Mosquito-specific microRNA-1890 targets the juvenile hormone-regulated serine protease JHA15 in the female mosquito gut

Abstract

Females of the hematophagous mosquito species require a vertebrate blood meal to supply amino acids and other nutrients necessary for egg development, serving as the driving force for the spread of many vector-borne diseases in humans. Blood digestion utilizes both early and late phase serine proteases (SPs) that are differentially regulated at the transcriptional and post-transcriptional level. To uncover the regulatory complexity of SPs in the female mosquito midgut, we investigated involvement of miRNAs in regulating the juvenile hormone (JH)-controlled chymotrypsin-like SP, JHA15. We identified regulatory regions complementary to the mosquito-specific miRNA, miR-1890, within the 3' UTR of JHA15 mRNA. The level of the JHA15 transcript is highest post eclosion and drastically declines post blood meal (PBM), exhibiting an opposite trend to miR-1890 that peaks at 24 h PBM. Depletion of miR-1890 results in defects in blood digestion, ovary development and egg deposition. JHA15 mRNA and protein levels are elevated in female mosquitoes with miR-1890 inhibition. JHA15 RNA interference in the miR-1890 depletion background alleviates miR-1890 depletion phenotypes. The miR-1890 gene is activated by the 20-hydroxyecdysone pathway that involves the ecdysone receptor and the early genes, E74B and Broad Z2. Our study suggests that miR-1890 controls JHA15 mRNA stability in a stage- and tissue- specific manner.

Keywords: Mosquito; digestive protease; ecdysone; juvenile hormone; microRNA.

Figure 1.

JHA15 and miR-1890 display opposing expression profiles. (A) Dual Luciferase Reporter Assay for JHA15. Data represents the percent activity (Δ Fold Activity * 100) average ± SEM of triplicate samples. Percentage shown. (B) Relative expression profile of mature miR-1890 in the female mosquito midgut. Relative expression was analyzed at the following time-points: 0–6, 24, 48 and 72 h post eclosion (PE), and 6, 12, 24, 36, 48 h post blood meal (PBM). (C) Relative expression profile of JHA15 in the female mosquito midgut. Relative expression was analyzed at the following time-points: 0–6, 24, 48 and 72 h post eclosion (PE), and 6, 12, 24, 36, 48 h PBM. (B-C) Data represents 3 biological replicates with 3 technical replicates and are illustrated as average ± SEM.

Figure 2.

miR-1890 depletion results in increased JHA15 levels. (A) Percent relative expression of miR-1890 in the female mosquito midgut 24 h post blood meal (PBM). (B) Mature miR-275 percent relative expression in the female mosquito midgut 24 h PBM. (C) JHA15 transcript levels increases in miR-1890-depleted female mosquito midguts. (A-C) Data represents 3 biological replicates with 3 technical replicates and are illustrated as average ± SEM, * P< 0.05; ** P < 0.01. (D) Western blot analyses utilizing antibodies against JHA15 in 1890Ant-treated, MsAnt-treated and non-injected female mosquito midguts. Beta-actin was used as a loading control.

Figure 3.

miR-1890 depletion results in impaired blood digestion and egg development (A-C) Midguts 24 h post blood meal (PBM) of (A) miR-1890 antagomir (1890Ant)-treated, (B) MsAnt-treated, and (C) non-injected females. Images (A-C) obtained under the same conditions using the Leica M165FC stereo microscope. (D) Female mosquito ovaries 24 h PBM. Ovaries were visualized using the Leica M165FC stereo microscope. (E) Average follicle size of 1890Ant-treated, MsAnt-treated and non-injected mosquitoes 24 h PBM. Measurements were made using the Leica M165FC stereo microscope. (F) Egg numbers per female mosquito for 1890Ant-treated, MsAnt-treated and non-injected mosquitoes. (E-F) Data represents 3 biological replicates with 3 technical replicates and are illustrated as average ± SEM, * P< 0.05; ** P < 0.01.

Figure 4.

JHA15 RNAi partially rescues miR-1890 depletion phenotypes. (A) JHA15 RNAi alleviated ovary development phenotype in miR-1890 antagomir (1890Ant)-treated female mosquitoes. (B) JHA15 RNAi alleviated egg deposition phenotype in miR-1890Ant-treated female mosquitoes. Data represents 3 biological replicates with 3 technical replicates and are illustrated as average ± SEM, *P< 0.05; **P < 0.01.

Figure 5.

miR-1890 responds to 20E, while JHA15 is regulated by JH. (A) Mature miR-1890 expression from in vitro midgut. Midguts were isolated from non-blood fed female mosquitoes 72 post eclosion (PE) and incubated under indicated conditions in vitro for 6 h. (B) Mature miR-1890 expression in vivo in Ecdysone Receptor (EcR) and Insulin Receptor (iInR) RNAi treatments 24 h post blood meal (PBM). (C) Mature miR-1890 expression in vivo in E74B and Broad RNAi treatments PBM. (D) JHA15 expression in vivo in Methoprene Tolerant (iMet) RNAi-treated non-blood fed female mosquitoes. (B-D) RNAi for Luciferase (iLuc) served as a control. (A-D) Data represents 3 biological replicates with 3 technical replicates and are illustrated as average ± SEM, * P< 0.05; ** P < 0.01; *** P < 0.001.