Bluetongue outbreaks: Looking for effective control strategies against Culicoides vectors

Accepted Manuscript Bluetongue outbreaks: Looking for effective control strategies against Culicoides vectors

Giovanni Benelli, Luca Buttazzoni, Angelo Canale, Armando D'Andrea, Paola Del Serrone, Gavino Delrio, Cipriano Foxi, Susanna Mariani, Giovanni Savini, Chithravel Vadivalagan, Kadarkarai Murugan, Chiara Toniolo, Marcello Nicoletti, Mauro Serafini PII: DOI: Reference:

S0034-5288(17)30314-4 doi: 10.1016/j.rvsc.2017.05.023 YRVSC 3339

To appear in:

Research in Veterinary Science

Received date: Revised date: Accepted date:

16 March 2017 18 May 2017 19 May 2017

Please cite this article as: Giovanni Benelli, Luca Buttazzoni, Angelo Canale, Armando D'Andrea, Paola Del Serrone, Gavino Delrio, Cipriano Foxi, Susanna Mariani, Giovanni Savini, Chithravel Vadivalagan, Kadarkarai Murugan, Chiara Toniolo, Marcello Nicoletti, Mauro Serafini , Bluetongue outbreaks: Looking for effective control strategies against Culicoides vectors, Research in Veterinary Science (2017), doi: 10.1016/ j.rvsc.2017.05.023

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Review

Bluetongue outbreaks: looking for effective control strategies against

SC RI PT

Culicoides vectors

Giovanni Benelli 1*, Luca Buttazzoni 2, Angelo Canale 1, Armando D’Andrea 3, Paola Del Serrone 2, Gavino Delrio 4, Cipriano Foxi 4, Susanna Mariani 3, Giovanni Savini 5, Chithravel Vadivalagan 6, Kadarkarai Murugan 6, Chiara Toniolo 7, Marcello Nicoletti 7,

Department of Agriculture, Food and Environment, University of Pisa, Via del

ED

Borghetto 80, 56124 Pisa, Italy 2

MA

1

NU

Mauro Serafini 7

Council for Agricultural Research and Economics (CREA), Animal Production

3

C. R. ENEA Casaccia, SSPT-TECS-BIORISC, Via Anguillarese 301, 00123, S. M.

CE

Galeria, Roma, Italy

Dipartimento di Agraria, Sezione di Patologia Vegetale ed Entomologia, Università

AC

4

PT

Research Centre, Via Salaria 31, 00016 Monterotondo Scalo, Roma, Italy

degli Studi di Sassari, Via Enrico de Nicola, 07100 Sassari, Italy. 5

Istituto Zooprofilattico dell’Abruzzo e del Molise “G. Caporale”, Via Campo Boario, 64100 Teramo, Italy.

6

Division of Entomology, School of Life Sciences, Bharathiar University, Coimbatore641046, Tamil Nadu, India

1

ACCEPTED MANUSCRIPT

7

Department of Environmental Biology, Sapienza University, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy

* Corresponding author. Tel.: +39-0502216141. Fax: +39-0502216087. E-mail address:

AC

CE

PT

ED

MA

NU

SC RI PT

[email protected]

2

ACCEPTED MANUSCRIPT

Abstract

Several arthropod-borne diseases are now rising with increasing impact and risks for public health, due to environmental higher changes and resistance to pesticides

SC RI PT

currently marketed. In addition to community surveillance programs and a careful management of herds, a next-generation of effective products is urgently needed to control the spread of these diseases. Natural product research can afford alternative solutions. Recently, a re-emerging of bluetongue disease is ongoing in Italy. Bluetongue is a viral disease that affects ruminants and is spread through the bite of bloodsucking

NU

insects, especially Culicoides species. In this review, we focused on the importance of

MA

vector control programs for prevention or bluetongue outbreaks, outlining the lack of effective tools in the fight against Culicoides vectors. Then, we analyzed a field case

ED

study in Sardinia (Italy) concerning the utilization of the neem cake (Azadirachta indica), to control young instar populations of Culicoides biting midges, the vectors of

PT

bluetongue virus. Neem cake is a cheap and eco-friendly by-product obtained from the extraction of neem oil. Overall, we propose that the employ of neem extraction by-

CE

products as aqueous formulation, in muddy sites close to livestock grazing areas, may

AC

represent an effective tool in the fight against the spread of bluetongue virus in the Mediterranean areas.

Keywords: arthropod-borne diseases; biosafety; botanical; biting midge; Ceratopogonidae; neem cake

3

ACCEPTED MANUSCRIPT

1. Introduction

Currently, the control of arthropod-borne diseases represent one of the major challenges of medical and veterinary importance (WHO 2000; Benelli 2015a). Several

SC RI PT

factors are nowadays flowing into global and national emergencies, in particular for farm animals. After a long period dominated by intensive livestock farming, supported by massive use of insecticides and antibiotics production levels are nowadays in danger (Mehlhorn 2012). The indications are that several new situations, like international trade, globalization, and massive migrations, are important biotic factors that pose

NU

vector-borne animal diseases as a continuous threat to livestock economies worldwide

MA

(Ebers et al. 2015). Notably, pesticides currently marked for vector control have to face the rapid development of resistance, besides leading to important problems for human

ED

and animal health, as well as non-target effects on the environment (Hemingway and Ranson 2000; Isman et al. 2006; Naqqash et al. 2016; Pavela and Benelli 2016a,b).

PT

The rapid diffusion of arthropod-borne disease is the result of the cooccurrence of at least three factors: environmental changes, boosted vector efficiency,

CE

and selection of more aggressive pathogens and parasites, thanks to their rapid adaption

AC

to novel hosts (Bethan 2005). A reliable way to tackle these issues is to face the problem with an integrated multidisciplinary approach (Nicoletti et al. 2012b). As recently highlighted by the Centers for Disease Control and Prevention, to successfully fight arthropod-borne diseases, a One Health approach is important (Day 2011; DantasTorres et al. 2012). One Health pointed out that the human health is strongly connected to the health of animals and the environment care. One Health intends encourage the

4

ACCEPTED MANUSCRIPT

cooperation among multiple disciplines, protecting human health and the environment (Franco et al. 2014; Webster et al. 2016). Bluetongue is a viral disease that affects ruminants and is spread through the bite of bloodsucking insects (Mordue e al. 2007). This mechanism is common to many

SC RI PT

insect-borne diseases affecting humans, such as malaria, West Nile virus and the recent Zika virus, or animals, as the Schmallenberg virus, Usutu virus and viral hemorrhagic fever. In these cases, micro-organisms and other infectious agents develop in the cells of vertebrate organisms (Benelli and Mehlhorn 2016; Benelli et al. 2016a,b). Biting midges, belonging to the genus Culicoides (Diptera: Ceratopogonidae),

NU

can feed on viremic animals, transmitting the infection caused by the bluetongue virus

MA

to livestock, as well as wild ruminant populations (Wilson and Mellor 2009; Carpenter et al. 2013). These insects are bad fliers, but being lightweight, they can be carried by

ED

wind over long distances (Ducheye 2007; see also Bhasin and Mordue 2000 and Rasmussen et al. 2012). The spread of the disease can also occur with the transport of

PT

infected animals. However, the tentative of blocking the movements of receptive animals is not always able to prevent the spread of bluetongue. Furthermore, it affects

CE

the sale of the animals, creating serious socio-economic damages to farmers (Wilson

AC

and Mellor 2009).

In Italy, the bluetongue disease appeared in 2000, probably favored by wind

translocation of infected Culicoides vectors from North Africa (Wilson and Mellor 2009). Although bluetongue initially affected Sicily and Calabria (Southern Italy), then spread out in Sardinia, the second main island of Italy, where became virtually endemic and caused the death of hundreds of thousands of sheep of high economic value (Foxi and Delrio 2010).

5

ACCEPTED MANUSCRIPT

Despite the preventive actions taken, mainly consisting in restriction of movement of animals and massive vaccination program, the bluetongue infection continued to spread in the Italian peninsula, affecting the entire South and Central Italy, and rapidly spreading to the North of Italy, although Sardinia remained the most

SC RI PT

damaged (Nicoletti et al. 2014). Since 2006, the bluetongue, as bluetongue serotype 8, has also been reported in the Netherlands, Belgium, Germany, France, Bulgaria and United Kingdom, where it was particularly virulent on cattle (Elbers et al. 2008; Gloster et al. 2008), involving also the Northern regions of Europe, like Scotland (Hendry and Godwin 1988; Kettle 1951, 1995) with several negative effects (Blackwell et al. 2004;

NU

Carpenter et al. 2013).

MA

As a general trend, even cattle and goats become infected, but they are often asymptomatic. In sheep, however, the disease manifests itself in different clinical forms

ED

of hyper-acute, with a lethality of up to 30%, and sub-clinical, in which case the sick animals recover in a few days with complete remission of symptoms. The presence of

PT

viral particles in the blood of sheep, which reaches a peak 7-8 days after infection, usually does not exceed thirty days, whereas in cattle can persist for about 60 days

CE

(Schwartz-Cornil et al. 2008). In this period, the infected animal acts as reservoir of the

AC

virus, and is used by the insect vector as intermediate step to spread the disease (Schwartz-Cornil et al. 2008).

2. Current tools in the fight against bluetongue

In many countries, wherein the bluetongue disease and its insect vectors are present or expected, an effective monitoring system (Mands et al. 2004; Gerry et al.

6

ACCEPTED MANUSCRIPT

2009; Nielsen et al. 2010; Thompson et al. 2014; Murchie et al. 2016) can allow to identify new strains of the virus and carefully following the spread of serotypes already in the territory, as well as invasion of new regions. This is an aspect of fundamental importance, since the infection by a serotype does not protect against further infection

SC RI PT

by different viral serotypes (Perrin et al. 2007). The entomological surveillance national plan aims to identify the geographical distribution and seasonal dynamics of insect vectors through the placement of light traps on farms (Mordue et al. 2007). The insect catches are analyzed for quantization of total Culicoides and to determine the presence/absence of C. imicola, the main vector of

NU

bluetongue in sheep, both in the protection and surveillance zones and in the high-risk

MA

areas (Goffredo and Meiswinkel 2004). This vector is widespread in the warmer coastal areas of central and southern Italy. However, in recent years the presence of other

ED

vector species, such as C. obsoletus, C. scoticus, C. dewulfi, C. newsteadi, C. pulicaris and C. punctatus, has been reported, with a wider distribution also affecting northern

PT

areas (Goffredo et al. 2015; Foxi et al. 2016). In Italy, the National Veterinary Epidemiological Bulletin reported outbreaks of bluetongue in 2015 and the first quarter

CE

of 2016, as observed in several Italian regions (IZSM&A 2016a,b).

AC

In the future, the bluetongue outbreaks could spread more rapidly if compared to the past, due to climate change, with periods characterized by the persistence of high temperatures that accompany rains, and consequently high humidity. These conditions also favor early spring and late fall larval development, consequently leading to the increase in Culicoides vector populations and the risk of disease transmission. After a period of relative calm, in 2016-2017 a new upsurge of the bluetongue emerged in Sardinia. To counter the resurgence of the disease, more than a million vaccines were

7

ACCEPTED MANUSCRIPT

produced. However, to avoid a continuous recourse to vaccination in an illness that recurs periodically, this intervention must be accompanied by prevention and by combating vectors in periods of stasis (Foxi et al. 2016). The control of Culicoides is currently limited to the treatments with synthetic

SC RI PT

insecticides against adults on the stable walls or using treated nets (Narladkar et al. 2006; Mordue et al. 2007; Carpenter et al. 2008; Bauer et al. 2009; Del Rìo et al. 2014a,b, Baker et al. 2015; De Keyser et al. 2017), in potential synergy with animal housing (Baylis et al. 2010) and mechanical disturbance of Culicoides emergence from cowpats (Lühken et al. 2014, 2015). Notably, insecticidal treatments with synthetic

NU

pesticides are currently facing the possible development of resistance in targeted pests

MA

(Naqqash et al. 2016), which can strongly limit the effectiveness of control programs (De Keyser et al. 2017).

ED

A further option can be the treatment directly on animals with repellent products, with special reference to N, N-diethyl-meta-toluamide, (DEET), para-

PT

menthane-3, 8-diol (PMD) and citronella (Cymbopogon citratus L.) oil (Braverman et al. 1997, 2000, 2004; Calvete et a. 2010; Schmahl et al. 2008; Martínez-de la Puente et

CE

al. 2009; Page et al. 2009, 2014; Venter et al. 2011; González et al. 2014; Murchie et al.

AC

2016). However, the continuous protection of livestock with repellents is a difficult challenge, which lead to uncertain results (Narladkar et al. 2006; Carpenter et al. 2008). A good example has been reported by Page et al. (2009), in field assays testing 15% (DEET), 0.6% citronella oil, and 0.3% alpha-cyano-cypermethrin against Culicoides species. The three products were applied to polyester meshes fitted to down-draught suction 220V UV light traps operating overnight. Notably, no significant repellent effect against Culicoides was found for the citronella oil or the alpha-cyano-cypermethrin,

8

ACCEPTED MANUSCRIPT

while DEET had a significant repellent effect against Culicoides species and C. imicola for all catches made from after sunset to before sunrise (Page et al. 2009; see also Vente et al. 2014). Stuart et al. (2000) evaluated the repellent and antifeedant effect of derivatives

SC RI PT

of salicylic acid on the biting midge Culicoides impunctatus Goetghebeur. They noted that salicyluric acid strongly inhibited feeding. Following alkyl substitution of salicylic acid, it has been observed that o-thymotic and o-cresotic acids were also effective. Salicyluric acid indicated led to a marked protective effect in clinical trials; Stuart et al. (2000) hypothesized that this may result primarily from contact, since no repellent

NU

effects were achieved by salicyluric acid (Stuart et al. 2000).

MA

Later, González et al. (2014) studied the efficacy of 23 chemical and plantderived repellents against Culicoides obsoletus (Meigen) females, relying to Y-tube

ED

olfactometer assays. Therefore, the authors selected the ten most effective products for landing assay evaluation. The six most promising products were tested at 10% and 25%

PT

concentrations in field assays using the Centers for Disease Control (CDC) light traps. Notably, results varied according to the testing methodology. Indeed, while DEET at

CE

1  µg/µL was the most effective repellent in olfactometer assays, filter paper landing

AC

bioassays showed that plant essential oils, with special reference to lemon eucalyptus oil (see also Trigg 1996), achieved the best results, while 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, showing an efficacy comparable to DEET (González et al. 2014). While most of the researches on biting midge control targeted the adults, the control of young instar populations has been scarcely developed, since larval habitats of

9

ACCEPTED MANUSCRIPT

these vectors typically consisting of wet environments or frequent wet seeds livestock undertakings (Foxi and Delrio 2013; Zimmer et al. 2014). These larval development sites can be remediated with simple clearance operations carried out directly by the farmer, and industrial hygiene practices (Foxi et al. 2016). The larval populations could

SC RI PT

be also effectively managed relying to insecticides with low environmental impact. These new generation insecticides should be effective, readily soluble in water (without employing co-formulations and stabilizers), eco-friendly, with low risks for livestock and human health. They should be also selective against the targeted insect vectors. In this framework, natural product research can offer important innovations (Benelli et al.

MA

NU

2017a).

ED

3. Neem products in the fight against arthropod vectors

Currently, a wide number of botanicals have been tested against arthropod

PT

vectors (Lorentz 2013, Benelli et al. 2014). Among them, a first-rank role is covered by neem (Azadirachta indica A. Juss.) (Benelli et al. 2015b). Neem is a tree of Indian

CE

origin belonging to the family of Meliaceae, whose products are gaining increasing

AC

importance in the context of natural insecticides (National Research Council 1992). The insecticidal properties of neem oil, mechanically extracted from the kernels containing the seeds, have been tested and certified by numerous institutions and by a wide scientific literature (Benelli et al. 2017a; see also Boeke et al. 2004).). Neem seeds contain more than 200 bioactive chemicals, even if attention has been mainly focused on limonoids (chemically known as nortriterpenes, e.g. azadirachtins, nimbin, nimbidin and nimbolides) (Nicoletti and Murugan 2013; Senthil-Nathan et al. 2005). The US

10

ACCEPTED MANUSCRIPT

Environmental Protection Agency (EPA) stated the insecticide efficacy and the absence of any environmental toxicity (Office of Pesticide Programs, and Biopesticides Pollution Prevention Diseases-Biopesticides Registration Document-Action Cold Pressed Neem Oil PC Code 025006). Neem is also the only plant-borne biocide

SC RI PT

accepted by the U.E. normative (Directive 2012/15/EU) (Benelli et al. 2017a). Notably, the extract of neem kernels has been used to control a wide array of arthropod pests and vectors (Mulla and Su 1999; Semmler et al. 2010; Nicoletti et al. 2016; Benelli et al. 2017a). Good examples include mosquitoes (Rao et al. 1992, 1995; Su and Mulla 1998a,b, 1999; Senthil-Nathan et al. 2005; Dua et al. 2009; Nicoletti et al.

NU

2010, 2012a,b; Mariani and Nicoletti 2013; Benelli et al. 2014; Benelli et al. 2015a),

MA

sandflies (Sharma and Dhiman 1993; Chandramohan et al. 2016), Ixodes and Rhipicephalus ticks, house dust mites, cockroaches (Blatta, Blattella and

ED

Gromphadorhina), raptor bugs (Triatoma), cat fleas, bed bugs (Schmahl et al. 2010), biting and bloodsucking lice (Al-Quraishy et al. 2011, 2012; Abdel-Ghaffar et al. 2012;

PT

Mehlhorn et al. 2012), Sarcoptes scabiei mites infesting dogs (Abdel-Ghaffar et al. 2008), poultry mites (Abdel-Ghaffar et al. 2009; Locher et al. 2010) and even beetle

CE

larvae feeding on poultry plumage (Walldorf et al. 2012). In addition, it has been also

AC

proved that neem cake, a cheap by-product of neem oil extraction, is also effective against insect pests and vectors (Benelli et al. 2014, 2015b).

4. Effectiveness of neem against Culicoides

The females of Culicoides, after mating in swarms and their blood meal, laid eggs (about 100-200) in muddy areas. The dark brown eggs are elongate and banana-

11

ACCEPTED MANUSCRIPT

shaped. The larvae are vermiform, without legs and prolegs, easy to recognize for eellike movements. The pupae are light brown with a pair of respiratory horns. The development from egg to adult usually takes about 15 -25 days, depending on climatic conditions (Figure 1).

SC RI PT

Larval populations of C. imicola develop mainly in muddy environments that are formed near drinking troughs and/or on artificial lakes edges used as water resources in livestock farms (Figure 2). The water quality in the larval habitats is characterized by a high concentration of organic matter derived from animal droppings. The larvae of C. imicola live in the surface layer of the soil, to a depth of several centimeters. In artificial

NU

ponds, larval foci are found in banks, in the first 50 cm above the water line (Foxi and

MA

Delrio, 2010). The larval stages of C. newsteadi, C. pulicaris and C. punctatus suit also live in drains, low brackish ponds and stream banks, often covered with herbs and rich

ED

in organic matter decomposition. On the other hand, the larvae of C. obsoletus and C. scoticus develop preferably in moist accumulations of leaves, in shaded environments in

PT

wooded areas and in the sheep manure heaps, horse and cattle in the fields (Delrio et al. 2002; Carpenter et al. 2008; Foxi et al. 2010).

CE

We focused on several experiments designed to control Culicoides larvae in

AC

field. In these experiments, a by-product from neem kernel oil extraction was employed, i.e. neem cake. This material is cheap, if compared to neem kernel oil, while retaining much of the chemical constitution (Nicoletti et al. 2010; Nicoletti et al. 2013), and then the toxic properties against targeted vectors (Benelli et al. 2015; Nicoletti et al. 2016). However, the potential usefulness of neem cake still should be still explored and fully validated in the field. In this scenario, the biology of Culicoides larvae offers us a perfect niche where the efficacy of neem cake-based products can be evaluated.

12

ACCEPTED MANUSCRIPT

In October 2008, the Department of Agriculture, University of Sassari, in collaboration with the Department of Environmental Biology, University of Rome "Sapienza" and C.R. ENEA Casaccia, in the frame of a research project funded by the Lazio Region, started aresearch to control Culicoides larvae in an extensive breeding of

SC RI PT

sheep in the province of Sassari (Sardinia, Italy), affected in previous years by outbreaks of bluetongue. In this study, a previous sampling revealed high populations of Culicoides larvae in a pond used for watering the animals. Therefore, a commercial product was employed, the Green Neem Cake (NeemGreen, Virudhunagar, India; i.e. neem cake granules), was tested against the larvae of Culicoides breeding in the open

NU

pond. In vitro tests were performed to establish a scale of efficacy of a series of

MA

fractions obtained throughout extraction of biphasic mixtures with decreasing polarity, extracted into ethyl acetate defatted neem cake (Nicoletti et al. 2010, 2012a,b). The field assays were carried out treating the shores of an artificial lake with

ED

Green Neem cake at 100 g/m2. A randomized complete block design with four

PT

replications of the treated and untreated plots was used (Foxi and Delrio 2013). The individual plots of an area of 1 m2 were contiguous to the shoreline for the length of 2 m

CE

and 50 cm wide. For the estimation of the population of Culicoides, from each single

AC

plot a mud sample of about 800 cm3 was collected. Each mud sample was scraped from the soil surface at 20 cm above shoreline using a flat trowel and maintained in laboratory for 30 days for retrieval of emerging biting midges. The samples were taken the week before the treatment, and then on a weekly basis for a month (Foxi and Delrio 2013). Results showed that the neem cake achieved a significant effect on the development of the Culicoides larvae (Table 1), with particular reference to C. imicola

13

ACCEPTED MANUSCRIPT

(Table 2), in agreement with previous studies that reported the neem oil ability to inhibit the oviposition of Culicoides, and act as ovicidal and larvicidal agent, along with a marked repellent activity (Blackwell et al. 2004; Narladkar et al. 2006). In the tests conducted, the commercial neem cake formulation showed a higher larvicidal

SC RI PT

effectiveness, if compared to the individual fractions. Notably, Foxi and Delrio (2013) showed that a single treatment with neem cake showed a considerable residual effect for about a month. The Green Neem Cake compared in the laboratory with other natural products such as commercial OIKOS 25 plus (azadirachtin A + B, 25%) showed superior efficacy (Foxi and Delrio 2013).

NU

Furthermore, besides larvicidal treatment against Culicoides young instars, it

MA

should be also pointed out that the neem oil has been also reported for its promising repellent and antifeedant activity against Culicoides adults, relying to three

ED

complementary methods with serial dilutions (Blackwell et al. 2004). Indeed, electroantennograms revealed the sensitivity of the females of Culicoides nubeculosus

PT

(Meigen) to neem oil ≥ 0.10%. Moreover, the females of Culicoides impunctatus Goetghebuer can be effectively repelled by ≥ 1% of neem oil in Y-tube olfactometer

CE

assays. Lastly, using a membrane feeder for wild-caught parous females of C.

AC

impunctatus, the proportion blood-feeding was significantly reduced by topical applications of neem oil ≥ 0.10% concentrations, with blood-feeding completely prevented by ≥ 1% (Blackwell et al. 2004). Based on these findings, Blackwell et al. (2004) proposed a neem-based formulation with 2% of neem oil for personal protection from Culicoides biting activity (see also Cole et al. 2002). However, personal protection tools have limited efficacy and high costs for prevention of bluetongue on livestock,

14

ACCEPTED MANUSCRIPT

where the treatment of young instar breeding sites remains the most appropriate control strategy in rural areas.

SC RI PT

5. Conclusions and challenges for future research

Overall, the neem oil and derived extraction products are already used in other countries in integrated control programs of insect pests and vectors (Su and Mulla 1999; Cole et al. 2002; Blackwell et al. 2004; Nicoletti et al. 2012; Foxi and Delrio 2013; Sujarwo et al. 2016; Benelli et al. 2015, 2017a). This article reviewed current control

NU

tools for the management of Culicoides larval and adult populations. Besides

MA

appropriate monitoring programs, as well as the use of repellents and antifeedant products against adults, we highlighted the concrete possibility of using neem cake for

ED

control of Culicoides young instar populations in wet environments close to livestock breeding sites. After the industrial process for the extraction of neem kernel oil, this by-

PT

product still contains relevant quantities of active metabolites (Nicoletti et al. 2012b, 2016). The above-discussed evidence of efficacy against Culicoides in the field

CE

represents the first attempt to use neem for larval control of the bluetongue disease

AC

vectors. Notably, this product is cheap and shows a considerable residual effect for a long period of time, about a month (Foxi and Delrio 2013). Furthermore, a better knowledge of Culicoides larval breeding sites is strategic

to establish an effective control of the vector species. Interventions to control Culicoides larvae integrated with adult insecticide treatments and use of eco-friendly repellents on livestock (Cole et al. 2002; Blackwell et al. 2004; Pavela et al. 2016) could provide a help to reduce the probability of transmission of bluetongue virus.

15

ACCEPTED MANUSCRIPT

The neem-borne products can also be used against adults Culicoides for systemic disinfection of animal housing and surrounding environments and as repellents with repeated treatments of the animals. Numerous studies report the multiple action of the constituents of the neem in the control of pests and vectors, according to multiple

SC RI PT

mechanisms of action (Del Serrone et al. 2015): interference with post-embryonic development by simulating the action of juvenoid substances that inhibits the metamorphosis and blocks the synthesis of ecdysone, phago-repellent action, and reduction of female oviposition rates and egg fertility (Semmler et al. 2010; Benelli et al. 2014, 2017a).

NU

Moreover, assuming a possible absorption through the skin, at the Research

MA

Centre for Animal Production of the Council for Research in Agriculture and Agricultural Economy Analysis tests have been conducted to evaluate the effect of

ED

neem on peripheral blood mononuclear cells of goats treated with neem and no adverse effects have been reported (De Matteis et al. 2015).

PT

For its highly effective and environmental care properties, neem kernel oil and neem cake have been ranked among the most interesting active substances for use in

CE

organic farming, with special reference to the control of insect pests and vectors

AC

(Blackwell et al. 2004; Benelli et al. 2017a). The neem is already used in other countries in IPM programs for the control of crop pests and management of livestock, as indicated by EPA in USA (EPA 2012). In Italy, although increasing, the use of neem is still limited and scarcely known, in comparison with the still large-scale employing of traditional pesticides. Based on the findings analyzed in this review, we believe that the employ of neem extraction by-products as aqueous formulation in muddy sites close to livestock grazing areas may represent a promising tool in the fight against the spread of

16

ACCEPTED MANUSCRIPT

bluetongue virus in the Mediterranean areas (Foxi and Delrio 2013). Therefore, we hope that the knowledge summarized here would boost research on eco-friendly control of Culicoides vectors, a One Health perspective integrating basic information on vector biology and ecology, phytochemistry, natural product research, and the employ of neem

SC RI PT

cake in the IPM framework, due to its high efficacy and multiple mechanisms of action.

Acknowledgements

The authors are grateful to Dr. Paolo Pasquali, Dr. Krisztian Magori and the

MA

NU

anonymous reviewers for improving an earlier version of our manuscript.

ED

Conflict of Interest

AC

CE

PT

The Authors declare no competing interests.

17

ACCEPTED MANUSCRIPT

References

Abdel-Ghaffar F, Al-Quraishy S, Sobhy H, Semmler M (2008) Neem seed extract shampoo, Wash Away Louse®, an effective plant agent against Sarcoptes scabiei

SC RI PT

mite infesting dogs in Egypt. Parasitol Res 104:145–148 Abdel-Ghaffar F, Semmler M, Al-Rasheid KAS, Mehlhorn H (2009) In vitro efficacy of ByeMite® and Mite-Stop® on developmental stages of the red chicken mite Dermanyssus gallinae. Parasitol Res 105:469–471

Abdel-Ghaffar F, Al-Quraishy S, Al-Rasheid KAS, Mehlhorn H (2012) Efficacy of a

NU

single treatment of head lice with a neem seed extract: an in vivo and in vitro study

MA

on nits and motile stages. Parasitol Res 110:277–280 Al-Quraishy S, Abdel-Ghaffar F, Al-Rasheid KAS, Mehlhorn J, Mehlhorn H (2011)

ED

Effects of a neem seed extract (MiteStop®) on mallophages (featherlings) of chicken: in-vivo and in-vitro studies. Parasitol Res 110:617–622

PT

Al-Quraishy S, Abdel-Ghaffar F, Al-Rasheid KAS, Mehlhorn J, Mehlhorn H (2012) Observations on effects of a neem seed extract (MiteStop®) on biting lice

AC

339.

CE

(mallophages) and bloodsucking insects parasitizing horses. Parasitol Res 110:335–

Baker T, Carpenter S, Gubbins S, Newton R, Lo Iacono G, Wood J, Harrup LE (2015) Can insecticide-treated netting provide protection for equids from Culicoides biting midges in the United Kingdom? Parasit Vect 8(1):1–17 Bauer B, Jandowsky A, Schein E, Mehlitz D, Clausen P-H (2009) An appraisal of current and new techniques intended to protect bulls against Culicoides and other

18

ACCEPTED MANUSCRIPT

haematophagous nematocera: the case of Schmergow, Brandenburg, Germany. Parasitol Res 105(2):359–365 Baylis M, Parkin H, Kreppel K, Carpenter S, Mellor P, McIntyre K (2010) Evaluation of housing as a means to protect cattle from Culicoides biting midges, the vectors

SC RI PT

of bluetongue virus. Med Vet Entomol 24:38–45 Benelli G, Conti B, Garreffa R, Nicoletti M (2014) Shedding light on bioactivity of botanical by-products: neem cake compounds deter oviposition of the arbovirus vector Aedes albopictus (Diptera: Culicidae) in the field. Parasitol Res 113:933– 940.

MA

future. Parasitol Res 114:2801–2805.

NU

Benelli G (2015a) Research in mosquito control: current challenges for a brighter

Benelli G (2015b) Plant-borne ovicides in the fight against mosquito vectors of medical

ED

and veterinary importance: a systematic review. Parasitol Res 114:3201–3212 Benelli G, Mehlhorn H (2016) Declining malaria, rising dengue and Zika virus: insights

PT

for mosquito vector control. Parasitol Res 115:1747-54 Benelli G, Bedini S, Cosci F, Toniolo C, Conti B, Nicoletti M (2015a) Larvicidal and

CE

ovideterrent properties of neem oil and fractions against the filariasis vector Aedes

AC

albopictus (Diptera: Culicidae): a bioactivity survey across production sites. Parasitol Res 114:227–236 Benelli G, Murugan K, Panneerselvam C, Madhiyazhagan P, Conti B, Nicoletti M. (2015b) Old ingredients for a new recipe? Neem cake, a low-cost botanical byproduct in the fight against mosquito-borne diseases. Parasitol Res 114:391–397 Benelli G, Lo Iacono A, Canale A, Mehlhorn H (2016a) Mosquito vectors and the spread of cancer: an overlooked connection? Parasitol Res 115:2131-2137

19

ACCEPTED MANUSCRIPT

Benelli G, Pavela R, Canale A, Mehlhorn H (2016b) Tick repellents and acaricides of botanical origin: a green roadmap to control tick-borne diseases? Parasitol 115:2545–2560 Benelli G, Canale A, Toniolo C, Higuchi A, Murugan K, Pavela R, Nicoletti M (2017a)

SC RI PT

Neem (Azadirachta indica): towards the ideal insecticide? Nat Prod Res 31:369386

Benelli G, Chandramohan B, Murugan K, Madhiyazhagan P, Kovendan K, Panneerselvam C, Dinesh D, Govindarajan M, Higuchi A, Toniolo C, Canale A, Nicoletti M (2017b) Neem cake as a promising larvicide and adulticide against the

NU

rural malaria vector Anopheles culicifacies (Diptera: Culicidae): a HPTLC

MA

fingerprinting approach. Nat Prod Res 31:1185-1190. Bethan VP, Philip SM, David JR, Alan RS, Peter PCM, Matthew B (2005) Climate

ED

change and the recent emergence of bluetongue in Europe. Nature Reviews Microbiology 3, 171-181.

PT

Bhasin A, Mordue W (2000) Electrophysiological and behavioural identification of host kairomones as olfactory cues for Culicoides impunctatus and C. nubeculosus.

CE

Physiol Entomol 25:6-16

AC

Blackwell A, Evans KA, Strang RHC, Cole M (2004) Toward development of neem‐ based repellents against the Scottish Highland biting midge Culicoides impunctatus. Med Vet Entomol 18:449-452 Boeke SJ, Boersma MG, Alink GM, van Loon JJA, van Huis A, Dicke M, Rietjens IMCM (2004) Safety evaluation of neem (Azadirachta indica) derived pesticides. J Ethnopharmacol 94:25–41

20

ACCEPTED MANUSCRIPT

Braverman Y, Chizov-Ginzburg A (1997) Repellency of synthetic and plant-derived preparations for Culicoides imicola. Med Vet Entomol 11(4):355–360 Braverman Y, Wegis M, Mullens B (2000) Response of Culicoides sonorensis (Diptera: Ceratopogonidae) to 1-octen-3-ol and three plant-derived repellent formulation in

SC RI PT

the field. J Am Mosq Control Assoc 16(2):158–163 Braverman Y, Chizov-Ginzburg A, Pener H, Wilamowski A (2004) Susceptibility and repellency of Culicoides imicola and Culex pipiens to lambda-cyhalothrin. Vet Ital 40(3):336–339

Calvete C, Estrada R, Miranda MA, Del Rio R, Borrás D, Beldron FJ, Martínez A,

NU

Calvo AJ, Lucientes J (2010) Protection of livestock against bluetongue virus

Entomol 24(2):169–175.

MA

vector Culicoides imicola using insecticide-treated netting in open areas. Med Vet

ED

Carpenter S, Mellor PS, Torr SJ (2008) Control techniques for Culicoides biting midges and their application in the U.K. and northwestern Palaearctic. Med Vet Entomol

PT

22:175-187

Carpenter S, Groschup MH, Garros C, Felippe-Bauer ML, Purse BV (2013) Culicoides

CE

biting midges, arboviruses and public health in Europe. Antiviral Res 100:102-113

AC

Chandramohan B, Murugan K, Panneerselvam C, Madhiyazhagan P, Chandirasekar R, Dinesh D, Mahesh Kumar P, Kovendan K, Suresh U, Subramaniam J, Rajaganesh R, Aziz AT, Syuhei B, Saleh Alsalhi M, Devanesan S, Nicoletti M, Wei H, Benelli G (2016) Characterization and mosquitocidal potential of neem cake-synthesized silver nanoparticles: genotoxicity and impact on predation efficiency of mosquito natural enemies. Parasitol Res 115(3):1015-1025

21

ACCEPTED MANUSCRIPT

Cole M, Blackwell A, Evans A, Strang RHC (2002) The repellent action of neem oil (Azadirachta indica) against the Scottish biting midge (Culicoides impunctatus). Focus Altern Complem Ther 7:90-91 Dantas-Torres F, Chomel BB, Otranto D (2012) Ticks and tick-borne diseases: a One

SC RI PT

Health perspective. Tr Parasitol 28(10):437-446 Day MJ (2011) One health: the importance of companion animal vector-borne diseases. Parasit Vect 4(1):49

De Keyser R, Cassidy C, Laban S, Gopal P, Pickett JA, Reddy YK et al. (2017) Insecticidal effects of deltamethrin in laboratory and field populations of Culicoides

NU

species: how effective are host-contact reduction methods in India? Parasit & Vect

MA

10(1):54

De Matteis G, Rufrano D, Claps S, Veneziano V, Di Sotto A, Nicoletti M, Del Serrone

ED

P (2015) Assessment of Neem oil effect on hematological profile and towards peripheral blood mononuclear cells of goat. Adv Appl Sci Res 6:46-54,

PT

doi:10.6088/ijaser

Del Río R, Barceló C, Lucientes J, Miranda MA (2014a) Detrimental effect of

CE

cypermethrin treated nets on Culicoides populations (Diptera; Ceratopogonidae)

AC

and non-targeted fauna in livestock farms. Vet Parasitol 199:230–234. Del Río R, Barcelo C, Paredes-Esquivel C, Lucientes J, Miranda MA (2014b) Susceptibility of Culicoides species biting midges to deltamethrin-treated nets as determined under laboratory and field conditions in the Balearic Islands, Spain. Med Vet Entomol 28:414–420

22

ACCEPTED MANUSCRIPT

Delrio G, Deliperi S, Foxi C, Pantaleoni RA, Piras S (2002) Osservazioni in Sardegna sulla dinamica di popolazione di Culicoides imicola Kieffer vettore della Bluetongue. Atti XIX Congresso Nazionale Italiano di Entomologia pp. 1089-1094. Del Serrone P, De Matteis G, Rufrano D, Veneziano V, Claps S, Nicoletti M (2015)

SC RI PT

Goat Wound-Contaminating-Batteria Restraint by Neem Oil. SOJ Microbiol Infect Dis 3(2):1-10

Dua VK, Pandey AC, Raghavendra K, Gupta A, Sharma T, Dash A (2009) Larvicidal activity of neem oil (Azadirachta indica) formulation against mosquitoes. Malar J 8:124

NU

Ducheyne E., De Deken R., Becu S., Codina B., Nomikou K., Mangana-Vougiaki O.,

MA

Georgiev G., Purse B.V. & Hendrickx G. (2007). Quantifying the wind dispersal of Culicoides species in Greece and Bulgaria. Geospat. Hlth 2:177–189

ED

Elbers AR, Backx A, Meroc E, Gerbier G, Staubach C, Hendrickx G, van der Spek A, Mintiens K (2008) Field observations during the bluetongue serotype 8 epidemic in

PT

2006. I. Detection of first outbreaks and clinical signs in sheep and cattle in Belgium, France and the Netherlands. Prev Vet Med 87:21-30. doi: 10.1016/j

CE

Elbers ARW, Koernaadt CJM, Meiswintel R (2015) Mosquitoes and Culicoides biting midges: vector range and the influence of climate changes. Rev Sci Tech Off Ind

AC

Epiz 34(1): 123-137. EPA (US Environmental Protection Agency). (2012). Biopesticide Registration Action Document. Office of Pesticide Programs. Cold Pressed Neem Oil. PC Code 025006. Foxi C, Delrio G (2010) Larval habitats and seasonal abundance of Culicoides biting midges found in association with sheep in northern Sardinia, Italy. Med Vet Entomol 24:199-209.

23

ACCEPTED MANUSCRIPT

Foxi C, Delrio G (2013) Efficacy of a neem cake for the control of Culicoides biting midges larvae. Pharmacologyonline 3:110 – 114 Foxi C, Delrio G, Falchi G, Marche MG, Satta G, Ruiu L (2016) Role of different Culicoides vectors (Diptera: Ceratopogonidae) in bluetongue virus transmission

SC RI PT

and overwintering in Sardinia (Italy). Parasite Vector 9:440, doi: 10.1186/s13071016-1733-9

Franco AO, Gomes MGM, Rowland M, Coleman PG, Davies CR (2014) Controlling malaria using livestock-based interventions: a One Health approach. PloS One, 9(7):e101699.

NU

Gerry AC, Monteys, VSI, Vidal JOM, Francino O, Mullens BA (2009) Biting rates of

MA

Culicoides midges (Diptera: Ceratopogonidae) on sheep in northeastern Spain in relation to midge capture using UV light and carbon dioxide-baited traps. J Med

ED

Entomol 46:615-624

González M, Venter GJ, López S, Iturrondobeitia JC, Goldarazena A (2014) Laboratory

PT

and field evaluations of chemical and plant‐derived potential repellents against Culicoides biting midges in northern Spain. Med Vet Entomol 28:421-431

CE

Gloster J, Burgin L, Witham C, Athanassiadou M, Mellor PS (2008) Bluetongue in the

AC

United Kingdom and northern Europe in 2007 and key issues for 2008. Vet Rec 162: 298-302.

Goffredo M, Meiswinkel R (2004) Entomological surveillance of bluetongue in Italy: methods of capture, catch analysis and identification of Culicoides biting midges. Vet Ital 40:260-265

24

ACCEPTED MANUSCRIPT

Goffredo M, Catalani M, Federici V, Portanti O, Marini V, Mancini G, Quaglia M, Santilli A, Teodori L, Savini G (2015) Vector species of Culicoides midges implicated in the 2012-2014 Bluetongue epidemics in Italy. Vet Ital 51:131-8 Hemingway J, Ranson H (2000) Insecticide resistance in insect vectors of human

SC RI PT

disease. Annu Rev Entomol 45:371-391 Hendry G, Godwin G (1988) Biting midges in Scottish forestry: a costly irritant or a trivial nuisance? Scottish For 42:113-119

Isman MB (2006) Botanical insecticides, deterrents and repellents in modern agriculture and an increasingly regulated world. Annu Rev Entomol 51:45–66.

NU

IZSA&M (Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise). 2016a.

MA

Bollettino Epidemiologico Nazionale Veterinario, n. 23 Gennaio 2016. http://www.izs.it/BENV_NEW/Engine/RAServePG.php/P/299910040605/T/NumeriPrecedenti.

ED

IZSA&M (Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise). 2016b.

PT

Bollettino Epidemiologico Nazionale Veterinario, n. 24 Aprile 2016. http://www.izs.it/BENV_NEW/Engine/RAServePG.php/P/299910040605/T/Numer

CE

i-Precedenti.

AC

Locher N, Al-Rasheid KAS, Abdel-Ghaffar F, Mehlhorn H (2010) In vitro and field studies on the contact and fumigant toxicity of a neem-product (Mite-Stop®) against the developmental stages of the poultry red mite Dermanyssus gallinae. Parasitol Res 107:417–423 Lorentz ML, Mara MF, Moore SJ (2013) The use of Pants in Vector Control. In Biological and Environmental Control of Disease Vectors. Cameroon MH, Lorentz LM (Ed). Cabi Int., Oxfordshire, UK.

25

ACCEPTED MANUSCRIPT

Lühken R, Kiel E, Steinke S (2014) Impact of mechanical disturbance on the emergence of Culicoides from cowpats. Parasitol Res 113:1283–1287 Lühken R, Kiel E, Steinke S, Fladung R (2015) Topsoil conditions correlate with the emergence rates of Culicoides chiopterus and Culicoides dewulfi (Diptera:

SC RI PT

Ceratopogonidae) from cowpats. Parasitol Res 114:1113-1117 Mands V, Kline DL, Blackwell A (2004) Culicoides midge trap enhancement with animal odour baits in Scotland. Med Vet Entomol 18:336-342

Mariani S, Nicoletti M (2013) Antilarval activity of neem cake extracts against Aedes albopictus. Pharmacologyonline 3:137–140

NU

Martínez-de la Puente J, Merino S, Lobato E, Rivero-de Aguilar J, del Cerro S, Ruiz-de-

MA

Castañeda R (2009) Testing the use of a citronella-based repellent as an effective method to reduce the prevalence and abundance of biting flies in avian nests.

ED

Parasitol Res 104:1233–1236

Mehlhorn H (ed) (2012) Arthropods as vectors of emerging diseases. Parasitol Res

PT

Monographs Vol 3, Springer, Heidelberg Mehlhorn H, Walldorf V, Abdel-Ghaffar F, Al-Quraishy S, Al-Rasheid KAS, Mehlhorn

CE

J (2012) Biting and bloodsucking lice of dogs—treatment by means of a neem seed

AC

extract (MiteStop®, Wash Away Dog). Parasitol Res 110:769–773 Mordue AJ, Dallas JF, Nolan DV, Logan JG (2007) Culicoides midge as vector of emerging diseases in Emerging Pests and Vector-borne Disease in Europe. p. 306325. Tallen W and Knols BGJ (Ed). Academic Publisher. Wageningen. Mulla MS, Su T (1999) Activity and biological effects of neem products against arthropods of medical and veterinary importance. J Am Mosq Control Assoc 15:133–152

26

ACCEPTED MANUSCRIPT

Murchie AK, Clawson S, Rea I, Forsythe IWN, Gordon AW, Jess S (2016) DEET (N, N-diethyl-meta-toluamide)/PMD (para-menthane-3, 8-diol) repellent-treated mesh increases Culicoides catches in light traps. Parasitol Res 115:3543-3549 Naqqash MN, Gökçe A, Bakhsh A, Salim M (2016) Insecticide resistance and its

SC RI PT

molecular basis in urban insect pests. Parasitol Res 115:1363-1373 Narladkar BW, Deshpande PD, Vaniprasad V, Shivpuje PR, Deshpande AR (2006) Integrated management of Culicoides sp. of domesticated animals. J Vet Parasitol 20:125-128

National Research Council (1992) Neem: a tree for solving global problems. In: Report

NU

of an ad hoc panel of the Board on Science and Technology for International

MA

Development. National Academy Press, Washington Nicoletti M, Murugan K (2013) Neem the tree of XXI century. PharmacologyOnline 3:115–121

ED

Nicoletti M, Serafini M, Aliboni A, D’Andrea A, Mariani S (2010) Toxic effects of

PT

neem cake extracts on Aedes albopictus larvae. Parasitol Res 107:89–84. Nicoletti M, Mariani S, Maccioni O, Coccioletti T, Murugan K (2012a) Neem cake:

CE

chemical composition and larvicidal activity on Asian tiger mosquito. Parasitol Res

AC

111:205–2013

Nicoletti M, Maccioni O, Coccioletti T, Mariani S, Vitali F (2012b) Neem tree (Azadiractha indica A. Juss) as source of bioinsecticides. In: Perveen (Ed.) Insecticides—advances in integrated pest manage ment, InTech, Rijeka, Croatia, pp. 411–428

27

ACCEPTED MANUSCRIPT

Nicoletti M, Toniolo C, Gallo FR, Multari G, Palazzino G (2013) Traceability in multiingredient botanicals by HPTLC fingerprint approach. J Plan Chromatogr 26:243– 247. Nicoletti M, Murugan K, Del Serrone (2014) Current mosquito-borne disease

SC RI PT

emergencies in Italy and climate changes. The Neem opportunity. Trends Vector Res Parasitol 1:2

Nicoletti M, Murugan, K, Canale A, Benelli G (2016) Neem-borne molecules as ecofriendly control tools against mosquito vectors of economic importance. Curr Org Chem 20:2681-2689

NU

Nielsen SA, Nielsen BO, Chirico J (2010) Monitoring of biting midges (Diptera:

MA

Ceratopogonidae: Culicoides Latreille) on farms in Sweden during the emergence of the 2008 epidemic of bluetongue. Parasitol Res 106:1197–1203

ED

Page PC, Labuschagne, K, Nurton JP, Venter GJ, Guthrie AJ (2009) Duration of repellency of N, N-diethyl-3-methylbenzamide, citronella oil and cypermethrin

PT

against Culicoides species when applied to polyester mesh. Vet Parasitol 163:105109

CE

Page PC, Labuschagne K, Venter GJ, Schoeman JP, Guthrie AJ (2014) Field and in

AC

vitro insecticidal efficacy of alphacypermethrin-treated high density polyethylene mesh against Culicoides biting midges in South Africa. Vet Parasitol 203:184–188 Pavela R (2016) History, presence and perspective of using plant extracts as commercial botanical insecticides and farm products for protection against insects–a review. Plant Prot Sci 52:229-241

28

ACCEPTED MANUSCRIPT

Pavela R, Benelli G (2016a) Ethnobotanical knowledge on botanical repellents employed in the African region against mosquito vectors - a review. Exp Parasitol 167:103–108 Pavela R, Benelli G (2016b) Essential oils as eco-friendly biopesticides? Challenges

SC RI PT

and constraints. Tr Plant Sci 21:1000-1007 Pavela R, Canale, A, Mehlhorn H, Benelli G (2016) Application of ethnobotanical repellents and acaricides in prevention, control and management of livestock ticks: a review. Res Vet Sci 109:1-9

Perrin A, Albina E, Bréard E, Sailleau C, Promé S, et al. (2007) Recombinant

NU

capripoxviruses expressing proteins of bluetongue virus: evaluation of immune

MA

responses and protection in small ruminants. Vaccine 25:6774-6783 Rao DR, Reuben R, Venugopal MS, Nagasampagi BA, Schmutterer H (1992)

ED

Evaluation of neem, Azadirachta indica, with and without water management, for the control of culicine mosquito larvae in rice-fields. Med Vet Entomol 6:318–324

PT

Rao DR, Reuben R, Nagasampagi BA (1995) Development of combined use of neem (Azadirachta indica) and water management for the control of Culicinae mosquito

CE

in rice fields. Med Vet Entomol 9:25–33

AC

Rasmussen LD, Kristensen B, Kirkeby C et al. (2012) Culicoids as vectors of Schmallenberg virus. Emerg Infect Dis 18:1204–1205 Schmahl G, Walldorf V, Klimpel S, Al-Quraishy S, Mehlhorn H (2008) Efficacy of Oxyfly™ on Culicoides species—the vectors of Bluetongue virus—and other insects. Parasitol Res 103(5):1101–1103

29

ACCEPTED MANUSCRIPT

Schmahl G, Al-Rasheid KAS, Abdel-Ghaffar F, Klimpel S, Mehlhorn H (2010) The efficacy of neem seed extracts (Tresan®, MiteStop®) on a broad spectrum of pests and parasites. Parasitol Res 107:261–269 Schwartz-Cornil I, Mertens PPC, Contreras V, Hemati B, Pascale F, Bréard E, Mellor

SC RI PT

PS, MacLachlan NJ, Zientara S (2008) Bluetongue virus: virology, pathogenesis and immunity. Vet Res 39:46. doi: 10.1051/vetres:2008023

Semmler M, Abdel-Ghaffar F, Al-Rasheid KAS, Mehlhorn H (2010) Nature helps: from research to products against blood sucking parasites. Parasitol Res 105:1483–1487 Semmler M, Abdel-Ghaffar F, Schmidt J, Mehlhorn H (2014) Evaluation of biological

NU

and chemical insect repellents and their potential adverse effects. Parasitol Res

MA

113:185–188

Sharma VP, Dhiman RC (1993) Neem oil as a sand fly (Diptera: Psychodidae)

ED

repellent. J Am Mosq Control Assoc 9:364–366 Senthil Nathan S, Kalaivani K, Murugan K (2005) Effects of neem limonoids on the

PT

malaria vector Anopheles stephensi Liston (Diptera: Culicidae). Acta Trop 96:47– 55

CE

Stuart AE, Brooks CJW, Prescott RJ, Blackwell A (2000) Repellent and antifeedant

AC

activity of salicylic acid and related compounds against the biting midge, Culicoides impunctatus (Diptera: Ceratopogonidae). J Med Entomol 37:222-227 Su T, Mulla MS (1998a) Antifeedancy of neem products containing Azadirachtin against Culex tarsalis and Culex quinquefasciatus (Diptera: Culicidae). J Vector Ecol 23:114–122

30

ACCEPTED MANUSCRIPT

Su T, Mulla MS (1998b) Ovicidal activity of neem products (azadirachtin) against Culex tarsalis and Culex quinquefasciatus (Diptera; Culicidae). J Am Mosq Control Assoc 14:204–209 Su T, Mulla MS (1999) Oviposition bioassay responses of Culex tarsalis and Culex

91:337–345

SC RI PT

quinquefasciatus to neem products containing azadirachtin. Entomol Exp Appl

Sujarwo W, Keim AP, Caneva G, Toniolo C, Nicoletti M (2016). Ethnobotanical uses of neem (Azadirachta indica A. Juss.; Meliaceae) leaves in Bali (Indonesia) and the Indian subcontinent in relation with historical background and phytochemical

NU

properties. J Ethnopharmacol 189:186-193

MA

Thompson GM, Jess S, Gordon AW, Murchie AK (2014) Sticky-trapping biting midges (Culicoides spp.) alighting on cattle and sheep: effects of trap colour and evidence

ED

for host preference. Parasitol Res 113:3085–3094 Trigg JK (1996) Evaluation of a eucalyptus-based repellent against Culicoides

12(2):329–330

PT

impunctatus (Diptera: Ceratopogonidae) in Scotland. J Am Mosq Control Assoc

CE

Venter GJ, Labuschagne K, Boikanyo SNB, Morey L, Snyman MG (2011) The

AC

repellent effect of organic fatty acids on Culicoides midges as determined with suction light traps in South Africa. Vet Parasitol 181(2–4):365–369 Venter GJ, Labuschagne K, Boikanyo SN, Morey L (2014) Assessment of the repellent effect of citronella and lemon eucalyptus oil against South African Culicoides species. J South Afric Vet Assoc 85:01-05

31

ACCEPTED MANUSCRIPT

Walldorf V, Mehlhorn H, Al-Quraishy S, Al-Rasheid KAS, Abdel-Ghaffar F, Mehlhorn J (2012) Treatment with a neem seed extract (MiteStop®) of beetle larvae parasitizing the plumage of poultry. Parasitol Res 110:623–627 Webster JP, Gower CM, Knowles SC, Molyneux DH, Fenton A (2016) One Health–an

SC RI PT

ecological and evolutionary framework for tackling Neglected Zoonotic Diseases. Evol Appl 9(2):313-333

Wilson A J, Mellor PS (2009) Bluetongue in Europe: past, present and future. Phil Trans R Soc B 364: 2669–2681. doi: 10.1098/rstb.2009.0091

Zimmer J-Y, Brostaux Y, Haubruge E, Francis F (2014) Larval development sites of the

NU

main Culicoides species (Diptera: Ceratopogonidae) in northern Europe and

MA

distribution of coprophilic species larvae in Belgian pastures. Vet Parasitol

AC

CE

PT

ED

205:676–686

32

ACCEPTED MANUSCRIPT

Figure 1. Life cycle of Culicoides imicola Kieffer (Diptera: Ceratopogonidae), a vector of the bluetongue virus and the African horse sickness virus.

SC RI PT

Female

I instar larva emerging from an egg

MA

NU

Pupa

Egg

AC

CE

PT

ED

IV instar larva

33

ACCEPTED MANUSCRIPT

Figure 2. Main larval habitats of Culicoides imicola in Sardinia (Italy): muddy environments formed in proximity of livestock drinking troughs (a), as well as on artificial lakes edges (b)

(a)

AC

CE

PT

ED

MA

NU

SC RI PT

representing water resources for livestock farms.

(b)

34

ACCEPTED MANUSCRIPT

Table 1. Mean number (±SE) of Culicoides adults emerging from mud samples taken weekly in control and treated plots in Sassari (Sardinia, Italy) during October 2008. Within a column, means followed by the same letter are not significantly different

SC RI PT

(one-way repeated measures ANOVA, P=0.05) (Foxi and Delrio 2013).

Post-treatment

Treatment Pre-treatment 7 days Control

27.25±9.51 a

21 days

28 days

44.13±5.81 a 30.58±8.42 a 23.92±4.20 a 22.42±1.85 a 3.88±1.69 b

4.00±1.47 b

3.75±1.16 b

9.25±1.86 b

AC

CE

PT

ED

MA

NU

Neem cake 17.63±3.56 a

14 days

35

ACCEPTED MANUSCRIPT

Table 2. Mean number (±SE) of C. cataneii, C. circumscriptus, C. festivipennis and C. imicola emerging during 4 weeks from mud samples in control and treated plots in Sassari (Sardinia, Italy) during October 2008. Within a column, means followed by the same letter are not significantly different (one-way repeated measures ANOVA,

C. cataneii

SC RI PT

P=0.05) (Foxi and Delrio 2013).

C. circumscriptus C. festivipennis

52.71±12.45 a

21.29±3.01 a

32.38±8.95 a

12.29±2.23 a

Neem cake

4.63±1.55 b

4.08±1.82 b

6.88±2.89 b

1.75±0.66 b

F1,6

28.27

16.58

6.52

36.59

P

0.0018

0.0066

0.0433

0.0009

AC

CE

PT

ED

MA

NU

Control

C. imicola

36

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC RI PT

Graphical abstract

37

ACCEPTED MANUSCRIPT

Highlights

Bluetongue virus affects ruminants and is spread through the bites of Culicoides vectors



Effective vector control is crucial for prevention of bluetongue outbreaks



Culicoides larvae develop in muddy areas, their control is really challenging



Aqueous neem cake formulations in muddy sites represents a key tool to control

SC RI PT



bluetongue vectors

CE

PT

ED

MA

NU

In the field, residual efficacy of a single treatment with neem cake was >30 days

AC



38