Abstract
For successful transmission, the malaria parasite must traverse tissue epithelia and survive attack from the insect’s innate immune system. Hemocytes play a multitude of roles in mosquitoes, including defense against invading pathogens. Here, we show that hemocytes of the major malaria vector Anopheles gambiae promote Plasmodium falciparum infection by maintaining midgut epithelial integrity by controlling cell proliferation upon blood feeding. The mosquito’s hemocytes also control the midgut microbiota and immune gene expression. Our study unveils novel hemocyte functions that are exploited by the human malaria parasite to evade the mosquito’s immune system.
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Introduction
Malaria remains one of the most devastating global public health burdens and has plagued mankind for at least 5000 years, causing 2–5% of all deaths worldwide in the 20th century1. Malaria control that is mainly based on strategies targeting its mosquito vectors has achieved a considerable reduction in the number of cases and deaths since 2000. Nevertheless, progress has been eroded mainly by the vector’s insecticide resistance and the parasite’s drug resistance2. Therefore, novel vector control methods are needed, and those based on targeting the malaria parasite in the vector have recently gained increasing interest3. Development of such strategies, however, will require a better understanding of mosquito-parasite interactions and infection biology to enable the identification of potent transmission-blocking targets3,4,5,6.
Mosquitoes defend themselves against bacteria, fungi, viruses, and protists by means of their innate immune system7,8. Hemocytes (macrophage-like cells in invertebrates) are specialized immune cells that carry out a variety of immune defense processes such as phagocytosis, melanization, production of antimicrobial peptides (AMPs) and complement-like factors9,10,11. Vertebrate macrophages also play other roles in addition to their involvement in immune defense, such as eliminating dead cells, promoting tissue regeneration, controlling the differentiation of other cell types, and other functions12.
Drosophila melanogaster hemocytes also play multiple roles in morphogenesis and tissue repair, including regulating cell proliferation in tissues such as the ovaries and midgut13, suggesting that hemocytes in mosquitoes may have similar functions. Several studies have shown that Anopheles hemocytes restrict the rodent malaria parasite Plasmodium berghei in the mosquito14,15,16,17,18,19,20; however, the mosquito’s immune defenses against the human malaria parasite P. falciparum have been shown to be quite different from those against P. berghei21,22,23,24,25,26, and the role of hemocytes in anti-P. falciparum defenses is poorly understood.
In addition to surviving the immune responses, Plasmodium parasites need to traverse several physical barriers to reach the salivary glands in order to render the mosquito infective. Upon ingestion of an infectious blood meal, the ookinete-stage parasites penetrate the peritrophic matrix that protects the midgut epithelial cells from direct contact with ingested blood27. Next, the ookinetes traverse the midgut epithelium to reach the basal side, where they develop into oocysts28,29. This invasion process causes substantial damage to the midgut epithelium, triggering an immune response involving the production of reactive oxygen and nitrogen species that kill ookinetes and recruit hemocytes to mediate complement anti-Plasmodium defenses15,30.
In this work, we provide evidence of several phagocytic hemocyte functions related to mosquito physiology and immunity that affect P. falciparum infection. We show that phagocytic hemocytes are agonists of the early stages of P. falciparum infection and maintain midgut epithelial integrity by regulating intestinal cell proliferation. We also show that these cells influence midgut microbiota load, most likely by influencing the expression of immune genes in the midgut tissue. These findings reveal new non-canonical functions of hemocytes that also affect mosquito vector competence for P. falciparum.
Results
Phagocytic hemocytes are agonists of early midgut-stage P. falciparum infection
Mosquito hemocytes produce several immune factors that can protect mosquitoes from a broad range of pathogens, including protozoa. Many of these factors, such as c-type lectin 4 (CTL4) and thioester-containing protein 1 (TEP1), have agonist and antagonist roles against Plasmodium, respectively25,31,32. Hence, we hypothesize that hemocytes could play diverse roles during Plasmodium infection of the midgut. To test this hypothesis, we chemically depleted the phagocytic hemocytes, as previously described by Kwon and Smith17, by injecting clodronate-containing liposomes (clodrosomes [CLDs]) into the hemocoel, while empty liposomes (LPSMs)-injected and non-injected mosquitoes (naive mosquitoes [NT]) were used as controls (Supplementary Fig. 1a). We evaluated the efficiency of clodronate liposome -mediated depletion of circulating (Circulating hemocytes were quantified according to methodology established by Castillo et al.33) and sessile (hemocytes attached to the tissues) phagocytes (Supplementary Fig. 1b). The number of circulating and sessile phagocytes in the CLD-injected mosquitoes was reduced by approximately 90% when compared to the LPSMs-injected and NT control mosquitoes (Supplementary Fig. 1c–f).
After phagocyte depletion, we infected mosquitoes with P. falciparum by allowing the mosquitoes to feed on human blood with either a low or high gametocyte concentration. Infection intensity and prevalence were evaluated by counting the number of oocysts on the midgut epithelium at 7 days after the infectious blood meal. The results showed that phagocyte-type immune cell depletion had no impact on P. falciparum infection at the low infection intensity but resulted in a significant reduction in oocyst numbers at the high infection intensity, indicating that phagocytes act as agonist of P. falciparum midgut infection (Fig. 1a). Furthermore, phagocyte-depleted mosquitoes exhibited substantial mortality at both low- and high-infection intensities (Fig. 1a), suggesting that phagocytic hemocytes mediate mosquito tolerance to infection and/or blood feeding. We also investigated whether phagocytes could influence later-stage P. falciparum infection (i.e., the sporozoite stage) by injecting mosquitoes with CLDs or LPSMs at 10 days after the consumption of an infectious blood meal, followed by enumeration of sporozoites in the salivary glands. We did not observe any significant difference in the number of sporozoites per salivary gland between phagocyte-depleted and control mosquito cohorts, indicating that phagocytic hemocytes do not control sporozoite-stage infection (Fig. 1b).
a P. falciparum infection intensity, prevalence, and mortality in NT, LPSM-injected, and CLD-injected mosquitoes fed on blood with high or low gametocytemia, measured at 7 dpi. b P. falciparum sporozoites in salivary glands (SGs) measured at 14 dpi. Mosquitoes were injected with LPSMs or CLDs or not injected (NT), and sporozoites were counted 4 days later (at 10 dpi). c Model of the role of A. gambiae hemocytes during P. falciparum infection. Hemocytes play an agonistic role in the early stages of P. falciparum but have no effect against sporozoites. Each experiment consisted of three biological replicates, and data were pooled to generate the graphs. Data are presented as boxes and whiskers with medians with interquartile ranges; each dot indicates the number of oocysts/sporozoites per midgut/salivary glands. Mortality rates are presented as percentages in a bar graph. Prevalence is presented as a percentage. The n value for each condition is indicated in the subfigures. Statistical analysis of infection intensity was performed using the Kruskal–Wallis test with Dunn´s post-test. *p < 0.05; ***p > 0.0005; ns not significant. Statistical analysis of prevalence and mortality rates were performed with Fisher´s exact test, ****p < 0.0001; ns not significant. Detailed statistical analysis and source data are provided as a Source Data file. Schematic illustrations of the experimental strategy in Fig. (1a, b), and the model of Fig. 1c were created by Victor Cardoso-Jaime using Microsoft PowerPoint Version 16.92.
Phagocytes mediate midgut epithelial integrity upon blood feeding
To determine whether the high mortality observed in infected phagocyte-depleted mosquitoes was related to P. falciparum infection or blood feeding, we fed phagocyte-depleted mosquitoes on uninfected blood and monitored their survival over the course of 14 days. We found no differences in mortality between the NT and LPSMs-injected cohorts, whereas CLDs-injected mosquitoes exhibited high mortality (Fig. 2a), indicating that hemocytes play a vital role upon blood-feeding. We frequently observed blood leakage from the midgut into the hemocoel of phagocyte-depleted (CLDs-injected) mosquitoes, which probably resulted from midgut rupture (Fig. 2b) and is a likely reason for their elevated mortality.
a Survival curve of NT, LPSM-injected, and CLD-injected mosquitoes fed on blood. Each experiment consisted of at least three biological replicates pooled together to generate the graph. Data are represented as percentage ± SE. n = 35 (NT), n = 25 (LPSMs), n = 31 (CLDs). Statistical analysis was performed using the log-rank (Mantel–Cox) test. ****p < 0.0001; ns not significant. b Mosquito phenotype 48 h after blood feeding. Note that NT and LPSM-injected mosquitoes have a normal abdomen, with a regular shape of the midgut containing a blood bolus. Some CLD-injected mosquitoes have blood spread along the abdomen, indicating a rupture of the midgut epithelium. c, d Smurf assay to measure the midgut epithelium integrity. c Smurf phenotype rate and mortality of NT, LPSMs-injected, and CLDs-injected mosquitoes. Data were generated from three biological replicates; they are represented as percentages in a bar graph. n = 84 (NT), n = 89 (LPSMs), n = 77 (CLDs). Statistical analyses of smurf phenotype and mortality rates were performed with Fisher´s exact test, ****p < 0.0001; ns not significant. d Mosquito phenotype of Smurf (left) and Non-Smurf (Right). e Representative image of immunofluorescent staining detecting phosphorylation of serine 10 of histone H3 (PH3) in sugar- and blood-fed NT, LPSM-injected, and CLD-injected mosquitoes. Red stain indicates anti-PH3 antibody; blue, nuclei stained with DAPI; PH3+ cells are indicative of cell division, bar scale = 50 μm. f Index of PH3+ cells per midgut in NT, LPSM-injected, and CLD-injected mosquitoes at 24 h post-blood feeding. Data were generated from three biological replicates; each dot indicates the number of PH3+ cells per midgut, presented as mean ±SD in a bar graph. n = 11 (NT), n = 10 (LPSMs), n = 11 (CLDs). Statistical analysis of PH3+ cell index was performed by ANOVA test followed of Tukey´s multiple comparation test. *p < 0.05; **,p < 0.01; ns not significant (g) Heatmap of Log2 (-fold change) of cell proliferation-related genes in the whole mosquito and midguts. Basal expression is indicated by the color white, upregulation by red, and downregulation by blue; cells contain Log2 values with SD. Significant changes were determined by ANOVA test, followed of Tukey´s multiple comparation test. *p < 0.05; ns not significant, (n = 3). Data and detailed statistical analysis of the -fold changes are available in the Source Data file and Supplementary Figs. 7k–m, 8k–m. b All experiments were performed three times. Source data are provided as a Source Data file. Schematic illustrations of the experimental strategy in the Fig. (2a, c, g) were created by Victor Cardoso-Jaime using Microsoft PowerPoint Version 16.92.
To rule out the possibility that the mortality was caused by CLDs-induced side effects, we fed the mosquitoes on blood supplemented with CLDs and LPSMs and then monitored survival for 14 days. We observed no differences in mortality between the cohorts (Supplementary Fig. 2). Furthermore, we treated mosquitoes with liposomes containing DiO dye (LPSMs-DiO) to track hemocytes that engulfed liposomes (Supplementary Fig. 3a-b) and other non-target cells that may have internalized liposomes. We found that liposomes specifically target circulating and sessile phagocytes attached to the abdominal tissues, and about 90% of circulating phagocytes engulfed LPSMs-DiO (Supplementary Fig. 3c-d). Furthermore, we analyze the midguts from LPSMs-DiO-injected mosquitoes 24 h after blood feeding when midgut distention may have ruptured the basal lamina enabling internalization of liposomes by epithelial cells. We did not observe any presence of liposomes in midgut epithelial cells (Supplementary Fig. 4). Thus, liposomes are not taken by epithelial cells, which excludes the possibility that a non-specific effect of liposomes could produce epithelial damage. Clodronate liposomes are highly efficient in depleting phagocytic cells; no toxic effects or depletion of phagocytic cells or other cell types have been reported in non-encapsulated clodronate since cells are not permeable34,35. To test whether toxicity produced by clodronate could be responsible for some of the observed phenotypes, we injected mosquitoes from the same batches with encapsulated clodronate (CLDs) or non-encapsulated clodronate (clodronate in solution) and evaluated the effect on hemocyte depletion and mosquito survival. This assay showed that only mosquitoes injected with encapsulated clodronate (CLDs) experienced hemocyte depletion and elevated mortality (Supplementary Fig. 5a-b), while mosquitoes injected with non-encapsulated clodronate did not show any of these effects (Supplementary Fig. 5c-d).
We could not detect differences between treatments with CLDs diluted in either RPMI or PBS, both resulting in mortality (Supplementary Fig. 6), thus suggesting that the vehicles do not influence mortality rates after CLDs injection at 27 °C. All together, these results (Supplementary Figs. 2–6) support our hypothesis that phagocytic hemocytes are important for mosquito viability, most likely by maintaining midgut tissue integrity at human malaria transmission conditions.
Blood feeding has been shown to increase the number of hemocytes in the mosquito36,37, potentially increasing the amount of hemocyte-produced immune factors36. However, it is unclear whether P. falciparum infection influences the number of hemocytes in the mosquito. To investigate this possibility, we counted the number of hemocytes in sugar- and blood-fed mosquitoes, as well as in P. falciparum-infected mosquitoes at both 7 and 14 days post-infection (Supplementary Fig. 7). While the number of hemocytes was higher in blood-fed than in sugar-fed mosquitoes, there was no difference between the blood-fed and infected mosquitoes, further supporting the hypothesis that hemocytes play a primary role in blood feeding. Furthermore, since we observed an increase in the number of hemocytes upon a blood meal, we evaluated whether phagocyte population could be re-established in CLDs-injected mosquitoes after feeding on blood. The experiment showed that phagocytic hemocyte depletion was maintained for at least 7 days post-blood feeding (Supplementary Fig. 1d), indicating that the phagocyte population cannot be reestablished after depletion.
During blood feeding, the midgut experiences drastic changes, such as an increase in size and shape, and in processes related to blood digestion, including peritrophic matrix production, ROS synthesis, and proliferation of the microbiota38,39,40. To determine whether phagocyte depletion can alter midgut epithelial integrity during blood feeding, we performed “smurf assay” [described by Rera et al.]41, and by feeding mosquitoes on blood containing blue food dye (Fig. 2c, d), which diffuses across the ruptured midgut epithelium into the hemolymph (Fig. 2d). The phagocyte-depleted (CLDs-injected) mosquitoes showed a higher mortality and prevalence of the “smurf-phenotype” mosquitoes than did the controls. These results shows that phagocyte-depleted mosquitoes are more susceptible to midgut damage upon blood feeding (Fig. 2c).
Cell proliferation and differentiation are required for the maintenance of midgut epithelial integrity42,43,44. Since hemocytes influence the midgut epithelial integrity, it suggests their possible involvement in regulating these processes. To address this hypothesis, we studied the effect of phagocytic hemocyte depletion on midgut epithelial cell proliferation by measuring the expression of AURKA (a mitosis marker), Delta (intestinal stem cells markers), and Upd3 (proliferation cytokine) in whole mosquitoes and midguts of NT, LPSMs-injected, and CLDs-injected mosquitoes at 24 h post-blood feeding. Interestingly, we observed a significant down-regulation of AURKA in phagocyte-depleted whole mosquitoes and a slightly reduced expression of AURKA, Delta, and Upd3 in the midgut tissue (Fig. 2g and Supplementary Figs. 8k–m, 9k–m).
In addition, cellular proliferation was measured by assaying the phosphorylation of serine 10(PS10) of histone H3 (H3) in the nucleus of cells, a well-established mitosis marker in vertebrates and invertebrates, including several mosquito species45. We employed an anti-histone H3 antibody previously used in studies with An. albimanus which shares a 100% histone H3 sequence identity with An. gambiae. Histone H3 sequences are highly conserved across the animal kingdom, including mosquitoes, flies, and humans45 (Supplementary Fig. 10). As a positive control for mitosis, and to validate cross-reactivity and specificity of the anti-S10-PH3 antibody, we stained ovaries at 24 h post-blood feeding. Oogenesis is initiated by a blood meal and involves cell mitosis and endocycle for egg maturation. The primary egg chamber will terminate follicle cell mitosis at 24 h when the secondary egg chamber will initiate growth and follicle cell mitosis46. As expected, we observed a PH3 signal from the secondary egg chamber follicle cells at 24 h post-blood feeding, and no signal was observed in ovaries from sucrose-fed mosquitoes (Supplementary Fig. 11). We were also able to observe the segregation of chromosomes in the ovary PH3+ cells (Supplementary movies 1 and 2). These data show that that anti-S10-PH3 recognizes mitotic cells.
Mosquito intestinal stem cells have been suggested to represent the only proliferative/regenerative cell type in the midgut epithelium45,47. The Drosophila stem cells have smaller nuclei48, but similar morphology to those in mosquitoes49. Our results show that PH3+ cells of the midgut have nuclei of smaller size and present a similar staining pattern to that described in recent studies in Drosophila, An. gambiae, and An. albimanus (Supplementary Fig. 12)45,47,50,51. We stained the midguts of NT, LPSMs, and CLDs-treated mosquitoes at 24 h post-blood feeding to evaluate the effect of phagocyte depletion on the mitotic index of the midgut epithelium. This experiment showed that phagocyte-depleted mosquitoes had fewer PH3+ cells than did the controls (Fig. 2e, f, and Supplementary Fig. 12), confirming that phagocytic hemocytes modulate midgut cell proliferation. Together, these results suggest that phagocytes control multiple physiological functions that are essential to coping with the blood meal, and they indirectly affect Plasmodium infection.
Phagocytes control the midgut microbiota, but their influence on midgut epithelial integrity, and P. falciparum infection is independent of microbiota
The mosquito midgut microbiota has been shown to proliferate after a blood meal and to play a role in maintaining the integrity of the midgut epithelium. Thus, microbiota dysbiosis could result in midgut rupture and leakage of bacteria that would cause a systemic infection40. Since we found that phagocyte depletion compromised midgut epithelial integrity and caused high mortality, we hypothesized that alterations in microbiota homeostasis may be involved in both midgut rupture and mortality. We determined the bacterial load of the midguts of sugar-fed and blood-fed (24–48 h post-blood feeding) mosquitoes, both phagocyte-depleted and non-depleted (Fig. 3a). We observed a higher bacterial load in the midguts of phagocyte-depleted mosquitoes regardless of the meal (Fig. 3a), suggesting that phagocytic hemocytes influence the homeostasis of the midgut microbiota. The expanded midgut microbiota of phagocyte-depleted mosquitoes could be responsible for the disruption of the midgut epithelium, and bacterial leakage into the hemolymph could lead to systemic infection and eventual mortality. To test these hypotheses, we analyzed the bacterial load in the hemolymph of NT, LPSMs-injected, and CLDs-injected mosquitoes after blood feeding. Our results showed that phagocyte depletion increased the bacterial load in the hemolymph (Fig. 3b). Taken together, these results suggest that phagocytic hemocytes are essential for controlling microbiota homeostasis and maintaining midgut epithelial integrity to prevent systemic bacterial infection that can cause mortality (Fig. 3c).
a The midgut microbiota was analyzed in NT, LPSM-injected, and CLD-injected mosquitoes fed on sucrose or at 24 and 48 h post-blood feeding. b Bacterial load in the hemolymph of NT, LPSM-injected, and CLD-injected mosquitoes was measured at 24 and 48 h after blood feeding. c Model showing that hemocytes restrict the midgut microbiota load and control systemic infections produced by midgut microbiota leakage. Each experiment had three biological replicates; data were pooled to generate the graph. Data are represented as medians with maximum and minimum value. a Sucrose (n = NT, 32; LPSMs, 32; CLDS, 31), Blood (24 h) (NT, LPSMs, and CLDs, n = 36), Blood (48 h) (NT, LPSMs, and CLDs n = 32); b Blood (24 h) (n = NT, 20; LPSMs, 22; CLDs, 24), Blood (48 h) (n = NT, 17; LPSMs, 21; CLDs, 21). Statistical analysis was performed using the Kruskal–Wallis test with the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. Statistical differences: *p < 0.05; nd not discovery. Source data and detailed statistical analysis are provided as a Source Data file. Schematic illustrations of the experimental strategy in Fig. (3a, b), and the model of Fig. 3c were created by Victor Cardoso-Jaime using Microsoft PowerPoint Version 16.92.
To specifically determine whether the proliferation of the microbiota is linked to the compromised integrity of the midgut epithelium, we performed blood-feeding assays with phagocyte-depleted and non-depleted mosquitoes under aseptic conditions achieved through antibiotic treatment (Fig. 4a). Surprisingly, phagocyte depletion of aseptic mosquitoes also caused midgut leakage and mortality upon blood feeding, indicating that the over-proliferation of bacteria is not the main reason for the midgut epithelial disruption in phagocyte-depleted mosquitoes. Hence, phagocytic hemocytes appear to play a direct role in maintaining midgut epithelium integrity.
a Smurf assay of NT, LPSM-injected, and CLD-injected mosquitoes maintained on sugar containing antibiotics. The rates of smurf phenotype and mortality were evaluated at 48 h post-blood feeding. Each experiment had three experimental replicates. Data are presented as percentages in bar graph. (n = NT, 84; LPSMs, 89; CLDs, 77). Statistical analysis was performed using Fisher´s exact test. ****p < 0.0001, ns not significant. b Mosquitoes maintained on sugar containing antibiotics (NT, LPSM-injected, and CLD-injected mosquitoes) were fed on blood containing high P. falciparum gametocytemia. The intensity and prevalence of infection and the mortality rate were evaluated at 7 dpi. Data were obtained from three experiments, pooled together to generate the graph. Plasmodium infection data are presented as medians with interquartile range; each dot indicates the number of oocysts per midgut. Mortality rates and prevalences are expressed as percentages in bar and circular diagrams, respectively. Statistical analysis of intensity of infection was done using the Kruskal–Wallis test with Dunn´s post-test. **p < .005; ns not significant. Statistical analysis of mortality rate and prevalence were performed using Fisher´s exact test. ****p < .0001; ns not significant. Source data and detailed statistical analysis are provided in the Source Data file. Schematic illustrations of the experimental strategy in the Fig. (4a, b) were created by Victor Cardoso-Jaime, using Microsoft PowerPoint Version 16.92.
Next, we asked whether the increase in the midgut microbiota load upon phagocyte depletion affects permissiveness to P. falciparum infection. We infected antibiotic-treated aseptic CLDs-injected mosquitoes with P. falciparum by feeding them on high gametocytemic blood (Fig. 4b). The CLDs-treated mosquitoes showed a decreased infection intensity and a higher mortality rate than did the controls (Fig. 4b), paralleling what we had observed with septic mosquitoes (Fig. 1a). Since the antibiotic treatment did not prevent mortality of CLDs-treated mosquitoes, and midgut epithelium integrity is essential for establishing Plasmodium infections, we concluded that phagocytes likely facilitate P. falciparum infection indirectly by maintaining midgut epithelial integrity.
Phagocytes regulate immune responses and Plasmodium agonists
The influence of phagocytic hemocytes on the proliferation of the midgut microbiota and P. falciparum infection, and midgut epithelial integrity suggested a possible involvement in the regulation of immune factors, and agonists of Plasmodium infection.
To test this possibility, we analyzed the expression of immune genes in whole body and midguts of NT, LPSMs-injected, and CLDs-injected mosquitoes at 24 h post-blood feeding and found that phagocyte-depleted whole mosquitoes exhibited a reduction in the expression of the anti-Plasmodium immune factors FBN9, Def1, Cec1, TEP1, APL1C, and NOS when compared to the control mosquitoes (Fig. 5 and Supplementary Fig. 8a–j). However, only Cec1 and Def1 showed a down-regulation in midgut tissue of phagocyte-depleted mosquitoes (Fig. 5 and Supplementary Fig. 9a–j). We also analyzed the expression of the P. falciparum agonists CTL4, Lanα, LanB2, and CollIV and found that phagocyte-depleted mosquitoes (whole body) showed a significantly diminished CTL4 transcriptional expression, while Lanα, and CollIV were slightly down-regulated in the midgut tissue (Fig. 5). Upon Drosophila tissue damage, hemocytes have been shown to control intestinal stem cell proliferation and Drosomicyn-like genes expression in the intestine through up-regulating and secretion of Upd352. Similarly, our results show that phagocytic hemocytes control intestinal cell proliferation (Fig. 2e–g) and regulate AMPs expression (Fig. 5) in blood-fed mosquitoes; therefore, we explored whether Upd3 could be up-regulated after blood feeding. We analyzed the Upd3 expression in NT, LPSMs, and CLDs mosquitoes fed on sucrose (control) or blood, however, no differences were observed in any group or treatment (Supplementary Fig. 13). This suggests that Upd3 is not upregulated upon a blood meal, and its expression is not hemocyte dependent.
NT, LPSM-injected, and CLD-injected mosquitoes were fed on blood, and then samples (whole body and midgut) were collected at 24 h post-blood feeding to analysis gene expression and proliferation. Heatmap of Log2 (-fold change) of antagonist and agonist genes of P. falciparum infection in the whole mosquito and midguts. Basal expression is indicated by the color white, upregulation by red, and downregulation by blue. Data were obtained from three independent experiments pulled together to generate the graph. Data are represented as Log2 means with SD, (n = 3). Statistical analysis was performed by ANOVA test, followed of Tukey´s multiple comparation test for fold changes values. #p < 0.1; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Data and detailed statistical analysis of the -fold changes are available in the Source Data file and Supplementary Figs. 7a–j, 8a–j. Schematic illustrations of the experimental strategy were created by Victor Cardoso-Jaime, using Microsoft PowerPoint Version 16.92.
Discussion
Here we reveal multiple novel functions of phagocytic hemocytes of the malaria vector An. gambiae with regards to P. falciparum infection, midgut integrity upon blood feeding, microbiota, and immunity. We show that the phagocytes are essential for the maintenance of midgut epithelial integrity and cell dynamics upon blood feeding and facilitate P. falciparum infection. We also show that phagocytes influence the immune response regulation of midgut tissue, which is related to microbiota homeostasis.
Previous studies employing the rodent malaria parasite P. berghei have shown that mosquito hemocytes produce factors and exert mechanisms to suppress parasite infection. However, while P. berghei has been extensively used as an amenable experimental model for studying malaria transmission, its biology differs significantly from the human pathogen P. falciparum. The rodent parasite can only infect the mosquito midgut at a temperature 7 °C lower than that at which P. falciparum infections are typically performed, and it triggers a different immune response than does P. falciparum21,24. P. falciparum has been shown to evade the An. gambiae immune system by disrupting the JNK pathway in midgut cells, resulting in limited apoptosis, which is involved in activating the immune response. In contrast, P. berghei strongly activates this pathway, triggering the expression of hemocyte-derived anti-Plasmodium factors53. Furthermore, An. gambiae is not a natural vector of P. berghei, and this infection model is thus artificial, resulting in a different physiological response along with a higher fitness cost upon infection24,54. In contrast, P. falciparum has co-evolved with, and adapted to, its natural An. gambiae vector, resulting in a less extensive immune response activation and low, if any, fitness cost25,26,54.
Unlike the hemocyte effects on P. berghei15,17,31, we show here that phagocytic hemocytes are agonists for the early midgut infection stages of the human malaria-causing parasite P. falciparum. We also show that phagocytes do not influence the infection of the mosquito salivary glands by P. falciparum sporozoites. Our findings are in agreement with earlier studies showing that sporozoite phagocytosis by An. gambiae hemocytes is very rare55, similar to An. albimanus, which mounts a weak immune response (phagocytosis, encapsulation, melanization, and lysis) against P. vivax sporozoites56.
While we show that phagocyte depletion, by injecting clodrosomes, results in mortality after blood feeding, a previous study using a similar clodrosome-based approach did not show such effects17 after feeding on blood infected with the rodent malaria parasite P. berghei. This is likely due to the different infection models and environmental conditions used in the two studies. While we used P. falciparum and human blood at 27 °C57, the study by Kwon and Smith used P. berghei with mouse blood at 19 °C17. P. falciparum and P. berghei infections are known to differentially impacts mosquito physiology21,58. The host blood specificity can also influence a variety of mosquito physiological processes47,59,60,61,62. Another major difference is the temperature at which the experiments in the two studies were performed; 27 °C in our study versus 19 °C in the study by Kwon and Smith17. Temperature influences a variety of biological and physiological processes including blood digestion61,63.
Midgut epithelial cell dynamics and proliferation are crucial for resisting and tolerating infections42,43,45,47,64. Anopheles epithelial cell differentiation and proliferation have been shown to be activated by P. berghei infection64,65. In Drosophila, intestinal epithelial integrity is required to tolerate and survive infections, and hemocytes control intestinal stem cell activity through the secretion of factors that promote stem cell proliferation52,66. We show here, for the first time, that mosquito phagocytic hemocytes control intestinal stem cell proliferation and midgut epithelial integrity in the major malaria vector An. gambiae. Thus, they are directly involved in tissue regeneration and repair of the midgut epithelium. We show that the disruption of the midgut epithelium caused by phagocyte depletion results in mosquito mortality. Blood-sucking insects use the peritrophic matrix and detoxification enzymes to protect themselves against the toxic components of blood and by-products of digestion67. When the integrity of the midgut epithelium is disrupted, the mosquito becomes over-exposed to these toxins. This exposure is likely one of the reasons for the mortality observed in hemocyte-depleted mosquitoes. Furthermore, extensive injury and tissue damage can also directly contribute to tissue and systemic failure, also leading to death.
Drosophila hemocytes control stem cell proliferation and the expression of AMPs in the intestinal epithelium upon injury by up-regulating and releasing the cytokine-like protein Upd3. Mosquitoes’ midgut epithelium gets injured upon blood feeding, and we show that phagocytic hemocytes control intestinal cell proliferation, suggesting that, perhaps similarly to Drosophila, UPD3 could be involved in this process. However, no changes in the Upd3 expression upon blood-feeding was observed and our data suggest that phagocytes are not the primary source of Upd3. While mosquito cytokine biology is poorly understood, molecules such as ROS and NO30, the neuropeptide Allatotropin68, TNF-α/Eiger69, and Vago70 have been shown to act as signaling molecules that activate the immune response. Furthermore, genetic tools for conditional/tissue genetic manipulation are not well established in mosquitoes71, their creation will allow us to understand many of the mechanisms involved in the systemic immune response, where cytokines play a major role. Therefore, we cannot discard the possibility that Upd3, and other known or unknown cytokine-like genes expressed by hemocytes play a role in modulating systemic immune responses, tissue regeneration, and other functions.
Several studies have contributed to our understanding of Plasmodium invasion of the midgut epithelium. The time bomb model20 first suggested that ookinetes produce irreversible damage to invading midgut epithelial cells and that parasites must exit these cells before they undergo apoptosis, which was described as a defense mechanism against Plasmodium. This model suggested that the midgut epithelium can manage extensive damage in the absence of cell division and tissue regeneration22. Our results show that hemocytes promote midgut epithelium integrity by controlling stem cell proliferation and tissue regeneration. Moreover, our study demonstrates that this hemocyte-controlled process is important not only during parasite infection but also during blood feeding, which can pose stress by causing rupture of the midgut epithelium72. Epithelial rupture can result in systemic dissemination and infection by the midgut microbiota, eventually causing death40.
Here we show for the first time that mosquitoes naturally leak bacteria from the midgut into the hemolymph upon blood feeding, and subsequently the lack of phagocytes does not enable clearance of infection. The lower mRNA abundance of antimicrobial factors in phagocyte-depleted mosquitoes that contain high numbers of immune-stimulating bacteria in their hemolymph shows that a significant proportion of antimicrobial factors derive from phagocytic hemocytes and that the fat body cannot compensate for this loss. Similar studies in Drosophila have shown that hemocyte-mediated phagocytosis is required to control systemic infection caused by leaking intestinal bacteria73, and the hemocyte-less Drosophila mutant was shown to be leaking midgut microbiota into the hemolymph which it was unable to clear likely due to a lesser production of AMPs74.
Drosophila´s hemocytes are known to be involved in general wound healing, being quickly recruited to a wound to facilitate clot and scab formation75, and gut-associated hemocytes are required for intestinal stem cell proliferation66. Mosquito hemocytes are recruited to the midgut after feeding on bacteria or P. berghei-infected blood14,15 where they deliver vesicles that activate the complement-like system in the infected cells. Most likely hemocytes also release other components involved in tissue regeneration and immune response modulation. We now report a novel phagocytic hemocyte-dependent mechanism by which these cells control midgut damage upon blood feeding. In this way, P. falciparum parasites cross unnoticed through the midgut epithelium, increasing the probability of survival and establishing the infection. The integrity of the midgut epithelium, as well as wound healing, are both dependent on the basal lamina, which is comprised of a variety of proteins including laminin and collagen, both of which are produced by hemocytes76,77,78,79. The basal lamina that surrounds mosquito tissues mediates self-tolerance, and its disruption can trigger both immune response and repair mechanisms77. Interestingly, Plasmodium oocysts integrate laminin, collagen, and other hemocyte factors into their capsules to mask themselves from the mosquito’s immune system6. Phagocytic hemocytes’ protective effect on P. falciparum may be mediated at least partially by the secretion of such basal lamina factors onto the intestinal epithelium as an indiscriminate response to damage produced by blood feeding and parasite infection. However, further research is necessary to test this hypothesis.
The mosquito midgut microbiota contributes to the restriction of Plasmodium infection by priming the mosquito’s immune system and augmenting the expression of immune factors such as AMPs, TEPs, and Fibrinogens (FBNs), which can inhibit P. falciparum development80. We found that phagocyte-depleted mosquitoes had a higher midgut bacterial load, but we only observed a decrease in the number of oocysts in high-intensity infections, and a slight effect on infection prevalence in low-intensity infections after hemocyte depletion. Furthermore, rendering mosquitoes aseptic did not change the permissiveness to infection after phagocyte depletion, suggesting phagocytic hemocytes control microbiota and Plasmodium infection by independent mechanisms. Phagocyte-depleted mosquitoes only showed a slightly lower expression of complement-like anti-Plasmodium factors, but still had fewer P. falciparum oocysts than the control, likely due to immune effector expression in the midgut and fat body81. Furthermore, the APL1C immune factor which is mainly expressed by hemocytes and plays a major role in suppressing P. berghei but not P. falciparum82. This could partly explain why phagocyte depletion increases the number of P. berghei oocysts17 but reduces P. falciparum. On the other hand, phagocyte-depleted mosquitoes expressed lower levels of Cec1 and Def1 in the midgut, which may relate to the higher midgut microbiota load. Drosophila hemocytes have also been shown to modulate AMPs expression in the fat body and intestine52,83,84. While mosquito hemocytes have been shown to suppress pathogens directly though phagocytosis and the production of immune factors, here we reveal a new immunity-related role for mosquito phagocytic hemocytes as modulators of immune responses by controlling AMPs expression in the midgut tissue.
In summary, we show, for the first time, that An. gambiae phagocytic hemocytes act as P. falciparum agonists during midgut infection with that organism, and we demonstrate these cells have pleiotropic functions that affect several physiological processes, including the control of the microbiota, immune gene expression, maintenance of tissue integrity, and epithelial cell regeneration. Furthermore, all of these processes, in turn, influence mosquito survival (Fig. 6). Thus, P. falciparum utilizes the mosquito’s cellular immune response mechanisms for self-protection.
(1) Hemocytes control the expression of AMPs in the midgut epithelium, thereby regulating the microbiota load. (2) Epithelial wounds are quickly controlled by hemocyte clotting, preventing bacteria from leaking into the hemolymph. (3) Bacteria reach the hemolymph and are eliminated by the hemocytes’ immune response. (4) Tissue regeneration is modulated through remote control of intestinal stem cells proliferation and cell differentiation by hemocytes. (5) Hemocytes immune response kill parasites crossing the tolerance threshold. (6) Hemocytes protect the parasites that do not produce uncontrolled damage and coat them with lamina basal components, making the parasites invisible to the immune system. The Figure was created by Victor Cardoso-Jaime, using Microsoft PowerPoint Version 16.92.
Methods
Ethics statements
All animal procedures were performed in strict accordance with the recommendations in the Guide for Care and Use of Laboratory Animal of the National Institutes of Health (NIH), USA and those of the Animal Care and Use Committee of Johns Hopkins University (permit no. M006H300). Commercial anonymous human blood was used for parasite cultures and mosquito feeding; thus, informed consent was not required. The Institutional Animal Care and Use Committee (IACUC) approved the protocol.
Mosquito rearing
Anopheles gambiae (Keele strain) mosquitoes were obtained from the Johns Hopkins Malaria Research Institute Insectary Core Facility. Mosquito larvae were maintained at 27 °C with a 12-h day/night cycle and fed on ground fish food supplement and cat food pellets. Adult mosquitoes were kept at 27 °C and 80% relative humidity with a 12-h day/night cycle; a 10% sugar solution was used to maintain the mosquitoes, which were allowed to feed on anesthetized mice before egg production.
Hemolymph perfusion and hemocytes staining
To evaluate the efficiency of phagocytic hemocyte depletion, we counted the number of hemocytes in the mosquitoes according the following protocol: Mosquitoes were cold-anesthetized and then intra-abdominally injected with 5 μl buffer containing 60% Schneiders´s medium (Gibco, SKU: 21720-024), 10% fetal bovine serum (Sigma, SKU: 22L538), and 30% citrate buffer (98 mM NaOH, 186 mM NaCl, 1.7 mM EDTA, and 41 mM citric acid, at pH 4.5), as previously described by Castillo et al.33. In addition, we added Vybrant CM-DiI Cell-Labeling Solution (Invitrogen, SKU: V22888) [5 μl/ml of buffer]; and the mosquitoes were then incubated on ice for 20 min. The hemocytes were recovered by perfusion and then injected intrathoracically with 5 μl of the same buffer (CM-Dil less). The abdominal intersegmental membrane (6-7 segments) was cut, and a drop of diluted hemolymph containing the hemocytes was collected on a microscope slide (Fisherbrand, SKU: 22-267-104) and incubated for 30 min at room temperature in a wet chamber. At this point, the majority of the hemocytes had adhered to the glass surface; they were then fixed by incubation for 10 min in 4% formaldehyde in PBS. The microscope glass with the fixed hemocytes was washed three times with PBS, a drop of Fluoromont G with DAPI (Invitrogen SKU: 00-4959-52) was added, and a cover glass was placed on top of the cells, which were observed with a LEICA DM2500 microscope.
To study abdominal sessile hemocytes we used a established protocol described by Cardoso-Jaime et al.85. Samples were analyzed by confocal microscopy using a Zeiss LSM 700 confocal microscope. Images were analyzed using Fiji software86. The Z-stack data set was presented in a single 2D projection. Sessile hemocytes were indirectly quantified by measuring the intensity of fluorescence on the red channel (CM-Dil) in each 2D image.
Mosquito phagocyte depletion
Female mosquitoes (3 days old) were cold-anesthetized and then injected intra-abdominally between the membrane between 6-7 segments, as previously was reported by Cardoso-Jaime et al.87, with either a 69 nl suspension (1:5 dilution in RPMI 1640 medium (SKU: 21870-076, Gibco)) of clodrosomes (CLDs) or control liposomes (LPSMs), both obtained from a Standard Macrophage Depletion Kit (clodrosome + encapsome); SKU: CLD-8901-2ml, Encapsula Nanosciences. Injections were performed using needles made with 10-μl microdispenser replacement tubes (Drummond, SKU: 7690N70) and a Nanoject II Injector (Drummond Scientific). All the experiments were performed at 4 days post-injection.
Specificity and toxicity of clodronate liposomes
Mosquitoes female An. gambiae were intraabdominal injected with 69 nl suspension (1:5 dilution in RPMI 1640 medium, Gibco, or PBS) of Liposomes containing DiO dye (SKU: CLD-8908, Encapsula Nanosciences). Four days post-injection, hemocytes were obtained by perfusion as described in the Methods section “Hemolymph perfusion and hemocytes staining”; also, sessile hemocytes were analyzed as described in the same Method section.
Since we injected each mosquito with 69 nl of LPSMs or CLDs suspension, and Anopheles mosquitoes ingest 1–4 μl of blood upon feeding88, 69 μl of LPSMs or CLDs (1:5 dilution in RPMI 1640 medium) were added per milliliter of reconstituted blood (50% RBC, 49% Human blood serum, and 1% ATP solution). Thus, each mosquito was estimated to ingest at least 69 nl of LPSMs or CLDs suspension.
For toxicity assays, mosquitoes were injected with 69 nl of RMPI medium containing 10 mg/ml of clodronate (SKU: 233183-10MG, Sigma), which is the same concentration as in the clodrosomes. Control mosquitoes were injected with RPMI 1640 medium.
Blood feeding and Plasmodium infection
Mosquitoes were kept for 4 h under starvation conditions before blood feeding or infection. Then blood was reconstituted by mixing red blood cells [RBC] (O+), human serum (O+), and 100 mM ATP solution (50%, 49%, and 1% by volume, respectively). P. falciparum NF54 gametocyte cultures were obtained from the Johns Hopkins Malaria Research Institute Parasitology Core Facility and used in all experiments. The infections were carried out in accordance with a previous procedure outlined by Tripathi et al.57, and low and high dosages of infection were obtained by using 0.03% and 0.3% of the final concentration of gametocytes, respectively. At 7 days post-infection, the midguts of the mosquitoes were dissected out in a PBS drop, and the isolated midguts were then incubated in a 0.2% mercurochrome solution for 10 min. Finally, the stained midguts were mounted in a drop of PBS between a glass slide and a cover slide, and the oocysts were counted using a bright field microscope.
Smurf assay
Midgut epithelial integrity was assessed by the “smurf assay”, which was carried out using a modified version of the protocols of Rera et al.41; and Bottino-Rojas et al.41,89. Smurf solution was prepared by dissolving 25 g/l of FD&C blue dye #1 (Sigma, SKU: 861146-25 g) in buffered saline solution (150 mM NaCl, 10 m M NaHCO3, pH 7.5). A mixture containing 50% RBC, 48% human serum, 1% smurf solution, and 1% 100 mM ATP was made to mimic the effects of blood feeding. Four hours before feeding, mosquitoes were deprived of sugar solution; then, using glass feeders, they were fed on blood/smurf solution.
Immunohistochemical staining
Cold-anesthetized mosquitoes were dissected in 1% formaldehyde in TBS and midguts were isolated and transversally cut in the anterior portion of the midgut to remove the blood bolus. Clean midguts were incubated in 4% formaldehyde in TBS for 30 min at room temperature, followed by three washes with TBS for 10 min each. Fixed midguts were stained using the protocol supplied with the anti-histone H3 phospho S10 (PH3) primary antibody (Abcam-ab47297)[dilution 1:250] previously described by Maya-Maldonado et al.45,64. Also, during incubation with the secondary antibody goat anti-rabbit Alexa Fluor 568 (Invitrogen, SKU: A-11011) [dilution 1:200 in TBS]. Samples were mounted in Fluoromount G mounting medium with DAPI (Invitrogen, SKU: 00-4959-52). Images were obtained in a LSM 700 confocal microscope (Zeiss), and analyzed using Image J Fiji software86.
Quantification of midgut and hemolymph bacterial load
Mosquitoes were cold-anesthetized over ice, then sprayed with 70% ethanol for 1 min and washed twice in sterile PBS. Clean mosquitoes were dissected in a drop of sterile PBS, and midguts were individually collected in 200 μl of sterile PBS in a 1.6-ml tube containing glass beads. All the samples were kept on ice. Samples were homogenized in a bead beater, followed by centrifugation at 1000 × g for 30 s. To quantify the bacteria in hemolymph, cold-anesthetized mosquitoes were perfused with 10 μl of sterile PBS. The diluted hemolymph of each mosquito was placed in 200 μl of sterile PBS, and samples were kept on ice. Serial dilutions were made, and 20 μl of each dilution was plated twice in LB agar (Sigma, SKU: L2897). Plates were incubated at 28 °C for 24 h, then the CFU were determined.
qRT-PCR
Mosquitoes were cold-anesthetized over ice, then sprayed with 70% ethanol for 1 min and washed twice in sterile PBS. Ten mosquitoes or 20 midguts per condition were collected on 500 μl of TRIzol reagent (Invitrogen, SKU: 15596018), then RNA was isolated according to the method suggested by the manufacturer. The cDNA was generated using 1 μg of RNA previously treated with DNAse I (Invitrogen, SKU: 18068015) and using the M-MLV Reverse transcriptase (Promega, SKU: M1705), in a final reaction volume of 25 μl. The qPCR reactions were performed using SYBR Green PCR Master Mix (Applied biosystems, SKU: 4309155). Each reaction contained 1 μl of cDNA reaction, 1.5 μl of primer mix solution [1 μM each, Forward and Reverse] in final volume of 10 μl. Reactions were run in a StepOnePlus Real-Time PCR System. Results were analyzed following the 2-ΔΔCT method90, using the S7 gene and the NT conditions. The sequences of the primers are available in Supplementary Table 1.
Sequences alignment
The Histone H3 amino acid sequences of An. gambiae (ID: AGAP001813) and An. albimanus (ID: AALB002213) were obtained from VectorBase, and the sequences of human (ID: NP_003522.1) and Drosophila (ID: NP_001027387.1) were obtained from NCBI. Sequences were analyzed by multiple alignment program Clustal O using the Jalview software V2.11.4.1. Sequence alignment was visualized in ESPript3 server91.
Statistical analysis
All statistical analyses were performed using three biological replicates per experiment. The n values and statistical tests used for each experiment are indicated in the figure legends. Data were analyzed using the appropriate statistical test in GraphPad Prism V.10.1.1. Detailed statistical analyses of results are available in the Source data file.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The raw data generated in this study are provided in the Source Data file. Source data are provided with this paper.
References
Carter, R. & Mendis, K. N. Evolutionary and Historical Aspects of the Burden of Malaria. Clin. Microbiol. Rev. 15, 564–594 (2002).
Paaijmans, K. P. & Lobo, N. F. Gaps in protection: the actual challenge in malaria elimination. Malar. J. 22, 46 (2023).
Dong, S., Dong, Y., Simões, M. L. & Dimopoulos, G. Mosquito transgenesis for malaria control. Trends Parasitol. 38, 54–66 (2022).
Bottino-Rojas, V. & James, A. A. Use of Insect Promoters in Genetic Engineering to Control Mosquito-Borne Diseases. Biomolecules 13, 16 (2022).
Dong, Y., Simões, M. L. & Dimopoulos, G. Versatile transgenic multistage effector-gene combinations for Plasmodium falciparum suppression in Anopheles. Sci. Adv. 6, eaay5898 (2020).
Smith, R. C. & Barillas-Mury, C. Plasmodium Oocysts: Overlooked Targets of Mosquito Immunity. Trends Parasitol. 32, 979–990 (2016).
King, J. G. Developmental and comparative perspectives on mosquito immunity. Dev. Comp. Immunol. 103, 103458 (2020).
Kumar, A. et al. Mosquito Innate Immunity. Insects 9, 95 (2018).
Cardoso-Jaime, V., Tikhe, C. V., Dong, S. & Dimopoulos, G. The Role of Mosquito Hemocytes in Viral Infections. Viruses 14, 2088 (2022).
Eleftherianos, I. et al. Haemocyte-mediated immunity in insects: Cells, processes and associated components in the fight against pathogens and parasites. Immunology 164, 401–432 (2021).
Hillyer, J. F. & Strand, M. R. Mosquito hemocyte-mediated immune responses. Curr. Opin. Insect Sci. 3, 14–21 (2014).
Mosser, D. M., Hamidzadeh, K. & Goncalves, R. Macrophages and the maintenance of homeostasis. Cell Mol. Immunol. 18, 579–587 (2021).
Melcarne, C., Lemaitre, B. & Kurant, E. Phagocytosis in Drosophila: From molecules and cellular machinery to physiology. Insect Biochem. Mol. Biol. 109, 1–12 (2019).
Barletta, A. B. F. et al. Hemocyte differentiation to the megacyte lineage enhances mosquito immunity against Plasmodium. eLife 11, e81116 (2022).
Castillo, J. C., Ferreira, A. B. B., Trisnadi, N. & Barillas-Mury, C. Activation of mosquito complement antiplasmodial response requires cellular immunity. Sci. Immunol. 2, eaal1505 (2017).
Kwon, H., Mohammed, M., Franzén, O., Ankarklev, J. & Smith, R. C. Single-cell analysis of mosquito hemocytes identifies signatures of immune cell subtypes and cell differentiation. eLife 10, e66192 (2021).
Kwon, H. & Smith, R. C. Chemical depletion of phagocytic immune cells in Anopheles gambiae reveals dual roles of mosquito hemocytes in anti- Plasmodium immunity. Proc. Natl. Acad. Sci. USA 116, 14119–14128 (2019).
Raddi, G. et al. Mosquito cellular immunity at single-cell resolution. Science 369, 1128 (2020).
Rodrigues, J., Brayner, F. A., Alves, L. C., Dixit, R. & Barillas-Mury, C. Hemocyte Differentiation Mediates Innate Immune Memory in Anopheles gambiae Mosquitoes. Science 329, 1353–1355 (2010).
Smith, R. C. et al. Molecular Profiling of Phagocytic Immune Cells in Anopheles gambiae Reveals Integral Roles for Hemocytes in Mosquito Innate Immunity. Mol. Cell. Proteom. 15, 3373–3387 (2016).
Dong, Y. et al. Anopheles gambiae Immune Responses to Human and Rodent Plasmodium Parasite Species. PLoS Pathog. 2, e52 (2006).
Han, Y. S., Thompson, J., Kafatos, F. C. & Barillas-Mury, C. Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes. EMBO J. 19, 6030–6040 (2000).
Han, Y. S. & Barillas-Mury, C. Implications of Time Bomb model of ookinete invasion of midgut cells. Insect Biochem. Mol. Biol. 32, 1311–1316 (2002).
Jaramillo-Gutierrez, G. et al. Mosquito immune responses and compatibility between Plasmodium parasites and anopheline mosquitoes. BMC Microbiol. 9, 154 (2009).
Simões, M. L., Dong, Y., Mlambo, G. & Dimopoulos, G. C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites. PLoS Biol. 20, e3001515 (2022).
Simões, M. L., Mlambo, G., Tripathi, A., Dong, Y. & Dimopoulos, G. Immune Regulation of Plasmodium Is Anopheles Species Specific and Infection Intensity Dependent. mBio 8, e01631-17 (2017).
Hegedus, D., Erlandson, M., Gillott, C. & Toprak, U. New Insights into Peritrophic Matrix Synthesis, Architecture, and Function. Annu. Rev. Entomol. 54, 285–302 (2009).
Baton, L. A. & Ranford-Cartwright, L. C. Plasmodium falciparum ookinete invasion of the midgut epithelium of Anopheles stephensi is consistent with the Time Bomb model. Parasitology 129, 663–676 (2004).
Trisnadi, N. & Barillas-Mury, C. Live In Vivo Imaging of Plasmodium Invasion of the Mosquito Midgut. mSphere 5, e00692–20 (2020).
Herrera-Ortiz, A., Martínez-Barnetche, J., Smit, N., Rodriguez, M. H. & Lanz-Mendoza, H. The effect of nitric oxide and hydrogen peroxide in the activation of the systemic immune response of Anopheles albimanus infected with Plasmodium berghei. Dev. Comp. Immunol. 35, 44–50 (2011).
Blandin, S. et al. Complement-Like Protein TEP1 Is a Determinant of Vectorial Capacity in the Malaria Vector Anopheles gambiae. Cell 116, 661–670 (2004).
Lombardo, F. & Christophides, G. K. Novel factors of Anopheles gambiae haemocyte immune response to Plasmodium berghei infection. Parasites Vectors 9, 78 (2016).
Castillo, J. C., Robertson, A. E. & Strand, M. R. Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti. Insect Biochem. Mol. Biol. 36, 891–903 (2006).
Rooijen, N. V. & Sanders, A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J. Immunological Methods 174, 83–93 (1994).
Danenberg, H. D. et al. Macrophage Depletion by Clodronate-Containing Liposomes Reduces Neointimal Formation After Balloon Injury in Rats and Rabbits. Circulation 106, 599–605 (2002).
Bryant, W. B. & Michel, K. Blood feeding induces hemocyte proliferation and activation in the African malaria mosquito, Anopheles gambiae Giles. J. Exp. Biol. jeb.094573 https://doi.org/10.1242/jeb.094573 (2013).
Castillo, J., Brown, M. R. & Strand, M. R. Blood Feeding and Insulin-like Peptide 3 Stimulate Proliferation of Hemocytes in the Mosquito Aedes aegypti. PLoS Pathog. 7, e1002274 (2011).
Cázares-Raga, F. E. et al. Morphological and proteomic characterization of midgut of the malaria vector Anopheles albimanus at early time after a blood feeding. J. Proteom. 111, 100–112 (2014).
Okuda, K. et al. Morphological and enzymatic analysis of the midgut of Anopheles darlingi during blood digestion. J. Insect Physiol. 51, 769–776 (2005).
Rodgers, F. H., Gendrin, M., Wyer, C. A. S. & Christophides, G. K. Microbiota-induced peritrophic matrix regulates midgut homeostasis and prevents systemic infection of malaria vector mosquitoes. PLoS Pathog. 13, e1006391 (2017).
Rera, M., Clark, R. I. & Walker, D. W. Intestinal barrier dysfunction links metabolic and inflammatory markers of aging to death in Drosophila. Proc. Natl. Acad. Sci. USA 109, 21528–21533 (2012).
Maya-Maldonado, K., Lanz-Mendoza, H. & de la Cruz Hernandez-Hernandez, F. Cell Cycle Dynamics and Endoreplication In the Mosquito Midgut. AJBSR 5, 43–46 (2019).
Hixson, B., Taracena, M. L. & Buchon, N. Midgut Epithelial Dynamics Are Central to Mosquitoes’ Physiology and Fitness, and to the Transmission of Vector-Borne Disease. Front. Cell. Infect. Microbiol. 11, 653156 (2021).
Janeh, M., Osman, D. & Kambris, Z. Comparative Analysis of Midgut Regeneration Capacity and Resistance to Oral Infection in Three Disease-Vector Mosquitoes. Sci. Rep. 9, 14556 (2019).
Maya-Maldonado, K. et al. DNA synthesis increases during the first hours post-emergence in Anopheles albimanus mosquito midgut. Dev. Comp. Immunol. 112, 103753 (2020).
Valzania, L., Mattee, M. T., Strand, M. R. & Brown, M. R. Blood feeding activates the vitellogenic stage of oogenesis in the mosquito Aedes aegypti through inhibition of glycogen synthase kinase 3 by the insulin and TOR pathways. Dev. Biol. 454, 85–95 (2019).
Taracena-Agarwal, M. L. et al. The midgut epithelium of mosquitoes adjusts cell proliferation and endoreplication to respond to physiological challenges. BMC Biol. 22, 22 (2024).
Ohlstein, B. & Spradling, A. The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature 439, 470–474 (2006).
Baton, L. A. & Ranford-Cartwright, L. C. Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion. J. Invertebr. Pathol. 96, 244–254 (2007).
Guo, X., Wang, C., Zhang, Y., Wei, R. & Xi, R. Cell-fate conversion of intestinal cells in adult Drosophila midgut by depleting a single transcription factor. Nat. Commun. 15, 2656 (2024).
Shaw, R. L. et al. The Hippo pathway regulates intestinal stem cell proliferation during Drosophila adult midgut regeneration. Development 137, 4147–4158 (2010).
Chakrabarti, S. et al. Remote Control of Intestinal Stem Cell Activity by Haemocytes in Drosophila. PLoS Genet 12, e1006089 (2016).
Ramphul, U. N., Garver, L. S., Molina-Cruz, A., Canepa, G. E. & Barillas-Mury, C. Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells. Proc. Natl. Acad. Sci. USA 112, 1273–1280 (2015).
Shaw, W. R., Marcenac, P. & Catteruccia, F. Plasmodium development in Anopheles: a tale of shared resources. Trends Parasitol. 38, 124–135 (2022).
Hillyer, J. F., Barreau, C. & Vernick, K. D. Efficiency of salivary gland invasion by malaria sporozoites is controlled by rapid sporozoite destruction in the mosquito hemocoel. Int. J. Parasitol. 37, 673–681 (2007).
Hernández-Martínez, S., Lanz, H., Rodríguez, M. H., González-Ceron, L. & Tsutsumi, V. Cellular-Mediated Reactions to Foreign Organisms Inoculated into the Hemocoel of Anopheles albimanus (Diptera: Culicidae). J. Med. Entomol. 39, 61–69 (2002).
Tripathi, A. K., Mlambo, G., Kanatani, S., Sinnis, P. & Dimopoulos, G. P.lasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding. JoVE 61426, https://doi.org/10.3791/61426 (2020).
Kwon, H., Simões, M. L., Reynolds, R. A., Dimopoulos, G. & Smith, R. C. Additional Feeding Reveals Differences in Immune Recognition and Growth of Plasmodium Parasites in the Mosquito Host. mSphere 6, e00136–21 (2021).
Harrison, R. E., Brown, M. R. & Strand, M. R. Whole blood and blood components from vertebrates differentially affect egg formation in three species of anautogenous mosquitoes. Parasites Vectors 14, 119 (2021).
Muturi, E. J., Dunlap, C., Ramirez, J. L., Rooney, A. P. & Kim, C.-H. Host blood meal source has a strong impact on gut microbiota of Aedes aegypti. FEMS Microbiol. Ecol. https://doi.org/10.1093/femsec/fiy213 (2018).
Jian, X. et al. Effects of constant temperature and daily fluctuating temperature on the transovarial transmission and life cycle of Aedes albopictus infected with Zika virus. Front. Microbiol. 13, 1075362 (2023).
De Swart, M. M., Balvers, C., Verhulst, N. O. & Koenraadt, C. J. M. Effects of host blood on mosquito reproduction. Trends Parasitol. 39, 575–587 (2023).
Barr, J. S., Martin, L. E., Tate, A. T. & Hillyer, J. F. Warmer environmental temperature accelerates aging in mosquitoes, decreasing longevity and worsening infection outcomes. Immun. Ageing 21, 61 (2024).
Maya-Maldonado, K. et al. Plasmodium exposure alters midgut epithelial cell dynamics during the immune memory in Anopheles albimanus. Dev. Comp. Immunol. 133, 104424 (2022).
Maya-Maldonado, K. et al. Transcriptome analysis uncover differential regulation in cell cycle, immunity, and metabolism in Anopheles albimanus during immune priming with Plasmodium berghei. Dev. Comp. Immunol. 120, 104046 (2021).
Ayyaz, A., Li, H. & Jasper, H. Haemocytes control stem cell activity in the Drosophila intestine. Nat. Cell Biol. 17, 736–748 (2015).
Whiten, S. R., Eggleston, H. & Adelman, Z. N. Ironing out the Details: Exploring the Role of Iron and Heme in Blood-Sucking Arthropods. Front. Physiol. 8, 1134 (2018).
Hernández-Martínez, S. et al. Allatotropin: A pleiotropic neuropeptide that elicits mosquito immune responses. PLoS ONE 12, e0175759 (2017).
Samantsidis, G.-R., Kwon, H., Wendland, M., Fonder, C. & Smith, R. C. TNF signaling mediates cellular immune function and promotes malaria parasite killing in the mosquito Anopheles gambiae. Preprint at https://doi.org/10.1101/2024.05.02.592209 (2024).
Paradkar, P. N., Trinidad, L., Voysey, R., Duchemin, J.-B. & Walker, P. J. Secreted Vago restricts West Nile virus infection in Culex mosquito cells by activating the Jak-STAT pathway. Proc. Natl. Acad. Sci. USA 109, 18915–18920 (2012).
Kefi, M., Cardoso-Jaime, V., Saab, S. A. & Dimopoulos, G. Curing mosquitoes with genetic approaches for malaria control. Trends Parasitol. 40, 487–499 (2024).
Dong, S. et al. Chikungunya virus dissemination from the midgut of Aedes aegypti is associated with temporal basal lamina degradation during bloodmeal digestion. PLoS Negl. Trop. Dis. 11, e0005976 (2017).
Nehme, N. T. et al. A Model of Bacterial Intestinal Infections in Drosophila melanogaster. PLoS Pathog. 3, e173 (2007).
Braun, A., Hoffmann, J. A. & Meister, M. Analysis of the Drosophila host defense in domino mutant larvae, which are devoid of hemocytes. Proc. Natl. Acad. Sci. USA 95, 14337–14342 (1998).
Wang, L., Kounatidis, I. & Ligoxygakis, P. Drosophila as a model to study the role of blood cells in inflammation, innate immunity and cancer. Front. Cell. Infect. Microbiol. 3, 113 (2014).
Bunt, S. et al. Hemocyte-Secreted Type IV Collagen Enhances BMP Signaling to Guide Renal Tubule Morphogenesis in Drosophila. Dev. Cell 19, 296–306 (2010).
Kim, M. J. & Choe, K.-M. Basement Membrane and Cell Integrity of Self-Tissues in Maintaining Drosophila Immunological Tolerance. PLoS Genet 10, e1004683 (2014).
Van De Bor, V. et al. Companion Blood Cells Control Ovarian Stem Cell Niche Microenvironment and Homeostasis. Cell Rep. 13, 546–560 (2015).
Ramos-Lewis, W., LaFever, K. S. & Page-McCaw, A. A scar-like lesion is apparent in basement membrane after wound repair in vivo. Matrix Biol. 74, 101–120 (2018).
Dong, Y., Manfredini, F. & Dimopoulos, G. Implication of the Mosquito Midgut Microbiota in the Defense against Malaria Parasites. PLoS Pathog. 5, e1000423 (2009).
Volohonsky, G. et al. Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae. PLoS Pathog. 13, e1006113 (2017).
Zmarlak, N. M. et al. The Anopheles leucine-rich repeat protein APL1C is a pathogen binding factor recognizing Plasmodium ookinetes and sporozoites. PLoS Pathog. 20, e1012008 (2024).
Basset, A. et al. The phytopathogenic bacteria Erwinia carotovora infects Drosophila and activates an immune response. Proc. Natl Acad. Sci. USA 97, 3376–3381 (2000).
Charroux, B. & Royet, J. Elimination of plasmatocytes by targeted apoptosis reveals their role in multiple aspects of the Drosophila immune response. Proc. Natl. Acad. Sci. USA 106, 9797–9802 (2009).
Cardoso-Jaime, V., Maya-Maldonado, K., Tsutsumi, V. & Hernández-Martínez, S. Mosquito pericardial cells upregulate Cecropin expression after an immune challenge. Dev. Comp. Immunol. 147, 104745 (2023).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Cardoso-Jaime, V., Maya-Maldonado, K., Celestino-Montes, A., Tsutsumi, V. & Hernández-Martínez, S. Lysozyme c-1 gene is overexpressed in Anopheles albimanus pericardial cells after an immune challenge. Dev. Comp. Immunol. 114, 103830 (2021).
Graumans, W. et al. A mosquito feeding assay to examine Plasmodium transmission to mosquitoes using small blood volumes in 3D printed nano-feeders. Parasites Vectors 13, 401 (2020).
Bottino-Rojas, V. et al. Beyond the eye: Kynurenine pathway impairment causes midgut homeostasis dysfunction and survival and reproductive costs in blood-feeding mosquitoes. Insect Biochem. Mol. Biol. 142, 103720 (2022).
Livak, K. J. & Schmittgen, T. D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 25, 402–408 (2001).
Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).
Acknowledgements
We thank Dr. Deborah McClellan for editorial assistance, and the Johns Hopkins Malaria Research Institute Parasite and Insectary Core Facilities and the Johns Hopkins Institute for Basic Biomedical Sciences microscope facility for their support. This work has been supported by NIH R01AI12274, R21AI131574, R01AI170692, R01AI158615, and the Bloomberg Philanthropies.
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Conceptualization: V.C., G.D. Methodology: V.C. Investigation and data analysis: V.C. Visualization: V.C. Writing - original draft: V.C. Review & Editing: V.C., G.D.
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Cardoso-Jaime, V., Dimopoulos, G. Anopheles gambiae phagocytic hemocytes promote Plasmodium falciparum infection by regulating midgut epithelial integrity. Nat Commun 16, 1465 (2025). https://doi.org/10.1038/s41467-025-56313-y
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DOI: https://doi.org/10.1038/s41467-025-56313-y
