Malaria transmission to mosquitoes depends on the presence of mature gametocytes in human peripheral blood that are ingested by a mosquito during blood feeding. Ingested parasites undergo several developmental transformations in a process called sporogony. After ingestion, gametocytes transform into male and female gametes that fuse to form a zygote. The zygote differentiates into a motile ookinete that penetrates the midgut epithelium to form an oocyst. Multiple rounds of mitotic replication result in the formation of sporozoites inside an oocyst. Upon oocyst rupture, sporozoites are released into the haemocoel and invade the salivary glands (Mueller et al., 2010). These sporozoites penetrate the distal portion of the two lateral and medial lobes of the glands and accumulate extracellularly inside secretory cavities before entering the salivary ducts (Wells and Andrew, 2019). Despite the large number of sporozoites in the cavities, only a small proportion pass through the proximal part of the lobes where the salivary ducts become narrow (Frischknecht et al., 2004; Bradley et al., 2018), and only tens or low hundreds of sporozoites are assumed to be inoculated per mosquito bite (Graumans et al., 2020). Developmental bottlenecks during sporogony, as well as the size of the sporozoite inoculum, remain incompletely understood (Graumans et al., 2020).

The density of gametocytes in human peripheral blood is an important determinant of human to mosquito transmission. Though infections with parasite densities below the microscopic threshold for detection can infect mosquitoes (Schneider et al., 2007; Bousema et al., 2006), the likelihood and infection intensity increase with the number of ingested gametocytes (Bradley et al., 2018). Because of the abundance of low-density gametocyte carriers among infected populations, these are considered important drivers of malaria transmission (Andolina et al., 2021; Slater et al., 2019; Ouédraogo et al., 2016). Importantly, this conclusion is based on the assumption that all infected mosquitoes are equally infectious regardless of oocyst densities.

In apparent support of this assumption, single oocyst infections can result in thousands of salivary gland sporozoites (Rosenberg and Rungsiwongse, 1991; Pringle, 1965). A positive correlation between oocyst densities and salivary gland sporozoites was previously observed in Plasmodium falciparum (Vaughan et al., 1992; Miura et al., 2019) as well as in Plasmodium vivax (Solarte et al., 2011), suggesting that on average of 1000–2000 sporozoites reach the salivary glands per single ruptured oocyst. The few studies that quantified sporozoite inoculum by allowing mosquitoes to salivating into capillary tubes containing mineral oil, sucrose solution, or blood (Beier et al., 1991a; Beier et al., 1991b; Rosenberg et al., 1990) estimated median inocula ranging between 8 and 39 sporozoites with a minority of mosquitoes expelling >100 sporozoites (reviewed in Graumans et al., 2020).

While these studies provide some insights into sporozoite expelling and transmission dynamics, they do not reflect natural feeding conditions. Also, microscopy techniques used to quantify sporozoites may have underestimated the number of sporozoites (Medica and Sinnis, 2005). Subsequent studies with rodent Plasmodium models allowed natural mosquito probing through skin and showed a positive association between sporozoite density in salivary glands and expelled sporozoite numbers (Medica and Sinnis, 2005; Aleshnick et al., 2020). A recent study using a Plasmodium yoelii malaria model demonstrated that mosquitoes with over 10,000 salivary gland sporozoites were 7.5 times more likely to initiate a malaria infection in mice (Aleshnick et al., 2020). This finding has not been replicated for human malarias but is broadly consistent with post hoc analysis of infection likelihood in controlled human malaria infections (CHMI), where only mosquitoes that had >1000 P. falciparum salivary gland sporozoites remaining after probing (the residual sporozoite load) were capable of establishing an infection in malaria-naïve volunteers (Churcher et al., 2017).

If low oocyst/low sporozoite densities in mosquitoes are unlikely to initiate infections in humans, this may have profound consequences for our understanding of transmission (WHO, 2017). If mosquitoes with low-infection burdens have limited transmission potential, then the rationale for targeting low-density infections in humans that give rise to low-infection burdens in mosquitoes (Bradley et al., 2018) may be diminished.

Here, we examined the progression of sporozoite development and the number of sporozoites expelled into artificial skin by individual Anopheles mosquitoes infected either with P. falciparum gametocyte cultures or in experimental infections with naturally circulating parasite strains. We directly assessed the association between oocyst burden, salivary gland infection intensity, and the number of sporozoites expelled.

We examined P. falciparum sporogony in different mosquito species by employing sensitive molecular and staining techniques in conjunction with mosquito probing experiments on artificial skin. Through the visualization of ruptured oocysts and the simultaneous quantification of sporozoites that are expelled by individual mosquitoes, we observed that 95% of oocysts rupture to release sporozoites and that higher salivary gland sporozoite load is associated with shorter time to colonization of salivary glands and larger inoculum size.

The proportion of infectious mosquitoes is a central component of malariometric indices both in terms of quantifying the force of infection and the human infectious reservoir. The entomological inoculation rate (EIR) is defined as the number of infectious bites per person per time unit and is the product of human biting rate and the proportion of sporozoite-positive mosquitoes (Tusting et al., 2014; Shaukat et al., 2010). While EIR is a common measure of human malaria exposure, mosquito infection prevalence is used in this calculation and thus assumes all sporozoite-positive mosquitoes are equally infectious (Smith et al., 2012). Similarly, assessments of the human infectious reservoir for malaria typically take the number of oocyst-positive mosquitoes as a measure of transmission (Ouédraogo et al., 2016; Gonçalves et al., 2017) and thereby not only assume that all oocysts will lead to salivary gland sporozoites but also that all oocyst-positive mosquitoes have equal transmission potential. Recent work with a rodent malaria model challenged this central assumption (Aleshnick et al., 2020); we provide the first direct evidence for P. falciparum that sporozoite burden may indeed be a relevant determinant in efficient sporozoite expelling.

In this study, we assessed sporozoite expelling by mosquitoes carrying low- and high-infection burdens. Our findings confirm that the vast majority (~95%) of oocysts rupture to release sporozoites. This estimate is higher than a previous study with cultured gametocytes (~72%) (Stone et al., 2013) that did not provide a second bloodmeal that may accelerate oocyst maturation (Shaw et al., 2020); a second bloodmeal also better mimics natural feeding habits where multiple bloodmeals are taken within the period required for sporogony. Moreover, we observed a strong positive association between sporozoite salivary gland load and ruptured oocyst density in mosquitoes infected with both cultured and naturally circulating gametocytes, with similar median numbers of 4951 (IQR, 3016–8318) and 6350 (IQR, 4225–8475) salivary gland sporozoites per ruptured oocyst in An. stephensi and An. coluzzii, respectively. Previous studies have shown that despite the substantial release of sporozoites per oocyst, only a proportion of sporozoites successfully reach the salivary glands (Rosenberg and Rungsiwongse, 1991; Hillyer et al., 2007). In line with this, we observed considerably higher sporozoite estimates in intact oocysts (10,485–15,390 sporozoites per oocyst). Whilst these sporozoite estimates are higher than commonly reported (Graumans et al., 2020), an early study from the 1960s reported microscopy-detected sporozoite densities above 9000 sporozoites per oocyst (Pringle, 1965) and a recent study using qPCR similarly observed up to 12,583 sporozoites from a single oocyst (Wang et al., 2018). These high sporozoite numbers per oocyst also mean that, different from P. yoelii (Aleshnick et al., 2020) and P. berghei (Churcher et al., 2017), only a minority of infected mosquitoes had sporozoite densities below the threshold values previously reported in relation to a very low likelihood of achieving secondary infections. Even among infected An. stephensi mosquitoes with only one ruptured oocyst, 92.9% (26/28) had >1000 and 35.7% (10/28) had >10,000 sporozoites in their salivary glands. In our experiments in natural gametocyte carriers in Burkina Faso, only two mosquitoes were observed with single oocysts and both had >10,000 salivary gland sporozoites. Although P. falciparum sporozoite densities below these lower thresholds were thus uncommon, we observed that 39% of sporozoite-positive mosquitoes across a wide range of infection densities failed to expel sporozoites upon probing. This finding broadly aligns with an earlier study of Medica and Sinnis that reported that 22% of P. yoelii-infected mosquitoes failed to expel sporozoites (Medica and Sinnis, 2005). For highly infected mosquitoes, this inefficient expelling has been related to a decrease in apyrase in the mosquito saliva (Rossignol et al., 1984; Thiévent et al., 2019).

Importantly, we observed a positive association between salivary gland sporozoite density and the number of expelled sporozoites. For unknown reasons, this association was markedly stronger in experiments where An. coluzzii mosquitoes were infected using blood from gametocyte carriers who were naturally infected with P. falciparum in Burkina Faso compared to An. stephensi mosquitoes infected with cultured gametocytes. In our experiments with natural gametocyte carriers, sporozoite density appeared associated with both the likelihood of expelling any sporozoites and the inoculum size. The most heavily infected mosquitoes expelled ~14-fold higher sporozoite numbers compared to the lowest quartile. In addition, we found evidence that the EIP – the period required for a mosquito to become salivary gland sporozoite positive – is shorter for heavily infected mosquitoes. Previous studies have not found consistent effects of parasite burden on EIP (Guissou et al., 2023; Stopard et al., 2021) but, unlike our experiments, were also not specifically designed to examine this association across a broad range of infection intensities. Our associations of a shorter EIP in highly infected mosquitoes and, in separate experiments, a larger sporozoite inoculum size for highly infected mosquitoes make mosquito infection intensity a plausible factor in determining onward transmission potential to humans. Heavily infected mosquitoes may be infectious sooner and be more infectious. On the other side of the infection spectrum, it is conceivable that submicroscopic gametocyte carriers that typically result in low oocyst burdens in mosquitoes give rise to infected mosquitoes with a reduced transmission potential. This would greatly reduce their importance for sustaining malaria transmission and make it less important to identify individuals with low parasite densities for malaria elimination purposes (Slater et al., 2015). At the same time, we occasionally observed high sporozoite inocula from mosquitoes with low infection intensity. This argues against a clear threshold sporozoite or oocyst density below which mosquitoes are truly irrelevant for transmission. Moreover, the high number of sporozoites per oocyst makes it plausible that even the low oocyst burdens that are typically observed from asymptomatic parasite carriers (in the range of 1–5 oocysts/infected gut; Andolina et al., 2021; Gonçalves et al., 2017) would be sufficient to result in salivary gland sporozoite loads that are sufficient to result in secondary infections.

Our study leaves a number of questions and has several limitations. While the use of two mosquito and gametocyte sources was a relevant strength of our study, an uncertainty relates to the choice of artificial skin that has a realistic 1.33 mm thickness but is arguably less natural than microvascularized skin with all the natural cues for mosquito probing. Whilst genuine skin might have improved natural feeding behavior, probing and blood feeding were highly efficient in our model and we see no reasons to assume bias in the comparison between high- and low-infected mosquitoes. Our assessments of EIP and sporozoite expelling did not demonstrate the viability of sporozoites. Whilst the infectivity of sporozoites at different time points PI has been examined previously (Yang et al., 2017), these experiments have never been conducted with individual mosquitoes. In vitro experiments that aim to determine the infectivity of single mosquito bites would ideally retain the skin barrier that may be a relevant determinant for invasion capacity and use primary hepatocytes. These experiments were beyond the scope of the current work and would also not provide conclusive evidence on the likelihood of achieving secondary infections. Given striking differences in sporozoite burden between different Plasmodium species – low sporozoite densities appear considerably more common in mosquitoes infected with P. yoelii and P. berghei (Graumans et al., 2020; Churcher et al., 2017; Ahmed et al., 2021), and the inherent limitations of in vitro studies – the association between sporozoite inoculum size and the likelihood of achieving secondary infections may be best examined in controlled human infection studies. CHMI experiments can be specifically designed to estimate the likelihood that probing by (individual) high- and low-infected mosquitoes results in blood-stage infection in malaria-naïve volunteers. Whilst laborious, CHMI studies are unique in allowing definitive evidence on the possible differences in infectiousness between high- and low-infected mosquitoes.

In conclusion, we observed that the majority of oocysts rupture and contribute to salivary gland infection load. We further observe that this sporozoite load is highly variable and an important determinant of the number of sporozoites that is expelled into the skin upon probing.

All data generated or analysed during this study are deposited on Dryad: https://doi.org/10.5061/dryad.dbrv15f89.

1. 1. Andolina C
   2. Graumans W
   3. Guelbeogo M
   4. Gemert GJV
   5. Ramjith J
   6. Harouna S
   7. Soumanaba Z
   8. Stoter R
   9. Vegte-Bolmer M
   10. Pangos M
   11. Sinnis P
   12. Collins K
   13. Staedke SG
   14. Tiono AB
   15. Drakeley C
   16. Lanke K
   17. Bousema T

   (2024) Dryad Digital Repository

   Data from: Quantification of sporozoite expelling by Anopheles mosquitoes infected with laboratory and naturally circulating P. falciparum gametocytes.

   https://doi.org/10.5061/dryad.dbrv15f89

1. 1. Agostinis C
   2. Spazzapan M
   3. Vuerich R
   4. Balduit A
   5. Stocco C
   6. Mangogna A
   7. Ricci G
   8. Papa G
   9. Zacchigna S
   10. Bulla R

   (2021) Differential capability of clinically employed dermal regeneration scaffolds to support vascularization for tissue bioengineering

   Biomedicines 9:1458.

   https://doi.org/10.3390/biomedicines9101458

   • PubMed
   • Google Scholar
2. 1. Ahmed A
   2. Pignatelli P
   3. Elaagip A
   4. Abdel Hamid MM
   5. Alrahman OF
   6. Weetman D

   (2021) Invasive malaria vector anopheles stephensi mosquitoes in sudan, 2016-2018

   Emerging Infectious Diseases 27:2952–2954.

   https://doi.org/10.3201/eid2711.210040

   • PubMed
   • Google Scholar
3. 1. Aleshnick M
   2. Ganusov VV
   3. Nasir G
   4. Yenokyan G
   5. Sinnis P

   (2020) Experimental determination of the force of malaria infection reveals a non-linear relationship to mosquito sporozoite loads

   PLOS Pathogens 16:e1008181.

   https://doi.org/10.1371/journal.ppat.1008181

   • PubMed
   • Google Scholar
4. 1. Andolina C
   2. Rek JC
   3. Briggs J
   4. Okoth J
   5. Musiime A
   6. Ramjith J
   7. Teyssier N
   8. Conrad M
   9. Nankabirwa JI
   10. Lanke K
   11. Rodriguez-Barraquer I
   12. Meerstein-Kessel L
   13. Arinaitwe E
   14. Olwoch P
   15. Rosenthal PJ
   16. Kamya MR
   17. Dorsey G
   18. Greenhouse B
   19. Drakeley C
   20. Staedke SG
   21. Bousema T

   (2021) Sources of persistent malaria transmission in a setting with effective malaria control in eastern uganda: a longitudinal, observational cohort study

   The Lancet. Infectious Diseases 21:1568–1578.

   https://doi.org/10.1016/S1473-3099(21)00072-4

   • PubMed
   • Google Scholar
5. 1. Beier JC
   2. Davis JR
   3. Vaughan JA
   4. Noden BH
   5. Beier MS

   (1991a) Quantitation of Plasmodium falciparum sporozoites transmitted in vitro by experimentally infected Anopheles gambiae and Anopheles stephensi

   The American Journal of Tropical Medicine and Hygiene 44:564–570.

   https://doi.org/10.4269/ajtmh.1991.44.564

   • PubMed
   • Google Scholar
6. 1. Beier JC
   2. Onyango FK
   3. Koros JK
   4. Ramadhan M
   5. Ogwang R
   6. Wirtz RA
   7. Koech DK
   8. Roberts CR

   (1991b) Quantitation of malaria sporozoites transmitted in vitro during salivation by wild Afrotropical Anopheles

   Medical and Veterinary Entomology 5:71–79.

   https://doi.org/10.1111/j.1365-2915.1991.tb00523.x

   • PubMed
   • Google Scholar
7. 1. Boissière A
   2. Gimonneau G
   3. Tchioffo MT
   4. Abate L
   5. Bayibeki A
   6. Awono-Ambéné PH
   7. Nsango SE
   8. Morlais I

   (2013) Application of a qPCR assay in the investigation of susceptibility to malaria infection of the M and S molecular forms of an: gambiae S.S. in cameroon

   PLOS ONE 8:e54820.

   https://doi.org/10.1371/journal.pone.0054820

   • PubMed
   • Google Scholar
8. 1. Bousema JT
   2. Schneider P
   3. Gouagna LC
   4. Drakeley CJ
   5. Tostmann A
   6. Houben R
   7. Githure JI
   8. Ord R
   9. Sutherland CJ
   10. Omar SA
   11. Sauerwein RW

   (2006) Moderate effect of artemisinin-based combination therapy on transmission of Plasmodium falciparum

   The Journal of Infectious Diseases 193:1151–1159.

   https://doi.org/10.1086/503051

   • PubMed
   • Google Scholar
9. 1. Bradley J
   2. Stone W
   3. Da DF
   4. Morlais I
   5. Dicko A
   6. Cohuet A
   7. Guelbeogo WM
   8. Mahamar A
   9. Nsango S
   10. Soumaré HM
   11. Diawara H
   12. Lanke K
   13. Graumans W
   14. Siebelink-Stoter R
   15. van de Vegte-Bolmer M
   16. Chen I
   17. Tiono A
   18. Gonçalves BP
   19. Gosling R
   20. Sauerwein RW
   21. Drakeley C
   22. Churcher TS
   23. Bousema T

   (2018) Predicting the likelihood and intensity of mosquito infection from sex specific Plasmodium falciparum gametocyte density

   eLife 7:e34463.

   https://doi.org/10.7554/eLife.34463

   • PubMed
   • Google Scholar
10. 1. Briggs J
    2. Teyssier N
    3. Nankabirwa JI
    4. Rek J
    5. Jagannathan P
    6. Arinaitwe E
    7. Bousema T
    8. Drakeley C
    9. Murray M
    10. Crawford E
    11. Hathaway N
    12. Staedke SG
    13. Smith D
    14. Rosenthal PJ
    15. Kamya M
    16. Dorsey G
    17. Rodriguez-Barraquer I
    18. Greenhouse B

    (2020) Sex-based differences in clearance of chronic Plasmodium falciparum infection

    eLife 9:e59872.

    https://doi.org/10.7554/eLife.59872

    • PubMed
    • Google Scholar
11. 1. Brugman VA
    2. Kristan M
    3. Gibbins MP
    4. Angrisano F
    5. Sala KA
    6. Dessens JT
    7. Blagborough AM
    8. Walker T

    (2018) Detection of malaria sporozoites expelled during mosquito sugar feeding

    Scientific Reports 8:7545.

    https://doi.org/10.1038/s41598-018-26010-6

    • PubMed
    • Google Scholar
12. 1. Churcher TS
    2. Blagborough AM
    3. Delves M
    4. Ramakrishnan C
    5. Kapulu MC
    6. Williams AR
    7. Biswas S
    8. Da DF
    9. Cohuet A
    10. Sinden RE

    (2012) Measuring the blockade of malaria transmission--an analysis of the Standard Membrane Feeding Assay

    International Journal for Parasitology 42:1037–1044.

    https://doi.org/10.1016/j.ijpara.2012.09.002

    • PubMed
    • Google Scholar
13. 1. Churcher TS
    2. Sinden RE
    3. Edwards NJ
    4. Poulton ID
    5. Rampling TW
    6. Brock PM
    7. Griffin JT
    8. Upton LM
    9. Zakutansky SE
    10. Sala KA
    11. Angrisano F
    12. Hill AVS
    13. Blagborough AM

    (2017) Probability of transmission of malaria from mosquito to human is regulated by mosquito parasite density in naïve and vaccinated hosts

    PLOS Pathogens 13:e1006108.

    https://doi.org/10.1371/journal.ppat.1006108

    • PubMed
    • Google Scholar
14. 1. Conway DJ
    2. Fanello C
    3. Lloyd JM
    4. Al-Joubori BM
    5. Baloch AH
    6. Somanath SD
    7. Roper C
    8. Oduola AM
    9. Mulder B
    10. Povoa MM
    11. Singh B
    12. Thomas AW

    (2000) Origin of Plasmodium falciparum malaria is traced by mitochondrial DNA

    Molecular and Biochemical Parasitology 111:163–171.

    https://doi.org/10.1016/s0166-6851(00)00313-3

    • PubMed
    • Google Scholar
15. 1. Feldmann AM
    2. Ponnudurai T

    (1989) Selection of Anopheles stephensi for refractoriness and susceptibility to Plasmodium falciparum

    Medical and Veterinary Entomology 3:41–52.

    https://doi.org/10.1111/j.1365-2915.1989.tb00473.x

    • PubMed
    • Google Scholar
16. 1. Frischknecht F
    2. Baldacci P
    3. Martin B
    4. Zimmer C
    5. Thiberge S
    6. Olivo-Marin JC
    7. Shorte SL
    8. Ménard R

    (2004) Imaging movement of malaria parasites during transmission by Anopheles mosquitoes

    Cellular Microbiology 6:687–694.

    https://doi.org/10.1111/j.1462-5822.2004.00395.x

    • PubMed
    • Google Scholar
17. 1. Gonçalves BP
    2. Kapulu MC
    3. Sawa P
    4. Guelbéogo WM
    5. Tiono AB
    6. Grignard L
    7. Stone W
    8. Hellewell J
    9. Lanke K
    10. Bastiaens GJH
    11. Bradley J
    12. Nébié I
    13. Ngoi JM
    14. Oriango R
    15. Mkabili D
    16. Nyaurah M
    17. Midega J
    18. Wirth DF
    19. Marsh K
    20. Churcher TS
    21. Bejon P
    22. Sirima SB
    23. Drakeley C
    24. Bousema T

    (2017) Examining the human infectious reservoir for Plasmodium falciparum malaria in areas of differing transmission intensity

    Nature Communications 8:1133.

    https://doi.org/10.1038/s41467-017-01270-4

    • PubMed
    • Google Scholar
18. 1. Graumans W
    2. Tadesse FG
    3. Andolina C
    4. van Gemert GJ
    5. Teelen K
    6. Lanke K
    7. Gadisa E
    8. Yewhalaw D
    9. van de Vegte-Bolmer M
    10. Siebelink-Stoter R
    11. Reuling I
    12. Sauerwein R
    13. Bousema T

    (2017) Semi-high-throughput detection of Plasmodium falciparum and Plasmodium vivax oocysts in mosquitoes using bead-beating followed by circumsporozoite ELISA and quantitative PCR

    Malaria Journal 16:356.

    https://doi.org/10.1186/s12936-017-2011-9

    • PubMed
    • Google Scholar
19. 1. Graumans W
    2. Andolina C
    3. Awandu SS
    4. Grignard L
    5. Lanke K
    6. Bousema T

    (2019) Plasmodium falciparum gametocyte enrichment in peripheral blood samples by magnetic fractionation: gametocyte yields and possibilities to reuse columns

    The American Journal of Tropical Medicine and Hygiene 100:572–577.

    https://doi.org/10.4269/ajtmh.18-0773

    • PubMed
    • Google Scholar
20. 1. Graumans W
    2. Jacobs E
    3. Bousema T
    4. Sinnis P

    (2020) When is a plasmodium-infected mosquito an infectious mosquito?

    Trends in Parasitology 36:705–716.

    https://doi.org/10.1016/j.pt.2020.05.011

    • PubMed
    • Google Scholar
21. 1. Guissou E
    2. Da DF
    3. Hien D
    4. Yameogo KB
    5. Yerbanga SR
    6. Ouédraogo GA
    7. Dabiré KR
    8. Lefèvre T
    9. Cohuet A

    (2023) Intervention reducing malaria parasite load in vector mosquitoes: no impact on Plasmodium falciparum extrinsic incubation period and the survival of Anopheles gambiae

    PLOS Pathogens 19:e1011084.

    https://doi.org/10.1371/journal.ppat.1011084

    • PubMed
    • Google Scholar
22. 1. Hillyer JF
    2. Barreau C
    3. Vernick KD

    (2007) Efficiency of salivary gland invasion by malaria sporozoites is controlled by rapid sporozoite destruction in the mosquito haemocoel

    International Journal for Parasitology 37:673–681.

    https://doi.org/10.1016/j.ijpara.2006.12.007

    • PubMed
    • Google Scholar
23. 1. Holzschuh A
    2. Koepfli C

    (2022) Tenfold difference in DNA recovery rate: systematic comparison of whole blood vs dried blood spot sample collection for malaria molecular surveillance

    Malaria Journal 21:88.

    https://doi.org/10.1186/s12936-022-04122-9

    • PubMed
    • Google Scholar
24. 1. Matsuoka H
    2. Yoshida S
    3. Hirai M
    4. Ishii A

    (2002) A rodent malaria, Plasmodium berghei, is experimentally transmitted to mice by merely probing of infective mosquito, Anopheles stephensi

    Parasitology International 51:17–23.

    https://doi.org/10.1016/s1383-5769(01)00095-2

    • PubMed
    • Google Scholar
25. 1. Medica DL
    2. Sinnis P

    (2005) Quantitative dynamics of Plasmodium yoelii sporozoite transmission by infected anopheline mosquitoes

    Infection and Immunity 73:4363–4369.

    https://doi.org/10.1128/IAI.73.7.4363-4369.2005

    • PubMed
    • Google Scholar
26. 1. Miura K
    2. Swihart BJ
    3. Deng B
    4. Zhou L
    5. Pham TP
    6. Diouf A
    7. Fay MP
    8. Long CA

    (2019) Strong concordance between percent inhibition in oocyst and sporozoite intensities in a Plasmodium falciparum standard membrane-feeding assay

    Parasites & Vectors 12:206.

    https://doi.org/10.1186/s13071-019-3470-3

    • PubMed
    • Google Scholar
27. 1. Mueller AK
    2. Kohlhepp F
    3. Hammerschmidt C
    4. Michel K

    (2010) Invasion of mosquito salivary glands by malaria parasites: prerequisites and defense strategies

    International Journal for Parasitology 40:1229–1235.

    https://doi.org/10.1016/j.ijpara.2010.05.005

    • PubMed
    • Google Scholar
28. 1. Musiime AK
    2. Okoth J
    3. Conrad M
    4. Ayo D
    5. Onyige I
    6. Rek J
    7. Nankabirwa JI
    8. Arinaitwe E
    9. Kamya MR
    10. Dorsey G
    11. van Gemert GJ
    12. Staedke SG
    13. Drakeley C
    14. Bousema T
    15. Andolina C

    (2019) Is that a real oocyst? Insectary establishment and identification of Plasmodium falciparum oocysts in midguts of Anopheles mosquitoes fed on infected human blood in Tororo, Uganda

    Malaria Journal 18:287.

    https://doi.org/10.1186/s12936-019-2922-8

    • PubMed
    • Google Scholar
29. 1. Ouédraogo AL

    (2013) A protocol for membrane feeding assays to determine the infectiousness of P. falciparum naturally infected individuals to Anopheles gambiae

    PLOS ONE 7:0042821.

    https://doi.org/10.1371/journal.pone.0042821

    • Google Scholar
30. 1. Ouédraogo AL
    2. Gonçalves BP
    3. Gnémé A
    4. Wenger EA
    5. Guelbeogo MW
    6. Ouédraogo A
    7. Gerardin J
    8. Bever CA
    9. Lyons H
    10. Pitroipa X
    11. Verhave JP
    12. Eckhoff PA
    13. Drakeley C
    14. Sauerwein R
    15. Luty AJF
    16. Kouyaté B
    17. Bousema T

    (2016) Dynamics of the human infectious reservoir for malaria determined by mosquito feeding assays and ultrasensitive malaria diagnosis in burkina faso

    The Journal of Infectious Diseases 213:90–99.

    https://doi.org/10.1093/infdis/jiv370

    • PubMed
    • Google Scholar
31. 1. Ponnudurai T
    2. Lensen AH
    3. Leeuwenberg AD
    4. Meuwissen JH

    (1982) Cultivation of fertile Plasmodium falciparum gametocytes in semi-automated systems. 1. static cultures

    Transactions of the Royal Society of Tropical Medicine and Hygiene 76:812–818.

    https://doi.org/10.1016/0035-9203(82)90116-X

    • Google Scholar
32. 1. Ponnudurai T
    2. Lensen AH
    3. Van Gemert GJ
    4. Bensink MP
    5. Bolmer M
    6. Meuwissen JH

    (1989) Infectivity of cultured Plasmodium falciparum gametocytes to mosquitoes

    Parasitology 98 Pt 2:165–173.

    https://doi.org/10.1017/s0031182000062065

    • PubMed
    • Google Scholar
33. 1. Pringle G

    (1965) A count of the sporozoites in an ooecyst of Plasmodium falciparum

    Transactions of the Royal Society of Tropical Medicine and Hygiene 59:289–290.

    https://doi.org/10.1016/0035-9203(65)90009-x

    • PubMed
    • Google Scholar
34. 1. Rosenberg R
    2. Wirtz RA
    3. Schneider I
    4. Burge R

    (1990) An estimation of the number of malaria sporozoites ejected by a feeding mosquito

    Transactions of the Royal Society of Tropical Medicine and Hygiene 84:209–212.

    https://doi.org/10.1016/0035-9203(90)90258-g

    • PubMed
    • Google Scholar
35. 1. Rosenberg R
    2. Rungsiwongse J

    (1991) The number of sporozoites produced by individual malaria oocysts

    The American Journal of Tropical Medicine and Hygiene 45:574–577.

    https://doi.org/10.4269/ajtmh.1991.45.574

    • PubMed
    • Google Scholar
36. 1. Rossignol PA
    2. Ribeiro JM
    3. Spielman A

    (1984) Increased intradermal probing time in sporozoite-infected mosquitoes

    The American Journal of Tropical Medicine and Hygiene 33:17–20.

    https://doi.org/10.4269/ajtmh.1984.33.17

    • PubMed
    • Google Scholar
37. 1. Schneider P
    2. Bousema JT
    3. Gouagna LC
    4. Otieno S
    5. van de Vegte-Bolmer M
    6. Omar SA
    7. Sauerwein RW

    (2007)

    Submicroscopic Plasmodium falciparum gametocyte densities frequently result in mosquito infection

    The American Journal of Tropical Medicine and Hygiene 76:470–474.

    • PubMed
    • Google Scholar
38. 1. Shaukat AM
    2. Breman JG
    3. McKenzie FE

    (2010) Using the entomological inoculation rate to assess the impact of vector control on malaria parasite transmission and elimination

    Malaria Journal 9:122.

    https://doi.org/10.1186/1475-2875-9-122

    • PubMed
    • Google Scholar
39. 1. Shaw WR
    2. Holmdahl IE
    3. Itoe MA
    4. Werling K
    5. Marquette M
    6. Paton DG
    7. Singh N
    8. Buckee CO
    9. Childs LM
    10. Catteruccia F

    (2020) Multiple blood feeding in mosquitoes shortens the Plasmodium falciparum incubation period and increases malaria transmission potential

    PLOS Pathogens 16:e1009131.

    https://doi.org/10.1371/journal.ppat.1009131

    • PubMed
    • Google Scholar
40. 1. Slater HC
    2. Ross A
    3. Ouédraogo AL
    4. White LJ
    5. Nguon C
    6. Walker PGT
    7. Ngor P
    8. Aguas R
    9. Silal SP
    10. Dondorp AM
    11. La Barre P
    12. Burton R
    13. Sauerwein RW
    14. Drakeley C
    15. Smith TA
    16. Bousema T
    17. Ghani AC

    (2015) Assessing the impact of next-generation rapid diagnostic tests on Plasmodium falciparum malaria elimination strategies

    Nature 528:S94–S101.

    https://doi.org/10.1038/nature16040

    • PubMed
    • Google Scholar
41. 1. Slater HC
    2. Ross A
    3. Felger I
    4. Hofmann NE
    5. Robinson L
    6. Cook J
    7. Gonçalves BP
    8. Björkman A
    9. Ouedraogo AL
    10. Morris U
    11. Msellem M
    12. Koepfli C
    13. Mueller I
    14. Tadesse F
    15. Gadisa E
    16. Das S
    17. Domingo G
    18. Kapulu M
    19. Midega J
    20. Owusu-Agyei S
    21. Nabet C
    22. Piarroux R
    23. Doumbo O
    24. Doumbo SN
    25. Koram K
    26. Lucchi N
    27. Udhayakumar V
    28. Mosha J
    29. Tiono A
    30. Chandramohan D
    31. Gosling R
    32. Mwingira F
    33. Sauerwein R
    34. Paul R
    35. Riley EM
    36. White NJ
    37. Nosten F
    38. Imwong M
    39. Bousema T
    40. Drakeley C
    41. Okell LC

    (2019) The temporal dynamics and infectiousness of subpatent Plasmodium falciparum infections in relation to parasite density

    Nature Communications 10:1433.

    https://doi.org/10.1038/s41467-019-09441-1

    • PubMed
    • Google Scholar
42. 1. Smith DL
    2. Battle KE
    3. Hay SI
    4. Barker CM
    5. Scott TW
    6. McKenzie FE

    (2012) Ross, macdonald, and a theory for the dynamics and control of mosquito-transmitted pathogens

    PLOS Pathogens 8:e1002588.

    https://doi.org/10.1371/journal.ppat.1002588

    • PubMed
    • Google Scholar
43. 1. Solarte Y
    2. Manzano MR
    3. Rocha L
    4. Hurtado H
    5. James MA
    6. Arévalo-Herrera M
    7. Herrera S

    (2011) Plasmodium vivax sporozoite production in Anopheles albimanus mosquitoes for vaccine clinical trials

    The American Journal of Tropical Medicine and Hygiene 84:28–34.

    https://doi.org/10.4269/ajtmh.2011.09-0499

    • PubMed
    • Google Scholar
44. 1. Soontarawirat I
    2. Andolina C
    3. Paul R
    4. Day NPJ
    5. Nosten F
    6. Woodrow CJ
    7. Imwong M

    (2017) Plasmodium vivax genetic diversity and heterozygosity in blood samples and resulting oocysts at the Thai-Myanmar border

    Malaria Journal 16:355.

    https://doi.org/10.1186/s12936-017-2002-x

    • PubMed
    • Google Scholar
45. 1. Soumare HM
    2. Guelbeogo WM
    3. van de Vegte-Bolmer M
    4. van Gemert GJ
    5. Soumanaba Z
    6. Ouedraogo A
    7. Ouattara MS
    8. Abdullahi A
    9. Jadama L
    10. Camara MM
    11. Gaye PM
    12. Mendy M
    13. Davis N
    14. Tiono AB
    15. D’Alessandro U
    16. Drakeley C
    17. Bousema T
    18. Moreno M
    19. Collins KA

    (2021) Maintaining Plasmodium falciparum gametocyte infectivity during blood collection and transport for mosquito feeding assays in the field

    Malaria Journal 20:191.

    https://doi.org/10.1186/s12936-021-03725-y

    • PubMed
    • Google Scholar
46. 1. Stone WJR
    2. Eldering M
    3. van Gemert G-J
    4. Lanke KHW
    5. Grignard L
    6. van de Vegte-Bolmer MG
    7. Siebelink-Stoter R
    8. Graumans W
    9. Roeffen WFG
    10. Drakeley CJ
    11. Sauerwein RW
    12. Bousema T

    (2013) The relevance and applicability of oocyst prevalence as a read-out for mosquito feeding assays

    Scientific Reports 3:3418.

    https://doi.org/10.1038/srep03418

    • PubMed
    • Google Scholar
47. 1. Stopard IJ
    2. Churcher TS
    3. Lambert B

    (2021) Estimating the extrinsic incubation period of malaria using a mechanistic model of sporogony

    PLOS Computational Biology 17:e1008658.

    https://doi.org/10.1371/journal.pcbi.1008658

    • PubMed
    • Google Scholar
48. Software

    1. Team RC

    (2019) The R project for statistical computing

    Cran.

    https://www.r-project.org/
49. 1. Teirlinck AC
    2. Roestenberg M
    3. van de Vegte-Bolmer M
    4. Scholzen A
    5. Heinrichs MJL
    6. Siebelink-Stoter R
    7. Graumans W
    8. van Gemert GJ
    9. Teelen K
    10. Vos MW
    11. Nganou-Makamdop K
    12. Borrmann S
    13. Rozier YPA
    14. Erkens MAA
    15. Luty AJF
    16. Hermsen CC
    17. Sim BKL
    18. van Lieshout L
    19. Hoffman SL
    20. Visser LG
    21. Sauerwein RW

    (2013) NF135.C10: a new Plasmodium falciparum clone for controlled human malaria infections

    The Journal of Infectious Diseases 207:656–660.

    https://doi.org/10.1093/infdis/jis725

    • PubMed
    • Google Scholar
50. 1. Thiévent K
    2. Zilio G
    3. Hauser G
    4. Koella JC

    (2019) Malaria load affects the activity of mosquito salivary apyrase

    Journal of Insect Physiology 116:10–16.

    https://doi.org/10.1016/j.jinsphys.2019.04.003

    • PubMed
    • Google Scholar
51. 1. Tusting LS
    2. Bousema T
    3. Smith DL
    4. Drakeley C

    (2014) Measuring changes in Plasmodium falciparum transmission: precision, accuracy and costs of metrics

    Advances in Parasitology 84:151–208.

    https://doi.org/10.1016/B978-0-12-800099-1.00003-X

    • PubMed
    • Google Scholar
52. 1. van de Vegte-Bolmer M
    2. Graumans W
    3. Stoter R
    4. van Gemert GJ
    5. Sauerwein R
    6. Collins KA
    7. Bousema T

    (2021) A portfolio of geographically distinct laboratory-adapted Plasmodium falciparum clones with consistent infection rates in Anopheles mosquitoes

    Malaria Journal 20:381.

    https://doi.org/10.1186/s12936-021-03912-x

    • PubMed
    • Google Scholar
53. 1. Vaughan JA
    2. Noden BH
    3. Beier JC

    (1992)

    Population dynamics of Plasmodium falciparum sporogony in laboratory-infected Anopheles gambiae

    The Journal of Parasitology 78:716–724.

    • PubMed
    • Google Scholar
54. 1. Wang CYT
    2. McCarthy JS
    3. Stone WJ
    4. Bousema T
    5. Collins KA
    6. Bialasiewicz S

    (2018) Assessing Plasmodium falciparum transmission in mosquito-feeding assays using quantitative PCR

    Malaria Journal 17:249.

    https://doi.org/10.1186/s12936-018-2382-6

    • PubMed
    • Google Scholar
55. 1. Wells MB
    2. Andrew DJ

    (2019) Anopheles salivary gland architecture shapes plasmodium sporozoite availability for transmission

    mBio 10:e01238.

    https://doi.org/10.1128/mBio.01238-19

    • Google Scholar
56. Website

    1. WHO

    (2017) Meeting report of the WHO Evidence Review Group on Low-Density Malaria Infections

    Accessed July 17, 2023.

    https://www.who.int/publications/m/item/WHO-HTM-GMP-MPAC-2017.10
57. 1. Yang ASP
    2. Lopaticki S
    3. O’Neill MT
    4. Erickson SM
    5. Douglas DN
    6. Kneteman NM
    7. Boddey JA

    (2017) AMA1 and MAEBL are important for Plasmodium falciparum sporozoite infection of the liver

    Cellular Microbiology 19:2745.

    https://doi.org/10.1111/cmi.12745

    • PubMed
    • Google Scholar

Author details

1. Chiara Andolina

   Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft

   Contributed equally with

   Wouter Graumans

   Competing interests

   No competing interests declared

2. Wouter Graumans

   Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft

   Contributed equally with

   Chiara Andolina

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3952-6491

3. Moussa Guelbeogo

   Centre National de Recherche et de Formation sur le Paludisme, Ouagadougou, Burkina Faso

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

4. Geert-Jan van Gemert

   Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2740-5337

5. Jordache Ramijth

   Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

   Contribution

   Formal analysis, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

6. Soré Harouna

   Centre National de Recherche et de Formation sur le Paludisme, Ouagadougou, Burkina Faso

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

7. Zongo Soumanaba

   Centre National de Recherche et de Formation sur le Paludisme, Ouagadougou, Burkina Faso

   Contribution

   Writing – review and editing

   Competing interests

   No competing interests declared

8. Rianne Stoter

   Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

   Contribution

   Investigation

   Competing interests

   No competing interests declared

9. Marga Vegte-Bolmer

   Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

   Contribution

   Investigation

   Competing interests

   No competing interests declared

10. Martina Pangos

    Department of Plastic and Reconstructive Surgery, Azienda Ospedaliero Universitaria GiulianoIsontina Trieste, Trieste, Italy

    Contribution

    Resources, Methodology

    Competing interests

    No competing interests declared

11. Photini Sinnis

    Department of Molecular Microbiology and Immunology, Johns HopkinsBloomberg School of Public Health, Baltimore, United States

    Contribution

    Conceptualization, Writing – review and editing

    Competing interests

    No competing interests declared

12. Katharine Collins

    Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

    Contribution

    Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

13. Sarah G Staedke

    Liverpool School of Tropical Medicine, Liverpool, United Kingdom

    Contribution

    Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

14. Alfred B Tiono

    Centre National de Recherche et de Formation sur le Paludisme, Ouagadougou, Burkina Faso

    Contribution

    Supervision, Investigation, Writing – review and editing

    Competing interests

    No competing interests declared

15. Chris Drakeley

    Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, London, United Kingdom

    Contribution

    Conceptualization

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-4863-075X

16. Kjerstin Lanke

    Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

    Contribution

    Investigation, Methodology, Writing – original draft

    Competing interests

    No competing interests declared

17. Teun Bousema

    1. Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands
    2. Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, London, United Kingdom

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Writing – original draft, Writing – review and editing

    For correspondence

    teun.bousema@radboudumc.nl

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2666-094X

• 987

  views

• 121

  downloads

• 5

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.