Conference Agenda

Plasmodium parasites, the causative agents of malaria, must cycle between mosquito vectors and vertebrate hosts, encountering population bottlenecks at each transition. Parasite numbers can drop from billions in the bloodstream to only a few in the mosquito midgut after feeding, and again during transmission back to a host, where only a small fraction of sporozoites establish a liver infection. These bottlenecks shape parasite population structure and key selective pressures, influencing drug resistance and vaccine escape. However, the magnitude of these bottlenecks has not been precisely quantified.

Here, we use cellular barcoding to track parasite populations at high resolution across the lifecycle. We generated a library of P. berghei parasites containing over 1,000 unique DNA barcodes integrated into a neutral genomic locus. This diverse population was transmitted between mice and mosquitoes via natural bites or intravenous sporozoite injection, with samples collected across the lifecycle stages for barcode sequencing.

Our initial analyses quantify changes in barcode diversity throughout the lifecycle, identifying where parasite diversity is lost or maintained. These results define key transmission bottlenecks and highlight stages most susceptible to intervention. This framework can be extended to assess how drugs and vaccines impact parasite population dynamics, informing more precise malaria control strategies.

The LSHTM Human Malaria Transmission Facility provides access to human malaria parasite transmission both for research groups within the London School of Hygiene & Tropical Medicine and external collaborators worldwide. Our specialist team works with these collaborators to design and execute studies relating to the transmission of Plasmodium parasites using gametocytes grown in vitro in the laboratory or collected directly from clinical samples received by the UK HSA Malaria Reference Laboratory and fed to insectary-reared Anopheles mosquitoes. As a case study illustrating the support offered by the HMTF, we present new data indicating an important role for pyridoxal 5’-phosphate biosynthesis (vitamin B6; PLP) in mosquito stage development of P. falciparum. Enzymes in this pathway were found to be highly enriched in an analysis of the stage V gametocyte “translatome”, the subset of the total gametocyte proteome actively incorporating a pulse of labelled methionine in late-stage gametocytes. Disruption of PDX2, one of two subunits of pyridoxal 5’-phosphate synthase, reduced gametocyte infectivity to Anopheles coluzzi mosquitoes. This reduction was manifest in three measures: oocyst numbers per mosquito midgut, oocyst diameter and, most profoundly, number of salivary gland sporozoites per mosquito. All three negative outcomes were rescued by vitamin B6 supplementation.

Transmission-blocking vaccines aim to induce antibody responses that prevent human-to-mosquito malaria transmission by targeting the parasite inside the mosquito midgut. The furthest advanced transmission-blocking vaccine candidates are based on the Pfs230:Pfs48/45 complex. During the preparation of a cryo-EM structure of the endogenous Pfs230:Pfs48/45 complex, we found a previously unidentified protein that interacts with Pfs230. This protein is without described function and is conserved throughout all Plasmodium spp.. Based on structural homology modelling, we have tentatively dubbed this protein PfHAP2-novel (PfHAP2n). We generated a PfHAP2n^KO parasite line that is able to undergo gametocytogenesis and gametogenesis. However, just like Pfs230^KO parasites, male microgametes are no longer able to attach to erythrocytes and no longer form “exflagellation centres”. PfHAP2n^KO parasites have a strong, male-dependent reduction in mosquito infectivity. We have recombinantly produced PfHAP2n, and we are testing whether these constructs can elicit malaria transmission-blocking antibodies in immunization studies. Our results have uncovered a novel protein that is essential for malaria transmission, which might be a suitable target for future transmission-blocking vaccine development.

Plasmodium falciparum sporozoites must successfully invade Anopheles mosquito salivary glands and human liver hepatocytes to establish infection. While CERLI1 and CERLI2 are known critical mediators of blood-stage invasion, their potential roles during these sporozoite-specific events remain unexplored.

This study investigates CERLI1 and CERLI2 function during sporozoite transmission. Using CRISPR/Cas9, we generated transgenic parasite lines with inducible knockouts for CERLI1 and CERLI2. Phenotypic analyses revealed that inducing these knockouts significantly reduces the sporozoite load within the salivary glands of Anopheles mosquitoes. Furthermore, utilizing ultrastructure expansion microscopy (U-ExM), we demonstrated that this impaired salivary gland colonization correlates with structural defects in the rhoptry organelles.

Collectively, our findings establish CERLI1 and CERLI2 as essential, multi-stage mediators of the Plasmodium invasion machinery. Elucidating their function in the mosquito vector, alongside pending investigations into hepatocyte entry, provides vital insights into the fundamental biology of parasite transmission.

Light microscopy is the most widely used tool in the study of cell biology but many of the subcellular structures of parasites are too small to see even with the best light microscopes. Recently, a technique called expansion microscopy that physically enlarges parasites has revolutionised parasite cell biology. To date, expansion microscopy has only been applied on either parasites grown in vitro or from isolated host tissues. We wanted to perform expansion microscopy on whole mosquitoes, to simultaneously visualise the ultrastructure of both malaria parasites and their mosquito hosts, but the presence of the Chitin-rich mosquito cuticle prevents expansion. Here, we develop expansion microscopy for whole mosquitoes by first digesting the cuticle with enzymes. We validate that the mosquitoes expand as expected, show preservation of mosquito anatomy, and visualise it at exquisite detail. The application of this methodology allows for the co-visualisation of parasite and mosquito ultrastructure including microvilli, salivary glands, gut-microbiome and ovaries. Additionally, we have validated this technique for other arthropods: Ixodes holocyclus and Drosophila melanogaster, highlighting its versatility across arthropods. While developed to investigate cell biology, application of this technique could greatly improve the resolution of spatial omics techniques in the study of vector-parasite interactions.