Video playback versus live stimuli to assess quantity discrimination in angelfish (Pterophyllum scalare).

Video playback is a widely used technique for presentation of visual stimuli in animal behavior research. In the analysis of behavioral responses to social cues, presentation of video recordings of live conspecifics represents a consistently reproducible stimulus. However, video-recordings do not interact with the experimental subject, and thus this stimulus may be inferior in the social context. Here, we evaluated how angelfish (Pterophyllum scalare) respond to a video playback of conspecifics versus a live shoal of conspecifics. Using binary choice tests, subjects were presented different stimuli. Time spent close to one versus the other stimulus was considered an index of preference. We found angelfish to prefer a live shoal of conspecifics to an empty tank, and also the video playback of a shoal of conspecifics to a blank screen, although the level of preference in the latter was lower than in the former. These results indicate that video-playback of live conspecifics may be appropriate in angelfish, thus allowing manipulation of specific cues that angelfish may use in quantity discrimination. However, when we directly contrasted a live and a video recorded shoal, both having the same number of members, experimental fish preferred the live shoal. When the choice consisted of a live shoal of four conspecifics versus a video playback of a shoal of nine conspecifics no clear preference emerged. These results imply that video-playback has disadvantages in quantity discrimination studies with angelfish. Exploring procedural and/or technological parameters will verify the suitability of video-recording-based stimulus presentation for future use in angelfish.

Food Quantity Discrimination in Angelfish (Pterophyllum scalare): The Role of Number, Density, Size and Area Occupied by the Food Items.

Quantity discrimination, the ability to identify, process, and respond to differences in number, has been shown in a variety of animal species and may have fitness value. In fish, the ability to distinguish between numerically different shoals has been well studied. However, little work has been devoted to the investigation of such ability in a foraging context. Nevertheless, angelfish (Pterophyllum scalare) have been previously shown to be able to discriminate numerically different sets of food items, with variables such as size and density of the food items playing important roles in making the choice. Here, we examine the possible role of other numerical and non-numerical variables. Using a spontaneous binary choice task, we contrasted sets of food items differing in specifically controlled ways: (1) different numerical size but equal inter-item distance; (2) different numerical size and different inter-item distance; and (3) identical total contour length and area occupied but different individual food size and inter-food distance between the contrasted food sets. In Experiment 1, angelfish were found to prefer the sets with a large number of food items. In Experiment 2, they preferred the numerically smaller sets with clustered items to the numerically larger sets with scattered items, but only when the sets were in the large number range (10 vs. 5 food items). Finally, in Experiment 3 fish preferred numerically smaller sets with large-sized and scattered food items in the large number range sets. We conclude that food item number, density, and size may not be considered individually by angelfish, but instead, the fish respond to all these factors attempting to maximize energy gained from eating the food while minimizing energy expenditure collecting and/or protecting the food.

Food density and preferred quantity: discrimination of small and large numbers in angelfish (Pterophyllum scalare).

Many animal species share the ability to discriminate between sets with different quantity of food items. In fish, this ability has rarely been investigated, although findings have been obtained do indicate a preference, as in other animals, for sets with large over small quantities. The role played by food item size has also been found to be important in the discrimination. However, another potentially important non-numerical variable, food density, has not been investigated. In this study, we examined the influence of density (inter-item distance) in the decision-making process of food discrimination in angelfish (Pterophyllum scalare). In a binary choice task, we kept the number and size of food items constant, but contrasted a set containing food items spaced further apart (sparse set) to another set with food items spaced more closely (dense set). We conducted this analysis with sets in the small (3 vs 3 food items) and in the large number range (5 vs 5 food items) and also varied the specific spatial arrangements of the food items in the sets. Contrary to expectations, angelfish showed a preference for the sparse sets over the dense sets in the five vs five contrasts irrespective of the specific spatial arrangement, but exhibited no preference in case of the three vs three contrasts. Subsequently, we slightly lengthened the inter-item distance in the dense sets, and found preference for the dense over the sparse sets. Last, we further examined the potential effect of spatial configuration of the items in the sets, but found no effect of this latter factor. Overall, these results indicate that higher density of the contrasted food item sets significantly influences choice in angelfish, which prefer denser sets if a clear discriminability of each individual item within the sets is provided.

The role of item size on choosing contrasted food quantities in angelfish (Pterophyllum scalare).

Comparative studies on quantity discrimination in animals are important for understanding potential evolutionary roots of numerical competence. A previous study with angelfish has shown that they discriminate numerically different sets of same-sized food items and prefer the larger set. However, variables that covary with number were not controlled and choice could have been influenced by variables such as size or density of the food items rather than numerical attributes. Here using a recently developed approach, we examined whether contour length of the food items affects choice in a spontaneous binary choice task. In Experiment 1, a contrast of 1 vs. 1 food item was presented, but the ratio between the size (diameter) of the food items was varied. In Experiment 2, numerically different food sets were equated in overall size by increasing the size (diameter) of the items in the numerically small sets. In both Experiments, subjects showed a preference for the larger sized food items with a discrimination limit. These results show that item size plays a prominent role in foraging decisions in angelfish. Experiment 3 placed numerical and size attributes of the sets in conflict by presenting one larger-sized food item in the numerically smaller set that also had smaller overall size (diameter) of food items. Angelfish showed no preference in any of the contrasts, suggesting that they could not make optimal foraging decisions when these attributes were in conflict. Maximization of energy return is central to optimal foraging. Accordingly, here item size was also found to be a key feature of the sets, although the numerical attributes of the sets also influenced the choice.

Quantity discrimination in angelfish (Pterophyllum scalare) is maintained after a 30-s retention interval in the large but not in the small number range.

The ability to discriminate between sets that differ in the number of elements can be useful in different contexts and may have survival and fitness consequences. As such, numerical/quantity discrimination has been demonstrated in a diversity of animal species. In the laboratory, this ability has been analyzed, for example, using binary choice tests. Furthermore, when the different number of items first presented to the subjects are subsequently obscured, i.e., are not visible at the moment of making a choice, the task requires memory for the size of the sets. In previous work, angelfish (Pterophyllum scalare) have been found to be able to discriminate shoals differing in the number of shoal members both in the small (less than 4) and the large (4 or more) number range, and they were able to perform well even when a short memory retention interval (2-15 s) was imposed. In the current study, we increased the retention interval to 30 s during which the shoals to choose between were obscured, and investigated whether angelfish could show preference for the larger shoal they saw before this interval. Subjects were faced with a discrimination between numerically small shoals (≤4 fish) and also between numerically large (≥4 fish) shoals of conspecifics. We found angelfish not to be able to remember the location of larger versus smaller shoals in the small number range, but to exhibit significant memory for the larger shoal in the large number range as long as the ratio between these shoals was at least 2:1. These results, together with prior findings, suggest the existence of two separate quantity estimation systems, the object file system for small number of items that does not work with the longer retention interval and the analogue magnitude system for larger number of items that does.

Short-Term Memory Effects on Crossing the Boundary: Discrimination between Large and Small Quantities in Angelfish (Pterophyllum scalare).

Rudimentary quantification abilities are found in numerous animal species and in human infants all demonstrating the ability to discriminate between quantities differing in numerical size. An open question is whether individuals rely on different underlying systems to discriminate between large (analogue magnitude system (AMS) for number of items exceeding 3) and small quantities (object-file system (OFS) for number of items below 4), or they use only one system (AMS) for the entire number range. The two-system hypothesis has been supported by finding reduced ability to discriminate between quantities that cross the large-small boundary in several species. Recently, the role of cognitive representation, i.e., memory, in quantity discrimination has also been recognized. Here, we investigated whether angelfish can discriminate quantities across the boundary under two memory conditions. In a binary choice test, single angelfish were allowed to see groups (shoals) of conspecifics of different numerical size on the two sides of their test tank. In Experiment 1, their choice was recorded after a 2-sec retention interval during which shoal size information was unavailable. Angelfish were able to discriminate the larger shoal across the boundary when the shoals differed by a 2:1 or higher ratio, but not when the ratio was lower. In Experiment 2, however, with a 15-sec retention interval, angelfish could only detect a four-fold difference in ratio but failed to detect a three- or a two-fold difference across the boundary. These results suggest that angelfish can remember smaller differences for a short (2 sec) but not for a longer (15 sec) period. Together with previous findings, the current results support the idea that angelfish use two distinct systems for representing quantity, but they may recruit the AMS even for the small number range under some circumstances, e.g., when higher memory demand is imposed by a greater retention interval.

Angelfish (Pterophyllum scalare) discriminate between small quantities: a role of memory.

The ability to discriminate between sets of items differing in quantity has shown a growing interest in comparative studies as a diversity of animal species exhibit such quantitative competence. Previous studies with angelfish (Pterophyllum scalare) have demonstrated that this species is capable of spontaneously discriminating between fully visible groups (shoals) of conspecifics of different numerical size. In the present study, we investigated quantity discrimination in angelfish adopting a new procedure that we expected to make the task more difficult for the fish. During a pretest period, angelfish were allowed to fully see shoals of conspecifics of different numerical size, subsequently all fish but 1 in each stimulus shoal were hidden behind opaque barriers. Thus, during testing, experimental fish had to rely on their working memory, which implies a certain level of mental representation of the quantities or numbers discriminated. Angelfish chose the larger shoal with similar accuracy when 1 versus 2, 1 versus 3, 1 versus 4, 2 versus 3, and 2 versus 4 stimulus fish were contrasted, but failed to distinguish shoals when 3 versus 4, 4 versus 5, and 4 versus 6 fish were contrasted. Strong similarities were observed in relation with our previous procedure indicating the robustness of the quantity discrimination abilities of this species. Our results imply that angelfish form internal representations and demonstrate that these fish can make comparisons between small quantities of items while relying on their working memory alone.

Quantification abilities in angelfish (Pterophyllum scalare): the influence of continuous variables.

Previous studies investigating quantity discrimination have shown that angelfish are able to select the larger of two groups of conspecifics (shoals). The discrimination limits shown by angelfish were similar to those found for other vertebrates when large (≥4) and small quantities (<4) were presented. However, in these studies, no attempt was made to control for non-numerical features of the stimulus shoals and thus the question whether numerical or some quantitative attributes of the shoals were utilized for making the choices could not be answered. Here, we investigate whether angelfish can discriminate between shoals differing in numerical size using non-numerical attributes. We systematically manipulate density, inter-fish distance, and overall space occupied by the shoals, one factor at a time, and analyse the choices angelfish made between the contrasting stimulus shoals. The stimulus shoals consisted of contrasts between large (10 vs. 5) and small (3 vs. 2) number of conspecifics. We found density to be a sufficient condition for discrimination between large shoals as the test subjects preferred the more dense shoal. Manipulation of inter-fish distance indicated that this variable is not a necessary factor in discrimination at either shoal size contrast. Likewise, we found that the size of space occupied by the contrasted shoals also did not significantly influence discrimination. Sensitivity to the density of large shoals indicates that angelfish can discriminate shoal size using this non-numerical cue. Nevertheless, the factors we examined may represent only a subset of all possible non-numerical features upon which angelfish may base their discrimination. Thus, we suggest that further research is required to clarify whether and under what circumstances angelfish may use numerical or non-numerical features when discriminating between shoals of differing size.

The role of body surface area in quantity discrimination in angelfish (Pterophyllum scalare).

Although some fish species have been shown to be able to discriminate between two groups (shoals) of conspecifics differing in the number of members, most studies have not controlled for continuous variables that covary with number. Previously, using angelfish (Pterophyllum scalare) we started the systematic analysis of the potential influence of such continuous variables, and found that they play different roles in shoal discrimination depending on whether large (≥ 4 fish) or small (<4 fish) shoals were contrasted. Here, we examine the potential role of the overall body surface area of stimulus fish in shoal preference, a prominent variable not yet examined in angelfish. We report that both when numerically large (5 versus 10 fish) and when small (2 versus 3 fish) shoals were contrasted, angelfish were unable to discriminate the numerically different shoals as long as the surface area of the contrasted shoals was equated. Thus, we conclude that body surface may be an important continuous variable in shoal discrimination. This conclusion was further supported by the analysis of preference when shoals of the same numerical size but different body surface area were contrasted. We found subjects to spend significantly more time close to the shoals with the greater overall surface area. Last, we conducted an experiment in which we simultaneously controlled a set of continuous variables, including overall surface area, and found angelfish to use the number of shoal members as a cue only in large shoal contrasts but not in small shoal contrasts. This result suggests the potential existence of different processing systems for large and small numbers in fish.

Activity counts: the effect of swimming activity on quantity discrimination in fish.

Human infants and non-human animals can discriminate the larger of two sets of discrete items. This quantity discrimination may be based upon the number of items, or upon non-numerical variables of the sets that co-vary with number. We have demonstrated that angelfish select the larger of two shoals of conspecifics without using inter-fish distance or space occupied by the stimuli as cues. However, density appeared to influence the choice between large shoals. Here, we examine the role of another non-numerical cue, swimming activity of the stimulus fish, in quantity discrimination by angelfish. To control this variable, we varied the water temperature of the stimulus aquaria or restricted the space occupied by each fish in the stimulus shoals. We used the previously successfully discriminated contrasts consisting of large (10 vs. 5) and small (3 vs. 2) shoals. We also studied whether more active or less active shoals are preferred in case of equally sized shoals (10 vs. 10, 5 vs. 5, and 3 vs. 3). When differences in stimulus fish activity were minimized by temperature manipulation we found angelfish to prefer the larger shoal in the 3 vs. 2 comparison, but not in the 10 vs. 5 comparison. When activity was controlled by space restriction, angelfish preferred the larger shoal in both numerical contrasts. These results imply that the overall activity level of the contrasted shoals is not a necessary condition for small shoals discrimination in angelfish. On the other hand, the results obtained for the large shoals, together with results obtained in the control treatments (equal numerical contrasts and differing activity levels), suggest that activity is a sufficient condition for discrimination when large shoals are involved. Further experiments are needed to evaluate the influence of other continuous variables, and to assess whether the mechanisms underlying performance are comparable to those suggested for other animals.

Spontaneous discrimination of small quantities: shoaling preferences in angelfish (Pterophyllum scalare).

The ability to quantify, i.e. to estimate quantity, may provide evolutionary advantages in some contexts and has been demonstrated in a variety of animal species. In a prior study, we showed that angelfish (Pterophyllum scalare) were able to discriminate between groups (shoals) in which a large number of conspecifics swam preferring to join the larger of the two. Our results implied that angelfish can compare relative shoal sizes likely on the basis of some quantitative attributes of the shoal. Here, also using a binary preference test, we examined whether angelfish are able to discriminate between shoals of small numbers of conspecifics, and if so whether their performance reveals a comparable underlying mechanism to that proposed for discrimination of small quantities in human and non-human animals, namely the possible precursor of the ability to count. Our results demonstrate that fish reliably chose 4 versus 1, 3 versus 1, 2 versus 1 and 3 versus 2 individuals, but were at chance performance level when having to choose between 4 versus 3, 5 versus 4 and 6 versus 5. Findings also reveal that the density of the fish in the stimulus shoals did not significantly affect the performance of experimental angelfish. These results are compatible with the hypothesis of the existence of an object-file mechanism to discriminate small quantities in vertebrates and provide evidence for spontaneous discrimination of up to three elements in angelfish, a similar limit to that found in human and non-human animals. The findings add to the growing body of data, suggesting that the mechanisms underlying discrimination between different quantities of items may be shared across different taxa and have an evolutionary ancient origin.

Can angelfish (Pterophyllum scalare) count? Discrimination between different shoal sizes follows Weber's law.

The ability to discriminate between larger and smaller quantities has been demonstrated in several mammalian and avian species suggesting the possibility of evolutionary conservation of this characteristic. Preference for the larger of two groups has also been shown in fish species, although this ability has rarely been systematically studied in lower order vertebrates, and thus the mechanisms of such ability are not understood. Here, we exploit the tendency of angelfish to seek protection in an unfamiliar environment by joining a group of conspecifics, a behaviour called shoaling. Test fish were given a simultaneous choice between shoals varying both in terms of numerical ratios and absolute numbers of fish. Our results provide evidence for quantity discrimination in angelfish. In general, experimental subjects chose the larger of two shoals. Furthermore, in agreement with Weber's law, which holds that discrimination between two quantities depends on their ratio, the discrimination between shoals of different quantities of fish was more difficult when the shoal sizes became more similar. The limit of discrimination ratio was found to be below 2:1. Briefly, angelfish are able to discriminate between different quantities of conspecifics subject to a ratio limit, a finding that implies a fitness component in this behaviour similar to what has been demonstrated in higher order vertebrates.

Latent learning in zebrafish (Danio rerio).

The zebrafish may represent an excellent compromise between system complexity and practical simplicity for behavioral brain research. It may be particularly appropriate for large scale screening studies whose aim is to identify mutants with altered phenotypes or novel compounds with particular efficacy. For example, the zebrafish may have utility in the analysis of the biological mechanisms of learning and memory. Although learning and memory have been extensively studied and hundreds of underlying molecular mechanisms have been identified, this number may represent only the fraction of genes involved in these complex brain functions. Thus large scale mutagenesis screens may have utility. In order for such screens to succeed, appropriate screening paradigms must be developed. The first step in this research is the characterization of learning and memory capabilities of zebrafish and the development of automatable tasks. Here we show that zebrafish is capable of latent learning, i.e. can acquire memory of their environment after being allowed to explore it. For example, we found experimental zebrafish that experienced an open left tunnel or an open right tunnel of a maze during the unrewarded exploration phase of the test to show the appropriate side bias during a probe trial when they had to swim to a group of conspecifics (the reward). Given that exploration of the maze does not require the presence of the experimenter and the probe trial, during which the subjects are video-recorded and their memory is tested, is short, we argue that the paradigm has utility in high-throughput screening.

Recent social environment affects colour-assortative shoaling in juvenile angelfish (Pterophyllum scalare).

Theory predicts that fish should show colour-assortative shoaling in order to avoid the oddity effect whereby individuals that differ in some feature from the group majority appear to incur increased risk of predation. It has also been shown that early experience plays an important role in affecting social preferences in some fish species. In this study, the importance of colour phenotype in promoting assortative shoaling and the role played by the recent social environment on its expression were investigated in juvenile angelfish, Pterophyllum scalare. Individuals of the uniformly black and golden colour morphs were housed in a group with conspecifics of like and unlike body colour to themselves, as well as in mixed-colour groups for 4 weeks. Subsequently, they were subjected to a binary choice to shoal with a group of conspecifics composed of unfamiliar fish of either a like or unlike colour phenotype to themselves. The response of the individuals to the colour attributes of the shoals was related to their recent social environment. Fish in like- and mixed-colour previous treatments showed a preferential association with like colour conspecifics. In contrast, the shoaling behaviour exhibited by fish previously maintained with a group of unlike-coloured conspecifics (cross-housed treatment) indicated no significant preference for any of the shoals. The results suggest that angelfish use body colouration as an intraspecific shoaling cue and that learning, in the form of recent familiarization with a specific colour phenotype of conspecifics, can affect colour-assortative shoaling preferences in this species. This learning component of the choice need not be restricted to early developmental stages.

Imprinting in fish: a little explored phenomenon with possible implications for fish welfare

Imprinting, an early learning process that can determine later preferences for particular types of stimuli, is usually not a consideration inmany activities related to fish. However, disruption of such learning and/or absence of suitable conditions for the development of preferences are frequent despite the lack of appropriate knowledge regarding theeffects that these practices may have on later behaviour and health. The present study reviews the phenomenon of imprinting in fish and discusses its possible influence on fish welfare. Although there isalso potential for acoustic imprinting, here we discuss how sexual, filial and olfactory imprinting should be considered when addressing welfare issues in fish. The review focuses on the relatively few fish speciesin which this phenomenon has been mainly researched, the results often proving that it may affect some behaviour patterns and social preferences. A diversity of factors, including species, context, behavioural repertoire and social systems may affect the actual existence of the phenomenon, although conditions normally prevailing in husbandry and other systems are not adjusted to allow the emergence of the process. Likewise, the effects of imprinting (or its absence) on fish welfare have still to be demonstrated in manyfish species and should constitute a matter for further studies. The conclusion is drawn that much more needs to be learned. However, a better understanding of the relationship between this early learning processin fish and welfare is essential if a complete analysis of conditions promoting fish welfare is to be made.

Time-place learning in the cichlid angelfish, Pterophyllum scalare.

The ability of the cichlid angelfish, Pterophyllum scalare, to associate time and place to locate food, provided twice a day in two different places, was tested. Food was delivered daily in one corner of the tank in the morning and in the diagonally opposite corner in the afternoon, for a 3-week period, and the distribution of the fish in the tank was noted prior to and during feeding time. The results indicate that, in a fairly uniform environment and in the absence of external time cues, angelfish can discriminate and associate time and place to obtain a food reward. It is suggested that they do so by means of an endogenous timing mechanism.