Technical and conceptual considerations forusing animated stimuli in studies of animalbehavior

Rapid technical advances in the field of computeropened new avenues in animal behavior research.animation （CA） and virtual reality （VR） haveAnimated stimuli are powerful tools as theyoffer standardization, repeatability, and complete control over the stimulus presented, thereby＂reducing＂ and ＂replacing＂ the animals used, and ＂refining＂ the experimental design in line withthe 3Rs. However, appropriate use of these technologies raises conceptual and technical questions.In this review, we offer guidelines for common technical and conceptual considerations related tothe use of animated stimuli in animal behavior research. Following the steps required to create ananimated stimulus, we discuss （I） the creation, （11） the presentation, and （111） the validation of CAsand VRs. Although our review is geared toward computer-graphically designed stimuli, consider-ations on presentation and validation also apply to video playbacks. CA and VR allow both new be-havioral questions to be addressed and existing questions to be addressed in new ways, thus weexpect a rich future for these methods in both ultimate and proximate studies of animal behavior.

The virtual lover： variable and easily guided 3D fish animations as an innovative tool inmate-choice experiments with sailfin mollies-I,Design and implementation

Animal behavior researchers often face problems regarding standardization and reproducibility oftheir experiments. This has led to the partial substitution of live animals with artificial virtual stim-uli. In addition to standardization and reproducibility, virtual stimuli open new options for re-searchers since they are easily changeable in morphology and appearance, and their behavior canbe defined. In this article, a novel toolchain to conduct behavior experiments with fish is presentedby a case study in sailfin mollies Poecilia latipinna. As the toolchain holds many different and novelfeatures, it offers new possibilities for studies in behavioral animal research and promotes thestandardization of experiments. The presented method includes options to design, animate, andpresent virtual stimuli to live fish. The designing tool offers an easy and user-friendly way to definesize, coloration, and morphology of stimuli and moreover it is able to configure virtual stimuli ran-domly without any user influence. Furthermore, the toolchain brings a novel method to animatestimuli in a semiautomatic way with the help of a game controller. These created swimming pathscan be applied to different stimuli in real time. A presentation tool combines models and swim-ming paths regarding formerly defined playlists, and presents the stimuli onto 2 screens.Experiments with live sailfin mollies validated the usage of the created virtual 3D fish models inmate-choice experiments.

The virtual lover： variable and easily guided 3D fish animations as an innovative tool inmate-choice experiments with sailfinmollies-II. Validation

The use of computer animation in behavioral research is a state-of-the-art method for designing andpresenting animated animals to live test animals. The major advantages of computer animations are：（1） the creation of animated animal stimuli with high variability of morphology and even behavior; （2）animated stimuli provide highly standardized, controlled and repeatable testing procedures; and （3）they allow a reduction in the number of live test animals regarding the 3Rs principle. But the use of ani-mated animals should be attended by a thorough validation for each test species to verify that behaviormeasured with live animals toward virtual animals can also be expected with natural stimuli. Here wepresent results on the validation of a custom-made simulation for animated 3D sailfin mollies Poecilialatipinna and show that responses of live test females were as strong to an animated fish as to a videoor a live male fish. Movement of an animated stimulus was important but female response was stron-ger toward a swimming 3D fish stimulus than to a ＂swimming＂ box. Moreover, male test fish wereable to discriminate between animated male and female stimuli; hence, rendering the animated 3D fisha useful tool in mate-choice experiments with sailfin mollies.

Sex differences in color discrimination and serial reversal learning in mollies and guppies

Behavioral flexibility provides an individual with the ability to adapt its behavior in response to environmental changes. Studies on mammals, birds, and teleosts indicate greater behavioral flexibility in females. Conversely, males appear to exhibit greater behavioral persistenee. We, therefore, investigated sex differences in behavioral flexibility in 2 closely related molly species (Poecilia latipinna, P. mexicana) and their more distant relative, the guppy P. reticulata by comparing male and female individuals in a serial, visual reversal learning task. Fish were first trained in color discrimination, which was quickly learned by all females (guppies and mollies) and all molly males alike. Despite continued training over more than 72 sessions, male guppies did not learn the general test procedure and were, therefore, excluded from further testing. Once the reward contingency was reversed serially, molly males of both species performed considerably better by inhibiting their previous response and reached the learning criterion sign讦icantly faster than their respective con specific females. Moreover, Atlantic molly males clearly outperformed all other individuals (males and females) and some of them even reached the level of 1-trial learning. Thus, the apparently un iversal pattern of higher female behavioral flexibility seems to be in verted in the 2 examined molly species, although the evolutionary account of this pattern remains highly speculative. These findings were complemented by the observed lower neophobia of female sailfin mollies compared with their male con specifics. This sex differe nee was not observed in Atlantic mollies that were observed to be sigrdficantly less distressed in a novel situation than their consexuals. Hypothetically, sex differences in behavioral flexibility can possibly be explained in terms of the different roles that males and females play in mating competition, mate choice, and reproduction or, more gen erally, in complex social in teractio ns. Each of these characteristics clearly differed between the closely related mollies and the more distantly related guppies.

(Under)water love-linking mate choice and cognition in fish and frogs

Aims and Objectives This Special Column aims at complementing our knowledge and deepening our understanding of the complex processes involved in learning and neurobiological mechanisms in the context of sexual selection.

Virtual is the new reality

The ideal animal stimulus is under total control of the experimenter,has visual traits, and behavior patterns that can be varied in anyway, and it appears and behaves consistently between test trials thatcan be easily repeated many times and any time. Does this soundlike wishful thinking of a biologist？ Using and exploiting the poten-tial of artificial stimuli in animal behavior research is actually not anew idea.