Hemocyte‐specific FREP13 abrogates the exogenous bacterial population in the hemolymph and promotes midgut endosymbionts in Anopheles stephensi

Abstract

The immune blood cells "hemocytes" of mosquitoes impart a highly selective immune response against various microorganisms/pathogens. Among several immune effectors, fibrinogen-related proteins (FREPs) have been recognized as key modulators of cellular immune responses; however, their physiological relevance has not been investigated in detail. Our ongoing comparative RNAsequencing analysis identified a total of 13 FREPs originating from na€ ıve sugarfed, blood-fed, bacterial challenged and Plasmodium vivax-infected hemocytes in Anopheles stephensi. Transcriptional profiling of the selected seven FREP transcripts showed distinct responses against different pathophysiological conditions, where an exclusive induction of FREP12 after 10 days of P. vivax infection was observed. This represents a possible role of FREP12 in immunity against free circulating sporozoites and needs to be explored in the future. When challenged with live bacterial injection in the thorax, we observed a higher affinity of FREP13 and FREP65 toward Gram-negative and Grampositive bacteria in the mosquito hemocytes, respectively. Furthermore, we observed increased bacterial survival and proliferation, which is likely compromised by the downregulation of TEP1, in FREP13 messenger RNAdepleted mosquito hemolymph. In contrast, after blood-feeding, we also noticed a significant delay of 24 h in the enrichment of gut endosymbionts in the FREP13-silenced mosquitoes. Taken together, we conclude that hemocytespecific FREP13 carries the unique ability of tissue-specific regulation, having an antagonistic antibacterial role in the hemolymph, and an agonistic role against gut endosymbionts.

Molecular Dynamics of Mosquito-Plasmodium vivaxInteraction: A Smart Strategy of Parasitism

“…Novel molecules like FREP12 and FREP50 were predicted to be involved in the clearance of P. vivax sporozoites. Furthermore, storage proteins like ApolipophorinIII, Hexamerin were also found to be highly induced during P. vivax oocysts development, which further supported the previous evidence of host nutrient scavenging by the maturing oocysts [73][74][75].…”

Genetic changes of Plasmodium vivax tempers host tissue-specific responses in Anopheles stephensi

“…3 d). While, in our recent study we further observed that the expression of FREP12 significantly upregulated after 10 days of P. vivax infection, highlighting its possible role in immunity against free circulating sporozoites ( Chauhan et al., 2020 ).…”

A testis-expressing heme peroxidase HPX12 regulates male fertility in the mosquito Anopheles stephensi

“…To knockdown or silence the AsHPX12 mRNA expression, dsRNA primers carrying T7 overhang were synthesized as listed in the Supplemental Table 1 , following the protocols as described earlier 42 , 43 . The amplified PCR product was examined by agarose gel electrophoresis, purified (Thermo Scientific Gene JET PCR Purification Kit #K0701), quantified, and subjected to double-stranded RNA synthesis using Transcript Aid T7 high-yield transcription kit (Cat# K044, Ambion, USA).…”

“…The amplified PCR product was examined by agarose gel electrophoresis, purified (Thermo Scientific Gene JET PCR Purification Kit #K0701), quantified, and subjected to double-stranded RNA synthesis using Transcript Aid T7 high-yield transcription kit (Cat# K044, Ambion, USA). The bacterial dsr LacZ gene was used as a control 42 , 43 . Approximately ~ 69 nl (3 µg/µl) of purified dsRNA product was injected into the thorax of a cold anesthetized 1–2-day old male mosquito using a nano-injector (Drummond Scientific, CA, USA), as described earlier 44 , 45 .…”

“…The bacterial dsr LacZ gene was used as a control 42 , 43 . Approximately ~ 69 nl (3 µg/µl) of purified dsRNA product was injected into the thorax of a cold anesthetized 1–2-day old male mosquito using a nano-injector (Drummond Scientific, CA, USA), as described earlier 44 , 45 . The knockdown of the respective gene was confirmed by quantitative RT-PCR after 3–4-days of dsRNA injection.…”