S Doutreligne
email: doutreligne@ibpc.fr

C Gageat
email: gageat@ibpc.fr

Tristan Cragnolini
email: tristan.cragnolini@ibpc.fr

Antoine Taly
email: taly@ibpc.fr

Samuela Pasquali
email: samuela.pasquali@ibpc.fr

Philippe Derreumaux
email: philippe.derreumaux@ibpc.fr

Marc Baaden
email: baaden@smplinux.de

S Ébastien Doutreligne

C Édric Gageat

UnityMol: Interactive and Ludic Visual Manipulation of Coarse-Grained RNA and other Biomolecules

Keywords: I, 6, 6 [Simulation and Modeling]-Simulation Output Analysis; J, 3 [Computer Applications]: Life and Medical

HAL is

INTRODUCTION

Structural data for biomolecules is nowadays abundant. Yet we only start to understand the dynamical properties of these objects. Molecular simulation is key to extend our knowledge on the conformational landscape of a given static model. Interactive simulations in particular enable a scientist to directly probe and examine such properties. In our lab, we have a growing interest in studying RNA molecules, which lead to the development of original computational models. Here, we present a solution for their interactive manipulation based on the HireRNA physics engine and the UnityMol [START_REF] Lv | Game on, science -how video game technology may help biologists tackle visualization challenges[END_REF] visualization and interaction framework, based on the Unity3D game engine. Relevant aspects are reviewed in [START_REF] Hirst | Molecular simulations and visualization: introduction and overview[END_REF] providing a state of the art of advances in molecular simulation and visualization.

Visualization of molecular structures is commonly achieved by specialized software, among which UnityMol. It has an emphasis on real-time graphics with visual effects from the video game industry and the fast prototyping of new molecular representations, for example the HyperBalls ray-casting technique designed to render evolving atoms and bonds [START_REF] Chavent | GPU-accelerated atom and dynamic bond visualization using hyperballs: a unified algorithm for balls, sticks, and hyperboloids[END_REF]. Among similar software figures VMD, a widespread molecular visualization software [START_REF] Humphrey | VMD: visual molecular dynamics[END_REF]. VMD inspired feature extensions that we added to UnityMol such as the coupling with molecular dynamics simulations and the support for haptic devices.

Delalande et al. [START_REF] Delalande | Complex molecular assemblies at hand via interactive simulations[END_REF] used VMD together with the Gromacs molecular dynamics engine to manipulate a variety of biomolecular systems. This approach allows to interactively apply forces and investigate biomechanical effects in detail. In [START_REF] Gao | Steered molecular dynamics studies of titin i1 domain unfolding[END_REF], Gao et al. used steered molecular dynamics for the study of the all-atom Titin protein I1 domain. Stretching the molecule and measuring its extension allowed them to correlate the resilience of the domain to specific inter-atomic interactions. This virtual setup was used to interpret experimental Atomic Force Microscopy (AFM) data.

Here, our objective is to use a dedicated user-friendly graphical environment coupled with a coarse-grained physical interaction model for a similar purpose. Our contribution then is to show how our implementation can be applied to various use cases and validate our design choices. One of Gao's unfolding experiments uses a constant force, inspiring the conditions for our Use Case 3 (Section 3.3) for RNA molecules. Particularly, we show how preliminary virtual experiments were conducted in this environment. Our work demonstrates that such investigations are feasible with a mainstream game engine.

This paper is organized as follows. We first describe UnityMol's features: the rendering methods, the coupling with molecular dynamics simulations and the use of virtual reality devices. Next, we show how these features can support research in biology with multiple use cases. Application to education and pedagogy is also addressed. The last part discusses the performance of our application, current limitations and the next additions we intend for UnityMol.

PRESENTATION OF UNITYMOL

UnityMol is a molecular visualization software geared towards biology. Created in 2009, it enables us to quickly prototype and validate new molecular representations. UnityMol development is based on the Unity3D video game engine, a promising new way to design research software as previously discussed [START_REF] Lv | Game on, science -how video game technology may help biologists tackle visualization challenges[END_REF]. The next sections describe UnityMol capacities, first pre-existing ones that were customized, then additions implemented specifically for the present contribution.

Molecular Visualization

UnityMol includes both original and more traditional implementations of molecular graphics algorithms based on polygonal static and ray-casted dynamic rendering techniques.

Secondary Structures, SugarRibbons and other Polygonal Mesh Representations

Common tools are provided for a static view of the system. Among them, we can cite molecular surfaces based on a Gaussian convolution kernel, secondary structure ribbon depictions of proteins as introduced by Richardson and an innovative simplified representation for complex sugar molecules. The two first representations are standard in the field. The surface is an abstraction showing the exposed parts of the molecule, typically in contact with solvent or with a membrane. Secondary structures reflect regular atom position patterns induced by aligned interatomic hydrogen bonds. These depictions are illustrated in Figure1 for a particular molecule, a membrane-bound ion channel called GLIC rendered in UnityMol. GLIC is composed of five subunits spread around a symmetry axis, conferring a tubular core to the assembly. Atomic interactions at the entrance conduct cations inside the structure. This way, cations are propagated from one side of the membrane to the other -from top to bottom on the figure.

In addition to such relatively common visualizations, we implemented an innovative sugar representation [START_REF] Prez | Three-dimensional representations of complex carbohydrates and polysaccharidesSweet-UnityMol: A video game-based computer graphic software[END_REF]. This extension has two main purposes. Sugar molecules feature cycles detected by our algorithm. These cycles are filled with color in accordance to the underlying chemical composition. This way, specialists can quickly determine the molecule type in 3D. On the other hand, we take advantage of this geometry to generate simplified polygonal meshes and reduce the number of triangles to render. We improve the performance for the user to navigate, but also pass from a crowded all-atom view to a useful abstraction revealing the molecular morphology (See Figure 2).

HyperBalls Representation and Customization for

Coarse-Grained RNA Rendering

UnityMol comes with an HyperBalls GPU implementation. The heart of this ray-casting technique is the use of quadratic surface equations, perfectly compatible with a traditional rendering pipeline. HyperBalls elegantly render dynamic biomolecular events such as the formation of bonds and interactions.

To render an atom, a polygonal cube is first drawn. A fragment shader GPU program then traces a ray and solves the quadratic equation of a sphere. The normal is deduced for use in the lighting stage. The ray casting is clamped to the mesh boundaries, thus avoiding rays running toward infinity.

Atoms are lit using the "MatCap" shader found in Unity3D. The underlying principle is a simple texture lookup, relating the normal of each pixel to a texel. Visual feedback of molecular properties can be enhanced during this pass by using so-called "lit sphere" lighting. The procedure is the same for bonds, using a two-sheeted hyperboloid equation in this case.

UnityMol feature "metaphors". These are parameter presets corresponding to representations commonly found in the literature. An example of such a preset is the space-filling CPK representation named after the chemists Corey, Pauling, and Koltun, in which atoms are depicted as spheres.

For bigger molecules, a simplified molecular representation called coarse-grained (CG) might be used. We develop such CG models for biomolecules, among which HiRE-RNA, a model for DNA and RNA molecules [10] [3]. With such a method, several atoms are grouped together to form a pseudo-particle or grain. The amount of data to be rendered is significantly reduced, typically by an order of magnitude. The molecule parser is able to read CG models.

Furthermore, for the purposes of interactive simulations of RNA molecules, we extended the HyperBalls capabilities to render nucleo-bases as oriented ellipsoids instead of isotropic spheres. This is shown in Figure 3 on a tRNA molecule found in Escherichia coli (PDB ID 3L0U). The orientation of an ellipsoid is computed based on the plane formed by three grains constituting the nucleobase part. This geometry reflects how nucleobases must align to establish stacking interactions and form hydrogen bonded complementary pairs.

Live Simulation and Virtual Reality

To address the question of RNA and DNA folding, the HiRE-RNA physics engine computes the molecular dynamics based on a custom force field specifically developed for this model. All particles are free to move in these simulations. The next required step towards interactive visualization and manipulation of such RNA and DNA simulations was to couple UnityMol with the HiRE-RNA simulator. This coupling enables non-IT researchers to intuitively interact with the simulation and integrate their a priori knowledge about the system under study. This is a non-negligeable factor to increase the precision of such simulations.

Both the HiRE-RNA in-house simulator and UnityMol now use MDDriver, a light and generic framework for bridging molecular simulators with visualization software based on the IMD protocol [START_REF] Delalande | Complex molecular assemblies at hand via interactive simulations[END_REF]. The HiRE-RNA simulator calculates atoms positions over The simulation transmits information related to the molecule's energy. Among them figures the total energy of the system. The lower this energy, the more stable the molecule is. We provide this information to the user by the means of a time series plot (as shown later in Figure 5-right, subsection 3.2).

The protocol that was implemented supports sending forces to running simulations. Users are thus enabled to interact with the simulation by applying forces to atoms. Using the simplest possible interaction, with the mouse, the user can click an atom and grab it until the button is released.

To push this idea further, UnityMol supports special devices such as an haptic arm with force feedback and a 3D mouse with 6 degrees of freedom (d.o.f), three degrees of translation and three degrees of rotation, via the VRPN library. We came to implement several interaction models to obtain the best usability out of these peripherals.

When using the haptic arm, atoms act like magnets upon it. Once the arm is virtually sufficiently close to an atom, the user can feel a force attracting him toward the atom. For this purpose, we detect the closest atom. We substract the atom position vector with the pen position vector to determine the force direction. We use a parameterized gaussian function to relate the distance between the arm and the atom to the applied force. The further the arm is from the atom, the higher is the attracting force until the gaussian peak is passed. To bypass the physical limitation of the device workspace, we implemented a clutching feature. These functionalities and more are inspired by [START_REF] Tek | Advances in human-protein interaction -interactive and immersive molecular simulations[END_REF].

For a team to collaborate on the same dataset, complex and largescale data may be rendered on advanced displays such as the 33 megapixel 4,3x2,4 m display wall at Maison de la Simulation in Saclay used to validate our framework as illustrated in Figure 4.

When dealing with very large molecules, it becomes difficult to navigate in the scene. This is why we implemented an experimental guided navigation feature. The problem is to place the camera looking toward a target, while minimizing the occlusion; that is minimizing the number of obstacles between the camera and the target. For now, this feature is limited to the GLIC protein [START_REF] Trellet | Content-guided navigation in multimeric molecular complexes[END_REF]. We also exploit the symmetrical architecture of GLIC. We precompute trajectories around and outside the channel to constrain the camera. To further denote the molecule structure, the five sub-units can be moved away from the center. These principles are illustrated in Figure 4. See [START_REF] Trellet | Content-guided navigation in multimeric molecular complexes[END_REF]. Bottom: a picture of UnityMol running on a display wall and rendering the GLIC surface. The camera looks inside the channel thanks to the precomputed trajectory. The image is rendered across a 4x4 tiled display matrix at 7680 x 4320 pixel resolution.

APPLICATIONS

Interactive manipulation of biomolecules on the computer is a very useful technique [START_REF] Delalande | Complex molecular assemblies at hand via interactive simulations[END_REF] to probe their mechanical properties, build complex models, explore their interactions and complement experimental investigations. In our hands, we came to the conclusion that a CG model provides an excellent compromise between simulation speed and biological fidelity, being more robust with respect to user interactions than computations carried out at an all-atom level. Enriched by special screen-space effects such as motion blur or glowing atoms, the underlying dynamic molecular behavior is highlighted visually. Important properties may augment the view to inform the user about specific features such as hydrogen bonds or overall simulation energy.

In this section, we relate four use cases of these tools that we implemented, tested on selected scenarios and so far evaluated subjectively.

Use Case 1: Simulation Setup and Preparation

UnityMol has been used in the lab to prepare and validate molecular simulations. By executing a short interactive simulation, aberrations and errors in the simulation setup are easily spotted by a trained observer, whereas they may not show up immediately upon batch execution. This visual examination of the early simulation stages avoids the loss of CPU hours for erroneous runs. Very often, available structural data does not correspond to the exact conformation a numerical experiment is supposed to investigate. An interactive simulation helps to address this issue by starting a run with a molecule in an available initial conformation. Forces are ap-

Use Case 2: Immersive Interactive Exploration

The interactive approach opens up perspectives to guide simulations in an intuitive way via user input, within a dedicated graphical environment providing immersion within the scene. Peripherals such as a 6 d.o.f. 3D mouse ease the navigation in the virtual space. The user can change the point of view intuitively, and focus on the relevant part of the studied molecule. Haptic devices help to render accurate molecular models more real and tangible to the scientists. Hence, the user feels an immediate force feedback by a straightforward combination of molecular modeling and virtual reality leading to an intuitive understanding of the causal relationship between the theoretical model and its biologically relevant properties. Simpler devices such as the LeapMotion (for which experimental support was tested within Unity3D) offer less features and no haptic feedback but are easier to use. They suffice for navigation and exploration tasks.

A typical biological application that we implemented in relation to experimental nanomanipulation is to stretch an RNA hairpin on two ends, controlling and monitoring the successive detachment of its base pairs. When released, the structure may either progressively return to the initial hairpin state or feature an altered conformation, for instance induced by a base shift. This numerical experiment provides insight into the hairpin's resilience, similarly to what can be probed experimentally with optical tweezers. Figure 5 depicts a typical setup.

Use Case 3: Automated Unfolding of an RNA Triple Helix

We set up an experiment in which an RNA triple helix (PDB ID: 2K96) is stretched automatically from both ends (Figure 6). We measured the distance between atoms at each side over time. This procedure is analog to what happens in AFM experiments. We performed the resilience test at constant forces of 350 pico Newtons (pN), 700pN and 1400pN and stopped once the helix was completely straight. In such simulations and throughout our tests, we used an integration time step of 4 femtoseconds. The experiment at 350pN lasted about 11,000 time steps, providing us with 44 picoseconds of simulated time.

The observed end-to-end distance time series are shown in Figure 7 and show mostly a linear relationship between time and extension. A few cases of stable distances despite prolonged force application are observed at 350pN. We can match these distance stag- nations with the resilience of hydrogen bonds between base pairs, known to favor the stability of such molecular assemblies. Previous results are quantitative. We replicated a similar digital experiment and gathered more data. This time, a single end was pulled while the other was fixed (A is fixed, B is pulled; Figure 6). The applied force was 700pN. Measurements are reported in Figure 8.

We stored which bases formed hydrogen bonds at each timestep. Around 32 ps (marked by a red line in Figure 8), the further extension is directly related to the annihilation of the bond between nucleotides 1 and 27. We captured a video of the experiment, from which we extracted two pictures just before (Figure 9a) and right after (Figure 9b) this molecular event (Figure 9).

The applied forces are many orders of magnitude higher than in an experimental setup. This feature considerably reduces the simulation time. Furthermore, the particle of nucleotide 1, found to form an hydrogen bond with nucleotide 27, is spatially fixed in the simulation. How these simulation conditions impact the results has to be determined in the future and the numerical results should be correlated with experimental data.

Use Case 4: Education, Gamification and Ludic Research

UnityMol provides us with a ludic platform for teaching the chemistry and physics related to biomolecules. It has been used at Université Paris Diderot (Paris 7) and will be part of online courses. In addition, the curriculum includes theory and practice related to molecular dynamics simulations in general, as students acquire an insight about modelling molecular behaviour. For instance, they experience hands on how the combination of weak interactions favors stable structures in such biomolecular assemblies. We already extended the idea and set up a web app so that students can send their data to a web service. The teacher can gather their results from a centralized service to further rate them. The current implementation provides a first rudimentary basis to develop serious games with molecules. In addition to posting the interaction score on a server as currently implemented, future enhancements focusing on serious gaming concern among others simplified interaction, clear feedbacks and challenge pacing. As UnityMol is integrated in a game engine many such features can easily be used to develop a game. UnityMol features different representations potentially reusable for other projects. Also, the interactive aspect is non-negligeable for research and offers a ludic playground for visualizing biological data. The shape of complex molecules and their physical behavior can be difficult to comprehend. These aspects can be greatly clarified with interactive manipulations.

DISCUSSION

Here, we provide benchmarks under different conditions to carry out objective performance measurements. We further discuss the limitations of our application in its current state. Last but not least, we describe the focus of our future efforts.

Benchmarks

We benchmarked the performance of a compiled version of Uni-tyMol. We measured the number of frames generated per second (FPS). ticles. PDB-Ids 1Y26 and 3D0X are Metabolite-sensing mRNA, also referred to as "riboswitches". They respectively have 71 and 161 nucleotides for a total 459 and 1062 CG particles each.

The second test set includes four proteins, directly downloaded from the PDB website, selected based on their sizes. Values of both benchmarks are plotted in Figure 10.

We notice that the rendering rate remains acceptable for an interactive application around 1400 rendered atoms/particles (including bonds). Updating atom positions based on simulation data has a non-negligible impact on performance, and thus further limits our current implementation to relatively small biomolecules with simplified representations. The difference between RNA without simulation and protein might be due to the different bond count. To this date, we must figure out how to avoid bottlenecks for the application to scale better, but also optimize the costly bond rendering. Unlike the experiments reported in [START_REF] Delalande | Complex molecular assemblies at hand via interactive simulations[END_REF], it is currently not possible to simulate and interact with large biomolecules in our setup.

Limitations

Currently, we are using a simulation engine initially designed for batch execution on computation farms. This design feature may limit the deployment of the application to high-end computers with sufficient resources, in particular in terms of CPU power.

Exchanging data between the Hire-RNA simulation and the Uni-tyMol visualization is another bottleneck. The underlying MD-Driver library requires enhancements in terms of bandwidth management. More technically speaking, MDDriver integration and implementation within our simulation and visualization programs is mostly performed with mutexes. This procedure blocks the data transfer and leads to performance decrease with high simulation throughput.

Another important aspect is the user experience within interactive simulations. There is a need and much room for further improvements in UnityMol. Indeed, atoms are naturally vibrating due to thermal agitation. This relatively fast motion however impedes proper atom selection and force application, and adds jitter to the visualization. Reducing this effect visually, but also for interaction devices -in particular haptic ones -is challenging and requires further experimentation. Trivial approaches are likely to reduce the usefulness of the setup.

Feature Extension

In the next few months, we intend to organize a contest during which students will be asked to virtually interact with RNA molecules. A first set of exercices will consist in refolding molecules of known conformations. Students will pull on atoms until they obtain stable structures and then try to estimate the one of the lowest energy. They will be able to submit their structures to a web server for further evaluation and scoring, for instance using an RMSD calculation based on the known solution. A second dataset will consist in molecules of unknown structure. There, the scoring will be based on energy calculations, number of hydrogen bonds, base pair stacking etc. and it will be possible for the students to exchange and mutually improve their solution candidates. Our intention is to see how human beings may individually and collectively challenge molecular dynamics simulations in terms of time efficiency and of finding non-trivial solutions. This approach to the molecular conformation optimization problem follows a similar spirit as the Foldit puzzle game applied to protein folding [START_REF] Cooper | Predicting protein structures with a multiplayer online game[END_REF].

Rendering massive molecular data on very high resolution display systems is challenging. Furthermore we want to be able to interact fluidly with the simulated molecule. In the future, we plan to integrate the support for various Virtual Reality devices to allow an intuitive interaction with the model through immersion. The realism of interactive immersive exploration can be enhanced by pushing the illusion to be immersed within a virtual molecular world, notably when adding 3D stereoscopic rendering of the scene and head tracking, eventually wearing a head mounted display such as the Occulus rift. These extensions are not yet implemented in Uni-tyMol. In addition, this extension opens the way to collaborative investigations among researchers and a new era for presenting and sharing research results interactively.

We wish to extend the plotting functionalities to monitor the running simulation more exhaustively. We may watch the evolution of the mean distance between atoms, the number of weak bonds, the energy, etc., offering a visual dashboard for investigating molecular properties of biological systems.

Integrative biology takes advantage of available experimental data from various sources to accelerate simulations. Such data reduces the vast space of possible solutions to a subset of realistic ones. We want to extend the exchange protocol between simulations and visualization to manage constraints. When two parts of a molecule exhibit a high correlation in terms of atomic movements, the researcher would force the pairing. Such a feature would allow the further behavioral study under a priori knowledge.

There are molecules for which we know the structure of fragments, but not their structure as a whole. A very useful extension is to allow the user to place fragments in space, maybe constrained to an experimentally determined envelope. Various prepared setups could then be simulated to deduce the favorable ones. We recently started to explore such techniques [START_REF] Molza | Innovative interactive flexible docking method for multi-scale reconstruction elucidates dystrophin molecular assembly[END_REF] and are eager to test them within UnityMol.

Figure 1 :

 1 Figure 1: Three representations of the GLIC ion channel rendered in UnityMol. The five sub-units are depicted by different shades of blue and green.

  (a) All-atom representation of a sugar double-helix. Atoms are colored according to a classic convention: white for hydrogen, red for oxygen. For convenience, carbon is green instead of black. (b) Highlighting of cycles on top of a stick representation of the same molecule. The color denotes the sugar type; here, blue for glucose. Chains are differentiated by two colors; here, green and red.(c) Geometry simplification where cycles have an "internal" color to show their types. They are linked together by ribbons that can be colored according to chain or type. This can be changed by the user.

Figure 2 :

 2 Figure 2: Incremental simplification of a sugar molecule. This molecule has a double helix structure. The SugarRibbons abstraction makes this obvious.

  (a) Default coarse-grained rendering. (b) Ellipsoid rendering of bases.

Figure 3 :

 3 Figure 3: An RNA molecule of type tRNA in all-sphere and ellipsoid representations. Found in Escherichia coli, it has 73 nucleotides or 1561 atoms. With the CG HiRE-RNA model, the number of spheres to render is reduced to 475. PDB ID: 3L0U.

Figure 4 :

 4 Figure4: GLIC guided navigation through pre-computed trajectories on large-scale displays. Top: GLIC sub-units are highlighted in green, cyan, yellow, magenta and pink. The axis of symmetry is depicted as a thick green line. Red arrows show the different camera paths. Black arrows designate the camera orientation. See[START_REF] Trellet | Content-guided navigation in multimeric molecular complexes[END_REF]. Bottom: a picture of UnityMol running on a display wall and rendering the GLIC surface. The camera looks inside the channel thanks to the precomputed trajectory. The image is rendered across a 4x4 tiled display matrix at 7680 x 4320 pixel resolution.

Figure 5 :

 5 Figure 5: Left: A typical workspace. The user moves the camera with a 3D mouse (left hand) and manipulates an RNA hairpin (PDB ID 1F9L) with the haptic arm (right hand). Right: Screenshot of UnityMol with the same molecule next to an energy time plot as displayed during simulation.

Figure 6 :

 6 Figure 6: The triple helix and its extremities (PDB ID: 2K96). A: residue number 1; B: residue number 47

Figure 7 :

 7 Figure 7: Distance (in Angstrom) over time with various applied forces. The yellow curve features two plateaus, meaning distance stagnation, caused by the molecule's resilience to external forces. Yellow: 350 pico Newtons (pN), red: 700pN, blue:1400pN

Figure 8 :

 8 Figure 8: Distance over time with the second experiment. The bond annihiliation between base pair 1/27 is marked in red. It occurs at around 32 picoseconds (8000 timesteps) of simulation.

  (a) Bases of nucleotides 1 and 27 forming an hydrogen bond. (b) The bond has now disappeared.

Figure 9 :

 9 Figure9: Snapshots of our second experiment. We selected one particle of each nucleobase forming pairs. These are highlighted by yellow spheres. The gray boxes indicate information related to our selection.

Figure 10 :

 10 Figure 10: FPS for various systems with increasing atom counts

  The first test set comprises four CG RNA molecules of different structures for which we prepared simulation files. PDB-Id 1F9L is an hairpin featuring a GA mismatch. It has 22 nucleotides for 146 CG particles. PDB-Id 2K96 is the wild-type pseudoknot of human telomerase RNA. The molecule has 47 nucleotides for 303 CG par-

	The benchmarks were performed on a Mac Pro early
	2009 (4.1) model equipped with 2 x 2,93 GHz Quad-Core Intel
	Xeon CPUs and 32 Go of memory. The graphics card features an
	NVIDIA GeForce GT 630 GPU with 1024 Mo of memory. The
	operating system is Mac OS X Yosemite 10.10.2. The selected res-
	olution is 1920x1080 in windowed mode. Both atoms and bonds
	are rendered as HyperBalls.

CONCLUSION

We presented UnityMol as a platform for a full-blown virtual laboratory featuring graphical representations of molecular data coupled with interactive visual analysis tools. UnityMol takes advantage of Virtual Reality devices to ease the interaction with the model.

We showed how this computational tool can and will be applied to research and education. We ended with a performance benchmark, current limitations and the future expected features.


Albert Kollmann

Isabelle Touton

Agathe Brault

Michel Alvinerie

Pierre Galtier

Christian Mougin

Effect of the endectocide ivermectin on filamentous fungi

Keywords: Avermectin, Enzyme induction, Ivermectin, Fungi, Spore germination

We investigated the effect of ivermectin, at environmental concentrations and above, on several strains of filamentous fungi. Ivermectin did not show significant short-term effect on biomass production and germination rate of the spores. By contrast, the spore production was stimulated twofold in Phanerochaete chrysosporium and Mucor racemosus cultures, whereas that endpoint was reduced in Fusarium cultures. A long-term exposure of F. oxysporum to ivermectin reduced both production and germination of fungal spores. Ivermectin was not an inducer of exocellular oxidases.

Introduction

Avermectins are a class of veterinary medicines with insecticidal and anthelmintic properties. They have been developed for the protection of animals, human and crops. For instance, ivermectin is an avermectin widely used for cattle treatment. After treatment, most ivermectin is excreted in the faeces then is degraded in the dung. The decay process of dung involves the concomitant action of insects, annelids, and microorganisms. During this complex ecological process, no organism can substitute for another, and the lack of one specific activity induces a strong impact on dung decomposition.

In a previous paper [START_REF] Mougin | Fate of the veterinary medicine ivermectin in soil[END_REF], we observed that high levels of ivermectin were poorly degraded in the soil and could thus be stored for long periods. Despite a low bioavailability, ivermectin can alter mesofauna and microorganisms from dung and soil because high amounts of ivermectin enter the environment. Ivermectin reduces the decomposition of dung organic matter in soil [START_REF] Sommer | The influence of veterinary medicines on the decomposition of dung organic matter in soil[END_REF]. The effects of anthelmintics on dung-dwelling insects are now welldocumented, e.g. by [START_REF] Strong | Avermectins: a review of their impact on insects of cattle dung[END_REF] and [START_REF] Lumaret | Use of anthelmintics in herbivores and evaluation of risks for the non target fauna of pastures[END_REF]. In addition, there is a potential risk of ivermectin for springtails and earthworms, namely enchytraeids (Jensen et al. 2003) and Eisenia foetida (Halley et al. 1990). Halley et al. (1990) reported that ivermectin had no effect on soil microbes by studying effects on soil respiration and nitrification. They have also shown that ivermectin had no antifungal or antibacterial effects at environmental concentrations. Nevertheless, there is a lack of data on the potential impact of ivermectin on soil filamentous fungi. Here, we investigated the effect of ivermectin on filamentous fungi. Specifically, we evalu-ated the chemical impact on fungal growth, measured by biomass production, and on reproduction, by observing sporulation and germination of spores.

Experimental

Chemical reagents

High purity ivermectin, named 22,23-dihydroavermectin B1, and chemical reagents were purchased from Sigma-Aldrich.

Fungi

The fungal strains Fusarium oxysporum, a deuteromycete, Fusarium solani Martius Saccardo, an ascomycete, and Mucor racemosus Fresenius, a zygomycete, were isolated from soil samples. The white-rot basidiomycetes Phanerochaete chrysosporium Burdsall and Trametes versicolor (L.:Fr.) Pilat were obtained from ATCC, referenced as 24725 and 32745, respectively. They were stored at 4 °C on agar plates.

3 Liquid cultures, preparation of spore suspensions Fungal strains for suspensions of spores were grown on a culture medium containing glycerol (P. chrysosporium, [START_REF] Mougin | Biotransformation of the herbicide atrazine by the white rot fungus Phanerochaete chrysoporium[END_REF] or maltose (all other strains, [START_REF] Lesage-Meesen | A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus[END_REF] and ammonium tartrate as carbon and nitrogen sources. A mycelial mat on 10-mm-diameter agar plugs was inoculated into 10 ml of the culture medium in a 150-mL Erlenmeyer flask. Cultivation was carried out statically in the dark at 25 °C. After 8-12 days of growth, the spores were harvested by shaking the cultures with glass beads, counted and conditioned as liquid suspensions containing 2.5±0.5x10 6 spores ml -1 for inoculation. Spore counting was achieved by placing 200-µl aliquots of the medium into a Thomas counting chamber.

Short-term toxicity assessment

Culture media were supplemented with ivermectin concentrations ranging from 10 -3 to 10 -8 M immediately after inoculation (acetone solutions, 50 µl per Erlenmeyer) to assess the effect on biomass production. Fungal biomass was separated from the medium after 4, 8, and 12 days of culture and dried overnight at 105 °C for dry weight determination. The effect of ivermectin on spore germination was determined in the same experiments after a 24-h exposure by calculating the germinating rate. The effect of ivermectin on sporulation was estimated by counting the spores formed in the cultures treated with ivermectin at 10 -4 M.

Long-term toxicity assessment

Suspensions of spores were used to inoculate Erlenmeyers containing 10 ml liquid medium supplemented with 10 -5 M ivermectin, using a previously described protocol [START_REF] Kollmann | Effect of nonylphenol surfactants on fungi following the application of sewage sludge on agricultural soils[END_REF]. The cultures were then allowed to grow and sporulate in the dark at 25 °C for 10 days. Spores were harvested as described above and the experiments were repeated three times. Fungal biomass was measured on a dry weight basis. Germination and sporulation were expressed as a spore multiplication factor, which reflects the ratio between the amounts of spores produced by a culture versus the amounts of spores used for inoculation.

Peroxidase induction

Cultures of P. chrysosporium were carried out as described above in the presence of acetone solutions of ivermectin ranging from 10 -8 to 10 -3 M (50 µl solution per Erlenmeyer flask). Aliquots were assayed for peroxidase activity after 4, 8, and 12-day incubations, as previously reported by [START_REF] Mougin | Biotransformation of the herbicide atrazine by the white rot fungus Phanerochaete chrysoporium[END_REF].

Experimental design

All experiments were carried out in triplicate. Results are expressed as means ± standard deviation.

In several experiments, a Student's t-test was performed to determine significant differences.

Results and discussion

Short-term toxicity of ivermectin on fungi

We studied the dose-response relationship between ivermectin concentration ranging from 10 -3 to 10 -8 M and the development of fungi in liquid cultures. It is noteworthy that only three fungal strains, namely F. oxysporum, F. solani, and M. racemosus produced spores in our culture conditions, whereas T. versicolor was unable to sporulate. The three former strains were inoculated to media as spore suspensions whereas the latter was used as mycelial mats grown on agar plugs.

As shown on Fig. 1, ivermectin has no significant effect on biomass production, measured by weighting control and treated cultures after 4, 8, or 12 days, even at high drug concentrations.

Fungal biomass production in liquid cultures was slightly stimulated in the presence of the drug. However, the stimulation was not significant at the 0.05 level. Further, ivermectin did not effect spore germination (data not shown). As a consequence, we were unable to calculate a concentration decreasing the germination of spores by 50% after 24 h (IC50) compared with untreated controls.

In order to assess the impact of ivermectin on fungal sporulation, T. versicolor was replaced by a spore-producing-basidiomycete, P. chrysoporium. Ivermectin at 10 -4 M altered sporulation at different degrees depending on strain nature (Table 1). For instance, spore production by P. chrysosporium and M. racemosus was increased twofold, whereas it was reduced in both Fusarium strains.

Long-term toxicity of ivermectin on fungi

We further examined the effect of ivermectin on fungi following four cycles of biological events, namely spore germination, biomass formation and spore production, which may change the size of fungal populations. Two culture conditions were used: controls without ivermectin and assays under constant ivermectin concentration of 10-5 M. We calculated a spore multiplication factor (SMF) reflecting both the production and germination of spores [START_REF] Kollmann | Effect of nonylphenol surfactants on fungi following the application of sewage sludge on agricultural soils[END_REF].

The spore multiplication factor usually decreased in control cultures after successive exposure of spores to ivermectin (Fig. 2). The drug modified this endpoint according to the fungal strains.

Positive effects of the drug on the spore multiplication factor of P. chrysosporium and M. racemosus were significant at the end of the experiment. In contrast, a decreasing effect of ivermectin on spore multiplication factor was observed in F. solani and F. oxysporum cultures, in agreement with the negative effect of the drug noticed during short-term experiments on the sporulation of these strains.

As regards to F. oxysporum, the spore multiplication factor values were dramatically decreased in the presence of ivermectin after three and four cycles, suggesting a great sensitivity to the chemical during the reproduction stages. It is noteworthy that this strain responds differently to various chemicals, as the spore multiplication factor was highly stimulated after the treatment of the cultures with nonylphenol [START_REF] Kollmann | Effect of nonylphenol surfactants on fungi following the application of sewage sludge on agricultural soils[END_REF].

Biomass production in the control cultures of the four strains showed a slight decrease as the number of biological cycles increased (data not shown). There was no significant effect of ivermectin on that endpoint in P. chrysosporium, F. solani, and M. racemosus cultures. By contrast, the presence of ivermectin decreased the biomass production of F. oxysporum by 45-50% after three and four cycles. Concerning F. oxysporum, it appears that ivermectin, which was strongly affected the fungal reproduction after a long-term exposure, also decreased the production of biomass and then the size of the population.

Induction of exocellular oxidases by ivermectin

Various exocellular oxidases, namely lignin-peroxidases, manganese-dependent peroxidases and laccases, are produced by white-rot fungi. Here, the effect of ivermectin was assessed by measuring the activities of peroxidases in cultures of P. chrysosporium. At concentrations ranging from 10 -8 to 10 -3 M, ivermectin did not modify the activities of these oxidases in comparison with untreated controls after 4, 8, and 12 days of exposure (data not shown).

The effect of ivermectin on laccases produced by T. versicolor has already been studied by [START_REF] Mougin | Enhanced production of laccases by the fungus Trametes versicolor by the addition of xenobiotics[END_REF]. We showed that their activity was stimulated twofold after 3 days of exposure to the drug at 0.5x10 -3 M. In a similar way, treatment of fungal cultures with 8,9-Z and 10,11-Z isomers of ivermectin, formed through phototransformation processes at the soil surface [START_REF] Mougin | Fate of the veterinary medicine ivermectin in soil[END_REF], enhanced laccase activity by two and three, respectively. In the present study, higher concentrations (10-5 M) of these ivermectin isomers did not show additional effects.

Conclusions

Using fungal strains isolated from soil samples or originating from our collection, we showed that ivermectin affects the reproduction and growth of several strains of fungi. Nevertheless, these effects were observed after fungal exposure to amounts of the drug much higher than those bioavailable in the soil or in the dung. For that reason, we conclude that ivermectin at environmental concentration is without effect on the fungal endpoints studied here.

Fig. 1 .

 1 Fig. 1. Short-term effects of ivermectin on fungal biomass production in liquid cultures. Filled circle: Trametes versicolor; empty circle: Fusarium solani; inverted filled triangle: Mucor racemosus; empty triangle: Fusarium oxysporum

Table 1 .

 1 Short-term effect of ivermectin on fungal sporulation 9

Acknowledgements

This study was supported by INRA (AIP Ecotox) and PNETOX (contract no. 03177). The authors thank Annick Lacombe (INRA, Jouy en Josas) for attentively reading the manuscript.

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