The phylogenetic analysis of glucose transporters in Anopheles stephensi
There are three genes annotated as glucose transporter, ASTE005839, ASTE003001 and ASTE008063 in the database of An. stephensi gene (AsteS1.6). To investigate the phylogenetic relationships of these proteins between An. stephensi and other organisms, a phylogenetic tree was constructed based on the amino acid sequence of known glucose transporters from Anopheles gambiae, Anopheles stephensi, Aedes aegypti, Drosophila melanogaster and Homo sapiens by the maximum likelihood and Bayesian phylogenetic analyses (Fig. 1). The family of GLUT transporters known in humans 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)[29, 30, 31, 32]. Amino acid sequence of ASTE005839 has the greatest similarity to the GLUT1 of Drosophila melanogaster (FBpp0305693) and Homo sapiens (NP 006507.2) that belong to class1. We herein named it Asteglut1. While ASTE008063 was categorized into GLUT- class3, and ASTE003001 was not phylogenetically close to GLUT- class1 or GLUT- class 2, so we named it Asteglut3 and Asteglutx, respectively (Fig 1).
Expression pattern of Astegluts in An. stephensi
To determine the expression pattern of Astegluts in An. stephensi. We analyzed the expression level of these genes in the midgut and carcass 24 h before and after infectious blood meal by qPCR, respectively. Asteglut1 and Asteglut3 were mainly localized in the midgut tissue of An. stephensi (Fig. 2A and 2C). Asteglutx was distributed in both midgut and carcass (Fig. 2B). Infectious blood meal stimulated the expression of all three genes (Fig. 2A to C).
Knockdown of Asteglut1 facilitates Plasmodium berghei infection in An. stephensi
To investigate the role of Asteglut1, Asteglutx and Asteglut3 in the susceptibility of A. stephensi to P. berghei, double-stranded RNA (dsRNA)-mediated silencing was employed. The expression level of dsAsteglut1, dsAsteglutx and dsAsteglut3 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) and 65% (P=0.0002) comparing to dsGFP control, respectively (Fig 3A to C). Silencing Asteglut1 let to a significant increase in the number of oocysts of P. berghei, while dsAsteglutx and dsAsteglut3 treatments had no apparent effect on malaria infection (Fig 3D to F). Such effect was due to the specific knockdown of Asteglut1 instead of the compensatory expression of other Astegluts because silencing Asteglut1 didn’t change the expression level of either Asteglutx or AteGlut3 (Fig 3G & H).
Role of Asteglut1 in maintaining mosquito hemolymph glucose homeostasis
We next analyzed the influence of Asteglut1 on glucose level 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 before blood-feeding (Fig. 4A). However, its level in hemolymph is comparable to that in dsGFP (Fig. 4C). Knocking down of Asteglut1didn’t change the level trehalose either in midgut or in hemolymph (Fig. 4B&D). Thus, Asteglut1might play a role in maintenance of glucose homeostasis in mosquito midgut.
Transcriptional analysis of Asteglut1-knockdown mosquitoes
To explore the mechanism that could be responsible for the influence on P. berghei infection, we performed a transcriptome analysis of mosquito’s midgut microinjected with dsAsteglut1 and dsGFP 24 h post blood-meal, respectively. 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, Table S1) with 26 up-regulated and 20 down-regulated. These differentially expressed products were belong to the 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 mosquito [33]. Gene encoding peroxiredoxin was significantly up-regulated, peroxiredoxin is known to control cytokine-induced peroxide levels in cells [34]. However, DUOX, which has been widely reported to be associated with Plasmodium elimination, showed significant down-regulated expression [35].
A cluster containing numerous differentially expressed genes is belong to ‘proteolysis’ in dsAsteglut1 treated mosquitoes. The class of serine proteases, also known as CLIP family, was considered to be involved in immune response, among them two genes were found to be up-regulated while two were down-regulated. The down-regulated transcript genes under this GO term was the CLIPB2 and CLIPB19, while the upregulated gene was CLIPB3 [36, 37]. Two genes encoding allantoinase (ASTE005468 and ASTE014323) were significantly up-regulated, and these genes are involved in the process of nitrogenous waste in mosquitoes [38].
In the cluster associated with the immune response, caudal, the negative regulator of Imd pathway was significantly up-regulated [39]. There were 4 genes related to innate immune responses in mosquitoes, the peptidoglycan recognition proteins, pgrp-la, -lc, -ld, and the antimicrobial peptides, defensin were significantly down-regulated [23, 39, 40, 41, 42].