Genome-wide annotation of chemosensory and glutamate-gated receptors, and related genes in glossina morsitans morsitans tsetse fly

Abstract

Tsetse flies are the sole vectors of trypanosomes that cause nagana and sleeping sickness in animals and humans respectively in tropical Africa. Tsetse are unique: both sexes adults are exclusive blood-feeders, females are mated young and give birth to a single mature larva in sheltered habitats per pregnancy. Tsetse use chemoreception to detect and respond to chemical stimuli, helping them to locate hosts, mates, larviposition and resting sites. The detection is facilitated by chemoreceptors expressed on sensory neurons to cause specific responses. Specific molecular factors that mediate these responses are poorly understood in tsetse flies. This study aimed to identify and characterize genes that potentially mediate chemoreception in Glossina morsitans morsitans tsetse flies. These genes included sensory odorant (OR), gustatory (GR), ionotropic (IR), and related genes for odorant-binding (OBP), chemosensory (CSP) and sensory neuron membrane (SNMP) proteins. Synaptic transmission in higher brain sites may involve ionotropic glutamate-gated (iGluR) and metabotropic glutamate-gated (mGluR) receptors. The genes were annotated in G. m. morsitans genome scaffold assembly GMOY1.1 Yale strain using orthologs from D. melanogaster as query via TBLASTX algorithm at e-value below 1e-03. Positive blast hits were seeded as gene constructs in their respective scaffolds, and used as genomic reference onto which female fly-derived RNA sequence reads were mapped using CLC Genomics workbench suite. Seeded gene models were modified using RNA-Seq reads then viewed and re-edited using Artemis genome viewer tool. The genome was iteratively searched using the G. m. morsitans gene model sequences to recover additional similar hit sequences. The gene models were confirmed through comparisons against the NCBI conserved domains database (CDD) and non-redundant Swiss-Prot database. Trans-membrane domains and secretory peptides were predicted using TMHMM and SignalP tools respectively. Putative functions of the genes were confirmed via Blast2GO searches against gene ontology database. Evolutionary relationships amongst and between the genes were established using maximum likelihood estimates using best fitting amino acid model test in MEGA5 suite and PhyML tool. Expression profiles of genes were estimated using the RNA-seq data via CLCGenomics RNA-sequences analysis pipeline. Overall, 46 ORs, 14 GRs, and 19 IRs were identified, of which 21, 6 and 4 were manually identified for ORs, GRs, and IRs respectively. Additionally, 15 iGluRs, 6 mGluRs, 5 CSPs, 15 CD36-like, and 32 OBPs were identified. Six copies of OR genes (GmmOR41-46) were homologous to DmelOr67d, a single copy cis vacenyl acetate (cVA) receptor . Genes whose receptor homologs are associated with responses to CO2, GmmGR1-4, had higher expression profiles from amongst glossina GR genes. Known core-receptor homologs OR1, IR8a, IR25a and IR64a were conserved, and three species-specific divergent IRs (IR10a, IR56b and IR56d) were identified. Homologs of GluRIID, IR93a, and sweet taste receptors (Gr5a and Gr64a) were not identified in the genome. Homolog for LUSH protein, GmmOBP26, and sensory neuron membrane receptors SNMP1 and SNMP2 were conserved in the genome. Results indicate reduced repertoire of the chemosensory genes, and suggest reduced host range of the tsetse flies compared to other Diptera. Genes in multiple copies suggest their prioritization in chemoreception, which in turn may be tied to high specificity in host selection. Genes with high sequence conservation and expression profiles probably relate to their broad expression and utility within the fly nervous system. These results lay foundation for future comparative studies with other insects, provide opportunities for functional studies, and form the basis for re-examining new approaches for improving tsetse control tools and possible drug targets based on chemoreception.