing in stage IV gametocytes. Contrary to previous descriptions of this protein as male-specific in P. falciparum, α-tubulin II recognised both male and female gametocytes at stages I to IV, but evidence of differential expression levels of this protein in late stage male and female gametocytes was found. Using antibodies recognising PfG377 as the primary marker and α-tubulin II as a secondary marker, robust estimates of sex ratio in in vitro cultures were obtained for gametocytes at stage IV or later, and validated by light microscopic counts. However, sex ratio estimation was not possible for early stage gametocytes due to the promiscuity of α-tubulin II protein expression, and the relatively late accumulation of PfG377 during the development process.Conclusions/SignificanceThis approach is a feasible method for the evaluation of drug impacts on late-stage gametocyte sex ratio in in vitro studies. Additional sex-specific antigens need to be evaluated for sex ratio estimation in early stage gametocyte preparations.
Highlights
The propagation of malaria is a public health threat throughout the tropics
RT-PCR of mRNA from stage-specific preparations of synchronised developing gametocytes demonstrated that a-tubII transcripts were abundant throughout development (Fig. 1, 2nd panel), whereas pfg377 transcripts were barely detectable in stage I/IIa gametocytes, becoming very abundant in later stage preparations (Fig. 1, 3rd panel)
This lack of coordination between the two genes of interest suggests that quantitative measurements of a-tubII and pfg377 transcript accumulation during development would not provide stable estimates of sex ratio; other transcripts may be more suited to this purpose
Summary
The propagation of malaria is a public health threat throughout the tropics. Recent calls for intensification of the effort towards malaria elimination have emphasised the need for drugs and vaccines that target gametocytes, those of P. falciparum, in order to bring malaria transmission under control [1]. Recent renewed interest in the evaluation of the anti-gametocyte effects of therapeutic agents, both in vitro and in vivo, is very welcome [2,3]. These evaluations require establishment of novel methods for studying gametocytes in vitro. Drug or vaccine-elicited effects may differentially affect the relative abundance of mature male and female sexual forms, and this can be measured by estimation of sex ratios before and after intervention in vivo and in vitro.
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