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Additional file 3: Figure S1. DLP nano-feeder production by Nextdent 5100. DLP feeders were designed using 3DSprint software from 3Dsystems. A total of 23 nano-feeders were printed in 1 h by the Nextdent 5100 Beta printer by the Radboudumc 3D lab [22]. Nextdent SG (surgical guide) was used as resin, no support material was used. Each layer of printed liquid resin was hardened by UV beamer projection (xyz resolution 50 µm). In 30 minutes, post printing (B), feeders were two times ultrasonic cleaned in 96% ethanol and flushed with 96% ethanol before they were dried with compressed air and post-cured for 10 min in a UV box (Nextdent LC-3DPrint Box, UV-A 108 en UV-Blue 108).

Additional file 7: Table S1. Mosquito feeding rates after protocol optimization. To increase mosquito feeding rates the original nano-feeder protocol was optimised by (i) extension of the starvation period prior to feed (+ 12 h), (ii) stimulation of mosquitoes prior to feeding by placing a culture flask (canted neck 25 cm2) filled with warm water for 1 minute on top of cup, and (iii) plastic cages were changed to small paper cups to reduce distance between the mosquitoes and the feeder.

Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1.IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.

Protocol. Malaria antigen-specific IgG ELISA (S1). Isolation and cultivation of primary human hepatocytes (S2). In vitro sporozoite infectivity assay of primary human hepatocytes (S3). Immunofluorescent analysis of P. falciparum-infected primary human hepatocytes (S4). In vitro PBMC restimulation with PfRBCs and flow cytometry staining (S5) [11, 12, 14, 17, 42]. (DOCX 20 kb)

Protocol. Malaria antigen-specific IgG ELISA (S1). Isolation and cultivation of primary human hepatocytes (S2). In vitro sporozoite infectivity assay of primary human hepatocytes (S3). Immunofluorescent analysis of P. falciparum-infected primary human hepatocytes (S4). In vitro PBMC restimulation with PfRBCs and flow cytometry staining (S5) [11, 12, 14, 17, 42]. (DOCX 20 kb)

Additional file 5: Figure S3. Mosquito blood-meal size determined by eye. Mosquitoes were selected by eye and determined unfed (UF), partially blood-fed (PBF) or fully blood-fed (FBF) (from left to right).

Additional file 6: Figure S4. Mosquito feeding performance on 3D MJ-printed nano-feeders. In three independent experiments with three different blood donors, plastic cages with 5, 10, 15, 20 or 50 mosquitoes were fed on a 60 µl blood-meal to determine the feeding performance on the nano-feeder. Data from independent experiments were pooled per group for analysis; pie charts present the mean feeding rate in percentages of fully- (FBF), partially- (PBF) or unfed (UF) mosquitoes. In all conditions a number of mosquitoes were PBF or UF. No blood material in the feeder cavity was left for cups with 20 or 50 mosquitoes. For 10 mosquitoes per feeder, all visually FBF mosquitoes had a blood-meal > 2.5 µl, with a mean volume of 3.52 µl (range 2.6–4.0, n = 12) and feeding prevalence of 65% FBF.

Additional file 5: Figure S3. Mosquito blood-meal size determined by eye. Mosquitoes were selected by eye and determined unfed (UF), partially blood-fed (PBF) or fully blood-fed (FBF) (from left to right).

Additional file 6: Figure S4. Mosquito feeding performance on 3D MJ-printed nano-feeders. In three independent experiments with three different blood donors, plastic cages with 5, 10, 15, 20 or 50 mosquitoes were fed on a 60 µl blood-meal to determine the feeding performance on the nano-feeder. Data from independent experiments were pooled per group for analysis; pie charts present the mean feeding rate in percentages of fully- (FBF), partially- (PBF) or unfed (UF) mosquitoes. In all conditions a number of mosquitoes were PBF or UF. No blood material in the feeder cavity was left for cups with 20 or 50 mosquitoes. For 10 mosquitoes per feeder, all visually FBF mosquitoes had a blood-meal > 2.5 µl, with a mean volume of 3.52 µl (range 2.6–4.0, n = 12) and feeding prevalence of 65% FBF.

First-wave parasitemia after challenge. Parasitemia on day 7 post-challenge in immunized (open circles) and control (closed circles) volunteers. The line and error bars show the geometric mean and 95% CI interval. Figure S2. Inhibition of in vitro homologous and heterologous intra-hepatic sporozoite development in primary human hepatocytes by mAb 2A10. P. falciparum NF54 (blue), NF135.C10 (orange) and NF166.C8 (green) sporozoites in the presence of 10% heat-inactivated naive human control serum were pre-incubated with 3-fold serial dilutions of the 2A10 monoclonal antibody (0.027–20 μg/mL), targeting the repeat region of the circumsporozoite protein (CSP), and added to primary human hepatocyte cultures. Six days post-infection, the number of P. falciparum-infected hepatocytes was assessed as described in Additional file 1: S4 and S5. Figure S3. Amino acid changes in CSP. Table S1. Whole-genome sequencing statistics. Table S2. Mosquito salivary gland infectivity and sporozoite load of the three clones. Mean mosquito salivary gland infectivity and sporozoite load determined 1 day prior to challenge infection by dissecting a sample of 10 mosquitoes per strain. (DOCX 427 kb)

Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1.IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.

Additional file 4: Figure S2. Feeder temperature of glass mini-feeders and 3D MJ-printed nano-feeders. Two probes were used to simultaneously measure the temperature of the first (P1) and last (P2) feeder in a row of four. At 0 s, the water bath was turned on (dotted line, temperature set at 39 °C) and the blood-meal was injected in the feeder cavity of glass mini-feeders (circles) and nano-feeders (triangles). Both plastic and glass feeders reached a stable temperature quickly within 4.5 min after attaching to the water bath. Plastic feeders remained at a stable temperature on average 1.8 °C lower (mean = 35.9 °C) than glass feeders (mean = 37.7 °C).

First-wave parasitemia after challenge. Parasitemia on day 7 post-challenge in immunized (open circles) and control (closed circles) volunteers. The line and error bars show the geometric mean and 95% CI interval. Figure S2. Inhibition of in vitro homologous and heterologous intra-hepatic sporozoite development in primary human hepatocytes by mAb 2A10. P. falciparum NF54 (blue), NF135.C10 (orange) and NF166.C8 (green) sporozoites in the presence of 10% heat-inactivated naive human control serum were pre-incubated with 3-fold serial dilutions of the 2A10 monoclonal antibody (0.027–20 μg/mL), targeting the repeat region of the circumsporozoite protein (CSP), and added to primary human hepatocyte cultures. Six days post-infection, the number of P. falciparum-infected hepatocytes was assessed as described in Additional file 1: S4 and S5. Figure S3. Amino acid changes in CSP. Table S1. Whole-genome sequencing statistics. Table S2. Mosquito salivary gland infectivity and sporozoite load of the three clones. Mean mosquito salivary gland infectivity and sporozoite load determined 1 day prior to challenge infection by dissecting a sample of 10 mosquitoes per strain. (DOCX 427 kb)

Additional file 4: Figure S2. Feeder temperature of glass mini-feeders and 3D MJ-printed nano-feeders. Two probes were used to simultaneously measure the temperature of the first (P1) and last (P2) feeder in a row of four. At 0 s, the water bath was turned on (dotted line, temperature set at 39 °C) and the blood-meal was injected in the feeder cavity of glass mini-feeders (circles) and nano-feeders (triangles). Both plastic and glass feeders reached a stable temperature quickly within 4.5 min after attaching to the water bath. Plastic feeders remained at a stable temperature on average 1.8 °C lower (mean = 35.9 °C) than glass feeders (mean = 37.7 °C).