Quantitative trait loci concentrate in specific regions of the Mexican cavefish genome and reveal key candidate genes for cave-associated evolution.

A major goal of modern biology is connecting phenotype with its underlying genetic basis. The Mexican cavefish (Astyanax mexicanus), a characin fish species comprised of a surface ecotype and a cave-derived ecotype, is well suited as a model to study the genetic mechanisms underlying adaptation to extreme environments. Here we map 206 previously published quantitative trait loci (QTL) for cave-derived traits in A. mexicanus to the newest version of the surface fish genome assembly, AstMex3. These analyses revealed that QTL cluster in the genome more than expected by chance, and this clustering is not explained by the distribution of genes in the genome. To investigate whether certain characteristics of the genome facilitate phenotypic evolution, we tested whether genomic characteristics associated with increased opportunities for mutation, such as highly mutagenic CpG sites, are reliable predictors of the sites of trait evolution but did not find any significant trends. Finally, we combined the QTL map with previously collected expression and selection data to identify 36 candidate genes that may underlie the repeated evolution of cave phenotypes, including rgrb, which is predicted to be involved in phototransduction. We found this gene has disrupted exons in all non-hybrid cave populations but intact reading frames in surface fish. Overall, our results suggest specific regions of the genome may play significant roles in driving adaptation to the cave environment in Astyanax mexicanus and demonstrate how this compiled dataset can facilitate our understanding of the genetic basis of repeated evolution in the Mexican cavefish.

Repeated evolution of circadian clock dysregulation in cavefish populations.

Circadian rhythms are nearly ubiquitous throughout nature, suggesting they are critical for survival in diverse environments. Organisms inhabiting largely arrhythmic environments, such as caves, offer a unique opportunity to study the evolution of circadian rhythms in response to changing ecological pressures. Populations of the Mexican tetra, Astyanax mexicanus, have repeatedly invaded caves from surface rivers, where individuals must contend with perpetual darkness, reduced food availability, and limited fluctuations in daily environmental cues. To investigate the molecular basis for evolved changes in circadian rhythms, we investigated rhythmic transcription across multiple independently-evolved cavefish populations. Our findings reveal that evolution in a cave environment has led to the repeated disruption of the endogenous biological clock, and its entrainment by light. The circadian transcriptome shows widespread reductions and losses of rhythmic transcription and changes to the timing of the activation/repression of core-transcriptional clock. In addition to dysregulation of the core clock, we find that rhythmic transcription of the melatonin regulator aanat2 and melatonin rhythms are disrupted in cavefish under darkness. Mutants of aanat2 and core clock gene rorca disrupt diurnal regulation of sleep in A. mexicanus, phenocopying circadian modulation of sleep and activity phenotypes of cave populations. Together, these findings reveal multiple independent mechanisms for loss of circadian rhythms in cavefish populations and provide a platform for studying how evolved changes in the biological clock can contribute to variation in sleep and circadian behavior.

Course-based undergraduate research experiences (CURES) as a pathway to diversify science.

There is widespread recognition of the need to increase research opportunities in biomedical science for undergraduate students from underrepresented backgrounds. Here, we describe the implementation of team-based science combined with intensive mentoring to conduct a large-scale project examining the evolution of behavior. This system can be widely applied in other areas of STEM to promote research-intensive opportunities in STEM fields and to promote diversity in science.

Characterizing the genetic basis of trait evolution in the Mexican cavefish.

Evolution in response to a change in ecology often coincides with various morphological, physiological, and behavioral traits. For most organisms little is known about the genetic and functional relationship between evolutionarily derived traits, representing a critical gap in our understanding of adaptation. The Mexican tetra, Astyanax mexicanus, consists of largely independent populations of fish that inhabit at least 30 caves in Northeast Mexico, and a surface fish population, that inhabit the rivers of Mexico and Southern Texas. The recent application of molecular genetic approaches combined with behavioral phenotyping have established A. mexicanus as a model for studying the evolution of complex traits. Cave populations of A. mexicanus are interfertile with surface populations and have evolved numerous traits including eye degeneration, insomnia, albinism, and enhanced mechanosensory function. The interfertility of different populations from the same species provides a unique opportunity to define the genetic relationship between evolved traits and assess the co-evolution of behavioral and morphological traits with one another. To define the relationships between morphological and behavioral traits, we developed a pipeline to test individual fish for multiple traits. This pipeline confirmed differences in locomotor activity, prey capture, and startle reflex between surface and cavefish populations. To measure the relationship between traits, individual F2 hybrid fish were characterized for locomotor behavior, prey-capture behavior, startle reflex, and morphological attributes. Analysis revealed an association between body length and slower escape reflex, suggesting a trade-off between increased size and predator avoidance in cavefish. Overall, there were few associations between individual behavioral traits, or behavioral and morphological traits, suggesting independent genetic changes underlie the evolution of the measured behavioral and morphological traits. Taken together, this approach provides a novel system to identify genetic underpinnings of naturally occurring variation in morphological and behavioral traits.

Evolution of the acoustic startle response of Mexican cavefish.

The ability to detect threatening stimuli and initiate an escape response is essential for survival and under stringent evolutionary pressure. In diverse fish species, acoustic stimuli activate Mauthner neurons, which initiate a C-start escape response. This reflexive behavior is highly conserved across aquatic species and provides a model for investigating the neural mechanism underlying the evolution of escape behavior. Here, we characterize evolved differences in the C-start response between populations of the Mexican cavefish, Astyanax mexicanus. Cave populations of A. mexicanus inhabit an environment devoid of light and macroscopic predators, resulting in evolved differences in various morphological and behavioral traits. We find that the C-start is present in river-dwelling surface fish and multiple populations of cavefish, but that response kinematics and probability differ between populations. The Pachón population of cavefish exhibits an increased response probability, a slower response latency and speed, and reduction of the maximum bend angle, revealing evolved differences between surface and cave populations. Analysis of the responses of two other independently evolved populations of cavefish, revealed the repeated evolution of reduced angular speed. Investigation of surface-cave hybrids reveals a correlation between angular speed and peak angle, suggesting these two kinematic characteristics are related at the genetic or functional levels. Together, these findings provide support for the use of A. mexicanus as a model to investigate the evolution of escape behavior.

Dark world rises: The emergence of cavefish as a model for the study of evolution, development, behavior, and disease.

A central question in biology is how naturally occurring genetic variation accounts for morphological and behavioral diversity within a species. The Mexican tetra, Astyanax mexicanus, has been studied for nearly a century as a model for investigating trait evolution. In March of 2019, researchers representing laboratories from around the world met at the Sixth Astyanax International Meeting in Santiago de Querétaro, Mexico. The meeting highlighted the expanding applications of cavefish to investigations of diverse aspects of basic biology, including development, evolution, and disease-based applications. A broad range of integrative approaches are being applied in this system, including the application of state-of-the-art functional genetic assays, brain imaging, and genome sequencing. These advances position cavefish as a model organism for addressing fundamental questions about the genetics and evolution underlying the impressive trait diversity among individual populations within this species.

Comparative transcriptome analysis of wild and lab populations of Astyanax mexicanus uncovers differential effects of environment and morphotype on gene expression.

Studying how different genotypes respond to environmental variation is essential to understand the genetic basis of adaptation. The Mexican tetra, Astyanax mexicanus, has cave and surface-dwelling morphotypes that have adapted to entirely different environments in the wild, and are now successfully maintained in lab conditions. While this has enabled the identification of genetic adaptations underlying a variety of physiological processes, few studies have directly compared morphotypes between lab-reared and natural populations. Such comparative approaches could help dissect the varying effects of environment and morphotype, and determine the extent to which phenomena observed in the lab are generalizable to conditions in the field. To this end, we take a transcriptomic approach to compare the Pachón cavefish and their surface fish counterparts in their natural habitats and the lab environment. We identify key changes in expression of genes implicated in metabolism and physiology between groups of fish, suggesting that morphotype (surface or cave) and environment (natural or lab) both alter gene expression. We find gene expression differences between cave and surface fish in their natural habitats are much larger than differences in expression between morphotypes in the lab environment. However, lab-raised cave and surface fish still exhibit numerous gene expression changes, supporting genetically encoded changes in livers of this species. From this, we conclude that a controlled laboratory environment may serve as an ideal setting to study the genetic underpinnings of metabolic and physiological differences between the cavefish and surface fish.

Analysis of stress responses in Astyanax larvae reveals heterogeneity among different populations.

Stress responses are conserved physiological and behavioral outcomes as a result of facing potentially harmful stimuli, yet in pathological states, stress becomes debilitating. Stress responses vary considerably throughout the animal kingdom, but how these responses are shaped evolutionarily is unknown. The Mexican cavefish has emerged as a powerful system for examining genetic principles underlying behavioral evolution. Here, we demonstrate that cave Astyanax have reduced behavioral and physiological measures of stress when examined at larval stages. We also find increased expression of the glucocorticoid receptor, a repressible element of the neuroendocrine stress pathway. Additionally, we examine stress in three different cave populations, and find that some, but not all, show reduced stress measures. Together, these results reveal a mechanistic system by which cave-dwelling fish reduced stress, presumably to compensate for a predator poor environment.

Repeated evolution of eye loss in Mexican cavefish: Evidence of similar developmental mechanisms in independently evolved populations.

Evolution in similar environments often leads to convergence of behavioral and anatomical traits. A classic example of convergent trait evolution is the reduced traits that characterize many cave animals: reduction or loss of pigmentation and eyes. While these traits have evolved many times, relatively little is known about whether these traits repeatedly evolve through the same or different molecular and developmental mechanisms. The small freshwater fish, Astyanax mexicanus, provides an opportunity to investigate the repeated evolution of cave traits. A. mexicanus exists as two forms, a sighted, surface-dwelling form and at least 29 populations of a blind, cave-dwelling form that initially develops eyes that subsequently degenerate. We compared eye morphology and the expression of eye regulatory genes in developing surface fish and two independently evolved cavefish populations, Pachón and Molino. We found that many of the previously described molecular and morphological alterations that occur during eye development in Pachón cavefish are also found in Molino cavefish. However, for many of these traits, the Molino cavefish have a less severe phenotype than Pachón cavefish. Further, cave-cave hybrid fish have larger eyes and lenses during early development compared with fish from either parental population, suggesting that some different changes underlie eye loss in these two populations. Together, these data support the hypothesis that these two cavefish populations evolved eye loss independently, yet through some of the same developmental and molecular mechanisms.

Utilizing the blind cavefish Astyanax mexicanus to understand the genetic basis of behavioral evolution.

Colonization of novel habitats often results in the evolution of diverse behaviors. Comparisons between individuals from closely related populations that have evolved divergent behaviors in different environments can be used to investigate behavioral evolution. However, until recently, functionally connecting genotypes to behavioral phenotypes in these evolutionarily relevant organisms has been difficult. The development of gene editing tools will facilitate functional genetic analysis of genotype-phenotype connections in virtually any organism, and has the potential to significantly transform the field of behavioral genetics when applied to ecologically and evolutionarily relevant organisms. The blind cavefish Astyanax mexicanus provides a remarkable example of evolution associated with colonization of a novel habitat. These fish consist of a single species that includes sighted surface fish that inhabit the rivers of Mexico and southern Texas and at least 29 populations of blind cavefish from the Sierra Del Abra and Sierra de Guatemala regions of Northeast Mexico. Although eye loss and albinism have been studied extensively in A. mexicanus, derived behavioral traits including sleep loss, alterations in foraging and reduction in social behaviors are now also being investigated in this species to understand the genetic and neural basis of behavioral evolution. Astyanax mexicanus has emerged as a powerful model system for genotype-phenotype mapping because surface and cavefish are interfertile. Further, the molecular basis of repeated trait evolution can be examined in this species, as multiple cave populations have independently evolved the same traits. A sequenced genome and the implementation of gene editing in A. mexicanus provides a platform for gene discovery and identification of the contributions of naturally occurring variation to behaviors. This review describes the current knowledge of behavioral evolution in A. mexicanus with an emphasis on the molecular and genetic underpinnings of evolved behaviors. Multiple avenues of new research that can be pursued using gene editing tools are identified, and how these will enhance our understanding of behavioral evolution is discussed.

CRISPR mutagenesis confirms the role of oca2 in melanin pigmentation in Astyanax mexicanus.

Understanding the genetic basis of trait evolution is critical to identifying the mechanisms that generated the immense amount of diversity observable in the living world. However, genetically manipulating organisms from natural populations with evolutionary adaptations remains a significant challenge. Astyanax mexicanus exists in two interfertile forms, a surface-dwelling form and multiple independently evolved cave-dwelling forms. Cavefish have evolved a number of morphological and behavioral traits and multiple quantitative trait loci (QTL) analyses have been performed to identify loci underlying these traits. These studies provide a unique opportunity to identify and test candidate genes for these cave-specific traits. We have leveraged the CRISPR/Cas9 genome editing techniques to characterize the effects of mutations in oculocutaneous albinism II (oca2), a candidate gene hypothesized to be responsible for the evolution of albinism in A. mexicanus cave populations. We generated oca2 mutant surface A. mexicanus. Surface fish with oca2 mutations are albino due to a disruption in the first step of the melanin synthesis pathway, the same step that is disrupted in albino cavefish. Hybrid offspring from crosses between oca2 mutant surface and cavefish are albino, definitively demonstrating the role of this gene in the evolution of albinism in this species. This research elucidates the role oca2 plays in pigmentation in fish, and establishes that this gene is solely responsible for the evolution of albinism in multiple cavefish populations. Finally, it demonstrates the utility of using genome editing to investigate the genetic basis of trait evolution.

Temperature preference of cave and surface populations of Astyanax mexicanus.

Little is known about the genetic basis of behavioral choice, such as temperature preference, especially in natural populations. Thermal preference can play a key role in habitat selection, for example in aquatic species. Examining this behavior on a genetic level requires access to individuals or populations of the same species that display distinct temperature preferences. Caves provide a uniquely advantageous setting to tackle this problem, as animals colonizing caves encounter an environment that generally has a different, and far more stable, annual temperature than what is encountered on the outside. Here, we focus on cave and surface populations of Astyanax mexicanus, the Mexican tetra, and examine temperature preference and strength of temperature preference (reflected in the percent of time spent at the optimal temperature). We used a tank with a stable temperature gradient and automated tracking software to follow individual fish from each population. We found that distinct populations of A. mexicanus display differences in both temperature preference and strength of preference. Hybrid crosses established that these are multigenic traits that segregate independently from one another. Temperature preference in many aquatic animals is known to shift towards warmer temperatures following infection with parasites (akin to a fever response in humans). While surface fish infected by the ectoparasite Gyrodactylus turnbulli (a gill fluke) displayed a strong fever response, cavefish showed a significantly attenuated fever response. This work establishes A. mexicanus as a genetically tractable system in which differences in temperature preference can be studied in naturally evolved populations.

Convergence in feeding posture occurs through different genetic loci in independently evolved cave populations of Astyanax mexicanus.

When an organism colonizes a new environment, it needs to adapt both morphologically and behaviorally to survive and thrive. Although recent progress has been made in understanding the genetic architecture underlying morphological evolution, behavioral evolution is poorly understood. Here, we use the Mexican cavefish, Astyanax mexicanus, to study the genetic basis for convergent evolution of feeding posture. When river-dwelling surface fish became entrapped in the caves, they were confronted with dramatic changes in the availability and type of food source and in their ability to perceive it. In this setting, multiple independent populations of cavefish exhibit an altered feeding posture compared with their ancestral surface forms. We determined that this behavioral change in feeding posture is not due to changes in cranial facial morphology, body depth, or to take advantage of the expansion in the number of taste buds. Quantitative genetic analysis demonstrates that two different cave populations have evolved similar feeding postures through a small number of genetic changes, some of which appear to be distinct. This work indicates that independently evolved populations of cavefish can evolve the same behavioral traits to adapt to similar environmental challenges by modifying different sets of genes.

Loci associated with cave-derived traits concentrate in specific regions of the Mexican cavefish genome.

A major goal of modern evolutionary biology is connecting phenotypic evolution with its underlying genetic basis. The Mexican cavefish (Astyanax mexicanus), a characin fish species comprised of a surface ecotype and a cave-derived ecotype, is well suited as a model to study the genetic mechanisms underlying adaptation to extreme environments. Here we map 206 previously published quantitative trait loci (QTL) for cave-derived traits in A. mexicanus to the newest version of the surface fish genome assembly, AstMex3. This analysis revealed that QTL cluster in the genome more than expected by chance, and this clustering is not explained by the distribution of genes in the genome. To investigate whether certain characteristics of the genome facilitate phenotypic evolution, we tested whether genomic characteristics, such as highly mutagenic CpG sites, are reliable predictors of the sites of trait evolution but did not find any significant trends. Finally, we combined the QTL map with previously collected expression and selection data to identify a list of 36 candidate genes that may underlie the repeated evolution of cave phenotypes, including rgrb which is predicted to be involved in phototransduction. We found this gene has disrupted exons in all non-hybrid cave populations but intact reading frames in surface fish. Overall, our results suggest specific "evolutionary hotspots" in the genome may play significant roles in driving adaptation to the cave environment in Astyanax mexicanus and demonstrate how this compiled dataset can facilitate our understanding of the genetic basis of repeated evolution in the Mexican cavefish.

Mutations in the albinism gene oca2 alter vision-dependent prey capture behavior in the Mexican tetra.

Understanding the phenotypic consequences of naturally occurring genetic changes, as well as their impact on fitness, is fundamental to understanding how organisms adapt to an environment. This is critical when genetic variants have pleiotropic effects, as determining how each phenotype impacted by a gene contributes to fitness is essential to understand how and why traits have evolved. A striking example of a pleiotropic gene contributing to trait evolution is the oca2 gene, coding mutations in which underlie albinism and reductions of sleep in the blind Mexican cavefish, Astyanax mexicanus. Here, we characterize the effects of mutations in the oca2 gene on larval prey capture. We find that when conspecific surface fish with engineered mutations in the oca2 allele are hunting, they use cave-like, wide angle strikes to capture prey. However, unlike cavefish or surface fish in the dark, which rely on lateral line mediated hunting, oca2 mutant surface fish use vision when striking at prey from wide angles. Finally, we find that while oca2 mutant surface fish do not outcompete pigmented surface siblings in the dark, pigmented fish outcompete albino fish in the light. This raises the possibility that albinism is detrimental to larval feeding in a surface-like lighted environment, but does not have negative consequences for fish in cave-like, dark environments. Together, these results demonstrate that oca2 plays a role in larval feeding behavior in A. mexicanus. Further, they expand our understanding of the pleiotropic phenotypic consequences of oca2 in cavefish evolution.

Defining the Unseen: Population-Specific Markers for Astyanax mexicanus Blind Cavefish.

Astyanax mexicanus is an emerging model system used to study development, evolution, and behavior of multiple cavefish populations that have repeatedly evolved from conspecific surface fish. Although surface and cavefish live and breed in the laboratory, there are no rapid methods for distinguishing between different cavefish populations. We present 2 methods for genotyping fish for a total of 16 population-specific markers using methods that are easy and inexpensive to implement in a basic molecular biology laboratory. This resource will help researchers maintain independent stocks within the laboratory and distinguish between fish from different populations.

Postprandial sleep in short-sleeping Mexican cavefish.

Interaction between sleep and feeding behaviors are critical for adaptive fitness. Diverse species suppress sleep when food is scarce to increase the time spent foraging. Post-prandial sleep, an increase in sleep time following a feeding event, has been documented in vertebrate and invertebrate animals. While interactions between sleep and feeding appear to be highly conserved, the evolution of postprandial sleep in response to changes in food availability remains poorly understood. Multiple populations of the Mexican cavefish, Astyanax mexicanus, have independently evolved sleep loss and increased food consumption compared to surface-dwelling fish of the same species, providing the opportunity to investigate the evolution of interactions between sleep and feeding. Here, we investigate effects of feeding on sleep in larval and adult surface fish, and two parallelly evolved cave populations of A. mexicanus. Larval surface and cave populations of A. mexicanus increase sleep immediately following a meal, providing the first evidence of postprandial sleep in a fish model. The amount of sleep was not correlated to meal size and occurred independently of feeding time. In contrast to larvae, postprandial sleep was not detected in adult surface or cavefish, that can survive for months without food. Together, these findings reveal that postprandial sleep is present in multiple short-sleeping populations of cavefish, suggesting sleep-feeding interactions are retained despite the evolution of sleep loss. These findings raise the possibility that postprandial sleep is critical for energy conservation and survival in larvae that are highly sensitive to food deprivation.

Evolution: Out of the shadows and into the light.

While many species are active during specific time periods throughout the day, there is significant variation across species in preferred daily temporal niche. A new study investigates the molecular changes that occurred in a mammal that has evolved diurnality.