Low-Cost Alternative for Examining Social Preference in Zebrafish (Danio rerio) Using the 3-Chamber Open-Tank Free-Swim Task

Summary

Multi-chamber social preference tasks have been adapted from rodents to zebrafish, facilitating the study of social behavior in model organisms. This protocol validates a low-cost 3-chamber open-tank task using webcams and open-source software to measure zebrafish social preference, making it accessible to undergraduate research and teaching.

Abstract

Understanding social behavior is a core pursuit in neurobehavioral research, often studied using social preference tasks. These tasks, originally developed for rodents and later adapted for zebrafish, are widely used to examine social dynamics and responses. The 3-chamber open-tank free-swim task (OTFST) consists of a central tank flanked by two stimulus tanks with a camera positioned to record behavior of the subject in the central tank and polarizing film and backlighting to enhance video quality and validity. This protocol uses a low-cost setup with USB webcams and readily available materials. We feature the use of DeepLabCut, an open-source markerless pose estimation tool that enables accurate video tracking of animal movements and provides positional time-series data to be analyzed for various metrics. Pilot sessions validate the system’s ability to capture robust social preference behavior. This budget-friendly approach offers an accessible and scalable platform for labs investigating social interaction in zebrafish, and the versatility and relative simplicity of the 3-Chamber OTFST make it suitable for undergraduate teaching laboratories and research projects.

Introduction

Social preference is an individual’s tendency to engage, seek, or avoid social stimuli and is a fundamental aspect of social behavior that plays a critical role in establishing bonds and hierarchies within groups of animals, including humans^1,2,3,4. Understanding social preference is essential to characterizing the neural and behavioral mechanisms that underlie social interaction. Prior research indicates that deficits in social preferences can be associated with neurodevelopmental and psychiatric disorders⁵

Protocol

All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Christopher Newport University (Protocol #2025-23).

1. Animals

1. Obtain adult short-fin wild-type zebrafish^23,36,37, although other strains can be used. Alternatively, obtain embryos and rear them in-house.
2. House the fish in free-standing 10-gallon (38 L) tanks at a density of approximately two fish per gallon (3.8 L) of water. Equip tanks with active bio-filters, air stones, and submersible heaters. Alternati....

Results

In order to validate the low-cost methods outlined above, one condition from the experiments conducted in Velkey et. al.³³ was replicated using the present low-cost setup. Subjects (N = 23, 11 males and 12 females) were presented with the artificial moving mobile (low-fidelity) social stimuli on the left and the live shoal (high-fidelity) social stimuli on the right (Figure 2); as previous studies^33,34 have no.......

Discussion

The current protocol demonstrates that the present low-cost adaptation of the 3-Chamber OTFST can be used to reliably characterize social preference in adult zebrafish as measured by time spent in zone. Using inexpensive construction materials, a USB-connected webcam, and open-source acquisition and analysis software applications, the present system successfully replicated the finding that zebrafish prefer a shoal of live conspecifics over lower-fidelity moving models. These results validate the application of a low-cost.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10-Gallon Aquarium	Aqueon, Inc. Franklin, WI, USA	#100110010	purchased at local pet supply (1 to 4)
12 Pieces 1-inch T-Pipe Clamps	JoyHua Trade, China	ASIN #B0DF19DJF1	purchased from Amazon.com (1)
2-gang electrical box	Sigma Engineered Solutions, Raleigh, NC, USA	# 14350	purchased at local hardware store (1)
3/4" Silver-Metallic electrical metallic(EMT) Conduit - 10-foot	Wheatland Tube, Inc., Wheatland, PA, USA	#0550110000	purchased at local hardware store (3)
3-conductor cord	Utilitech, Inc., Charlotte, NC, USA	UT010710	purchased at local hardware store (1)
3-prong receptacle	Eaton, Inc., Beachwood, OH, USA	#270W-SP-LW	purchased at local hardware store (1)
3-way Air Valve	Penn-Plax, Inc. 35 Marcus Blvd. Hauppauge, NY, USA	Model VN3	purchased at local pet supply (1)
A4 Cardstock (white), laminated	varies	varies	purchased from campus bookstore (2)
Active bio-filter Penguin 100	Spectrum Brands Pet, LLC, Blacksburg, VA, USA	#PF00100B	purchased at local pet supply (1 to 4)
Air stones	Penn-Plax, Inc. 35 Marcus Blvd. Hauppauge, NY, USA	Model AS6Q	purchased at local pet supply (3)
Aquarium Air Tubing	Penn-Plax, Inc. 35 Marcus Blvd. Hauppauge, NY, USA	Model STD8	purchased at local pet supply (1)
Black Fabric Twin Sheets (167.6 cm X 243.8 cm)	Royale Linens, Inc., Melville, NY, USA	#00011342258875	purchased from Amazon.com (3)
Brio 4k Webcam	Logitech International S.A., Lausanne, Switzerland	960-001419	purchased from Amazon.com (1 to 3)
Computer (sufficient for running Open Broadcaster Studio & 1-3 Webcams)	Dell, Inc., Round Rock, TX, USA	OptiPlex Micro 7010	purchased from dell.com (1)
Dimmable light-emitting diode (LED) flat-panel light	Lithonia Lighting-Acuity Brands, Inc. Conyers, GA, USA	CTCL-153P-WH	purchased from Amazon.com (1)
E-Z Up Dust Containment Pole (2 Pack)	Trimaco, Inc., Cary, NC, USA	# 00047034547330	purchased from Amazon.com (1)
Feet Hardware Twine	varies	Varies	purchased at local hardware store (1)
Linear polarizing filters	B&H Foto & Electronics, Inc., New York, NY USA	Rosco #7300	purchased online (7)
Open Broadcaster Software	OBS Project	v32.0.0	download from https://obsproject.com (1)
Polycarbonate Sheet (0.38"/9.6mm thick)	Spectraglass, ltd., Perth, Scotland	UPC # 608037376817	purchased from Amazon.com (1)
polyvinyl chloride (PVC) Cement, 4 oz canister	The AZEK Co, Chicago, IL, USA	#ARAD0004OZ	purchased at local hardware store (1)
Rimless 5.5-Gallon Aquarium	Aqueon, Inc. Franklin, WI, USA	#100541424	purchased at local pet supply (3)
Self-adhesive white plastic film	CON-TACT Brand, La Miranda, CA, USA	#16F-C9A952-06	purchased at local hardware store (1)
Slider Dimmer switch	Lutron, Inc., Cooperburg, PA, USA	#LECL-150H-WH	purchased at local hardware store (1)
small pulley	varies	Varies	purchased at local hardware store (1)
Submersible 50W heaters (preset to 26C)	Aqueon, Inc. Franklin, WI, USA	#100106251	purchased at local pet supply (3 to 7)
Super Clamp Camera Mount	JINSUI, China	X0040GEYLR	purchased from Amazon.com (1 to 3)
Tetra Whisper 10 Aquarium Air Pump	Spectrum Pet, Home & Garden, Inc., Middleton, WI, USA	UPC #046798778516	purchased at local pet supply (1)
Weatherproof 2-gang electrical enclosure	Hubbell, Inc., Shelton, CT, USA	#MM2420C	purchased at local hardware store (1)
White PVC Sheet 1/4" x 24" 48"	Royal Building Products, Bristol, TN, USA	#3101	purchased at local hardware store (2)
White PVC Trim Board, nominal 1 x 2 (0.75" x 1/5" x 96")	Royal Building Products, Bristol, TN, USA	#7832-8	purchased at local hardware store (1)