Significant reduction in abundance of peridomestic mosquitoes (Culicidae) and Culicoides midges (Ceratopogonidae): an additional benefit of a novel attract-and-kill intervention applied against Leishmania infantum transmission, in

Background Following the long-term (42-month) evaluation of residual insecticide (co-located with sand fly pheromone) and insecticide-impregnated dog-collars in a randomised control trial (RCT) against leishmaniasis, we assessed the impact of these interventions on the peridomestic abundance and distribution of mosquitoes (Culicidae) and midges (Ceratopogonidae) in Western São Paulo, Brazil. Both of these Dipteran groups are vectors of diseases of medical and veterinary relevance to humans and domestic animals in Brazil. giving a total of 17 applications (four rounds per year).Thus, in this current study we evaluated the impact of the residual insecticide λ-cyhalothrin sprayed in chicken roosting sites (PI-arm) or the presence of deltamethrin-impregnated dog-collars (DC-arm) or no insecticide (C-arm) on the abundance and diversity of mosquitoes and biting midges in the chicken roosts, dog sleeping places and the interior of people’s houses (inside dwellings).


Abstract Background
Following the long-term (42-month) evaluation of residual insecticide (co-located with sand fly pheromone) and insecticide-impregnated dog-collars in a randomised control trial (RCT) against leishmaniasis, we assessed the impact of these interventions on the peridomestic abundance and distribution of mosquitoes (Culicidae) and midges (Ceratopogonidae) in Western São Paulo, Brazil. Both of these Dipteran groups are vectors of diseases of medical and veterinary relevance to humans and domestic animals in Brazil.

Methods
The interventions in the 3-arm RCT: pheromone + insecticide (PI) (chicken roosts sprayed with microencapsulated lambda-cyhalothrin), dog-collars (DC) (dogs fitted with deltamethrin-impregnated collars), and control (C) (unexposed to pyrethroids) were extended by 12 months. During that time adult mosquitoes and midges were sampled at three household locations (inside human dwellings, dog sleeping sites, chicken roosts).

Results
We collected 3,145 culicids (9 genera mostly Culex spp.) distributed relatively uniformly across all 3 arms; 43.5% inside houses, 36.2% at chicken roosts and 20.3% at dog sleeping sites. We collected 11,464 Culicoides (at least 15 species) found mostly at chicken roosting sites (84.7%) compared with dog sleeping sites (12.2%) or houses (3.1%). Mosquitoes and Culicoides were predominant during the hot and rainy reason. Increased daytime temperature was significantly associated with increased mosquito abundance (z = 1.97; P = 0.049) but marginally associated with Culicoides abundance (z = 1.71; P = 0.087). There was no significant association with average rainfall for either group. Household-level mosquito and midge numbers were both significantly reduced by the PI intervention 56% (Incidence Rate Ratio, IRR = 0.54 [95% C.I. 0.30, 0.97], P ≤ 0.05] and 53% (IRR = 0.47 [0.26, 0.85], P ≤ 0.05), respectively. The abundance of both Dipteran groups at dog sleeping sites was largely unaffected by the PI and DC interventions. The PI intervention significantly reduced abundance of mosquitoes inside houses (41%) and at chicken roosting sites (48%) and reduced midge abundance by 51% in chicken roosting sites.

Conclusions
Sprayed insecticide at chicken roosting sites reduced the abundance of mosquitoes and midges at the peridomestic level while dog collars had no effect on numbers for any group.

Background
In Brazil, mosquitoes (Diptera: Culicidae) are by far the most important and well-studied group of blood-sucking insects [1] and > 450 species have been described [2]. Some pathogens transmitted to humans and domestic animals have the potential to cause significant morbidity and mortality [3]. Aedes aegypti is the vector of several viruses, most notably dengue, yellow fever, chikungunya, Zika and the filarial roundworm Wuchereria bancrofti which causes lymphatic filariasis [4]. Culex quinquefasciatus transmits the heartworm causing microfilaria, Dirofilaria immitis, in some coastal cities [5] and is incriminated in the transmission of viruses such as Saint Louis, Mayaro, equine and Rocio encephalitis [3]. Culex spp. also cause significant discomfort and allergic responses through their nocturnal nuisance biting activity and present an increased risk of transmission of new arbovirus and pathogens from avian hosts to humans [6].
The genus Culicoides (Diptera: Ceratopogonidae) includes almost 150 species of biting midges in Brazil [7] and are responsible for the transmission of several viral diseases such as Oropouche virus (OROV), which affects humans in the Amazon Basin, and Bluetongue virus (BTV), which affects wild and domestic ruminants worldwide [8,9]. OROV is one of the most common human arbovirus infections in Brazil and more than 30 major outbreaks and half a million cases have been reported since it was first isolated in 1955 in Trinidad and Tobago [10]. Culicoides midges, e.g. C. paraensis, also cause a significant biting nuisance because of population size and their persistent biting activity [8,11].
Sand flies (Diptera: Psychodidae) are also widespread in Brazil and are found in the same peridomestic environment as mosquitoes and biting midges. There are approximately 285 sand fly species in Brazil and 13 of these are proven vectors of Leishmania spp. [12]. Lutzomyia longipalpis is the most widespread and important vector of the protist parasite Leishmania infantum (Kinetoplastida: Trypanosomatidae), which causes visceral leishmaniasis (VL) in humans and dogs [13].
The recommended VL control options in Brazil include the reactive application of insecticides in houses and animal sheds to reduce vector numbers; the euthanasia of seropositive domestic dogs; the diagnosis and treatment of human cases; and public education [14,15]. However, despite the efforts of the Ministry of Health, the burden of VL in Brazil more than doubled between 1990 and 2016 [16].
Recently, a new vector control approach using a synthetic version of a Lu. longipalpis sex pheromone (9methylgermacrene-B) co-located with microencapsulated λ-cyhalothrin to reduce vector densities and canine Leishmania infantum infection incidence in dogs was tested in a large-scale, long-term (42 month) stratified randomised control trial (sRCT) in the Araçatuba region of Western São Paulo State, Brazil. The trial which also investigated the use of Scalibor® deltamethrin impregnated-dog collars, an established sand fly control device, was carried out in 33 municipalities and 9 districts of Araçatuba [17,18].
As part of that study, we investigated for the first time the impact of these two insecticide-based interventions [sprayed residual insecticide (SRI) and insecticide-impregnated dog-collars (IIDC)] on two biting Diptera groups: mosquitoes and Culicoides biting midges, which are pests often found in abundance in chicken sheds, other animal shelters and inside human dwellings throughout Brazil along with Lu. longipalpis sand flies. In addition, the study also gave us the opportunity to assess the species richness, abundance, distribution, annual dynamics and influence of climatic conditions (temperature and rainfall) on mosquitoes and Culicoides midges in households.

Study area
Studies were conducted in the mesoregion of Araçatuba (ca. 11,250 km² and ca. 700,000 inhabitants) in northwest São Paulo State, Brazil. A total of 280 houses in 42 sRCT clusters were included in the Araçatuba region ( Fig. 1; Additional file 1: Table S1). The climate in this region is the Aw type (tropical sub-warm and subdry) according to the Köppen-Geiger classification [19] with two distinct seasons: a dry and cool season from April to September (autumn through to winter), and a hot and wet season from October to March (spring through to summer). The mean annual temperature was 23.8 ºC (min: 17.0, max: 30.6), total annual rainfall was 1309 mm, and the wettest months were January, February and December in decreasing order of rainfall. Climate data (rainfall and temperatures) were obtained from a weather station located at Araçatuba city from July-2015 to April-2016 [20].
All experiments were carried out within private households and within their yards either at the front or back of the house. The average number of hosts per household was (Min, Max; X ± SD): dogs (1, 12; 2.65 ± 1.80), chickens (1, 125; 24.51 ± 21.80) and humans (0, 10; 3.50 ± 1.83). Other poultry (geese, guinea fowls, ducks) and other animals (pigs, goats) were common and kept within the yard which may also have contained fruit trees, flowers or shrubs.

Study design and trapping
The study design followed that of the previously described sRCT [17,18] and collections of mosquitoes and biting midges were concurrently made when collecting sand flies.
The collections were made in each of the three arms of the trial; (1) synthetic pheromone + insecticide colocated in chicken roosting sites including chicken sheds (PI-arm); (2) deltamethrin-impregnated collars fitted to dogs (DC-arm); and (3) a placebo control (C-arm).
Within the PI-arm, microencapsulated λ-cyhalothrin (Demand CS; BASF PLC, Cheshire, UK. 20 mg a.i.m − 2 ) was sprayed using a hand-compression sprayer (GUARANY 441 − 10 compression sprayer, Guarany Industria e Comercio Ltda, SP) according to the guidelines of the Brazilian Ministry of Health of São Paulo State [14]. The pheromone lure containing 10 mg of synthetic pheromone for sand fly attraction, is known to be highly specific, with no attraction even to other subspecies of Lu. longipalpis sand flies [21], therefore we excluded any effect on mosquitoes and biting midges. The insecticidal activity of microencapsulated λ-cyhalothrin formulation offered outstanding residual control of a broad range of flying insects. Sprayed sites were mostly (i) variable size (open, close, semi-close) chicken sheds, (ii) roosting trees from ground level to 3 m up the roosting tree particularly on roosting branches, and into a lesser extent (iii) on walls adjacent to ground roosting chicken or similar unusual sites (3 m 2 area).
Within the DC-arm, each dog living in the dwelling was provided with a collar impregnated with 1.0 g of deltamethrin (Scalibor® Dog Collar, Intervet Productions S.A., France). Collars were replaced every 5-6 months according to the manufacturer instructions.
Control-arm (C), chicken shelters were sprayed with pure water rather than insecticide, and dogs received a placebo collar. Houses selected for the C-arm were described as insecticide-free by the householders as they had no previous residual insecticide application.
The time needed to complete a spraying round (insecticide arms + control households) was approximately 45 days. The insecticide application was carried out in three monthly periods between January 2012 to March 2016 giving a total of 17 applications (four rounds per year).Thus, in this current study we evaluated the impact of the residual insecticide λ-cyhalothrin sprayed in chicken roosting sites (PI-arm) or the presence of deltamethrin-impregnated dog-collars (DC-arm) or no insecticide (C-arm) on the abundance and diversity of mosquitoes and biting midges in the chicken roosts, dog sleeping places and the interior of people's houses (inside dwellings).

Sampling
Adult mosquitoes and Culicoides biting midges were collected with CDC miniature light-suction traps employing a standard incandescent bulb powered by a rechargeable 6V-battery [22]. Trapping was implemented for one night per round per household approximately every three months after the λ-cyhalothrin residual spraying or deltamethrin-dog collar application. After 13 rounds of insecticide intervention, we started trapping both biting Diptera groups in round 14 ( The three CDC traps per household were set from 18.00 to 08.30 h; one located close to a chicken roosting site (e.g. chicken shed or roosting tree), one at the dog sleeping site (e.g. a dog pen or kennel), and one within the house (e.g. a living room, kitchen or bathroom, to minimise disturbance of the residents). In the infrequent event of heavy rain or strong wind, the nights' collections were discarded, and trapping was repeated the following night.

Sample processing and species identification
The live collected insects were placed in a -20º C freezer for 20 min to kill them prior to being placed in 70% alcohol. They were stored until the culicids were separated from the Culicoides, sorted by sex and counted under a binocular stereomicroscope (Quimis Ltda., Sao Paulo) at x4 magnification.
In Culicidae, female morphological features were not conclusive because of their preservation in alcohol. Male culicids were identified to species level based on male genitalia morphology. Because of the large numbers of Culex specimens, only a subsample (ca. 30% of the total catches) were randomly selected from the three household locations) and slide-mounted for determination of species. Heavy-sclerotized male genitalia was first cleared (10% potassium hydroxide for 24 h), then dehydrated (ethanol series from 70-100%) and finally immersed in a clearing agent (eugenol) before being mounted in balsam and allowed to dry at room temperature for several days (adapted from Consoli and Lourenço-Oliveira et al. [1]. Specimens were identified in the Laboratorio de Transmissores de Hematozoários of the Institute Oswaldo Cruz (IOC, Rio de Janeiro, Brazil) using taxonomic keys [3,[23][24][25][26]. Culicoides species identification was based initially on wing pattern and then confirmed by mounting the specimens directly in Canadian Balsam on glass slides, allowed to dry at room temperature for several days and identified with the appropriate taxonomic keys [27][28][29] and with access to the reference collection of Neotropical Culicoides housed at the Museo de La Plata, Buenos Aires, Argentina. Voucher specimens of both Diptera groups are available upon request.

Statistical Analyses
Data were statistically analysed for impact of insecticide intervention (abundance and distribution) and climatic variables (temperature and rainfall). Household covariate data, the abundance of people, dogs and chickens were collected concurrently during alternative quarterly data collection but assumed to be representative of the household. Data were matched to Dipteran counts by household ID, and date. To assess the impact of the insecticide interventions, we compared changes in the total numbers (as well as numbers of males + females separately) of mosquitoes and biting midges captured per household, and at each of the described chicken, dog, and house capture sites.
Nightly Diptera trapping records per household were excluded from analysis where any Dipteran group or trap location within households were missing. Similarly, data were also excluded if household covariate data was missing. Outliers, such as households associated with unusually high host abundance (> 1000 chickens such as chicken farms) are also excluded from analyses. Dipteran count data were analysed by negative binomial regression and clustered on neighbourhood [30]. Seasonal variation between trapping rounds and household host abundances of humans, dogs, and chickens were expected to confound capture rates of biting Diptera, thus, we adjusted for these by inclusion of a priori predictors of trapping round and host abundance in all count analyses.
For both climatic variables (average rainfall and temperature), we analysed the trap count data of both Dipteran groups from the control arm as without the intervention effects it was considered to be the most indicative of seasonal trends. Climatic plots were constructed using Geometric-Williams (GW) means plus 95% CI to make a fairer comparison due to overdispersion over nightly Diptera capture rates. All data were analysed in STATA v.15 (StataCorp LP, College Station, TX).

Mosquitoes (Culicidae)
Species richness. Nine genera of Culicidae were trapped during this study (Table 2). Culex was the most abundant genus and comprised 2,754 specimens (87.6% of the catches), followed by Aedeomyia (105, 3.3%), while Anopheles, Aedes and Mansonia contributed < 7% (  Fig. 2A). Nine genera were recorded in chicken roosting sites, eight in dog sleeping sites and seven in houses.

Annual dynamics and climatic variables.
Mosquitoes were predominantly captured during the summer and early autumn (January and April 2016, rounds 16 and 17, respectively) and to a lesser extent in the early winter and spring (July and October 2015, rounds 14 and 15, respectively). The average daily temperature had a significant positive effect on the average number of mosquitoes (z = 1.97; P = 0.049) with a 0.10 factor change per degree increase in temperature. Rainfall average did not significantly affect mosquito abundance (z = 0.78; P = 0.437) (Fig. 3A). Up to 4x times more specimens were captured in April (the most abundant, GM = 7.9; 15.0-4.8) compared to October (the poorest, GM = 2.3; 3.1-1.8). Similar annual variation was seen in all the captured genera, peaking in summer-autumn (Additional file 1: Fig. 1).
The abundance of chickens was associated with high rates of capture at the household level but only accounted for a small fraction of the variation in the numbers caught (IRR = 1.0; CI 1.00, 1.02; P ≤ 0.1) ( Table 4).
Abundance and distribution. Culicoides (11,464 specimens) were trapped most frequently at chicken roosting sites (9,711, 84.7%), followed by dog sleeping sites (1,396, 12.2%), and to a minor extent houses (357, 3.1%) indicating an exophilic tendency of the species caught (Table 1). Thirteen species were recorded in chicken roosting sites and 11 in both dog sleeping sites and in houses. 2) compared to July, which had the lowest catch (GM = 1.7, 1.9-1.3) (Fig. 3B).

Annual dynamics and climatic variables. Adult
Differences in abundance of the three dominant species were observed throughout the year. Culicoides leopoldoi was present in substantial numbers throughout all four sampling periods with a peak of abundance in January-2016, whereas C. limai was absent in July-2015 but present since October-2015. Culicoides insignis was particularly abundant during the rainy season (January-April 2016) but almost absent over the remaining sampling periods. The other 13 less abundant species followed a similar pattern to C. leopoldoi (Additional file 1: Figure S1).

Impact of the insecticide interventions on
Culicoides abundance and distribution. Analysis of Culicoides abundance indicated that the use of λ-cyhalothrin in the PI-arm significantly reduced (53%) the number of Culicoides (females + males) across the total of all household captures compared to the control arm (IRR = 0.47; 95% CI 0.26, 0.85; P ≤ 0.05) ( Table 4; Fig. 2B). However, when the household trap sites were examined individually only the reduction of Culicoides in chicken roosting sites was significant (IRR = 0.48; 95% CI 0.27, 0.84; P ≤ 0.05) ( Table 4, Fig. 2B). Numbers of females alone followed a similar pattern with a significant reduction at the household level (IRR = 0.45; 95% CI 0.25, 0.81; P ≤ 0.05) and at chicken roosting sites (IRR = 0.47; 95% CI 0.26, 0.84; P ≤ 0.05) but not in houses or at dog sleeping sites (Additional file 2: Table 2).
The use of deltamethrin impregnated dog collars in the DC-arm did not significantly alter Culicoides capture rates at any of the peridomestic sites (Table 4; Fig. 2B).
All three rounds (15, 16 and 17) produced significant peaks of abundance (Table 4). The abundance of animal hosts was a significant predictor of Culicoides capture rates, and greater numbers of both dogs and chickens were associated with larger numbers of Culicoides midges (Table 4).

Discussion
Overall, the pheromone + insecticide intervention applied to control Lu. longipalpis in chicken roosting sites resulted in a reduction in the numbers of Culicidae (mosquitoes) (56%) and Culicoides (biting midges) (53%) in the peridomestic environment (chicken roosting sites + dog sleeping sites + in houses). By contrast, dog collars had no impact on the numbers of either mosquitoes or biting midges. It is likely that the reduction in numbers in the PI-arm was caused by increased mosquito and midge mortality near to chicken roosting sites where insecticide was applied to surfaces that can serve as resting places for blood-seeking/blood-fed Dipterans. In addition, the mortality around chicken roosting sites led to a reduction of mosquitoes (but not biting midges) in houses. A reduction in Lu. longipalpis sand fly abundance attributed to the insecticide + pheromone was also observed in the PI-arm (66% in females and 69% in males) [17]. This was slightly higher than the observed percent reductions in mosquitoes and biting midges. In addition, to the best of our knowledge, our study represents the first promising large-scale attempt to control poultry biters in peridomestic environments of Latin America.
The insecticide deployment had no significant effect on species richness and a few more species (all minor species < 0.1%) of both Diptera groups were found in traps located near chicken roosting sites than in the other locations, which is perhaps not surprising considering that wild-environments are prone to have higher diversity than other sites because they have high host availability, variable vegetation, resting places, and potential breeding sites [31,32].
In this study, Culicids were common and present in most of the sampled households. In particular Culex spp. were abundant and represented nearly 90% of the total catches. Culex quinquefasciatus, the most commonly collected species, is widely distributed in the equatorial, tropical and subtropical regions of Brazil [3,33]. This species is highly endophilic and opportunistic and the females might feed on humans, chickens or many other available hosts, i.e. dogs, horses, cattle, rodents, rabbits, and pigs [1,[34][35][36]. The second most abundant genus was Aedeomyia, represented by the sole tropical species Ad. squamipennis. This species is found throughout most of the American Tropics and is considered to be an important vector of various bird viruses, including Gamboa virus [37]. It is reported as an ornithophilic species, commonly found in association with chickens. Their affinity for light sources might explain the catches in traps located adjacent to dog sleeping sites and houses. Important dengue vectors Aedes aegypti and Ae. albopictus were uncommon in our light traps because they are daytime biters, but they were found mostly in traps in houses confirming their preference for feeding primarily on humans and resting indoors [38].
The study also revealed a rich and abundant midge fauna in peridomestic environments and most of the Culicoides species that have been reported widely in the Neotropics were recorded here. The most predominant species trapped near chickens was C. leopoldoi, a widely-distributed species that is associated with poultry and a wide range of mammals in Brazil [39][40][41]. Culicoides limai is a forest species with an adaptable feeding behaviour [39,42]. Other common species collected such as C. insignis and C. pusillus, are known to be potential vectors of BTV [9,43]. Culicoides insignis is a widespread species often associated with animals and commonly found in pasture environments with cattle and pigs [41,[44][45][46][47] and to a lesser extent attracted towards poultry [40]. In spite of the low numbers collected, the roles of C. paraensis involved in OROV and C. debilipalpis, another competent BTV vector [48], should be considered in future health surveillance programs both for their vectorial capacity and annoyance of humans [49].
Most Culicoides specimens were captured in outdoor traps, the small proportion trapped indoors, predominantly males, suggested an exophilic behaviour and reluctance to enter buildings to feed on humans. Although studies on the degree of exo/endophagy behaviour of Culicoides has not been reported previously in Brazil, it is assumed that most Culicoides species in farm environments are exophilic in tropical areas. Consequently, Culicoides outdoor activity is presumably associated with the presence of host availability (cattle and poultry) [50]. The high proportion of specimens collected in chicken shelters contrasts to the numbers collected inside human dwellings, supporting the hypothesis that outdoor animals (e.g. chickens) divert Culicoides away from entering houses.
Our study found that mosquitoes (mostly Culex) were present throughout the year although there was an increase in abundance from summer to early autumn. The relationship between mosquito abundance and meteorological conditions has been extensively reported on by different authors but the seasonality of peak mosquito numbers varies geographically [51]. These variations may be related to the interaction of availability of breeding sites and other unidentified ecological factors [52]. Other studies have reported high densities of Cx. quinquefasciatus in areas in which preferential breeding sites are scarce, suggesting the existence other elements related to intrinsic residential characteristics as important factors for maintaining the infestation of this mosquito species. Similarly, Culicoides were present all year although substantial higher numbers were recorded during the rainy season, although no significant effect of rainfall was observed in contrast to other studies [42,47,53,54]. Our catches also indicated different patterns of seasonal occurrence possibly related to different potential ecological requirements, i.e. water availability or land use. Culicoides leopoldoi was captured throughout the entire study period, although it was much more abundant in the rainy season [42]. By contrast, C. insignis was restricted to the wet season. This species has previously been captured during autumn and winter in Argentina [55] and during the rainy season in Brazil [47].
Methods to control adult mosquitoes over small areas most commonly include application of insecticide "barrier sprays" on vegetation and other structures where mosquitoes rest during the day [56]. However, mosquito control efficacy with insecticides is highly controversial and success depends on multiple elements [57]. Residual spraying of lambda-cyhalothrin against Cx. pipiens, Ae. albopictus [56], and Anopheles spp. [58] has been carried out in many regions of the world with variable degrees of entomological efficacy. Ground-applied space spray applications to control Culex and Aedes mosquitoes tend not be effective, partially because they tend to rest indoors on objects and other structures that are inaccessible or should not be sprayed (e.g. personal items) rather than on walls and ceilings [58,59]. Interestingly, our study showed that long-term insecticide spraying of poultry shelters targeted adult mosquito (Culex) resting sites and reduced the numbers found in human dwellings as a collateral effect.
There are few published evaluations of the impact of insecticide spraying in houses, animal shelters or poultry on Culicoides abundance. Most studies have focused on topical insecticide applications to livestock or physical barriers to improve animal welfare [60,61]. The impact of environmental spraying in and around sheep pens against Culicoides in Europe to reduce BTV was not conclusive [62,63]. The insecticide λ-cyhalothrin has both repellent and adulticide action against Culicoides spp. [61,64]; other organophosphates and pyrethroids have historically been evaluated against Culicoides with overall unsuccessful results in field trials [8,62]. Thus, the results presented in the current study are promising. The impact of insecticide could be further enhanced if used against adult resting sites and larval feeding sites [65]; in one recent study, a combination of adult insecticides applied outdoors on walls and roofs of animal shelters, combined with applying larvicides on Culicoides breeding sites, resulted in significant reductions in Culicoides abundance [66].
Our study suggested that Scalibor dog collars do not offer any protection against biting Diptera populations. Deltamethrin-impregnated collars have provided anti-feeding protection or insecticidal effects against mosquitoes (e.g. Culex pipiens pipiens) for up to 6 months under laboratory trials [67], making this device potentially an effective solution against common dirofilariasis given the proven feeding behaviour of Culex on dogs [68]. However, our results did not demonstrate effectiveness in reducing mosquito numbers. Similar experiments to test insecticide-impregnated collars against bites of Culicoides have not been reported perhaps because Culicoides do not readily feed on dogs [69,70].
Therefore, we attribute the reductions in the abundance of biting Diptera in the PI-arm to the residual activity of the insecticide sprayed at the chicken roosting sites, which are a likely blood source for both biting Diptera groups, and not to an additional effect of the synthetic sand fly pheromone. As the sex-aggregation pheromone is species specific for Lu. longipalpis it would only attract that species [21]. It is unsurprising that the reductions in the biting Diptera was related to the presence of insecticide; the current analyses demonstrate the potential additional benefit of such insecticidal interventions against sand flies on non-target hematophagous vectors of other important diseases. Such benefits will depend on the behaviour of the given Dipteran species, which may vary in their degree of zoophily and thus their likelihood of coming into contact with the insecticide.

Conclusions
This study demonstrates that spraying λ-cyalothrin has a beneficial effect against medically important Diptera adult populations in and around poultry roosts. From a vector control perspective, this intervention seems likely to be an effective control measure to reduce blood-feeding Diptera and thus, the feeding pressure and capacity to spread pathogens (other than Le. infantum), which present a substantial impact on poultry. The effect of any sustained insecticide spraying campaigns in triggering insecticide resistance and the environmental consequences on beneficial non-target insects, such as dung beetles and pollinators, warrants further investigation.

Abbreviations
RCT:randomised control trial; SRI:sprayed residual insecticide; IIDC:insecticide-impregnated dog-collars; Figure 1 Map of the study area in São Paulo state, Brazil. The region of study (11,250 km2) is showed in an orange rectangle located within the mesoregion of Araçatuba (red coloured area). The location of Araçatuba city is denoted by a black triangle and São Paulo city by a black circle (ArcGIS 10.4.1; layer sources: IBGE -Instituto Brasileiro de Geografía e Estatistica/Ocean Basemap).

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