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.

Fish maintenance and husbandry: A. mexicanus 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. A. mexicanus stocks were housed in the Florida Atlantic Universities Mexican tetra core facilities. A. mexicanus 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.

Dietary ingredients and feeding schedule: 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.

Standard length measurements: 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).

Novel Tank Assay: 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.

Schooling Assay: 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.

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