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Lungfishes belong to lobe-fined fish (Sarcopterygii) that, in the Devonian period, ‘conquered’ the land and ultimately gave rise to all land vertebrates, including humans 1 – 3 . Here we determine the chromosome-quality genome of the Australian lungfish ( Neoceratodus forsteri ), which is known to have the largest genome of any animal. The vast size of this genome, which is about 14× larger than that of humans, is attributable mostly to huge intergenic regions and introns with high repeat content (around 90%), the components of which resemble those of tetrapods (comprising mainly long interspersed nuclear elements) more than they do those of ray-finned fish. The lungfish genome continues to expand independently (its transposable elements are still active), through mechanisms different to those of the enormous genomes of salamanders. The 17 fully assembled lungfish macrochromosomes maintain synteny to other vertebrate chromosomes, and all microchromosomes maintain conserved ancient homology with the ancestral vertebrate karyotype. Our phylogenomic analyses confirm previous reports that lungfish occupy a key evolutionary position as the closest living relatives to tetrapods 4 , 5 , underscoring the importance of lungfish for understanding innovations associated with terrestrialization. Lungfish preadaptations to living on land include the gain of limb-like expression in developmental genes such as hoxc13 and sall1 in their lobed fins. Increased rates of evolution and the duplication of genes associated with obligate air-breathing, such as lung surfactants and the expansion of odorant receptor gene families (which encode proteins involved in detecting airborne odours), contribute to the tetrapod-like biology of lungfishes. These findings advance our understanding of this major transition during vertebrate evolution. A chromosome-quality genome of the lungfish Neoceratodus fosteri sheds light on the development of obligate air-breathing and the gain of limb-like gene expression in lobed fins, providing insights into the water-to-land transition in vertebrate evolution.

The harbor porpoise populations of the North and Baltic Seas are highly impacted by human activities, including underwater-radiated noise, fisheries and pollution. These cumulative stressors can have various detrimental effects, such as reduced foraging success, altered behavior and an impaired immune system. Harbor porpoises especially suffer from diseases of the respiratory tract which are partly caused or exacerbated by high parasitic prevalence in the lungs that may ultimately affect diving ability and competitiveness due to insufficient oxygen uptake and supply to the locomotor musculature. To investigate pathophysiological mechanisms and potential compensatory adaptations to pathogenic insults, we employed transcriptomics and compared lungs and muscles of harbor porpoises with compromised respiratory health to healthy individuals. Additionally, a transcriptome assembly was generated to identify transcripts that may be involved in immune-related responses. Non-healthy harbor porpoises showed a distinct host-pathogen defense reaction in the lung, suggesting similarities to immune responses of humans suffering from lung diseases, which may be conserved along the mammalian lineage despite vastly different habitats. However, the lung transcriptomes did not indicate a Th2 immune response which is typically activated upon parasitic insults. Severely infected harbor porpoises may be overwhelmed or weakened by prolonged parasitic exposure and immune activation, possibly affecting simultaneous pathogenic clearance and tissue repair. The muscles of non-healthy harbor porpoises exhibited enhanced stress signaling and tightly regulated tissue degradation/regeneration, potentially reflecting a chronic inflammation state. Higher expression of hypoxia- and oxidative stress-associated transcripts in the muscle were consistent with hypoxia-induced transcriptional patterns and suggest a systemic pathological challenge. The assembly identified significantly dysregulated non-coding RNAs in the lung and muscle which may be associated with regulatory processes. Several transcripts of the assembly remained unidentified, thus their putative function needs to be elucidated. In marine mammals, the understanding of molecular immune responses still remains incomplete. This is the first study to describe the lung transcriptome of wild harbor porpoises in regard to pathophysiology. These insights contribute to the understanding of the interaction between anthropogenic impacts, infectious diseases and molecular immune responses in cetaceans, thus supporting cetacean health assessments and conservation efforts.

Food webs in estuarine ecosystems serve as important biological indicators. The feeding ecology of four keystone fish species, pikeperch (Sander lucioperca L.), smelt (Osmerus eperlanus L.), ruffe (Gymnocephalus cernua L.) and flounder (Platichthys flesus L.), in the Elbe and Odra estuaries was analyzed using stomach content analyses. Important prey of pikeperch were fishes and mysids in both estuaries. Amphipods were especially important as prey for smelt in the Elbe estuary, whereas smelt caught in the Odra estuary mainly consumed mysids. Ruffe fed mainly on amphipods in the Elbe estuary, while annelids (lower section) and insect larvae (upper section) were the most important prey in the Odra estuary. Flounder favored copepods as prey in the Elbe estuary, while bivalves were preferred in the Odra estuary. Higher dietary overlaps were found in the Elbe estuary between smelt vs. ruffe, pikeperch vs. ruffe, and pikeperch vs. smelt. In the Elbe estuary, a shift in the diet composition of pikeperch, smelt, and ruffe was observed from 2021 to 2022 compared to food analyses from the 1990s. These shifts included an increased consumption of amphipods, while mysids and copepods had recently decreased in their diets. These changes indicate a restructuring of the food web, potentially linked to environmental changes, which highlights the sensitivity of estuarine ecosystems.

The only native cetacean in German waters, the harbor porpoise ( Phocoena phocoena ), is impacted by numerous pathological lesions in the respiratory tract mainly caused by parasites or bacteria. Although harbor porpoises have been observed to not use their complete lung volume, it has not been studied whether this insufficiency leads to lower oxygen uptake, impaired diving ability, and, ultimately, reduced foraging success. This project aims to analyze whether harbor porpoises developed novel molecular adaptations to compensate impairments in oxygen supply, thus remaining viable and competitive despite the high parasitic load. Here, initial comparative transcriptome RNA sequencing (NextSeq 2000, Illumina) was performed on muscles of harbor porpoises with a respiratory tract considered as healthy and of harbor porpoises that suffered from more severe lesions and parasitic infestations in the respiratory tract. Our findings suggest an elevated response to oxidative stress in the muscles of parasitic infested harbor porpoises compared with that of healthy animals. Higher antioxidant and antiapoptotic gene expression in the muscles of non-healthy harbor porpoises might function as a compensatory effect to enhanced reactive oxygen species production and accumulation in the muscles. Simultaneously enhanced selective proteasomal degradation and myogenesis suggest a tightly controlled, finely tuned switch of the intrinsic muscle response to stress. Lipid metabolism pathways and rate-limiting transcripts involved in glycolysis were upregulated and may uphold muscle energy supply for tissue function and energy-consuming regenerative and biosynthetic processes. These preliminary results hint at a defined response of the muscle to oxidative stress that may be caused by lung tissue with more severe pathological lesions and may indicate a possible adaptation in cetaceans.

The brain of diving mammals tolerates low oxygen conditions better than the brain of most terrestrial mammals. Previously, it has been demonstrated that the neurons in brain slices of the hooded seal (Cystophora cristata) withstand hypoxia longer than those of mouse, and also tolerate reduced glucose supply and high lactate concentrations. This tolerance appears to be accompanied by a shift in the oxidative energy metabolism to the astrocytes in the seal while in terrestrial mammals the aerobic energy production mainly takes place in neurons. Here, we used RNA-Seq to compare the effect of hypoxia and reoxygenation in vitro on brain slices from the visual cortex of hooded seals. We saw no general reduction of gene expression, suggesting that the response to hypoxia and reoxygenation is an actively regulated process. The treatments caused the preferential upregulation of genes related to inflammation, as found before e.g. in stroke studies using mammalian models. Gene ontology and KEGG pathway analyses showed a downregulation of genes involved in ion transport and other neuronal processes, indicative for a neuronal shutdown in response to a shortage of O2 supply. These differences may be interpreted in terms of an energy saving strategy in the seal's brain. We specifically analyzed the regulation of genes involved in energy metabolism. Hypoxia and reoxygenation caused a similar response, with upregulation of genes involved in glucose metabolism and downregulation of the components of the pyruvate dehydrogenase complex. We also observed upregulation of the monocarboxylate transporter Mct4, suggesting increased lactate efflux. Together, these data indicate that the seal brain responds to the hypoxic challenge by a relative increase in the anaerobic energy metabolism.

Hypoxia has gained ecological importance during the last decades, and it is the most dramatically increasing environmental factor in coastal areas and estuaries. The gills of fish are the prime target of hypoxia and other stresses. Here we have studied the impact of the exposure to hypoxia (1.5 mg O2/l for 48 h) on the protein expression of the gills of two estuarine fish species, the ruffe (Gymnocephalus cernua) and the European flounder (Platichthys flesus). First, we obtained the transcriptomes of mixed tissues (gills, heart and brain) from both species by Illumina next-generation sequencing. Then, the gill proteomes were investigated using two-dimensional gel electrophoresis and mass spectrometry. Quantification of the normalized proteome maps resulted in a total of 148 spots in the ruffe, of which 28 (18.8%) were significantly regulated (> 1.5-fold). In the flounder, 121 spots were found, of which 27 (22.3%) proteins were significantly regulated. The transcriptomes were used for the identification of these proteins, which was successful for 15 proteins of the ruffe and 14 of the flounder. The ruffe transcriptome dataset comprised 87,169,850 reads, resulting in an assembly of 72,108 contigs (N50 = 1,828 bp). 20,860 contigs (26.93%) had blastx hits with E < 1e-5 in the human sequences in the RefSeq database, representing 14,771 unique accession numbers. The flounder transcriptome with 78,943,030 reads assembled into 49,241 contigs (N50 = 2,106 bp). 20,127 contigs (40.87%) had a hit with human proteins, corresponding to 14,455 unique accession numbers. The regulation of selected genes was confirmed by quantitative real-time RT-PCR. Most of the regulated proteins that were identified by this approach function in the energy metabolism, while others are involved in the immune response, cell signalling and the cytoskeleton.

Animals living at high or temperate latitudes are challenged by extensive changes in environmental conditions over seasons. Djungarian hamsters ( ) are able to cope with extremely cold ambient temperatures and food scarcity in winter by expressing spontaneous daily torpor. Daily torpor is a circadian controlled voluntary reduction of metabolism that can reduce energy expenditure by up to 65% when used frequently. In the past decades it has become more and more apparent, that the hypothalamus is likely to play a key role in regulating induction and maintenance of daily torpor, but the molecular signals, which lead to the initiation of daily torpor, are still unknown. Here we present the first transcriptomic study of hypothalamic gene expression patterns in Djungarian hamsters during torpor entrance. Based on Illumina sequencing we were able to identify a total number of 284 differentially expressed genes, whereby 181 genes were up- and 103 genes down regulated during torpor entrance. The 20 most up regulated group contained eight genes coding for structure proteins, including five collagen genes, and , as well as the procoagulation factor . In a proximate approach we investigated these genes by quantitative real-time PCR (qPCR) analysis over the circadian cycle in torpid and normothermic animals at times of torpor entrance, mid torpor, arousal and post-torpor. These qPCR data confirmed up regulation of , and during torpor entrance, but a decreased mRNA level for all other investigated time points. This suggests that gene expression of structure genes as well as the procoagulation factor are specifically initiated during the early state of torpor and provides evidence for protective molecular adaptions in the hypothalamus of Djungarian hamsters including changes in structure, transport of biomolecules and coagulation.

Comparative genomic studies have led to the recent identification of several novel globin types in the Metazoa. They have revealed a surprising evolutionary diversity of functions beyond the familiar O2 supply roles of hemoglobin and myoglobin. Here we report the discovery of a hitherto unrecognized family of proteins with a unique modular architecture, possessing an N-terminal calpain-like domain, an internal, circular permuted globin domain, and an IQ calmodulin-binding motif. Putative orthologs are present in the genomes of many metazoan taxa, including vertebrates. The calpain-like region is homologous to the catalytic domain II of the large subunit of human calpain-7. The globin domain satisfies the criteria of a myoglobin-like fold but is rearranged and split into two parts. The recombinantly expressed human globin domain exhibits an absorption spectrum characteristic of hexacoordination of the heme iron atom. Molecular evolutionary analyses indicate that this chimeric globin family is phylogenetically ancient and originated in the common ancestor to animals and choanoflagellates. In humans and mice, the gene is predominantly expressed in testis tissue, and we propose the name “androglobin” (Adgb). Expression is associated with postmeiotic stages of spermatogenesis and is insensitive to experimental hypoxia. Evidence exists for increased gene expression in fertile compared with infertile males.

Background: During long dives, the brain of whales and seals experiences a reduced supply of oxygen (hypoxia). The brain neurons of the hooded seal (Cystophora cristata) are more tolerant towards low-oxygen conditions than those of mice, and also better survive other hypoxia-related stress conditions like a reduction in glucose supply and high concentrations of lactate. Little is known about the molecular mechanisms that support the hypoxia tolerance of the diving brain. Results: Here we employed RNA-seq to approach the molecular basis of the unusual stress tolerance of the seal brain. An Illumina-generated transcriptome of the visual cortex of the hooded seal was compared with that of the ferret (Mustela putorius furo), which served as a terrestrial relative. Gene ontology analyses showed a significant enrichment of transcripts related to translation and aerobic energy production in the ferret but not in the seal brain. Clusterin, an extracellular chaperone, is the most highly expressed gene in the seal brain and fourfold higher than in the ferret or any other mammalian brain transcriptome. The largest difference was found for S100B, a calcium-binding stress protein with pleiotropic function, which was 38-fold enriched in the seal brain. Notably, significant enrichment of S100B mRNA was also found in the transcriptomes of whale brains, but not in the brains of terrestrial mammals. Conclusion: Comparative transcriptomics indicates a lower aerobic capacity of the seal brain, which may be interpreted as a general energy saving strategy. Elevated expression of stress-related genes, such as clusterin and S100B, possibly contributes to the remarkable hypoxia tolerance of the brain of the hooded seal. Moreover, high levels of S100B that possibly protect the brain appear to be the result of the convergent adaptation of diving mammals.

Hemocyanin transports oxygen in the hemolymph of many arthropod species. Within the crustaceans, this copper-containing protein was thought to be restricted to Malacostraca, while other crustacean classes were assumed to employ hemoglobin or lack any respiratory protein. Only recently it has become evident that hemocyanins also occur in Remipedia and Ostracoda. Here we report for the first time the identification and characterisation of hemocyanin in the fish louse Argulus , which belongs to the class of Branchiura. This finding indicates that hemocyanin was the principal oxygen carrier in the stem lineage of the pancrustaceans, but has been lost independently multiple times in crustacean taxa. We obtained the full-length cDNA sequences of two hemocyanin subunits of Argulus foliaceus by a combination of RT-PCR, RACE and Illumina sequencing of the transcriptome. In addition, one full-length and one partial cDNA sequence were derived from the transcriptome data of Argulus siamensis. Western blot analysis confirmed the presence of at least two hemocyanin subunits in A. foliaceus , which are expressed at the mRNA level at a 1:3.5 ratio. The addition to the branchiuran hemocyanin subunits to a multiple sequence alignment of arthropod, hemocyanins improved the phylogenetic resolution within the pancrustacean hemocyanins. Malacostracan, ostracod and branchiuran hemocyanins are distinct from the hexapod and remipede hemocyanins, reinforcing the hypothesis of a close relationship of Remipedia and Hexapoda. Notably, the ostracod hemocyanins are paraphyletic with respect to the branchiuran hemocyanins, indicating ancient divergence and differential loss of distinct subunit types.