Jérémy S P Froidevaux
email: jeremy.froidevaux@stir.ac.uk

Gareth Jones

Christian Kerbiriou

Kirsty Park

Acoustic activity of bats at power lines correlates with relative humidity: a potential role for corona discharges

Keywords: Chiroptera, corona effect, electromagnetic fields, foraging behaviour, light, noise

With the ever-increasing dependency on electric power, electrical grid networks are expanding worldwide. Bats exhibit a wide diversity of foraging and flight behaviours, and their sensitivity to anthropogenic stressors suggests this group is very likely to be affected by power lines in a myriad of ways. Yet the effects of power lines on bats remains unknown. Here we assessed the responses of insectivorous bats to very high voltage power lines (>220 kV, VHVPL). We implemented a paired sampling design and monitored bats acoustically at 25 pairs, one pair consisting of one forest edge near to VHVPL matched with one control forest edge. Relative humidity mediates the effects of power lines on bats: we detected bat attraction to VHVPL at high relative humidity levels and avoidance of VHVPL by bats at low relative humidity levels. We argue that the former could be explained by insect attraction to the light emitted by VHVPL due to corona discharges while the latter may be due to the physical presence of pylons/cables at foraging height and/or because of electromagnetic fields. Our work highlights the response of bats to power lines at foraging habitats, providing new insight into the interactions between power lines and biodiversity.

Introduction

With the ever-increasing dependency on electric power in modern societies and the recent expanding focus on electrification as part of climate change mitigations [START_REF] Zhang | The role of transport electrification in global climate change mitigation scenarios[END_REF][START_REF] Sugiyama | Climate change mitigation and electrification[END_REF], electrical grid networks are expanding worldwide. Very high-voltage power lines (≥220 kV, VHVPL) traverse over 300,000 km in Europe and the network is expected to grow further. In addition to collision and electrocutions [START_REF] Bernardino | Bird collisions with power lines: State of the art and priority areas for research[END_REF][START_REF] D'amico | Bird on the wire: Landscape planning considering costs and benefits for bird populations coexisting with power lines[END_REF], power lines may negatively affect biodiversity through various mechanisms, ranging from habitat loss and fragmentation [START_REF] Biasotto | Power lines and impacts on biodiversity: A systematic review[END_REF][START_REF] Richardson | A review of the impact of pipelines and power lines on biodiversity and strategies for mitigation[END_REF] to the effects of electromagnetic fields [START_REF] Shepherd | Extremely low frequency electromagnetic fields impair the cognitive and motor abilities of honey bees[END_REF][START_REF] Burda | Extremely low-frequency electromagnetic fields disrupt magnetic alignment of ruminants[END_REF]. In contrast, some species may benefit from the presence of power lines, possibly as a consequence of the altered environmental conditions or the management conducted under power lines [START_REF] Villemey | Can linear transportation infrastructure verges constitute a habitat and/or a corridor for insects in temperate landscapes? A systematic review[END_REF][START_REF] Steinert | Different cutting regimes improve species and functional diversity of insect-pollinated plants in power-line clearings[END_REF]. To date, however, information on the interactions between power lines and biodiversity remains largely limited to birds [START_REF] Biasotto | Power lines and impacts on biodiversity: A systematic review[END_REF].

Because of the wide diversity of foraging and flight behaviours exhibited by bats [START_REF] Denzinger | Bat guilds, a concept to classify the highly diverse foraging and echolocation behaviors of microchiropteran bats[END_REF] and their sensitivity to anthropogenic stressors [START_REF] Jones | Carpe noctem: the importance of bats as bioindicators[END_REF], this taxa is very likely to be affected (either negatively or positively) by power lines in a myriad of ways. Large species, species flying at height of the wires (typical height for VHVPL: ~10-50 m above ground, but varies with topography), and species foraging in open habitats are the most susceptible to barrier effects from VHVPL, which include mortality by collision and electrocution, and site avoidance. For instance, Tella, Hernández-Brito [START_REF] Tella | Urban sprawl, food subsidies and power lines: An ecological trap for large frugivorous bats in Sri Lanka?[END_REF] recently documented the electrocution of 300 Indian flying foxes (Pteropus giganteus) in Sri Lanka while Kahnonitch, Lubin [START_REF] Kahnonitch | Insectivorous bats in semi-arid agroecosystems-effects on foraging activity and implications for insect pest control[END_REF] revealed power line avoidance by the open-space and highflying forager Tadarida teniotis in Israel. Studies assessing the effects of forest logging (e.g. clearcutting) on bats also indicate that habitat modification during the installation and maintenance of power lines could benefit open-and edge-space specialists because of increased habitat availability [START_REF] Kirkpatrick | Responses of bats to clear fell harvesting in Sitka Spruce plantations, and implications for wind turbine installation[END_REF][START_REF] Tena | Mind the gap: effects of canopy clearings on temperate forest bat assemblages[END_REF] but could also negatively affect clutter-adapted species that mainly forage within forest. Furthermore, bats could be affected by less perceptible abiotic impacts of power lines such as corona discharges and electromagnetic fields. Corona dischargean electric discharge produced by the ionization of atmospheric air surrounding the conductorsmainly occurs during wet conditions (relative humidity level >80-90%) with low wind speed (< 2 m/s) [START_REF] Matthews | The effect of weather on corona ion emission from AC high voltage power lines[END_REF]. It results in the production of a hissing noise (see power spectra and spectrograms of the hissing sounds in electronic supplementary material S1) [START_REF] Engel | Audible noise of transmission lines caused by the corona effect: analysis, modelling, prediction[END_REF] and the emission of blue and ultraviolet light over the entire conductor all along the span length (the spark generating these lights occurs at each voltage peak, i.e. ca. 100 times per second in 50 Hz VHVPL with alternative current (AC)) and on insulators [START_REF] Grum | Spectral emission of corona discharges[END_REF] (see spectral composition of corona discharge emission in air in electronic supplementary material S1). Noise may disrupt bat foraging behaviour and deter bats from approaching power lines either because of avoidance [START_REF] Luo | How anthropogenic noise affects foraging[END_REF][START_REF] Finch | Traffic noise playback reduces the activity and feeding behaviour of free-living bats[END_REF], noise-induced distraction [START_REF] Allen | Noise distracts foraging bats[END_REF] and/or auditory masking [START_REF] Siemers | Hunting at the highway: traffic noise reduces foraging efficiency in acoustic predators[END_REF][START_REF] Gilmour | Acoustic deterrents influence foraging activity, flight and echolocation behaviour of free-flying bats[END_REF]. Masking may be more pronounced in species that rely on listening for prey-generated sounds to glean prey from substrates, especially if low frequency noise overlaps with the frequency hearing sensitivity of the bats. In contrast, corona discharges produce blue and UV light that can attract insects sensitive to these short wavelengths [START_REF] Barghini | UV radiation as an attractor for insects[END_REF][START_REF] Brehm | Moths are strongly attracted to ultraviolet and blue radiation[END_REF][START_REF] Van Langevelde | Effect of spectral composition of artificial light on the attraction of moths[END_REF]. Insect attraction may, in turn, attract 'light tolerant' insectivorous bats to power lines, as is the case for streetlamps, especially those that emit short wavelength light [START_REF] Lewanzik | Transition from conventional to light-emitting diode street lighting changes activity of urban bats[END_REF]. Species that use magnetic cues are particularly affected by EMF generated by power lines [START_REF] Barnes | Handbook of biological effects of electromagnetic fields[END_REF] and this is the case of many bat species which use Earth's magnetic fields for homing, roosting and foraging [START_REF] Holland | Bat orientation using Earth's magnetic field[END_REF][START_REF] Tian | A magnetic compass guides the direction of foraging in a bat[END_REF][START_REF] Wang | Bats respond to polarity of a magnetic field[END_REF]. Power lines generate extremely low frequency EMFs (50-60 Hz) but also EMFs at higher frequency (mainly between 150 kHz to 30 MHz) when corona discharges occur [START_REF] Santos | Electromagnetic Spectrum of the Corona Discharge and Their Fundamental Frequency[END_REF]. EMFs could exert avoidance responses in bats and disrupt foraging behaviour as documented for other mammals [START_REF] Balmori | The incidence of electromagnetic pollution on wild mammals: A new "poison" with a slow effect on nature?[END_REF] and as also observed in bats at the much higher frequencies emitted by radar [START_REF] Nicholls | Bats avoid radar installations: could electromagnetic fields deter bats from colliding with wind turbines[END_REF][START_REF] Nicholls | The aversive effect of electromagnetic radiation on foraging bats-a possible means of discouraging bats from approaching wind turbines[END_REF] (but see [START_REF] Gilmour | Comparing acoustic and radar deterrence methods as mitigation measures to reduce human-bat impacts and conservation conflicts[END_REF]).

Since bats are expected to respond either positively or negatively to power lines depending on foraging guild, the net effect of power lines on bat communities is not obvious and has not been assessed. In this study, we examined the responses of insectivorous bats to VHVPL in the field. The aim was to assess the potential effects of VHVPL on bat activity and foraging intensity while controlling for the landscape context. We tested the hypothesis that bats would avoid power lines (i.e. lower bat foraging activity at foraging habitats near power lines). Four potential non-exclusive mechanisms for avoidance are (i) the physical presence of these structure (pylons and cables) at foraging height which may affect high-flying species and open-space foragers; (ii) exposure to electromagnetic fields that may disrupt foraging behaviour; (iii) noise caused by corona discharges, especially for passive-listening bats, and/or (iv) corona light that may deter light-sensitive species due to high perceived predation risk (Table 1). We also tested a contrasting hypothesis that light emitted by VHVPL due to corona discharge would attract light-tolerant bats to VHVPL (Table 1). More specifically, we predicted higher bat activity and foraging intensity near VHVPL for light-tolerant bat species during wet conditions (i.e. when corona discharges occur) because of insect aggregation. This is the first study we are aware of to examine the response of bats to power lines, providing new insight into the interactions between power lines and biodiversity.

Methods

(a) Sampling design

We applied a paired sampling design to investigate the effects of VHVPL on bat activity and foraging intensity. The study was conducted in the Eastern part of France, in Doubs and Jura counties (electronic supplementary material S1). We monitored bats between June and August (i.e. seasonal peak of bat activity) at 25 pairs of sites over two years (2017: N=10; 2021: N=15). Each pair consisted of one forest edge near to VHVPL (< 10 m; hereafter referred to as 'treatment site') matched with one control forest edge (hereafter referred to as 'control site'). Forest edges were adjacent to an agricultural field (pasture or meadow). We selected forest edges as our sampling sites since they are used frequently as foraging and commuting habitats for a wide range of bat species in the study area. Treatment and control sites within each pair were matched at the local scale in terms of altitude and forest composition and at larger scales in terms of landscape composition, configuration and diversity (electronic supplementary material S2). We aimed at selecting pairs that were separated by a minimum distance of 1000 m from each other (median of minimum distances between pairs: 2793 m, range: 972-7371 m). Sites within pairs were separated by distances between 300 and 1500 m (median: 581 m). Control sites were at least >200 m from any VHVPL (range: 241-981 m). Pairs were located along six aerial transmission AC power lines, including one with maximum voltage of 225 kV (N=2 pairs) and five of 400 kV (N=23). General information on electromagnetic field levels generated by power lines as well as sound measurements and spectral emission of corona discharges can be found in electronic supplementary material S1.

(b) Acoustic analysis

We sampled bats acoustically using SM2BAT+ recorders (sampling rate: 384 kHz; Wildlife Acoustics, Concord, USA; electronic supplementary material S3). Sites within each pair were sampled simultaneously during two to three consecutive nights, from 30 min before sunset to 30 min after sunrise. We sampled between one and four pairs per night simultaneously, representing a total of 17 and 16 sampling nights in 2017 and 2021, respectively. Sampling took place during warm nights (> 10°C) with low wind speed (< 10 km/h) and no rain but with varying relative humidity levels, ranging from 52 to 99% (electronic supplementary material S4). Weather conditions were retrieved from the nearest weather station (<10 km; https://www.meteociel.fr/) and averaged over the entire night.

As the aim of the study was to assess the effects of VHVPL on both bat activity and foraging intensity, we used bat sound recordings to calculate these response metrics. More specifically, we used the number of bat passes recorded per night as a measure of bat activity and used the bat sequence duration to get information on bat foraging intensity [START_REF] Kerbiriou | Potential of bat pass duration measures for studies of bat activity[END_REF]. We defined a bat pass as one or more echolocation calls recorded during a fixed interval of five seconds [START_REF] Millon | Bat activity in intensively farmed landscapes with wind turbines and offset measures[END_REF][START_REF] Stahlschmidt | Bats as bioindicators-the need of a standardized method for acoustic bat activity surveys[END_REF][START_REF] Barré | Accounting for automated identification errors in acoustic surveys[END_REF]. The fixed interval allowed to standardize the measure of bat activity among bat species. A bat sequence duration was calculated as the duration of a series of echolocation calls with interpulse intervals <2 s within one or several consecutive bat passes of the same species or group of species (electronic supplementary material S5).

We automatically identified each bat pass to the lowest taxonomic level (i.e. species or species group) using the Tadarida toolbox [START_REF] Bas | Tadarida: A toolbox for animal detection on acoustic recordings[END_REF] which provides a confidence index associated to each bat sequence identification. We then followed recommendations from Barré et al. Barré,Le Viol [41] to account for potential automated identification errors. Thus, we used the confidence index to retain two separate datasets: (i) one dataset of bat passes with a score ≥ 0.90 (i.e. with maximum error risk tolerance of 10%); and (ii) another dataset of bat passes with a score ≥ 0.50 (i.e. with maximum error risk tolerance of 50%). The former threshold is conservative and minimises the inclusion of false positives while the latter is less cautious but retains a larger quantity of data. We conducted the statistical analyses on the dataset of bat passes with a score ≥ 0.50 and checked for result consistency and robustness with the other dataset [START_REF] Barré | Accounting for automated identification errors in acoustic surveys[END_REF].

We computed the community weighted mean bat sequence duration (CWMBSD)a metric related to foraging intensity at the bat community levelas follows:

(1

)
where n is the total number of species or species group recorded, a ij is the number of bat sequences of the species or species group at a given site-night combination j, and MBSD ii is the mean bat sequence duration of the species or species group at a given site-night combination j. Beforehand, a ij and MBSD ii were scaled with minimum = 0 and maximum = 1 as these metrics are not directly comparable on their original scales between species or species group (notably because detection and abundance vary among species). Longer bat sequences (i.e. higher values of CWMBSD) would indicate that a bat is foraging while shorter bat sequences would suggest that a bat is commuting [START_REF] Kerbiriou | Potential of bat pass duration measures for studies of bat activity[END_REF].

The CWMBSD provided a single metric that can inform about overall bat foraging intensity and that is not correlated with other response variables such as bat activity (electronic supplementary material S6).

(c) Landscape analysis

Landscape composition, configuration and diversity are key drivers of bat activity at local scales [START_REF] Fuentes-Montemayor | Species mobility and landscape context determine the importance of local and landscape-level attributes[END_REF][START_REF] Froidevaux | Bat responses to changes in forest composition and prey abundance depend on landscape matrix and stand structure[END_REF][START_REF] Put | Bats respond negatively to increases in the amount and homogenization of agricultural land cover[END_REF][START_REF] Monck-Whipp | Farmland heterogeneity benefits bats in agricultural landscapes[END_REF][START_REF] Froidevaux | Tree size, microhabitat diversity and landscape structure determine the value of isolated trees for bats in farmland[END_REF]. We therefore included landscape variables in our models to control for residual variations. Since bats respond to landscape variables at different spatial scales [START_REF] Fuentes-Montemayor | Species mobility and landscape context determine the importance of local and landscape-level attributes[END_REF][START_REF] Lacoeuilhe | Effects of hedgerows on bats and bush crickets at different spatial scales[END_REF], we created ten buffers of 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, and 5000 m radii around each sampling site using ArcGIS Desktop v10 (ESRI, Redlands, CA, USA). The large scales represent the mean maximum daily foraging movement of European bat species [START_REF] Laforge | Landscape composition and life-history traits influence bat movement and space use: Analysis of 30 years of published telemetry data[END_REF] whereas the small ones allow us to describe the near environment of the sampling sites. Within each buffer, we calculated the amount of deciduous forest, coniferous forest, grassland, cropland and urban area (CES OSO land cover data 2018, 10 m resolution), and computed the density of hedgerows and rivers (IGN BD Haie and BD Carthage, respectively) and distance to the nearest river. We used the "landscapemetrics" R-package to calculate the edge density (landscape configuration) and the Shannon diversity of habitats (landscape diversity).

(d) Statistical analysis

We conducted a series of (generalized) linear mixed-effect models (GLMMs; "glmmTMB" package) to assess the effects of VHVPL on bat activity and foraging intensity. The eleven response variables were the number of bat passes per night for species or group of species (i.e. species-specific bat activity and composite bat activity, ten response variables), as well as the community weighted mean bat sequence duration per night (i.e. bat foraging intensity, one response variable). Composite bat activity refers to the inclusion of species-specific bat activity in a single model to investigate the overall response of bats to VHVPL. Models for bat activity were fitted with a negative binomial error distribution due to over-dispersion and coupled with a logit link function while models for bat foraging intensity were fitted with a Gaussian distribution. We considered site identity nested within pair as random effects because bats were surveyed for several nights and to account for the pairedsampling design. Moreover, we followed recommendations from Oberpriller, de Souza Leite [START_REF] Oberpriller | Fixed or random? On the reliability of mixed-effects models for a small number of levels in grouping variables[END_REF] and added the sampling year as an additional random effect, except for CWMBSD because of model nonconvergence (whether sampling year was included as random or fixed effect). Species identity was added as a random factor in models for composite bat activity to account for non-independence of observations corresponding to the same species [START_REF] Kerbiriou | Disentangling effects of local and landscape variables on attractiveness of restored gravel-sand pits for bat foraging activities[END_REF].

For each response variable we built 10 candidate models (including the null one). We considered three blocks of variables (A: experiment, i.e. VHVPL vs control (categorical variable), B: weather variables (continuous variables), C: landscape variables (continuous variables)) that we included independently (A, B, C), in combination (A+B, A+C, B+C, A+B+C), or in interaction (between blocks A and B only, i.e. A*B, A*B+C) into the models. More specifically, weather variables (block B) comprised the mean temperature at night to account for its well-known positive effect on bat (foraging) activity and relative humidity at night given that corona discharges occur at wet conditions (relative humidity levels >80-90%) with low wind speed (< 7.2 km/h) [START_REF] Matthews | The effect of weather on corona ion emission from AC high voltage power lines[END_REF]. Since bat sampling took place in calm conditions (see section (b) Acoustic analysis and electronic supplementary material S2) we did not consider wind speed as a covariate. Among the nine landscape variables computed at ten spatial scales, only the two most informative ones at their most relevant scale were considered in block C (see electronic supplementary material S7 for landscape variable selection). We only restrained this selection to two landscape variables to avoid collinearity issues and model overparameterization. To test the effects of corona discharges, we included the interaction between the experiment and relative humidity into the models (interaction between blocks A and B).

Thus, the full models were written as follows:

(2)

(3) (4)
All continuous, explanatory variables were standardized prior to their inclusion within the full models so that the regression coefficients were comparable in magnitude. We then applied an information-theoretic approach using the AICc to select the most parsimonious models [START_REF] Burnham | A practical information-theoretic approach. Model selection and multimodel inference[END_REF] and accounted for model uncertainty by computing model averaged predictions and standard errors across best models (ΔAICc < 6) [START_REF] Grueber | Multimodel inference in ecology and evolution: challenges and solutions[END_REF][START_REF] Richards | Model selection and model averaging in behavioural ecology: the utility of the IT-AIC framework[END_REF][START_REF] Burnham | AIC model selection and multimodel inference in behavioral ecology: some background, observations, and comparisons[END_REF]. We determined statistical significance using effect size statistics and their confidence intervals (CIs) [START_REF] Nakagawa | Effect size, confidence interval and statistical significance: a practical guide for biologists[END_REF]. In line with Muff, Nilsen [START_REF] Muff | Rewriting results sections in the language of evidence[END_REF], we considered as weak, moderate and strong evidence when the 85, 95 and 98% CIs did not overlap zero, respectively. We checked for model assumptions, assessed collinearity among predictors and spatial autocorrelation of model's residuals, and validated our models (see details in the electronic supplementary material S8).

Finally, when the interaction between the experiment and relative humidity was significant, we tested the bat activity-relative humidity relationship at control sites, and at power line sites independently, using the "emmeans" package (on the full model). From the same package, we then conducted pairwise comparison of bat activity and foraging intensity between control and power line sites at each extreme value of the relative humidity gradient sampled (i.e. at 52% and 98%) at which we expect absence and presence of corona discharges, respectively. All analyses were conducted in R v4.1.1 [START_REF]R: a language and environment for statistical computing[END_REF] and references of packages used are presented in the electronic supplementary material S9.

Results

(a) Bat sampling

We recorded a total of 87,940 bat passes along 50 forest edges surveyed (117 detector-nights). The most detected species (or species groups) were Pipistrellus pipistrellus with 68,360 bat passes (77.7% of the total bat activity), followed by Eptesicus serotinus (7.7%), small Myotis bats (4.3%, hereafter referred to as 'Myotis spp.' which includes M. alcathoe, M. bechsteinii, M. brandtii, M. daubentonii, M. emarginatus, M. mystacinus, and M. nattereri), Pipistrellus nathusii/kuhlii (4.3%), Pipistrellus pygmaeus/Miniopterus schreibersii (1.6%), Barbastella barbastellus (1.6%), Nyctalus spp. (1.6%), Rhinolophus hipposideros (0.6%), and Myotis myotis/blythii (0.5%, large Myotis bats). We recorded <100 bat passes of Rhinolophus ferrumequinum and Plecotus spp. and therefore disregarded these species for the analysis.

(b) Effects of power lines on bat activity and foraging intensity

We found evidence that eight out of the 11 response variables investigated in this study (i.e. species-specific bat activity, composite bat activity and CWMBSD, a metric related to foraging intensity at the bat community level) responded to VHVPL. For each response variable, between three and ten models were considered as best models after model selection. The null model was, however, retained amongst best models for Myotis spp. activity, composite bat activity and CWMBSD (electronic supplementary material S10).

The interaction between the experiment (VHVPL vs control) and mean relative humidity at night was retained in all sets of best candidate models after model selection (electronic supplementary material S10). Our models revealed a significant interaction (with varying strength of evidence) between mean relative humidity at night and the presence of power lines on bats for 7 out 11 of our response variables (Table 2). Overall, there was lower bat activity and CWMBSD at control sites with increasing relative humidity, but at power line sites these relationships were stable or even positive. This general pattern was especially supported by our results on B. barbastellus and P. pipistrellus activity, composite bat activity and CWMBSD (Figure 1; electronic supplementary materials S11 and S12) as well as on E. serotinus, M. myotis/blythii and P. pygmaeus/M. schreibersii activity, though only with weak support (Table 2, Figure 2, electronic supplementary material S12).

The pairwise comparison of bat activity and CWMBSD between control sites and power lines at the extreme values of the relative humidity gradient further indicated that relative humidity mediates the effects of power lines on bats. For a low relative humidity level (here 52%) at which no corona discharges are expected we found avoidance of power lines by batsi.e. lower bat activity and CWMBSD at power lines compared to control sites (Figure 3). Conversely, for a high relative humidity level (here 98%) at which corona discharges occur, we found bat attraction to power lines with higher activity CWMBSD at power lines compared to control sites (Figure 3). This general pattern was detected for most response variables but with varying strength of evidence (Figure 3). Furthermore, we found moderate evidence that Nyctalus spp. activity was negatively affected by the presence of very high voltage power lines (Table 2, Figure 4), irrespective of meteorological conditions. Nyctalus spp. activity was almost twofold lower (i.e. 46% reduction) at forest edges located near power lines compared to matched control sites. No evidence for an effect of power lines on Myotis spp., P. nathusii/kuhlii, and R. hipposideros activity was detected (Table 2).

Finally, when comparing model outputs between the two bat datasets (i.e. datasets with acoustic data identified at the 50% and 10% error risk tolerance, respectively), influential variables showed consistent patterns (electronic supplementary material S13). We are therefore confident in our results as they are not sensitive to the rate of error risk tolerance.

(c) Effects of landscape variables on bat activity and foraging intensity

Landscape variables were retained in all most parsimonious models (electronic supplementary material S9) and had significant effects on species-specific bat activity but no effect on CWMBSD (a metric related to foraging intensity at the bat community level) and composite bat activity (electronic supplementary material S13). The effect of landscape compositional heterogeneity (i.e. Shannon diversity of habitats) at broad spatial scale (≥ 2 km radius scale) was always positively associated with bat activity, including E. serotinus, M. myotis/blythii, P. nathusii/kuhlii, and P. pipistrellus. The density of hedgerows was the most selected landscape variable in models on species-specific bat activity (present in six out of nine models) but had contrasting effects: the activity of B. barbastellus, R. hipposideros, and P. pygmaeus/M. schreibersii increased with hedgerow density while the opposite was true for M. myotis/blythii and Nyctalus spp. and no significant effect was found for Myotis spp.

Additionally, the amount of coniferous forest at 4 km radius scale had a negative effect on both B. barbastellus and P. pipistrellus nathusii/kuhlii activity whereas R. hipposideros was less active with increasing deciduous forest cover at the small spatial scale (50 m radius scale). The density of river at 5 km radius scale was positively related with the activity P. pipistrellus and P. pygmaeus/M. schreibersii. Surprisingly, grassland cover had negative effects on open-space forager activity (E. serotinus and Nyctalus spp.).

Discussion

Our field experiment shows conclusively that bat activity and foraging intensity at foraging habitats are affected by the presence of very high voltage power lines (VHVPL). Overall, our results indicate that relative humidity mediates the effects of power lines on bats as we detected bat attraction to power lines at high relative humidity levels (i.e. when corona discharges occur) and avoidance of power lines by bats at low relative humidity levels (i.e. when no corona discharges are expected).

While the underlying mechanisms remain to be tested, the former result is consistent with expectations from our hypothesis that light emitted by VHVPL due to corona discharges would attract insects and therefore increase bat foraging intensity near VHVPL. From the four potential nonexclusive mechanisms that could explain power line avoidance by bats, our results suggest that any negative effects of VHVPL on bats are most likely due to the physical presence of the power lines and/or exposure to extremely low frequency electromagnetic fields. Noise, light and high frequency EMFs arising from corona discharges seemed to play no role in explaining avoidance of power lines by bats.

We found that relative humidity exacerbated bat activity and community-level foraging intensity at foraging habitats near power lines compared to control sites. Among many other factors, corona discharges at power lines mainly occur during wet conditions [START_REF] Matthews | The effect of weather on corona ion emission from AC high voltage power lines[END_REF] and result in the emission of UV and blue light with peaks within the range of 230-440 nm [START_REF] Grum | Spectral emission of corona discharges[END_REF]. The so-called 'corona light' has shown to be responsible of power lines avoidance by reindeer [START_REF] Tyler | Cryptic impact: visual detection of corona light and avoidance of power lines by reindeer[END_REF], but given its spectrum it may attract nocturnal insects [START_REF] Barghini | UV radiation as an attractor for insects[END_REF][START_REF] Brehm | Moths are strongly attracted to ultraviolet and blue radiation[END_REF] and thus bats. Interestingly, we found attraction to power lines in both light-tolerant (e.g. P. pipistrellus) and light-sensitive (e.g. B. barbastellus) bat species and no attraction or avoidance in other light-sensitive bats present in our study area including species (e.g. Myotis spp.) that may perceive UV and short wavelength blue light [START_REF] Gorresen | Ultraviolet vision may be widespread in bats[END_REF][START_REF] Jones | From the ultrasonic to the infrared: molecular evolution and the sensory biology of bats[END_REF]. While we did not specifically test the corona effect on insect prey, previous studies have highlighted clear positive relationships between insect abundance and bat (foraging) activity [START_REF] Kolkert | Insectivorous bats foraging in cotton crop interiors is driven by moon illumination and insect abundance, but diversity benefits from woody vegetation cover[END_REF][START_REF] Froidevaux | Managing hedgerows for nocturnal wildlife: do bats and their insect prey benefit from targeted agri-environment schemes[END_REF][START_REF] Blažek | Bat aggregational response to pest caterpillar emergence[END_REF]. Furthermore, our results not only suggest changes in bat activity but also changes in bat behaviour with increased foraging intensity near power lines when corona discharges occur (i.e. at high relative humidity levels), thus implying that changes in bat activity mirror bat responses to their insect prey. Direct measurements of insect abundance in relation to corona discharges are however needed to confirm the process involved. Indeed, other factors such as high frequency EMFs due to corona discharges could also be at play (e.g. by disrupting sensory orientation of bats), but so far the only studies assessing the effects of electromagnetic fields on bats have suggested either a negative effect on bat activity and foraging intensity [START_REF] Nicholls | Bats avoid radar installations: could electromagnetic fields deter bats from colliding with wind turbines[END_REF][START_REF] Nicholls | The aversive effect of electromagnetic radiation on foraging bats-a possible means of discouraging bats from approaching wind turbines[END_REF] or no impact [START_REF] Gilmour | Comparing acoustic and radar deterrence methods as mitigation measures to reduce human-bat impacts and conservation conflicts[END_REF], albeit at much higher frequencies. Further research is also required to identify the distance at which this cascading effect operates to fully appreciate the mechanisms involved.

Corona discharges can also cause a hissing noise and we predicted power lines avoidance by passive-listening batshere Myotis myotis/blythii [START_REF] Russo | Echolocation and passive listening by foraging mouse-eared bats Myotis myotis and M. blythii[END_REF] and Plecotus spp. [START_REF] Anderson | Feeding behaviour of captive brown long-eared bats, Plecotus auritus[END_REF] during wet conditions. We could not test this hypothesis with Myotis spp. in a robust way as this species group includes both active-(e.g. M. nattereri) and passive-(e.g. M. bechsteinii) listening bat species [START_REF] Siemers | Differences in sensory ecology contribute to resource partitioning in the bats Myotis bechsteinii and Myotis nattereri (Chiroptera: Vespertilionidae)[END_REF], even though the former is more likely to forage at forest edges than the later. However, our results on M. myotis/blythii refute the power lines avoidance hypothesis due to "noise disruption" since its activity at forest edges near power lines was less affected by relative humidity compared to control sites. We did not have enough records of Plecotus spp. to conduct the analysis on its activity or occurrence and the potential effects of noise produced by VHVPL on this species group cannot be excluded for two reasons. First, Plecotus spp. cease echolocating during the hovering phase of gleaning attacks [START_REF] Swift | Gleaning as a foraging strategy in Natterer's bat Myotis nattereri[END_REF] and are therefore more likely to be disrupted by noise. Second, Plecotus spp.

have exceptionally high hearing sensitivity with a threshold of -20 dB sound pressure level for hearing frequencies between 12-19 kHz [START_REF] Coles | Frequency sensitivity and directional hearing in the gleaning bat, Plecotus auritus (Linnaeus 1758)[END_REF], i.e. in the high frequency range of sound produced by the power lines.

Our prediction on power line avoidance by high-flying and open-space foragers due to the physical structure (pylons and cables) of the power lines was supported by our results on Nyctalus spp. which showed significantly higher activity at control sites than near VHVPL regardless of the weather conditions. The Nyctalus species group includes N. noctula and N. leisleri, two open-space forager species that forage at height [START_REF] Roemer | Bat sonar and wing morphology predict species vertical niche[END_REF]. Our results for Nyctalus spp. corroborates those of Kahnonitch, Lubin [START_REF] Kahnonitch | Insectivorous bats in semi-arid agroecosystems-effects on foraging activity and implications for insect pest control[END_REF] who revealed that the activity of the open-space and high-flying forager T. teniotis decreases closer to 161 KV power lines. Overhead wires greatly vary in height depending on topography (from 10 m to >50 m, especially in our hilly study area) and may overlap with the flight height of Nyctalus spp., thus potentially representing obstacles while foraging/commuting. However, we also observed power line avoidance by other bat species (B. barbastellus, E. serotinus, P. pipistrellus, and P. pygmaeus/M. schreibersii) and reduced composite activity and foraging intensity at power lines, but only at low relative humidity levels. Wing morphology of these bat species makes them more manoeuvrable than Nyctalus spp. [START_REF] Denzinger | Bat guilds, a concept to classify the highly diverse foraging and echolocation behaviors of microchiropteran bats[END_REF] and it seems unlikely that overhead wires represent physical barriers to movement for these species. As our sampling design does not disentangle the effects of extremely low frequency electromagnetic fields (50 Hz) generated by the VHVPL and the physical presence of VHVPL on bats, the potential negative effect of electromagnetic fields on bats cannot be excluded. The mechanisms underlying the avoidance of power lines by bats merits further investigation and further behavioural experiments are therefore needed to assert our findings.

To conclude, our work highlights the response of bats to power lines at foraging habitats, providing new insight into the interactions between power lines and biodiversity. We found that the effects of VHVPL on bats results from a range of potential mechanisms, with (i) corona discharges being one of the most likely factors responsible for bat attraction to power lines and (ii) the physical presence of power lines and extremely low frequency electromagnetic fields generated by power lines the main reasons explaining power lines avoidance by bats. VHVPL traverse over 300,000 km in Europe and power lines avoidance by bats could result in large-scale loss, alteration and fragmentation of foraging habitat, as observed with other anthropogenic structures [START_REF] Azam | Evidence for distance and illuminance thresholds in the effects of artificial lighting on bat activity[END_REF][START_REF] Barré | Estimating habitat loss due to wind turbine avoidance by bats: Implications for European siting guidance[END_REF][START_REF] Claireau | Major roads have important negative effects on insectivorous bat activity[END_REF][START_REF] Leroux | Distance to hedgerows drives local repulsion and attraction of wind turbines on bats: Implications for spatial siting[END_REF]. This is especially true in more arid areas where bats will not benefit from potential insect aggregation near power lines. Given that power lines can have significant conservation consequences for these protected species in Europe, these infrastructures should be considered in appropriate planning legislation and policy. We therefore highlight the crucial need of mitigating any negative impact that power lines may cause to bats in arid areas by applying the mitigation hierarchy with the ambition of no-net-loss, for instance by avoiding siting new power lines near important foraging habitats and offsetting habitat loss (e.g. by restoring/creating new habitats) caused by existing power lines.

Figure 1 .

 1 Figure 1. Predicted bat responses with the 95% confidence interval to relative humidity at forest

Figure 2 .

 2 Figure 2. Predicted bat responses with the 95% confidence interval to relative humidity at forest

Figure 3 .

 3 Figure 3. Pairwise comparisons of bat activity and foraging intensity (CWMBSD) between control

Figure 4 .

 4 Figure 4. Boxplot of Nyctalus spp. activity (number of bat passes per night on a logarithm scale to the

Table 1 .

 1 Summary of a priori hypotheses regarding the potential effects of very high voltage power lines on bats investigated in this study. (+) indicates positive association expected, and (-) negative association.

	Potential effects investigated	Expected responses
	Physical presence of power lines (pylons and cables) High-flying and open-space species (-)
	Electromagnetic fields	All species (-)
	Corona discharges: noise	Passive-listening species (-)
	Corona discharges: light	Light-tolerant species (+)
		Light-sensitive species (-)

Table 2 .

 2 Standardized, model-averaged parameter estimates with associated standards errors (SE) and 85, 95% and 98% confidence intervals (CIs) of the best (G)LMMs (ΔAICc < 6) relating the effects of very high voltage power lines, humidity, and their interaction on bat taxon-specific activity, composite bat activity and community weighted mean bat sequence duration (CWMBSD). CIs that do not overlap zero are represented in bold red (85% CI: weak evidence; 95% CI: moderate evidence; 98% CI: strong evidence). Description of the most parsimonious models can be found in electronic supplementary material S10 and full results of the models in the electronic supplementary material S12.

	Variable		Barbastella barbastellus	Eptesicus serotinus	Myotis myotis/blythii	Myotis spp.	Nyctalus spp.	Pipistrellus nathusii/kuhlii	Pipistrellus pipistrellus	Pipistrellus pygmaeus /Miniopterus schreibersii	Rhinolophus hipposideros	activity Composite bat	CWMBSD
	Power line vs control	Est. ± SE	0.31 ± 0.31	-0.05 ± 0.30	0.29 ± 0.32	-0.05 ± 0.36 -0.63 ± 0.28	0.41 ± 0.35	-0.16 ± 0.30 -0.61 ± 0.46	0.39 ± 0.43	-0.25 ± 0.21	0.02 ± 0.03
		85% CI	-0.13, 0.75	-0.48, 0.39	-0.18, 0.75	-0.56, 0.46	-1.03, -0.22	-0.10, 0.92	-0.60, 0.28	-1.26, 0.05	-0.22, 1.01	-0.55, 0.05	-0.02, 0.05
		95% CI	-0.29, 0.91	-0.64, 0.55	-0.35, 0.92	-0.75, 0.65	-1.18, -0.08	-0.29, 1.10	-0.76, 0.44	-1.5, 0.29	-0.45, 1.23	-0.66, 0.16	-0.03, 0.07
		98% CI	-0.41, 1.03	-0.76, 0.66	-0.47, 1.04	-0.88, 0.78	-1.28, 0.03	-0.42, 1.24	-0.88, 0.56	-1.67, 0.46	-0.61, 1.39	-0.74, 0.24	-0.04, 0.08
	Relative humidity	Est. ± SE	-0.18 ± 0.25	-0.53 ± 0.24 -0.41 ± 0.24	0.05 ± 0.15	0.32 ± 0.20	-0.41 ± 0.23 -0.38 ± 0.19 -0.35 ± 0.24	0.11 ± 0.22	-0.17 ± 0.13 -0.03 ± 0.02
		85% CI	-0.53, 0.18	-0.88, -0.18	-0.76, -0.06	-0.17, 0.28	0.03, 0.61	-0.75, -0.08	-0.66, -0.10	-0.69, 0.00	-0.20, 0.42	-0.36, 0.03	-0.06, 0.00
		95% CI	-0.66, 0.31	-1.00, -0.05	-0.89, 0.07	-0.25, 0.36	-0.08, 0.72	-0.87, 0.05	-0.76, 0.00	-0.82, 0.12	-0.32, 0.54	-0.43, 0.10	-0.07, 0.01
		98% CI	-0.75, 0.40	-1.09, 0.04	-0.98, 0.16	-0.30, 0.41	-0.16, 0.80	-0.96, 0.13	-0.83, 0.07	-0.90, 0.21	-0.40, 0.62	-0.48, 0.15	-0.08, 0.02
	Power line vs control :	Est. ± SE	0.73 ± 0.29	0.48 ± 0.29	0.49 ± 0.31	0.19 ± 0.26	-0.26 ± 0.26	0.39 ± 0.30	0.59 ± 0.22	0.68 ± 0.36	0.34 ± 0.32	0.44 ± 0.16	0.06 ± 0.02
	Relative humidity	85% CI	0.32, 1.14	0.06, 0.89	0.05, 0.93	-0.19, 0.56	-0.64, 0.11	-0.03, 0.82	0.25, 0.92	0.17, 1.20	-0.12, 0.80	0.22, 0.67	0.03, 0.10
		95% CI	0.17, 1.29	-0.10, 1.05	-0.12, 1.09	-0.33, 0.70	-0.78, 0.25	-0.19, 0.97	0.12, 1.05	-0.02, 1.39	-0.29, 0.97	0.13, 0.75	0.01, 0.11
		98% CI	0.06, 1.40	-0.20, 1.16	-0.23, 1.21	-0.43, 0.80	-0.88, 0.35	-0.30, 1.08	0.04, 1.13	-0.16, 1.52	-0.41, 1.09	0.08, 0.81	0.00, 0.12

Acknowledgements

We are grateful to the landowners for granting permission to access their lands. We thank James Mathews (University of Bristol) and François Dechamps (RTE) for sharing their expertise. We thank CC-IN2P3 and PCIA-MNHN for providing computing and storage facilities of acoustic data, and Yves and Didier Bas for their help in this process.

Funding

Funding was provided by the Leverhulme Trust through an early-career fellowship (Award Reference: ECF-2020-571).

Data accessibility

Data collected for this study and codes will be available to download on Zenodo. For the review process, data and codes have been uploaded as part of the supplementary material. Acoustic recordings are archived and available via the French citizen science programme "Vigie-Chiro" (http://vigienature.mnhn.fr/page/participer-vigie-chiro), at the portal http://vigiechiro.herokuapp.com/.

Ethics

Ethical approval was given by University of Stirling Biological and Environmental Sciences Ethical Committee.

Authors' contributions

JSPF secured the funding, designed the set-up of the study, collected, processed and analysed the data and wrote the initial draft of the manuscript. KP and GJ supervised the project. CK developed the CWMBSD equation. All authors contributed critically to the final version of the manuscript.

Competing interests

We declare we have no competing interests.


Abdennour Abbas

Philippe Supiot

Vianney Mille

Didier Guillochon

Bertrand Bocquet
email: bertrand.bocquet@univ-lille1.fr

Phlippe Supiot

Capillary microchannel fabrication using plasma polymerized TMDS for fluidic MEMS technology

Keywords: 42, 82, Cr Fabrication techniques; lithography, pattern transfer -85, 85, Va Micromachining -64, 70, km Polymers -85, 85, +j Micro-and nano-electromechanical systems (MEMS/NEMS) and devices

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Over the last decade, the need for rapid and low cost channel microfabrication methods is becoming increasingly important. Microchannels found their utility in three main topics: the integrated circuit technology, through the use of air gaps in interconnect structures for the reduction of the dielectric constant [START_REF] Kohl | Air-Gaps for Electrical Interconnections[END_REF], the microfluidic cooling of high density integrated circuits [START_REF] Dang | Wafer-level microfluidic cooling interconnects for GSI[END_REF], the investigation of fluid mechanics on the microscale [START_REF] Zhao | Surface-Directed Liquid Flow Inside[END_REF], and their applications in microfluidic devices including bio-and chemical-MEMS, biosensors and lab-on-chip [START_REF] Delamarche | Patterned Delivery of Immunoglobulins to Surfaces Using Microfluidic Networks[END_REF], [START_REF] Treizebré | Nanometric metal wire as a guide for THz investigation of living cells[END_REF].

Many different techniques have been reported for the fabrication of microfluidic circuits. The most prevalent are the etching techniques [START_REF] Boer | Micromachining of Buried Micro Channels in Silicon[END_REF], [START_REF] Dwivedi | Fabrication of very smooth walls and bottoms of silicon microchannels for heat dissipation of semiconductor devices[END_REF], replication methods based essentially on molding [START_REF] Mcdonald | Poly(dimethylsiloxane) as a material for fabricating microfluidic devices[END_REF], [START_REF] Chie | Micromolding of biochip devices designed with microchannels Sens[END_REF] and sacrificial material approach [START_REF] Peeni | Sacrificial layer microfluidic device fabrication methods[END_REF]. The main drawback common to both the etching methods and polymer molding techniques is the small flexibility for different substrate materials, the need of several photolithographic mask which multiply time consuming steps and the need of aligning and bounding process of different fabricated layers. This step usually requires thermal or chemical treatment and can lead to wafer-to-wafer misalignments [START_REF] Chatterjee | Three dimensional chip stacking using a wafer-to-wafer integration International Interconnect Technology Conference[END_REF]. A unique technology that permits a rapid and easy fabrication of microchannels on one side of the silicon wafer is the sacrificial material approach. In this method, microchannel fabrication begins with deposition of the sacrificial layer and patterning into the desired shape. The structural material is then deposited over the micropatterned sacrificial layer, except in a small area which will provide an access to the sacrificial material. The later is then dissolved chemically or thermally or by plasma etching, leaving a covered channel. Several materials have been used as sacrificial layer, including photosensitive materials such as electronic photoresists [START_REF] Walsh | Photoresist as a sacrificial layer by dissolution in acetone The 14th[END_REF], thermally decomposable materials such as polycarbonates [START_REF] Jayachandran | Air-Channel Fabrication for Microelectromechanical Systems via Sacrificial Photosensitive Polycarbonates[END_REF], polyoxymethylene [START_REF] Gleason | Hot filament chemical vapor deposition of polyoxymethylene as a sacrificial layer for fabricating air gaps[END_REF], polynorbornene [START_REF] Bhusari | Fabrication of air-channel structures for microfluidic, microelectromechanical, and microelectronic applications[END_REF], [START_REF] Wu | Fabrication of Microchannels Using Polynorbornene Photosensitive Sacrificial Materials[END_REF], Parylene [START_REF] Noha | Rapid, low-cost fabrication of parylene microchannels for microfluidic applications The 12th International Conference on Solid State Sensors[END_REF], and water soluble materials like poly(acrylic acid) and polyvinylalcohol [START_REF] Linder | Water-Soluble Sacrificial Layers for Surface Micromachining[END_REF]. Different sacrificial materials were widely investigated in the literature, while the amount of studies exploring new methods or materials for the overcoat layer remains relatively very low. Today, the most used technique to fabricate the overcoat material is a chemical vapour deposition (CVD) of silicon dioxide (SiO2), silicon nitride (SiN x ) [START_REF] Joseph | Improved fabrication of micro air-channels by incorporation of a structural barrier[END_REF], [START_REF] Peeni | Planar thin film device for capillary electrophoresis[END_REF], silicon carbide (SiC) or different metals such as aluminium (Al), nickel (Ni) and titanium (Ti) [START_REF] Papautsky | A low-temperature IC-compatible process for fabricating surfacemicromachined metallic microchannels[END_REF], [START_REF] Bagolini | Polyimide sacrificial layer and novel materials for post-processing surface micromachining[END_REF]. These materials are deposited at relatively high temperatures (≥ 200 °C). This requires the use of materials with high thermal stability and high glass transition temperatures. It also implies the use of adhesion layers. These constraints limit dramatically the number of materials that can be used. Here, we describe the realization and characterization of a simple process for buried air-microchannel fabrication. An alternative technology called Plasma Polymerization on Sacrificial Layer (PPSL) is introduced. It refers to the use of plasma polymerization to deposit the overcoat material in a process at ambient temperature.

The Plasma polymerization considered here is essentially a Remote Plasma Enhanced Chemical Vapour Deposition (RPECVD). It consists in the conversion of organic and/or organometallic monomers into highly cross-linked polymers through partial decomposition activated by energetic plasma species. This polymerization is accompanied by a subsequent deposition of thin films on the substrate. The primary advantage of the present plasma polymerization is that it can occur at moderate temperatures compared to conventional plasma deposition methods. This allows the use of a large panel of materials as sacrificial or encapsulating layers. The benefits of plasma polymerized films arise from their high biocompatibility, good adhesion on different substrates and very interesting surface properties either for microfluidic flows or for functionalization [START_REF] Tsai | Application of plasma-polymerized films for isoelectric focusing of proteins in a capillary electrophoresis chip[END_REF], [START_REF] Muguruma | Plasma-polymerized films for biosensors TrAC[END_REF].

Experimental procedure

1. Microchannel fabrication

The fabrication process starts with a standard photolithography, with a two-step spin-coating process (Figure 1). First, the sacrificial material consisting of a positive photoresist (AZ9260, Hoechst) is spun-coated in two steps on the p-doped silicon wafer (100). The spin parameters and the viscosity of the polymer solution control the thickness of the deposited layer which determines the height of the air-channel (Table 1). The deposited photoresist is then cured to evaporate the solvent. This step is followed by UV exposure at 365 nm for a dose of 10 mJ.cm -2 , through a high-resolution photomask featuring the microchannels. Areas not exposed to UV light are dissolved by immersion of the wafer in a basic solution, such as AZ 351B, leaving only a patterned sacrificial layer on the substrate. The microchannels were designed as 10 to 700 µm wide, 10 to 25 µm high and 3 mm long.

The following step is the deposition of the overcoat layer over the micropatterned surface. For the first, a plasma polymerization technology was integrated to a microelectronic fabrication process for the deposition of the encapsulating material. The wafer with desired structures was introduced into the reaction chamber of a home-built microwave (2.45 GHz) plasma reactor [START_REF] Quédé | Characterization of organosilicon films synthesized by N2-PACVD. Application to fire retardant properties of coated polymers Surf[END_REF].

Then, the organosilicon monomer 1,1,3,3,TetraMethylDiSiloxane (TMDS) was used as the precursor to synthetize and deposit a polymer organosilicon film by a remote afterglow PECVD. The deposited polymer, so called plasma polymerized TMDS (ppTMDS) is used as the structural material of the microchannel. Figure 2 shows the principal species composing the polymer. It was deposited at room temperature at a rate of 14 nm.s -1 . This technique is described in more details elsewhere [START_REF] Supiot | Growth and Modification of Organosilicon Films in PECVD and remote Afterglow Reactors Plasma Process[END_REF], and the optimized conditions are summarized in Table 2.

In the last step, the sacrificial layer is removed by immersion of the substrates into AZ351B developer (1:4) prepared with deionized water. The sacrificial photoresist decomposes into volatile and soluble products leaving behind a hollow channel. The resulting microchannels are optically transparent. Typical ppTMDS channels on Si substrate are shown in Figure 3. The released structures are rinsed in deionized water then underwent a supercritical CO 2 drying to avoid the sticking effect caused by capillary forces.

MEMS design and fabrication

PPSL technique was used to fabricate a BioMicroElectro Mechanical System (BioMEMS). This fabrication aims to demonstrate the feasibility of the direct integration of microfluidic circuits onto a substrate supporting electromagnetic components, here a coplanar wave guide (CPW). The later is used in high frequency circuits as propagation lines for dielectric and TeraHrertz spectroscopies [START_REF] Facer G R, Notterman | Dielectric spectroscopy for bioanalysis: From 40 Hz to 26.5 GHz in a microfabricated wave guide[END_REF], [START_REF] Mille | Proc. of the 28th IEEE-EMBS International Conference. New technology for high throughput THz BioMEMS[END_REF]. Figure 4 shows the proposed fabrication process. The CPW fabrication requires three steps. First, a thin layer of 200 nm Chromium (Cr) was sputtered over a high resistivity (5-10 Ω.cm -1 ) Si substrate as adhesion layer, before the deposition of 1 µm gold layer using Plassys MP 450S. Next, the substrate underwent a standard photolithography to transfer CPW features onto the gold layer. Then, the exposed gold areas are removed by ion polishing leaving the desired waveguides. The substrate is finally cleaned by oxygen plasma treatment.

The integration of the microfluidic circuits is achieved by the plasma polymerization technique discussed above. The microchannels were designed as 130 to 700 µm wide, 25 µm deep and 3 mm long. Two channel ends were broadened to 1 mm to form open reservoirs for easier fluidic access (Figure 5). These reservoirs are masked during the plasma polymerization of the cover layer. There are subsequently used as etch holes of the sacrificial layer.

3. Water filling experiments

The wetting properties of different materials and their surface energies are controlled by angle contact measurements using a GBX Digidrop. Experiments to study the filling of the microchannels were carried out with DI water and phosphate buffer (0.02 M, pH 6.8) as test fluids. To allow flow visualization, the microchannel system was mounted on an optical microscope equipped with a CCD camera. Because ppTMDS is transparent, liquid flow inside microchannel may be observed and captured as digital movie files. The fluidic features were determined by manual frame-to-frame comparisons.

Results and discussion

1. Plasma polymerization

The ability to form a buried air microchannel requires specific properties for both the overcoat and the sacrificial materials. Here, PPSL technique was performed using plasma polymerized TMDS (ppTMDS) as the encapsulating material, and the positive photoresist AZ9260 as the sacrificial one. The ppTMDS deposit presents a good mechanical strength to span large air-gaps without collapsing during or after fabrication. Figure 6 shows a channel of 700 µm wide and 20 µm high without pillars. A variety of aspect ratios and channel sizes can be achieved with this method. This is made possible not only because of the mechanical strength of the polymer but also because of the special polymerization mode of the TMDS monomer at the edges of the micropatterned structures. In fact, it can be seen on Figure 7 that the film surface is formed by smooth and spherical hilltype morphologies of few micrometers size and the microchannel is surrounded by self-formed support structures. The laters play an important role to stabilize very large channels without pillars and also could explain the formation of periodic constrictions in the interior surface of ppTMDS channel side walls (Figure 8). These observations suggest that (i) the polymerization mechanism can be affected by surface micropatterning, leading to the appearance of some interesting features of the deposited films and (ii) the film grows by forming small spherical clusters on the surface. The polymerization parameters (input power, pressure, monomer flow rate) determine the size of these clusters which in turn probably determine the deposition resolution discussed below. Further investigations are needed to understand ppTMDS growth mechanism on patterned surface and its related properties. Besides its topographical aspects, ppTMDS offers several advantages related to its physical and chemical properties, such as transparency in the visible wavelengths, chemical stability against solvents used in lithography process and a relative low permittivity (ε r ≈ 4) with low losses in the millimeter wave and terahertz spectrum [START_REF] Bocquet | Design Of Silicon-PPTMDS Bio-MEMS By Cold ird-00403290[END_REF]. The latter is of great interest for MEMS based on electromagnetic waveguides. Finally, unlike many polymers, ppTMDS is not selffluorescent which enables fluorescent detection. The photoresist AZ9260 allows the achievement of a good aspect ratio and the deposition of film thickness up to 150 µm, corresponding to the achievable channel height [START_REF] Conédéra | Potentialities of a new positive photoresist for the realization of thick moulds[END_REF]. However, PPSL is presently limited by the decomposition rate of this sacrificial material. Removing a polymerized AZ 9260 layer is the most time-consuming step, because the film polymerized during UV irradiation and becomes relatively highly cross-linked. The sacrificial polymer decomposes with initial rate of 7 µm/min into volatile and soluble products. This rate depends strongly on the channel dimensions, and decreases over time to 2 µm.min -1 because of the saturation of the etching solution. As a result, 10 hours are needed to release the fluidic circuit displayed in Figure 5. Longer decomposition times are required for longer and thicker microchannels. However, because of the chemical etching is a diffusion-limited process, there is a limitation in the length of the channel that can be processed. This hindrance can be overcome by the use of other sacrificial materials that can decompose within a reasonable time or that can be etched by plasma or thermal process. Video 1 in supplemental material shows the meniscus shape of water evaporating in the microchannel. The observed curvature confirms the hydrophobic properties of the channel wall after etching of the sacrificial layer, and ensures that there are negligible residues after decomposition. Figure 8 confirms that the microchannels are clean with no visible particulates contaminating the surface.

The direct plasma polymerization allows a good adhesion on a large variety of substrates, thus avoiding the use of additional adhesion layer. Furthermore, the deposition resolution (distance between two patterns that allows the deposition of the film on the support material, denoted R d ) of this method is estimated to be around 4 µm. This property permits the fabrication of small channels (< 4µm width) without need of sacrificial materials. As we can see in Figure 1, the plasma polymerization on predefined micropatterns leads to the appearance of hydrophobic closed channels whether the distance between these patterns is slightly higher than R d . It is important to note that the deposition resolution of the polymer could also be used to create small accesses in the sacrificial layer patterns (Figure 1, when L≤R d ) to allow the entering of the etching solution and thus permit a faster decomposition.

2. Microfluidic characterization

The ppTMDS microchannel is 2 mm long, 20 µm high and has a total volume of 23 nL. We designed two reservoirs at the ends of the channel. In addition to their role as access for the etching solution and as inlet/outlet ports, these reservoirs provide a surface tension that permits the filling of the channel by capillary force. The final device is shown in Figure 8. The system was filled by adding a droplet to the channel reservoir. Capillary forces dominate largely in microfluidic systems due to the large surface-tovolume ratio. Surface tension of a liquid meniscus provides sufficient pressure to push water into the channel. Water and buffer was drawn into the device by capillarity from one reservoir to another. Liquid flow inside micrometer-sized channels is essentially laminar (Reynolds number < 5 for the channel used here). In spite of the hydrophobic feature of ppTMDS, there is no need to treat the surface to fill the channels, unlike other hydrophobic polymers such as PDMS based channels which need a prior hydrophilization treatment to allow positive capillary action [START_REF] Bodas | Hydrophilization and hydrophobic recovery of PDMS by oxygen plasma and chemical treatment-An SEM investigation Sens[END_REF]. This might be explained by three main factors:

(1) The capillary pressure produced by the liquid meniscus at the channel access reservoir allows water entering the microchannel. This pressure is described as follows [START_REF] White | Fluid Mechanics[END_REF], [START_REF] Yang | Marching velocity of capillary meniscuses in microchannels The Fifteenth[END_REF]:

                        + + + + = = = = w h 1 1 2 p δ ( 1)
Where w is the width of the channel, h its height, and the surface tension δ (H2O) = 7,36.10 -2 N.m -1 at 20 °C.

As the width of the reservoir is much larger than its thickness, equation ( 1) can be simplified to p = 2δ/h. The pressure difference at the entrance of the microchannel used here is then: p = 8.096. 10 7 Pa. This pressure draws water with initial velocity of V= 4.4 cm.s -1 . The filling speed is reduced at the end to V= 2.18 mm.s -1 , due to the loss of pressure and viscosity effects. The filling speed is determined by the channel geometries, its surface energy and the liquid features. (2) The internal walls of the ppTMDS channel present periodic constrictions of 1 to 2 µm width and height. These constrictions may favor capillarity effects and then promote the displacement of waterbased solutions along channel surface [START_REF] Simoes | Study of preconcentration of non-polar compounds in microchannels with constrictions Sens[END_REF]. This also can be useful in channel functionalization as these constrictions cause small velocities near the surface which in turn increases resident time and surface interactions, and thus promote adsorption and condensation of molecules.

(3) The inner walls of the microchannels are nonwettable. The only Si part is wettable, with the static contact angles θ Si = 58° ± 3° and θ ppTMDS = 95° + 3°.

Total surface energies are 41.8 mJ.m -2 (dispersive component γ d = 24.1 mJ.m -2 , polar component γ p = 17.7) and 22.3 mJ.m -2 (γ d = 20.4 mJ.m -2 , γ p = 1.9 mJ.m - 2 ) for the hydrophilic and hydrophobic surfaces respectively. Internal ppTMDS wall generates a positive pressure acting against filling, while surface tension force due to bottom hydrophilic silicon surface reduce the ppTMDS effect and assists the water to flow inside the microchannel. Other factors are already known to affect slip, including surface roughness and the reduction in channel width which acts as passive valves.

The water filling operation can be seen in Supplemental material (Video 2). Capillary pressure is used here as the simplest way to draw liquids on the circuits. However, PPSL method can easily be applied to pressure driven flow systems and cavity based MEMS.

Conclusion

A novel fabrication method of closed microchannels for microfluidic systems is reported. It is based on a direct plasma polymerization of the encapsulating material, here the organosilicon polymer ppTMDS, on a photopatterned resist. The latter, used as a sacrificial layer, was then decomposed to release the desired channels. Using this technology, microchannels with various shapes and dimensions and with different surface features can be performed easily. In addition, this simple method allows the direct and rapid integration of fluidic circuits onto various sensors such as microelectrodes and waveguides.

Applying the concept of plasma polymerization to the fluidic MEMS fabrication is particularly attractive for several reasons: (1) it avoids the bonding step in the fabrication protocol; (2) it uses ambient temperature process; (3) the large panel of organic or organosilicon materials that can be deposited, (4) the good biocompatibility of these materials and their easy functionalization; (5) the processing onto different material substrates without the need of adhesion layer; [START_REF] Boer | Micromachining of Buried Micro Channels in Silicon[END_REF] this process can be scaled up to industrial production relatively easily. Beside this, ppTMDS films enable the fabrication of very large cavities (> 700 µm) without pillars and small channels (< 5 µm width) without using a sacrificial material. One of our further challenges in this issue is the creation of microfluidic networks by the direct plasma polymerization of both sacrificial and overcoat layer to achieve the whole process in the same plasma reactor.

Figure 1 .

 1 Figure 1. Principle of the PPSL process using two methods. Method 1 requires three main steps: (a) Micropatterning of the sacrificial material. The mask and the dose of UV exposure determine the shape of the patterns, (b) plasma polymerization of TMDS on the sacrificial layer, and (c)The decomposition of the sacrificial material leading to the release of a desired channel. Method 2 allows the fabrication of small channels without etching the sacrificial material. The distance between two patterns (L) governs the shape and the type of the microchannel obtained. It is to note that the SEM image (at the right) represent two hydrophobic microchannels, while the corresponding scheme show only one.

Figure 2 .

 2 Figure 2. Infrared spectrum of plasma polymerized TMDS. (a) Semi developed formula of TMDS monomer.

Figure 3 .

 3 Figure 3. SEM micrographs of the cross-sectional view of microchannels resulting from the PPSL process before (a1, b1) and after (a2, b2) the etching of the sacrificial layer. (c) Circular-shape microchannel. (d) Top view optical micrograph of two parallel microchannels during the sacrificial material decomposition. The gray part represents the remaining photoresist and the clear area is the released channel after 30 min of etching.

Figure 4 .

 4 Figure 4. Schematic representation of the microfabrication process of a fluidic MEMS. (A) photolithography of the coplanar waveguides on a silicon wafer: (1) deposition of a gold layer (2) Spin coating of the photoresist, (3) standard photolithography, (4, 5) Ion polishing and oxygen plasma treatment, (B) PPSL technique: starting with surface micropatterning of a sacrificial photoresist (steps 1 and 2), followed by the direct polymerization of TMDS monomer (step 3) and the decomposition of the sacrificial material (step 4).

Figure 5 .

 5 Figure 5. SEM micrograph of a cross-sectional profile of the largest microchannel obtained by PPSL process. It presents dimensions of 700 µm width and 20 µm height.

Figure 6 .

 6 Figure 6. External aspect of a microchannel obtained by PPSL. The ppTMDS film surface is organized in spherical hill-type morphologies of few micrometers size. Special features can be seen at the microchannel edges.

Figure 7 .

 7 Figure 7. SEM micrographs of the (a) fluidic MEMS, (b) Enlarged view of the partially abrased region, showing the electromagnetic sensor (CPW) and the self-formed support structures, (c) Interior surface of the ppTMDS channel side wall. We can see the periodic constrictions formed during polymerization.

Table 1 .

 1 Process conditions of the photoresist AZ9260.

	Process step	Conditions
	Spin coating 1	Speed: 2000 rpm, Acceleration: 5000 rpm, 40
	(AZ 9260)	sec
		Stabilization: 10 min.
		Soft-bake: 100 °C , 3 min
		Water reabsorption: 15 min at room
		temperature
	Spin coating 2	Speed:2000 rpm, Acceleration: 5000 rpm, 40
	(AZ 9260)	sec
		Stabilization: 10 min.
		Soft-bake 3 min at 100 °C
		Water reabsorption: 15 min
	UV exposure	10,48 mW/cm 2 @ 365 nm (I-line), 45 sec
	Development	AZ 351 b (30 %), 90 sec
	Soft-bake	1 min at 100 °C
	Film thickness	~ 25 µm

Table 2 .

 2 Process parameters of the Plasma polymerization of TMDS.

	Base pressure	0.028 mbar
	Operating pressure	5.6 mbar
	Power	200 W
	Plasmagenic gas	N2 : 1.8 slpm a
	carrier gas	O2 : 25 sccm b
	Pre-treatment	N2 Plasma during 4 min,
		followed by N2/O2 plasma
		during 3min.
	Treatment	TMDS Plasma : 5 sccm
	a slpm: standard liter per minute,
	b sccm: standard cubic centimeter per minute

Acknowledgements

This work is supported by the National Research Agency funds (contract ANR-05-NT05-2_46562) and the Interdisciplinary Research Program of Lille University (PPF BioMEMS 2006 N° 1803).