The authors acknowledge funding from the National Institutes of Health (NH089934, DK108495). F. Leal and A. Powers are acknowledged for editing.

<ol>
	<li>Mitchell, R. W., Russel, W. H., Elliot, 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=Mexican+eyeless+characin+fishes,+Genus+Astyanax:+Environment,+Distribution,+and+Evolution.">Mexican eyeless characin fishes, Genus Astyanax: Environment, Distribution, and Evolution.</a> Special Publications, the Museum Texas Tech University. 12, (1977).</li><li>Elliott, 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=Cave+Exploration+and+Mapping+in+the+Sierra+de+El+Abra+Region.">Cave Exploration and Mapping in the Sierra de El Abra Region.</a> Biology and Evolution of the Mexican Cavefish. , 9-40 (2015).</li><li>Herman, A., 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=The+role+of+gene+flow+in+rapid+and+repeated+evolution+of+cave-related+traits+in+Mexican+tetra,+Astyanax+mexicanus.">The role of gene flow in rapid and repeated evolution of cave-related traits in Mexican tetra, Astyanax mexicanus.</a> Molecular Ecology. 27, 4397-4416 (2018).</li><li>Rohner, N., 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=Cryptic+variation+in+morphological+evolution:+HSP90+as+a+capacitor+for+loss+of+eyes+in+cavefish.">Cryptic variation in morphological evolution: HSP90 as a capacitor for loss of eyes in cavefish.</a> Science (80). 342, 1372-1375 (2013).</li><li>McGaugh, S. E., 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=The+cavefish+genome+reveals+candidate+genes+for+eye+loss.">The cavefish genome reveals candidate genes for eye loss.</a> Nature Communications. 5, (2014).</li><li>Lyon, A., Powers, A. K., Gross, J. B., O'Quin, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+-+Three+loci+control+scleral+ossicle+formation+via+epistasis+in+the+cavefish+astyanax+mexicanus.">Two - Three loci control scleral ossicle formation via epistasis in the cavefish astyanax mexicanus.</a> PLoS One. 12, (2017).</li><li>Atukorala, A. D. S., Franz-Odendaal, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+linkage+between+altered+tooth+and+eye+development+in+lens-ablated+Astyanax+mexicanus.">Genetic linkage between altered tooth and eye development in lens-ablated Astyanax mexicanus.</a> Developmental Biology. , (2018).</li><li>Gore, A. V., 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=An+epigenetic+mechanism+for+cavefish+eye+degeneration.">An epigenetic mechanism for cavefish eye degeneration.</a> Nature Ecology &amp; Evolution. 2, 1155-1160 (2018).</li><li>Sumner-Rooney, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kingdom+of+the+blind:+disentangling+fundamental+drivers+in+the+evolution+of+eye+loss.">The kingdom of the blind: disentangling fundamental drivers in the evolution of eye loss.</a> Integrative and Comparative Biology. , (2018).</li><li>Protas, M. E., 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=Genetic+analysis+of+cavefish+reveals+molecular+convergence+in+the+evolution+of+albinism.">Genetic analysis of cavefish reveals molecular convergence in the evolution of albinism.</a> Nature Genetics. 38, 107-111 (2006).</li><li>Protas, M., Conrad, M., Gross, J. B., Tabin, C., 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>Protas, M., 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=Multi-trait+evolution+in+a+cave+fish,+Astyanax+mexicanus.">Multi-trait evolution in a cave fish, Astyanax mexicanus.</a> Evolution &amp; Development. 10, 196-209 (2008).</li><li>Stahl, B. A., Gross, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+Mc1r+gene+expression+are+associated+with+regressive+pigmentation+in+Astyanax+cavefish.">Alterations in Mc1r gene expression are associated with regressive pigmentation in Astyanax cavefish.</a> Development Genes and Evolution. 225, 367-375 (2015).</li><li>McCauley, D. W., Hixon, E., 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=Evolution+of+pigment+cell+regression+in+the+cavefish+Astyanax:+A+late+step+in+melanogenesis.">Evolution of pigment cell regression in the cavefish Astyanax: A late step in melanogenesis.</a> Evolution &amp; Development. 6, 209-218 (2004).</li><li>Dubou&#233;, E. R., Keene, A. C., Borowsky, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+convergence+on+sleep+loss+in+cavefish+populations.">Evolutionary convergence on sleep loss in cavefish populations.</a> Current Biology. 21, 671-676 (2011).</li><li>Yoshizawa, M., 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=Distinct+genetic+architecture+underlies+the+emergence+of+sleep+loss+and+prey-seeking+behavior+in+the+Mexican+cavefish.">Distinct genetic architecture underlies the emergence of sleep loss and prey-seeking behavior in the Mexican cavefish.</a> BMC Biology. 13, 15 (2015).</li><li>Jaggard, J., 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=The+lateral+line+confers+evolutionarily+derived+sleep+loss+in+the+Mexican+cavefish.">The lateral line confers evolutionarily derived sleep loss in the Mexican cavefish.</a> Journal of Experimental Biology. 220, 284-293 (2017).</li><li>Jaggard, J. B., 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=Hypocretin+underlies+the+evolution+of+sleep+loss+in+the+Mexican+cavefish.">Hypocretin underlies the evolution of sleep loss in the Mexican cavefish.</a> Elife. 7, (2018).</li><li>Dubou&#233;, E. R., Borowsky, R. L., Keene, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-adrenergic+signaling+regulates+evolutionarily+derived+sleep+loss+in+the+mexican+cavefish.">&#946;-adrenergic signaling regulates evolutionarily derived sleep loss in the mexican cavefish.</a> Brain, Behavior and Evolution. 80, 233-243 (2012).</li><li>Aspiras, A. C., Rohner, N., Martineau, B., Borowsky, R. L., Tabin, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanocortin+4+receptor+mutations+contribute+to+the+adaptation+of+cavefish+to+nutrient-poor+conditions.">Melanocortin 4 receptor mutations contribute to the adaptation of cavefish to nutrient-poor conditions.</a> Proceedings of the National Academy of Sciences of the United States of America. 112, 9668-9673 (2015).</li><li>Riddle, M. R., 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=Insulin+resistance+in+cavefish+as+an+adaptation+to+a+nutrient-limited+environment.">Insulin resistance in cavefish as an adaptation to a nutrient-limited environment.</a> Nature. 555, 647-651 (2018).</li><li>Xiong, S., Krishnan, J., Peu&#223;, R., 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=Early+adipogenesis+contributes+to+excess+fat+accumulation+in+cave+populations+of+Astyanax+mexicanus.">Early adipogenesis contributes to excess fat accumulation in cave populations of Astyanax mexicanus.</a> Developmental Biology. , (2018).</li><li>Yoshizawa, M., Gori&#269;ki, &. #. 3. 5. 2. ;., Soares, D., 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=Evolution+of+a+behavioral+shift+mediated+by+superficial+neuromasts+helps+cavefish+find+food+in+darkness.">Evolution of a behavioral shift mediated by superficial neuromasts helps cavefish find food in darkness.</a> Current Biology. 20, 1631-1636 (2010).</li><li>Franz-Odendaal, T. A., Hall, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modularity+and+sense+organs+in+the+blind+cavefish,+Astyanax+mexicanus.">Modularity and sense organs in the blind cavefish, Astyanax mexicanus.</a> Evolution and Development. 8, 94-100 (2006).</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=Sensory+evolution+in+blind+cavefish+is+driven+by+early+embryonic+events+during+gastrulation+and+neurulation.">Sensory evolution in blind cavefish is driven by early embryonic events during gastrulation and neurulation.</a> Development. 143, 4521-4532 (2016).</li><li>Blin, M., 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+evolution+and+developmental+plasticity+of+the+olfactory+epithelium+and+olfactory+skills+in+Mexican+cavefish.">Developmental evolution and developmental plasticity of the olfactory epithelium and olfactory skills in Mexican cavefish.</a> Developmental Biology. , (2018).</li><li>Lloyd, E., 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=Evolutionary+shift+towards+lateral+line+dependent+prey+capture+behavior+in+the+blind+Mexican+cavefish.">Evolutionary shift towards lateral line dependent prey capture behavior in the blind Mexican cavefish.</a> Developmental Biology. 441, 328-337 (2018).</li><li>Powers, A. K., Boggs, T. E., Gross, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canal+neuromast+position+prefigures+developmental+patterning+of+the+suborbital+bone+series+in+Astyanax+cave-+and+surface-dwelling+fish.">Canal neuromast position prefigures developmental patterning of the suborbital bone series in Astyanax cave- and surface-dwelling fish.</a> Developmental Biology. 441, 252-261 (2018).</li><li>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="Out+of+the+Dark"+Cavefish+Are+Entering+Biomedical+Research.">"Out of the Dark" Cavefish Are Entering Biomedical Research.</a> Zebrafish, Medaka, and Other Small Fishes. , 253-268 (2018).</li><li>Yoshizawa, M., 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=The+evolution+of+a+series+of+behavioral+traits+is+associated+with+autism-risk+genes+in+cavefish.">The evolution of a series of behavioral traits is associated with autism-risk genes in cavefish.</a> BMC Evolutionary Biology. 18, 89 (2018).</li><li>Stockdale, W. T., 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=Heart+Regeneration+in+the+Mexican+Cavefish.">Heart Regeneration in the Mexican Cavefish.</a> Cell Reports. 25, 1997-2007 (2018).</li><li>Peu&#223;, R., 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=Gamete+Collection+and+In+Vitro+Fertilization+of+Astyanax+mexicanus.">Gamete Collection and In Vitro Fertilization of Astyanax mexicanus.</a> Journal of Visualized Experiments. , (2019).</li><li>Luc, H., Sears, C., Raczka, A., Gross, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wholemount+In+Situ+Hybridization+for+Astyanax+Embryos.">Wholemount In Situ Hybridization for Astyanax Embryos.</a> Journal of Visualized Experiments. , (2019).</li><li>Riddle, M., Martineau, B., Peavey, M., Tabin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raising+the+Mexican+Tetra+Astyanax+mexicanus+for+Analysis+of+Post-larval+Phenotypes+and+Whole-mount+Immunohistochemistry.">Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry.</a> Journal of Visualized Experiments. , (2018).</li><li>Stahl, B. A., 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=Manipulation+of+Gene+Function+in+Mexican+Cavefish.">Manipulation of Gene Function in Mexican Cavefish.</a> Journal of Visualized Experiments. , (2019).</li><li>Dubou&#233;, E. R., Keene, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+the+Evolution+of+Sleep+in+the+Mexican+Cavefish.">Investigating the Evolution of Sleep in the Mexican Cavefish.</a> Biology and Evolution of the Mexican Cavefish. , 291-308 (2015).</li><li>Fr&#248;land Steindal, I. A., Beale, A. D., Yamamoto, Y., Whitmore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Astyanax+mexicanus+circadian+clock+and+non-visual+light+responses.">Development of the Astyanax mexicanus circadian clock and non-visual light responses.</a> Developmental Biology. , (2018).</li><li>Carlson, B. M., Gross, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+comparison+of+activity+profiles+exhibited+by+the+cave+and+surface+morphotypes+of+the+blind+Mexican+tetra,+Astyanax+mexicanus.">Characterization and comparison of activity profiles exhibited by the cave and surface morphotypes of the blind Mexican tetra, Astyanax mexicanus.</a> Comparative Biochemistry and Physiology Part C: Toxicology &amp; Pharmacology. 208, 114-129 (2018).</li><li>Jaggard, J. B., Lloyd, E., Lopatto, A., Duboue, E. R., Keene, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+Measurements+of+Sleep+and+Locomotor+Activity+in+Mexican+Cavefish.">Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish.</a> Journal of Visualized Experiments. , (2019).</li><li>Kowalko, J. E., 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=Convergence+in+feeding+posture+occurs+through+different+genetic+loci+in+independently+evolved+cave+populations+of+Astyanax+mexicanus.">Convergence in feeding posture occurs through different genetic loci in independently evolved cave populations of Astyanax mexicanus.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 16933-16938 (2013).</li><li>Worsham, M., 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=Behavioral+Tracking+and+Neuromast+Imaging+of+Mexican+Cavefish.">Behavioral Tracking and Neuromast Imaging of Mexican Cavefish.</a> Journal of Visualized Experiments. , (2019).</li></ol>

The authors have nothing to disclose.

Despite a longstanding interest in the origins of animal diversity, we have a limited understanding of the genetic changes that underlie the evolution of morphology, physiology, and behavior. The Mexican tetra, Astyanax mexicanus, has emerged as a powerful model to further our understanding of how traits evolve. This species of fish exists as two morphotypes, an eyed river-dwelling morph (surface fish) that is abundant in rivers across Mexico and Southern Texas, and eyeless cave-dwelling morphs (cavefish) that thrive in the perpetually dark limestone caves within the Sierra del Abra region in Northeastern Mexico1. There are 29 known cave populations that have originated from at least two independent surface fish invasions2,3. The cave and river-morphs have remained interfertile and are easy to breed in the laboratory allowing researchers to genetically map the traits that evolve in response to the unique selective pressures in the cave. For example, absence of light has led to regression of eyes4,5,6,7,8,9, pigment10,11,12,13,14, and sleep15,16,17,18,19; nutrient limitation has selected for overeating20, weight gain21, and fat storage22; and navigating in the dark has promoted increased ability to sense the environment23,24,25,26,27,28. A growing number of researchers are harnessing this fish to explore the genetic basis of evolution. Because some traits that are adaptive in cavefish, are detrimental in humans, the cavefish has also become attractive for biomedical research21,29,30,31. With the goal of promoting reproducibility and providing a platform for new investigators, this JoVE Methods Collection provides techniques for husbandry, trait measurement, and examination of gene activity.
As a starting point, the in vitro fertilization procedure demonstrated by Peu&#223; et al.32 serves multiple purposes as it provides a method to obtain 1) cave/surface hybrids that may be difficult to breed, 2) one-cell stage embryos for microinjections, and 3) stage-matched samples for analysis of embryonic phenotypes. A. mexicanus typically spawn during the night. Peu&#223; et al. describe a system to alter light-dark cycle and obtain fish that are primed for gamete collection during normal working hours. Their visual demonstration of gamete collection is particularly helpful as this step requires careful technique. Their article is essential for a number of downstream applications including comparison of gene expression patterns during embryogenesis.
Luc et al.33 provide a robust in situ hybridization protocol optimized for visualizing gene expression in A. mexicanus embryos. They simplify this multi-day procedure using a checklist (provided as a supplementary file) and share guidelines for troubleshooting. Complementary to this method, Riddle et al.34 show how to examine gene expression and protein localization in hatched fish using wholemount immunohistochemistry. To make meaningful comparisons between morphotypes after hatching and feeding begin, it is necessary to control the growth conditions. They show how to breed adults through natural spawning and raise larvae at fixed densities on a rotifer diet that has considerable advantages in nutrient content and cost compared to other food sources. Their immunostaining protocol successfully recognizes antigens in the brain, gut, and pancreas as late as 12-days post hatching allowing late stages of organ development and function to be interrogated. Importantly, they provide a list of antibodies that have been successfully used in A. mexicanus. The combined methods of Luc et al. and Riddle et al. are useful to analyze the effects of genetic manipulation.
In this Methods Collection, Stahl et al.35 describe multiple ways to manipulate gene function in A. mexicanus including: morpholino microinjection, CRISPR-Cas9-mediated mutagenesis, and Tol2-mediated transgenesis. In addition to demonstrating pico-injection and screening of injected embryos, they provide helpful guidelines for designing morpholinos, guide RNAs, and transgenes. They share results that validate each of these techniques. For example, targeting Hypocretin/orexin (HCRT) using a splice-blocking morpholino reduces locomotor activity and increases sleep in cavefish; editing oca2 (the causative locus for cavefish albinism) in surface fish using CRISPR/Cas9 produces albino surface fish; and inserting a genetically encoded calcium indicator driven by a pan-neuronal zebrafish promotor using Tol2 transgenesis achieves near-ubiquitous neuronal expression in the Mexican tetra. They point out that many of the genetic tools developed in zebrafish can be directly transferred to cavefish because of their similar genomes. Their article is an essential resource for researchers performing functional studies in A. mexicanus and complements the additional articles focused on measuring phenotypes, including behavior.
Cavefish have dramatically reduced sleep and altered circadian rhythm36,37,38. This presents an exciting opportunity to investigate the contribution of genetics and environment to variation in sleep in a vertebrate. Jaggard et al.39 show how to build a system from inexpensive materials that can be used to record sleep at different life stages and under different contexts. Further, they describe how to automate fish tracking in this set-up and obtain quantitative measurements for sleep and locomotion. Their technique is applicable for high-throughput drug and genetic screening as well as traditional genetic mapping studies.
In addition to reduced sleep, cavefish have evolved differences in locomotor activities linked to foraging, including altered feeding angle and vibration attraction16,30,40. Central to understanding how these activities evolved is linking them to developmental changes. Worsham et al.41 present methods for recording behaviors and correlating them with changes in neuromasts; external mechanosensory structures that respond to water flow. They first show how to create chambers to record vibration attraction and sleep, and provide a detailed description of how to quantify the behaviors using free software and customizable scripts. Next, they demonstrate how to visualize neuromasts in living adult fish using a fluorescent mitochondrial stain and again, using free software, how to quantify the number of neuromasts in a semi-automated fashion. Vibration attraction and neuromast number are correlated23; their method provides a step by step protocol to interrogate this relationship. The methods of Jaggard and Worsham can be used to uncover additional behaviors and can also be applied to other species of fish. These methods produce quantitative and consistent results that are essential to discovering the genetic basis of behavioral evolution.
Modern techniques to measure and manipulate gene function have pushed evolutionary studies from comparative to mechanistic. Combined, the articles in this Methods Collection serve as a guidebook to carry out investigations of gene function in A. mexicanus. While researchers have uncovered fascinating differences in morphology, physiology, and behavior between surface and cave morphs, there are many traits that have yet to be explored in detail, and likely more to discover. As more work is done, this little fish will continue to have a big impact on our understanding of evolution.

little fish, big questions: a collection of modern techniques for mexican tetra research

Articles Discussed:
Stahl, B. A. et al. Manipulation of Gene Function in Mexican Cavefish. Journal of Visualized Experiments. (146) (2019).
Peu&#223;, R. et al. Gamete Collection and In Vitro Fertilization of Astyanax mexicanus. Journal of Visualized Experiments. (147) (2019).
Worsham, M. et al. Behavioral Tracking and Neuromast Imaging of Mexican Cavefish.Journal of Visualized Experiments. (147) (2019).
Jaggard, J.B., Lloyd, E., Lopatto, A., Duboue, E.R., Keene, A.C. Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish. Journal of Visualized Experiments. (145) (2019).
Luc, H., Sears, C., Raczka, A., Gross, J.B. Wholemount In Situ Hybridization for Astyanax Embryos. Journal of Visualized Experiments. (145) (2019).
Riddle, M., Martineau, B., Peavey, M., Tabin, C. Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry. Journal of Visualized Experiments. (142) (2018).

Watch this Scientific Journal Video about Little Fish, Big Questions: A Collection of Modern Techniques for Mexican Tetra Research at JoVE.com

Little Fish, Big Questions: A Collection of Modern Techniques for Mexican Tetra Research

Articles Discussed:
Stahl, B. A. et al. Manipulation of Gene Function in Mexican Cavefish. Journal of Visualized Experiments. (146) (2019).
Peu&#223;, R. ...

little-fish-big-questions-collection-modern-techniques-for-mexican

Without Section

4.4K Views.  Harvard Medical School. Little Fish, Big Questions: A Collection of Modern Techniques for Mexican Tetra Research

Video: Little Fish, Big Questions: A Collection of Modern Techniques for Mexican Tetra Research

August 2012: This Month in JoVE

Traditional microscopy requires lens objectives to magnify specimens, and can involve numerous optical components like additional objectives, filters, and mirrors to refract and direct light to optical sensors. The August 2012 issue of JoVE (Journal of Visualized Experiments) is marked by the third publication from the Ozcan Lab (University of California, Los Angeles) on their lens free lens-free "on-chip" microscopy platform, which they have pioneered.
Back in 2009, the Ozcan lab published their first article, which demonstrated the use of a lens-free imaging platform that could image cells within whole blood. This imaging technique involves placing a sample directly on an optoelectric sensor array, like the CCD or CMOS chips found in the standard camcorder. Then, LED illumination from above the sample casts shadows on the chip, which are recorded as digital holograms that can be subjected to image processing. Not only does this lensless imaging system allow for an extremely large field of view (FOV of 0.6-8 cm2), the lack of bulky optical components makes it excellent for microfluidic applications. The Ozcan group demonstrates this in their follow-up 2011 JoVE article, in which fluorescently labeled white blood cells are imaged within a microfluidic chip.
This August in JoVE, the Ozcan group demonstrates further advancements to their lens-free microscopy platform by applying it to tomographic imaging of the nematode, C. elegans. By changing the angle of illumination, using a motorized rotation stage, the Ozcan group shows how multiple holographic slices may be generated for a sample-which, following processing, can be combined into tomograms. The Ozcan group confirms that their 3D reconstruction of the worm is successful by citing that the pharyngeal tube, a 5-micron-thick structure, is only visible in a single slice and in a 3D reconstructed cross section.
The compact and inexpensive nature of the lens-free imaging systems - designed and assembled by the Ozcan group - make them well suited for point-of-care diagnostic applications, or even cell phone based microscopy that can be performed in the field.
At the Ernest Gallo Clinic and Research Center at University of California, San Francisco JoVE learns how to visualize morphological changes in single cells using three-dimensional fluorescence microscopy and computer analysis. In this procedure, cells are given agonist, antagonist plus agonist, or no treatment, and prepared for immunofluorescence microscopy. Three-dimensional images are acquired with the help of a two-photon laser system, and the fluorescence data are processed on the computer using Imaris and Matlab.
To demonstrate the power of this technique, our authors quantify single-cell phenotypic changes in transfected HEK293 cells. Three-dimensional morphometric analysis shows dramatic changes in cells treated with agonist. These changes are not always apparent when using conventional 2D analysis. This technique allows morphological changes in single cells to be quantified and characterized in response to pharmacologic stimuli, offering a powerful new assessment tool to the arsenal of drug discovery research.
This month, in JoVE Applied Physics, we travel to South Carolina to visit the School of Materials Science and Engineering at Clemson University. Here, researchers show us how to construct lithium batteries for laboratory research.
The basic components of a lithium coin cell are the working electrode, the lithium electrolyte, and a lithium foil counter electrode. The different components of the coin cell are carefully assembled and sealed with a crimping machine. The finished coin cell can then be characterized in a battery tester. Using their homemade coin cells, our researchers can experiment with using different materials to make the cathodes of lithium coin cells.
This month in JoVE Immunology and Infection, we head to Basel, Switzerland to witness two techniques for diagnosing helminth infections. We also see how investigators perform the shuttle run test in children, in order to assess the effects of these infections on physical fitness.
Helminths are parasitic worms that can live inside humans and animals. Many species infect the intestinal tract, and may be transmitted through soil. Our authors demonstrate the Kato-Katz technique, which is used for diagnosing several common soil-transmitted helminths. They also demonstrate the Baermann technique, which uses light to stimulate the migration of certain types of phototactic helminths out of sample preparations. The various types of helminth species can subsequently be determined based on morphology.
Our authors then demonstrate how to set up and perform a 20-meter shuttle run test for children, and to measure the impact of helminth infections on physical fitness. This method can help determine the true burden of soil-transmitted helminth infections on overall health.
This brief summary highlights but a few notable video-articles that will be released in August. We'll also feature articles that demonstrate how to express genes in the midbrain of the chick embryo, study neutrophil transmigration in parallel flow chambers lined with endothelial cells, and precisely measure proteolytic degradation of the extracellular matrix.

Traditional microscopy requires lens objectives to magnify specimens, and can involve numerous optical components like additional objectives, filters, and mirrors to refract and direct light to optical sensors. The August 2012 issue of JoVE (Journal of Visualized Experiments) is marked by the third publication from the Ozcan Lab (University of California, Los Angeles) on their lens-free "on-chip" microscopy platform, which they have pioneered.

Traditional microscopy requires lens objectives to magnify specimens, and can involve numerous optical components like additional objectives, filters, and ...

Research

JoVE Journal

Biology

2012: A Year In Review

Here's a look at some of the milestones and highlights of the year 2012 in Journal of Visualized Experiments (JoVE).
At the start of 2012, JoVE reached a major milestone by publishing our 1500th article. In this publication, in JoVE Bioengineering, Yu et al. described a method for analyzing the free radical composition of cigarette smoke. This method simulated cigarette puffing with a single-port smoking device, and combined it with electron spin resonance (ESR) spectroscopy to measure the degree to which antioxidant compounds can scavenge free radicals in cigarette smoke. A possible outcome of this research is the creation of less harmful cigarettes.
JoVE Bioengineering also contained the most-viewed article of 2012. Owczarczak et al. described a method for hacking a standard inkjet printer, loading ink cartridges with a cell-suspension "bioink," and printing lines of live cells onto glass slides. The thermal inkjet printing process creates temporary pores in the cell membrane, visualized by the incorporation of fluorescently labeled actin monomers. This concept, called "bioprinting," has many potential applications in cell and tissue engineering.
Also in 2012, JoVE Bioengineering featured a protocol by Hsia et al. for isolating and purifying spider silk protein, spinning it into fibers, and assessing the fiber strength. This protocol for laboratory-scale production of spider silk, which is stronger than tensile steel, can potentially be extended to large-scale manufacturing.
In JoVE Clinical and Translational Medicine, we featured a number of articles related to stroke, one of which. In one article, M&ouml;bius-Winkler et al. demonstrated placement of the WATCHMAN left atrial appendage occlusion device from Boston Scientific. This device is designed to prevent strokes by trapping clots within the left atrial appendage of the heart before they exit. Our authors demonstrated how to insert the guide wire through the femoral artery into the heart, advance the device into the left atrium, and deploy the device in the left atrial appendage.
Another article focused on stroke, Hamel et al. described the use of a driving simulator to study how patients compensate for visual field defects following stroke. By analyzing the compensatory gaze behavior of patients as they navigated through virtual driving courses with varying degrees of complexity, our authors see great potential for the use of driving simulators in stroke rehabilitation.
In JoVE Immunology and Infection, we published an article by Keyel et al. describing the real-time kinetics of immune cell responses to bacterial toxins using live cell microscopy. Combined with high-speed 3D confocal microscopy, this technique can also visualize the cellular repair response.
Also in JoVE Immunology and Infection, we featured an article by Yap et al. describing methods for diagnosing helminth infections in children. Helminths are parasitic worms that can infect the intestinal tract of humans and animals, and may be distinguished based on their morphology. The same article demonstrated how to measure the impact of helminth infections on physical fitness in children, which is a measure of overall health.
In JoVE Neuroscience, we featured a method by Hoffmann et al., who used tiny headphones to alter the auditory feedback in songbirds, and analyzed the computational and neurophysiological basis of vocal learning in birds when they adjust their singing in response to altered acoustic signals.
Also in JoVE Neuroscience, we moved from birds to bees with a method that assesses associative and non-associative tactile learning in honeybees. This method, by Mujagi&#263; et al., allowed bees to scan different metal surfaces with their antennae, then conditioned them to expect sugar water when their antennae touched particular surfaces, and analyzed the corresponding changes in antennal movement.
In 2012, we launched a brand-new section: JoVE Applied Physics, which features articles on subjects ranging from plasma physics to material science. The Applied Physics section also contained JoVE's 2000th video article, filmed in a synchrotron radiation facility, where Borisenko et al. showed how to determine the electronic structure of complex materials using angle-resolved photoemission spectroscopy.
In the JoVE General section, we published an article by Artioli et al. demonstrating the use of a portable gas analyzer to measure oxygen consumption during complex exercise - such as judo. By analyzing oxygen consumption and collecting blood for measuring plasma lactate concentration, this method can determine the relative contributions of different energy systems to specific aspects of complex exercise.
Finally, JoVE followed Dolhi et al. to a permanently ice-covered saline lake in Antarctica. There, scientists drilled through the ice to study single-celled microorganisms called protists that live in the harsh conditions of Antarctica.
This Year in Review was just a brief glimpse of a few of over 600 video articles that JoVE offered in 2012. Browse the JoVE archives to see thousands of other videos, and stay tuned for what's coming up this year in JoVE: The Journal of Visualized Experiments.

Here's a look at some of the milestones and highlights of the year 2012 in Journal of Visualized Experiments (JoVE).

 
 
Here's a look at some of the milestones and highlights of the year 2012 in Journal of Visualized Experiments (JoVE).
At the start of 2012, JoVE ...