Allelic diversification after transposable element exaptation promoted gsdf as the master sex determining gene of sablefish.

Concepts of evolutionary biology suggest that morphological change may occur by rare punctual but rather large changes, or by more steady and gradual transformations. It can therefore be asked whether genetic changes underlying morphological, physiological, and/or behavioral innovations during evolution occur in a punctual manner, whereby a single mutational event has prominent phenotypic consequences, or if many consecutive alterations in the DNA over longer time periods lead to phenotypic divergence. In the marine teleost, sablefish (Anoplopoma fimbria), complementary genomic and genetic studies led to the identification of a sex locus on the Y Chromosome. Further characterization of this locus resulted in identification of the transforming growth factor, beta receptor 1a (tgfbr1a) gene, gonadal somatic cell derived factor (gsdf), as the main candidate for fulfilling the master sex determining (MSD) function. The presence of different X and Y Chromosome copies of this gene indicated that the male heterogametic (XY) system of sex determination in sablefish arose by allelic diversification. The gsdfY gene has a spatio-temporal expression profile characteristic of a male MSD gene. We provide experimental evidence demonstrating a pivotal role of a transposable element (TE) for the divergent function of gsdfY By insertion within the gsdfY promoter region, this TE generated allelic diversification by bringing cis-regulatory modules that led to transcriptional rewiring and thus creation of a new MSD gene. This points out, for the first time in the scenario of MSD gene evolution by allelic diversification, a single, punctual molecular event in the appearance of a new trigger for male development.

Environmental DNA provides quantitative estimates of Pacific hake abundance and distribution in the open ocean.

All species inevitably leave genetic traces in their environments, and the resulting environmental DNA (eDNA) reflects the species present in a given habitat. It remains unclear whether eDNA signals can provide quantitative metrics of abundance on which human livelihoods or conservation successes depend. Here, we report the results of a large eDNA ocean survey (spanning 86 000 km2 to depths of 500 m) to understand the abundance and distribution of Pacific hake (Merluccius productus), the target of the largest finfish fishery along the west coast of the USA. We sampled eDNA in parallel with a traditional acoustic-trawl survey to assess the value of eDNA surveys at a scale relevant to fisheries management. Despite local differences, the two methods yield comparable information about the broad-scale spatial distribution and abundance. Furthermore, we find depth and spatial patterns of eDNA closely correspond to acoustic-trawl estimates for hake. We demonstrate the power and efficacy of eDNA sampling for estimating abundance and distribution and move the analysis eDNA data beyond sample-to-sample comparisons to management relevant scales. We posit that eDNA methods are capable of providing general quantitative applications that will prove especially valuable in data- or resource-limited contexts.

A mobile sex-determining region, male-specific haplotypes and rearing environment influence age at maturity in Chinook salmon.

Variation in age at maturity is an important contributor to life history and demographic variation within and among species. The optimal age at maturity can vary by sex, and the ability of each sex to evolve towards its fitness optimum depends on the genetic architecture of maturation. Using GWAS of RAD sequencing data, we show that age at maturity in Chinook salmon exhibits sex-specific genetic architecture, with age at maturity in males influenced by large (up to 20 Mb) male-specific haplotypes. These regions showed no such effect in females. We also provide evidence for translocation of the sex-determining gene between two different chromosomes. This has important implications for sexually antagonistic selection, particularly that sex linkage of adaptive genes may differ within and among populations based on chromosomal location of the sex-determining gene. Our findings will facilitate research into the genetic causes of shifting demography in Chinook salmon as well as a better understanding of sex determination in this species and Pacific salmon in general.

Functional Annotation of All Salmonid Genomes (FAASG): an international initiative supporting future salmonid research, conservation and aquaculture.

We describe an emerging initiative - the 'Functional Annotation of All Salmonid Genomes' (FAASG), which will leverage the extensive trait diversity that has evolved since a whole genome duplication event in the salmonid ancestor, to develop an integrative understanding of the functional genomic basis of phenotypic variation. The outcomes of FAASG will have diverse applications, ranging from improved understanding of genome evolution, to improving the efficiency and sustainability of aquaculture production, supporting the future of fundamental and applied research in an iconic fish lineage of major societal importance.

Migration-related phenotypic divergence is associated with epigenetic modifications in rainbow trout.

Migration is essential for the reproduction and survival of many animals, yet little is understood about its underlying molecular mechanisms. We used the salmonid Oncorhynchus mykiss to gain mechanistic insight into smoltification, which is a morphological, physiological and behavioural transition undertaken by juveniles in preparation for seaward migration. O. mykiss is experimentally tractable and displays intra- and interpopulation variation in migration propensity. Migratory individuals can produce nonmigratory progeny and vice versa, indicating a high degree of phenotypic plasticity. One potential way that phenotypic plasticity might be linked to variation in migration-related life history tactics is through epigenetic regulation of gene expression. To explore this, we quantitatively measured genome-scale DNA methylation in fin tissue using reduced representation bisulphite sequencing of F2 siblings produced from a cross between steelhead (migratory) and rainbow trout (nonmigratory) lines. We identified 57 differentially methylated regions (DMRs) between smolt and resident O. mykiss juveniles. DMRs were high in magnitude, with up to 62% differential methylation between life history types, and over half of the gene-associated DMRs were in transcriptional regulatory regions. Many of the DMRs encode proteins with activity relevant to migration-related transitions (e.g. circadian rhythm pathway, nervous system development, protein kinase activity). This study provides the first evidence of a relationship between epigenetic variation and life history divergence associated with migration-related traits in any species.

Ontogenetic changes in embryonic and brain gene expression in progeny produced from migratory and resident Oncorhynchus mykiss.

Little information has been gathered regarding the ontogenetic changes that contribute to differentiation between resident and migrant individuals, particularly before the onset of gross morphological and physiological changes in migratory individuals. The aim of this study was to evaluate gene expression during early development in Oncorhynchus mykiss populations with different life histories, in a tissue known to integrate environmental cues to regulate complex developmental processes and behaviours. We sampled offspring produced from migrant and resident parents, collecting whole embryos prior to the beginning of first feeding, and brain tissue at three additional time points over the first year of development. RNA sequencing for 32 individuals generated a reference transcriptome of 30 177 genes that passed count thresholds. Differential gene expression between migrant and resident offspring was observed for 1982 genes. The greatest number of differentially expressed genes occurred at 8 months of age, in the spring a full year before the obvious physiological transformation from stream-dwelling parr to sea water-adaptable smolts begins for migrant individuals. Sex and age exhibited considerable effects on differential gene expression between migrants and resident offspring. Differential gene expression was observed in genes previously associated with migration, but also in genes previously unassociated with early life history divergence. Pathway analysis revealed coordinated differential expression in genes related to phototransduction, which could modulate photoperiod responsiveness and variation in circadian rhythms. The role for early differentiation in light sensitivity and biological rhythms is particularly intriguing in understanding early brain processes involved in differentiation of migratory and resident life history types.

Novel Cadmium Responsive MicroRNAs in Daphnia pulex.

Daphnia pulex is a widely used toxicological model and is known for its sensitivity to cadmium (Cd). Recent research suggests that microRNAs (miRNAs) play a critical role in animal responses to heavy metals. To investigate the functions of D. pulex miRNAs under Cd exposure, we analyzed the miRNA profiles of D. pulex after 48 h using miRNA microarrays and validated our findings by q-PCR. miRNA dpu-let-7 was identified as a stably expressed gene and used as a reference. We identified 22 and 21 differentially expressed miRNAs under low (20 µg/L CdCl2) and high-exposure (40 µg/L CdCl2) concentrations compared to controls, respectively. Cellular functions of predicted miRNA target Cd-responsive genes included oxidative stress, ion transport, mitochondrial damage, and DNA repair. An insulin-related network was also identified in relation to several Cd-responsive miRNAs. The expression of three predicted target genes for miR-71 and miR-210 were evaluated, and expression of two of them (SCN2A and SLC31A1) was negatively correlated with the expression of their regulator miRNAs. We show miR-210 is hypoxia-responsive in D. pulex and propose Cd and hypoxia induce miR-210 via a same HIF1α modulated pathway. Collectively, this research advances our understanding on the role of miRNAs in response to heavy-metal exposure.

Mapping and Expression of Candidate Genes for Development Rate in Rainbow Trout (Oncorhynchus mykiss).

Development rate has important implications for individual fitness and physiology. In salmonid fishes, development rate correlates with many traits later in life, including life-history diversity, growth, and age and size at sexual maturation. In rainbow trout (Oncorhynchus mykiss), a quantitative trait locus for embryonic development rate has been detected on chromosome 5 across populations. However, few candidate genes have been identified within this region. In this study, we use gene mapping, gene expression, and quantitative genetic methods to further identify the genetic basis of embryonic developmental rate in O. mykiss Among the genes located in the region of the major development rate quantitative trait locus (GHR1, Clock1a, Myd118-1, and their paralogs), all were expressed early in embryonic development (fertilization through hatch), but none were differentially expressed between individuals with the fast- or slow-developing alleles for a major embryonic development rate quantitative trait locus. In a follow-up study of migratory and resident rainbow trout from natural populations in Alaska, we found significant additive variation in development rate and, moreover, found associations between development rate and allelic variation in all 3 candidate genes within the quantitative trait locus for embryonic development. The mapping of these genes to this region and associations in multiple populations provide positional candidates for further study of their roles in growth, development, and life-history diversity in this model salmonid.

Contrasting effect of hybridization on genetic differentiation in three rockfish species with similar life history.

Hybridization can provide evolutionary benefits (e.g., population resilience to climate change) through the introduction of adaptive alleles and increase of genetic diversity. Nevertheless, management strategies may be designed based only on the parental species within a hybrid zone, without considering the hybrids. This can lead to ineffective spatial management of species, which can directly harm population diversity and negatively impact food webs. Three species of rockfish (Brown Rockfish (Sebastes caurinus), Copper Rockfish (S. auriculatus), and Quillback Rockfish (S. maliger)) are known to hybridize within Puget Sound, Washington, but genetic data from these species are used to infer population structure in the entire genus, including in species that do not hybridize. The goal of this project was to estimate the hybridization rates within the region and determine the effect of hybridization on geographic patterns of genetic structure. We sequenced 290 Brown, Copper, and Quillback rockfish using restriction-site associated DNA sequencing (RADseq) from four regions within and outside Puget Sound, Washington. We show that (i) hybridization within Puget Sound was asymmetrical, not recent, widespread among individuals, and relatively low level within the genome, (ii) hybridization affected population structure in Copper and Brown rockfish, but not in Quillback Rockfish and (iii) after taking hybridization into account we found limited directional dispersal in Brown and Copper rockfish, and evidence for two isolated populations in Quillback Rockfish. Our results suggest that rockfish population structure is species-specific, dependent on the extent of hybridization, and cannot be inferred from one species to another despite similar life history.

Affordable de novo generation of fish mitogenomes using amplification-free enrichment of mitochondrial DNA and deep sequencing of long fragments.

Biomonitoring surveys make use of metabarcoding tools to describe the community composition. These studies match their sequencing results against public genomic databases to identify the species. However, mitochondrial genomic reference data are yet incomplete, only a few genes may be available, or the suitability of existing sequence data is suboptimal for species level resolution. Here, we present a dedicated and cost-effective workflow with no DNA amplification for generating complete fish mitogenomes for the purpose of strengthening fish mitochondrial databases. Two different strategies using long fragment sequencing with Oxford Nanopore technology coupled with mitochondrial DNA enrichment were used. One where the enrichment is achieved by preferential isolation of mitochondria followed by DNA extraction and nuclear DNA depletion ("mitoenrichment"). A second enrichment approach takes advantage of the CRISPR Cas9 targeted scission on previously dephosphorylated DNA ("targeted mitosequencing"). The sequencing results varied between tissue, species, and integrity of the DNA. The mitoenrichment method yielded 0.17%-12.33% of sequences on target and a mean coverage ranging from 74.9 to 805-fold. The targeted mitosequencing experiment from native genomic DNA yielded 1.83%-55% of sequences on target and a 38 to 2123-fold mean coverage. These produced complete mitogenomes of species with homopolymeric regions, tandem repeats, and gene rearrangements. We demonstrate that deep sequencing of long fragments of native fish DNA can be achieved with low computational resources in a cost-effective manner, opening the discovery of mitogenomes of nonmodel or understudied fish taxa to a broad range of laboratories worldwide.

Los estudios de biomonitoreo utilizan herramientas de caracterización genética (metabarcoding) para describir la composición de la comunidad. Estos estudios contrastan las secuencias obtenidas con bases de datos genómicas públicas para así identificar la especie. Sin embargo, las bases de datos mitocondriales de referencia distan mucho de estar completas. En la mayor parte de los casos solo hay unos pocos genes disponibles o los datos existentes no ofrecen resolución hasta el nivel de especie. En este estudio presentamos un método dedicado a generar mitogenomas de peces completos de forma rentable y sin necesidad de amplificación del ADN, con el objeto de ampliar las bases de datos mitocondriales de peces. Para ello se utilizaron dos enfoques diferentes de secuenciación de fragmentos largos utilizando secuenciación Oxford Nanopore y enriquecimiento de ADN mitocondrial. Uno en el que el enriquecimiento se logra mediante el aislamiento preferencial de mitocondrias seguido de extracción del ADN y la eliminación del ADN nuclear ("mitoenriquecimiento"). En el segundo enfoque se aprovecha la capacidad de escisión dirigida por la endonucleasa CRISPR-Cas9 sobre ADN previamente desfosforilado ("mitosecuenciación dirigida"). Los resultados difirieron con el tejido, la especie y la integridad del ADN. El método de mitoenriquecimiento produjo un 0,17%-12,33% de secuencias objetivo y una cobertura media entre 74,9 y 805 secuencias. El experimento de mitosecuenciación dirigida a partir de ADN genómico nativo produjo entre 1,83 y 55% de secuencias objetivo y una cobertura media de 38 a 2123 secuencias. Este estudio permitió completar mitogenomas de diferentes especies que incluyen regiones homopoliméricas, repeticiones en tándem y reorganización de genes. Demostramos que la secuenciación intensiva de fragmentos largos de ADN de peces es posible, se puede lograr con bajos recursos informáticos de una manera económica, superando el método generalizado de secuenciación genómica de baja cobertura y permitiendo el descubrimiento de mitogenomas de taxones de peces no modelo o poco estudiados a una amplia gama de laboratorios en todo el mundo.

Comparative genomics of rainbow trout (Oncorhynchus mykiss): Is the genetic architecture of migratory behavior conserved among populations?

Rainbow trout (Oncorhynchus mykiss) are a partially migratory species wherein some individuals undergo long-distance anadromous migrations, and others stay as residents in their native freshwater streams. The decision to migrate is known to be highly heritable, and yet, the underlying genes and alleles associated with migration are not fully characterized. Here we used a pooled approach of whole-genome sequence data from migratory and resident trout of two native populations-Sashin Creek, Alaska and Little Sheep Creek, Oregon-to obtain a genome-wide perspective of the genetic architecture of resident and migratory life history. We calculated estimates of genetic differentiation, genetic diversity, and selection between the two phenotypes to locate regions of interest and then compared these associations between populations. We identified numerous genes and alleles associated with life history development in the Sashin Creek population with a notable area on chromosome 8 that may play a critical role in the development of the migratory phenotype. However, very few alleles appeared to be associated with life history development in the Little Sheep Creek system, suggesting population-specific genetic effects are likely important in the development of anadromy. Our results indicate that a migratory life history is not controlled by a singular gene or region but supports the idea that there are many independent ways for a migratory phenotype to emerge in a population. Therefore, conserving and promoting genetic diversity in migratory individuals is paramount to conserving these populations. Ultimately, our data add to a growing body of literature that suggests that population-specific genetic effects, likely mediated through environmental variation, contribute to life history development in rainbow trout.

Correction: Fraik et al. The Impacts of Dam Construction and Removal on the Genetics of Recovering Steelhead (Oncorhynchus mykiss) Populations across the Elwha River Watershed. Genes 2021, 12, 89.

In the original publication [...].

Differential gene expression in male and female rainbow trout embryos prior to the onset of gross morphological differentiation of the gonads.

BACKGROUND: There are large differences between the sexes at the genetic level; these differences include heterogametic sex chromosomes and/or differences in expression of genes between the sexes. In rainbow trout (Oncorhynchus mykiss) qRT-PCR studies have found significant differences in expression of several candidate sex determining genes. However, these genes represent a very small fraction of the genome and research in other species suggests there are large portions of the transcriptome that are differentially expressed between the sexes. These differences are especially noticeable once gonad differentiation and maturation has occurred, but less is known at earlier stages of development. Here we use data from a microarray and qRT-PCR to identify genes differentially expressed between the sexes at three time points in pre-hatch embryos, prior to the known timing of sexual differentiation in this species. RESULTS: The microarray study revealed 883 differentially expressed features between the sexes with roughly equal numbers of male and female upregulated features across time points. Most of the differentially expressed genes on the microarray were not related to sex function, suggesting large scale differences in gene expression between the sexes are present early in development. Candidate gene analysis revealed sox9, DMRT1, Nr5a1 and wt1 were upregulated in males at some time points and foxl2, ovol1, fst and cyp19a1a were upregulated in females at some time points. CONCLUSION: This is the first study to identify sexual dimorphism in expression of the genome during embryogenesis in any fish and demonstrates that transcriptional differences are present before the completion of gonadogenesis.

Differential gene expression associated with behavioral variation in ecotypes of Lake Superior brook trout (Salvelinus fontinalis).

Associations between behaviors and the development of different life history tactics have been documented in several species of salmon, trout, and charr. While it is well known that such behaviors are heritable the genes and molecular pathways connected to these behaviors remain unknown. We used an RNA-seq approach to identify genes and molecular pathways differentially regulated in brain tissue between "shy" and "bold" brook trout (Salvelinus fontinalis). A small number of genes were differentially expressed between the behavioral types at several months after hatching and two years of age. Pathway analysis revealed that EIF2 signaling differed consistently between shy and bold individuals suggesting large-scale differences in protein synthesis between behavioral types in the brain. Additionally, the RNA-seq data were used to find polymorphisms within the brook trout genome and a GWAS approach was used to test for statistical associations between genetic variants and behavior type. One allele located in a transcription factor (TSHZ3) contained a protein-coding non-synonymous SNP suggesting that functional variation within TSHZ3 is connected to the development of different behaviors. These results suggest that the molecular basis of behavioral development is complex and due to the differential expression of many genes involved in a wide-range of different molecular pathways.

The Impacts of Dam Construction and Removal on the Genetics of Recovering Steelhead (Oncorhynchus mykiss) Populations across the Elwha River Watershed.

Dam construction and longitudinal river habitat fragmentation disrupt important life histories and movement of aquatic species. This is especially true for Oncorhynchus mykiss that exhibits both migratory (steelhead) and non-migratory (resident rainbow) forms. While the negative effects of dams on salmonids have been extensively documented, few studies have had the opportunity to compare population genetic diversity and structure prior to and following dam removal. Here we examine the impacts of the removal of two dams on the Elwha River on the population genetics of O. mykiss. Genetic data were produced from >1200 samples collected prior to dam removal from both life history forms, and post-dam removal from steelhead. We identified three genetic clusters prior to dam removal primarily explained by isolation due to dams and natural barriers. Following dam removal, genetic structure decreased and admixture increased. Despite large O. mykiss population declines after dam construction, we did not detect shifts in population genetic diversity or allele frequencies of loci putatively involved in migratory phenotypic variation. Steelhead descendants from formerly below and above dammed populations recolonized the river rapidly after dam removal, suggesting that dam construction did not significantly reduce genetic diversity underlying O. mykiss life history strategies. These results have significant evolutionary implications for the conservation of migratory adaptive potential in O. mykiss populations above current anthropogenic barriers.

The rise and fall of the ancient northern pike master sex-determining gene.

The understanding of the evolution of variable sex determination mechanisms across taxa requires comparative studies among closely related species. Following the fate of a known master sex-determining gene, we traced the evolution of sex determination in an entire teleost order (Esociformes). We discovered that the northern pike (Esox lucius) master sex-determining gene originated from a 65 to 90 million-year-old gene duplication event and that it remained sex linked on undifferentiated sex chromosomes for at least 56 million years in multiple species. We identified several independent species- or population-specific sex determination transitions, including a recent loss of a Y chromosome. These findings highlight the diversity of evolutionary fates of master sex-determining genes and the importance of population demographic history in sex determination studies. We hypothesize that occasional sex reversals and genetic bottlenecks provide a non-adaptive explanation for sex determination transitions.

Strong population differentiation in lingcod (Ophiodon elongatus) is driven by a small portion of the genome.

Delimiting intraspecific genetic variation in harvested species is crucial to the assessment of population status for natural resource management and conservation purposes. Here, we evaluated genetic population structure in lingcod (Ophiodon elongatus), a commercially and recreationally important fishery species along the west coast of North America. We used 16,749 restriction site-associated DNA sequencing (RADseq) markers, in 611 individuals collected from across the bulk of the species range from Southeast Alaska to Baja California, Mexico. In contrast to previous population genetic work on this species, we found strong evidence for two distinct genetic clusters. These groups separated latitudinally with a break near Point Reyes off Northern California, and there was a high frequency of admixed individuals in close proximity to the break. F-statistics corroborate this genetic break between northern and southern sampling sites, although most loci are characterized by low FST values, suggesting high gene flow throughout most of the genome. Outlier analyses identified 182 loci putatively under divergent selection, most of which mapped to a single genomic region. When individuals were grouped by cluster assignment (northern, southern, and admixed), 71 loci were fixed between the northern and southern cluster, all of which were identified in the outlier scans. All individuals identified as admixed exhibited near 50:50 assignment to northern and southern clusters and were heterozygous for most fixed loci. Alignments of RADseq loci to a draft lingcod genome assembly and three other teleost genomes with chromosome-level assemblies suggest that outlier and fixed loci are concentrated on a single chromosome. Similar genomic patterns have been attributed to chromosomal inversions in diverse taxonomic groups. Regardless of the evolutionary mechanism, these results represent novel observations of genetic structure in lingcod and designate clear evolutionary units that could be used to inform fisheries management.

The genetic basis of smoltification-related traits in Oncorhynchus mykiss.

The timing and propensity for migration between fresh- and seawater is a key theme in the diversity of life histories within the salmonid fishes. Across salmonid species, life-history strategies range from wholly freshwater-resident populations, to migratory and nonmigratory variation within populations, to populations and species that are primarily migratory. Despite the central theme of migration to the evolution of these fishes, the genetic architecture of migration-related processes is poorly understood. Using a genetic cross of clonal lines derived from migratory and nonmigratory life-history types of Onchorhynchus mykiss (steelhead and rainbow trout, respectively), we have dissected the genetic architecture of the complex physiological and morphological transformation that occurs immediately prior to seaward migration (termed smoltification). Quantitative trait loci (QTL) analyses were used to identify the number, effects, and genomic location of loci associated with smoltification-related traits, including growth and condition factor, body coloration, morphology, and osmoregulatory enzymes during the smoltification period. Genetic analyses revealed numerous QTL, but one locus in particular is associated with multiple traits in single and joint analyses. Dissecting the genetic architecture of this highly complex trait has profound implications for understanding the genetic and evolutionary basis of life-history diversity within and among migratory fishes.

A large-scale chromosomal inversion is not associated with life history development in rainbow trout from Southeast Alaska.

In studying the causative mechanisms behind migration and life history, the salmonids-salmon, trout, and charr-are an exemplary taxonomic group, as life history development is known to have a strong genetic component. A double inversion located on chromosome 5 in rainbow trout (Oncorhynchus mykiss) is associated with life history development in multiple populations, but the importance of this inversion has not been thoroughly tested in conjunction with other polymorphisms in the genome. To that end, we used a high-density SNP chip to genotype 192 F1 migratory and resident rainbow trout and focused our analyses to determine whether this inversion is important in life history development in a well-studied population of rainbow trout from Southeast Alaska. We identified 4,994 and 436 SNPs-predominantly outside of the inversion region-associated with life history development in the migrant and resident familial lines, respectively. Although F1 samples showed genomic patterns consistent with the double inversion on chromosome 5 (reduced observed and expected heterozygosity and an increase in linkage disequilibrium), we found no statistical association between the inversion and life history development. Progeny produced by crossing resident trout and progeny produced by crossing migrant trout both consisted of a mix of migrant and resident individuals, irrespective of the individuals' inversion haplotype on chromosome 5. This suggests that although the inversion is present at a low frequency, it is not strongly associated with migration as it is in populations of Oncorhynchus mykiss from lower latitudes.

Uncovering mechanisms of global ocean change effects on the Dungeness crab (Cancer magister) through metabolomics analysis.

The Dungeness crab is an economically and ecologically important species distributed along the North American Pacific coast. To predict how Dungeness crab may physiologically respond to future global ocean change on a molecular level, we performed untargeted metabolomic approaches on individual Dungeness crab juveniles reared in treatments that mimicked current and projected future pH and dissolved oxygen conditions. We found 94 metabolites and 127 lipids responded in a condition-specific manner, with a greater number of known compounds more strongly responding to low oxygen than low pH exposure. Pathway analysis of these compounds revealed that juveniles may respond to low oxygen through evolutionarily conserved processes including downregulating glutathione biosynthesis and upregulating glycogen storage, and may respond to low pH by increasing ATP production. Most interestingly, we found that the response of juveniles to combined low pH and low oxygen exposure was most similar to the low oxygen exposure response, indicating low oxygen may drive the physiology of juvenile crabs more than pH. Our study elucidates metabolic dynamics that expand our overall understanding of how the species might respond to future ocean conditions and provides a comprehensive dataset that could be used in future ocean acidification response studies.