Mosquito colony was maintained in insectory at 28°C, 80% relative humidity, and 12 h light–dark cycle as described before (12). Larvae were fed on a 1:1 mixture of dog food (PetLover’s crunch milk biscuit, India) and fish food (Gold Tokyo, India) as before (11, 12). Adult mosquitoes were fed on 10% sugar solution ad libitum. For colony propagation, 3–4 days old, starved females were fed on anesthetized mice. The eggs laid by these blood-fed (BF) females were collected in moist condition, and the hatched larvae were floated in water to continue the cycle.

The transgenic P. berghei ANKA strain that expresses GFP (PbGFP) in all developmental stages (13) was a gift from Dr. Agam Prasad Singh, National Institute of Immunology, New Delhi, India. The Plasmodium strain was maintained in Swiss albino mice following the standard protocols as before (6, 14, 15). The parasitemia of the infected mice was determined from Giemsa-stained blood smears as mentioned before (14, 15). For blood stage passages, 100–150 μl of blood from an infected mouse (containing ~10–15% parasitemia) was injected intraperitoneal into the healthy mice. Parasitemia and potential infectivity of the Plasmodium to the mosquitoes was determined by exflagellation assays as before (16). In brief, 2 μl blood from the tail of the infected mouse was mixed with 20 μl of exflagellation buffer that was prepared by mixing equal parts of solution A (10mM Tris-Cl, 150mM NaCl, and 10mM glucose, pH 8) and heat-inactivated fetal bovine serum. In all the experiments, mice containing ~5–7% parasitemia and 2–3 exflagellations per field under 40× objective were used to infect the mosquitoes.

The 4- to 5-day-old and overnight-starved 200 female mosquitoes were allowed to feed on an anesthetized Swiss albino mouse infected with the GFP expressing P. berghei. The mosquitoes fed on an uninfected mouse served as control. The unfed mosquitoes were removed and only fully engorged females were maintained at 21°C and 80% humidity, the permissive conditions for P. berghei development as discussed before (6, 11, 15, 17). Midguts samples were collected from 20 mosquitoes at different time points (3, 6, 12, 18, and 24 h) as mentioned below.

A 218-bp fragment of the lacZ gene was amplified using the following primers (5′ to 3′) Fw-GAGTCAGTGAGCGAGGAAGC and Rev-TATCCGCTCACAATTCCACA and cloned into the pCRII-TOPO vector as before (18). In parallel, a 428-bp cDNA fragment of AsHPX15 gene that reported in our previous publication (10) was also cloned in the same vector. These recombinant plasmids were used individually as a template for in vitro transcription. The recombinant plasmid already had a T7 promoter site at M13F primer end, thus, a T7 promoter site was added to the other end of the fragment through amplifying M13R primer. The sequences (5′-3′) for these primers are following: M13F-GTAAAACGACGGCCAGT and T7-M13R-CTCGAGTAATACGACTCACTATAGGGCAGGAAACAGCTATGAC. PCR amplification using M13F and M13R primers was started with 94°C for 5 min followed by 40 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, and final extension was carried at 72°C for 10 min. Amplicons were purified with the QIA quick PCR purification kit (Qiagen, Valencia, CA, USA). PCR-purified amplicons tailed with T7 promoter sequences were used to synthesize dsRNAs with the MEGAscript kit (Cat No. AM1626, Ambion, Austin, TX, USA) following the manufacturer’s instructions. dsRNA was further purified using a Microcon YM-100 filter (Millipore) and finally concentrated to 3 μg/μl in DNase and RNase free water.

The 1- to 2-day-old female mosquitoes were injected with 69 nl of 3 μg/μl dsAsHPX15 RNA (finally 207 ng/mosquito). Control mosquitoes were injected with dsLacZ RNA in the same manner. The gene silencing efficiency of the method was analyzed in sugar fed (SF), blood fed (BF), and P. berghei-infected midguts through qPCR against the respective controls. In these samples, on an average, we could achieve 98% silencing. To evaluate the effect of gene silencing on Plasmodium development, 4 days post dsRNA injected females were fed on transgenic GFP-P. berghei-infected mice, and the number of oocysts per midgut was determined after 7 days post infection. Furthermore, the midguts were dissected in Ashburner’s PBS (3mM sodium chloride, 7mM disodium hydrogen phosphate, and 3mM sodium dihydrogen phosphate, pH 7.2), fixed for 15 min with 4% paraformaldehyde, washed thrice in PBS, and mounted on glass slides in Vectashield mounting medium. The numbers of green fluorescent oocysts were counted in each midgut under a fluorescent microscope (Olympus).

Midguts from 20 uninfected or P. berghei-infected blood fed mosquitoes were dissected at different time points (3, 6, 12, 18, and 24 h) after the blood feeding. The dissected midguts or carcasses (rest of the body except midgut) were kept in RNAlater solution (Qiagen) and stored at −80°C. The midguts from randomly collected 20 SF mosquitoes were also dissected in similar way.

Total RNA from the midgut samples was isolated using RNeasy Mini Kit (Qiagen Cat no. 74104) following manufacturer’s instructions. First-strand cDNA was synthesized from total RNA using QuantiTect Reverse Transcription kit (Qiagen Cat no. 205311). Expression profile of different genes was carried through semiquantitative real-time PCR using SYBR Green supermix in a CFX Connect™ real-time PCR detection system (Bio-Rad) where ribosomal protein subunit S7 mRNA was used as internal loading control for normalization as described before (11, 19). The PCR primer pairs used for different genes are mentioned in Table 1. The PCR started with an initial denaturation at 95°C for 3 min and followed by 35 additional cycles at 94°C for 10 s, 57°C for 30 s, and 72°C for 50 s. Fluorescence for the PCR products was read at 72°C after each cycle. A final extension at 72°C for 10 min was completed before deriving a melting curve. Each experiment was performed in three independent biological replicates. Relative mRNA levels were calculated using ^ΔΔC_t method by dividing the technical replicates in the control or test groups by the mean of the control group. This adjusted the control groups to a value of 1.0 as discussed before (6, 20–22).

Statistical analysis was performed using GraphPad Prism 3.0 software (25). All these data were expressed as mean ± SD. Differences between test samples and their respective controls were evaluated by unpaired Student’s t-test and considered to be significant if the p-Value was less than 0.05. Each experiment was performed at least thrice to validate the findings.

To understand the organ-specific expression of AsHPX15 gene, we compared its relative mRNA levels in different body compartments. For that, we collected 24 h post uninfected or P. berghei-infected BF midguts and carcasses separately from 20 mosquitoes and analyzed the mRNA levels of AsHPX15 gene. We selected the 24 h post fed samples because it corresponds to the time when ookinetes invade the midgut epithelium (1, 6). Results shown in Figure 1 revealed that in SF midguts the basal levels of AsHPX15 mRNA were ~40-fold higher than carcasses. Furthermore, its expression in 24 h BF midguts was induced ~17-folds against SF midguts (Figure 1). However, the expression of AsHPX15 gene was downregulated approximately twofold in P. berghei-infected midguts when compared to the uninfected BF controls (p = 0.008, Figure 1). Interestingly, the relative mRNA levels of AsHPX15 gene were indifferent in the carcass of SF or uninfected BF mosquitoes and also remained unaffected after Plasmodium infection (Figure 1). From these results, we concluded that AsHPX15 is a blood induced midgut-specific gene and suppressed during the Plasmodium ookinete invasion of the midgut epithelium. These findings are in agreement with the previous reports where A. gambiae AgHPX15 gene, an ortholog of AsHPX15, is induced in BF midguts and negatively regulated after malaria parasite infection (6, 26).

In parallel, we also analyzed the kinetics of AsHPX15 expression in P. berghei-infected midguts to understand its regulation during various stages of malaria parasite development. For that, the control or P. berghei-infected BF midguts were collected at different time points (3, 6, 12, 18, and 24 h) after the feeding and expression levels of AsHPX15 gene were analyzed through qPCR. Results presented in Figure 2 revealed that the relative mRNA levels of AsHPX15 gene were similar in control and infected midguts for first 12 h (p = 0.328, p = 0.091, and p = 0.945 at 3, 6, and 12 h post blood feeding, respectively). However, there was a significant reduction of AsHPX15 mRNA levels in P. berghei-infected midguts at 18 h (p < 0.0001) and 24 h (p = 0.0054) post feeding when compared to the BF controls (Figure 2). These results indicated that the expression of AsHPX15 gene remains unaffected during the initial hours (up to 12 h) in infected midguts when the pre-ookinete stages of Plasmodium development predominate in the blood bolus (1). However, at later time points, the expression of this gene is suppressed when the mature ookinetes start invading the midgut epithelium (Figure 2).

Our previous findings revealed that A. gambiae heme peroxidase AgHPX15 catalyzes the cross-linking of a mucin layer at the luminal side of the midgut epithelium (6). This cross-linked mucin barrier does not allow the bolus antigens, especially, the naturally acquired microbes, to interact with the immune reactive midgut epithelium that subsequently suppresses the activation of innate immunity in this body compartment (6, 7). Thus, we hypothesized that the reduction of AsHPX15 mRNA levels through gene-silencing approaches might accelerate the recognition and killing of Plasmodium by the mosquito innate immunity. To test these assumptions, we injected a group of mosquitoes with dsLacZ (controls) or dsAsHPX15 (silenced) RNA and, after 5 days, these mosquitoes were fed on P. berghei-infected mice. After 24 h post infected blood feeding, our analysis revealed 98% reduction of AsHPX15 mRNA levels in silenced midguts when compared to the sham-treated controls (Figure 3A).

Subsequently, we counted the number of developing oocysts in the above-mentioned control and silenced midguts after 7 days post blood feeding. Results presented in Figure 3B revealed that the variable number of oocysts were observed in controls (oocysts range 0–1,800) and silenced (oocysts range 0–600) midguts. However, the median value for the oocysts numbers in controls and AsHPX15 silenced midguts was 329 and 89, respectively. This indicated that the numbers of developing oocysts were reduced significantly in the silenced midguts against controls (Figure 3B, p = 0.027). Based on these data, we concluded that AsHPX15 is a natural agonist that positively regulates Plasmodium development inside the mosquito midgut. These findings are in agreement with the previous reports where silencing of the AgHPX15 gene in A. gambiae also reduced Plasmodium survival (6).

The development of Plasmodium was suppressed in AsHPX15-silenced midguts (Figure 3). We assumed that the induction of an antiplasmodial immunity in the silenced midgut might be responsible for the negative regulation of Plasmodium development. This may be simply due to the reduced or defective cross-linking of AsHPX15-mediated mucin barrier that, in turn, allowed the interaction and recognition of parasites by the immune reactive midgut epithelium. These assumptions were confirmed through expression analysis of various known antiplasmodial genes (6, 27) in the silenced and P. berghei-infected BF midguts that are mentioned in Figure 3A. Our analyses revealed that the relative mRNA levels of thioester-containing protein 1 (TEP1), an antiplasmodial immune gene (27), were similar in controls and dsHPX15-injected (silenced) mosquito midguts (Figure 4, p = 0.106). In addition, the relative levels of TEP1 mRNA were also indifferent between uninfected or P. berghei-infected BF midguts (Figure 5, p = 0.091).

Furthermore, the comparative analysis of another antiplasmodial immune gene nitric oxide synthase (NOS) in above samples revealed that this gene was induced ~13-folds in the silenced midguts against the controls (Figure 4, p = 0.005). These findings suggested that the induced NOS is most probably playing an antiplasmodial role in HPX15 silenced midguts as reported before (6, 28, 29). NOS catalyzed the formation of nitric oxide, a highly diffusable and reactive immune molecule that modifies and inactivates the biological marcomolecules (23). In mosquitoes, the induction of NOS gene is regulated by the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. Previous studies revealed that the suppressor of cytokinin signaling (SOCS) is also induced in parallel to the NOS and regulates the over activation of JAK/STAT pathway (6, 15, 28, 29). Thus, we also analyzed the induction of SOCS gene in the above-mentioned AsHPX15 silenced midguts to understand the activation of NOS through JAK/STAT pathway. Results presented in Figure 4 revealed that the SOCS gene was induced approximately twofold in the silenced midguts against controls (p = 0.001). Thus, the induction of NOS, an effector gene, and SOCS, a suppressor gene, indicated that the activation of JAK/STAT pathway might regulate antiplasmodial immunity in AsHPX15 silenced midguts. In addition, both NOS and SOCS genes were also induced twofold and eightfold, respectively in Plasmodium-infected unsilenced midguts when compared to the BF controls (Figure 5, p = 0.011 for NOS and p < 0.0001 for SOCS). These results collectively indicated that, in general, the JAK/STAT pathway is induced in Plasmodium-infected midguts (Figure 5). However, an additional induction of the same pathway in HPX15-silenced midguts seems to be the major regulator of Plasmodium development (Figure 4).

In conclusion, the absence of HPX15-catalyzed mucin barrier in infected midgut provides a better opportunity for the innate immune system to recognize the malaria parasite that, in turn, activates JAK/STAT pathway to regulate Plasmodium development negatively.