E93 confers steroid hormone responsiveness of digestive enzymes to promote blood meal digestion in the midgut of the mosquito Aedes aegypti

Abstract

Anautogenous female mosquitoes obtain the nutrients needed for egg development from vertebrate blood, and consequently they transmit numerous pathogens of devastating human diseases. Digestion of blood proteins into amino acids that are used for energy production, egg maturation and replenishment of maternal reserves is an essential part of the female mosquito reproductive cycle. However, the regulatory mechanisms underlying this process remain largely unknown. Here, we report that the transcription factor E93 is a critical factor promoting blood meal digestion in adult females of the major arboviral vector Aedes aegypti in response to the steroid hormone 20-hydroxyecdysone (20E). E93 was upregulated in the female mosquito midgut after a blood meal, and RNA interference (RNAi)-mediated knockdown of E93 inhibited midgut blood digestion. E93 RNAi depletion repressed late trypsin (LT), serine protease I (SPI), SPVI and SPVII, and activated early trypsin (ET) expression in the female mosquito midgut after a blood meal. Injection of 20E activated E93, LT, SPI, SPVI and SPVII, and repressed ET expression, whereas RNAi knockdown of the ecdysone receptor (EcR) repressed E93, LT, SPI, SPVI and SPVII, and activated ET expression in the midgut. Furthermore, E93 depletion resulted in a complete loss of 20E responsiveness of LT, SPVI and SPVII. Our findings reveal important mechanisms regulating blood meal digestion in disease-transmitting mosquitoes.

Keywords: 20-Hydroxyecdysone; Blood meal digestion; E93; Mosquito; Serine protease; Steroid hormone.

Fig. 1.. E93 is upregulated and essential for blood digestion in the midgut of female mosquitoes after a blood meal.

(A) Relative mRNA levels of E93 in the midgut, fat body, ovary and leftover tissue of female mosquitoes at 72 h PE and at 24 and 48 h PBM. (B) Relative mRNA levels of E93 in the female mosquito midgut analyzed at 72 and 96 h PE and at 6, 24, 36, 48, 72 and 96 h PBM using qPCR. Fold changes are relative to the expression of E93 at 72 h PE, arbitrarily set to 1. Mean ± SEM from three independent experiments. (C) Schematic representation of the study design. (D) Effect of E93 knockdown on blood meal digestion in the midgut observed at 24 and 48 h PBM. Midguts were visualized under the Leica M165FC stereo microscope. Scale bars, 1 mm. Images are representative of three independent experiments, with a total of 40 females analyzed.

Fig. 2.. Knockdown of E93 disturbs midgut SP expression after a blood meal.

(A–G) Relative mRNA levels of SPs in the midgut of iLuc and iE93 female mosquitoes at 6, 24 and 36 h PBM. (H) Trypsin-like activity in the midgut of iLuc and iE93 female mosquitoes at 6, 24 and 36 h PBM. (A–H) Mean ± SEM from three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 (independent-samples t-test).

Fig. 3.. EcR-mediated 20E signaling activates E93 and PBM SP expression in the midgut.

(A and B) Relative mRNA levels of E93 (A) and PBM SPs (B) in the midguts of 20E- or ethanol (solvent)-injected female mosquitoes at 96 h PE. (C and D) Relative mRNA levels of E93 (C) and PBM SPs (D) in the midguts dissected from female mosquitoes at 96 h PE and incubated in medium containing 20E (10 μM) or ethanol (solvent) for 6 h. (A–D) Fold changes are relative to expression in the solvent treatment, arbitrarily set to 1. (E and F) Relative mRNA levels of E93 (E) and PBM SPs (F) in the midguts of iLuc and iEcR female mosquitoes at 6 h (SPI) or 24 h (E93, LT, SPVI and SPVII) PBM. Fold changes are relative to expression in the iLuc control, arbitrarily set to 1. (A–F) Mean ± SEM from three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 (independent-samples t-test).

Fig. 4.. E93 is required for 20E-dependent activation of PBM SP expression in the midgut.

(A) Relative mRNA levels of PBM SPs in the midguts dissected from female mosquitoes at 96 h PE and cultured for 6 h in medium under indicated conditions. Fold changes are relative to expression in the solvent treatment, arbitrarily set to 1. (B) Relative mRNA levels of PBM SPs in the midguts of iLuc and iE93 female mosquitoes after 20E or solvent injection at 96 h PE. Fold changes are relative to expression in the iLuc females after solvent treatment, arbitrarily set to 1. (A and B) Mean ± SEM from three independent experiments; n.s., not significant, *P < 0.05, **P < 0.01 (independent-samples t-test).

Fig. 5.. 20E acts independently of the insulin pathway to activate PBM SP expression.

(A) Relative mRNA levels of PBM SPs in the midguts of bovine insulin- or solvent-injected female mosquitoes at 96 h PE. Fold changes are relative to expression in the solvent-treated animals, arbitrarily set to 1. (B) Relative mRNA levels of PBM SPs in the midguts of iLuc and iInR female mosquitoes at 24 h PBM. Fold changes are relative to expression in the iLuc control, arbitrarily set to 1. (C) Relative mRNA levels of PBM SPs in the midguts of iLuc and iInR female mosquitoes after 20E or solvent injection at 96 h PE. Fold changes are relative to expression in the iLuc females after solvent treatment, arbitrarily set to 1. (A–C) Mean ± SEM from three independent experiments; n.s., not significant, *P < 0.05, **P < 0.01 (independent-samples t-test).

Fig. 6.. The role of JH signaling in regulating PE SP expression.

(A and B) Relative mRNA levels of Hairy (A) and PE SPs (B) in the midguts of methoprene (Meth)- or acetone (solvent)-treated female mosquitoes at 9 h PE. Female mosquitoes were treated topically on the abdomen with Meth (1 ng/insect) or acetone within 1 h PE and dissected at 9 h PE for qPCR analysis. (C and D) Relative mRNA levels of Met (C) and PE SPs (D) in the midguts of iLuc and iMet female mosquitoes at 96 h PE. Fold changes are relative to expression in the iLuc control, arbitrarily set to 1. (E and F) Relative mRNA levels of Hairy (E) and PE SPs (F) in the midguts of Meth- or solvent-treated female mosquitoes at 24 h PBM. Female mosquitoes were treated topically on the abdomen with Meth (10 ng/insect) or acetone within 1 h PBM and dissected at 24 h PBM for qPCR analysis. (A–B and E–F) Fold changes are relative to expression in the solvent-treated animals, arbitrarily set to 1. (A–F) Mean ± SEM from three independent experiments; n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001 (independent-samples t-test).

Fig. 7.. The role of 20E signaling in regulating PE SP expression.

(A) Relative mRNA levels of PE SPs in the midguts of 20E- or ethanol (solvent)-injected female mosquitoes at 96 h PE. Fold changes are relative to expression in the solvent-treated animals, arbitrarily set to 1. (B) Relative mRNA levels of PE SPs in the midguts of iLuc and iEcR female mosquitoes at 24 h PBM. Fold changes are relative to expression in the iLuc control, arbitrarily set to 1. (A and B) Mean ± SEM from three independent experiments; n.s., not significant, *P < 0.05 (independent-samples t-test).