Female Culicoides Research Articles

Culicoides biting midges feeding behaviour as a key for understanding avian Haemoproteus transmission in Lithuania.

Investigations of host feeding behaviour in haematophagous insects are critical to assess transmission routes of vector-borne diseases. Understanding if a certain species has ornithophilic or mammalophilic feeding behaviour can facilitate future studies focused on pathogens transmission to and from certain host species. Culicoides Latreille (Diptera: Ceratopogonidae) are vectors of several pathogens, which include arboviruses, bacteria and parasites to a considerable diversity of vertebrate hosts. However, most of the studies focused on feeding habits target Culicoides species that could transmit the Bluetongue virus, consequently with a mammalophilic feeding behaviour, leaving aside the Culicoides species that are involved in the transmission of vector-borne parasites to birds, such as Haemoproteus Kruse (Haemosporida: Haemoproteidae). This study aimed to investigate the source of blood meals of wild-caught Culicoides using molecular-based methods and to correlate our findings with the reports of Haemoproteus parasites in Culicoides species. Engorged Culicoides females were collected using ultraviolet (UV)-light traps at seven different localities in Lithuania in 2021-2023. Biting midges were dissected, and the abdomens of engorged females were used for molecular investigation of the blood meal source. A polymerase chain reaction (PCR) protocol that amplifies a fragment of the Cytochrome B gene of vertebrates was used. Obtained sequences were compared to available information in GenBank database to confirm the source of the blood meal. In total, 258 engorged Culicoides females, representing nine different species, were analysed. The source of blood meal was identified in 29.1% of them with most of the insects having fed on birds (74.7%) and the remaining on mammals (25.3%). Culicoides segnis Campbell, Pelham-Clinton was the only species to feed exclusively on birds; Culicoides from the Obsoletus group, C. pallidicornis Kieffer and C. punctatus Latreille were found to feed exclusively on mammals; C. festivipennis Kieffer, C. kibunensis Tokunaga and C. pictipennis Staeger had an opportunistic feeding behaviour, with the first two preferably feeding on birds. Due to their feeding behaviour and the presence of Haemoproteus parasites reported in the literature, C. festivipennis, C. kibunensis, C. pictipennis, and C. segnis play an important role in the transmission of those avian vector-borne parasite in the wild. These Culicoides species were already confirmed as being able to support the development of several Haemoproteus species and lineages. Future studies focused on understanding the epidemiology of avian pathogens transmitted by Culicoides should target these species.

Discovery of Novel Viruses in Culicoides Biting Midges in Chihuahua, Mexico.

Biting midges (Culicoides) are vectors of many pathogens of medical and veterinary importance, but their viromes are poorly characterized compared to certain other hematophagous arthropods, e.g., mosquitoes and ticks. The goal of this study was to use metagenomics to identify viruses in Culicoides from Mexico. A total of 457 adult midges were collected in Chihuahua, northern Mexico, in 2020 and 2021, and all were identified as female Culicoides reevesi. The midges were sorted into five pools and homogenized. An aliquot of each homogenate was subjected to polyethylene glycol precipitation to enrich for virions, then total RNA was extracted and analyzed by unbiased high-throughput sequencing. We identified six novel viruses that are characteristic of viruses from five families (Nodaviridae, Partitiviridae, Solemoviridae, Tombusviridae, and Totiviridae) and one novel virus that is too divergent from all classified viruses to be assigned to an established family. The newly discovered viruses are phylogenetically distinct from their closest known relatives, and their minimal infection rates in female C. reevesi range from 0.22 to 1.09. No previously known viruses were detected, presumably because viral metagenomics had never before been used to study Culicoides from the Western Hemisphere. To conclude, we discovered multiple novel viruses in C. reevesi from Mexico, expanding our knowledge of arthropod viral diversity and evolution.

High Abundance of Haemoproteus Parasites in Culicoides (Diptera, Ceratopogonidae), with a Confirmation of Culicoides reconditus as a New Vector of These Avian Blood Parasites.

Haemoproteus parasites are the most diverse among Haemosporida. However, their natural vectors (Culicoides) are still poorly investigated and were identified for only a few parasite species and lineages. The application of an integrative approach (insect dissection, microscopic analysis, and molecular-based methods) is necessary in these studies, which have been carried out by a few research groups, mainly in Europe. The aim of this study was (i) to determine the Culicoides species that are naturally infected by Haemoproteus parasites, and which can support its complete sporogonic development, and (ii) to investigate the prevalence of Culicoides species and Haemoproteus parasite lineages in different study sites. In total, 1953 parous Culicoides females, from 11 species, were collected in four different localities in Lithuania and were dissected and analyzed using an integrative approach. The most abundant was C. pictipennis (30.3%). Parasite DNA was found in 7.9% of all investigated Culicoides, of which ~30% had sporozoites in their salivary glands, confirming their vector competence for these parasites. The Botanical Garden presented the highest number of Culicoides parous females, Culicoides species, and parasite lineages, as well as the highest positivity for sporozoites. Culicoides reconditus was confirmed as a natural vector of Haemoproteus parasites, sporozoites of six Haemoproteus lineages were reported for the first time, and 12 new interactions between Haemoproteus parasite lineages and Culicoides species were identified. Haemoproteus parasites seem to be transmitted by a high number of Culicoides species, with C. kibunensis, C. pictipennis, and C. segnis being the most important vectors.

Natural Vector of Avian Haemoproteus asymmetricus Parasite and Factors Altering the Spread of Infection.

Avian haemosporidians (Apicomplexa, Haemosporida) are widespread blood protists, often causing severe haemosporidiosis, pathology, or even mortality in their hosts. Migrant birds regularly bring various haemosporidian parasites from wintering grounds to European breeding areas. Some haemosporidian parasites are prevalent in breeding sites and complete their life cycles in temperate climate zones and can be transmitted, but others do not. The factors altering the spread of these haemosporidians are not fully understood. Culicoides biting midges (Diptera: Ceratopogonidae) play an important role in the transmission of worldwide distributed avian haemosporidian parasites belonging to the genus Haemoproteus, but this information is particularly scarce and insufficient. The key factors limiting the spread of these pathogens in temperate climate zones, which we suspect and aim to study, are the absence of susceptible vectors and the ecological isolation of birds from vectors during the breeding period when transmission occurs. The primary objective of this study was to evaluate how the habitats of biting midges and bird breeding sites influence parasite transmission while also seeking to expand our understanding of the natural vectors for these parasites. Biting midges were collected using UV traps on the Curonian Spit, Lithuania, in different habitats, such as woodland and reeds, from May to September. Parous Culicoides females were identified, dissected, and investigated for the presence of Haemoproteus parasites using both microscopy and PCR-based tools. Among the dissected 1135 parous Culicoides females, the sporozoites of Haemoproteus asymmetricus (genetic lineage hTUPHI01) have been detected for the first time in the salivary glands of Culicoides festivipennis. The sporozoites of four Haemoproteus lineages were detected in Culicoides segnis, C. festivipennis, and Culicoides kibunensis biting midges. PCR-based screening showed that the females of seven Culicoides species were naturally infected with Haemoproteus parasites. The DNA of the parasite of owls, Haemoproteus syrnii (hSTAL2), was detected for the first time in Culicoides punctatus. The highest abundance of collected Culicoides females was in June, but the highest prevalence of Haemoproteus parasites in biting midges was in July. The abundance of Culicoides was higher in the woodland compared with reeds during the season. The acquired findings indicate the varied abundance and diversity of biting midges throughout the season and across distinct habitats. This variability could potentially impact the transmission of Haemoproteus parasites among birds with diverse breeding site ecologies. These outcomes hold the potential to enhance our understanding of the epizootiology of Haemoproteus infections within temperate climatic zones.

Visualisation of Bluetongue Virus in the Salivary Apparatus of Culicoides Biting Midges Highlights the Accessory Glands as a Primary Arboviral Infection Site

BackgroundArthropods transmit a wide range of pathogens of importance for the global health of humans, animals, and plants. One group of these arthropod vectors, Culicoides biting midges (Diptera: Ceratopogonidae), is the biological vector of several human and animal pathogens, including economically important livestock viruses like bluetongue virus (BTV). Like other arthropod-borne viruses (arboviruses), Culicoides-borne viruses must reach and replicate in the salivary apparatus, from where they can be transmitted to susceptible hosts through the saliva during subsequent blood feeding. Despite the importance of the salivary gland apparatus for pathogen transmission to susceptible animals from the bite of infected Culicoides, these structures have received relatively little attention, perhaps due to the small size and fragility of these vectors.ResultsIn this study, we developed techniques to visualize the infection of the salivary glands and other soft tissues with BTV, in some of the smallest known arbovirus vectors, Culicoides biting midges, using three-dimensional immunofluorescence confocal microscopy. We showed BTV infection of specific structures of the salivary gland apparatus of female Culicoides vectors following oral virus uptake, related visualisation of viral infection in the salivary apparatus to high viral RNA copies in the body, and demonstrated for the first time, that the accessory glands are a primary site for BTV replication within the salivary apparatus.ConclusionsOur work has revealed a novel site of virus-vector interactions, and a novel role of the accessory glands of Culicoides in arbovirus amplification and transmission. Our approach would also be applicable to a wide range of arbovirus vector groups including sand flies (Diptera: Psychodidae), as well as provide a powerful tool to investigate arbovirus infection and dissemination, particularly where there are practical challenges in the visualization of small size and delicate tissues of arthropods.

Topical, contact, and oral susceptibility of adult Culicoides biting midges (Diptera: Ceratopogonidae) to fluralaner

BackgroundCulicoides biting midges (Diptera: Ceratopogonidae) are economically important blood-feeding pests closely associated with livestock production. They are the principal vectors of two hemorrhagic disease viruses affecting both wild and domestic ruminants within the US: bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). BTV impacts the US agriculture sector through direct commodity loss and strict international livestock trade restrictions. Yet, despite posing a considerable threat to US livestock, Culicoides are understudied, and management strategies are lacking. Current control tools for Culicoides are limited to synthetic chemicals, predominantly pyrethroids. With limited products available for livestock producers, proper pesticide rotation is difficult. The present study investigates the efficacy of fluralaner, an isoxazoline insecticide, beyond its current labeled use as an ectoparasiticide in anticipation of adding a new class of pesticides into rotation for use against biting midges.MethodsThe efficacy of fluralaner was evaluated by conducting contact, topical, and oral toxicity bioassays on adult female Culicoides sonorensis. Contact toxicity was assessed by using a modified WHO cone assay, which simulates exposure through landing on an insecticide-treated surface. A modified WHO topical toxicity assay, in which fluralaner dilutions were administered to the lateral thorax, was used to assess topical toxicity. For evaluation of oral toxicity, females were offered a blood meal spiked with fluralaner in an artificial membrane feeding system to simulate a systemic insecticide.ResultsContact exposure of fluralaner did not cause extensive or consistent mortality. Even the highest concentration tested (100 mg/ml) resulted in an average of only 24.3% mortality at 24 h, and mortality did not significantly differ between exposed and control midges at any concentration. One hundred percent mortality was consistently achieved at concentrations of 1 mg/ml when fluralaner was applied topically. The LC50 for topical exposure to fluralaner at 24 h was estimated to be 0.011 mg/ml. Oral exposure to fluralaner through ingestion of a spiked blood meal proved to be the most effective exposure method, significantly increasing mortality in a dose-dependent manner at 1 h post-exposure. The LC50 at 24 h following ingestion was 14.42 ng/ml.ConclusionOur results suggest that fluralaner is a viable candidate for use as an insecticide against adult biting midges if exposed orally, such as in a systemic given to livestock. As withdrawal period requirements for meat animals present unique yet definitive challenges, pharmacokinetic studies of isoxazoline drugs need to be pursued and finalized for livestock before fluralaner may be used as a management strategy in this manner. Alternatively, livestock not raised for consumption, such as hair sheep, would directly benefit from administering oral fluralaner as a component of a BTV disease management program.Graphical abstract

Culicoides jiangchengensis, a new species of the subgenus Sinocoides (Diptera, Ceratopogonidae) based on integrative taxonomy from China.

Biting midges of the genus Culicoides are important in both medicine and veterinary medicine because their blood-feeding regime enable them to transmit a variety of pathogens. In this study, the morphological characteristics of the new species of Culicoides (Sinocoides) jiangchengensis Wang et Liu sp. nov are described and compared with the other species of female Culicoides in the subgenus Sinocoides. Three morphological characteristics of C. jiangchengensis, such as without sensory pit in 3rd palpus segment, sensilla coeloconica on flagellomeres 1,9-13, and m1 and m2 cell of the wings with pale spots, were different from the other nine species of culicoides in subgenus Sinocoides. Genetically, C. jiangchengensis are most closely related to C. malipoensis, but they were located in different branches and the minimum interspecific distance between them was 12.6%. In addition, a checklist of 10 species in the subgenus Sinocoides Chu, 1983 (Diptera: Ceratopogonidae: Culicoides) in China, including the new species C. jiangchengensis Wang et Liu sp. nov., is provided, and an updated key to species of the subgenus Sinocoides Chu, 1983 was presented.

Trypanosomatids in Bloodsucking Diptera Insects (Ceratopogonidae and Simuliidae) Wild-Caught at Raptor Bird Nests in Temperate Forests

Trypanosomatids are a huge group of vertebrate, invertebrate and plant parasites that can cause severe illnesses in hosts. Although avian trypanosomes are widely spread throughout the world, information about their transmission and vector–host relationships is still scarce. This study aimed to investigate Trypanosoma prevalence in bloodsucking Diptera collected at tree-nesting raptor birds’ nests. Insects were collected in temperate forests of Eastern-Central Europe, in Lithuania, using UV light traps hung near common buzzard (Buteo buteo) and lesser spotted eagle (Clanga pomarina) nests at about 15 m height above the ground. A total of 1248 Culicoides (Ceratopogonidae) females and 3 blackflies (Simuliidae) were collected and tested for the presence of trypanosomatids using PCR-based methods. The blood of 85 nestlings, belonging to three different species (Buteo buteo, Clanga pomarina and Haliaeetus albicilla) was collected and tested using the same methods. We found that 11.1% of the investigated insects (one Simulium female and Culicoides biting midges belonging to five species) were infected with Trypanosoma parasites (Trypanosoma sp., T. bennetti group, T. avium) and monoxenous trypanosomatids (Crithidia sp., Obscuromonas sp.). Only one common buzzard nestling was found to be infected with Trypanosoma avium. The phylogenetic relationships of detected parasites were determined. Our findings supplement information on the ornithophilic behavior of Culicoides females, potential vector species of avian Trypanosoma, and produce some new information on the detection of monoxenous trypanosomatids (Crithidia sp. and Obscuromonas sp.) in Culicoides.

Molecular Identification of Culicoides Species and Host Preference Blood Meal in the African Horse Sickness Outbreak-Affected Area in Hua Hin District, Prachuap Khiri Khan Province, Thailand.

African horse sickness (AHS) was reported as an outbreak in Thailand in 2020. Hematophagous insects from the genus Culicoides are the suspected vector responsible for AHS transmission. Horses in Hua Hin district, Prachuab Khiri Khan province, Thailand, were affected and died from AHS in 2020. However, the potential Culicoides species and its host preference blood meal in the affected areas are unknown. To investigate the potential vectors of AHS, Culicoides were collected using ultraviolet light traps placed near horse stables. Six horse farms, including five farms with AHS history and one farm without AHS history, were included in this study. Morphological and molecular identification of the Culicoides species was performed. Polymerase chain reaction (PCR) targeting the cytochrome b oxidase I (COXI) gene for confirmation of the Culicoides species, identification of the prepronociceptin (PNOC) gene for host preference blood meal, and bidirectional sequencing were conducted. Consequently, 1008 female Culicoides were collected, consisting of 708 and 300 samples captured at positions A and B at a distance of <2 and >5 m from the horse, respectively. Twelve Culicoides species identified by morphology were noted, including C. oxystoma (71.92%), C. imicola (20.44%), C. actoni (2.28%), C. flavipunctatus (1.98%), C. asiana (0.99%), C. peregrinus (0.60%), C. huffi (0.60%), C. brevitarsis (0.40%), C. innoxius (0.30%), C. histrio (0.30%), C. minimus (0.10%), and C. geminus (0.10%). The PCR detection of the Culicoides COXI gene confirmed Culicoides species in 23 DNA samples. PCR targeting the PNOC gene revealed that the Culicoides collected in this study fed on Equus caballus (86.25%), Canis lupus familiaris (6.25%), Sus scrofa (3.75%), and Homo sapiens (3.75%) for their blood meal. Human blood was identified from two samples of C. oxystoma and a sample of C. imicola. Three dominant species including C. oxystoma, C. imicola, and C. actoni that were reported in the Hua Hin area prefer to feed on horse blood. Moreover, C. oxystoma, C. imicola, and C. bravatarsis also feed on canine blood. This study revealed the species of Culicoides in Hua Hin district, Thailand, after the AHS outbreak.

The first detection of avian haemosporidia from Culicoides biting midges in Japan, with notes on potential vector species and the transmission cycle.

Biting midges (Ceratopogonidae) are capable of transmitting a variety of pathogens including viruses, trypanosomes and haemosporidia. The majority of Haemoproteus parasites are transmitted by biting midges predominantly of the genus Culicoides and are known to cause significant physical and reproductive impacts on both wild and domestic birds. In Japan, Haemoproteus had been detected from various avian hosts, but not from arthropod vectors. In this study, we investigated the prevalence of avian haemosporidia at an educational forest in central Japan in attempt to reveal possible vector species of Haemoproteus, which would help to better understand the transmission cycle of Haemoproteus within Japan and to develop preventative measures for captive and domestic birds. Biting midges were caught using UV light traps from 2016 to 2018. The collected samples were morphologically identified, and haemosporidian parasites were detected using PCR-based methods. The detected lineages were phylogenetically analyzed and compared with lineages previously detected from birds. Bloodmeal analyses were also carried out for part of the blood-fed individuals. Six Haemoproteus lineages were detected from 17 of 1042 female Culicoides (1.63%), including three species (C. sigaensis, C. arakawae, and C. pictimargo) in which Haemoproteus was detected for the first time. All detected lineages were placed in the subgenus Parahaemoproteus clade and were previously detected from crows of central Japan, strongly suggesting that parasites of these genetic lineages are transmitted between Culicoides and crows. Two Plasmodium lineages were also detected but are thought to be transmitted between Culex mosquitoes and birds of the educational forest based on previous detections. No amplifications were seen in bloodmeal analysis, possibly due to insufficient amount of blood, denaturation via digestion, or insufficient detectability of the used protocol. Haemoproteus DNA was detected from Culicoides for the first time in Japan, suggesting that transmission is possible within the country. These findings highlight the necessity to investigate Culicoides populations and Haemoproteus infections dynamics in Japan. However, vector competence could not be confirmed in this study and further studies are anticipated.

The use of path analysis to determine effects of environmental factors on the adult seasonality ofCulicoides(Diptera: Ceratopogonidae) vector species in Spain

Culicoides biting midges (Diptera: Ceratopogonidae) are the main vectors of livestock diseases such as bluetongue (BT) which mainly affect sheep and cattle. In Spain, bluetongue virus (BTV) is transmitted by several Culicoides taxa, including Culicoides imicola, Obsoletus complex, Culicoides newsteadi and Culicoides pulicaris that vary in seasonality and distribution, affecting the distribution and dynamics of BT outbreaks. Path analysis is useful for separating direct and indirect, biotic and abiotic determinants of species' population performance and is ideal for understanding the sensitivity of adult Culicoides dynamics to multiple environmental drivers. Start, end of season and length of overwintering of adult Culicoides were analysed across 329 sites in Spain sampled from 2005 to 2010 during the National Entomosurveillance Program for BTV with path analysis, to determine the direct and indirect effects of land use, climate and host factor variables. Culicoides taxa had species-specific responses to environmental variables. While the seasonality of adult C. imicola was strongly affected by topography, temperature, cover of agro-forestry and sclerophyllous vegetation, rainfall, livestock density, photoperiod in autumn and the abundance of Culicoides females, Obsoletus complex species seasonality was affected by land-use variables such as cover of natural grassland and broad-leaved forest. Culicoides female abundance was the most explanatory variable for the seasonality of C. newsteadi, while C. pulicaris showed that temperature during winter and the photoperiod in November had a strong effect on the start of the season and the length of overwinter period of this species. These results indicate that the seasonal vector-free period (SVFP) in Spain will vary between competent vector taxa and geographic locations, dependent on the different responses of each taxa to environmental conditions.

First Evidence of Co-Circulation of Emerging Leishmania martiniquensis, Leishmania orientalis, and Crithidia sp. in Culicoides Biting Midges (Diptera: Ceratopogonidae), the Putative Vectors for Autochthonous Transmission in Southern Thailand.

Since 1996, autochthonous cases of emerging leishmaniasis caused by Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis have been more frequently reported, especially in the northern and southern parts of Thailand. However, the accurate identification of their natural vectors and reservoirs remains unconfirmed. Previous studies have suggested that these emerging parasites might be transmitted by other non-phlebotomine vectors. Herein, we speculated that Culicoides biting midges might act as the competent vectors responsible for autochthonous leishmaniasis in southern Thailand. In this research, 187 non-engorged, parous and gravid Culicoides females and 47 blood-engorged ones were trapped from the residences of two recently diagnosed visceral leishmaniasis patients in Sadao District and the unaffected site in Rattaphum District, Songkhla Province, southern Thailand. Species diversity and abundance of biting midges varied among the trapping sites. Using ITS1-PCR and BLASTn analysis, L. martiniquensis was predominantly detected in several Culicoides species, including C. peregrinus, C. oxystoma, C. mahasarakhamense, and C. huffi from the vicinity of patients' houses; and in C. fordae and C. fulvus from the unaffected site. L.&amp;nbsp;orientalis was also co-circulated in C. peregrinus and C. oxystoma caught near the second patient's house. Additionally, Crithidia sp. were also detected using SSU rRNA-PCR across Culicoides spp. Host blood meal analysis of eight different Culicoides species from the unaffected site also revealed that all trapped Culicoides had fed on cows and goats, indicating the possible role of these mammalian species as reservoir hosts. Essentially, this study is the first entomological investigation, revealing the co-circulation of emerging trypanosomatids among several species of Culicoides biting midges and strongly supporting the potential role of this insect group as the main vectors responsible for the epidemiology of autochthonous leishmaniasis in southern Thailand.

Assigning Culicoides larvae to species using DNA barcoding of adult females and phylogenetic associations

BackgroundOrbivirus-induced hemorrhagic diseases cause high mortality in wild and captive white-tailed deer in North America. The role of different Culicoides species in Orbivirus transmission outside of areas of intensive animal production has not been established. At our study location, bluetongue virus (BTV) RNA-positive female Culicoides debilipalpis pools have been detected annually since 2012 when BTV transmission was noted in a captive deer herd. Identifying specific larval habitats of suspected vectors at active transmission sites is crucial both for identifying the source of the vectors and for subsequently planning intervention actions. Since C. debilipalpis larvae are known to develop in tree holes, this study was designed to use DNA barcoding to identify larvae collected from tree holes.MethodsAdult female Culicoides were collected using light or emergence traps and morphologically identified to 11 species. Culicoides sonorensis were also obtained from a laboratory colony. Substrate was collected from tree holes and flooded with water to harvest floating larvae. Total DNA from three to seven adult females per species and 19 larvae was extracted. Two loci of the nuclear 18S ribosomal RNA (rRNA) gene, one locus each of the mitochondrial cytochrome oxidase subunit I (COI) gene and the nuclear 28S rRNA gene were amplified using loci-specific primers.ResultsAll 61 adults were sequenced at each of the four loci under study. Since no single locus delineated all putative species and the COI locus yielded unreliable pseudogenes for two individuals of C. arboricola, sequences of all four loci were concatenated to maximize species separation and allow for larval association with identified adults. Sixteen larvae were clearly assigned to species based on DNA barcoding and phylogenetic results. Multiple larvae were assigned to each of the C. debilipalpis clade, the C. villosipennis clade, the C. arboricola clade and the C. nanus clade.ConclusionsOf the approximately 62 species described in the southeast USA, 21 have now been barcoded and sequences are publicly available. In this study, we constructed a database composed of species-specific sequences of adult Culicoides and then identified larvae to species by matching their corresponding sequences with adults. Since Culicoides larvae are difficult to identify, using DNA barcoding to facilitate larval habitat surveys can be a valuable tool.Graphical

Comparison of surveillance trapping methods to monitor Culicoides biting midge activity in Trinidad, West Indies.

Culicoides biting midges (Diptera: Ceratopogonidae) are biting nuisances and arbovirus vectors of both public health and veterinary significance in Trinidad. We compared sampling methods to define the behaviour and bionomics of adult Culicoides populations at a commercial dairy goat farm. Three static trap designs were compared: (a) Centre for Disease Control (CDC) downdraft UV trap; (b) CDC trap with an incandescent bulb and (c) CDC trap with semiochemical lure consisting of R-(-)-1-octen-3-ol and CO2 (no bulb). Sweep netting was used to define diel periodicity. A total of 30,701 biting midges were collected using static traps, dominated by female Culicoides furens (>70% of trap collections across all three designs). There was no significant difference in the Margalef's index between the three traps; however, trap designs A and C collected a significantly greater number of individuals than trap B, and trap C gained highest species richness. The greatest species richness and abundance of Culicoides collected by sweep net was observed between 6:00 and 6:15 pm and notable differences in the crepuscular activity pattern of several species were identified. Comparative data on Culicoides species richness, abundance, sex and reproductive status is discussed and can be used to improve surveillance strategies, research designs and risk management.

Habitat-dependent Culicoides species composition and abundance in blue tit (Cyanistes caeruleus) nests.

Wild birds are hosts of Culicoides from as early on as the nesting stage when constrained to their nests. However, the environmental factors which determine the abundance and composition of Culicoides species within each bird nest are still understudied. We sampled Culicoides from Eurasian blue tit (Cyanistes caeruleus) nests found in 2 types of forests located in southern Spain. Firstly, we monitored the abundance of Culicoides species in bird nests from a dry Pyrenean oak deciduous forest and a humid mixed forest comprising Pyrenean and Holm oaks throughout 2 consecutive years. During the 3rd year, we performed a cross-fostering experiment between synchronous nests to differentiate the role of rearing environment conditions from that of the genetically determined or maternally transmitted cues released by nestlings from each forest. We found 147 female Culicoides from 5 different species in the birds' nests. The abundance of Culicoides was higher in the dry forest than in the humid forest. Culicoides abundance, species richness and prevalence were greater when the nestlings were hatched later in the season. The same pattern was observed in the cross-fostering experiment, but we did not find evidence that nestling's features determined by the forest of origin had any effect on the Culicoides collected. These results support the notion that habitat type has a strong influence on the Culicoides affecting birds in their nests, while some life history traits of birds, such as the timing of reproduction, also influence Culicoides abundance and species composition.

Culicoides segnis and Culicoides pictipennis Biting Midges (Diptera, Ceratopogonidae), New Reported Vectors of Haemoproteus Parasites.

As bloodsuckers of birds, Culicoides biting midges (Diptera, Ceratopogonidae) play an important role in the transmission of avian haemosporidian (Haemoproteus) parasites, which are prevalent in many bird populations and cause disease, pathology, or even mortality in their hosts. Information about the role of the various Culicoides species in the transmission of Haemoproteus parasites remains insufficient. This presents an obstacle for the better understanding of the epizootiology of haemoproteosis. The aim of this study was to determine new Culicoides species involved in the transmission of Haemoproteus parasites in the wild. Biting midges were collected using UV traps on the Curonian Spit, Lithuania. Only parous Culicoides females were investigated: they were identified and were diagnosed for the presence of Haemoproteus parasites using both microscopy and PCR-based methods. We collected and dissected 420 parous Culicoides females. PCR-based screening showed that 28 parous Culicoides biting midges were infected with avian Haemoproteus parasites. Haemoproteid DNA was detected in Culicoides kibunensis, Culicoides pictipennis, Culicoides festivipennis, Culicoides segnis, Culicoides pallidicornis, and Culicoides obsoletus biting midges. The DNA of Haemoproteus palloris, genetic lineage hWW1, was found for the first time in C. pallidicornis. Haemoproteus sporozoites were detected in the salivary glands of two Culicoides segnis biting midges. According to the PCR results, one female contained Haemoproteus tartakovskyi (genetic lineage hHAWF1) DNA and another Haemoproteus majoris (genetic lineage hCCF5) DNA. The sporozoites of Haemoproteus parasites were also detected in the salivary glands of four C. pictipennis biting midges using microscopy, and this finding was confirmed by PCR as Haemoproteus parabelopolskyi DNA (genetic lineage hSYAT02) was detected in three out of the four biting midges. The obtained results supplement existing information about Culicoides biting midges as natural vectors of Haemoproteus spp. and add two new Culicoides species to the vector list, showing the low specificity of these parasites for the invertebrate hosts.

DNA-based blood meal analysis of Culicoides (Diptera: Ceratopogonidae) species from Jamari National Forest, Southwestern Amazon, Brazil

Culicoides biting midges are insects involved in the transmission of filarial nematodes, protozoans, and viruses. Greater knowledge of Culicoides blood meal sources could improve our understanding of parasite transmission cycles. Our study used molecular tools to evaluate the blood meal sources of Culicoides biting midges from sylvatic environments. This study was conducted in Jamari National Forest, Rondônia, Brazil. Culicoides were captured using HP light traps positioned at ground level (1.5 m above ground) and in canopy (15 m above ground). To identify blood meal sources, females were subjected to DNA extraction and PCR targeting the cytb gene fragment, and the obtained sequences were analyzed and compared with sequences from GenBank. DNA extraction and PCR were performed on 455 Culicoides females, and blood meal sources were identified in 186 females. Thirty Culicoides specimens were collected from the Potosi trail and 156 were collected from the Santa Maria trail. A total of 22 species were captured; all 22 species were collected in canopy (100%) but only three species were collected at ground level (13.6%). The cytb fragment was amplified in 162 of 186 samples. Sample sequencing identified cytb DNA from nine blood-meal sources: Pauxi sp., Psophia viridis, Ramphastos tucanus tucanus, Choloepus didactylus, Choloepus hoffmanni, Tamandua tetradactyla, Ateles chamek, Homo sapiens and Pithecia irrorata. We observed that several different blood meal sources were utilized by a high diversity of Culicoides species. The abundance of Culicoides in the canopy may be related to the fact that the majority of blood meal hosts feed in treetops. We observed that C. (Hoffmania) sp. and C. coutinhoi tend to be more generalist, feeding on a range of mammals and piciform, gruiform and galliform birds. This data improves our knowledge of the feeding profile of biting midges from forest environments and should serve as a future basis for defining zoonotic transmission cycles.

The flying activity of biting midges (Ceratopogonidae: Culicoides) in Verkiai Regional Park, southeastern Lithuania.

Culicoides biting midges are small dipterous insects (Diptera: Ceratopogonidae) which are known to be vectors of arboviruses, bacteria, protozoan and helminth parasites that can cause disease and mortality in livestock and poultry globally. Detailed knowledge of the Culicoides species composition and biology is essential to assess the risk of the introduction and transmission of pathogens. The aim of this study was to obtain data on Culicoides species composition and flying activity in southeastern Lithuania and to determine the meteorological variables related to the abundance of Culicoides biting midges. Biting midges were collected in Verkiai Regional Park, southeastern Lithuania, using an Onderstepoort trap once a week from April to October 2016 and 2018, and from April to July 2019; 7332 Culicoides females belonging to 22 species were identified. Both morphology and DNA barcoding were used for identification. The number of specimens trapped was highest for the Obsoletus Group, followed by Culicoides kibunensis and Culicoides impunctatus. The highest relative abundance and diversity of biting midges were found in May and June. The number of trapped biting midges correlated positively with the mean air temperature. The first biting midges in spring were caught when the mean daily temperature rose higher than 10 °C. No Culicoides were detected when the air temperature dropped below 5 °C in autumn. Wind speed and air humidity had no statistically significant effect on Culicoides abundance.

The utilisation of CytB and COI barcodes for the identification of bloodmeals and Culicoides species (Diptera: Ceratopogonidae) reveals a variety of novel wildlife hosts in South Africa.

Biting midges in the genus Culicoides (Diptera; Ceratopogonidae) are vectors of pathogens that can cause diseases of major economic importance in humans and animals. Identifying host ranges of these biting midges might aid in understanding the complex epidemiology of such diseases, often involving reservoir hosts and multiple species. In this study, we aim to identify bloodmeal origin from engorged female Culicoides biting midges. All bloodfed females were opportunistically collected as part of an ongoing surveillance programme using Onderstepoort light traps in two provinces in South Africa. DNA of individuals was extracted and subjected to PCR targeting the cytochrome B (CytB) gene region of mammals and avians as well as cytochrome oxidase I (COI) for species identification. In total, 21 new reference barcodes were generated for C. bedfordi, C imicola, C. leucosticus, C. magnus, and C. pycnostictus. Seventy-four blood meals were identified, originating from 12 mammal and three avian species. COI sequence data performed well for species delimitation and 54 Culicoides specimens were identified with C. imicola the predominant species identified (41.8%). Generally, Culicoides species feed on a variety of hosts and host availability might be an important factor when selecting a host. Culicoides species thus appear to be opportunistic feeders rather than specialists. This implicates Culicoides as transfer vectors and demonstrates possible transmission routes of arboviruses and other pathogens from wildlife onwards to domestic animals and humans.

Culicoides biting midges involved in transmission of haemoproteids

BackgroundCulicoides biting midges (Diptera, Ceratopogonidae) are known vectors of avian Haemoproteus parasites. These parasites cause diseases, pathology and even mortality in birds. The diversity of biting midges in Europe is great, but only four Culicoides species are known to be vectors of avian Haemoproteus parasites. In general, our knowledge about the role of the particular Culicoides species in the transmission of Haemoproteus parasites remains insufficient. Information gaps hinder a better understanding of parasite biology and the epizootiology of parasite-caused diseases. The aim of this study was to determine new Culicoides species involved in the transmission of Haemoproteus parasites.MethodsBiting midges were collected using a UV trap as well as sticky traps installed in bird nest boxes. Individual parous females were diagnosed for the presence of haemoproteids using both PCR-based and microscopic methods.ResultsWe collected and dissected 232 parous Culicoides females from 9 species using a UV trap and 293 females from 11 species from bird nest boxes. Culicoides obsoletus was the dominant species collected using a UV trap, and Culicoides kibunensis dominated among midges collected in nest boxes. PCR-based screening showed that 5.2% of parous biting midges collected using a UV trap and 4.4% of midges collected from nest boxes were infected with avian haemosporidian parasites. Haemoproteid DNA was detected in C. kibunensis, Culicoides pictipennis, Culicoides punctatus, Culicoides segnis and Culicoides impunctatus females. The sporozoites of Haemoproteus minutus (genetic lineages hTURDUS2 and hTUPHI01) were detected in the salivary glands of two C. kibunensis females using microscopy, and this finding was confirmed by PCR.ConclusionsCulicoides kibunensis was detected as a new natural vector of Haemoproteus minutus (hTURDUS2 and hTUPHI01). Haemoproteid DNA was detected in females from five Culicoides species. This study contributes to the epizootiology of avian Haemoproteus infections by specifying Culicoides species as vectors and species that are likely to be responsible for the transmission of haemoproteids in Europe.Graphical