The effects of trial number and individual differences in exploration on fish performance in an object location recognition task
Data files
Jun 26, 2026 version files 45.51 KB
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README.md
5.63 KB
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SandCPhaseData_ForPublic.xlsx
28.99 KB
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SLR_Habituation_AnalysisCode.R
10.89 KB
Abstract
Frequent exposure to stimuli and testing procedures during repeat experimental trials can enhance the ability of animals to detect and respond to acute environmental change. This is especially relevant for assays which incorporate manipulation of novel objects or test object location recognition. In the spontaneous location recognition (SLR) test, animals are typically given one sample trial where they first encounter identical objects, and then a choice phase where discrimination ability is measured via differences in preferences for an object in a novel location versus one in a familiar location. This preference is based on the animal’s propensity to explore and be attracted to novelty. The SLR and similar tests, afford rapid assays of cognition, however, a fundamental question remains outstanding for many species: is there an optimal number of sample trials to ensure learning and discrimination of object locations in the choice phase while also capturing meaningful individual variation within groups? This study investigated the behavioral response of common minnows (Phoxinus phoxinus) to repeated exposures of the SLR’s sample phase, and the influence of individual behavioral type (measured as time spent in the center of the open field test) on cognitive performance. Increasing exposure to sample trials increased object exploration and movement in the spatial task. The group exposed to the most sample trials were the only group that learned the task showing significant discrimination between familiar and unfamiliar object locations. Interestingly, there was a preference for the object in the familiar location. Within group variation in discrimination ability was explained by individual differences in exploration in an open field test. Our results highlight the importance of considering multiple sample trials during SLR testing for this fish species, and more generally how increasing the number of trials and the role individual variation play in cognitive performance.
This repository contains two files: 1. An excel file of the raw data (SandCPhaseData_ForPublic.xlsx) and the R code used to analyze the data (SLR_Habituation_AnalysisCode.R)
File 1: SandCPhaseData_ForPublic.xlsx
SHEET 1: "SampleData" - Each row = one sample trial for one fish (N=103 total). Contains behavioural data from habituation trials only.
Key Variables, those used in analysis for publication:
Acclimation_Group: Treatment: 1, 3, or 5 sample trials.
TrialNumber: Sample trial number (1-5) (same as trial below).
FishID: Individual fish ID (matches Fish in ChoiceData).
Meanvelocity: Average swimming speed (cm/s) within the sample phases.
EncounterFrequency: Sum of all object encounters (left, right, other) during trial.
Other Variables measured to calculate key variables or those not used in publication analysis:
Fish: Order of fish run per group through Ethovision (a combination of Acclimation group and Fish number).
SamplePhase: How many sample trials the fish had experienced.
Trial: Trial order was captured and labelled by Ethovision according to the camera ( 4 trials were run simultaneously).
Encounter Frequency: number of times a fish encountered an object (i.e. came within 10cm of it). We labelled three objects, ‘Left’, ‘Right’, and ‘other’ during sample phase trials.
Encounter Duration: as above: duration of time (seconds) a fish spent near an object (i.e. within 10cm of it). We labelled three objects, ‘Left’, ‘Right’, and ‘other’ during sample phase trials.
Encounter Latency to First: First time the fish encountered an object (i.e. came within 10cm of it) in seconds. We labelled three objects, ‘Left’, ‘Right’, and ‘other’ during sample phase trials. A value of 'Null' indicates the fish did not ever encounter the object.
Mobility: Mean percentage time the fish spent actively swimming.
Total Encounter: Sum of all encounter durations (left, right, other) for all three objects in seconds.
SHEET 2: "ChoiceData " - Each row = one fish (N=35: 12 per treatment group), however one fish ( fish 8) was excluded in acclimation group 1 as Ethovision could not track it consistently. The data shows choice phase test results and Open Field Test (OFT) scores.
Key Variables, those used in analysis for publication:
FishID: Individual fish ID, calculated by combining Trial and Fish.
Acclimation_Group: Treatment: 1, 3, or 5 sample trials.
DiscriminationRatio: Preference score (duration-based) - calculated using Familiar and novel durations ((Novel duration - Familiar Duration)/(Novel Duration + Familiar Duration)).
DiscriminationRatioEncounter: Preference score (encounter-based) - calculated using Familiar encounter and novel encounter durations ((Novel Duration Encounter - Familiar Duration Encounter)/(Novel Duration Encounter + Familiar Duration Encounter)).
InsideCumulativeDuration: Time spent within the central zone during the Open Field Test (seconds).
Other Variables measured to calculate key variables or not used in publication analysis:
Trial: Trial order was captured and labelled by Ethovision according to the camera (trials were run simultaneously).
Fish: Order of fish run per group through Ethovision (a combination of Acclimation group and Fish number).
Familiar Frequency: number of times a fish approached the familiar object (came within 10cm).
Novel Frequency: number of times a fish approached the novel object (came within 10cm).
Familiar Duration: amount of time (seconds) a fish spent within the zone containing the familiar object.
Novel Duration: amount of time (seconds) a fish spent within the zone containing the novel object.
Familiar Encounter Duration: amount of time (seconds) a fish spent within 10cm from the familiar object.
Novel Encounter Duration: amount of time (seconds) a fish spent within 10cm from the novel object.
Meanvelocity: Average swimming speed (cm/s) within the choice phase.
File 2: R code: SLR_Habituation_AnalysisCode.R
The R script is split into 3 different analysis sections:
- Behavioral changes across repeated habituation trials
- Object discrimination performance across acclimation treatments
- The relationship between discrimination ability and individual behavioral traits
The following packages were used:
- readxl – data import from Excel files
- ggplot2 – data visualization
- lme4 – generalized linear mixed-effects models (GLMMs)
- car – ANOVA and statistical testing
- emmeans – estimated marginal means (if needed)
- dplyr – data manipulation
- DHARMa – model diagnostics and residual simulation
Data Inputs
The script uses a single Excel file (File 1: SandCPhaseData_ForPublic.xlsx) containing multiple sheets:
- SampleData – habituation (sample phase) dataset
- ChoiceData – main experimental dataset (object discrimination phase)
Key Variables
SampleData:
- FishID: individual identifier
- TrialNumber: habituation trial number
- Meanvelocity: average swimming speed
- EncounterFrequency: number of object exploration events
- Acclimation_Group: treatment group
ChoiceData:
- Fish: individual ID
- Trial: trial identifier
- Acclimation_Group: number of habituation trials (1, 3, or 5)
- DiscriminationRatio: duration-based discrimination measure
- DiscrminationRatioEncounter: encounter-based discrimination measure
- InsideCumulativeDuration: time spent in the center zone during Open Field Test (OFT)