Immunohistochemical mapping of the adipokinetic hormone/corazonin-related peptide and insights into its receptor specificity using receptor chimera in Aedes aegypti mosquito

Aedes aegypti are an important research model as they are a major vector responsible for several arboviral diseases such as Yellow fever and Zika virus. The adipokinetic hormone/corazonin-related peptide (ACP) is an insect neuropeptide structurally intermediate between corazonin (CRZ) and adipokinetic hormone (AKH), which all demonstrate homology to the vertebrate gonadotropin-releasing hormone (GnRH). Several studies have characterized the AKH and CRZ signalling systems within diverse insect species, and most notable are the energy mobilization and cardio-acceleratory functions, respectively. In contrast, the role of ACP and its receptor (ACPr) remains unclear. Firstly, we aimed to map the distribution of ACP in A. aegypti using immunohistochemistry combined with in situ hybridization. Adult mosquitoes displayed ACP immunostaining in two pairs of lateral neurosecretory cells in the brain and 2-3 cells in the thoracic ganglia. Extensive ACP-immunoreactive axonal projection emanating within each abdominal ganglia was also observed. These provide some evidence that ACP might act as a neurotransmitter or neuromodulator. Secondly, we aimed to determine the specific regions of the ACPr most critical for ligand fidelity and specificity by creating ACPr chimera by singly replacing the native ACPr extracellular loops (ECL1, ECL2, and ECL3) and incorporating those from the AKH receptor. Interestingly, it was determined that the three ACPr-ECL mutants receptors showed no response to either ACP or AKH using a heterologous functional assay. These results suggest that complete replacement of each extracellular loop is detrimental for ligand binding and ongoing research is aimed at discerning specific residues critical for functional ligand-binding.