Decision letter: Hemocyte differentiation to the megacyte lineage enhances mosquito immunity against Plasmodium

Field: Immunology and Microbiology (Life Sciences)

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Activation of Toll signaling in Anopheles gambiae by silencing Cactus, a suppressor of this pathway, enhances local release of hemocyte-derived microvesicles (HdMv), promoting activation of the mosquito complement-like system, which eliminates Plasmodium ookinetes. We uncovered the mechanism of this immune enhancement. Cactus silencing triggers a Rel1-mediated differentiation of granulocytes to the megacyte lineage, a new subpopulation of giant cells, resulting in a dramatic increase in the proportion of circulating megacytes. Megacytes are very plastic cells that are massively recruited to the basal midgut surface in response to Plasmodium infection. We show that Toll signaling modulates hemocyte differentiation and that megacyte recruitment to the midgut greatly enhances mosquito immunity against Plasmodium. Editor's evaluation This article reveals a mechanism by which Toll pathway activation lead to protection against Plasmodium in Anopheles mosquitoes by promoting the differentiation of hemocytes. https://doi.org/10.7554/eLife.81116.sa0 Decision letter eLife's review process eLife digest Malaria causes hundreds of thousands of deaths each year. This devastating disease is caused by Plasmodium parasites, which are transmitted to people through female Anopheles gambiae mosquitos. Mosquitos become infected with Plasmodium when they ingest blood containing these malaria-causing parasites. However, Plasmodium must avoid the mosquito immune system to survive and spread. The mosquito immune system is made up of several types of immune cells, including cells known as granulocytes. Granulocytes can further develop into additional cell subtypes, such as megacytes and antimicrobial granulocytes, but it is not clear how these types of cells work to protect mosquitos against infections. In the mosquitos that transmit malaria, a cell signaling pathway called Toll helps control immune responses to disease-causing microbes, such as Plasmodium. When Toll signaling is strongly triggered in mosquitos, Plasmodium infection is eliminated because immune cell responses are enhanced – which results in lower levels of transmission to humans. But what is the underlying mechanism through which high levels of Toll signaling eradicate Plasmodium infection? To find out, Barletta et al. collected cell samples from A. gambiae mosquitos and analyzed what happened when Toll signaling was strongly activated. They observed a large increase in the proportion of megacytes in these mosquitos (from 2% to 80% of all granulocytes). Toll signaling also caused megacytes to become bigger, cluster together, and have higher plasticity – meaning they could adopt different shapes. Barletta et al. used microscopy to show that these megacytes were releasing large mitochondria-like structures and membrane vesicles , which may be the trigger activating the mosquito’s immune system. In live mosquitos, megacytes move towards the area of the Plasmodium infection and release microvesicles. These microvesicles are known to activate a part of the the mosquito’s immune system called the complement-like system, destroying the parasites and preventing mosquito infection and disease transmission. These findings show how strong Toll signaling triggers the mosquito immune system to eliminate Plasmodium infections. Understanding how the mosquito immune system tackles Plasmodium infection may help reveal ways to reduce or block transmission. Introduction Ookinete traversal of the Anopheles gambiae midgut disrupts the barriers that normally prevent bacteria of the gut microbiota from coming in direct contact with epithelial cells (Kumar et al., 2010), and this attracts hemocytes to the basal surface of the midgut (Barletta et al., 2019). Plasmodium ookinetes also cause irreversible damage to the cells they invade and trigger a strong caspase-mediated nitration response (Han et al., 2000; Oliveira et al., 2012, Trisnadi and Barillas-Mury, 2020). When hemocytes come in contact with a nitrated midgut surface, they undergo apoptosis and release hemocyte-derived microvesicles (HdMv) (Castillo et al., 2017). Local HdMv release promotes activation of thioester containing-protein 1 (TEP1) (Castillo et al., 2017), a major final effector of the mosquito complement-like system that binds to the parasite’s surface and forms a complex that lyses the ookinete (Blandin et al., 2004). Mosquito hemocytes are classified into three cell types, prohemocytes, oenocytoids, and granulocytes, based on their morphology. However, single-cell RNA sequencing (sc-RNAseq) analysis of A. gambiae hemocytes identified several novel subpopulations of granulocytes based on their transcriptional profiles and defined molecular markers specific for hemocyte subpopulations (Raddi et al., 2020). Furthermore, Lineage analysis revealed that regular granulocytes derive from prohemocytes and can further differentiate into distinct cell types, including dividing granulocytes, and two final effector cells, megacytes and antimicrobial (AM) granulocytes (Raddi et al., 2020). Silencing Cactus, a negative regulator of Toll signaling in A. gambiae mosquitoes, elicits a very strong TEP1-mediated immune response that eliminates Plasmodium berghei ookinetes (Frolet et al., 2006). This phenotype can be rescued by co-silencing Cactus with either TEP1 or the Rel1 transcription factor, indicating that parasite elimination is mediated by activation of Toll signaling, with TEP1 as a final effector (Frolet et al., 2006). Later studies showed that hemocytes mediate this enhanced immune response, as transfer of Cactus-silenced hemocytes into naïve mosquitoes recapitulates the phenotype of systemic Cactus silencing (Ramirez et al., 2014). Furthermore, Cactus silencing also increases HdMv release in response to ookinete midgut invasion (Castillo et al., 2017), indicating that hemocytes are more reactive to Plasmodium infection. However, the nature of the functional changes in Cactus-silenced hemocytes that enhance immunity against Plasmodium are not known. Here, we explore the effect of Cactus silencing on circulating hemocyte populations and their response to infection of mosquitoes with bacteria and Plasmodium. Results Effect of Cactus silencing on mRNA markers of granulocyte populations The effect of silencing Cactus, a suppressor of Toll signaling, on hemocyte differentiation was explored. Hemocytes that adhere to glass (mostly granulocytes) or that remain in suspension (mostly prohemocytes and oenocytoids) were collected 4 days post-injection from dsLacZ control and dsCactus-injected females. Bulk sequencing of cDNA libraries generated between 16.2 and 25.3 million fragments that mapped to the A. gambiae AgamP4.9 transcriptome. Only transcripts with 10 or more reads were included in the analysis, resulting in a total of 9421 unique transcripts (https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-11252). Glass-bound and unbound hemocyte samples were analyzed together, because the differences in expression between dsLacZ versus dsCactus-silenced hemocytes explained 81% of the variance between the four experimental groups (Figure 1—figure supplement 1A and B). Differential expression (DE) analysis of Cactus-silenced hemocytes using the DESeq2 software identified 1071 differentially expressed genes (Q value <0.001), of which 407 were upregulated (log2 fold change >2), while 664 were downregulated (log2 fold change <−2) (Figure 1A). Figure 1 with 1 supplement see all Download asset Open asset Effect of Toll pathway activation on mRNA markers of granulocyte populations. (A) Differential expression (DE) of Cactus dsRNA knockdown. From a total of 9421 filtered genes. Volcano plot of DE genes in Cactus-silenced hemocytes compared to LacZ control filtered for log2 fold change >2 and Q value <0.001. Dark blue dots on the right represent upregulated DE genes and on the left the downregulated ones. Red dots show genes that are hemocyte-specific markers. Complete list of up- and downregulated genes is listed in Supplementary file 1 and Supplementary file 2. (B) Percentage of granulocyte subpopulation markers up- and downregulated in Cactus-silenced hemocytes. Complete list of up- and downregulated genes for each hemocyte subpopulation is in Supplementary file 1 and Supplementary file 2. Reg Gran: regular granulocytes; Div Gran: dividing granulocytes; AM Gran: antimicrobial granulocytes; Mega: megacytes. The effect of Cactus silencing on expression of the transcripts that define the different hemocyte clusters established by sc-RNAseq (Raddi et al., 2020) was analyzed (Supplementary file 1 and Supplementary file 2), to establish whether there was a significant effect on the relative abundance of specific hemocyte subpopulations. Overall, 23 oenocytoid markers, 2 from prohemocytes, and 57 from granulocytes were differentially expressed between dsLacZ and dsCactus hemocytes (Supplementary file 1 and Supplementary file 2). Most differentially expressed oenocytoid markers 22/23 (95%) were downregulated, while one of the prohemocyte markers was upregulated and the other one was downregulated (Figure 1B). The number of downregulated granulocyte markers 28/57 (49%) was very similar to that of upregulated ones 29/57 (51%). However, detailed analysis of granulocyte subpopulations revealed that most upregulated markers 18/29 (62%) correspond to megacytes, while most downregulated markers correspond to regular granulocytes 21/28 (75%). This suggests that dsCactus silencing increases the proportion of circulating megacytes, at the expense of a reduction in regular granulocytes. As expected, expression of several genes involved in Toll signaling or final effector this pathway, such as Toll-like receptors, CLIP proteases, Serpins, C-type Lectins, and Defensin are upregulated in dsCactus hemocytes (Supplementary file 3). Cactus silencing promotes granulocyte differentiation into megacytes Cactus silencing did not significantly increase the proportion of total circulating granulocytes, based on hemocyte counts by light microscopy (Figure 2—figure supplement 1), suggesting that the observed enhanced immune response could be due to functional changes in hemocytes. The morphology of hemocytes perfused from Cactus-silenced females was analyzed using fluorescent probes to stain the actin cytoskeleton and the nucleus. Cactus silencing dramatically increased the proportion of large granulocytes (diameter >40 µm), presumably megacytes, from 5.3% to 79.2% (p < 0.0001, X2 test) (Figure 2A, B), in agreement with the observed increase in upregulated megacyte-specific markers in the transcriptomic analysis of Cactus-silenced hemocytes (Figure 1B). Interestingly, megacytes from Cactus-silenced mosquitoes (Figure 2A) are even larger (average diameter of 47 µm after spreading in a glass surface) than megacytes from dsLacZ controls (average diameter of 30 µm) (Figure 2C, D). In situ RNA hybridization of dsCactus granulocytes with a fluorescent probe for the megacyte-specific marker TM7318, confirmed that the proportion of TM7318-positive granulocytes was much higher (80%) in Cactus-silenced females than in dsLacZ controls (4%) (p < 0.0001, X2 test) (Figure 2E, F), providing direct evidence that overactivation of Toll signaling triggers a dramatic increase in the proportion of circulating megacytes. Expression analysis of the TM7318 marker in perfused hemocyte samples confirmed that mRNA levels were 42-fold higher in dsCactus hemocytes than the dsLacZ control group (p < 0.001, T-test) (Figure 2G), while a modest increase (2.8-fold) in FBN50 mRNA, a marker of AM effector granulocytes, was observed (p < 0.0001, T-test). Conversely, expression of FBN11228, a marker of regular granulocytes, decreased by 30-fold in circulating hemocytes of Cactus-silenced mosquitoes (Figure 2G). The changes in the relative abundance of mRNAs from cell-specific markers in dsCactus hemocytes agrees with the observed changes in hemocyte morphology and the in situ hybridization (ISH) and transcriptomic data (Figure 2G). Figure 2 with 2 supplements see all Download asset Open asset Cactus silencing promotes granulocyte differentiation into megacytes. (A) A. gambiae hemocytes in LacZ control and Cactus attached to a glass surface. Actin is shown in green and nuclei in blue. Scale bar: 40 µm. (B) Percentage of megacytes among all granulocytes in dsLacZ and dsCactus mosquitoes. Percentages were compared using X2 test. **** = p ≤ 0.0001. (C) Megacyte in control LacZ mosquitoes (upper) and in Cactus-silenced mosquitoes (lower). Actin is showing in green, and nuclei is in blue. Scale bar: 10 µm. (D) Diameter of megacytes from LacZ control and Cactus-silenced mosquitoes. Error bars represent mean ± standard error of the mean (SEM). Unpaired T-test. **** = p ≤ 0.0001. (E) RNA in situ hybridization for megacyte-specific marker TM7318. Actin is shown in green (phalloidin), TM7318 mRNA in red, and the nuclei in blue (Hoechst). Scale bar: 40 µm. (F) Percentage of TM7318-positive cells in LacZ and Cactus-silenced granulocytes. Percentages were compared using X2 test. **** = p ≤ 0.0001. (G) Relative mRNA expression of hemocyte-specific markers in LacZ control and Cactus hemocytes for transcriptome validation. Megacyte marker (TM7318), antimicrobial granulocytes (FBN50), and regular granulocytes (FBN11228). Gene expression was normalized using RpS7 expression. Error bars represent mean ± SEM. Unpaired T-test, ***=p ≤0.001, = p ≤ 0.0001. Two independent experiments with 3 replicates each experiment, a total of 6 pools of 15 mosquitoes each. The relative increase in megacytes in Cactus-silenced A. gambiae females could be due to enhanced megacyte proliferation or to increased differentiation of regular granulocytes into megacytes. A total hemocyte count revealed that the number of circulating hemocytes was not significantly different between dsLacZ (mean 17,151/mosquito, n = 14) and dsCactus mosquitoes (mean 23,477/mosquito, n = 14, Mann–Whitney T-test) (Figure 2—figure supplement 1A) 2 days post-injection. The mean number of total hemocytes was lower 4 days post-silencing, but there was also no significant difference between dsLacZ (mean 8725/mosquito, n = 14) and dsCactus mosquitoes (mean 10,836/mosquito, n = 14, Mann–Whitney T-test) (Figure 2—figure supplement 1A). Furthermore, there was no difference in the proportion granulocytes at 2 days, dsLacZ (3.62%) and dsCactus females (3.48%, Mann–Whitney T-test), and 4 days, dsLacZ (3.91%) and dsCactus females (5.47%, Mann–Whitney T-test), post-injection. In contrast, a significant increase in the proportion of megacyte was already apparent 2 days post-injection in Cactus-silenced mosquitoes (2.08% of all hemocytes, p < 0.0001, Mann–Whitney T-test), relative to dsLacZ controls (0.07%) (Figure 2—figure supplement 1B); with a corresponding decrease in the proportion of other granulocytes from 3.5% in dsLacZ controls to 1.4% in dsCactus (Figure 2—figure supplement 1B). At 4 days post-injection, the differences were more pronounced, with the proportion of megacytes reaching 3.7% in Cactus-silenced mosquitoes (p < 0.0001, Mann–Whitney T-test) relative to dsLacZ controls (0.07%) (Figure 2—figure supplement 1C), with a corresponding decrease in other granulocytes from 3.8% in dsLacZ to 1.8% in dsCactus mosquitoes (Figure 2—figure supplement 1C). Taken together, these data indicate that, although the total number of hemocytes and the percentage of total granulocytes remained unchanged in response to dsCactus silencing, the proportion of megacytes increased at the expense of other granulocytes. The effect of Cactus silencing on granulocyte proliferation was evaluated by quantitating the proportion of hemocytes that incorporated Bromodeoxyuridine/5-bromo-2′-deoxyuridine (BrdU), a thymidine analog. The proportion of BrdU+ hemocytes that adhered to glass (mostly granulocytes) in dsCactus mosquitoes (51%, n = 694 cells) is not significantly different from dsLacZ controls (52%, n = 410 cells) (Figure 2—figure supplement 2A, B). BrdU fluorescence intensity (RFU) is also not significantly different between dsLacZ and dsCactus hemocytes (Figure 2—figure supplement 2C). However, the ratio of BrdU fluorescence intensity to nuclear volume is significantly lower in dsCactus hemocytes (Figure 2—figure supplement 2D). This indicates that the increase in nuclear volume in megacytes does not involve DNA replication. However, BrdU labeling can be lost over time, making it hard to establish when DNA replication occurred. The proportion of hemocytes undergoing mitosis after Cactus silencing was directly evaluated using phospho-Histone H3 (PHH3) staining, which only labels mitotically active cells. Two days post-injection, the proportion of PHH3+ hemocytes that adhered to glass (mostly granulocytes) in dsCactus mosquitoes (0.7%, n = 896) was small, and not significantly different from dsLacZ controls (0.6%, n = 718 cells) (Figure 2—figure supplement 1D). At 4 days the proportions were also similar, with very few hemocytes positive for PHH3 staining both in dsLacZ (0.5%, n = 799 cells) and dsCactus (0.3%, n = 1039) (Figure 2—figure supplement 1D). Moreover, the few hemocytes that were positive for PHH3 in dsCactus mosquitoes did not have the characteristic size or morphology of megacytes (Figure 2—figure supplement 1D). These observations, together with the increase in the proportion of megacytes in dsCactus females, at the expense of other regular granulocytes (Figures 1B and 2G), indicate that Cactus silencing promotes differentiation of granulocytes to the megacyte lineage. Cell dynamics of mosquito granulocytes Megacytes are about twice as large as regular granulocytes. Regular granulocytes (Figure 3A) reach an average diameter of 14.2 μm (Figure 3B) when they spread over a glass surface, while the average diameter of megacytes is 28.6 μm (p < 0.0001, unpaired T-test) (Figure 3A, B). Granulocyte cellular dynamics was evaluated by live imaging of perfused hemocytes in vitro as they adhered and spread on a glass surface. Hemocytes were labeled in vivo, through systemic injection of adult females with a red lipophilic dye (Vybrant CM-DiI) that accumulates on intracellular vesicles. After perfusion, a green, fluorescent probe (Cell Mask) was added to label the plasma membrane. Both regular granulocytes and megacytes attached to the glass surface and spread fully within 1 hr (Videos 1–4). Megacytes already have a larger cell diameter when they first attach to glass (Figure 3C, upper panel and Video 3), and exhibit a peripheral ‘halo’, corresponding to an area of extended thin cytoplasm, almost devoid of vesicles (Figure 3C, upper panel and Video 3). Lateral views revealed that, initially, megacytes have a large nucleus and a voluminous cytoplasm in the central region of the cell that flattens dramatically as the cell ‘spreads’ over the glass surface (Figure 3C, lower panel and Video 4). In contrast, the central region of regular granulocytes remains mostly unchanged (Figure 3D, lower panel and Video 2) and the periphery of the cell exhibits a modest increase in diameter as the cell spreads along the surface (Figure 3D, upper panel and Video 1). Figure 3 Download asset Open asset Snapshots of megacyte and granulocyte cell dynamics. (A) Regular granulocytes and megacytes from A. gambiae females spread on a glass surface. Actin, green (phalloidin) and nuclei, blue (Hoechst). Scale bar: 20 µm. (B) Granulocyte diameter of sugar-fed mosquitoes after spreading on a glass surface. Error bars represent mean ± standard error of the mean (SEM). Unpaired T-test. ****p ≤ 0.0001. (C) Live imaging time-lapse of a megacyte spreading in a glass surface for 30 min. Plasma membrane stained in green and microvesicles in red. Top (XY) and lateral view (XZ) of a megacyte. Scale bars: 10 and 5 µm, respectively. (D) Live imaging time-lapse of a granulocyte spreading on a glass surface for 30 min. Top (XY) and lateral view (XZ) of a regular granulocyte. Scale bars: 10 and 5 µm, respectively (see Videos 1–4). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Top view (XY) of a regular granulocyte from A. gambiae mosquito female. Showing in red is the microvesicle staining and in green the plasma membrane. Scale bar: 10 µm. Hemocyte was imaged for 1 hr in intervals of 5 min. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Side view (XZ) of a regular granulocyte from A. gambiae mosquito female. Showing in red is the microvesicle staining and in green the plasma membrane. Scale bar: 5 µm. Hemocyte was imaged for 1 hr in intervals of 5 min. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Top view (XY) of a megacyte from A. gambiae mosquito female. Showing in red is the microvesicle staining and in green the plasma membrane. Scale bar: 10 µm. Hemocyte was imaged for 1 hr in intervals of 5 min. Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Side view (XZ) of a megacyte from A. gambiae mosquito females. Showing in red is the microvesicle staining and in green the plasma membrane. Scale bar: 5 µm. Hemocyte was imaged for 1 hr in intervals of 5 min. Characterization of megacyte in vivo dynamics and ultrastructure The effect of Cactus silencing on granulocyte dynamics was evaluated in vivo, through live imaging of hemocytes circulating in adult female mosquitoes. Female mosquitoes were imaged for 2 hr, 1 day after blood feeding on a healthy mouse. Hemocytes were visualized by systemic injection of Vybrant CM-DiI, a fluorescent lipophilic dye that is preferentially taken up by granulocytes. Circulating hemocytes in dsLacZ females (presumably normal granulocytes) have a smaller diameter than those of dsCactus females (Videos 5 and 6; Figure 3A, B), and they seldom come in contact with each other as they patrol the basal surface of the midgut (Video 5). Hemocytes from dsCactus females (presumably megacytes) are larger and have a spindle shape (Figure 3A, B; Videos 3 and 4). They appear to have higher plasticity, as they can readily stretch their cytoplasm and often come into contact with each other (Video 6). The plasticity of dsCactus megacytes was confirmed by in vitro live imaging of perfused hemocytes labeled by systemic injection of Vybrant CM-DiI and green Cell Mask. Some megacytes from dsCactus mosquitoes projected long thin filopodia toward other megacytes (Video 7). This process was not observed in regular granulocytes or in megacytes from the dsLacZ controls. Taken together, our live imaging data indicate that, in addition to their larger diameter (Figure 2C, dsCactus megacytes are also more have increased plasticity as they patrol the midgut (Video and to with each other and clusters (Videos 6 and 7). Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg In vivo hemocyte in dsLacZ mosquitoes. Hemocytes stained in red were imaged through the of the mosquito for 1 hr and 20 min. Scale bar: 30 µm. Video 6 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg In vivo hemocyte in dsCactus mosquitoes. Hemocytes stained in red were imaged through the of the mosquito for 1 hr and 20 min. Scale bar: 30 µm. Video Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg In vitro dynamics of dsCactus megacytes. hemocytes from dsCactus mosquitoes. Plasma membrane is showing in green, microvesicles in red, and nuclei in blue. Scale bar: 20 µm. The detailed ultrastructure of megacytes was using transmission microscopy Hemocytes from Cactus-silenced females were collected by perfusion, in and to As expected, the diameter of hemocytes while in suspension was smaller than when they were to spread on a glass surface. However, regular granulocytes were still significantly smaller µm) than megacytes µm), with nuclei that are also smaller (Figure B). are observed in the nuclei of megacytes, corresponding to the of that are as as an (Figure B). of perfused hemocytes was further using of regular granulocytes have a with strong staining on (Figure In contrast, megacytes exhibit a more and (Figure is that large mitochondria-like structures and vesicles are often observed from the surface of dsCactus megacytes (Figure but not from regular granulocytes (Figure Figure 4 Download asset Open asset of megacytes in Cactus-silenced mosquitoes. (A) microscopy of regular granulocytes from Cactus-silenced mosquitoes. Scale bar: 2 µm. (B) of megacytes from Cactus-silenced mosquitoes. Scale bar: 5 µm. (C) giant mitochondria-like structures of a mitochondria-like Scale bars: 2 and 1 µm. were cytoplasm is shown in in red, and nuclei in (D) staining in regular granulocytes. Scale bar: 20 µm. (E) staining of Cactus-silenced megacytes. Actin is stained in green (phalloidin), in red and nuclei in blue (Hoechst). indicates an of a megacyte. Scale bar: 20 µm. Megacytes with the basal surface of the midgut in response to feeding We have shown that direct contact of bacteria with epithelial cells, the is triggers release and attracts hemocytes to the basal surface of the midgut (Barletta et al., 2019). Hemocyte recruitment to the midgut in dsCactus females was by providing a containing As expected, feeding hemocytes to the midgut surface in both dsCactus and dsLacZ control females (Figure B). However, there are differences in hemocyte recruitment between In dsLacZ females, hemocytes attach to the midgut basal or in (Figure while hemocytes from dsCactus females large clusters on the basal midgut surface, with hemocytes in very (Figure dsCactus hemocytes on the midgut surface have the characteristic morphology of megacytes, with a larger cytoplasm and nuclei than those from dsLacZ hemocytes (Figure of actin was often observed in the hemocytes from dsCactus females come in direct contact with each other as they clusters (Figure Figure 5 Download asset Open asset feeding increases megacyte to the midgut basal surface. (A) Effect of feeding in controls on hemocytes to the midgut basal surface. (B) Effect of Cactus silencing on the hemocytes to the basal surface of the midgut 4 hr Scale bar: 30 µm. Hemocyte cluster attached to the midgut surface in Cactus-silenced mosquitoes 4 hr Scale bar: 15 µm. actin is shown in green (phalloidin), hemocytes with Vybrant CM-DiI) in red, and nuclei in blue (Hoechst). (E) Relative mRNA levels of effector hemocyte markers in the midgut 4 hr after feeding in LacZ and Cactus-silenced mosquitoes. Scale bar: 15 µm. Error bars in (E) represent mean ± standard error of the mean (SEM). Unpaired T-test, ≤ ≤ Two independent experiments with 3 replicates for each experiment, a total of 6 pools of 15 mosquitoes each. The recruitment of granulocyte subpopulations to the midgut of dsCactus females in response feeding was confirmed by of mRNAs transcripts of markers expressed in specific hemocyte subpopulations. TM7318 mRNA levels increased