DOI: 10.5061/dryad.44j0zpchs
title: Novel husbandry practices result in rapid rates of growth and sexual maturation without impacting adult behavior in the blind Mexican cavefish

description: The development of animal model systems is dependent on the
 standardization of husbandry protocols that increase fecundity and reduce
 generation time. The blind Mexican tetra, Astyanax mexicanus, is an
 emerging genetic vertebrate model for evolution and biomedical research.
 Surface and cave populations of A. mexicanus have independently evolved,
 providing a model system for studying the genetic basis of divergent
 biological traits. While a rapid increase in the use of A. mexicanus has
 led to the generation of genetic tools including gene-editing and
 transgenesis, a slow and inconsistent growth rate remains a major
 limitation to the expanded application of A. mexicanus. The optimization
 of husbandry protocols that maximize high-nutrient feed, smaller tank
 densities, and larger tank sizes across development, would facilitate
 faster growth and expand the use of this model. Here, we describe
 standardized husbandry practices that optimize growth through a
 high-protein diet, increased feeding, growth sorting of larvae and
 juveniles, and tank size transitions based on standard length. These
 changes to husbandry had a significant effect on growth rates and
 decreased the age of sexual maturity in comparison to our previous
 protocols. To determine whether our nutritional change and increased
 feeding impacted behavior, we tested fish in exploration and schooling
 assays. We found that a change in diet had no effect on the behaviors we
 tested, suggesting that increased feeding and rapid growth will not impact
 the natural variation in behavioral traits. Taken together, this
 standardized husbandry protocol will accelerate the development of A.
 mexicanus as a genetic model.
description: <strong>Fish maintenance and husbandry</strong>:
 <em>A. mexicanus</em> were cared for in accordance with NIH
 guidelines and all experiments were approved by the Florida Atlantic
 University Institutional Care and Use Committee protocol #A1929.
 <em>A. mexicanus</em> stocks were housed in the Florida
 Atlantic Universities Mexican tetra core facilities. <em>A.
 mexicanus</em> fish lines used for this study; Pachón cavefish
 stocks were initially derived from Richard Borowsky (NYU); Surface fish
 stocks were derived from Rio Choy wild populations.
 <strong>Dietary ingredients and feeding
 schedule</strong>: Control fish stocks were fed using pet store
 flake food with nutritional content of 40% crude protein, 5% crude fat, 5%
 crude fiber, 9% moisture, and 9% ash. Fish fed under our new protocol
 changed diet during development; Gemma as larvae, ground blood worms and
 Zeigler pellets as juveniles, and blood worms and Zeigler pellets as
 adults. Brine shrimp - 60% protein, 24% fat, 4.4% ash, and 8.5% moisture -
 was used as a supplement at all stages. Gemma (100-500 pellet size)
 nutritional content; 59% protein, 14% oil, 14% ash, 0.2% fiber and 1.3%
 phosphorus. Zeigler pellets nutritional content; 45% protein, 16% fat, 2%
 fiber, 12% moisture, and 8% ash. Fish were fed to satiation and tank
 cleaning was performed every other week to promote excellent water quality
 and high oxygenation. <strong>Standard length
 measurements</strong>: Videos were collected with rulers placed at
 the front and back of the tank. Frames were analyzed in Fiji by measuring
 the standard length of fish swimming against the ruler’s edge. The scale
 was coded by setting the pixel length of 1 cm line segment against the
 ruler. Five fish per tank were measured for each time point and continued
 measuring through 5 cm in standard length.  A standard length of 50 mm or
 5 cm was chosen as a target because previous laboratory members have
 reported successful breeding of 4-5 cm standard length surface fish and
 cavefish (data not shown). <strong>Novel Tank
 Assay</strong>: Adult fish were transported to the adult behavioral
 room to acclimate for 1 hour. Following acclimation, individual fish were
 added singly to 5L plastic zebrafish tanks (Aquaneering Inc., San Diego,
 CA, USA) and recorded using a FLIR Grasshopper®3, GS3-U3-23S6M-C 1/1.2
 (Teledyne FLIR LLC., Wilsonville, OR, USA) at 15 frames per second for 15
 minutes. Videos were then analyzed using Noldus EthoVision® Software XT14
 (Noldus Inc., Wageningen, Netherlands, EU) to track and measure time spent
 at the top and bottom half of the tank.
 <strong>Schooling Assay</strong>: Fish were fed at
 least 1 hour prior to being assayed and then carried to a designated
 behavior room in a 2.5 L carrier tank. Pairs of fish were then gently
 netted into the experimental arena and allowed to acclimate for 10
 minutes. All individuals that were assayed together were from the same
 home tank. Experiments were conducted in a round tank (111 cm diameter x
 66 cm height) filled to a depth of 9 cm with system water. A Genius
 WideCam F100 video camera (Dongguan Gaoying Computer Products Co.,
 Guangdong, China) was affixed to a custom-built PVC stand that allowed
 recording from above the center of the tank. Lighting was provided via
 four white 75-watt equivalent halogen light bulbs (Philips A19 Long Life
 Light Bulb, Amsterdam, Netherlands) mounted in clamp lights with 5.5 in
 shades (HDX, The Home Depot, Georgia, United States) to diffuse light.
 Videos were collected at 30 fps using OBS Studio (Open Broadcaster
 Software). Automated tracking was done using EthoVision
 XT v. 13.0.1220 and data were exported as .xlsx files containing the X and
 Y coordinates of each fish. The median pair distance between and median
 centroid speed was found for each trial using the Pandas and NumPy
 libraries in Python 3.9.5. Pair distance was calculated as the distance
 between the center points of each fish and centroid speed was calculated
 as the movement speed of the center point directly between individuals.
 The Shapiro-Wilk test was performed to assess the normality of pair
 distance and centroid speed medians. Data were subsequently compared using
 unpaired t-tests. All statistical analyses were performed using GraphPad
 Prism 9.2.1.
description: Microsoft Excel