We thank David Martasian, Diedre Heyser, Amy Parkhurst, Craig Foote, and Mandy Ng for valuable contributions to the Jeffery laboratory A. mexicanus culture facility. The research in the Jeffery laboratory is currently supported by NIH grant EY024941.

This procedure has been approved by Institutional Animal Care guidelines of the University of Maryland, College Park (Currently IACUC 469 #R-NOV-18-59; Project 1241065-1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61708/61708fig1.jpg" /> 
Figure 1. Calendars during a breeding week and a non-breeding week. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61708/61708fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Monday
<ol>
	<li>At 9-10 AM, perform water tests and steps 1.1.1-4 below.
	<ol>
		<li>Record the room, tank, and reservoir temperatures using a thermometer.</li>
		<li>Record the ammonia, nitrate, and nitrate levels with a colorimetric test kit.</li>
		<li>Record the pH from the monitoring system as well as the colorimetric test kit.</li>
		<li>Record the conductivity from the carboy monitor and the main system monitor.</li>
	</ol>
	</li>
	<li>Beginning at 10 AM, feed all fish.
	<ol>
		<li>Feed all fish in the adult system with tetra flakes, crushing the flakes in the tanks with young fish. Feed only as many flakes as a tank of fish can consume completely in 3-5 minutes, about a &#8220;finger pinch&#8221;.</li>
	</ol>
	</li>
	<li>Check the incubator used to house fingerbowls of developing embryos and change the water if needed.
	<ol>
		<li>Open the embryo incubator and check the water levels in all reservoirs. If they are running low on water, add system water. Check incubator temperature settings. Raise embryos at 73-77 &#176;F (23-25 &#176;C).</li>
	</ol>
	</li>
	<li>Clean live feed.
	<ol>
		<li>To clean the blackworms, remove the uncovered Tupperware basins containing their cultures from the live feed refrigerator and pour off the excess water above the worm clusters into the sink. Using distilled water, suspend and rinse the worms repeatedly until the pour-off water is clear.</li>
		<li>Add enough clean water so that the worm clusters are about half covered. Replace remaining worms in the live feed refrigerator, uncovered.</li>
	</ol>
	</li>
	<li>Feed fish.
	<ol>
		<li>At least 30 min after the first feeding, feed fish in the tanks in which breeding is desired with egg yolk flakes, blackworms, or both. Add a &#8220;fingerpinch&#8221; of yolk flakes per tank. Add enough blackworm clusters to allow each fish in the tank to consume about 5-10 worms.</li>
	</ol>
	</li>
	<li>At 10 AM- 1 PM, set the water temperature to 74 &#176;F.</li>
	<li>Scrub the breeding tanks as needed and set the breeding nets.</li>
	<li>Clean the tanks and place the nets at least one hour after the last feeding. Clean all tanks that nets are placed in beforehand. Set breeding nets carefully so as not to interfere with the air supply to the tank.</li>
</ol>
2. Tuesday
<ol>
	<li>Collect the embryos and wash all breeding nets.
	<ol>
		<li>At 9-10 AM, remove the breeding nets from the bottom of adult system tanks. Gently rinse the embryos into a hand-held net using the hose attached to the carboy, and invert the hand-held net into a fingerbowl of clean system water to expel the embryos.</li>
		<li>Collect and wash each set of embryos, and then place in a fingerbowl of clean system water containing 0.00003% methylene blue (blue water). If there is an exceptionally large number of embryos from a single tank, separate them into multiple bowls. The concentration of living embryos should be about 100 per 200 mL of blue water in each fingerbowl.</li>
		<li>Estimate the time of fertilization by staging the embryos under a microscope using the published A. mexicanus developmental time table17.</li>
		<li>Monitor the fingerbowls containing embryos frequently. Remove dead or deformed embryos and debris, such as uneaten food or feces, with a Pasteur pipette. Change the blue water in fingerbowls frequently.</li>
		<li>Place the fingerbowls in an incubator for 5-7 days. At this time, the yolk has been used up and feeding cultures with living brine shrimp is necessary for further development.</li>
	</ol>
	</li>
	<li>Take spawning data.
	<ol>
		<li>For every tank that drops embryos, record the following information.
		<ol>
			<li>Record the date and the tank number.</li>
			<li>Record the approximate number&#160;of embryos dropped (Figure 2): 
			High (500+) 
			Medium (200-500) 
			Low(&#60;200)</li>
			<li>Record the quality of embryos dropped (Figure 2): 
			High (&#62;75% alive) 
			Medium (25-50% alive) 
			Low (&#60;25% alive)</li>
			<li>Estimate the original spawning time by consulting the Astyanax mexicanus staging table17.</li>
			<li>Record the temperature that the system was set at when the fish spawned.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Feed all fish.</li>
	<li>Set the water temperature to 76 &#176;F.</li>
	<li>Prepare live feed.</li>
	<li>Feed fish #2.</li>
	<li>Scoop excess food and debris from tanks and scrub before resetting nets.</li>
</ol>
3. Wednesday
<ol>
	<li>Repeat steps 2.1-2.2. Collect embryos and wash all breeding nets.</li>
	<li>Take spawning data as before.</li>
	<li>Perform water tests as before.</li>
	<li>Feed all fish.</li>
	<li>Set the water temperature to 78 &#176;F.</li>
	<li>Prepare the live feed.</li>
	<li>Check on embryos in the incubator.
	<ol>
		<li>Clean and change the water of the embryos in fingerbowls that will eventually be used to replenish the general adult breeding stock. Use methylene blue-treated system water.</li>
	</ol>
	</li>
	<li>Feed fish again.</li>
	<li>Clean tanks as needed and reset nets.</li>
</ol>
4. Thursday
<ol>
	<li>Repeat steps 2.1-2.2. Collect embryos and wash and store breeding nets.</li>
	<li>Take spawning data as before.</li>
	<li>Set water temperature to 76&#176;F.</li>
	<li>Clean the live feed.</li>
	<li>Clean all individual tanks.</li>
	<li>Check on embryos in the incubator.</li>
	<li>Feed fish #2.</li>
</ol>
5. Friday
<ol>
	<li>Feed all fish.</li>
	<li>Set water temperature to 74 &#176;F.</li>
	<li>Clean the live feed.</li>
	<li>Check on embryos in the incubator.</li>
</ol>
6. Saturday
<ol>
	<li>Feed fish.</li>
</ol>
7. Sunday
<ol>
	<li>Feed fish.</li>
</ol>

<ol>
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J., Borowsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regressive+evolution+in+the+Mexican+cave+tetra,+Astyanax+mexicanus.">Regressive evolution in the Mexican cave tetra, Astyanax mexicanus.</a> Current Biology. 17, 452-454 (2007).</li><li>O'Quin, K. E., Yoshizawa, M., Doshi, P., Jeffery, W. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Astyanax+caves+of+Mexico.+Cavefishes+of+Tamaulipas,+San+Luis+Potosi,+and+Guerrero.">The Astyanax caves of Mexico. Cavefishes of Tamaulipas, San Luis Potosi, and Guerrero.</a> Association for Mexican Cave Studies Bulletin. 26, 1 (2018).</li><li>Luo, S., Wu, B., Xiong, X., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+total+hardness+and+calcium:magnesium+ratio+of+water+during+early+stages+of+rare+minnows+(Gobiocypris+rarus).">Effects of total hardness and calcium:magnesium ratio of water during early stages of rare minnows (Gobiocypris rarus).</a> Comparative Medicine. 66, 181-187 (2016).</li><li>Balasch, J. C., Tort, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netting+the+stress+responses+in+fish.">Netting the stress responses in fish.</a> Frontiers in Endocrinology. 10, 62 (2019).</li><li>Borowsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Determining the sex of adult Astyanax mexicanus. , (2008).</li><li>Paschos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+clocks,+feeding+time,+and+metabolic+homeostasis.">Circadian clocks, feeding time, and metabolic homeostasis.</a> Frontiers in Pharmacology. 6, 112 (2015).</li><li>Scheiermann, C., Kunisaki, Y., Frenette, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+control+of+the+immune+system.">Circadian control of the immune system.</a> Nature Reviews Immunology. 13, 190-198 (2013).</li><li>Williams, M. B., Watts, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+basis+and+future+directions+of+zebrafish+nutrigenomics.">Current basis and future directions of zebrafish nutrigenomics.</a> Genes &amp; Nutrition. 14, 34 (2009).</li><li>Harper, C., Wolf, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+effects+of+the+stress+response+in+fish.">Morphologic effects of the stress response in fish.</a> ILAR Journal. 50, 387-396 (2009).</li><li>Neubauer, P., Andersen, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+performance+in+fish+is+explained+by+an+interplay+between+physiology,+behavior+and+ecology.">Thermal performance in fish is explained by an interplay between physiology, behavior and ecology.</a> Conservation Physiology. 7 (1), 025 (2019).</li><li>Hinaux, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+staging+table+for+Astyanax+mexicanus.">Developmental staging table for Astyanax mexicanus.</a> Zebrafish. 8 (4), (2011).</li><li>Borowsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In vitro fertilization of Astyanax mexicanus. , (2008).</li><li>Simon, V., Hyacinthe, C., R&#233;taux, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+behavior+in+the+blind+Mexican+cavefish+and+its+river-dwelling+conspecific.">Breeding behavior in the blind Mexican cavefish and its river-dwelling conspecific.</a> PLoS One. 14 (2), 0212591 (2019).</li><li>Harvey, B. J., Carolsfield, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+Breeding+in+Tropical+Fish+Culture.">Induced Breeding in Tropical Fish Culture.</a> International Development Research Centre. , (1993).</li><li>Ma, L., Parkhurst, A., Jeffery, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+a+lens+survival+pathway+including+sox2+and+aA-crystallin+in+the+evolution+of+cavefish+eye+degeneration.">The role of a lens survival pathway including sox2 and aA-crystallin in the evolution of cavefish eye degeneration.</a> EvoDevo. 5, 28 (2014).</li><li>Krishnan, J., Rohner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cavefish+and+the+basis+for+eye+loss.">Cavefish and the basis for eye loss.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 5 (372), 20150487 (2017).</li><li>Biland&#382;ija, H., Abraham, L., Ma, L., Renner, K., Jeffery, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+changes+controlled+by+catecholaminergic+systems+explain+recurrent+loss+of+pigmentation+in+cavefish.">Behavioral changes controlled by catecholaminergic systems explain recurrent loss of pigmentation in cavefish.</a> Proceedings of the Royal Society. 285, (2018).</li><li>Ma, L., Jeffery, W. R., Essner, J. J., Kowalko, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+using+TALENs+in+blind+Mexican+cavefish.">Genome editing using TALENs in blind Mexican cavefish.</a> PLoS ONE. 1093, 0119370 (2015).</li><li>Klaassen, H., Wang, Y., Adamski, K., Rohner, N., Kowalko, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+mutagenesis+confirms+the+role+of+oca2+in+melanin+pigmentation+in+Astyanax+mexicanus.">CRISPR mutagenesis confirms the role of oca2 in melanin pigmentation in Astyanax mexicanus.</a> Developmental Biology. 441, 313-318 (2018).</li></ol>

The authors have nothing to disclose.

Astyanax mexicanus is a novel biological model that spawns frequently and can be bred easily in the laboratory1,2. Because we are interested in the developmental mechanisms that underlie evolutionary changes in A. mexicanus cave fish, the production and use of embryos is vital to our research goals. The primary purpose of maintaining an adult stock of fish is the production of embryos and young fry for use in developmental experiments and for re...

The teleost Astyanax mexicanus has an eyed surface-dwelling (surface fish) form and many different blind cave-dwelling (cave fish) forms1,2. Cave fish have evolved in perpetual darkness and under food limitations, resulting in the appearance of novel constructive and regressive traits3. The constructive traits include increases in taste buds and jaw size, sensory organs of the lateral line, and fat reserves. The regressive traits include the loss or reduction of melanin pigmentation, eyes, and behaviors, such as sleep, schooling, and aggression. An attribute of the Astyanax system is complete fertility between the two forms, allowing the use of quantitative trait loci (QTL) mapping to determine the genomic region(s) associated with constructive and regressive evolution4,5,6,7. A. mexicanus offers an advantageous system to study development because it can be induced to spawn frequently in the laboratory. The embryos of A. mexicanus are translucent, slightly larger than those of zebrafish, produced in large quantities, and develop into sexually mature adults in approximately 8-12 months. Their period of maximal spawning capacity is about 5 years. This protocol describes the workflow needed in an A. mexicanus culture facility during a typical breeding week and includes the details of fish system maintenance and the temperature control regime for maximal spawning.
A. mexicanus is a tropical fish that lives in rivers originating in limestone plateaus (surface fish) and in pools in limestone caves (cave fish)8. Limestone dissolves to produce hard water, and A. mexicanus thrives in hard water. Fish adapted to hard water conditions can tolerate a range of salty conditions, but generally breed in specific ones9. Induction of spawning behavior is accomplished by a combination of factors. Because fish are cold-blooded and rely on their environment to maintain homeostasis, their metabolism is sensitive to environmental changes and they react more quickly to stressors10. A. mexicanus should be cultured in aquatic systems under carefully regulated conditions of water flow, pH, conductivity, osmotic pressure, lighting, and water temperatures.
In the Jeffery laboratory, fish are maintained in two running water systems: (1) a &#8220;baby system&#8221; for young adult fish prior to sexual maturity and (2) an adult (or main) system for sexually mature, breeding adults. The &#8220;baby system&#8221; consists of 8 L and 15 L tanks supplied with running water. The &#8220;baby system&#8221; is seeded by fry and young metamorphosized juveniles grown from larvae in smaller (1-10 L) tanks, in which water is exchanged weekly. Larvae, fry, and juveniles are extremely food dependent and must be fed live food (brine shrimp) once a day to ensure a high rate of survival. Young juveniles from the &#8220;baby system&#8221; are placed into the adult system after about 1-1.5 years. At first, they are fed pulverized tetra flakes, and after further growth they are transferred to the regular adult feeding regime. Sexual maturity can be assessed by abdominal volume in females, and methods for determining sex have been described11. In the adult system, water is exchanged automatically in 42 L tanks 3 times per 24-hour period. The adult system is monitored daily by visual inspection and automatic temperature, pH, and conductivity readings from probes. The optimal pH is around 7.4 and can range between 6.8-7.5, the base temperature of the system is 72/73 &#176;F, and the ideal conductivity ranges between 600-800 mS. Automatic readings are displayed on a controller screen, and visual checks of water pressure are read at flow meters distributed throughout the system. Independent checks on water quality are made weekly by testing temperature and measuring water quality parameters for pH, ammonia, and nitrate using a colorimetric test. Ammonia and nitrate levels are kept at or close to zero by adding beneficial bacteria (e.g., Nutafin Cycle) to the system. Room lighting is controlled by a timer adjusted to 14-hour light and 10-hour dark periods. In addition to the overall water quality parameters mentioned above, the following considerations need special attention during a breeding week.
The first consideration is photoperiod, as fish (even cave fish in the laboratory) depend on light cycles to set their circadian clock. Circadian rhythms can affect everything from breeding and feeding to immune system health12,13 and must be consistent for maximum health benefits. Fish are maintained in a running-water system on a 14-hour light and 10-hour dark photoperiod. The surface fish generally begin spawning an hour after the system has been darkened, and light introduced during this period can interfere with and terminate spawning. The spawning of blind cave fish is less disturbed by light. Compared to surface fish spawning, cave fish spawning is delayed, usually beginning four to five hours after the system has been darkened.
The second consideration is nutrition. Adult fish are normally fed a diet of tetra flakes once a day. Prior to spawning, fish are fed a protein-rich diet supplemented with extra amounts of tetra flakes and other food: egg yolk flakes and occasionally living California blackworms (Lumbriculus variegatus) to compensate for protein loss due to egg production during the previous spawning cycle. During the breeding week, fish are fed twice per day, once in the morning and again in the afternoon/evening. Fish feeding only once a day but with a single very large portion of food should be avoided, as this can cause malnutrition14.
The third consideration is space. The space requirements are based on the average body mass of an adult as well as behavioral considerations, such as whether the fish have schooling behavior or aggressive behavior. Over or under-crowding tanks can lead to heightened aggression and constant stress, making fish vulnerable to injury from their tank mates and reluctant to participate in spawning15. We typically house 10-20 fish per 42 L tank.
The fourth consideration is temperature. As mentioned above, fish are cold-blooded animals and rely on the environment to maintain body temperature. Because temperature has a direct effect on metabolic processes, changes in temperature can trigger behavioral alterations in fish16. This breeding program consists of two-week cycles in temperature: the first week introduces a temperature spike to 78 &#176;F, and the next week maintains a static temperature of 72 &#176;F. During the first (breeding) week, plastic-edged breeding nets are placed at the bottom of the tanks each evening. The breeding nets serve as a barrier between the fish in the tanks and the spawned eggs, which would otherwise be consumed. The temperature is raised by 2 &#176;F per day to a maximum of 78 &#176;F by mid-week, and spawning is induced according to the light cycle in the first 2-3 evenings of this week. The temperature is then lowered by 2 &#176;F increments to 72 &#176;F during the remaining days of the week, and base temperature is maintained until the beginning of the next breeding week. Breeding is usually stimulated no more than twice a month to allow the fish time to recover.
Overall, this method allows for the spawning of large&#160;quantities of the highest quality embryos over a longer period of time.

<table><tbody><tr><td>Blackworms</td><td>Eastern Aquatics, Lancaster, PA</td><td>None</td><td></td></tr><tr><td>Breeding Nets</td><td>Custom made</td><td></td><td></td></tr><tr><td>Brine shrimp eggs</td><td>AquaCave Lake Forest, IL.</td><td>None</td><td></td></tr><tr><td>Colorimetric test kit</td><td>Petco</td><td>SKU:11916</td><td>API Freshwater pH Test Kit</td></tr><tr><td>Egg yolk flakes</td><td>Pentair, Minneapolis, MN</td><td>None</td><td></td></tr><tr><td>Fingerbowls</td><td>Carolina Biological Supply</td><td>741004</td><td>Culture dishes, 4.5 in, 250 mL</td></tr><tr><td>Hand held nets</td><td>Any Pet Store</td><td></td><td></td></tr><tr><td>Incubator for embryos</td><td>Fisher Scientific</td><td>51-029-321HPM</td><td>405 L</td></tr><tr><td>Instant Ocean sea salts</td><td>Spectrum Brands, Blacksburg, VA</td><td>None</td><td></td></tr><tr><td>Methylene Blue</td><td>Sigma-Aldrich, St. Louis, MO</td><td>M9140</td><td></td></tr><tr><td>Pasteur Pipettes</td><td>Fisher Scientific</td><td>13-678-20</td><td>5.75 in.</td></tr><tr><td>Net soaking solution</td><td>Any Pet Store</td><td></td><td></td></tr><tr><td>Nutrafin Cycle</td><td>Amazon</td><td>None</td><td>Bacterial boost</td></tr><tr><td>Refrigerator for live feed</td><td>Any source</td><td></td><td></td></tr><tr><td>Stereomicroscope</td><td>Any source</td><td></td><td></td></tr><tr><td>Thermometer</td><td>Any source</td><td></td><td></td></tr><tr><td>Tetra Tropical Crisps</td><td>Spectrum Brands, Blacksburg, VA</td><td>None</td><td></td></tr></tbody></table>

incremental temperature changes for maximal breeding and spawning in astyanax mexicanus

The Mexican tetra, Astyanax mexicanus, is an emerging model system for studies in development and evolution. The existence of eyed surface (surface fish) and blind cave (cave fish) morphs in this species presents an opportunity to interrogate the mechanisms underlying morphological and behavioral evolution. Cave fish have evolved novel constructive and regressive traits. The constructive changes include increases in taste buds and jaws, lateral line sensory organs, and body fat. The regressive changes include loss or reduction of eyes. melanin pigmentation, schooling behavior, aggression, and sleep. To experimentally interrogate these changes, it is crucial to obtain large numbers of spawned embryos. Since the original A. mexicanus surface fish and cave fish were collected in Texas and Mexico in the 1990s, their descendants have been routinely stimulated to breed and spawn large numbers of embryos bimonthly in the Jeffery laboratory. Although breeding is controlled by food abundance and quality, light-dark cycles, and temperature, we have found that incremental temperature changes play a key role in stimulating maximal spawning. The gradual increase of temperature from 72 &#176;F to 78 &#176;F in the first three days of a breeding week provides two-three consecutive spawning days with maximal numbers of high-quality embryos, which is then followed by a gradual decrease of temperature from 78 &#176;F to 72 &#176;F during the last three days of the spawning week. The procedures shown in this video outline the workflow before and during a laboratory breeding week for incremental temperature stimulated spawning.

We generally breed and spawn the descendants of surface fish originally collected at Nacimiento del Rio Choy in San Luis Potosi, Mexico (Rio Choy surface fish) and San Solomon Springs in Balmorhea State Park, Texas (Texas surface fish) and cave fish derived from Cueva de El Pach&#243;n (Pach&#243;n cave fish) in Tamaulipas, Mexico, and Cueva de los Sabinos (Los Sabinos cave fish) and Sotano de la Tinaja (Tinaja cave fish) in San Luis Potosi, Mexico. &#160;
Throughout a breeding week, data is c...

Watch this Scientific Journal Video about Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus at JoVE.com

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus

This article outlines the basic laboratory conditions and protocols for an incremental temperature regime to stimulate maximal spawning in the Mexican tetra Astyanax mexicanus, which is an emerging model for developmental and evolutionary studies.

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus

The Mexican tetra, Astyanax mexicanus, is an emerging model system for studies in development and evolution. The existence of eyed surface (surface fish) ...

incremental-temperature-changes-for-maximal-breeding-spawning

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

3.9K Views.  University of Maryland. This article outlines the basic laboratory conditions and protocols for an incremental temperature regime to stimulate maximal spawning in the Mexican tetra Astyanax mexicanus, which is an emerging model for developmental and evolutionary studies.

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Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical data that may be used in predictive modelling to forecast evolutionary responses of natural ecosystems to future environmental changes. However, excluding a few exceptional cases, long-term studies are scarce because of logistic difficulties associated with accessing temporal samples. Temporal dynamics are frequently studied in the laboratory or in controlled mesocosm experiments with exceptional studies that reconstruct the evolution of natural populations in the wild.
Here, a standard operating procedure (SOP) is provided to revive or resurrect dormant Daphnia magna, a widespread zooplankton keystone species in aquatic ecosystems, to dramatically advance the state-of-the-art longitudinal data collection in natural systems. The field of Resurrection Ecology was defined in 1999 by Kerfoot and co-workers, even though the first attempts at hatching diapausing zooplankton eggs date back to the late 1980s. Since Kerfoot&#39;s seminal paper, the methodology of resurrecting zooplankton species has been increasingly frequently applied, though propagated among laboratories only via direct knowledge transfer. Here, an SOP is described that provides a step-by-step protocol on the practice of resurrecting dormant Daphnia magna eggs.
Two key studies are provided in which the fitness response of resurrected Daphnia magna populations to warming is measured, capitalizing on the ability to study historical and modern populations in the same settings. Finally, the application of next generation sequencing technologies to revived or still dormant stages is discussed. These technologies provide unprecedented power in dissecting the processes and mechanisms of evolution if applied to populations that have experienced changes in selection pressure over time.

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies are essential to understanding the process of evolution and the mechanisms of adaptation. Generally, these studies require commitments beyond the life-time of researchers. Here, a powerful method is described that dramatically advances state-of-the-art data collection to generate longitudinal data in natural systems.

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical ...

Environment

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

Genetics

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.&#160;

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a series of basic techniques are described to enable observations of the reproductive behavior of a wild-caught gobiid fish, as a model, kept in an aquarium. The method focuses on three steps: collection, transport, and observations of reproductive ecology of a substrate spawner. Essential aspects of live fish collection and transport are (1) preventing injury to the fish, and (2) careful acclimation to the aquarium. Preventing harm through injuries such as scratches or a sudden change of water pressure is imperative when collecting live fish, as any physical damage is likely to negatively affect the survival and later behavior of the fish. Careful acclimation to aquaria decreases the incidence death and mitigates the shock of transport. Observations during captive rearing include (1) the identification of individual fish and (2) monitoring spawned eggs without negative effects to the fish or eggs, thereby enabling detailed investigation of the study species' reproductive ecology. The subcutaneous injection of a visible implant elastomer (VIE) tag is a precise method for the subsequent identification of individual fish, and it can be used with a wide size range of fish, with minimal influence on their survival and behavior. If the study species is a substrate spawner that deposits adhesive eggs, an artificial nest site constructed from polyvinyl chloride (PVC) pipe with the addition of a removable waterproof sheet will facilitate counting and monitoring the eggs, lessening the investigator's influence on the nest-holding and egg-guarding behavior of the fish. Although this basic method entails techniques that are seldom mentioned in detail in research articles, they are fundamental for undertaking experiments that require the captive rearing of a wild fish.

A series of basic methods to enable the study of the reproductive ecology of fish kept in aquaria are described. These are useful protocols for collecting fish using SCUBA, transporting live fish, and observing the reproductive behavior of wild-caught fish kept in aquaria.

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a ...

Behavior

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as axolotls, to regenerate entire limbs as adults. Here, we outline techniques used to generate chimeric axolotls with Cas9-mutagenized haploid forelimbs that can be used for exploring gene function and the fidelity of limb regeneration. We combine several embryological and genetic techniques, including haploid generation via in vitro activation, CRISPR/Cas9 mutagenesis, and tissue grafting into one protocol to produce a unique system for haploid genetic screening in a model organism of regeneration. This strategy reduces the number of animals, space, and time required for the functional analysis of genes in limb regeneration. This also permits the investigation of regeneration-specific functions of genes that may be required for other essential processes, such as organogenesis, tissue morphogenesis, and other essential embryonic processes. The method described here is a unique platform for conducting haploid genetic screening in a vertebrate model system.

This goal of this protocol is to produce chimeric axolotls with haploid forelimbs derived from Cas9-mutagenized donor tissue using embryonic tissue grafting techniques.

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as ...

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

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.

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.

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among ...

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs.  Towards this end, much recent ...

Biology

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus

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Resurrection of Dormant Daphnia magna: Protocol and Applications

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

Manipulation of Gene Function in Mexican Cavefish

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Wholemount In Situ Hybridization for Astyanax Embryos

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus