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21 pages, 10 figures.-- This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License, Toll-like receptors (TLRs) are the most widespread class of membrane-bound innate immune receptors, responsible of specific pathogen recognition and production of immune effectors through the activation of intracellular signaling cascades. The repertoire of TLRs was analyzed in 85 metazoans, enriched on molluscan species, an underrepresented phylum in previous studies. Following an ancient evolutionary origin, suggested by the presence of TLR genes in Anthozoa (Cnidaria), these receptors underwent multiple independent gene family expansions, the most significant of which occurred in bivalve molluscs. Marine mussels (Mytilus spp.) had the largest TLR repertoire in the animal kingdom, with evidence of several lineage-specific expanded TLR subfamilies with different degrees of orthology conservation within bivalves. Phylogenetic analyses revealed that bivalve TLR repertoires were more diversified than their counterparts in deuterostomes or ecdysozoans. The complex evolutionary history of TLRs, characterized by lineage-specific expansions and losses, along with episodic positive selection acting on the extracellular recognition domains, suggests that functional diversification might be a leading evolutionary force. We analyzed a comprehensive transcriptomic data set from Mytilus galloprovincialis and built transcriptomic correlation clusters with the TLRs expressed in gills and in hemocytes. The implication of specific TLRs in different immune pathways was evidenced, as well as their specific modulation in response to different biotic and abiotic stimuli. We propose that, in a similar fashion to the remarkable functional specialization of vertebrate TLRs, the expansion of the TLR gene family in bivalves attends to a functional specification motivated by the biological particularities of these organisms and their living environment, This research was funded by Ministerio de Ciencia e Innovación (PID2021-124955OB-I00) and by Agencia Galega de Innovación (IN607B 2022/13). A.S. was supported by a Spanish AEI/EU-FSE predoctoral contract PRE2019-090760

In this thesis RNA-sequencing tools are applied on some representatives of the antarctic trophic chain exposed to a non lethal temperature increase, as to understand and model their response to the increase in temperature expected to happen in this decade, in order to extend eventual functional conclusions to other temperate stenotherm organisms such as those living at extreme depths. RNA-sequencing consists in a set of powerful and sensible tools that allow qualitative and quantitative measurements in gene expression, pairing molecular analyses with state-of-the-art statistics and computer science. The aforementioned sensitivity, in synergy with a proper experimental design, allowed studying not only the response to a temperature increase, but also to the experimental condition itself (i.e. stabling of wild animals in tanks). The notothenioid fish Trematomus bernacchii (from which muscle, gills and brain were sampled), the scallop Adamussium colbecki (from which gills, mantle and digestive gland were sampled), the amphipod Pseudorchomene plebs and the tardigrade Acutuncus antarcticus are the models of choice of this work. In particular T. bernacchii was the most responsive of the bunch, specially in the brain tissues, where the temperature (+1.5°C increase) induced upregulation of genes involved in immune response and inflammatory state after 7 days of exposure. After 20 days of heat exposure many pathways related to synapse function and structure were altered, suggesting an on-going profound alterations in the tissue, which may also be the result of the combined effect of heat and the impossibility to avoid it in captivity. Even more profound effects were observed in the response to time in experimental tanks, as an important shift in gene expression was observed in the brain, suggesting profound changes in the neural pathways, including those involved in specific stress perception. The scallop A. colbecki did not show significant change in gene expression patterns in response to heat, but a small change was observed in the digestive gland after 20 days of experimental captivity, showing alterations in genes whose functions are mainly involved in feeding ability. Samples from the amphipod P. plebs were challenging to prepare and to analyze, as many were lost due to unfreezing during transport. Moreover the presence of a bycatch of Eusirus cf. giganteus juvenile and an high degree of parasitism from a dinoflagellate of the genus Hematodinium in all samples made it impossible to study the response of the amphipod to a warmer environment. Nevertheless, the transcriptomic data allowed the identification of an high expression of hemocyanin in E. giganteus suggesting a possible cold adaptation trait, the classification of our samples as a Orchomenid cryptic species, and the first identification of active Hematodinium parasitism in the antarctic ocean. Finally the tardigrade A. antarcticus displayed a striking sample variability, that may underlie an never described genomic variability and/or an high level of heterozygosity. This fact reduced the number of samples in the differential expression analysis, which nevertheless showed a response to short term exposure to heat with a pattern proportional to temperature in terms of fold changes of gene expression levels. The differentially expressed genes suggested that mitochondrial oxidative metabolism increased and hinted the activation of pathways related to resistance forms typical of the Tardigrada phylum. Such a response was not observed in a long term exposure to the higher temperatures, suggesting that the changes observed in short term are compensatory. ​

Many stenotherm marine species live in the cold and stable environment of the Antarctic Ocean which could be impacted by climate change in the upcoming years. To investigate this issue, gene expression analysis was carried out on Trematomus bernacchii specimens caught near Mario Zucchelli Station. Brain, gill and muscle tissues were sampled from naïve animals and from those kept in control (- 0.9°C) and experimental (+0.6°C) tanks for six hours, seven and twenty days, post acclimation. RNAseq data showed that the temperature increase mostly affected the brain, with a time dependent response. Immune response was up-regulated at mid-term; protein turnover and energy management were down-regulated and neuronal remodelling related genes were up-regulated at 20 days, where the highest number of differentially expressed genes was observed. Gill tissue showed a mild response after 20 days, mostly involving DNA replication. A great early-starting response to stabling was also observed across the entire experiment in brain and gills, with the latter being the most affected. The expression pattern clustering analysis showed that many synapse-related genes were down-regulated and energy related genes were up-regulated, while several binding processes were up-regulated at the latest time point in brain. The response in gills was milder and mostly involved cytoskeleton and glycolysis. No significant change was observed in muscle. These results identify the brain as the tissue most affected by both heat and stabling, evidencing its great sensitivity to the slightest environmental change, and highlight the importance of a careful experimental design when working with captive wild organisms.

Lysozyme is one of the most important anti-bacterial effectors in the innate immune system of animals. Besides its direct antibacterial enzymatic activity, lysozyme displays other biological properties, pointing toward a significant anti-inflammatory effect, many aspects of which are still elusive. Here we investigate the perturbation of gene expression profiles induced by lysozyme in a monocyte cell line in vitro considering a perspective as broad as the whole transcriptome profiling. The results of the RNA-seq experiment show that lysozyme induces transcriptional modulation of the TNF-alpha /IL-1 beta pathway genes in U937 monocytes. The analysis of transcriptomic profiles with IPA((R)) identified a simple but robust molecular network of genes, in which the regulation trends are fully consistent with the anti-inflammatory activity of lysozyme. This study provides the first evidence in support of the anti-inflammatory action of lysozyme on the basis of transcriptomic regulation data resulting from the broad perspective of a whole-transcriptome profiling. Such important effects can be achieved with the supplementation of relatively low concentrations of lysozyme, for a short time of exposure. These new insights allow the potential of lysozyme in pharmacological applications to be better exploited.

Additional file 3. Review history.

Additional file 1. Manuscript data notes. This file includes additional data notes 1–24, along with all the supplementary figures and most supplementary tables referenced in the main text.

Additional file 1. Manuscript data notes. This file includes additional data notes 1–24, along with all the supplementary figures and most supplementary tables referenced in the main text.

Additional file 3. Review history.

Although the major SARS-CoV-2 omicron lineages share over 30 non-synonymous substitutions in the spike glycoprotein, they show several unique mutations that were acquired after their ancestral split. One of the most intriguing mutations associated with BA.1 is the presence of the inserted tripeptide Glu-Pro-Glu within the N-terminal domain, at a site that had previously independently acquired short insertions in several other SARS-CoV-2 lineages. Although the functional implications of the small nucleotide sequences found at this insertion hotspot, named RIR1, are still unclear, we have previously hypothesized that they may play a compensatory role in counterbalancing minor fitness deficits associated with other co-occurring spike non-synonymous mutations. Here, we show that similar insertion events have independently occurred at RIR1 at least 20 times in early 2022 within the BA.2 lineage, being occasionally associated with significant community transmission. One of these omicron sublineages, characterized by a Ser-Gly-Arg insertion in position 212, has been responsible for over 4000 documented COVID-19 cases worldwide between January and July 2022, for the most part concentrated in Denmark, where it reached a national prevalence close to 4% (10% in the Nordjylland region) in mid-May. Although the concurrent spread of the BA.2.12.1, BA.4 and BA.5 lineages led to the rapid decline of this BA.2 sublineage, the independent acquisition of several other RIR1 insertions on a BA.2 genomic background suggests that these events may provide a slight fitness advantage. Therefore, they should be carefully monitored in the upcoming months in other emerging omicron-related lineages, including BA.5.

The Antarctic continent is one of the most pristine environments on planet Earth, yet one of the most fragile and susceptible to the effects of the ongoing climate change. The overwhelming majority of the components of Antarctic marine trophic chain are stenotherm organisms, highly adapted to the extreme, but extremely stable, freezing temperatures of the Antarctic ocean, which have not changed significantly during the past fifteen million years. Notothenioid fishes are the most abundant representatives of ichthyofauna at these latitudes, being ubiquitously found in coastal areas across the entire continent. While different Antarctic fish species have been previously subjected to studies aimed at defining their range of thermal tolerance, or at studying the response to acute thermal stress, just a handful of authors have investigated the effects of the exposure to a moderate increase of temperature, falling within the expected forecasts for the next few decades in some areas of the Antarctica. Here, the emerald rockcod Trematomus bernacchii was used as a model species to investigate the effects of a 20-day long exposure to a +1.5 °C increase in the brain, gills and skeletal muscle, using a RNA-sequencing approach. In parallel, the experimental design also allowed for assessing the impact of stabling (including acclimation, the handling of fishes and their confinement in tanks during the experimental phase) on gene expression profiling. The results of this study clearly identified the brain as the most susceptible tissue to heat stress, with evidence of a time-dependent response dominated by an alteration of immune response, protein synthesis and folding, and energy metabolism-related genes. While the gills displayed smaller but still significant alterations, the skeletal muscle was completely unaffected by the experimental conditions. The stabling conditions also had an important impact on gene expression profiles in the brain, suggesting the presence of significant alterations of the fish nervous system, possibly due to the confinement to tanks with limited water volume and of the restricted possibility of movement. Besides providing novel insights in the molecular mechanisms underlying thermal stress in notothenioids, these findings suggest that more attention should be dedicated to an improved design of the experiments carried out on Antarctic organism, due to their extreme susceptibility to the slightest environmental alterations.

As complete genomes become easier to attain, even from previously difficult-to-sequence species, and as genomic resequencing becomes more routine, it is becoming obvious that genomic structural variation is more widespread than originally thought and plays an important role in maintaining genetic variation in populations. Structural variants (SVs) and associated gene presence-absence variation (PAV) can be important players in local adaptation, allowing the maintenance of genetic variation and taking part in other evolutionarily relevant phenomena. While recent studies have highlighted the importance of structural variation in Mollusca, the prevalence of this phenomenon in the broader context of marine organisms remains to be fully investigated.Here, we describe a straightforward and broadly applicable method for the identification of SVs in fully assembled diploid genomes, leveraging the same reads used for assembly. We also explain a gene PAV analysis protocol, which could be broadly applied to any species with a fully sequenced reference genome available. Although the strength of these approaches have been tested and proven in marine invertebrates, which tend to have high levels of heterozygosity, possibly due to their lifestyle traits, they are also applicable to other species across the tree of life, providing a ready means to begin investigations into this potentially widespread phenomena.

Caudata is an order of amphibians with great variation in genome size, which can reach enormous dimensions in salamanders. In this work, we analysed the activity of transposable elements (TEs) in the transcriptomes obtained from female and male gonads of the Chinese fire-bellied newt, Cynops orientalis, a species with a genome about 12-fold larger than the human genome. We also compared these data with genomes of two basal sarcopterygians, coelacanth and lungfish. In the newt our findings highlighted a major impact of non-LTR retroelements and a greater total TE activity compared to the lungfish Protopterus annectens, an organism also characterized by a giant genome. This difference in TE activity might be due to the presence of young copies in newt in agreement also with the increase in the genome size, an event that occurred independently and later than lungfish. Moreover, the activity of 33 target genes encoding proteins involved in the TE host silencing mechanisms, such as Ago/Piwi and NuRD complex, was evaluated and compared between the three species analysed. These data revealed high transcriptional levels of the target genes in both newt and lungfish and confirmed the activity of NuRD complex genes in adults. Finally, phylogenetic analyses performed on PRDM9 and TRIM28 allowed increasing knowledge about the evolution of these two key genes of the NuRD complex silencing mechanism in vertebrates. Our results confirmed that the gigantism of the newt genomes may be attributed to the activity and accumulation of TEs.

The striped venus Chamelea gallina is a bivalve mollusc that represents one of the most important fishery resources of the Adriatic Sea. In this work, we investigated for the first time the ability of this species to modulate the expression of genes encoding proteins involved in biomineralization process in response to biotic and abiotic factors. We provided the first comprehensive transcriptome from the mantle tissue of clams collected in two sampling sites located along the Italian Adriatic coast and characterized by different environmental features. Moreover, the assessment of environmental parameters, scanning electron microscopy (SEM), and X-ray diffraction (XRD) measurements on valves were conducted to better contextualize RNA sequencing (RNA-Seq) data. Functional annotation of differentially expressed genes (DEGs) and SEM observations highlighted a different shell mineralization behaviour in C. gallina clams collected from two selected sites characterized by diverse environmental parameters.

Fish are an interesting taxon comprising species adapted to a wide range of environments. In this work, we analyzed the transcriptional contribution of transposable elements (TEs) in the gill transcriptomes of three fish species exposed to different salinity conditions. We considered the giant marbled eel Anguilla marmorata and the chum salmon Oncorhynchus keta, both diadromous, and the marine medaka Oryzias melastigma, an euryhaline organism sensu stricto. Our analyses revealed an interesting activity of TEs in the case of juvenile eels, commonly adapted to salty water, when exposed to brackish and freshwater conditions. Moreover, the expression assessment of genes involved in TE silencing mechanisms (six in heterochromatin formation, fourteen known to be part of the nucleosome remodeling deacetylase (NuRD) complex, and four of the Argonaute subfamily) unveiled that they are active. Finally, our results evidenced for the first time a krüppel-associated box (KRAB)-like domain specific to actinopterygians that, together with TRIM33, might allow the functioning of NuRD complex also in fish species. The possible interaction between these two proteins was supported by structural prediction analyses.

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.

Cryonotothenioidea is the main group of fishes that thrive in the extremely cold Antarctic environment, thanks to the acquisition of peculiar morphological, physiological and molecular adaptations. We have previously disclosed that IgM, the main immunoglobulin isotype in teleosts, display typical cold-adapted features. Recently, we have analyzed the gene encoding the heavy chain constant region (CH) of the IgT isotype from the Antarctic teleost Trematomus bernacchii (family Nototheniidae), characterized by the near-complete deletion of the CH2 domain. Here, we aimed to track the loss of the CH2 domain along notothenioid phylogeny and to identify its ancestral origins. To this end, we obtained the IgT gene sequences from several species belonging to the Antarctic families Nototheniidae, Bathydraconidae and Artedidraconidae. All species display a CH2 remnant of variable size, encoded by a short Cτ2 exon, which retains functional splicing sites and therefore is included in the mature transcript. We also considered representative species from the three non-Antarctic families: Eleginopsioidea (Eleginops maclovinus), Pseudaphritioidea (Pseudaphritis urvillii) and Bovichtidae (Bovichtus diacanthus and Cottoperca gobio). Even though only E. maclovinus, the sister taxa of Cryonotothenioidea, shared the partial loss of Cτ2, the other non-Antarctic notothenioid species displayed early molecular signatures of this event. These results shed light on the evolutionary path that underlies the origins of this remarkable gene structural modification.

The CS-&alpha, &beta, architecture is a structural scaffold shared by a high number of small, cationic, cysteine-rich defense peptides, found in nearly all the major branches of the tree of life. Although several CS-&alpha, peptides involved in innate immune response have been described so far in bivalve mollusks, a clear-cut definition of their molecular diversity is still lacking, leaving the evolutionary relationship among defensins, mytilins, myticins and other structurally similar antimicrobial peptides still unclear. In this study, we performed a comprehensive bioinformatic screening of the genomes and transcriptomes available for marine mussels (Mytilida), redefining the distribution of mytilin-like CS-&alpha, peptides, which in spite of limited primary sequence similarity maintain in all cases a well-conserved backbone, stabilized by four disulfide bonds. Variations in the size of the alpha-helix and the two antiparallel beta strand region, as well as the positioning of the cysteine residues involved in the formation of the C1&ndash, C5 disulfide bond might allow a certain degree of structural flexibility, whose functional implications remain to be investigated. The identification of mytilins in Trichomya and Perna spp. revealed that many additional CS-&alpha, AMPs remain to be formally described and functionally characterized in Mytilidae, and suggest that a more robust scheme should be used for the future classification of such peptides with respect with their evolutionary origin.