Sensibilité olfactive à l’octénol, aux phénols et à l’urine de cheval

Résumé

Suite aux résultats obtenus lors de l’étude précédente, nous avons souhaité approfondir nos investigations sur l’attractivité des urines en focalisant notre attention sur l’urine de cheval, la plus attractive, et ses composés volatiles. Compte-tenu des contraintes imposées par l’expérimentation de terrain, les tests se sont limités à l’urine et à trois de ses composants : l’octénol, le 4-méthylphénol (4MP) et le 3-n-propylphénol (3PP), seuls ou combinés.

Tout d’abord, nous avons analysé la composition de l’urine de cheval, préalablement vieillie pendant trois semaines. Les composés volatils de l’urine de cheval ont été captés par microextraction sur phase solide à l’aide de fibres SPME (Solide-Phase MicroExtraction). L’analyse de ces composés a été faite par chromatographie en phase gazeuse et spectrométrie de masse. 29 composés ont ainsi été identifiés notamment des cétones (11), des alcools gras (6) et des phénols (5). L’octénol et le 3PP ont été détectés en petite quantité (<2% du mélange) tandis que le 4MP était le volatil le plus abondant (79%). La concentration en 4MP dans l’urine de cheval a ensuite été dosée par étalonnage externe grâce à une solution tampon à différentes concentrations. Elle était de 0,42 mg/L.

Les enregistrements EAG ont été réalisés dans les mêmes conditions que dans l’article 4 à l’exception de l’hexane utilisé à la place du dichlorométhane pour la dilution des différents composés et comme témoin négatif. Pour T. bromius et A. quadrifarius, les réponses antennaires ont été doses-dépendantes avec l’octénol, le 4MP, le 3PP et une combinaison 4MP:3PP (16:1) (Figure 21). Le 4MP provoque des réponses EAG significativement très supérieures à celles obtenues avec l’octénol ou le 3PP. Les phénols en mélange donnent des réponses EAG significativement supérieures à celles déclenchées par le 4MP seul surtout aux faibles concentrations.

Article 5 : Olfactory responses of tabanids to octenol, phenols and aged horse urine F. Baldacchino, S. Manon, L. Puech, B. Buatois, L.Dormont & P. Jay-Robert.

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Figure 21. Réponses EAG de (A) T. bromius et (B) A. quadrifarius

Les antennes ont été stimulées par des doses croissantes de 1-octen-3-ol (OCT), 3-propylphénol (3PP), 4-methylphénol (4MP) et un mélange de phénols (4MP:3PP, 16:1). L’octénol 1 mg/µL est utilisé comme valeur de référence (100%) pour la normalisation des réponses EAG. Les différences significatives entre deux doses successives sont notées avec des lettres différentes (test de rang de Wilcoxon, P<0,05).

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La réponse à un mélange de composés n’est pas une fonction simple liée aux réponses des composés testés séparément (Laing &Francis, 1989). L’interaction entre ces composés peut se faire au niveau des récepteurs périphériques ou bien au niveau du lobe antennaire (Carlsson & Hansson, 2002). Seuls des enregistrements électrophysiologiques sur la sensille (SCR-EAG, Single Cell Recording EAG) permettent de démontrer des interactions au niveau de l’antenne. D’après nos résultats EAG, nous pouvons toutefois suggérer l’existence d’une synergie entre les phénols, en particulier aux quantités très faibles qui peuvent être captées par l’insecte dans son environnement naturel.

Nous avons également enregistré des réponses EAG très fortes avec l’urine de cheval, en particulier pour A. quadrifarius. Les réponses EAG provoquées par l’urine (dans laquelle la concentration en 4MP = 0,42.10^-6 mg/µL) sont significativement supérieures aux réponses EAG provoquées par le 4MP à la plus faible dose testée (1,10^-4 mg/µL) et similaires à celles provoquées par les doses plus fortes. La forte activité de l’urine sur les récepteurs des antennes de taons semble donc liée à la présence de différents composés actifs qui pourraient agir en synergie. Parallèlement à la caractérisation des réponses physiologiques, l’attractivité de différentes odeurs a été évaluée en Crau. Nous avons testé les combinaisons suivantes : urine de cheval seule, octénol + ammoniaque, phénols (4MP:3PP, 16:1) + ammoniaque, ammoniaque seule. Nous avons utilisé de l’ammoniaque car Mihok & Lange (2012) ont démontré un effet synergique entre l’ammoniaque et les phénols.

Les résultats comportementaux contrastent légèrement par rapport aux réponses physiologiques. Si les pièges avec attractifs augmentent les collectes de T. bromius (x1,8 à 2,2) et d’A. quadrifarius (x2,7 à 4,1), pour T. bromius, aucune différence n’a été observée entre les trois combinaisons. En revanche, pour A. quadrifarius, l’urine de cheval (x4,1) attire davantage que l’octénol (x2,8) et que les phénols (x2,7). Pour ces tests, l’urine a été placée dans un flacon avec une ouverture de 2,2 cm de diamètre. Le taux d’évaporation de l’urine a été estimé à 6,8 mL/jour soit pour le 4MP présent dans l’urine 0,12.10^-3 mg/h. L’octénol et les phénols ont diffusé à travers des sachets en polyéthylène. Le taux d’évaporation du 4MP à travers ce type de sachet est de 0,38 mg/h d’après Torr et al. (1997). L’attractivité de l’urine pour A. quadrifarius ne s’explique donc

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pas par la quantité de 4MP émise mais certainement par la présence d’autres composés qui restent à identifier.

Comme chez les insectes phytophages, la reconnaissance des hôtes par les taons dépend probablement de la détection de molécules volatiles communes présentes dans l’air à un ratio donné plutôt que de la détection ou non de molécules particulières (Bruce et al. 2005). Par exemple, chez l’eudémis, L. botrana, un ravageur de la vigne, la localisation des grappes est codée par un ratio spécifique de trois terpénoïdes communs dans les émissions volatiles des plantes (Tasin et al. 2006).

Nos observations confirment également les différences interspécifiques qui existent chez les Tabanidae vis-à-vis des attractifs. Au regard des réponses EAG et des collectes de terrain, il semblerait qu’A. quadrifarius soit plus sensible aux urines et à leurs constituants phénoliques que

T. bromius. En Croatie, des pièges Canopy appâtés avec de l’urine d’âne attirent significativement plus d’A. loewianus qu’un mélange octénol:acétone:ammoniaque (5:3:2) alors qu’il n’y a pas de différence entre ces deux attractifs pour Tabanus spp. (Krčmar et al. 2010). Comme chez les glossines, certaines espèces de taons pourraient être moins sensibles aux stimuli olfactifs mais plus réceptives aux stimuli visuels (Torr & Solano 2010). Ceci expliquerait pourquoi nous n’avons pas observé de différences entre les combinaisons d’odeurs chez T. bromius avec les pièges Nzi.

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Article 5 : Olfactory responses of tabanids to octenol, phenols and aged horse urine F. Baldacchino, S. Manon, L. Puech, B. Buatois, L.Dormont & P. Jay-Robert.

Olfactory and behavioural responses of tabanids to octenol, phenols and aged horse urine

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Olfactory and behavioural responses of tabanids to octenol,

phenols and aged horse urine

F. BALDACCHINO¹, S. MANON¹, L. PUECH¹, B. BUATOIS², L. DORMONT¹ AND P. JAY-ROBERT¹

1Unité Mixte de Recherche (UMR) 5175, Centre d’Ecologie Fonctionnelle et Evolutive (CEFE), Université Paul-Valéry (UM3), 34199, route de Mende, Montpellier cedex 5, France and ² UMR 5175, CEFE, Centre National de la Recherche Scientifique (CNRS), 1919 route de Mende, 34293 Montpellier cedex 5, France

Abstract.Electrophysiological and behavioural responses of females of two tabanid species (Tabanus bromius and Atylotus quadrifarius) to ammonia, octenol (1-octen-3-ol), phenols and aged horse urine were compared. EAG responses of both species to octenol, 4-methylphenol (4MP), 3-propylphenol (3PP) and a phenol mixture (4MP and 3PP at a ratio of 16:1) increased in a dose-dependent fashion. The most effective stimulus was 4MP, and synergism between the two phenols might exist. Aged horse urine also elicited strong EAG responses in both species. Using GC-MS analysis, we identified 29 compounds in horse urine; in particular, ketones, fatty alcohols and phenols, among which 4MP was the most abundant component (~80%). Trapping

experiments were carried out using Nzi traps baited with various odours. Octenol and the phenol mixture in combination with ammonia increased catches of tabanids 1.8 to 2.8 times relative to ammonia on its own. Aged horse urine increased catches of T. bromius and A. quadrifarius 2.2 and 4.1 times, respectively.The high attractiveness of aged horse urine, especially for A. quadrifarius, is likely not to be due to 4MP alone, but to the mixture of various active compounds involved in host location.

Key words.Tabanidae, baits, electroantennography, horse flies, host odours, Nzi trap, octenol, urine phenols.

Introduction

Locating a vertebrate host is crucial for blood-sucking insects to fulfil their nutritional and reproductive requirements. Typically, biting Diptera must locate a distant, mobile food source, which is frequently difficult to find and which has evolved defences against insect attack (Gibson & Torr, 1999). In general, insects use visual and olfactory stimuli, aided by anemotactic and optomotor responses (Paynter & Brady, 1996; Gibson & Torr, 1999; Horvath & Varju, 2004) and chemical signals are the principal cues with which they identify their hosts (Takken & Knols, 2010). A better understanding of the involvement of kairomones in host location for

haematophagous Diptera is of interest since identifying the chemical compounds that affect vector–host interactions should help in more effectively luring target insects to sampling devices and/or removing them from the environment using pest management methods (Spath, 1997; Bernier et al., 2007; Logan & Birkett, 2007; Harrup et al., 2012; Pinto et al., 2012).

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Biting flies generally show marked activation and attraction to carbon dioxide (CO2), which is present in mammalian breath, but are also attracted to other cues emanating from hosts (Birkett et al., 2004). The role of natural host odours in host location was demonstrated with tsetse flies in Zimbabwe by Vale (1974). Since then, numerous studies have identified the semiochemicals involved in host location and selection for tsetse flies; for example, octenol (1-octen-3-ol), a component of ruminant breath, and phenols, especially 4-methylphenol (4MP) and

3-propylphenol (3PP), known to be the key compounds in urine that elicit attraction of tsetse flies (Hall et al., 1984; Owaga et al., 1988). P-cresol or 4MP is also commonly found in rumen fluid odour collections (Harraca et al., 2009) and in dung volatiles (Dormont et al., 2010).

In contrast, the semiochemicals used by tabanids have received little attention. Horse flies are a serious nuisance to livestock and can mechanically transmit several animal pathogens (Mullen & Durden, 2002). In Europe, recent epizootics have focused on the role of tabanids as mechanical vectors in the transmission of equine infectious anaemia and besnoitiosis, considered emerging diseases (EFSA, 2010; Jacquiet et al., 2010; ANSES, 2012; Gentile et al., 2012; Maresca et al., 2012).

Previous behavioural studies in the field have focused on tsetse attractants such as octenol, phenols and urine. Octenol has been proved to be an effective attractant, as traps baited with octenol caught 2–9 times more tabanids compared to unbaited traps (Gibson & Torr, 1999; S. Krcmar et al., 2005; Mihok & Carlson, 2007). Reports on the use of phenols as bait have been more conflicting. In Croatia, canopy traps baited with 4MP collected 16 times more tabanids than unbaited traps (Krčmar, 2007), whereas in Canada, catches in Nzi traps baited with phenols did not collect more than unbaited traps, though it should be considered that Nzi traps are much more effective without bait than canopy traps (S. Krcmar, 2007; Mihok & Mulye, 2010).Aged animal urine has also been shown to be a good attractant in the field for Tabanus spp., Atylotus spp. and

Hybomitra spp. in North America and in Europe (Stjepan Krcmar et al., 2006; Mihok & Mulye, 2010; Baldacchino et al., 2012). The synergism between ammonia and phenols demonstrated for

Hybomitra spp. may explain the attractiveness of aged urine (Mihok & Lange, 2012). To determine the activity of mammalian kairomones on horse flies, electrophysiological responses to urine volatiles have been recently reported for two species (Baldacchino et al., 2012). Tabanus bromius Linnaeus 1758and Atylotus quadrifarius (Loew 1874) were both found to be sensitive to urine volatiles, with electroantennogram (EAG) responses similar to or greater than those for octenol. Moreover, cow and horse urine elicited stronger EAG responses than did sheep urine, which is consistent with the biology of horse flies as they feed predominantly on large mammals. These results suggest that tabanids may perceive and react to species-specific compounds, or specific ratios of ubiquitous compounds, to recognize a host. However, there were no significant differences in tabanid catches in the field using these urines as baits.

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Two phenols (4MP and 3PP) are thought to account for most of the attractive properties of cattle urine, and binary mixtures of the two significantly increase catches of tsetse flies (Owaga et al., 1988; Vale et al., 1988). However, it may be that other unidentified active components are involved in the attractiveness of urine, as combinations of these two kairomones appear to be less than 50% as attractive as natural cattle odour in some circumstances (Gikonyo et al., 2003). For temperate tabanids, the involvement of phenols in the attractiveness of aged urine remains unclear.

The goal of this study was to evaluate the response of females of two species of tabanids (T. bromius and A. quadrifarius) to phenols (4MP and 3PP) in comparison to aged horse urine by using electroantennogram recordings and field experiments. Our specific questions were: Is the urine blend more active and attractive than the two phenols? Do 4MP and 3PP principally

account for the attractiveness of urine for tabanids? Do urine volatiles act together synergistically on tabanid responses? Do EAG responses reflect behavioural responses by tabanids to phenols and urine volatiles?

Materials and methods

Odour stimuli

All chemicals used in this study were purchased from Sigma-Aldrich Chemie Gmbh (Buchs, Switzerland). These included 1-octen-3-ol (98%), 4-methylphenol (98%), 3-propylphenol

(>99%), dioctyl phthalate (≥99.5%) and ammonium hydroxide solution (5.0 M). The ammonium solution was diluted to obtain a 5% ammonia solution. A phenol binary mixture was made using 16 parts 4MP to 1 part 3PP. Dilutions were prepared using hexane (0.1, 0.01, 0.001 and 0.0001 mg/µL). Octenol at 0.1 mg/µL was used as a positive control, and hexane was used as a negative control in the electrophysiological recordings.

Horse urine was collected directly from the bladders of 10 mares and one gelding. The urine was collected from adults as young cattle have lower levels of phenols in their urine (Torr et al., 2006). The urine was kept for 3 weeks at room temperature (20–22 °C) in a 1.5 L plastic bottle and was then stored at -20 °C in 250 mL bottles.

Gas chromatography-mass spectrometry (GC-MS) of horse urine volatiles

The atmospheric concentrations of urine phenols were estimated using solid-phase

microextraction (SPME) with two 65µm polydimethylsiloxane/divinylbenzene (PDMS/DVB) Stable Flex fibers (Supelco, Sigma-Aldrich, Bellefonte, PA, USA). The two fibers were conditioned in a GC inlet kept at 250 °C for 30 minutes before sampling and were used simultaneously to collect volatiles over two samples of the same urine. 100 µL of aged horse urine (3 weeks old) was deposited in two 20 mL amber glass vials. One SPME fiber was exposed in each vial for 20-minutes at 25 °C.

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The quantity of 4MP in horse urine was assessed by the external standard method. 4MP was dissolved with 3PP at a ratio of 24:1 in ammonium hydroxide solution (0.5%). Acetic acid was used to produce a solution with a pH similar to that of urine (pH ~8). A calibration curve was obtained at three concentrations (0.048, 0.48 and 4.8 mg/L). Sampling was done using the same conditions as for the aged horse urine with the same SPME fibers. A logarithmic curve was obtained for the total peak area, and the regression coefficient provided R²>0.94. The calculated concentration of 4MP was based on the established standard curve.

GC-MS analyses of SPME extracts were performed using a Shimadzu GCMS-QP2010 Plus quadrupole mass spectrometer (Shimadzu Scientific Instruments, Kyoto, Japan). GC-MS analyses were conducted with a potential of 70 eV for ionization by electron impact, with the transfer line heated to 270 °C and the temperature of the ion source programmed at 250 °C. The mass spectrometer was used in scan mode, from a 38 to 600 m/z ratio. The Shimadzu QP2010 Plus was equipped with a 30 m x 0.25 mm x 0.25 µm SLB™-5ms (Supelco) fused silica capillary column. The injector temperature was 250 °C (splitless mode). The column temperature was programmed at 40 °C (for 5 minutes) then increased to 220 °C at 5 °C/min and to 250 °C at 10 °C/min. The carrier gas was helium, with a constant flow rate set to 1.0 mL/min. Compounds were identified by matching the mass spectra with data from the NIST 2005 and Wiley (9ed) MS software libraries and a personal laboratory bank of mass spectra. Compound identification was confirmed by comparing the retention indices (RI) with data from libraries and published data (Adams, 2007), and also by comparison with the retention time of standards. The relative amount of each compound was expressed with respect to the total volatiles in order to compare the volatile profiles of the samples.

Electroantennogram recordings

EAG recordings from antennae of female T. bromius and A. quadrifarius were recorded as in Baldacchino et al. (2012) with an EAG recording device (Electrode holders, EAG combi probe internal gain x10, stimulus controller CS-55 and signal acquisition controller IDAC-2; Syntech, Hilversum, the Netherlands). Recordings were made using electrolyte-filled (0.1 m KCl) glass capillary electrodes, with Ag/AgCl wires making contact with the EAG recording device. The antenna was maintained in a humidified charcoal-filtered air stream delivered at 13.5 mL/s through a metal tube. Each stimulus was prepared by depositing 10 µL of the test solution on a strip of filter paper (20 × 5 mm) placed in a glass Pasteur pipette and the solvent was allowed to evaporate for 15 minutes before first use.

The tip of the pipette was connected to the metal tube, and the stimulus was delivered to the antenna using an air pulse (20 mL/s for 0.8 s). We used one antenna per compound at the

different doses (corresponding to less than 10 min recordings per antenna). Stimuli were released successively in random order at 60-second intervals to avoid antennal receptor cell saturation.

Olfactory and behavioural responses of tabanids to octenol, phenols and aged horse urine

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The field experiment was conducted in the Coussouls de Crau Nature Reserve in southeastern France. This study site is characterized by open steppe and high populations of tabanids in the early summer. The trapping site was at the Peau de Meau sheepfold (43°33’N, 04°50’E; elevation 10 m a.s.l., area 160 ha). This region is influenced by a Mediterranean climate with high

interannual variability, low rainfall (400–500 mm/year) with maximum precipitation in spring and autumn, long hot summers, and mild winters (mean annual temperature 14 °C). On average, the sun shines 3000 hours per year, and a very strong predominant wind (the mistral) blows from the northwest 334 days per year, thus increasing evapotranspiration (Blight et al., 2011). During the study, no sheep were present in the Peau de Meau sheepfold, but flocks were present in other sheepfolds at a distance of less than1 km.

Field site meteorological data

Tabanid activity is influenced by meteorological variables such as wind velocity, temperature, relative humidity (RH) or evaporation, atmospheric pressure, and sky radiation or cloud cover (Van Hennekeler et al., 2011). Daily climate data were obtained from the meteorological station at Istres, which is located 20 km from the study site. The weather conditions were relatively good during the study period, with only a few cloudy days. Maximum daily temperatures ranged from 22.4 °C to 34.1 °C (mean = 28.4°C), and RH at midday ranged from 28% to 82% (mean = 46%). There was no precipitation except on 11 June 2012 (11.8 mm), 1 July 2012 (6 mm) and 5 July 2012 (0.6 mm). The maximum wind speeds were in the range of 7.8–20.6 m/s (mean = 13.1 m/s) with very variable wind directions.

Field experimental design

New Nzi traps were used as standard device as it is a multipurpose trap for biting flies designed by Mihok (2002). The efficiency of an odour-baited Nzi trap was estimated at 20-30% for tsetse flies and stable flies in Kenya (Mihok et al., 2007), and at 91% (45% when unbaited) for three species of Sudanese tabanids (Mohamed-Ahmed et al., 2007). No experiments have been conducted to estimate the efficiency of Nzi traps for temperate tabanids. However, these traps have previously shown to be very efficient for tabanids in the same study site (Baldacchino et al., 2012). The blue and black components of the traps were made from the same fabric (SuperMaine 300 g cotton/polyester 65/35%; TDV Industries, Laval, France). The cone and the back of the trap were made of polyester mosquito netting. The experiment consisted of setting up five 4 × 4 Latin squares that were in place from 5 June 2012 to 5 July 2012.

Four Nzi traps were set up in the open facing west at 200-m intervals along a line running northeast to southwest, perpendicular to the predominant wind. One trap was baited with

ammonia and was used as a control, a second was baited with octenol plus ammonia, a third with the phenol mixture plus ammonia, and a fourth with aged horse urine.

The phenol mixture consisted of a formulation of 4MP and 3PP at a ratio of 16:1. Such combinations of phenols have been used to attract tabanids in Canada, but at a ratio of 8:1

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(Mihok & Mulye, 2010). The ratio used in this study was more consistent with ratios of these products in animal urines (Baldacchino, unpublished data). The phenol mixture (3.05 mL of 4MP