20-hydroxyecdysone (20E) primes innate immune responses that limit bacteria and malaria parasite survival in Anopheles gambiae

Blood-feeding is an integral behavior of mosquitoes to acquire nutritional resources needed for reproduction. This requirement also enables mosquitoes to serve as efficient vectors to acquire and potentially transmit a multitude of mosquito-borne diseases, most notably malaria. Recent studies suggest that mosquito immunity is stimulated following a blood meal, independent of infection status. Since blood-feeding results in the increased production of the hormone 20-hydroxyecdysone (20E), we hypothesized that 20E may play an important role in priming the immune response for pathogen challenge. Herein, we examine the immunological effects of priming in Anopheles gambiae with 20E prior to pathogen infection, demonstrating a significant reduction in bacteria and Plasmodium berghei survival in the mosquito host. RNA-seq analysis following 20E treatment identifies several known 20E-regulated genes, as well as several immune genes with previously reported function in anti-pathogen defense. This includes the anti-microbial peptide cecropin 3, which we demonstrate its role as an antagonist of bacteria and Plasmodium in Anopheles gambiae and provide support that these responses are under temporal regulation. Together, these data demonstrate that 20E influences cellular immune function and anti-pathogen immunity following mosquito blood-feeding, arguing the importance of hormones in the regulation of mosquito innate immune function.


Introduction 24
Blood-feeding behavior evolved in mosquitoes to provide nutritional resources required 25 for egg development. While providing benefits for reproduction, blood-feeding also 26 exposes mosquitoes to a myriad of blood-borne pathogens that can ultimately be 27 transmitted to a new host through an additional blood meal. For this reason, mosquitoes 28 are arguably the deadliest animals on the planet, causing hundreds of millions of 29 infections and over 500,000 deaths every year. Of mosquito-borne diseases, malaria 30 continues to be the most deadly, where more than 400,000 people die annually from 31 Plasmodium parasite infection transmitted by female Anopheles mosquitoes [1]. 32 Following a blood meal, signals initiated in the brain produce ovarian ecdysteroidogenic 33 hormone (OEH) and insulin-like peptides (ILPs), which trigger ecdysone production by 34 the ovaries [2][3][4]. Ecdysone is then converted into 20-hydroxyecdysone (20E) by 35 hydroxylation in the fat body, stimulating the production of yolk protein precursors (YPPs) 36 in a process known as vitellogenesis [2,5-7]. Reaching peak levels approximately 18-24 37 hours after blood-feeding [8,9], 20E expression also coincides with Plasmodium ookinete 38 invasion of the midgut epithelium [10]. While the influence of 20E is well-established with 39 respect to insect development [11], mating [12,13], reproduction [7,14,15], and vectorial 40 capacity [16,17], the role of 20E expression on mosquito immunity remains relatively 41

unexplored. 42
Evidence from other insect systems suggests that 20E is an important regulator of host 43 innate immune responses [18][19][20][21][22]. In Drosophila, 20E initiates immune signaling through 44 the IMD pathway via regulation of the peptidoglycan receptor PGRP-LC, or through 45 PGRP-LC-independent mechanisms, to directly regulate a subset of antimicrobial genes 46 for anti-bacterial defense [20]. However, only a limited number of studies have addressed 47 were quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific) and 112 ~2ug of total RNA was used as a template for cDNA synthesis using the RevertAid First 113 Strand cDNA Synthesis kit (Thermo Fisher). Gene expression was measured by qRT-114 PCR using gene-specific primers (Table S1) and PowerSYBR Green (Invitrogen). The 115 results were normalized to an S7 reference gene and quantified using the 2 -ΔΔCT method 116 as previously [39]. Samples were run in triplicate for each experiment. 117 dsRNA synthesis and gene-silencing 118 T7 primers for GFP and cecropin 3 (cec3) were used to amplify cDNA prepared from 119 whole An. gambiae mosquitoes and cloned into a pJET1.2 vector using a CloneJET PCR 120 Cloning Kit (Thermo Fisher). The resulting plasmids were used as a template for 121 amplification using the corresponding T7 primers (Table S2). PCR products were purified 122 using the DNA Clean and Concentrator kit (Zymo Research) and used as a template for 123 dsRNA synthesis as previously described [30,31]. The resulting dsRNA was resuspended 124 in RNase-free water to a concentration of 3 µg/uL. For gene-silencing experiments, 69nL 125 of dsRNA was injected per mosquito and evaluated by qRT-PCR to establish gene 126 knockdowns at 1-5 days post-injection. Time points with the highest efficiency of gene-127 silencing were chosen for downstream experiments. 128

Phagocytosis Assays 129
In vivo phagocytosis assays were performed as previously described [25,31]. Briefly, 130 mosquitoes were injected with 69nL of red fluorescent FluoSpheres (1 μm, Molecular 131 Probes) at a 1:10 dilution in 1x PBS. Following injection, mosquitoes were allowed to 132 recover for two hours at 19°C, then perfused onto a multi-test slide. Samples were fixed 133 using 4% paraformaldehyde for 30 minutes, washed three times using 1X PBS, then 134 blocked in 1% BSA for 30 minutes at room temperature. Hemocytes were stained using

Blood-feeding and 20E injection increase phagocytic activity
To determine the effects of 20E on mosquito immune function, we first explored whether 170 blood-feeding and 20E influenced mosquito cellular immunity. Immune cells, known as 171 hemocytes, serve as primary immune sentinels that recognize and destroy invading 172 pathogens by phagocytosis or the production of humoral immune factors [43,44]. To 173 examine how blood-feeding and 20E influence these phagocytic properties, we injected 174 fluorescent beads to evaluate phagocytosis under different physiological conditions as 175 previously [25,31]. Similar to Kwon and Smith [25], we demonstrate that blood-feeding, 176 independent of pathogen challenge, significantly increased the percentage of phagocytic 177 cells ( Figure 1A) and phagocytic activity ( Figure 1B). We therefore hypothesized that the 178 increase in phagocytic activity may be influenced in part by the increased levels of 20E 179 post-blood-feeding. To address this question, we injected 20E into mosquitoes prior to 180 challenge with fluorescent beads. Similarly, we observed a significant increase in the 181 proportion of phagocytic cells ( Figure 1C) and phagocytic activity ( Figure 1D) 24 hours 182 after 20E injection, suggesting that 20E activates mosquito cellular immunity. 183

Blood-feeding and 20E limit bacterial infection 184
Based on our observations that blood-feeding and 20E increase phagocytic activity, we 185 next looked to determine the role of blood-feeding and 20E priming on bacterial challenge. 186 To approach this question, we challenged naïve and blood-fed mosquitoes (~24 hours Interestingly, this peak effect on anti-bacterial immunity occurs ~12 hours post 20E-198 priming. This corresponds to the approximate timing of peak 20E production in the mosquito host, where 20E is induced ~12 hours post-blood meal and peaks between 18-200 24 hours post-blood feeding [45]. 201

20E priming limits Plasmodium survival 202
Since 20E influences phagocytosis and anti-bacterial immunity (Figures 1 and 2), we 203 wanted to explore whether 20E priming similarly influences malaria parasite numbers in 204 the mosquito host. To examine this question, we primed mosquitoes with 20E, then 205 challenged with P. berghei. We found the injection of 20E 24 hours before Plasmodium 206 infection significantly reduced parasite survival ( Figure 3A) and the prevalence of infection 207 ( Figure 3B), suggesting that 20E initiates anti-Plasmodium immune responses that prime 208 the mosquito host. 209

20E-regulation of mosquito immunity 210
To better understand how 20E primes immune responses to bacteria and malaria 211 parasites, we applied 20E to An. gambiae Sua 4.0 cells to identify genes under the direct 212 or indirect regulation of 20E. Our analysis identified 128 differentially regulated genes, 213 including 80 up-regulated genes and 48 down-regulated genes (Table S3). Gene ontology 214 analysis demonstrates that 20E application up-regulates transcripts associated with 215 transport, immunity, and transcription, while down-regulating transcripts with predicted 216 function in metabolism and redox metabolism ( Figure 4A). This includes the upregulation 217 of several previously described 20E-induced "early genes", HR3, HR4, E75A/B, and 218 multiple broad complex isoforms ( Figure 4B, Table S3), implicated in canonical 20E 219 signaling [46]. 220 In addition, 20E application up-regulated 12 genes associated with mosquito immune 221 function ( Figure 4B, Table S1). PPO3 is a known Plasmodium antagonist We validated our RNA-seq results using a subset of differentially regulated genes in vitro 231 ( Figure 4C, Figure S1) and in vivo using whole mosquito samples following 20E injection 232 ( Figure 4D Figure S1). 237 However, we cannot exclude that 20E may significantly influence the expression of these 238 transcripts in specific mosquito tissues. 239

Cecropin 3 (CEC3) limits bacterial and malaria parasite survival 240
Since several of the immune genes under 20E regulation are previously described, we  survival.

258
Blood-feeding is an essential behavior for An. gambiae and other hematophagous insects 259 to acquire nutrient resources for egg production. This requires the synthesis of yolk 260 protein precursors in a process known as vitellogenesis that is regulated by the production 261 of 20E shortly after a blood-meal [7,15,56]. In addition, the male transfer of 20E during 262 mating further contributes to oogenesis to maximize female fecundity [12,13,57]. Given the role of 20E priming on bacteria and malaria parasites, 20E likely triggers both 292 cellular and humoral immune components that limit pathogen survival. We demonstrate 293 that 20E increases the phagocytic activity of mosquito immune cells, the production of 294 AMPs, and the production of other immune elicitors, yet the precise mechanisms and 295 tissues involved in these 20E immune priming responses remain unknown. In addition to 296 changes in cellular immunity, humoral components produced by hemocytes or the fat 297 body and secreted into the hemolymph may also act on bacteria or invading malaria 298 parasites. Interestingly, our data suggest 20E priming differs temporally between 299 pathogens, potentially providing additional resolution into the mechanisms of 20E-300 regulated immunity. The effects of 20E on E. coli appear more transient, with peak activity 301 12 to 18 hours post-priming. This contrasts with the longer-lasting influence of 20E 302 immune priming on anti-Plasmodium immunity, where P. berghei challenge occurred 24 303 hours post-priming and persisted more than 18 hours post-infection after the onset of 304 ookinete invasion. One potential explanation is that anti-bacterial responses are primarily 305 cellular-mediated, while the immune responses that limit parasite development are 306 humoral. This is supported by the transient activation of mosquito hemocytes following 307 blood-feeding [26,27], which juvenile hormone (JH) may quickly suppress as described 308 in Drosophila [19]. This is in contrast to protein-based humoral components, such as 309 TEP1, which are stable in circulation in the mosquito hemolymph where they are integral 310 to the recognition and lysis of invading ookinetes [10]. 311 The establishment that 20E regulates mosquito immune responses raises additional 312 considerations into the potential trade-offs between reproduction and immunity. Both 313 physiological processes are energetically costly, where 20E production may serve as a 314 limiting factor for the allocation of resources towards egg production or immunity [59]. 315 Evidence suggests reduced mosquito fitness at the cost of anti-Plasmodium immunity 316 [60,61], arguing a competition for resources in the mosquito host. This is supported by 317 fitness experiments in transgenic mosquito lines expressing anti-Plasmodium effector 318 genes which display increased fecundity compared to wild-type following Plasmodium 319 challenge, presumably by enabling more resources for egg production [62,63]. However, 320 recent studies argue P. falciparum infection intensity positively correlates with increased 321 egg production and levels of 20E, where the production of lipids during vitellogenesis are 322 used by P. falciparum to increase survival and optimize transmission [17]. Therefore, the 323 effects of 20E on mosquito immunity and reproductive fitness potentially depend on 324 specific host-pathogen interactions that define An. gambiae immunity to P. berghei and 325 P. falciparum parasites [49,64] that require further study. 326 In summary, our findings demonstrate the role of the hormone, 20E, in priming mosquito 327 innate immunity to both bacteria and malaria parasites. These effects are mediated in 328 part through an activation of cellular immunity and likely involve a number of humoral 329 factors, including that of cecropin 3 described herein, that make mosquitoes more 330 resistant to pathogen infection. As a result, these data provide novel insights into the 331 hormonal regulation of the mosquito immune system, yet require further investigation to 332 better understand the regulation and tissue-specific contributions of 20E immune priming.  Figure 6. Multi-modal effects of blood-feeding and 20E on mosquito reproduction 590 and immunity. Blood-feeding stimulates the production of 20E that can initiate 591 vitellogeneis and influence mosquito fecundity, as well as prime mosquito immune 592 responses that limit bacteria and malaria parasite survival. 593