https://orcid.org/0000-0003-0245-2265
Figures
Abstract
Tumor Necrosis Factor-α (TNF-α) is a proinflammatory cytokine and a master regulator of immune cell function in vertebrates. While previous studies have implicated TNF signaling in invertebrate immunity, the roles of TNF in mosquito innate immunity and vector competence have yet to be functionally explored. Herein, we confirm the identification of a conserved TNF-α pathway in Anopheles gambiae consisting of the TNF-α ligand, Eiger, and its cognate receptors Wengen and Grindelwald. Through gene expression analysis, RNAi, and in vivo injection of recombinant TNF-α, we provide direct evidence for the requirement of TNF signaling in regulating mosquito immune cell function by promoting granulocyte midgut attachment, increased granulocyte abundance, and oenocytoid rupture. Moreover, our data demonstrate that TNF signaling is an integral component of anti-Plasmodium immunity that limits malaria parasite survival. Together, our data support the existence of a highly conserved TNF signaling pathway in mosquitoes that mediates cellular immunity and influences Plasmodium infection outcomes, offering potential new approaches to interfere with malaria transmission by targeting the mosquito host.
Author summary
Mosquito innate immunity is a major determinant of vector competence with significant implications in malaria transmission. During infection with the Plasmodium parasite, mosquitoes mount a sequence of immune signals originating from the mosquito midgut that stimulate the activation of mosquito immune cells (hemocytes) to limit parasite survival. Here, we provide compelling evidence that Tumor Necrosis Factor (TNF) signaling, a well-characterized immune pathway in vertebrates, is directly involved in mosquito hemocyte function and Plasmodium killing. We demonstrate that mosquito TNF signaling via the TNF ortholog, Eiger, requires the concerted function of the receptors Wengen and Grindelwald to control several aspects of mosquito immune cell biology and that ultimately limits malaria parasite survival. These data provide novel mechanistic insight into previously undescribed roles of mosquito TNF signaling in anti-Plasmodium immunity that offer potential new molecular targets for malaria control.
Citation: Samantsidis G-R, Kwon H, Wendland M, Fonder C, Smith RC (2025) TNF signaling mediates cellular immune function and promotes malaria parasite killing in the mosquito Anopheles gambiae. PLoS Pathog 21(7): e1013329. https://doi.org/10.1371/journal.ppat.1013329
Editor: Jeffrey D. Dvorin, Boston Children's Hospital, UNITED STATES OF AMERICA
Received: May 6, 2024; Accepted: June 26, 2025; Published: July 3, 2025
Copyright: © 2025 Samantsidis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by R21AI144705 and R21AI166857 to RCS from the National Institutes of Health, National Institute of Allergy and Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Anopheles mosquitoes serve as the primary vectors of Plasmodium parasites, which cause malaria and impose substantial burdens on public health across the globe [1]. While there has been a significant reduction in malaria cases over the last twenty years due to improved vector control strategies, the continued effectiveness of these strategies has been jeopardized by increased insecticide resistance [2–4], which highlights the need to develop alternative approaches for malaria control. With recent advancements in gene-drive systems offering significant promise for population modification [5–7], the potential that these genetic techniques can be used to manipulate the vector competence of mosquito populations to impair malaria transmission has become a reality. However, to fully leverage these genetic approaches, we require a better understanding of the molecular mechanisms that define Plasmodium infection in the mosquito host.
In response to Plasmodium infection, mosquitoes mount a series of sequential immune signals initiated by the midgut in response to ookinete invasion [8–12], which are further processed by the mosquito immune cells (hemocytes) [13–15] to promote malaria parasite killing [16–19]. As a result, hemocytes serve as integral immune mediators that directly contribute to ookinete recognition [16,17] or that promote humoral responses to limit oocyst survival [15,17,19]. Mosquito hemocytes have traditionally been classified into three main cell types based on morphological and biochemical properties: granulocytes, oenocytoids, and prohemocytes [20], with more recent single-cell studies expanding on the complexity of these cell populations [21,22]. Macrophage-like granulocytes are phagocytic and behave as immune sentinels either in circulation in the hemolymph or as sessile cells attached to the midgut or other mosquito tissues [20,23]. Previous studies have demonstrated that granulocytes respond to stimuli resulting from ookinete midgut invasion to mediate both early- and late-phase immune responses against Plasmodium [15–18]. Oenocytoids have primarily been associated with the expression of prophenoloxidases (PPOs) [20], which are key enzymes in the melanization pathway and have been previously implicated in oocyst survival [19]. Lastly, prohemocytes are presumed precursors [20,24] that give rise to granulocyte and oenocytoid populations under infective conditions [13–15].
Mosquito hemocyte populations are highly heterogenic [21,22], with their composition tightly regulated by a variety of signaling pathways in response to different physiological conditions. This includes previous studies that have demonstrated the ability of hemocytes to proliferate in response to blood-feeding [24,25], a process regulated by the release of insulin-like peptides and subsequent activation of the PI3K/AKT and MAPK/ERK signaling pathways [26–29]. In addition, the Signal Transducer and Activator of Transcription (STAT) [14,15], the LPS-induced TNF-alpha factor (LITAF)-like transcription factor 3 (LL3) [15,30], c-Jun N-terminal kinase (JNK) [14], and Toll [14,30] pathways have been associated with hemocyte differentiation and parasite attrition. Furthermore, eicosanoid signaling pathways have been implicated in hemocyte function, differentiation, and Plasmodium killing [18,19,31,32], and are central to the establishment of innate immune memory that confers increased resistance to infection [18,31]. Yet, despite these advances, our understanding of the immune signals that modulate mosquito hemocytes remains limited.
Tumor Necrosis Factor-α (TNF-α) is one of the most important regulators of immune function in vertebrates, acting as a proinflammatory cytokine and critical mediator of immune cell regulation [33,34]. Across vertebrate systems, components of TNF signaling have remained conserved, consisting of a TNF-α ligand and two receptors: the TNFR1 and TNFR2 [34]. Previous phylogenetic analyses have revealed the presence of orthologous TNF pathways in invertebrates [35,36], however few studies have examined TNF signaling beyond Drosophila. In Drosophila, the TNF pathway is comprised of an analogous TNF ligand, Eiger, and its receptors, Wengen (Wgn) and Grindelwald (Grnd) [37]. Together, these TNF signaling components regulate multiple physiological processes in Drosophila, including tissue growth regulation, cellular proliferation, development, and host defense [38]. This includes significant contributions from hemocytes in mediating these physiological functions [39,40]. Under both homeostatic or infected conditions, Eiger modulates Drosophila hemocyte function to enhance survival through its actions as a chemoattractant [41], to promote cell death [42], or as a regulator of phagocytosis [43–45]. While recent studies have expanded our knowledge of how TNF signaling influences the function of immune cells in other insects [46], the role of TNF signaling in the mosquito innate immune system remains unclear. While orthologs of Anopheles TNF signaling components have previously been identified and support the presence of a functional pathway that influences mosquito vectorial capacity [36], direct investigations of TNF signaling have not previously been performed.
In this study, we establish integral roles of mosquito TNF signaling in mediating anti-Plasmodium immune responses that limit malaria parasite survival in the mosquito host. While gene expression analysis indicates that Eiger is induced in response to blood-feeding regardless of infection status in midgut and hemocyte tissues, downstream experiments clearly demonstrate the roles of mosquito TNF signaling in cellular immune function and immune responses that promote malaria parasite killing. Together, our data provide novel mechanistic insight into the function of TNF signaling in mosquito immune cell regulation and anti-Plasmodium immunity.
Results
Expression analyses of mosquito TNF signaling pathway components
To better understand TNF signaling in Anopheles gambiae we examined the expression of the TNF ligand, Eiger, and two TNF receptors, Wgn and Grnd (Fig 1A), across tissues (midgut, hemocytes, and carcass) and physiological conditions (naïve, blood-fed, and Plasmodium berghei infection). Under naïve conditions, Eiger displayed comparable expression levels between hemocytes and the carcass, with reduced levels of expression in the midgut (Fig 1B). A similar expression pattern was observed for Wgn, although higher levels of Wgn expression were found in carcass tissues (Fig 1C). In contrast, Grnd was enriched in both midgut and carcass, with the lowest expression in hemocytes (Fig 1D). Of note, Eiger expression was generally increased across tissues in response to blood-feeding and infection, and was significantly induced in hemocytes regardless of infection status (Fig 1B). In contrast, the different feeding conditions had little effect on Wgn and Grnd expression, although Wgn displayed significantly reduced levels of expression in the carcass following P. berghei infection (Fig 1C), suggesting the potential down-regulation of TNF signaling in the carcass following infection. While both TNF and TNF receptors (TNFRs) are influenced by posttranscriptional modifications in Drosophila [47–49] and other vertebrate systems, the observed patterns of Eiger expression support potential roles of TNF signaling in mosquito immunity and cellular immune function.
(A) Schematic representation of the mosquito TNF signaling pathway. The expression of Eiger (B), Wgn (C), and Grnd (D) was examined by qPCR in pooled mosquito midgut, hemolymph, and fat body samples under naive, blood-fed (24 h post-feeding) or P. berghei-infected (24 h post-infection) conditions. Expression data are displayed relative to rpS7 expression with bars representing the mean ± SE of three to six independent biological replicates (black dots). Each independent biological replicate consists of pooled tissues from ~10-15 individual mosquitoes for midgut and carcass samples, or hemocyte perfusions from ~30 individual mosquitoes. Data were analyzed using a two-way ANOVA with a Tukey’s multiple comparisons test to determine significance. Asterisks denote significance (*P < 0.05, **P < 0.01). Summary figure created with BioRender.com.
Influence of TNF signaling on mosquito survival
With TNF signaling components implicated in a broad range of physiological responses in other insects [38], we wanted to examine the potential that targeting these genes by RNA interference (RNAi) could potentially impact An. gambiae survival. After confirming that the injection of dsRNA successfully promoted the RNAi-mediated silencing of Eiger, Wgn, and Grnd (S1 Fig), we examined mosquito survival in each genetic background at different temperatures and following infection with the rodent malaria parasite Plasmodium berghei (Fig 2). When examined in insectary conditions at either 19°C or 27°C, the silencing of Eiger, Wgn, or Grnd had no effect on mosquito survival when compared to GFP-silenced control mosquitoes maintained on a 10% sucrose solution, although we did observe significant effects of temperature (19°C vs. 27°C) on mosquito survival independent of the genetic background (Fig 2A). Additional experiments were performed in which survival was examined post-silencing in mosquitoes infected with P. berghei (Fig 2B). While the silencing of Eiger or Wgn had no effect on survival when compared to similarly infected GFP-silenced control mosquitoes, Grnd-silencing resulted in significantly increased mosquito mortality when compared to each of the other silenced backgrounds (Fig 2B). This suggests that TNF components are not essential to mosquito survival, although under certain physiological conditions, such as infection, that TNF does contribute to mosquito fitness.
An. gambiae survival was monitored over 12 days following the RNAi-mediated knockdown of Eiger (dsEiger), Wgn (dsWgn), Grnd (dsGrnd), and compared to dsGFP controls. Survival was monitored under naïve conditions at 19°C and 27°C (A) or was examined following P. berghei infection (B). In A, survival curves of each genetic background were compared for each temperature condition (significance displayed in figure) or compared within the same genetic background between temperatures (significance displayed in figure legend). Statistical analysis was performed using a Log-rank (Mantel-Cox) test, with significance displayed by asterisks: *P < 0.05, ****P < 0.0001. Error bars represent standard error of the mean (SEM). ns, not significant. n = number of individual mosquitoes in each genetic background. Data from three independent biological experiments (N = 3) are displayed for each graph.
Mosquito TNF signaling limits P. berghei development
To examine the influence of TNF signaling on malaria parasite infection, we first injected adult female mosquitoes with 50 ng of recombinant human TNF-α (rTNF-α) one day before challenging with P. berghei. When malaria parasite numbers were evaluated 8 days post-infection (dpi), mosquitoes primed with rTNF-α significantly reduced P. berghei oocyst numbers and the prevalence of infection (Fig 3A). Conversely, when Eiger is silenced via RNAi (S1 Fig), oocyst numbers and infection prevalence were significantly increased (Fig 3B). Similarly, when the TNFRs Wgn and Grnd were silenced (S1 Fig), mosquitoes from both Wgn- and Grnd-silenced backgrounds displayed significant increases in P. berghei oocyst numbers (Fig 3B and 3C). Together, these findings demonstrate the importance of mosquito TNF signaling in the innate immune response against P. berghei and support integral roles of Eiger, Wgn, and Grnd in this process.
(A) Adult female mosquitoes were injected with 1X PBS (control) or 50 ng of rTNF-α prior to infection with P. berghei. Oocyst numbers and infection prevalence were evaluated at 8 days post-infection (dpi). Additional RNAi experiments were performed to evaluate the contributions of the TNF signaling components Eiger and Wgn (B), or Grnd (C) in the context of P. berghei infection. Oocyst numbers and infection prevalence were similarly evaluated at 8 dpi. Mosquitoes injected with dsGFP served as control in all experiments. For each graph, dots correspond to the number of oocysts identified in individual midguts, with the median represented by a red horizontal line. Infection prevalence (% infected/total) is depicted as pie charts pies below each figure. Data were combined from three or more independent experiments. Statistical significance was determined using a Mann-Whitney test or Kruskal-Wallis with a Dunn’s post-test to assess oocyst numbers, while a Fisher’s Exact test was performed to measure differences in infection prevalence. Asterisks denote statistical significance (* P < 0.05, **P < 0.01). ns, not significant. n = numbers of individual mosquitoes examined.
Wgn and Grnd are required to promote TNF-mediated anti-Plasmodium immunity
To confirm that the responses limiting P. berghei survival following the injection of rTNF-α (Fig 3A) are mediated by mosquito TNF signaling components, we examined the influence of rTNF-α in Wgn- and Grnd-silenced backgrounds (Fig 4A). Two days after the injection of dsRNA to promote RNAi, mosquitoes from each genetic background were injected with either rTNF-α or 1X PBS as a control, then 24 hours later challenged with a P. berghei infection. While rTNF-α injection significantly reduced P. berghei oocyst numbers in the GFP-silenced (control) background when examined 8 dpi, the injection of rTNF-α in either the Wgn- or Grnd-silenced backgrounds had no effect (Fig 4A), suggesting that both Wgn and Grnd are required for the TNF-mediated signals that confer anti-Plasmodium immunity. It is also of note that we did not see significant differences in oocyst numbers between the GFP-, Wgn-, and Grnd-silenced backgrounds in these double-injection experiments (Fig 4A), suggesting that the additional injection post-RNAi may physically interfere with canonical immune signaling in these genetic backgrounds.
(A) RNAi experiments were performed to examine the requirement of Wgn and Grnd in rTNF-α-mediated responses that limit P. berghei oocyst numbers. After RNAi, mosquitoes were injected with either 1X PBS (-) or 50 ng of rTNF-α (+), then challenged with P. berghei one day later. (B) Similar RNAi experiments were performed to evaluate the combined contributions of the TNF signaling components Wgn, and Grnd in the context of P. berghei infection. For all experiments, oocyst numbers and infection prevalence were evaluated at 8 dpi in the specific gene-silenced backgrounds. Dots correspond to the number of oocysts identified in individual midguts, with the median represented by a red horizontal line. Infection prevalence (% infected/total) is depicted as pie charts pies below each figure. Data were combined from three or more independent experiments. Statistical significance was determined using Kruskal-Wallis with a Dunn’s multiple comparison test to assess oocyst numbers, while a Fisher’s Exact test was performed to measure differences in infection prevalence. Asterisks denote statistical significance (* P < 0.05, **P < 0.01). ns, not significant; n = numbers of individual mosquitoes examined.
In vertebrate systems, TNF signaling can initiate distinct cellular responses based on the interactions of TNF-α with its cognate TNFR1 and TNFR2 receptors [33,34]. With both Wgn and Grnd serving as antagonists to Plasmodium development (Fig 3), and data suggesting that the loss of either Wgn or Grnd eliminated the effects of rTNF-α on P. berghei survival (Fig 4A), we wanted to examine whether TNF signaling via Wgn or Grnd produced functionally dependent or independent responses to promote parasite killing. Similar to our results in Fig 3B and 3C, the independent silencing of Wgn or Grnd resulted in increased P. berghei oocyst numbers (Fig 4B). However, when both Wgn and Grnd were silenced, the infection intensity did not further increase (Fig 4B). This suggests that Wgn and Grnd are functionally dependent, working together in a singular pathway to promote P. berghei killing, where the loss of either component abrogates TNF signaling.
Mosquito TNF signaling modulates granulocyte and oenocytoid populations
Previous studies have demonstrated that mosquito hemocyte populations undergo significant changes in response to different physiological conditions [13–19,24,25,29] and have established their significant roles in malaria parasite killing [14–19]. Based on the upregulation of Eiger in perfused hemocyte samples (Fig 1) and the importance of TNF signaling associated with immune cell regulation in other systems [33,34,42,46], we wanted to explore the effects of TNF signaling on mosquito hemocyte populations.
To better understand the roles of TNF signaling in mosquito hemocytes, we first examined the expression patterns of the receptors, Wgn and Grnd, in hemocyte subtypes using previously published single-cell data for An. gambiae hemocyte populations [22]. While this transcriptional data suggests that Wgn is enriched in both granulocyte and oenocytoid populations, Grnd is expressed only in oenocytoids (S2 Fig). These data enable a testable model in which granulocytes express Wgn, while oenocytoids express both Wgn and Grnd (Fig 5A). In the absence of antibodies to confirm these expression patterns, we performed additional experiments using clodronate liposomes (CLD) to deplete phagocytic granulocyte populations [17,22,50–52]. Following CLD treatment, Wgn displayed a significant reduction in expression (Fig 5B), while Grnd expression increased (Fig 5C). Together, these data demonstrate that Wgn is sensitive to CLD treatment, further supporting that Wgn is predominantly expressed in phagocytic granulocyte populations (S2 Fig). In contrast, CLD treatment did not negatively impact Grnd expression, supporting its expression in non-phagocytic oenocytoid populations similar to other oenocytoid-specific genes [19,22] (S2 Fig).
(A) Previous single cell transcriptomic data [22] support the expression of Wgn in both granulocytes and oenocytoids, whereas Grnd is enriched specifically in oenocytoids. The expression levels of Wgn (B) and Grnd (C) were examined in mosquitoes treated with either clodronate liposomes (CLD) to deplete mosquito granulocytes or control liposomes to functionally demonstrate the specificity of Grnd to oenocytoids using qPCR. Data from three independent experiments were analyzed by an unpaired Students’ t-test. (D) Injection with rTNF-α (50 ng or 200 ng) increases the granulocyte proportions at 24 h post-injection compared to 1XPBS controls. Similar experiments performed in GFP-, Wgn-, and Grnd-silenced backgrounds demonstrate the importance of both Wgn and Grnd for the increase in granulocytes following rTNF-α (50 ng) injection (E). For both D and E, the percentage of granulocytes of the total hemocytes are represented as mean ± SE of three independent biological replicates with statistical significance determined by Mann-Whitney to compare the effects of rTNF-α versus the 1XPBS control mosquitoes. The injection of 50 ng rTNF-α reduces the percentage of oenocytoids (of total hemocytes) (F) and the expression of oenocytoid specific genes (PPO1, PPO8, and PGE2R) (G), which together suggest that TNF signaling promotes oenocytoid lysis. The granulocyte marker Eater was used as a negative control. Data from three independent experiments were analyzed by an unpaired Students’ t-test. (H) Additional experiments were performed to determine the effects of rTNF-α on the phenoloxidase (PO)-activity of mosquito hemolymph (n = 20). Six measurements (OD490) were taken for DOPA conversion assays at 5-min intervals. Bars represent mean ± SE of three independent biological experiments with statistical significance determined with a two-way repeated-measures ANOVA followed by Sidak’s multiple comparison test. (I) Silencing the expression of Wgn and Grnd impaired the rTNF-α induced phenotypes on PPO1 and PPO8 expression when tested in whole adult mosquitoes. Results are presented as a heatmap displaying the log2 fold change (FC) and indicate differences gene expression as measured by qPCR following treatment with rTNF-α or 1X-PBS (control). Data represent the mean fold change expression of three independent biological replicates, with significance determined using an unpaired Students’ t-test. Asterisks indicate significance (* P < 0.05, **P < 0.01, **** P < 0.0001). ns, not significant; n = numbers of individual mosquitoes examined. Summary figure created with BioRender.com.
To determine the effects of mosquito TNF signaling on hemocyte subpopulations, we first injected mosquitoes with rTNF-α and examined the effects on granulocyte numbers. The injection of mosquitoes with either 50 ng or 200 ng of rTNF-α caused a substantial increase in the proportion of granulocytes, with the 50 ng and 200 ng concentrations able to influence granulocytes at comparable levels (Fig 5D). Based on patterns of Wgn and Grnd expression in hemocyte subtypes (Figs 5A-C and S2), we wanted to examine the ability of rTNF-α to influence granulocyte populations in the Wgn- and Grnd-silenced backgrounds. While the injection of rTNF-α increased the percentage of granulocytes in the control GFP-silenced background as expected, both Wgn- and Grnd-silencing negated the effects of rTNF-α on granulocyte proportions (Fig 5E), suggesting that both Wgn and Grnd are required for the rTNF-α-mediated increase in the percentage of granulocytes. Based on evidence suggesting that Grnd is not expressed in granulocytes (Figs 5A-C and S2), this suggests that the effects of rTNF-α on granulocyte populations are indirectly mediated by oenocytoid function or other humoral factors.
With evidence in Drosophila demonstrating that TNF signaling promotes melanization through crystal cell lysis [42,44], the equivalent of mosquito oenocytoid immune cell populations, we wanted to explore if TNF signaling might similarly promote oenocytoid lysis. To address this question, we injected mosquitoes with rTNF-α and examined the percentage of oenocytoids present in circulating hemolymph. Following rTNF-α injection, we observed a significant reduction in oenocytoids (Fig 5F), suggesting that TNF signaling may similarly promote oenocytoid lysis/rupture. For this reason, we examined the expression levels of PPO1, PPO8, and PGE2R, genes enriched in oenocytoids [19,22], which have previously been used to illustrate oenocytoid cell rupture [19]. At 24 hrs post-injection, PPO1, PPO8, and PGE2R each displayed significantly reduced gene expression when compared to controls (Fig 5G), suggestive of oenocytoid lysis. In contrast, the expression of the granulocyte marker, Eater, remained unchanged following rTNF-α injection (Fig 5G). With the rupture of oenocytoids believed to release prophenoloxidases and other cellular contents into the hemolymph, we performed dopa conversion assays to measure hemolymph phenoloxidase (PO) activity following rTNF-α injection as an additional measure to quantify oenocytoid rupture [19]. As expected, rTNF-α treatment significantly increased hemolymph PO activity when compared to control mosquitoes (Fig 5H), providing further evidence that TNF signaling promotes oenocytoid lysis/rupture. Additional injection experiments with rTNF-α performed in Grnd- or Wgn-silenced backgrounds negated the rTNF-α-mediated down-regulation of PPO1 and PPO8 expression as seen in control dsGFP mosquitoes (Fig 5I), suggesting that both Wgn and Grnd are required to promote oenocytoid lysis via mosquito TNF signaling. When paired with our infection experiments demonstrating that the loss of either Wgn or Grnd impaired the TNF-mediated responses that promote parasite killing (Fig 4), these data similarly suggest that both receptors are required to initiate the TNF-mediated signals that promote cellular immune function and oenocytoid lysis.
TNF-mediated Plasmodium killing is not dependent on granulocyte function
Previous studies have shown that both granulocytes and oenocytoids have central roles in anti-Plasmodium immunity [16,17,19,53], which given the effects of TNF signaling on granulocyte numbers and oenocytoid lysis (Fig 5), corroborate our observations regarding the influence of mosquito TNF signaling on Plasmodium infection (Figs 3 and 4). Since granulocytes play pivotal roles in ookinete recognition [16,17], we examined the effects of TNF signaling on early oocyst numbers. Similar to the results presented in Fig 3A performed at 8 dpi, rTNF-α injection significantly reduced P. berghei early oocyst numbers by 2 dpi (Fig 6A), suggesting that TNF signaling may enhance ookinete recognition. This is further supported by the increased attachment of mosquito hemocytes to the mosquito midgut following rTNF-α injection (Fig 6B), which suggests that TNF signaling contributes to early-phase anti-Plasmodium killing responses. However, although TEP1 and mosquito complement are often implicated in the recognition and killing of the ookinete stage [16,17,54], the injection of rTNF-α did not significantly alter TEP1 expression (S3 Fig). To further examine the role of granulocytes in TNF-mediated parasite killing, we again employed the use of clodronate liposomes to deplete mosquito granulocyte populations [17,22,50,51]. To approach this question, we first depleted phagocytic granulocytes using clodronate liposomes, then treated mosquitoes with rTNF-α prior to challenge with P. berghei (Fig 6C). Before infection experiments, hemolymph was perfused and the granulocyte proportions were examined under each experimental condition to confirm granulocyte depletion. Similar to Fig 5D, the injection of rTNF-α in the control liposome background resulted in increased granulocyte numbers (Fig 6D). However, in clodronate liposome-treated mosquitoes which displayed a reduced percentage of granulocytes, the injection of rTNF-α had no effect (Fig 6D), thereby providing a methodology to examine the TNF-mediated contributions of granulocytes to anti-Plasmodium immunity. Similar to previous experiments (Figs 3A and 6A), the injection of rTNF-α reduced the parasite load as compared to the injection of 1X PBS in mosquitoes with a control liposome-treated background (Fig 6E). Also as expected [17], P. berghei oocyst numbers were significantly increased in PBS treated mosquitoes in the granulocyte-depleted background (Fig 6E). Of note, when rTNF-α injection was performed in the granulocyte-depleted background, parasite numbers were significantly reduced when compared to those treated with 1X PBS (Fig 6E). However, oocyst numbers were higher in mosquitoes injected with rTNF-α in the clodronate liposome background as compared to the control liposomes, suggesting that while granulocytes may contribute to TNF-mediated mechanisms that promote parasite killing, granulocyte depletion does not fully impair these immune responses. As a result, other TNF-mediated effects on oenocytoid immune function which influence hemolymph PO activity (Fig 5) and that have previously described roles in oocyst killing responses [17,19] may also contribute to malaria parasite killing (Fig 7). In addition, we also cannot rule out the potential that TNF signaling initiates humoral immune responses produced by the fat body or other tissues to limit parasite survival.
(A) Adult female mosquitoes were injected with 1XPBS (control) or 50 ng of rTNF-α prior to infection with P. berghei. Oocyst numbers and infection prevalence were evaluated at 2 days post infection to measure early oocyst numbers and the success of ookinete invasion. (B) Immunofluorescence images of DiI-stained hemocytes (red) attached to the mosquito midgut (counterstained with phalloidin, green) approximately 24 hrs post-treatment with 1xPBS (control) or 50 ng of rTNF-α. The number of attached hemocytes was quantified for each respective treatment with the dots corresponding to the number of hemocytes attached to each individual midgut examined. To address the role of granulocytes in TNF-mediated parasite killing, mosquitoes were first injected with either clodronate liposomes (CLD) to deplete granulocytes or control liposomes (LP), then 24 hours later surviving mosquitoes were treated with 50 ng rTNF-α or 1X-PBS (C). Each group was then challenged with P. berghei and oocyst numbers were examined on Day 8 post-infection. (D) Before infection, the effects of clodronate treatment on the percentage of granulocytes was examined in the presence or absence of rTNF-α to confirm our experimental approach. (E) Infection outcomes following granulocyte depletion and rTNF-α treatment, with oocyst numbers and infection prevalence evaluated at 8 days post infection. For both D and E, “+” denotes treatment with rTNF-α, while “-“ indicates treatment with 1X-PBS. For all experiments, the dots represent the respective measurements from an individual mosquito. The red horizontal lines represent the median oocysts numbers, while infection prevalence (% infected/total) is depicted as chart pies below each figure containing infection data. Data were combined from three or more independent experiments. Statistical significance was determined using either Mann-Whitney (individual comparisons) or Kruskal-Wallis with a Dunn’s multiple comparison test (multiple comparisons) to assess oocyst numbers, the number of attached hemocytes, or the percentage of granulocytes. A Fisher’s Exact test was performed to measure differences in infection prevalence. Asterisks denote statistical significance (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). ns, not significant; n = numbers of individual mosquitoes examined. Figure created in part with BioRender.com.
Figure created with BioRender.com.
Discussion
Mosquito innate immunity is an integral component of vector competence [55], therefore understanding the immune mechanisms that influence Plasmodium survival is essential for ongoing efforts to limit malaria transmission. While several conserved immune signaling pathways, such as Toll, IMD, and JAK/STAT, have been previously implicated in mosquito vector competence [56–59], here we provide direct evidence for the role of TNF signaling in mosquito immune function.
Our expression analysis of the mosquito TNF signaling components, Eiger, Wgn, and Grnd, suggests that TNF signaling is ubiquitous across mosquito tissues, with expression detected across midgut, hemocyte, and fat body tissues. While the expression of the receptors remained relatively constant across physiological conditions, the mosquito TNF ortholog Eiger was more responsive to blood-feeding or P. berghei infection, displaying significant induction in hemocytes. While this implies that TNF signaling is further amplified by mosquito hemocytes, potential post-translational modifications, which require cleavage to release TNF-α/Eiger in its active soluble form [60], complicate our ability to make broad sweeping conclusions from these data.
While our RNA interference (RNAi) experiments suggest that the loss of TNF signaling does not influence adult mosquito fitness under naïve conditions, these survival experiments do not exclude the potential that TNF components might still contribute to several important aspects of mosquito physiology or development. TNF signaling has been previously implicated in Drosophila as a master regulator of midgut homeostasis regulating lipid metabolism and intestinal stem cell (ISC) proliferation to maintain tissue integrity [48]. In addition, Eiger is expressed in Drosophila hemocytes in response to the midgut-derived reactive oxygen species (ROS), which ultimately triggers ISC proliferation [39]. Since mosquito blood-feeding represents a significant physiological event causing the expansion of the midgut microbiota, as well as distention and damage to the midgut epithelium, the repair of the midgut epithelium may require similar roles of intestinal stem cell differentiation [61]. This is supported by previous studies that highlight the involvement of stem cells in mosquito midgut homeostasis in response to blood-feeding, oxidative stress, and infection [62,63]. Considering the function of Eiger/Wgn signaling in Drosophila epithelial turnover [39,40], there is potential that the observed increase in Eiger expression following blood-feeding and infection could similarly stimulate ISC proliferation to promote midgut homeostasis, a process that may potentially involve hemocyte function. In addition, it is unclear how the mosquito midgut microbiome could also influence this process. For example, the presence and expansion of specific microbes may contribute to dysbiosis and inflammatory-like responses that lead to Eiger activation that influence midgut damage and repair. Therefore, the observed increase in Eiger expression following blood feeding and infection may not only be a direct response to these physiological conditions, but also an indirect consequence of microbe-mediated modulation of TNF signaling. However, at present, direct roles of TNF signaling in mosquito midgut regeneration have yet to be determined.
Using the paired approach of rTNF-α injection and RNAi to address the potential roles of mosquito TNF signaling, we demonstrate that TNF signaling is an integral component of anti-Plasmodium immunity that limits P. berghei infection in Anopheles gambiae. While rTNF-α injection (and presumably overexpression of the pathway) results in reduced malaria parasite survival, loss of Eiger, Wgn, or Grnd prior to P. berghei challenge each cause an increase in Plasmodium oocyst numbers. While in vertebrates, TNF signaling can initiate distinct cellular responses mediated by TNFR1 and TNFR2 [64], our knockdown experiments suggest that both Wgn and Grnd mediate TNF signaling responses that are required to initiate anti-Plasmodium immunity. At present, it is unclear if Wgn and Grnd act as a heterodimeric receptor to promote TNF signaling, despite the lack of support from other systems. Alternatively, based on recent studies [65], Wgn and Grnd may differ in their subcellular localization and functional roles in the processing of TNF-mediated signals.
Although TNF-α is a well-established proinflammatory cytokine known for its role in regulating various aspects of macrophage function in vertebrates [66,67], our understanding of TNF signaling on insect immune cells has been limited. Previous studies have implicated Eiger/TNF in phagocytosis and host survival to pathogen infection [44–46], supporting the conservation of the TNF signaling pathway across insect taxa. Moreover, TNF signaling has been implicated in regulating phagocytic immune cell populations in solitary locusts [46] and in promoting crystal cell rupture in Drosophila [42]. Here, we provide evidence that TNF signaling similarly regulates mosquito immune cell function by increasing the percentage of circulating granulocyte populations and in driving oenocytoid immune cell lysis in adult female mosquitoes. While our RNA-mediated knockdowns of TNF pathway components did not influence the basal percentages of immune cell subtypes, these experiments were performed transiently in adult mosquitoes and may not fully capture potential roles of TNF signaling on hematopoiesis and immune cell maturation that may occur at earlier stages of mosquito development. As a result, further experiments are required to fully examine roles of TNF signaling on immune cell development, immune activation, and cell lysis.
As important immune sentinels, granulocytes are the primary phagocytic immune cells in the mosquito, either circulating in the open hemolymph or attached to various mosquito tissues [20]. While data support the increase in the percentage of granulocytes following rTNF-α treatment, our limited understanding of mosquito hemocyte biology and lack of genetic tools makes this a challenging phenotype to address. As a result, it remains unclear if TNF signaling via Wgn/Grnd promotes differences in cell adherence (from sessile cells to in circulation), granulocyte activation, or the differentiation of precursor cells to granulocytes. This is further complicated by the requirement of Grnd for the rTNF-α-mediated effects on granulocyte populations, which based on the data presented here and previous single-cell studies [22], suggest that Grnd is not expressed in granulocytes. As a result, we speculate that the TNF-mediated increase in granulocytes is indirect, and potentially caused by the release of other molecules resulting from oenocytoid lysis or the production of humoral factors from other tissues such as the fat body.
Similar to previous studies in Drosophila demonstrating the role of Eiger in crystal cell lysis [42], our data suggest that rTNF-α promotes the lysis/rupture of mosquito oenocytoids, the equivalent of Drosophila crystal cells. With evidence that Eiger is required for the release of prophenoloxidase [42] and melanization activity [43] in Drosophila, our data displaying reduced oenocytoid percentages following the injection of rTNF-α provide support for a similar mechanism in mosquitoes. These morphological observations are also supported by complementary molecular studies demonstrating that the injection of rTNF-α reduced the expression of PPO1, PPO8, and PGE2R, genes enriched in oenocytoid populations [22], while promoting increased hemolymph PO activity. When placed in the context of previously published studies examining mosquito oenocytoid lysis [19], the reduced percentage of oenocytoids, reduced expression of oenocytoid markers, and increased PO activity are highly suggestive that TNF signaling regulates oenocytoid lysis/rupture. This, in turn, is believed to promote the release of prophenoloxidases that have been previously implicated in mosquito anti-bacterial [19] and anti-Plasmodium immunity [17,19], and potentially other immune factors that may influence mosquito innate immune function.
Previous studies indicate that both granulocytes and oenocytoids contribute to limiting malaria parasite survival in the mosquito host [16,17,19,53]. Since both immune cell subtypes display phenotypes associated with TNF signaling, we further investigated the potential roles of mosquito hemocytes in TNF-mediated parasite killing. Considering that Plasmodium killing in mosquitoes is multimodal, with distinct immune responses targeting either invading ookinetes or immature oocysts [15,17,54,68], we examined early (day 2) oocyst numbers as a proxy to determine the success of ookinete invasion [15,68]. Our results demonstrate a reduction in early oocyst numbers in mosquitoes injected with rTNF-α, suggesting that TNF signaling enhances P. berghei ookinete killing. Given that granulocytes are integral to ookinete recognition by mosquito complement [16,17], this implies that the TNF regulation of granulocyte function is central to early-phase immune responses targeting the ookinete. This is further supported by our observations of increased hemocyte attachment to the midgut following rTNF-α treatment. However, our cursory analysis of TEP1 expression following rTNF-α injection was inconclusive in implicating direct roles of mosquito complement in TNF-mediated killing responses. Moreover, through the use of clodronate liposomes to deplete phagocytic granulocyte populations [17,22,50,51], we confirm the involvement of granulocytes in TNF-mediated parasite killing. However, granulocyte depletion did not fully abrogate parasite killing following rTNF-α treatment, suggesting that additional components contribute to limiting parasite survival. It remains unclear if these TNF-mediated responses are produced by granulocyte populations not influenced by clodronate depletion [17] or if these immune responses are produced by other immune cell subtypes or tissues. Given that rTNF-α also influences oenocytoid rupture and PO activity, similar to known late-phase immune responses limiting oocyst survival via prostaglandin signaling [19], we propose a model where TNF signaling influences anti-Plasmodium immunity by involving both granulocyte and oenocytoid immune functions (summarized in Fig 7). Alternatively, we acknowledge the potential that TNF-mediated parasite killing responses may also be mediated in part by humoral responses produced by the fat body. Yet, due to the systemic nature of RNAi, we currently lack the genetic tools to examine the cell- or tissue-specific contributions of TNF signaling in An. gambiae.
In addition, an unexplored aspect of our study is the potential that blood meal-derived levels of TNF-α in host blood may be able to initiate immune signaling in the mosquito host. This is supported by previous studies which have shown that other blood meal- derived components such as insulin-like growth factor 1 (IGF1) and transforming growth factor (TGF)-beta1 are able to influence Anopheles mosquito vector competence to Plasmodium infection [69–73]. Given that acute and chronic inflammatory conditions in humans produce increased levels of TNF-α in blood [74], it remains to be seen whether certain hosts for mosquito blood meals may promote mosquito immune responses through the uptake of TNF-α in ingested blood.
While our experiments examine malaria parasite infection using the rodent malaria model, P. berghei, it remains to be explored if TNF pathway components similarly influence infection outcomes with the human malaria parasite, P. falciparum. In addition to differences in incubation temperature which may influence host physiology, there is evidence that P. falciparum has evolved mechanisms to evade immune recognition [75–77]. While hemocyte-mediated immune responses have been implicated in killing responses that limit both P. berghei and P. falciparum survival [15,53], it remains unclear if the TNF-mediated responses influencing cellular immune function or other humoral factors that promote P. berghei killing will also attenuate P. falciparum. As a result, further experiments are required to define the exact mechanisms by which TNF signaling influences malaria parasite survival and whether these responses are conserved across Plasmodium species.
In summary, our findings provide important new insights into the roles of TNF signaling in An. gambiae, demonstrating the effects of TNF signaling in limiting malaria parasite survival and immune cell regulation. While further study is required to fully determine the influence of TNF signaling on mosquito physiology and immune function, there is significant evidence, in addition to that provided herein, that TNF signaling is a central component that defines mosquito vectorial capacity and the susceptibility to Plasmodium infection in natural mosquito populations [36]. As a result, our study represents an important contribution to our understanding of the mechanisms of malaria parasite killing and the collective efforts to develop novel approaches for malaria control.
Materials and methods
Ethics statement
All protocols and experimental procedures regarding vertebrate animal use were approved by the Animal Care and Use Committee at Iowa State University (IACUC-21–143).
Mosquito rearing and plasmodium infection
Anopheles gambiae mosquitoes (Keele strain) were reared at 27°C and 80% relative humidity, with a 14:10 h light: dark photoperiod cycle. Larvae were fed on commercialized fish flakes (Tetramin, Tetra), while adults were maintained on a 10% sucrose solution and fed on commercial sheep blood (Hemostat) for egg production.
Female Swiss Webster mice were used for mosquito blood-feeding and infections with a Plasmodium berghei (P. berghei) transgenic strain expressing mCherry [15,17]. For P. berghei infections, mice were inoculated via intraperitoneal injection using either fresh or frozen parasite stocks, then respectively monitored for the presence of exflagellating microgametes at three- or four-days post-infection as previously described [11,15]. To enable mosquito feeding, infected mice were anaesthetized via an intraperitoneal injection of ketamine:xylazine before mosquito challenge. After challenge, blood-fed mosquitoes were sorted on ice, incubated at 19°C. At either two days or eight days post-infection, individual mosquito midguts were dissected in 1X PBS, then examined by fluorescence under a compound fluorescent microscope (Nikon Eclipse 50i; Nikon) to determine parasite loads. All data are displayed in S1 Table.
RNA extraction and gene expression analyses
Total RNA was extracted from pooled whole mosquito samples (~10 individuals) or dissected tissues (~10–15 individual mosquitoes for midgut and carcass samples, ~ 30 for hemocyte perfusions) using Trizol (Invitrogen, Carlsbad, CA). RNA from perfused hemolymph samples was isolated using the Direct-Zol RNA miniprep kit (Zymo Research). Two micrograms of non-hemolymph-derived or 200 ng of hemolymph-derived total RNA were used for first-strand synthesis with the RevertAid reverse transcriptase kit (Thermo Fisher Scientific). Gene expression analysis was performed with quantitative real-time PCR (qPCR) using PowerUp SYBRGreen Master Mix (Thermo Fisher Scientific) as previously described [17]. qPCR results were calculated using the 2-ΔCt formula and standardized by subtracting the Ct values of the target genes from the Ct values of the internal reference, rpS7. All primers used in this study are summarized in S2 Table.
Gene identification and silencing
The mosquito orthologs of known Drosophila TNF-α signaling components, Eiger (AGAP006771), Wengen (Wgn, AGAP000728), and Grindelwald (Grnd, AGAP008399), were identified using the OrthoDB database [78]. To address gene function, T7 primers specific to each candidate gene (S2 Table) were used to amplify DNA templates from whole female mosquito cDNA for dsRNA production and RNAi as previously [15,17]. PCR products were purified using the DNA Clean & Concentrator kit (Zymo Research) following gel electrophoresis to test for target specificity. dsRNA synthesis was performed using the MEGAscript RNAi kit (Thermo Fisher Scientific), with the concentration of the resulting dsRNA adjusted to 3 μg/μl. For RNAi, adult female mosquitoes (3–5 days old) were anesthetized on a cold block and injected intrathoracically with 69 nl of dsRNA targeting each gene or GFP as a negative control. Co-silencing of Wgn and Grnd was accomplished by injecting mosquitoes with a solution consisting of equal parts of the dsRNA suspensions targeting each gene. Injections were performed using Nanoject III manual injector (Drummond Scientific). To assess gene-silencing, groups of ten mosquitoes were used to analyze the efficiency of dsRNA-mediated silencing at 2 days post-injection via qPCR.
Mosquito survival experiments
To determine the effects of silencing TNF signaling components on mosquito survival, mosquitoes were injected with dsRNA targeting GFP (control), Eiger, Wgn, or Grnd as described above and maintained on a 10% sucrose solution under insectary conditions at 19°C or 27°C. Similar experiments were performed in which gene-silencing was first performed, then mosquitoes were challenged with P. berghei two days later. Fully engorged mosquitoes were then maintained on 10% sucrose under insectary conditions at 19°C. Mosquito survival was monitored every 24 h over a period of 12 days. All data are displayed in S1 Table.
Injection of human recombinant TNF-α
Recombinant human TNF-a (rTNF-α; Sigma #H8916) was resuspended in 1X PBS to a stock solution of 0.72 ng/nl. Naive or dsRNA-injected mosquitoes were anesthetized and intrathoracically injected with either 69 nl of 1X PBS (control) or the stock solution of rTNF-α to administer 50 ng of protein per individual. Following injection, mosquitoes were maintained at 27°C for 24 hrs then used for downstream infection experiments or hemocyte analysis.
Hemocyte counting
Mosquito hemolymph was collected by perfusion using an anticoagulant buffer of 60% v/v Schneider’s Insect medium, 10% v/v Fetal Bovine Serum, and 30% v/v citrate buffer (98 mM NaOH, 186 mM NaCl, 1.7 mM EDTA, and 41 mM citric acid; buffer pH 4.5) as previously described [15,17,53]. For perfusions, incisions were performed on the posterior abdomen, then anticoagulant buffer (~10 μl) was injected into the thorax. Collected perfusate from individual mosquitoes were placed in a Neubauer Improved hemocytometer and observed under a light microscope (Nikon Eclipse 50i; Nikon) to distinguish hemocyte subtypes by morphology and determine the proportion of granulocytes out of the total number hemocytes in the sample. All data are displayed in S1 Table.
Hemocyte gene expression analysis
Following mosquito injections with rTNF-α, perfused hemolymph from at least 20 mosquitoes was used for RNA extraction, cDNA synthesis, and qPCR to estimate the expression levels of hemocyte subtype gene markers (PPO1, PPO8, and PGE2R; oenocytoid-specific, and Eater; granulocyte-specific) [19,22]. All data are displayed in S1 Table.
Characterization of hemolymph PO activity
To determine the effects of TNF-α on phenoloxidase (PO) activity, naïve mosquitoes were injected with either 1X PBS (control) or rTNF-α. At 24 h post-injection, hemolymph was perfused from 15 mosquitoes using nuclease-free water as previously described [17,19,79]. The perfusate (10 μl) was added to a 90 μl suspension of 3, 4-Dihydroxy-L-phenylalanine (L-DOPA, 4 mg/ml), then incubated at room temperature for 10 min prior to measurements of PO activity using a microplate reader at 490 nm. Samples were measured using six independent measurements at 5 min intervals. All data are displayed in S1 Table.
Plasmodium infections following rTNF-α injection
After the injection of rTNF-α as described above, both control and experimental groups were challenged on a P. berghei-infected mouse. After selecting for blood-fed mosquitoes on ice, mosquitoes were kept at 19°C until oocyst survival was assessed at either 2- or 8- days post-infection.
To determine the TNF-α-mediated responses against Plasmodium in a granulocyte-depleted background, 3-day-old mosquitoes were first injected with either control or clodronate liposomes as previously [17]. At 24 h post-injection, each group was treated with 50 ng of rTNF-α or 1X PBS as control. Following an additional 24 h incubation, surviving mosquitoes were challenged with P. berghei, with oocyst numbers examined from dissected midguts at either 2- or 8-days post-infection. All data are displayed in S1 Table.
Analysis of Wgn and Grnd expression in hemocyte subtypes
To determine the expression of TNF signaling components in mosquito hemocyte subpopulations, the expression of Wgn and Grnd was referenced with previous single-cell transcriptomic data for An. gambiae hemocytes [22]. Further validation was performed using methods of granulocyte depletion via clodronate liposomes as described above [17] to confirm the presence/absence of Wgn and Grnd expression in granulocyte populations using qPCR. All data are displayed in S1 Table.
Immunofluorescent analysis of hemocytes attached to midguts
Hemocyte attachment to mosquito midguts in response to rTNF-α treatment was examined by immunofluorescence analysis as previously described [18] with slight modification. Two days after treatment with either rTNF-α or 1XPBS, mosquitoes were injected with 69 nl of 100 μM Vybrant CM-DiI cell labeling solution (ThermoFisher) and allowed to recover for 30 min at 27°C. Mosquitoes were injected with 200 nl of 16% paraformaldehyde (PFA), then the entire mosquito was immediately submerged/ incubated in a solution of 4% PFA for 40 sec prior to transfer in ice-cold 1XPBS for midgut dissection. Dissected midguts were incubated overnight in 4% PFA at 4°C for fixation. The following day, midguts were washed with ice-cold 1XPBS three times and permeabilized with 0.1% TritonX-100 for 10 minutes at room temperature. After washing three times with 1XPBS, tissues were blocked with 1% BSA in 1XPBS for 40 minutes at room temperature and stained with Phalloidin-iFluor 405 Reagent (1:400 in PBS; abcam, ab176752) for 1 hour to visualize actin filaments. Midguts were washed with 1XPBS to remove excess staining, placed on microscope slides and mounted with ProLong Diamond Antifade Mountant (ThermoFisher). Samples were imaged by fluorescence microscopy using a Zeiss Axio Imager 2 and analyzed to determine the number of hemocytes attached to individual midguts from each experimental condition.
Supporting information
S1 Fig. Silencing efficiency of Eiger, Grnd, and Wgn genes in An. gambiae.
Naïve adult female mosquitoes were injected with dsRNA targeting GFP (control), Eiger, Wgn, or Grnd. Two days post-injection, whole-body mosquitoes (10–15 total) were collected for RNA extraction, followed by gene expression analysis using qPCR. Data from three or more independent experiment were examined for statistical significance using an unpaired student’s t-test. Asterisks indicate significance (* P < 0.05, **** P < 0.0001).
https://doi.org/10.1371/journal.ppat.1013329.s001
(TIF)
S2 Fig. Expression of Eiger, Wgn, and Grnd in mosquito hemocyte populations.
Using previously published single-cell RNA-seq data of An. gambiae hemocytes [22], the expression of Eiger, Wgn, and Grnd was evaluated for each mosquito immune cell subtype. Each dot represents a cell-specific gene expression value. RPKM, Reads Per Kilobase Per Million.
https://doi.org/10.1371/journal.ppat.1013329.s002
(TIF)
S3 Fig.
TEP1 expression following the injection of rTNF-α. The influence of TNF signaling on TEP1 expression was assessed in whole mosquitoes, 24 hours after the injection of 1X PBS (control) or rTNF-α injection (50 ng/mosquito). rTNF-α. Data were collected from four independent experiments, each involving 10 female mosquitoes per replicate. Statistical analysis was performed using an unpaired Students’ t-test. ns, not significant.
https://doi.org/10.1371/journal.ppat.1013329.s003
(TIF)
S2 Table. List of primers used for gene expression and RNAi.
Small letters indicate the T7 promoter sequence.
https://doi.org/10.1371/journal.ppat.1013329.s005
(PDF)
References
- 1.
WHO. Word Malaria Report 2021. Geneva: World Health Organization; 2021.
- 2. Dahmana H, Mediannikov O. Mosquito-Borne Diseases Emergence/Resurgence and How to Effectively Control It Biologically. Pathogens. 2020;9(4):310. pmid:32340230
- 3. Shaw WR, Catteruccia F. Vector biology meets disease control: using basic research to fight vector-borne diseases. Nat Microbiol. 2019;4(1):20–34. pmid:30150735
- 4. Kleinschmidt I, Bradley J, Knox TB, Mnzava AP, Kafy HT, Mbogo C, et al. Implications of insecticide resistance for malaria vector control with long-lasting insecticidal nets: a WHO-coordinated, prospective, international, observational cohort study. Lancet Infect Dis. 2018;18(6):640–9. pmid:29650424
- 5. Hoermann A, Tapanelli S, Capriotti P, Masters EKG, Habtewold T, Christophides GK. Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement. Elife. 2021;10:e58791.
- 6. Hoermann A, Habtewold T, Selvaraj P, Del Corsano G, Capriotti P, Inghilterra MG, et al. Gene drive mosquitoes can aid malaria elimination by retarding Plasmodium sporogonic development. Sci Adv. 2022;8(38):eabo1733. pmid:36129981
- 7. Hammond A, Pollegioni P, Persampieri T, North A, Minuz R, Trusso A, et al. Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field. Nat Commun. 2021;12(1):4589. pmid:34321476
- 8. Han YS, Thompson J, Kafatos FC, Barillas-Mury C. Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes. EMBO J. 2000;19(22):6030–40. pmid:11080150
- 9. Vlachou D, Schlegelmilch T, Christophides GK, Kafatos FC. Functional genomic analysis of midgut epithelial responses in Anopheles during Plasmodium invasion. Curr Biol. 2005;15(13):1185–95. pmid:16005290
- 10. Oliveira G de A, Lieberman J, Barillas-Mury C. Epithelial nitration by a peroxidase/NOX5 system mediates mosquito antiplasmodial immunity. Science. 2012;335(6070):856–9. pmid:22282475
- 11. Smith RC, Eappen AG, Radtke AJ, Jacobs-Lorena M. Regulation of anti-Plasmodium immunity by a LITAF-like transcription factor in the malaria vector Anopheles gambiae. PLoS Pathog. 2012;8(10):e1002965. pmid:23093936
- 12. Ramphul UN, Garver LS, Molina-Cruz A, Canepa GE, Barillas-Mury C. Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells. Proc Natl Acad Sci U S A. 2015;112(5):1273–80. pmid:25552553
- 13. Rodrigues J, Brayner FA, Alves LC, Dixit R, Barillas-Mury C. Hemocyte differentiation mediates innate immune memory in Anopheles gambiae mosquitoes. Science. 2010;329(5997):1353–5. pmid:20829487
- 14. Ramirez JL, Garver LS, Brayner FA, Alves LC, Rodrigues J, Molina-Cruz A. The role of hemocytes in anopheles gambiae antiplasmodial immunity. J Innate Immun. 2014;6:119–28.
- 15. Smith RC, Barillas-Mury C, Jacobs-Lorena M. Hemocyte differentiation mediates the mosquito late-phase immune response against Plasmodium in Anopheles gambiae. Proc Natl Acad Sci. 2015;112:E3412-20.
- 16. Castillo JC, Ferreira ABB, Trisnadi N, Barillas-Mury C. Activation of mosquito complement antiplasmodial response requires cellular immunity. Sci Immunol. 2017;2(7):eaal1505. pmid:28736767
- 17. Kwon H, Smith RC. Chemical depletion of phagocytic immune cells in Anopheles gambiae reveals dual roles of mosquito hemocytes in anti-Plasmodium immunity. Proc Natl Acad Sci U S A. 2019;116(28):14119–28. pmid:31235594
- 18. Barletta ABF, Trisnadi N, Ramirez JL, Barillas-Mury C. Mosquito midgut prostaglandin release establishes systemic immune priming. iScience. 2019;19:54–62.
- 19. Kwon H, Hall DR, Smith RC. Prostaglandin E2 Signaling Mediates Oenocytoid Immune Cell Function and Lysis, Limiting Bacteria and Plasmodium Oocyst Survival in Anopheles gambiae. Front Immunol. 2021;12:680020. pmid:34484178
- 20. Hillyer JF, Strand MR. Mosquito hemocyte-mediated immune responses. Curr Opin Insect Sci. 2014;3:14–21. pmid:25309850
- 21. Raddi G, Barletta ABF, Efremova M, Ramirez JL, Cantera R, Teichmann SA, et al. Mosquito cellular immunity at single-cell resolution. Science. 2020;369(6507):1128–32. pmid:32855340
- 22. Kwon H, Mohammed M, Franzén O, Ankarklev J, Smith RC. Single-cell analysis of mosquito hemocytes identifies signatures of immune cell subtypes and cell differentiation. Elife. 2021;10:e66192. pmid:34318744
- 23. King JG, Hillyer JF. Spatial and temporal in vivo analysis of circulating and sessile immune cells in mosquitoes: hemocyte mitosis following infection. BMC Biol. 2013;11:55. pmid:23631603
- 24. Castillo JC, Robertson AE, Strand MR. Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti. Insect Biochem Mol Biol. 2006;36(12):891–903. pmid:17098164
- 25. Baton LA, Robertson A, Warr E, Strand MR, Dimopoulos G. Genome-wide transcriptomic profiling of Anopheles gambiae hemocytes reveals pathogen-specific signatures upon bacterial challenge and Plasmodium berghei infection. BMC Genomics. 2009;10:257. pmid:19500340
- 26. Martinson EO, Chen K, Valzania L, Brown MR, Strand MR. Insulin-like peptide 3 stimulates hemocytes to proliferate in anautogenous and facultatively autogenous mosquitoes. J Exp Biol. 2022;225.
- 27. Castillo J, Brown MR, Strand MR. Blood feeding and insulin-like peptide 3 stimulate proliferation of hemocytes in the mosquito Aedes aegypti. PLoS Pathog. 2011;7(10):e1002274. pmid:21998579
- 28. Bryant WB, Michel K. Anopheles gambiae hemocytes exhibit transient states of activation. Dev Comp Immunol. 2016;55:119–29. pmid:26515540
- 29. Bryant WB, Michel K. Blood feeding induces hemocyte proliferation and activation in the African malaria mosquito, Anopheles gambiae Giles. J Exp Biol. 2014;217(Pt 8):1238–45. pmid:24363411
- 30. Barletta ABF, Saha B, Trisnadi N, Talyuli OAC, Raddi G, Barillas-Mury C. Hemocyte differentiation to the megacyte lineage enhances mosquito immunity against Plasmodium. Elife. 2022;11:e81116. pmid:36052991
- 31. Ramirez JL, de Almeida Oliveira G, Calvo E, Dalli J, Colas RA, Serhan CN, et al. A mosquito lipoxin/lipocalin complex mediates innate immune priming in Anopheles gambiae. Nat Commun. 2015;6:7403. pmid:26100162
- 32. Kwon H, Smith RC. Inhibitors of Eicosanoid Biosynthesis Reveal that Multiple Lipid Signaling Pathways Influence Malaria Parasite Survival in Anopheles gambiae. Insects. 2019;10(10):307. pmid:31547026
- 33. van Loo G, Bertrand MJM. Death by TNF: a road to inflammation. Nat Rev Immunol. 2023;23:289–303.
- 34. Dostert C, Grusdat M, Letellier E, Brenner D. The TNF family of ligands and receptors: Communication modules in the immune system and beyond. Physiol Rev. 2019;99:115–60.
- 35. Wiens GD, Glenney GW. Origin and evolution of TNF and TNF receptor superfamilies. Dev Comp Immunol. 2011;35(12):1324–35. pmid:21527275
- 36. Srinivasan S, Ghosh C, Das S, Thakare A, Singh S, Ganesh A, et al. Identification of a TNF-TNFR-like system in malaria vectors (Anopheles stephensi) likely to influence Plasmodium resistance. Sci Rep. 2022;12(1):19079. pmid:36351999
- 37. Moreno E, Yan M, Basler K. Evolution of TNF Signaling Mechanisms. Curr Biol. 2002;12:1263–8.
- 38. Igaki T, Miura M. The Drosophila TNF ortholog Eiger: Emerging physiological roles and evolution of the TNF system. Semin Immunol. 2014;26:267–74.
- 39. Amcheslavsky A, Lindblad JL, Bergmann A. Transiently “Undead” Enterocytes Mediate Homeostatic Tissue Turnover in the Adult Drosophila Midgut. Cell Rep. 2020;33(8):108408. pmid:33238125
- 40. Fogarty CE, Diwanji N, Lindblad JL, Tare M, Amcheslavsky A, Makhijani K, et al. Extracellular Reactive Oxygen Species Drive Apoptosis-Induced Proliferation via Drosophila Macrophages. Curr Biol. 2016;26(5):575–84. pmid:26898463
- 41. Mirzoyan Z, Valenza A, Zola S, Bonfanti C, Arnaboldi L, Ferrari N, et al. A Drosophila model targets Eiger/TNFα to alleviate obesity-related insulin resistance and macrophage infiltration. Dis Model Mech. 2023;16(11):dmm050388. pmid:37828911
- 42. Bidla G, Dushay MS, Theopold U. Crystal cell rupture after injury in Drosophila requires the JNK pathway, small GTPases and the TNF homolog Eiger. J Cell Sci. 2007;120(Pt 7):1209–15. pmid:17356067
- 43. Mabery EM, Schneider DS. The Drosophila TNF ortholog eiger is required in the fat body for a robust immune response. J Innate Immun. 2010;2(4):371–8. pmid:20505310
- 44. Schneider DS, Ayres JS, Brandt SM, Costa A, Dionne MS, Gordon MD, et al. Drosophila eiger mutants are sensitive to extracellular pathogens. PLoS Pathog. 2007;3(3):e41. pmid:17381241
- 45. Caravello G, Franchet A, Niehus S, Ferrandon D. Phagocytosis Is the Sole Arm of Drosophila melanogaster Known Host Defenses That Provides Some Protection Against Microsporidia Infection. Front Immunol. 2022;13:858360. pmid:35493511
- 46. Wang Y, Tong X, Yuan S, Yang P, Li L, Zhao Y, et al. Variation of TNF modulates cellular immunity of gregarious and solitary locusts against fungal pathogen Metarhizium anisopliae. Proc Natl Acad Sci U S A. 2022;119(6):e2120835119. pmid:35110413
- 47. de Vreede G, Morrison HA, Houser AM, Boileau RM, Andersen D, Colombani J. A Drosophila Tumor Suppressor Gene Prevents Tonic TNF Signaling through Receptor N-Glycosylation. Dev Cell. 2018;45:595-605.e4.
- 48. Loudhaief R, Jneid R, Christensen CF, Mackay DJ, Andersen DS, Colombani J. The Drosophila tumor necrosis factor receptor, Wengen, couples energy expenditure with gut immunity. Sci Adv. 2023;9(23):eadd4977. pmid:37294765
- 49. Kauppila S, Maaty WSA, Chen P, Tomar RS, Eby MT, Chapo J, et al. Eiger and its receptor, Wengen, comprise a TNF-like system in Drosophila. Oncogene. 2003;22(31):4860–7. pmid:12894227
- 50. Kumar RJ, Smith JP, Kwon H, Smith RC. Use of clodronate liposomes to deplete phagocytic immune cells in Drosophila melanogaster and Aedes aegypti. Front Cell Dev Biol. 2021;9:627976.
- 51. Adegoke A, Ribeiro JMC, Brown S, Smith RC, Karim S. Rickettsia parkeri hijacks tick hemocytes to manipulate cellular and humoral transcriptional responses. Front Immunol. 2023;14:1094326. pmid:36845157
- 52. Mameli E, Samantsidis G, Viswanatha R, Kwon H, Hall D, Butnaru M. A genome-wide CRISPR screen in Anopheles mosquito cells identifies essential genes and required components of clodronate liposome function. bioRxiv. 2024.
- 53. Smith RC, King JG, Tao D, Zeleznik OA, Brando C, Thallinger GG, et al. Molecular Profiling of Phagocytic Immune Cells in Anopheles gambiae Reveals Integral Roles for Hemocytes in Mosquito Innate Immunity. Mol Cell Proteomics. 2016;15(11):3373–87. pmid:27624304
- 54. Smith RC, Barillas-Mury C. Plasmodium Oocysts: Overlooked Targets of Mosquito Immunity. Trends Parasitol. 2016;32(12):979–90. pmid:27639778
- 55. Smith RC, Vega-Rodríguez J, Jacobs-Lorena M. The Plasmodium bottleneck: malaria parasite losses in the mosquito vector. Mem Inst Oswaldo Cruz. 2014;109(5):644–61. pmid:25185005
- 56. Xi Z, Ramirez JL, Dimopoulos G. The Aedes aegypti toll pathway controls dengue virus infection. PLoS Pathog. 2008;4:e1000098.
- 57. Garver LS, Dong Y, Dimopoulos G. Caspar controls resistance to Plasmodium falciparum in diverse anopheline species. PLoS Pathog. 2009;5(3):e1000335. pmid:19282971
- 58. Souza-Neto JA, Sim S, Dimopoulos G. An evolutionary conserved function of the JAK-STAT pathway in anti-dengue defense. Proc Natl Acad Sci U S A. 2009;106(42):17841–6. pmid:19805194
- 59. Clayton AM, Dong Y, Dimopoulos G. The Anopheles innate immune system in the defense against malaria infection. J Innate Immun. 2014;6(2):169–81. pmid:23988482
- 60. Agrawal N, Delanoue R, Mauri A, Basco D, Pasco M, Thorens B, et al. The Drosophila TNF Eiger Is an Adipokine that Acts on Insulin-Producing Cells to Mediate Nutrient Response. Cell Metab. 2016;23(4):675–84. pmid:27076079
- 61. Hixson B, Taracena ML, Buchon N. Midgut Epithelial Dynamics Are Central to Mosquitoes’ Physiology and Fitness, and to the Transmission of Vector-Borne Disease. Front Cell Infect Microbiol. 2021;11:653156. pmid:33842397
- 62. Cui Y, Franz AWE. Heterogeneity of midgut cells and their differential responses to blood meal ingestion by the mosquito, Aedes aegypti. Insect Biochem Mol Biol. 2020;127:103496. pmid:33188922
- 63. Taracena ML, Bottino-Rojas V, Talyuli OAC, Walter-Nuno AB, Oliveira JHM, Angleró-Rodriguez YI, et al. Regulation of midgut cell proliferation impacts Aedes aegypti susceptibility to dengue virus. PLoS Negl Trop Dis. 2018;12(5):e0006498. pmid:29782512
- 64. Gough P, Myles IA. Tumor necrosis factor receptors: pleiotropic signaling complexes and their differential effects. Front Immunol. 2020;11:585880.
- 65. Palmerini V, Monzani S, Laurichesse Q, Loudhaief R, Mari S, Cecatiello V, et al. Drosophila TNFRs Grindelwald and Wengen bind Eiger with different affinities and promote distinct cellular functions. Nat Commun. 2021;12(1):2070. pmid:33824334
- 66. Parameswaran N, Patial S. Tumor necrosis factor-α signaling in macrophages. Crit Rev Eukaryot Gene Expr. 2010;20:87–103.
- 67. Jang D-I, Lee A-H, Shin H-Y, Song H-R, Park J-H, Kang T-B, et al. The Role of Tumor Necrosis Factor Alpha (TNF-α) in Autoimmune Disease and Current TNF-α Inhibitors in Therapeutics. Int J Mol Sci. 2021;22(5):2719. pmid:33800290
- 68. Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE. The STAT Pathway Mediates Late-Phase Immunity against Plasmodium in the Mosquito Anopheles gambiae. Cell Host Microbe. 2009;5:498–507.
- 69. Pakpour N, Corby-Harris V, Green GP, Smithers HM, Cheung KW, Riehle MA, et al. Ingested human insulin inhibits the mosquito NF-κB-dependent immune response to Plasmodium falciparum. Infect Immun. 2012;80(6):2141–9. pmid:22473605
- 70. Drexler A, Nuss A, Hauck E, Glennon E, Cheung K, Brown M, et al. Human IGF1 extends lifespan and enhances resistance to Plasmodium falciparum infection in the malaria vector Anopheles stephensi. J Exp Biol. 2013;216(Pt 2):208–17. pmid:23255191
- 71. Drexler AL, Pietri JE, Pakpour N, Hauck E, Wang B, Glennon EKK, et al. Human IGF1 regulates midgut oxidative stress and epithelial homeostasis to balance lifespan and Plasmodium falciparum resistance in Anopheles stephensi. PLoS Pathog. 2014;10(6):e1004231. pmid:24968248
- 72. Luckhart S, Crampton AL, Zamora R, Lieber MJ, Dos Santos PC, Peterson TML, et al. Mammalian transforming growth factor beta1 activated after ingestion by Anopheles stephensi modulates mosquito immunity. Infect Immun. 2003;71(6):3000–9. pmid:12761076
- 73. Surachetpong W, Singh N, Cheung KW, Luckhart S. MAPK ERK signaling regulates the TGF-beta1-dependent mosquito response to Plasmodium falciparum. PLoS Pathog. 2009;5(4):e1000366. pmid:19343212
- 74. Popa C, Netea MG, van Riel PLCM, van der Meer JWM, Stalenhoef AFH. The role of TNF-alpha in chronic inflammatory conditions, intermediary metabolism, and cardiovascular risk. J Lipid Res. 2007;48(4):751–62. pmid:17202130
- 75. Molina-Cruz A, DeJong RJ, Ortega C, Haile A, Abban E, Rodrigues J, et al. Some strains of Plasmodium falciparum, a human malaria parasite, evade the complement-like system of Anopheles gambiae mosquitoes. Proc Natl Acad Sci U S A. 2012;109(28):E1957-62. pmid:22623529
- 76. Molina-Cruz A, Garver LS, Alabaster A, Bangiolo L, Haile A, Winikor J, et al. The human malaria parasite Pfs47 gene mediates evasion of the mosquito immune system. Science. 2013;340(6135):984–7. pmid:23661646
- 77. Molina-Cruz A, Canepa GE, Alves E Silva TL, Williams AE, Nagyal S, Yenkoidiok-Douti L, et al. Plasmodium falciparum evades immunity of anopheline mosquitoes by interacting with a Pfs47 midgut receptor. Proc Natl Acad Sci U S A. 2020;117(5):2597–605. pmid:31969456
- 78. Kuznetsov D, Tegenfeldt F, Manni M, Seppey M, Berkeley M, Kriventseva EV. OrthoDB v11: annotation of orthologs in the widest sampling of organismal diversity. Nucleic Acids Research. 2023;51:D445–51.
- 79. Loghry HJ, Kwon H, Smith RC, Sondjaja NA, Minkler SJ, Young S, et al. Extracellular vesicles secreted by Brugia malayi microfilariae modulate the melanization pathway in the mosquito host. Sci Rep. 2023;13(1):8778. pmid:37258694