Characterization of Anopheles gambiae (African Malaria Mosquito) Ferritin and the Effect of Iron on Intracellular Localization in Mosquito Cells

Abstract

Ferritin is a 24-subunit molecule, made up of heavy chain (HC) and light chain (LC) subunits, which stores and controls the release of dietary iron in mammals, plants, and insects. In mosquitoes, dietary iron taken in a bloodmeal is stored inside ferritin. Our previous work has demonstrated the transport of dietary iron to the ovaries via ferritin during oogenesis. We evaluated the localization of ferritin subunits inside CCL-125 [Aedes aegypti Linnaeus (Diptera: Culicidae), yellow fever mosquito] and 4a3b [Anopheles gambiae Giles (Diptera: Culicidae), African malaria mosquito] cells under various iron treatment conditions to further elucidate the regulation of iron metabolism in these important disease vectors and to observe the dynamics of the intracellular ferritin subunits following iron administration. Deconvolution microscopy captured 3D fluorescent images of iron-treated mosquito cells to visualize the ferritin HC and LC homologue subunits (HCH and LCH, respectively) in multiple focal planes. Fluorescent probes were used to illuminate cell organelles (i.e., Golgi apparatus, lysosomes, and nuclei) while secondary probes for specific ferritin subunits demonstrated abundance and co-localization within organelles. These images will help to develop a model for the biochemical regulation of ferritin under conditions of iron exposure, and to advance novel hypotheses for the crucial role of iron in mosquito vectors.

Description of a Second Ferritin Light Chain Homologue From the Yellow Fever Mosquito (Diptera: Culicidae)

“…Larvae were kept in water-filled trays for approximately 7 d until pupation where they were transferred into water-filled cups inside small cages. From the onset of pupation, adult mosquitoes emerged approximately 1–2 d later and were provided 10% sucrose solution ad libitum; animals were kept at 27°C, 70–80% relative humidity with a photoperiod of 16:8 (L:D) h. Mated female mosquitoes, 4–5 d postemergence, were fasted for 12 h with sterile water ad libitum and then membrane-fed a diet of warmed (37°C), defibrinated porcine blood supplemented with 5-mM ATP (Sigma) for 2 h in glass feeders ( Zhou et al 2007 , Geiser et al 2015 ).…”

“…As a control cohort, cold anesthetized sugar-fed (S) females (15) were collected on each of days 6 (24 hS) and 8 (72 hS) postemergence. Collection of animals for developmental stage protein analysis was performed as previously described ( Zhou et al 2007 , Geiser et al 2015 ) with modifications. Briefly, 900 eggs (E), 900 first instar larvae (1L), 45 fourth instar larvae (4L), 45 pupae (P), cold anesthetized 45 males (M), and 45 females (nonblood fed, 0 h) at day 4 postemergence were collected.…”

Ferritin Assembly in Enterocytes of Drosophila melanogaster

“…These regulatory relationships are conserved in other insects besides Drosophila melanogaster [ 30 , 31 ]; Fourth, iron loading into ferritin critically depends on transport from the cytosol to the endoplasmic reticulum, a function likely mediated by the zinc regulated and iron regulated transporter 13 (Zip13) [ 25 ]; Fifth, the two subunits Fer1HCH and Fer2LCH have been detected in distinct vesicular compartments at the initial stages of the ferritin biosynthetic process, one hour post-feeding on iron-containing media, suggesting that subunit-specific trafficking and post-translational modifications may precede the formation of the ferritin complex [ 21 ]. A recent complementary effort in mosquito cells is likely to provide independent information for the ferritin assembly and secretion processes [ 32 ].…”

How micronutrients influence the physiology of mosquitoes

“…aegypti triggers a wide range of pleiotropic changes in the expression of genes associated to antioxidant activities, energy metabolism, cell cycle, cellular signaling, immunity and blood meal digestion [35]. Ferritin is an iron-storage protein found free in hemolymph, as well as intracellularly in most tissues [36,37]. It is transcriptionally induced after a blood meal, and contributes to sequester iron released from heme digestion, preventing oxidative damage [38,39].…”