Functional disruption of Transferrin expression alters reproductive physiologyin Anopheles culicifacies

Abstract

Iron metabolism is crucial to maintain optimal physiological homeostasis of every organism and any alteration of the iron concentration (i.e. deficit or excess) can have adverse consequences. Transferrins are glycoproteins that play important role in iron transportation and have been widely characterized in vertebrates, and insects, but poorly studied in blood-feeding mosquitoes. Here, we characterized a 2102 bp long transcript AcTrf1a encoding putative transferrin homolog protein from mosquito An. culicifacies. A detailed in silico analysis predicts AcTrf1a (ACUA023913-RA) encodes 624 amino acid (aa) long polypeptide that carries transferrin domain. AcTrf1a also showed a putative N-linked glycosylation site, a characteristic feature of most of the mammalian transferrins and certain non-blood feeding insects. Structure modelling prediction confers the presence of an iron binding site at the N-terminal lobe of the transferrin. Our spatial and temporal expression analysis under altered pathophysiological conditions showed that AcTrf1a abundantly express in the fat-body, ovary, and its response is significantly altered (enhanced) after blood meal uptake, and exogenous bacterial challenge. Additionally, a non-heme iron supplementation of FeCl3 at 1 mM concentration not only augmented the AcTrf1a transcript expression in fat-body, also enhanced the reproductive fecundity of gravid adult female mosquitoes. RNAi mediated knockdown of AcTrf1a causes a significant reduction in the egg laying/fecundity, confirmed important role of transferrin in oocyte maturation. Further detailed characterization may help to select this transcript as a unique target to impair the mosquito reproductive outcome. HighlightsO_LIInsect transferrins are mostly glycoprotein of about 60-80 kDa molecular weight, involved in myriad physiological events and serve as a major iron transport protein. C_LIO_LIHere, we identified and characterized a 2102 bp long transcript encoding putative transferrin homolog of 624 aa long peptide, carrying only one fully functional transferrin domain at N-terminal from An. culicifacies. C_LIO_LISpatial and temporal expression analysis of AcTrf1a highlights an enriched expression in fat-body and ovary during vitellogenesis. C_LIO_LIIron supplementation and dsRNA mediated knockdown experiments together confer that AcTrf1a may have key role in the iron homeostasis regulation during oogenesis, and egg maturation in the gravid female mosquitoes. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/448311v1_fig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@a87ec0org.highwire.dtl.DTLVardef@197cecorg.highwire.dtl.DTLVardef@1588092org.highwire.dtl.DTLVardef@16110b4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig 1:C_FLOATNO Schematic presentation of iron transport from midgut to ovary by transferrin1 and oocyte reduction after AcTrf1a knockdown. Mosquito acquires iron either from blood meal or iron supplementation in sugar meal. Fat-body derived transferrin proceed towards the gut surface, load iron in its N-terminal iron-binding pocket and deliver iron to ovary. This blood meal iron is required by adult female for completion of gonotrophic cycle. (a) limited iron availability in sugar meal does not support the ovary development and hence no oogenesis; (b) when sugar meal is replaced by blood meal upregulation of transferrin protein results in rapid iron transport to various organs including ovary results in healthy ovarian growth; (c) RNAi mediated knockdown of this transporter protein transferrin in fat-body followed by blood meal, may cause reduced iron transport to ovary and consequently declines in oocyte load. C_FIG