Anti-malarial contact dependent blocking of transmission of Plasmodium vivax by Anopheles darlingi mosquito vector

Jéssica E A Kassupá; Alice O Andrade; Alessandra S Bastos; Gabriel L L Moura; Marina L Rocha; Leandro N Martinez; Elisandra K S Afonso; Wallyson J Costa; Daniel Y Bargieri; Carolina B G Teles; Jansen F Medeiros; Nirbhay Kumar; Ana Carolina R Guimarães; Douglas G Paton; Flaminia Catteruccia; Maisa S Araujo

doi:10.1371/journal.ppat.1013531

. 2026 Jul 2;22(7):e1013531. doi: 10.1371/journal.ppat.1013531

Anti-malarial contact dependent blocking of transmission of Plasmodium vivax by Anopheles darlingi mosquito vector

Jéssica E A Kassupá ^1,², Alice O Andrade ^1,³, Alessandra S Bastos ¹, Gabriel L L Moura ^1,⁴, Marina L Rocha ^1,⁴, Leandro N Martinez ⁵, Elisandra K S Afonso ⁵, Wallyson J Costa ⁵, Daniel Y Bargieri ⁶, Carolina B G Teles ^1,^2,⁵, Jansen F Medeiros ^1,⁵, Nirbhay Kumar ⁷, Ana Carolina R Guimarães ⁸, Douglas G Paton ⁹, Flaminia Catteruccia ^10,¹¹, Maisa S Araujo ^1,^2,^3,^4,^5,^12,^*

Editor: Kenneth Vernick¹³

¹Fundação Oswaldo Cruz, Fiocruz, Production and Infection of Malaria Vectors Platform (PIVEM)/Entomology, Porto Velho, Rondônia, Brazil

²Programa de Pós-Graduação em Biodiversidade e Biotecnologia da Amazônia Legal – BIONORTE, Porto Velho, Brazil

³Programa de Pós-Graduação em Saúde Pública, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil

⁴Programa de Pós-Graduação em Conservação e uso de Recursos Naturais – PPGReN, Fundação Universidade Federal de Rondônia, Porto Velho, Rondônia, Brazil

⁵Programa de Pós-Graduação em Biologia Experimental, Fundação Universidade Federal de Rondônia, FIOCRUZ Rondônia, Brazil

⁶Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

⁷Department of Global Health, George Washington University, Washington D.C., United States of America

⁸Instituto Oswaldo Cruz, IOC/FIOCRUZ, Laboratório de Genômica Aplicada e Bioinovações, Rio de Janeiro, Brazil

⁹Department of Infectious Diseases, University of Georgia, Athens, Georgia, United States of America

¹⁰Department of Immunology and Infectious Diseases, Harvard T. H. Chain School of Public Health, Boston, Massachusetts, United States of America

¹¹Howard Hughes Medical Institute, Boston, Massachusetts, United States of America

¹²Centro de Pesquisa em Medicina Tropical (CEPEM), Laboratório de Pesquisa Translacional e Clínica, Porto Velho, Rondônia, Brazil

¹³Institut Pasteur, FRANCE

The authors have declared that no competing interests exist.

^✉

* E-mail: maisa.araujo@fiocruz.br, maisaraujo@gmail.com

Roles

Jéssica E A Kassupá: Formal analysis, Investigation, Methodology, Validation, Writing – original draft

Alice O Andrade: Investigation, Methodology

Alessandra S Bastos: Methodology

Gabriel L L Moura: Methodology

Marina L Rocha: Methodology

Leandro N Martinez: Methodology

Elisandra K S Afonso: Methodology

Wallyson J Costa: Methodology

Daniel Y Bargieri: Investigation

Carolina B G Teles: Investigation

Jansen F Medeiros: Funding acquisition, Investigation

Nirbhay Kumar: Funding acquisition, Investigation, Writing – review & editing

Ana Carolina R Guimarães: Data curation, Formal analysis, Investigation

Douglas G Paton: Conceptualization, Formal analysis, Investigation, Writing – review & editing

Flaminia Catteruccia: Conceptualization, Formal analysis, Investigation, Writing – review & editing

Maisa S Araujo: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing

Kenneth Vernick: Editor

PMCID: PMC13327285 PMID: 42391120

Abstract

Malaria, caused by protozoa of the genus Plasmodium and transmitted to humans through the bite of mosquitoes of the genus Anopheles, remains a public health problem. Long-Lasting Insecticide -treated Bed Nets (LLINS) and Indoor Residual Spraying (IRS) represent the main vector control measures for malaria prevention. However, to address the concerns of mosquito resistance to pyrethroids, other malaria control strategies are being explored for effectively blocking malaria transmission by eliminating or reducing the parasite in the vector. This study evaluated the use of antimalarials through tarsal contact of female Anopheles darlingi infected with Plasmodium vivax via a Direct Membrane Feeding Assay (DMFA). Female An. darlingi were exposed tarsally using Petri dishes impregnated with antimalarials at 1 mmol/m² for exposure times of 6 or 60 minutes. Among the antimalarials evaluated were Atovaquone (ATQ), Tafenoquine (TQ), Chloroquine (CQ), Mefloquine (MQ), Primaquine (PQ), and the compound Nanchangmycin (NCG). Atovaquone was the only antimalarial evaluated before and after DMFA at exposure times of 60 min and 6 min. The results demonstrate complete elimination of P. vivax in female An. darlingi exposed to ATQ by tarsal contact 60 min before infection. ATQ was also effective 6 min before or after infection, reducing infection prevalence. In addition, MQ also significantly reduced infection intensity, but there was no difference in infection prevalence. No significant differences were observed for the other antimalarials.

Author summary

Malaria caused by Plasmodium vivax is the most prevalent in the Amazon region, with Anopheles darlingi as its main vector. Mosquito resistance to pyrethroid insecticides, already described in African countries, also raises an alarm for areas endemic for vivax malaria. Given this scenario, strategies that involve blocking parasite transmission have proven effective. Our study involved the transmission-blocking potential of antimalarials administered via tarsal contact to An. darlingi infected with P. vivax. Tarsal exposure involves the direct contact of the mosquitoes’ tarsi with surfaces impregnated with antimalarials. We speculate that in a real-world setting, this approach could be translated by treating surfaces like bed nets, eaves, or resting sites with the compounds, exploiting the natural resting and host-seeking behaviors of mosquitoes that bring their tarsi into contact with these treated substrates. If the drugs/compounds penetrate the cuticle, they could impact the parasite’s biological cycle within the vector. Our results confirmed the potential of this approach. Atovaquone eliminated or reduced P. vivax in An. darlingi midguts following different exposure times, demonstrating successful uptake after contact with treated surface. Mefloquine also reduced parasite intensity via tarsal contact. These findings reinforce the potential of this approach as a complementary tool in malaria control.

Introduction

Malaria is a disease caused by Plasmodium parasites transmitted to humans through bites of infected Anopheles mosquitoes. In 2024, malaria accounted for 282 million cases and 610,000 deaths worldwide, representing a persistent public health challenge [1]. The Global Technical Strategy for Malaria (GTS) through 2030 outlines malaria elimination measures targeting both parasites and vectors. These efforts seek to interrupt local transmission by reducing the human parasite reservoir and addressing outdoor transmission including chemotherapeutic interventions to block the malaria transmission cycle. In this context, research is expected to lead to new interventions such as vaccines, new and more effectives drugs and combinations, novel insecticides or combinations, repellents, toxic baits for vectors and other innovations in vector control [2].

Vector control, primarily through Indoor Residual Spraying (IRS) and Long-Lasting Insecticide -treated Bed Nets (LLINs), is an important malaria prevent strategy. Until recently, LLINs relied solely on a single class of insecticide, pyrethroids [1,3]. However, emergence of pyrethroids resistance has presented a primary threat to the long-term viability of LLINs [4,5], driven by its rapid geographic spread [2,4]. This highlights the need for new strategies targeting to control malaria transmission with novel active approaches [6–10] to address pyrethroid resistance and enhance the effectiveness of current malaria control strategies [8,9]. To mitigate the resistance issue includes two new classes of dual active-ingredient: pyrethroid-clorfenapyr, which combine pyrethroid and pyrrole insecticide to enhance the net’s lethality [11], and pyrethroid-pyriproxyfen nets, which pair a pyrethroid with an insect growth regulator (IGR) [12] have been developed for impregnating LLINs. Both combinations aim to improve efficacy against pyrethroid-resistant mosquitoes [8]. To contribute to these strategies, Paton et al. [13] generated a new strategy that at least partially overcomes the challenge of insecticide resistance in LLINs by blocking parasite transmission by the Anopheles mosquito. Their study exposed An. gambiae (s.s.), a primary malaria vector in Africa, to the antimalarial atovaquone (ATQ) prior to infection with P. falciparum. This direct contact assay eliminated parasites from the mosquitoes’ midguts and reduced both the intensity and prevalence of P. falciparum infection in pyrethroid-resistant An. coluzzi [14]. In a recent study, they screened additional compounds with the ability to block infections, identifying a compound combination that retained full anti-plasmodial activity even after incorporation into bed net-like substrates. Overall, these studies validate this approach as a promising malaria control tool [15].

The strategy of exposing Anopheles females to antimalarials before and also after Plasmodium infection [13–15] is based on their tendency to feed at night, when people sleep under mosquito nets. Moreover, after feeding, females rest on internal walls, likely to regain flight capacity and/or digest the blood meal before reaching a gravid state [16]. This feeding and resting behavior is characteristic of certain Anopheles species that exhibit more endophagic and endophilic [17] as well as anthropophilic traits, such as An. darlingi, a primary malaria vector in the Amazon region [18,19]. Reorienting the use of LLINs and IRS to deliver antimalarial through tarsal contact addresses key challenges associated with drug resistance in parasites and insecticide resistance in mosquitoes [14,20]. Additionally, this approach offers novel opportunities for vector targeted drug delivery [9], disrupting sporogonic development, eliminating the parasites within the mosquito, and thereby blocking transmission to humans. In this context, the present study investigates this transmission-blocking strategy by evaluating the impact of antimalarials and other compounds using the P. vivax-An. darlingi model through direct contact assay, a malaria species that is comparatively neglected, harder to study due to challenges in continuous in vitro culture, and likely to be more difficult to eliminate than P. falciparum.

Results

Exposure to Atovaquone substantially reduces infection of Anopheles darlingi with Plasmodium vivax isolates

To test whether ATQ, a parasite cytochrome-b inhibitor, could inhibit P. vivax development in mosquitoes, we allowed An. darlingi females to rest on a glass substrate coated with ATQ immediately before P. vivax infection via direct membrane feeding assay (DMFA). Exposing An. darlingi to ATQ at 1 mmol/m² for 60 min resulted in 100% inhibition of P. vivax oocysts development after the infectious blood meal, whereas control mock-exposed mosquitoes exhibited a high prevalence and intensity of infection (Fig 1A). In a subsequent experiment, the exposure time of mosquitoes to ATQ was reduced to 6 min. Although this did not completely block transmission, both prevalence and intensity of infection were significantly reduced (See Fig 1B). The transmission reduction activity (TRA) was 97.49%, while the transmission blocking activity (TBA) was above 73.97% (S1A Table).

Consistent with our previous DMFA study using P. vivax [21], neither parasitemia nor gametocytemia influenced TBA or TRA. The observed block/reduction is likely related to the ATQ exposure time, as the 60-min exposure group blocked transmission completely even with high gametocytemia, while the 6-min group did not fully block transmission even with low or zero gametocytemia (S1B Table).

In additional experiments, tarsal exposure to ATQ for 6 min before infection again impaired oocyst survival (Fig 1C) and also significantly reduced sporozoites intensity and prevalence (Fig 1D).

Parasite prevalence and intensity of P. vivax infection were also significantly reduced when mosquitoes were exposed to ATQ 24h before (Fig 2A) or 12h after infection (Fig 2B). These findings indicate that ATQ can suppress P. vivax development in the female mosquitoes both before and after an infected blood meal.

Consistent with Paton et al. [13], we confirmed that in mosquitoes exposed to ATQ for 6 min before infection, P. vivax parasites were killed during the early zygote-ookinete transition, as determined by immunofluorescence assay of infected midguts (Fig 2C). Additionally, ATQ-treated female mosquitoes had fewer parasites in the blood bolus when compared to controls (Fig 2C).

No effect on Plasmodium vivax development in mosquitoes exposed to antimalarials used in Brazil

Considering the positive results of the direct contact assay using the antimalarial ATQ, we also tested antimalarials used in Brazil against P. vivax, such as primaquine (PQ), tafenoquine (TQ), chloroquine (CQ) and mefloquine (MQ) [22]. These were evaluated at the maximum exposure time of 60 minutes and at the same concentration as ATQ. The antimalarials PQ, TQ and CQ did not achieve blocking or reduction of infection (Fig 3A, 3B and 3C). Mefloquine-exposed mosquitoes had a significant reduction in oocyst intensity compared to the control (P = 0.0002), but the prevalence of infection was not affected (Fig 3D).

Fig 3 — A) Primaquine (PQ): n = 107, prevalence 93.1%, degrees of freedom (df) = 1, χ2 = 0.06161, P = 0.8040, ns. Oocyst intensity P = 0.085, n = 99, U = 970.5, ns; B) Tafenoquine (TQ): n = 97, prevalence 95.9%, degrees of freedom (df) = 1, χ2 = 0.3231, P = 0.5698, ns. Oocyst intensity P = 0.5658, n = 94, U = 1028, ns; C) Chloroquine (CQ): n = 136, prevalence 97.3%, degrees of freedom (df) = 1, χ2 = 1.045, P = 0.3067; Oocyst intensity P = 0.7328, U = 2021, n = 130, ns; and D) Mefloquine (MQ): n = 123, prevalence 96.7%, degrees of freedom = 1, χ2 = 0.3937, P = 0.5303, n = 120, ns; Oocyst intensity ***P = 0.0002, U = 1102, significant. The results shown are from three biological replicates (see S1B and S1C Table). Median values are indicated for all tests.

To investigate whether the physical barrier formed by the mosquito cuticle hindered the uptake of MQ after tarsal exposure, the drug was directly added to the infected blood meal at a final concentration of 10 μM and then offered to An. darlingi females. Atovaquone was used as a positive control. When added to the infected blood prior to infection, ATQ blocked/reduced oocysts development at the 7^th day post-DMFA (Fig 4A), and sporozoite infection at the 14^th day post-DMFA (Fig 4B). Mefloquine significantly reduced the oocyst and sporozoite intensity of infection (Fig 4C and 4D), showing that MQ can reduce the transmission as in the tarsal exposition (Fig 3D) – highlighting its potential, albeit less potent than ATQ, as a candidate for mosquito-target transmission-blocking strategies. Note that our methodology for P. vivax infection is designed to generate much higher infection intensities with extremely high parasite loads (high median oocyst and sporozoite counts). This allows detection of smaller biological effects, and reduces the need for replication and additional human volunteers. There is a general consensus that oocyst loads in the wild are rather much lower, with each infected wild mosquito carrying less than five oocysts [23,24].

Fig 4 — A) Atovaquone (ATQ) added to the blood meal completely blocked *P. vivax* oocysts in the midguts and sporozoites in the salivary glands (B) of female mosquitoes. Oocyst prevalence: two-sided chi-squared test, n = 180, degrees of freedom (df) = 1, χ2 = 176.0, ^****P < 0.0001. Intensity: two-sided Mann-Whitney U test, n = 179, U = 0, ^****P < 0.0001. B) Sporozoite prevalence: two-sided chi-squared test, n = 224, degrees of freedom (df) = 1, χ2 = 124.8, ^****P < 0.0001. Intensity: two-sided Mann-Whitney U test, n = 126, U = 10, ^****P < 0.0001. C) The presence of Mefloquine (MQ) in the *P. vivax* blood meal partially reduced oocysts in the midguts of female mosquitoes and sporozoites in the salivary glands **(D)**. Oocyst prevalence **(C)**: two-sided chi-squared test, n = 180, degrees of freedom (df) = 1, χ2 = 0.3390, P = 0.5604. Intensity: two-sided Mann-Whitney U test, n = 177, U = 2714, ^***P = 0.0004. D) Sporozoite prevalence: two-sided chi-squared test, n = 233, degrees of freedom (df) = 1, χ2 = 2.459 P = 0.1168. Intensity: two-sided Mann-Whitney U test, n = 231, U = 4841, ^**P = 0.0005. The results shown are from three biological replicates (see S1 B and S1C Table). Medians are indicated.

Tarsal exposure of Anopheles darlingi to Nanchangmycin (NCG) does not reduce infection with Plasmodium vivax

Nanchangmycin (NCG) is a polyketide antibiotic [25] and was previously shown to block P. vivax development in An. darlingi when added to the infected blood prior to mosquito feeding [26]. Tarsal exposure of NGC to mosquito however did not significantly reduce the transmission of P. vivax to the vector (Fig 5).

Fig 5 — n = 111, prevalence 88.14%. Prevalence (Prev.): two-sided chi-squared test, n = 111, degrees of freedom (df) = 1, χ² = 1.253, P = 0.2630, ns. Intensity: two-sided Mann-Whitney U test, df = 1, U = 1082, n = 101, P = 0.1931, ns. The results shown are from three biological replicates (see S1 B and S1C Table). Medians are indicated.

Antimalarials and Nanchangmycin (NCG) do not affect the survival of Anopheles darlingi females after tarsal exposure

Since some compounds, in addition to affecting Plasmodium development, may also impact the survival or overall fitness of mosquitoes, we also assessed the survival of mosquitoes following direct contact assay. The results indicated that mosquito survival until 7^th day post-DMFA was not affected in any of the experimental groups exposed to tarsal treatment (Fig 6, Table 1).

Fig 6 — A) Atovaquone (ATQ) for 60 min. Two-sided Log-rank. (Mantel-Cox), X² = 0.08946, *P = 0.7649*. B) Atovaquone (ATQ) for 6 min. Log-rank test (Mantel-Cox), X² = 0.7326, *P = 0.3921*. C) Atovaquone for 6 min. 24 hours before infection (24h.a.i). Log-rank test (Mantel-Cox), X² = 0.1230, *P = 0.7258*. D) Atovaquone for 6 min. 12 hour after infection (12h.d.i). Log-rank test (Mantel-Cox), X² = 2.461, *P = 0.1167.* E) Chloroquine for 60min. Log-rank test (Mantel-Cox), X² = 0.5646, *P = 0.4524*. F) Mefloquine for 60 min. Log-rank test (Mantel-Cox), X² = 0.1207, *P = 0.7283*. G) Primaquine for 60 min. Log-rank test (Mantel-Cox), X² = 0.2012, *P = 0.6538*. H) Tafenoquine for 60 min. Log-rank test (Mantel-Cox), X² = 0.6015, *P = 0.4380.* I) Nanchangmycin for 60 min. Log-rank test (Mantel-Cox), X² = 0.4474, *P = 0.5036*. Survival results were not significant for all antimalarial drugs evaluated (see Tables 1 and S1C).

Table 1. Hazard ratio estimated by Cox model until 7 days post infection.

Reference in the model
	Control	p-value
	HR (CI 95%)	p-value
ATQ_60m	1.1568 (0.4566-2.931)	0.8
ATQ_6m	1.8133 (0.4535-7.25)	0.4
ATQ_6m_(24h.a.i.)	1.4001 (0.2339-8.379)	0.7
ATQ_6m_(12h.d.i.)	0.2112 (0.02467-1.808)	0.1
CQ_60m	1.6681 (0.4172-6.67)	0.5
MQ_60m	0.8418 (0.3135-2.26)	0.7
PQ_60m	0.8262 (0.3508-1.945)	0.7
TQ_60m	0.6169 (0.1806-2.107)	0.4
NCG_60m	1.6102 (0.3848-6.738)	0.5

Open in a new tab

Hazard ratios were estimated using a Cox proportional hazards model, considering independent controls for each antimalarial and compound tested. Values are presented as hazard ratio (HR) with 95% confidence intervals (95% CI). ATQ: Atovaquone; CQ: Chloroquine; MQ: Mefloquine; NCG: Nanchangmycin; PQ: Primaquine; TQ: Tafenoquine.

Comparative structural modeling and molecular docking identify a conserved ATQ-binding site in Plasmodium vivax cytochrome b

To further explore ATQ’s mechanism, we investigated its interaction with the cytb target using molecular docking. Although an experimental structure of P. vivax cytb is unavailable, we used the Alphafold-predicted model (AF-O63696-F1-v4). Structural alignment of this model with the Saccharomyces cerevisiae cytbc1 complex (PDB ID: 4pd4), which contains a co-crystallized ATQ model, yielded a Root Mean Square Deviation (RMSD) of 0.824 Â, indicating high structural similarity (S1 Fig).

The docking validation was performed by redocking ATQ into the S. cerevisiae Qo site, confirming the method’s reliability. Subsequent docking simulations on the P. vivax cytb model using the same parameters revealed ATQ binding to a conserved hydrophobic pocket (S2 Fig). The binding pose involved key residues, including Phe123, Met133, Trp136, Gly137, Val140, Ile258, Leu285, Leu288, Pro260, Phe264, Tyr268, Leu271, Ile141, Phe267, and Val284). Notably Met133, Tryr268, and Val284 have been associated with ATQ resistance in P. falciparum, and P. berghei [15,27,28]. The calculated binding energy for ATQ in the P. vivax model was – 9.825 kcal/mol. Electrostatic surface analysis further demonstrated physicochemical compatibility between ATQ and the binding site in P. vivax.

Discussion

Previous studies of Paton et al. [13] garnered significant attention within the scientific community with a landmark study demonstrating that incorporating ATQ into a glass substrate - on which blood-fed Anopheles mosquitoes rested - effectively eliminated P. falciparum parasites within the mosquitoes’ midgut blood meal. This novel approach, which delivers an antimalarial compound through surface contact during the mosquito’s resting phase pre- or post-blood-feeding, represents a highly innovative strategy to disrupt the Plasmodium transmission cycle and offers numerous advantages [29]. However, the effect of ATQ on the sporogonic development of P. vivax had yet to be evaluated. Here, we observed that the P. vivax development in An. darlingi mosquitoes was significantly impaired when mosquitoes were exposed to ATQ before or shortly after infection.

Numerous chemical compounds, including ATQ, have demonstrated efficacy against P. falciparum parasites, as well as against P. berghei and P. yoelii during sporogony [15,20,30–32]. However, data on the effects of ATQ on the sporogonic development of P. vivax remain limited, as do studies on other antimalarials [33,34]. Atovaquone is well-known for its dual activity against both the initial liver and the pathogenic erythrocytic stages of P. falciparum and P. vivax [28,35]. Our in vitro experiments have shown that ATQ effectively eliminates asexual stages (S2 Table) and ookinetes (S3 Table) of P. vivax clinical isolates obtained from patients. Due to its strong activity, ATQ has been used as a positive control in some of our in vitro assays.

Notably, ATQ was the only antimalarial among those tested that was capable of penetrating the cuticle of mosquito legs via tarsal contact, directly reaching midgut via the hemolymph and blocking P. vivax oocyst development in An. darlingi. For this strategy to work, compounds/antimalarials must overcome the exoskeleton barrier to access internal tissues where the parasite develops. To achieve this, the compounds should possess specific characteristics [15]. First, it must be lipophilic; with a positive logP value, as lipophilicity is a critical factor in a compound’s absorption, distribution, membrane penetration, and overall pharmacokinetic properties (ADME: absorption, distribution, metabolism, and excretion). Lipophilicity is a key parameter used in pharmaceutical and biotech industries to evaluate drug efficacy. Second, the polar surface area (PSA) of the compound should not be excessively large, as larger molecules may have to overcome cuticle penetration resistance [13]. Moreover, the compound must exhibit intrinsic antiplasmodial activity.

Atovaquone, a ubiquinone analog, inhibits the mitochondrial electron transport chain by displacing ubiquinone, thereby disrupting ATP synthesis and de novo pyrimidine biosynthesis, ultimately leading to parasite growth inhibition [36]. Given the observed effectiveness of ATQ, future studies will evaluate a dose-response effect and analytically determine time kinetics of ATQ dissemination through the cuticle and persistence in the midgut and salivary glands. Furthermore, ATQ is highly lipophilic, with a positive logP and a PSA of 54.4 Â² (S4 Table). Based on immunofluorescence assays using anti-Pvs25 [37,38], our results suggest that ATQ also targets P. vivax during the early zygote-ookinete transition, as previously shown for P. falciparum [13]. This is consistent with the parasite’s development window, 18–24 hours post-infection, when ookinetes are typically formed in the midgut (Fig 2C).

To further explore ATQ’s mechanism, our comparative modeling and approach a structure conservation of the Qo binding site between the S. cerevisiae crystal structure and the P. vivax Alphafold model, particularly with the ATQ-binding pocket, reinforces the validity of our findings. The docking results support that ATQ binds to a conserved hydrophobic pocket in P. vivax cytb model with high affinity, as indicated by the favorable binding energy. This binding mode is consistent with the mechanism described in other systems by Birth et al. [39], despite noted differences in residues numbering and the identity of four residues between the P. vivax and S. cerevisiae proteins (S3 Fig). The influence of these specific residue differences on the binding dynamics and affinity of ATQ remains to be fully elucidated and represents a key direction for future work, ideally through molecular dynamics simulations. The strong and electrostatic conservation supports the use of P. vivax cytb model for the future in silico screening of novel transmission-blocking compounds.

Mefloquine, another antimalarial tested, affected oocyst and sporozoite intensity but not infection prevalence (Fig 4C and 4D). Commonly used for prophylaxis and combination therapies [40], MQ is a 4-methanolquinoline structurally related to quinine [40,41]. Its proposed mechanism involves inhibition of heme detoxification [42–44]. Interestingly, a study on the inhibition of esophageal carcinoma cell growth in vitro observed the downregulation of protein expression in all subunits involved in oxidative phosphorylation. Proteomic analysis indicated that mitochondria are particularly affected by MQ [45], including the bc1 complex, pointing to similarities to the mode of action of ATQ. A previous study conducted by Li et al. [46] also highlighted MQ’s ability to inhibit mitochondrial respiration. Mefloquine exhibits stage-specificity action similar to quinine, primarily targeting large ring and trophozoite stage asexual parasites [40,42,44]. There is also some evidence for sporontocidal activity. For instance, Coleman et al. [47] showed dose-dependent effects on P. berghei ANKA sporogony in An. stephensi, with changes in oocyst numbers and the extent of sporozoite invasion into salivary glands. Further research is needed to fully elucidate the mechanisms underlying MQ’s effects on parasite development. Mefloquine’s physicochemical properties permit cuticle penetration (S4 Table), unlike TQ, PQ and NCG, all of which have PSA > 60 Â².

Interestingly, NCG completely blocked transmission of P. vivax in An. darlingi and P. falciparum in An. stephensi during DMFA and SMFA assays, respectively [26] (Calit et al. 2023), but had no effect when administered to mosquitoes via tarsal exposure likely due to its higher PSA (S3 Table). Nanchangmycin is a polyether ionophore antibiotic produced by Streptomyces nanchangensis [25] and has known insecticidal properties against silkworms and anti-bacterial activity in vitro [48,49]. Alternative delivery methods, such as attractive toxic sugar baits (ATSBs), could be explored for such compounds [20].

Chloroquine, although possessing a low PSA (S4 Table), which likely allows it to penetrate the cuticle of mosquito legs via tarsal contact and reach the hemolymph, did not show any evidence of sporontocidal activity. Measuring compound concentrations in mosquito hemolymph using HPLC after tarsal exposure could clarify pharmacokinetics profile and drug-Plasmodium interactions in mosquitoes [50,51]. Paton et al. [13] demonstrated that the cytochrome b inhibitor decoquinate (DEC) and the known transmission-blocking antifolate pyrimethamine (PYR) were ineffective in blocking Plasmodium development in exposed mosquitoes, likely due to higher PSA. Recently, harmane – a small, hydrophobic β-carboline secreted by Delfitia tsuruhatensis - was found to fully inhibit P. falciparum development via tarsal penetration [52]. Harmane has small PSA (28,7 Â²; PubChem, 2024), which likely supports this trans-cuticle uptake.

Regarding potential mosquito fitness costs, Paton et al. [13] reported no effect in survival rate and fecundity of Anopheles mosquitoes at 48 hours after 60 minutes of exposure to concentrations up to 1 mmol/m² via tarsal contact. In our experiments, mosquito survival was evaluated until the 7^th day post-contact and P. vivax infection, and no reductions in survival were observed for any compounds tested. These results suggest that ATQ, and even MQ, selectively target P. vivax without compromising mosquito viability.

Despite the promising effects of ATQ and MQ on P. vivax development in An. darlingi, it is crucial to recognize the limitations of applying human-use antimalarials for vector-targeted control. Malaria parasites have evolved resistance to nearly all antimalarial used in humans, thus, it would be naïve to assume that compounds targeting parasites during sporogony will not face any resistance evolution, however the vast differences in parasite density between the human and mosquito hosts does make this less likely. As Kamiya et al. [20] proposed, using different compounds in humans and mosquitoes could reduce selective pressure for resistance and drug combination therapy has been shown to be highly effective against mosquito stages of P. falciparum [15]. Furthermore, it is important to evaluate the potential impact of this vector control strategy in a specific context like the Amazon Basin. The resting and feeding behaviour of An. darlingi – which is often plastic, exophagic, and not exclusively endophagic – presents a challenge. Deploying ATSB in this region could be challenging, as mosquitoes have access to many other sugar sources.

Our proof-of-concept study showing that with ATQ blocking P. vivax in An. darlingi provides a foundation for identifying compounds with structural and chemical similarities to ATQ for further evaluation. Using dedicated compounds to target parasites during sporogony offers an additional control strategy, which could reduce the reliance on human antimalarials for suppressing transmission. This approach represents a promising avenue for integrated malaria control, combining interventions in both humans and mosquitoes to achieve sustainable transmission reduction.

Materials and methods

Ethics statement

Individuals who participated in the study were selected from patients diagnosed with vivax malaria through Giemsa-stained blood smears collected at the Center for Tropical Medicine Research (CEPEM) in Porto Velho, Rondônia, an endemic region in the Brazilian Amazon (under protocol approval number #28176720.9.0000.0011). Written and verbal informed consent was obtained from each participant prior to blood collection.

Anopheles darlingi Direct Membrane-Feeding Assays (DMFA)

Females An. darlingi mosquitoes were obtained from the colony established at the Malaria Vectors Production and Infection Platform (PIVEM), located at FIOCRUZ-RO, Brazil, as described by Araujo et al. [53]. The colony, initiated in 2017 [54], has been maintained without the introduction of wild mosquitoes. Mosquitoes were reared at a temperature of 26°C ± 1°C and a relative humidity of 70 ± 10%, being fed a 15% honey solution.

For the experiments, 10–50 female An. darlingi mosquitoes, 3–5 days old, were used for each experimental and control group. Sucrose food was removed the day before the DMFA.

Tarsal exposure test

For tarsal exposure, the compound or antimalarial were diluted in an appropriate volatile vehicle. Solutions were prepared considering the area of the Petri dish and the molecular weight of the compound or antimalarial (S4 Table) [13].

One milliliter of the prepared solution at a concentration of 1 mmol per $m^{2}$ ( $m g / m^{2})$ was pipetted onto the Petri dish. The volatile vehicle used for compound dilution was used as control. The plates were then kept overnight on an orbital shaker at 25°C and 100 rpm to coat the entire area of the plate. A transparent plastic container of the same diameter as the Petri dish was chosen to allow the two parts to fit together. This container was then adapted to introduce the mosquitoes, ensuring that their tarsi were in contact with the compound or antimalarial-impregnated surface of the plates. These procedures were performed as described by Paton et al. [13].

The antimalarials tested in this tarsal exposure assay were atovaquone (ATQ), primaquine (PQ), chloroquine (CQ), mefloquine (MQ), tafenoquine (TQ), and the compound nanchangmycin (NCG), with an initial exposure time of 60 minutes before infection (60 m.b.i.). Direct membrane feeding assay was then performed as described below.

When the exposure time of 60 min was efficient to block mosquito infection, the time was reduced to 6 minutes. It also was tested mosquito exposure to the compounds 24 h before or 12 h after infection.

Blood collection from patients with diagnosed Plasmodium vivax infection

Volunteers were selected based on the following criteria: monoinfection with P. vivax by thick blood smear (parasitemia > 2000 parasites/ µL), age between 18 and 85 years, without signs or symptoms of severe malaria or concomitant diseases, with or without a previous history of malaria, no pregnant, and agreed to the study procedures.

Direct membrane-feeding assays

Patient’s venous blood was collected in lithium heparin tubes (10 mL, Vacutainer, BD) and then transported in a thermal bottle at 37°C from CEPEM to PIVEM. The heparinized tube was centrifuged at 1,500 rpm for 10 minutes. Only the red blood cells were used. Prior to mosquito feeding, 500 µL of inactive AB⁺ serum was mixed with 500 µL of parasitized red blood cells obtained [55].

The prepared blood was offered to the female mosquitoes of each group for 30 minutes using glass feeders attached to a water bath or disc with the Hemotek device, as previously described [53]. After this period, unfed or partially fed mosquitoes were removed, leaving only fully engorged mosquitoes in the experimental cages for subsequent examination of sporogonic development. A cotton wad soaked in a 15% honey solution was regularly provided and changed every two days until mosquito dissection. The mosquito survival was evaluated daily until the 7^th day after blood feeding, when the midgut was dissected. To determinate the sporozoite intensity of infection, salivary glands were dissected at the 14^th day post-DMFA.

For dissection, mosquitoes were anesthetized on ice, immersed in 70% ethanol, and transferred to phosphate-buffered saline (1X PBS). The midguts were stained with 0.2% mercurochrome solution and examined for the presence of oocysts by microscopy.

For experiments involving the addition of antimalarials to P. vivax-infected blood, which was then offered orally to female An. darlingi, the antimalarials were first mixed with the inactivated AB⁺ serum (5 μL of antimalarial + 495 μL of serum). Patient-derived red blood cells (separated by centrifugation, as previously described) were then admixed, resulting in final volume of 1,000 μL with 10μM of the antimalarial.

Ookinete immunofluorescence assays – IFA

Twenty-one hours after an infectious blood meal, ten female mosquitoes from control and ATQ groups were aspirated into 1x PBS at 4° C. Midguts with blood bolus were isolated and transferred to 20 μL of 1X PBS on ice. Guts were disrupted by pipetting and the crude isolate homogenized by vortexing briefly (about 5 seconds), and 10 μL of the homogenate was spotted onto a poly-L-lysine-coated slide and air dried. Once dry, the tissues were fixed by incubation with 4% paraformaldehyde (PFA) for 10 minutes [13]. Slides were then rinsed three times with 5–10 μL of 1x PBS, to blocked with 1% BSA in 1 x PBS for 1 h and then rinsed again three times with 5–10 μL 1x PBS. Ookinetes were stained with mouse antibody raised against the P. vivax surface protein Pvs25 (100 μg/mL) for 1 h in a humid box, at room temperature [37,38]. Secondary staining was carried out with 1:100 dilution of Alexa Fluor 488 goat anti mouse IgG (Invitrogen) for 1 h a dark humid box. After several washes with 1 x PBS, the cells were counter stained with Hoechst 33342 (10 μg/mL), washed and then the tissues were mounted in Everbrite mounting medium (Biotium). Slides were examined by fluorescence microscopy (Nikon Eclipse 80i) with 100x oil immersion objective, and the images were captured using the software Nikon Nis Elements.

Statistics analysis

Sample sizes were determined via a priori power analysis to determine the sample size required to detect a 50% reduction in oocyst intensity (n = 21 for power 0.9, μa 20, μb 10, SD 10, equal variance assumed) and to detect a 50% reduction in prevalence (n = 18, power = 0.9, PA = 0.9, PB = 0.45) based on a conservative estimate of the expected effect of ATQ in these experiments. Data of infection prevalence and the proportion of parasite life stages (zygote, retort and ookinete) were analyzed using the Chi-square test. In experiments in which both treatment groups had individuals that produced > 0 oocysts, differences in median oocyst burden between groups (intensity of infection) was analyzed using Mann-Whitney mean ranks test. Experiments in which the controls presented means lower than 2.5 oocysts per mosquito and infection prevalence below 60% were not taken into consideration in the statistical analyses (S1A and S1B Table) as described by [33,53,56]. The transmission-reducing assay (TRA) was measured as the percentage of reduction in mean oocyst density, using the formula: TRA = [(Mean oocyst count in control group – Mean oocyst count in test group)/Mean oocyst count in control group] x 100, including zero oocyst mosquitoes. The transmission-blocking assay (TBA) was evaluated as the percentage inhibition in infection prevalence, calculated as: TBA = [(Prevalence in group – prevalence in test group)/ Prevalence in control group] x 100 [56,57] (S1A Table).

Female mosquitoes exposed to antimalarials and the NCG compound were monitored until the seventh day after infection to verify survival with the Kaplan-Meier survival curve and the survival rate was compared using the Log-rank test. Cox proportion hazards regression was employed to estimate the hazard ratio, and the likehood-ratio test was performed to assess overall significance of the model.

All infection experiments included two to seven biological replicates (S1C Table). Statistical analyses were conducted in R program (version 3.6.3, R Foundation for Statistical Computing, Austria), and GraphPad Prism software (version 9.3.1).

Supporting information

S1 Table. Individual and summary data from Direct Membrane Feeding Assays (DMFA).

A) Transmission-reducing activity (TRA, %) and transmission blocking activity (TBA, %) for each DMFA. B) Individual DMFA results. C) Summary of DMFA data.

(XLSX)

ppat.1013531.s001.xlsx^{(108.2KB, xlsx)}

S2 Table. Characteristics of Plasmodium vivax obtained from patients and used in ex vivo assay with Atovaquone (ATQ).

(DOCX)

ppat.1013531.s002.docx^{(29.6KB, docx)}

S3 Table. Characteristics of Plasmodium vivax obtained from patients and used in ookinete inhibition assay with Atovaquone (ATQ).

(DOCX)

ppat.1013531.s003.docx^{(26.5KB, docx)}

S4 Table. Chemical properties from all compound’s testes in this study – atovaquone, primaquine, tafenoquine, chloroquine, mefloquine and nanchangmycin.

(DOCX)

ppat.1013531.s004.docx^{(28KB, docx)}

S1 Fig. Structural alignment.

Structural superposition of Plasmodium vivax cytochrome b (light pink, AlphaFold model AF-O63696-F1-v4) and the C-chain of Saccharomyces cerevisiae cytochrome bc1 complex (green, PDB ID: 4pd4). Atovaquone (ATQ) is shown in yellow sticks as positioned in the crystallographic structure of Saccharomyces cerevisiae. The Root Mean Square Deviation (RMSD) between the two models was 0.824 Å, indicating high structural similarity. Structural visualization was performed in ChimeraX.

(TIFF)

ppat.1013531.s005.tiff^{(1.7MB, tiff)}

S2 Fig. Docking result: Atovaquone (ATQ) binding site on the cytochrome b (AlphaFold ID: AF-O63696-F1-v4).

A) Tertiary structure of the cytochrome b (AlphaFold ID: AF-O63696-F1-v4) shown as a light pink ribbon. A transparent surface highlights the overall shape of the protein. ATQ is represented in yellow sticks. B) Close-up of ATQ-binding residues shown in ball-and-stick representation with residue names labeled (Phe123, Met133, Trp136, Gly137, Val140, Ile258, Leu285, Leu288, Pro260, Phe264, Tyr268, Leu271, Ile141, Phe267, and Val284). C) Electrostatic surface potential of the ATQ binding pocket. Red indicates negatively charged regions, blue indicates positively charged regions, and white indicates neutral areas. Structural visualization was performed in ChimeraX.

(TIFF)

ppat.1013531.s006.tiff^{(8.9MB, tiff)}

S3 Fig. Sequence alignment.

Sequence alignment of Plasmodium vivax cytb proteins (AF-O63696) and Saccharomyces cerevisiae Cytb C chain (PDB ID: 4pd4), performed using Clustalw. Residues highlighted in yellow correspond to those involved in atovaquone (ATQ) binding. Green arrows indicate residues that changed between proteins. Image made in ESPript 3.0.

(TIFF)

ppat.1013531.s007.tiff^{(8.9MB, tiff)}

S1 File. Description of methods to obtain data from S1 to S3 Fig.

(DOCX)

ppat.1013531.s008.docx^{(23.6KB, docx)}

Acknowledgments

We thank the staff of the Malaria Outpatient Clinic at the Centro de Pesquisa de Medicina Tropical de Rondônia (CEPEM) in Porto Velho/Rondônia/Brazil for recruiting study participants and collecting blood samples, as well as the volunteers who donated blood for this study.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This research was funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico CNPq/MCTI/FNDCT Nº 19/2024 – Centros Avançados em Áreas Estratégicas para o Desenvolvimento Sustentável da Região Amazônica - Pro- Amazônia [N° 444882/2024-3] (J.F.M and M.S.A), Programa de Excelência em Pesquisa – PROEP (PRES-028-FIO-24-2-11 to J.F.M) and NIH grant R01-AI183533 to N.K. The facilities (Rede de Plataformas Tecnológicas Fiocruz: RPT) used in this study were funded by Fundação Oswaldo Cruz (M.S.A). J.M.F. is CNPq productivity fellows (process number: 304830/2022-4). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.World Health Organization. World malaria report 2025: addressing inequity in the global malaria response. Geneva: World Health Organization. 2025. https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2025 [Google Scholar]
2.World Health Organization. Global technical strategy for malaria 2016-2030, 2021 update. Geneva. 2021. https://www.who.int/publications/i/item/9789240031357 [Google Scholar]
3.Hougard JM, Corbel V, N’Guessan R, Darriet F, Chandre F, Akogbéto M, et al. Efficacy of mosquito nets treated with insecticide mixtures or mosaics against insecticide resistant Anopheles gambiae and Culex quinquefasciatus (Diptera: Culicidae) in Côte d’Ivoire. Bull Entomol Res. 2003;93(6):491–8. doi: 10.1079/ber2003261 [DOI] [PubMed] [Google Scholar]
4.Sherrard-Smith E, Winskill P, Hamlet A, Ngufor C, N’Guessan R, Guelbeogo MW, et al. Optimising the deployment of vector control tools against malaria: a data-informed modelling study. Lancet Planet Health. 2022;6(2):e100–9. doi: 10.1016/S2542-5196(21)00296-5 [DOI] [PubMed] [Google Scholar]
5.Hemingway J, Ranson H, Magill A, Kolaczinski J, Fornadel C, Gimnig J, et al. Averting a malaria disaster: will insecticide resistance derail malaria control? Lancet. 2016;387(10029):1785–8. doi: 10.1016/S0140-6736(15)00417-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Goodman CD, Buchanan HD, McFadden GI. Is the mitochondrion a good malaria drug target? Trends in Parasitology. 2017;33(3):185–93. doi: 10.1016/j.pt.2016.10.002 [DOI] [PubMed] [Google Scholar]
7.Plouffe D, Brinker A, McNamara C, Henson K, Kato N, Kuhen K, et al. In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen. Proc Natl Acad Sci U S A. 2008;105(26):9059–64. doi: 10.1073/pnas.0802982105 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.World Health Organization. WHO publishes recommendations on two new types of insecticide-treated nets. https://www.who.int/news/item/14-03-2023-who-publishes-recommendations-on-two-new-types-of-insecticide-treated-nets Accessed 2023 October 1. [Google Scholar]
9.Bargieri DY, Williamson KC. Targeting Malaria in Flight. Cell Host Microbe. 2019;25(5):631–2. doi: 10.1016/j.chom.2019.04.007 [DOI] [PubMed] [Google Scholar]
10.Parker JEA, Angarita-Jaimes N, Abe M, Towers CE, Towers D, McCall PJ. Infrared video tracking of Anopheles gambiae at insecticide-treated bed nets reveals rapid decisive impact after brief localised net contact. Sci Rep. 2015;5:13392. doi: 10.1038/srep13392 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.N’Guessan R, Odjo A, Ngufor C, Malone D, Rowland M. A Chlorfenapyr Mixture Net Interceptor® G2 Shows High Efficacy and Wash Durability against Resistant Mosquitoes in West Africa. PLoS One. 2016;11(11):e0165925. doi: 10.1371/journal.pone.0165925 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Tiono AB, Ouédraogo A, Ouattara D, Bougouma EC, Coulibaly S, Diarra A, et al. Efficacy of Olyset Duo, a bednet containing pyriproxyfen and permethrin, versus a permethrin-only net against clinical malaria in an area with highly pyrethroid-resistant vectors in rural Burkina Faso: a cluster-randomised controlled trial. Lancet. 2018;392(10147):569–80. doi: 10.1016/S0140-6736(18)31711-2 [DOI] [PubMed] [Google Scholar]
13.Paton DG, Childs LM, Itoe MA, Holmdahl IE, Buckee CO, Catteruccia F. Exposing Anopheles mosquitoes to antimalarials blocks Plasmodium parasite transmission. Nature. 2019;567(7747):239–43. doi: 10.1038/s41586-019-0973-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Paton DG, Probst AS, Ma E, Adams KL, Shaw WR, Singh N, et al. Using an antimalarial in mosquitoes overcomes Anopheles and Plasmodium resistance to malaria control strategies. PLoS Pathog. 2022;18(6):e1010609. doi: 10.1371/journal.ppat.1010609 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Probst AS, Paton DG, Appetecchia F, Bopp S, Adams KL, Rinvee TA, et al. In vivo screen of Plasmodium targets for mosquito-based malaria control. Nature. 2025;643(8072):785–93. doi: 10.1038/s41586-025-09039-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Burrows J, Slater H, Macintyre F, Rees S, Thomas A, Okumu F, et al. A discovery and development roadmap for new endectocidal transmission-blocking agents in malaria. Malar J. 2018;17(1):462. doi: 10.1186/s12936-018-2598-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Strode C, Donegan S, Garner P, Enayati AA, Hemingway J. The impact of pyrethroid resistance on the efficacy of insecticide-treated bed nets against African anopheline mosquitoes: systematic review and meta-analysis. PLoS Med. 2014;11(3):e1001619. doi: 10.1371/journal.pmed.1001619 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Carlos BC, Rona LDP, Christophides GK, Souza-Neto JA. A comprehensive analysis of malaria transmission in Brazil. Pathog Glob Health. 2019;113(1):1–13. doi: 10.1080/20477724.2019.1581463 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Tadei WP, Dutary Thatcher B. Malaria vectors in the Brazilian amazon: Anopheles of the subgenus Nyssorhynchus. Rev Inst Med Trop Sao Paulo. 2000;42(2):87–94. doi: 10.1590/s0036-46652000000200005 [DOI] [PubMed] [Google Scholar]
20.Kamiya T, Paton DG, Catteruccia F, Reece SE. Targeting malaria parasites inside mosquitoes: ecoevolutionary consequences. Trends Parasitol. 2022;38(12):1031–40. doi: 10.1016/j.pt.2022.09.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Santos NAC, Andrade AO, Santos TC, Martinez LN, Ferreira AS, Bastos AS, et al. Evaluation of sustainable susceptibility to Plasmodium vivax infection among colonized Anopheles darlingi and Anopheles deaneorum. Malar J. 2022;21(1):163. doi: 10.1186/s12936-022-04204-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Brasil M da S, Secretaria de Vigilância em Saúde, Departamento de Imunização e Doenças Transmissíveis. Guia de tratamento da malária no Brasil. Brasília: Ministério da Saúde. 2020. https://www.gov.br/saude/pt-br/centrais-de-conteudo/publicacoes/svsa/malaria/tratamento/guia_tratamento_malaria_2nov21_isbn_site.pdf/view [Google Scholar]
23.Carr AL, Rinker DC, Dong Y, Dimopoulos G, Zwiebel LJ. Transcriptome profiles of Anopheles gambiae harboring natural low-level Plasmodium infection reveal adaptive advantages for the mosquito. Sci Rep. 2021;11(1):22578. doi: 10.1038/s41598-021-01842-x [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Rosenberg R. Malaria: some considerations regarding parasite productivity. Trends Parasitol. 2008;24(11):487–91. doi: 10.1016/j.pt.2008.07.009 [DOI] [PubMed] [Google Scholar]
25.Wollesen M, Mikkelsen K, Tvilum MS, Vestergaard M, Wang M, Meyer RL, et al. Polyether Ionophore Antibiotics Target Drug-Resistant Clinical Isolates, Persister Cells, and Biofilms. Microbiol Spectr. 2023;11(4):e0062523. doi: 10.1128/spectrum.00625-23 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Calit J, Araújo JE, Deng B, Miura K, Gaitán XA, Araújo M da S, et al. Novel Transmission-Blocking Antimalarials Identified by High-Throughput Screening of Plasmodium berghei Ookluc. Antimicrob Agents Chemother. 2023;67(4):e0146522. doi: 10.1128/aac.01465-22 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Goodman CD, Siregar JE, Mollard V, Vega-Rodríguez J, Syafruddin D, Matsuoka H, et al. Parasites resistant to the antimalarial atovaquone fail to transmit by mosquitoes. Science. 2016;352(6283):349–53. doi: 10.1126/science.aad9279 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Balta VA, Stiffler D, Sayeed A, Tripathi AK, Elahi R, Mlambo G, et al. Clinically relevant atovaquone-resistant human malaria parasites fail to transmit by mosquito. Nat Commun. 2023;14(1):6415. doi: 10.1038/s41467-023-42030-x [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Burrows J, Fidock DA, Miller RS, Rees S. Blocking Plasmodium Development in Mosquitoes: A Powerful New Approach for Expanding Malaria Control Efforts. Am J Trop Med Hyg. 2019;101(4):734–5. doi: 10.4269/ajtmh.19-0318 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Delves M, Plouffe D, Scheurer C, Meister S, Wittlin S, Winzeler EA, et al. The activities of current antimalarial drugs on the life cycle stages of Plasmodium: a comparative study with human and rodent parasites. PLoS Med. 2012;9(2):e1001169. doi: 10.1371/journal.pmed.1001169 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Butcher GA, Sinden RE. Persistence of atovaquone in human sera following treatment: inhibition of P. falciparum development in vivo and in vitro. Am J Trop Med Hyg. 2003. doi: 10.4269/ajtmh.2003.68.111 [DOI] [PubMed] [Google Scholar]
32.Butcher GA. Antimalarial drugs and the mosquito transmission of Plasmodium. Int J Parasitol. 1997;27(9):975–87. doi: 10.1016/s0020-7519(97)00079-9 [DOI] [PubMed] [Google Scholar]
33.Andrade AO, Santos NAC, Bastos AS, Pontual JDC, Araújo JE, Silva AMV, et al. Transmission-blocking activity of antimalarials for Plasmodium vivax malaria in Anopheles darlingi. PLoS Negl Trop Dis. 2023;17(6):e0011425. doi: 10.1371/journal.pntd.0011425 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Penna-Coutinho J, da Silva Araújo M, Campos Aguiar AC, Sá PM, Rios CT, Medeiros JF, et al. MEFAS, a hybrid of artesunate-mefloquine active against asexual stages of Plasmodium vivax in field isolates, inhibits malaria transmission. Int J Parasitol Drugs Drug Resist. 2021;17:150–5. doi: 10.1016/j.ijpddr.2021.09.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Mavrogordato A, Lever AML. A cluster of Plasmodium vivax malaria in an expedition group to Ethiopia: prophylactic efficacy of atovaquone/proguanil on liver stages of P. vivax. J Infect. 2012;65(3):269–74. doi: 10.1016/j.jinf.2012.04.015 [DOI] [PubMed] [Google Scholar]
36.Painter HJ, Morrisey JM, Vaidya AB. Mitochondrial electron transport inhibition and viability of intraerythrocytic Plasmodium falciparum. Antimicrob Agents Chemother. 2010;54(12):5281–7. doi: 10.1128/AAC.00937-10 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Cao Y, Hayashi CTH, Araujo M da S, Tripathi AK, Andrade AO, Medeiros JF, et al. Evaluation of combination vaccines targeting transmission of Plasmodium falciparum and P. vivax. Vaccine. 2024;42(21):126140. doi: 10.1016/j.vaccine.2024.07.041 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Bansal GP, Araujo M da S, Cao Y, Shaffer E, Araujo JE, Medeiros JF, et al. Transmission-reducing and -enhancing monoclonal antibodies against Plasmodium vivax gamete surface protein Pvs48/45. Infect Immun. 2024;92(3):e0037423. doi: 10.1128/iai.00374-23 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Birth D, Kao W-C, Hunte C. Structural analysis of atovaquone-inhibited cytochrome bc1 complex reveals the molecular basis of antimalarial drug action. Nat Commun. 2014;5:4029. doi: 10.1038/ncomms5029 [DOI] [PubMed] [Google Scholar]
40.World Health Organization. Global technical strategy for malaria 2016-2030. 2015. https://www.who.int/docs/default-source/documents/global-technical-strategy-for-malaria-2016-2030.pdf [Google Scholar]
41.Rathod GK, Jain M, Sharma KK, Das S, Basak A, Jain R. New structural classes of antimalarials. Eur J Med Chem. 2022;242:114653. doi: 10.1016/j.ejmech.2022.114653 [DOI] [PubMed] [Google Scholar]
42.Vangapandu S, Jain M, Kaur K, Patil P, Patel SR, Jain R. Recent advances in antimalarial drug development. Med Res Rev. 2007;27(1):65–107. doi: 10.1002/med.20062 [DOI] [PubMed] [Google Scholar]
43.Dorn A, Vippagunta SR, Matile H, Jaquet C, Vennerstrom JL, Ridley RG. An assessment of drug-haematin binding as a mechanism for inhibition of haematin polymerisation by quinoline antimalarials. Biochem Pharmacol. 1998;55(6):727–36. doi: 10.1016/s0006-2952(97)00510-8 [DOI] [PubMed] [Google Scholar]
44.Palmer KJ, Holliday SM, Brogden RN. Mefloquine. Drugs. 1993;45(3):430–75. doi: 10.2165/00003495-199345030-00009 [DOI] [PubMed] [Google Scholar]
45.Xie Y, Zhang J, Lu B, Bao Z, Zhao J, Lu X, et al. Mefloquine Inhibits Esophageal Squamous Cell Carcinoma Tumor Growth by Inducing Mitochondrial Autophagy. Front Oncol. 2020;10:1217. doi: 10.3389/fonc.2020.01217 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Li H, Jiao S, Li X, Banu H, Hamal S, Wang X. Therapeutic effects of antibiotic drug mefloquine against cervical cancer through impairing mitochondrial function and inhibiting mTOR pathway. Can J Physiol Pharmacol. 2017;95(1):43–50. doi: 10.1139/cjpp-2016-0124 [DOI] [PubMed] [Google Scholar]
47.Coleman RE, Vaughan JA, Hayes DO, Hollingdale MR, Do Rosario VE. Effect of mefloquine and artemisinin on the sporogonic cycle of Plasmodium berghei ANKA in Anopheles stephensi mosquitoes. Acta Leiden. 1988;57(1):61–73. [PubMed] [Google Scholar]
48.Ouyang L, Tu G, Gao Y, Zhang P, Xie X. Ouyang: Two insecticidal antibiotics produced by Streptomyces nanchangensis. J Jiangxi Agricul Univ 15:148–53. https://scholar.google.com/scholar_lookup?title=Two+insecticidal+antibiotics+produced+by+Streptomyces+nanchangensis+&author=L.+Ouyang&author=G.+Tu&author=Y.+Gao&author=P.+Zhang&author=X.+Xie&journal=J+Jiangxi+Agricul+Univ&volume=15&pages=148-153&publication_year=1993& [Google Scholar]
49.Sun Y, Zhou X, Liu J, Bao K, Zhang G, Tu G, et al. “Streptomyces nanchangensis”, a producer of the insecticidal polyether antibiotic nanchangmycin and the antiparasitic macrolide meilingmycin, contains multiple polyketide gene clusters. Microbiology (Reading). 2002;148(Pt 2):361–71. doi: 10.1099/00221287-148-2-361 [DOI] [PubMed] [Google Scholar]
50.Duthaler U, Weber M, Hofer L, Chaccour C, Maia M, Müller P, et al. The pharmacokinetics and drug-drug interactions of ivermectin in Aedes aegypti mosquitoes. PLoS Pathog. 2021;17(3):e1009382. doi: 10.1371/journal.ppat.1009382 [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Jacobs S, Wang L, Rosales Rosas AL, Van Berwaer R, Vanderlinden E, Failloux A-B, et al. Favipiravir Does Not Inhibit Chikungunya Virus Replication in Mosquito Cells and Aedes aegypti Mosquitoes. Microorganisms. 2021;9(5):944. doi: 10.3390/microorganisms9050944 [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Huang W, Rodrigues J, Bilgo E, Tormo JR, Challenger JD, De Cozar-Gallardo C, et al. Delftia tsuruhatensis TC1 symbiont suppresses malaria transmission by anopheline mosquitoes. Science. 2023;381(6657):533–40. doi: 10.1126/science.adf8141 [DOI] [PubMed] [Google Scholar]
53.Araujo MS, Andrade AO, Bastos AS, Santos NAC, Pontual JDC, Araújo JE, et al. From colonization to high production and Plasmodium vivax infection of Anopheles darlingi and Anopheles deaneorum: a platform for malaria research. Bio-Protoc. 2025;15(9):e5302. doi: 10.21769/BioProtoc.5302 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Araujo M da S, Andrade AO, Santos NACD, Pereira DB, Costa G da S, Paulo PFM de, et al. Brazil’s first free-mating laboratory colony of Nyssorhynchus darlingi. Rev Soc Bras Med Trop. 2019;52:e20190159. doi: 10.1590/0037-8682-0159-2019 [DOI] [PubMed] [Google Scholar]
55.Andrade AO, Santos NAC, Bastos AS, Pontual JDC, Araújo CS, Lima AS, et al. Optimization of Plasmodium vivax infection of colonized Amazonian Anopheles darlingi. Sci Rep. 2023;13(1):18207. doi: 10.1038/s41598-023-44556-y [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Miura K, Swihart BJ, Fay MP, Kumpitak C, Kiattibutr K, Sattabongkot J, et al. Evaluation and modeling of direct membrane-feeding assay with Plasmodium vivax to support development of transmission blocking vaccines. Sci Rep. 2020;10(1):12569. doi: 10.1038/s41598-020-69513-x [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Delves M, Lafuente-Monasterio MJ, Upton L, Ruecker A, Leroy D, Gamo F-J, et al. Fueling Open Innovation for Malaria Transmission-Blocking Drugs: Hundreds of Molecules Targeting Early Parasite Mosquito Stages. Front Microbiol. 2019;10:2134. doi: 10.3389/fmicb.2019.02134 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS Pathog. doi: 10.1371/journal.ppat.1013531.r001

Decision Letter 0

Kenneth Vernick, Jeffrey D Dvorin

4 Nov 2025

PPATHOGENS-D-25-02262

Anti-malarial contact dependent blocking of transmission of Plasmodium vivax by Anopheles darlingi mosquito vector

PLOS Pathogens

Dear Dr. Araujo,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript within 60 days Jan 03 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Kenneth Vernick

Academic Editor

PLOS Pathogens

Jeffrey Dvorin

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

1) We do not publish any copyright or trademark symbols that usually accompany proprietary names, eg ©, ®, or TM (e.g. next to drug or reagent names). Therefore please remove all instances of trademark/copyright symbols throughout the text, including:

- ® on page: 22.

2) Some material included in your submission may be copyrighted. According to PLOSu2019s copyright policy, authors who use figures or other material (e.g., graphics, clipart, maps) from another author or copyright holder must demonstrate or obtain permission to publish this material under the Creative Commons Attribution 4.0 International (CC BY 4.0) License used by PLOS journals. Please closely review the details of PLOSu2019s copyright requirements here: PLOS Licenses and Copyright. If you need to request permissions from a copyright holder, you may use PLOS's Copyright Content Permission form.

Please respond directly to this email and provide any known details concerning your material's license terms and permissions required for reuse, even if you have not yet obtained copyright permissions or are unsure of your material's copyright compatibility. Once you have responded and addressed all other outstanding technical requirements, you may resubmit your manuscript within Editorial Manager.

Potential Copyright Issues:

i) Figures 1A, 1C, 2A, 2B, 3, 4, and 5. Please confirm whether you drew the images / clip-art within the figure panels by hand. If you did not draw the images, please provide (a) a link to the source of the images or icons and their license / terms of use; or (b) written permission from the copyright holder to publish the images or icons under our CC BY 4.0 license. Alternatively, you may replace the images with open source alternatives. See these open source resources you may use to replace images / clip-art:

- https://commons.wikimedia.org

- https://openclipart.org/.

3) Please amend your detailed Financial Disclosure statement. This is published with the article. It must therefore be completed in full sentences and contain the exact wording you wish to be published.

1) State the initials, alongside each funding source, of each author to receive each grant. For example: "This work was supported by the National Institutes of Health (####### to AM; ###### to CJ) and the National Science Foundation (###### to AM)."

2) State what role the funders took in the study. If the funders had no role in your study, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

3) If any authors received a salary from any of your funders, please state which authors and which funders..

If you did not receive any funding for this study, please simply state: u201cThe authors received no specific funding for this work.u201d

4) Please ensure that the funders and grant numbers match between the Financial Disclosure field and the Funding Information tab in your submission form. Note that the funders must be provided in the same order in both places as well.

Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors demonstrate the successful adaptation of the treatment of Anopheles darlingii mosquitoes with ATQ to reduce or eliminate their infection with Plasmodium vivax parasites. This is an important finding that has implications in malaria control strategies, especially in South America where this work was conducted. Moreover, it is encouraging that one of the same lead compounds identified by Paton et al (ATQ) works similarly for both of the major human-infectious malaria species.

Reviewer #2: This proof-of-concept paper expands the potential use of antimalarials to impair P. vivax development within one of the natural vectors, An. darlingi, by tarsal exposure, which was previous reported for P. falciparum- An. gambiae s.s. model. The validation of the use of antimalarials to blocking malaria transmission is crucial to and brings new insights into transmission dynamics in the Americas.

Experiments were robustly designed to test the TBA and TBR in several antimalarial compounds, including Atovaquone, Mefloquine, Tafenoquine and Cloroquine (some of them of extended use in Brazil as antimalarial treatment) via mosquito feeding experiments with natural P. vivax infected-blood (blood donors). One of the key findings is that Atovaquone inhibits the development of the parasite in the midgut completely after 60min exposure, and a significantly decrease after 6 min exposure to the compound (which is more realistic in mosquito behaviour).

The results obtained from these robustly designed and executed experiments supported very well their conclusion. Heterogeneity in the experiments, particularly the source of vivax gametocytes and the infectiousness between individuals, however, needs to be taken into account when evaluating the results. Furthermore, it would be good to mention the potential impact of this vector control strategy in a context as the Amazon and in An. darlingi, in terms of resting behaviour (very plastic and usually not indoors, so the delivery of this methodology could be challenging with certain mosquito populations) and/feeding behaviour (ATSB in the Amazon context could be challenging as potentially many other sugar sources are available for mosquitoes).

Reviewer #3: The effect of mosquito exposure to antimalarial compounds (particularly atovaquone) on Plasmodium development has been recently demonstrated in Anopheles gambiae and An. coluzzii infected with P. falciparum. The present study represents an important contribution to the field of malariology by showing that atovaquone (and, to a lesser extent, mefloquine) similarly disrupts the development of P. vivax in Anopheles darlingi. Overall, this is a clear, well-organized, and well-written manuscript.

I have four major comments, followed by several minor remarks below.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: There are no mentions of how many biological or technical replicates were conducted for each experimental type in the materials and methods, results, or figure legend. Because of this, it is unclear how rigorous this work is and how the statistical tests were run. Please include this information in a revised manuscript so that reviewers can understand and appropriately interpret the experiments and results of these statistical tests.

Reviewer #2: As in some experiments the number of mosquitoes is low, it would be good to know whether there was any difference in the blood-fed females (% fed-females) between control and antimalarials experiments.

As is generally the case when using DMFA with P. vivax, controlling for the gametocytemia in these kinds of experiments is extremely complicated, as compared with P. falciparum with available cultures. It would be good whether the authors could explain whether this could be a limitation to interpret the results, particularly in prevalence and oocyst density.

Also, in some experiments with the ATQ60mins, the number of mosquitoes exposed/tested was really low (e.g. Exp1) and it would be difficult to draw any conclusions. The number of replicates by antimalarials and experiments is only in the Supplemental information. I would recommend including this information in the text or in the Figures, as it is not easy to disentangle if the figures in the graphs originated from single experiments (in general, the number of mosquitoes tested/dissected per DMFA is low). Also, for clarification purposes, it would be good to know if the same blood donor (same gametocytemia) was used across several experiments, for instance, in Fig 4. experiments in AB and CD.

The authors explore the ATQ mechanism in the parasite development via in silico analysis. Part of this could be moved to the results sections, to make the discussion clearer.

Reviewer #3: 1/ Throughout the text (e.g. line 75, 108, 128), the authors refer to the exposure/administration route as “tarsal contact". However, as in similar studies (e.g. Paton et al. 2019), the experimental setup involves placing whole mosquitoes on a treated surface, meaning that several body parts (tarsi, ventral abdomen, thorax, proboscis) may be in contact with the compound. The exclusive involvement of tarsal uptake has not been demonstrated. I guess what the author means by "tarsal contact" is kind of a standardized exposure via resting on a treated surface, not an mechanistic proof exclusive absorption through tarsi.

I suggest rephrasing to indicate that the full body of mosquitoes were exposed (by resting on treated surfaces), rather than implying that absorption occurs solely through the tarsi. Clarifying this point would avoid overstating the mechanistic interpretation of the exposure route.

2/ The authors use the term “intensity of infection” to describe the number of parasites (oocysts) per mosquito, including individuals with zero oocysts. According to standard parasitological definitions (Bush et al. 1997, J. Parasitol. 83:575–583), intensity refers specifically to the number of parasites per infected host, whereas abundance refers to the number of parasites per examined host, including uninfected individuals. In Figure 1A, since mosquitoes with zero oocysts are included (as indicated by data points at y = 0), the metric plotted corresponds to parasite abundance, not intensity.

Moreover, because abundance integrates both infected and uninfected mosquitoes, the information it conveys partly overlaps with that shown in the prevalence pie charts below (in all figures). Both parasite abundance (upper part of the pannels) and prevalence (pie chart) reflect the proportion of uninfected mosquitoes (zeros) in the dataset, meaning that they are not independent metrics. Presenting both can therefore be misleading, as the same underlying information is represented twice, once as a binary infection outcome and once as zeros within the count data.

This overlap is precisely why two-part (hurdle) models are commonly applied in parasitological analyses. These models treat infection as a two-step process:

- a binomial component (infection status: 0 = uninfected, 1 = infected), which models prevalence;

- a zero-truncated count component (e.g., negative binomial or Poisson), which models intensity among infected hosts only.

From a visual and analytical standpoint, it would be clearer to align the figures with this framework by either:

- presenting prevalence (binary outcome, analyzed with a binomial GLMM) and intensity (counts excluding zeros, analyzed with a zero-truncated negative binomial GLMM) as separate but complementary panels; or

- presenting abundance (counts including zeros, analyzed with a zero-inflated negative binomial GLMM) alone (in which case the prevalence plot becomes redundant).

Note that in the stat analyses incorporating replicate/DMFA ID and/or parasite isolate ID as random effects is required.

This distinction also carries important biological meaning: prevalence can reflects a qualitative resistance trait, the ability of the mosquito or compound to prevent parasite establishment (and/or the inability of the parasite to establish); whereas intensity reflects a quantitative resistance trait, the ability to limit parasite development once infection is established.

Adopting these definitions and ensuring consistent terminology throughout the manuscript would improve both the biological interpretation and the statistical rigor of the study.

Finally, in the same vein, please ensure that TRA is correctly estimated. TRA should be calculated based on intensity (excluding zeros), not abundance (which includes zeros), since using abundance would confound the effects of TRA with those of TBA.

3/ Mosquito survival, Table 1. Although overall mosquito survival was relatively high (>85%), reporting only the percentage of dead mosquitoes at day 7 provides limited insight into the dynamics of survival over time. A Kaplan–Meier survival curve, showing the proportion of surviving mosquitoes each day from day 1 to day 7, would be far more informative. This approach would also allow for a proper statistical comparison between treatments using standard survival analysis methods (e.g., the Cox proportional hazards model). Presenting only day-7 mortality may mask subtle differences that occur earlier or later in the observation period.

4/ Clarity on parasite isolates and experimental design. It is not clear from the Results, Figures, or Materials and Methods whether results presented in the different figures are based on the same or different P. vivax isolates. The correspondence between figure panels, isolate numbers, and experimental replicates is confusing. More generally, it would be helpful to specify how many isolates were used for each assay or experiment. In the Materials and Methods (lines 418–419), the authors state that “25–50 female An. darlingi mosquitoes, 3–5 days old, were used for each experimental and control group.” However, in e.g. Fig 1A, the sample sizes indicated are n = 80 (control) and n = 76 (ATQ_60 group). This suggests that data from several DMFA replicates or parasite isolates may have been pooled, but this is not explicitly stated. If this interpretation is correct, it would be important to clarify it in the Materials and Methods section and/or in the figure legends. Currently, it seems that one has to search through the Supplementary Tables to infer this information.

In addition, I could not find any information about the gametocytemia or parasitemia of the volunteer donors from whom the P. vivax isolates were obtained. These data are important, as both TBA and TRA can be influenced by the initial parasite exposure level. Reporting this information would strengthen the interpretation of the results, for example, by allowing the reader to assess whether ATQ is more effective at low versus high gametocytemia (= more effective at low vs. high infection level in the mosquito provided there is a positive relationship between gametocytemia and infection levels).

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Lines 186-187: Please revise this statement as it does not agree with your findings.

Lines 226-230: Please clarify this statement, as MQ doesn’t retain partial transmission.

Clarify the group identifiers in Table 1

Italicize gene names and Latin words throughout

Reviewer #2: -Please, review consistencies in Figures (font size, height graphs)

-S2 and S3 Table: check references

Reviewer #3: - line 76: "Mefloquine was also reduced parasite intensity via tarsal contact". Shouldn't this read "Mefloquine also reduced parasite intensity via tarsal contact" ?

-Lines 194-197: It is unclear whether this immunflorescence assay was conducted on mosquito exposed to ATQ, pre- or post-infection?

- Line 205: "The antimalarials PQ, TQ and CQ and the compound did not achieve blocking or reduction of infection (Fig 3A, B and C)" what the term compound refer to? should this read: "The antimalarials PQ, TQ and CQ and MQ did not achieve blocking or reduction of infection (Fig 3A, B and C)". Or do you mean NGC? please clarify.

- Line 225: "When added to the infected blood prior to infection, blocked/reduced oocysts development at the 7th day post-DMFA (Fig 4A), and sporozoite infection at the 14th day post-DMFA (Fig 4B). Shouldn't this read: "When added to the infected blood prior to infection, ATQ blocked/reduced oocysts development at the 7th day post-DMFA (Fig 4A), and sporozoite infection at the 14th day post-DMFA (Fig 4B).

- Line 263: "Since some compounds, in addition to affecting Plasmodium development, may also impact the survival or overall fitness of mosquitoes. Therefore, we also assessed the...", shouldn't this read: "Since some compounds, in addition to affecting Plasmodium development, may also impact the survival or overall fitness of mosquitoes, we also assessed the..."

- Line 374: "Alternative delivery methods, such as sugar baits (ATSBs), could be explored such compounds" should read: "Alternative delivery methods, such as sugar baits (ATSBs), could be explored for such compound"

- Lines 375–376: The mention of nanchangmycin feels somewhat abrupt and disconnected from the surrounding text. It is not clear how this sentence fits within the current flow of the discussion. The authors should clarify the purpose of introducing nanchangmycin at this point, what specific point are they trying to make with this? If relevant, this part should be further developed and ideally presented as a separate paragraph, since, as currently written, it appears unrelated to the preceding discussion

Lines 415–416: “Female An. darlingi mosquitoes were obtained from the colony established at the Malaria Vectors Production and Infection Platform (PIVEM), located at FIOCRUZ-RO, Brazil, as described by Araujo et al.”. Beyond the cited reference (Araujo et al. [48)), it would be useful to provide additional information about this colony: How old is it, and is it maintained as an outbred population? Furthermore, to what extent does the genetic diversity of this laboratory colony reflect that of natural An. darlingi populations? Is it sympatric with the parasite isolates? Clarifying these points would help readers assess how representative the experimental mosquitoes are of field populations and how generalizable the findings may be.

-Lines 499-501: "Experiments in which the controls presented medians lower than 2.5 oocysts per mosquito and infection prevalence below 60% were not taken into consideration in the statistical analyses (S1A and S1B Table)." The rationale for this exclusion criterion is not explained. Why were experiments with lower infection levels discarded, and how is this decision justified? Excluding data based on control infection intensity or prevalence could introduce bias or affect the robustness of the analysis. Please clarify the biological or statistical reasoning behind this threshold.

-Fig4A: "control" instead of "controle"

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure resubmission:

While revising your submission, we strongly recommend that you use PLOS’s NAAS tool (https://ngplosjournals.pagemajik.ai/artanalysis) to test your figure files. NAAS can convert your figure files to the TIFF file type and meet basic requirements (such as print size, resolution), or provide you with a report on issues that do not meet our requirements and that NAAS cannot fix.

After uploading your figures to PLOS’s NAAS tool - https://ngplosjournals.pagemajik.ai/artanalysis, NAAS will process the files provided and display the results in the "Uploaded Files" section of the page as the processing is complete. If the uploaded figures meet our requirements (or NAAS is able to fix the files to meet our requirements), the figure will be marked as "fixed" above. If NAAS is unable to fix the files, a red "failed" label will appear above. When NAAS has confirmed that the figure files meet our requirements, please download the file via the download option, and include these NAAS processed figure files when submitting your revised manuscript.

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

PLoS Pathog. 2026 Jul 2;22(7):e1013531. doi: 10.1371/journal.ppat.1013531.r002

Author response to Decision Letter 1

26 Dec 2025

Attachment

Submitted filename: Comments from the Editor.docx

ppat.1013531.s010.docx^{(34KB, docx)}

PLoS Pathog. doi: 10.1371/journal.ppat.1013531.r003

Decision Letter 1

Kenneth Vernick, Jeffrey D Dvorin

5 Mar 2026

PPATHOGENS-D-25-02262R1

Anti-malarial contact dependent blocking of transmission of Plasmodium vivax by Anopheles darlingi mosquito vector

PLOS Pathogens

Dear Dr. Araujo,

Please submit your revised manuscript by May 04 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Kenneth Vernick

Academic Editor

PLOS Pathogens

Jeffrey Dvorin

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments :

The two reviewers still have several issues, which can be addressed without need for new experimental work. Please address all of the points raised, but the major ones are: Rev1, clarification between whether you present biological replicates or technical replicates; and Rev3, present infection metrics that do not conflate effects on prevalence (TBA) and effects on parasite development (TRA), and improve the presentation of mosquito survival data.

Journal Requirements:

- ® on page: 22.

Note: If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors have considered and responded to many of the comments and requests from the reviewers, but several issues remain to be resolved so that the readers of this work can understand and confidently interpret the results.

Reviewer #3: While the authors have satisfactorily addressed two of my previous main comments (comments 1 and 4), I find that the responses to the remaining two comments (initially comments 2 and 3) remain insufficient and require further clarification and revision (see below part III minor issues)

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #1: 1. It remains unclear if what is shown are biological replicates or technical replicates. They are listed as "independent replicates" or "Number of Replications" in the supplement. Please define how these replicates were done, and whether they are biological or technical replicates. If they are biological replicates, please note how many technical replicates were done per biological replicate. This is essential so that the reader knows how rigorous the work is, and what the statistical tests can or cannot say. If PLoS has standard definitions for biological and technical replicates (other journals do), please use the PLoS definitions.

2. The median values for Figure 4A, C, and D have changed, but the N and prevalence of infection have not. Why?

3. Figure 4C and 4D: the differences between control and treated samples are not even 2-fold different. While the appropriate statistical test was used (as the data was not shown to be normally distributed), and there was a statistically significant difference between groups, it is unlikely that there is a biologically meaningful difference between groups. The statements made about these differences should be revisited, as a strong conclusion was given and this compound is unlikely to be a viable intervention.

4. All of the supplemental files should be reviewed and revised to better describe what is being listed so that the reader can unambiguously know what each value means. For instance, I am assuming that in Supplemental Table 1A that "M" stands for an individual mosquito that was accessed. If true, some of these experiments have a very low number of mosquitoes that were assessed (and retained in the final analyses), as noted previously by Reviewer 3. Because this experiment is weighted the same as other experiments with more mosquitoes (but still not with a large number of mosquitoes), there is an outsized effect on the final calculation by just these 4 individual mosquitoes. I'm not sure that this has been adequately addressed in the revision.

Reviewer #3: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: I'll defer to the editor on whether the material and methods and references found on Supp Tables 3, 4, and 5 should be incorporated into the manuscript, or if they can remain in the table itself (once revised to remove mark-ups).

Reviewer #3: Previous comment 2/

First, the justification for including zeros in the calculation of transmission-reducing activity (TRA) is not supported by the cited reference. The quotation from Sagaria et al. (2023) explicitly states that oocysts were counted per infected mosquito. There is no indication in this definition or formula that uninfected mosquitoes (zeros) were included in the calculation of mean oocyst counts. On the contrary, the wording indicates that the metric refers to parasite intensity among infected mosquitoes only. Consequently, this reference cannot be used to justify the inclusion of zeros without redefining the biological meaning of TRA, which would then inherently conflate effects on prevalence (TBA) and effects on parasite development (TRA).

I acknowledge that some earlier studies have indeed estimated TRA using parasite abundance (i.e. including uninfected mosquitoes), a practice that can appear tempting when transmission-blocking activity (TBA) is strong, as few infected mosquitoes may remain to estimate “strict” TRA based on intensity alone. However, this methodological choice has important consequences for biological interpretation. When zeros are included, the resulting metric inherently conflates effects on parasite establishment (TBA) with effects on parasite development among infected mosquitoes (TRA), for the reasons outlined in my initial comment. If the authors choose to retain this approach, it is therefore essential that it is stated explicitly in the Methods and that readers are clearly warned that, under this definition, TRA should not be interpreted independently from TBA.

Second, the terminology used in the manuscript and figures is still problematic. The data shown include mosquitoes with zero oocysts, which corresponds to parasite abundance, not intensity as defined in standard parasitology. Even if the term “intensity” has sometimes been used loosely in the malaria literature, clear definitions exist, and adopting them would substantially improve clarity and biological interpretation. I therefore strongly recommend replacing “oocyst intensity” with “oocyst abundance” throughout the manuscript wherever zeros are included.

Previous comment 3/

I appreciate the authors’ clarification and the addition of a Cox proportional hazards analysis. However, the response does not fully address my initial concern. While the authors state that Kaplan–Meier analyses were performed, the corresponding survival curves are not presented in the manuscript, and survival is still primarily summarized as percentage mortality at day 7 in Table 1.

Reporting survival at a single time point provides limited information and may mask temporal differences between treatments. If survival was analyzed using Kaplan–Meier curves and Cox models, these results should be presented in the main figures to allow readers to visualize survival dynamics over time and to ensure consistency between data presentation and statistical analyses. I therefore reiterate that Kaplan–Meier survival curves (day 1–7), together with the associated statistical comparison, should be included in the manuscript rather than relying solely on day-7 mortality values.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

Figure resubmission:

Reproducibility:

PLoS Pathog. 2026 Jul 2;22(7):e1013531. doi: 10.1371/journal.ppat.1013531.r004

Author response to Decision Letter 2

13 May 2026

Attachment

Submitted filename: Comments from the Editor_20260310.docx

ppat.1013531.s011.docx^{(26.1KB, docx)}

PLoS Pathog. doi: 10.1371/journal.ppat.1013531.r005

Decision Letter 2

Kenneth Vernick, Jeffrey D Dvorin

19 May 2026

Dear Dr Araujo,

We are pleased to inform you that your manuscript 'Anti-malarial contact dependent blocking of transmission of Plasmodium vivax by Anopheles darlingi mosquito vector' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kenneth Vernick

Academic Editor

PLOS Pathogens

Jeffrey Dvorin

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

PLoS Pathog. doi: 10.1371/journal.ppat.1013531.r006

Acceptance letter

Kenneth Vernick, Jeffrey D Dvorin

Dear Dr Araujo,

We are delighted to inform you that your manuscript, "Anti-malarial contact dependent blocking of transmission of Plasmodium vivax by Anopheles darlingi mosquito vector," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

For Research Articles, you will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Individual and summary data from Direct Membrane Feeding Assays (DMFA).

A) Transmission-reducing activity (TRA, %) and transmission blocking activity (TBA, %) for each DMFA. B) Individual DMFA results. C) Summary of DMFA data.

(XLSX)

ppat.1013531.s001.xlsx^{(108.2KB, xlsx)}

S2 Table. Characteristics of Plasmodium vivax obtained from patients and used in ex vivo assay with Atovaquone (ATQ).

(DOCX)

ppat.1013531.s002.docx^{(29.6KB, docx)}

S3 Table. Characteristics of Plasmodium vivax obtained from patients and used in ookinete inhibition assay with Atovaquone (ATQ).

(DOCX)

ppat.1013531.s003.docx^{(26.5KB, docx)}

S4 Table. Chemical properties from all compound’s testes in this study – atovaquone, primaquine, tafenoquine, chloroquine, mefloquine and nanchangmycin.

(DOCX)

ppat.1013531.s004.docx^{(28KB, docx)}

S1 Fig. Structural alignment.

(TIFF)

ppat.1013531.s005.tiff^{(1.7MB, tiff)}

S2 Fig. Docking result: Atovaquone (ATQ) binding site on the cytochrome b (AlphaFold ID: AF-O63696-F1-v4).

(TIFF)

ppat.1013531.s006.tiff^{(8.9MB, tiff)}

S3 Fig. Sequence alignment.

(TIFF)

ppat.1013531.s007.tiff^{(8.9MB, tiff)}

S1 File. Description of methods to obtain data from S1 to S3 Fig.

(DOCX)

ppat.1013531.s008.docx^{(23.6KB, docx)}

Attachment

Submitted filename: Comments from the Editor.docx

ppat.1013531.s010.docx^{(34KB, docx)}

Attachment

Submitted filename: Comments from the Editor_20260310.docx

ppat.1013531.s011.docx^{(26.1KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[ppat.1013531.ref001] 1.World Health Organization. World malaria report 2025: addressing inequity in the global malaria response. Geneva: World Health Organization. 2025. https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2025 [Google Scholar]

[ppat.1013531.ref002] 2.World Health Organization. Global technical strategy for malaria 2016-2030, 2021 update. Geneva. 2021. https://www.who.int/publications/i/item/9789240031357 [Google Scholar]

[ppat.1013531.ref003] 3.Hougard JM, Corbel V, N’Guessan R, Darriet F, Chandre F, Akogbéto M, et al. Efficacy of mosquito nets treated with insecticide mixtures or mosaics against insecticide resistant Anopheles gambiae and Culex quinquefasciatus (Diptera: Culicidae) in Côte d’Ivoire. Bull Entomol Res. 2003;93(6):491–8. doi: 10.1079/ber2003261 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref004] 4.Sherrard-Smith E, Winskill P, Hamlet A, Ngufor C, N’Guessan R, Guelbeogo MW, et al. Optimising the deployment of vector control tools against malaria: a data-informed modelling study. Lancet Planet Health. 2022;6(2):e100–9. doi: 10.1016/S2542-5196(21)00296-5 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref005] 5.Hemingway J, Ranson H, Magill A, Kolaczinski J, Fornadel C, Gimnig J, et al. Averting a malaria disaster: will insecticide resistance derail malaria control? Lancet. 2016;387(10029):1785–8. doi: 10.1016/S0140-6736(15)00417-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref006] 6.Goodman CD, Buchanan HD, McFadden GI. Is the mitochondrion a good malaria drug target? Trends in Parasitology. 2017;33(3):185–93. doi: 10.1016/j.pt.2016.10.002 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref007] 7.Plouffe D, Brinker A, McNamara C, Henson K, Kato N, Kuhen K, et al. In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen. Proc Natl Acad Sci U S A. 2008;105(26):9059–64. doi: 10.1073/pnas.0802982105 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref008] 8.World Health Organization. WHO publishes recommendations on two new types of insecticide-treated nets. https://www.who.int/news/item/14-03-2023-who-publishes-recommendations-on-two-new-types-of-insecticide-treated-nets Accessed 2023 October 1. [Google Scholar]

[ppat.1013531.ref009] 9.Bargieri DY, Williamson KC. Targeting Malaria in Flight. Cell Host Microbe. 2019;25(5):631–2. doi: 10.1016/j.chom.2019.04.007 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref010] 10.Parker JEA, Angarita-Jaimes N, Abe M, Towers CE, Towers D, McCall PJ. Infrared video tracking of Anopheles gambiae at insecticide-treated bed nets reveals rapid decisive impact after brief localised net contact. Sci Rep. 2015;5:13392. doi: 10.1038/srep13392 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref011] 11.N’Guessan R, Odjo A, Ngufor C, Malone D, Rowland M. A Chlorfenapyr Mixture Net Interceptor® G2 Shows High Efficacy and Wash Durability against Resistant Mosquitoes in West Africa. PLoS One. 2016;11(11):e0165925. doi: 10.1371/journal.pone.0165925 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref012] 12.Tiono AB, Ouédraogo A, Ouattara D, Bougouma EC, Coulibaly S, Diarra A, et al. Efficacy of Olyset Duo, a bednet containing pyriproxyfen and permethrin, versus a permethrin-only net against clinical malaria in an area with highly pyrethroid-resistant vectors in rural Burkina Faso: a cluster-randomised controlled trial. Lancet. 2018;392(10147):569–80. doi: 10.1016/S0140-6736(18)31711-2 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref013] 13.Paton DG, Childs LM, Itoe MA, Holmdahl IE, Buckee CO, Catteruccia F. Exposing Anopheles mosquitoes to antimalarials blocks Plasmodium parasite transmission. Nature. 2019;567(7747):239–43. doi: 10.1038/s41586-019-0973-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref014] 14.Paton DG, Probst AS, Ma E, Adams KL, Shaw WR, Singh N, et al. Using an antimalarial in mosquitoes overcomes Anopheles and Plasmodium resistance to malaria control strategies. PLoS Pathog. 2022;18(6):e1010609. doi: 10.1371/journal.ppat.1010609 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref015] 15.Probst AS, Paton DG, Appetecchia F, Bopp S, Adams KL, Rinvee TA, et al. In vivo screen of Plasmodium targets for mosquito-based malaria control. Nature. 2025;643(8072):785–93. doi: 10.1038/s41586-025-09039-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref016] 16.Burrows J, Slater H, Macintyre F, Rees S, Thomas A, Okumu F, et al. A discovery and development roadmap for new endectocidal transmission-blocking agents in malaria. Malar J. 2018;17(1):462. doi: 10.1186/s12936-018-2598-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref017] 17.Strode C, Donegan S, Garner P, Enayati AA, Hemingway J. The impact of pyrethroid resistance on the efficacy of insecticide-treated bed nets against African anopheline mosquitoes: systematic review and meta-analysis. PLoS Med. 2014;11(3):e1001619. doi: 10.1371/journal.pmed.1001619 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref018] 18.Carlos BC, Rona LDP, Christophides GK, Souza-Neto JA. A comprehensive analysis of malaria transmission in Brazil. Pathog Glob Health. 2019;113(1):1–13. doi: 10.1080/20477724.2019.1581463 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref019] 19.Tadei WP, Dutary Thatcher B. Malaria vectors in the Brazilian amazon: Anopheles of the subgenus Nyssorhynchus. Rev Inst Med Trop Sao Paulo. 2000;42(2):87–94. doi: 10.1590/s0036-46652000000200005 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref020] 20.Kamiya T, Paton DG, Catteruccia F, Reece SE. Targeting malaria parasites inside mosquitoes: ecoevolutionary consequences. Trends Parasitol. 2022;38(12):1031–40. doi: 10.1016/j.pt.2022.09.004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref021] 21.Santos NAC, Andrade AO, Santos TC, Martinez LN, Ferreira AS, Bastos AS, et al. Evaluation of sustainable susceptibility to Plasmodium vivax infection among colonized Anopheles darlingi and Anopheles deaneorum. Malar J. 2022;21(1):163. doi: 10.1186/s12936-022-04204-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref022] 22.Brasil M da S, Secretaria de Vigilância em Saúde, Departamento de Imunização e Doenças Transmissíveis. Guia de tratamento da malária no Brasil. Brasília: Ministério da Saúde. 2020. https://www.gov.br/saude/pt-br/centrais-de-conteudo/publicacoes/svsa/malaria/tratamento/guia_tratamento_malaria_2nov21_isbn_site.pdf/view [Google Scholar]

[ppat.1013531.ref023] 23.Carr AL, Rinker DC, Dong Y, Dimopoulos G, Zwiebel LJ. Transcriptome profiles of Anopheles gambiae harboring natural low-level Plasmodium infection reveal adaptive advantages for the mosquito. Sci Rep. 2021;11(1):22578. doi: 10.1038/s41598-021-01842-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref024] 24.Rosenberg R. Malaria: some considerations regarding parasite productivity. Trends Parasitol. 2008;24(11):487–91. doi: 10.1016/j.pt.2008.07.009 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref025] 25.Wollesen M, Mikkelsen K, Tvilum MS, Vestergaard M, Wang M, Meyer RL, et al. Polyether Ionophore Antibiotics Target Drug-Resistant Clinical Isolates, Persister Cells, and Biofilms. Microbiol Spectr. 2023;11(4):e0062523. doi: 10.1128/spectrum.00625-23 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref026] 26.Calit J, Araújo JE, Deng B, Miura K, Gaitán XA, Araújo M da S, et al. Novel Transmission-Blocking Antimalarials Identified by High-Throughput Screening of Plasmodium berghei Ookluc. Antimicrob Agents Chemother. 2023;67(4):e0146522. doi: 10.1128/aac.01465-22 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref027] 27.Goodman CD, Siregar JE, Mollard V, Vega-Rodríguez J, Syafruddin D, Matsuoka H, et al. Parasites resistant to the antimalarial atovaquone fail to transmit by mosquitoes. Science. 2016;352(6283):349–53. doi: 10.1126/science.aad9279 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref028] 28.Balta VA, Stiffler D, Sayeed A, Tripathi AK, Elahi R, Mlambo G, et al. Clinically relevant atovaquone-resistant human malaria parasites fail to transmit by mosquito. Nat Commun. 2023;14(1):6415. doi: 10.1038/s41467-023-42030-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref029] 29.Burrows J, Fidock DA, Miller RS, Rees S. Blocking Plasmodium Development in Mosquitoes: A Powerful New Approach for Expanding Malaria Control Efforts. Am J Trop Med Hyg. 2019;101(4):734–5. doi: 10.4269/ajtmh.19-0318 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref030] 30.Delves M, Plouffe D, Scheurer C, Meister S, Wittlin S, Winzeler EA, et al. The activities of current antimalarial drugs on the life cycle stages of Plasmodium: a comparative study with human and rodent parasites. PLoS Med. 2012;9(2):e1001169. doi: 10.1371/journal.pmed.1001169 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref031] 31.Butcher GA, Sinden RE. Persistence of atovaquone in human sera following treatment: inhibition of P. falciparum development in vivo and in vitro. Am J Trop Med Hyg. 2003. doi: 10.4269/ajtmh.2003.68.111 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref032] 32.Butcher GA. Antimalarial drugs and the mosquito transmission of Plasmodium. Int J Parasitol. 1997;27(9):975–87. doi: 10.1016/s0020-7519(97)00079-9 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref033] 33.Andrade AO, Santos NAC, Bastos AS, Pontual JDC, Araújo JE, Silva AMV, et al. Transmission-blocking activity of antimalarials for Plasmodium vivax malaria in Anopheles darlingi. PLoS Negl Trop Dis. 2023;17(6):e0011425. doi: 10.1371/journal.pntd.0011425 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref034] 34.Penna-Coutinho J, da Silva Araújo M, Campos Aguiar AC, Sá PM, Rios CT, Medeiros JF, et al. MEFAS, a hybrid of artesunate-mefloquine active against asexual stages of Plasmodium vivax in field isolates, inhibits malaria transmission. Int J Parasitol Drugs Drug Resist. 2021;17:150–5. doi: 10.1016/j.ijpddr.2021.09.003 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref035] 35.Mavrogordato A, Lever AML. A cluster of Plasmodium vivax malaria in an expedition group to Ethiopia: prophylactic efficacy of atovaquone/proguanil on liver stages of P. vivax. J Infect. 2012;65(3):269–74. doi: 10.1016/j.jinf.2012.04.015 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref036] 36.Painter HJ, Morrisey JM, Vaidya AB. Mitochondrial electron transport inhibition and viability of intraerythrocytic Plasmodium falciparum. Antimicrob Agents Chemother. 2010;54(12):5281–7. doi: 10.1128/AAC.00937-10 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref037] 37.Cao Y, Hayashi CTH, Araujo M da S, Tripathi AK, Andrade AO, Medeiros JF, et al. Evaluation of combination vaccines targeting transmission of Plasmodium falciparum and P. vivax. Vaccine. 2024;42(21):126140. doi: 10.1016/j.vaccine.2024.07.041 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref038] 38.Bansal GP, Araujo M da S, Cao Y, Shaffer E, Araujo JE, Medeiros JF, et al. Transmission-reducing and -enhancing monoclonal antibodies against Plasmodium vivax gamete surface protein Pvs48/45. Infect Immun. 2024;92(3):e0037423. doi: 10.1128/iai.00374-23 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref039] 39.Birth D, Kao W-C, Hunte C. Structural analysis of atovaquone-inhibited cytochrome bc1 complex reveals the molecular basis of antimalarial drug action. Nat Commun. 2014;5:4029. doi: 10.1038/ncomms5029 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref040] 40.World Health Organization. Global technical strategy for malaria 2016-2030. 2015. https://www.who.int/docs/default-source/documents/global-technical-strategy-for-malaria-2016-2030.pdf [Google Scholar]

[ppat.1013531.ref041] 41.Rathod GK, Jain M, Sharma KK, Das S, Basak A, Jain R. New structural classes of antimalarials. Eur J Med Chem. 2022;242:114653. doi: 10.1016/j.ejmech.2022.114653 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref042] 42.Vangapandu S, Jain M, Kaur K, Patil P, Patel SR, Jain R. Recent advances in antimalarial drug development. Med Res Rev. 2007;27(1):65–107. doi: 10.1002/med.20062 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref043] 43.Dorn A, Vippagunta SR, Matile H, Jaquet C, Vennerstrom JL, Ridley RG. An assessment of drug-haematin binding as a mechanism for inhibition of haematin polymerisation by quinoline antimalarials. Biochem Pharmacol. 1998;55(6):727–36. doi: 10.1016/s0006-2952(97)00510-8 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref044] 44.Palmer KJ, Holliday SM, Brogden RN. Mefloquine. Drugs. 1993;45(3):430–75. doi: 10.2165/00003495-199345030-00009 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref045] 45.Xie Y, Zhang J, Lu B, Bao Z, Zhao J, Lu X, et al. Mefloquine Inhibits Esophageal Squamous Cell Carcinoma Tumor Growth by Inducing Mitochondrial Autophagy. Front Oncol. 2020;10:1217. doi: 10.3389/fonc.2020.01217 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref046] 46.Li H, Jiao S, Li X, Banu H, Hamal S, Wang X. Therapeutic effects of antibiotic drug mefloquine against cervical cancer through impairing mitochondrial function and inhibiting mTOR pathway. Can J Physiol Pharmacol. 2017;95(1):43–50. doi: 10.1139/cjpp-2016-0124 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref047] 47.Coleman RE, Vaughan JA, Hayes DO, Hollingdale MR, Do Rosario VE. Effect of mefloquine and artemisinin on the sporogonic cycle of Plasmodium berghei ANKA in Anopheles stephensi mosquitoes. Acta Leiden. 1988;57(1):61–73. [PubMed] [Google Scholar]

[ppat.1013531.ref048] 48.Ouyang L, Tu G, Gao Y, Zhang P, Xie X. Ouyang: Two insecticidal antibiotics produced by Streptomyces nanchangensis. J Jiangxi Agricul Univ 15:148–53. https://scholar.google.com/scholar_lookup?title=Two+insecticidal+antibiotics+produced+by+Streptomyces+nanchangensis+&author=L.+Ouyang&author=G.+Tu&author=Y.+Gao&author=P.+Zhang&author=X.+Xie&journal=J+Jiangxi+Agricul+Univ&volume=15&pages=148-153&publication_year=1993& [Google Scholar]

[ppat.1013531.ref049] 49.Sun Y, Zhou X, Liu J, Bao K, Zhang G, Tu G, et al. “Streptomyces nanchangensis”, a producer of the insecticidal polyether antibiotic nanchangmycin and the antiparasitic macrolide meilingmycin, contains multiple polyketide gene clusters. Microbiology (Reading). 2002;148(Pt 2):361–71. doi: 10.1099/00221287-148-2-361 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref050] 50.Duthaler U, Weber M, Hofer L, Chaccour C, Maia M, Müller P, et al. The pharmacokinetics and drug-drug interactions of ivermectin in Aedes aegypti mosquitoes. PLoS Pathog. 2021;17(3):e1009382. doi: 10.1371/journal.ppat.1009382 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref051] 51.Jacobs S, Wang L, Rosales Rosas AL, Van Berwaer R, Vanderlinden E, Failloux A-B, et al. Favipiravir Does Not Inhibit Chikungunya Virus Replication in Mosquito Cells and Aedes aegypti Mosquitoes. Microorganisms. 2021;9(5):944. doi: 10.3390/microorganisms9050944 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref052] 52.Huang W, Rodrigues J, Bilgo E, Tormo JR, Challenger JD, De Cozar-Gallardo C, et al. Delftia tsuruhatensis TC1 symbiont suppresses malaria transmission by anopheline mosquitoes. Science. 2023;381(6657):533–40. doi: 10.1126/science.adf8141 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref053] 53.Araujo MS, Andrade AO, Bastos AS, Santos NAC, Pontual JDC, Araújo JE, et al. From colonization to high production and Plasmodium vivax infection of Anopheles darlingi and Anopheles deaneorum: a platform for malaria research. Bio-Protoc. 2025;15(9):e5302. doi: 10.21769/BioProtoc.5302 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref054] 54.Araujo M da S, Andrade AO, Santos NACD, Pereira DB, Costa G da S, Paulo PFM de, et al. Brazil’s first free-mating laboratory colony of Nyssorhynchus darlingi. Rev Soc Bras Med Trop. 2019;52:e20190159. doi: 10.1590/0037-8682-0159-2019 [DOI] [PubMed] [Google Scholar]

[ppat.1013531.ref055] 55.Andrade AO, Santos NAC, Bastos AS, Pontual JDC, Araújo CS, Lima AS, et al. Optimization of Plasmodium vivax infection of colonized Amazonian Anopheles darlingi. Sci Rep. 2023;13(1):18207. doi: 10.1038/s41598-023-44556-y [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref056] 56.Miura K, Swihart BJ, Fay MP, Kumpitak C, Kiattibutr K, Sattabongkot J, et al. Evaluation and modeling of direct membrane-feeding assay with Plasmodium vivax to support development of transmission blocking vaccines. Sci Rep. 2020;10(1):12569. doi: 10.1038/s41598-020-69513-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1013531.ref057] 57.Delves M, Lafuente-Monasterio MJ, Upton L, Ruecker A, Leroy D, Gamo F-J, et al. Fueling Open Innovation for Malaria Transmission-Blocking Drugs: Hundreds of Molecules Targeting Early Parasite Mosquito Stages. Front Microbiol. 2019;10:2134. doi: 10.3389/fmicb.2019.02134 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Anti-malarial contact dependent blocking of transmission of Plasmodium vivax by Anopheles darlingi mosquito vector

Jéssica E A Kassupá

Alice O Andrade

Alessandra S Bastos

Gabriel L L Moura

Marina L Rocha

Leandro N Martinez

Elisandra K S Afonso

Wallyson J Costa

Daniel Y Bargieri

Carolina B G Teles

Jansen F Medeiros

Nirbhay Kumar

Ana Carolina R Guimarães

Douglas G Paton

Flaminia Catteruccia

Maisa S Araujo

Roles

Abstract

Author summary

Introduction

Results

Exposure to Atovaquone substantially reduces infection of Anopheles darlingi with Plasmodium vivax isolates

Fig 1. Anopheles darlingi exposure to Atovaquone (ATQ) affects Plasmodium vivax development.

Fig 2. Atovaquone (ATQ) reduced Plasmodium vivax infection when mosquitoes were exposed for six minutes, either before or after infection.

No effect on Plasmodium vivax development in mosquitoes exposed to antimalarials used in Brazil

Fig 3. Other antimalarials did not significantly affect Plasmodium vivax parasite development in Anopheles darlingi mosquitoes were exposed to the following antimalarials.

Fig 4. Antimalarials ATQ and MQ were added to blood infected with Plasmodium vivax and offered to female Anopheles darlingi mosquitoes via the Direct Membrane Feeding Assays (DMFA).

Tarsal exposure of Anopheles darlingi to Nanchangmycin (NCG) does not reduce infection with Plasmodium vivax

Fig 5. Tarsal exposure using Nanchangmycin (NCG) compound (1mmol per m2 for 60 min).

Antimalarials and Nanchangmycin (NCG) do not affect the survival of Anopheles darlingi females after tarsal exposure

Fig 6. Survival of Anopheles darlingi submitted to direct contact assay and infected with Plasmodium vivax until 7th day post-DMFA.

Table 1. Hazard ratio estimated by Cox model until 7 days post infection.

Comparative structural modeling and molecular docking identify a conserved ATQ-binding site in Plasmodium vivax cytochrome b

Discussion

Materials and methods

Ethics statement

Anopheles darlingi Direct Membrane-Feeding Assays (DMFA)

Tarsal exposure test

Blood collection from patients with diagnosed Plasmodium vivax infection

Direct membrane-feeding assays

Ookinete immunofluorescence assays – IFA

Statistics analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Kenneth Vernick

Jeffrey D Dvorin

Roles

Author response to Decision Letter 1

Decision Letter 1

Kenneth Vernick

Jeffrey D Dvorin

Roles

Author response to Decision Letter 2

Decision Letter 2

Kenneth Vernick

Jeffrey D Dvorin

Roles

Acceptance letter

Kenneth Vernick

Jeffrey D Dvorin

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Fig 5. Tarsal exposure using Nanchangmycin (NCG) compound (1mmol per m² for 60 min).

Fig 6. Survival of Anopheles darlingi submitted to direct contact assay and infected with Plasmodium vivax until 7^th day post-DMFA.