Comparative genomics of a novel Erwinia species associated with the Highland midge (Culicoides impunctatus).

Erwinia (Enterobacterales: Erwiniaceae) are a group of cosmopolitan bacteria best known as the causative agents of various plant diseases. However, other species in this genus have been found to play important roles as insect endosymbionts supplementing the diet of their hosts. Here, I describe Candidatus Erwinia impunctatus (Erwimp) associated with the Highland midge Culicoides impunctatus (Diptera: Ceratopogonidae), an abundant biting pest in the Scottish Highlands. The genome of this new Erwinia species was assembled using hybrid long and short read techniques, and a comparative analysis was undertaken with other members of the genus to understand its potential ecological niche and impact. Genome composition analysis revealed that Erwimp is similar to other endophytic and ectophytic species in the genus and is unlikely to be restricted to its insect host. Evidence for an additional plant host includes the presence of a carotenoid synthesis operon implicated as a virulence factor in plant-associated members in the sister genus Pantoea. Unique features of Erwimp include several copies of intimin-like proteins which, along with signs of genome pseudogenization and a loss of certain metabolic pathways, suggests an element of host restriction seen elsewhere in the genus. Furthermore, a screening of individuals over two field seasons revealed the absence of the bacteria in Culicoides impunctatus during the second year indicating this microbe-insect interaction is likely to be transient. These data suggest that Culicoides impunctatus may have an important role to play beyond a biting nuisance, as an insect vector transmitting Erwimp alongside any conferred impacts to surrounding biota.

Floral Odors and the Interaction between Pollinating Ceratopogonid Midges and Cacao.

Most plant species depend upon insect pollination services, including many cash and subsistence crops. Plants compete to attract those insects using visual cues and floral odor which pollinators associate with a reward. The cacao tree, Theobroma cacao, has a highly specialized floral morphology permitting pollination primarily by Ceratopogonid midges. However, these insects do not depend upon cacao flowers for their life cycle, and can use other sugar sources. To understand how floral cues mediate pollination in cacao we developed a method for rearing Ceratopogonidae through several complete lifecycles to provide material for bioassays. We carried out collection and analysis of cacao floral volatiles, and identified a bouquet made up exclusively of saturated and unsaturated, straight-chain hydrocarbons, which is unusual among floral odors. The most abundant components were tridecane, pentadecane, (Z)-7-pentadecene and (Z)-8-heptadecene with a heptadecadiene and heptadecatriene as minor components. We presented adult midges, Forcipomyia sp. (subgen. Forcipomyia), Culicoides paraensis and Dasyhelea borgmeieri, with natural and synthetic cacao flower odors in choice assays. Midges showed weak attraction to the complete natural floral odor in the assay, with no significant evidence of interspecific differences. This suggests that cacao floral volatiles play a role in pollinator behavior. Midges were not attracted to a synthetic blend of the above four major components of cacao flower odor, indicating that a more complete blend is required for attraction. Our findings indicate that cacao pollination is likely facilitated by the volatile blend released by flowers, and that the system involves a generalized odor response common to different species of Ceratopogonidae.

A cream containing omega-3-fatty acids, humectants and emollients as an aid in the treatment of equine Culicoides hypersensitivity.

BACKGROUND: Topical application of polyunsaturated fatty acids (PUFAs) has shown satisfactory results in dogs and humans with allergic skin diseases. Urea and glycolic acid act as keratolytics and moisturizers. Culicoides hypersensitivity is the most common equine hypersensitivity disorder and only limited treatment options exist. OBJECTIVES: To evaluate the effect of a cream containing topical PUFAs, humectants and emollients on clinical signs of equine Culicoides hypersensitivity. ANIMALS: Privately owned horses (n = 28) with clinical signs of Culicoides hypersensitivity. METHODS AND MATERIALS: For a period of four weeks, one half of the horse's body (left or right) was treated with a cream containing concentrated fish oil and several moisturizing and emollient ingredients in a randomized, single-blinded fashion to evaluate the influence of the treatment on skin lesions. In the subsequent four weeks, the lesional areas of the entire body were treated to assess the treatment effect on pruritus. Additionally, the quality of the hair coat, an overall assessment and adverse effects were recorded. RESULTS: Twenty-one horses completed the study. Skin lesions on the treated side improved significantly between days 0 and 28 (P < 0.0001) in comparison to the untreated side. Neither pruritus scores nor coat quality improved significantly between days 0 and 56. Overall condition improved during the study. Five horses showed adverse effects. CONCLUSIONS AND CLINICAL IMPORTANCE: The cream improved Culicoides-induced skin lesions in affected horses, but anti-pruritic effects were less prominent.

Dermatite alérgica à picada de Culicoides em muar: relato de caso / Allergic dermatitis due to bite of Culicoides in muar: case report

A dermatite alérgica à picada de ectoparasitos é uma enfermidade alergoparasitária bastante comum entre animais domésticos, sendo relatada principalmente em pequenos ruminantes e em animais de companhia. Contudo, a doença é pouco diagnosticada na clínica de equídeos devido a similaridades nosológicas com outras dermatopatias. Objetivou-se, com este relato de caso, descrever a síndrome clínica, o plano diagnóstico e a conduta terapêutica de um muar acometido por essa enfermidade. Atendeu-se, no Hospital Veterinário da Universidade Federal Rural do Pernambuco, uma mula de oito anos de idade, que apresentava lesões cutâneas pápulo-crostosas e pruriginosas com evolução clínica de dois anos. Em três situações anteriores, a doença havia sido tratada como dermatite fúngica por outros médicos veterinários. Para o diagnóstico, foram solicitados exame citopatológico e parasitológico de pele, cultivo bacteriológico e fúngico, análise histopatológica e hemograma. Os exames demonstraram uma dermatite superficial perivascular eosinofílica crônica, sendo indicada a terapia tópica com dimetilsufóxido, sulfadiazina, ureia e vitamina A. O protocolo terapêutico mostrou-se satisfatório, permitindo completa remissão do quadro clínico. Este trabalho relatou achados clínicos e patológicos da dermatite alérgica à picada de Culicoides spp. em muar, além de alertar sobre a importância de exames complementares para a realização do diagnóstico diferencial e para o direcionamento terapêutico adequado.(AU)

Allergic dermatitis to ectoparasite bites is a common parasitic disease among domestic animals, being reported mainly in small ruminants and companion animals. However, the disease is poorly diagnosed in equine clinics due to nosological similarities with other skin diseases. The aim of this case report was to describe the clinical syndrome, the diagnostic plan and the therapeutic management of a mule affected by this disease. An 8-year-old mule was observed at Universidade Federal Rural de Pernambuco, presenting papular-crusted and pruritic cutaneous lesions with clinical evolution of two years. In three previous situations, the disease had been treated as fungal dermatitis by other veterinarians. For the diagnosis, cytopathological and parasitological examination of the skin, bacteriological and fungal culture, histopathological analysis and blood count were performed. The exams showed a chronic eosinophilic perivascular superficial dermatitis. A topical therapy with dimethyl sulfoxide, sulfadiazine, urea, and vitamin A was indicated. The therapeutic protocol was satisfactory, allowing complete remission of the clinical condition. This work reported clinical and pathological findings of allergic dermatitis to the bites of Culicoides spp. in muar, in addition to alerting about the importance of complementary examinations for the accomplishment of the differential diagnosis and adequate therapeutic orientation.(AU)

DEET (N,N-diethyl-meta-toluamide)/PMD (para-menthane-3,8-diol) repellent-treated mesh increases Culicoides catches in light traps.

Biting midges (Culicoides spp.) are vectors of bluetongue and Schmallenberg viruses. Treatment of mesh barriers is a common method for preventing insect-vectored diseases and has been proposed as a means of limiting Culicoides ingression into buildings or livestock transporters. Assessments using animals are costly, logistically difficult and subject to ethical approval. Therefore, initial screening of test repellents/insecticides was made by applying treatments to mesh (2 mm) cages surrounding Onderstepoort light traps. Five commercial treatments were applied to cages as per manufacturers' application rates: control (water), bendiocarb, DEET/p-menthane-3,8-diol (PMD) repellent, Flygo (a terpenoid based repellent) and lambda-cyhalothrin. The experimental design was a 5 × 5 Latin square, replicated in time and repeated twice. Incongruously, the traps surrounded by DEET/PMD repellent-treated mesh caught three to four times more Obsoletus group Culicoides (the commonest midge group) than the other treatments. A proposed hypothesis is that Obsoletus group Culicoides are showing a dose response to DEET/PMD, being attracted at low concentrations and repelled at higher concentrations but that the strong light attraction from the Onderstepoort trap was sufficient to overcome close-range repellence. This study does not imply that DEET/PMD is an ineffective repellent for Culicoides midges in the presence of an animal but rather that caution should be applied to the interpretation of light trap bioassays.

Cuticular and internal chemical composition of biting midges Culicoides spp. (Diptera: Ceratopogonidae), potential vectors of viral diseases.

The chemical profile of the cuticle and internal tissues of four species of Culicoides have been studied for the first time by gas chromatography-mass spectrometry. The chemical composition of females of C. obsoletus s.l. and C. lupicaris, vectors of diverse viral diseases, have been compared with that of other biting midges, such as C. kibunensis and C. fascipennis, and the non-biting midge Forcipomyia bipunctata. A total of 61 compounds belonging to 8 major chemical classes were identified in cuticular and internal tissues in n-hexane extracts. The compounds include carboxylic acids (CAs) (C6-C20), with C16:0, C16:1 and C18:1 being dominant, branched hydrocarbons (C29 to C38 mono/di/trimethylalkanes), linear hydrocarbons (C15 to C33, mainly odd chain carbons), terpenes (geranylacetone, geranylgeraniol acetate, squalene, terpenic alcohol), steroids (cholesterol), aldehydes (C9-C10 and even chain C20 to C30), and esters. The chemical profile depends on the species and whether the extracts are external (cuticle) or internal. The contents of linear and branched hydrocarbons and aldehydes was high in cuticular extracts but practically absent in internal tissues, which were, in contrast, rich in CAs, terpenes and steroids. The results are discussed and compared with other Culicoides midges and mosquito-related species.

Laboratory and field evaluations of chemical and plant-derived potential repellents against Culicoides biting midges in northern Spain.

The efficacy of 23 compounds in repelling Culicoides biting midges (Diptera: Ceratopogonidae), particularly Culicoides obsoletus (Meigen) females, was determined by means of a Y-tube olfactometer. The 10 most effective compounds were further evaluated in landing bioassays. The six most promising compounds (including chemical and plant-derived repellents) were evaluated at 10% and 25% concentrations in field assays using Centers for Disease Control (CDC) light traps. At least three compounds showed promising results against Culicoides biting midges with the methodologies used. Whereas olfactometer assays indicated DEET at 1 µg/µL to be the most effective repellent, filter paper landing bioassays showed plant-derived oils to be better. Light traps fitted with polyester mesh impregnated with a mixture of octanoic, decanoic and nonanoic fatty acids at 10% and 25% concentrations collected 2.2 and 3.6 times fewer midges than control traps and were as effective as DEET, which is presently considered the reference standard insect repellent. The best plant-derived product was lemon eucalyptus oil. Although these have been reported as safe potential repellents, the present results indicate DEET and the mixture of organic fatty acids to be superior and longer lasting.

Assessment of the repellent effect of citronella and lemon eucalyptus oil against South African Culicoides species.

The use of insect repellents to reduce the attack rate of Culicoides species (Diptera:Ceratopogonidae) should form part of an integrated control programme to combat African horse sickness and other diseases transmitted by these blood-feeding midges. In the present study the repellent effects of a commercially available mosquito repellent, a combination of citronella and lemon eucalyptus oils, on Culicoides midges was determined. The number of midges collected with two 220 V Onderstepoort traps fitted with 8 W 23 cm white light tubes and baited with peel-stick patches, each containing 40 mg of active ingredient, was compared with that of two unbaited traps. Two trials were conducted and in each trial the four traps were rotated in two replicates of a 4 x 4 randomised Latin square design. Although more midges were collected in the baited traps, the mean number in the baited and unbaited traps was not significantly different. This mosquito repellent did not influence either the species composition or the physiological groups of Culicoides imicola Kieffer. The higher mean numbers in the baited traps, although not statistically significant, may indicate that this mosquito repellent might even attract Culicoides midges under certain conditions.

Chemical composition of silage residues sustaining the larval development of the Culicoides obsoletus/Culicoides scoticus species (Diptera: Ceratopogonidae).

Culicoides (Diptera: Ceratopogonidae) are biological vectors of bluetongue virus (BTV). Bluetongue is a viral disease that affects domestic and wild ruminants. Since its recent emergence in northern Europe, this disease has caused considerable economic losses to the sheep and cattle industry. The biotopes, and more particularly the chemical characteristics which are suitable for larval development of the main vector species, are still relatively unknown. This study shows that the larvae of biting midges belonging to the species Culicoides obsoletus and Culicoides scoticus are able to breed in different types of silage residue (maize, grass, sugar beet pulp and their combinations). The chemical composition of substrates strongly influences the presence of the immature stages of these biting midges. Higher lignin and insoluble fibre contents seem to favour their presence and could play the role of a physical support for semi-aquatic larvae. In contrast, higher concentrations of magnesium and calcium are negatively correlated with the presence of these two species. These data will help to locate and monitor the breeding sites of these species and could contribute to the control of these insects on farms.

[Field efficacy of repellent formulation containing para-menthane-3,8-diol and lemongrass against Culicoides pachymerus (Diptera: Ceratopogonidae) in Colombia]. / Eficacia en campo de un repelente a base de para-mentano-3,8-diol y aceite de limonaria contra Culicoides pachymerus (Diptera: Ceratopogonidae) en Colombia.

INTRODUCTION: Culicoides pachymerus is a major pest species for the inhabitants of the western Boyacá province of Colombia. OBJECTIVE: The effect of a repellent lotion based on p-menthane-3,8-diol (16%) and lemongrass oil (2%) was evaluated against the bites of C. pachymerus. MATERIALS AND METHODS: The repellent lotion was compared simultaneously with a control (no treatment) by human landing catches of C. pachymerus on the forearms of paired volunteers situated near human dwellings. Protection percentage and protection time for 3 to 6 h after repellent application was calculated. The test was repeated ten times. RESULTS: Only two females of C. pachymerus were collected on arms with the repellent treatment. In contrast, the mean biting rate in the untreated control was 47.7 midges/person/10 min. Mean protection percentage of the repellent was 100% up to 4 h and 99.5% up to 5 h. Protection time was 332.2 and 338.2 min in the two replicates where bites of C. pachymerus were confirmed. In the remaining eight replicates protection time exceeded the test duration. CONCLUSION: The repellent showed high efficacy against C. pachymerus, up to 5 h post-application.

The effect of a supplement containing sunflower oil, vitamins, amino acids, and peptides on the severity of symptoms in horses suffering insect bite hypersensitivity.

Insect bite hypersensitivity (IBH) is most often caused by Culicoides spp., although other insect species are also thought to play a role in causing this disease. The disease has been reported to occur in most countries, with a prevalence of up to 28% in some countries in some breeds. The only truly effective therapy currently available is to prevent horses from coming into contact with Culicoides midges, which can be achieved by stabling horses during certain times of the day or by covering them with insect blankets. The effect of a new dietary supplement containing vitamins, polypeptides, and amino acids on IBH severity was tested at the start of the Culicoides season (April 2009). Fifty horses participated in this placebo-controlled, double-blinded study: 25 horses received the supplement and 25 received placebo for 30 days; thereafter all horses received the supplement for a further 30 days. The investigators scored the severity of IBH in all horses, based on clinical evaluation and digital photographs taken before and after the first 30 days of the trial. The owners were asked to assess the severity of the signs prior to the investigation and after the first and second 30-day periods. For each period, IBH clinical symptoms were classified as increased, decreased, or the same. Results showed that investigator-assessed symptoms became worse in more horses receiving placebo than in horses receiving supplement, indicating a positive effect of the supplement. However, there were no treatment-group differences when symptom severity was scored by the horse owners. No side-effects were observed.

Duration of repellency of N,N-diethyl-3-methylbenzamide, citronella oil and cypermethrin against Culicoides species when applied to polyester mesh.

The repellent efficacy of 15% N,N-diethyl-3-methylbenzamide (DEET), 0.6% citronella oil, and 0.3% alpha-cyano-cypermethrin against Culicoides species was compared in three 5x5 Latin squares (15 replicates) under South African field conditions. DEET, citronella oil or alpha-cyano-cypermethrin were applied to polyester meshes that were fitted to down-draught suction 220V UV light traps which were operated overnight. No significant repellent effect against Culicoides was found for the citronella oil or the alpha-cyano-cypermethrin. DEET had a significant repellent effect against Culicoides species and C. imicola for all catches made from after sunset to before sunrise.

Testing the use of a citronella-based repellent as an effective method to reduce the prevalence and abundance of biting flies in avian nests.

Here, we validate the use of a citronella (natural oil) based repellent to reduce the abundance of flying blood-sucking insects in avian nests. These insects are important parasites of birds affecting them as blood feeders and as vectors of a diversity of pathogens. When nestling were 10 days old, we assigned wild great tit Parus major nests to one of two treatments, control and fumigated nests. The abundance of biting midges and blackflies captured during 3 days following the treatment application were lower in fumigated nests with respect to control ones. By contrast, the abundance of blowfly pupae measured when nestlings left their nests was not affected by the treatment. Although many experimental studies modify the abundance of nest-dweller ectoparasites, to our knowledge, this is the first one describing an easy, safe, and effective method, reducing the total abundance of both biting midges and blackflies in wild avian nests. Our results could be used in future conservation projects and experimental studies on host-parasite evolution affecting the abundance of flying blood-feeder insects under natural conditions.

Field test of a lemon eucalyptus repellent against Leptoconops biting midges.

We tested a lemon eucalyptus-based repellent against the biting midge Leptoconops carteri Hoffman in the Central Valley of California. This relatively new active ingredient has demonstrated high efficacy in a number of studies with mosquitoes. Ten subjects tested spray and lotion formulations on 2 consecutive days, along with a deet-positive control and an untreated control, with 6 h of continuous exposure per treatment. Half of the eucalyptus subjects received no bites, and the true median protection time probably exceeded the test duration.

Midgut and salivary gland transcriptomes of the arbovirus vector Culicoides sonorensis (Diptera: Ceratopogonidae).

Numerous Culicoides spp. are important vectors of livestock or human disease pathogens. Transcriptome information from midguts and salivary glands of adult female Culicoides sonorensis provides new insight into vector biology. Of 1719 expressed sequence tags (ESTs) from adult serum-fed female midguts harvested within 5 h of feeding, twenty-eight clusters of serine proteases were derived. Four clusters encode putative iron binding proteins (FER1, FERL, PXDL1, PXDL2), and two clusters encode metalloendopeptidases (MDP6C, MDP6D) that probably function in bloodmeal catabolism. In addition, a diverse variety of housekeeping cDNAs were identified. Selected midgut protease transcripts were analysed by quantitative real-time PCR (q-PCR): TRY1_115 and MDP6C mRNAs were induced in adult female midguts upon feeding, whereas TRY1_156 and CHYM1 were abundant in midguts both before and immediately after feeding. Of 708 salivary gland ESTs analysed, clusters representing two new classes of protein families were identified: a new class of D7 proteins and a new class of Kunitz-type protease inhibitors. Additional cDNAs representing putative immunomodulatory proteins were also identified: 5' nucleotidases, antigen 5-related proteins, a hyaluronidase, a platelet-activating factor acetylhydrolase, mucins and several immune response cDNAs. Analysis by q-PCR showed that all D7 and Kunitz domain transcripts tested were highly enriched in female heads compared with other tissues and were generally absent from males. The mRNAs of two additional protease inhibitors, TFPI1 and TFPI2, were detected in salivary glands of paraffin-embedded females by in situ hybridization.

Flaxseed (Linum usitatissimum) supplementation associated with reduced skin test lesional area in horses with Culicoides hypersensitivity.

The purpose of this study was to quantify the effect of flaxseed (Linum usitatissimum) supplementation on the skin test response of atopic horses. Six horses that displayed a positive skin test for allergy to extract from Culicoides sp. participated in the 42-day, placebo-controlled, double-blind, cross-over trial. Results showed that supplementation with flaxseed for 42 days in our experimental horses reduced the mean skin test response to Culicoides sp. This observation was concurrent with a significant decrease in the long-chain saturated fatty acids; behenic acid (22:0) and lignoceric acid (24:0), in the hair of horses receiving flaxseed. There was also a significant decrease in aspartate aminotransferase, and increase in serum glucose in the treatment animals at specific sampling points. It was concluded that; in this small pilot study, flaxseed was able to reduce the lesional area of the skin test response of atopic horses, alter the fatty acid profile of the hair, reduce inflammation, and did not elicit any negative side-effects in the experimental horses.

Response of Culicoides sonorensis (Diptera: Ceratopogonidae) to 1-octen-3-ol and three plant-derived repellent formulations in the field.

The potential attractant 1-octen-3-ol and 3 potential repellents were assayed for activity for Culicoides sonorensis, the primary vector of bluetongue virus in North America. Collections using octenol were low, but numbers in suction traps were greater in the high-octenol treatment (11.5 mg/h) than in the low-octenol treatment (1.2 mg/h) or unbaited control for both sexes. Collections using high octenol, CO2 (approximately 1,000 ml/min), or both showed octenol alone to be significantly less attractive than either of the CO2 treatments and that octenol did not act synergistically with this level of CO2. A plant-derived (Meliaceae) extract with 4.5% of active ingredient (AI) (Ag1000), heptanone solvent, Lice free (2% AI from plant extracts in water), Mosi-guard with 50% Eucalyptus maculata var. citriodora Hook extract, and N,N-diethyl-m-toluamide (deet) were applied to polyester-cotton coarse mesh nets and deployed in conjunction with suction light traps plus CO2. Collections in the trap with deet were 66% lower (P < 0.05) than the heptanone and 56% (P > 0.05) less than the untreated (negative) control. Relative to deet, collections in the traps with the lice repellent, Ag1000, and Mosi-guard were reduced by 15, 34, and 39%, respectively (P > 0.05). The method has promise for field screening of potential repellents before on-animal testing.

Duration of repellency of various synthetic and plant-derived preparations for Culicoides imicola, the vector of African horse sickness virus.

Objectives of the study were threefold: to find a safer and longer lasting repellent of the biting midge Culicoides imicola than di-ethyl toluamide (DEET), to examine whether the current recommendations in Israel for application of repellents during an outbreak of C. imicola-borne pathogens are justified; and to examine whether plant-derived preparations that have no known detrimental side effects are potential replacements of synthetic repellents. Of the seven repellents tested, those inferior to DEET were: oregano and Herbipet which showed a slight non-significant repellency for 2 h and 1 h respectively and Stomoxin which showed significant (P < 0.05) repellency for only 1 h. As the active ingredient of Stomoxin is permethrin, this suggests that recommendations to spray animals with this insecticide to prevent the spread of C. imicola-borne pathogens are not useful. Tri-Tec14 showed significant (P < 0.05) repellency with respect to controls for 2 h only, but performed similarly to, or slightly better than DEET. The repellents clearly superior to DEET were: the plant-derived material Ag1000 that repelled significantly (P < 0.05) with respect to controls for up to 4 h following a similar pattern to but somewhat more strongly than DEET, and pyrethroid-T which exerted significant (P < 0.05) repellency for 9 h. Pyrethroid-T proved to be the best repellent tested and if sprayed nightly it might provide protection from C. imicola-borne pathogens.

Evaluation of a eucalyptus-based repellent against Culicoides impunctatus (Diptera:Ceratopogonidae) in Scotland.

A eucalyptus-based insect repellent (PMD) was evaluated against Culicoides impunctatus in Scotland in comparison with deet. In human landing catches, both repellents still afforded 98% protection from biting 8 h after application of 0.5 ml to the forearm. A second trial looking at protection between 8 and 10 h after repellent application showed 99.5% protection for PMD and 97% for deet as compared with controls.