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.


Introduction
Line 79 the equipment can be described at one with mention of "linked" twice in a sentence. Line 90 remove comma in 4thylphenols Line 103, in the last part of the introduction, its not clear how they authors hypothesized that increasing attractants consisting of (εnonalactone, nonanoic acid and 2-nonanone) would have specific number of folds to attraction to savanna species.

Materials and methods
Three replicates of these experiments where done and all the work was performed according to the recommended. However, the choice of 30 flies of three day teneral tsetse flies is not justified in line 139. Can the authors justify. It's not very clear how the control experiment without any treatment was done Line 139, there is cage (1), the authors need to clarify on this. Line 152 to 154, the sentence, "we conducted these experiments and recorded the observation three times for each treatment (Odor and concentration). The sentence could be re-structured to mention the experiment was replicated three times. Secondly, the word "Odor" change to the lower case.
Line 166 to 167 the formula used, the authors need to quote the reference for this formula. Line 176, the sentence, "number of generations of the flies that had been colonized which could affected their behavioral phenotype. The authors know that on average tsetse flies can have about 8 generations per year (Krafsur et al 2008). The authors could clarify on how this justify this sentence especially on the behavior and the time this experiment was performed during the year.

Field bioassay validations of wind tunnel for behavioral responses of G. m. morsitans and G. pallidipes
From line 175 to 223, there is no control or mention of control included in this part of the field experiment. The authors need to mention what controls where used much as they are presented in the result section but could be capture in the materials and methods section.

Results
Line 237 to 241 starting sentence " the structure of ε-nonalactone sounds more of the mention description than results. Possibly this can be taken to the methods and summary results of these techniques summaried. Line 259 to 262, how was the significant tested?? Line 269 to line 273, in each treatment, there was significant difference which the authors should mention as indicated by the standard error bars in these results description.
Figure, 3B Line 278, I was wondering why the response was increasing gradually in the different treatment? Could the authors clarify why this graph is performing in this way?
Line 503, Table 4: Am wondering why G.m morsitans data for the same treatment of G. pallidipes is not presented. Could the authors clarify?

Discussion
Line 305, G. pallidipes or G.m morsitans, authors could use "and" Line 308 authors could describe more why they are no significant difference in G.pallidpes and G.m. morsitans. General comment on the discussion Much of the results are presented in this paper could be discussed more. I find three paragraph discussion very limiting as compared to the data presented