Determinants of the current and future distribution of the West Nile virus mosquito vector Culex pipiens in Spain.

Changes in environmental conditions, whether related or not to human activities, are continuously modifying the geographic distribution of vectors, which in turn affects the dynamics and distribution of vector-borne infectious diseases. Determining the main ecological drivers of vector distribution and how predicted changes in these drivers may alter their future distributions is therefore of major importance. However, the drivers of vector populations are largely specific to each vector species and region. Here, we identify the most important human-activity-related and bioclimatic predictors affecting the current distribution and habitat suitability of the mosquito Culex pipiens and potential future changes in its distribution in Spain. We determined the niche of occurrence (NOO) of the species, which considers only those areas lying within the range of suitable environmental conditions using presence data. Although almost ubiquitous, the distribution of Cx. pipiens is mostly explained by elevation and the degree of urbanization but also, to a lesser extent, by mean temperatures during the wettest season and temperature seasonality. The combination of these predictors highlights the existence of a heterogeneous pattern of habitat suitability, with most suitable areas located in the southern and northeastern coastal areas of Spain, and unsuitable areas located at higher altitude and in colder regions. Future climatic predictions indicate a net decrease in distribution of up to 29.55%, probably due to warming and greater temperature oscillations. Despite these predicted changes in vector distribution, their effects on the incidence of infectious diseases are, however, difficult to forecast since different processes such as local adaptation to temperature, vector-pathogen interactions, and human-derived changes in landscape may play important roles in shaping the future dynamics of pathogen transmission.

Micro-computed tomography visualization of the vestigial alimentary canal in adult oestrid flies.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage as they acquire all necessary nutrients during the parasitic larval stage. The adult mouthparts and digestive tract are therefore frequently vestigial; however, morphological data on the alimentary canal in adult oestrid flies are scarce and a proper visualization of this organ system within the adult body is lacking. The present work visualizes the morphology of the alimentary canal in adults of two oestrid species, Oestrus ovis L. and Hypoderma lineatum (de Villiers), with the use of non-invasive micro-computed tomography (micro-CT) and compares it with the highly developed alimentary canal of the blow fly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). Both O. ovis and H. lineatum adults showed significant reductions of the cardia and the diameter of the digestive tract, an absence of the helicoidal portion of the midgut typical of other cyclorrhaphous flies, and a lack of crop and salivary glands. Given the current interest in the alimentary canal in adult dipterans in biomedical and developmental biology studies, further understanding of the morphology and development of this organ system in adult oestrids may provide valuable new insights in several areas of research.

Colonization of the Mediterranean basin by the vector biting midge species Culicoides imicola: an old story.

Understanding the demographic history and genetic make-up of colonizing species is critical for inferring population sources and colonization routes. This is of main interest for designing accurate control measures in areas newly colonized by vector species of economically important pathogens. The biting midge Culicoides imicola is a major vector of orbiviruses to livestock. Historically, the distribution of this species was limited to the Afrotropical region. Entomological surveys first revealed the presence of C. imicola in the south of the Mediterranean basin by the 1970s. Following recurrent reports of massive bluetongue outbreaks since the 1990s, the presence of the species was confirmed in northern areas. In this study, we addressed the chronology and processes of C. imicola colonization in the Mediterranean basin. We characterized the genetic structure of its populations across Mediterranean and African regions using both mitochondrial and nuclear markers, and combined phylogeographical analyses with population genetics and approximate Bayesian computation. We found a west/east genetic differentiation between populations, occurring both within Africa and within the Mediterranean basin. We demonstrated that three of these groups had experienced demographic expansions in the Pleistocene, probably because of climate changes during this period. Finally, we showed that C. imicola could have colonized the Mediterranean basin in the Late Pleistocene or Early Holocene through a single event of introduction; however, we cannot exclude the hypothesis involving two routes of colonization. Thus, the recent bluetongue outbreaks are not linked to C. imicola colonization event, but rather to biological changes in the vector or the virus.

Sensitivity of Culicoides obsoletus (Meigen) (Diptera: Ceratopogonidae) to deltamethrin determined by an adapted WHO standard susceptibility test.

Bluetongue is a disease of major economic concern in Europe. Its causative agent, bluetongue virus (BTV), is transmitted by several Culicoides species (mainly Culicoides imicola and Culicoides obsoletus in Europe). The application of insecticides on animals may reduce transmission of BTV, however, no formulation is currently licensed specifically against Culicoides midges. The present study assesses the susceptibility of C. obsoletus to deltamethrin using an adapted World Health Organization (WHO) susceptibility test. Midges were exposed to different dosages of deltamethrin for 1 h, and mortality after 1 h and 24 h was recorded. Results indicated that deltamethrin is highly toxic to C. obsoletus since a dose of 1·33×10(-4)% was enough to kill 50% of the population (LD50) in 24 h. The deltamethrin concentration needed to kill 90% of the population (LD90) was 5·55×10(-4)%. The results obtained in the present work could help to create a system that can be used to assess insecticide resistance and susceptibility of Culicoides biting midges.

Susceptibility of Culicoides species biting midges to deltamethrin-treated nets as determined under laboratory and field conditions in the Balearic Islands, Spain.

Culicoides Latreille (Diptera: Ceratopogonidae) are vectors of several arboviruses, including bluetongue virus (BTV) and African horse sickness virus (AHSV), which cause diseases in, respectively, sheep and cattle, and horses, and have economic repercussions mainly as a result of trade restrictions. Insecticides can be used to reduce vector populations and hence the spread of disease. Despite the economic importance of these diseases, relatively few studies have evaluated the efficacy of commercially available insecticides and the effectiveness of treated nets against Culicoides species. The aim of the present study was to evaluate the insecticidal effect of commercially available polyethylene nets (ZeroVector(®) ) treated with deltamethrin (4.4 g/kg ± 15%) on Culicoides species. Laboratory and field trials were conducted in Culicoides populations collected in Majorca in the Balearic Islands, Spain. The present study shows that deltamethrin-treated nets provoke high and rapid mortality (90-100%) in Culicoides midges under laboratory conditions and increase mortality by 13% when deployed in the field.

Survey of flea infestation in cats in Spain.

Fleas are a common cause of feline skin disorders as well as vectors of zoonotic diseases. This study evaluated the flea species infesting domestic cats in Spain and assessed factors influencing their distribution. Fleas from 217 cats from 57 localities in Spain were identified and associations between abundance, and host-dependent, host habitat and environmental factors were examined. Variations in infracommunity and component community structure were also explored. Three species were present, of which Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae) was the most abundant (98.4%), followed by Ctenocephalides canis (Curtis) (1.1%) and Pulex irritans (L.) (Siphonaptera: Pulicidae) (0.5%). Overall abundance and abundances of both C. felis and C. canis were higher on farms than in apartments, but overall flea abundance and abundances of both C. felis and C. canis were lower in rural than urban environments. Overall abundance and C. felis abundance were lower during the warmest months, and mean annual rainfall was positively correlated with overall, C. felis and C. canis abundances. No relationship between the number of species per cat and any host, habitat or physiographical variable was found. Species richness was not correlated with mean annual temperature or rainfall. Flea abundance was mainly associated with host habitat and environmental factors.

Description of two gynandromorphs of Culex theileri from Extremadura, Spain.

Two gynandromorphs of Culex theileri are described. The individuals were caught in the Centers for Disease Control and Prevention miniature light traps in the municipality of Alcollarin, Cáceres, Spain, in 2011. One of the specimens presented polar gynandromorphism, whereas the other showed a combination of polar and transverse gynandromorphic features. Details are given of the main characteristics of both mosquitoes, with particular emphasis that this gynandromorphic finding is only the second reported for this mosquito species worldwide.

Recovery rates of bluetongue virus serotypes 1, 2, 4 and 8 Spanish strains from orally infected Culicoides imicola in South Africa.

Bluetongue (BT) is an infectious disease of ruminants that has spread northwards in Europe during the last decade. The aetiological agent of the disease is an arbovirus [bluetongue virus (BTV)] that belongs to the genus Orbivirus (family Reoviridae). The virus is transmitted by certain species of biting midge within the genus Culicoides (Diptera: Ceratopogonidae). Information on the vector status of the Culicoides species in a specific area will be essential to predict the risk for BTV incursion. Field-collected Culicoides (Avaritia) imicola Kieffer from South Africa were fed on blood containing several Spanish isolates of BTV. Despite the high virus concentrations in the bloodmeal (5.1-6.4 log(10) TCID(50) /mL of blood), virus was recovered from <1% of midges assayed after incubation. Virus concentrations >2.5 log(10) TCID(50) /midge in individual infected C. imicola suggest virus replication with possible risk for transmission to susceptible vertebrate hosts in the field for at least two of the serotypes assayed (BTV-1 and BTV-2). A third serotype (BTV-4) was very close to the estimated threshold for transmission. The relatively low to near refractory status of C. imicola compared with other vector species such as Culicoides bolitinos supports previous results, indicating that Culicoides species other than C. imicola may play a more important role in the epidemiology of BTV.

Host feeding patterns of Culicoides species (Diptera: Ceratopogonidae) within the Picos de Europa National Park in northern Spain.

Blood meal identification can provide information about the natural host-feeding patterns or preferences of Culicoides species. Such information could indirectly provide data indicating which reservoirs are significant in associated vector-borne diseases. We positively identified the host species through DNA sequencing of the cytochrome b gene in 144 of the 170 (84.7%) blood meal specimens tested. In the remaining samples, identification of the blood-meal source was unsuccessful, possibly due to the post-ingestion time prior to sampling or the availability of the species-specific cytochrome b gene sequences in the database. The majority of identified blood meals were derived from mammalian blood (95.8%), and only six contained chicken blood. We identified five species as mammalian hosts for Culicoides spp.: sheep (87.7%), human (6.5%), cattle (3.7%) and Savi's Pine Vole (Micrototus savii) (2.1%). The results suggested that large mammals, specifically ruminants, were most frequently fed upon by biting midges (Culicoides spp.), but evidence of opportunistic feeding behaviour was also found. Host feeding behaviour of Culicoides species may also be influenced by the relative abundance of a particular host species in the area being studied. In this sense, Savi's Pine Vole, a wild species, was found to be a locally relevant host and a putative reservoir for viruses transmitted by species of biting midges belonging to the Culicoides genus. Finally, feeding on multiple potential host species was observed. One midge acquired blood meals from human and chicken hosts, while four other midges fed on two different sheep.

Experimental Study of the Mechanical Transmission of Rabbit Hemorrhagic Disease Virus (RHDV2/b) by Aedes albopictus (Diptera: Culicidae) and Phlebotomus papatasi (Diptera: Psychodidae).

Rabbit hemorrhagic disease (RHD) is caused by a lagovirus mainly affecting European rabbits (Oryctolagus cuniculus), although other European and North American lagomorph species are also susceptible to fatal infection by the new viral variant RHDV2/b. In the present work, direct mechanical transmission of the rabbit hemorrhagic disease virus (RHDV2/b variant) by the hematophagous Diptera Aedes albopictus (Skuse) (Diptera: Culicidae) and the sand fly Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) was tested. For each species, six and three laboratory rabbits were exposed to bites of dipterous females partially fed on RHDV2/b viral suspension 2 h and 24 h prior to exposure, respectively. The rabbits were then monitored for clinical changes and mortality for 35 d, and seroconversion was assessed by indirect ELISA. No rabbit died or showed clinical signs of disease, and seroconversion was recorded in two rabbits challenged with P. papatasi females fed the viral suspension 2 h prior to exposure. The number of RHDV2/b RNA copies/female was higher in Ae. albopictus than in P. papatasi but the decrease over time of RNA load in Ae. albopictus was greater than that in P. papatasi. The results of this study suggest the inability of Ae. albopictus to serve as a direct mechanical vector of RHDV2/b, but sand flies could play a role in the local transmission of RHD.

The susceptibility of Culicoides imicola and other South African livestock-associated Culicoides species to infection with bluetongue virus serotype 8.

In 2006, a strain of bluetongue virus serotype 8 (BTV-8) of sub-Saharan origin was responsible for the first outbreaks in recorded history of clinical bluetongue disease (BT) in northern Europe. In this study, we examine the oral susceptibility of Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae) and other livestock-associated Culicoides species from southern Africa to infection with several strains of BTV-8. Following feeding using an artificial membrane-based method and incubation, virus was found in <1% of C. imicola individuals tested. Higher rates of susceptibility were found, however, for a variety of other South African species, including Culicoides (Avaritia) bolitinos Meiswinkel. Although these results do not preclude the role of C. imicola as a vector of BTV-8, its low susceptibility to BTV indicates that other less abundant Culicoides species may have the potential to play decisive roles in the epidemiology of this virus and should not be excluded from risk assessment studies.

First Findings and Molecular Data of Phlebotomus mascittii (Diptera: Psychodidae) in the Cantabrian Cornice (Northern Spain).

Phlebotomus (Transphlebotomus) mascittii Grassi, 1908 (Diptera: Psychodidae) has been found in several European countries. In Spain, sporadic records were reported in the early '80s in Catalonia (Northeast Spain), and it was never detected again. Recent entomological surveys carried out between 2004 and 2020 revealed the presence of several specimens of P. mascittii in Spain. The species identification was confirmed by both morphological and molecular analyses. The analyzed specimens belonged to the haplotype (COI_2) defined by one polymorphic site compared to other European specimens. Phlebotomus mascittii was found in low population densities in rural areas associated with livestock farms and in an urban cemetery during the summer season. This study provides the first records of this species in various localities along the Cantabrian cornice (Northern Spain) and represents its westernmost observation in the Palearctic region. The implications of the finding of this uncommon species are discussed at different levels, with emphasis on its suspected role in the transmission of leishmaniosis.

Protection of livestock against bluetongue virus vector Culicoides imicola using insecticide-treated netting in open areas.

The protection of livestock against Culicoides species (Diptera: Ceratopogonidae) using physical barriers or chemically treated barriers is difficult owing to the small size of these biting midges and animal welfare concerns associated with the reduction of air flow. Culicoides imicola Kieffer is the main bluetongue virus vector in the Mediterranean basin, including the southern Iberian peninsula, where livestock is mainly housed in open pens or sheds which offer no physical protection against C. imicola. In this study we assessed the efficacy of surrounding yearling ewe pens with a canvas barrier or a cypermethrin-treated canvas barrier in reducing the entry of Culicoides spp. and C. imicola. Analyses were based on comparisons of Culicoides catches in traps in pens with and without barriers, and in traps located outside pens. Although there was no clear reduction in the abundance of Culicoides other than C. imicola in pens with either barrier, the C. imicola presence was markedly reduced by the insecticide-treated barrier compared with the untreated barrier; the latter did not reduce the abundance of this species in pens. Estimates of the protection conferred against C. imicola by the treated barrier differed depending on whether catch comparisons were based on outside traps or on traps located inside no-barrier pens. The results suggest that the use of insecticide-treated barriers may reduce contact between livestock and C. imicola in open areas or sheds. More research is necessary to assess the degree of protection as a function of barrier height, C. imicola abundance, and the size of the area to be protected.

Entry of bluetongue vector Culicoides imicola into livestock premises in Spain.

Culicoides imicola Kieffer is considered to be the main vector of bluetongue disease (BT) and African horse sickness (AHS) in the Mediterranean basin. It has been assumed that this midge species is exophilic and, consequently, that stabling of livestock should provide effective protection against these diseases. This study presents the results of sampling surveys for C. imicola carried out both inside and outside stables on three farms in mainland Spain. The number of C. imicola captured varied as a function of the populations sampled and trap location (inside vs. outside). The daily mean number captured inside during the sampling of each farm population was directly correlated with the daily mean number captured outside, but daily correlation of captures was not observed. By contrast with previous studies, the mean catch of C. imicola inside was consistently higher than that outside. No clear effect of stable characteristics on the degree of entry was detected. In addition, proportions of males and age-graded female groups varied among populations and with trap location. Proportionately more males and fewer engorged females were captured outside than inside, although the proportions varied among stables. These results contrast with those of previous studies, and with the assumed pronounced exophilic behaviour of C. imicola, and raise important questions about the vector activity of this species in the study area and its implications for the epidemiology of BT and/or AHS.

Variations in the mitochondrial cytochrome c oxidase subunit I gene indicate northward expanding populations of Culicoides imicola in Spain.

Culicoides imicola is the main vector for bluetongue (BT) and African horse sickness (AHS) viruses in the Mediterranean basin and in southern Europe. In this study, we analysed partial mitochondrial cytochrome c oxidase subunit I (COI) gene to characterize and confirm population expansion of Culicoides imicola across Spain. The data were analysed at two hierarchical levels to test the relationship between C. imicola haplotypes in Spain (n = 215 from 58 different locations) and worldwide (n = 277). We found nineteen different haplotypes within the Spanish population, including 11 new haplotypes. No matrilineal subdivision was found within the Spanish population, while western and eastern Mediterranean C. imicola populations were very structured. These findings were further supported by median networks and mismatch haplotype distributions. Median networks demonstrated that the haplotypes we observed in the western Mediterranean region were closely related with one another, creating a clear star-like phylogeny separated only by a single mutation from eastern haplotypes. The two, genetically distinct, sources of C. imicola in the Mediterranean basin, thus, were confirmed. This type of star-like population structure centred around the most frequent haplotype is best explained by rapid expansion. Furthermore, the proposed northern expansion was also supported by the statistically negative Tajima's D and Fu's Fs values, as well as predicted mismatch distributions of sudden and spatially expanding populations. Our results thus indicated that C. imicola population expansion was a rapid and recent phenomenon.

Initial distribution assessment of Aedes albopictus (Diptera: Culicidae) in the Barcelona, Spain, area.

The invasive species Aedes (Stegomyia) albopictus (Skuse 1894) (Diptera: Culicidae) has reached several European countries, including Albania, Belgium, Bosnia and Herzegovina, Croatia, France, Greece, Israel, Italy, Montenegro, Serbia, Slovenia, Switzerland, The Netherlands, and recently Spain (Med. Vet. Entomol. 20: 150-152, 2006). Here, we present the initial characterization of the distribution of Ae. albopictus in the municipality of Sant Cugat del Vallès, Barcelona, Spain, where it was found for the first time in the Iberian Peninsula. An ovitrap sampling campaign was developed from September to December 2004 to assess the spatial distribution and abundance of Ae. albopictus to evaluate the potential of an eradication attempt. The population of Ae. albopictus in the whole area was shown to be widespread within the municipality, and it included at least another one neighboring town, so authorities were advised to develop large-scale control measures. Some indirect evidence was collected on the introduction means and date.

Fleas parasitizing domestic dogs in Spain.

In addition to their importance to veterinary clinical practice as ectoparasites, fleas of domestic dogs are of special concern because they can be vectors of disease, including zoonoses. Flea assemblages parasitizing domestic dogs usually comprise several flea species whose distribution is determined by factors acting at several scales. Knowledge of these factors will aid in assessment of the distribution patterns of flea parasitism, and is an important tool in developing control strategies and in evaluation of flea-borne disease risk in dogs and humans. In this survey we used data from 744 domestic dogs from 79 localities in Spain to explore the associations between the abundance of flea species, host-dependent factors (sex and age), and host habitat factors including abode (farm, house with garden, apartment), location (urban or rural), the presence of other pets, and dog activity (measured as the frequency with which dogs left their abode). We also considered environmental factors including the time of year and mean annual temperature and rainfall. Variations in flea community structure at infracommunity and component community levels were also explored. Four flea species were found parasitizing dogs. Ctenocephalides felis was the most abundant (88.02% of fleas identified), followed by Ctenocephalides canis (10.38%), Pulex irritans (1.47%) and Echidnophaga gallinacea (0.13%). Overall flea abundance was higher on dogs living on farms than in apartments, as was the abundance of Ct. felis, Ct. canis and P. irritans. Ct. felis was more abundant on dogs living in houses than in apartments, but the reverse was found for P. irritans. Overall flea abundance and Ct. canis abundance were highest in rural areas, whereas the presence of other pets sharing the abode was associated with higher overall flea abundance and Ct. felis abundance. Only P. irritans abundance was positively related to the activity of dogs. Ct. canis and P. irritans abundances were higher during the warm period of the year. Mean annual temperature was negatively correlated with overall, Ct. canis and P. irritans abundances, but positively related to Ct. felis abundance. Annual rainfall was negatively correlated with Ct. canis and P. irritans abundances. Variations in the number of flea species found on a dog reflected the abundance distribution patterns for each species and their associations with host habitat and environmental factors. At the component community level, flea species richness was inversely related to annual mean temperature. The structure of flea assemblages on dogs was mainly associated with host habitat and environmental variables, and not with host-dependent variables. However, a large amount of variation in flea abundance remained unexplained, suggesting the effect of other non-controlled factors.

A survey of mosquitoes breeding in used tires in Spain for the detection of imported potential vector species.

The used tire trade has facilitated the introduction, spread, and establishment of the Asian tiger mosquito, Aedes albopictus, and other mosquito species in several countries of America, Africa, Oceania, and Europe. A strategy for detecting these imported mosquito vectors was developed in Spain during 2003-2004 by EVITAR (multidisciplinary network for the study of viruses transmitted by arthropods and rodents). A survey in 45 locations found no invasive species. Eight autochthonous species of mosquitoes were detected in used tires, including Culex pipiens, Cx. hortensis, Cx. modestus, Anopheles atroparvus, An. claviger, Culiseta longiareolata, Cs. annulata, and Aedes caspius. Dominant species were Cx. pipiens and Cs. longiareolata. Aedes caspius was found in only once, near its natural breeding habitat. Considering the recent discovery of an established population of Ae. albopictus in Catalonia, the increasing commerce of used tires in Spain for recycling, storage, and recapping might greatly contribute to the rapid spread of this species across the Iberian Peninsula.

Kinetics of oestrus ovis infection and activity of adult flies.

Oestrus Ovis is a common sheep parasite in the Mediterranean region. This study was carried out in the Ebro River Valley near Zaragoza (northeast Spain) using tracer animals to describe the seasons when infestation is more likely. Based on that information and an analysis of the evolution of the parasite within the host, we suggest the most appropriate time for treatment. Adult instars appeared in May until November and there was a diapause beginning in October-November and as least until February, so it is suggested than sheep be treated with larvicide in December.

Short report: one case of nasal human myiasis caused by third stage instar larvae of Oestrus ovis.

This is a report of a case of human nasal myiasis caused by third instar larvae of the sheep nasal bot fly, Oestrus ovis. Female flies rarely deposit first instar larvae in the eye, nostrils, and external auditory canal of humans, where they usually survive only a few days without further development. One human infestation by third instar larvae of O. ovis has been recorded in an patient infected with human immunodeficiency virus (HIV) in the United Kingdom. Our report describes a new case of nasal myiasis caused by the third instar larvae of the sheep bot fly in a patient without HIV or signs of any other disease.