Assessment of the Hemotek® system for the in vitro feeding of field-collected Culicoides imicola (Diptera: Ceratopogonidae) in South Africa.

The optimising and standardisation of in vitro blood feeding protocols for field-collected Culicoides species (Diptera: Ceratopogonidae) will be of essence for the comparison of the vector competencies of various populations of viruses of veterinary importance and the establishment of laboratory colonies of putative vector species. A custom-made feeding chamber to accommodate the small size of Culicoides imicola Kieffer was designed for the commercially available Hemotek® system and compared to existing membrane and cotton pledge feeding methods. High feeding rates coupled to higher mean blood meal volume than that of the existing OVI device indicated that the Hemotek system will be suitable for the feeding of field-collected Culicoides. The Hemotek system was subsequently used to identify factors that may affect feeding success in the laboratory. Evaluated factors were the source (host) and temperature of the blood meal, time of the day of feeding, the position of the blood reservoir in relation to the midges and exposure time to the blood. While only feeding orientation and the temperature of the blood source seems to significantly affect the feeding rate, all the factors did influence the volume of blood consumed.

Development and lifespan of Culicoides obsoletus s.s. (Meigen) and other livestock-associated species reared at different temperatures under laboratory conditions.

Culicoides Latreille (Diptera: Ceratopogonidae) transmit arboviruses affecting wild and domestic ruminants such as bluetongue (BTV) and Schmallenberg virus (SBV). The sub-adult development and lifespan of Culicoides obsoletus s.s. (Meigen), Culicoides circumscriptus Kieffer and Culicoides paolae Boorman were examined at three different temperatures under laboratory conditions. Insects were collected from field between spring and autumn 2015 in two livestock farms located in Majorca (Spain). Gravid females were held individually at 18, 25 or 30 °C. Low temperatures increased the adult lifespan, time to oviposit and rate of development, whereas high temperatures increased the number of eggs, successful pupation and adult emergence as well as the larvae growth rate. The results showed that C. obsoletus s.s. have optimum development at 18 °C, whereas the optimal rearing temperature for C. circumscriptus and C. paolae was under warmer conditions of 25-30 °C. Variations in temperature/humidity and assays with different materials and substrates for oviposition should be considered in future studies. Understanding the requirements of the different species of Culicoides optimizing the results should be of special interest for predicting environmental change effects on these species, in addition to determining the rearing conditions for candidate Culicoides vectors.

Identification of a cyanidin-3-O-ß-galactoside in gall tissue induced by midges on Japanese beech (Fagus crenata Blume).

Insect gall structures have many characteristic forms and colors, which are distinguishable from host plants. In this study, we identified an anthocyanin from red color insect galls and revealed that the anthocyanin biosynthesis of plants was induced by the gall extracts. The galling insects presumably regulate the anthocyanin biosynthesis of host plants to protect their larvae from environmental stresses.

Sampling Considerations for Adult and Immature Culicoides (Diptera: Ceratopogonidae).

Developing sampling programs for Culicoides can be challenging due to variation in ecology and behavior of the numerous species as well as their broad distributions and habitats. In this paper, we emphasize the need to clearly define research goals to select appropriate sampling methods. This includes not just the choice of sampling device, but also choice of attractant, site, number of traps per site, the duration and frequency of sampling, and the number of traps per unit area. Animal-baited trapping using enclosure traps and direct animal aspiration is more labor-intensive but yields information on species attracted to specific hosts as well as their biting rates. Sampling immatures is discussed with respect to choosing collection sites in semiaquatic mud, soil, and rich organic habitats. Sorting and extracting larvae using emergence traps, flotation, and Berlese funnels is also discussed.

Latitudinal diversity of biting midge species within the Obsoletus group across three habitats in Europe.

Culicoides species from the Obsoletus group are important vectors of bluetongue and Schmallenberg virus. This group consists of several species that cannot easily be identified using morphological characteristics. Therefore, limited information is available about their distribution and habitat preferences. In this study, we aimed to elucidate the species composition of the Obsoletus group in three habitat types at climatically different latitudes across Europe. Traps were placed in three habitat types in three countries at different latitudes. After DNA extraction, biting midges were identified using PCR and gel electrophoresis. Extraction of DNA using Chelex proved to be a cost and time efficient method for species identification. A latitudinal effect on the relative abundance of species from the Obsoletus group was found. Species composition was unique for most country-habitat combinations. The majority of biting midges were either C. obsoletus s.s. or C. scoticus, and both species were found at all latitudes and habitats. Their wide distribution and their high abundance at livestock farms make these species likely candidates for rapid farm-to-farm transmission of pathogens throughout Europe. Our results emphasize the need to differentiate Obsoletus group species to better understand their ecology and contribution to pathogen transmission.

The Interaction between Vector Life History and Short Vector Life in Vector-Borne Disease Transmission and Control.

Epidemiological modelling has a vital role to play in policy planning and prediction for the control of vectors, and hence the subsequent control of vector-borne diseases. To decide between competing policies requires models that can generate accurate predictions, which in turn requires accurate knowledge of vector natural histories. Here we highlight the importance of the distribution of times between life-history events, using short-lived midge species as an example. In particular we focus on the distribution of the extrinsic incubation period (EIP) which determines the time between infection and becoming infectious, and the distribution of the length of the gonotrophic cycle which determines the time between successful bites. We show how different assumptions for these periods can radically change the basic reproductive ratio (R0) of an infection and additionally the impact of vector control on the infection. These findings highlight the need for detailed entomological data, based on laboratory experiments and field data, to correctly construct the next-generation of policy-informing models.

Impact of experimental flooding on larvae and pupae of dung-breeding Culicoides.

Culicoides biting midges (Diptera: Ceratopogonidae) spend the greatest part of their life in the larval stage. However, knowledge on the immature stages and the impact of abiotic factors on their development is still poor. Therefore, we investigated the effect of flooding on the larvae and pupae of Culicoides chiopterus (Meigen, 1830) and C. dewulfi Goetghebuer, 1936. In water, the larvae of both species showed head-to-tail flexions and sinuous flexions, at slow rates, but were not able to swim. Flooding of larvae for 24 h did not affect the number of emerging adults; flooding of pupae significantly reduced the emergence rate of C. chiopterus, compared to the control group, while C. dewulfi was not affected. Pupae were not able to float and no pupae survived flooding for 10 days. After flooding of larvae for 10 days, 50 % of C. chiopterus and 4 % of C. dewulfi completed the pre-adult development. During this treatment, 84 % of C. chiopterus and 48 % of C. dewulfi larvae pupated in water.

Laboratory rearing of immature Culicoides peregrinus Kieffer, a potential vector of bluetongue virus.

Culicoides peregrinus (Kieffer) (Diptera: Ceratopogonidae) was reared from egg to adult using four different combinations of food and substrate (T1: absorbent cotton, mud broth with 2% yeast; T2: 1% agar, mud broth with 2% yeast; T3: absorbent cotton, nutrient broth; T4: absorbent cotton, 2% yeast). Field-collected engorged females exhibited mean fecundity of 82.45 ± 4.00. The highest rate of emergence and largest adults were obtained in T1, and the lowest rate of emergence was observed in T4. Two-way analyses of variance with post hoc Tukey tests showed significant differences in age at pupation, pupal weight and wing length among the various treatments, although the sex ratio was 1 : 1 in all food/substrate combinations. The successful rearing of immature C. peregrinus is an encouraging step towards the establishment of a laboratory colony of this prevalent species associated with livestock.

Species Diversity and Seasonal Distribution of Culicoides spp. (Diptera: Ceratopogonidae) in Jeju-do, Republic of Korea.

Biting midges belonging to the genus Culicoides (Diptera: Ceratopogonidae) were collected by Mosquito Magnet(®) and black light traps at 5 sites on Jeju-do, Republic of Korea (Korea), from May-November 2013 to determine species diversity and seasonal distribution. A total of 4,267 specimens were collected, of which 99.9% were female. The most common species was Culicoides tainanus (91.8%), followed by C. lungchiensis (7.2%) and C. punctatus (0.6%), while the remaining 4 species accounted for <0.5% of all Culicoides spp. that were collected. High numbers of C. tainanus were collected in May, followed by decreasing numbers through August, and then increasing numbers through November when surveillance was terminated. Peak numbers of C. lungchiensis were collected during September, with low numbers collected from May-August and October-November. The presence of C. lungchiensis in Korea was confirmed by morphological and molecular analyses.

Principal climatic and edaphic determinants of Culicoides biting midge abundance during the 2007-2008 bluetongue epidemic in the Netherlands, based on OVI light trap data.

Palaearctic Culicoides midges (Diptera: Ceratopogonidae) represent a vital link in the northward advance of certain arboviral pathogens of livestock such as that caused by bluetongue virus. The effects of relevant ecological factors on weekly Culicoides vector abundances during the bluetongue virus serotype 8 epidemics in the Netherlands in 2007 and 2008 were quantified within a hurdle modelling framework. The relative role of meteorological parameters showed a broadly consistent association across species, with larger catches linked to temperature-related variables and lower wind speed. Moreover, vector abundance was found to be influenced by edaphic factors, likely related to species-specific breeding habitat preferences that differed markedly amongst some species. This is the first study on Culicoides vector species in the Netherlands identified during an entomological surveillance programme, in which an attempt is made to pinpoint the factors that influence midge abundance levels. In addition to providing key inputs into risk-mitigating tools for midge-borne pathogens and disease transmission models, the adoption of methods that explicitly address certain features of abundance datasets (frequent zero-count observations and over-dispersion) helped enhance the robustness of the ecological analysis.

Impact of mechanical disturbance on the emergence of Culicoides from cowpats.

The outbreaks of Bluetongue virus and Schmallenberg virus between 2006 and 2012 highlighted the need for control methods for north-western Palaearctic biting midge species. There is especially a demand for alternative control methods without application of insecticides. Therefore, the objective of this paper was to evaluate the impact of mechanical disturbance as a control method for Culicoides biting midges in cowpats. We used a trash rake to destroy the compact structure of cowpats and spread the dung over the ground. We expected the disturbance to cause a faster alteration of physical characteristics and drying of the dung, resulting in lower emergence from disturbed cowpats. Emerging biting midges were collected with emergence traps. The number of emerged Culicoides was compared between treated and control cowpats. A total of 12,979 biting midges emerged, all belonging to the Obsoletus group. The majority of the 6,758 male individuals were identified as Culicoides chiopterus (Meigen, 1830) (91.4%), followed by Culicoides dewulfi (Goetghebuer, 1936) (6.5%), and a small number of Culicoides scoticus (Downes & Kettle, 1952) (1.2%). Our results showed no significant differences between the emergence of Culicoides (males and females) from disturbed and control cowpats. The lack of differences is discussed in light of the time period chosen for the experiment and the climatic conditions (especially low temperatures) during the study period. The climatic conditions during the study did not favour desiccation effects and therefore did not cause a severe alteration of the dung and the biotic parameters. In conclusion, immature Culicoides showed a high tolerance against mechanical disturbance.

Culicoides biting midge density in relation to the position and substrate temperature in a cattle dung heap.

Dung heaps offer warm breeding sites, which might be suitable for a continuing development or even emergence at low air temperatures in winter. Therefore, this study collected substrate samples from the outer surface and core of a cattle dung heap at the beginning of the winter period. We aimed to analyze the density of immature Culicoides in relation to substrate position and temperature. We took samples from the outer layer and core of the dung heap at different heights. Floatation was used to extract Culicoides larvae from the dung heap samples. In order to rear larvae individually, we separated them in glass tubes. A total of 229 Culicoides larvae were extracted from the dung heap samples. Highest densities (99.1% of all larvae) were recorded for the outer layers of the dung heap but hardly any in the core (0.9% of all individuals). While the density of larvae was negatively correlated with increasing substrate temperatures, Culicoides larvae were found in a temperature range between 7.9 and 38.0 °C (mean 16.6 °C). Extracted larvae were reared to adults. All male individuals were identified as Culicoides obsoletus (Meigen), 1818 and all female individuals as C. obsoletus/Culicoides scoticus. It can be concluded that dung heaps offer temperature conditions, which allow the survival and probably also the development to adults for immature Culicoides also under harsh climate conditions in winter.

Community analysis of biting midges (Culicoides Latr.) on livestock farms in Denmark.

This study presents descriptive statistics and community analysis of adult biting midges trapped at 16 livestock farms by means of light traps on Zealand and Lolland-Falster, Denmark. A total of 9,047 male and female Culicoides divided into 24 species, were caught. Biotic and abiotic factors ranging from presence of different host species (cattle or sheep/goats), presence of small woody areas or wetlands in the surrounding landscape, and agricultural practice (organic or conventional) were included in the community analysis. Only differences in the Culicoides communities between conventional and organic practices were tested significantly different. Total numbers of Culicoides individuals were higher on the organic farms than on the conventional farms. The larger loads of biting midges on the organic farms may be due to free-ranging animals that attracted the midges on pastures and carried them to the stable environment (the cattle of the conventional farms were held inside the stables). Presence of deciduous trees within 500 m of the farms resulted in higher numbers of Culicoides obsoletus s.s., while presence of wetlands increased the numbers of Culicoides punctatus and Culicoides pulicaris. Furthermore, Culicoides riethi and Culicoides puncticollis (subgenus Monoculicoides) were recorded in high numbers on individual farms. C. puncticollis was found for the first time in Denmark and so far only recorded from Zealand.

Seasonal abundance of Culicoides (Diptera: Ceratopogonidae) collected by mosquito Magnet® in Northern Gyeonggi-do (Province), Korea.

Biting midges (Culicoides: Ceratopogonidae) were collected by Mosquito Magnet® traps at the Neutral Nations Supervisory Commission (NNSC) camp and Daeseongdong village inside the demilitarized zone (DMZ) and near the military demarcation line (MDL) separating North and South Korea and at Warrior Base (US Army training site) and Tongilchon 3 km south of the DMZ in northern Gyeonggi Province, Republic of Korea (ROK), from May-October 2010-2012, to determine their seasonal distributions. A total of 18,647 Culicoides females (18,399; 98.7%) and males (248; 1.3%) comprising 16 species were collected. Overall, the most commonly collected species was Culicoides nipponensis (42.9%), followed by C. erairai (29.2%), C. punctatus (20.3%), C. arakawae (3.3%), C. pallidulus (1.8%), and C. circumscriptus (1.4%), while the remaining 10 species accounted for only 1.1% of all Culicoides spp. collected. The seasonal distribution of C. nipponensis was bimodal, with high numbers collected during May-June and again during September. C. erairai was more frequently collected during June-July, followed by sharply decreased populations from August-October. C. punctatus was collected in low numbers from May-September with high numbers collected during October. C. erairai was predominantly collected from the NNSC camp (85.1% of all C. erairai collected) located adjacent to the MDL at Panmunjeom in the northernmost part of Gyeonggi-do (Province), while other sites yielded low numbers of specimens.

Molecular identification of field-collected Culicoides larvae in the southern part of Japan.

Although Culicoides biting midges act as a vector of important human and domestic animal diseases, their ecology is poorly understood. The lack of proper identification systems of Culicoides larvae is one of the main obstacles to progress in research. Based on mitochondrial sequences of 19 Japanese Culicoides species, we designed a universal primer set to amplify the partial sequence of the mitochondrial cytochrome c oxidase I (cox 1). The polymerase chain reaction product amplified from extracted DNA of Culicoides larvae using the primer set was directly sequenced, and species identification based on the variation at cox1 was conducted. Using the molecular identification system, we sorted 243 specimens of field-collected larvae from the southern part of Japan into 10 species including Culicoides arakawae (Arakawa), Culicoides oxystoma Kieffer, and Culicoides brevitarsis Kieffer, which are regarded as vectors of important livestock animal diseases. Eight species of Culicoides larvae, including C. arakawae and C. oxystoma, were recovered from active paddy fields and an abandoned paddy field. The result suggests that paddy fields contribute to breeding a variety of Culicoides species and maintenance and spread of Culicoides-borne pathogens. In contrast, larvae of C. brevitarsis were collected from cattle dung in pastures. The molecular identification system described herein using nucleotide sequences successfully achieved larval identification and will be useful for a better understanding of larval habitats of Culicoides biting midges.

A new species of Dasyhelea Kieffer (Diptera: Ceratopogonidae) from Brazilian Amazonia.

A new species from Manaus, Amazonas, Brazil, Dasyhelea eloyi Díaz & Ronderos, is described, illustrated and photomicrograph as larva, pupa and male and female adults based on examination with binocular and scanning electron microscopes. This new species, a typical member of the Dasyhelea grisea species group, is compared with the most similar congeners D. necrophila Spinelli & Rodriguez and the Nearctic D. pseudoincinsurata Waugh & Wirth.

New contributions to the knowledge of the immatures of Stilobezzia punctulata Lane and Stilobezzia fiebrigi Kieffer (Diptera: Ceratopogonidae).

The fourth instar larva of Stilobezzia punctulata Lane is described for the first time and the one of Stilobezzia fiebrigi Kieffer is redescribed. They are illustrated and photomicrographed from material collected in different aquatic environments of the province of Corrientes, Argentina. Both species show features typical to carnivorous-predatory larva.

Morphological description of the fourth instar larva: Culicoides cataneii and Culicoides sahariensis (Diptera: Ceratopogonidae).

This study was carried out of the region of Monastir in Central Tunisia, between July and August 2010. Larvae were collected using a floatation technique with magnesium sulfate in mud samples. The fourth instar larva of Culicoides cataneii Clastrier, 1957 and Culicoides sahariensis Callot, Kremer, Bailly-Choumara, 1970 are described, illustrated and drawn. Measurements of instars IV are also presented. This is the first record of Culicoides cataneii and Culicoides sahariensis (Diptera: Ceratopogonidae) to Tunisia.

Impacts of climate, host and landscape factors on Culicoides species in Scotland.

Culicoides biting midges (Diptera: Ceratopogonidae) vector a wide variety of internationally important arboviral pathogens of livestock and represent a widespread biting nuisance. This study investigated the influence of landscape, host and remotely-sensed climate factors on local abundance of livestock-associated species in Scotland, within a hierarchical generalized linear model framework. The Culicoides obsoletus group and the Culicoides pulicaris group accounted for 56% and 41%, respectively, of adult females trapped. Culicoides impunctatus Goetghebuer and C. pulicaris s.s. Linnaeus were the most abundant and widespread species in the C. pulicaris group (accounting for 29% and 10%, respectively, of females trapped). Abundance models performed well for C. impunctatus, Culicoides deltus Edwards and Culicoides punctatus Meigen (adjusted R(2) : 0.59-0.70), but not for C. pulicaris s.s. (adjusted R(2) : 0.36) and the C. obsoletus group (adjusted R(2) : 0.08). Local-scale abundance patterns were best explained by models combining host, landscape and climate factors. The abundance of C. impunctatus was negatively associated with cattle density, but positively associated with pasture cover, consistent with this species' preference in the larval stage for lightly grazed, wet rush pasture. Predicted abundances of this species varied widely among farms even over short distances (less than a few km). Modelling approaches that may facilitate the more accurate prediction of local abundance patterns for a wider range of Culicoides species are discussed.

Sediment organic tin contamination promotes impoverishment of non-biting midge species communities in the Archipelago Sea, S-W Finland.

Chironomid species are a vital component in many benthic and terrestrial food webs; they have an important role in the detritus cycle, and are an important source of food for many species. We studied how tributyltin (TBT) in brackish water sediments affect the composition of chironomid species communities. Emergence traps were used at selected sites on a TBT gradient in the Archipelago Sea, S-W Finland. Increased sediment TBT concentration was associated with significant chironomid species turnover, which in turn was related to decreased species diversity (number of species and genera). However, the overall number of individuals did not decrease markedly with increasing TBT contamination. This suggests that the ecological role of chironomids in the food web may be preserved even under severe impoverishment of the chironomid community due to organic tin contamination. The increased prevalence of more TBT tolerant species can potentially lead to a transport of organic tin compounds between aquatic and terrestrial food webs. Furthermore, the reduced diversity of an ecologically influential group might lower the resistance of the entire food web to other environmental hazards and perturbations.