This work was supported by the Deutsche Forschungsgemeinschaft DFG (WI 1531/12-1 to KW and SG and KU 689/11-1 to KDK, KM and JMH). We sincerely thank the DAAD RISE Germany program for providing and organizing an undergraduate research internship between SG and DB (Funding-ID: 57346313). We are grateful to Mitacs for funding DB with a RISE-Globalink Research Internship Award (FR21213). We kindly thank Aaron Berard for inviting us to introduce FishSim to the JoVE readership and Alisha DSouza as well as three anonymous reviewers for their valuable comments on a previous version of the manuscript.

The performed experiments and handling of the fish were in line with the German Animal Welfare legislation (Deutsches Tierschutzgesetz), and approved by the internal animal welfare officer Dr. Urs Gie&#223;elmann, University of Siegen, and the regional authorities (Kreisveterin&#228;ramt Siegen-Wittgenstein; Permit number: 53.6 55-05).
1. Virtual Fish Design
Note: Find a list of the required hardware and software in the supplementary materials list. A...

The gravid spot in sailfin molly females was previously described to serve as a means of fertility advertisement towards conspecific males59,60. Whether a gravid spot may also provide information to conspecific females in the context of mate choice had not been tested so far. In the present case study, we investigated the potential role of a gravid spot as a source of public information for observing conspecific females in the context of MCC. Our study shows that...

Recently, utilizing modern techniques for the creation of artificial stimuli, such as computer animations and virtual reality, has garnered popularity in research1. These methods provide several advantages compared to classic experimental approaches with live stimulus animals1,2. Computer animation enables non-invasive manipulation of the appearance (size, color) and behavior of virtual stimulus animals used in experiments. For example, the surgical removal of the sword in male green swordtails (Xiphophorus helleri) to test mate preferences in females3 was rendered unnecessary by using computer animation in a later study on this species4. Furthermore, computer animations can create phenotypes that are only rarely encountered in nature5. Morphological features of virtual animals may even be altered beyond the natural range of that species4. Particularly, the possible systematic manipulation of behavior is one major advantage of computer animation, since it is almost impossible with live animals6,7.
Various techniques exist to date for creating computer animations. Simple two-dimensional (2D) animations typically derive from a picture of a stimulus moving in only two dimensions and can be created with common software like MS PowerPoint8 or Adobe After Effects9. Three-dimensional (3D) animations, which require more sophisticated 3D graphics modelling software, enable the stimulus to be moved in three-dimensions, increasing possibilities for realistic and complex physical movement6,7,10,11,12. Even virtual reality designs that simulate a 3D environment where live animals navigate have been used13,14. In a recent review Chouinard-Thuly et al. 2 discuss these techniques one by one and highlight advantages and disadvantages on their implementation in research, which notably depends on the scope of the study and the visual capacities of the test animal (see &#8220;Discussion&#8221;). Additionally, Powell and Rosenthal15 give advice on appropriate experimental design and what questions may be addressed by employing artificial stimuli in animal behavior research.
Since creating computer animation may be difficult and time consuming, the need for software to facilitate and standardize the process of animation design arose. In this study, we introduce the free and open-source FishSim Animation Toolchain16 (short: FishSim; https://bitbucket.org/EZLS/fish_animation_toolchain/), a multidisciplinary approach combining biology and computer science to address these needs. Similar to the earlier published tool anyFish17,18, the development of the toolchain followed the goal to provide researchers with an easy-to-use method for implementing animated 3D stimuli in experiments with fish. Our software consists of a set of tools that can be used to: (1) create 3D virtual fish (FishCreator), (2) animate the swimming paths of the virtual fish with a video game controller (FishSteering), and (3) organize and present prerecorded animations on monitors to live focal fish (FishPlayer). Our toolchain provides various features that are especially useful for testing in a binary choice situation but also applicable to other experimental designs. Moreover, the possible animation of two or more virtual fish enables the simulation of shoaling or courtship. Animations are not bound to a specific stimulus but may be replayed with other stimuli making it possible to change the appearance of a stimulus but keep its behavior constant. The open-source nature of the toolchain, as well as the fact that it is based on the robot operation system ROS (www.ros.org), provide high modularity of the system and offer nearly endless possibilities to include external feedback devices (as the controller or a tracking system) and to adapt the toolchain to one&#8217;s own needs in research. In addition to the sailfin molly, four other species are currently usable: the Atlantic molly Poecilia mexicana, the guppy Poecilia reticulata, the three-spined stickleback Gasterosteus aculeatus and a cichlid Haplochromis spp. New species can be created in a 3D graphics modelling tool (e.g., Blender, www.blender.org). To exemplify the workflow with FishSim and to provide a protocol on how to conduct a mate-choice copying experiment with computer animation, we performed a case study with sailfin mollies.
Mate choice is one of the most important decisions animals make in their life history. Animals have evolved different strategies for finding the best mating partners. They may rely on personal information when evaluating potential mating partners independently, possibly according to predetermined genetic preferences for a certain phenotypic trait19, 20. However, they may also observe the mate choice of conspecifics and thereby utilize public information21. If the observer then decides to choose the same mate (or the same phenotype) as the observed conspecific &#8212; the &#8220;model&#8221; &#8212; chosen previously, this is termed mate-choice copying (hereafter abbreviated as MCC)22,23. Mate-choice copying is a form of social learning and, hence, a non-independent mate-choice strategy24, which has been observed in both vertebrates25,26,27,28,29 and invertebrates30,31,32. So far, MCC was predominantly studied in fish and is found both under laboratory conditions33,34,35,36,37,38 and in the wild39,40,41,42. Mate-choice copying is especially valuable for an individual if two or more potential mating partners are apparently similar in quality, and a &#8220;good&#8221; mate choice &#8212; in terms of maximizing fitness &#8212; is difficult to make43. The quality of a model female herself can affect whether focal females copy her choice or not44,45,46,47. Respectively, &#8220;good&#8221; or &#8220;bad&#8221; model female quality has been attributed to her being more or less experienced in mate choice, for example with regard to size and age44,45,46, or by her being a conspecific or a heterospecific47. In sailfin mollies that copy the mate choice of conspecifics39,48,49,50,51, it was found that focal females even copy the rejection of a male52. Since MCC is considered to play an important role in the evolution of phenotypic traits as well as speciation and hybridization21,23,53,54, the consequences of copying a &#8220;false&#8221; choice may be tremendous in reducing the fitness of the copier55. If an individual decides to copy the choice of another individual, it is important to evaluate if the observed model is a reliable source of information, i.e., that the model itself is making a &#8220;good&#8221; choice due to him or her being well experienced in mate choice. Here the question arises: what visual features may characterize a reliable model to copy from in sailfin molly females?
A distinct visual feature in female sailfin mollies and other Poeciliids is the gravid spot (also known as &#8216;anal spot&#8217;, &#8216;brood patch&#8217; or &#8216;pregnancy spot&#8217;). This darkly pigmented area in their anal region derives from melanization of the tissue lining the ovarian sac56. The size and presence of the gravid spot are variable across conspecific females and may further individually change during the progression of ovarian cycles56,57. Gravid spots may serve to attract males and facilitate gonopodial orientation for internal insemination58 or as a means of advertising fertility59,60. Considering the link between the gravid spot and a female&#8217;s reproductive status, we predicted that the gravid spot serves as a sign of model female quality by providing information on her current reproductive state to observing focal females. We investigated two alternate hypotheses. First, if the gravid spot is a general sign for maturity, as predicted by Farr and Travis59, it denotes a presumably reliable and experienced model compared to an immature model (without the spot). Here, focal females are more likely to copy the choice of a model with a spot but not that of a model without a spot. Second, if the gravid spot marks non-receptivity due to already developing broods, as predicted by Sumner et al.60, the model is presumably less reliable since non-receptive females would be considered less choosy. In this case, focal females will not copy their choice but that of models without spot. So far, the role of the gravid spot for MCC in sailfin molly females has never been tested, nor experimentally manipulated.
We used FishSim to perform an MCC experiment by presenting virtual stimulus males and virtual model females on computer monitors instead of using live stimulus and model fish as used in the classic experimental procedure49,50,51,61. The general usability of our software has previously been validated for testing hypotheses about mate choice in sailfin mollies12. Here, we tested whether the absence or presence of a gravid spot in virtual model females affects the mate choice of observing live focal females. We first let focal females acclimate to the test tank (Figure 1.1) and let them choose between two different virtual stimulus males in a first mate-choice test (Figure 1.2). Afterwards, during the observation period, the prior non-preferred virtual male was presented together with a virtual model female (Figure 1.3). In a subsequent second mate-choice test, focal females chose again between the same males (Figure 1.4). We analyzed whether focal females had copied the mate choice of the observed model female by comparing her mate-choice decision in the first and second mate-choice test. We performed two different experimental treatments in which we visually manipulated the quality of the virtual model female. During the observation period, we either presented the prior non-preferred virtual male (1) together with a virtual model female with a gravid spot (&#8220;spot&#8221; treatment); or (2) together with a virtual model female without a gravid spot (&#8220;no spot&#8221; treatment). Additionally, in a control without any model female, we tested whether focal females chose consistently when no public information was provided.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58435/58435fig1.jpg" /> 
Figure 1. General overview of the most important experimental steps for a MCC experiment using virtual fish stimuli. (1) Acclimatization period. (2) First mate-choice test: live focal female chooses between virtual stimulus males. (3) Observation period: focal female watches the prior non-preferred male together with a virtual model female with gravid spot. (4) Second mate-choice test: the focal female again chooses between virtual stimulus males. In this example, she copies the choice of the model. <a href="https://www.jove.com/files/ftp_upload/58435/58435fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>Hardware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x 19&#34; Belinea LCD displays</td>
			<td>Belinea GmbH, Germany</td>
			<td>Model 1970 S1-P</td>
			<td>1280 x 1024 pixels resolution</td>
		</tr>
		<tr>
			<td>1x 24&#34; Fujitsu LCD display</td>
			<td>Fujitsu Technology Solutions GmbH, Germany</td>
			<td>Model B24-8 TS Pro</td>
			<td>1920 x 1080 pixels resolution</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td></td>
			<td></td>
			<td>Intel Core 2 Quad CPU Q9400 @ 2.66GHz x 4, GeForce GTX 750 Ti/PCIe/SSE2, 7.8 GiB memory, 64-bit, 1TB; keyboard and mouse</td>
		</tr>
		<tr>
			<td>SONY Playstation 3 Wireless Controller</td>
			<td>Sony Computer Entertainment Inc., Japan</td>
			<td>Model No. CECHZC2E</td>
			<td>USB-cable for connection to computer</td>
		</tr>
		<tr>
			<td>Glass aquarium</td>
			<td></td>
			<td></td>
			<td>100 cm x 40 cm x 40 cm (L x H x W)</td>
		</tr>
		<tr>
			<td>Plexiglass cylinder</td>
			<td>custom-made</td>
			<td></td>
			<td>49.5 cm height, 0.5 cm thickness, 12 cm diameter; eight small holes (approx. 5 mm diameter) drillt close to the end of the cylinder lower the amount of water disturbance while releasing the fish</td>
		</tr>
		<tr>
			<td>Gravel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x OSRAM L58W/965</td>
			<td>OSRAM GmbH, Germany</td>
			<td></td>
			<td>Illumination of the experimental setup</td>
		</tr>
		<tr>
			<td>2x Stopwatches</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ubuntu 16.04 LTS</td>
			<td></td>
			<td></td>
			<td>Computer operating system; Download from: https://www.ubuntu.com/</td>
		</tr>
		<tr>
			<td>FishSim Animation Toolchain v.0.9</td>
			<td></td>
			<td></td>
			<td>Software download and user manual (PDF) from: https://bitbucket.org/EZLS/fish_animation_toolchain</td>
		</tr>
		<tr>
			<td>GIMP Gnu Image Manipulation Program (version 2.8.22)</td>
			<td></td>
			<td></td>
			<td>Download from: https://www.gimp.org/</td>
		</tr>
	</tbody>
</table>

using the fishsim animation toolchain to investigate fish behavior: a case study on mate-choice copying in sailfin mollies

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the appearance and behavior of visual stimuli, compared to manipulating live animals. Here, we present the FishSim Animation Toolchain, a software framework developed to provide researchers with an easy-to-use method for implementing 3D computer animations in behavioral experiments with fish. The toolchain offers templates to create virtual 3D stimuli of five different fish species. Stimuli are customizable in both appearance and size, based on photographs taken of live fish. Multiple stimuli can be animated by recording swimming paths in a virtual environment using a video game controller. To increase standardization of the simulated behavior, the prerecorded swimming path may be replayed with different stimuli. Multiple animations can later be organized into playlists and presented on monitors during experiments with live fish.
In a case study with sailfin mollies (Poecilia latipinna), we provide a protocol on how to conduct a mate-choice copying experiment with FishSim. We utilized this method to create and animate virtual males and virtual model females, and then presented these to live focal females in a binary choice experiment. Our results demonstrate that computer animation may be used to simulate virtual fish in a mate-choice copying experiment to investigate the role of female gravid spots as an indication of quality for a model female in mate-choice copying.
Applying this method is not limited to mate-choice copying experiments but can be used in various experimental designs. Still, its usability depends on the visual capabilities of the study species and first needs validation. Overall, computer animations offer a high degree of control and standardization in experiments and bear the potential to &#39;reduce&#39; and &#39;replace&#39; live stimulus animals as well as to &#39;refine&#39; experimental procedures.

Following the protocol, we used FishSim to create computer animations of virtual sailfin molly males and females. We further used the toolchain to present animations to live focal females in a binary choice situation to perform an MCC experiment according to the experimental procedure described in Figure 1 and step 5 of the protocol.
In order to determine whether focal females copied the ch...

Watch this Scientific Journal Video about Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies at JoVE.com

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Using the novel FishSim Animation Toolchain, we present a protocol for non-invasive visual manipulation of public information in the context of mate-choice copying in sailfin mollies. FishSim Animation Toolchain provides an easy-to-use framework for the design, animation and presentation of computer-animated fish stimuli for behavioral experiments with live test fish.