Medical and Veterinary Entomology (2014) 28, 421–431

doi: 10.1111/mve.12081

Laboratory and field evaluations of chemical and plant-derived potential repellents against Culicoides biting midges in northern Spain M. G O N Z Á L E Z 1 , G. J. V E N T E R 2 , S. L Ó P E Z 3 , J. C. I T U R R O N D O B E I T I A 4 and A. G O L D A R A Z E N A 1,5 1

Entomology Laboratory, Basque Institute of Agricultural Research and Development, NEIKER-TECNALIA, Vitoria, Spain, ARC-Onderstepoort Veterinary Institute, Onderstepoort, South Africa, 3 Department of Biological Chemistry and Molecular Modelling, IQAC-CSIC, Barcelona, Spain, 4 Department of Zoology and Animal Cell Biology, University of Basque Country, Leioa, Spain and 5 Department of Pest Management and Vector Control, Institute of Ecology (INECOL), Xalapa, Veracruz, Mexico 2

Abstract. 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. Key words. Culicoides, Culicoides obsoletus, landing assays, light traps, plant extracts, repellents, synthetic repellents, Y-tube olfactometer.

Introduction Biting midges of the genus Culicoides have significant impact on the health of livestock. In addition to representing a severe biting nuisance in some parts of the world, certain members of this genus are implicated in the transmission of at least 66 different viruses, 15 protozoans and 26 filarial worms worldwide (Meiswinkel et al., 2004; Borkent, 2005). The veterinary importance of these midges in Spain was highlighted when bluetongue virus (BTV), a devastating orbiviral disease affecting ruminants and transmitted by Culicoides midges, entered the

Balearic islands in 2000 and subsequently spread across almost the whole of the Iberian peninsula from 2004 to 2008 (Pérez de Diego et al., 2013). After the detection of BTV in northwest Europe in 2006, the virus spread rapidly throughout northern Europe (Wilson & Mellor, 2009). In 2011 a novel orthobunyavirus, Schmallenberg virus (SBV), also apparently transmitted by Culicoides midges (Veronesi et al., 2013), was detected in Germany. It has caused severe economic losses to sheep and cattle breeders in Europe (Hoffmann et al., 2012). Of the 81 species of Culicoides recorded in Spain (Alarcón-Elbal & Lucientes, 2012), Culicoides obsoletus is

Correspondence: Arturo Goldarazena, Department of Pest Management and Vector Control, Institute of Ecology (INECOL), Xalapa 91070, Veracruz, Mexico. Tel.: + 52 228 842 1800; Fax: + 52 228 842 1801; E-mail: [email protected] © 2014 The Royal Entomological Society

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considered the most abundant livestock-associated species in northern Spain (Romón et al., 2012; González et al., 2013a). This species is widely distributed throughout the western Palaearctic (Carpenter et al., 2008) and is considered a potential vector of BTV and SBV throughout northern Europe (Wilson & Mellor, 2009; Veronesi et al., 2013). The effective control and monitoring of Culicoides midges are hampered by their small size (< 3 mm), predominantly nocturnal activity and the wide, and mostly unknown, range of habitats that can be utilized as larval development sites (González et al., 2013b; Harrup et al., 2013). These factors may have contributed to the failure of large-scale programmes using insecticides to control midges in the past (Satta et al., 2004). Nevertheless, the use of insecticides should be developed in the future and should not be limited to specific behavioural and habitat conditions. The effective use of repellents to reduce the biting rate of Culicoides midges may contribute to the more effective, sustainable integrated control of the pathogens transmitted. This approach could support current control strategies and may be particularly relevant in contexts in which appropriate vaccines are not available, such as in areas at risk for SBV or during outbreak situations (Mellor & Hamblin, 2004). Available insect repellents can be classified as either synthetic or plant-derived. Amongst synthetic products, DEET (N,N-diethyl-3-methylbenzamide) is considered one of the most efficient insect repellents worldwide (Katz et al., 2008). Although the activity of DEET in mosquitoes and other insects is widely documented, information regarding its effectiveness against Culicoides midges is limited to a few studies focusing on the Afrotropical Culicoides imicola and North American Culicoides sonorensis (Braverman & Chizov-Ginzburg, 1997, 1998; Page et al., 2009). Preliminary studies in the Basque Country showed significant repellent effects of DEET against C. obsoletus/Culicoides scoticus, Culicoides lupicaris and Culicoides achrayi (P. Romón et al., unpublished data, 2010). Plant-derived repellents consist of a wide range of natural compounds that can act as fumigants, contact insecticides or anti-feedants (Isman, 2000). Of thousands of plant-derived essential oils that have been evaluated as potential repellents, relatively few have demonstrated the broad effectiveness and long-lasting protection provided by DEET (Fradin, 1998). Some essential oils derived from Meliaceae and Myricaceae plants, such as neem oil and lemon eucalyptus oil have, however, demonstrated repellence for some Culicoides species in Scotland (Blackwell et al., 2004). Despite the apparent efficiency and widespread use of DEET, there are still some unresolved controversies concerning its safety and mechanisms of action (Corbel et al., 2009). In addition, it was shown that Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae) can become desensitized following exposure, with a subsequent decrease in the effectiveness of DEET (Stanczyk et al., 2013). The continuous development and evaluation of synthetic or chemical formulations to replace or complement DEET-based repellents will be essential to the sustainable integrated control of the pathogens transmitted by insect vectors. Because of the medical importance of mosquitoes, most studies involving repellents focus on their applicability against mosquitoes. Given

the veterinary importance of Culicoides midges, it will be necessary to expand these studies to include these species. In the present study 23 repellents were selected for testing based on literature showing their effectiveness against mosquitoes or other Culicoides spp. Laboratory and field results relative to their roles as potential insect repellents against the genus Culicoides were compared and discussed. Materials and methods Compounds evaluated The categories, chemical compositions and/or formulae and purity of 23 compounds or mixtures previously shown to repel either mosquitoes or Culicoides midges are shown in Table 1. Of the 23 products evaluated, 12 were of chemical or synthetic origin, and 11 of botanical origin (Table 1). The potential synergistic effects of four mixtures of these products were also determined (Table 1). All compounds, except KBR3023 and IR3535, which were obtained from commercial chemists, were purchased from Sigma-Aldrich Química SL (Madrid, Spain). After screening with Y-tube olfactometer and landing assays, the most promising products were evaluated under field conditions using light traps.

Origin and collection of test insects For olfactometer assays, newly emerged nulliparous C. obsoletus females were obtained from heaps of old manure on a farm near Barrundia, Elguea (42∘ 55′ 59′′ N, 02∘ 30′ 51′′ E; 754 m a.s.l.) in the north of Alava province (Basque Country), northern Spain, as previously described (Foxi & Delrio, 2010). Previous studies indicated that more than 99% of the specimens emerging from these manure heaps were C. obsoletus (González et al., 2013b). After emergence in the laboratory, C. obsoletus were aspirated and transferred to acclimatization cages and maintained at 22–26 ∘ C and 50–60% relative humidity (RH) on a 15% sucrose solution provided on cottonwool pledgets until used. For landing bioassays, C. obsoletus females were collected alive using suction light traps (CDC blacklight 4W model 1212; Entomopraxis, Barcelona, Spain) positioned near sheep. The collected females were anaesthetized with carbon dioxide and identified to species level prior to the Y-tube olfactometer and landing behaviour assays.

Y-tube olfactometer assays Repellence responses of C. obsoletus females were determined with a Y-tube olfactometer [Analytical Research Systems (ARS), Inc., Micanopy, FL, U.S.A.] adapted to accommodate smaller size midges. The main tube was 150 mm long and 20 mm in diameter. The arms were 100 mm long and 15 mm in diameter with a 90∘ angle between them. Regulated air flow was maintained at 0.3 L/min with a filtered charcoal-humidified air stream connected to the two glass chambers containing the test compound located at the proximal ends of the short arms.

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Table 1. Compounds and/or mixtures evaluated against Culicoides obsoletus in laboratory assays (two-choice olfactometer and landing bioassays). N

Commercial name

Category

Composition/formula

Purity

1 2 3

Cypermethrin DEET Geranyl acetone

Pyrethroid Organochloride Synthetic

99% ≥ 97% ≥ 97%

4 5 6 7 8 9 10 11 12 13 14

Picaridin/BayRepel (KBR3023) IR3535 Decanal Nonanal Octanal 6-Methyl-5-hepten-2-one Neem (azadirachtin) oil Limonene 4-Propylphenol Lavender oil Rhodinol (lemon grass) oil

Synthetic Synthetic Aldehyde Aldehyde Aldehyde Natural Plant-derived Terpene Organic compound Plant-derived Plant-derived

15 16 17

Lemon eucalyptus oil Rosemary oil Jasmine oil

Plant-derived Plant-derived Plant-derived

18

Balm leaves oil

Plant-derived

19 Mix 1

Lemon oil Mix: 1 : 1 : 1 (jasmine + lavender + rosemary) Mix: 1 : 1 (geranyl acetone + 6-methyl-5-hepten-2-one) Mix: 1 : 1 : 1 (octanoic + nonanoic + decanoic) Mix: 1 : 1 : 1 : 1 (balm + rhodinol + lemon eucalyptus + lemon oil)

Plant-derived Plant-derived

C22 H19 Cl2 NO3 N,N-diethyl-3-methyl-benzamide [(E)-6,10-dimethyl-5,9-undecadien-2-one], mixture isomers 1-piperidine carboxilic acid 3-[N-butyl-N-acetyl] aminopropionic acid ethyl ester CH3 (CH2 )8 CHO CH3 (CH2 )7 CHO CH3 (CH2 )6 CHO (CH3 )2 C = CHCH2 CH2 COCH3 Azadirachta indica oil C10 H16 (sum of enantiomers) C9 H12 O Lavandula angustifolia oil Cymbopogon citratus ct rhodinol (mixture of L-citronellol and geraniol) Eucalyptus maculata var. citriodora oil (PMD) Rosmarinus officinalis oil Jasminum grandiflorum oil (mix eugenol 2.1%, farnesol 0.1%, geraniol 0.1% and linalol 4%) Melissa officinalis oil (citral, citronellol, geraniol and linalool) Citrus limon oil (citronellol) Mixture of natural plant extracts

Mixture

Mixture of human odour constituents

≥ 90%

Acids

Mixture of fatty acids (C8, C9, C10)

96–98%

Plant-derived

Mixture of four components with citronellol as constituent

≥ 90%

Mix 2 Mix 3 Mix 4

The olfactometer was installed in a light-tight box. To instigate movement in the insects, a cold light source (LED) was placed at a position equidistant from each of the arms (Blackwell et al., 1994). Each of the 23 compounds was evaluated at dilutions of 1.0, 0.1 and 0.01 μg/μL in hexane. Quantities of 10 μL of the relevant compound were placed on Whatman filter papers (10 mm diameter), which were air-dried for 30 s before being inserted into one of the short arms of the olfactometer. An identical paper with pure hexane was used as a control in the second arm. For each replicate, 10–12 insects were immobilized at − 4 ∘ C for 30 s before they were inserted into the main tube of the olfactometer. Ten replicates were performed for each compound. To avoid directional bias, the arms were reversed after five replicates. The behavioural responses of the midges were observed after 4 min and were considered to be positive if midges advanced at least 4 cm into one of the arms, and negative if midges showed uplight orientation and/or inactivation. Assays were conducted from May to July 2012. Tests were performed between 07.00 hours and 10.00 hours. Each insect was tested only once until a maximum of 3 days after emergence. After 4 min the percentage repellence was calculated as 100 × N/T, where N = number of Culicoides repelled (i.e. the

50% 50% 95% 95% 99% ≥ 98% ≥ 90% 90% 99% ≥ 90% ≥ 90% ≥ 90% ≥ 90% ≥ 90% ≥ 90% ≥ 90% ≥ 90%

number of midges which advanced at least 4 cm into the arm containing the control) and T = number of midges which advanced at least 4 cm into either one of the two arms.

Filter paper landing bioassays The 10 repellents determined as most effective in olfactometer tests were subsequently evaluated in filter paper landing bioassays. Six conical, opaque plastic tubes measuring 150 mm in length and 50 mm in diameter were inserted 150 mm apart into one of the vertical sides of a gauze mosquito cage (600 × 600 × 600 mm). To encourage movement towards these tubes, the mosquito cage was lined with black paper and the tubes homogeneously illuminated with a small infrared light LED bulb. Circular Whatman filter papers with the same diameter as the tubes were impregnated with 100 μL of the relevant solution at a concentration of 1 μg/μL. Papers were air-dried for 30 s and then attached to the conical outlets of three of the six tubes. The remaining three tubes were loaded with filter papers impregnated with 100 μL hexane as controls. The positions of the six tubes were rotated in six replicates in a randomized Latin square design. Freshly impregnated filter papers were

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used for each replicate. Batches of 20–30 C. obsoletus females anaesthetized with carbon dioxide were released into the cage opposite the tubes. The number of midges on the filter papers was counted 2 s and 5 min after release. A fresh batch of midges was used in each trial and only one repellent was evaluated per day in order to avoid residual air contamination. Mosquito gauze in contact with products was washed. The percentage of repellence for each time period was calculated as 100 × N/T, where N = total number of Culicoides sitting on the three control papers and T = total number of Culicoides on all six filter papers (control + test). Assays were conducted in August 2012.

Statistical analyses of findings in field trials were carried out using systat Version 13.0 (Systat Software, Inc., San Jose, CA, U.S.A.). A P-value of ≤ 0.05 was considered to indicate statistical significance. If the data were not normally distributed, square root transformations were performed prior to conducting statistical analyses. If zero counts were observed, all data were (1 + x) transformed. Analyses of variance (anova) were used to differentiate between the trap treatment effects; means were compared using Fisher’s protected t-test of least significant difference (LSD). Results Olfactometer tests

Light trap evaluation Compounds tested and test procedure. The chemical compounds evaluated with light traps under field conditions were: (a) a 1 : 1 mixture of geranyl acetone and 6-methyl-5-hepten-2-one (Mix 2); (b) a 1 : 1 : 1 mixture of octanoic, nonanoic and decanoic fatty acids (Mix 3); (c) DEET, and (d) 99% ethanol (control) (Table 1). The chemicals were evaluated at concentrations of 10% and 25%. Trapping was conducted using CDC traps with white-light tubes for 24 nights in the first part of the summer in 2013. Each concentration was evaluated in three replicates (12 nights) of a 4 × 4 randomized Latin square design (Page et al., 2009; Venter et al., 2011). Plant extracts and compounds evaluated were: (a) lemon eucalyptus oil; (b) a 1 : 1 : 1 mixture of jasmine, lavender and rosemary oils (Mix 1); (c) lavender oil; (d) DEET, and (e) 99% ethanol (control) (Table 1). The extracts were evaluated at 10% and 25% concentrations for 20 nights at the end of the summer in 2013. Each concentration was evaluated in two replicates of a 5 × 5 randomized Latin square design. The traps were located 12 m apart in front of a stable housing approximately 150 sheep. An hour before sunset, pieces of polyester mesh (surface area 0.15 m2 , mesh size 2 mm) were immersed for 30 min in the relevant test solution. The pieces of mesh were air-dried for 30 min before being attached to the traps with Velcro strips. The midges did not need to contact the netting in order to pass through it to be collected, although some larger midges might graze the net. An automatic time switch ensured that traps were operational for 5 h after sunset. Midges were collected into water to which a drop of odourless detergent was added to break the surface tension. The catches were retrieved each morning and the mesh discarded. All Culicoides midges were counted, sexed and identified to species level using appropriate keys (Delécolle, 1985; González & Goldarazena, 2011). The males of all species were pooled.

Statistical analysis Chi-squared tests were conducted to determine if the numbers of C. obsoletus in the two arms of the olfactometer differed significantly (𝛼 = 0.05) from the expected 50 : 50 distribution in the absence of any stimuli. Similar analyses were performed with the data from filter paper bioassays.

Three concentrations of each of 23 compounds were evaluated with the Y-tube olfactometer (Table 2). Relatively high numbers of midges responded positively towards one of the olfactometer arms. Except for cypermethrin (50.4%), Mix 3 (62.8%) and nonanal (66.7%), this positive response was > 70% for all compounds (Table 2). The percentage of repellence for C. obsoletus ranged from 97.8% (DEET) to 45.4% (neem oil) at 1 μg/μL, from 91.3% (4-propyl-phenol) to 36.8% (KBR3023) at 0.1 μg/μL and from 86.6% (jasmine) to 45.8% (rosemary) at 0.01 μg/μL (Table 2). At 1 μg/μL concentrations, except in the tests of limonene and neem, statistically significantly greater numbers of midges were found in the control arm than in the arm containing the potential repellent (Table 2). At 0.1 μg/μL concentrations, only 12 of these compounds showed significant repellence and at concentrations of 0.01 μg/μL only eight compounds did so (Table 2). The essential oils exhibited a higher degree of repellence than all chemical compounds except DEET. DEET elicited the highest efficacy at 1 μg/μL (97.8%), 4-propyl-phenol at 0.1 μg/μL (91.3%) and jasmine at 0.01 μg/μL (86.3%) (Table 2). In general, relatively high percentages of repellence were observed with plant-derived oils such as jasmine oil (93.9%), lavender oil (93.7%), and a mixture of these two with rosemary oil (Mix 1) (94.4%) at 1 μg/μL (Table 2). Likewise, the mixture of fatty acids (Mix 3) gave relatively high repellence (Table 2). By contrast, neem oil and limonene had low repellent properties against C. obsoletus at all doses (Table 2). Filter paper landing bioassays Percentages of repellence after 2 s and 5 min for each of the 10 compounds are depicted in Fig. 1. For all 10 compounds significantly more midges were found on control papers than on papers containing the potential repellent (Fig. 1). The compound with the highest repellent activity at 2 s after release was lemon eucalyptus. At 2 s 100% of midges were found on the control paper and none on the paper with lemon eucalyptus (Fig. 1). Lemon eucalyptus was followed by lavender (96.1%), Mix 4 (92–94%) and jasmine oil (90.0%) (Fig. 1). Surprisingly DEET elicited only 76.1% repellence and Mixes 1 and 3 (which achieved the best results in the olfactometer tests) showed repellence of < 75% (Fig. 1).

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Table 2. Percentage repellence obtained with 19 single repellents and four mixtures, at 1, 0.1 and 0.01 μg/μL, against Culicoides obsoletus as determined with a Y-tube olfactometer. 1 μg/μL

0.1 μg/μL

0.01 μg/μL

Compound

+

C

P-value

C

P-value

C

P-value

DEET Mixture 1 Jasmine Lavender 4-Propyl-phenol Eucalyptus oil Mixture 3 Balm leaves Mixture 2 Mixture 4 Rhodinol Geranyl acetone Decanal Octanal Nonanal IR3535 Lemon oil KBR3023 Rosemary Cypermethrin 6-Methyl-5-hepten-2-one Limonene Neem (azadirachtin)

76.0 85.7 90.0 87.2 92.7 81.5 62.8 87.1 78.9 74.2 81.7 80.6 85.6 90.8 66.7 84.3 84.8 76.7 89.5 50.4 88.2 91.5 81.4

97.8 94.4 93.9 93.7 91.0 90.5 89.5 88.4 87.5 85.7 85.7 85.0 82.6 81.6 81.6 80.8 72.7 71.4 70.0 66.7 66.6 57.9 45.5

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.005 0.001 0.020 0.006 0.135 0.460

86.0 78.1 86.6 74.3 91.3 48.1 73.8 80.4 76.3 80.7 86.9 76.3 67.5 54.9 46.7 58.3 62.5 36.8 53.4 50.0 60.8 56.2 52.7

0.000 0.000 0.000 0.000 0.000 0.785 0.002 0.000 0.001 0.000 0.000 0.001 0.003 0.414 0.460 0.157 0.083 0.104 0.550 1.000 0.140 0.317 0.637

66.0 71.4 86.4 61.4 57.2 66.7 55.0 65.9 71.7 66.6 68.0 78.6 57.1 52.5 54.5 52.7 53.1 53.8 45.8 69.3 52.2 48.4 50.0

0.023 0.001 0.000 0.055 0.127 0.089 0.527 0.034 0.000 0.006 0.010 0.000 0.354 0.654 0.490 0.738 0.617 0.579 0.479 0.011 0.252 0.799 1.000

Results are expressed as the percentage of midges found in the control arm (C) calculated from choice responses (+, positive). P-values were obtained with the chi-squared test (𝛼 = 0.05); n = 100–120 specimens per replicate.

At 5 min after release, 100% repellence was found for lemon eucalyptus oil, lavender oil, Mix 4, jasmine oil, 4-propyl-phenol and balm leaves (Fig. 1). For DEET, Mix 1, Mix 2 and Mix 3, the numbers of midges on the control papers did not differ from those observed after 2 s (Fig. 1). Light trap evaluation The numbers of midges and species breakdown of the 48 collections made at 10% and 25% concentrations of the chemicals are shown in Table 3. At both concentrations fewer midges were collected in the three baited traps than in the control trap (Table 3). At the 10% concentration, the mean ± standard deviation number of midges collected with the mixture of fatty acids (Mix 3) (285.5 ± 408.6) was significantly (P = 0.030) lower than those collected with the control (641.8 ± 872.4) or the trap with Mix 2 (459.7 ± 588.1) (Table 3). The lower mean numbers collected with Mix 2 and DEET (313.8 ± 385.5) were not significantly different from the control collection (Table 3). In addition, at the 25% concentration, numbers collected with Mix 3 (198.8 ± 125.5) and DEET (298.2 ± 144.2) were significantly (P = 0.000) lower than that collected with the control (716.8 ± 223.6) (Table 3). At this higher concentration, the mean number collected with the DEET trap was significantly (P = 0.000) lower than that collected with the Mix 2 trap (581.8 ± 381.7) (Table 3). Whereas 18 Culicoides species were collected in the control trap at the 10% concentration, only 13 species were collected

by each of the DEET and Mix 3 traps (Table 3). The dominant species, which accounted for 49.1% (control) to 33.7% (DEET) of collections in all four treatments was Culicoides brunnicans (Table 3). The proportional representation of C. brunnicans differed significantly between treatments (𝜒 2 = 270.92, d.f. = 3, P < 0.001). In a pattern reflecting that of total mean collections, the three treated traps collected fewer C. brunnicans than the control traps (Table 3). In addition, only the number of C. brunnicans collected with the Mix 3 trap (122.0 ± 213.3) was significantly (P = 0.021) lower than that collected by the control (315.4 ± 509.7) (Table 3). Similar results were obtained for C. obsoletus and C. scoticus (hereafter grouped together according to the morphological similarity of the females) and the subgenus Oecacta (Table 3). For other species and males, the numbers collected, although lower, did not differ significantly from those collected by the control (Table 3). Data for six species that represented < 4% of the collections were not analysed. In the 48 collections made with the 25% concentrations, 20 species were collected with the control and 16 by each of the Mix 3 and DEET traps (Table 3). Culicoides lupicaris and Culicoides pulicaris, as a group, replaced C. brunnicans as the dominant species (Table 3). Except for the C. obsoletus/C. scoticus and the subgenera Silvaticulicoides and Oecacta collections, the mean numbers of the different species collected with the DEET and Mix 3 traps, although not statistically different from one another, were lower than those collected by the control (Table 3). Significantly (P < 0.001) lower numbers of C. obsoletus/C. scoticus were collected with Mix 3 (24.7 ± 17.1)

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Fig. 1. Findings in Culicoides obsoletus exposed to spatial repellent chemicals within a mosquito cage at doses of 1 μg/μL. Results are expressed as the percentage of midges that alighted on filter papers after 2 s ( ) or 5 min ( ) of exposure (the positive axis). The negative axis ( , ) represents the percentage of midges that did not alight on papers (i.e. midges that were repelled by the treatments). Specimens per replicate: n =20; replicates per repellent: n = 6. Compounds are ordered by strength of repellency in the first trial. Mix 1: jasmine + lavender + rosemary oil; Mix 2: geranyl acetone + 6-methyl-5-hepten-2-one; Mix 3: octanoic + nonanoic + decanoic acids; Mix 4: rhodinol + lemon eucalyptus + lemon oil. *P < 0.05; **P < 0.01; ***P < 0.001 (chi-squared test).

than with DEET (56.5 ± 30.3) (Table 3). Numbers of the subgenus Silvaticulicoides collected did not differ significantly (P < 0.001) between treatments (Table 3). Numbers of midges and species breakdowns of 50 collections made at 10% and 25% concentrations of plant repellents are shown in Table 4. At 10% concentrations, significantly (P = 0.002) fewer midges were collected in the four treated traps compared with the control (112.1 ± 48.6) (Table 4). At 25% concentrations, the lower mean numbers of Culicoides midges collected with Mix 1 (32.8 ± 24.7) and lavender oil (49.9 ± 41.1) did not differ significantly from the number collected by the control (60.0 ± 43.6) (Table 4). At 10% concentrations, DEET and eucalyptus oil each captured 12 species, whereas 16 species were collected in the control trap (Table 4). The dominant species collected, which accounted for 36.7% (control) to 23.7% (lavender oil) of collections, was C. lupicaris (Table 4). Proportional representation differed significantly between treatments (𝜒 2 = 41.524, d.f. = 4, P < 0.001). Similar to the mean numbers, significantly (P = 0.008) fewer midges were collected in the treatment traps than in the control trap (Table 4). In addition, members of C. obsoletus/C. scoticus (P = 0.004) and the subgenus Oecacta (P = 0.026) were collected in significantly lower numbers in the treated than the control traps (Table 4). Members of C. obsoletus/C. scoticus were also collected in significantly lower numbers with DEET (27.0 ± 13.9) than with eucalyptus oil (18.2 ± 6.0) (Table 4). Because of a prolonged drought, the numbers and species collected at the 25% concentration were lower than in the previous evaluations (Table 4). Although C. lupicaris remained the dominant species in control trap and lavender oil trap

collections, C. obsoletus/C. scoticus represented the dominant species in the other three traps (Table 4). The mean numbers of C. lupicaris collected in the control (24.5 ± 26.5) were significantly higher than those collected with DEET (6.5 ± 5.9), eucalyptus oil (7.1 ± 5.4) and Mix 1 (9.5 ± 8.6) (P = 0.002) (Table 4). Mean numbers of C. obsoletus/C. scoticus collected with DEET (8.2 ± 12.1) were significantly (P = 0.038) lower than those collected with Mix 1, lavender oil and the control trap (Table 4).

Discussion Of the 23 compounds evaluated, 13 were previously shown to be effective against various Culicidae mosquitoes (Amer & Mehlhorn, 2006; Logan et al., 2010a; Nerio et al., 2010; Campbell et al., 2011; Maia & Moore, 2011; Kazembe & Chaivba, 2012; Phasomkusolsil & Soonwera, 2012). Previous studies also showed that cypermethrin (Braverman & Chizov-Ginzburg, 1998; Calvete et al., 2010), DEET (Braverman & Chizov-Ginzburg, 1997, 1998; Page et al., 2009), neem oil (Braverman & Chizov-Ginzburg, 1998; Braverman et al., 1999), lemon eucalyptus (Braverman et al., 1999), rosemary (Braverman & Chizov-Ginzburg, 1998) and the fatty acids mixture (Venter et al., 2011) will repel C. imicola. Compounds previously shown to repel C. sonorensis were neem oil and lemon eucalyptus (Trigg & Hill, 1996; Braverman et al., 2000) and cypermethrin (Papadopoulos et al., 2009, 2010). Picaridin (Carpenter et al., 2005), neem oil (Trigg, 1996; Blackwell et al., 2004) and 6-methyl-5-hepten-2-one (Bhasin et al., 2001) were shown to repel Culicoides impunctatus. Martinez-de la Puente et al. (2009) showed that IR3535 will repel Culicoides species

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Treatment

© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, 28, 421–431 13 3 766 (18.4)ab 313.8 (385.8) 1271 (13.8)ab 106.0 (141.7) 33.7 978 (22.3) 81.5 (111.8) 26.0 295 (28.7) 24.6 (39.6) 7.8 144 (21.4)ab 12.0 (8.7) 3.8 709 (23.3) 59.1 (76.1) 18.8 238 (17.1)ab 19.8 (11.3) 6.3 124 (17.2) 10.3 (16.9) 3.3

DEET 17 5 516 (27.0)a 459.7 (588.1) 2638 (28.8)a 219.8 (342.6) 47.8 1138 (25.9) 94.8 (126.4) 20.6 187 (18.2) 15.6 (14.2) 3.4 219 (32.6)a 18.3 (11.8) 4.0 723 (23.8) 60.3 (75.1) 13.1 392 (28.2)a 32.7 (18.9) 7.1 214 (29.8) 17.8 (24.6) 3.9

Mix 2 13 3 426 (16.8)b 285.5 (408.6) 1464 (16.0)b 122.0 (213.3) 42.7 757 (17.2) 63.1 (95.1) 22.1 164 (16.0) 13.7 (17.1) 4.8 93 (13.9)b 7.8 (6.9) 2.7 638 (21.0) 53.2 (70.5) 18.6 199 (14.3)b 16.6 (15.4) 5.8 107 (14.9) 8.9 (12.2) 3.1

Mix 3 18 7 702 (37.7)a 641.8 (872.4) 3785 (41.3)a 315.4 (509.7) 49.1 1511 (34.4) 125.9 (174.0) 19.6 380 (37.0) 31.7 (39.9) 4.9 216 (32.1)a 18.0 (10.2) 2.8 965 (31.8) 80.4 (92.9) 12.5 557 (40.1)a 46.4 (45.6) 7.2 272 (37.9) 22.7 (34.6) 3.5

Control — 0.030 — 0.021 — — 0.382 — — 0.324 — — 0.002 — — 0.551 — — 0.017 — — 0.056 — —

16 3 578 (16.6)b 298.2 (144.2) 73 (8.3)b 6.1 (5.5) 2.0 308 (16.1)b 25.7 (90.5) 8.6 1245 (16.0)b 103.8 (87.3) 34.8 678 (20.0)b 56.5 (30.3) 18.9 738 (16.0) 61.5 (33.3) 20.6 339 (19.3)ab 28.3 (32.2) 9.5 95 (16.0)b 7.9 (9.1) 2.7

P-value DEET

25% (w/w)

19 6 982 (32.4)a 581.8 (381.7) 278 (31.9)a 23.2 (20.3) 4.0 624 (32.7)a 52.0 (28.8) 8.9 2690 (34.7)a 224.2 (203.7) 38.5 1086 (32.6)a 90.5 (57.9) 15.5 1380 (30.0) 115.0 (92.0) 19.8 473 (26.9)a 39.4 (32.9) 6.8 178 (30.0)a 14.8 (9.3) 2.5

Mix 2 16 2 386 (11.1)b 198.8 (125.5) 78 (8.9)b 6.5 (6.4) 3.3 217 (11.3)b 18.1 (41.5) 9.1 896 (11.5)b 74.7 (74.1) 37.6 297 (8.9)c 24.7 (17.1) 12.4 505 (10.9) 42.1 (30.2) 21.7 260 (14.8)b 21.7 (30.2) 10.9 50 (8.4)b 4.2 (3.6) 2.1

Mix 3

20 8 601 (39.9)a 716.8 (223.6) 442 (50.7)a 36.8 (30.8) 5.1 760 (40.0)a 63.3 (26.5) 8.8 2928 (37.7)a 244.0 (170.3) 34.0 1266 (38.0)a 105.5 (48.8) 14.7 1976 (42.9) 164.7 (70.6) 22.8 683 (38.9)a 56.9 (53.9) 7.9 269 (45.4)a 22.4 (18.1) 3.1

Control

— 0.000 — 0.000 — — 0.000 — — 0.004 — — 0.000 — — 0.554 — — 0.020 — — 0.000 — —

P-value

P–values of > 0.05 indicate no statistical difference. ∗Includes mainly Culicoides achrayi and Culicoides picturatus and to a lesser extent Culicoides fascipennis, Culicoides pallidicornis and Culicoides subfascipennis. †Includes mainly Culicoides simulator and to a lesser extent Culicoides santonicus, Culicoides poperinghensis, Culicoides festivipennis, Culicoides pictipennis, Culicoides shaklawensis, Culicoides vexans, Culicoides tauricus, Culicoides kibunensis, Culicoides furcillatus and Culicoides kurensis. ‡Males pooled together. The other six Culicoides species were discarded. Numbers per row for the same concentrations followed by different letters in bold denote statistically differences at the 5% level. Mix 2, mixture of 1 : 1 of geranyl acetone + 6- methyl-5-hepten-2-one; Mix 3, mixture of 1 : 1 : 1 of octanoic + nonanoic + decanoic fatty acids. SD, standard deviation.

Species collected, n Total Culicoides collected, n (%) Mean collected (SD) C. brunnicans Total collected Mean collected (SD) % total of Culicoides C. punctatus Total collected Mean collected (SD) % total of Culicoides C. lupicaris + C. pulicaris Total collected Mean collected (SD) % total of Culicoides C. obsoletus + C. scoticus Total collected Mean collected (SD) % total of Culicoides Sub. Silvaticulicoides∗ Total collected Mean collected (SD) % total of Culicoides Sub. Oecacta† Total collected Mean collected (SD) % total of Culicoides Males‡ Total collected Mean collected (SD) % total of Culicoides

Species

10% (w/w)

Table 3. Culicoides midges collected with four traps impregnated with three different chemical treatments at 10% and 25% (w/w) during the summer of 2013 at a sheep farm in the Basque Country, Spain. Twelve collections were made with each repellent.

Field and laboratory trials with repellents in Culicoides species 427

Treatment 12 485 (13.2)b 48.5 (22.7) 148 (13.5)b 14.9 (10.4) 30.5 93 (10.5)c 9.3 (5.3) 19.2 41 (11.0) 4.1 (2.8) 8.5 109 (15.8) 10.9 (8.3) 22.5 46 (19.6)b 3.8 (3.4) 9.5 39 (13.7) 4.0 (4.3) 8.0 8 (8.8) 0.7 (1.0) 1.6

DEET

10% (w/w)

12 613 (16.7)b 61.3 (21.3) 150 (13.6)b 15.0 (8.0) 24.5 182 (20.4)b 18.2 (6.0) 29.7 79 (21.6) 7.9 (10.2) 12.9 103 (14.8) 10.3 (6.8) 16.8 31 (13.0)b 2.5 (2.3) 5.1 47 (16.4) 4.7 (3.8) 7.7 22 (23.4) 1.8 (2.3) 3.6

Eucalypt. 14 751 (20.5)b 75.1 (34.5) 233 (21.2)b 23.3 (16.1) 31.0 170 (19.1)bc 17.0 (8.8) 22.6 112 (30.5) 11.2 (10.5) 14.9 150 (21.6) 15.0 (11.9) 20.0 25 (10.7)b 2.1 (1.3) 3.3 50 (17.5) 5.1 (2.3) 6.7 12 (12.2) 0.9 (1.0) 1.6

Mix 1 14 693 (18.9)b 69.3 (30.6) 164 (14.9)b 16.5 (30.5) 23.7 174 (19.5)bc 17.4 (7.1) 25.1 65 (17.5) 6.5 (10.8) 9.4 153 (22.0) 15.3 (12.4) 22.1 39 (16.7)b 3.3 (2.4) 5.6 75 (26.0) 7.5 (5.2) 10.8 24 (25.3) 1.9 (3.1) 3.5

Lavender 16 1121 (30.6)a 112.1 (48.6) 403 (36.7)a 40.3 (31.5) 36.7 270 (30.4)a 27.0 (13.9) 24.1 71 (12.3) 7.1 (6.8) 6.3 179 (25.7) 17.9 (6.9) 16.0 94 (40.1)a 7.9 (8.9) 8.4 76 (26.4) 7.6 (4.1) 6.8 28 (30.1) 2.3 (2.9) 2.5

Control

25% (w/w)

— 0.002 — 0.008 — — 0.004 — — 0.492 — — 0.456 — — 0.026 — — 0.577 — — 0.439 — —

4 182 (9.9)b 18.2 (15.1) 65 (9.5)c 6.5 (5.9) 35.7 82 (11.8)b 8.2 (12.1) 45.1 0 0 0 0 0 0 35 (8.5) 3.5 (3.9) 19.2 0 0 0 0 0 0

P-value DEET 5 219 (12.0)b 21.9 (15.5) 71 (10.4)c 7.1 (5.4) 32.4 92 (14.5)ab 9.2 (6.4) 42.0 0 0 0 0 0 0 47 (11.5) 4.7 (5.3) 21.5 8 (25.8) 0.8 (0.6) 3.7 1 (8.0) 0.1 (0.3) 0.5

Eucalypt. 5 328 (17.9)ab 32.8 (24.7) 95 (13.9)bc 9.5 (8.6) 29.0 145 (22.9)a 14.5 (13.6) 44.2 0 0 0 0 0 0 80 (19.5) 8.0 (7.8) 24.4 6 (19.3) 0.6 (0.5) 1.8 2 (17.2) 0.2 (0.5) 0.6

Mix 1

7 499 (27.3)a 49.9 (41.1) 207 (30.3)ab 20.7 (25.1) 41.5 159 (25.1)a 15.9 (9.9) 31.8 0 0 0 0 0 0 119 (29.0) 11.9 (11.1) 23.8 10 (32.3) 1 (0.7) 2.0 4 (33.1) 0.4 (0.7) 0.8

Lavender

9 600 (32.8)a 60 (43.6) 245 (35.9)a 24.5 (26.5) 40.8 214 (33.7)a 21.4 (14.9) 35.7 0 0 0 0 0 0 129 (31.5) 12.9 (12.8) 21.5 7 (22.6) 0.7 (0.6) 1.2 5 (42.4) 0.5 (1.3) 0.8

Control

— 0.019 — 0.002 — — 0.038 — — — — — — — — 0.127 — — NA — — NA — —

P-value

P-values of > 0.05 indicate no statistical difference. ∗Includes mainly Culicoides achrayi and Culicoides picturatus and to a lesser extent Culicoides pallidicornis, Culicoides fascipennis and Culicoides subfascipennis. †Includes mainly Culicoides simulator and to a lesser extent Culicoides brunnicans, Culicoides kibunensis, Culicoides furcillatus, Culicoides festivipennis, Culicoides vexans, Culicoides gejgelensis, Culicoides kurensis and Culicoides alazanicus. ‡Includes another six Culicoides species. §Males pooled together. Numbers per row for the same concentrations followed by different letters in bold denote statistically differences at the 5% level. Mix 1, mixture of 1 : 1 : 1 of jasmine + lavender + rosemary. NA, not analysed; SD, standard deviation.

Species collected, n Total Culicoides collected (%) Mean collected (SD) C. lupicaris Total collected Mean collected (SD) % total of Culicoides C. obsoletus + C. scoticus Total collected Mean collected (SD) % total of Culicoides C. punctatus Total collected Mean collected (SD) % total of Culicoides Sub. Silvaticulicoides∗ Total collected Mean collected (SD) % total of Culicoides Sub. Oecacta† Total collected Mean collected (SD) % total of Culicoides Other‡ Total collected Culicoides spp. Mean collected (SD) % total of Culicoides Males§ Total collected Mean collected (SD) % total of Culicoides

Species

Table 4. Culicoides midges collected with four traps impregnated with three different plant-derived treatments at 10% and 25% (w/w) during the summer of 2013 at a sheep farm in the Basque Country, Spain. Ten collections were made with each repellent.

428 M. González et al.

© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, 28, 421–431

Field and laboratory trials with repellents in Culicoides species in general. Although some studies showed that the oil of Eucalyptus maculata var. citriodora will repel C. impunctatus from humans in the field (Trigg, 1996) and Culicoides variipennis from humans in the laboratory (Trigg & Hill, 1996), Braverman et al. (1999) found that it can attract C. imicola. The comprehensive evaluation of repellents should be based on laboratory as well as field data (Logan et al., 2010a). Although olfactometer and other laboratory-based assays are effective in determining the responses of biting insects to odour stimuli under controlled conditions, field studies are considered more realistic and natural. However, olfactometry assays may evoke unnatural behaviour (e.g. insects may respond differently to the same odour stimuli when walking as opposed to flying), and elicit chemotactic and chemokinetic responses (Kennedy, 1977). Logan et al. (2010a) demonstrated the results of laboratory and field trials to be comparable. A comparison of the results obtained with the two laboratory assays used in the present study showed some discrepancies. Whereas the olfactometer assays indicated DEET and Mix 3 to be superior to the essential oils of lemon eucalyptus and lavender and Mixes 2 and 4, the results of the landing assays were rather disappointing (Table 2, Fig. 1). This apparent discrepancy may be attributable to the fact that midges in the olfactometer were in a confined space, whereas midges in the landing assays had the opportunity to fly. The compounds evaluated in the field were selected based on two different laboratory assays and included products previously shown to repel mosquitoes. In both laboratory assays, repellence was determined for relatively short exposure periods (2 s to 5 min). During these first seconds and/or minutes after application, materials showed marked efficacy for a relatively short time. This initial repellent effect seems to have become less effective over time, as was shown with light traps. A similar observation was made with Mix 2 (chemical formulation) in the field. Although it repelled > 85% of midges in the olfactometer and landing bioassays, it appeared to be rather ineffective in the light trap study. Similar results were obtained by Logan et al. (2010b) with mosquitoes using arm-in-cage studies. Those experiments showed Mix 2 to be relatively effective over short time periods and that improvements to achieve a slow release over longer periods will be required. It should also be noted that whereas the laboratory assays were conducted with C. obsoletus, the light trap studies involved a range of different species. In assays using nonanal, Mix 3 and cypermethrin, < 70% of midges moved towards either of the two arms within the allocated 4 min (Table 2). This may be attributable to a potential toxic effect of these compounds, as was recorded for Culicoides nubeculosus with the insecticide cypermethrin (Papadopoulos et al., 2010). Despite some limitations, light traps are appropriate for screening putative repellents before they are tested on animals, especially in contexts in which these products may cause adverse allergic reactions such as skin irritation. The use of livestock may be expensive, have welfare implications and may present some difficulties in relation to animal management. Although light traps are an artificial collection method and their results do not always reflect the true biting rate on animals (Viennet et al., 2011), the mixture of fatty acids was as effective as DEET in repelling Culicoides species from the traps. Findings also

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showed that lemon eucalyptus extracts may be as effective as DEET in repelling Culicoides midges. The majority of species or groups studied here responded similarly to the repellents tested. However, some groupings, such as pooled males, Culicoides punctatus and particularly the subgenus Silvaticulicoides, which comprises Culicoides picturatus (65% of total captures), C. achrayi (32%) and, to a lesser extent Culicoides fascipennis, Culicoides pallidicornis and Culicoides subfasciipennis (3%), seemed to be more resistant to some of the repellents. These species will feed on mammals (Ninio et al., 2010; Lassen et al., 2012; Santiago-Alarcon et al., 2012; Pettersson et al., 2013), but their roles as disease vectors are still unknown. More relevant are the promising results obtained with C. obsoletus/C. scoticus and C. lupicaris/C. pulicaris as both of these groups are of immense importance as potential vectors of arboviral diseases (Mellor et al., 2000; Carpenter et al., 2008). Recent studies show that C. punctatus, an abundant and ubiquitous midge in Europe, may play a greater role in the epidemiology of both SBV and BTV in Europe than was previously suspected (Larska et al., 2013; Meiswinkel et al., 2013). Over the last 60 years, DEET has been considered the most effective and has been the most commonly used insect repellent (Fradin, 1998). The efficiency of DEET at 15% concentrations against C. imicola can last from a few hours (Braverman & Chizov-Ginzburg, 1997; Braverman et al., 2000) to as long as 8 h (Page et al., 2009). The results obtained with plant-based repellents suggest that the oil of lemon eucalyptus will be more effective at a higher concentration (25%), at which it reduces collections by 2.7 times compared with controls. Although numbers of midges collected in traps using plant-derived repellents at concentrations of 10% were statistically different from and were 1.5–1.8 times lower than those collected with the control trap, DEET collected 2.3 times fewer midges than the control trap. The other plant extracts in the present study caused statistically significant reductions in collections, but seemed to be less effective. The active ingredient of E. maculata var. citriodora [p-menthane-3,8 diol (PMD)] tends to be less volatile than some other natural plant derivatives and can provide protection for several hours (Fradin & Day, 2002; Maia & Moore, 2011).

Conclusions Of the 23 compounds evaluated, at least three showed promising results with the methodologies used. Among defined chemicals, the widely used insect repellent DEET and the mixture of fatty acids (octanoic, nonanoic and decanoic organic acids) were shown to be effective. Among plant-derived repellents, lemon eucalyptus oil (E. maculata var. citriodora) was better in the field. The discrepancies between laboratory and field results highlight some of the problems and factors that can influence results. Despite some discrepancies, laboratory-based assays can serve as a preliminary method of screening potential repellents to be evaluated in the field. Given the apparent repellent effects of the mixture of fatty acids, it is recommended that this product be further evaluated. Skin reactions in animals, in vivo testing, synergistic formulations, new concentrations and release rates are issues that remain

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to be determined. New formulation technologies with synergistic effects that may result in higher bioactivity, such as might be produced, for example, by mixing essential oils with synthetic blends, are also required.

Acknowledgements The authors thank the Department of Education, Universities and Research of the Basque Country for granting a PhD fellowship to MG. Patxi Saenz de Urturi and Ander Isasmendi, NEIKER-TECNALIA, are thanked for technical assistance. Pedro Echeverria Beltrán, shepherd at Elguea, is thanked for allowing the authors to conduct experiments on his farm. Dr Truuske Gerdes, Department of Entomology, University of Pretoria, South Africa, is thanked for improving the English language of the manuscript, as are two anonymous reviewers for improving the manuscript with their suggestions and comments.

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© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, 28, 421–431

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...
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