Genome-Wide Comparative Analysis of Chemosensory Gene Families in Five Tsetse Fly Species

Abstract

For decades, odour-baited traps have been used for control of tsetse flies (Diptera; Glossinidae), vectors of African trypanosomes. However, differential responses to known attractants have been reported in different Glossina species, hindering establishment of a universal vector control tool. Availability of full genome sequences of five Glossina species offers an opportunity to compare their chemosensory repertoire and enhance our understanding of their biology in relation to chemosensation. Here, we identified and annotated the major chemosensory gene families in Glossina. We identified a total of 118, 115, 124, and 123 chemosensory genes in Glossina austeni, G. brevipalpis, G. f. fuscipes, G. pallidipes, respectively, relative to 127 reported in G. m. morsitans. Our results show that tsetse fly genomes have fewer chemosensory genes when compared to other dipterans such as Musca domestica (n>393), Drosophila melanogaster (n = 246) and Anopheles gambiae (n>247). We also found that Glossina chemosensory genes are dispersed across distantly located scaffolds in their respective genomes, in contrast to other insects like D. melanogaster whose genes occur in clusters. Further, Glossina appears to be devoid of sugar receptors and to have expanded CO2 associated receptors, potentially reflecting Glossina's obligate hematophagy and the need to detect hosts that may be out of sight. We also identified, in all species, homologs of Ir84a; a Drosophila-specific ionotropic receptor that promotes male courtship suggesting that this is a conserved trait in tsetse flies. Notably, our selection analysis revealed that a total of four gene loci (Gr21a, GluRIIA, Gr28b, and Obp83a) were under positive selection, which confers fitness advantage to species. These findings provide a platform for studies to further define the language of communication of tsetse with their environment, and influence development of novel approaches for control.

Fig 1. Phylogeny of classic odorant binding proteins.

Insect classic OBPs are characterized by six conserved cysteine residues. Different symbols depict OBPs from the different species at the terminal nodes: Glossina austeni (red*), Glossina brevipalpis (purple*), Glossina fuscipes fuscipes (pink*), Glossina morsitans morsitans (dark blue*), Glossina pallidipes (light orange*), Drosophila melanogaster (black*), Ceratitis capitata (sky blue*) and Musca domestica (lime green*). The symbol * represents the name of the specific OBP. Sequence alignment was performed using MuSCLE v3.8.31 and phylogeny relationship was inferred using RAxML v8 with best fitting Wheelan and Goldman (WAG) model and 1000 bootstrap iterations.

Fig 2. Phylogeny of Minus-C odorant binding proteins.

The minus-C OBPs have less than six conserved cysteine residues (Missing C1or C2 and/or C5). Different symbols depict OBPs from the different species at the terminal nodes: Glossina austeni (red*), Glossina brevipalpis (purple*), Glossina fuscipes fuscipes (pink*), Glossina morsitans morsitans (dark blue*), Glossina pallidipes (light orange*), Drosophila melanogaster (Dm*), Ceratitis capitata (sky blue*) and Musca domestica (lime green*). The symbol * represents the name of the specific OBP. Sequence alignment was performed using MuSCLE v3.8.31 and phylogeny relationship was inferred using RAxML v8 with best fitting Wheelan and Goldman (WAG) model and 1000 bootstrap iterations.

Fig 3. Phylogeny of Plus-C and Classic-Dimer odorant binding proteins.

The Plus-C OBPs are characterized by having more than six cysteines and a conserved proline residue. The Classic-dimers have two conserved domains of classic sub-family. Different symbols depict OBPs from the different species at the terminal nodes: Glossina austeni (red*), Glossina brevipalpis (purple*), Glossina fuscipes fuscipes (pink*), Glossina morsitans morsitans (dark blue*), Glossina pallidipes (light orange*), Drosophila melanogaster (black*), Ceratitis capitata (sky blue*) and Musca domestica (lime green*). The symbol * represents the name of the specific OBP. Sequence alignment was performed using MuSCLE v3.8.31 and phylogeny relationship was inferred using RAxML v8 with best fitting Wheelan and Goldman (WAG) model and 1000 bootstrap iterations.

Fig 4. Phylogeny of chemosensory proteins.

Clade A shows duplication of ejaculatory bulb protein 3 (Ejbp3 in four tsetse species). Clade B shows expansion of A10p—like homologs in An. gambiae while clades C and D depicts conservation of Pherokine-3 and CSP1 across the species compared, respectively. Different symbols depict CSPs from the different species at the terminal nodes: Glossina austeni (red*), Glossina brevipalpis (purple*), Glossina fuscipes fuscipes (pink*), Glossina morsitans morsitans (dark blue*), Glossina pallidipes (light orange*), Drosophila melanogaster (black*), Anopheles gambiae (sky blue*) and Musca domestica (lime green*). The symbol * represents the name of the specific CSP. Sequence alignment was performed using MuSCLE v3.8.31 and phylogeny relationship was inferred using RAxML v8 with best fitting Wheelan and Goldman (WAG) model and 1000 bootstrap iterations.

Fig 5. Phylogeny of sensory neuron membrane proteins.

Both clades I and II show one to one orthology of the specific SNMP from different insect species. Different symbols depict SNMPs from the different species at the terminal nodes: Glossina austeni (red*), Glossina brevipalpis (purple*), Glossina fuscipes fuscipes (pink*), Glossina morsitans morsitans (dark blue*), Glossina pallidipes (light orange*), Drosophila melanogaster (black*), Anopheles gambiae (sky blue*) and Musca domestica (lime green*). The symbol * represents the name of the specific SNMP. Sequence alignment was performed using MuSCLE v3.8.31 and phylogeny relationship was inferred using RAxML v8 with best fitting Wheelan and Goldman (WAG) model and 1000 bootstrap iterations. Phylogenetic relationships of GRs identified in Glossina genes and their homologs in An. gambiae, D. melanogaster and M. domestica are shown in Fig 6. In all the tsetse species, there was expansion of Gr21a, associated with CO₂ detection in fruit fly and mosquitoes [64,65]. Similarly, expansion of CO2 receptors was noted in An. gambiae which has expanded Gr63a, a protein co-expressed with Gr21a and involved in CO₂ detection [65]. No homologs to sugar receptors in D. melanogaster [66] were identified in any of the five Glossina species (Fig 6). Similarly, D. melanogaster Gr43a, implicated in internal fructose sensing [67] was absent in all tsetse species.

Fig 6. Phylogeny of gustatory receptors.

Gustatory receptors responsible for CO₂ detection show expansion in Glossina species and Musca domestica relative to Drosophila. On the contrary, all receptors responsible for sugar detection are found to be absent in Glossina. Different symbols depict GRs from the different species at the terminal nodes: Glossina austeni (red*), Glossina brevipalpis (purple*), Glossina fuscipes fuscipes (pink*), Glossina morsitans morsitans (dark blue*), Glossina pallidipes (light orange*), Drosophila melanogaster (black*), Anopheles gambiae (sky blue*) and Musca domestica (lime green*). The symbol * represents the name of the specific GR. Sequence alignment was performed using MuSCLE v3.8.31 and phylogeny relationship was inferred using RAxML v8 with best fitting Wheelan and Goldman (WAG) model and 1000 bootstrap iterations.

Fig 7. Phylogeny of odorant receptors.

Expansion of cis-Vaccenyl acetate receptor (Or67d), 4-Methylphenol receptor (Or46a) and aggregation-linked receptor (Or7a) is observed in Glossina species and Musca domestica relative to Drosophila. Sequence alignment was performed using MuSCLE v3.8.31 and phylogeny relationship was inferred using RAxML v8 with best fitting Wheelan and Goldman (WAG) model and 1000 bootstrap iterations. Different symbols and colors were used to depict ORs from the different species at the terminal nodes: Glossina austeni (red*), Glossina brevipalpis (purple*), Glossina fuscipes fuscipes (pink*), Glossina morsitans morsitans (dark blue*), Glossina pallidipes (light orange*), Drosophila melanogaster (black*) and Musca domestica (lime green*). The symbol * represents the name of the specific OR.

Fig 8. Phylogeny of antennal ionotropic receptors.

Antennal IRs are primarily expressed at the antenna of the insect. Sequence alignment was performed using MuSCLE v3.8.31 and phylogeny relationship inferred using RAxML v8 with best fitting Wheelan and Goldman (WAG) model and 1000 bootstrap iterations. Different symbols and colors were used to depict IRs from the different species at the terminal nodes: Glossina austeni (red*), Glossina brevipalpis (purple*), Glossina fuscipes fuscipes (pink*), Glossina morsitans morsitans (dark blue*), Glossina pallidipes (light orange*), Drosophila melanogaster (black*), Musca domestica (lime green*) and Anopheles gambiae (sky blue*). The symbol * represents the name of the specific IR.

Fig 9. Phylogeny of divergent ionotropic receptors.

Sequence alignment was performed using MuSCLE v3.8.31 and phylogeny relationship inferred using RAxML v8 with best fitting Wheelan and Goldman (WAG) model and 1000 bootstrap iterations. Different symbols and colors were used to depict IRs from the different species at the terminal nodes: Glossina austeni (red*), Glossina brevipalpis (purple*), Glossina fuscipes fuscipes (pink*), Glossina morsitans morsitans (dark blue*), Glossina pallidipes (light orange*), Drosophila melanogaster (black*), Musca domestica (lime green*) and Anopheles gambiae (sky blue*). The symbol * represents the name of the specific IR.

Fig 10. Phylogeny of ionotropic glutamate receptors and kainate receptors.

Sequence alignment was performed using MuSCLE v3.8.31 and phylogeny relationship inferred using RAxML v8 with best fitting Wheelan and Goldman (WAG) model and 1000 bootstrap iterations. Different symbols and colors were used to depict IGluRs from the different species at the terminal nodes: Glossina austeni (red*), Glossina brevipalpis (purple*), Glossina fuscipes fuscipes (pink*), Glossina morsitans morsitans (dark blue*), Glossina pallidipes (light orange*), Drosophila melanogaster (black*), Musca domestica (lime green*) and Anopheles gambiae (sky blue*). The symbol * represents the name of the specific IGluR.