Localization and Expression Profiles of Odorant Receptor Genes in Non-olfactory Tissues of Male and Female Tsetse Fly Glossina morsitans morsitans

Abstract

Glossina morsitans morsitans, a savannah species of tsetse fly, are vectors of trypanosome which cause African trypanosomiasis. Tsetse flies rely on olfaction in identifying hosts, mates, larviposition site, and escape from predators. Olfactory proteins located in the tsetse antennae are involved in perceiving odors from the external environment, processing and signal transduction. Odorant receptors (ORs) primarily located in the antennae’s olfactory receptor neuron have a vital role in the signal transduction process. Recent evidence in the mosquito, Anopheles gambiae, and fruit fly Drosophila melanogaster suggests expression of ORs in insect’s non-olfactory tissues implying other physiological functions that could aid in vector control. The aim was this study was therefore to study the expression of Ors in non-olfactory tissues in males and females G. m. morsitans. Herein, ORs expression in the brain, gut, and reproductive tissues of male and female G. m. morsitans was determined. Total RNA was extracted from brain, gut, and reproductive tissues (testis, and accessory, spermathecae and ovary) and used to synthesize cDNA. 46 OR genes identified in G. m. morsitans were screened in individual tissues using conventional PCR. Amplicons were sequenced and searched against VectorBase and their protein sequences used to identify homologs in D. melanogaster and other Glossina spp genomes in FlyBase and VectorBase, respectively. ORs domains and the number of transmembrane helices were confirmed using the Conserved Domain Database and TMHMM, respectively. Gene ontology functions of the identified D. melanogaster homologs were inferred from FlyBase. Tissue expression of homologs in D. melanogaster was checked in Fly Atlas 2. Real-time PCR was used to determine the expression profile of 46 ORs in male and female G. m. morsitans brain, gut and reproductive tissues. Results revealed the localization of Ors in the male brain (41), gut (38) and reproductive tissue (39) and similarly, female brain (44), gut (23) and reproductive tissues (40) respectively. Sequence identity between G. m. morsitans and other Glossina genomes ranged from 21.7% to 99.4% while for D. melanogaster it ranged from 22.4 to 77.5%. All sequenced ORs belonged to the seven transmembrane odorant receptors (7tm_6) family and varied in number of transmembrane helices. Gene ontology identified signal transduction and response to stimuli as the main biological processes. The membrane was identified as the main cellular component, while odorant receptor activity and odorant binding were the main molecular function. The presence of transmembrane helices and biological process such as signal transduction suggests ORs act as receptors for endogenous ligands. Fly Atlas results revealed expression of G. m. morsitans orthologs in D. melanogaster including DmelOR85f (GmmOR28) and DmelOR67d (GmmOR42) in the male brain, DmelOR94a (GmmOR12) in the male gut, and DmelOR67c (GmmOR27) and DmelOR13a (GmmOR32) in the male testis. DmelOR67c (GmmOR27) was also present in female brain and DmelOR49b (GmmOR33) in female gut. Quantitative real time PCR revealed four significantly highly expressed OR genes in male G. m. morsitans brain (GmmOR19, GmmOR28, GmmOR30, GmmOR31),six in the gut (GmmOR7, GmmOR12, GmmOR32, GmmOR34, GmmOR42, GmmOR44) and three ORs in the testis and accessory glands (GmmOR32, GmmOR44, GmmOR46). In females, two ORs were highly expressed the brain (GmmOR25, GmmOR27), three in the gut (GmmORr17, GmmOR33, GmmOR35) and three in the reproductive tissues (GmmOR9, GmmOR22, GmmOR27). OR expression in non-olfactory tissues suggests unique physiological roles in the brain, gut and reproductive tissues in male and female G. m. morsitans. Thus, GmmOR28 expression in the male and female brain may suggest a role in brain related functions while presence of GmmOR32 and GmmOR44 in the male gut and reproductive tissue and GmmOR27 in the female brain and reproductive tissue suggests conserved non-olfactory functions across different tissues. The conserved odorant receptor, Orco was expressed in male reproductive tissue and may suggest male reproductive roles such as spermatozoa activation. The findings provide insight on the localization and expression profile of OR genes in non-olfactory organs (brain, gut and reproductive tissues) in male and female G. m. morsitans and open avenues to investigate functional roles of significantly expressed ORs for the control of Glossina and trypanosomiasis.