DHA gavage in mice prevented P. yoelii infection in An. stephensi
To understand whether taking blood from malaria patients treated with artemisinin drugs affects the development of malaria parasites in Anopheles, Plasmodium-infected mice were orally administered DHA or physiological saline via gavage. Anopheles were allowed to feed on DHA or control mice 1.5 h after gavage (Fig. 1A). Oocysts were counted 8 days after infection. Infection intensities and rates were compared between the two groups. The results showed that the infection intensity of DHA group was significantly lower than that of the control group (P = 0.046) (Fig. 1B and C) but no significant difference was found in infection rates (χ2 = 2.456, df = 1, P = 0.117). These results suggest that taking blood from DHA-treated mice inhibited Plasmodium development in An. stephensi, indicating a reduced susceptibility of Anopheles to Plasmodium.
Blood-feeding in vitro with DHA inhibited P. yoelii infection in An. stephensi
To exclude the effect of DHA on gametophytes in mice, which might affect the development of parasites in mosquitoes, a blood feeding in vitro method was used. The blood of P. yoelii infected mice was obtained and added with DHA to a final concentration of 50 µM. The mosquitoes were then fed blood and the oocysts were counted eight days after infection (Fig. 2A). The comparison of infection intensities and rates showed that the oocyst counts in the DHA group were significantly lower than those in the control group (P = 0.016) (Fig. 2B and C) and there was no significant difference in infection rates (χ2 = 0.149, df = 1, P = 0.699). This indicates that in vitro blood feeding with DHA inhibited the development of oocysts in An. stephensi.
Feeding An. stephensi with DHA containing sugar inhibited P. yoelii oocyst development
To eliminate the influence of DHA metabolites on parasite development in the mosquitoes, An. stephensi were challenged with P. yoelii after feeding them a sugar solution containing DHA or DMSO for 3 consecutive days (Fig. 3A). Engorged females were dissected 8 days after infection and the oocysts were counted. Infection rates and intensities were compared between the two groups. The results showed that the infection intensity in the DHA group was significantly lower than that in the control group (P = 0.002) (Fig. 3B and C). There was no significant difference in the infection rates between the control group and the DHA treatment group (χ2 = 0.523, df = 1, P = 0.469) (Fig. 3D). These results indicate that direct feeding of DHA inhibited the development of malaria parasites in mosquitoes, which might decrease the malarial vector competence of An. stephensi.
Toll signaling pathway was essential in DHA-mediated inhibition of P. yoelii development in An. stephensi
Thioester-containing protein 1 (TEP1) plays a key role in killing Plasmodium parasites by forming complexes with the complement system of leucine-rich repeat proteins (LRIM1 and APL1) [20]. To understand whether these three molecules are involved in the inhibitory effect of DHA on Plasmodium development in Anopheles, the transcriptional levels of TEP1, LRIM, and APL1 were measured. The results showed that TEP1, LRIM1, and APL1C were significantly increased by DHA treatment at 24 and 72 h post-infection (hpi) compared with those in the control group (Fig. 4A). As the expression of TEP1 can be regulated by the Toll signaling pathway [21], some key molecules of the Toll signaling pathway were detected using quantitative PCR. The results showed that DHA treatment triggered the upregulation of myeloid differentiation primary-response gene 88(MyD88), Tube, and the NF-κB transcription factors Rel1 at 24 or 72 hpi and inhibited factor Cactus down-regulation at 24 hpi compared with the control group (Fig. 4A). These results suggest that the Toll signaling pathway was activated in DHA-treated mosquitoes. Activation of the Toll pathway allows Rel1 to enter the nucleus and induces the expression of antimicrobial peptide (AMP) genes such as DEF1 [22–23]. To further confirm that DHA can activate the Toll signaling pathway, the transcriptional level of DEF1 was determined using qPCR. The expression of DEF1 was upregulated by DHA treatment at 24 and 72 hpi compared with that in the control group (Fig. 4A). To better reflect the expression levels, western blotting was conducted to detect the proteins TEP1 and Rel1 in mosquitoes. Both proteins were upregulated by DHA treatment at 24 and 72 hpi compared with those in the control group (Fig. 4B). Overall, these results indicate that DHA treatment could induce high expression of TEP1 and AMP by activating the Toll signaling pathway, effectively impairing oocyst development and inhibiting the susceptibility of An. stephensi to P. yoelii.
Vector competence of An. stephensi was recovered by interference of Rel1 expression
To confirm the role of the Toll signaling pathway in the effect of DHA treatment on P. yollii development in Anopheles, the expression of Rel1, a key molecule in the Toll signaling pathway, was silenced using RNAi. The knockdown efficiency of Rel1 and expression levels of TEP1 were examined by qPCR. The silencing efficiency of Rel1 was 62.8% and 47.4% in the control and DHA-treated groups, respectively (Fig. 5A). Meanwhile, DHA treatment led to a significant increase in the expression of TEP1 compared to the untreated group in dsGFP mosquitoes, whereas the increase was eliminated when Rel1 was silenced by RNAi (Fig. 5B). These results indicate that changes in TEP1 transcription levels induced by DHA are regulated by Rel1. To further ascertain the role of Rel1 in the DHA-induced anti-Plasmodium defense against P. yoelii infection, we measured oocyst development after Rel1 RNAi in An. stephensi. Oocyst counts were compared between the dsRel1 and dsGFP groups in the presence or absence of DHA. There were more oocysts in the dsRel1 group than in the dsGFP group, whereas the inhibitory effect of DHA on oocyst development disappeared when Rel1 was knocked down by RNAi (Fig. 5C, D). In summary, these results indicate that the activation of the Toll signaling pathway by DHA significantly enhances the defense of An. stephensi against malaria parasites.