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

Mosquitoes transmit pathogens that cause numerous infectious diseases including dengue, yellow fever, and malaria. These diseases exact a high cost on millions of lives and are the cause of abundant morbidity in endemic regions (WHO 2013a,b,2014). Infection occurs when a female blood feeds on a host to acquire sufficient nutrients for egg production (Telang et al. 2013; Jason Pitts et al. 2014). Although transmission rates are low, numbers of infected individuals are very high reflecting the numbers of disease vectors. The bloodmeal is iron rich (Zhou et al. 2007). The majority of iron absorbed from the diet is transported to the ovaries for egg production (Zhou et al. 2007), and egg numbers and progeny survivorship may fall when the insects receive an iron limited meal (Kogan 1990; Jason Pitts et al. 2014). We are interested in the potential for interference with iron metabolism or iron limitation as an insect control strategy. We previously reported that following dietary iron intake, mosquitoes produce ferritin that is secreted into hemolymph from the midgut loaded with iron, and that ferritin, rather than transferrin, is the primary iron transport protein in these animals (Zhou et al. 2007). Earlier work in the yellow fever mosquito, Aedes aegypti Linnaeus (Diptera: Culicidae), demonstrated the presence of two ferritin subunits, heavy and light chain homologues (HCH and LCH, respectively) that are similar to the heavy and light chains subunits of mammals and other animals (Pham et al. 2000; Geiser et al. 2003). Synthesis of either homologue is subject to positive transcriptional control by iron (Dunkov et al. 2002; Pham et al. 2003; Pham and Chavez 2005), while translation of the HCH is subject to positive control by iron via an iron responsive element (IRE) found in the message (Zhang et al. 2002). In contrast to mammals, no IRE is found in the message for the LCH (Geiser et al. 2003). In mammals, ferritin is found in cell cytoplasm, increases in response to iron exposure and serves as the primary iron storage protein (Arosio and Levi 2010; Linder 2013). Ferritin also is the primary iron storage protein in mosquitoes and expression increases in response to iron (Zhou et al. 2007; Geiser et al. 2009). However, unlike mammalian ferritin that is localized to the cytoplasm, Ae. aegypti ferritin is found primarily in the membranes of animal tissues and larval epithelial CCL-125 cells (Geiser et al. 2007). Cells show a linear uptake of iron in direct proportion to iron level of the culture medium (Geiser et al. 2006). The majority of the iron is stored in the membranes and membrane iron exceeds cytoplasmic iron by an order of magnitude. Iron exposure will increase both cytoplasmic and membrane ferritin. However, membrane ferritin plateaus because CCL-125 cells secrete iron-loaded ferritin into the culture medium, and thereby, limit cellular iron levels. Iron can participate in the Fenton and Haber–Weiss reactions producing toxic free radicals. Ferritin is considered cytoprotective against oxidative stress because it converts ferrous to ferric and stores ferric in a complex that prevents free radical formation (Arosio and Levi 2010). Because female mosquitoes receive a high iron load in the bloodmeal, while males of the species survive on nectars with little iron, we are studying how mosquitoes cope with a high iron load. In addition to the studies in larval CCL-125 cells, we evaluated the effects of iron exposure in a second mosquito cell line, Anopheles gambiae Giles [(Diptera: Culicidae), African malaria mosquito] 4a3b cells. This cell line originates from larvae and is thought to be derived from hemocytes, immune-like phagocytic cells found in the open circulatory system of insects (Muller et al. 1999). Unlike our previous studies in CCL-125 cells, 4a3b cells accumulate high levels of iron and do not secrete iron-loaded ferritin to limit cellular iron accumulation (Geiser et al. 2009).