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.
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Method Article
Behavioral Tracking and Neuromast Imaging of Mexican Cavefish
In This Article
Summary
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 interfertile2,3. 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....
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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 modifying a USB webcam. To modify a USB webcam, see a detailed description presented by the Keene Lab in this cavefish issue at JoVE (From this A. ....
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Results
The results presented herein are representative examples of what can be acquired with the presented methods. Therefore, results can deviate slightly from the ones presented here for both cavefish and surface fish depending on the experimental conditions.
Vibration attraction behavior
Representative results for VAB can be found in F.......
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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.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank all the members of the Yoshizawa lab including N. Cetraro, N. Simon, C. Valdez, C. Macapac, J. Choi, L. Lu, J. Nguyen, S. Podhorzer, H. Hernandes, J. Fong, J. Kato, and I. Lord for fish care on the experimental fish used in this manuscript. We also thank A. Keene lab members including P. Masek to train MY to assemble IR CCD camera. Lastly, we would like to thank the Media Lab - College of Social Sciences - School of Communications at the University of Hawai'i Mānoa for their invaluable help with making the video, especially B. Smith, J. Lam, and S. White. This work was supported by Hawaiian Community Foundation (16CON-78919 and 18CON-90818) and Natio....
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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/ |
References
- Keene, A. C., Yoshizawa, M., McGaugh, S. E. Biology and Evolution of the Mexican Cavefish. Biology and Evolution of the Mexican Cavefish. , Elsevier Inc. Amsterdam. (2015).
- Mitchell, R. W., Russell, W. H., Elliott, W. R.
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