A zone-of-inhibition assay to screen for humoral antimicrobial activity in mosquito hemolymph

Abstract

In insects, antibacterial immunity largely depends on the activation of downstream signaling and effector responses, leading to the synthesis and secretion of soluble effector molecules, such as antimicrobial peptides (AMPs). AMPs are acute infection response peptides secreted into the hemolymph upon bacterial stimulation. The transcription of innate immunity genes encoding for AMPs is highly dependent on several signaling cascade pathways, such as the Toll pathway. In the African malaria mosquito, Anopheles gambiae, AMPs hold a special interest as their upregulation have been shown to limit the growth of malaria parasites, bacteria, and fungi. Most of the current knowledge on the regulation of insect AMPs in microbial infection have been obtained from Drosophila. However, largely due to the lack of convenient assays, the regulation of antimicrobial activity in mosquito hemolymph is still not completely understood. In this study, we report a zone of inhibition assay to identify the contribution of AMPs and components of the Toll pathway to the antimicrobial activity of A. gambiae hemolymph. As a proof of principle, we demonstrate that Micrococcus luteus challenge induces antimicrobial activity in the adult female mosquito hemolymph, which is largely dependent on defensin 1. Moreover, by using RNAi to silence Cactus, REL1, and MyD88, we showed that Cactus kd induces antimicrobial activity in the mosquito hemolymph, whereas the antimicrobial activity in REL1 kd and MyD88 kd is reduced after challenge. Finally, while injection itself is not sufficient to induce antimicrobial activity, our results show that it primes the response to bacterial challenge. Our study provides information that increases our knowledge of the regulation of antimicrobial activity in response to microbial infections in mosquitoes. Furthermore, this assay represents an ex vivo medium throughput assay that can be used to determine the upstream regulatory elements of antimicrobial activity in A. gambiae hemolymph.

Keywords: ZOI (zone of inhibition); antimicrobial peptide (AMP); defensin 1; innate immunity; mosquito; toll pathway.

Figure 1

The antimicrobial activity of Anopheles gambiae hemolymph is induced by bacterial challenge. (A) Top: M. luteus-seeded plate showing the zone of inhibition (ZOI) produced by mosquito hemolymph collected 24 h after challenge with E coli, S. aureus, and M. luteus, respectively. The hemolymph of uninjected and phosphate-buffered-saline-injected mosquitoes acted as negative controls. Bottom: Drawing of the plate generated in Fiji is Image J to quantify the zone of inhibition (ZOI) for each treatment. (B) Zones of bacterial growth inhibition (mm²) were measured using customized macros in Fiji is Image J (C) The temporal variation of antimicrobial activity induction with M. luteus challenge was determined by collecting hemolymph at 12, 24, 48, 60, and 72 h after challenge. The ZOI was then measured as in (B). In (B) (C), data are shown as means with ±1 standard error (n = 6). One-way ANOVA, followed by Bonferroni’s multiple-comparison post-test, was performed to calculate the statistical significance (P < 0.05). Means with the same letter are not significantly different.

Figure 2

Defensin 1 is mainly responsible for the antimicrobial activity against M. luteus. (A) Top: M. luteus-seeded plate showing the zone of inhibition (ZOI) produced by the hemolymph of dsDEF1- and dsCEC1-injected mosquitoes. The mosquitoes were challenged 48 h after dsRNA injection with E coli, S. aureus, and M. luteus, respectively. Hemolymph was collected 24 h after challenge. Mosquitoes that were uninjected and injected with dsGFP prior to phosphate-buffered saline acted as negative controls. Bottom: Drawing of the plate generated in Fiji is Image J to quantify the ZOI for each treatment. (B) Zones of bacterial growth inhibition (mm²) were measured using customized macros in Fiji is Image J Data are shown as means with ±1 standard error (n = 6). One-way ANOVA, followed by Bonferroni’s multiple-comparison post-test, was performed to calculate the statistical significance (P < 0.05). Means with the same letter are not significantly different.

Figure 3

The antimicrobial activity of Anopheles gambiae hemolymph is regulated by the Toll pathway. (A) Overview of the Toll signal transduction pathway showing the known components that act as positive and negative regulators of the transcription of AMP genes in Anopheles gambiae. (B) Zone of inhibition produced by the hemolymph of dsREL1-injected (B) and dsMyD88-injected (C) mosquitoes. The mosquitoes were challenged 48 h after dsRNA injection with E coli, S. aureus, and M. luteus, respectively. Hemolymph was collected 24 h after challenge. Mosquitoes that were uninjected (UC) and injected with dsGFP prior to phosphate-buffered saline acted as negative controls, whereas those which had dsGFP injection prior to E coli, S. aureus, and M. luteus challenges acted as the corresponding positive controls. (D) The temporal variation of antimicrobial activity induction after CACT kd was determined by collecting hemolymph at 24, 48, 60, and 72 h after dsCACT injection. In (B), (C), and (D), data are shown as means ±1 standard error (n = 6). One-way ANOVA, followed by Bonferroni’s multiple-comparison post-test, was performed to calculate the statistical significance (P < 0.05). Means with the same letter are not significantly different.

Figure 4

DsREL1 injection partially reverses the antimicrobial activity induced by CACT kd. (A) Zone of inhibition (ZOI) produced by the hemolymph of mosquitoes injected with dsREL1/dsGFP, dsCACT/dsGFP, and dsCACT/dsREL1. Uninjected and dsGFP-injected mosquitoes acted as negative controls. (B) ZOI produced by the hemolymph of mosquitoes injected with dsMyD88/dsGFP, dsCACT/dsGFP, and dsCACT/dsMyD88. Uninjected (UC) and dsGFP-injected mosquitoes acted as negative controls. Data are shown as means ±1 standard error (n = 6). One-way ANOVA, followed by Bonferroni’s multiple-comparison post-test, was performed to calculate the statistical significance (P < 0.05). Means with the same letter are not significantly different.

Figure 5

Intrathoracic injection enhances the efficacy of bacterial growth inhibition after bacterial challenge. The graph shows the zone of inhibition (ZOI) produced by the hemolymph of mosquitoes injected with sterile deionized water (H₂O) or dsGFP, followed by phosphate-buffered saline injection, M. luteus challenge only, and H₂O and dsGFP followed by M. luteus challenge. The mosquitoes were challenged 48 h after dsRNA or H₂O injection. The hemolymph of uninjected mosquitoes acted as negative controls. Data are shown as means ±1 standard error (n = 6). One-way ANOVA, followed by Bonferroni’s multiple-comparison post-test, was performed to calculate the statistical significance (P < 0.05). Means with the same letter are not significantly different.