Cold Adaptation in Antarctic Notothenioids: Comparative Transcriptomics Reveals Novel Insights in the Peculiar Role of Gills and Highlights Signatures of Cobalamin Deficiency

Abstract

Far from being devoid of life, Antarctic waters are home to Cryonotothenioidea, which represent one of the fascinating cases of evolutionary adaptation to extreme environmental conditions in vertebrates. Thanks to a series of unique morphological and physiological peculiarities, which include the paradigmatic case of loss of hemoglobin in the family Channichthyidae, these fish survive and thrive at sub-zero temperatures. While some of the distinctive features of such adaptations have been known for decades, our knowledge of their genetic and molecular bases is still limited. We generated a reference de novo assembly of the icefish Chionodraco hamatus transcriptome and used this resource for a large-scale comparative analysis among five red-blooded Cryonotothenioidea, the sub-Antarctic notothenioid Eleginops maclovinus and seven temperate teleost species. Our investigation targeted the gills, a tissue of primary importance for gaseous exchange, osmoregulation, ammonia excretion, and its role in fish immunity. One hundred and twenty genes were identified as significantly up-regulated in Antarctic species and surprisingly shared by red- and white-blooded notothenioids, unveiling several previously unreported molecular players that might have contributed to the evolutionary success of Cryonotothenioidea in Antarctica. In particular, we detected cobalamin deficiency signatures and discussed the possible biological implications of this condition concerning hematological alterations and the heavy parasitic loads typically observed in all Cryonotothenioidea.

Keywords: Antarctica; Cryonotothenioidea; RNA-seq; cold adaptation; transcobalamin.

Figure 1

External morphology of an adult Chionodraco hamatus specimen, with the assembly and annotation statistics obtained for gill transcriptome.

Figure 2

Hierarchical clustering of a set of representative genes (achieving a TPM value > 1000 in at least one tissue), based on the gene expression levels, i.e., Log10(TPM+1), of skeletal muscle (M), hearth (H), head kidney (HK), liver (L), and gills (G). Genes were clustered with an average linkage method, based on Pearson distances. Colors represent the Z-scores, calculated for each row based on the maximum and minimum gene expression level observed. Gills-specific genes are boxed, and the top 20 most highly expressed gill-specific protein-coding transcripts, defined by a fold change value > 10 in all the pairwise comparisons with the other available tissues, are shown in a table. TPM = Transcripts Per Million. * target genes for validation by qRT-PCR, see Supplementary Data Note 2.

Figure 3

Hierarchical clustering of the six Cryonotothotenioidea and eight additional Eupercaria species considered in this study, based on gills gene expression profiles. An average linkage clustering method was used, based on Euclidean distance, which considered the expression levels of the 33 orthologous genes displaying the highest consistency score of differential expression between the two groups (≥42). For graphical representation, each gene’s square root-transformed TPM values were first standardized on the maximal level observed across species, and subsequently subtracted with the median standardized square root-transformed TPM value of all species. Ch: Chionodraco hamatus; Dl: Dicentrarchus labrax; Dm: Dissostichus mawsoni; Em: Eleginops maclovinus; Ga: Gasterosteus aculeatus; Lg, Ljutanus guttatus; Lm: Lateolabrax maculatus; Lp: Larimichthys polyactis; Pb: Pagothenia borchgrevinki; Pc: Parachaenichthys charcoti; Pf: Perca fluviatilis; Tb: Trematomus bernacchii; Tn: Trematomus newnesi; Tr: Takifugu rubripes.

Figure 4

Panel (A): Multiple sequence alignment of C-terminal α domain from the C: hamatus transcobalamin-like protein (TCNL) and the human transcobalamin-1, -2 and gastric intrinsic factor. Residues involved in hydrophobic interactions with cobalamin are shown in a box; residues involved in forming hydrogen bonds with cobalamin are shaded (from Wuerges et al., 2007). Panels (B–E): comparative expression levels of transcobalamin-like (TCNL), bleomycin hydrolase (BLMH), methenyltetrahydrofolate synthetase (MTHFSD), and guanosine monophosphate reductase (GMPR) orthologs in Cryonotothenioidea and non-Antarctic Eupercaria.

Figure 5

Panel (A): Bayesian phylogeny of carbonic anhydrases from Eupercaria, based on a WAG+G+I+F model of molecular evolution and on the multiple sequence alignment of the CA domain (the two domains of CA4C proteins were both included). For methodological details, see the Materials and Methods section. The carbonic anhydrase-related proteins (CARP) clade was used as an outgroup to root the tree. Posterior probability support values for the primary nodes are shown. Nodes supported by poor posterior probability values (<0.5) were collapsed. Sequences from Cryonotothenioidea are indicated with red branches, and those from C. hamatus are marked with a yellow circle. Sequences from Homo sapiens, included to allow orthology assessment, are indicated with blue branches. The domain architecture of each CA group is schematically displayed; transmembrane domains are indicated by a helix. PTX: pentraxin. Panel (B): heat map summarizing gene expression levels of carbonic anhydrases in different tissues of C. hamatus. Gene expression levels are shown as log₂TPM. Panel (C): cumulative gene expression levels of all carbonic anhydrase genes in the 14 Eupercaria species considered in the present study. Gene expression levels are shown as the relative contribution to the total gene expression of the gill tissue, calculated on the BUSCO orthologous gene set.

Figure 6

Panel (A): schematic representation of proteasome subunits, indicating those showing increased expression levels in Cryonotothenioidea in red. Panel (B): comparative expression levels of peptidylprolyl isomerase D (PPID), poly(ADP-ribose) polymerase family, member 16 (PARP16), pyrroline-5-carboxylate reductase (PYCR3), ornithine aminotransferase (OAT), oligosaccharyltransferase complex non-catalytic subunit (OSTC), and FK506 binding protein 3 (FKBP3) and orthologs in Cryonotothenioidea and non-Antarctic Eupercaria. * = q-value < 0.05; ** = q-value < 1 × 10⁻⁵.