Host macrophages/monocytes promote malaria transmission by modulating mosquito microbiota via SR-A-mediated phagocytosis

Domain: neuroscience
Published: 2026-03-25
Editorial outcome: minor_revision

Abstract
Blood-stage malaria infection significantly increases the number of immune cells in the peripheral blood of vertebrate hosts, yet their roles in malaria transmission by mosquitoes remain poorly understood. In this study, we demonstrate that macrophages/monocytes—rather than neutrophils or NK/NKT cells—play a crucial role in enhancing malaria transmission to mosquitoes, both in rodent models and human. Mechanistically, these macrophages/ monocytes that migrate into the mosquito midgut suppress the growth of transmission-blocking microbiota, including E. anophelis, S. sonnei, and K. oxytoca, primarily through non-opsonic phagocytosis mediated by scavenger receptor A (SR-A). Importantly, either the depletion of macrophages/monocytes or the use anti-SR-A neutralizing antibodies significantly enhances the transmission-blocking efficacy of anti-Pfs25 monoclonal antibodies by increasing the abundance of these bacteria. This combination approach can lead to the complete blockade of transmission of Pfs25-transgenic Plasmodium berghei (P. berghei-pfs25). These findings reveal a previously unrecognized mechanism that the parasite co-opts host immune cells to eliminate transmission-blocking bacteria, suggesting opportunities for using anti-SR-A antibodies to inhibit malaria transmission and improve the efficacy of anti-Pfs25 monoclonal antibodies.

Introduction
Malaria continues to be one of the deadliest infectious diseases worldwide1. Although the use of insecticides and antimalarial drugs has significantly reduced malaria infections over the past few decades, the number of cases has plateaued and even increased in recent years2. Consequently, there is an urgent need for new intervention strategies to combat this infectious disease3. Malaria is transmitted through the bite of infected female mosquitoes. After a bite, both female and male gametocytes enter the mosquito's midgut, where they fertilize to form zygotes. These zygotes then develop into ookinetes, which traverse the midgut epithelium and mature into oocysts. Once matured, the oocysts rupture, releasing sporozoites into the hemolymph. Finally, the sporozoites invade the salivary glands and are injected into the skin when the infected mosquito bites a person4. Thus, targeting mosquito infection represents a promising strategy to disrupt malaria transmission.

Following penetration of the midgut epithelium and subsequent development into oocysts, malaria parasites largely evade detection by the mosquito immune system, facilitating their maturation into sporozoites within the salivary glands5,6. In contrast, a substantial loss of malaria parasites occurs during their development in the midgut, rendering this stage a critical target for transmission-blocking interventions4. Several factors contribute to parasite attrition within the midgut. For instance, mosquito midgut microbiota can directly eliminate malaria sexual stages through the secretion of lipases or the generation of reactive oxygen species7-9. Additionally, mosquito midgut microbiota can also indirectly impede parasite development by modulating peritrophic matrix formation10 or mosquito immune responses11,12. Furthermore, the invasion of the midgut epithelium by ookinetes elicits both localized and systemic immune responses, leading to parasite destruction13-16.

Emerging evidence indicates that host-derived serum factors, including gametocyte-targeted antibodies and transforming growth factor (TGF)-β1, also enter the mosquito midgut during natural malaria transmission and can modulate malaria transmission17,18. It has been shown that malaria blood-stage infections induces a significant expansion of immune cells in the peripheral blood of mice19. However, it remains largely unknown whether immune cells that are taken into the mosquito midgut during bites from malaria patients can also influence malaria transmission20,21.

Here, we revealed that innate immune cells were expanded in the peripheral blood of the infected mice, and macrophages/monocytes enhanced malaria transmission by reducing the abundance of transmission-blocking microbiota, including E. anophelis, S. sonnei and K. oxytoca in the mosquito midgut via SR-A-mediated phagocytosis.

Results
Malaria infection primarily expands innate immune cells in peripheral blood of mice
Mosquitoes acquire malaria parasites by biting infected hosts. During these bites, immune cells from the host's peripheral blood, along with the parasites, are ingested into the mosquito's midgut. However, it remains unclear whether these ingested immune cells influence the development of the parasites within the mosquito. To explore the impact of vertebrate host-derived immune cells on malaria transmission, we first investigated whether blood-stage malaria infections could trigger the expansion of immune cells in the peripheral blood.

Four days post-infection with P. yoelii or P. berghei, blood samples were collected from the tails of infected mice, and the frequencies of innate immune cells, including DCs, macrophages, monocytes, NK/NKT Cells and neutrophils, as well as adaptive immune cells, such as CD4+/CD8+ T cells, B cells and DCs, were analyzed using flow cytometry (FACS) (Figs. 1a, Supplementary Fig 1). Notably, we observed a significant increase in the frequency of neutrophils (CD11b⁺Ly6G⁺), macrophages (CD11b⁺F4/80⁺), and monocytes (CD11b⁺F4/80⁻) in the peripheral blood of parasite-infected mice. Initially, the frequency of DCs (CD11c⁺MHC II⁺) were also found to increase in response to either P. yoelii or P. berghei infection, but no significant change was observed for P. berghei infection after three replications (Figs. 1b-e). The frequencies of NK/NKT cells (NK1.1⁺), CD4⁺ T cells (CD3α⁺CD4α⁺), and B cells (CD19⁺CD220⁺) did not change in the peripheral blood of mice infected with either P. yoelii or P. berghei. However, parasite infection reduced the frequency of CD8⁺ T cells (CD3α⁺CD8α⁺) (Figs. 1f-i). These findings suggest that malaria infection primarily expands specific immune cell populations in the peripheral blood of mice.

Macrophages/monocytes, but not neutrophils and NK/NKT cells, significantly promote both rodent and human malaria transmission
To investigate the role of expanded innate immune cells in malaria transmission, we analyzed innate immune cell populations in the mosquito midgut three hours after the mosquitoes fed on parasite-infected mice using FACS. All major innate immune cell types were detected, with particularly high levels of NK/NKT cells, monocytes and neutrophils (Fig. 2a), suggesting a potential role for these cells in influencing malaria transmission.

Since gametocytes are the only stage of the malaria parasite that can develop further in mosquitoes, factors influencing gametocytemia in mice are potentially important for malaria transmission. We monitored gametocytemia in mice treated with anti-Ly6G (to deplete neutrophils), anti-NK1.1 (to deplete NK/NKT cells), or clodronate liposomes (CL) (to deplete monocytes and macrophages). We found no significant changes in gametocytemia in either P. yoelii or P. berghei-infected mice pre-treated with anti-Ly6G, anti-NK1.1, or CL (Supplementary Figs. 2a-b). Furthermore, the in vitro transformation of gametocytes from anti-Ly6G, anti-NK1.1, and CL-treated infected mice into ookinetes was comparable to that of gametocytes from control mice (Supplementary Figs. 2c-d). This indicates that the depletion of neutrophils, NK/NKT cells, or macrophages/monocytes does not significantly impact gametocyte function in mice.

Next, mice infected with P. yoelii for three days were treated with anti-Ly6G, or anti-NK1.1. Twenty-four hours later, mosquitoes were allowed to feed on these mice, and oocyst numbers were quantified 7 days post-blood meal (Fig. 2b). No significant difference in either infection intensity or prevalence was observed in mosquitoes fed on P. yoelii-infected mice depleted of neutrophils (anti-Ly6G-treated) or NK/NKT cells (anti-NK1.1-treated) compared to the control (Figs. 2c-d, Supplementary Fig. 3a-d). These results suggest that neither neutrophils nor NK/NKT cells significantly influence malaria transmission.

Then, mice were treated with a high dose of CL to selectively deplete monocytes and macrophages in peripheral blood. FACS confirmed that CL treatment significantly reduced the frequency of macrophages/monocytes but had no significant effect on CD4+ T cells, CD8+ T cells, or B cells (Supplementary Figs. 3e-h). These results demonstrate the specificity of CL-mediated depletion for macrophages/monocytes. Interestingly, we found that mosquitoes feed on CL-treated mice displayed significantly reduced expression of secreted ookinete adhesive protein (SOAP) and von Willebrand factor A domain-related protein (WARP), key markers of ookinete development22,23, at 12 hours post-infection (Figs. 2e-f). This suggests that ookinete numbers are lower in mosquitoes fed on CL-treated mice compared to controls. In addition, the depletion of macrophages/monocytes using CL substantially reduced both infection intensity and prevalence of mosquitoes, regardless of whether they had fed on P. yoelii- or P. berghei-infected mice (Figs. 2g-h). By day 18, only a few sporozoites were detectable in mosquito salivary glands that had fed on P. yoelii-infected mice pretreated with CL (Fig. 2i), while no sporozoites were found in the salivary glands of mosquitoes feed on the CL-treated P. berghei-infected mice (Fig. 2j). Furthermore, the percentage of mice infected with P. yoelii was significantly reduced, and even no mice were infected with P. berghei following bites from mosquitoes that had fed on the infected mice pretreated with CL (Figs. 2k-l).

Evidence has shown that monocytes frequency in peripheral blood was also upregulated upon P. falciparum infection24. So, monocytes from healthy adult peripheral blood were isolated, and then added into in vitro cultured P. falciparum gametocyte. The influence of human monocytes on the transmission of P. falciparum was evaluated by standard membrane feeding assay (SMFA). SMFA revealed that the addition of human monocytes significantly increased both the intensity and prevalence of P. falciparum infection (Fig. 2m). These results demonstrate that macrophages/monocytes, rather than neutrophils and NK/NKT cells, play a crucial role in facilitating both rodent and human malaria transmission.

Macrophages/monocytes promote malaria transmission by reducing microbiota abundance in mosquito midgut
The depletion of macrophages/monocytes has no significant effect on gametocyte generation in mice, indicating that macrophages/monocytes exert their function in mosquito to facilitate malaria transmission. A previous study showed that monocytes present in the mosquito midgut might phagocytose and kill developing gametes25. However, our findings indicate that the depletion of macrophages/monocytes does not promote, but rather inhibits, malaria transmission (Figs. 2e-m). Therefore, the impact of monocytes on malaria transmission is not due to their direct action on malaria parasites within the mosquito midgut.

Recent evidence suggests that microbiota in the mosquito midgut can modulate malaria transmission by directly killing the parasite or inducing anti-malarial immune responses in mosquitoes26. This raises the question of whether macrophages/monocytes influence the microbial composition of the mosquito midgut. We found that mosquitoes fed on CL-treated mice exhibited a higher abundance of microbiota at 1-, and 12-hours post-blood meal, by colony-forming units (CFUs) quantification assay (Figs. 3a-b) and 16S rDNA qPCR (Fig. 3c). As microbiota proliferation after the blood meal has been found to affect mosquito lifespan27, we tested whether the increased microbial abundance in mosquitoes feed on CL-treated mice would reduce their survival. As expected, mosquitoes that fed on CL-treated mice exhibited a significantly lower survival rate compared to those that fed on control mice (Fig. 3d). To further investigate the role of midgut microbiota in the enhanced parasite infection observed in mosquitoes feeding on CL-treated mice, we pretreated mosquitoes with antibiotics to eliminate their native microbiota. Subsequently, mosquitoes with or without microbiota were fed on either control or CL-treated mice. Following antibiotic-mediated clearance of the midgut microbiota, oocysts numbers were significantly higher than in control mosquitoes that did not receive antibiotic treatment—a finding consistent with previous study7. However, there was no significant difference in the number of oocysts or their prevalence between mosquitoes without microbiota, regardless of whether they were fed on the control or CL-treated mice (Fig. 3f). These findings suggest that macrophage/monocytes not only promote malaria transmission, but also enhance their longevity by reducing microbiota abundance in mosquito midgut.

The depletion of macrophages/monocytes significantly increases the abundance of bacteria with transmission-blocking activity in mosquito midgut
The composition of the gut microbiota plays a crucial role in malaria infection. So, which specific bacterial differences contribute to this phenomenon? To address this question, midguts were collected from mosquitoes fed on control and CL-treated mice, and 16S rDNA sequencing were performed and compared. The midgut microbiota composition of mosquitoes fed on the CL-treated mice differed significantly from mosquitoes fed on the control mice (Fig. 4a). Notably, the abundances of E. anophelis—the dominant species in our laboratory An. stephensi strain—as well as S. sonnei and K. oxytoca, were significantly higher in mosquitoes that fed on CL-treated mice compared to controls. The increased abundance of E. anophelis, S. sonnei, and K. oxytoca in mosquitoes that fed on CL-treated mice was confirmed by qPCR (Figs. 4b-d). However, no significant changes were observed for other bacteria, such as Dietzia natronolimnaea, Nesteren salmonea, and Asaia spathodeae, which were also verified by qPCR (Supplementary Figs. 4a-c). This prompted us to investigate whether macrophages/monocytes enhance malaria transmission by decreasing the abundance of E. anophelis, S. sonnei or K. oxytoca in the mosquito midgut.

The endogenous microbiota in mosquitoes was eliminated by antibiotic treatment. Following this, the mosquitoes were fed a sugar solution supplemented with isolated E. anophelis, or S. sonnei or K. oxytoca for their colonization in the midgut. After colonization, mosquitoes were fed on the infected mice pretreated with CL (Figs. 4e, Supplementary Fig. 4d-g). E. anophelis, or S. sonnei or K. oxytoca colonization significantly reduced both infection intensity and prevalence in mosquitoes, demonstrating their suppressive effect on parasite development. These effects were further enhanced in mosquitoes fed on CL-treated mice (Figs. 4f-g). We also investigated whether these bacteria impair parasite development in the mosquito midgut individually or synergistically. Mosquitoes were colonized with each of the three bacterial species individually or in combination and then allowed to feed on the infected mice. However, we did not observe an additive or synergistic effect in mosquitoes colonized with multiple bacterial species compared to those harboring a single bacterium (Fig. 4h). Therefore, we conclude that macrophages/monocytes contribute to malaria transmission by reducing the abundance of E. anophelis, as well as S. sonnei and K. oxytoca, in the mosquito midgut, but no synergistical inhibitory effect was found for those three bacteria in malaria transmission. Noticeably, there is difference between the gut microbiota of long-term laboratory-raised Anopheles mosquitoes and that of wild Anopheles mosquitoes. S. marcescens, a naturally occurring symbiotic bacterium found in field-caught mosquitoes, exhibits antimalarial activity in mosquito12. Consequently, we colonized the midguts of laboratory-reared Anopheles mosquitoes with this bacterium to evaluate whether CL treatment of infected mice could similarly influence malaria transmission. Consistent with our hypothesis, CL-treatment also significantly reduced both infection intensity and prevalence in S. marcescens-colonized mosquitoes (Figs. 4i, Supplementary Fig. 4h). These findings suggested that the enhancement of malaria transmission by macrophages/monocytes may extend beyond specific microbial communities, highlighting a broader ecological relevance.

Macrophages/monocytes phagocytose microbiota in mosquito midgut dependent on SR-A
To investigate whether ingested macrophages/monocytes reduce midgut microbiota via phagocytosis, we first cleared the mosquito midgut microbiota with antibiotics and then colonized it with GFP-tagged E. anophelis. Mosquitoes were allowed to fed on P. yoelii-infected mice pre-treated with or without CL. Three hours post-feeding, midguts were dissected and analyzed by FACS to detect GFP-positive macrophages/monocytes. CL treatment significantly reduced the number of GFP-positive macrophages/monocytes (Fig. 5a). To distinguish between phagocytosed bacteria and surface-adherent GFP-tagged E. anophelis, extracellular bacteria were removed using antibiotic treatment as previously described28. Subsequent CFUs assays revealed that macrophages/monocytes from mosquitoes fed on CL-treated mice phagocytosed significantly fewer bacteria than those from control-fed mosquitoes (Fig. 5b). We also assessed the survival of macrophages/monocytes in the mosquito midgut by FACS. A high frequency of macrophages/monocytes was detected at 1 and 3 hours post–blood meal, but their numbers declined sharply by 12 hours and were nearly undetectable by 24 hours (Fig. 5c). These results indicate that macrophages/monocytes are present only transiently in the midgut and likely engage in active phagocytosis of midgut microbiota during the early phase following the blood meal.

Since microbiota in the mosquito midgut do not induce the generation of antibodies, their phagocytosis is unlikely to be mediated by opsonization through Fc receptors on macrophages/monocytes. Furthermore, in the absence of antibodies, the classical pathway cannot activate C3. However, host-derived C3 ingested into the mosquito midgut may be activated via the alternative pathway to generate C3b29. This suggests that macrophages/monocytes might phagocytose bacteria opsonized by C3b. Notably, the depletion of macrophages/monocytes in C3-/- mice treated with CL still reduces malaria transmission (Fig. 5d). Therefore, it appears that macrophages/monocytes phagocytose bacteria independent on complement.

SR-A is one of the non-opsonic receptors involved into the phagocytosis of bacteria by macrophages/monocytes30. The treatment of parasite-infected mice with 10 ug anti-SR-A could significantly reduce malaria transmission (Fig. 5e), and 50 μg anti-SR-A could even reduce malaria transmission to a level comparable to CL treatment (Fig. 5f). We also observed the persistent blocking effect of anti-SR-A. The infected mice were pre-treated with or without anti-SR-A at -1, or 1 day, then allowed to fed to mosquitoes. As a result, the neutralization of SR-A at 1 day before infection (5 days before the blood meal) did not affect the number of oocysts in the mosquito midgut but reduced oocyst prevalence. In contrast, the administration of anti-SR-A antibody at 1-day post-infection (3 days before the blood meal) achieved a level of transmission reduction comparable to that observed in the CL-treated group (Fig. 5g), suggesting that antibody-mediated neutralization of SR-A can suppress malaria transmission for at least three days. Taken together, our data suggest that SR-A was responsible for the non-opsonic phagocytosis of bacteria in mosquito midgut by macrophages/monocytes, thereby promoting malaria transmission.

Anti-Pfs25 antibodies completely block malaria transmission in the presence of anti-SR-A antibodies
Malaria transmission blocking vaccines are designed to induce antibody against surface proteins on sexual stages31. Pfs25, one of the leading transmission blocking vaccines, is thought to induce anti-Pfs25 antibodies to block the penetration of ookinetes through the midgut epithelium32,33. However, the regulation of the blocking efficacy of anti-Pfs25 antibodies is still largely unknown. Since monocyte/macrophage depletion suppressed natural malaria transmission by increasing transmission-blocking bacteria, we investigated whether it could also enhance the transmission-blocking efficacy of anti-Pfs25. Mice infected with P. berghei-pfs25 (Pfs25-transgenic P. berghei) were treated with or without CL and then administered anti-Pfs25 antibodies before mosquito feeding (Fig. 6a). While anti-Pfs25 antibodies alone significantly reduced transmission, complete blocking was achieved only when combined with CL-mediated monocyte/macrophage depletion (Figs. 6b-e). Furthermore, anti-Pfs25 antibodies also completely block the transmission of P. berghei-pfs25 in the presence of anti-SR-A antibodies (Figs. 6f-j). This effect was closely linked to the reduced burden of E. anophelis, S. sonnei and K. oxytoca in the midgut of mosquitoes feed on the infected mice pretreated with anti-SR-A antibodies (Figs. 6k-m). Therefore, we demonstrated that the transmission of P. berghei-pfs25 can be completely blocked by anti-Pfs25 antibodies following the inhibition of SR-A-mediated phagocytosis of transmission-blocking bacteria by macrophages/monocytes.

Methods
Ethics Statement
Human blood was collected from healthy donors who provided written informed consent at Southwest Hospital of Army Medical University. Anonymous human blood and serum used for P. falciparum culture were obtained from the Chongqing Blood Bank. The in vitro culture of P. falciparum and subsequent infection of mosquitoes with the parasite were conducted in BSL-2+ laboratories, with oversight and approval from the Ethics Committee of the Army Medical University Institute of Medical Research (KY2025153).

Mosquito, parasites, and mice
An. stephensi (Hori strain) were maintained were reared and maintained under standard laboratory conditions at 27°C with 75% RH (relative humidity) on a 12-hour light-dark cycle. Rodent malaria parasites P. yoelii, P. berghei, P. berghei-pfs25 transgenic parasites (kindly donated from Xiamen University by professor Jian Li) and the P. falciparum NF54 strain (kindly donated from Chinese Academy of Sciences by professor Lubin Jiang) were used in this study. Female C57BL/6J mice, aged 6-8 weeks, were obtained from GemPharmat Animal Institute (Nanjing, China). C3−/− mice with C57BL/6J background were purchased from Jackson Laboratory. The mice were housed in a controlled environment with regulated temperature and humidity and maintained on a 12-hour light/dark cycle, with lights off at 18:00 hours. All animal procedures were reviewed and approved by the Animal Ethics Committee of the Army Medical University Institute of Medical Research under protocol number AMUWEC20218022.

Mice infection
C57BL/6J mice female mice aged at 6-8 weeks were intravenously (i.v) injected with 1 × 106 (4 × 106 for P. berghei-pfs25) P. yoelii, P. berghei or P. berghei-pfs25 parasitized red blood cells (pRBCs). Parasitemia and gametocytemia were checked before fed to mosquitos by examining Giemsa-stained blood films under a light microscope.

Mosquito infection
An. stephensi were deprived of sugar meal for 24 hours, then allowed to feed on P. yoelii-infected mice for 30 min. Not fully engorged mosquitoes were removed. Mosquitoes were maintained at 23-24 °C environment, with 70 % RH. At 7 days (P. yoelii oocysts), 9 days (P. berghei oocysts) or 18 days (P. yoelii and P. berghei salivary gland sporozoites) post infection, mosquitoes were dissected, and oocysts and salivary gland sporozoites were counted, respectively.

Flow cytometry analysis
Peripheral blood of mice infected with P. yoelii or P. berghei was collected from orbital vein and RBCs were lysis by Gey’s solution. Sample collected from mosquito midgut were gently homogenate with RPMI-1640 (Gibco, USA), by 1 ml pipette to release blood from its midgut, then RBCs were lysis by Gey’s solution after filter with a 70-micron mesh sieve. Then cells were incubated with anti-CD16/CD32 antibodies (Biolegend, USA) for 10 min to block Fc receptors and then incubated with the antibodies of interest for 45 minutes. Antibodies used for flow cytometry analysis were as followed: anti-CD11b, anti-Ly6G, anti-MHC II, anti-CD3a, anti-CD4, anti-CD19, anti- CD8a, anti-CD3, and anti-NK1.1. Dead cells were excluded using LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen, USA) according to the manufacturer’s instructions. Cells were collected on a BD FACS Canto II cytometer (BD Biosciences, USA) and analyzed by Flow Jo software v10.8 .

Depletion of macrophages, NK/NK T cells, and neutrophils.
After infected with parasites for three days, mice were i.v injected with 200 μl CL (Liposoma, Netherlands), 200 μg anti-NK1.1 (Bio X Cell, USA) or 200 μg anti-Ly6G ((Bio X Cell, USA) mono-antibody to deplete macrophages, NK/NKT cells, and neutrophils, respectively. The depletion efficacy was monitored by FACS analysis.

In vitro culture of ookinetes
After mice were infected with P. yoelii or P. berghei for 3 d, mice were i.v injected with 200 μl CL or control liposomes, 24 h later blood was collected from orbit and immediately added to the ookinete culture medium which contains: RPMI-1640 (Gibco, USA), 10% fetal calf serum (Gibco, USA), 100 μM xanthurenic acid (MedChemExpress, USA), 25 mM Hepes (Sigma, USA); pH 8.0, 100 μg/ml streptomycin (Sangon, China), 100 U/ml penicillin (Sangon, China). The gametocytes were cultured at 19°C (for P. berghei) 23 °C (for P. yoelii) for 24 hours to allow gametogenesis, fertilization, and ookinete differentiation. The generated ookinetes were calculated per 1000 RBCs in Giemsa-staining thin blood smears based on morphology, at least 50 field were counted for each sample.

Detection of bacteria load in mosquito midgut.
Colony forming unites (CFUs) assay were used to detect the bacteria load of mosquito’s midgut. Briefly, Mosquitoes were sterilization by spray 75 % ethyl alcohol, then washed three times with sterile PBS. Midguts were dissected under sterile conditions and homogenized. The homogenate was then diluted 10-fold and cultured on LB medium at 28 °C for 24 hours. CFUs was calculated by the following equation:
CFU (per plate)×dilution times
(1) CFUs=
number of moquito midgut

Antibiotics treatment of mosquitoes
Three days old female mosquitoes were fed with 10 % sugar solution supplement with 10 U/ml penicillin (Sangon, China), 15 μg/ml streptomycin (Sangon, China), and 15 μg/ml gentamicin (Gibco, USA). Four days later, the bactericidal efficacy of antibiotics was accessed by CFUs method.

Detection of bacterial phagocytosis by macrophages/monocytes.
Method was midfield from described before28. Mice were infected with P. yoelii or for 3 days, followed by i.v injection of 200 µl of CL or control liposomes. Twenty-four hours post-injection, mosquitoes were allowed to feed on the mice for 30 minutes. One hour after blood feeding, midguts were dissected, homogenized in 1 ml of PBS, and centrifuged (14,000 g, 5 min) to pellet bacteria and blood cells. The pellet was resuspended in 1 ml of PBS containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 µg/ml gentamicin to eliminate extracellular bacteria. After 3 hours of incubation, the homogenate was centrifuged (14,000 g, 5 min) and washed twice with sterile PBS to remove antibiotics. Macrophages and monocytes were lysed with 0.1% Triton X-100 (Sigma, USA) to release intracellular bacteria. Lysates were plated on LB agar, and bacterial CFUs were quantified after 24 hours of incubation at 37°C.

P. falciparum culture and mosquito infection
The P. falciparum NF54 strain was used to culture mature gametocytes, as described previously48. Matured gametocytes were diluted to 0.5% by adding fresh RBC, then resuspended with equal volume of O+ serum and fed to mosquitoes by SMFA. To investigate the influence of human-derived monocytes on P. falciparum infection of mosquitoes, peripheral blood was collected from health individual, and mononuclear cells were isolated by Percoll method. Monocytes were then purified by Dynabeads™ Untouched™ Human Monocytes kit (Invitrogen, USA) according to the manufacture’s instruction. Isolated monocytes (5×105/ml) were added to in vitro cultured mature P. falciparum gametocytes. Nine days post blood meal, midguts of mosquitoes were dissected and oocysts number was counted after stained with 0.1% mercurochrome (MedChemExpress, USA) for 10 min.

Mosquito bite challenge
Mice infected with P. yoelii or P. berghei for 3 days were treated with CL or control liposomes, and then were allowed to fed to mosquitoes. Eighteen days later, 5 infected mosquitoes were fed on naïve mice, fully engorged mosquito based on its abdomen was considered as a successful bite. Three days post mosquitoes’ bites, Giemsa-stained blood smears were made daily to check the on-set of blood stage infection for 12 days.

DNA Extraction and 16S rRNA Gene Amplicon Sequencing
Mosquitoes were surface-sterilized with 75% ethanol for 5 minutes and rinsed three times with sterile 1× PBS. Midguts were dissected aseptically, pooled into groups of 20 midguts per biological replicate, and homogenized. Total bacterial genomic DNA was isolate using the DNeasy PowerFecal Pro DNA Kit (QIAGEN, USA) according to the manufacture’s protocol. DNA purity and concentration were determined spectrophotometrically (NanoDrop One, Thermo Fisher Scientific, USA) by measuring absorbance ratios at 260/280 nm and 260/230 nm. The hypervariable V3–V4 region of the bacterial 16S rRNA gene was amplified using universal primers flanked by Illumina adapter and sample-specific barcode sequences. Primary PCR products were purified with VAHTS™ DNA Clean Beads (Vazyme Biotech, China), and libraries were quantified fluorometrically using the Quant-iT™ dsDNA High-Sensitivity Assay Kit (Thermo Fisher Scientific, USA). Equimolar amounts of purified amplicons were pooled and subjected to paired-end sequencing (2 × 250 bp) on the Illumina NovaSeq 6000 platform (Illumina, USA). Absolute abundance was calculated by times the relative change of CFUs between Control and CL-treated group.

Quantitative real-time PCR (qPCR)
To quantify the bacterial load, total DNA was extracted from mosquitoes that had fed on P. yoelii-infected mice treated with CL or control liposomes, using the DNeasy PowerFecal Pro DNA Kit (QIAGEN, USA) according to the manufacturer’s protocol. One hundred nanograms of DNA per sample were used as the template for qPCR to detect total and specific bacterial loads. For quantify SOAP and WARP expression of ookinetes, RNA from mosquito midgut was extracted 12 h post blood meal, cDNA was obtained by using revers transcription kit (Takara Bio Inc., Japan) following the manufacture’s protocol. Amplifications were performed with TB Green® Premix Ex Taq™ II (Takara Bio Inc., Japan) on a CFX96 Real-Time PCR Detection System (Bio-Rad, USA). All reactions included no-template controls to rule out contamination. Relative quantification was performed using the mosquito ribosomal protein S7 (S7) gene as an endogenous control. Data are expressed as fold-changes normalized to S7. Primers used in this study were listed in Supplemental Table 1.

Mosquito survival assay
Mice were infected with P. yoelii, three days later mice were i.v injected with 200 μl CL or control liposomes, 24 hours later, mosquitoes were allowed to fed on mice for 30 mins. Not fully engorged mosquitoes were removed. Mosquitoes were maintained at 23-24 °C environment, with 70 % RH. Survival condition was record daily for 7 days.

Bacteria isolation and identification
Midguts were dissected, homogenized, and serially diluted (10-fold) on LB agar for 28°C/24-h culture. Colonies underwent morphology-based classification (shape/color/size). Bacterial 16S rDNA was amplified via PCR (universal primers), purified, and sequenced (ABI 3730, USA). Sequences were BLAST-aligned against GenBank for identification.

Colonization of bacteria in mosquito midgut
After clearing the symbiont bacteria in the midgut with antibiotics, mosquitoes were fed a sugar solution containing 1 × 106/mL isolated E. anopheles, S. sonnei, K. oxytoca, or S. maecescens (kindly donated from Chinese Academy of Sciences by professor Sibao Wang) for 2 days to colonize E. anophelis into mosquito midgut. A successful colonization was confirmed by qPCR (See S table 1 for primer information) or using fluorescence microscope (for eGFP-E. anophelis) as previously described47.

Neutralization of SR-A receptor
Three days after P. yoelii infection, mice were i.v injected with 10, or 50 μg neutralizing anti-SR-A monoclonal antibody (R & D, USA). Twenty hours later, mice were fed to mosquitoes. To observe the longevity of SR-A blocking effect, the infected mice were pre-treated with 50 μg anti-SR-A at 1 day before infection, or 1 day post infection, and treated with CL 3 days post infection, 7 days post blood meal, oocysts number in mosquitoes were counted and compared.

Anti-Pfs25 transmission blocking assay
Anti-Pfs25 antibody was customized by GenScript (Shanghai, China) as previously described47. To evaluate the ability of anti-Pfs25 antibody to block P. berghei-pfs25 transmission, mice were i.v injected with 4 × 106 P. berghei-pfs25 pRBCs; Three days later, mice were i.v injected with 200 μl CL, 24 hours later, 20 μg of anti-Pfs25 antibody was i.v injected 1 hour before mosquito feeding. To determine the enchantment of anti-Pfs25 antibody block P. berghei-pfs25 transmission efficacy in the present of anti-SR-A, mice were i.v injected with 4 × 106 P. berghei-pfs25 pRBCs; Three days later, mice were i.v injected with 50 μg anti-SR-A, 24 hours later, 20 μg of anti-Pfs25 antibody was i.v injected 1 hour before mosquito feeding. Nine days later, mosquitoes were dissected, and the oocysts were counted and compared.

Transmission reduction activity (TRA) and transmission blocking activity (TRB) were calculated based on oocysts intensity and prevalence by following formula:
𝑂𝑜𝑐𝑦𝑠𝑡𝑠 𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝−𝑂𝑜𝑦𝑠𝑡𝑠 𝑖𝑛 𝑡𝑒𝑠𝑡 𝑔𝑟𝑜𝑢𝑝
(2) TRA %=100∗( )
𝑂𝑜𝑐𝑦𝑠𝑡𝑠 𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝
𝑃𝑟𝑒𝑣𝑎𝑙𝑒𝑛𝑐𝑒 𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝−𝑃𝑟𝑒𝑣𝑎𝑙𝑒𝑛𝑐𝑒 𝑖𝑛 𝑡𝑒𝑠𝑡 𝑔𝑟𝑜𝑢𝑝
(3) TBA %=100∗( )
𝑃𝑟𝑒𝑣𝑎𝑙𝑒𝑛𝑐𝑒 𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝

Statistics analysis
Analyses were performed using GraphPad Prism 10.0 (GraphPad Software, USA) and R studio (version, 2024.12.1+563). Differences of oocyst number between treatment and control groups were analyzed using the (Generalized Linear Mixed Models) GLMMs test. Specifically, if overdispersion is detected, a negative binomial regression was employed. Subsequently, the DHARMa package was used to test for zero inflation; if present, a zero-inflated model was applied for the final analysis. Fisher’s exact test (two-sided) was applied to compare infection rates. Kaplan-Meier test was used for comparing the ability of infected mosquito to transmit malaria and mosquito survival rate. Pooled data sets were determined by unpaired t-test (two-tailed). Specific analysis methods were detailed in each figure legend. Number between each group represent the P value, P<0.05 was consider as statistically significant.

Data availability
The 16S rDNA sequence data generated in this study have been deposited in the National Center of Biotechnology Information Sequence Read Archive database under accession code PRJNA1209247 [PRJNA1209247 - SRA - NCBI]. Source data are provided as a Source Data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, He et al report a very interesting study showing that mouse monocytes and/or macrophages enhance transmission of murine malaria via phagocytosing bacteria of the mosquito microbiota. Overall, the study is well-conducted and the main conclusions would definitely be worth a publication in Nature Communications. I have however a few comments to be fully convinced by this study.\nMajor comments:\nThe main point is that some experiments have been performed only twice, I think that a third replicate in all the experiments presented in the main figures would be required for publication in Nature communications, especially that the overall numbers are sometimes reasonable but low. Also, it would be interesting to see the n of each replicate below charts instead of the total amount of mosquitoes.\nSecondly, I wonder if the authors have pointed survival of their mosquitoes at least until dissection time. Microbiota proliferation after the blood meal has been found to affect mosquito lifespan (Gendrin et al, 2015) and I imagine that a reduction in phagocytosis, besides reducing vector competence, may as well reduce mosquito survival. Whether log-rank or a simple one-time point observation at the time of dissection, such survival data would make the study more convincing.\nConsidering stats, I would like to see GLMMs used instead of Mann Whitney U test, to take into account random effects of replicates. When the authors make a point on the non significance (after antibiotic treatment), it would like to see the actual p-value rather than a \"ns\". This \"ns\" may be close to significance, but only 2 replicates have been done to test this point so a third one can clarify this.\nThe manuscript requires extensive English and latin proofreading, checks on the use of italics. Besides mistakes the main text, I spotted some in titles or axis titles in Fig 1B,D, 2A, S2B,D,G; S3A,C,E.\nMinor points:\nLine 255 \"in the control bacteria\" please clarify this sentence.\nS4E: are both pictures two representative pictures in each condition? Don't the authors have a larger sample size to show?\nLine 509 : are there differences between batches in the % of not fully engorged mosquitoes?\nIn my opinion, at least some items of figure 3 should be used in a main figure, because it makes an important point (maybe removing some data from figure 1 e.g. choose one parasite for main and one for supplements).\nIn sum, I am excited by this study, but I know that I have been disappointed by very beautiful stories that have not been confirmed. I am hesitating between this excitement and a lack of trust, and I hope that revision will help me decide what I think about it...", "reviewer_2": "The manuscript by He et al examines the effects of mammalian immune cells on infection outcomes in the mosquito vector using rodent models of malaria infection. The study is simple, yet elegant, in its design and is clear in its presentation that macrophage/monocyte populations in the mosquito blood bolus influence the bacteria in the mosquito gut to influence malaria infection outcomes. The results are therefore very interesting and provide new insight into a previously unexplored aspect of malaria transmission. My comments are very minor and only look to increase the clarity of the presented study.\nMy comments are as follows:\n-Please edit the abstract to improve readability and clarity, particularly for lines 16-24.\n-There are several instances where more detailed editing is required that includes missing words, changes to improve clarity, and multiple times where Anopheles should be italicized.\n-Line 73-74: Please revise for clarity.", "reviewer_3": "This manuscript by He et al. reports a novel and mechanistically detailed finding: that macrophages and monocytes taken up during mosquito blood feeding facilitate Plasmodium transmission by suppressing gut microbiota through SR-A-mediated phagocytosis. The authors demonstrate that this immunological interaction promotes ookinete development and enhances transmission for rodent malaria parasites. They show that this process undermines the efficacy of transmission-blocking antibodies (anti-Pfs25) against a P. berghei line expressing P. falciparum Pfs25, an effect that can be rescued by macrophage/monocyte depletion or SR-A neutralization. This represents an important contribution to our understanding of host-vector-parasite interactions.\nThe work is technically sound, conceptually novel, and of high potential interest to the malaria transmission, immunology, and vector biology communities. It is supported by a comprehensive series of in vivo experiments and microbial/molecular analyses in rodent and humanised models. However, several scientific, mechanistic, and editorial issues require attention. These range from clarifications of the main claims and better contextualisation, to refinements in data interpretation, presentation, terminology, and writing.\nMajor comments\n• The authors repeatedly state that macrophages and monocytes “promote transmission.” This should be clarified throughout as an indirect effect, mediated via suppression of anti-Plasmodium bacteria, rather than any direct facilitation of parasite development.\n• The role of SR-A in bacterial phagocytosis is convincingly demonstrated, but it is not clear whether this is the sole receptor involved. The possible contributions of other receptors (e.g., CD36, DC-SIGN, MR) are mentioned in the discussion but should be more explicitly addressed or acknowledged as a limitation.\n• The finding that SR-A blockade restores the full efficacy of anti-Pfs25 antibodies is compelling, but the presentation could be improved. For example, clarifying that these are parallel and synergistic mechanisms (i.e., microbiota-mediated blocking and antibody-mediated blocking), rather than interacting ones. Also, include time-course data to address how long SR-A remains blocked after antibody injection and how long macrophages/monocytes persist in the mosquito midgut.\n• The human data (addition of monocytes in SMFA) is an important proof of concept but limited to a single experimental system. It would strengthen the manuscript to show whether similar monocyte expansions are observed in naturally infected individuals or gametocyte carriers.\n• The translational implications of this work, particularly for transmission-blocking vaccines or antibody combinations, deserve stronger emphasis in both the abstract and discussion. Currently, relevant statements in the abstract are unclear or overly compressed.\n• The roles of E. anophelis, S. sonnei, K. oxytoca, and S. marcescens are central to the proposed mechanism. However, their relative contributions to parasite suppression are not disentangled. Do they act independently, additively, or synergistically? This should be discussed more clearly.\n• While the manuscript shows that gametocytemia and ookinete development are unaffected by clodronate liposome (CL) treatment, this is presented too late. These controls should be moved earlier in the Results and shown in main figures to reassure the reader that effects on transmission are not due to altered gametocyte production.\n• Similarly, depletion of neutrophils and NK/NKT cells is shown to have no effect on transmission, but the results and conclusions (lines 120-129) should be supported by replication and earlier gametocyte data.\n• The effect of CL treatment on systemic cytokine responses (e.g., IL-10, IFN-γ), which may influence parasite development or gametocyte activation, is not addressed. Even if not measured, this should be acknowledged as a limitation.\n• The claim that “macrophages and monocytes actively phagocytose microbiota during early blood digestion, while ookinetes opsonised by anti-Pfs25 antibodies might evade phagocytosis through Fc receptors” is intriguing but speculative. Further discussion or qualification is needed.\n• More broadly, the relevance to natural P. falciparum transmission remains indirect. Additional data or discussion is needed to support extrapolation from the rodent model to the human system.\n• Lines 117-118 and 125-129: The authors should show that CL treatment does not affect gametocyte levels at these stages, not only later in the manuscript.\n• Lines 120-124: Are the results in the figure from a single replicate? Three replicates are referenced but what is shown in the figure can’t be from all.\n• Line 221-223: Were these experiments (with and without antibiotics) performed in parallel? There appears to be a baseline difference, which should be clarified, replicated, and discussed.\n• Lines 252-257: Please indicate the number of replicates and mosquito batches tested. Are the results consistent?\nMinor comments\n• The title could be rephrased to reflect the indirect nature of the effect. For example: “Host monocytes/macrophages promote malaria transmission by modulating mosquito microbiota via SR-A-mediated phagocytosis.”\n• Avoid phrases like “enhance” or “promote transmission” unless immediately followed by the mechanistic qualifier (e.g., “by reducing gut bacteria”).\n• Statistical significance symbols should be consistently defined in all figure legends.\n• There is no need to explain species name abbreviations; this is a standard convention.\n• Line 22 (abstract): The phrase “depletion of SR-A” is misleading. The authors refer to SR-A blockade. Please rephrase.\n• Abstract: The term “immune evasion strategy” is vague. Spell out the mechanism: that the parasite co-opts host immune cells to eliminate transmission-blocking bacteria.\n• Lines 54-56: Of the three references cited, only the last one supports modulation of the peritrophic matrix. Please correct.\n• Lines 67-68: This sentence suggests that NK/NKT cells were previously implicated in transmission. If so, cite appropriate references. If not, rephrase for clarity.\n• Lines 74-76: The start of the Results section is abrupt. Consider rewriting or referencing appropriately.\n• Line 143-146: The mention of the P. falciparum SMFA appears abruptly. Better contextualisation is needed.\n• Line 148: The term “anti-malarial bacteria” may be confusing. Consider “bacteria with transmission-blocking activity” or similar.\n• Line 309 and onward: A microscopy image showing intracellular phagocytosed bacteria would strengthen the evidence.\n• Line 335 and throughout: Replace “ug” with the correct symbol for micrograms (μg)."}, "author_response": "Dear editor and reviewers,\nThank you for the opportunity to revise our manuscript and for your instructive comments,\nwhich will significantly enhance its quality. We have addressed each of the reviewers' points\nindividually. We hope that you will be satisfied with our responses.\nResponse to reviewer 1’ comments:\nReviewer #1 (Remarks to the Author):\nIn this manuscript, He et al report a very interesting study showing that mouse monocytes and/or\nmacrophages enhance transmission of murine malaria via phagocytosing bacteria of the mosquito\nmicrobiota. Overall, the study is well-conducted and the main conclusions would definitely be\nworth a publication in Nature Communications. I have however a few comments to be fully\nconvinced by this study.\nResponse: Thanks a lot for your appreciation.\nMajor comments:\nThe main point is that some experiments have been performed only twice, I think that a third\nreplicate in all the experiments presented in the main figures would be required for publication in\nNature communications, especially that the overall numbers are sometimes reasonable but low.\nAlso, it would be interesting to see the n of each replicate below charts instead of the total amount\nof mosquitoes.\nResponse: We have now conducted a third round of experiments, compiling data from three\nindependent studies to reinforce the findings presented in the main figures and supporting\ninformation. The number of replicates is indicated in the pie chart and figure legends. In response\nto your suggestion, we re-analyzed the pooled data using Generalized Linear Mixed Models\n(GLMMs). Most of our results align closely with those reported in the preliminary manuscript, with\na few notable exceptions detailed in Figures 1e, 1g, 1h, and 2l.\nIn our previous Figure 1e, we reported a significant increase in the frequency of DCs in the\nperipheral blood of mice infected with P. yoelii or P. berghei. However, our recent data show that\nonly P. yoelii infection significantly increased DC frequency. In Figure 1g, we initially indicated a\nsignificant increase in CD4+ T cell frequency in the peripheral blood of mice infected with either\nstrain. Yet, our pooled data analysis did not demonstrate a significant difference in CD4+ T cell\nfrequency between the two infections. Furthermore, in our earlier Figure 1h, we observed no\nsignificant reduction in CD8+ T cell frequency following P. berghei infection. In contrast, our current\ndata reveal that P. berghei infection significantly decreases CD8+ T cell frequency in the peripheral\nblood. We hypothesize that the notable increase in innate immune cell frequency may contribute\nto the observed reduction in CD8+ T cells following malaria infection. Since we did not examine the\nroles of CD4+ and CD8+ T cells, or DCs, in malaria transmission, this discrepancy does not alter our\nconclusions. Figures 1e, 1g, and 1h, along with the relevant context, have been updated accordingly\n(lines 85-94).\nIn our previous Figure 2l, we stated that 100% of control mice were infected with P. berghei after\nmosquito bites. However, during the third replication, two control mice were not infected, likely\ndue to low sporozoite production in the mosquitoes’ salivary glands. After pooling data from all\nthree replicates, we found that only 90% of control mice were infected with P. berghei post-bite\n(lines 155). This finding has been updated in the new Figure 2l. Please refer to the revised\nmanuscript for additional details.\nSecondly, I wonder if the authors have pointed survival of their mosquitoes at least until dissection\ntime. Microbiota proliferation after the blood meal has been found to affect mosquito lifespan\n(Gendrin et al, 2015) and I imagine that a reduction in phagocytosis, besides reducing vector\ncompetence, may as well reduce mosquito survival. Whether log-rank or a simple one-time point\nobservation at the time of dissection, such survival data would make the study more convincing.\nResponse: Thanks a lot for your suggestion. Now, the effect of CL treatment on the survival of\nmosquitoes has been observed. As you expected, mosquito fed on CL-treated mice has a lower\nsurvival rate compared with mosquito fed on control mice, and the data of which has been\npresented as Fig3 d. The relevant context has been updated accordingly (lines220-224).\nConsidering stats, I would like to see GLMMs used instead of Mann Whitney U test, to take into\naccount random effects of replicates. When the authors make a point on the non significance (after\nantibiotic treatment), it would like to see the actual p-value rather than a \"ns\". This \"ns\" may be\nclose to significance, but only 2 replicates have been done to test this point so a third one can\nclarify this.\nResponse: Thanks a lot for your comments. We have re-analyze our data by GLMMs analysis as the\ndata of mosquito oocysts often follows a Poisson or negative binomial distribution, and zero-\ninflation was also taken into consideration. Now, the actual p-value rather than ns have been added.\nPlease refer to the revised manuscript. We are sincerely appreciating for your professional advice.\nAll figure legends and method has been revised. Please refers to our updated manuscript.\nThe manuscript requires extensive English and latin proofreading, checks on the use of italics.\nBesides mistakes the main text, I spotted some in titles or axis titles in Fig 1B, D, 2A, S2B,D,G;\nS3A,C,E.\nResponse: We have carefully read thoroughly our manuscript, and tried our best to correct spelling\nand grammar errors. In addition, we have also corrected the mistakes in the titles or axis titles in\nFig 1B, D, 2A, S2B, D, G; S3A, C, E, as you suggested. Please refer to the revised manuscript.\nMinor points:\nLine 255 \"in the control bacteria\" please clarify this sentence.\nResponse: Thanks. \"in the control bacteria\" has been revised as \"for other bacteria\".\nS4E: are both pictures two representative pictures in each condition? Don't the authors have a\nlarger sample size to show?\nResponse: Yes. Both pictures are two representative pictures in each condition, now we have added\nup to four representative pictures of midguts colonized with eGFP-E. anophelis from three\nindependent experiments (line 950). Please refer to the revised manuscript.\nLine 509: are there differences between batches in the % of not fully engorged mosquitoes?\nResponse: We did not find the significant difference between batches in the % of not fully engorged\nmosquitoes.\nIn my opinion, at least some items of figure 3 should be used in a main figure, because it makes an\nimportant point (maybe removing some data from figure 1 e.g. choose one parasite for main and\none for supplements).\nResponse: Thanks a lot for your suggestion. We have re-organized our manuscript, the influence of\nCL, anti-Ly6G, and anti-NK1.1 have been displayed before we mention the influence of their roles\nin mosquito stage transmission (lines 117-128).\nIn sum, I am excited by this study, but I know that I have been disappointed by very beautiful stories\nthat have not been confirmed. I am hesitating between this excitement and a lack of trust, and I\nhope that revision will help me decide what I think about it...\nResponse: We understand what you worried about. Now, we have added a third repeat for the\nexperiments presented in the main figures. As a result, our repeated data supports our conclusion\nin this manuscript. Hopefully, you will be satisfied with our revision.\nResponse to reviewer 2’ comments:\nReviewer #2 (Remarks to the Author):\nThe manuscript by He et al examines the effects of mammalian immune cells on infection\noutcomes in the mosquito vector using rodent models of malaria infection. The study is simple, yet\nelegant, in its design and is clear in its presentation that macrophage/monocyte populations in the\nmosquito blood bolus influence the bacteria in the mosquito gut to influence malaria infection\noutcomes. The results are therefore very interesting and provide new insight into a previously\nunexplored aspect of malaria transmission. My comments are very minor and only look to increase\nthe clarity of the presented study.\nResponse: Thanks a lot for your appreciation.\nMy comments are as follows:\n-Please edit the abstract to improve readability and clarity, particularly for lines 16-24.\nResponse: Thanks for your comments. Now, the abstract has been modified to improve readability\nand clarity (lines 15-27).\n-There are several instances where more detailed editing is required that includes missing words,\nchanges to improve clarity, and multiple times where Anopheles should be italicized.\nResponse: We have carefully read thoroughly our manuscript, and tried our best to correct spelling\nand grammar errors. The word of “Anopheles” has been checked and italicized.\n-Line 73-74: Please revise for clarity.\nResponse: This sentence has been revised as “Mosquitoes acquire malaria parasites by biting\ninfected hosts. During these bites, immune cells from the host's peripheral blood, along with the\nparasites, are ingested into the mosquito's midgut. However, it remains unclear whether these\ningested immune cells influence the development of the parasites within the mosquito.” (lines 73-\n76). Hopefully, it has been clear now.\nResponse to reviewer 3’ comments:\nReviewer #3 (Remarks to the Author):\nThis manuscript by He et al. reports a novel and mechanistically detailed finding: that macrophages\nand monocytes taken up during mosquito blood feeding facilitate Plasmodium transmission by\nsuppressing gut microbiota through SR-A-mediated phagocytosis. The authors demonstrate that\nthis immunological interaction promotes ookinete development and enhances transmission for\nrodent malaria parasites. They show that this process undermines the efficacy of transmission-\nblocking antibodies (anti-Pfs25) against a P. berghei line expressing P. falciparum Pfs25, an effect\nthat can be rescued by macrophage/monocyte depletion or SR-A neutralization. This represents an\nimportant contribution to our understanding of host-vector-parasite interactions.\nResponse: Thanks a lot for your appreciation of our study.\nThe work is technically sound, conceptually novel, and of high potential interest to the malaria\ntransmission, immunology, and vector biology communities. It is supported by a comprehensive\nseries of in vivo experiments and microbial/molecular analyses in rodent and humanised models.\nHowever, several scientific, mechanistic, and editorial issues require attention. These range from\nclarifications of the main claims and better contextualisation, to refinements in data interpretation,\npresentation, terminology, and writing.\nResponse: Thanks a lot for your comments. We have thoroughly read our manuscript, and\nespecially pay attention on the scientific, mechanistic, and editorial issues as you mentioned. Now,\nwe have been tried our best to make our data interpretation and main claims clearer, and improve\nour writing. Please refer to our revised manuscript.\nMajor comments\n• The authors repeatedly state that macrophages and monocytes “promote transmission.” This\nshould be clarified throughout as an indirect effect, mediated via suppression of anti-Plasmodium\nbacteria, rather than any direct facilitation of parasite development.\nResponse: The indirect effect of macrophages and monocytes on malaria transmission has been\nclarified thoroughly for our revised manuscript.\n• The role of SR-A in bacterial phagocytosis is convincingly demonstrated, but it is not clear\nwhether this is the sole receptor involved. The possible contributions of other receptors (e.g., CD36,\nDC-SIGN, MR) are mentioned in the discussion but should be more explicitly addressed or\nacknowledged as a limitation.\nResponse: Thanks a lot for your suggestion. The possible contributions of other receptors (e.g.,\nCD36, DC-SIGN, MR) has been more explicitly addressed or acknowledged as a limitation (lines\n514-518).\n• The finding that SR-A blockade restores the full efficacy of anti-Pfs25 antibodies is compelling,\nbut the presentation could be improved. For example, clarifying that these are parallel and\nsynergistic mechanisms (i.e., microbiota-mediated blocking and antibody-mediated blocking),\nrather than interacting ones. Also, include time-course data to address how long SR-A remains\nblocked after antibody injection and how long macrophages/monocytes persist in the mosquito\nmidgut.\nResponse: As you suggested, we have presented the data on microbiota-mediated blocking and\nantibody-mediated blocking in relation to TRA and TRB percentages to clarify their relationship.\nThe new data indicate that these two mechanisms are not synergistic.\nRegarding the persistence of macrophages and monocytes in the mosquito midgut, we used flow\ncytometry (FACS) to analyze their frequency at 1-, 3-, 12-, and 24-hours post-feeding on infected\nmice. Our results show that macrophages and monocytes ingested by mosquitoes can survive for\nat least 3 hours, but their numbers dramatically decrease at 12 hours post-blood meal, becoming\ndifficult to detect by 24 hours. This data is presented in Figure 5c, and the corresponding context\nhas been updated accordingly (lines 346-350).\nIn response to your suggestion, we also investigated the longevity of the SR-A blocking effect. We\nfound that neutralizing SR-A 1 day before infection (5 days before the blood meal) did not affect\nthe number of oocysts in the mosquito midgut but did reduce oocyst prevalence. Additionally,\ninjecting anti-SR-A 1 day post-infection (3 days before the blood meal) resulted in a reduction in\ntransmission comparable to that observed in the CL-treated group. This indicates that\nneutralization of SR-A by antibody can effectively reduce malaria transmission for at least 3 days.\nThis data is presented in Figure 5h, and the corresponding context has been updated accordingly\n(lines 368-374).\n• The human data (addition of monocytes in SMFA) is an important proof of concept but limited\nto a single experimental system. It would strengthen the manuscript to show whether similar\nmonocyte expansions are observed in naturally infected individuals or gametocyte carriers.\nResponse: Literatures about the expansions of monocytes in naturally infected individuals or\ngametocyte carriers have been cited and discussed (lines 156-157).\n• The translational implications of this work, particularly for transmission-blocking vaccines or\nantibody combinations, deserve stronger emphasis in both the abstract and discussion. Currently,\nrelevant statements in the abstract are unclear or overly compressed.\nResponse: Thanks for your suggestion. Both abstract and discussion have been revised to\nemphasize the translational implications of this work (lines 24-29, and lines 531-539).\n• The roles of E. anophelis, S. sonnei, K. oxytoca, and S. marcescens are central to the proposed\nmechanism. However, their relative contributions to parasite suppression are not disentangled. Do\nthey act independently, additively, or synergistically? This should be discussed more clearly.\nResponse: Thanks for your suggestion. The independent, additive, or synergistical effect of E.\nanophelis, S. sonnei, K. oxytoca on malaria transmission has been performed. As a result, we did\nnot observe a superimposed effect when fed mosquito with multiple bacteria compared with solely\ncolonization, indicating there is no synergistical effect of E. anophelis, S. sonnei, K. oxytoca on\nmalaria transmission. This data has been presented as Figure 4h, and the relevant context has been\nupdated accordingly (lines 284-289).\n• While the manuscript shows that gametocytemia and ookinete development are unaffected by\nclodronate liposome (CL) treatment, this is presented too late. These controls should be moved\nearlier in the Results and shown in main figures to reassure the reader that effects on transmission\nare not due to altered gametocyte production.\nResponse: As you suggested, the result of CL treatment on gametocytemia and ookinete\ndevelopment has been moved to the S Figure 2, and mentioned before their effects on\ntransmission. Please refer to our revised manuscript.\n• Similarly, depletion of neutrophils and NK/NKT cells is shown to have no effect on transmission,\nbut the results and conclusions (lines 120-129) should be supported by replication and earlier\ngametocyte data.\nResponse: Thanks. The depletion of neutrophils and NK/NKT cells on transmission has been\nrepeated for three times and their effect on gametocytemia has been included (S Fig 2a, b).\n• The effect of CL treatment on systemic cytokine responses (e.g., IL-10, IFN-γ), which may\ninfluence parasite development or gametocyte activation, is not addressed. Even if not measured,\nthis should be acknowledged as a limitation.\nResponse: Thanks. The effect of CL treatment on systemic cytokine responses (e.g., IL-10, IFN-γ),\nwhich may influence parasite development or gametocyte activation, has been addressed in the\ndiscussion (lines 510-518).\n• The claim that “macrophages and monocytes actively phagocytose microbiota during early\nblood digestion, while ookinetes opsonised by anti-Pfs25 antibodies might evade phagocytosis\nthrough Fc receptors”is intriguing but speculative. Further discussion or qualification is needed.\nResponse: Thanks. Further discussion has been added (lines 497-501).\n• More broadly, the relevance to natural P. falciparum transmission remains indirect. Additional\ndata or discussion is needed to support extrapolation from the rodent model to the human system.\nResponse: Thanks. As we did not provide the direct evidence that the neutralization of SR-A to\nreverse the enhanced effect of human monocytes on P. falciparum transmission, we have added\na sentence of “However, the direct evidence whether the neutralization of SR-A could reverse the\npromoted effect of human monocytes on P. falciparum transmission is still required for the\nextrapolation from our findings in rodent model to human” in discussion to show the gap of\nextrapolation from the rodent model to the human system (lines 487-489).\n• Lines 117-118 and 125-129: The authors should show that CL treatment does not affect\ngametocyte levels at these stages, not only later in the manuscript.\nResponse: The result that CL treatment does not affect gametocyte levels has been moved forward\nas you suggested.\n• Lines 120-124: Are the results in the figure from a single replicate? Three replicates are\nreferenced but what is shown in the figure can’t be from all.\nResponse: Sorry for that, we only showed a single replicate in previously manuscript. Now, we have\nre-analyzed the pooled data from three independent experiments, and showed the consistent\nresults. Please refers to our revised manuscript.\n• Line 221-223: Were these experiments (with and without antibiotics) performed in parallel?\nThere appears to be a baseline difference, which should be clarified, replicated, and discussed.\nResponse: Yes, those experiments were performed in parallel, and replicated. There are two\ncontrol, one control is treated with antibiotics, and another is not. After clearance of microbiota\nby antibiotics treatment, the oocysts number was much higher than that of the control mosquitoes\nnot treated with antibiotics, which is consistent with previous study\n(doi:10.1371/journal.ppat.1000423). Also, we have discussed in our updated manuscript (lines\n228-231).\n• Lines 252-257: Please indicate the number of replicates and mosquito batches tested. Are the\nresults consistent?\nResponse: Thanks for your comments. Now, we have updated our figure legend, number of each\nexperiment has been indicated, and the results were consistent. Twenty mosquito midguts were\npooled as one biological replicate, and three biological replicates were performed. Three\nindependent experiments were performed for each experiment and the data were pooled. Please\nrefers to our revised manuscript.\nMinor comments\n• The title could be rephrased to reflect the indirect nature of the effect. For example: “Host\nmonocytes/macrophages promote malaria transmission by modulating mosquito microbiota via\nSR-A-mediated phagocytosis.”\nResponse: The title has been revised as you suggested (lines 1-2).\n• Avoid phrases like “enhance” or “promote transmission” unless immediately followed by\nthe mechanistic qualifier (e.g., “by reducing gut bacteria”).\nResponse: It has been revised as your suggestion.\n• Statistical significance symbols should be consistently defined in all figure legends.\nResponse: We have re-done all the statistical analyze, now the actual p-value has been added in\neach figure, and the sentence of “P<0.05 was considered as statistically significant” was\nconsistently added in all figure legends.\n• There is no need to explain species name abbreviations; this is a standard convention.\nResponse: Thanks for your suggestion. Now, the name abbreviations have been removed.\n• Line 22 (abstract): The phrase “depletion of SR-A” is misleading. The authors refer to SR-A\nblockade. Please rephrase.\nResponse: Yes, the phrase of “depletion of SR-A” has been revised as SR-A neutralization in the\nabstract (line 22).\n• Abstract: The term “immune evasion strategy” is vague. Spell out the mechanism: that the\nparasite co-opts host immune cells to eliminate transmission-blocking bacteria.\nResponse: Thanks for your suggestion. The term “immune evasion strategy”has been specified\nas your suggestion (line26 ).\n• Lines 54-56: Of the three references cited, only the last one supports modulation of the\nperitrophic matrix. Please correct.\nResponse: Yes. You are right. There is only one reference supporting modulation of the peritrophic\nmatrix, the other two references are supporting the modulatory role of microbiota in the mosquito\nimmune responses. To make it clear, the references have been separately cited to support the\nmodulation of PM or immune response by microbiota, respectively (line 56).\n• Lines 67-68: This sentence suggests that NK/NKT cells were previously implicated in transmission.\nIf so, cite appropriate references. If not, rephrase for clarity.\nResponse: The sentence seems to be confusing, misleading the known role of NK/NKT cells in\ntransmission. Now, it has been removed for clarity (lines 66-69).\n• Lines 74-76: The start of the Results section is abrupt. Consider rewriting or referencing\nappropriately.\nResponse: Now, the start of the Results section has been modified (lines 73-76).\n• Line 143-146: The mention of the P. falciparum SMFA appears abruptly. Better contextualisation\nis needed.\nResponse: The mention of the P. falciparum SMFA has been modified (lines 156-160).\n• Line 148: The term “anti-malarial bacteria” may be confusing. Consider “bacteria with\ntransmission-blocking activity” or similar.\nResponse: The term “anti-malarial bacteria” has been modified as “bacteria with transmission-\nblocking activity”. We have cheeked thought out our manuscript, and all the “anti-malarial bacteria”\nhas been re-phased with “bacteria with transmission-blocking activity” or similar as you suggested\n(line 260, line 306, and lines 533-534).\n• Line 309 and onward: A microscopy image showing intracellular phagocytosed bacteria would\nstrengthen the evidence.\nResponse: The microscopy image showing intracellular phagocytosed bacteria now has been\nincluded in Fig 5b of the revised manuscript (line 378).\n• Line 335 and throughout: Replace “ug” with the correct symbol for micrograms (μg).\nResponse: Thanks for your careful review, “ug” has been replaced with “μg” (line 364).\nDear editor and reviewer,\nThank you very much for your valuable comments, which have significantly enhanced our\nmanuscript. We have thoroughly reviewed both the previous rebuttal and the main article file,\nremoving statements made by the reviewers and rephrasing relevant sections in our own words.\nFurthermore, we have acknowledged the use of AI in the Acknowledgments section of the\nmanuscript. All revisions have been highlighted in yellow. Below, you will find our point-by-point\nresponse to Reviewer 3’s comments. We hope our responses meet your expectations.\nResponse to Reviewer #3:\nThe manuscript has improved in several important respects. However, a number of issues remain\nthat should be addressed before further consideration.\nResponse: Yes. Now, we have fixed these issues.\nThe addition of a third independent replicate across the main figures is welcome and was necessary.\nThe re-analysis using GLMMs is more appropriate and statistically sound. However, it is important\nto note that the inclusion of the third replicate altered several previously reported findings (e.g.,\nimmune cell frequencies in Fig. 1 and infection prevalence in Fig. 2). While the authors report these\nchanges transparently in the rebuttal, the fact that conclusions shifted with the addition of one\nreplicate indicates that the original dataset was underpowered and that some earlier conclusions\nwere not robust.\nResponse: Yes. This result is also out of our anticipation, and this also reminds us to repeat the\nimportant experiments for three times in our future study.\nAt present, I have no independent means of verifying the robustness of the revised dataset beyond\ntrusting the statistical analyses, which appear appropriately conducted. Nonetheless, it would\nstrengthen the manuscript if the authors explicitly acknowledge in the Results section that certain\nimmune cell changes did not remain significant after full replication.\nResponse: Thanks. It has been explained as you suggestion (line 85-87).\nDespite this concern, it is my view that the study remains mechanistically interesting and\nconceptually novel, and the additional replication and statistical re-analysis substantially\nstrengthen the scientific core of the work.\nResponse: Thanks a lot for your appreciation of the statistical re-analysis.\nThe clarification that macrophages/monocytes influence transmission indirectly via modulation of\nthe mosquito microbiota improves conceptual clarity. The addition of time-course data, SR-A\nblocking duration experiments, and bacterial colonisation experiments strengthens the\nmechanistic argument. However, causal phrasing still occasionally overreaches and should be\nmoderated to avoid implying direct pro-parasitic effects where the mechanism is indirect.\nResponse: Thanks. Now, we have rephrased some sentences to avoid implying direct pro-parasitic\neffects where the mechanism is indirect. Please see line 17, 22, 66,180, 279-280, 291-292, 301,\nand 315-316.\nThere are instances where the rebuttal states that certain corrections were made, but the revised\nmanuscript does not fully reflect these claims. For example:\n- The authors state that unnecessary species name explanations were removed, yet explanatory\nphrasing remains in places.\nResponse: We have checked the manuscript thoroughly, and removed all explanatory phrasing for\nspecies. Please see line 78, 141, 189, 216, 239, 383, 385 and 398.\n- Several stylistic and grammatical issues persist.\nResponse: Thanks. We have read the manuscript carefully and corrected stylistic and grammatical\nissues as possible as we can. Please refers to the highlighted words or sentences.\n- The description of the GLMM model structure remains insufficiently detailed in the Methods\nsection.\nResponse: Now, the detailed description of the GLMM model structure has been supplemented in\nthe Methods section, lines 553-555.\nIn several sections, portions of reviewer comments appear to have been incorporated into the\nmanuscript with minimal or no modification, at times retaining phrasing that reads as external\ncritique rather than as integrated narrative.\nWhile it is entirely appropriate to respond to reviewer suggestions, revised text should be fully\nintegrated into the authors’ voice and the structure of the manuscript. Direct or near-direct\nreproduction of reviewer phrasing without careful adaptation may give the impression of reactive\nediting rather than coherent revision.\nThe manuscript would benefit from careful stylistic harmonisation to ensure that the text reads as\na unified scientific narrative rather than a patchwork of responses.\nResponse: Thanks for your comments. We have carefully checked the manuscript, especially the\nrevised parts, and rephrased in our own words instead of statements made by reviewers and kept\nstylistic harmonization of the whole manuscript. Please see the line 349-351.\nFinally, the writing style in parts of the revised manuscript suggests the possible use of generative\nAI tools for drafting or rephrasing. The use of such tools is increasingly common and not inherently\nproblematic; however, best practice and many journal policies require transparent disclosure if\ngenerative AI has been used in manuscript preparation. If AI-assisted drafting or rewriting was used,\nthis should be clearly stated in the appropriate section in accordance with journal policy.\nResponse: Thanks for the reminding. We did use the AI to help us to rephase our manuscript, which\nhas been stated in the acknowledgement section (line 726-727)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I congratulate the authors for taking all my comments into account, I am more convinced about the manuscript as it stands now. I agree with them that the differences observed after the third replicate are not contradicting the main message, and think that the confirmation of the other results after proper analyses strengthens the story.\nHappy to see that the survival of mosquitoes was affected as I suspected.\nBeautiful work, contratulations...", "reviewer_3": "The manuscript has improved in several important respects. However, a number of issues remain that should be addressed before further consideration.\nThe addition of a third independent replicate across the main figures is welcome and was necessary. The re-analysis using GLMMs is more appropriate and statistically sound. However, it is important to note that the inclusion of the third replicate altered several previously reported findings (e.g., immune cell frequencies in Fig. 1 and infection prevalence in Fig. 2). While the authors report these changes transparently in the rebuttal, the fact that conclusions shifted with the addition of one replicate indicates that the original dataset was underpowered and that some earlier conclusions were not robust.\nAt present, I have no independent means of verifying the robustness of the revised dataset beyond trusting the statistical analyses, which appear appropriately conducted. Nonetheless, it would strengthen the manuscript if the authors explicitly acknowledge in the Results section that certain immune cell changes did not remain significant after full replication.\nDespite this concern, it is my view that the study remains mechanistically interesting and conceptually novel, and the additional replication and statistical re-analysis substantially strengthen the scientific core of the work.\nThe clarification that macrophages/monocytes influence transmission indirectly via modulation of the mosquito microbiota improves conceptual clarity. The addition of time-course data, SR-A blocking duration experiments, and bacterial colonisation experiments strengthens the mechanistic argument. However, causal phrasing still occasionally overreaches and should be moderated to avoid implying direct pro-parasitic effects where the mechanism is indirect.\nThere are instances where the rebuttal states that certain corrections were made, but the revised manuscript does not fully reflect these claims. For example:\n- The authors state that unnecessary species name explanations were removed, yet explanatory phrasing remains in places.\n- Several stylistic and grammatical issues persist.\n- The description of the GLMM model structure remains insufficiently detailed in the Methods section.\nIn several sections, portions of reviewer comments appear to have been incorporated into the manuscript with minimal or no modification, at times retaining phrasing that reads as external critique rather than as integrated narrative.\nWhile it is entirely appropriate to respond to reviewer suggestions, revised text should be fully integrated into the authors’ voice and the structure of the manuscript. Direct or near-direct reproduction of reviewer phrasing without careful adaptation may give the impression of reactive editing rather than coherent revision.\nThe manuscript would benefit from careful stylistic harmonisation to ensure that the text reads as a unified scientific narrative rather than a patchwork of responses.\nFinally, the writing style in parts of the revised manuscript suggests the possible use of generative AI tools for drafting or rephrasing. The use of such tools is increasingly common and not inherently problematic; however, best practice and many journal policies require transparent disclosure if generative AI has been used in manuscript preparation. If AI-assisted drafting or rewriting was used, this should be clearly stated in the appropriate section in accordance with journal policy."}, "author_response": "Dear editor and reviewer,\nThank you very much for your valuable comments, which have significantly enhanced our\nmanuscript. We have thoroughly reviewed both the previous rebuttal and the main article file,\nremoving statements made by the reviewers and rephrasing relevant sections in our own words.\nFurthermore, we have acknowledged the use of AI in the Acknowledgments section of the\nmanuscript. All revisions have been highlighted in yellow. Below, you will find our point-by-point\nresponse to Reviewer 3’s comments. We hope our responses meet your expectations.\nResponse to Reviewer #3:\nThe manuscript has improved in several important respects. However, a number of issues remain\nthat should be addressed before further consideration.\nResponse: Yes. Now, we have fixed these issues.\nThe addition of a third independent replicate across the main figures is welcome and was necessary.\nThe re-analysis using GLMMs is more appropriate and statistically sound. However, it is important\nto note that the inclusion of the third replicate altered several previously reported findings (e.g.,\nimmune cell frequencies in Fig. 1 and infection prevalence in Fig. 2). While the authors report these\nchanges transparently in the rebuttal, the fact that conclusions shifted with the addition of one\nreplicate indicates that the original dataset was underpowered and that some earlier conclusions\nwere not robust.\nResponse: Yes. This result is also out of our anticipation, and this also reminds us to repeat the\nimportant experiments for three times in our future study.\nAt present, I have no independent means of verifying the robustness of the revised dataset beyond\ntrusting the statistical analyses, which appear appropriately conducted. Nonetheless, it would\nstrengthen the manuscript if the authors explicitly acknowledge in the Results section that certain\nimmune cell changes did not remain significant after full replication.\nResponse: Thanks. It has been explained as you suggestion (line 85-87).\nDespite this concern, it is my view that the study remains mechanistically interesting and\nconceptually novel, and the additional replication and statistical re-analysis substantially\nstrengthen the scientific core of the work.\nResponse: Thanks a lot for your appreciation of the statistical re-analysis.\nThe clarification that macrophages/monocytes influence transmission indirectly via modulation of\nthe mosquito microbiota improves conceptual clarity. The addition of time-course data, SR-A\nblocking duration experiments, and bacterial colonisation experiments strengthens the\nmechanistic argument. However, causal phrasing still occasionally overreaches and should be\nmoderated to avoid implying direct pro-parasitic effects where the mechanism is indirect.\nResponse: Thanks. Now, we have rephrased some sentences to avoid implying direct pro-parasitic\neffects where the mechanism is indirect. Please see line 17, 22, 66,180, 279-280, 291-292, 301,\nand 315-316.\nThere are instances where the rebuttal states that certain corrections were made, but the revised\nmanuscript does not fully reflect these claims. For example:\n- The authors state that unnecessary species name explanations were removed, yet explanatory\nphrasing remains in places.\nResponse: We have checked the manuscript thoroughly, and removed all explanatory phrasing for\nspecies. Please see line 78, 141, 189, 216, 239, 383, 385 and 398.\n- Several stylistic and grammatical issues persist.\nResponse: Thanks. We have read the manuscript carefully and corrected stylistic and grammatical\nissues as possible as we can. Please refers to the highlighted words or sentences.\n- The description of the GLMM model structure remains insufficiently detailed in the Methods\nsection.\nResponse: Now, the detailed description of the GLMM model structure has been supplemented in\nthe Methods section, lines 553-555.\nIn several sections, portions of reviewer comments appear to have been incorporated into the\nmanuscript with minimal or no modification, at times retaining phrasing that reads as external\ncritique rather than as integrated narrative.\nWhile it is entirely appropriate to respond to reviewer suggestions, revised text should be fully\nintegrated into the authors’ voice and the structure of the manuscript. Direct or near-direct\nreproduction of reviewer phrasing without careful adaptation may give the impression of reactive\nediting rather than coherent revision.\nThe manuscript would benefit from careful stylistic harmonisation to ensure that the text reads as\na unified scientific narrative rather than a patchwork of responses.\nResponse: Thanks for your comments. We have carefully checked the manuscript, especially the\nrevised parts, and rephrased in our own words instead of statements made by reviewers and kept\nstylistic harmonization of the whole manuscript. Please see the line 349-351.\nFinally, the writing style in parts of the revised manuscript suggests the possible use of generative\nAI tools for drafting or rephrasing. The use of such tools is increasingly common and not inherently\nproblematic; however, best practice and many journal policies require transparent disclosure if\ngenerative AI has been used in manuscript preparation. If AI-assisted drafting or rewriting was used,\nthis should be clearly stated in the appropriate section in accordance with journal policy.\nResponse: Thanks for the reminding. We did use the AI to help us to rephase our manuscript, which\nhas been stated in the acknowledgement section (line 726-727)."}]

Decision letter