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Cave animals, compared to surface-dwelling relatives, tend to have reduced eyes and pigment, longer appendages, and enhanced mechanosensory structures. Pressing questions include how certain cave-related traits are gained and lost, and if they originate through the same or different genetic programs in independent lineages. An excellent system for exploring these questions is the isopod, Asellus aquaticus. This species includes multiple cave and surface populations that have numerous morphological differences between them. A key feature is that hybrids between cave and surface individuals are viable, which enables genetic crosses and linkage analyses. Here, we advance this system by analyzing single animal transcriptomes of Asellus aquaticus. We use high throughput sequencing of non-normalized cDNA derived from the head of a surface-dwelling male, the head of a cave-dwelling male, the head of a hybrid male (produced by crossing a surface individual with a cave individual), and a pooled sample of surface embryos and hatchlings. Assembling reads from surface and cave head RNA pools yielded an integrated transcriptome comprised of 23,984 contigs. Using this integrated assembly as a reference transcriptome, we aligned reads from surface-, cave- and hybrid- head tissue and pooled surface embryos and hatchlings. Our approach identified 742 SNPs and placed four new candidate genes to an existing linkage map for A. aquaticus. In addition, we examined SNPs for allele-specific expression differences in the hybrid individual. All of these resources will facilitate identification of genes and associated changes responsible for cave adaptation in A. aquaticus and, in concert with analyses of other species, will inform our understanding of the evolutionary processes accompanying adaptation to the subterranean environment.

In this study, we report evidence of a novel duplication of Melanocortin receptor 1 (Mc1r) in the cavefish genome. This locus was discovered following the observation of excessive allelic diversity in a ~820 bp fragment of Mc1r amplified via degenerate PCR from a natural population of Astyanax aeneus fish from Guerrero, Mexico. The cavefish genome reveals the presence of two closely related Mc1r open reading frames separated by a 1.46 kb intergenic region. One open reading frame corresponds to the previously reported Mc1r receptor, and the other open reading frame (duplicate copy) is 975 bp in length, encoding a receptor of 325 amino acids. Sequence similarity analyses position both copies in the syntenic region of the single Mc1r locus in 16 representative craniate genomes spanning bony fish (including Astyanax) to mammals, suggesting we discovered tandem duplicates of this important gene. The two Mc1r copies share ~89% sequence similarity and, within Astyanax, are more similar to one another compared to other melanocortin family members. Future studies will inform the precise functional significance of the duplicated Mc1r locus and if this novel copy number variant may have adaptive significance for the Astyanax lineage. Key words: cavefish, brown mutation, Guerrero, copy number variation. Dans ce travail, les auteurs rapportent des evidences pour une duplication inedite du recepteur 1 de la melanocortine (Mc1r) au sein du genome d'un poisson cavernicole. Ce locus a ete decouvert a la suite de l'observation d'une diversite allelique excessive au sein d'un fragment de ~820 pb de Mc1r amplifie au moyen d'amorces degenerees a partir d'une population naturelle de l'Astyanax aeneus provenant de Guerrero au Mexique. Le genome revele la presence de deux cadres de lectures Mc1r tres similaires separes l'un de l'autre par une region intergenique de 1,46 kb. Un cadre de lecture correspond au recepteur Mc1r rapporte anterieurement, tandis que l'autre cadre de lecture (copie dupliquee) mesure 975 pb et code pour un recepteur de 325 acides amines. Une analyse de similarite place les deux copies au sein de la region syntenique de l'unique locus Mc1r present chez 16 genomes representatifs des Craniata allant des poisson osseux (incluant l'Astyanax) jusqu'au mammiferes, ce qui suggere la decouverte d'un premier cas de duplication de ce gene important. Les deux copies de Mc1r sont similaires a ~89% et, au sein des Astyanax, sont plus semblables l'une a l'autre que les autres membres de la famille des melanocortines. De futures etudes pourront determiner l impact fonctionnel precis decoulant de la duplication du locus Mc1r et si ce variant de nombre de copies se veut adaptative au sein des Astyanax. [Traduit par la Redaction] Mots-cles: poisson cavernicoles, mutation brown, Guerrero, variation du nombre de copies., Introduction The gene melanocortin receptor 1 (Mc1r) encodes a seven-transmembrane domain G-protein coupled receptor expressed in melanin-producing cells (Tsatmali et al. 2002). This protein receptor binds [alpha]-melanophore stimulating hormone, resulting [...]

Background: Astyanax mexicanus is a well‐established fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. Despite important progress in the deployment of new technologies, deep mechanistic insights into the genetic basis of evolution, development, and behavior have been limited by a lack of transgenic lines commonly used in genetic model systems. Results: Here, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficient Tol2 system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter expresses enhanced green fluorescent protein ubiquitously throughout development in a surface population of Astyanax. To define specific cell‐types, a Cntnap2‐mCherry construct labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish. Conclusion: The lines provide proof‐of‐principle for the application of Tol2 transgenic technology in A. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype‐phenotype gap. Key Findings: We have generated two new stable transgenic lines in Astyanax mexicanus using the Tol2 transposase system.We characterize in detail both lines, one expresses ubiquitously GFP and the other expresses mCherry cell‐type specific in the lateral line and the brain.We purposely used previously published zebrafish constructs in order to validate cross‐reactivity between the evolutionary divergent promoters. [ABSTRACT FROM AUTHOR]

RNA sequencing is a popular next‐generation sequencing technique for assaying genome‐wide gene expression profiles. Nonetheless, it is susceptible to biases that are introduced by sample handling prior gene expression measurements. Two of the most common methods for preserving samples in both field‐based and laboratory conditions are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid nitrogen can be impractical, particularly for field collections. RNAlater is a solution for stabilizing tissue for longer‐term storage as it rapidly permeates tissue to protect cellular RNA. In this study, we assessed genome‐wide expression patterns in 30‐day‐old fry collected from the same brood at the same time point that were flash‐frozen in liquid nitrogen and stored at −80°C or submerged and stored in RNAlater at room temperature, simulating conditions of fieldwork. We show that sample storage is a significant factor influencing observed differential gene expression. In particular, genes with elevated GC content exhibit higher observed expression levels in liquid nitrogen flash‐freezing relative to RNAlater storage. Further, genes with higher expression in RNAlater relative to liquid nitrogen experience disproportionate enrichment for functional categories, many of which are involved in RNA processing. This suggests that RNAlater may elicit a physiological response that has the potential to bias biological interpretations of expression studies. The biases introduced to observed gene expression arising from mimicking many field‐based studies are substantial and should not be ignored. [ABSTRACT FROM AUTHOR]

SUMMARY Animals that colonize dark and nutrient-poor subterranean environments evolve numerous extreme phenotypes. These include dramatic changes to the craniofacial complex, many of which are under genetic control. These phenotypes can demonstrate asymmetric genetic signals wherein a QTL is detected on one side of the face but not the other. The causative gene(s) underlying QTL are difficult to identify with limited genomic resources. We approached this task by searching for candidate genes mediating fragmentation of the third suborbital bone (SO3) directly inferior to the orbit of the eye. We integrated positional genomic information using emerging Astyanax resources, and linked these intervals to homologous (syntenic) regions of the Danio rerio genome. We identified a discrete, approximately 6 Mb, conserved region wherein the gene causing SO3 fragmentation likely resides. We interrogated this interval for genes demonstrating significant differential expression using mRNA-seq analysis of cave and surface morphs across life history. We then assessed genes with known roles in craniofacial evolution and development based on GO term annotation. Finally, we screened coding sequence alterations in this region, identifying two key genes: transforming growth factor β3 ( tgfb3) and bone morphogenetic protein 4 ( bmp4). Of these candidates, tgfb3 is most promising as it demonstrates significant differential expression across multiple stages of development, maps close (<1 Mb) to the fragmentation critical locus, and is implicated in a variety of other animal systems (including humans) in non-syndromic clefting and malformations of the cranial sutures. Both abnormalities are analogous to the failure-to-fuse phenotype that we observe in SO3 fragmentation. This integrative approach will enable discovery of the causative genetic lesions leading to complex craniofacial features analogous to human craniofacial disorders. This work underscores the value of cave-dwelling fish as a powerful evolutionary model of craniofacial disease, and demonstrates the power of integrative system-level studies for informing the genetic basis of craniofacial aberrations in nature. [ABSTRACT FROM AUTHOR]

Numerous organisms around the globe have successfully adapted to subterranean environments. A powerful system in which to study cave adaptation is the freshwater characin fish, Astyanax mexicanus. Prior studies in this system have established a genetic basis for the evolution of numerous regressive traits, most notably vision and pigmentation reduction. However, identification of the precise genetic alterations that underlie these morphological changes has been delayed by limited genetic and genomic resources. To address this, we performed a transcriptome analysis of cave and surface dwelling Astyanax morphs using Roche/454 pyrosequencing technology. Through this approach, we obtained 576,197 Pachón cavefish-specific reads and 438,978 surface fish-specific reads. Using this dataset, we assembled transcriptomes of cave and surface fish separately, as well as an integrated transcriptome that combined 1,499,568 reads from both morphotypes. The integrated assembly was the most successful approach, yielding 22,596 high quality contiguous sequences comprising a total transcriptome length of 21,363,556 bp. Sequence identities were obtained through exhaustive blast searches, revealing an adult transcriptome represented by highly diverse Gene Ontology (GO) terms. Our dataset facilitated rapid identification of sequence polymorphisms between morphotypes. These data, along with positional information collected from the Danio rerio genome, revealed several syntenic regions between Astyanax and Danio. We demonstrated the utility of this positional information through a QTL analysis of albinism in a surface x Pachón cave F2 pedigree, using 65 polymorphic markers identified from our integrated assembly. We also adapted our dataset for an RNA-seq study, revealing many genes responsible for visual system maintenance in surface fish, whose expression was not detected in adult Pachón cavefish. Conversely, several metabolism-related genes expressed in cavefish were not detected in surface fish. This resource will enable powerful genetic and genomic analyses in the future that will better clarify the heritable genetic changes governing adaptation to the cave environment. [ABSTRACT FROM AUTHOR]

Numerous organisms around the globe have successfully adapted to subterranean environments. A powerful system in which to study cave adaptation is the freshwater characin fish, Astyanax mexicanus. Prior studies in this system have established a genetic basis for the evolution of numerous regressive traits, most notably vision and pigmentation reduction. However, identification of the precise genetic alterations that underlie these morphological changes has been delayed by limited genetic and genomic resources. To address this, we performed a transcriptome analysis of cave and surface dwelling Astyanax morphs using Roche/454 pyrosequencing technology. Through this approach, we obtained 576,197 Pachón cavefish-specific reads and 438,978 surface fish-specific reads. Using this dataset, we assembled transcriptomes of cave and surface fish separately, as well as an integrated transcriptome that combined 1,499,568 reads from both morphotypes. The integrated assembly was the most successful approach, yielding 22,596 high quality contiguous sequences comprising a total transcriptome length of 21,363,556 bp. Sequence identities were obtained through exhaustive blast searches, revealing an adult transcriptome represented by highly diverse Gene Ontology (GO) terms. Our dataset facilitated rapid identification of sequence polymorphisms between morphotypes. These data, along with positional information collected from the Danio rerio genome, revealed several syntenic regions between Astyanax and Danio. We demonstrated the utility of this positional information through a QTL analysis of albinism in a surface x Pachón cave F2 pedigree, using 65 polymorphic markers identified from our integrated assembly. We also adapted our dataset for an RNA-seq study, revealing many genes responsible for visual system maintenance in surface fish, whose expression was not detected in adult Pachón cavefish. Conversely, several metabolism-related genes expressed in cavefish were not detected in surface fish. This resource will enable powerful genetic and genomic analyses in the future that will better clarify the heritable genetic changes governing adaptation to the cave environment. [ABSTRACT FROM AUTHOR]