Characterization of Carbonic Anhydrase 9 in the Alimentary Canal of Aedes aegypti and Its Relationship to Homologous Mosquito Carbonic Anhydrases

1. Introduction

In the mosquito midgut, luminal pH regulation is important for the digestion of food. Digestive proteases are secreted into the midgut lumen to process detritus in mosquito larvae and the blood meals in female adults. These proteases are highly active at neutral and alkaline pH [1]. The midgut lumen of mosquito larvae and adults is alkaline [2,3], and the major buffer molecule in the midgut lumen of mosquitoes is bicarbonate (HCO₃⁻). Luminal HCO₃⁻ and carbonate (CO₃²⁻) levels are regulated by a network of proteins in mosquitoes which may include carbonic anhydrase (CA), a zinc metaloenzyme that hydrates carbon dioxide (CO₂) to form HCO₃⁻ and protons (H⁺) [4,5]. In this report, we characterized the fourteen isoforms of Aedes aegypti CA via bioinformatic gene sequence analysis, data mining of transcriptomes [6], and molecular phylogenetics. We also analyzed Aedes aegypti CA9 (AeCA9), one of the highly expressed cytoplasmic CAs, using molecular biology and immunohistochemical analyses. More specifically, AeCA9 mRNA expression and protein localization were analyzed in the different life stages along with adult digestive and ion regulatory tissues.

There are six different evolutionary gene families of CA: alpha, beta, gamma, delta, zeta, and eta [7,8] Also, there are five different types of CAs, each with different spatial localizations: soluble cytoplasmic, soluble secreted, integral membrane, mitochondrial, and glycosylphosphatidyl inositol (GPI)-anchored [5]. CA isozymes also have different activity levels due to differences in amino acid sequence, active site structure, and post-translational modifications [9,10,11]. Despite catalyzing the same chemical reaction, different isoforms are associated with a vast array of physiological processes [12,13,14]. Various solute carriers and ion transporters such as members of the SLC4 protein family and energizing transporters such as H+ V-ATPase and metabolic enzymes such as the CAs are hypothesized to influence gut luminal pH and maintain ion homeostasis in larval and adult mosquito guts [3,4,15,16,17,18,19].

In the larvae, the midgut is described as having three regions: the gastric caeca (GC), anterior midgut (AMG), and posterior midgut (PMG) [20] (Figure 1A). The GC are eight lobed extensions of the midgut epithelium which occupy much of the internal space of the thorax of the larvae and are the gut epithelial extensions just posterior to...