Effect of zebra skin-derived compounds on field catches of the human African trypanosomiasis vector Glossina fuscipes fuscipes.

The riverine tsetse fly Glossina fuscipes fuscipes is a major vector of trypanosome pathogens causing African trypanosomiasis. This fly species uses a combination of olfactory and visual cues to locate its hosts. Previously, traps and targets baited with visual cues have been used in vector control, but the development of olfactory-based tools has been challenging. Recently, repellents have shown promise as olfactory-based tools in tsetse vector control. Here, we evaluated a three-component blend comprising 6-methyl-5-hepten-2-one, acetophenone and geranyl acetone (blend K), previously identified as a repellent for savannah tsetse flies in zebra skin odor, on G. f. fuscipes populations. Using a series of 6 × 6 randomized Latin square-designed experiments, G. f. fuscipes catches in biconical traps were monitored on four islands of Lake Victoria in western Kenya between July and September 2019, after the long rainy season. Traps were baited with blend K and individual components of this blend. The known tsetse repellent blend WRC (waterbuck repellent compounds) and trap alone were included as controls. Daily catch data in thirty-six replicate trials were analyzed using generalized linear model with negative binomial error structure using the package "MASS" in R. Treatment, day and site were set as predictor variables. Our results showed that, blend K significantly reduced G. f. fuscipes catches by 25.6% (P < 0.01) compared to the control trap alone but was not significantly different from WRC which reduced catches by 20.7% (P < 0.05). Of the individual compounds, geranyl acetone solely significantly reduced catches by 29.1% (P < 0.01) which did not differ from blend K or WRC. We conclude that geranyl acetone accounts for the repellent effect of blend K on the riverine tsetse fly, G. f. fuscipes, demonstrating the ecological importance of animal skin odors in the host-seeking behavior of medically-important tsetse fly vectors.

Responses of Glossina fuscipes fuscipes to visually attractive stationary devices baited with 4-methylguaiacol and certain repellent compounds in waterbuck odour.

BACKGROUND: A blend of compounds (pentanoic acid, guaiacol, δ-octalactone and geranylacetone) identified in waterbuck (Kobus defassa) body odour referred to as waterbuck repellent compounds (WRC) and a synthetic repellent 4-methylguaiacol have previously been shown to repel tsetse flies from the morsitans group. However, these repellents have not been evaluated on palpalis group tsetse flies. In this study, we evaluated the effect of these repellents on catches of Glossina fuscipes fuscipes (major vector of human sleeping sickness) in biconical traps and on sticky small targets which are visually attractive to palpalis group flies. The attractive devices were baited with different doses and blends of the repellent compounds. We also assessed the effect of removal of individual constituents in the synthetic blend of WRC on catches of G. f. fuscipes. METHODOLOGY/PRINCIPAL FINDINGS: The study was conducted in western Kenya on four islands of Lake Victoria namely Big Chamaunga, Small Chamaunga, Manga and Rusinga. The tsetse fly catches from the treatments were modeled using a negative binomial regression to determine their effect on catches. In the presence of WRC and 4-methylguaiacol (released at ≈2 mg/h and ≈1.4 mg/h respectively), catches of G. f. fuscipes were significantly reduced by 33% (P<0.001) and 22% (P<0.001) respectively in biconical traps relative to control. On sticky small targets the reduction in fly catches were approximately 30% (P<0.001) for both 4-methylguiacol and WRC. In subtractive assays, only removal of geranylacetone from WRC significantly increased catches (by 1.8 times; P <0.001) compared to the complete blend of WRC. CONCLUSIONS/SIGNIFICANCE: We conclude that WRC and 4-methylguaiacol reduce catches of G. f. fuscipes at stationary visually attractive traps and suggest that they may serve as broad spectrum repellents for Glossina species. We recommend further studies to investigate the effects of these compounds on reduction of G. f. fuscipes attracted to human hosts as this may lead to development of new strategies of reducing the prevalence and incidence of sleeping sickness.

Zebra skin odor repels the savannah tsetse fly, Glossina pallidipes (Diptera: Glossinidae).

BACKGROUND: African trypanosomosis, primarily transmitted by tsetse flies, remains a serious public health and economic challenge in sub-Saharan Africa. Interventions employing natural repellents from non-preferred hosts of tsetse flies represent a promising management approach. Although zebras have been identified as non-preferred hosts of tsetse flies, the basis for this repellency is poorly understood. We hypothesized that zebra skin odors contribute to their avoidance by tsetse flies. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the effect of crude zebra skin odors on catches of wild savannah tsetse flies (Glossina pallidipes Austen, 1903) using unbaited Ngu traps compared to the traps baited with two known tsetse fly management chemicals; a repellent blend derived from waterbuck odor, WRC (comprising geranylacetone, guaiacol, pentanoic acid and δ-octalactone), and an attractant comprising cow urine and acetone, in a series of Latin square-designed experiments. Coupled gas chromatography-electroantennographic detection (GC/EAD) and GC-mass spectrometry (GC/MS) analyses of zebra skin odors identified seven electrophysiologically-active components; 6-methyl-5-hepten-2-one, acetophenone, geranylacetone, heptanal, octanal, nonanal and decanal, which were tested in blends and singly for repellency to tsetse flies when combined with Ngu traps baited with cow urine and acetone in field trials. The crude zebra skin odors and a seven-component blend of the EAD-active components, formulated in their natural ratio of occurrence in zebra skin odor, significantly reduced catches of G. pallidipesby 66.7% and 48.9% respectively, and compared favorably with the repellency of WRC (58.1%- 59.2%). Repellency of the seven-component blend was attributed to the presence of the three ketones 6-methyl-5-hepten-2-one, acetophenone and geranylacetone, which when in a blend caused a 62.7% reduction in trap catch of G. pallidipes. CONCLUSIONS/SIGNIFICANCE: Our findings reveal fundamental insights into tsetse fly ecology and the allomonal effect of zebra skin odor, and potential integration of the three-component ketone blend into the management toolkit for tsetse and African trypanosomosis control.

Sticky small target: an effective sampling tool for tsetse fly Glossina fuscipes fuscipes Newstead, 1910.

BACKGROUND: Small targets comprising panels of blue and insecticide-treated black netting material each 0.25 × 0.25 m have been shown to attract and kill Glossina fuscipes fuscipes Newstead, 1910 (Diptera: Glossinidae) thereby reducing its population density by over 90% in field trials. However, their attractive ability has not been fully exploited for sampling purposes. Therefore, in this study we assessed the effectiveness of using sticky small targets as sampling tools for G. f. fuscipes in western Kenya. We also determined the influence of colour on the landing response of female and male flies on sticky small targets. METHODS: Using a series of randomised block experiments, the numbers of tsetse flies caught with sticky small targets were compared with those caught with biconical traps. A negative binomial regression was used to model fly catches. Odds ratios as measures of association between the landing response on the blue or black panel of the sticky small target and the sex of flies were obtained from a multiple logistic regression. RESULTS: The results showed that sticky small targets caught 13.5 and 3.6 times more female and male tsetse flies than biconical traps (Z = 9.551, P < 0.0001 and Z = 5.978, P < 0.0001, respectively). Females had a 1.7 times likelihood of landing on the black panel than males (Z = 2.25, P = 0.025). CONCLUSION: This study suggests that sticky small targets are an effective sampling tool for G. f. fuscipes. Therefore, we recommend the use of sticky small targets as an alternative to biconical traps for observational and experimental investigations of G. f. fuscipes.

Effects of vector control on the population structure of tsetse (Glossina fuscipes fuscipes) in western Kenya.

Displacement rates of tsetse affect performance of targets during vector control. Fly size, one of the indicators of population structure usually obtained from wing measurement, is among the determinants of displacement rates. Although recovery of tsetse in previous intervention areas has been widely reported, the population structure of tsetse that recover is rarely evaluated despite being associated with displacements rates. Previously, intervention trials had reduced tsetse densities by over 90% from >3 flies/trap/day to <1fly/trap/day on Big Chamaunga and Manga islands of Lake Victoria in western Kenya. In this study, we assessed the recovery in densities of Glossina fuscipes fuscipes on the two islands and evaluated the effects vector control might have on the population structure. A before and after intervention study was undertaken on four islands of Lake Victoria in western Kenya; Small and Big Chamaunga, Manga and Rusinga Islands, two of which tsetse control intervention had previously been undertaken. Three years after intervention average G. f. fuscipes catches in biconical traps were estimated on each island. Wing centroid size (CS) (a measurement of fly size) and shape, indicators of the population structure of flies from the four islands were compared using geometric morphometric analyses. CS and shape of available female but not male tsetse wings obtained before the intervention trial on Big and Small Chamaunga islands were compared with those from the same islands after the intervention trial. G. f. fuscipes apparent density on the previous intervention islands were>9 flies/trap/day. Irrespective of sex, wing shape did not isolate tsetse based on their islands of origin. The fly size from Big and Small Chamaunga did not differ significantly before intervention trials (P = 0.728). However, three years after the intervention flies from Big Chamaunga were significantly smaller than those from Small Chamaunga (P < 0.003). Further, there was an increase in the divergence of wing morphology between flies collected from Big Chamaunga and those from Small Chamaunga after tsetse control. In conclusion, even though populations are not isolated, vector control could influence the population structure of tsetse by exerting size and wing morphology differential selection pressures. Therefore, we recommend further studies to understand the mechanism behind this as it may guide future vector control strategies.

Protecting cows in small holder farms in East Africa from tsetse flies by mimicking the odor profile of a non-host bovid.

BACKGROUND: For the first time, differential attraction of pathogen vectors to vertebrate animals is investigated for novel repellents which when applied to preferred host animals turn them into non-hosts thereby providing a new paradigm for innovative vector control. For effectively controlling tsetse flies (Glossina spp.), vectors of African trypanosomosis, causing nagana, repellents more powerful than plant derived, from a non-host animal the waterbuck, Kobus ellipsiprymnus defassa, have recently been identified. Here we investigate these repellents in the field to protect cattle from nagana by making cattle as unattractive as the buck. METHODOLOGY/PRINCIPAL FINDINGS: To dispense the waterbuck repellents comprising guaiacol, geranylacetone, pentanoic acid and δ-octalactone, (patent application) we developed an innovative collar-mounted release system for individual cattle. We tested protecting cattle, under natural tsetse challenge, from tsetse transmitted nagana in a large field trial comprising 1,100 cattle with repellent collars in Kenya for 24 months. The collars provided substantial protection to livestock from trypanosome infection by reducing disease levels >80%. Protected cattle were healthier, showed significantly reduced disease levels, higher packed cell volume and significantly increased weight. Collars >60% reduced trypanocide use, 72.7% increase in ownership of oxen per household and enhanced traction power (protected animals ploughed 66% more land than unprotected). Land under cultivation increased by 73.4%. Increase in traction power of protected animals reduced by 69.1% acres tilled by hand per household per ploughing season. Improved food security and household income from very high acceptance of collars (99%) motivated the farmers to form a registered community based organization promoting collars for integrated tsetse control and their commercialization. CONCLUSION/SIGNIFICANCE: Clear demonstration that repellents from un-preferred hosts prevent contact between host and vector, thereby preventing disease transmission: a new paradigm for vector control. Evidence that deploying water buck repellents converts cattle into non-hosts for tsetse flies-'cows in waterbuck clothing'.

Repellency of tsetse-refractory waterbuck (Kobus defassa) body odour to Glossina pallidipes (Diptera: Glossinidae): assessment of relative contribution of different classes and individual constituents.

Our earlier studies on the comparative behavioural responses of caged savanna tsetse (Glossina morsitans morsitans and Glossina pallidipes) on a preferred host (ox) and a non-host (waterbuck) suggested the presence of allomonal constituents on the latter. Follow up comparison of the compositions of odours of waterbuck with those of ox and buffalo led to the identification of a series of compounds (15) specific to waterbuck, including straight chain carboxylic acid (C5-C10), phenols (guaiacol and carvacrol), 2-alkanone homologues (C8-C12), geranylacetone and δ-octalactone. Behavioural studies in a windtunnel in the laboratory suggested that G. m. morsitans was repelled by a synthetic blend of waterbuck-specific constituents. In the present study, the effects of different blends of these compounds on catches of mixed sexes of G. pallidipes in attractant-baited NG2G traps were evaluated in the field. Each multicomponent class of constituents (acids, ketones and phenols) was found to reduce fly catches, but a 14-component blend of all these compounds was more effective (reduced catches by 79-85%), indicating that each of these classes of compounds contributes incrementally to the repellency of the waterbuck odour. However, subtractive assays showed some redundancy within each class of compounds, with some even demonstrating attractive properties. Addition of (RS)-δ-octalactone to the 14-component significantly increased the repellency of the resulting blend. A 5-component blend of compounds selected on the basis of their relative performance in subtractive assays (δ-octalactone, guaiacol, geranylacetone, hexanoic and pentanoic acid) showed substantial reduction in fly catches (84%) relative to the baited control. In separate sets of experiments involving an ox tethered in the middle of an incomplete ring of electric screens in the presence or absence of 15-component or 5-component blends, comparable levels in the reduction of fed flies (94 and 96%, respectively) were obtained with the two blends. The chemo-ecological significance and practical implication of these results are highlighted.

Responses of Glossina morsitans morsitans to blends of electroantennographically active compounds in the odors of its preferred (buffalo and ox) and nonpreferred (waterbuck) hosts.

In a previous study, comparison of the behavior of teneral Glossina morsitans morsitans on waterbuck, Kobus defassa (a refractory host), and on two preferred hosts, buffalo, Syncerus caffer, and ox, Bos indicus, suggested the presence of allomones in the waterbuck odor. Examination of the volatile odors by coupled gas chromatography-electroantennographic detection showed that the antennal receptors of the flies detected constituents common to the three bovids (phenols and aldehydes), as well as a series of compounds specific to waterbuck, including C8-C13 methyl ketones, delta-octalactone, and phenols. In this study, behavioral respones of teneral G. m. morsitans to different blends of these compounds were evaluated in a choice wind tunnel. The flies' responses to known or putative attractant blends (the latter comprising EAG-active constituents common to all three animals and those common to buffalo and ox, excluding the known tseste attractants, 4-methylphenol and 3-n-propylphenol), and to putative repellent (the blend of EAG-active compounds specific to the waterbuck volatiles), were different. A major difference related to their initial and final behaviors. When a choice of attractant blends (known or putative) and clean air was presented, flies initially responded by flying upwind toward the odor source, but later moved downwind and rested on either side of the tunnel, with some preference for the side with the odor treatments. However, when presented with a choice of waterbuck-specific blend (putative repellent) and clean air, the flies' initial reaction appeared random; flies flew upwind on either side, but eventually settled down on the odorless side of the tunnel. Flies that flew up the odor plume showed an aversion behavior to the blend. The results lend further support to previous indications for the existence of a tsetse repellent blend in waterbuck body odor and additional attractive constituents in buffalo and ox body odors.