Characterization and expression analysis of gene encoding heme peroxidase HPX15 in major Indian malaria vector Anopheles stephensi (Diptera: Culicidae)

Silencing of Anopheles stephensi Heme Peroxidase HPX15 Activates Diverse Immune Pathways to Regulate the Growth of Midgut Bacteria

“…Thus, the heavy load of exogenous bacteria in the midgut had no effect on the early phase of AsHPX15 gene expression ( Figure 2 ). This may be simply due to the regulation of this gene by ecdysone hormone, which is induced in blood fed mosquitoes as reported before (Kajla et al, 2016a). We also believed that AsHPX15 catalyzed mucin barrier might be completed or its formation started within the first 12 h of post blood feeding because the presence of mucin and AgHPX15 protein is detected at the luminal surface of A. gambiae midgut epithelium by this time (Kumar et al, 2010; Injera et al, 2013).…”

“…This effect might be due to the SOCS mediated suppression of STAT pathway ( Figure 4 ). The presence of STAT binding site in the regulatory region of AsHPX15 gene supported this assumption (Kajla et al, 2016a). We also believed that the suppression of AsHPX15 gene expression in bacteria fed midguts might be least effective on the mucin barrier formation because the luminal secretion of mucins and localization of HPX15 protein occur by this time, which is well evident in the case of A. gambiae midguts (Kumar et al, 2010; Injera et al, 2013).…”

“…Previously, we found that the heme peroxidase HPX15 gene is highly induced in A. gambiae or A. stephensi blood fed midguts (Kumar et al, 2004; Kajla et al, 2016a). This molecule is important to suppress the immune activation and facilitating the growth of endogenous bacteria in the blood fed midguts (Kumar et al, 2010; Kajla et al, 2015a).…”

“…The expression of various genes (as mentioned in the Table 1 ) was analyzed through qPCR using Sybr Green Master Mix (Biorad Cat no. 170-8882) and normalized against the constitutively expressed S7 ribosomal protein gene as before (Kajla et al, 2016a). The PCR was initiated with denaturation at 95°C for 3 min followed by 35 additional cycles at 94°C for 10 s, 57°C for 30 s and 72°C for 50 s. The final extension was carried at 72°C for 10 min and ΔΔ Ct method was used to calculate the relative expressions of the target genes in the samples against the controls as discussed before (Livak and Schmittgen, 2001).…”

“…In addition, the reduction of Plasmodium development was also evident in these silenced midguts. It is also noteworthy to mention that HPX15 is a unique anopheline-lineage specific gene and its orthologs are absent in insects and human, however, they are present in the genome of nineteen worldwide distributed species of Anopheles mosquito and share 65–99% amino acids identity (Kajla et al, 2015b, 2016a). …”