A "Dock-Work" Orange: A Dual-Receptor Biochemical Theory on the Deterrence Induced by Citrusy Aroma on Elephant Traffic Central to a Conservation Effort.

Conservation of elephants requires physical, chemical, and biological approaches to ensure the protection of these gargantuan pachyderms. One such approach is using orange plants (as biofencing) for the repellence of elephants, which precludes catastrophic events related to the encroachment of elephants into human habitats. Elephants have sensitive olfactory discrimination of plant volatile compounds for foraging and other behavior using G-protein-coupled receptors (GPCRs). However, 2 such receptors are the A2A and A2B receptors mediating olfaction elicited by a host of ligands, including limonene, the main volatile compound in citrus plants, which is hypothesized to be the chief repelling agent. Bioinformatics at the protein and mRNA levels (BLAST/Multiple Sequence Alignments) were employed to explore the multiple expression products of A2B receptors, namely full-length and truncated proteins produced by isoform mRNAs translated from multiple methionines, while the comparison of the limonene-binding pockets of human and elephant A2B receptors and prediction servers [Netphos 3.1; Protter] was used to focus, respectively, on the contacts limonene binding entails and the post-translational modifications that are involved in cell signaling. Finally, the link between limonene and antifeedant activity was explored by considering limonene content on trees that are preferentially foraged or avoided as part of the feeding behavior by elephants. The African bush elephant (Loxodonta africana) possesses a full-length A2A receptor but unlike most mammals, expresses a highly truncated A2B receptor isoform possessing only transmembrane helices 5, 6, and 7. Truncation may lead to higher traffic and expression of the A2B receptor in olfactory interfaces/pathways and aid stronger activation. In addition, all residues in the putative limonene-binding cleft are perfectly conserved between the human and African bush elephant A2B receptors, both full length and truncated. Shallow activation sites require micromolar affinity and fewer side-chain interactions, which is speculated to be the case for the truncated A2B receptor. An N-terminal extremity N-glycosylation motif is indicative of membrane localization of the truncated A2B receptor following accurate folding. A combination of truncation, indels, substitutions, and transcript isoforms are the attributed roles in the evolution of the L. africana A2B receptor, out of which limonene receptivity may be the key. It is also inferred how limonene may act as a dietary repellent/antifeedant to a generalist herbivore, with the documented limonene content being absent in some dietary favorites including the iconic Sclerocarya birrea.