Blocking endogenous retinoic acid degradation induces oral tooth formation in zebrafish.

According to Dollo's Law of irreversibility in evolution, a lost structure is usually considered to be unable to reappear in evolution due to the accumulation over time of mutations in the genes required for its formation. Cypriniform fish are a classic model of evolutionary loss because, while they form fully operational teeth in the ventral posterior pharynx, unlike other teleosts, they do not possess oral teeth. Paleontological data show that Cypriniforms, a clade of teleost fish that includes the zebrafish, lost their oral teeth 50 to 100 Mya. In order to attempt to reverse oral tooth loss in zebrafish, we block the degradation of endogenous levels of retinoic acid (RA) using a specific inhibitor of the Cyp26 RA degrading enzymes. We demonstrate the inhibition of endogenous RA degradation is sufficient to restore oral tooth induction as marked by the re-appearance of expression of early dental mesenchyme and epithelium genes such as dlx2b and sp7 in the oral cavity. Furthermore, we show that these exogenously induced oral tooth germs are able to be at least partly calcified. Taken together, our data show that modifications of signaling pathways can have a significant effect on the reemergence of once-lost structures leading to experimentally induced reversibility of evolutionary tooth loss in cypriniforms.

Control of cardiac contractions using Cre-lox and degron strategies in zebrafish.

Cardiac contractions and hemodynamic forces are essential for organ development and homeostasis. Control over cardiac contractions can be achieved pharmacologically or optogenetically. However, these approaches lack specificity or require direct access to the heart. Here, we compare two genetic approaches to control cardiac contractions by modulating the levels of the essential sarcomeric protein Tnnt2a in zebrafish. We first recombine a newly generated tnnt2a floxed allele using multiple lines expressing Cre under the control of cardiomyocyte-specific promoters, and show that it does not recapitulate the tnnt2a/silent heart mutant phenotype in embryos. We show that this lack of early cardiac contraction defects is due, at least in part, to the long half-life of tnnt2a mRNA, which masks the gene deletion effects until the early larval stages. We then generate an endogenous Tnnt2a-eGFP fusion line that we use together with the zGRAD system to efficiently degrade Tnnt2a in all cardiomyocytes. Using single-cell transcriptomics, we find that Tnnt2a depletion leads to cardiac phenotypes similar to those observed in tnnt2a mutants, with a loss of blood and pericardial flow-dependent cell types. Furthermore, we achieve conditional degradation of Tnnt2a-eGFP by splitting the zGRAD protein into two fragments that, when combined with the cpFRB2-FKBP system, can be reassembled upon rapamycin treatment. Thus, this Tnnt2a degradation line enables non-invasive control of cardiac contractions with high spatial and temporal specificity and will help further understand how they shape organ development and homeostasis.

Rapid physiological and transcriptomic changes associated with oxygen delivery in larval anemonefish suggest a role in adaptation to life on hypoxic coral reefs.

Connectivity of coral reef fish populations relies on successful dispersal of a pelagic larval phase. Pelagic larvae must exhibit high swimming abilities to overcome ocean and reef currents, but once settling onto the reef, larvae transition to endure habitats that become hypoxic at night. Therefore, coral reef fish larvae must rapidly and dramatically shift their physiology over a short period of time. Taking an integrative, physiological approach, using swimming respirometry, and examining hypoxia tolerance and transcriptomics, we show that larvae of cinnamon anemonefish (Amphiprion melanopus) rapidly transition between "physiological extremes" at the end of their larval phase. Daily measurements of swimming larval anemonefish over their entire early development show that they initially have very high mass-specific oxygen uptake rates. However, oxygen uptake rates decrease midway through the larval phase. This occurs in conjunction with a switch in haemoglobin gene expression and increased expression of myoglobin, cytoglobin, and neuroglobin, which may all contribute to the observed increase in hypoxia tolerance. Our findings indicate that critical ontogenetic changes in the gene expression of oxygen-binding proteins may underpin the physiological mechanisms needed for successful larval recruitment to reefs.

Structural and Functional Characterization of a Fish Type I Subgroup d IFN Reveals Its Binding to Receptors.

Teleost fish type I IFNs and the associated receptors from the cytokine receptor family B (CRFB) are characterized by remarkable diversity and complexity. How the fish type I IFNs bind to their receptors is still not fully understood. In this study, we demonstrate that CRFB1 and CRFB5 constitute the receptor pair through which type I subgroup d IFN (IFNd) from large yellow croaker, Larimichthys crocea, activates the conserved JAK-STAT signaling pathway as a part of the antiviral response. Our data suggest that L. crocea IFNd (LcIFNd) has a higher binding affinity with L. crocea CRFB5 (LcCRFB5) than with LcCRFB1. Furthermore, we report the crystal structure of LcIFNd at a 1.49-Å resolution and construct structural models of LcIFNd in binary complexes with predicted structures of extracellular regions of LcCRFB1 and LcCRFB5, respectively. Despite striking similarities in overall architectures of LcIFNd and its ortholog human IFN-ω, the receptor binding patterns between LcIFNd and its receptors show that teleost and mammalian type I IFNs may have differentially selected helices that bind to their homologous receptors. Correspondingly, key residues mediating binding of LcIFNd to LcCRFB1 and LcCRFB5 are largely distinct from the receptor-interacting residues in other fish and mammalian type I IFNs. Our findings reveal a ligand/receptor complex binding mechanism of IFNd in teleost fish, thus providing new insights into the function and evolution of type I IFNs.

The origin of blinking in both mudskippers and tetrapods is linked to life on land.

Blinking, the transient occlusion of the eye by one or more membranes, serves several functions including wetting, protecting, and cleaning the eye. This behavior is seen in nearly all living tetrapods and absent in other extant sarcopterygian lineages suggesting that it might have arisen during the water-to-land transition. Unfortunately, our understanding of the origin of blinking has been limited by a lack of known anatomical correlates of the behavior in the fossil record and a paucity of comparative functional studies. To understand how and why blinking originates, we leverage mudskippers (Oxudercinae), a clade of amphibious fishes that have convergently evolved blinking. Using microcomputed tomography and histology, we analyzed two mudskipper species, Periophthalmus barbarus and Periophthalmodon septemradiatus, and compared them to the fully aquatic round goby, Neogobius melanostomus. Study of gross anatomy and epithelial microstructure shows that mudskippers have not evolved novel musculature or glands to blink. Behavioral analyses show the blinks of mudskippers are functionally convergent with those of tetrapods: P. barbarus blinks more often under high-evaporation conditions to wet the eye, a blink reflex protects the eye from physical insult, and a single blink can fully clean the cornea of particulates. Thus, eye retraction in concert with a passive occlusal membrane can achieve functions associated with life on land. Osteological correlates of eye retraction are present in the earliest limbed vertebrates, suggesting blinking capability. In both mudskippers and tetrapods, therefore, the origin of this multifunctional innovation is likely explained by selection for increasingly terrestrial lifestyles.

FABP1 induces lipogenesis by regulating the processing of SREBP1 in hepatocytes of large yellow croaker (Larimichthys crocea).

Fatty acid-binding protein 1 (FABP1) plays an important role in regulating fatty acid metabolism in liver, which is a potential therapeutic target for diseases such as non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanisms are not well defined. Using complementary experimental models, we discovered FABP1 induction in hepatocytes as a primary mediator of lipogenesis when exposed to fatty acids, especially saturated fatty acids (SFAs). In the feeding trial, palm oil led to excess lipid accumulation in the liver of large yellow croaker (Larimichthys crocea), accompanied by significant induction of FABP1. In cultured cells, palmitic acid (PA), a kind of SFA, triggered the fabp1 expression and increased triglyceride (TG) contents. Knockdown of FABP1 dampened PA-induced TG accumulation through mitigated lipogenesis. The overexpression of FABP1 showed the opposite result. Furthermore, the inactivation of FABP1 led to induction in insulin-induced gene 1 (INSIG1) expression, which attenuated the processing of sterol regulatory element-binding protein 1 (SREBP1) by down-regulating the nuclear-localized SREBP1. These results revealed a previously unrecognized function of FABP1 in response to PA, providing additional evidence for targeting FABP1 in the treatment of NAFLD caused by SFA.

Modelling how cleaner fish approach an ephemeral reward task demonstrates a role for ecologically tuned chunking in the evolution of advanced cognition.

What makes cognition "advanced" is an open and not precisely defined question. One perspective involves increasing the complexity of associative learning, from conditioning to learning sequences of events ("chaining") to representing various cue combinations as "chunks." Here we develop a weighted graph model to study the mechanism enabling chunking ability and the conditions for its evolution and success, based on the ecology of the cleaner fish Labroides dimidiatus. In some environments, cleaners must learn to serve visitor clients before resident clients, because a visitor leaves if not attended while a resident waits for service. This challenge has been captured in various versions of the ephemeral reward task, which has been proven difficult for a range of cognitively capable species. We show that chaining is the minimal requirement for solving this task in its common simplified laboratory format that involves repeated simultaneous exposure to an ephemeral and permanent food source. Adding ephemeral-ephemeral and permanent-permanent combinations, as cleaners face in the wild, requires individuals to have chunking abilities to solve the task. Importantly, chunking parameters need to be calibrated to ecological conditions in order to produce adaptive decisions. Thus, it is the fine-tuning of this ability, which may be the major target of selection during the evolution of advanced associative learning.

METTL3-Mediated m6A Modification of TRIF and MyD88 mRNAs Suppresses Innate Immunity in Teleost Fish, Miichthys miiuy.

Methyltransferase (METTL3), the most important N6-methyladenosine (m6A) writer, plays a vital role in regulating immune-related signaling pathways. However, the underlying mechanism of METTL3 action remains largely unknown, especially in lower vertebrates. The results of this study show that METTL3 inhibits innate immune response and promotes the infection of miiuy croaker, Miichthys miiuy, by Siniperca chuatsi rhabdovirus and Vibrio anguillarum. Significantly, the function of METTL3 in inhibiting immunity depends on its methylase activity. Mechanistically, METTL3 increases the methylation level of trif and myd88 mRNA, rendering them sensitive to degradation by the YTHDF2/3 reader proteins. By contrast, we found that the YTHDF1 reader protein promotes the translation of myd88 mRNA. In summary, these results indicate that METTL3-mediated m6A modification of trif and myd88 mRNAs suppresses innate immunity by inhibiting the TLR pathway, unveiling a molecular mechanism by which RNA methylation controls innate immunity to pathogens in the teleost fish.

Structural basis of DNA polymerase θ mediated DNA end joining.

DNA polymerase θ (Pol θ) plays an essential role in the microhomology-mediated end joining (MMEJ) pathway for repairing DNA double-strand breaks. However, the mechanisms by which Pol θ recognizes microhomologous DNA ends and performs low-fidelity DNA synthesis remain unclear. Here, we present cryo-electron microscope structures of the polymerase domain of Lates calcarifer Pol θ with long and short duplex DNA at up to 2.4 Šresolution. Interestingly, Pol θ binds to long and short DNA substrates similarly, with extensive interactions around the active site. Moreover, Pol θ shares a similar active site as high-fidelity A-family polymerases with its finger domain well-closed but differs in having hydrophilic residues surrounding the nascent base pair. Computational simulations and mutagenesis studies suggest that the unique insertion loops of Pol θ help to stabilize short DNA binding and assemble the active site for MMEJ repair. Taken together, our results illustrate the structural basis of Pol θ-mediated MMEJ.

Co-habiting ants and silverfish display a converging feeding ecology.

BACKGROUND: Various animal taxa have specialized to living with social hosts. Depending on their level of specialization, these symbiotic animals are characterized by distinct behavioural, chemical, and morphological traits that enable close heterospecific interactions. Despite its functional importance, our understanding of the feeding ecology of animals living with social hosts remains limited. We examined how host specialization of silverfish co-habiting with ants affects several components of their feeding ecology. We combined stable isotope profiling, feeding assays, phylogenetic reconstruction, and microbial community characterization of the Neoasterolepisma silverfish genus and a wider nicoletiid and lepismatid silverfish panel where divergent myrmecophilous lifestyles are observed. RESULTS: Stable isotope profiling (δ13C and δ15N) showed that the isotopic niches of granivorous Messor ants and Messor-specialized Neoasterolepisma exhibit a remarkable overlap within an ant nest. Trophic experiments and gut dissections further supported that these specialized Neoasterolepisma silverfish transitioned to a diet that includes plant seeds. In contrast, the isotopic niches of generalist Neoasterolepisma silverfish and generalist nicoletiid silverfish were clearly different from their ant hosts within the shared nest environment. The impact of the myrmecophilous lifestyle on feeding ecology was also evident in the internal silverfish microbiome. Compared to generalists, Messor-specialists exhibited a higher bacterial density and a higher proportion of heterofermentative lactic acid bacteria. Moreover, the nest environment explained the infection profile (or the 16S rRNA genotypes) of Weissella bacteria in Messor-specialized silverfish and the ant hosts. CONCLUSIONS: Together, we show that social hosts are important determinants for the feeding ecology of symbiotic animals and can induce diet convergence.

Gill ionocyte remodeling mediates blood pH regulation in rockfish (Sebastes diploproa) exposed to environmentally relevant hypercapnia.

Marine fishes excrete excess H+ using basolateral Na+-K+-ATPase (NKA) and apical Na+/H+ exchanger 3 (NHE3) in gill ionocytes. However, the mechanisms that regulate H+ excretion during exposure to environmentally relevant hypercapnia (ERH) remain poorly understood. Here, we explored transcriptomic, proteomic, and cellular responses in gills of juvenile splitnose rockfish (Sebastes diploproa) exposed to 3 days of ERH conditions (pH ∼7.5, ∼1,600 µatm Pco2). Blood pH was fully regulated at ∼7.75 despite a lack of significant changes in gill 1) mRNAs coding for proteins involved in blood acid-base regulation, 2) total NKA and NHE3 protein abundance, and 3) ionocyte density. However, ERH-exposed rockfish demonstrated increased NKA and NHE3 abundance on the ionocyte plasma membrane coupled with wider apical membranes and greater extension of apical microvilli. The observed gill ionocyte remodeling is consistent with enhanced H+ excretion that maintains blood pH homeostasis during exposure to ERH and does not necessitate changes at the expression or translation levels. These mechanisms of phenotypic plasticity may allow fishes to regulate blood pH during environmentally relevant acid-base challenges and thus have important implications for both understanding how organisms respond to climate change and for selecting appropriate metrics to evaluate its impact on marine ecosystems.NEW & NOTEWORTHY Splitnose rockfish exposed to environmentally relevant hypercapnia utilize existing proteins (rather than generate additional machinery) to maintain homeostasis.

Role of cold shock proteins B and D in Aeromonas salmonicida subsp. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus).

Cold shock proteins (Csp) are pivotal nucleic acid binding proteins known for their crucial roles in the physiology and virulence of various bacterial pathogens affecting plant, insect, and mammalian hosts. However, their significance in bacterial pathogens of teleost fish remains unexplored. Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is a psychrotrophic pathogen and the causative agent of furunculosis in marine and freshwater fish. Four csp genes (cspB, cspD, cspA, and cspC) have been identified in the genome of A. salmonicida J223 (wild type). Here, we evaluated the role of DNA binding proteins, CspB and CspD, in A. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus). A. salmonicida ΔcspB, ΔcspD, and the double ΔcspBΔcspD mutants were constructed and characterized. A. salmonicida ΔcspB and ΔcspBΔcspD mutants showed a faster growth at 28°C, and reduced virulence in lumpfish. A. salmonicida ΔcspD showed a slower growth at 28°C, biofilm formation, lower survival in low temperatures and freezing conditions (-20°C, 0°C, and 4°C), deficient in lipopolysaccharide synthesis, and low virulence in lumpfish. Additionally, ΔcspBΔcspD mutants showed less survival in the presence of bile compared to the wild type. Transcriptome analysis revealed that 200, 37, and 921 genes were differentially expressed in ΔcspB, ΔcspD, and ΔcspBΔcspD, respectively. In ΔcspB and ΔcspBΔcspD virulence genes in the chromosome and virulence plasmid were downregulated. Our analysis indicates that CspB and CspD mostly act as a transcriptional activator, influencing cell division (e.g., treB), virulence factors (e.g., aexT), and ultimately virulence.

De novo assembly and annotation of the Patagonian toothfish (Dissostichus eleginoides) genome.

BACKGROUND: Patagonian toothfish (Dissostichus eleginoides) is an economically and ecologically important fish species in the family Nototheniidae. Juveniles occupy progressively deeper waters as they mature and grow, and adults have been caught as deep as 2500 m, living on or in just above the southern shelves and slopes around the sub-Antarctic islands of the Southern Ocean. As apex predators, they are a key part of the food web, feeding on a variety of prey, including krill, squid, and other fish. Despite its importance, genomic sequence data, which could be used for more accurate dating of the divergence between Patagonian and Antarctic toothfish, or establish whether it shares adaptations to temperature with fish living in more polar or equatorial climes, has so far been limited. RESULTS: A high-quality D. eleginoides genome was generated using a combination of Illumina, PacBio and Omni-C sequencing technologies. To aid the genome annotation, the transcriptome derived from a variety of toothfish tissues was also generated using both short and long read sequencing methods. The final genome assembly was 797.8 Mb with a N50 scaffold length of 3.5 Mb. Approximately 31.7% of the genome consisted of repetitive elements. A total of 35,543 putative protein-coding regions were identified, of which 50% have been functionally annotated. Transcriptomics analysis showed that approximately 64% of the predicted genes (22,617 genes) were found to be expressed in the tissues sampled. Comparative genomics analysis revealed that the anti-freeze glycoprotein (AFGP) locus of D. eleginoides does not contain any AFGP proteins compared to the same locus in the Antarctic toothfish (Dissostichus mawsoni). This is in agreement with previously published results looking at hybridization signals and confirms that Patagonian toothfish do not possess AFGP coding sequences in their genome. CONCLUSIONS: We have assembled and annotated the Patagonian toothfish genome, which will provide a valuable genetic resource for ecological and evolutionary studies on this and other closely related species.

Mitochondrial genome structure and composition in 70 fishes: a key resource for fisheries management in the South Atlantic.

BACKGROUND: Phylogenetic gaps of public databases of reference sequences are a major obstacle for comparative genomics and management of marine resources, particularly in the Global South, where economically important fisheries and conservation flagship species often lack closely-related references. We applied target-enrichment to obtain complete mitochondrial genomes of marine ichthyofauna from the Brazilian coast selected based on economic significance, conservation status and lack of phylogenetically-close references. These included sardines (Dorosomatidae, Alosidae), mackerels (Scombridae) croakers (Sciaenidae), groupers (Epinephelidae) and snappers (Lutjanidae). RESULTS: Custom baits were designed to enrich mitochondrial DNA across a broad phylogenetic range of fishes. Sequencing generated approximately 100k reads per sample, which were assembled in a total of 70 complete mitochondrial genomes and include fifty-two new additions to GenBank, including five species with no previous mitochondrial data. Departures from the typical gene content and order occurred in only three taxa and mostly involved tRNA gene duplications. Start-codons for all genes, except Cytochrome C Oxidase subunit I (COI), were consistently ATG, whilst a wide range of stop-codons deviated from the prevailing TAA. Phylogenetic analysis confirmed assembly accuracy and revealed signs of cryptic diversification within the Mullus genus. Lineage delimitation methods using Sardinella aurita and S. brasiliensis mitochondrial genomes support a single Operational Taxonomic Unit. CONCLUSIONS: Target enrichment was highly efficient, providing complete novel mitochondrial genomes with little sequencing effort. These sequences are deposited in public databases to enable subsequent studies in population genetics and adaptation of Latin American fish species and serve as a vital resource for conservation and management programs that rely on molecular data for species and genus-level identification.

Genome-wide identification, characterization, and expression analysis of the transient receptor potential gene family in mandarin fish Siniperca chuatsi.

BACKGROUND: Temperature is a crucial environmental determinant for the vitality and development of teleost fish, yet the underlying mechanisms by which they sense temperature fluctuations remain largely unexplored. Transient receptor potential (TRP) proteins, renowned for their involvement in temperature sensing, have not been characterized in teleost fish, especially regarding their temperature-sensing capabilities. RESULTS: In this study, a genome-wide analysis was conducted, identifying a total of 28 TRP genes in the mandarin fish Siniperca chuatsi. These genes were categorized into the families of TRPA, TRPC, TRPP, TRPM, TRPML, and TRPV. Despite notable variations in conserved motifs across different subfamilies, TRP family members shared common structural features, including ankyrin repeats and the TRP domain. Tissue expression analysis showed that each of these TRP genes exhibited a unique expression pattern. Furthermore, examination of the tissue expression patterns of ten selected TRP genes following exposure to both high and low temperature stress indicated the expression of TRP genes were responsive to temperatures changes. Moreover, the expression profiles of TRP genes in response to mandarin fish virus infections showed significant upregulation for most genes after Siniperca chuatsi rhabdovirus, mandarin fish iridovirus and infectious spleen and kidney necrosis virus infection. CONCLUSIONS: This study characterized the TRP family genes in mandarin fish genome-wide, and explored their expression patterns in response to temperature stress and virus infections. Our work will enhance the overall understanding of fish TRP channels and their possible functions.

Whole genome sequence and characterisation of Streptococcus suis 3112, isolated from snakeskin gourami, Trichopodus pectoralis.

BACKGROUND: Streptococcus suis (S. suis) is an important swine and human pathogen. A recent study reported the first isolate of S. suis capable of infecting fish, designated as S. suis strain 3112. The bacterium was isolated from snakeskin gourami (Trichopodus pectoralis), an economically important fish species native to Southeast Asia, and it was previously shown that it can infect and cause lethal streptococcosis in the fish. RESULTS: In this study, we present the complete genome of S. suis 3112. Molecular sequence analysis revealed that it belongs to serotype 6, sequence type 2340. Phylogenetic analysis showed that the bacterium clustered with healthy-pig S. suis isolates, suggestive of an ultimate swine (as opposed to human) origin of the bacterium. Two fluoroquinolone resistance genes are present in the bacterial genome, namely patA and patB. Our results showed that both genes are expressed in our bacterium, and the bacterium is resistant to norfloxacin, but is still sensitive to other fluoroquinolones, including ciprofloxacin, enrofloxacin, and sparfloxacin. Additionally, the bacterium is sensitive to ß-lactams, tetracyclines, sulphonamides, and an aminoglycoside. CONCLUSIONS: This study reports and describes the complete genome of S. suis 3112, the first isolate of S. suis known to infect fish, and provides further insights into the bacterial isolate, particularly regarding its drug resistance profile. These results will facilitate further investigations of the comparative genomics and pathogenic characteristics of S. suis, as well as the development of control strategies against this newly-identified fish pathogen.

A Chromosome-Level Genome Assembly of the Reef Stonefish (Synanceia verrucosa) Provides Novel Insights into Stonustoxin (sntx) Genes.

Reef stonefish (Synanceia verrucosa) is one of the most venomous fishes, but its biomedical study has been restricted to molecular cloning and purification of its toxins, instead of high-throughput genetic research on related toxin genes. In this study, we constructed a chromosome-level haplotypic genome assembly for the reef stonefish. The genome was assembled into 24 pseudo-chromosomes, and the length totaled 689.74 Mb, reaching a contig N50 of 11.97 Mb and containing 97.8% of complete BUSCOs. A total of 24,050 protein-coding genes were annotated, of which metalloproteinases, C-type lectins, and stonustoxins (sntx) were the most abundant putative toxin genes. Multitissue transcriptomic and venom proteomic data showed that sntx genes, especially those clustered within a 50-kb region on the chromosome 2, had higher transcription levels than other types of toxins as well as those sntx genes scatteringly distributed on other chromosomes. Further comparative genomic analysis predicted an expansion of sntx-like genes in the Percomorpha lineage including nonvenomous fishes, but Scorpaenoidei species experienced extra independent sntx duplication events, marking the clear-cut origin of authentic toxic stonustoxins. In summary, this high-quality genome assembly and related comparative analysis of toxin genes highlight valuable genetic differences for potential involvement in the evolution of venoms among Scorpaeniformes fishes.

Cold-Driven Hemoglobin Evolution in Antarctic Notothenioid Fishes Prior to Hemoglobin Gene Loss in White-Blooded Icefishes.

Expression of multiple hemoglobin isoforms with differing physiochemical properties likely helps species adapt to different environmental and physiological conditions. Antarctic notothenioid fishes inhabit the icy Southern Ocean and display fewer hemoglobin isoforms, each with less affinity for oxygen than temperate relatives. Reduced hemoglobin multiplicity was proposed to result from relaxed selective pressure in the cold, thermally stable, and highly oxygenated Antarctic waters. These conditions also permitted the survival and diversification of white-blooded icefishes, the only vertebrates living without hemoglobin. To understand hemoglobin evolution during adaptation to freezing water, we analyzed hemoglobin genes from 36 notothenioid genome assemblies. Results showed that adaptation to frigid conditions shaped hemoglobin gene evolution by episodic diversifying selection concomitant with cold adaptation and by pervasive evolution in Antarctic notothenioids compared to temperate relatives, likely a continuing adaptation to Antarctic conditions. Analysis of hemoglobin gene expression in adult hematopoietic organs in various temperate and Antarctic species further revealed a switch in hemoglobin gene expression underlying hemoglobin multiplicity reduction in Antarctic fish, leading to a single hemoglobin isoform in adult plunderfishes and dragonfishes, the sister groups to icefishes. The predicted high hemoglobin multiplicity in Antarctic fish embryos based on transcriptomic data, however, raises questions about the molecular bases and physiological implications of diverse hemoglobin isoforms in embryos compared to adults. This analysis supports the hypothesis that the last common icefish ancestor was vulnerable to detrimental mutations affecting the single ancestral expressed alpha- and beta-globin gene pair, potentially predisposing their subsequent loss.

Genomics of Secondarily Temperate Adaptation in the Only Non-Antarctic Icefish.

White-blooded Antarctic icefishes, a family within the adaptive radiation of Antarctic notothenioid fishes, are an example of extreme biological specialization to both the chronic cold of the Southern Ocean and life without hemoglobin. As a result, icefishes display derived physiology that limits them to the cold and highly oxygenated Antarctic waters. Against these constraints, remarkably one species, the pike icefish Champsocephalus esox, successfully colonized temperate South American waters. To study the genetic mechanisms underlying secondarily temperate adaptation in icefishes, we generated chromosome-level genome assemblies of both C. esox and its Antarctic sister species, Champsocephalus gunnari. The C. esox genome is similar in structure and organization to that of its Antarctic congener; however, we observe evidence of chromosomal rearrangements coinciding with regions of elevated genetic divergence in pike icefish populations. We also find several key biological pathways under selection, including genes related to mitochondria and vision, highlighting candidates behind temperate adaptation in C. esox. Substantial antifreeze glycoprotein (AFGP) pseudogenization has occurred in the pike icefish, likely due to relaxed selection following ancestral escape from Antarctica. The canonical AFGP locus organization is conserved in C. esox and C. gunnari, but both show a translocation of two AFGP copies to a separate locus, previously unobserved in cryonotothenioids. Altogether, the study of this secondarily temperate species provides an insight into the mechanisms underlying adaptation to ecologically disparate environments in this otherwise highly specialized group.

Multi-omics Investigation of Freeze Tolerance in the Amur Sleeper, an Aquatic Ectothermic Vertebrate.

Freeze tolerance, the ability of an organism to survive internal ice formation, is a striking survival strategy employed by some ectotherms living in cold environments. However, the genetic bases of this remarkable adaptation are largely unknown. The Amur sleeper (Perccottus glenii), the only known freeze-tolerant fish species, can overwinter with its entire body frozen in ice. Here, we sequenced the chromosome-level genome of the Amur sleeper and performed comparative genomic, transcriptomic, and metabolomic analyses to investigate its strategies for surviving freezing. Evolutionary analysis suggested that the Amur sleeper diverged from its closest non-cold-hardy relative about 15.07 million years ago and has experienced a high rate of protein evolution. Transcriptomic and metabolomic data identified a coordinated and tissue-specific regulation of genes and metabolites involved in hypometabolism, cellular stress response, and cryoprotectant accumulation involved in freezing and thawing. Several genes show evidence of accelerated protein sequence evolution or family size expansion were found as adaptive responses to freezing-induced stresses. Specifically, genetic changes associated with cytoskeleton stability, cryoprotectant synthesis, transmembrane transport, and neuroprotective adaptations were identified as potentially key innovations that aid in freezing survival. Our work provides valuable resources and opportunities to unveil the molecular adaptations supporting freeze tolerance in ectothermic vertebrates.