Aspirin inhibition of prostaglandin synthesis impairs egg development across mosquito taxa

Several endocrine signals are known to mediate mosquito egg development including insulin-like peptide, 20-hydroxyecdysone, and juvenile hormone. The objective of this study was to determine the effects of prostaglandin E2 (PGE2) as an additional mediator of oogenesis in the mosquitoes, Aedes albopictus and Anopheles gambiae. The injection of aspirin (an inhibitor of cyclooxygenase) shortly after blood-feeding significantly inhibited egg development at choriogenesis in a dose-dependent manner in Ae. albopictus. Moreover, oral administration of aspirin to An. albopictus and An. gambiae also inhibited egg production. The aspirin treatment suppressed expression of the genes (Yellow-g and Yellow-g2) associated with exochorion darkening and led to the production of a malformed egg shell in Ae. albopictus. These inhibitory effects of aspirin on egg development were rescued by the addition of PGE2, confirming the specificity of aspirin in inhibiting prostaglandin production. To validate these results, we identified a putative PGE2 receptor (Aa-PGE2R) in Ae. albopictus. Aa-PGE2R expression was highly inducible in adult ovary after blood-feeding. RNA interference of Aa-PGE2R expression resulted in the significant suppression of choriogenesis similar to aspirin treatment, where the addition of PGE2 to Aa-PGE2R-silenced females failed to rescue egg production. Together, these results suggest that PG synthesis and signaling are required for egg development across diverse mosquito taxa. Author Summary Progstaglandins (PGs) play crucial roles in mediating various physiological processes in insects. Aspirin (ASP) inhibits PG biosynthesis and has been used as an anti-inflammatory drug. ASP injection or feeding to mosquitoes of Aedes albopictus or Anopheles gambiae significantly inhibits egg production at chorion formation. This led to significant reduction in fecundity and egg hatchability. PG signal is interrupted by RNA interference (RNAi) of PGE2 receptor. The RNAi treatment also gave a similar damage to females in egg production as seen in ASP treatment. Thus, PG signal is required for egg production of these mosquitoes. Data Availability Statement All relevant data are within the manuscript and its Supporting Information file.

in 10% sucrose solution or 10% sucrose solution (control). After three days female mosquitoes 139 were fed on an anaesthetized female Swiss Webster mouse and continually provided with either 140 sucrose solution before dissection 72 h post BF (PBF).

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Counting oocytes at different developmental stages 143 For oocyte counting, females were allowed to feed blood for 1 h in the afternoon (2-6 pm) at 3 144 days PE. Females were mated with males of the same age after BF and reared with 10% sugar Measurements of mosquito fecundity following aspirin treatment 154 To explore aspirin's effect on mosquito oviposition and fecundity, individual female An. 155 gambiae mosquitoes were transferred to 50 mL conical tubes containing a moistened filter 156 paper at 48 h PBF. Oviposition rate was determined by observing the presence or absence of larvae and one adult both from male and female) of mosquitoes for developmental expression 185 analysis. To analyze adult body parts, females at 8 days PE were used to isolate head, thorax, 186 ovary, and abdomen. To collect total RNAs from blood-fed ovaries, 5 days old females were 187 allowed to feed blood. From these samples, total RNAs were extracted using Trizol reagent 188 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. After RNA 189 extraction, RNA was resuspended in nuclease-free water and quantified using a    For RNAi treatment, 1 g of gene-specific dsRNA in 1 L was injected to 5 days PE females at 10 min before BF. After BF, females were reared with 10% sugar solution. To rescue RNAi-231 treated females, 1 L of PGE 2 (10 g) was injected to females at 12 h after dsRNA injection.

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Control RNAi ('dsCON') was injected at the same concentration. Females at 72 h after BF 233 were used to count the total number of oocytes under a stereo microscope (Stemi SV11, Zeiss, 234 Germany). For fecundity test, three females were mated with one male in a replication. Each 235 treatment was replicated three times. dose-dependent manner. When ASP was injected to females, only 10 ng of ASP per female 288 was required to significantly (P < 0.05) reduce choriogenesis (Fig 3A). At 1 μg of ASP, about 289 80% oocytes failed to be chorionated compared to control. Oral administration of ASP also 290 inhibited choriogenesis in a dose-dependent manner, although its inhibitory effect was much 291 less than that of the injection method (Fig 3B). Even at 1,000 ppm of ASP, only 30% oocytes 292 did not form chorions.

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Aspirin effect on egg production of An. gambiae 295 Similar to the detrimental effects on reproduction and larval hatching by oral administration of 296 ASP in Ae. albopictus, we observed that the administration of ASP at 10 mg/mL by oral feeding 297 in An. gambiae also resulted in the attenuated choriogenesis of the ovarian follicle at 72 h PBF 298 (Fig 4A). ASP feeding significantly reduced the number of chorionated oocytes in An. gambiae 299 as compared to sucrose control (Fig 4B). Moreover, ASP negatively influenced mosquito fecundity, causing a reduction in the number of oviposited eggs (Fig 4C) as well as a decrease 301 in larval hatching (Fig 4D) at 144 h PBF.

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Aspirin suppresses Yellow genes associated with chorion formation 304 To understand a molecular action of ASP to inhibit choriogenesis of mosquitoes, expression of 305 Yellow-g and Yellow-g2, which are required for exochorion darkening and subsequent rigidity 306 in Ae. albopictus [25], was assessed after inhibitor treatment (Fig 5). Most eggs collected from   6). Aa-PGE 2 R encoded 398 amino acid residues and seven transmembrane domains (Fig 6A). 316 Its predicted amino acid sequence was clustered with other EP4 receptors (Fig 6B). Aa-PGE 2 R 317 was expressed in all developmental stages. It was highly expressed in adult females (Fig 7A). 318 In female adults, it was highly expressed in ovary tissue (Fig 7B). Expression of Aa-PGE 2 R in 319 ovaries increased along with increasing number (r = 0.9972; P < 0.0001) of chorionated oocytes 320 (Fig 7C).
Double-stranded RNA specific to Aa-PGE 2 R significantly (P < 0.05) suppressed its expression 324 level when it was injected to teneral female adults (Fig 8A). RNAi-treated adults failed to 325 develop chorionated oocytes as compared to control females (Fig 8B). PGE 2 addition to RNAi-326 treated females did not alter the reduction in choriogenesis.

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In insects, the first report on the physiological role of PG was regarding the egg laying behavior    aspirin: ASP), and LOX inhibitor (naproxen: NAP) were used to treat 5 days old females before 576 blood-feeding ('BF'). At 10 min before BF, the inhibitor was injected to females at a dose of 1 577 g/individual. Dimethylsulfoxide used to dissolve inhibitors was injected as a control ('CON').  ('dsPGE 2 R') or control dsRNA ('dsCON') to 5 days old females at 10 min before blood-652 feeding (BF). Changes in mRNA levels were assessed at 72 h after injecting dsPGE 2 R or 653 dsCON by RT-qPCR using actin as a reference gene to normalize target gene expression level.

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Each treatment was replicated three times. (B) Influence of RNAi on choriogenesis. At 12 h 655 after dsRNA injection, 1 µL of PGE 2 (10 g per female) was injected to RNAi-treated adults.
At 72 h after BF, total number of chorionated oocytes was counted under a stereomicroscope.

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Each treatment used 10 females. Different letters above standard deviation bars indicate 658 significant difference among means at Type I error = 0.05 (LSD test).