Daniel Rouland

Denis Legrand

Mikhail Zhizhin

Sylvie Vergniolle

Automatic Detection and Discrimination of Volcanic Tremors and Tectonic Earthquakes : An Application to Ambrym Volcano, Vanuatu

Keywords: automatic detector and discriminator, Ambrym volcano, long duration tremors

An algorithm of automatic detection and discrimination of volcanic tremors, defined as events containing only P-type wave, and tectonic earthquakes, containing both P-and S-

Introduction

Numerous event-detection schemes have been developed using single-station, vertical component seismic data. Those are effective in detecting teleseismic P phases, but their efficiency decreases for regional and local events due to lower incidence angles of the Pwave at short epicentral distances. Magotra et al. (1989) underlines at first the advantage to (1989) was first proposed by [START_REF] Zhizhin | Rapid estimation of earthquake source parameters from pattern analysis of waveforms recorded at a single 3component station[END_REF], and is further updated in this paper in order to discriminate volcanic tremors from tectonic earthquakes.

The code consists in detecting P-waves and, if any, S-waves. If P-and S-waves are detected, the corresponding signal is identified as a tectonic event (which may eventually be a volcano-tectonic event). If no S-wave has been detected, the event is labelled as a volcanic tremor. The present study deals with the analysis of finite-duration signals recorded at Ambrym volcano, Vanuatu, on a 3-component STS-2 seismometer, in the 3-9 Hz frequency domain, using this automatic detection algorithm. Additional processing is incorporated to automatically calculate basic source parameters, such as duration-and amplitude-magnitudes.

The frequency-size distribution is then worked out from these automatic determinations of both volcanic tremors and tectonic earthquakes.

Ambrym volcano and the July-November 2000 seismic experiment

Ambrym volcano (Vanuatu Archipelago), located at 168.1°E; 16.3°S, is a 35 x 50 km wide basaltic volcano, rising about 1800 m from the adjacent ocean floor (Fig. 1). Nearly 800 m above sea level, the edifice is truncated by a 12 km-diameter caldera. Details on the Ambrym

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volcano structure, the historical eruptions and the time-frequency analysis of the continuous seismic data (short-period volcanic signals and very long period tremors) has already been discussed [START_REF] Carniel | Spectral and dynamical transitions on the hours-to-days timescale[END_REF][START_REF] Legrand | Interpretation of Very Long Period Tremors at Ambrym volcano, Vanuatu, as quasi-Static Displacement Field related to two distinct magmatic sources[END_REF]. Since the 1988-89 eruption, which produced extensive lava flows on the caldera floor, two lava lakes are observed inside

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Methodology

Since the volcanic activity of Ambrym mainly consists in the bursting of large bubbles, we expect that most of volcanic signals will be related to the rise and the bursts of these bubbles acting as pressure sources. Hence the degassing will solely induce P-waves at the source before being potentially converted, called here volcanic tremors. In order to automatically discriminate these volcanic tremors from tectonic earthquakes, an algorithm, further called Pand S-wave detector, has been developed. This algorithm is based on the detection of Pwaves and, if any, S-waves according to a modified version [START_REF] Zhizhin | Rapid estimation of earthquake source parameters from pattern analysis of waveforms recorded at a single 3component station[END_REF] of the Component Energy Comparison Method (CECM) proposed by [START_REF] Nagano | Automatic algorithm for triaxial hodogram source location in downhole acoustic emission measurement[END_REF]. The candidate events are defined by the signal-to-noise ratio (SNR) 5 dB threshold with short gaps (< 20 s) removed by closing operator from mathematical morphology [START_REF] Serra | Image Analysis and Mathematical Morphology[END_REF]. If a signal of P-wave type is found near the rise of SNR together with an S-wave, the detected signal is considered as a tectonic earthquake; if no S-wave is detected, the signal is interpreted as a volcanic tremor. In the first case, searching for the arrival time of S-wave is done by a statistical segmentation of the amplitude distributions before and after S-arrival.

An estimation of the signal duration is obtained from either the P-arrival or the SNR rise until the SNR decays below the same threshold.

P-and S-wave detector

In order to obtain a robust and sensitive P-wave detector, several different methods have been tried, including the STA/LTA detector (Vanderbulk et al., 1965), the linear polarization detector, and the multi-resolution detector using wavelet-transform (Anant and Dowla, 1997).

The best results are obtained when using a modified version of the Component Energy

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Comparison Method (CECM), proposed, for automatic detection of P-arrivals, in [START_REF] Nagano | Automatic algorithm for triaxial hodogram source location in downhole acoustic emission measurement[END_REF], for the location of acoustic emissions.

The simple idea behind CECM is that in any P-wave, the 3-D particle motion occurs along one direction, and there is no particle motion in the plane perpendicular to this direction.

Thus, a correlation of energy dissipating in the direction of the P-wave motion with the energy dissipated in other directions will be close to zero. On the other hand, in case of random motion in seismic noise we have, on average, the same amount of energy dissipated in all directions.

If we assume that the seismic noise is a multivariate Wiener stochastic process, then the energy dissipated in each channel will be proportional to the recording time, [START_REF] Wentzell | A course in the theory of stochastic processes[END_REF]. Thus, the energy will be correlated between channels. Deterministic component in the stochastic signal (in our case the P-wave arrival) will temporarily destroy the established cross-channel correlation.

( ) t t E x ~
In our version of the CECM algorithm, we correlate the energy dissipated in a moving time window ∆T = 25 sec between the three components of the seismic record. The total energy dissipated in each direction (e.g. along discrete time channel ( )

i x ) is defined by: ( ) ( ) ∑ = > = t i x t i x t E 1 2 1 , . (1)
The choice of the initial time t=1 may seem arbitrary: the only requirement is that it is "fixed" long before the P-wave onset at each hour boundary. To be less sensitive to the

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direction of the P-wave arrival, we analyze a product of the energy correlation coefficients between x-z and y-z channels (z is the vertical component of the motion; x and y are in the horizontal plane; x is North-South; y is West-East):

( ) ( ) ( )

T t R T t R T t R yz xz ∆ ∆ = ∆ , , , , (2)
where the energy correlation between x-z channels is defined by

( ) ( ) ( ) ( ) ( ) ∑ ∑ ∑ ∆ + = ∆ + = ∆ + = = ∆ T t t i z T t t i x T t t i z x xz i E i E i E i E T t R 2 2 , , (3) and
( )

T t R yz ,
is defined by the same formula by changing x into y. The P-wave arrival t 0 is preliminarily determined as the time at which the coefficient ( )

T t R ,
reaches a local minimum, as shown in Fig. 3. To reduce the number of false detections, we introduce an experimentally determined threshold for the candidate local minima values: ( )

6 . 0 , ≤ T t R
.

The P-wave arrival time t p is more precisely determined by fitting a parabola to several values of ( )

T t R ,
before t 0 and finding the time when the fitted parabola intersects time axis.

Hence we pick the first statistical evidence of the P-arrival before the well-developed wavetrain at t 0 .

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Our S-wave detector is based on two assumptions: first, the probability density functions for the distribution of seismic signal amplitudes ( ) ; second, the signals within each of the wavetrains can be considered as quasi-stationary (see [START_REF] Nagano | Automatic algorithm for triaxial hodogram source location in downhole acoustic emission measurement[END_REF]. Then arrival time t s of the S-wave will divide the signal in the time window between P-arrival t p and maximum degree of polarization t max (which for regional seismograms occurs within the S-wave) into two homogeneous segments

max t t t s p ≤ ≤ with the likelihood ( ) ( ) ( ) ( ) ( ) ∏ ∏ = + = = s p s t t i t t i i s i p s t a p t a p t L max 1 log . (4)
For normal distribution functions ( ) ( )

t a p p and ( ) ( ) t a p s
, the likelihood formula will be

( ) ( ) [ ] ( ) [ ] ∑ ∑ = = - - - - - - - - ≈ max 2 max max 1 2 1 log 2 1 log 2 t t i s i s s t i p i p s p s s s s t a t t t t t a t t t t t L µ µ , (5)
where

( ) ( ) ( ) ( ) ( ) ( ) ∑ ∑ = = - = - = + + = max max 1 2 2 2 1 , 1 , t t i i s s t i i p s p i s s t a t t t a t t t z t y t x t a µ µ . (6) A C C E P T E D M A N U S C R I P T

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The optimal S-wave arrival time t s should maximize the value of the likelihood function ( ) s t L (Fig. 3).

Tremors discrimination

Following [START_REF] Nagano | Automatic algorithm for triaxial hodogram source location in downhole acoustic emission measurement[END_REF], we examine the SNR in a time window T centred at time t and defined by the formula ( ) (  )

) ( / ) ( log 20 t P t P t SNR noise signal = , ( 7
)
where the average amplitudes of signal and noise are defined as

( ) ( ) ( ) ( ) ∑ + - = + + = 2 2 2 2 2 1 T t T t i signal i z i y i x T t P , ( 8
) ( ) ( ) ( ) ( ) ∑ = + + = t t i noise i z i y i x t t P 0 2 2 2 1 . ( 9
)
The arbitrary starting point 0 t used for accumulation of the average noise amplitude should be taken some time before the beginning of the tremor (we take 0 t at the hour boundary). A tremor is detected when its amplitude is above the noise level threshold

( ) 5 > t SNR dB .
To remove small gaps in the tremor detection when the SNR is just around the threshold, we apply a closing operator from mathematical morphology [START_REF] Serra | Image Analysis and Mathematical Morphology[END_REF]. It is a 1-D version of the, widely one used in image processing, composition of the basic morphological operators,

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dilation and erosion, both of them using the same structural element -a segment with fixed duration of 20 sec. As a result of the closing, any two detected tremors with a less-than-20sec time gap between them will be merged in a single tremor without gap.

The SNR threshold detection is usually good for detection of the beginning of the tremor, but it is biased by the embedded signal when used for detection of the tremor end. To estimate the tremor duration, we have to recalculate the SNR starting from the beginning of the tremor ( 0 t in Eq. 9 equals the current tremor start time). The "unbiased" tremor end is reached when the noise level gets below the same threshold

( ) 5 < t SNR dB
We use a simple discrimination rule between the tectonic earthquakes and volcanic tremors:

if a signal of P-wave type is detected near the rise of SNR tremor (within 8 sec), the detected signal is considered as a tectonic earthquake; otherwise the signal is associated to a volcanic tremor.

Data processing

The permanent signal is generated by the superposition of the intrinsic volcanic and tectonic activity and the local (and regional) meteorological conditions. Hence, the data processing is strongly dependent on the choice of the frequency window. It has been shown that station noise levels strongly vary according to the frequency, and the local site effects, especially on an island like Ambrym. [START_REF] Stutzmann | Geoscope station noise levels[END_REF] show numerous observations of noise levels in Geoscope stations. A minimum of the noise level is frequently observed at short periods,

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between 1.0 sec and 0.10 sec, for stations located on islands or close to a coast, such as at stations PPT, French Polynesia, or KIP, Hawaii). Fig. 4 shows raw and filtered data for different fourth-order Butterworth band-pass filters, with a good resolution in the 3-9 and 3-18 Hz frequency band. A significant increase of SNR for both tremors and local (and regional) earthquakes is also observed in these frequency domains. In the 9-27 Hz frequency range, numerous signals are generated by human near-activities and therefore we do not process the data in this frequency range. We have finally selected the fourth-order Butterworth 3-9Hz band-pass filter for the data processing: an example of half-a-day record (Fig. 5) clearly displays both regional tectonic earthquakes, identified on the local IRD (Institut pour la Recherche et le Développement, Centre de Nouméa, Nouvelle Calédonie) earthquakes catalogue and volcanic tremors.

Results

During the 4.5 months of measurements, during which the volcano was relatively quiet, 1959 tectonic earthquakes with epicentral distances (estimated from S-P interval time) ranging from a few kilometres to about 800 km (but most of them with distances < 200km), have been detected, as well as 14942 volcanic tremors with duration ranging from 10 to 2000 seconds.

Tectonic earthquakes

We have removed 150 events from the initial set of the 1959 detected earthquakes because a visual inspection had shown that they mainly correspond to an identification of a PP-wave instead of an S-wave. Records from selected tectonic earthquakes are presented in Fig. 6,

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corresponding to two short-distance hypocenters (top), with an epicentral distance lower than 4 km, and two other regional earthquakes (bottom) with respectively 34 and 67 km epicentral distances. In Fig. 7, the whole set of detected events is plotted in a chronological sequence according to their epicentral distances (in km): each distance is roughly estimated (no account of the depth of the event is taken) by multiplying by a factor of 8 the difference of the arrival times, in seconds, between S-and P-waves (this empirical formula being deduced from a standard crust model, e.g. [START_REF] Jeffreys | Times of transmission of earthquake waves[END_REF].

Most of the regional seismicity is well detected with this unique station. Three swarms of seismic activity corresponding to 3 large earthquakes which occurred in Vanuatu show up as dense dot clusters: the first swarm corresponds to the 9 August 2000, Mw = 6.0, earthquake which occurred at about 70 km, northward, from the station. The second swarm corresponds to the 4 October 2000, Mw = 6.9, earthquake located in the north-west direction from the station, at about 150 km, and the third one corresponds to the 15 November 2000, Mw = 5.7, earthquake located off the coast of Espiritu-Santo Island, at a distance of about 250 km. A persistent seismic activity is also observed following the 3 November 2000, mb = 4.9, local event located off the coast of Ambrym at a distance of about 50 km. The number of events at distances greater than 300 km decreases rapidly, partially due to the high-frequency range (3-9Hz) used for data filtering.

The cumulative number of the entire tectonic earthquakes displayed as a function of the amplitude-magnitude (Fig. 8) follows the Gutenberg-Richter law. Fig. 8 also shows that the catalogue is almost complete above local amplitude-magnitude 2.4. This supports the hypothesis that our code can automatically detect almost all the local and regional

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earthquakes. Obviously we don't take into account the largest events because of the short time duration of the survey.

In Fig. 9a, the cumulative number of the whole set of tectonic earthquakes is plotted according to their duration-magnitude and is very similar to that one displayed in Fig 8 : this means that for tectonic earthquakes the magnitudes calculated from the maximum amplitude or from the duration are almost equivalent. In Fig. 9b, we selected only the local events (at a distance less than 100 km). Here again, the curve follows the Gutenberg-Richter law, which means that the catalogue is also complete for local earthquakes.

Hence, the cumulative number of tectonic events follows a Gutenberg-Richter law whatever the magnitude we use (amplitude or duration-magnitudes).

Volcanic tremors

14942 volcanic tremors of duration greater than 10 s have been detected during the entire period of measurements. The cumulative numbers of volcanic tremors are discussed hereafter as a function of their duration-magnitude and of their relative amplitude-magnitude and compared to tectonic earthquakes. The log-log plot frequency-duration of tremor distribution (Fig. 10a) clearly shows two regimes, with a transition at duration of 100 sec (Fig. 10a). The number of long duration (>100sec) tremors (hereafter called LDT) is much larger than those predicted by the extrapolation based on the distribution of the short duration (< 100 sec) tremors (hereafter called SDT). The deviation from the Gutenberg-Richter law of the volcanic tremors of duration greater than 100 sec suggests that an additional mechanism is at work inside the volcanic edifice and might correspond to two different physical

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processes for example related to some cooling of the lava lake as observed at Kilauea [START_REF] Chouet | Sources of seismic events in the cooling lava lake of Kilauea Iki, Hawaii[END_REF]. in Figures 12a and12b respectively. We can see that the LDTs do not follow a regular time distribution (Fig. 12b) in contrast to the distribution of all tremors including SDTs (Fig. 12a), revealing one more time very particular physical processes for these LDTs that are not well understood. Therefore we analyze hereafter the tremors with respect to their duration magnitude instead of using the amplitude magnitude (several tremors have the same amplitude with a completely different duration).

Analysis of the long duration tremors (LDT):

Numerous long duration tremors (LDT) are very well observed on some daily records of the vertical component filtered between 3 and 9 Hz (Fig. 13). We observe that the quiescence time, here called intermittency, between two consecutive LDTs varies between two main values, around one and two hours. This intermittency of the total number of LDTs presents four distinct peaks (Fig. 14). The first one at 77 +/-17 min is composed of three values, around 60, 85 and 95 min, whose differences may or may not be significant; the second one

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at 165 +/-10 min, the third one at 565 +/-50 min and the fourth at 1065 +/-50 min. This

shows that these LDTs have a tendency to occur regularly, with four different intermittencies, suggesting that the system must restore its energy before emitting new LDT.

Hence the generation of LDTs seems to be quasi-periodic with four characteristic time scales.

Furthermore, the four intermittencies are proportional, within the error bar, to the lowest intermittency of 77 min by a factor of 1, 2, 7 and 13 respectively.

Our definition of a volcanic tremor as being a sole P-wave suggests looking for a mechanism related to a local source of pression. One obvious candidate on a basaltic volcano is related to gas because of the very compressible nature of the gas. Furthermore bursting bubbles have already thought to be responsible for the tremor at Stromboli but they can also radiate significant amount of seismic and acoustic energy during explosions [START_REF] Ripepe | Evidence for gas influence on volcanic seismic signals recorded at Stromboli[END_REF][START_REF] Vergniolle | Strombolian explosions: A large bubble breaking at the surface of a lava column as a source of sound[END_REF][START_REF] Chouet | Broadband measurements of the sources of explosions at Stromboli volcano, Italy[END_REF]2003). The seismic energy at Stromboli is explained by the pressurisation of the volcanic conduit associated with the ascent of a slug [START_REF] Chouet | Source mechanisms of explosions at Stromboli volcano, Italy, determined from moment-tensor inversions of very-long-period data[END_REF]. Visual observations at Niri Mbwelsu Tatem of large bubbles bursting while radiating sound waves (P Vetsch, pers. comm.; M Lardy, pers. comm.) indeed suggest associating the lowest intermittency of 77 min to the regular occurrence of large bubbles.

Furthermore this intermittency is similar to that observed at the northeast vent at Stromboli [START_REF] Ripepe | Evidence for gas influence on volcanic seismic signals recorded at Stromboli[END_REF][START_REF] Vergniolle | Strombolian explosions: A large bubble breaking at the surface of a lava column as a source of sound[END_REF]. Although, at Stromboli, the duration of explosions recorded on seismometers is usually shorter than 100 s [START_REF] Chouet | Broadband measurements of the sources of explosions at Stromboli volcano, Italy[END_REF]2003), a mechanism related to the ascent of a slug is also likely for explaining the long duration of the LDTs at Ambrym. It is then tempting to relate each of the three similar intermittencies, 60 min, 85 min and 95 min, to the occurrence of large bubbles in each of the three lava lakes. Furthermore the proportionality of the four main intermittencies, 77 min, Since a sole three-component broadband seismometer has been installed, we cannot locate the source generating these tremors. Moreover, the particle motion associated with these

LDTs is erratic and cannot be used for source location purposes. Nevertheless, we have to mention that these LDTs are very well recorded by the vertical short period instrument installed on the rim of the Benbow's vent. For example, on 26 th July, an intense LDT's activity is observed both at Lalinda broad-band station and at the short-period station located on the rim itself. The sudden increase, limited in time to one day, of both signals may be associated to a strong reactivation of the lava lake.

Analysis of short duration tremors (SDT) :

The cumulative number of SDTs (duration between 10 and 100sec) has been shown on Figure 10a. The SDTs follow a classical Gutenberg-Richter law, both for duration-magnitude (Fig 10a) and for amplitude-magnitude (Fig. 10b). This was not the case for LDTs (Fig. 10a)

for which the source time function may be longer and more complex than that expected for a classical tectonic event controlled by a short and simple source time function.

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Concluding comments

We have developed a code to automatically discriminate tectonic earthquakes from volcanic tremors, both recorded on a volcano. The algorithm is applied to the seismic data collected during a four-and-a-half-month survey at Ambrym volcano (Vanuatu). We found that the Gutenberg-Richter law is satisfied for both the tectonic earthquakes and the volcanic tremors:

this means that the physics generating these tectonic and volcanic events are auto-organized processes. It shows also that the code is efficient to select all the events and that very few may have been missed. This has been possible, despite the sole use of a single station, only because of the use of an automatic detection and discrimination code applied on a large amount of data.

The time series associated to LDT show the existence of four intermittencies, from one hour to less than a day: the one hour period could be related to the occurrence of large bubbles rising in the conduit and bursting at the vent, while the three others are likely to result from other degassing process within the magma reservoir. The depth of the lava lake is probably 140 m below the surface.

  components to outperform a single-channel detector, and developed a detection scheme working in a manner similar to beam steering.[START_REF] Ohrnberger | Seismic event detection and source location using single-station (three-component) data[END_REF] has applied the Hidden Markov Models for discrimination of volcanic tremors in a vertical component of the continuous seismic record. The use of 3-component data became prevalent in the 90's. As the beginning of volcanic tremors is generally weak and emergent, automatic detection must be strongly reinforced by analysing three-component records. A modified version of the Component Energy Comparison Method (CECM) developed by Nagano et al.

  Benbow and Marum craters. A third one, called Niri Mbwelesu Tatem, the smallest and newest, is present on the flank of the Marum crater. Large bubbles, a few meters in diameter, are quite often seen and heard bursting at the surface, such as on August 7 1997 at Niri-Mbwelesu-Tatem (Fig.2, P Vetsch, pers. comm. and Steven Clegg, Kelby Hicks, http://www.volcan.org, Global Volcanism Program: http://www.volcano.si.edu).During the July-November 2000 experiment, two seismic sensors (a vertical short-period Mark Product L4C and a 3-component Streckeisen STS-2 broad-band seismometer) have been deployed on Ambrym volcano. The short period instrument (16.257°S, 168.108°E) was installed near the eastern rim of Benbow crater (Fig.1) and operated from 15 July to 11 August 2000, while the broad-band STS-2 seismometer (16.326°S and 168.058°E) was installed near Lalinda village (Fig.1) on the southwestern flank of the main cone, at about 9 km from the Benbow crater and operated from 14 July to 26 November 2000. Both seismometers were connected to a GEOSTAR 16-bit data acquisition system. Continuous signals were sampled at a 75 Hz rate, the GPS-time being recorded on a separate channel.The complete data set was stored on a 2Gb hard disk, allowing an independent, continuous acquisition for about 4.5 months. During the 137-day data field acquisition, only 55 hours (9 November late to 11 November early) have been lost for the broad-band instrument, due to power-supply problems.

  But if we consider now the amplitude instead of the duration (Fig 10b), the double slope of the Gutenberg-Richter observed on Fig 10a disappears. This suggests that no clear relation exists between duration and amplitude for volcanic tremors as shown on Figure 11 and in contrast to tectonic events. The relation between amplitude and duration (Fig. 11) is not clear for SDT and small amplitudes (< 100 in relative amplitudes), whereas most of the LDTs have a quasi-constant amplitude. The number of tremors/day (the whole set with relative amplitude > 10) and the number of tremors/day with duration > 100 sec are displayed

  565 min and 1065 min with the lowest one may point towards a mechanism more widely related to degassing within a magma reservoir. Fig 15 shows the time distribution of LDTs as a function of amplitude (a) and duration (b). Despite the dispersion of the data, a delay of about thirteen days separate most of the bursts of LDTs, whose duration exceeds 100 s (Fig 15b). This time scale, although not as apparent on the time evolution of the amplitude (Fig 15a) could indicate that another characteristic time may also exist. But in the absence of longer time series, the existence of intermittency at 13 +/-2 days is still not formally shown.

Fig. 1 .

 1 Figure captions

Fig. 2 .

 2 Fig.2. Large bubbles, probably a few meters in diameter, quite often seen and heard bursting

Fig. 3 .

 3 Fig. 3. Example of P and S detections: top and 2 nd rows : vertical and transverse components with P and S arrival marks and

Fig. 4 .Fig. 5 .

 45 Fig. 4. Examples of fourth-order Butterworth band-pass filtered seismic signals: each subset

Fig. 8 .

 8 Fig. 8. Cumulative number of the whole set of detected tectonic earthquakes as a function of

Fig. 11 .Fig. 14 .

 1114 Fig. 11. Amplitude versus duration of the tremors.

Fig. 15 .

 15 Fig. 15. Time distribution of the LDTs as a function of amplitude (a) and duration (b).

Acknowledgements

We are very grateful to the people of Lalinda village, Vanuatu, for their help in the field operations, to M. Frogneux and the technical support of the Laboratory of Seismology, EOST, Strasbourg, France, the Department of Geology, Mines and Water Resources, Port Vila, Vanuatu, J.Ph. Caminade, from I.R.D, in place at Port Vila, who organized successfully the whole logistic of the survey and R. Carniel, D. Charlie, M. Fulle, D. Nakedau, M.


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Imen Amine Khendek

Ben Ammar

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Sylvain Milla
email: sylvain.milla@univ-lorraine.fr

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Y Ledoré

Żarski Roche

Ben Khendek

Broquard, Depp, Ledoré, Policar, Fontaine Milla Ammar

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Keywords: pikeperch, ovulation, dopamine, antagonist, sex steroid Implications

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Pikeperch, Sander lucioperca, is a species of interest for aquaculture diversification. It is a highly valuable economic and commercial species partly thanks to its flesh quality and recreational value [START_REF] Kestemont | Biology and culture of percid fishes: principles and practices[END_REF]. Despite an increase in inland aquaculture production over the past decades, declining captures remain today the main supply to the increasing market demand (Food and Agriculture Organization of the United Nations, 2017). Consequently, great efforts are made to develop pikeperch aquaculture in order to respond to consumer demand. To achieve this objective, controlling the production of high-quality offspring in a synchronous and predictable way, year after year, is a crucial step in the culture process [START_REF] Zarski | Artificial reproduction of percid fishes[END_REF].

Controlled fish spawning is usually supported by the application of hormonal treatment, which allows synchronizing the ovulation process and optimizing egg collection while minimizing the handling and stress to the fish [START_REF] Zohar | Endocrine manipulations of spawning in cultured fish: from hormones to genes[END_REF]. In pikeperch, various hormonal treatments have already been tested. The most widely used, human chorionic gonadotropin (hCG), was found to be highly effective in triggering ovulation [START_REF] Zarski | Artificial reproduction of percid fishes[END_REF]. However, the spawning effectiveness observed in this species was highly variable, with frequently reported ovulation rates between 75% and 100% and embryonic survival rates between 50% and 90% [START_REF] Zarski | Artificial reproduction of percid fishes[END_REF]. Besides, some authors demonstrated that application of hCG induces immune [START_REF] Zohar | Endocrine manipulations of spawning in cultured fish: from hormones to genes[END_REF] and stress responses [START_REF] Falahatkar | Effects of hormonal manipulation on stress responses in male and female broodstocks of pikeperch Sander lucioperca[END_REF], which could alter the subsequent reproductive operations (i.e. need to use higher doses of hCG or ineffectiveness of the treatment) [START_REF] Zohar | Endocrine manipulations of spawning in cultured fish: from hormones to genes[END_REF]. For these reasons, analogues of the gonadotropin-releasing hormone (GnRHa) were applied as an alternative treatment in finfish reproduction [START_REF] Mylonas | Broodstock management and hormonal manipulations of fish reproduction[END_REF][START_REF] Zarski | Artificial reproduction of percid fishes[END_REF]. Gonadotropin-releasing hormone acts directly at the pituitary level of the hypothalamicpituitary-gonadal axis stimulating the release of gonadotropin (LH), sex-steroid secretion and finally progression of the final stages of gametogenesis (oocyte meiotic maturation and ovulation). However, both basal and GnRH-stimulated LH secretion are under dopaminergic inhibition [START_REF] Yaron | Endocrine regulation of fish reproduction[END_REF]. Thus, the combination of dopamine (DA) receptor antagonists with GnRHa is usually used [START_REF] Mylonas | Broodstock management and hormonal manipulations of fish reproduction[END_REF] partly because antagonists would enhance the reproductive effectiveness of GnRHa therapy [START_REF] Zarski | Artificial reproduction of percid fishes[END_REF]. Usually, the D2 DA receptor antagonists (metoclopramide (MCP), domperidone or pimozide) have been used. The application of D2 DA receptor antagonists alone had positive effects on spawning in crucian carp, Carassius carassius [START_REF] Cejko | Application of dopaminergic antagonist: metoclopramide, in reproduction of crucian carp Carassius carassius (L.) under controlled conditions[END_REF]. By contrast, in Senegalese sole, Solea senegalensis, these treatments were found to be ineffective [START_REF] Guzman | Effects of in vivo treatment with the dopamine antagonist pimozide and gonadotropinreleasing hormone agonist (GnRHa) on the reproductive axis of Senegalese sole (Solea senegalensis)[END_REF]. In some perciforms, the effects of these antagonists on the reproductive effectiveness are highly contradictory and reduce the significance of the DA effect in this fish order [START_REF] Dufour | Neuroendocrine control by dopamine of teleost reproduction[END_REF][START_REF] Zarski | Artificial reproduction of percid fishes[END_REF]. Thus, the effectiveness of DA antagonists seems to be species-specific and its use should be verified for each species separately.

In vertebrates, DA effects are mediated through the binding to two receptor families: D1 and D2 receptor families (Cardinaud et al., 1997; Dufour et al., 2010). Interestingly, unlike D2 receptors, there is a lack of data concerning the existence and the role of D1 receptors in fish reproduction, although they were suggested to be involved in the control of GnRH release [START_REF] Yu | Adrenergic and dopaminergic regulation of gonadotropinreleasing hormone release from goldfish preoptic-anterior hypothalamus and pituitary in vitro[END_REF][START_REF] Kapsimali | Distribution of the mRNA encoding the four dopamine D1 receptor subtypes in the brain of the European eel (Anguilla anguilla): comparative approach to the function of D1 receptors in vertebrates[END_REF]. In addition, in vivo studies showed that the DA/D1 receptor complex regulates the decrease in serum LH levels and aromatase B transcript levels in the hypothalamus of the goldfish, Carassius auratus [START_REF] Popesku | Rapid dopaminergic modulation of the fish hypothalamic transcriptome and proteome[END_REF]2012). These data suggest that species specificity of DA may be associated with different involvement of D1 and D2 receptors in the overall DA-related processes. The use of DA antagonists specific to D1 or D2 receptor family alone has never been tested in pikeperch. In addition, the combination of the D1 receptor antagonist with GnRHa has never been studied to date. This, together with the unclear role of DA in perciform reproduction [START_REF] Dufour | Neuroendocrine control by dopamine of teleost reproduction[END_REF][START_REF] Zarski | Artificial reproduction of percid fishes[END_REF], creates the need for a detailed investigation of this mechanism which could form the basis for pikeperch-specific hormonal treatment protocols.

Given the lack of data about the effects of DA receptor antagonists alone (D1 and D2) or in combination with GnRHa (D1) and the unknown involvement of receptors D1 in pikeperch reproduction, we aimed to investigate the in vivo physiological (sex steroid) and zootechnical responses (gonado-somatic index (GSI), progress of the oocyte meiotic maturation, ovulation rate, latency time, survival, hatching and malformation rates) to D1 and D2 DA receptor antagonists in pikeperch.

Material and methods

This study was split into two independent in vivo experiments. The first experiment was performed as a preliminary study to describe the effects of two DA antagonists specific to D1 or D2 receptor, SCH23390 (SCH) or MCP, respectively, applied alone on pikeperch reproduction. The second experiment was dedicated to point out the effects of the two antagonists combined with a GnRHa on pikeperch reproduction. Both experiments were performed according to the European and French legislation for fish welfare and approved by the institutional Ethics Committee (APA-FIS3073-2016022913149909). Fish were handled after anaesthesia by immersion in a bath containing 150 mg/l of ethyl 3-aminobenzoate (MS-222; Sigma-Aldrich, Lyon, France).

Broodstock management Experiment 1. On 1 May 2015, 36 mature females (origin: production pond, Fishery Nove Hrady Ltd, Czech Republic; age: 3 to 4 years old; mean BW: 1.07 ± 0.06 kg) were transported to an outdoor recirculating system (La Bouzule, Laneuvelotte, France). On 4 May 2015, all the fish were individually tagged (ID-100A Microtransponder; Dorset Group BV, Aalten, The Netherlands). Fish were maintained in sub-squared tanks (3000 l, 1-m deep), fed to satiation with forage fish and exposed to natural photoperiod (Nancy, France) and temperature conditions (mean temperature: 17.3 ± 2.7°C) throughout the 2 weeks of experiment. Once a week, pH, and ammonia and nitrite concentrations in the water were measured using a WTW 340i pH meter and a CARY I spectrophotometer, respectively. All values remained above 7.5 for pH and below 1 mg/l for nitrites and ammonia.

Experiment 2. On 15 January 2016, 47 mature females reared in captivity (origin: Czech Republic; age: 4 to 5 years old; mean BW: 2.39 ± 0.48 kg) were used in the facilities of the fish farm Asialor (Pierrevillers, France). Tagged fish (FDX-B transponder; Biolog-ID, Bernay, France), were maintained in two 8000 l tanks in a recirculating aquaculture system under 20 lux of light intensity at the water surface. Before the experiment, all fish were subjected to an increase in photoperiod and temperature to reach 14 h light-10 h dark and 12.5°C, respectively. Throughout the experiment, fish were kept under automatically controlled photoperiod (from 14 h light-10 h dark to 15 h light-9 h dark) and temperature (12.9 ± 0.14°C) mimicking the environmental conditions prevailing during spawning. Dissolved oxygen (>6 mg/l) and pH (7.8 ± 0.2) were monitored daily. Ammonia and nitrite concentrations in the water were measured using a colourimetric method once a week and remained below 0.5 mg/l. Evaluation of oocyte maturation stages For both experiments, each female was catheterized for evaluation of the oocyte maturation stages at different sampling times (as described in the sampling strategy) according to the classification by [START_REF] Zarski | A new classification of pre-ovulatory oocyte maturation stages in pikeperch, Sander lucioperca (L.), and its application during artificial reproduction[END_REF]. In brief, oocytes were sampled using a catheter (CH06; 1.2 mm internal and 2 mm external diameter) and placed in Serra's solution (ethanol/formalin/glacial acetic acid, 6 : 3 : 1 v/v/v). After mixing slowly oocytes in Serra's solution and waiting (about 5 min) until the cytoplasm of the oocyte will become clarified, the oocyte maturation stage was evaluated under binocular microscope, magnification 4× (Motic ® SFC-11 Series, Motic Asia, Hong Kong, China). In pikeperch, the final stages of maturation were divided into seven morphological stages, from stage I to stage VII (ovulation; [START_REF] Zarski | A new classification of pre-ovulatory oocyte maturation stages in pikeperch, Sander lucioperca (L.), and its application during artificial reproduction[END_REF]. This allowed following the progression of the oocyte meiotic maturation (hereinafter termed final oocyte maturation (FOM)) until ovulation.

Hormonal treatments Experiment 1. On 4 May 2015, oocyte maturation stages were evaluated for each female. Females between stages II and IV were randomized, sampled for blood (0 h) and injected intraperitoneally with one of the following treatments:

(1) negative control with saline solution, the vehicle of all molecules (NaCl 0.9%, n = 5); (2) positive control with hCG (500 IU/kg, n = 5; Sigma-Aldrich); a D2 DA receptor antagonist; (3) MCP 4 (4 mg/kg, n = 6; Sigma-Aldrich) or (4) MCP 20 (20 mg/kg, n = 7); a D1 DA receptor antagonist, (5) SCH 0.8 (0.8 mg/kg, n = 6; Abcam, Paris, France) or (6) SCH 4 (4 mg/kg, n = 7). The doses applied for hCG and MCP 20 were the most commonly used in controlled reproduction of percids [START_REF] Zarski | Artificial reproduction of percid fishes[END_REF]. For SCH, the doses applied were chosen to be consistent with the literature and to get a common dose between MCP and SCH.

Experiment 2. On 15 January 2016, after determination of the oocyte maturation stage, all the females at stage I were randomized and injected intraperitoneally with one of the following treatments: (1) negative control with NaCl (0.9%, n = 9); (2) a first positive control with hCG (500 IU/kg, n = 7);

(3) a second positive control with a salmon-GnRH analogue ((sGnRHa), 25 µg/kg, n = 7; Pyr-His-Trp-Ser-Tyr-D-Arg-Trp-Leu-Pro-NHEt acetate salt; Syndel Laboratories Ltd, Nanaimo, Canada); (4) MCP 4 or (5) MCP 20 in combination with sGnRHa (25 µg/kg, n = 6 and n = 5, respectively); (6) SCH 0.8 or (7) SCH 4 in combination with sGnRHa (25 µg/kg, n = 7 and n = 6, respectively). Before injection, 10 females were randomly sampled for blood (0 h). The doses applied in both positive control groups were the most commonly used in controlled reproduction of percids [START_REF] Zarski | Artificial reproduction of percid fishes[END_REF].

Sampling strategy Experiment 1. Blood and oocytes were sampled 0 and 24 h after injection and at the time of ovulation or 7 days after injection (final sampling time, if the female did not ovulate). Blood was sampled from the caudal vein and placed in tubes containing heparin (28 mg/ml; 100 kU Sigma-Aldrich). Plasma was obtained by centrifugation (15 min at 10 000 r.p.m.) and stored at -80°C until further steroid hormone analysis. At the final sampling time, the fish were killed by overexposure to anaesthetics MS-222 (240 mg/l) and the whole gonads were cut out and weighed.

The following parameters were recorded: the progression of FOM ( = number of oocyte meiotic maturation stages between injection and the time of ovulation (or the end of the experiment)); the GSI ( = 100 × gonad weight/total fish weight); the ovulation rate for each treatment group ( = 100 × number of ovulating females/total number of females); and the latency time ( = time interval between injection and ovulation).

Experiment 2. Blood and oocytes were sampled 0, 24 and 48 h after injection and at the time of ovulation or 14 days after injection (final sampling time). Then, plasma was recovered and stored at -80°C until further steroid hormone analysis. From 48 h, if the females did not reach stage VI, oocyte maturation stages were determined every 2 days. At stage VI, the genital papilla was sewn (as described by [START_REF] Zarski | Artificial reproduction of percid fishes[END_REF] in order to prevent spontaneous releasing of eggs into the tank. From stage VI, ovulation control was performed every 6 h by gentle massage of the abdomen. At the time of ovulation, eggs were collected in dry plastic containers, weighed and then kept tightly covered at 11°C for no longer than 30 min until fertilization.

For in vitro fertilization, sperm was collected from 30 males of pikeperch (origin: Czech Republic; age: 4 to 5 years old; mean BW: 2.39 ± 0.36 kg). For each spawn, sperm from three males, injected with hCG (250 IU/kg) at 0 h, was collected in a dry syringe. For each fertilization procedure, only freshly collected sperm (15 min maximum before fertilization), with a motility rate above 80% evaluated under a light microscope (magnification 400×; Motic ® B3 Series, Motic, Hong Kong, China) [START_REF] Cejko | Influence of the length of time after hormonal stimulation on selected parameters of milt of ide Leuciscus idus L[END_REF] was used. For each female, three egg samples (~50 to 100 eggs each) were placed in three glass Petri dishes containing 5 ml of hatchery water. Simultaneously, 50 µl of pooled sperm was added in each dish. After vigorous agitation for 15 s, each dish was incubated in plastic cups containing 500 ml of water at 12°C.

The progression of FOM, the ovulation rate and the latency time were recorded. The GSI was not calculated due to the necessity to keep the fish alive. Additional zootechnical parameters were recorded: the embryo survival rate at 72 h post fertilization ( = 100 × number of viable eggs/total number of eggs), the hatching rate ( = 100 × number of larvae/total number of viable eggs) and the malformation rate ( = 100 × number of larvae showing malformations (lordosis, cardiac oedema, kyphosis, fragmentation of oil droplet, yolk sac oedema, spinal curvature, scoliosis, C-shaped larvae)/total number of larvae).

Levels of sex-steroid hormones in blood plasma For both experiments, sex-steroid hormones (17β estradiol (E 2 ), testosterone (T), 11 ketotestosterone (11KT) and 17,20β-dihydroxy-4-pregnen-3-one (DHP)) were measured in plasma using commercially available competitive ELISA kits. The kits for E 2 (KAP0621) and T (KAPD1559) were obtained from Diasource (Louvain-La-Neuve, Belgium), the kit for 11KT (582751) from Cayman Chemical (Ann Arbor, MI, USA) and the kit for DHP (MBS2602842) from MyBiosource (San Diego, CA, USA). The sensitivity limit, and the intra-and interassay CV were, respectively, 0.005 ng/ml (range: 0 to 0.935 ng/ml), <4% and <5% for E 2 ; 0.083 ng/ml (range: 0 to 16 ng/ml), <10% and <9% for T; 1.3 pg/ml (range: 0.78 to 100 pg/ml), <9% and <13% for 11KT; and 0.06 ng/ml (range: 0.312 to 20 ng/ml), <9% and <13% for DHP.

Statistical analysis

Statistical analyses were performed using the free software R version 3.3.1. For all dependent variables, homogeneity of variances was tested using Levene test (leveneTest, package 'car', [START_REF] Fox | An R companion to applied regression[END_REF]. For sex steroids, GSI, latency time, survival rate, hatching rate and malformation rate, data were analyzed by a linear mixed model (lmer, package 'lme4, [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF] with hormonal treatment and sampling time as fixed effects, and either the fish and the maturation stage at P0 (experiment 1) or only the fish (experiment 2) as random effects: model = lmer(Y ~treatment × sampling_time + (1|fish) + (1|maturation_ stage) with Y: dependent variable. For model validation, residuals were tested for homogeneity and normality using residual v. fitted value and sample v. theoretical quantile (Q-Q) plots, respectively (plotresid, package 'RVAideMemoire, [START_REF] Hervé | RVAideMemoire: diverse basic statistical and graphical functions[END_REF]. If necessary, data were log transformed, root square transformed or arcsin root square transformed (only for data expressed in percentage). When the model was validated, an ANOVA table was performed to calculate F-tests (Anova, package 'car, [START_REF] Fox | An R companion to applied regression[END_REF] followed by a least-squares means (predicted marginal means) multiple comparison between treatments, sampling times and/or their interaction as post hoc test (lsmeans, package 'lsmeans', [START_REF] Lenth | Least-squares means: the R package lsmeans[END_REF]. When data, even transformed, did not meet the assumptions for the linear mixed model, we used the aligned rank transformation for non-parametric factorial analysis (aligned.rank.transform, package 'ART, [START_REF] Villacorta | ART: Aligned rank transform for nonparametric factorial analysis[END_REF] followed by a pairwise comparison using Dunn test (posthoc.kruskal.dunn.test, package 'PMCMR, [START_REF] Pohlert | The Pairwise Multiple Comparisons of Mean Ranks package (PMCMR), R package[END_REF]. For the ovulation rate, data were analyzed with a χ 2 test (chisq.test, package 'MASS, [START_REF] Venables | Modern applied statistics with S, 4th edition[END_REF]. Data are expressed as mean ±SEM. The level of significance used in all tests was P < 0.05.

Results

Experiment 1 Effect of dopamine receptor antagonists alone on reproductive performance. In fish treated with hCG, 100% of ovulation was recorded associated with higher GSI and progression of FOM (3 to 5 stages) than in the other groups. In the latter, similar GSI and progression of FOM (0 to 3) were noted. Consequently, MCP and SCH failed to induce ovulation and did not trigger a significant progression of FOM (Table 1).

Effect of dopamine receptor antagonists alone on plasma steroid concentrations. Plasma T and 11KT levels varied significantly as a function of the interaction between hormonal treatments and sampling times (P < 0.05; Figure 1). Only females injected with hCG showed a significant decrease in T and 11KT concentrations between 24 h and the final sampling time (P < 0.001). However, no significant difference in plasma androgen levels was observed between the groups at each sampling time.

Only a time effect was monitored for E 2 with a drop observed at the final sampling time compared with 0 h and 24 h (P < 0.01; Figure 2a). No significant difference related to hormonal treatments and sampling times was obtained for plasma DHP levels (Figure 2b).

For all steroid levels, high variability was observed.

Experiment 2 Effect of dopamine receptor antagonists in combination with salmon gonadotropin-releasing hormone analogue on reproductive performance. Only the negative control group did not promote the progression of FOM and ovulation. All the other treatments triggered 100% of ovulation. The latency time and zootechnical parameters (survival rate, hatching rate, malformation rate) were similar in all the groups (Table 2).

Effect of dopamine receptor antagonists in combination with salmon gonadotropin-releasing hormone analogue on plasma steroid concentrations. Plasma T concentrations showed significant differences related to the interaction between hormonal treatments and sampling times (P < 0.001; Figure 3a). All hormonal treatments induced a marked increase in T compared with the negative control at 24 h (P < 0.05). At 48 h, the SCH 0.8, SCH 4, MCP 4 and hCG treatments were still effective in maintaining higher T levels compared with the negative control (P < 0.05). At the final sampling time, all hormonal treatments reduced the T concentrations (P < 0.05). The comparison between sGnRHa alone and the combination of DA receptor antagonists with sGnRHa showed that T levels increased with SCH 4 at 48 h (P < 0.01). Concerning 11KT, significant differences were observed related to the interaction between hormonal treatments and sampling times (P < 0.001; Figure 3b). All hormonal treatments induced an increase in 11KT levels at 24 h and Figure 1 Effect of hormonal treatments on plasmatic concentrations of (a) testosterone (T) and (b) 11 ketotestosterone (11KT) in pikeperch. Fish were injected with NaCl (0.9%, n = 5), human chorionic gonadotropin (hCG) (500 IU/kg, n = 5), MCP 4 or MCP 20 (metoclopramide at 4 mg/kg (n = 6) or 20 mg/kg (n = 7), respectively) and SCH 0.8 or SCH 4 (SCH23390 at 0.8 mg/kg (n = 6) or 4 mg/kg (n = 7), respectively). Blood was sampled at three sampling times: 0 and 24 h after injection and at the time of ovulation or 7 days after injection (if ovulation did not occur; final). Values are means ± SEM. a,b,c,d Different letters indicate significant differences for the interaction between hormonal treatments and sampling times (P < 0.05).

48 h (P < 0.001), while at the final sampling time they were significantly abolished but only after SCH and MCP 20 treatments (P < 0.001). However, for each sampling time, the DA receptor antagonist failed to change the 11KT level compared with the sGnRHa treatment alone.

For E 2 , concentrations varied significantly as a function of the interaction between hormonal treatments and sampling times (P < 0.001; Figure 4a). All hormonal treatments induced an increase in E 2 levels at 24 h and 48 h (P < 0.05) except for MCP 4 and SCH 4 at 24 h, and sGnRHa and MCP 4 at 48 h. At the final sampling time, all treatments decreased E 2 levels (P < 0.01). Here also, there was not any significant difference between sGnRHa alone and the combination of DA receptor antagonists with sGnRHa except with SCH 0.8 at 48 h which increased E 2 levels (P < 0.01).

No significant difference related to hormonal treatments and sampling times was obtained for plasma DHP levels (Figure 4b).

Mild variability was observed for all steroid levels.

Discussion

This study investigated, for the first time, the potential regulatory effects of DA receptor antagonists alone on reproductive mechanisms in pikeperch. Our results first confirmed the high effectiveness of hCG in inducing the progression of FOM and ovulation in this species [START_REF] Zarski | Artificial reproduction of percid fishes[END_REF]. During FOM, oocytes undergo a phenomenon of hydration inducing, in turn, an increase in follicle weight [START_REF] Mañanós | Reproduction and control of ovulation, spermiation and spawning in cultured fish[END_REF]. After hCG treatment, the rise in GSI may thus stem from the hydration process even if the latter remains to be demonstrated in pikeperch. Contrary to this positive control, no antagonist treatments were found to induce either GSI increase or ovulation. The latter finding is in accordance with previous reports on the Senegalese sole [START_REF] Guzman | Effects of in vivo treatment with the dopamine antagonist pimozide and gonadotropinreleasing hormone agonist (GnRHa) on the reproductive axis of Senegalese sole (Solea senegalensis)[END_REF] and the common tench, Tinca tinca [START_REF] Podhorec | The effects of GnRHa with and without dopamine antagonist on reproductive hormone levels and ovum viability in tench Tinca tinca[END_REF]. In contrast, [START_REF] Cejko | Application of dopaminergic antagonist: metoclopramide, in reproduction of crucian carp Carassius carassius (L.) under controlled conditions[END_REF] showed that the injection of MCP induces ovulation in the crucian carp. This inter-species difference may be due to variable potency of the DA inhibition as already suggested [START_REF] Mañanós | Reproduction and control of ovulation, spermiation and spawning in cultured fish[END_REF][START_REF] Zarski | Artificial reproduction of percid fishes[END_REF]. Consequently, we hypothesize that DA receptor antagonists applied alone would be ineffective in inducing ovulation in pikeperch because of a weakness in the DA inhibition during the spontaneous progression of FOM. Alternatively, we might speculate that the doses chosen were not sufficient to generate a GnRH and/or LH endogenous surge. However, when combined with sGnRHa, these DA receptor antagonists do not prevent sGnRHa from triggering ovulation. They do not thus appear as inhibitors of the final stages of gametogenesis in pikeperch. Conversely, DA receptor antagonists would not by themselves allow the reproductive performance to be improved or impaired.

The DA receptor antagonists alone did not induce significant changes in the sex-steroid levels. These results are in accordance with prior studies in striped bass, Morone saxatilis [START_REF] King | Plasma levels of gonadal steroids during final oocyte maturation of striped bass, Morone saxatilis[END_REF], and in Senegalese sole [START_REF] Guzman | Effects of in vivo treatment with the dopamine antagonist pimozide and gonadotropinreleasing hormone agonist (GnRHa) on the reproductive axis of Senegalese sole (Solea senegalensis)[END_REF] in which application of the D2 DA receptor antagonist did not modify the T and E 2 levels in plasma. The attempt to block the D1 receptor family did not succeed in Figure 4 Effect of hormonal treatments on plasmatic concentrations of (a) 17β estradiol (E 2 ) and (b) 17,20β-dihydroxy-4-pregnen-3-one (DHP) in pikeperch. Fish were injected with NaCl (0.9%, n = 9), human chorionic gonadotropin (hCG) (500 IU/kg, n = 7), salmon gonadotropin-releasing hormone analogue (sGnRHa) (25 µg/kg, n = 7), sGnRHa (25 µg/kg) in combination with MCP 4 or MCP 20 (metoclopramide at 4 mg/kg (n = 6) or 20 mg/kg (n = 5), respectively) or with SCH 0.8 or SCH 4 (SCH23390 at 0.8 mg/kg (n = 7) or 4 mg/kg (n = 6), respectively). Blood was sampled at four sampling times: 0, 24 and 48 h after injection and at the time of ovulation or 14 days after injection (if ovulation did not occur; final). Values are means ± SEM. A,B,C Different capital letters indicate significant differences between hormonal treatments for the same sampling time (P < 0.05). a,b Different lowercase letters indicate significant differences between sampling times for the same hormonal treatment (P < 0.05) altering those hormonal levels as well. The ineffectiveness of DA receptor antagonists alone in changing the sex-steroid secretion is consistent with the absence of effect on the ovulation and progression in oocyte maturation. In addition, this would indicate absence of or weakness in the dopaminergic inhibition by the application of D1 or D2 receptor antagonist at the tested doses during the non-hormonally manipulated oocyte maturation process in pikeperch.

Interestingly, we observed that the hCG treatment was unequally effective in inducing changes in the sex-steroid levels. These results are quite surprising considering that this molecule is a common substitute for natural (endogenous) fish LH, which induces fish spawning by direct action on gonads and sex-steroid levels [START_REF] Mylonas | Broodstock management and hormonal manipulations of fish reproduction[END_REF]. Considering our results from experiment 1, we noticed that at the same sampling time (e.g. 24 h), fish exhibited different stages of oocyte maturation, contrary to the synchronous stages in experiment 2. This high inter-individual variability may have induced a larger range in steroid concentrations among the fish, which could potentially explain statistically irrelevant endocrine response following hCG treatment. However, other differences between the two populations (e.g. environmental conditions, stress status, final number of individuals) may also explain this difference in sensitivity to hCG. However, some features of the experimental setup, notably the realization of two independent experiments, the low number of hormonal treatments but repeated over time, the usual number of individuals per group (King et al., 1994; Barry et al., 1995; Guzman et al., 2011), lead us to think that the statistical power of the tests were sufficient to detect some potential effects.

The sGnRHa treatment stimulated the production of sexsteroids confirming the widely observed activation of the gonadotropic axis in finfishes after such a treatment [START_REF] Yaron | Endocrine regulation of fish reproduction[END_REF]. Interestingly, T and E 2 production were stimulated when sGnRHa was complemented with SCH depending on the dose. These results lead us to hypothesize that SCH would boost the sGnRHa effect on T and E 2 secretion. To our knowledge, this is the first report of plasma sexsteroid change after exposure to D1 receptor family antagonist in fish. Either this antagonist would directly block the D1 receptors at the gonad level as shown in rats (Vene- gas-Meneses et al., 2015), which in turn would stimulate the ovarian steroidogenesis. Or, the blockage of those receptors in the brain would disrupt the aromatase activity and the further metabolism of T and E 2 [START_REF] Marsh | Aromatase immunoreactivity in the bluehead wrasse brain, Thalassoma bifasciatum : immunolocalization and co-regionalization with arginine vasotocin and tyrosine hydroxylase[END_REF][START_REF] Popesku | Meta-type analysis of dopaminergic effects on gene expression in the neuroendocrine brain of female goldfish[END_REF]. Also, the blockage of D1 receptors could induce a surge in blood LH [START_REF] Popesku | Rapid dopaminergic modulation of the fish hypothalamic transcriptome and proteome[END_REF] which would stimulate the sex-steroid production. Finally, we cannot rule out some indirect effects of SCH through other metabolic factors. Further studies would be needed to check the presence of those receptors in the ovary and to test these regulations by investigating the aromatase expression and activity in brain and oocytes as well as LH in the blood plasma.

Plasma DHP concentrations remained basal and stable over time in all the treatments. In many teleosts, the steroid DHP is the maturation-inducing steroid (MIS) [START_REF] Nagahama | Regulation of oocyte maturation in fish[END_REF]. Progression of FOM was found to be linked to a significant increase in DHP levels in walleye, Sander vitreus [START_REF] Barry | Effects of selected hormones and male cohorts on final oocyte maturation, ovulation, and steroid production in walleye (Stizostedion vitreum)[END_REF], and in striped bass (Mylonas et al., 1997). However, in our study, even after application of hCG or sGnRHa with which all the fish ovulated, no peak of DHP concentration was observed. Surprisingly, these results are not consistent with GnRH commonly known effects on the gonadotrope axis [START_REF] Yaron | Endocrine regulation of fish reproduction[END_REF] and with a prior study in which hCG induced an increase in plasma DHP levels during the progression of FOM in white perch, Morone americana, and white bass, Morone chrysops [START_REF] King | Involvement of gonadal steroids in final oocyte maturation of white perch (Morone americana) and white bass (M. chrysops): in vivo and in vitro studies[END_REF]. Several hypotheses may explain these findings. First, as reported in walleye, the DHP could be rapidly removed from the plasma after its conjugation to a non-immunodetectable molecule such as 17,20-P-sulphate or 17,20-P-glucuronate [START_REF] Scott | 17α,20β-Dihydroxy-4-pregnen-3-one 20-sulphate: a major new metabolite of the teleost oocyte maturation-inducing steroid[END_REF]. Second, in a closely related species, the Eurasian perch, Perca fluviatilis, Migaud et al. (2003) found very low DHP levels in plasma and suggested that these levels may be higher in the early morning than at other times. Due to experimental conditions, our sampling times were performed at the beginning of the afternoon, so we may have missed the peak of detectable DHP in the blood plasma. Third, a close hormone, the 17α,20β, 21-trihydroxy-4-pregnen-3-one (20β-S or 17,20, 21-P), identified as MIS in other perciforms [START_REF] Nagahama | Regulation of oocyte maturation in fish[END_REF] could also play this role in pikeperch. That would explain the mild level of DHP in this species, even after injection with hCG or sGnRHa. Nevertheless, although [START_REF] Barry | Effects of selected hormones and male cohorts on final oocyte maturation, ovulation, and steroid production in walleye (Stizostedion vitreum)[END_REF] demonstrated that 20β-S was not detectable in walleye, others studies conducted with an other perciform, the European sea bass, Dicentrarchus labrax, suggested that DHP and 20β-S could be both considered as MIS [START_REF] Sorbera | In vitro oocyte maturation in the sea bass: effects of hCG, pituitary extract and steroids[END_REF][START_REF] Asturiano | Hormonal regulation of the European sea bass (Dicentrarchus labrax, L.) reproductive cycle: an individualized female approach[END_REF]. From this latter study, it appears that DHP would be involved in the initiation of the oocyte maturation, just before our first sampling time, potentially explaining the absence of DHP detection in our study. From a general point of view, the absence of progestin peaks in the plasma can be due to their local and transitory actions into the gonad rendering them difficult to detect using the blood analyses. This indicates that both methodical and physiological studies on MIS in pikeperch should be reconsidered in the future. In any case, by considering DHP as the primary MIS in pikeperch, the lack of DA receptor antagonist effects (positive or negative) on FOM progression would support the absence of DHP regulation by these treatments.

In conclusion, this study, in the current experimental conditions, showed the ineffectiveness of DA receptor antagonist treatments alone, whatever the receptor family (D1 or D2), in inducing sex-steroid changes, FOM and ovulation in pikeperch. Combined with sGnRHa, these DA receptor antagonists did not prevent sGnRHa from triggering ovulation. However, in this combination with sGnRHa, SCH but not MCP proved efficiency to increase sGnRHastimulated steroid levels. Thus, only SCH would be involved in the regulation of sex-steroids indicating a putative Dopamine receptors affect pikeperch reproduction potentiator effect of sGnRHa through D1 DA receptor blockage. The use of in vitro biological tests of organ culture (e.g. brain, pituitary and ovary) could be useful in the future to pinpoint these endocrine mechanisms.

Figure 2

 2 Figure2Effect of hormonal treatments on plasmatic concentrations of (a) 17β estradiol (E 2 ) and (b) 17,20β-dihydroxy-4-pregnen-3-one (DHP) in pikeperch. Fish were injected with NaCl (0.9%, n = 5), human chorionic gonadotropin (hCG) (500 IU/kg, n = 5), MCP 4 or MCP 20 (metoclopramide at 4 mg/kg (n = 6) or 20 mg/kg (n = 7), respectively) and SCH 0.8 or SCH 4 (SCH23390 at 0.8 mg/kg (n = 6) or 4 mg/kg (n = 7), respectively). Blood was sampled at three sampling times: 0 h and 24 h after injection and at the time of ovulation or 7 days after injection (if ovulation did not occur; final). Values are means ± SEM. a,b Different letters indicate significant differences between sampling times (P < 0.05).

Figure 3

 3 Figure3Effect of hormonal treatments on plasmatic concentrations of (a) testosterone (T) and (b) 11 ketotestosterone (11KT) in pikeperch. Fish were injected with NaCl (0.9%, n = 9), human chorionic gonadotropin (hCG) (500 IU/kg, n = 7), salmon gonadotropin-releasing hormone analogue (sGnRHa) (25 µg/kg, n = 7), sGnRHa (25 µg/kg) in combination with MCP 4 or MCP 20 (metoclopramide at 4 mg/kg (n = 6) or 20 mg/kg (n = 5), respectively) or with SCH 0.8 or SCH 4 (SCH23390 at 0.8 mg/kg (n = 7) or 4 mg/kg (n = 6), respectively). Blood was sampled at four sampling times: 0, 24 and 48 h after injection and at the time of ovulation or 14 days after injection (if ovulation did not occur; final). Values are means ± SEM. A,B,C,D Different capital letters indicate significant differences between hormonal treatments for the same sampling time (P < 0.05). a,b Different lowercase letters indicate significant differences between sampling times for the same hormonal treatment (P < 0.05).

FOM

  prog = final oocyte maturation progression; OR = ovulation rate; LT = latency time (time between injection and ovulation); SR = survival rate; HR = hatching rate; MR = malformation rate; NaCl = saline solution; hCG = human chorionic gonadotropin; sGnRHa = analogue of gonadotropin-releasing hormone; MCP 4 or MCP 20 = metoclopramide at 4 mg/kg or 20 mg/kg; SCH 0.8 or SCH 4 = SCH23390 at 0.8 mg/kg or 4 mg/kg. 1 Reproductive performance parameters are FOM prog, OR, LT, SR, HR and MR. 2 Controls include negative control (NaCl) and positive controls (hCG and sGnRHa).

Table 1

 1 Effect of hormonal treatments on reproductive performance 1 in pikeperch females

							Treatments
			Controls 2					Antagonist groups 3
	Parameters	NaCl	SEM	hCG	SEM	MCP 4	SEM	MCP 20	SEM	SCH 0.8	SEM	SCH 4	SEM	P-value
	GSI (%)	13.5 A	0.3	19.5 B	1.3	11.8 A	0.8	11.6 A	1.4	11.5 A	0.8	10.4 A	0.4	<0.001
	FOM prog	0 to 1		3 to 5		0 to 1		0 to 3		0 to 1		0 to 1
	OR (%)	0 A		100 B		0 A		17 A		0 A		0 A		<0.001
	LT (h)			53.4	6.2			47.1

GSI = gonado-somatic index; FOM prog = final oocyte maturation progression; OR = ovulation rate; LT = latency time (time between injection and ovulation); NaCl = saline solution; hCG = human chorionic gonadotropin; MCP 4 or MCP 20 = metoclopramide at 4 mg/kg or 20 mg/kg; SCH 0.8 or SCH 4 = SCH23390 at 0.8 mg/kg or 4 mg/kg.

Table 2

 2 Effect of hormonal treatments on reproductive performance 1 in pikeperch females

								Treatments
				Controls 2					Antagonist groups with GnRH 3
	Parameters	NaCl	hCG	SEM sGnRHa SEM MCP 4 SEM MCP 20 SEM SCH 0.8 SEM SCH 4 SEM	P-value
	FOM prog	0 to 1	6		6		6		6		6		6
	OR (%)	0 A	100 B		100 B		100 B		100 B		100 B		100 B		< 0.001
	LT (h)		129.7	5.4	136.9	20.0	168.5	32.6	208.3	17.0	187.8	27.1 159.7 37.6	> 0.05
	SR (%)		46.8	8.1	15.0	10.7	28.3	7.7	44.0	15.2	41.5	10.2	24.7 18.0	> 0.05
	HR (%)		32.0	8.3	11.8	10.5	21.0	7.0	37.0	13.4	36.5	12.8	9.0	5.5	> 0.05
	MR (%)		22.6	4.1	40.7	26.9	34.5	15.5	14.3	3.2	35.5	3.9	38.5 28.5	> 0.05

Antagonist groups with sGnRHa are treatments combining GnRH with one dopamine receptor antagonist (SCH or MCP).A,BMeans within the same row with different superscripts differ significantly at P < 0.001.Dopamine receptors affect pikeperch reproduction

Acknowledgements

This study was partly supported by the Eurostars project (E!9390 TRANSANDER), the Lorraine region, and the Ministry of Education, Youth and Sports of the Czech Republic, projects CENAKVA (No. CZ.1.05/2.1.00/01.0024) and CENAKVA II (No. LO1205 under the NPU I programme).

Declaration of interest

None.

Ethics statement

Fish were handled according to the European and French legislation for fish welfare and approved by the institutional Ethics Committee (APAFIS-2016022913149909).

Software and data repository resources

Data are not deposited in an official repository.


Kacem Klai

Christian Jelsch

Emmanuel Wenger

Cherif Frederic Lefebvre

Ben Nasr

Kamel Kaabi
email: kamel_kaabi@yahoo.fr

Synthesis, crystal structure, Hirshfeld surface analysis and characterization of a new Cd

Keywords: Cadmium (II) complex, X-ray structure, Hirshfeld surface, contact enrichment ratio, DFT calculations

Introduction

Cadmium (II) complexes have attracted considerable attention during the past decade thanks to their functional properties such as luminescent potential [1], urease inhibitory activity [2], corrosion inhibitory effect [3], photocatalytic activity [4], etc. Cadmium (II) cation with d 10 configuration can adopt many coordination geometries, which may include tetrahedral, trigonal bipyramidal, square pyramidal and octahedral and are particularly useful for the development of coordination frameworks [5]. When it comes to organic ligands, 2,2'-bipyridines have enjoyed considerable success as ligands in metal complexes, because of their ability to coordinate a spread of metal ions in several oxidation states [6]. Pyridine rings present in bipyridines increase the electron delocalization and conformational rigidity upon coordination to the metal ion, and therefore improve π-conjugated systems and tune the optical, electrochemical, catalytic and biological properties of the complexes [7].

In this paper, we report the synthesis, Hirshfeld surface analysis and physicochemical characterization of a new Cd(II) complex with the bidentate ligand 4,4'-dimethyl-2,2'dipyridine [CdI2(DMDPy)2].

Experimental Chemical preparation

The synthesis of the [CdI2(DMDPy)2] complex was carried out by addition of a solution of 4,4'dimethyl-2,2'-dipyridine (0.4 mmol) in ethanol (20 mL) to a solution of CdI2 (0.2 mmol) in water (5 mL). After stirring for 45 min, the mixture was filtered and the resultant solution was allowed to evaporate at room temperature. Transparent prismatic single crystals of the title compound, which remained stable under normal conditions of temperature and humidity, were isolated after several days and subjected to X-ray diffraction analysis (yield 69%

X-ray single crystal structural analysis

A light green crystal of the material with dimensions 0.067 × 0.079 × 0.087 mm 3 was selected and mounted on a glass fiber. Diffraction data were obtained at 100 K on a Bruker D8 Venture diffractometer equipped with a Photon III CMOS area detector and with a Mo-Kα radiation (λ = 0.71073 Å) X-ray microfocus source. The X-ray intensities were corrected using multi-scan method applied by SADABS [START_REF] Bruker | version 2016/2[END_REF]. The crystal structure was solved by use of the ShelXT program using intrinsic phasing method and refined by full-matrix least-square techniques on F 2 using the ShelXL-2014 program [START_REF] Sheldrick | [END_REF]10]. All non-hydrogen atoms were refined anisotropically. The positions of all hydrogen atoms were introduced at ideal position and refined as riding atoms with isotropic displacement parameters ADPs (UisoH = 1.2UeqCArom; 1.2UeqN; 1.5UeqCmeth).

Theoretical calculations

All calculations were done with Gaussian 09 [START_REF] Frisch | Gaussian 09,Revision B.01[END_REF], the B3LYP method and the 6-31+G * basis for all atoms except for cadmium and iodine for which the LanL2DZ pseudopotential was used.

In order to understand why the two aromatic rings of aromatic molecules are not in the same plane (the dihedral angle between them is 8.91 °) two calculations were made:

-Optimization of hydrogen atoms only, the other atoms being in the positions defined by the X-rays in order to account for the properties in the solid state; -Full optimization of the isolated complex in order to account for the properties in solution or in the isolated state.

Each time the infrared spectrum has been calculated (in the case of full optimization it makes it possible to verify that the obtained structure corresponds to a minimum) as well as the HOMO-LUMO and MEP. Mulliken charges are also given.

Infrared spectroscopy

The IR spectra were recorded in the 4000-400 cm -1 range with a ''Perkin-Elmer FTIR'' spectrophotometer 1000 using samples dispersed in spectroscopically pure KBr pressed into a pellet.

Hirshfeld surface

The fingerprint plots of contacts around the organic molecule were generated with the CrystalExplorer3.1 software [START_REF] Mckinnon | [END_REF]. The analysis of contact types and their enrichment [13] were computed with the MoProViewer program [14]. In order to obtain, in one step, an integral Hirshfeld surface around the two independent ligands, the Cd(II) cation and the two iodine anions, it was computed around an ensemble of moieties which are not in contact with each other in the crystal.

Results and discussion

X-ray diffraction study

The main geometrical features of the different chemical entities of the new cadmium complex [CdI2(DMDPy)2] are reported in Tables 2 and3. X-ray crystal structure analysis reveals that the complex crystallizes in the orthorhombic space group Pbcn. The asymmetric unit of the compound is shown on The geometric characteristics of the bonds in the CdI2N4 octahedron are shown in Table 2.

Quantum mechanical study

Quantum chemical calculations were performed from the crystal data by using the Gaussian 09 program [START_REF] Frisch | Gaussian 09,Revision B.01[END_REF]. Full optimization of the complex results in a great decrease of the angle between the two aromatic rings (to ca. 2° instead of ca. 9°) showing that this twist is due to the structural arrangement in the solid, the solution structure being less distorted.

The HOMO and LUMO of the complex are shown in Fig. 6 for proton positions optimization only and for full optimization. In both cases, the highest occupied orbital (HOMO) is localized on the iodide anions bounded to the cadmium cation, while the lowest unoccupied orbital (LUMO) is localized on one of the organic ligands. The energy gaps of 2.34 and 2.80 eV, respectively, are relatively low, indicating that this material has a semiconductor behavior.

Molecular Electrostatic Potential analysis

The Molecular Electrostatic Potential (MEP) surface of this compound is depicted in Fig. 7. The MEP is used to determine the nuclear and electronic charge distribution of a given molecule. The maps were obtained at the B3LYP/6-31+G*-LANL2DZ level of theory.

Moreover, blue and red colors indicate the positive and negative potentials, respectively. As it can be seen, the electrostatic potential maps are color-coded and are subdivided into many regions. There is no noticeable difference between the two calculations. The most negative zone is located at one of the aromatic carbons, while the positive zones are on the two nitrogen atoms of the aromatic rings.

Mulliken population analysis

The Mulliken charge distribution in the title compound of all atoms, obtained with the same method as above, is given in Table 3. All atoms in the asymmetric unit are listed. The atoms of the organic molecule are numbered as follows:

Infrared spectroscopy

Fig. 8-a shows the IR spectrum of [CdI2(DMDPy)2]. The most representative and characteristic vibratory modes of this compound can be compared with those of analogous compounds [22,23]. In the high frequency region, the wide and intense band between 3600 and 2700 cm -1 corresponds to valence vibrations ν(C-H). The bands observed between 1700 and 1100 cm -1 are assigned to the valence vibrations ν(C=C) of the aromatic ring and ν(C-C) and ν(C-N) of the organic ligand. The vibration bands ranging between 1000 and 500 cm -1 are attributed to the out-of-plane γ(C-H), γ(C-C) and γ(C-N) deformation modes [24].

The calculated IR spectrum is given in Fig. 8-b. DFT calculations of the frequencies were made on the geometry obtained after optimization of the protons. This spectrum is very similar to the experimental one. A close agreement between the experimental and theoretical wavenumbers (R 2 = 0.998) is mostly achieved in the fingerprint region as shown in Fig. 9. Thus, the precision is sufficient to assign the experimental frequencies and to confirm the attributions proposed above.

Hirshfeld surface and contacts enrichment ratio

The Hirshfeld surface is a region in space surrounding the molecules where the chemical nature of their intermolecular contacts can be analyzed. The contact enrichment, derived from the Hirshfeld surface, is computed from the ratio of the actual contacts Cxy in the crystal with those computed as if all types of contacts had the same probability to form [25,26]. The contact enrichment ratio is a powerful tool to deduce which type of contacts are over or underrepresented in the crystal packing. An enrichment ratio larger than unity for a given pair of chemical species X…Y indicates that these contacts are favored (over-represented) in the crystal. The analysis of contact types and their enrichment were computed with the MoProViewer program [14]. The chemical nature of contacts and their enrichment ratios in the title compound are shown in Table 4 and Fig. 11. The Hirshfeld surface of the ensemble is constituted by as much as 41.8% of hydrogen atoms followed by carbon at 26.1%, I (18.5%), Cd(7.8%) and N(5.8%).

The major contacts are constituted by H…I, C…H, H…H and C…C contacts followed by

Cd…N and Cd…I coordination. The Cd(II) cation is coordinated by four nitrogen atoms resulting in a high enrichment ratio ECdN = 8.2. The metallic cation is also coordinated by two iodine atoms (ECdI = 2.1). The C…C contacts display E ratio around 1.5, which highlights the stacking interactions between the aromatic rings. The C…H contacts, corresponding to C-H…, represent 26.4% of the total area and are also slightly over represented at ECH = 1.3. DFT calculations allow the attribution of the experimental IR bands.

Fig. 1 .

 1 It contains a bidentate 4,4'-dimethyl-2,2'-dipyridine organic ligand, one iodine ion and a bivalent metallic cation of cadmium, in a special position, coordinated by four nitrogen atoms N1, N2, N1 i and N2 i (i: -x+1, y, -z+ 3 /2) from two organic molecules and two atoms of iodine I and I i to form a slightly distorted CdI2N4 octahedron. The dihedral angle between the planes containing the aromatic rings is 8.91° (Fig.2).

Fig. 1 Fig. 2

 12 Fig.1ORTEP of the title compound with displacement ellipsoids drawn at the 40% probability

  Figs. 3 and 4 respectively.

Fig. 3 Fig. 4

 34 Fig. 3 Projection along the a-axis of the structure of [CdI2(DMDPy)2]. Dotted lines indicate

Fig. 5

 5 Fig. 5 Intermolecular interactions C-H…π between hydrogen atoms and the neighboring

Fig. 6

 6 Fig. 6 Frontier orbitals in [CdI2(DMDPy)2]. (a) Optimization of protons, (b) full optimization

Fig. 7

 7 Fig. 7 Molecular Electrostatic Potential maps of [CdI2(DMDPy)2]. (a) Optimization of

Fig. 8 Fig. 9

 89 Fig. 8 (a) Experimental Infrared absorption spectrum. (b) Calculated infrared absorption spectrum of [CdI2(DMDPy)2]

Fig. 10

 10 Fig. 10 Hirshfeld surface fingerprint plots of the contacts around the 4,4'-dimethyl-2,2'dipyridine ligand

Fig. 11

 11 Fig. 11 View of the Hirshfeld surface at the interface between the organic shell and the CdI2 layer at x = n + ½. The view is a crystallographic autostereogram [14]. The first shell of organic molecules in front of the surface is shown in the central zone. At the top and at the bottom of the view, only the CdI2 ions are shown. The surface is coloured according to the atom type which is behind. blue: nitrogen, light grey: hydrogen, dark grey: carbon. Cadmium: green spheres, iodide: purple spheres.

ConclusionA

  new Cd(II) complex with the 4,4'-dimethyl-2,2'-dipyridine ligand has been prepared at room temperature. In the title compound, the organic entity behaves as a bidentate ligand and Cd(II) adopts a slightly distorted octahedron coordination. The Cd(II) metal coordination complex with four N ligands and two iodine anions results in the formation of the organic molecular dimer in the crystal. The contact enrichment ratios derived from the Hirshfeld surface analysis show that besides the coordination, the crystal packing is also stabilized by aromatic stacking between neighbor aromatic rings and by C-H… interactions well confirmed by overrepresented C...C and C…H hydrophobic contacts. The band gap energy between HOMO and LUMO indicates that the title compound has a semiconductor behavior. The Molecular Electrostatic Potential analysis reveals that the most negative zone is located at one of the aromatic carbons, while the positive zones are on the two nitrogen atoms of the aromatic rings.

  ). Anal. Calc.: C, 19.46; H, 2.26; N, 15.90. Found: C, 19.15; H, 2.57; N, 15.69.

Table 1

 1 Experimental details of [CdI2(DMDPy)2]

	Crystal data
	Chemical formula	C24H24CdI2N4
	Mr	734.68
	Crystal system, space group	Orthorhombic, Pbcn
	Temperature (K)	100
	a, b, c (Å)	16.248 (3), 10.099 (2), 15.380 (3)
	V (Å 3 )	2523.7 (8)
	Z	4
	Radiation type	Mo Kα
	µ (mm -1 )	3.33
	Crystal size (mm)	0.13 × 0.12 × 0.07
	Data collection
	Diffractometer	D8Venture
	Absorption correction	Multi-scan
	Tmin, Tmax	0.662, 0.746
	No. of measured, independent and	21666, 2898, 2282
	observed [I > 2σ(I)] reflections
	Rint	0.057
	(sin θ/λ)max (Å -1 )	0.650
	Refinement
	R[F 2 > 2σ(F 2 )], wR(F 2 ), S	0.025, 0.045, 1.07
	No. of reflections	2898
	No. of parameters	143
	H-atom treatment	H-atom parameters constrained
	Δρmax, Δρmin (e Å -3 )	0.58, -0.61

Table 2

 2 Selected

	Cd-N1	2.394 (2)	Cd -N1 i	2.394 (2)
	Cd-N2	2.394 (2)	Cd -N2 i	2.394 (2)
	Cd-I1	2.8648 (5)	Cd -I1 i	2.3949 (4)
	N2 -Cd-I1	163.27 (5)	N2 i -Cd-N1	97.78 (8)
	N2 i -Cd -I1 i	163.27 (5)	N1-Cd-I1 i	93.29 (6)
	N2 -Cd -I1 i	89.78 (6)	N1 i -Cd-I1	93.29 (6)
	N2 i -Cd -I1	89.78 (6)	N1-Cd-I1	97.58 (6)
	N2 i -Cd -N2 i	82.60 (11)	N1 i -Cd-I1 i	97.58 (6)
	N2-Cd-N1 i	97.78 (8)	N1-Cd-N1 i	162.84 (11)
	N2-Cd-N1	68.93 (8)	I1-Cd-I1 i	101.188 (19)

bond distances and angles (Å, º) in [CdI2(DMDPy)2]

The bond lengths around the cadmium cation are Cd-N1 = 2.394 (2), Cd-N2 = 2.393

(2)

and Cd-I = 2.8648 (5) Å. The bond angles around cadmium vary from 68.93 (8)° to 163.27 (5)°

Table 3

 3 Mulliken charge distribution of all atoms in [CdI2(DMDPy)2]The atomic charge distribution shows that the cadmium ion has a positive charge of 0.178 and 0.535 for the protons and full optimizations, respectively, while the iodide atoms have negative charges of -0.435 and -0.432. For the organic entity, as depicted by the Molecular

	Atom	Optimization of protons	Full optimization
	Cd	0.178	0.535
	I	-0.435	-0.432
	N	0.371 0.264	0.292 0.191
	C1-C7	0.168 0.401	0.159 0.32
	C3-C8	-0.761 -0.899	-0.726 -0.848
	H(C3,C8)	0.177 0.182	0.176 0.176
	C5-C10	0.294 0.024	0.227 0.074
	C6-C12	-0.904 -0.857	-0.95 -0.917
	H(C6,C12)	0.197 0.192	0.197 0.189
	C4-C11	0.517 0.425	0.627 0.511
	H(C4,C11)	0.236 0.204	0.23 0.203
	C13-C14	-0.594 -0.641	-0.64 -0.676
		0.241	0.241
		0.211	0.21
	H(C13,C14)	0.223 0.244	0.224 0.24
		0.223	0.226
		0.208	0.208
	Total	0	0

Table 4

 4 Nature of contacts at the ensemble of Hirshfeld surfaces around organic molecule, iodide and cadmium ions: chemical content on the surface, proportion of contact types and contacts enrichment ratios. Reciprocal contacts X-Y and Y-X were merged. The major contacts CXY are in bold characters. The enrichment values EXY larger than unity are in bold and highlight overrepresented interactions. The lower part of the table shows the major contacts on the Hirshfeld surface around the ensemble of an organic dimer and a CdI2 moiety.It is worth noting that, as can be seen in Fig.3, the CdI2 moieties are in cavities located in layers at x = n + ½ and x = n; and so they do not interact with the surrounding CdI2 groups, as the closest distance is d(Cd,I)=6.84 Å. The analysis performed on the Hirshfeld surface around the ensemble of the coordination dimer and CdI2 moiety gives another perspective of the contacts in the crystal packing (Table4). From this point of view, the C-H…I weak hydrogen bonds are and most enriched contact type. The other main contacts are H…C, H…H and C…C which are all over represented, except H…H.

	atom	H	C	N	Cd	I
	surface %	41.8	26.1	5.8	7.8	18.5
	H	11.4		%	contacts
	C	26.4	9.2
	N	1.5	0.4	0.1
	Cd	0.7	1.5	7.7	0
	I	29.1	3.0	0.9	7.7	0.4
	H	0.71			enrichment
	C	1.3	1.5
	N	0.35	0.1	0.34
	Cd	0.09	0.33	8.2	0
	I	1.7	0.29	0.39	2.1	0.10
	(dimer_formed)	H...I	H...C	H...H	C...C
	% contact%		35.2	32.5	18	11.8
	enrichment		1.8	1.07	0.67	1.39

Acknowledgment

The authors are grateful to the Tunisian Ministry of Higher Education and Scientific Research for financial support.

Supplementary data

Crystallographic data for the structural analysis have been deposited at the Cambridge Crystallographic Data Centre, CCDC No 2044251. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK: fax: (+44) 01223-336-033; e-mail: deposit@ccdc.cam.ac.