Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Summary

Here, we present methods for high-throughput study of a series of the Mexican cavefish behaviors and vital staining of a mechanosensory system. These methods use free-software and custom-made scripts, providing a practical and cost-effective method for the studies of behaviors.

Abstract

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

Introduction

The Mexican tetra, Astyanax mexicanus (Teleostei: Characidae), is unique among fishes for having two radically distinct alternative morphs - a sighted, surface-dwelling morph and a blind, cave-dwelling morph comprised of several distinct populations¹. Although different in morphology and physiology, they are still interfertile^2,3. These interfertile morphs appear to have evolved rapidly (~20,000 years)⁴, which makes them an ideal model system for the study of rapid adaptation. Cavefish are known to have a suite of divergent morphological and behavioral t....

Protocol

All procedures are performed following the guidelines described in "Principles of Laboratory Animal Care" (National Institute of Health publication no. 85-23, revised 1985) and the approved by University of Hawai'i at Manoa Institutional Animal Care and Use Committee animal protocol 17-2560-3.

1. Vibration attraction behavior (VAB) assay ( ≤ 10 min for entire recording procedure)

NOTE: Use an infrared sensitive camera or build an infrared camera by m.......

Discussion

These presented methods are easy-to-access but can be complicated to perform due to the nature of its freeware origins. Therefore, it is highly recommended to perform trial assays and analyses before any actual experimentation.

The rate of data generation can be rapid once the experimental and analytical framework are established. Once established, it is possible to record two fish in 7 min for the VAB assay, 30 fish in 24 h for the activity/sleep assay, and one fish in 2.5 to 3 min for neurom.......

Materials

Name	Company	Catalog Number	Comments
4-Di-1-ASP (4-(4-(dimethylaminostyryl)-1-methylpyridinium iodide)	MilliporeSigma	D3418
880 nm wave length black light	Advanced Illumination	BL41192-880
avfs	freeware	Version 1.0.0.6	http://turtlewar.org/avfs/
Avisynth	freeware	Version 2.6.0	http://avisynth.nl/index.php/Main_Page
Cygwin	freeware	Version 2.11.0	https://www.cygwin.com/
Cylindrical assay chamber (Pyrex 325 ml glass dish)	Corning	3140-100	10 cm diameter 5 cm high
Ethovision XT	Noldus Information  Technology, Wageningen, The Netherlands	Version 14	https://www.noldus.com/animal-behavior-research/products/ethovision-xt
Fish Aquarium Cylinder Soft Sponge Stone Water Filter, Black	Jardin (through Amazon.com)	NA	Sponge filter for Sleep/hyperactivity recording system
Grade A Brine shrimp eggs	Brine shrimp direct	BSEA16Z
ImageJ	freeware	Version 1.52e	https://imagej.nih.gov/ij/
macro 1.8/12.5-75mm C-mount zoom lens	Toyo	NA	Attach to USB webcam by using c-mount, which is printed in 3-D printer
Neutral Regulator	Seachem	NA
Optical cast plastic IR long-pass filter	Edmund optics	43-948	Cut into a small piece to fit in the CCD of USB webcam
pfmap	freeware	Build 178	http://pismotec.com/download/ (at “Download Archive” link at the bottom)
Reef Crystals Reef Salt	Instant Ocean	RC15-10
SwisTrack	freeware	Version 4	https://en.wikibooks.org/wiki/SwisTrack
USB webcam (LifeCam Studio 1080p HD Webcam)	Microsoft	Q2F-00013	Cut 2-2.5 cm of the front
WinAutomation	freeware	Version 8	https://www.winautomation.com/ (free stand-alone app for this procedure)
Windows operating system	Microsoft	7, 8 or 10	https://www.microsoft.com/en-us/windows
x264vfw	freeware	NA	https://sourceforge.net/projects/x264vfw/