The phylogenetic analysis of glucose transporters in Anopheles stephensi
There are four genes annotated as glucose transporter, ASTE005839, ASTE003001, ASTE006385 and ASTE008063 in the database of An. stephensi (AsteS1.6). To investigate the relationships of these genes between An. stephensi and other organisms, a phylogenetic tree was constructed based on the amino acid sequence of Anopheles gambiae, Anopheles stephensi, Aedes aegypti, Drosophila melanogaster and Homo sapiens by the maximum likelihood and Bayesian phylogenetic analyses (Fig. 1). The humans GLUT transporters can be divided into three classes, class 1 (GLUT1, GLUT2, GLUT3, GLUT4 and GLUT14), class 2 (GLUT5, GLUT7, GLUT9, and GLUT11) and class 3 (GLUT6, GLUT8, GLUT10 and GLUT12)[31, 32, 33, 34]. Due to the high similarity between An. stephensi ASTE005839, Drosophila melanogaster GLUT1 (FBpp0305693) and Homo sapiens GLUT1 (NP 006507.2), we named ASTE005839 Asteglut1. While ASTE008063 and ASTE006385 were categorized into GLUT- class3, so we named them Asteglut3 and Asteglut4, respectively. ASTE003001 was not phylogenetically related to any GLUT- classes, so we named it Asteglutx (Fig 1).
Expression of Astegluts in An. stephensi
To determine the expression pattern of Astegluts in An. stephensi. We analyzed the expression levels of these genes in the head, salivary glands, midgut, ovary and carcass 24 hours before blood meal by qPCR, respectively. Asteglut1, Asteglut3 and Asteglut4 were mainly localized in the midgut tissue of An. stephensi (Fig. 2A, C, D). In addition to the midgut, Asteglut1 and Asteglut4 were also expressed in the head and salivary glands (Fig. 2A, D). Asteglutx was distributed in all five tissues (Fig. 2B). We next investigated the influence of parasite infection on the four Astegluts expression in the midgut. Astegluts were differentially regulated by P. berghei 24 hours post infection. (Fig. 2E). P. berghei infection significantly decreased the expression of Asteglut1 and Asteglut4, while increased the expression of Asteglutx compared to those in normal blood feeding mosquitoes. No influence on Asteglut3 expression was observed during parasite infection (Fig. 2E).
Knockdown of Asteglut1 facilitates Plasmodium berghei infection in An. stephensi
To investigate the role of Asteglut1, Asteglutx, Asteglut3 and Asteglut4 in the capability of An. stephensi to transmit P. berghei, double-stranded RNA (dsRNA)-mediated silencing strategy was employed. The expression level of dsAsteglut1, dsAsteglutx, dsAsteglut3 and dsAsteglut4 was examined two days post dsRNA treatment. The expression level of these genes were significantly decreased by 57.8% (P=0.02), 40% (P<0.0001), 65% (P=0.0002) and 80% (P=0.0013) compared to dsGFP control, respectively (Fig 3A, B, C, D). However, only knockdown of Asteglut1 significantly increased oocysts number of P. berghei. The dsAsteglutx, dsAsteglut3 and dsAsteglut4 treatments had no apparent effect on the intensity of P. berghei infection (Fig 3F, G, H). No significant difference of infection prevalence was observed between dsGFP and any dsAsteglut treated mosquitoes (Fig 3E, F, G, H). We next analyzed the knocking down specificity of Asteglut1 and found this gene was knocked down specifically (Fig. 3I). Thus, the increasing susceptibility of An. stephensi to P. berghei infection was due to the knocking down of Asteglut1, instead of the compensatory effects of other Astegluts (Fig 3I).
Knockdown of Asteglut1 significantly elevates glucose level in mosquito midgut
We next analyzed the influence of Asteglut1 on sugar transportation in An. stephensi. The glucose and trehalose levels in the midgut and hemolymph of dsRNA treated mosquitoes were examined. The glucose level of Asteglut1-knockdown group was significantly higher than that in dsGFP controls 24 hours prior to blood-feeding (Fig. 4A). However, its level in hemolymph is comparable to that in dsGFP (Fig. 4C). There was no significant difference in sugar levels in the midgut or hemolymph either right before (0 hour) or 24 hours post blood-feeding (Fig 4). Knocking down of Asteglut1 didn’t change the level of trehalose either in the midgut or in hemolymph (Fig. 4B, D). Thus, Asteglut1 might play a role in transportation of glucose but not trehalose in mosquito midgut.
Transcriptional analysis of Asteglut1-knockdown mosquitoes
To explore how Asteglut1 regulated P. berghei infection, we performed a transcriptome analysis of mosquito’s midgut treated with dsAsteglut1 and dsGFP 24 hours post blood-meal, respectively. A total of 6 G PE clean sequences was generated by Illumina HiSeq ´10 (Additional file 1: Table. S1). Principal Component Analysis (PCA) showed a clear separation between dsAsteglut1 and dsGFP treatents (Additional file 2: Fig. S1). The Venn diagram shows that the expression of 10240 genes were overlapped in the two groups (Fig. 5A). A total 46 genes were differentially expressed (Fig. 5B, Additional file 3: Table. S2) with 26 up-regulated and 20 down-regulated. These differentially expressed genes were belong to the multiple functional clusters including cytoskeletal and structural, immunity, metabolism, proteolysis, redox, transport and unknown function (Fig. 5C).
Among the ‘redox’ functional cluster, five genes encoding cytochrome P450 (CYP450) were upregulated, indicating that the detoxification mechanism was activated in mosquitoes [35]. Gene encoding peroxiredoxin that controls cytokine-induced peroxide levels in mammalian cells was also significantly up-regulated, but the role of this gene in parasite control in mosquitoes is still unknown. [36]. We also observed DUOX that is involved in Plasmodium elimination significant down-regulated in dsAsteglut1 treated mosquitoes [37]. It is highly possible that the reduction of DUOX expression might render mosquitoes more permissive to P. berghei infection.
The CLIP family are involved in the melanization of P. berghei in An. gambiae [38]. Two CLIP genes, clip2 and clip9, were significantly down regulated in dsAsteglut1 treated mosquitoes compared to dsGFP ones, while clipb3 was upregulated [38, 39]. We next examined whether the increasing parasite infection could be due to the dysregulation in mosquito melanization. Midguts of mosquitoes treated with dsRNA 8 days post infection were collected and melanization was visualized microscopically. We found that the number of melanized ookinete increased with the number of oocysts (Fig. 5D). Thus there was no significant difference in the melanization rate between dsAsteglut1 and dsGFP group.
Five immune related genes were differentially regulated. Caudal, the negative regulator of Imd pathway was significantly up-regulated [40], while the peptidoglycan recognition proteins, pgrp-la, -lc, -ld, and the antimicrobial peptides, defensin were significantly down-regulated [25, 40, 41, 42, 43]. These results indicate that AsteGlut1 might control parasite infection by regulating mosquito immune responses.