Cryogenically preserved RBCs support gametocytogenesis ofPlasmodium falciparuminvitroand gametogenesis in mosquitoes

Abstract

21Background 22 The malaria Eradication Research Agenda (malERA) has identified human-to-mosquito 23 transmission of Plasmodium falciparum as a major target for eradication. The cornerstone for 24 identifying and evaluating transmission in the laboratory is small membrane feeding assays 25(SMFAs) where mature gametocytes of P. falciparum generated in vitro are offered to 26 mosquitoes as part of a blood-meal. However, propagation of "infectious" gametocytes requires 27 10-12 days with considerable physico-chemical demands imposed on host RBCs and 28 thus, "fresh" RBCs that are ≤1-week old post-collection are generally recommended. However, 29 in addition to the costs, physico-chemical characteristics unique to RBC donors may confound 30 reproducibility and interpretation of SMFAs. Cryogenic storage of RBCs (cryo-preserved RBCs 31 herein) is approved by the European and US FDAs as an alternative to refrigeration (4°C) for 32 preserving RBC quality and while cryo-preserved RBCs have been used for in vitro cultures of 33 other Plasmodia and the asexual stages of P. falciparum, none of the studies required RBCs to 34 support parasite development for >4 days. 35 Results 36Using the standard laboratory strain, P. falciparum NF54, we first demonstrate that cryo-37 preserved RBCs preserved in the gaseous phase of liquid nitrogen and thawed after storage for 38 1, 4, 8 and 12 weeks, supported gametocytogenesis in vitro and subsequent gametogenesis 39 in Anopheles stephensi mosquitoes. Using data from 11 SMFAs and RBCs from 4 separate 40 donors with 3 donors re-tested following various periods of cryo-preservation, we show that 41 overall levels of sporogony in the mosquito, as measured by oocyst prevalence and burdens in 42 the midguts and sporozoites in salivary glands, were similar or better than using ≤1-week old 43 3 refrigerated RBCs. Additionally, the potential for cryo-preserved RBCs to serve as a universal 44 substrate for SMFAs is shown for a Cambodian isolate of P. falciparum. 45 Conclusions 46Considering the suitability of cryo-preserved RBCs for P. falciparum SMFAs, we suggest 47 guidelines for their use and how they can be integrated into an existing laboratory/insectary 48 framework with the potential to significantly reduce running costs and provide greater reliability. 49Finally, we discuss scenarios where cryo-preserved RBCs may be especially useful in 50 enhancing our understanding and/or providing novel insights into the patterns and process 51 underlying human-to-mosquito transmission. 52Background 56Incidence of malaria due to Plasmodium falciparum has seen a steady decline in the last 15-20 57 years, enabled primarily by the synergy between drug-combination therapies for case/disease 58 management and vector control programs [1, 2]. Since transmission to the mosquito is thought 59 to be a severe bottleneck in the parasite's life history, considerable effort has been channeled 60 towards identifying parasite and vector traits influencing transmission to the mosquito with the 61 aim of designing and te...