Cryogenically preserved RBCs support gametocytogenesis of Plasmodium falciparum in vitro and gametogenesis in mosquitoes

Abstract

BackgroundThe malaria Eradication Research Agenda (malERA) has identified human-to-mosquito transmission of Plasmodium falciparum as a major target for eradication. The cornerstone for identifying and evaluating transmission in the laboratory is standard membrane feeding assays (SMFAs) where mature gametocytes of P. falciparum generated in vitro are offered to mosquitoes as part of a blood-meal. However, propagation of “infectious” gametocytes requires 10–12 days with considerable physico-chemical demands imposed on host RBCs and thus, “fresh” RBCs that are ≤ 1-week old post-collection are generally recommended. However, in addition to the costs, physico-chemical characteristics unique to RBC donors may confound reproducibility and interpretation of SMFAs. Cryogenic storage of RBCs (“cryo-preserved RBCs”) is accepted by European and US FDAs as an alternative to refrigeration (4 °C) for preserving RBC “quality” and while cryo-preserved RBCs have been used for in vitro cultures of other Plasmodia and the asexual stages of P. falciparum, none of the studies required RBCs to support parasite development for > 4 days.ResultsUsing the standard laboratory strain, P. falciparum NF54, 11 SMFAs were performed with RBCs from four separate donors to demonstrate that RBCs cryo-preserved in the gaseous phase of liquid nitrogen (− 196 °C) supported gametocytogenesis in vitro and subsequent gametogenesis in Anopheles stephensi mosquitoes. Overall levels of sporogony in the mosquito, as measured by oocyst and sporozoite prevalence, as well as oocyst burden, from each of the four donors thawed after varying intervals of cryopreservation (1, 4, 8, and 12 weeks) were comparable to using ≤ 1-week old refrigerated RBCs. Lastly, the potential for cryo-preserved RBCs to serve as a suitable alternative substrate is demonstrated for a Cambodian isolate of P. falciparum across two independent SMFAs.ConclusionsBasic guidelines are presented for integrating cryo-preserved RBCs into an existing laboratory/insectary framework for P. falciparum SMFAs with significant potential for reducing running costs while achieving greater reliability. Lastly, scenarios are discussed where cryo-preserved RBCs may be especially useful in enhancing the understanding and/or providing novel insights into the patterns and processes underlying human-to-mosquito transmission.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2612-y) contains supplementary material, which is available to authorized users.

Field relevant variation in ambient temperature modifies the density-dependent establishment of Plasmodium falciparum in mosquitoes: implications for the infectious reservoir and beyond?

“…For estimating gametocyte density, smears were prepared from the blood-228 meal corresponding to the 1:2 dilution for Giemsa staining. Mosquitoes were returned to the respective 229 temperature regimes and starved for a further 48 hours to eliminate any partial or non-blood fed 230 individuals after which they were provided cotton pads soaked in 5% dextrose (w/v) and 0.05% para-231 aminobenzoic acid (w/v) for the remainder of the study, as described previously (Pathak et al, 2018). 232…”

“…Tests for overdispersion were performed using a predetermined threshold ratio of squared Pearson 269 residuals over the residual degrees of freedom (overdispersion ratio= <1.5) and a Chi-squared distribution 270 of the squared Pearson residuals with p-value >0.05, as described previously (Pathak et al, 2018). Once 271 overdispersion was accounted for, the marginal means estimated by each model were then used to 272 perform pairwise comparisons between parasite densities nested within each temperature regime, using 273…”

“…Specifically, midguts and salivary glands, respectively, were dissected to assess vector competence on the 237 following days: 20 DTR 9⁰C, 15-17 dpi and 29 dpi; 24 DTR 9⁰C, 9-11 dpi and 17 dpi; and 28 DTR 9⁰C, 238 7-9 dpi and 13 dpi. At each time point, ~25-30 mosquitoes were vacuum aspirated directly into 70% 239 ethanol and vector competence measured as described previously (Pathak et al, 2018). Briefly, midguts 240…”

Field Relevant Variation in Ambient Temperature Modifies Density-Dependent Establishment of Plasmodium falciparum Gametocytes in Mosquitoes

“…stephensi from the same cohort were sorted into nine groups/cups with each temperature regime receiving three cups each, 24 h prior to the day of infection. On the day of infection, mature gametocytes of P. falciparum NF54 generated in vitro (Pathak et al, 2018) were serially diluted 3-fold with naïve RBCs to obtain blood-meals representing three final parasite densities with a 9-fold difference between the highest and lowest densities (~an order of magnitude). Aliquots of the three blood-meals with the varying gametocyte density were offered to the three cups, respectively, at each temperature regime.…”

“…Routine asexual cultures of P. falciparum NF54 and induction of gametocytogenesis in vitro were performed with cryo-preserved red blood cells (RBCs) as described in detail previously (Pathak et al, 2018). Gametocytogenesis of P. falciparum NF54 in vitro was monitored with Giemsa staining until 12 days post-culture, after which we assessed cultures for infectiousness through daily assays of male gametogenesis (Pathak et al, 2018). Giemsa stained slides were visualized at a 1000x magnification with an oil immersion lens while gametogenesis was assessed in vitro at 400x magnification under differential interference contrast setting on a Leica DM2500 upright microscope.…”