J F Mariscal
email: mariscal@latmos.ipsl.fr

N Rouanet

J L Maria

E Quémerais

P O Mine

P Zuppella

M Suman

P Nicolosi

M G Pelizzo

I Yoshikawa

K Yoshioka

G Murakami

PROBING OF HERMEAN EXOSPHERE BY ULTRAVIOLET SPECTROSCOPY: INSTRUMENT PRESENTATION, CALIBRATION PHILOSOPHY AND FIRST LIGHTS RESULTS

PHEBUS (Probing of Hermean Exosphere by Ultraviolet Spectroscopy) is a double spectrometer for the Extreme Ultraviolet range (55-155 nm) and the Far Ultraviolet range (145-315 nm) dedicated to the characterization of Mercury's exosphere composition and dynamics, and surface-exosphere connections. PHEBUS is part of the ESA BepiColombo cornerstone mission payload devoted to the study of Mercury. The BepiColombo mission consists of two spacecrafts: the Mercury Magnetospheric Orbiter (MMO) and the Mercury Planetary Orbiter (MPO) on which PHEBUS will be mounted. PHEBUS is a French-led instrument implemented in a cooperative scheme involving Japan (detectors), Russia (scanner) and Italy (ground calibration). Before launch, PHEBUS team want to perform a full absolute calibration on ground, in addition to calibrations which will be made in-flight, in order to know the instrument's response as precisely as possible. Instrument overview and calibration philosophy are introduced along with the first lights results observed by a first prototype.

I. INTRODUCTION

A. Instrument overview

The instrument is basically composed of two Ultra-Violet spectrophotometers and one scanning mirror with a single axis of rotation. This movable mirror will collect the light coming from the exosphere above the limb onto the entrance slit of the spectrometer with a minimum number of reflections in order to maximize the UV count rate on the micro-channel plate detectors. The mirror is protected from straylight by an entrance baffle characterized by a good rejection capability. Each detector has a specific range of wavelengths: the EUV (Extreme UV) channel spreads from 55 to 155 nm, and the FUV (Far UV) channel from 145 to 315 nm. A couple of photomultipliers receive two additional wavelengths in the Near UltraViolet range (NUV) at 404.7 and 422.8 nm. The main distinctiveness of PHEBUS will be to probe Mercury's exosphere in the spectral band (55 -110 nm) for the first time.

B. Measurement objectives

The atmosphere of Mercury is very tenuous, with a pressure of a fraction of picobar. It results from a complex interplay of the solar wind, its planetary magnetic field and its rocky surface. It is nearly noncollisional, and is highly variable with time and space, characterized by a global asymmetry between dayside and nightside and rapid temporal variations, possibly related to varying magnetospheric activity.

The core scientific objectives of PHEBUS [START_REF] Chassefière | PHEBUS: A double ultraviolet spectrometer to observe Mercury's exosphere[END_REF], are oriented toward a better understanding of the coupled surface-exosphere-magnetosphere system. The main measurement objectives are the following: -To detect new species, including metallic species (Si, Mg, Fe, …), atoms (C, N, S, …), molecules and radicals (H 2 O, H 2 , OH, CO), noble gases (Ar, Ne), ions (He + , Na + , Mg + , …), in addition to already detected species (Na, K, Ca, O, H, He). -To measure an average exosphere (densities of constituents, vertical structure), with as much as possible species monitored together, at different positions of Mercury around the Sun. -To measure sharp local and temporal variations of the exosphere content, at specific times and places of interest.

-To search for albedo variations of Mercury's nightside surface, lighted by the interplanetary H Ly-α glow, at 121.6 nm, in order to exhibit possible signatures of surface ice layers (H 2 O, SO 2 , N 2 , CO 2 ,) in high latitude polar craters.

ICSO 2010

Rhodes, Greece International Conference on Space Optics 4 -8 October 2010

II. INSTRUMENT DESIGN

A. Instrument concept PHEBUS (Fig. 1) is a double spectrometer for the Extreme UltraViolet (EUV) range (55-155 nm) and the Far UltraViolet (FUV) range (145-315 nm) with an extension for some extra visible emission lines (Potassium and Calcium at 404.7 nm and 422.8 nm respectively). The optical configuration of PHEBUS can be divided into two independent parts. The collecting part consists of a straylight rejection baffle, an off axis parabolic mirror and an entrance slit, allowing to scan Mercury's exosphere thanks to a rotating mechanism. This movable mirror collects the light from the exosphere above the limb and focuses it to the entrance slit. The parameters of the mirror were calculated so as to have a 170 mm effective focal length and a folding angle of 100°. This part determines the Field of View (FoV) and the Light of Sight (LoS). The spectrometer part determines the spectral resolution of the instrument and is composed by the entrance slit, two holographic gratings and the detectors. The spectrum detection is based on the photon counting method and is done using Micro-Channel Plate (MCP) detectors with Resistive Anode Encoder (RAE). Photocathodes are CsI for the EUV range, and CsTe for the FUV range. The size of the detectors active area is 40×25 mm² equivalent to a matrix of 1024×512 virtual pixels (spectral × spatial). Furthermore Calcium and Potassium lines are selected by the FUV grating. These extra visible lines are monitored using photomultipliers (PM) with bialkali photocathode also used in photon counting mode. The main advantage of the MCP+RAE detectors is their very high sensitivity mainly due to a very low dark current. Thus photon counting is easily achievable on typical experiment temperature range (from -20°C to +40°C), avoiding mass and power expensive devices to cool the detectors. Five to six orders of magnitude for the detection are then a typical value and offer the monitoring of a wide range of emission. Moreover PHEBUS is a very flexible instrument due to the rotating scan mirror at the entrance. The instrument is then quite independent from the spacecraft on an observation point of view, avoiding spacecraft slew for specific pointing requests. This scanning mirror is also very helpful to maintain the LoS close to the limb during long integrations, to make the search and monitoring geometry less dependent on orbit and to extend the vertical range of scanning.

B. Optical specifications

The wavelength ranges are 55-155 nm for the EUV and 145-315 nm for the FUV. The spectral resolution is defined in terms of Full Width at Half Maximum (FWHM) and Full Width at 1% of maximum (FW1%). The required spectral resolution is 1 nm for EUV and 1.5 nm for FUV. These values are to be compared with the result of the optical design optimization: the FWHM is about 0.5 nm on EUV, and 0.8 nm on FUV. Furthermore, the FW1% is about 0.9 nm on EUV, and 1.5 nm on FUV. These calculated values do not include any spreading effects due to scattering by gratings. The paraxial Field of View of the instrument is about 2° by 0.1°, and the straylight attenuation baffle is designed to protect from bright sources outside a guard angle of 8.3°. High level of attenuation is obtained by combining a very dark black treatment inside the entrance baffle and a superpolished entrance mirror (roughness: 0.5 nm RMS) compatible with EUV range. The two gratings of the PHEBUS instrument are aberration corrected holographic gratings. The mean groove density is ~1600 grooves/mm for the FUV and ~ 2700 grooves/mm for the EUV. Groove profile is laminar ion-etched optimized for the respective spectral range. AFM measurements on real prototypes show a micro-roughness of about 1nm RMS. The absolute efficiency can be deduced from AFM measurement of the groove profile via electromagnetic theory software codes: about 6% for EUV and 13% for FUV. The gratings are made of aluminium with a reflective platinum coating. Their active area size is 42 mm by 15 mm. A radiometric model of the PHEBUS instrument has been implemented in order to simulate recorded spectrum. The mean sensitivity of PHEBUS is in the order of 0.1 count per second per Rayleigh of emission, and the mean detection limit can be estimated to about 0.1 Rayleigh for EUV, and about 0.2 Rayleigh for FUV.

III. GROUND CALIBRATION PHILOSOPHY

A. Overview

We decided to perform an instrument's absolute ground calibration as complete as possible. The error made on the in-flight measurements will depend on the quality of calibration made on ground: a precise calibration is essential to process relevant scientific data. Before knowing if ground calibrations are realistic on an experimental point of view, we put forward a theoretical calibration plan. PHEBUS is a spectrophotometer, its objective is to determine the radiance L (ph.s -1 .m -2 sr -1 ) of an extended source (with respect to the source's wavelength) measuring a count rate R. The relation ( )

L f R =
between what we measure (R) and what we want to know (L) is the main purpose of the calibration. In the case of punctual source like a star, the calibration relation becomes ( )

E f R =
where E is the irradiance of the source (ph.s -1 .m -2 ). Ground calibration will be done at subsystem and system levels and we will distinguish two kinds of calibration, the radiometric calibration and the spectral calibration.

B. Instrument radiometric calibration

The radiometric performance of the instrument is evaluated through the knowledge of the Efficiency (η), the Etendue (G) and the Effective Etendue (G eff ). In a general way (1) defines the efficiency of the system:

G Etendue the inside photons incoming of Number counts of Number = η (1)
The Etendue is the geometric set of beam elements which enter inside the instrument and reach the detector without being subject to vignetting effect. In addition to these diaphragm losses, the Effective Etendue takes into account the efficiency of each optical element and thus the reflectivity losses. By definition, and assuming the approximation that the efficiency does not depend on the ray path we can write:

G eff G . η = (2)
This can also be seen as a averaging of η over all the ray paths. We can see that the Effective Etendue given by (2) includes both Efficiency and Etendue. These three parameters depend on several variables linked to the conditions of the measurement such as the wavelength, the polarization state, the scan angle, the type of source, the position of the slit. Wavelength will have to vary from 55 nm until 315 nm with two extra lines at 404.7 nm and 422.8 nm. It is worth pointing out that for the EUV and FUV channels, measurements made in the spectral range [55 -315 nm] corresponds to the nominal diffraction order (k=1) of the spectrometer. The overlapping diffraction order at k=2 must also be studied, so we need to use a spectral range two times smaller: [27 -158 nm]. Regarding the step, it will have to be sufficient in order to be significant (at least ten values per spectral range without wide gap). It would be also relevant to perform measurements out of the working spectral range in order to estimate the level of stray light inside the spectrometer.

As the irradiance of the entrance pupil projection on the gratings is not homogeneous due to the off-axis entrance parabola, the illumination pattern on the gratings and the signal distribution on the two channels will vary as a function of the scan angle. Therefore we must perform measurements for several angular positions of

ICSO 2010

Rhodes, Greece International Conference on Space Optics 4 -8 October 2010 the scanner mechanism. At least two points are required in order to evaluate the extreme cases, but to determine the variation between these two points others values are necessary.

In addition and to be rigorous, we must use two types of sources (uniform extended source and punctual source) and we must perform measurements with the two positions of the slit (nominal position and star position).

To describe the behavior of the instrument with respect to the incident polarization, we use the Stokes formalism and the Mueller matrix. Given M PHEBUS the (4×4) PHEBUS Mueller matrix, since the information provided by the instrument is a number of counts (i.e. the total output intensity which is corresponds to the first component of the output Stokes vector: S 0 ), we can make some simplifications and we can conclude that we only need the first four parameters

03 02 , 01 , 00 , M M M M of M PHEBUS .
Finally, these four parameters will completely define the photometric response of the system. It means that the radiometric response of the instrument is no longer a simple scalar value G eff but becomes this set of four scalars.

Given the Stokes vector of the source ( )

3 2 , 1 , 0 , L L L L
in terms of radiance, the count rate is:

3 03 2 02 1 01 0 00 L M L M L M L M R + + + = (3)
If we normalize (3) we can bring out a corrective factor to obtain:

( )

0 00 0 3 3 2 2 1 1 00 0 , , 1 L L L M M m L m L m L m M L R i i i i = ′ = ′ + ′ + ′ + = (4)
The instrument measure R, we must know a priori the polarization state of the source ( )

3 2 1 , , L L L ′ ′ ′
, the calibration give us the values M 00 and m i , thus we can deduce the value of interest L 0 .

The term M 00 can be assimilated to the Effective Etendue G eff for unpolarized light. The term inside the brackets of (4) represents a correction factor of G eff taking into account the incident polarization state and the polarimetric response of the instrument. It can also be seen as an error factor if the polarization effect is not taken into account.

C. Instrument spectral calibration

In addition to the radiometric calibration we must evaluate the Instrument Spectral Response Function (ISRF), characterizing the line profile at a detector level, for a given alignment of the whole system. It involves to know the position and orientation of each line with respect to the wavelength, but also to measure the resolution in terms of FWHM and FW1%. Since we want to characterize the spectral image, these tests must be done on both detectors EUV and FUV. Regarding the two photomultipliers, we must perform measurements of the spectral bandwidth imposed by the size of each deflection mirrors which work as slits.

D. Subsystem radiometric calibration

A calibration at subsystem level is important in term of redundancy and also to be able to assess the source of any problem that may affect the overall operation of the instrument. Regarding subsystems, only the radiometric calibration are needed and we will proceed in the same way as for the system calibration, by representing each subsystem by its Mueller matrix. Theoretically, if we know individually each subsystem Mueller matrix we should be able to obtain the Mueller matrix of the whole instrument M PHEBUS . We introduce here the approach only for the main subsystems.

Entrance mirror

The mirror is characterized by its Mueller matrix M mirror (5) which contains sixteen elements of any value a priori. But according to classical model of mirror reflectance this matrix can be simplified as: r and ⊥ r are respectively the reflection coefficients (possibly complex) in terms of amplitude for the parallel component of the electric field and for the perpendicular component of the electric field. Thus only two complex parameters have to be calibrated. Measurements must be made at a fixed incidence angle which is the working incidence angle of the mirror: 50°. Each measurement must be done by varying wavelength from 55 nm to 315 nm and at 404.7 nm and 422.8 nm.

( ) ( ) ( ) ( )                   - + - - + = ⊥ ⊥ ⊥ ⊥ ⊥ ⊥ ⊥ ⊥ r r

Gratings

Gratings are also represented by their own Mueller matrix M grating composed by sixteen elements. Measurements must be perform in the spectral range of each grating for the nominal working order k=1 and also in the spectral range corresponding to the diffraction order k=2.

Detectors (EUV & FUV)

In terms of radiometry, we must measure the dark count rate with respect to the temperature (-20°C, +40°C) and also regarding the high voltage (HV) of the MCP: the goal is to increase the HV during flight in order to compensate the ageing of the detectors. Solar blindness of EUV detector will also be verified, by using a visible calibrated source of variable intensity. The spatial uniformity of the detectors response will be tested with flat field illumination. Detector Efficiency will be measured at low signal level for photocathode characterisation, by using a few UV lamps covering the spectral range of the two UV detectors. Non-linearity mainly due to limited electronic sampling frequency will be estimated. Finally, along the whole life of the detectors, all the illuminations undergone by the detectors will be reported into a single document, in order to determine the ageing of the response. In terms of geometry, an evaluation of the useful area of the detector (i.e. the area where the response is nominal) will be done. The spatial resolution depends on HV and it has to be evaluated by PSF (Point Spread Function) or MTF (Modulation Transfer Function). The evaluation of the distortion of the detector is needed. The distortion may depend on HV and is evaluated by calculating:

( ) ( ) ( ) y x y x , , - ′ ′ = ∆
, where ( ) y x, is the real impact point of the photon on the photocathode and ( )

y x ′ ′,
is the position of the photon as read by the detector.

IV. FIRST LIGHTS FROM PHEBUS

An optical prototype of PHEBUS is currently used to calibrate detectors down to the extreme UV, and also to enable the adjustment of the gratings and the slit. The optical breadboard is representative from an optical point of view but operated with a fixed baffle orientation. In order to have a first approach of the instrument behaviour, first measurements were performed with the EUV detector and using a facility under vacuum composed by a EUV radiofrequency flow lamp as source, a grazing incidence monochromator to select the wavelength and a test chamber. We fill the lamp with different gases such as Ne, He, Ar, N and O in order to emit quasi-monochromatic sharp lines typical of the gas used. The EUV detector is provided with its own software allowing to control detector parameters such as the high voltage but also to acquire data coming out of the detector. We introduce here results obtained with Argon gas and using the zero order position of the monochromator in order to record the full spectrum between 55-155 nm. Data were processed by numerical computing software in order to display the real image seen by the detector (Fig. 2) and to plot a spectrum as shown in Fig. 3. Knowledge of the Argon theoretical emission lines allows us to calibrate the x-axis by replacing pixel values by wavelength values (calibration only valid for a given alignment of the components). The wavelengths written in Fig. 3 correspond to peak values of the detector data (and not the wavelengths of emission lines). We can clearly identify the two Argon doublets 104.8 & 106.6 nm (Ar I) and 92.0 & 93.2 nm (Ar II). Others Argon emission lines may be identified such as: 87.6 nm (Ar I), 72.3 nm (Ar II), 67.1 nm (Ar II), 57.7 nm, 54.3 nm, 51.9 nm (Ar II). We can also observe other lines maybe due to contamination such as H I at 121.6 nm, the lines seen at 113.6 nm seems to be N I triplet around 113.4 nm, and the one read at 130.8 nm seems to be O I triplet around 130.4 nm. The spectral resolution in terms of FWHM was calculated at the doublet 104.8 nm -106.6 nm levels and the value is 0.72 nm instead of 0.48 nm expected. However, it is a first calibration which allowed us to obtain the first lights from the instrument and to suggest improvements to optimize the measurements and alignments. Among others, a toroidal mirror will be put between the monochromator and the test chamber in order to shape the incident beam (either by collimating or by focusing according to the need). Alignments must be improved also and by the time this paper is written, calibration of the facility (EUV lamp, monochromator) is in progress. The PHEBUS instrument, due to its wide spectral range, high sensitivity and high straylight rejection rate is well suited for the study of Mercury exosphere. A full ground calibration will be made in order to know in the best possible way the response of the instrument. The starting point of the calibration was to evaluate all the parameters which are feature of the instrument and to list all the variables that can vary these parameters. Some calibrations on optical subsystems prototype measurements were already performed [START_REF] Zuppella | PHEBUS spectrometer : characterization of optical subsystems prototype[END_REF] in collaboration with the University of Padova (LUXOR laboratory) and an optical breadboard allows us to perform preliminaries calibrations and alignments. The next steps will be to know if all the calibration requirements are feasible or not. On an experimental point of view, we can already say that the measurement of the elements of Mueller matrices will be performed mainly by judicious rotations between the source and the instrument while knowing the Stokes vector of the source.

Fig. 1 .

 1 Fig. 1. a: PHEBUS instrument, b: Optical configuration 1 -Entrance pupil; 2 -Entrance mirror; 3 -Slit; 4 -FUV grating; 5 -EUV grating; 6 -FUV detector; 7 -EUV detector

Fig. 1 .

 1 Fig. 1. EUV detector Image for Argon gas: HV = -3436 V, this image corresponds to a cumulated integration time of 10 min. The count rate is about 1500 counts per second. (Levels are in log 10 -scale)

Fig. 2 .

 2 Fig. 2. EUV spectrum from the image Fig.2 (Levels are in log 10 -scale)


C V Long
email: c.v.long@lboro.ac.uk

J A Flint

P A Lepper

Insect attraction to wind turbines: does colour play a role?

Keywords: Wind power, Wildlife mortality, Insects, Colour, Turbines

Insect attraction to wind turbines: does colour play a

role?

Introduction

In recent years, concern has been growing over the phenomenon of avian and bat mortality at wind turbine installations, an issue that could potentially be an obstacle to the growth of the wind industry sector, particularly at sites known to be inhabited by rare or protected species. While both insectivorous birds and bats are involved with turbine mortality, in some cases the number of bat carcasses retrieved considerably outnumbers those of birds (e.g. [START_REF] Kerns | A study of bird and bat collision fatalities at the Mountaineer Wind Energy Centre, Tucker County, West Virginia[END_REF][START_REF] Piorkowski | Breeding bird habitat use and turbine collisions of birds and bats located at a wind farm in Oklahoma mixed-grass prairie[END_REF]). Whilst it is still unclear why bats frequent wind turbine installations, recent research has shown that bats appear to actively investigate turbine rotors [START_REF] Horn | Behavioural responses of bats to operating wind turbines[END_REF]. Some species may be assessing them as potential roost sites [START_REF] Cryan | Mating behavior as a possible cause of bat fatalities at wind turbines[END_REF], however there is also some evidence of foraging behaviour around turbines [START_REF] Horn | Behavioural responses of bats to operating wind turbines[END_REF][START_REF] Ahlén | Wind turbines and bats-a pilot study[END_REF]. Bats tend to be concentrated in areas of high insect density [START_REF] Nicholls | Bats avoid radar installations: could electromagnetic fields deter bats from colliding with wind turbines?[END_REF] and are much more likely to begin hunting when large numbers of insects are congregating [START_REF] Griffin | The echolocation of flying insects by bats[END_REF]. Reports into bat-turbine interactions frequently state the importance of investigation into the possibility of insect attraction to turbines (e.g. [START_REF] Johnson | Bat ecology related to wind development and lessons learned about impacts on bats from wind development[END_REF][START_REF] Ahlén | Wind turbines and bats-a pilot study[END_REF][START_REF] Nicholls | Bats avoid radar installations: could electromagnetic fields deter bats from colliding with wind turbines?[END_REF][START_REF] Rodrigues | Wind turbines and bats: guidelines for the planning process and impact assessments[END_REF], particularly since the recent loss of feeding habitats may be pressuring bats to feed in alternative areas [START_REF] Wickramasinghe | Bat activity and species richness on organic and conventional farms: impact of agricultural intensification[END_REF]. Turbine colour may play an important part in insect attraction [START_REF] Ahlén | Wind turbines and bats-a pilot study[END_REF], although to date this has not been closely investigated. Turbines are mostly painted white [START_REF] Johnson | Bat ecology related to wind development and lessons learned about impacts on bats from wind development[END_REF] or shades thereof; the reasoning behind painting turbines in light colours appears to be connected with making turbines 'visually unobtrusive' against the skyline, to make them 'blend well into the landscape', or to make them easier to locate for meteorological purposes (Danish Wind Industry Association DWIA 2003). In the case of offshore turbines, light colours minimize visibility from shore (Department of Trade and Industry (DTI) 2005). Offshore turbines are also required to be painted yellow from the level of 'highest astronomical tide' up to 15 m (Department of Trade and Industry DTI 2005) to ensure they are visible to ships. Turbines are typically coloured by paint mixed to RAL colour specifications; RAL is an established firm which has been producing colour standards for more than 70 years. The two most common turbine paint shades are 'Pure White' (RAL 9010) or 'Light Grey' (RAL 7035) (e.g. Vestas Wind Systems 2006). Turbine colour is usually dependent on local building regulations and occasionally turbines are painted in other shades, for example 'Squirrel Grey' (RAL 7000) (Bracknell Forest Borough Council 2007), a colour which was chosen as it was deemed to 'blend in better with a rural landscape and the UK skyline', and was 'less visually obtrusive' than green, brown or black (Bracknell Forest Borough Council 2007). The effect of the colour selection on bird and bat mortality was not stated to have been considered in this case. To date, there has been one notable study to investigate effects of turbine colour, by [START_REF] Young | Comparison of avian responses to UV-light-reflective paint on wind turbines[END_REF], with regard to painting turbines with ultraviolet (UV) reflective paint as a visual warning for birds. The results indicated an increase in the number of avian fatalities around UV painted turbines (68% at UV turbines; 15% at non-UV turbines). It could be speculated that the UV paint would increase the attraction of insects to the turbine, and thus the attraction of insectivorous birds; this theory is supported by the fact that the vision of many insects is shifted towards the UV portion of the spectrum [START_REF] Briscoe | The evolution of color vision in insects[END_REF]) (e.g. members of the fly genus Drosophila are consistently attracted to UV reflecting objects, a trait frequently exploited by webmaking spiders [START_REF] Craig | Insect attraction to ultraviolet-reflecting spider webs and web decorations[END_REF]). In this study, the insect attraction to specific turbine colours is systematically investigated in order to identify whether this might need to be considered for future wildlife-turbine mitigation.

Methods

Study area

The location for experimental work was a meadow at the base of a 13-m, three-blade turbine (light grey), approximately 5 m from the base. This turbine was situated in an area of public parkland, at GPS coordinates 52°35′16.70″N, 1°05′06.28″W in Oadby, Leicestershire, UK. Deciduous woodland and hedgerow (see Fig. 1) surrounded the meadow on all sides; there was an abundance of local bird and bat activity (although no documented mortality incidents) in the vicinity.

Data collection

A selection of ten reference colour cards from the RAL colour range were used, each measuring 215 by 303 mm and impregnated on one side with the specified RAL colour by the manufacturer (RAL gemeinnützige GmbH, Germany). The colours chosen were as follows; RAL 9010 'Pure White' (white), RAL 7035 'Light Grey' (light grey), RAL 7000 'Squirrel Grey' (dark grey), RAL 5015 'Sky Blue' (blue), RAL 3020 'Traffic Red' (red), RAL 4001 'Red Lilac' (purple), RAL 1023 'Traffic Yellow' (yellow), RAL 8025 'Pale Brown' (brown), RAL 6026 'Opal Green' (green) and RAL 9005 'Jet Black' (black). Colours were selected in such a way as to produce a varied spectrum of samples, in addition to the common turbine colours (white and light grey), a less common turbine colour (dark grey), and black. Some of the other additional colours were chosen for various reasons; several flower visiting insects express an innate colour preference [START_REF] Lunau | Innate colour preferences of flower visitors[END_REF] with many insects being attracted to yellow colours (560-590 nm) [START_REF] Prokopy | Visual detection of plants by herbivorous insects[END_REF], including Diptera (flies) and Lepidoptera (butterflies and moths) [START_REF] Kevan | Floral colors through the insect eye: what they are and what they mean[END_REF]. Blue flowers (400-500 nm) have been observed to be particularly attractive to Hymenoptera (bees) [START_REF] Kevan | Floral colors through the insect eye: what they are and what they mean[END_REF], while pink and red flowers (650-700 nm) are frequently visited by Lepidoptera [START_REF] Kevan | Floral colors through the insect eye: what they are and what they mean[END_REF]. In order to protect colour cards and to prevent discolouration by moisture contamination and other debris, each card was laminated inside a transparent plastic pocket. An empty laminated pocket was included in the study as a transparent control. To assess the relative 'attractiveness' of each colour card to insects, cards were laid out, face up, in two rows of six and five cards, respectively, approximately 2 cm apart. Each card was then observed, in turn, for a set period of time, during which the number of flying insects present on or within approximately 10 cm from the card's surface was recorded. After 5 s, the next card along the grid was observed, and so on in an anti-clockwise pattern, over a total of 10 min. By assessing cards in this manner, it is quite possible to re-count the same insect on the same card several times; this is not problematic as it effectively weights the count to account for a greater attraction to any one colour card. After this 10-min period, the cards were gathered, carefully cleaned, shuffled and randomly re-distributed throughout the grid in order to avoid potential bias caused by the proximity of any one colour to another. This was repeated three to five times in succession and the insect species present noted at the end. For sunset observations only, a low power torch (300 lx peak luminous emittance at 300 mm) was used to illuminate the card under observation in order to facilitate insect identification. While certain nocturnal insect species, such as moths, are known to be attracted to light sources, the torch was used consistently for observations and turned off between readings in order to minimise any bias caused by its use. Each set of measurements were taken between the months of June to October, corresponding with the months of peak bat activity, over a 3year period. In year 1, readings were taken at midday only, in year 2, readings were taken 1 h after sunset only, and in year 3, readings were taken both at midday and 1 h after sunset on the same day. Before each measurement, the time and weather conditions were noted and temperature, relative humidity (RH) and wind speed readings taken from an HHF81 digital 4-in-1 meter (OMEGA Engineering, Inc., Stamford, CT, USA). In addition, the wavelength spectral reflectance peaks (within a range of 900-300 nm) of all cards used in the experiment were measured using a UV/VIS Lambda Bio 40 Spectrometer (Perkin-Elmer, Inc., Waltham, MA, US).

Statistical analysis

Data were analysed using the GLM ANCOVA module of Statistica 5.1 (StatSoft, Inc., Tulsa, OK, USA). 'Insect count per 10 min period' was selected as the dependent variable in all cases, while either 'colour', 'month', 'time of day', 'year', 'weather conditions', 'temperature', 'relative humidity' or 'wind speed' were chosen as independent variables. Since these factors also varied in conjunction with the independent variables, in all cases the relevant variables from 'colour', 'month', 'time of day', 'year', 'weather conditions', 'temperature', 'relative humidity' and 'wind speed' were selected as covariates. In order to assess the importance of spectral peak percentage reflection, peak UV reflection and peak infrared (IR) reflection, these were selected as independent variables with 'insect count per 10 min period' as dependent variable and 'colour', 'month', 'year', 'weather conditions', 'temperature', 'relative humidity' and 'wind speed' selected as covariates. Because UV and IR measurements taken represented reflection in peak light conditions, this analysis was applied only to midday measurements with weather conditions of 'sunny' and 'sunny spells'.

Results

A total of 2012 insect observations were made over the 3year course of the study, from fifty-nine 10-min sessions. Note that approximately 90% of insects counted had landed on the surface of the colour cards.

Effects of colour on insect attraction

Colour was found to have an overall significant effect on insect count (p<0.0005; F [10,631] =3.25); Fig. 2 demonstrates the total mean insect count for each colour tested. Of these colours, yellow was the most attractive overall (p<0.00005; F [2,631] =11.09); however, the turbine colours white and light grey were significantly more attractive than all other colours (excluding yellow) tested (p<0.05; F [2,631] =4.34). Purple attracted significantly fewer insects overall (p<0.05; F [2,631] =4.63) than any of the other colours tested.

Effects of spectral reflectance on insect count

Spectral reflectance peaks were found to vary between each colour card tested, both in the visible, UV and IR spectrum (Fig. 3). Overall, peak spectral reflectance had a significant effect on insect count per 10 min period (p<0.005; F [9,225] = 3.0) for midday (sunny and sunny spells) measurements. Both peak UV reflectance and peak IR reflectance significantly influenced insect count (p<0.005; F [9,225] =3.0; p< 0.05; F [8,226] =2.82) with higher spectral reflectance in these ranges attracting more insects. The card colours yellow and white gave the highest IR reflectance peak, while the transparent 'control' card, interestingly, gave the highest UV peak (see Fig. 3).

Seasonal, annual and meteorological effects on insect count

Time of day was found to significantly influence insect numbers; total insect counts were significantly lower after sunset than at midday (p<0.00005; F [1,640] =18.44), although this phenomenon did vary according to colour (see Fig. 2). Insect counts varied significantly according to month (p<0.00005; F [4,637] =10.76) over the course of the data collection period. The month of July had the highest total mean count and October the lowest, as shown in Fig. 4. Relative insect attraction to each colour was found not to be consistent between months or times of day, as demonstrated in Fig. 5. Table 1 describes the insect orders observed each month during the data collection period. Weather conditions had an overall significant effect on insect count (p<0.005; F [4,637] =3.82) with fewer insects observed in 'clear' and 'cloudy' weather than other conditions ('overcast', 'sunny spells', 'sunny'). Figure 6 demonstrates the effect of wind speed, temperature and RH on total mean insect counts per 10 min period; temperature significantly influenced insect count (p<0.00005; F [10,631] = 8.31) with the highest activity observed at 15°C and 16°C. RH also significantly influenced insect activity (p<0.05; F [3,638] =2.93) with lowest activity at 60-69% RH, as did wind speed (p<0.00005; F [5,636] =1.75) with speeds of 2.5-4.4 ms -1 showing the highest counts.

Discussion

The finding that the common turbine colours white and light grey were amongst the most attractive colours to insects, independent of time of day, is of significant importance. Insects attracted to a turbine mast and rotor present a foraging opportunity to local insectivores, and thus this is likely to greatly increase the time spent in the vicinity of the turbine, which in turn increases the risk of fatal interaction with operational rotors. Furthermore, it is conceivable that flying insects attracted to white turbine structures could then become trapped in the rotor wake vortices [START_REF] Arnett | Patterns of bat fatalities at wind energy facilities in North America[END_REF][START_REF] Johnson | Bat ecology related to wind development and lessons learned about impacts on bats from wind development[END_REF]. It is Fig. 3 Spectral reflectance data for each experimental card, demonstrating peaks in reflectance for wavelengths between 900 (infrared) to 300 (ultraviolet) nm. Note that the reflectance peak for the transparent card is more than 100% due to fluorescence in the UV range Fig. 2 Mean insect count per 10-min period for each colour tested. Grey circles indicate total means with ± standard deviation whiskers; white and black boxes indicate means for midday and sunset +1 h counts, respectively not entirely clear why these light white and near-white colours are attractive to insects; [START_REF] Prokopy | Visual detection of plants by herbivorous insects[END_REF] noted that foliar reflectance occurs about 350-650 nm, which accompanied by a lower saturation via an increase in UV and blue reflectance results in a 'whitish' appearance. It could therefore be that insects mistake the white colour of the turbine for foliage and are drawn to it for foraging, mating or resting opportunities. White flowers have also been found to be highly visually attractive to a wide range of insects [START_REF] Kevan | Floral colors through the insect eye: what they are and what they mean[END_REF]. It therefore appears important to consider alternative turbine colours for future installations, particularly in areas known to be high in insectivore activity. Whilst white and light grey are by far the most common choices for turbine colour, dark grey is also known to be used; dark grey appeared to be slightly less attractive overall than light grey (Fig. 2), although this trend was not significant (F [2,631] =1.73).

Several other colours had marked effects on overall relative insect attraction. The fact that one colour demonstrated the lowest overall insect count (purple) indicates there are also likely to be other suitable candidates for turbine colour that are not highly attractive to insects independent of season, and these need to be further investigated for mitigation purposes. Whilst the high attractiveness of the colour yellow found in the experiment agreed with expectations (given the link between the yellow colour of many pollen types at the centre of flowers where nectar is located, and the spectral peak at which green leaves reflect most light [START_REF] Prokopy | Response of European cherry fruit flies to coloured rectangles[END_REF]), the high insect counts for the colour black were somewhat surprising. It may be speculated that the heat-absorbing properties of this colour acted as a 'thermal lure' for some insects observed. Black attracted more insects in the colder months of September and October during midday observations (Fig. 5), and was also the only colour to attract more insects, on average, after sunset than at midday (Fig. 2). Many insects are influenced by thermal gradients; tabanid flies, for example, have an exceptionally strong attraction to heat [START_REF] Thorsteinson | The orientation of horse flies and deer flies (Tabunidae, Diptera): I. The attraction of heat to Tabanids[END_REF], and so it is important to consider the thermal properties of the turbine structure in addition to colour. Both [START_REF] Ahlén | Wind turbines and bats-a pilot study[END_REF] and [START_REF] Horn | Behavioural responses of bats to operating wind turbines[END_REF] have used thermal imaging cameras on operational turbines to investigate this effect, finding the top portion of the tower, the blades and nacelle to appear warmer than the surrounding air. Insects may be attracted to the warmer air around the nacelle, particularly in autumn [START_REF] Dürr | Bat deaths and wind turbines-a review of current knowledge, and of the information available in the database for Germany[END_REF], which requires further investigation in conjunction with colour effects.

The transparent 'control' was found not to differ significantly in attractiveness as compared to all other colours (F [2,631] =1.69), which may have been due to the reflection of UV light from the card during midday readings. The results of the spectral reflectance tests (Fig. 3) confirm that this card reflected strongly in the UV region under peak light conditions, even fluorescing above 100% reflectance. This theory is supported by the higher midday insect counts for June and July (Fig. 5), months more likely to experience weather conditions of 'sunny' or 'sunny spells' in the dataset and UV index is highest.

It is interesting to note that the UV reflectance of the colour cards was markedly reduced as compared to the transparent laminate, despite all cards being laminated inside identical plastic pockets. This suggested that the coloured paints have UV absorbing properties, although the extent to which UV is absorbed did vary between colours. Because paint colours reflecting more UV light attracted significantly more insects under strong light conditions (even when the transparent card was not included in analysis (p<0.05; F [8,573] =2.66)), it may be important to select paint colours which do not have strong UV reflectance peaks for turbine installations. Similarly, the fact that paint colours with strong IR reflectance attracted significantly more insects (the turbine paint colour white had an IR peak identical with that of yellow; Fig. 3) indicates IR reflective properties may need to be taken into consideration in addition to visible colour spectrum alone.

As the least attractive colour, purple, had a UV reflectance of 10% and an IR reflectance of 60%, this could provide a useful comparison for turbine paint colour selection in relation to insect attraction under strong light conditions. Seasonal variation in the relative attraction of insects to specific colours (as shown in Fig. 5) could be affected by the seasonality of the different insect species observed (see Table 1). For example, while small and large Diptera, Boxes indicate means with ± standard deviation whiskers. In all cases n represents the number of 10-min sessions performed at that range common prey items for insectivorous wildlife, were present throughout all monthly midday counts and most sunset counts, species belonging to the order Tipulidae were present only in August and September. The variation in attraction of different colours between different insect species was not a factor that was considered in this study, but may account for some degree of seasonal variation. Monthly variation is also likely to be linked with changes in temperature, weather conditions and RH which all have significant interplay with insect activity levels (e.g. [START_REF] Willmer | Thermal constraints on activity patterns in nectarfeeding insects[END_REF][START_REF] Mccall | Influence of flower characteristics, weather, time of day, and season on insect visitation rates in three plant communities[END_REF], which our own data support. It is particularly interesting to note the relationship between total insect count and wind speed, with low 3-4 m s -1 speeds showing the highest activity levels. Previous studies have identified that bat mortality in particular is highest on low wind speed nights (less than 6 ms -1 ) [START_REF] Arnett | Patterns of bat fatalities at wind energy facilities in North America[END_REF][START_REF] Horn | Behavioural responses of bats to operating wind turbines[END_REF]) which could well be correlated with improved foraging opportunities at these lower wind speeds.

It is suggested that future studies investigate the effects of moving colours such as colour cards and painted blades/ turbines on insect attraction. A logical next step would be to paint turbines at known sites of wildlife interaction and investigate frequency of interaction between turbines and insectivores.

In conclusion, our results indicate that the common turbine colours 'Pure White' (RAL 9010) and 'Light Grey' (RAL 7035) may be having a significant influence on the attraction of insects to wind turbine installations, which could in turn be providing foraging opportunities for both diurnal and nocturnal insectivores. Because some colours were demonstrated to be less attractive to insects than others it may therefore be possible to contribute to reducing insect attraction to wind turbines by altering paint colours, both in the visible spectrum and in the UV and IR spectrum. However, it should be made clear that modifying turbine colour alone may not be enough to mitigate the problem of wildlife-turbine interaction and that further research into other aspects such as thermal generation is needed.

Fig. 1

 1 Fig. 1 Local habitat of the study site: a photograph of the site (left) taken in July at midday, and an aerial plan diagram of the immediate area (right), where 'X' denotes the precise location of measurements

Fig. 4

 4 Fig.4Mean total insect count per 10 min period according to month of observation, at both midday and 1 h after sunset

Fig. 5

 5 Fig. 5 Variation in mean insect count for each colour per 10 min period according to month, for both midday (a) and 1 h after sunset (b) observations

Fig. 6

 6 Fig. 6 Effect of environmental conditions on total mean insect count per 10 min period, including wind speed (a), temperature (b) and relative humidity (c). Boxes indicate means with ± standard deviation whiskers. In all cases n represents the number of 10-min sessions performed at that range

Table 1

 1 Insect species observed over the data collection period

	Month	Insect species (midday)	Insect species (sunset)
	June	Small Diptera (body size <5 mm), large Diptera (body size ≥5 mm),	Orthoptera, Hemiptera, Coleoptera
		Hemiptera, Orthoptera
	July	Small Diptera (<5 mm), large Diptera (≥5 mm), Thysanoptera,	Small Diptera (<5 mm), Hemiptera
		Coleoptera, Lepidoptera, Hymenoptera, Hemiptera, Orthoptera
	August	Small Diptera (<5 mm), large Diptera (≥5 mm), Thysanoptera,	Small Diptera (<5 mm), large Diptera (≥5 mm), Coleoptera,
		Lepidoptera, Hymenoptera, Hemiptera	Lepidoptera, Thysanoptera, Hemiptera, Tipulidae
	September Small Diptera (<5 mm), large Diptera (≥5 mm), Tipulidae,	Small Diptera (<5 mm), large Diptera (≥5 mm), Hemiptera,
		Thysanoptera, Hemiptera, Hymenoptera, Coleoptera	Thysanoptera, Coleoptera, Tipulidae
	October	Small Diptera (<5 mm), large Diptera (≥5 mm), Hemiptera	Small Diptera (<5 mm), large Diptera (≥5 mm), Hemiptera,
			Coleoptera

Eur J Wildl Res (2011) 57:323-331

Acknowledgements The authors would like to thank Brocks Hill Environment Centre and Oadby and Wigston Borough Council for granting permission to use the study area. We are also grateful to Dr. S. Williams-Worrall of the Department of Chemistry, Loughborough University, for assistance with spectrometer measurements.


Radka Ležalová-Piálková

R.Ležalová-Piálková 1 MOLECULAR EVIDENCE FOR EXTRA-PAIR PATERNITY AND INTRASPECIFIC BROOD PARASITISM IN THE BLACK-HEADED GULL

Keywords: Black-headed Gull, genetic mating system, extra-pair paternity, intraspecific brood parasitism, Larus ridibundus Zusammenfassung Molekularer Nachweis von Fremdvaterschaft und intraspezifischem Brutparasitismus bei der Lachmöwe Larus ridibundus Soziale Monogamie ist bei Vögeln weitverbreitet, während genetische Monogamie vermutlich selten ist untersucht, bei der, wie bei Seevögeln und den meisten anderen langlebigen und sozial R.Ležalová-Piálková 3 anderen verwandten Arten mittlere ISBP-Rate und eine hohe EPP-Rate erkennen und zeigen, dass Lachmöwen erfolgreich Kopulationen außerhalb des Paarbundes eingehen

Social monogamy is common among birds, while genetic monogamy is supposed to be rare. I investigated the genetic mating system of the Black-headed Gull Larus ridibundus, where, as in seabirds and most other long-lived and socially monogamous birds, extrapair paternity (EPP) is typically rather infrequent. Parentage was determined using six microsatellite markers for 79 chicks from 30 broods. In this study population, I found evidence of allelic inconsistencies between putative parents and chicks in 43% of nests, resulting from both EPP and intraspecific brood parasitism (ISBP). Extra-pair paternity was detected in 33 % (10/30) of broods, and 20% (16/79) of all nestlings were sired by extra-pair males. Furthermore, 9% (7/79) of chicks out of 5 nests (17%) were not the offspring of either member of the pair, indicating ISBP. These findings reveal a moderate rate of ISBP and a high rate of EPP compared with other related species, and shows that Black-headed Gulls successfully participate in extra-pair copulations.

Introduction

The application of molecular techniques to avian mating systems has revealed striking variation in the incidence of alternative reproductive strategies such as extra-pair paternity (EPP) and intraspecific brood parasitism (ISBP) [START_REF] Birkhead | Sperm competition and sexual selection[END_REF]Petrie and Kempenaers 1998;[START_REF] Arnold | Extra-pair paternity and egg dumping in birds: life history, parental care and the risk of retaliation[END_REF]. Even though many hypotheses have been proposed to explain this variation (Petrie and Kempenaers 1998;Møller and Ninni 1998;[START_REF] Arnold | Extra-pair paternity and egg dumping in birds: life history, parental care and the risk of retaliation[END_REF][START_REF] Griffith | Extra pair paternity in birds: a review of interspecific variation and adaptive function[END_REF]Westneat and Stewart 2003;Neudorf 2004), there is still inconsistency in the results. The strongest correlation with EPP was found phylogenetic. High EPP rates are associated with high rates of adult mortality and reduced parental care, while high ISBP rates are associated with high fecundity rates [START_REF] Arnold | Extra-pair paternity and egg dumping in birds: life history, parental care and the risk of retaliation[END_REF][START_REF] Bennett | Evolutionary Ecology of Birds Oxford[END_REF]). Variation at the population or individual level is more likely to be based on differences in current ecological and genetic factors (Petrie and Kempenaers 1998). Of ecological factors, breeding density may be an important factor at the species level (Westneat and Sherman 1997). Colonially nesting species are predicted to have high EPP rates because proximity to potential partners is high (Møller and Birkhead 1993).

The Black-headed Gull, like other gulls, is a long lived, colonially breeding species with high adult survivorship [START_REF] Cramp | The birds of the western palearctic[END_REF]. The standard clutch size of this species is three eggs. Both females and males participate in parental care throughout incubation and chick-rearing, which leads to social monogamy in this species [START_REF] Cramp | The birds of the western palearctic[END_REF]. Given these traits, a low frequency of EPP and ISBP could be expected.

The major goal of this study was to describe the rates of extra-pair parentage and intraspecific parasitism in the Black-headed Gull.

Materials and methods

Field methods and sampling

The study was carried out in a colony of about 3 000 pairs of Black-headed Gulls at the Jarohněvický pond in the south-eastern part of the Czech Republic (48˚55΄N 17˚05΄E).

Thirty families (chicks and both parents) were sampled during the breeding season 2004.

Adults were trapped with "walk-in-nest-traps" at randomly chosen nests from different parts of the colony, including both the centre and edges, and ringed with metal rings for identification. A sample of blood (approximately 50 μl) was taken from the tarsal vein of each trapped individual and mixed with 150 μl of storage buffer (0.5M EDTA), for later sex determination and paternity analysis.

The distribution of clutch sizes in the thirty sampled nests size was: 4 eggs (n=1), 3 eggs (n=20), 2 eggs (n=7) and one egg (n=2). Eggs were individually marked with waterproof ink on the shells, according to laying sequence. Each of the studied nests was enclosed within a 1.6 cm mesh wire circle about 1 m in diameter and 40 cm high. This fence prevented the escape of young chicks but allowed adults to access the nests.

On the first day after hatching, approximately 25 μl of blood was obtained from the brachial or tarsal vein of chicks and mixed with 150 μl of storage buffer (0.5M EDTA).

Samples of embryos were collected from unhatched eggs. All samples were kept at -20°C until DNA extraction.

Molecular determination of sex

The sex of social parents was determined by a polymerase chain reaction that amplifies an intron of the CHD1 genes on the sex chromosomes [START_REF] Griffiths | A DNA test to sex most birds[END_REF].

Genomic DNA was isolated from 10 μl of blood sample using the DNA Lego Kit (Top Bio) following the manufacturer's protocol. The avian sexing primers 2550 F and 2718 R [START_REF] Fridolfsson | A simple and universal method for molecular sexing of non-ratite birds[END_REF] were used in 10 μl PCR reactions. The PCR conditions were as follows: 1μl of 10X PCR buffer (TaKaRa), 2 μM of each dNTP, 2.5 pmol of each primer and 0.5 U of Taq polymerase (TaKaRa). About 150 ng of genomic DNA was used as template. PCR was performed according to [START_REF] Griffiths | A DNA test to sex most birds[END_REF] in a T3 Biometra thermal cycler. The products were separated by electrophoresis for 45-60 min at 7-10 V/cm using a 3% agarose gel stained with ethidium bromide.

Parentage analyses

Microsatellite typing was used to reveal the genetic relationships between parents and young. Parentage was analysed using 6 polymorphic dinucleotide microsatellite loci (RBG 13,RBG 18,RBG 20,RBG 27,RBG 28,RBG 29) originally developed for the Red-billed Gull Larus novaehollandiae scopulinus [START_REF] Given | Isolation of polymorphic microsatellite loci from the red-billed gull (Larus novaehollandiae scopulinus) and amplification in related species. R.Ležalová-Piálková 11[END_REF]. These six loci exhibited little or no evidence of null alleles and showed sufficient levels of polymorphism (mean heterozygosity = 0.77, mean number of alleles = 11.3), making them suitable for paternity assessment in this species (combined exclusion probability: first parent = 0.98, second parent = 0.998) (Table.1).

Genomic DNA was extracted from 10 μl of blood sample or from 25-35 mg of embryo tissue (depending on type of tissue) from unhatched eggs. The 10 μl PCR reactions consisted of about 100 ng of template DNA, 0.4 units of Taq Polymerase (TaKaRa), 1 μl of 10x PCR buffer (TaKaRa), 2.5 μM dNTPs and 2.5 pmol of each primer.

The forward primer was fluorescently labelled. The PCR conditions followed the protocol described by [START_REF] Given | Isolation of polymorphic microsatellite loci from the red-billed gull (Larus novaehollandiae scopulinus) and amplification in related species. R.Ležalová-Piálková 11[END_REF]. Amplification products were run on an ABI Prism 3100 automatic sequencer, and Gene Mapper software (Applied Biosystems Inc) was used to size the alleles. The number of alleles, observed and expected heterozygosity, parentage exclusion probability for the first and second parents, and theoretical frequency of null alleles for each locus were estimated with CERVUS 3.0.3 software (Kalinowski et al. 2007) (Table. 1). Allele frequencies were used to calculate the cumulative probability of resemblance P RCum for specific parent-offspring cases where ambiguous mismatches occurred [START_REF] Ibarguchi | Male-biased mutation rates and the overestimation of extrapair paternity: Problem, solution, and illustration using thick-billed murres (Uria lomvia, Alcidae)[END_REF]).

Results

I assessed paternity for 79 chicks and 60 adults from 30 broods. Offspring that possessed a microsatellite allele that did not match the putative father at two or more loci were considered extra-pair young (EPY), and offspring that did not share an allele with either of their social parents were considered to be a case of intraspecific brood parasitism. In 17 out of 30 sampled nests (57%), I found no case of allelic mismatch between the offspring and their putative parents. Thus, all social parents were true genetic parents in these nests. However, in 13/30 (43%) nests, allelic inconsistencies between putative parents and chicks were detected, resulting from both EPP and ISBP.

More specifically, in 10/30 (33 %) nests I found that 17/79 (21.5%; 95% c.i.: 0.13 -0.32) chicks shared the same allele at each locus with their mother, but were mismatched with the social father in at least two loci (Table .2). The number of mismatching loci in extra-pair chicks ranged from two to five loci (Table .3). In three cases, where the chick and its putative father differed only in two loci, I estimated the likelihood that mismatched loci were caused by mutation. Cumulative probabilities of resemblance (P Rcum ; [START_REF] Ibarguchi | Male-biased mutation rates and the overestimation of extrapair paternity: Problem, solution, and illustration using thick-billed murres (Uria lomvia, Alcidae)[END_REF]) were calculated for these three chicks, excluding loci with the mismatched alleles. In two cases, the probabilities of sharing alleles by chance were so high (0.2x10 -3 and 1.3x10 -3 ) that mismatches due to mutation could be ruled out. In the third case, the cumulative probability of resemblance was much lower (5x10 -6 ), suggesting that this was a case of mutation and the chick could be reassigned as legitimate. I considered the remaining 16/79 (20%; 95% c.i.: 0.12-0.31) chicks to be extra-pair, since in all cases social mothers were also true genetic mothers.

Moreover, in two of the ten nests with EPY and in three other nests I found chicks which did not match either of their social parents. Altogether 7/79 (9%; 95% c.i.: 0.04-0.17) chicks out of 5/30 (17%) nests were considered to result from intraspecific brood parasitism (Table .2). The frequency of mismatching loci in these parasitic chicks ranged from 3 to 6 loci (Table .3).

Finally, in another five chicks I detected differences between chicks and parents at a single locus; in four cases chicks differed from their social father and in one case the chick mismatched at one locus with their mother. The cumulative probabilities of resemblance were calculated for these five chicks, excluding loci with the mismatched alleles. In four of five cases, probabilities of sharing alleles by chance were so low (4.08 x 10 -6 to 7.01x10 -7 ) that mutation is the most probable reason for the mismatches. In the last case, the probability of sharing alleles was higher (1 x 10 -2 ) due to the male and chick sharing common alleles (Table .3). All those five chicks were considered as within pair offspring.

Discussion

Black-headed Gulls in the study population successfully participate in extra-pair copulations -33% of nests contained at least one extra-pair young (EPY). This level of strategy of gulls. In five (17%) out of 30 sampled nests, at least one chick was found that did not match either of their social parents. This rate of ISBP should not be affected by possible chick adoption, since the studied nests were enclosed with mesh wire and chicks were sampled within a couple of hours after hatching. Multiple parentage in these clutches most probably occurred through nest parasitism. The possibility that some of the mixed clutches represented female-female pairs or quasi-parasitism can be excluded, since the social parents trapped at each nest were always a male and female, and neither of them matched potentially parasitic chicks.

Among 61 species of gulls, intraspecific parasitism has been documented in another four species (Larus argentatus, L. canus, L. delawarensis and L. minutus;in Yom-Tov 2001 and[START_REF] Duda | Multiple maternity in blackheaded gull Larus ridibundus clutches as revealed by protein fingerprinting[END_REF] as well as in the Black-headed Gull (Duda et al. 2008, Ležalová-Piálková and[START_REF] Ležalová-Piálková | Responses of black-headed gulls Larus ridibundus to conspecific brood parasitism[END_REF]. [START_REF] Duda | Multiple maternity in blackheaded gull Larus ridibundus clutches as revealed by protein fingerprinting[END_REF] revealed the maternity in a population of Black-headed Gulls in Poland by protein fingerprinting, and found multiple maternity in 34% of nests with >1 egg. These results are much higher then those obtained from a previous study of ISBP based on the regular monitoring of nests in the same Black-headed Gull population as the current study, [START_REF] Ležalová-Piálková | Responses of black-headed gulls Larus ridibundus to conspecific brood parasitism[END_REF]. In this previous analysis, we found a parasitism rate of 10%, which now seems to be an underestimation. These studies allow an interesting comparison between three different methodological approaches to the question of ISBP, showing that a molecular approach is essential for finding the real frequencies of ISBP in birds [START_REF] Grønstøl | The importance of genetic evidence for identifying intra-specific brood parasitism[END_REF].

  Mills JA (1994) Extra-pair copulations in the red-billed gull-females with high quality, attentive males resist. Behaviour 128:41-64 Møller AP, Birkhead TR (1993) Cuckoldry and sociality: a comparative study of birds. Am Nat 142:118-131 Møller AP, Ninni P (1998) Sperm competition and sexual selection: a meta-analysis of paternity studies of birds. Behav Ecol Sociobiol 43:345-358 Neudorf DLH (2004) Extra pair paternity in birds: understanding variation among species. Auk 121:302-307 Petrie M, Kempenaers B (1998) Extra-pair paternity in birds: explaining variation between species and populations. Trends Ecol Evol 13:52-58 Westneat DF, Sherman PW (1997) Density and extra-pair fertilization in birds: a comparative study. Behav Ecol Sociobiol 41:205-215 Westneat DF, Stewart IRK (2003) Extra pair paternity in birds: Causes, Correlates and Conflict. Ann Rev Ecol Evol Syst 34: 365-396 Yom-Tov Y (2001) An update list and some comments on the occurrence of intraspecific nest parasitism in birds. Ibis 143:133 -143

Table 1

 1

Table . 1

 . Polymorphism data for six microsatellite markers in the Black-headed Gull.Values are based on the genotypes of 60 parents and calculated using CERVUS 3.0.3. kthe number of alleles, size (bp)-range of allele size in base-pairs, He -expected heterozygosity, Ho -observed heterozygosity, F null -estimated frequency of the null allele, NE-1P -non-exclusion probability for the first parent, NE-2P -non-exclusion probability for the second parent.

							R.Ležalová-Piálková
	RBG 18	6	170-180	0.39	0.35	0.09	0.93	0.8
	RBG20	16	169-203	0.92	0.9	0.01	0.29	0.17
	RBG 27	21	185-235	0.91	0.89	0	0.33	0.2
	RBG 28	7	159-173	0.76	0.75	0	0.64	0.46
	RBG 29	9	125-141	0.81	0.9	0.06	0.56	0.38
	Locus	k	size (bp)	He	Ho	Fnull	NE-1P	NE-2P
	RBG 13	9	205-223	0.82	0.86	0.02	0.54	0.36
				13

Table 2 Table . 2

 2. Distribution of chicks with extra-pair paternity (EPP) and parasitic chicks (ISBP) across the nests. n = number of analysed nests.

					R.Ležalová-Piálková
		No. of chicks with extra-	Total	n
		pair parentage in nest	(chick/nests)
		1	2	3
	No. of nests with determined EPP	6	2	2	16/10	30
	ISBP	4	-	1	7/5	30
	Table 3
		14

Table . 3

 . Frequency of chicks with different number of mismatched loci. PR cumcumulative probability of resemblance of sharing alleles by chance for parent-offspring cases with low number of mismatching loci, nd -not defined.

	No. of mismatched loci	No. of chicks PR cum	Classified as
	1	5	1 x 10 -2 to 7.01 x 10 -7	true parentage
	2	1	5 x 10 -6	true parentage
	2	2	0.2 x 10 -3 and 1.3 x 10 -3	EPP
	> 3 (max. 5)	14	nd	EPP
	> 3 (max. 6)	7	nd	ISBP

Acknowledgments

This research was funded by GAJU 51/2003/P-BF and MSM 6007665801 grants and was also supported by GAČR 206/05/H012. This study was carried out under permission 2KCDW 28/0/2003 and in accordance with the laws and ethical guidelines of R.Ležalová-Piálková

EPP is relatively high in comparison to related species and other colonially breeding seabirds (e.g. [START_REF] Mauck | Monogamy in Leach's storm-petrel: DNAfingerprinting evidence[END_REF][START_REF] Gilbert | No evidence of extra-pair paternity in the western gull[END_REF][START_REF] Lorentsen | Molecular evidence for extrapair paternity and female-female pairs in antarctic petrels[END_REF][START_REF] Griggio | No evidence of extra-pair paternity in a colonial seabird, the common tern (Sterna hirundo)[END_REF]). Previously measured variation in EPF rates for Laridae species has been low, ranging from zero in the Western Gull (Larus occidentalis; [START_REF] Gilbert | No evidence of extra-pair paternity in the western gull[END_REF], Blacklegged Kittiwake (Rissa tridactyla; [START_REF] Helfenstein | Low frequency of extra-pair paternity and high frequency of adoption in black-legged kittiwakes[END_REF]), Common Tern (Sterna hirundo; [START_REF] Griggio | No evidence of extra-pair paternity in a colonial seabird, the common tern (Sterna hirundo)[END_REF]) and Little Auk (Alle alle; Lifjeld et al. 2005), to 8.3 % in Common Gull (Larus canus;Bukacinska et al. 1998). These findings are in line with the prediction of low EPP rates in species with a long reproductive lifespan, such as seabirds.

In contrast, my data suggest that Black-headed Gulls have an unexpectedly high rate of EPP. Extra-pair paternity could arise by two means, either as the result of extrapair copulations (EPC), or rapid mate-switching [START_REF] Birkhead | Sperm competition and sexual selection[END_REF]Møller, 1992, Martins et al. 2002). In gulls, EPCs are common (Mills 1994, Bukacinska et al. 1998). In Black-headed Gulls, copulation activity has not yet been studied in detail; [START_REF] Frantová | Mating activity of the black headed-gull (Larus ridibundus)[END_REF] found a low frequency of forced copulations (1.8%), but did not distinguish between within-pair and unforced extra-pair copulations.

I collected no behavioural data on mate-switching, so that I can not exclude the possibility that the two nests where all three chicks were found to be sired by other than the social father (Table .2) could be cases of mate-switching or, alternatively, cases of putative father infertility. The replacement of a mate might happen rapidly due to a large supply of potential mates and strong competition for mates during the early stage of breeding. In the remaining 8 nests, one (5 nests) or two (3 nests) EPYs were detected, probably resulting from successful EPCs (Table .2). However, to unequivocally explain this high rate of EPP, other, especially behavioural studies will be necessary. Furthermore, the accuracy of the estimate could be affected by used sample size [START_REF] Griffith | Extra pair paternity in birds: a review of interspecific variation and adaptive function[END_REF]. [START_REF] Griffith | Extra pair paternity in birds: a review of interspecific variation and adaptive function[END_REF] suggest that approximately 200 offspring is a reasonable sample size limiting the magnitude of error around the estimate. The magnitude of error around my estimate of EPP level in studied population is 19%, which suggest that further sampling would increased the accuracy of the estimate. Moreover, I found a quite high rate of ISBP, especially considering the life-history the Czech Republic. I thank M. Honza for field assistance, J. Bryja for advice in the lab, D. Hardekopf for English corrections and anonymous referees for valuable comments on the manuscript.


Edit Schumacher

Éva Vigh
email: eva.vigh@aok.pte.hu

Valéria Molnár

Péter Kenyeres

Gergely Fehér

Gábor Késmárky

Kálmán Tóth

János Garai

Thrombosis preventive potential of chicory coffee consumption: a clinical study

Keywords: caffeic acid, chicory coffee, hemorheological parameters, macrophage migration inhibitor factor, platelet aggregation caffeic acid, chicory coffee, hemorheological parameters, macrophage migration inhibitor factor, platelet aggregation

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Diverse biological effects of plant polyphenols have been revealed in countless papers published throughout the last decades. Epidemiological studies suggest that consumption of polyphenol-rich food might impede the onset and the progression of certain human diseases [START_REF] Kris-Etherton | Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer[END_REF]. Phenolic compounds are abundant in fruits, vegetables, coffee, wine and olive oil [START_REF] Huang | Phenolic acid content of food plants and possible nutritional implications[END_REF]. The favourable effects of moderate red wine consumption on certain cardiovascular risk factors have been documented [START_REF] Fuhrman | Consumption of red wine with meals reduces the susceptibility of human plasma and low-density lipoprotein to lipid peroxidation[END_REF]. The possible benefits of coffee, the other widely popular polyphenol-rich beverage, still await clarification. The risk of acute coronary syndromes was found to follow a J-shaped trend dependent on the dose of coffee consumed per day [START_REF] Panagiotakos | The J-shaped effect of coffee consumption on the risk of developing acute coronary syndromes: The CARDIO2000 case-control study[END_REF]. The upsurge with higher doses might be attributable to elevated blood pressure caused by the caffeine content of coffee brewed from Coffea arabica beans. Nevertheless, moderate consumption conferred decreased risk compared with non-consumers. The antithrombotic effect of coffee has been reported to be independent from its caffeine, but rather to be attributable to its phenolic acids able to incorporate into platelets [START_REF] Natella | Effect of coffee drinking on platelets: inhibition of aggregation and phenols incorporation[END_REF]. The influence of coffee consumption on lifetime cardiovascular risk has been analyzed recently in two large cohorts. A dose dependent protective effect has been found that seems to be more pronounced in female consumers [START_REF] Lopez-Garcia | The relationship of coffee consumption with mortality[END_REF][START_REF] Lopez-Garcia | Coffee consumption and risk of stroke in women[END_REF]. These reports also found the protective effect independent from caffeine.

A cup of coffee is an abundant source of chlorogenic acid, i.e. caffeic acid esterified with quinic acid. Caffeic acid becomes available for absorption following hydrolysis. Known to be absorbed almost totally (~95%) from the small intestine caffeic 4 acid reaches its peak blood level within 2 hours after oral intake [START_REF] Olthof | Chlorogenic acid and caffeic acid are absorbed in humans[END_REF][START_REF] Nardini | Absorption of phenolic acids in human after coffee consumption[END_REF].

Macrophage migration inhibitory factor (MIF) has been the first nonimmunoglobulin immune-mediator described [START_REF] Bloom | Mechanism of a reaction in vitro associated with delayedtype hypersensitivity[END_REF][START_REF] David | Delayed hypersensitivity in vitro: its mediaton by cell-free substances formed by lymphoid cell-antigen interaction[END_REF].

The basal serum concentration of this proinflammatory cytokine is 3-5 ng/mL [START_REF] Metz | Role of macrophage migration inhibitory factor in the regulation of the immune response[END_REF]. Its level rises in infectious or non-infectious inflammatory conditions such as sepsis [START_REF] Bernhagen | MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia[END_REF] or rheumatoid arthritis [START_REF] Onodera | High expression of macrophage migration inhibitory factor in the synovial tissues of rheumatoid joints[END_REF]. Enzymatic tautomerase [START_REF] Rosengren | The immunoregulatory mediator macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction[END_REF][START_REF] Rosengren | The macrophage migration inhibitory factor MIF is a phenylpyruvate tautomerase[END_REF] and thiol-protein oxidoreductase (Kleemann et al., 1998) activities of MIF have been revealed recently. Certain antiinflammatory phytochemicals inhibit the tautomerase activity in a concentration dependent manner [START_REF] Molnar | Plant-derived anti-inflammatory compounds affect MIF tautomerase activity[END_REF]. Among these molecules caffeic acid exhibits one of the best inhibitory potential concerning either phenylpyruvate-or dopachrome-tautomerase activities [START_REF] Molnar | Plant-derived anti-inflammatory compounds affect MIF tautomerase activity[END_REF][START_REF] Senter | Inhibition of macrophage migration inhibitory factor (MIF) tautomerase and biological activities by acetaminophen metabolites[END_REF]. Although the exact role of the enzymatic activity of this cytokine remains to be delineated MIF tautomerase has already attained a reputation as a promising pharmacologic target in inflammatory conditions [START_REF] Lubetsky | The tautomerase active site of macrophage migration inhibitory factor is a potential target for discovery of novel antiinflammatory agents[END_REF]. Caffeic acid and its derivatives have been reported to inhibit platelet aggregation in vitro and in vivo [START_REF] Hung | Evaluation of caffeic acid amide analogues as anti-platelet aggregation and anti-oxidative agents[END_REF][START_REF] Hsiao | Characterization of a novel and potent collagen antagonist, caffeic acid phenethyl ester, in human platelets: In vitro and in vivo studies[END_REF] -an effect confirmed in our laboratory as well.

Chicory (Cichorium intybus) is one of the richest dietary sources of caffeic acid and its derivatives e.g. chlorogenic acid. An alcoholic (40%) extract (1:5) of the whole plant has been reported to contain 12.98 ± 0.06 g% of caffeic acid derivatives [START_REF] Kocsis | Effects of chicory on pancreas status of rats in experimental dislipidemia[END_REF]. Of note, coffee brewed from ground roasted chicory roots does not contain caffeine and has a long history being used as coffee substitute or admixture [START_REF] Galasko | The caffeine contents of non-alcoholic beverages[END_REF]. In our present study we have addressed the clinical effects of chicory coffee

MATERIALS AND METHODS

Study population

Our clinical study has been approved by the local Regional Ethical Committee according to the actual amended version of the World Medical Association Declaration of Helsinki. Written informed consent has been obtained from all participants.

In our self-controlled study 27 healthy volunteers (13 women, 14 men) were recruited (mostly university students). The average age was 23 ± 0.4 years. Five of them were smokers and 17 of them were habitual coffee drinkers. The volunteers were asked to refrain from consuming (Arabic) coffee or tea one week before and during the whole period of the study, because these also contain caffeic acid. If it became unavoidable caffeine pills were provided. No vitamins or drugs were allowed to be taken one week before and during the study. The volunteers were also asked to refrain from alcohol consumption.

Intervention and sample collection

The volunteers consumed 300 mL coffee in the morning each day prepared from 20g (3 tablespoons) ground chicory coffee throughout the one week study period. We aimed to observe the effects of both, a single dose and a week long daily consumption of chicory coffee on the following parameters: platelet aggregation, hemorheological factors, fibrinogen and MIF concentration. Blood samples were collected from 27 6 volunteers on the first day just before and 2 hours after the first coffee consumption in fasting conditions. 24 volunteers also donated blood samples on the eighth day following one week daily chicory coffee consumption. The remaining 3 participants were dropouts. They were unable to consume daily the requested amount of coffee because of taste aversion.

Statistical analysis

Results are expressed as mean ± SEM. All parameters were normally distributed.

Statistical comparisons between 2 time points were performed by one-way Student's ttest. The ANOVA Repeated Measures analysis was applied if the parameter was measured at 3 different time points during the study. p-values < 0.05 were accepted as statistically significant.

Platelet aggregation

Blood samples were collected from cubital veins in tubes containing 3.8% sodium citrate, and then incubated at 37°C for 20 minutes. Platelet-rich plasma was prepared from venous blood by centrifugation at 900g for 10 minutes. After carefully removing platelet-rich plasma, the remaining specimen was further centrifuged at 3600g for 10 minutes to obtain platelet-poor plasma. Platelet aggregation was measured in Carat TX-4 platelet aggregometer (Carat Ltd., Hungary). Samples were incubated at 37°C and continuously stirred at 1000 rpm during the measurement. 50 µL of adenosine diphosphate (ADP) (5 and 10 µM), collagen (2 µg/mL) or epinephrine (10 µM) was added to the platelet-rich plasma to induce platelet aggregation. All measurements were carried out within 2 hours after vein puncture. [START_REF] Toth | Hemorheological and hemodynamical parameters in patients with ischemic heart disease at rest and at peak exercise[END_REF]. Plasma fibrinogen concentration was determined by von Clauss's method.

Red blood cell deformability

Red blood cell (RBC) deformability was determined at various shear stresses by laser diffraction analysis, using an ektacytometer, LORCA (Laser-assisted Optical Rotational Cell Analyzer, RR Mechatronics, Hoorn, Netherland). The system has been described elsewhere in details [START_REF] Hardeman | Laser-assisted optical rotational cell analyzer (LORCA). 1. A new instrument for measurement of various structural hemorheological parameters[END_REF]. Twenty five µl of EDTAanticoagulated blood was suspended in a PBS solution of PVP (Poly-Vinyl-Pyrrolidone 360 kD, Sigma-Aldrich, Budapest, Hungary). The viscosity of the PVP solution was 30 ± 2 mPa x s, all measurements were performed at 37°C [START_REF] Johnson | Ektacytometry of red blood cells. Methods Enzymol 173: 35-54. macrophage migration inhibitory factor (MIF) as thiol-protein oxidoreductase[END_REF]. Elongation indexes were calculated from the diffraction patterns for shear stresses between 0.3-30 Pa: a higher elongation index indicates greater red blood cell deformation.

Serum MIF levels

RESULTS

Caffeic acid inhibits platelet aggregation in vitro [START_REF] Hung | Evaluation of caffeic acid amide analogues as anti-platelet aggregation and anti-oxidative agents[END_REF]. This finding has been confirmed by us as well: 1.2 mM caffeic acid decreased collagen induced platelet aggregation in vitro by 50% (data not shown). 10 µM ADP induced platelet aggregation has increased after chicory coffee consumption at 2 hours and on the 8 th day as well (Fig. 1A). 5 µM ADP induced platelet aggregation did not change significantly (Fig. 1B). Collagen-induced platelet aggregation decreased from baseline at 2 hours (Fig. 1C), but epinephrine-induced aggregation increased (Fig. 1D).

No changes in basic hematologic parameters like hematocrit or red blood cell counts were detected (Table 1). Whole blood viscosity significantly decreased after one week daily chicory coffee consumption (Fig. 2A). At all fluid shear stresses lower than 30 Pa significant improvements were seen there in RBC deformability, an acknowledged determinant of whole blood viscosity (Table 2). A significant decrease in plasma viscosity was evident after one week of chicory coffee consumption (Fig. 2B), while no changes were detected in fibrinogen levels, one of the main determinants of plasma viscosity (Table 1).

Daily consumption of chicory coffee for one week significantly decreased serum MIF levels (Fig. 3). unlikely to be attributable to only one single compound, but rather to an array of agents absorbed from chicory coffee. The inhibitory effect of chicory consumption on platelet aggregation seems to vary with the inductor used. This might be attributable to a particular mechanism by which agents in chicory coffee interfere with platelet aggregation. Nevertheless, in vivo antiplatelet activity was found much weaker than expected from preceding in vitro studies with caffeic acid, one of the most abundant plant phenolic of this beverage. Consumption of chicory coffee for one week, however, significantly improved hemorheological parameters, like plasma-and whole blood viscosity and red blood cell deformability. These changes indicate that daily chicory coffee consumption might be preventive against certain microcirculatory pathologies.

The decrease in whole blood viscosity might largely be attributable to the increased red blood cell deformability, however, the modest but significant decrease in plasma viscosity in the face of unchanged fibrinogen levels appears perplexing. Further studies are warranted to clarify the exact mechanisms and to identify the compounds behind these effects of chicory coffee. [START_REF] Baskurt | Red blood cell deformability in sepsis[END_REF] found significantly decreased RBC deformability in septic states at fluid shear stresses less than 5 Pa in humans and rats. Elevated MIF levels are acknowledged indicators of septic state [START_REF] Bernhagen | MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia[END_REF]. Potato peel extract rich in caffeic acid has been reported to protect erythrocytes against oxidative damage, a known cause of impaired cell deformability [START_REF] Singh | Antioxidant-mediated protective effect of potato peel extract in erythrocytes against oxidative damage[END_REF]. Daily for one week duration substantially decreased serum MIF levels of healthy volunteers in our study in parallel with the improved red blood cell deformability. To decide whether these phenomena are related or are merely coincidental further studies are needed.

Notably MIF upregulates adhesion molecules on endothelial cells [START_REF] Amin | Migration inhibitory factor up-regulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 via Src, PI3 kinase, and NFkappaB[END_REF], hence suppression of MIF levels could be regarded preventive against monocyte adhesion and emigration, a key element in initiation of the atherosclerotic process.

In conclusion, our study constitutes an encouraging starting point to investigate further the antithrombotic, antiinflammatory and beneficial hemorheologic effects of phenolic compounds from chicory coffee.

F

  aggregation, on hemorheological factors and on serum MIF levels.

  Blood samples were collected in tubes containing lithium heparin as anticoagulant. Plasma and whole blood viscosity values were determined with Hevimet 40 capillary viscometer (Hemorex Ltd; Hungary) at 37°C within 2 hours after blood sampling. In this viscosimeter the flow of whole blood (or plasma) is detected optoelectronically along the capillary tube. Apparent whole blood viscosity values were obtained at 90 1/s shear rate

  Blood samples were collected in tubes without anticoagulant. Serum MIF levels were assessed by ELISA with a Duo set ELISA Development System from R&D , MN, US according to the manufacturer's instructions. The amount of MIF was expressed as pmol/mL.

  no side effects during the study. Changes in hemorheological parameters detected in the course of this dietary intervention are

  coffee rich in antiinflammatory phenols (including caffeic acid)

Figure 1 .

 1 Figure 1. Effect of chicory coffee consumption on platelet aggregation. A: Platelet aggregation induced by 10 µM ADP. B: Platelet aggregation induced by 5 µM ADP. C: Platelet aggregation induced by 2 µg/ml collagen. D: Platelet aggregation induced by 10 µM epinephrine. Mean values ± SEM. *p < 0.05, ** p <0.01 1134x815mm (150 x 150 DPI)

Table 1 . Effect of chicory coffee consumption on basic hematologic parameters.

 1

	o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 .

 2 Effect of chicory coffee consumption on red blood cell deformability. The reported elongation indexes represent RBC deformability at different shear stresses.

	Mean values ± SEM.
	Shear stress (Pa)	Control (n=27)	2 hours (n=27)	1 week (n=24)
	0.3	0.0545 ± 0.0018	0.0532 ± 0.0017	0.0594 ± 0.0018** ##
	0.53	0.1094 ± 0.0027	0.1089 ± 0.0024	0.1141 ± 0.0030* #
	0.95	0.2067 ± 0.0027	0.2065 ± 0.0029	0.2120 ± 0.0033** #
	1.69	0.3176 ± 0.0027	0.3177 ± 0.0027	0.3235 ± 0.0028* #
	3	0.4194 ± 0.0019	0.4186 ± 0.0023	0.4240 ± 0.0024* #
	5.33	0.4974 ± 0.0014	0.4966 ± 0.0020	0.5015 ± 0.0019** ##
	9.49	0.5497 ± 0.0013	0.5493 ± 0.0018	0.5533 ± 0.0016** ##
	16.87	0.5961 ± 0.0013	0.5957 ± 0.0016	0.5985 ± 0.0015** ##
	30	0.6276 ± 0.0008	0.6277 ± 0.0011	0.6290 ± 0.0011
	*difference from baseline p < 0.05, **difference from baseline p < 0.01
	#difference from 2 hours p < 0.05, ##difference from 2 hours p < 0.01

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Acknowledgement

We are indebted to the volunteers participating in the study. The complimentary sample of ground roasted chicory provided to this study by Multi-Cikória Kft. Hungary is gratefully acknowledged. The authors have no financial or commercial conflict of interest.