An Integrated Pest Management Strategy Approach for the Management of the Stable Fly Stomoxys calcitrans (Diptera: Muscidae).

Stable flies, Stomoxys calcitrans, stand as formidable pests with a global impact, inflicting significant economic losses on the livestock sector. Larval development occurs in diverse substrates, including decomposing plant material and manure, while emerged adults pose a threat through blood-feeding on both animals and humans. Conventional chemical control methods, predominantly reliant on insecticides, not only pose environmental risks but also face challenges of resistance among stable fly populations. To address this pressing issue, we propose an integrated pest management (IPM) strategy for stable fly control. This approach involved a combination of sanitary-cultural practices, animal protection, the release of natural enemies targeting immature stages, and a specialized trapping system for adults. The Stomoxycc® trap, designed for mass trapping of adult Stomoxys, was employed alongside the release of the predatory mite Macrocheles robustulus and two wasp parasitoids, Spalangia cameroni and Muscidifurax raptor (under the commercial brands Biomite® and Biowasp®) on animal bedding as a key component of this IPM strategy. The implementation of this initiative has been undertaken at a significant sanctuary for donkeys and mules in western Spain. In this publication, we present the application and results of the IPM strategy utilized and provide insights into its use as a sustainable and environmentally friendly option for controlling stable fly populations.

Mosquitoes in urban green spaces and cemeteries in northern Spain.

BACKGROUND: Mosquitoes inhabiting urban green spaces and cemeteries in Europe represent a crucial facet of public health concern and contribute to the ecological balance. As urbanization intensifies, these areas increasingly serve as vital habitats for various mosquito species, fostering breeding grounds and increasing the risk of disease transmission. METHODS: A study was conducted in the three main cities (inland, coastal, and estuarine) of the Basque Country, northern Spain, to investigate the species composition, abundance, dynamic populations, larval habitats, and host preferences of mosquitoes in urban green spaces and cemeteries. CDC traps and dipping were used to collect mosquitoes for 2 years (2019-2020). RESULTS: A total of 21 mosquito species were identified, with Culex pipiens s.l. being the most abundant and widespread. The three ecological forms of Cx. pipiens were found, and Cx. pipiens pipiens was the most common in both green areas and cemeteries. Morphological identification together with molecular tools identified 65 COI sequences with high homology. The highest species richness was found in the inland city, followed by the coastal city and the estuarine city. Mosquito abundance was significantly higher in green areas compared to cemeteries and in the coastal and estuarine cities compared to the inland city. The investigation of larval breeding sites highlighted the dominance of Cx. pipiens s.l., particularly in semi-artificial ponds, diverse water-holding containers (tyres and buckets) and drainage systems in green areas; in cemeteries, most of the larvae were found in flowerpots and funerary urns. Seasonal activity exhibited variable peaks in mosquito abundance in the different cities, with a notable increase in July or August. Additionally, blood meal analysis revealed that Cx. pipiens s.l. fed on several common urban avian species. CONCLUSIONS: Studies on mosquitoes are essential to understand their role in disease transmission and to design targeted and sustainable management strategies to mitigate the associated risks.

Surveillance and screening of Stomoxyinae flies from Mallorca Island (Spain) reveal the absence of selected pathogens but confirm the presence of the endosymbiotic bacterium Wolbachia pipientis.

Adult brachycera biting flies can significantly impact livestock through both direct effects (reduction of food intake, disturbance, painful bites, and blood loss) and indirect effects (pathogen transmission), leading to substantial economic losses and production damage. This study aimed to assess the presence of blood-sucking flies in six mixed-animal farm environments on the island of Mallorca (Balearic Islands, Spain) by employing multiple trapping methods. Additionally, distribution maps of brachycera biting fly species recorded in Spain were created, based on data extracted thorough review of scientific literature and citizen digital databases. Investigation of several pathogens, including equine infectious anemia virus (EIAV), Anaplasmataceae bacteria, and piroplasm protozoa, was carried out using different PCR targets (18S rRNA, 16S rRNA, groESL, and tat genes). Citizen science databases and literature review corroborated the consistent distribution trend for two Stomoxyinae species, underscoring the importance of citizen collaboration as a complement to traditional entomological surveillance. Our study confirmed the presence of two biting Stomoxyinae species: the prevalent stable fly Stomoxys calcitrans across all sampled farms, and the horn fly Haematobia irritans, which turned out to be less abundant. DNA barcoding techniques validated the identification of the two species. Neither EIAV nor bacterial/protozoan pathogens were detected using the selected PCR targets in either fly species. However, Wolbachia pipientis (clustered in the supergroup A together with the only sequence of W. pipientis from the USA) was identified through PCR targeting 16S rRNA, groESL and wsp genes in all pools of H. irritans (n = 13) collected from two of the examined farms. This study represents the first attempt to investigate pathogens in Stomoxyinae biting flies in Spain. The discovery of the endosymbiotic Wolbachia organism in H. irritans represents the first record in Spain and the second from Europe. This finding holds significant implications for future research on the applications of this bacterium in biocontrol programs.

Reconstructing the post-glacial spread of the sand fly Phlebotomus mascittii Grassi, 1908 (Diptera: Psychodidae) in Europe.

Phlebotomine sand flies (Diptera: Phlebotominae) are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae). In Central Europe, Phlebotomus mascittii is the predominant species, but largely understudied. To better understand factors driving its current distribution, we infer patterns of genetic diversity by testing for signals of population expansion based on two mitochondrial genes and model current and past climate and habitat suitability for seven post-glacial maximum periods, taking 19 climatic variables into account. Consequently, we elucidate their connections by environmental-geographical network analysis. Most analyzed populations share a main haplotype tracing back to a single glacial maximum refuge area on the Mediterranean coasts of South France, which is supported by network analysis. The rapid range expansion of Ph. mascittii likely started in the early mid-Holocene epoch until today and its spread possibly followed two routes. The first one was through northern France to Germany and then Belgium, and the second across the Ligurian coast through present-day Slovenia to Austria, toward the northern Balkans. Here we present a combined approach to reveal glacial refugia and post-glacial spread of Ph. mascittii and observed discrepancies between the modelled and the current known distribution might reveal yet overlooked populations and potential further spread.

First report of Culicoides caucoliberensis in Spain: Exploring molecular phylogeny, host-feeding behaviour and avian haemosporidian parasites.

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of pathogens that affect wildlife and livestock. Understanding the composition and distribution of vector species is crucial for implementing control strategies and preventing the spread of infectious diseases. This study provides a morphological and molecular characterisation of Culicoides caucoliberensis, which represents the first record for Spain, increasing the number of Culicoides species in the country to 85. A total of 213 specimens were collected using Onderstepoort-ultraviolet down-draught light traps on a rocky coastline in the Balearic Islands during two sampling periods in 2022. Phylogenetic analysis showed that C. caucoliberensis forms a monophyletic cluster within the Maritimus group. Host preferences were determined for the first time and showed propensity to feed on the European shag (Phalacrocorax aristotelis). The vector role of C. caucoliberensis for haemosporidian transmission remains unclear since molecular detection of Haemosporidians (Haemoproteus and Plasmodium) was negative for all the pools of parous and engorged females analysed. This study emphasises the importance of conducting entomofauna studies in lesser-known Mediterranean islet landscapes and highlights the need for research on vectors within the One Health framework.

Mosquito community composition in two major stopover aquatic ecosystems used by migratory birds in northern Spain.

Mosquitoes (Diptera: Culicidae) are common bloodsucking Diptera frequently found in aquatic environments, which are valuable ecosystems for many animal species, particularly migrating birds. Therefore, interactions between these animal species and mosquitoes may play a critical role in pathogen transmission. During 2018-2019, mosquitoes were collected from two aquatic ecosystems in northern Spain using different methodologies and identified using classical morphology and molecular tools. A total of 1529 males and females of 22 native mosquito species (including eight new records for the region) were trapped using CO2 -baited Centers for Disease Control and Prevention (CDC) traps and sweep netting. Among the blood-fed female mosquitoes, 11 vertebrate host species-six mammals and five birds-were identified using DNA barcoding. The developmental sites of eight mosquito species were determined across nine microhabitats, and 11 mosquito species were caught landing on humans. The flight period varied among mosquito species, with some peaking in the spring and others in the summer. Our study highlights the advantages of mosquito sampling using various techniques to comprehensively characterise species composition and abundance. Information on the trophic preferences, biting behaviour and influence of climatic variables on the ecology of mosquitoes is also provided.

First record of the mermithid nematode worm Isomermis lairdi parasitizing black flies in Spain.

Mermithid nematodes are considered a promising biological control agent to reduce the population density of different blood-feeding vectors, i.e. black flies (Diptera: Simuliidae), which are important pests of medical and veterinary interest worldwide. Immature larvae of black flies were collected in a rill from La Rioja (Northern Spain) in the summer of 2016. Isomermis lairdi Mondet, Poinar & Bernadou, 1977 (Nematoda: Mermithidae) was found parasitizing eleven specimens of Simulium cryophilum s.l. (Rubtsov, 1959) (prevalence of 52%), which represent the first record of this nematode for Spain and the second for Europe. The confirmation of the nematode and the black fly species was carried out by both morphological and molecular approaches using the 18S ribosomal RNA and the cytochrome c oxidase subunit I genes. Phylogenetic analyses indicated that the collected specimens were Isomermis lairdi (99.4-99.9% identity with homologues from Africa) with a sequence divergence of 0.2%. The role of Isomermis lairdi as an alternative tool in the biological control of black flies in Spain should be further explored.

Culicoides species community composition and feeding preferences in two aquatic ecosystems in northern Spain.

BACKGROUND: Aquatic ecosystems provide breeding sites for blood-sucking insects such as Culicoides biting midges (Diptera: Ceratopogonidae), but factors affecting their distribution and host choice are poorly understood. A study was undertaken at two nature reserves in northern Spain to examine the abundance, species composition, population dynamics and feeding patterns of biting midges between 2018 and 2019. METHODS: Culicoides were captured by light suction traps baited with CO2 and by sweep netting vegetation. Blood meals and species identification of blood-fed specimens were determined using cytochrome c oxidase I subunit (COI) DNA barcoding. Multivariate generalized linear models were used to evaluate the associations between the abundance of Culicoides, the species richness and other parameters. RESULTS: The 4973 identified specimens comprised 28 species of Culicoides. These included two species reported for the first time in northern Spain, thus raising to 54 the number of Culicoides species described in the region. Specimens of all 28 species and 99.6% of the total specimens collected were caught in suction traps, while sweep netting vegetation revealed just 11 species and 0.4% of the total specimens. Midge abundance peaked in June/early July, with five species comprising > 80% of the captures: Culicoides alazanicus (24.9%), Culicoides griseidorsum (20.3%), Culicoides poperinghensis (16.2%), Culicoides kibunensis (10.7%) and Culicoides clastrieri (9.6%). DNA barcode analysis of blood meals from eight Culicoides species revealed that they fed on 17 vertebrate species (3 mammals and 14 birds). Species in the subgenus Avaritia were primarily ornithophilic, except for C. griseidorsum and C. poperinghensis. Host DNA from blood meals was successfully amplified from 75% of blood-fed females. A pictorial blood meal digestion scale is provided to accurately assess the blood-fed status of female Culicoides. CONCLUSIONS: The large number of different blood meal sources identified in the midges captured in this study signals the likely importance of wild birds and mammals (e.g. red deer and wild boar) as reservoir/amplifying hosts for pathogens. Available hosts are more exposed to being bitten by biting midge populations in aquatic ecosystems in late spring and early summer.

Severe Skin Lesions Caused by Persistent Bites of the Stable Fly Stomoxys calcitrans (Diptera: Muscidae) in a Donkey Sanctuary of Western Spain.

Animal sanctuaries are important organisations that promote animal welfare and health as well as social awareness. Following the appearance of several donkeys with severe and distinctive skin lesions in a sanctuary from western Spain in 2021, a multicomponent study was performed to rule out the possible causes. The lesions were mainly concentrated on the extremities and, to a lesser extent, on the face and/or chest. The use of cotton leggings on their extremities as an external barrier to prevent them from bites and treatments with antiparasitic, antiseptic washes and dermatitis lotion showed to be effective measures to improve the donkey lesions. Skin scraping was negative for any relevant causative agents. Histopathological examination of the lesions showed an inflammatory infiltrate at the superficial dermal level, compatible with diffuse chronic dermatitis. A field entomological study was conducted from May to October 2021 to ascertain if any Diptera was responsible for these lesions. Considerable numbers of the stable fly Stomoxys calcitrans were recorded in both sticky traps and decomposing straw bedding. Several species of hematophagous Diptera were also recorded in lower numbers in other traps placed on the farm facilities. According to the entomological data, the location of the bites and clinical signs, it was concluded that S. calcitrans was the main cause of the skin lesions in the donkeys. To our best knowledge, this is the first clinical case of donkeys affected by the stable fly in Spain. For differential diagnosis, a brief discussion of the evidence caused by other biting Diptera groups is provided.

A Survey on Native and Invasive Mosquitoes and Other Biting Dipterans in Northern Spain.

PURPOSE: Haematophagous Diptera, such as mosquitoes (Culicidae), biting midges (Ceratopogonidae), and black flies (Simuliidae), are important insects for public and animal health due to their capacity to bite and transmit pathogens. Outdoor recreation areas are usually affected by biting species and provide suitable habitats to both adult and immature stages. This study aimed to determine the species diversity and larval sites of these Diptera groups in two golf courses. METHODS: A multi-method collection approach using ultraviolet-CDC traps, human landing catches, collection in breeding sites, and ovitraps was implemented during summer 2020 in northern Spain. Insects were determined by morphological features accompanied by DNA barcoding. RESULTS: A total of ten native mosquito species were recorded either as adults or as larval stages. The invasive species Aedes japonicus was collected only at egg or pupa stage in ovitraps. Culex pipiens s.l. and Culex torrentium were both common mosquito species accounting for 47.9% of the total larval site collections and their larvae might be found in a wide range of natural and artificial sites. Culiseta longiareolata specimens were also prominent (30.1% of the total) and occurred exclusively in man-made water-filled containers. A total of 13 Culicoides species were identified, 10 of which were captured by ultraviolet-CDC traps, particularly members of the Obsoletus complex (Culicoides obsoletus/Culicoides scoticus, 74.9%) and seven species by emergence traps, being the two most abundant C. kibunensis (44.8%) and C. festivipennis (34.9%). Simulium cryophilum was also collected hovering around the operator under field sampling. CONCLUSION: A comprehensive representation of the blood-sucking Diptera fauna and their larval sites was obtained by the multi-method approach in two Spanish golf courses.

Diversity and abundance of tabanids in Northern Spain.

Tabanids (Diptera: Tabanidae) are large haematophagous flies that cause both direct (by biting nuisance) and indirect (primarily by mechanical transmission of diseases) damage to host species. Research studies on this family have received little attention in some parts of Europe. Our aims were to characterise the species richness, abundance, and peak of activity of tabanid fly species in a region of Northern Spain. Home-made canopy traps, sweep nets, and Malaise traps were employed for the collection of tabanids across four cattle farms, two equestrian centres, and two golf courses during a 3-month period in the summer of 2020. A total of 300 specimens of 27 tabanid species belonging to eight genera were identified. The most prevalent species were Haematopota pluvialis (23.3%), Tabanus eggeri (20.0%), and Tabanus bromius (8.0%). The former species was recorded biting humans and therefore should be considered of relevance to public health. Tabanids were more diverse and abundant in scrubland and grazing pastures [relative abundance (RA) = > 10%; species richness (S) = 8-12; Shannon-Index (H´) = 1.5 - 2.1] compared to crop landscapes (RA = < 1%; S = 0-1; H´ = 0) according to canopy traps. The tabanid population dynamics was determined to be short, with the greatest abundance and diversity concentrated in mid-late July. This study updates the checklist of this Diptera group in the Northern Spain from nine known extant species to 31 species, providing the first data on the summer peaks of activity of tabanids for this region.

At the tip of an iceberg: citizen science and active surveillance collaborating to broaden the known distribution of Aedes japonicus in Spain.

BACKGROUND: Active surveillance aimed at the early detection of invasive mosquito species is usually focused on seaports and airports as points of entry, and along road networks as dispersion paths. In a number of cases, however, the first detections of colonizing populations are made by citizens, either because the species has already moved beyond the implemented active surveillance sites or because there is no surveillance in place. This was the case of the first detection in 2018 of the Asian bush mosquito, Aedes japonicus, in Asturias (northern Spain) by the citizen science platform Mosquito Alert. METHODS: The collaboration between Mosquito Alert, the Ministry of Health, local authorities and academic researchers resulted in a multi-source surveillance combining active field sampling with broader temporal and spatial citizen-sourced data, resulting in a more flexible and efficient surveillance strategy. RESULTS: Between 2018 and 2020, the joint efforts of administrative bodies, academic teams and citizen-sourced data led to the discovery of this species in northern regions of Spain such as Cantabria and the Basque Country. This raised the estimated area of occurrence of Ae. japonicus from < 900 km2 in 2018 to > 7000 km2 in 2020. CONCLUSIONS: This population cluster is geographically isolated from any other population in Europe, which raises questions about its origin, path of introduction and dispersal means, while also highlighting the need to enhance surveillance systems by closely combining crowd-sourced surveillance with public health and mosquito control agencies' efforts, from local to continental scales. This multi-actor approach for surveillance (either passive and active) shows high potential efficiency in the surveillance of other invasive mosquito species, and specifically the major vector Aedes aegypti which is already present in some parts of Europe.

Diversity and distribution of larval habitats of mosquitoes (Diptera: Culicidae) in northern Spain: from urban to natural areas.

Studies of the biodiversity of mosquito larval habitats are important for vector-borne disease control programs and help to improve vector distribution maps. This study was designed to investigate the geographical distribution of mosquito species and their larval habitats in urban, rural, and natural areas in northern Spain. Pre-imaginal stages were collected over two sampling periods (spring and summer) in 2019. In the laboratory, immature specimens were reared to the adult stage for species identification by morphological taxonomy and/or molecular methods. In total, 2,182 specimens belonging to 13 different native mosquito species of five genera were collected from 135 sampling points of which 59.2% harbored larvae. Culex pipiens s.l. was the most abundant species (45.1%), followed by Culex torrentium (12.3%), Anopheles maculipennis s.l. (10.2%), Culex hortensis (9.5%), and nine other species with lower frequencies that accounted for less than 25%. By molecular identification, An. maculipennis s.s. was identified as the only species within the An. maculipennis species complex and Cx. pipiens pipiens as the predominant subspecies of the Cx. pipiens species complex. Margins in large sunlit water bodies were the most suitable sites for An. maculipennis s.l., whereas Cx. pipiens s.l. was present in both natural and artificial habitats. The larval site index, species richness, and relative abundance of the mosquitoes were determined based on the characteristics of the sites where they were collected. The public health importance and ecology of some identified mosquitoes is also discussed.

Significant reduction in abundance of peridomestic mosquitoes (Culicidae) and Culicoides midges (Ceratopogonidae) after chemical intervention in western São Paulo, Brazil.

BACKGROUND: We assessed the impact of two sand fly insecticide interventions (insecticide spraying and insecticide-impregnated dog collars) on the peridomestic abundance and distribution of mosquitoes (Culicidae) and biting midges (Ceratopogonidae) in western São Paulo (Brazil) in a long-term (42-month) evaluation. 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 stratified randomised control trial were: pheromone + insecticide (PI) (chicken roosts were sprayed with microencapsulated lambda-cyhalothrin; pheromone lure has no effect on the Diptera pests studied here); 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 along 280 households at three household locations (inside human dwellings, dog sleeping sites and chicken roosts). RESULTS: We collected 3145 culicids (9 genera, 87.6% Culex spp.) distributed relatively uniformly across all 3 arms: 41.9% at chicken roosts; 37.7% inside houses; and 20.3% at dog sleeping sites. We collected 11,464 Culicoides (15 species) found mostly at chicken roosting sites (84.7%) compared with dog sleeping sites (12.9%) or houses (2.4%). Mosquitoes and Culicoides were most abundant during the hot and rainy season. Increased daytime temperature was marginally associated with increased mosquito abundance (Z = 1.97, P = 0.049) and Culicoides abundance (Z = 1.71, P = 0.087). There was no significant association with daily 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% CI: 0.30-0.97), P ≤ 0.05] and 53% [IRR = 0.47 (95% CI: 0.26-0.85), P ≤ 0.05], respectively, compared to the control intervention. 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.

A survey of tire-breeding mosquitoes (Diptera: Culicidae) in the Dominican Republic: Considerations about a pressing issue. / Estudio de mosquitos (Diptera: Culicidae) que se desarrollan en neumáticos en República Dominicana: consideraciones sobre un problema acuciante

INTRODUCTION: Discarded vehicle tires represent a serious threat both to the environment and to public health as they have the potential to harbor important mosquito (Diptera: Culicidae) vectors. OBJECTIVE: To assess the importance of used vehicle tires as larval habitats for mosquito fauna that colonize these artificial reservoirs in Jarabacoa, Dominican Republic. MATERIALS AND METHODS: Used tires were sampled with pipettes at specialized tire fitting shops and scattered stockpiles of tires between June and August, 2018. RESULTS: We sampled 396 tires; 57 (Container Index=14.4%) were positive for immature stages and contained 2,400 specimens, 11 species, and four genera (Anopheles, Aedes, Culex, and Toxorhynchites). The most abundant species was Aedes albopictus (42.3%) followed by Aedes aegypti (34.3%), and Culex quinquefasciatus (14.0%) while other species (9.4%) were less abundant. The container index varied significantly among the different tire sizes (χ2=13.4; p≤0.05). The highest infestation levels were found in the largest tires. A low positive correlation (r=0.38, n=396; p≤0.001) between the tire size and the prevalence of immature stages was recorded. The presence of organic matter had an overall positive effect on the infestation levels (U=11,430.0; p≤0.001). CONCLUSIONS: These rubber residues, usually located nearby human populations, represent suitable breeding sites for arboviruses vectors such as dengue, chikungunya, Zika, and West Nile.

Introducción. Los neumáticos usados representan una grave amenaza para el medio ambiente y la salud pública, ya que tienen el potencial de albergar mosquitos (Diptera: Culicidae) vectores de enfermedades. Objetivo. Evaluar la importancia de los neumáticos usados como hábitats larvarios de mosquitos que colonizan estos recipientes artificiales en Jarabacoa, República Dominicana. Materiales y métodos. Se muestrearon mediante pipetas neumáticos usados en tiendas de venta y reparación y en vertederos entre junio y agosto de 2018. Resultados. Se muestrearon 396 neumáticos; 57 (índice de contenedor=14,4 %) albergaban estados inmaduros que contenían 2.400 especímenes de 11 especies y cuatro géneros (Anopheles, Aedes, Culex y Toxorhynchites). La especie más abundante fue Aedes albopictus (42,3 %) seguida de Aedes aegypti (34,3 %) y Culex quinquefasciatus (14,0 %), entre otras (9,4 %) de menor abundancia. El índice de contenedor varió significativamente según el tamaño del neumático (χ2=13,4; p≤0,05) y los mayores niveles de infestación se registraron en los neumáticos de mayor tamaño. Se observó una baja correlación positiva (r=0,38; n=396; p≤0,001) entre el tamaño de neumático y la prevalencia de estadios inmaduros. La presencia de materia orgánica tuvo un efecto positivo sobre los niveles de infestación de mosquitos (U=11430,0; p≤0,001). Conclusiones. Estos residuos de caucho, generalmente almacenados cerca de las poblaciones humanas, son propicios para la reproducción de vectores de arbovirus como el dengue, el chikungunya, el Zika y el West Nile.

Synthetic sex-aggregation pheromone of Lutzomyia longipalpis, the South American sand fly vector of Leishmania infantum, attracts males and females over long-distance.

BACKGROUND: In South America the sand fly Lutzomyia longipalpis is the predominant vector of Leishmania infantum, the parasite that causes canine and human visceral leishmaniasis. Co-location of synthetic male sex-aggregation pheromone with an insecticide provided protection against canine seroconversion, parasite infection, reduced tissue parasite loads, and female sand fly densities at households. Optimising the sex-aggregation pheromone + insecticide intervention requires information on the distance over which female and male Lu. longipalpis would be attracted to the synthetic pheromone in the field. METHODOLOGY/PRINCIPAL FINDINGS: Wild Lu. longipalpis were collected at two peridomestic study sites in Governador Valadares (Minas Gerais, Brazil). Sand flies were marked with coloured fluorescent powder using an improved protocol and then released into an existing domestic chicken shed at two independent sites, followed by recapture at synthetic-pheromone host-odour baited traps placed up to 30 metres distant from the release point. In total 1704 wild-caught Lu. longipalpis were released into the two chicken sheds. Overall 4.3% of the marked flies were recaptured in the pheromone baited experimental chicken sheds compared to no marked flies recaptured in the control sheds. At the first site, 14 specimens (10.4% of the marked and released specimens) were recaptured at 10m, 36 (14.8%) at 20m, and 15 (3.4%) at 30m. At the second site, lower recapture rates were recorded; 8 marked specimens (1.3%) were recaptured at 5 and 10m and no marked specimens were recaptured at 15m. Approximately 7x more marked males than females were recaptured although males were only 2x as common as females in the released population. 52% of the marked Lu. longipalpis were collected during the first night of sampling, 32% on the second night, and 16% on the third night. CONCLUSIONS/SIGNIFICANCE: The study established that both male and female sand flies can be attracted to the synthetic sex-aggregation pheromone in the presence of host odour over distances up to at least 30m in the field depending on local environmental and meterological conditions.

A survey of tire-breeding mosquitoes (Diptera: Culicidae) in the Dominican Republic: Considerations about a pressing issue / Estudio de mosquitos (Diptera: Culicidae) que se desarrollan en neumáticos en República Dominicana: consideraciones sobre un problema acuciante

Introducción. Los neumáticos usados representan una grave amenaza para el medio ambiente y la salud pública, ya que tienen el potencial de albergar mosquitos (Diptera: Culicidae) vectores de enfermedades. Objetivo. Evaluar la importancia de los neumáticos usados como hábitats larvarios de mosquitos que colonizan estos recipientes artificiales en Jarabacoa, República Dominicana. Materiales y métodos. Se muestrearon mediante pipetas neumáticos usados en tiendas de venta y reparación y en vertederos entre junio y agosto de 2018. Resultados. Se muestrearon 396 neumáticos; 57 (índice de contenedor=14,4 %) albergaban estados inmaduros que contenían 2.400 especímenes de 11 especies y cuatro géneros (Anopheles, Aedes, Culex y Toxorhynchites). La especie más abundante fue Aedes albopictus (42,3 %) seguida de Aedes aegypti (34,3 %) y Culex quinquefasciatus(14,0 %), entre otras (9,4 %) de menor abundancia. El índice de contenedor varió significativamente según el tamaño del neumático (χ2=13,4; p≤0,05) y los mayores niveles de infestación se registraron en los neumáticos de mayor tamaño. Se observó una baja correlación positiva (r=0,38; n=396; p≤0,001) entre el tamaño de neumático y la prevalencia de estadios inmaduros. La presencia de materia orgánica tuvo un efecto positivo sobre los niveles de infestación de mosquitos (U=11430,0; p≤0,001). Conclusiones. Estos residuos de caucho, generalmente almacenados cerca de las poblaciones humanas, son propicios para la reproducción de vectores de arbovirus como el dengue, el chikungunya, el Zika y el West Nile.

Introduction: Discarded vehicle tires represent a serious threat both to the environment and to public health as they have the potential to harbor important mosquito (Diptera: Culicidae) vectors. Objective: To assess the importance of used vehicle tires as larval habitats for mosquito fauna that colonize these artificial reservoirs in Jarabacoa, Dominican Republic. Materials and methods: Used tires were sampled with pipettes at specialized tire fitting shops and scattered stockpiles of tires between June and August, 2018. Results: We sampled 396 tires; 57 (Container Index=14.4%) were positive for immature stages and contained 2,400 specimens, 11 species, and four genera (Anopheles, Aedes,Culex, and Toxorhynchites). The most abundant species was Aedes albopictus (42.3%) followed by Aedes aegypti (34.3%), and Culex quinquefasciatus (14.0%) while other species (9.4%) were less abundant. The container index varied significantly among the different tire sizes (χ2=13.4; p≤0.05). The highest infestation levels were found in the largest tires. A low positive correlation (r=0.38, n=396; p≤0.001) between the tire size and the prevalence of immature stages was recorded. The presence of organic matter had an overall positive effect on the infestation levels (U=11,430.0; p≤0.001). Conclusions: These rubber residues, usually located nearby human populations, represent suitable breeding sites for arboviruses vectors such as dengue, chikungunya, Zika, and West Nile.

Sand fly synthetic sex-aggregation pheromone co-located with insecticide reduces the incidence of infection in the canine reservoir of visceral leishmaniasis: A stratified cluster randomised trial.

OBJECTIVE: To evaluate the efficacy of a synthetic sex-aggregation pheromone of the sand fly vector Lu. longipalpis, co-located with residual insecticide, to reduce the infection incidence of Leishmania infantum in the canine reservoir. METHODS: A stratified cluster randomised trial was designed to detect a 50% reduction in canine incident infection after 24 months in 42 recruited clusters, randomly assigned to one of three intervention arms (14 cluster each): synthetic pheromone + insecticide, insecticide-impregnated dog collars, or placebo control. Infection incidence was measured by seroconversion to anti-Leishmania serum antibody, Leishmania parasite detection and canine tissue parasite loads. Changes in relative Lu. longipalpis abundance within households were measured by setting three CDC light traps per household. RESULTS: A total 1,454 seronegative dogs were followed-up for a median 15.2 (95% C.I.s: 14.6, 16.2) months per cluster. The pheromone + insecticide intervention provided 13% (95% C.I. 0%, 44.0%) protection against anti-Leishmania antibody seroconversion, 52% (95% C.I. 6.2%, 74·9%) against parasite infection, reduced tissue parasite loads by 53% (95% C.I. 5.4%, 76.7%), and reduced household female sand fly abundance by 49% (95% C.I. 8.2%, 71.3%). Variation in the efficacy against seroconversion varied between trial strata. Equivalent protection attributed to the impregnated-collars were 36% (95% C.I. 14.4%, 51.8%), 23% (95% C.I. 0%, 57·5%), 48% (95% C.I. 0%, 73.4%) and 43% (95% C.I. 0%, 67.9%), respectively. Comparison of the two interventions showed no statistically consistent differences in their efficacies; however, the errors were broad for all outcomes. Reductions in sand fly numbers were predominant where insecticide was located (chicken and dog sleeping sites), with no evidence of insecticide-induced repellence onto humans or dogs. CONCLUSION: The synthetic pheromone co-located with insecticide provides protection particularly against canine L. infantum parasite transmission and sand fly vector abundance. The effect estimates are not dissimilar to those of the insecticide-impregnated collars, which are documented to reduce canine infection incidence, human infection and clinical VL disease incidence, in different global regions. The trialled novel lure-and-kill approach is a low-cost potential vector control tool against ZVL in the Americas.

Limited risk of Zika virus transmission by five Aedes albopictus populations from Spain.

BACKGROUND: Aedes albopictus, the Asian tiger mosquito, is an exotic invasive species in Europe. It has substantial public health relevance due to its potential role in transmitting several human pathogens. Out of the European countries, Spain has one of the highest risk levels of autochthonous arbovirus transmission due to both the high density of Ae. albopictus and the extensive tourist influx from vector-endemic areas. This study aims to investigate the susceptibility of five Ae. albopictus populations from mainland Spain and the Balearic Islands to a Brazilian Zika virus (ZIKV) strain. METHODS: The F1 generation of each Ae. albopictus population was orally challenged with a ZIKV-infected blood meal (1.8 × 106 PFU/ml). At 7 and 14 days post-infection (dpi), mosquito bodies (thorax and abdomen) and heads were individually analysed through RT-qPCR to determine the infection rate (IR) and dissemination rate (DR), respectively. The saliva of infected mosquitoes was inoculated in Vero cells and the transmission rate was assessed by plaque assay or RT-qPCR on ~33 individuals per population. RESULTS: The IR and DR ranged between 12-88%, and 0-60%, respectively, suggesting that ZIKV is capable of crossing the midgut barrier. Remarkably, no infectious viral particle was found in saliva samples, indicating a low ability of ZIKV to overcome the salivary gland barrier. A subsequent assay revealed that a second non-infective blood meal 48 h after ZIKV exposure did not influence Ae. albopictus vector competence. CONCLUSIONS: The oral experimental ZIKV infections performed here indicate that Ae. albopictus from Spain become infected and disseminate the virus through the body but has a limited ability to transmit the Brazilian ZIKV strain through biting. Therefore, the results suggest a limited risk of autochthonous ZIKV transmission in Spain by Ae. albopictus.

Susceptibility of wild-caught Lutzomyia longipalpis (Diptera: Psychodidae) sand flies to insecticide after an extended period of exposure in western São Paulo, Brazil.

BACKGROUND: In Brazil, members of the sand fly species complex Lutzomyia longipalpis transmit Leishmania infantum, a protist parasite that causes visceral leishmaniasis. Male Lu. longipalpis produce a sex pheromone that is attractive to both females and males. During a cluster randomised trial, to determine the combined effect of synthetic sex-aggregation pheromone and insecticide on Le. infantum transmission Lu. longipalpis had been continuously exposed to insecticide for 30 months. The objective of this study was to determine the effect of continuous exposure to the insecticides used in the trial on the susceptibility of Lu. longipalpis population. METHODS: During the trial the sand flies had been exposed to either lambda-cyhalothrin [pheromone + residual insecticide spray (PI)], deltamethrin [dog collars (DC)] or no insecticide [control (C)], for 30 months (November 2012 to April 2015). The insecticide treatment regime was kept in place for an additional 12 months (May 2015-April 2016) during this susceptibility study. Sand flies collected from the field were exposed to WHO insecticide-impregnated papers cyhalothrin (0.05%), deltamethrin (0.5%) and control (silicone oil) in a modified WHO insecticide exposure trial to determine their susceptibility. RESULTS: We collected 788 Lu. longipalpis using CDC-light traps in 31 municipalities across the three trial arms. Probit analysis showed that the knockdown times (KDTs) of Lu. longipalpis collected from the lambda-cyhalothrin exposed PI-arm [KDT50: 31.1 min, confidence interval (CI): 29.6-32.6 and KDT90: 44.2 min, CI: 42.1-46.7] were longer than the KDTs from the non-insecticide-treated C-arm (KDT50: 26.3 min, CI: 25.1-27.6 and KDT90: 38.2, CI: 36.5-40.2) (no-overlapping 95% CIs). KDTs of Lu. longipalpis collected from the deltamethrin exposed DC-arm had similar values (KDT50: 13.7 min, CI: 10.1-16.2 and KDT90: 26.7 min, CI: 21.8-30.6) to those for the C-arm (KDT50: 13.5 min; CI: 12.2-14.8 and KDT90: 23.2 min, CI: 21.4-25.4) (overlapping CIs). The wild-caught unexposed Lu. longipalpis (C-arm), took approximately twice as long to knock down as laboratory-colonised specimens for both insecticides. CONCLUSIONS: Our study reveals slight changes in KDT, in sand flies after prolonged exposure to lambda-cyhalothrin in the presence of pheromone. These changes are not considered to have reached the reference levels indicative of resistance in sand flies suggesting that pheromone and insecticide treatment at the level indicated in this study do not constitute a significant risk of increased insecticide resistance. Prolonged exposure to deltamethrin in dog collars did not result in changes to KDT.