Characterization of a composite with enhanced attraction to savannah tsetse flies from constituents or analogues of tsetse refractory waterbuck (Kobus defassa) body odor.

Savannah tsetse flies avoid flying toward tsetse fly-refractory waterbuck (Kobus defassa) mediated by a repellent blend of volatile compounds in their body odor comprised of δ-octalactone, geranyl acetone, phenols (guaiacol and carvacrol), and homologues of carboxylic acids (C5-C10) and 2-alkanones (C8-C13). However, although the blends of carboxylic acids and that of 2-alkanones contributed incrementally to the repellency of the waterbuck odor to savannah tsetse flies, some waterbuck constituents (particularly, nonanoic acid and 2-nonanone) showed significant attractive properties. In another study, increasing the ring size of δ-octalactone from six to seven membered ring changed the activity of the resulting molecule (ε-nonalactone) on the savannah tsetse flies from repellency to attraction. In the present study, we first compared the effect of blending ε-nonalactone, nonanoic acid and 2-nonanone in 1:1 binary and 1:1:1 ternary combination on responses of Glossina pallidipes and Glossina morsitans morsitans tsetse flies in a two-choice wind tunnel. The compounds showed clear synergistic effects in the blends, with the ternary blend demonstrating higher attraction than the binary blends and individual compounds. Our follow up laboratory comparisons of tsetse fly responses to ternary combinations with different relative proportions of the three components showed that the blend in 1:3:2 proportion was most attractive relative to fermented cow urine (FCU) to both tsetse species. In our field experiments at Shimba Hills game reserve in Kenya, where G. pallidipes are dominant, the pattern of tsetse catches we obtained with different proportions of the three compounds were similar to those we observed in the laboratory. Interestingly, the three-component blend in 1:3:2 proportion when released at optimized rate of 13.71mg/h was 235% more attractive to G. pallidipes than a combination of POCA (3-n-Propylphenol, 1-Octen-3-ol, 4-Cresol, and Acetone) and fermented cattle urine (FCU). This constitutes a novel finding with potential for downstream deployment in bait technologies for more effective control of G. pallidipes, G. m. morsitans, and perhaps other savannah tsetse fly species, in 'pull' and 'pull-push' tactics.

Blending studies with selected waterbuck odor constituents or analogues in the development of a potent repellent blend against savannah tsetse.

Previous comparison of the body odors of tsetse-refractory waterbuck and those of tsetse-attractive ox and buffalo showed that a blend of 15 EAG-active compounds specific to waterbuck, including C5-C10 straight chain carboxylic acid homologues, methyl ketones (C8-C12 straight chain homologues and geranyl acetone), phenols (guaiacol and carvacrol) and δ-octalactone, was repellent to tsetse. A blend of four components selected from each class of compounds (δ-octalactone, pentanoic acid, guaiacol, and geranylacetone) showed repellence that is comparable to that of the 15 components blend and can provide substantial protection to cattle (more than 80%) from tsetse bites and trypanosome infections. Structure-activity studies with the lactone and phenol analogues showed that δ-nonalactone and 4-methylguaiacol are significantly more repellent than δ-octalactone and guaiacol, respectively. In the present study, we compared the responses of Glossina pallidipes and Glossina morsitans to i) blends comprising of various combinations of the most active analogues from each class of compounds, and ii) a four-component blend of δ-nonalactone, heptanoic acid, 4-methylguaiacol and geranyl acetone in different ratios in a two-choice wind-tunnel, followed by a field study with G. pallidipes population in a completely randomized Latin Square Design set ups. In the wind tunnel experiments, the blend of the four compounds in 6:4:2:1 ratio was found to be significantly more repellent (94.53%) than that in 1:1:1:1 proportion and those in other ratios. G. m. morsitans also showed a similar pattern of results. In field experiments with G. pallidipes population, the 6:4:2:1 blend of the four compounds also gave similar results. The results lay down useful groundwork in the large-scale development of more effective 'push' and 'push-pull' control tactics of the tsetse flies.

Expansions of chemosensory gene orthologs among selected tsetse fly species and their expressions in Glossina morsitans morsitans tsetse fly.

Tsetse fly exhibit species-specific olfactory uniqueness potentially underpinned by differences in their chemosensory protein repertoire. We assessed 1) expansions of chemosensory protein orthologs in Glossina morsitans morsitans, Glossina pallidipes, Glossina austeni, Glossina palpalis gambiensis, Glossina fuscipes fuscipes and Glossina brevipalpis tsetse fly species using Café analysis (to identify species-specific expansions) and 2) differential expressions of the orthologs and associated proteins in male G. m. morsitans antennae and head tissues using RNA-Seq approaches (to establish associated functional molecular pathways). We established accelerated and significant (P<0.05, λ = 2.60452e-7) expansions of gene families in G. m. morsitans Odorant receptor (Or)71a, Or46a, Ir75a,d, Ionotropic receptor (Ir) 31a, Ir84a, Ir64a and Odorant binding protein (Obp) 83a-b), G. pallidipes Or67a,c, Or49a, Or92a, Or85b-c,f and Obp73a, G. f. fuscipes Ir21a, Gustatory receptor (Gr) 21a and Gr63a), G. p. gambiensis clumsy, Ir25a and Ir8a, and G. brevipalpis Ir68a and missing orthologs in each tsetse fly species. Most abundantly expressed transcripts in male G. m. morsitans included specific Or (Orco, Or56a, 65a-c, Or47b, Or67b, GMOY012254, GMOY009475, and GMOY006265), Gr (Gr21a, Gr63a, GMOY013297 and GMOY013298), Ir (Ir8a, Ir25a and Ir41a) and Obp (Obp19a, lush, Obp28a, Obp83a-b Obp44a, GMOY012275 and GMOY013254) orthologs. Most enriched biological processes in the head were associated with vision, muscle activity and neuropeptide regulations, amino acid/nucleotide metabolism and circulatory system processes. Antennal enrichments (>90% of chemosensory transcripts) included cilium-associated mechanoreceptors, chemo-sensation, neuronal controlled growth/differentiation and regeneration/responses to stress. The expanded and tsetse fly species specific orthologs includes those associated with known tsetse fly responsive ligands (4-methyl phenol, 4-propyl phenol, acetic acid, butanol and carbon dioxide) and potential tsetse fly species-specific responsive ligands (2-oxopentanoic acid, phenylacetaldehyde, hydroxycinnamic acid, 2-heptanone, caffeine, geosmin, DEET and (cVA) pheromone). Some of the orthologs can potentially modulate several tsetse fly species-specific behavioral (male-male courtship, hunger/host seeking, cool avoidance, hygrosensory and feeding) phenotypes. The putative tsetse fly specific chemosensory gene orthologs and their respective ligands provide candidate gene targets and kairomones for respective downstream functional genomic and field evaluations that can effectively expand toolbox of species-specific tsetse fly attractants, repellents and other tsetse fly behavioral modulators.