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Novel insecticides are urgently needed to control insecticide-resistant populations of Anopheles malaria vectors. Broflanilide acts as a non-competitive antagonist of the gamma-aminobutyric acid receptor and has shown prolonged effectiveness as an indoor residual spraying product (VECTRON T500) in experimental hut trials against pyrethroid-resistant vector populations. This multi-centre study expanded upon initial discriminating concentration testing of broflanilide, using six Anopheles insectary colonies (An. gambiae Kisumu KCMUCo, An. gambiae Kisumu NIMR, An. arabiensis KGB, An. arabiensis SENN, An. coluzzii N’Gousso and An. stephensi SK), representing major malaria vector species, to facilitate prospective susceptibility monitoring of this new insecticide; and investigated the potential for cross-resistance to broflanilide via the A296S mutation associated with dieldrin resistance (rdl). Across all vector species tested, the discriminating concentration for broflanilide ranged between LC99 × 2 = 1.126–54.00 μg/ml or LC95 × 3 = 0.7437–17.82 μg/ml. Lower concentrations of broflanilide were required to induce complete mortality of An. arabiensis SENN (dieldrin-resistant), compared to its susceptible counterpart, An. arabiensis KGB, and there was no association between the presence of the rdl mechanism of resistance and survival in broflanilide bioassays, demonstrating a lack of cross-resistance to broflanilide. Study findings provide a benchmark for broflanilide susceptibility monitoring as part of ongoing VECTRON T500 community trials in Tanzania and Benin.

Novel insecticides are urgently needed to control insecticide resistant populations of Anopheles malaria vectors. Broflanilide acts as a non-competitive antagonist of the gamma-aminobutyric acid (GABA) receptor and has shown prolonged effectiveness as an indoor residual spraying (IRS) product (VECTRON™ T500) in experimental hut trials against pyrethroid-resistant vector populations. This multi-centre study expanded upon initial discriminating concentration testing of broflanilide, using six Anopheles insectary colonies (An. gambiae Kisumu KCMUCo, An. gambiae Kisumu NIMR, An. arabiensis KGB, An. arabiensis SENN, An. coluzzii N’Gousso and An. stephensi SK), representing major malaria vector species, to facilitate prospective susceptibility monitoring of this new insecticide; and investigated the potential for cross-resistance to broflanilide via the A296S mutation associated with dieldrin resistance (rdl). Across all vector species tested, the discriminating concentration for broflanilide ranged between LC99x2=1.126 - 54.00 μg/ml or LC95x3=0.7437 - 17.82 μg/ml. Lower concentrations of broflanilide were required to induce complete mortality of An. arabiensis SENN (dieldrin-resistant), compared to its susceptible comparator, An. arabiensis KGB, and there was no association between the presence of the rdlmechanism of resistance and survival in broflanilide bioassays, demonstrating a lack of cross-resistance to broflanilide. Study findings provide a benchmark for broflanilide susceptibility monitoring as part of ongoing VECTRON™ T500 community trials in Tanzania and Benin.

Novel insecticides are urgently needed to control insecticide-resistant populations of Anopheles malaria vectors. Broflanilide acts as a non-competitive antagonist of the gamma-aminobutyric acid receptor and has shown prolonged effectiveness as an indoor residual spraying product (VECTRON T500) in experimental hut trials against pyrethroid-resistant vector populations. This multi-centre study expanded upon initial discriminating concentration testing of broflanilide, using six Anopheles insectary colonies (An. gambiae Kisumu KCMUCo, An. gambiae Kisumu NIMR, An. arabiensis KGB, An. arabiensis SENN, An. coluzzii N'Gousso and An. stephensi SK), representing major malaria vector species, to facilitate prospective susceptibility monitoring of this new insecticide; and investigated the potential for cross-resistance to broflanilide via the A296S mutation associated with dieldrin resistance (rdl). Across all vector species tested, the discriminating concentration for broflanilide ranged between LC

Background: Optimising insecticide use and managing insecticide resistance are important to sustain gains against malaria using long-lasting insecticidal nets (LLINs). Restricting insecticides to where mosquitoes are most likely to make multiple contacts could reduce insecticide needed to treat nets. Previous studies have shown that nets partially treated with a pyrethroid insecticide in experimental huts with human sleepers gave equivalent mortality to a fully treated net. This remains to be demonstrated in LLINs with dual active ingredients developed to control pyrethroid resistant vectors. Here, the efficacy of Interceptor®G2 (IG2; a dual active LLIN containing alpha-cypermethrin and chlorfenapyr) netting in different roof and side combinations with untreated netting was compared against wild free-flying Anopheles arabiensis in terms of mortality, blood-feeding inhibition, exophily and deterrence using the following treatment arms: untreated nets, roof-treated IG2 (IG2 on roof and untreated sides), side-treated IG2 (IG2 on sides and untreated roof) and IG2 (standard fully treated net).Methods: The study was conducted in cow baited experimental huts, Moshi Tanzania, using a 4-arm Latin square design. Test nets had 30 holes cut in panels to simulate a typical net after 2-3 years use. Analysis used grouped logistic regression with random effects to analyse proportions and negative binomial regression to analyse count data.Results: The roof-treated IG2, side-treated IG2 and IG2 nets significantly increased the likelihood of mortality by 51-fold, 138-fold and 223-fold (PP=0.009) compared to the roof-treated IG2 net. While mortality was not significantly different between IG2 and side-treated IG2 (P>0.05) nets, there was a trend of more area covered the higher the mortality. There was no significant difference in the likelihood of blood feeding or exiting between untreated net arm and the treatment arms. Conclusion: The results show that although the roof-treated IG2 net induced greater mortality compared to untreated nets, its efficacy was reduced compared to IG2 nets. Further investigations are required to explore how attractant used (human or animal), hut type, mosquito species, and properties of active ingredient can influence the effectiveness of restricted treatment on Dual-AI nets.

Monitoring the effectiveness of tsetse fly control interventions that aim to reduce transmission of African trypanosomiasis requires highly efficient sampling tools that can catch flies at low densities. The sticky small target (StS-target) has previously been shown to be more effective in sampling Glossina fuscipes fuscipes compared to the biconical trap. However, its efficiency in terms of the proportion of flies it catches out of those that visit it has not been reported. Furthermore, there are no reports on whether tsetse samples caught using the StS-target can be used for subsequent processes such as molecular tests. In this study, we evaluated the efficiency of the biconical trap and targets for sampling G. f. fuscipes. All targets were tiny (0.25 × 0.50 m) but varied in their capture system. We used targets with sticky surface (StS-targets) and those with an electrified surface (ES-targets). We also assessed the suitability of flies caught on the StS-target for molecular tests by amplifying DNA of bacterial communities. Randomized block design experiments were undertaken in Mbita area and Manga Island on Lake Victoria of western Kenya. Fly catches of each sampling tool were compared to those of the sampling tool flanked by electric (E) nets and analyzed using a negative binomial regression. The total catch for each sampling tool alone was divided by the total catch of the sampling tool flanked by two E-nets to obtain its efficiency expressed as a percentage. A proportion of flies caught on the StS-target was preserved for molecular tests. Overall, the efficiencies of the biconical trap, ES-target and StS-target were 7.7%, 13.3% and 27.0%, respectively. A higher proportion of females (69 to 79%) than males approached all the sampling tools, but the trap efficiency was greater for male G. f. fuscipes than females. Furthermore, sequencing the 16S rRNA gene from fly samples caught on the StS-target revealed the presence of Spiroplasma. Our results indicate that the SS-target is the most efficient trap to monitor G. f. fuscipes population during interventions, with the biconical trap being the least efficient, and samples collected from StS-targets are suitable for molecular studies.

Introduction A variety of techniques have been used to control tsetse with varying degrees of success. In a study on the population structure of Glossina fuscipes fuscipes that recovered after a previous vector control trial on two Kenyan islands, it was reported that the average fly size on the intervention islands was significantly smaller than on the none intervention islands and also compared to the size before the intervention. The conclusion was that vector control using tiny targets exerted size selection pressure on the population. The study recommended for further studies and suggested that this phenomenon could be among the reasons why targets used as a sole control method have rare reports of successful elimination of tsetse populations. Therefore, in this paper we report on a study of body size of tsetse flies caught in epsilon traps (as a stationary device) and black screen fly rounds (as a mobile trapping device). Materials and methods The study was carried out in eastern Zambia to test the hypothesis that the body size (measured as wing length) of G. m. morsitans males or females, captured by epsilon traps and fly rounds is the same. Results A total of 1442 (489 females and 953 males) wing length measurements of G. m. morsitans were used in the analysis. It was established that tsetse flies caught by epsilon traps are on average larger than those caught by fly rounds. The likelihood of a large female or male fly being caught by traps, relative to a small one, significantly increased by 5.088 times (95% CI: 3.138–8.429) and by 2.563 times (95% CI: 1.584–4.148), respectively, p Conclusion This study showed that epsilon traps capture significantly larger G. m. morsitans than fly rounds do. Therefore, further research is recommended to verify i) whether the predilection of traps to capture larger flies has an effect on the process of tsetse elimination when targets are used e.g. targets may take longer to reach elimination than if the predilection was not there, ii) whether different results can be obtained on ecogeographic distribution of different sizes of the species if fly rounds are used for sampling instead of epsilon traps. The results from such studies could influence the strategies used in future control operations.

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.

Tsetse flies (Diptera: Glossinidae) are considered primary cyclical vectors that transmit pathogenic trypanosomes in Africa. They harbour a variety of microbes including Wolbachia, Sodalis and the salivary gland hypertrophy virus (SGHV) which are all vertically transmitted. Knowledge on tsetse microbiome and their interactions may identify novel strategies for tsetse fly and trypanosomiasis control. Area-wide application of such strategies requires an understanding of the natural microbiome frequency in the different species and subspecies of Glossina in their geographical populations. Consequently, this study determined the prevalence of Sodalis, Wolbachia, SGHV and trypanosome infections in Glossina morsitanscentralis from two sites of Western Zambia. We also explored possible associations of the microbes with trypanosome infections. Male G. morsitanscentralis samples were collected from two sites (Lyoni and Lusinina) in Western Zambia. The age structure of the flies at each site was determined using the wing fray method. DNA was extracted from the samples and analyzed for Wolbachia, Sodalis, SGHV and trypanosome presence using PCR. Associations and measures of associations between trypanosome infection and microbes in the fly were determined. The flies from the two locations (Lusinina, n=45 and Lyoni, n=24) had a similar age structure with their median fray category not being significantly different (p=0.698). The overall prevalence of Wolbachia was 72.5% (95% CI: 61.6-83.3%), Sodalis was 15.9% (95% CI: 7.1-24.8%), SGHV was 31.9% (95% CI: 20.6-43.2%) and Trypanosoma species was 23.2% (95% CI: 13-33.4%). The prevalence of Wolbachia was significantly higher in Lusinina than Lyoni (p=0.000). However this was not the case for Sodalis, SGHV and Trypanosoma species. Despite the low number of flies that were positive for both trypanosome and Sodalis (6; 8.7%), a statistically significant association (p=0.013; AOR 6.2; 95% CI: 1.5-25.8) was observed in G. morsitanscentralis. The study showed that the prevalence of microbiota may vary within the same species of the tsetse depending on the geographical location as was the case of Wolbachia. Further it showed that infection with Sodalis could affect vector competence. The study concludes that Sodalis could be an ideal candidate for symbiont-mediated trypanosomiasis control interventions in G. morsitanscentralis.

Trypanocides will continue to play an important role in the control of tsetse fly transmitted trypanosomosis now and in the near future. The drugs are mostly administered by farmers without any veterinary supervision leading to misuse and under dosing of medication, and these could be factors that promote trypanocidal drug resistance (TDR) development. In order to delay or prevent TDR, the Food and Agriculture Organization (FAO) recommended guidelines on trypanocide use. It is not known if these recommended guidelines are adhered to in Itezhi tezhi district of Zambia. A survey was undertaken to examine how socio-economic and environmental factors were associated with adherence to the recommended guidelines on trypanocide use in Itezhi tezhi, Central Zambia. Ninety farmers who use trypanocides were interviewed using a questionnaire to collect their socio-economic characteristics (age, education in years, cattle herd size, competence on trypanocide use and their access to extension on trypanocide use) and trypanocide usage practices while crush pens which they use were stratified according to location, whether in the Game Management Area (GMA) (Mutenda, Itumbi, Kapulwe and Banachoongo) or non-GMA (Iyanda, New Ngoma and Shinampamba) as an environmental factor. Associations and measures of associations to adherence of FAO guidelines were determined. The results showed that 25.6% of the farmers adhered to guidelines by FAO on trypanocide use and that none of the socio-economic factors under investigation were significantly associated with it. Further the farmers that used crush pens that were in the GMA had an 80% reduction in the likelihood of adhering to the FAO guidelines on trypanocide use than those that used crush pens in the non-GMA (AOR 0.20, 95% CI: 0.05–0.81, P = 0.02). There was low adherence to the recommended FAO guidelines on trypanocide use and it was associated with the location of the crush pen whether in the GMA or not, as an environmental factor. With farmers in the GMA less likely to adhere to FAO guidelines than those in the non-GMA, we recommend an integrated approach of measures to control trypanosomosis in the GMA of Itezhi tezhi to lessen overuse of trypanocides by the farmers.

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% from3 flies/trap/day to1fly/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 were9 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.

Background The measure of anaemia status using packed cell volume (PCV) can be a reliable indicator of African trypanosomosis (AT) in the absence of other anaemia-causing conditions. However, studies that have estimated prevalence of anaemia in cattle from AT endemic areas have rarely reported the prevalence of the disease in the anaemic cattle. Therefore we investigated the prevalence of AT in anaemic cattle at sites that had recently reported the disease in Itezhi tezhi district of central Zambia. Methods During a survey, blood samples were collected from 564 randomly selected cattle for anaemia determination from seven crush pens (Mutenda, Kapulwe, Banachoongo, Itumbi, Iyanda, New Ngoma and Shinampamba). At a PCV- value cut off of 26 %, all samples positive for anaemia were subjected to both parasitological examination on thick and thin blood smears and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for detection of trypanosome DNA. Fisher’s exact test and a mixed effect logistic regression analyses were used to determine and measures associations, respectively. Results Of 564 cattle screened, 58 (10.3 %; 95 % CI: 7.8–12.8 %) had anaemia. PCR-RFLP results showed that 17 (29.3 %; 95 % CI; 17.2–41.4 %) anaemic cattle were positive for pathogenic trypanosomes compared to 1 (1.7 %; 95 % CI: 0.0–5.2 %) on parasitological examination using thick smears. The infections were caused by Trypanosoma congolense and Trypanosoma vivax. Fisher’s exact test showed a strong association between PCV and pathogenic trypanosome infection (P = 0.004). A mixed effect multivariate logistic regression showed that a one unit increase in PCV reduced the likelihood of detecting AT with PCR-RFLP by 24.7 % (95 % CI: 4.6–40.6 %; P = 0.019) in anaemic cattle, taking into account their age and sex, with random effects for crush pen. Conclusion These results suggest that T. congolense and T. vivax could be important causes of anaemia in cattle reared in AT endemic areas of Itezhi tezhi in Central Zambia. This also suggests that even though pathogenic trypanosomal infection was strongly associated with PCV, it could only account for up to 41 % of the anaemia in cattle. Therefore further investigation to ascertain other factors responsible for anaemia in AT endemic areas of Itezhi tezhi in Central Zambia is needed.