Medical and Veterinary Entomology (2014), doi: 10.1111/mve.12072
Susceptibility of Culicoides species biting midges to deltamethrin-treated nets as determined under laboratory and field conditions in the Balearic Islands, Spain R. D E L RÍ O 1 , C. B A R C E LÓ 1 , C. P A R E D E S- E S Q U I V E L 1 , J. L U C I E N T E S 2 and M. A. M I R A N D A 1 1
Laboratory of Zoology, University of the Balearic Islands, Palma de Mallorca, Spain and 2 Department of Animal Pathology, Faculty of Veterinary Science, University of Zaragoza, Zaragoza, Spain
Abstract. Culicoides Latreille (Diptera: Ceratopogonidae) are vectors of several arboviruses, including bluetongue virus (BTV) and African horse sickness virus (AHSV), which cause diseases in, respectively, sheep and cattle, and horses, and have economic repercussions mainly as a result of trade restrictions. Insecticides can be used to reduce vector populations and hence the spread of disease. Despite the economic importance of these diseases, relatively few studies have evaluated the efficacy of commercially available insecticides and the effectiveness of treated nets against Culicoides species. The aim of the present study was to evaluate the insecticidal effect of commercially available polyethylene nets (ZeroVector® ) treated with deltamethrin (4.4 g/kg ± 15%) on Culicoides species. Laboratory and field trials were conducted in Culicoides populations collected in Majorca in the Balearic Islands, Spain. The present study shows that deltamethrin-treated nets provoke high and rapid mortality (90–100%) in Culicoides midges under laboratory conditions and increase mortality by 13% when deployed in the field. Key words. Culicoides imicola, Culicoides obsoletus, impregnated nets, insecticides,
pyrethroids.
Introduction Bluetongue (BT) is a disease of ruminants caused by an Orbivirus [bluetongue virus (BTV)] of the family Reoviridae. Some species of the genus Culicoides can transmit BTV, as well as other viruses such as African horse sickness virus (AHSV), epizootic haemorrhagic disease virus (EHDV) and the recently discovered Schmallenberg virus (SBV), to susceptible animals (Mellor et al., 1990; Paweska et al., 2005; De Regge et al., 2012). Sheep and, to some extent, cattle manifest the most severe clinical symptoms of the disease. Although wild ruminants are susceptible to and can become infected with BTV, most will show no clinical symptoms and may act as silent carriers or amplifying hosts (Neitz, 1933; Backx et al., 2007; Linden et al.,
2008). Amongst the viruses transmitted by Culicoides midges, BTV and SBV are of particular interest in Europe. The recent introduction of these two viruses into northern Europe indicates the possibility that other Culicoides-transmitted diseases (e.g. African horse sickness) may enter the continent (De Vos et al., 2012). To date, the most effective way to control the spread of BTV is by using vaccination and restricting the movement of animals. However, the emergence of new serotypes of BTV (Carpenter et al., 2009) and the costs associated with treatments call for the development of additional, efficient and cost-effective control strategies. The susceptibility of Culicoides species to insecticides is poorly documented. Only a few publications describe the responses of some Culicoides species to organochlorines,
Correspondence: Ricardo del Río, Laboratory of Zoology, University of the Balearic Islands, Cra. Valldemossa Km. 7.5, Palma de Mallorca 07122, Spain. Tel.: + 34 971 173352; Fax: + 34 971 173184; E-mail: [email protected] © 2014 The Royal Entomological Society
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2 R. Del Río et al. organophosphates, carbamates and pyrethroids (Hill & Roberts, 1947; Kline & Roberts, 1981; Floore, 1985; Mehlhorn et al., 2008a, 2008b; Schmahl et al., 2009a, 2009b; Papadopoulos et al., 2010). In the present study, the susceptibility of Culicoides species to polyethylene nets impregnated with deltamethrin, a pyrethroid ester insecticide, was determined. Assessments were performed in the laboratory, using adapted World Health Organization (WHO) test kits (WHO, 1981), and under field conditions. The use of these nets in stables or transport vehicles may offer protection to susceptible animals and avoid infection and the spread of diseases transmitted by Culicoides species. Materials and methods Polyethylene nets (ZeroVector® Durable Lining; Dart Association, Lausanne, Switzerland) impregnated with 4.4 g/kg ± 15% of deltamethrin are currently used in malaria endemic zones for mosquito control. The active ingredients of the insecticide are effective on the nets for 3–5 years. These nets have never been tested against Culicoides species. Laboratory trial Culicoides midges were collected at a dairy farm named C’as Boter (39∘ 30′ N, 03∘ 07′ E) located in Majorca in the Balearic Islands, Spain, between 15 June and 17 July 2011. Insects were collected from dusk to dawn using ARC-OVI (Agricultural Research Council–Onderstepoort Veterinary Institute, Pretoria, South Africa) and mini Centers for Disease Control (CDC) (John W. Hock Co., Gainesville, FL, U.S.A.) light traps. Collected insects were transported to the insectary early in the morning (06.00–07.00 hours) inside thermal isolation containers and maintained in pools of 30–100 specimens inside WHO chambers (consisting of clear plastic cylinders 4.5 cm in diameter and 12.0 cm in length) (WHO, 1981). Culicoides were maintained at 25 ± 2 ∘ C and 75% relative humidity (RH) in dim light on a 5% sucrose solution for 12–24 h before each bioassay. Insecticide bioassays were conducted in WHO chambers lined with pieces (12 × 25 cm) of deltamethrin-treated net (ZeroVector® ). Culicoides midges were transferred to the WHO chambers and exposed to the treated nets for 6, 2, 1 min or 30 s. During the bioassay, WHO chambers were placed horizontally to facilitate contact with the nets. To accurately regulate the time of interaction with the treated nets at exposure times of ≤ 30 s, Culicoides midges were immobilized by refrigeration (− 20 ∘ C) before exposure. Once immobilized, insects were placed in contact with the treated net for 30, 20 or 10 s. In each bioassay, WHO chambers lined with polyethylene nets without insecticide were used as controls. After exposure, insects were transferred to the original WHO chambers without insecticide and the time to observe an effect to the treatment was recorded. Field trial The study was conducted on a farm in Spain producing horses (30–40 horses) for consumption (Ca Sa Manescala,
39∘ 25′ 18.86′′ N, 03∘ 00′ 22.31′′ E) over 12 nights during 12–28 October 2011. An Onderstepoort light trap was hung at a height of 1.5 m inside a cylinder (1.5 m in diameter, 1.5 m in height) made of metallic mesh (mesh size: 5 × 5 cm). The cylinder was covered with deltamethrin-treated net (ZeroVector® ; approximately 24 holes/cm2 ). The same trapping system but without the insecticide treatment was used as a control. To assess the effects of the impregnated nets on mortality in the Culicoides population and non-target fauna, the soil surface at the base of each structure (treatment and control) was covered with a 2 × 2 m piece of white polyethylene fabric from which all insects were aspirated the morning after each sampling. To minimize interference between the light traps, treatment and control structures were placed 10 m apart. Collections were obtained near the horses’ enclosures. Traps were operated from dusk until dawn (from 20.00 to 08.00 hours). Captures were collected early in the morning. After the collection of samples, the structures and traps were rotated for the next sampling to avoid the occurrence of any bias in collections caused by differences in the locations of traps. In the laboratory, living and dead midges were separated and levels of mortality were compared between treatment and control traps. All midges in this trial were identified according to their wing pattern (Rawlings, 1996) into species or species group (when morphological discrimination amongst species was difficult), and separated by sex and gonotrophic condition (Dyce, 1969).
Statistical analysis The mortality rates of immobilized and non-immobilized Culicoides midges were compared at 30 s exposure. As data were not normally distributed, the Mann–Whitney U test was applied to compare the medians of the two samples at 95% confidence levels using the statistics program R Version 2.15.1 (R Foundation for Statistical Computing, Vienna, Austria). Mean captures and mortality rates were compared between control and treatment groups under field conditions using t-tests in statgraphics Plus Version. 3.0 (Statpoint Technologies, Inc., Warrenton, VA, U.S.A.). Differences between treatments were considered to be significant if they achieved a P-value of < 0.05 (see File S1).
Results Laboratory trial A total of 1401 Culicoides, belonging to at least nine species (Obsoletus complex, Culicoides cataneii/gejgelensis, Culicoides circumscriptus Kieffer 1918, Culicoides imicola Kieffer 1913, Culicoides jumineri/kurensis, Culicoides longipennis/sahariensis, Culicoides newsteadi Austen 1921, Culicoides nubeculosus/puncticollis and Culicoides paolae Boorman 1996) were assayed. Of these, 34 (2.4%) were males and 1367 (97.6%) were females. The females consisted of 541 (39.6%) nulliparous, 176 (12.9%) parous, 630 (46.1%) gravid and 20 (1.5%) engorged specimens.
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12072
Susceptibility of Culicoides to deltamethrin nets
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Table 1. Effect of deltamethrin-impregnated nets on Culicoides spp. after exposures of 10 s to 6 min inside World Health Organization (WHO) test tubes. One group of Culicoides midges were transferred directly to the WHO chambers with the treated net (not immobilized), and the other was cold-immobilized before the transfer (immobilized). Control
Not immobilized
Immobilized
Treatment
Exposure
N
Alive
Killed
N
Total
6 min 2 min 1 min 30 s 30 s 20 s 10 s
1 1 1 3 1 1 2
19 13 15 85 52 33 27
0 0 0 0 0 0 0
2 2 3 12 3 3 5
44 63 80 465 108 150 252
Alive Mean ± SD
Killed Mean ± SD
Mortality, %
0 0 0 0 2.7 ± 3.1 2.0 ± 2.6 3.6 ± 3.4
22.0 ± 2.0 31.5 ± 13.5 26.7 ± 1.7 38.5 ± 12.6 33.3 ± 12.2 48.0 ± 18.0 46.8 ± 13.8
100% 100% 100% 100% 92.6% 96.0% 92.9%
N indicates the number of replicates. SD, standard deviation.
The most abundant species tested belonged to the Obsoletus complex (55.7%, n = 781), C. circumscriptus (18.7%, n = 262), C. cataneii/gejgelensis (10.8%, n = 152) and C. paolae (7.7%, n = 108). The remaining species represented < 10% of the total. Midges not immobilized before contact with insecticide (contact times of 30 s to 6 min) The mortality rate in midges that were not immobilized before contact with the deltamethrin was 100%. Insects died within 6–17 min after contact with the treated net, irrespective of exposure time. At 6 min post-exposure, 73.7–100% of the Culicoides midges exhibited clear symptoms of intoxication (great difficulty in crawling or flying) for both short (30 s) and long (6 min) exposures to the insecticide. At 9 min post-exposure, 100% of insects exhibited intoxication signs, and at 17 min post-exposure 100% mortality was observed (Table 1).
Midges immobilized before contact with insecticide (contact times of 10–30 s) Mortality rates in midges immobilized prior to 30, 20 and 10 s of exposure to the treated nets were 92.9, 96.0 and 92.6%, respectively (Table 1). Culicoides midges showed signs of intoxication and died later (> 17 min and < 40 min, respectively) than those not immobilized. After 30 s exposure, the mortality rate was significantly (P = 0.015) lower in midges that were immobilized before contact compared with those that were not. Intoxication also took significantly (P = 0.020) longer to become evident in midges that were immobilized. A survival rate of 100% was found in the control trials.
Field trial Eleven nights were sampled with the treatment trap and 12 with the control trap. A failure in the electricity supply during one of the nights altered the collections of one trap and the results from that night were therefore discarded.
A total of 819 Culicoides midges belonging to at least 13 different species (Obsoletus complex, C. cataneii/gejgelensis, C. circumscriptus, C. imicola, C. jumineri/kurensis, C. longi pennis/sahariensis, Culicoides maritimus Kieffer 1924, C. newsteadi, C. nubeculosus/puncticollis, C. paolae, Culicoides parroti/stigma, Culicoides pictipennis (Staeger 1939) and Culicoides univittatus Vimmer 1932) were collected with the traps. The most abundant species collected were C. imicola (33.2%) and C. circumscriptus (33.0%). Numbers of the species of the Obsoletus complex were relatively low (4.3%) compared with their abundance during spring (Table 2). The mean number of midges collected in the treated trap (26.1 ± 8.6) was not significantly (P = 0.405) lower than that in the control trap (46.0 ± 38.2). The mean ± standard deviation (SD) mortality rate of Culicoides collected in the treated traps (84.9 ± 10.5%) was significantly (P = 0.0020) higher than that of Culicoides collected in the control traps (72.3 ± 5.9%) (Fig. 1). The polyethylene sheets at the base of each of the structures were aspirated on 4 nights. The number of Culicoides collected on the polyethylene sheets was low compared with the captures of the traps. Mean ± SD numbers of Culicoides collected per night were 2.3 ± 1.7 at the base of each control trap, and 2.3 ± 1.9 at the base of each treatment trap. With regard to non-target fauna, 32 718 arthropods belonging to 12 orders and at least 25 families were collected (Table 3). Most of the non-target insects collected were Diptera (98.0%), and more specifically belonged to the Cecidomyiidae family (66.7%). Most of the specimens found dead at the polyethylene bases of control traps belonged to the Sphaeroceridae family (40.1%), whereas most of the specimens found dead at the bases of treatment structures belonged to the Cecidomyiidae family (54.1%) (Table 3). Significant differences (P < 0.05) between the treatment and control traps were observed in the ratio of floor to trap collections in some insects (Drosophilidae, Sphaeroceridae, Chironomidae and Lepidoptera) (Table 3). The higher numbers of live arthropods collected in the control trap did not differ significantly (P = 0.142) from that in the treatment trap. Differences among the arthropods collected dead in traps were also not significant (P = 0.959).
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12072
4 R. Del Río et al. Table 2. Culicoides species collected during 12 nights on a horse farm using a trap surrounded by a deltamethrin-impregnated net (treatment) and a control trap surrounded by a non-treated net (control). Control
Treatment
Species
Dead, n (%)
Alive, n (%)
Dead, n (%)
Alive, n (%)
C. cataneii/gejgelensis C. circumscriptus C. imicola C. jumineri/kurensis C. longipennis/sahariensis C. maritimus C. newsteadi C. paolae C. parroti/stigma C. pictipennis C. nubeculosus/puncticollis C. univittatus Obsoletus complex Total
45 (12.1%) 120 (32.3%) 116 (31.2%) 3 (0.8%) 3 (0.8%) 8 (2.2%) 21 (5.6%) 22 (5.9%) 10 (2.7%) 1 (0.3%) 0 1 (0.3%) 22 (5.9%) 372 (100%)
13 (9.7%) 59 (44.0%) 36 (26.9%) 3 (2.2%) 0 2 (1.5%) 6 (4.5%) 6 (4.5%) 3 (2.2%) 0 3 (2.2%) 0 3 (2.2%) 134 (100%)
31 (11.7%) 65 (24.5%) 104 (39.2%) 0 4 (1.5%) 18 (6.8%) 16 (6.0%) 10 (3.8%) 8 (3.0%) 0 0 0 9 (3.4%) 265 (100%)
0 26 (54.2%) 16 (33.3%) 0 0 2 (4.2%) 1 (2.1%) 1 (2.1%) 0 0 1 (2.1%) 0 1 (2.1%) 48 (100%)
Comparisons of percentages between traps indicate the relative abundance of each species for each treatment. No statistical differences between treatments were observed in the mortality of the vector species (C. imicola and C. obsoletus).
Fig. 1. Mortality rates of Culicoides midges collected in two ultraviolet light traps, one surrounded by a deltamethrin-treated net (Treatment) and the other by a non-treated net (Control). Numbers above bars indicate the total number of Culicoides (dead and alive) collected in each trap.
Discussion This work reports on the response of Culicoides midges to deltamethrin-treated nets (4.4 g/kg ± 15%) in laboratory and field conditions. Results demonstrate the highly toxic effect of these nets on Culicoides species. In the laboratory trial, exposure attained mortality rates of 100% in Culicoides midges not immobilized and > 90% in midges immobilized before exposure. Under field conditions, although the insecticide did not stop midges from passing through the net, the mortality rate of Culicoides collected in the treatment trap was significantly higher than that of midges collected in the control trap. Mean numbers of Culicoides collected did not differ significantly between the treatment and control traps. This may indicate that the insecticide did not have a significant repellent effect on the midges. However, the strong attraction of the light traps may have interfered with any possible repellence of the insecticide.
Despite the fact that the treated nets were shown to be toxic to a number of non-target arthropods, no significant impact on pollinating or predator insects was recorded. In addition, as a result of the non-discriminatory attraction of the light trap, a relatively large number of non-target organisms were attracted to the treated nets. If these nets were to be used in stables or during transport in the absence of light, the number of non-target or non-blood-feeding insects attracted would be much lower. The differences in mortality and in time required to kill observed between non-anaesthetized and anaesthetized Culicoides midges in the laboratory trial probably reflected a reduced interaction between the immobilized midges and the insecticide. In poikilothermic organisms oxygen consumption and chemical reactions decrease at lower temperatures (Precht et al., 1973; Taylor, 1977). This decrease in metabolic activity in the cold immobilized Culicoides midges may have led to a lower absorption of the toxin through the insect cuticle and hence to lower mortality. Mortality percentages obtained after 30 s and 10 s of exposure indicate that with 4.4 g/kg of deltamethrin, contact of 10 s (or less) with the treated net should be sufficient to kill Culicoides midges in < 1 h under laboratory conditions. The fact that different Culicoides populations were assayed in the field and laboratory trials renders the direct comparison of the present results problematic. Despite this possible shortcoming, it seems that the deltamethrin-treated net was less effective when tested in the field than in the laboratory. Although mortality was higher in the treatment trap, significant numbers of midges managed to pass through the net and live long enough to potentially bite and transmit a virus to a possible host. It has been shown previously that nets impregnated with deltamethrin and cypermethrin will not repel Culicoides midges from light traps (Bauer et al., 2009; Page et al., 2009; del Río et al., 2013). However, in a similar study, Rohrmann (2010) observed that the number of blood-fed females captured after passing through
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12072
Susceptibility of Culicoides to deltamethrin nets
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Table 3. Non-target fauna collected alive or dead from the control and treatment traps and from the floor around the control and deltamethrin-treated nets. Control trap Order
Family
Alive, n
Acarina Araneae Coleoptera Collembola Diptera
– – – – Camillidae Drosophilidae Muscidae Sepsidae Sphaeroceridae Ceratopogonidae Cecidomyiidae Chironomidae Culicidae Psychodidae Sciaridae Tipulidae Cicadellidae Miridae Formicidae Ichneumonidae – – – – –
2 1 53 – 4 651 33 – 1770∗ 6 134 21 2 243 26 – 7 2 – 4 44∗ 1 2 – 5 3011
Hemiptera Himenoptera Lepidoptera Neuroptera Psocoptera Strepsiptera Tisanoptera Total
Treatment trap Dead, n 2 5 177 – 8 281 39 – 746∗ 12 9531 271 12 1273 108 2 4 7 2 10 42∗ – – – 13 12 545
Alive, n – 1 4 – 8 576 1 – 388∗ 3 59 10 2 188 16 – 5 1 – – 17∗ – 2 – – 1281
Dead, n 4 2 58 7 – 204 16 – 1268∗ 2 9766 225 5 664 54 – 31 10 – 7 39∗ – 8 4 2 12 376
Control floor
Treatment floor
Dead, n
Dead, n
3 2 43 2 – 4† 20 – 117† 1 30 10† 1 36 – – 1 2 13 3 4† – – – – 292
– 3 28 6 1 440† 16 21 365† 3 1739 115† 6 347 42 2 18 6 25 – 28† – – – 2 3213
∗Differences in mortality rates between treatment and control were close to significance (Sphaeroceridae P = 0.056; Lepidoptera P = 0.092). †Statistically significant differences between control and treatment in the collection of dead insects from the floor compared with the total number of insects collected.
an insecticide-treated net was lower than in a control treatment. Furthermore, similarly to the present results, Calvete et al. (2010) found that mortality in insects which passed through an insecticide-treated net increased in comparison with that in a control treatment. These nets may therefore offer a certain degree of protection to livestock as a significant proportion of Culicoides midges will die soon after contact with an impregnated net. Accordingly, the results of the current study show that midges which have been affected by passing through an impregnated net are very unlikely to have time to bite a host and, if they do, will die before they can take a second bite, thus limiting the number of potential infective bites and avoiding the transmission of disease between hosts. A study conducted in Australia demonstrated that even supposedly impenetrable physical barriers (tarpaulins) will not stop Culicoides midges from entering protected zones (Doherty et al., 2004). It may therefore be better to impregnate a wider net with insecticide than to install insect-proof stables which may compromise the comfort and well-being of livestock (Porter, 1959). Although insecticide-treated nets will not completely reduce the risk for infection when used on their own, they can be used in combination with methods such as treated ear-tags, pour-on insecticides, and good farming practice to successfully lower
the attack rate of Culicoides midges on livestock (Mullens et al., 2000; Doherty et al., 2004; Mehlhorn et al., 2008a, 2008b). The results obtained in the present study confirm the insecticidal capacity of deltamethrin-treated nets on Culicoides midges. These findings promote the use of pyrethroids in Culicoides control programmes in view of their longlasting residual activity and their lower impact on the natural environment in comparison with other organic or synthesized insecticides. The use of insecticide-treated nets inside stables may protect animals in areas in which vector species are thought to be endophylic (e.g. Culicoides obsoletus in northern Europe) (Baldet et al., 2008; Meiswinkel et al., 2008). For the control of exophylic vector species (e.g. C. imicola) (Barnard et al., 1998; Meiswinkel et al., 2000), treated nets could be deployed as outer fences to alter Culicoides activity. However, this protection alone is likely to be insufficient (Calvete et al., 2010) and should be combined with other prophylactic measures such as those mentioned above. Insecticide-treated nets may also be useful in livestock transport vehicles to reduce the possibility of infection during the transport of animals (Doherty et al., 2004). The present study showed that deltamethrin-impregnated nets cause high and rapid mortality in exposed Culicoides midges. The effectiveness of these nets should be demonstrated in further studies conducted under field conditions to determine the
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, DOI: 10.1111/mve.12072
6 R. Del Río et al. potential of this measure for the control of Culicoides spp. and the diseases associated with them.
Supporting Information Additional Supporting Information may be found in the online version of this article under the DOI reference: DOI: 10.1111/mve.12072 File S1. Statistical analysis of the collections and mortality rates of Culicoides midges and associated arthropods with deltamethrin treated nets in field and laboratory trials.
Acknowledgements This study was partially funded by European Union (EU) grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext207 (http://www.edenext.eu). The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. The study also received partial funding by the EU as part of a project entitled ‘Surveillance Network of Reoviruses, Bluetongue and African Horse Sickness in the Mediterranean basin and Europe’ (MedReoNet) (contract no. 044285). The authors thank Vestergaard Frandsen, Lausanne, Switzerland, for the donation of the treated nets, and the Conselleria d’Innovació, Interior i Justicia and Ministerio de Agricultura, Alimentación y Medio Ambiente for financial support. Finally, we also thank the farm owners J. Capó and B. Figuerola for allowing us the use of their facilities.
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Susceptibility of Culicoides to deltamethrin nets citronella oil and cypermethrin against Culicoides species when applied to polyester mesh. Veterinary Parasitology, 163, 105–109. Papadopoulos, E., Rowlinson, M., Bartram, D., Carpenter, S., Mellor, P. & Wall, R. (2010) Treatment of horses with cypermethrin against the biting flies Culicoides nubeculosus, Aedes aegypti and Culex quinquefasciatus. Veterinary Parasitology, 169, 165–171. Paweska, J.T., Venter, G.J. & Hamblin, C. (2005) A comparison of the susceptibility of Culicoides imicola and C. bolitinos to oral infection with eight serotypes of epizootic haemorrhagic disease virus. Medical and Veterinary Entomology, 19, 200–207. Porter, J.L. (1959) Some effects of screens in retarding entry of the common salt-marsh sandfly Culicoides furens (Poey) (Diptera: Heleidae). Mosquito News, 19, 159–163. Precht, H., Christopherson, J., Hensel, H. & Larcher, W. (1973) Temperature and Life. Springer, Berlin. Rawlings, P. (1996) A key, based on wing patterns of biting midges (genus Culicoides Latreille – Diptera: Ceratopogonidae) in the Iberian Peninsula, for use in epidemiological studies. Graellsia, 52, 57–71. Rohrmann, K.M.A. (2010) The effectiveness of insecticide-treated nets for the protection of cattle from biting midges and insect pests on dairy cattle farms. PhD Thesis. Free University of Berlin, Berlin.
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Schmahl, G., Klimpel, S., Walldorf, V., Schumacher, B., Jatzlau, A., Al-Quraishy, S. & Mehlhorn, H. (2009a) Effects of permethrin ® ® ® (Flypor ) and fenvalerate (Acadrex 60, Arkofly ) on Culicoides species – the vector of bluetongue virus. Journal of Parasitology Research, 104, 815–820. Schmahl, G., Mehlhorn, H., Abdel-Ghaffar, F., Al-Rasheid, K., Schumacher, B., Jatzlau, A. & Pohle, H. (2009b) Does rain reduce the efficacy of butox 7.5 pour-on (deltamethrin) against biting midges (Culicoides specimens)? Journal of Parasitology Research, 105, 1763–1765. Taylor, P. (1977) The respiratory metabolism of tsetse flies, Glossina species, in relation to temperature, bloodmeal size and pregnancy cycle. Physiological Entomology, 2, 317–322. World Health Organization (1981) Instructions for Determining the Susceptibility or Resistance of Adult Blackflies, Sandflies and Biting Midges to Insecticides. Mimeographed Document (WHO/VBC/ 81.810). WHO, Geneva. Accepted 5 March 2014
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12072
Susceptibility of Culicoides species biting midges to deltamethrin-treated nets as determined under laboratory and field conditions in the Balearic Islands, Spain R. D E L RÍ O 1 , C. B A R C E LÓ 1 , C. P A R E D E S- E S Q U I V E L 1 , J. L U C I E N T E S 2 and M. A. M I R A N D A 1 1
Laboratory of Zoology, University of the Balearic Islands, Palma de Mallorca, Spain and 2 Department of Animal Pathology, Faculty of Veterinary Science, University of Zaragoza, Zaragoza, Spain
Abstract. Culicoides Latreille (Diptera: Ceratopogonidae) are vectors of several arboviruses, including bluetongue virus (BTV) and African horse sickness virus (AHSV), which cause diseases in, respectively, sheep and cattle, and horses, and have economic repercussions mainly as a result of trade restrictions. Insecticides can be used to reduce vector populations and hence the spread of disease. Despite the economic importance of these diseases, relatively few studies have evaluated the efficacy of commercially available insecticides and the effectiveness of treated nets against Culicoides species. The aim of the present study was to evaluate the insecticidal effect of commercially available polyethylene nets (ZeroVector® ) treated with deltamethrin (4.4 g/kg ± 15%) on Culicoides species. Laboratory and field trials were conducted in Culicoides populations collected in Majorca in the Balearic Islands, Spain. The present study shows that deltamethrin-treated nets provoke high and rapid mortality (90–100%) in Culicoides midges under laboratory conditions and increase mortality by 13% when deployed in the field. Key words. Culicoides imicola, Culicoides obsoletus, impregnated nets, insecticides,
pyrethroids.
Introduction Bluetongue (BT) is a disease of ruminants caused by an Orbivirus [bluetongue virus (BTV)] of the family Reoviridae. Some species of the genus Culicoides can transmit BTV, as well as other viruses such as African horse sickness virus (AHSV), epizootic haemorrhagic disease virus (EHDV) and the recently discovered Schmallenberg virus (SBV), to susceptible animals (Mellor et al., 1990; Paweska et al., 2005; De Regge et al., 2012). Sheep and, to some extent, cattle manifest the most severe clinical symptoms of the disease. Although wild ruminants are susceptible to and can become infected with BTV, most will show no clinical symptoms and may act as silent carriers or amplifying hosts (Neitz, 1933; Backx et al., 2007; Linden et al.,
2008). Amongst the viruses transmitted by Culicoides midges, BTV and SBV are of particular interest in Europe. The recent introduction of these two viruses into northern Europe indicates the possibility that other Culicoides-transmitted diseases (e.g. African horse sickness) may enter the continent (De Vos et al., 2012). To date, the most effective way to control the spread of BTV is by using vaccination and restricting the movement of animals. However, the emergence of new serotypes of BTV (Carpenter et al., 2009) and the costs associated with treatments call for the development of additional, efficient and cost-effective control strategies. The susceptibility of Culicoides species to insecticides is poorly documented. Only a few publications describe the responses of some Culicoides species to organochlorines,
Correspondence: Ricardo del Río, Laboratory of Zoology, University of the Balearic Islands, Cra. Valldemossa Km. 7.5, Palma de Mallorca 07122, Spain. Tel.: + 34 971 173352; Fax: + 34 971 173184; E-mail: [email protected] © 2014 The Royal Entomological Society
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2 R. Del Río et al. organophosphates, carbamates and pyrethroids (Hill & Roberts, 1947; Kline & Roberts, 1981; Floore, 1985; Mehlhorn et al., 2008a, 2008b; Schmahl et al., 2009a, 2009b; Papadopoulos et al., 2010). In the present study, the susceptibility of Culicoides species to polyethylene nets impregnated with deltamethrin, a pyrethroid ester insecticide, was determined. Assessments were performed in the laboratory, using adapted World Health Organization (WHO) test kits (WHO, 1981), and under field conditions. The use of these nets in stables or transport vehicles may offer protection to susceptible animals and avoid infection and the spread of diseases transmitted by Culicoides species. Materials and methods Polyethylene nets (ZeroVector® Durable Lining; Dart Association, Lausanne, Switzerland) impregnated with 4.4 g/kg ± 15% of deltamethrin are currently used in malaria endemic zones for mosquito control. The active ingredients of the insecticide are effective on the nets for 3–5 years. These nets have never been tested against Culicoides species. Laboratory trial Culicoides midges were collected at a dairy farm named C’as Boter (39∘ 30′ N, 03∘ 07′ E) located in Majorca in the Balearic Islands, Spain, between 15 June and 17 July 2011. Insects were collected from dusk to dawn using ARC-OVI (Agricultural Research Council–Onderstepoort Veterinary Institute, Pretoria, South Africa) and mini Centers for Disease Control (CDC) (John W. Hock Co., Gainesville, FL, U.S.A.) light traps. Collected insects were transported to the insectary early in the morning (06.00–07.00 hours) inside thermal isolation containers and maintained in pools of 30–100 specimens inside WHO chambers (consisting of clear plastic cylinders 4.5 cm in diameter and 12.0 cm in length) (WHO, 1981). Culicoides were maintained at 25 ± 2 ∘ C and 75% relative humidity (RH) in dim light on a 5% sucrose solution for 12–24 h before each bioassay. Insecticide bioassays were conducted in WHO chambers lined with pieces (12 × 25 cm) of deltamethrin-treated net (ZeroVector® ). Culicoides midges were transferred to the WHO chambers and exposed to the treated nets for 6, 2, 1 min or 30 s. During the bioassay, WHO chambers were placed horizontally to facilitate contact with the nets. To accurately regulate the time of interaction with the treated nets at exposure times of ≤ 30 s, Culicoides midges were immobilized by refrigeration (− 20 ∘ C) before exposure. Once immobilized, insects were placed in contact with the treated net for 30, 20 or 10 s. In each bioassay, WHO chambers lined with polyethylene nets without insecticide were used as controls. After exposure, insects were transferred to the original WHO chambers without insecticide and the time to observe an effect to the treatment was recorded. Field trial The study was conducted on a farm in Spain producing horses (30–40 horses) for consumption (Ca Sa Manescala,
39∘ 25′ 18.86′′ N, 03∘ 00′ 22.31′′ E) over 12 nights during 12–28 October 2011. An Onderstepoort light trap was hung at a height of 1.5 m inside a cylinder (1.5 m in diameter, 1.5 m in height) made of metallic mesh (mesh size: 5 × 5 cm). The cylinder was covered with deltamethrin-treated net (ZeroVector® ; approximately 24 holes/cm2 ). The same trapping system but without the insecticide treatment was used as a control. To assess the effects of the impregnated nets on mortality in the Culicoides population and non-target fauna, the soil surface at the base of each structure (treatment and control) was covered with a 2 × 2 m piece of white polyethylene fabric from which all insects were aspirated the morning after each sampling. To minimize interference between the light traps, treatment and control structures were placed 10 m apart. Collections were obtained near the horses’ enclosures. Traps were operated from dusk until dawn (from 20.00 to 08.00 hours). Captures were collected early in the morning. After the collection of samples, the structures and traps were rotated for the next sampling to avoid the occurrence of any bias in collections caused by differences in the locations of traps. In the laboratory, living and dead midges were separated and levels of mortality were compared between treatment and control traps. All midges in this trial were identified according to their wing pattern (Rawlings, 1996) into species or species group (when morphological discrimination amongst species was difficult), and separated by sex and gonotrophic condition (Dyce, 1969).
Statistical analysis The mortality rates of immobilized and non-immobilized Culicoides midges were compared at 30 s exposure. As data were not normally distributed, the Mann–Whitney U test was applied to compare the medians of the two samples at 95% confidence levels using the statistics program R Version 2.15.1 (R Foundation for Statistical Computing, Vienna, Austria). Mean captures and mortality rates were compared between control and treatment groups under field conditions using t-tests in statgraphics Plus Version. 3.0 (Statpoint Technologies, Inc., Warrenton, VA, U.S.A.). Differences between treatments were considered to be significant if they achieved a P-value of < 0.05 (see File S1).
Results Laboratory trial A total of 1401 Culicoides, belonging to at least nine species (Obsoletus complex, Culicoides cataneii/gejgelensis, Culicoides circumscriptus Kieffer 1918, Culicoides imicola Kieffer 1913, Culicoides jumineri/kurensis, Culicoides longipennis/sahariensis, Culicoides newsteadi Austen 1921, Culicoides nubeculosus/puncticollis and Culicoides paolae Boorman 1996) were assayed. Of these, 34 (2.4%) were males and 1367 (97.6%) were females. The females consisted of 541 (39.6%) nulliparous, 176 (12.9%) parous, 630 (46.1%) gravid and 20 (1.5%) engorged specimens.
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12072
Susceptibility of Culicoides to deltamethrin nets
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Table 1. Effect of deltamethrin-impregnated nets on Culicoides spp. after exposures of 10 s to 6 min inside World Health Organization (WHO) test tubes. One group of Culicoides midges were transferred directly to the WHO chambers with the treated net (not immobilized), and the other was cold-immobilized before the transfer (immobilized). Control
Not immobilized
Immobilized
Treatment
Exposure
N
Alive
Killed
N
Total
6 min 2 min 1 min 30 s 30 s 20 s 10 s
1 1 1 3 1 1 2
19 13 15 85 52 33 27
0 0 0 0 0 0 0
2 2 3 12 3 3 5
44 63 80 465 108 150 252
Alive Mean ± SD
Killed Mean ± SD
Mortality, %
0 0 0 0 2.7 ± 3.1 2.0 ± 2.6 3.6 ± 3.4
22.0 ± 2.0 31.5 ± 13.5 26.7 ± 1.7 38.5 ± 12.6 33.3 ± 12.2 48.0 ± 18.0 46.8 ± 13.8
100% 100% 100% 100% 92.6% 96.0% 92.9%
N indicates the number of replicates. SD, standard deviation.
The most abundant species tested belonged to the Obsoletus complex (55.7%, n = 781), C. circumscriptus (18.7%, n = 262), C. cataneii/gejgelensis (10.8%, n = 152) and C. paolae (7.7%, n = 108). The remaining species represented < 10% of the total. Midges not immobilized before contact with insecticide (contact times of 30 s to 6 min) The mortality rate in midges that were not immobilized before contact with the deltamethrin was 100%. Insects died within 6–17 min after contact with the treated net, irrespective of exposure time. At 6 min post-exposure, 73.7–100% of the Culicoides midges exhibited clear symptoms of intoxication (great difficulty in crawling or flying) for both short (30 s) and long (6 min) exposures to the insecticide. At 9 min post-exposure, 100% of insects exhibited intoxication signs, and at 17 min post-exposure 100% mortality was observed (Table 1).
Midges immobilized before contact with insecticide (contact times of 10–30 s) Mortality rates in midges immobilized prior to 30, 20 and 10 s of exposure to the treated nets were 92.9, 96.0 and 92.6%, respectively (Table 1). Culicoides midges showed signs of intoxication and died later (> 17 min and < 40 min, respectively) than those not immobilized. After 30 s exposure, the mortality rate was significantly (P = 0.015) lower in midges that were immobilized before contact compared with those that were not. Intoxication also took significantly (P = 0.020) longer to become evident in midges that were immobilized. A survival rate of 100% was found in the control trials.
Field trial Eleven nights were sampled with the treatment trap and 12 with the control trap. A failure in the electricity supply during one of the nights altered the collections of one trap and the results from that night were therefore discarded.
A total of 819 Culicoides midges belonging to at least 13 different species (Obsoletus complex, C. cataneii/gejgelensis, C. circumscriptus, C. imicola, C. jumineri/kurensis, C. longi pennis/sahariensis, Culicoides maritimus Kieffer 1924, C. newsteadi, C. nubeculosus/puncticollis, C. paolae, Culicoides parroti/stigma, Culicoides pictipennis (Staeger 1939) and Culicoides univittatus Vimmer 1932) were collected with the traps. The most abundant species collected were C. imicola (33.2%) and C. circumscriptus (33.0%). Numbers of the species of the Obsoletus complex were relatively low (4.3%) compared with their abundance during spring (Table 2). The mean number of midges collected in the treated trap (26.1 ± 8.6) was not significantly (P = 0.405) lower than that in the control trap (46.0 ± 38.2). The mean ± standard deviation (SD) mortality rate of Culicoides collected in the treated traps (84.9 ± 10.5%) was significantly (P = 0.0020) higher than that of Culicoides collected in the control traps (72.3 ± 5.9%) (Fig. 1). The polyethylene sheets at the base of each of the structures were aspirated on 4 nights. The number of Culicoides collected on the polyethylene sheets was low compared with the captures of the traps. Mean ± SD numbers of Culicoides collected per night were 2.3 ± 1.7 at the base of each control trap, and 2.3 ± 1.9 at the base of each treatment trap. With regard to non-target fauna, 32 718 arthropods belonging to 12 orders and at least 25 families were collected (Table 3). Most of the non-target insects collected were Diptera (98.0%), and more specifically belonged to the Cecidomyiidae family (66.7%). Most of the specimens found dead at the polyethylene bases of control traps belonged to the Sphaeroceridae family (40.1%), whereas most of the specimens found dead at the bases of treatment structures belonged to the Cecidomyiidae family (54.1%) (Table 3). Significant differences (P < 0.05) between the treatment and control traps were observed in the ratio of floor to trap collections in some insects (Drosophilidae, Sphaeroceridae, Chironomidae and Lepidoptera) (Table 3). The higher numbers of live arthropods collected in the control trap did not differ significantly (P = 0.142) from that in the treatment trap. Differences among the arthropods collected dead in traps were also not significant (P = 0.959).
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12072
4 R. Del Río et al. Table 2. Culicoides species collected during 12 nights on a horse farm using a trap surrounded by a deltamethrin-impregnated net (treatment) and a control trap surrounded by a non-treated net (control). Control
Treatment
Species
Dead, n (%)
Alive, n (%)
Dead, n (%)
Alive, n (%)
C. cataneii/gejgelensis C. circumscriptus C. imicola C. jumineri/kurensis C. longipennis/sahariensis C. maritimus C. newsteadi C. paolae C. parroti/stigma C. pictipennis C. nubeculosus/puncticollis C. univittatus Obsoletus complex Total
45 (12.1%) 120 (32.3%) 116 (31.2%) 3 (0.8%) 3 (0.8%) 8 (2.2%) 21 (5.6%) 22 (5.9%) 10 (2.7%) 1 (0.3%) 0 1 (0.3%) 22 (5.9%) 372 (100%)
13 (9.7%) 59 (44.0%) 36 (26.9%) 3 (2.2%) 0 2 (1.5%) 6 (4.5%) 6 (4.5%) 3 (2.2%) 0 3 (2.2%) 0 3 (2.2%) 134 (100%)
31 (11.7%) 65 (24.5%) 104 (39.2%) 0 4 (1.5%) 18 (6.8%) 16 (6.0%) 10 (3.8%) 8 (3.0%) 0 0 0 9 (3.4%) 265 (100%)
0 26 (54.2%) 16 (33.3%) 0 0 2 (4.2%) 1 (2.1%) 1 (2.1%) 0 0 1 (2.1%) 0 1 (2.1%) 48 (100%)
Comparisons of percentages between traps indicate the relative abundance of each species for each treatment. No statistical differences between treatments were observed in the mortality of the vector species (C. imicola and C. obsoletus).
Fig. 1. Mortality rates of Culicoides midges collected in two ultraviolet light traps, one surrounded by a deltamethrin-treated net (Treatment) and the other by a non-treated net (Control). Numbers above bars indicate the total number of Culicoides (dead and alive) collected in each trap.
Discussion This work reports on the response of Culicoides midges to deltamethrin-treated nets (4.4 g/kg ± 15%) in laboratory and field conditions. Results demonstrate the highly toxic effect of these nets on Culicoides species. In the laboratory trial, exposure attained mortality rates of 100% in Culicoides midges not immobilized and > 90% in midges immobilized before exposure. Under field conditions, although the insecticide did not stop midges from passing through the net, the mortality rate of Culicoides collected in the treatment trap was significantly higher than that of midges collected in the control trap. Mean numbers of Culicoides collected did not differ significantly between the treatment and control traps. This may indicate that the insecticide did not have a significant repellent effect on the midges. However, the strong attraction of the light traps may have interfered with any possible repellence of the insecticide.
Despite the fact that the treated nets were shown to be toxic to a number of non-target arthropods, no significant impact on pollinating or predator insects was recorded. In addition, as a result of the non-discriminatory attraction of the light trap, a relatively large number of non-target organisms were attracted to the treated nets. If these nets were to be used in stables or during transport in the absence of light, the number of non-target or non-blood-feeding insects attracted would be much lower. The differences in mortality and in time required to kill observed between non-anaesthetized and anaesthetized Culicoides midges in the laboratory trial probably reflected a reduced interaction between the immobilized midges and the insecticide. In poikilothermic organisms oxygen consumption and chemical reactions decrease at lower temperatures (Precht et al., 1973; Taylor, 1977). This decrease in metabolic activity in the cold immobilized Culicoides midges may have led to a lower absorption of the toxin through the insect cuticle and hence to lower mortality. Mortality percentages obtained after 30 s and 10 s of exposure indicate that with 4.4 g/kg of deltamethrin, contact of 10 s (or less) with the treated net should be sufficient to kill Culicoides midges in < 1 h under laboratory conditions. The fact that different Culicoides populations were assayed in the field and laboratory trials renders the direct comparison of the present results problematic. Despite this possible shortcoming, it seems that the deltamethrin-treated net was less effective when tested in the field than in the laboratory. Although mortality was higher in the treatment trap, significant numbers of midges managed to pass through the net and live long enough to potentially bite and transmit a virus to a possible host. It has been shown previously that nets impregnated with deltamethrin and cypermethrin will not repel Culicoides midges from light traps (Bauer et al., 2009; Page et al., 2009; del Río et al., 2013). However, in a similar study, Rohrmann (2010) observed that the number of blood-fed females captured after passing through
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12072
Susceptibility of Culicoides to deltamethrin nets
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Table 3. Non-target fauna collected alive or dead from the control and treatment traps and from the floor around the control and deltamethrin-treated nets. Control trap Order
Family
Alive, n
Acarina Araneae Coleoptera Collembola Diptera
– – – – Camillidae Drosophilidae Muscidae Sepsidae Sphaeroceridae Ceratopogonidae Cecidomyiidae Chironomidae Culicidae Psychodidae Sciaridae Tipulidae Cicadellidae Miridae Formicidae Ichneumonidae – – – – –
2 1 53 – 4 651 33 – 1770∗ 6 134 21 2 243 26 – 7 2 – 4 44∗ 1 2 – 5 3011
Hemiptera Himenoptera Lepidoptera Neuroptera Psocoptera Strepsiptera Tisanoptera Total
Treatment trap Dead, n 2 5 177 – 8 281 39 – 746∗ 12 9531 271 12 1273 108 2 4 7 2 10 42∗ – – – 13 12 545
Alive, n – 1 4 – 8 576 1 – 388∗ 3 59 10 2 188 16 – 5 1 – – 17∗ – 2 – – 1281
Dead, n 4 2 58 7 – 204 16 – 1268∗ 2 9766 225 5 664 54 – 31 10 – 7 39∗ – 8 4 2 12 376
Control floor
Treatment floor
Dead, n
Dead, n
3 2 43 2 – 4† 20 – 117† 1 30 10† 1 36 – – 1 2 13 3 4† – – – – 292
– 3 28 6 1 440† 16 21 365† 3 1739 115† 6 347 42 2 18 6 25 – 28† – – – 2 3213
∗Differences in mortality rates between treatment and control were close to significance (Sphaeroceridae P = 0.056; Lepidoptera P = 0.092). †Statistically significant differences between control and treatment in the collection of dead insects from the floor compared with the total number of insects collected.
an insecticide-treated net was lower than in a control treatment. Furthermore, similarly to the present results, Calvete et al. (2010) found that mortality in insects which passed through an insecticide-treated net increased in comparison with that in a control treatment. These nets may therefore offer a certain degree of protection to livestock as a significant proportion of Culicoides midges will die soon after contact with an impregnated net. Accordingly, the results of the current study show that midges which have been affected by passing through an impregnated net are very unlikely to have time to bite a host and, if they do, will die before they can take a second bite, thus limiting the number of potential infective bites and avoiding the transmission of disease between hosts. A study conducted in Australia demonstrated that even supposedly impenetrable physical barriers (tarpaulins) will not stop Culicoides midges from entering protected zones (Doherty et al., 2004). It may therefore be better to impregnate a wider net with insecticide than to install insect-proof stables which may compromise the comfort and well-being of livestock (Porter, 1959). Although insecticide-treated nets will not completely reduce the risk for infection when used on their own, they can be used in combination with methods such as treated ear-tags, pour-on insecticides, and good farming practice to successfully lower
the attack rate of Culicoides midges on livestock (Mullens et al., 2000; Doherty et al., 2004; Mehlhorn et al., 2008a, 2008b). The results obtained in the present study confirm the insecticidal capacity of deltamethrin-treated nets on Culicoides midges. These findings promote the use of pyrethroids in Culicoides control programmes in view of their longlasting residual activity and their lower impact on the natural environment in comparison with other organic or synthesized insecticides. The use of insecticide-treated nets inside stables may protect animals in areas in which vector species are thought to be endophylic (e.g. Culicoides obsoletus in northern Europe) (Baldet et al., 2008; Meiswinkel et al., 2008). For the control of exophylic vector species (e.g. C. imicola) (Barnard et al., 1998; Meiswinkel et al., 2000), treated nets could be deployed as outer fences to alter Culicoides activity. However, this protection alone is likely to be insufficient (Calvete et al., 2010) and should be combined with other prophylactic measures such as those mentioned above. Insecticide-treated nets may also be useful in livestock transport vehicles to reduce the possibility of infection during the transport of animals (Doherty et al., 2004). The present study showed that deltamethrin-impregnated nets cause high and rapid mortality in exposed Culicoides midges. The effectiveness of these nets should be demonstrated in further studies conducted under field conditions to determine the
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, DOI: 10.1111/mve.12072
6 R. Del Río et al. potential of this measure for the control of Culicoides spp. and the diseases associated with them.
Supporting Information Additional Supporting Information may be found in the online version of this article under the DOI reference: DOI: 10.1111/mve.12072 File S1. Statistical analysis of the collections and mortality rates of Culicoides midges and associated arthropods with deltamethrin treated nets in field and laboratory trials.
Acknowledgements This study was partially funded by European Union (EU) grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext207 (http://www.edenext.eu). The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. The study also received partial funding by the EU as part of a project entitled ‘Surveillance Network of Reoviruses, Bluetongue and African Horse Sickness in the Mediterranean basin and Europe’ (MedReoNet) (contract no. 044285). The authors thank Vestergaard Frandsen, Lausanne, Switzerland, for the donation of the treated nets, and the Conselleria d’Innovació, Interior i Justicia and Ministerio de Agricultura, Alimentación y Medio Ambiente for financial support. Finally, we also thank the farm owners J. Capó and B. Figuerola for allowing us the use of their facilities.
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