ABSTRACT. Aim Organisms detect environmental stimuli using an array of receptors, and the duplication and divergence of these receptors provide evolutionary opportunities for species expansion into new ecological niches. Pteriomorphian bivalves have a myriad of light sensing organs, including eyes, that have evolved multiple times across, and within, lineages. Recent work indicates that eyes may be the result of co-option: sharing and/or diversification of preexisting genes that were once used in other light-sensing tissues. Thus, understanding the origin of eyes can provide insight into how complex phenotypes can arise through redeployment and elaboration of genetic pathways.

Methods To examine the origin of eye types in pteriomorphian bivalves, we examined gene family expansion of opsin, the protein component of photopigments. We compared genomic content across Mollusca and spatiotemporal gene expression across developmental stages of bivalve species. Using a heterologous gene expression system, we are testing light-sensitivity of these opsin proteins.

Results After an analysis of 80 molluscan genome assemblies, we found extensive fluctuations in opsin number which appear to be limited to bivalves and gastropods. Surprisingly, eyeless bivalves have the highest numbers of opsins, indicating that neither the presence nor the complexity of eyes are tied to an increase in gene copy number. When comparing eyed lineages, we found that opsin content and expression patterns vary among eye types.

Main conclusion At the genetic level, eyed lineages do not follow the same evolutionary trajectory to achieve photosensitivity. Furthermore, the extensive diversification of opsins in eyeless species may indicate some opsins are multisensory receptors with light-independent functions. This hypothesis will require functional assays of the protein and allow for direct testing of adaptation.

ABSTRACT. Aim The competition between sexes to gain control over the fertilization process represents a crucial challenge for all organisms, but particularly for external fertilizers. In this context, marine organisms showcase a variety of male and female adaptations aimed at securing this control. Growing evidence shows that the non-gametic components released by both males and females can play a major role in influencing the outcomes of fertilization. Seminal fluid was shown to influence paternity success by affecting female mating behaviour and rival males’ sperm performance. In turn, female reproductive fluid can differentially influence ejaculates of different males and bias fertilization towards specific partners. However, no one so far has ever considered the consequences of these fluids intermingling for fertilization and sexual selection. In the study I will present we explored this scenario using the grass goby (Zosterisessor ophiocephalus), an externally fertilizing fish of the Venice lagoon with territorial-sneaker reproductive tactics, where sneaker males can exploit the territorials’ seminal fluid while penalizing territorial sperm performance with their own fluid.

Methods To test whether female reproductive fluid can rebalance the ejaculate competition in favour of territorial males (the preferred phenotype at the pre-mating level), we used in vitro fertilization with a seminal fluid mixture (territorial + sneaker), and increasing concentrations of female reproductive fluid, to simulate the natural conditions that sperm encounter when swimming towards the eggs.

Results Our findings revealed a female-driven directional process favouring only territorial males in presence of high concentrations of female reproductive fluid, seemingly mediated by the interaction with the seminal fluids of the different male competitors.

Main conclusion Through this process, females of the grass goby can discriminate among ejaculates of the different male phenotypes and influence the sperm competition outcome, ultimately reinforcing their pre-mating preference for territorial males and regaining control over the fertilization environment.

ABSTRACT. Aim Transposable elements constitute one of the main components of eukaryotic genomes. Due to their transposition ability, TEs can also be responsible for genome instability, and thus silencing mechanisms were evolved. However, TEs are not merely silenced rather, they can be domesticated for the regulation of host-coding gene expression, permitting species adaptation and resilience. Therefore, the aim of this work was to investigate the impact of TEs in genome composition and how their activity and related silencing mechanisms respond to variation of abiotic factors in teleosts. Ray-finned fish are adapted to different ecological niches and are responsive to environmental changes. Methods Bioinformatic analyses were performed to investigate the transcriptional activity of TEs and genes involved in silencing mechanisms. Results We investigated the transcriptional contribution of TEs in the gill transcriptomes of three fish species exposed to different salinity conditions. We considered Anguilla marmorata and Oncorhynchus keta, both diadromous, and 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, an activity of TEs was recorded also in the liver of the stenothermal fish Puntius tetrazona, after exposure at 13 °C indicating this tissue as target organ for this kind of stress. Our results evidenced for the first time the activity of NuRD complex, a TE silencing mechanism typical of sarcopterygians, also in actinopterygians. For its functioning, we proposed an interaction between a krüppel‐associated box (KRAB)‐like protein specific to actinopterygians and TRIM33 tested by structural prediction and CoIP analyses. Main conclusion Therefore, our results highlight the importance of exploiting silencing mechanisms for TE control to rewire gene expression networks allowing species adaptation and resilience, challenging the general view of TEs as threatening elements.

ABSTRACT. Adaptation to local conditions is known to occur in seagrasses, however, knowledge of the genetic basis underlying this phenomenon remains scarce. Here, we analyzed Posidonia oceanica from six sites within and around the Stagnone di Marsala, a semi-enclosed coastal lagoon where salinity and temperature exceed the generally described tolerance thresholds of the species. Sea surface temperatures (SSTs) were measured and plant samples were collected for the assessment of morphology, flowering rate and for screening genome-wide polymorphisms using double digest restriction-site-associated DNA sequencing. Results demonstrated more extreme SSTs and salinity levels inside the lagoon than the outer lagoon regions. Morphological results showed significantly fewer and shorter leaves and reduced rhizome growth of P. oceanica from the inner lagoon and past flowering events were recorded only for a meadow farthest away from the lagoon. Using an array of 51,329 SNPs, we revealed a clear genetic structure among the study sites and confirmed the genetic isolation and high clonality of the innermost site. Fourteen outlier loci were identified and annotated with several proteins including those relate to plant stress response, protein transport and regulators of plant-specific developmental events. Especially, five outlier loci showed maximum allele frequency at the innermost site, likely reflecting adaptation to the extreme temperature and salinity regimes, possibly due to the selection of more resistant genotypes and the progressive restriction of gene flow. Overall, this study helps us to disentangle the genetic basis of seagrass adaptation to local environmental conditions and may support future works on assisted evolution in seagrasses.

ABSTRACT. Aim Evidences of mitochondrially driven RNA interference (RNAi) are recently emerging. Recently, we suggested a new class of small non coding RNAs (sncRNAs) of mitochondrial origin called smithRNAs (Small MITochondrial Highly-transcribed RNAs) in the Manila clam, Ruditapes philippinarum, a bivalve species with Doubly Uniparental Inheritance (DUI) of mitochondrial DNA. Among other possible functions, those smithRNAs were predicted to regulate nuclear gene expression in R. philippinarum gonad formation, a possibility which has never been suggested before. However, all evidences for smithRNAs were in silico predictions, up to now.

Methods We experimentally tested for significant modifications in methylation/acetylation levels in specimens treated with smithRNAs predicted to target nuclear enzymes connected with such regulatory functions.

Results SmithRNAs are conserved in the Manila clam and we present significant clues of their functionality in vivo. Moreover, it is conceivable that smithRNAs will be found in other eukaryotes as well, maybe linked to functions other than gonad development: in fact, we present evidences that putative smithRNA genes are present in other metazoansʼ mtDNAs.

Main conclusion SmithRNAs are good candidates to evolve new functions in the compact mtDNA, because hairpin structures in mitochondrial intergenic regions (needed for correct RNA cleavage) could easily be exapted to evolve RNAi. SmithRNAs make mtDNA a much more complex genome than previously thought, and they represent a new mito-nuclear coevolutionary arena. Actually, the possibility that mtDNA may act as a reservoir of RNAi opens a plethora of new ways for it to interact with the nucleus and significantly raises the level of complexity of mito-nuclear coevolution: for instance, smithRNAs may play a role in reproductive isolation between closely related species.

ABSTRACT. The increase of CO2 into the atmosphere, as a result of human activities, is mitigated by ocean’s absorption processes, which lead to a gradual decrease of ocean pH. This phenomenon, known as Ocean Acidification (OA), has a significant impact on the biology of various marine species, particularly those that build calcium-based structures. However, some species seem more resistant than others in copying with acidic conditions, showing a high adaptation potential. In this context, it is fundamental to understand what are the biological mechanisms determining this resistance. This study focused on exploring how chronic and acute exposure to acidic conditions affects the physiology and behavior of the sea urchin Arbacia lixula, studying populations which resides near the natural CO2 vents in Ischia, Italy.Specimens of adult sea urchins were sampled from two distinct populations along the pH gradient generated by the vents: S1 (mean pH 8.1; representing the ambient area) and S2 (mean pH 7.7; representing acidified area). The experimental setup involved exposures of animals from both S1 and S2 populations to both tested pH, using seawater collected on-site. Physiological aspects such as respiration and excretion, along with behavioral traits including righting and sheltering time, were assessed. All trials were replicated four times during the year.The findings reveal a persistent impact of the area on the respiration rate of mature sea urchins, specifically observed during the second and third sampling periods. The excretion rate was influenced by pH conditions only in October. Both pH and area factors influenced behavioral aspects across all temporal replicates, with a significant effect of their interaction on righting time. The variations in responses observed at different sampling times suggest the potential involvement of additional factors, beyond acidification, in the modification of the species' physiology and behavior. Nevertheless, our results offer initial insights into the direct effects of acidification on A. lixula within their natural habitat.

ABSTRACT. Venomous animals, such as snakes, spiders, or scorpions, represent a fascinating example of diversity in both taxonomy and function, found across at least 101 independent lineages. Despite their varied origins, these animals have evolved specialized structures for producing, storing, and delivering venom—a complex mixture of toxins primarily composed of proteins and peptides. These toxins serve diverse functions acting synergistically to incapacitate their target organisms. One group of diverse predatory marine organisms, the Neogastropoda, displays an impressive range of feeding strategies, including envenomation, asphyxia, and vampirism. However, little is known about the link between the diversity of these compounds and the hyperdiversification of neogastropod species, and how genome evolution is related to both the compounds and species diversities. Only eight neogastropod genomes have been sequenced, and there is uneven quality assembly among the 45 gastropod genomes sequenced so far. To address this gap, we have generated high-quality chromosome-level assemblies of two species: Monoplex corrugatus (tonnoidean) and Stramonita haemastoma (neogastropod). These genomes provide valuable references for their respective taxa and facilitate the identification of genome-level processes that contribute to the evolutionary success of predatory neogastropods. Through the analysis of syntenic blocks, Hox gene cluster duplication and synonymous substitutions distribution pattern, we inferred the occurrence of a whole genome duplication event in both genomes. As these species are known to release venom, toxins were annotated, but few of them were found in homologous chromosomes. Using transcriptomes data from two tissues in S. haemastoma, a comparison of the expression of ohnolog genes (orthologs found in homologous chromosomes in each species), where both copies were differentially expressed, showed that most of them had similar expression profiles. By conducting this study, we shed light on the relationship between genome evolution, the diversity of venom compounds, and the hyperdiversification of neogastropod species.

ABSTRACT. Aim In the Era of Climate change, organisms are experiencing intense and rapid environmental changes worldwide. Coastal marine ecosystems, such as seagrass meadows, are among the most important in the world for providing human services and benefits. However, the ongoing rapid environmental changes are increasing seagrass loss rates, placing them among the most threatened ecosystems on Earth. Seagrasses can reproduce sexually (with flowers and seeds), similarly to their terrestrial relatives, or clonally, producing new identical vegetative individuals. Exploring stress memory in seagrasses through assisted evolution approaches, such as the priming treatment, and integrating multi-level assessments, could revolutionize the restoration plans implemented to date for seagrasses by combining the conservation of these important marine ecosystems with new assisted evolution technologies.

Methods The thermo-priming treatment was the approach used to assess stress memory in seagrasses. This approach was performed experimentally using indoor aquaria (mesocosms), where plants were treated with first heat stress to induce the priming stimulus. Seagrass performance was analyzed using a multi-level assessment that embraces photo-physiology, transcriptomics, and epigenetics. Global DNA methylation and differentially methylated cytosines were explored for the first time in seagrasses using the reduced representation bisulfite sequencing (RRBS) approach.

Results The approach was useful to primed P. oceanica seedlings as they respond better to the re-occurrence heat stress event. The acquisition of the priming status was accompanied by the largest transcriptomic regulation with respect to not-primed seedlings. DNA methylation results and their integration of transcriptomics could outline key genes involved in the regulation of stress memory.

Main conclusion Exploring stress memory in seagrasses by using assisted evolution approaches can be advantageous on the one hand to set up specific experimental conditions for enhancing stress tolerance. Integrative responses obtained by merging the NGS with phenotypic responses are essential to investigate specific stress-memory-related genes, including epigenetic regulators.

Thematic Areas (choose two among Macroevolution, Populations and Species, Adaptation, Biodiversity, Conservation)  Adaptation, Conservation

ABSTRACT. Aim Calcified algae are less common or absent in intertidal regions because they are particularly vulnerable to desiccation. Among corallinaceae, Ellisolandia elongata (J. Ellis & Solander) inhabits intertidal rock pools where large fluctuations in physic-chemical variables occur daily and represent a suitable model to study the capability of this species to cope with environmental constraints. To pursue this aim, individuals of E. elongata were sampled in the Marine Protected Area of the Gaiola Underwater Park (Gulf of Naples, Italy) during a prolonged low tide event occurring in early February to assess the photosynthetic efficiency and the occurrence of stress-defense ecological strategies in both submerged and exposed thalli.

Methods Photosynthetic-light curves (LRC) were performed in vivo in exposed semi-bleached and submerged thalli utilizing a pulse amplitude-modulated fluorometer (Junior-PAM, Walz Gmbh, Effeltrich, Germany). On samples, the expression of the D1 protein of photosystem II, photosynthetic pigments, and antioxidant activity were also evaluated to study the adaptive response of E. elongata photosynthetic apparatus.

Results At over-saturating irradiances, the prolonged low tide event determined, surprisingly, an increase of photosynthetic efficiency and a decline of non-photochemical thermal dissipation processes in exposed compared to submerged thalli together with a reduction of maximum photochemical efficiency, D1 protein expression, photosynthetic pigment content, and antioxidant activity.

Main conclusion The prolonged exposure of submerged thalli to multiple stresses (i.e., high irradiance and desiccation) may have triggered a regulation strategy by which photosystem II is down-regulated to avoid photoinhibition of photosynthetic apparatus. The decreased antioxidant activity confirmed scavenger molecules' crucial role in lowering oxidative stress at the cellular level, contributing to photoprotection in E. elongata.

ABSTRACT. Aim The regenerative capacity of Scyphozoans (Phylum Cnidaria) has been relatively understudied. The model organism Cassiopea xamachana hosts photosynthetic dinoflagellate symbionts in the host’s motile amoebocyte cells. A handful of studies have reported regeneration in the polyps of C. xamachana, but the mechanisms underlying regeneration have not been fully explored. Despite undergoing drastic developmental changes when symbiotic, the effect of symbiont presence and species on host regeneration has never been explored. Methods To explore the influence of photosymbionts, C. xamachana polyps were decapitated when aposymbiotic, and symbiotic with both a homologous and a heterologous symbiont species. Observations were carried out with a fluorescence stereo microscope. We counted daily 1) fully detached buds for each polyp, 2) survival and 3) regeneration of the cut stalks and heads. Confocal microscopy was also carried out for counting symbiont numbers. EdU labeling was performed to observe patterns of cell proliferation in regenerating polyps. Results Decapitated polyp stalks regenerated a head, but heads did not regenerate the stalk; instead, they produced planuloid buds. In most cases, first signs of regeneration appeared at Day 4 and were completed by Day 14. Both symbiont species and symbiotic state of the host contributed to significant differences in bud count between treatments. No difference in number or location of EdU labeled, cells was observed between algal symbiont or time point. Main conclusion The presence of symbionts increased likelihood to regenerate, yet symbiont species did not affect success of regeneration. No blastema or dividing cells were observed, implying cell proliferation is not the primary mechanism behind regeneration in polyps of C. xamachana.

ABSTRACT. Aim The Antarctic krill Euphausia superba is a pelagic organism with a central role in the Southern Ocean ecosystem. During the ages, this crustacean developed daily and seasonal rhythms in its physiology and behaviour which are regulated by an endogenous molecular clock. The main components of the krill’s circadian clock have been characterized, revealing an ancestral clock sharing both mammalian and insect features. Recently, the rhythmic expression of krill’s orthologue of D. melanogaster transcription factor VRI has been reported, suggesting the possible conservation of a previously characterized modulation loop consisting of two transcriptional regulators, VRI and PDP1. In Drosophila, these factors are known to modulate CLOCK expression by binding to a V/P-box located in its gene promoter.

Methods Using D. melanogaster homologues as inputs, the coding sequences of three different isoforms of pdp1 were identified in the krill’s transcriptome database KrillDB2. These sequences were cloned into specific vectors for the expression in Drosophila cells. Finally, a luciferase assay was performed to investigate the interaction of VRI and PDP1 with the V/P-box of Drosophila clock gene promoter. In addition, vrille and pdp1 isoforms expression levels were assessed in five different krill tissues through qRT-PCR.

Results The luciferase assay revealed the opposite action of these two factors, with VRI as inhibitor and PDP1 as activator of clock transcription. This result was achieved with all the tested isoforms with different degrees of action. At the same time, we characterized the expression patterns of vrille and pdp1 in different krill’s tissues in order to test the different isoforms tissue specificity.

Main conclusion We discovered and characterized a new component of the Antarctic krill clock, a feedback loop composed by the transcriptional factors PDP1 and VRI, regulating CLOCK expression. This study will pave the way to complete the functional characterization of krill’s clock machinery.

ABSTRACT. Aim Sexual selection is a powerful evolutionary agent responsible for the extraordinary variability in reproductive phenotypes ranging from fascinating and unusual displays to cryptic traits whose function is only uncovered by careful study. Pre- and post-mating episodes of sexual selection combine and interact to determine overall male reproductive fitness, and emerging evidence has been emphasising the role of sexual selection in influencing the capacity of a population to withstand environmental change, such as temperature variation. However, we still know very little about how thermal ecology influences sexual selection and, in particular, patterns of interaction between pre- and post-mating sexual selection episodes. Our study aims to fill this gap, focusing on one of the most abundant fish species in the rocky littoral environment of the Mediterranean Sea, the damselfish Chromis chromis.

Methods We explored the influence of temperature variation across the reproductive season on: nest density and competition for mating (through scuba visual census and behavioural observations); expression of male traits that are important in pre- and post-mating episodes of sexual selection (through morphological measures and physiological analyses on the ejaculate); the resulting pre- and postmating reproductive success (through molecular paternity analyses).

Results Here we show our preliminary results on the relation between temperature variation across season and nest density at different depths, and how this influences the intensity of male-male competition and the expression and relative importance of pre- and post-copulatory sexual traits in determine overall male reproductive fitness.

Main conclusion Our study contributes to extend the comprehension of evolutionary interaction between thermal ecology and sexual selection under changing environmental conditions and to better predict potential adaptation to future scenarios.

ABSTRACT. Abstract Aim: Microalgae are photosynthetic microorganisms adapted to live in very different environments and exhibit enormous biochemical and genetic diversity. Their long evolutionary and adaptive diversification to a multitude of habitats and extreme conditions makes them good candidates for the discovery of new molecules with potential human applications. Recently, many studies are focusing on the investigation of the enzymatic pathways involved in the biosynthesis of marine compounds of market interest in order to clarify their synthesis and suggest key genes for gene editing approaches, heterologous and homologous expression. Methods: We used an RNA-sequencing approach to de novo sequence the full transcriptome of two strains, A4 and FE355, of the diatom A. thurstonii, sampled from two different locations, and cultured in both control and phosphate starvation conditions. Differential expression analysis was also performed. Results: We in silico identified transcripts coding L-asparaginase I, polyketide cyclase/dehydrase, bifunctional polyketide phosphatase/kinase, 1-deoxy-D-xylulose-5-phosphate synthase (fragment), inositol polyphosphate 5-phosphatase INPP5B/F, catechol O-Methyltransferase, digalactosyldiacylglyc-erol synthase (DGD1), 1,2-diacylglycerol-3-beta-galactosyltransferase and glycerolphosphodiester phosphodiesterase. These are enzymes potentially involved in the synthesis/degradation of compounds with anti-cancer and immunomodulatory properties. Some of them were differentially expressed as well. Main conclusion: These data give new insights on the annotation of diatom genes, enzymatic pathways involved in the generation of bioactive molecules and possible exploitation of Asterionellopsis thurstonii.

ABSTRACT. Aim Despite the growing interest in the impact of microbial communities on host ecology, evolution, and fitness, very little is known on the reproductive microbiome. Microbes can affect sperm performance and fertilising efficiency and this effect is expected to be particularly pronounced in polyandrous species, where any reduction in fertilising efficiency can be exploited by rival ejaculates. In this pioneering study, we delve into the largely uncharted domain of ejaculate microbiomes in model fish exhibiting guard-sneaker male mating tactics, the grass goby Zosterisessor ophiocephalus. The grass goby gave the first evidence of tactic-related mechanisms of either rival ejaculate exploitation or impairment mediated by seminal fluid. Considering the impact of microbes on sperm performance observed in other vertebrates, we expect a role of the microbiome of seminal fluid in the outcome of competing interaction between rival ejaculates observed in the grass goby.

Methods We set up a method to extract bacterial DNA from the viscous seminal fluid, obtained by male seminal vesicles (i.e., accessory structures to the testes) during the reproductive season. High-throughput DNA sequencing allowed us to define the microbial composition and community structure in the seminal fluid of the two male phenotypes.

Results Our results revealed a highly biodiverse composition, despite the low biomass, with 176 families from 22 different phyla. α- and β-diversity metrics revealed a significant difference in microbial composition between territorial and sneaker males. Notably, a bacterium belonging to the family Mycoplasmataceae showed a significant difference in relative abundance between the two groups, being a promising candidate for further in vitro experiments.

Main conclusion Our preliminary findings suggest a potential role of the ejaculate microbiome in the competition for fertilisation. Future studies are warranted to uncover the mechanisms underlying these observed differences and to elucidate the potential adaptive benefits associated with these unique microbiomes.

ABSTRACT. Aim With specific reference to marine ecotoxicology, a crucial problem still to be resolved is understanding how a changing ecosystem could alter the existent toxicity of pollutants. Paracentrotus lividus has historically been used as model organism for scientific investigation, and to evaluate the toxicity of environmental matrices in Italian legislation. Our last published work (https://doi.org/10.1016/j.marpolbul.2023.115274) allowed us to demonstrate how the natural population of Livorno changed its sensitivity towards Cu under climate change conditions in the last 20 years.

Methods We measured Cu EC50 values obtained over approximately two decades, using wild specimens of P. lividus and their correlations with environmental variables, calculated using satellite observations (Copernicus). Animals have always been collected in the same natural site, unaffected by important anthropogenic activities and exposed to the same transport, preparation, and testing protocols.

Results From the correlation analysis conducted between EC50 and all the environmental variables considered, it can be inferred a stronger association relative the last 6 years compared to the other time interval. For all environmental variables, the cause-effect relationship with EC50 exacerbated in the second period studied. Among them, CO2 and pH are the factors which vary the most.

Main Conclusion The trend of EC50 values recorded in the past two decades has been projected until 2040, revealing a general deterioration of sea urchins' ability to withstand copper toxicity, but also a possible recovery of the health of the population under the most optimistic scenario. As a final consideration, it is still uncertain whether P. lividus will be able to adapt to changing climatic conditions or not, and if natural populations will be able to evolve plastic metabolic responses to climate stressors.

Thematic areas: Adaptation, Populations & Species

ABSTRACT. Aim Extreme weather events related to the global warming occur more and more frequently and are progressively damaging ecosystems biodiversity. Climate change is slowly disrupting the ecosystems equilibrium also in marine environment, affecting the global distribution of seagrasses. This group of marine angiosperms plays a key role in the coastal areas providing different essential services. Due to their fundamental roles for the structure and conservation of marine biodiversity, studying the strategies adopted by different seagrasses species and how they respond to the environmental factors is essential for predicting their fate in the face of the current environmental changes.

Methods We analysed the gene expression features related to different populations of two seagrasses (i.e. Posidonia oceanica and Cymodocea nodosa) coming from different geographical sites in the Mediterranean bioregion with a bioinformatics and comparative approach. Specifically, we provided a comparative transcriptomics analysis using publicly available RNA-seq data and not yet publicly genomic resources. In details, we merged the results obtained from the comparative and transcriptomic analyses to investigate about the expression profile of orthologs and paralogs included in a selected subset of networks.

Results The differentially expressed genes (DEGs) and the subsequent GO enrichment analyses underlined the involvement in the local adaptation of different biological processes connected with the secondary metabolism (in Cymodocea) and the photosynthesis (in Posidonia). Moreover, the study of the expression profiles of genes in a specific subset of networks including the ortholog DEGs between the two species, suggested that plant's transcriptional profiles have been more deeply influenced by the latitudinal origins rather than local thermal environments.

Conclusion The findings imply that latitudinal distribution of populations plays a critical role in influencing plant-environment interaction, although remarkable differences exist in the transcriptional and metabolic processes associate to environmental responses between the different species.

ABSTRACT. Animals exploit environmental cues to orchestrate key events in their life-cycles. However, this fundamental synchronization is challenged by climate change and anthropogenic impact, making the understanding of the underlying mechanisms paramount for species and ecosystems survival. We use the genetically-tractable marine bristleworm Platynereis dumerilii to investigate how sexual maturation and reproduction are properly timed. This species coordinates such processes with the lunar cycle, a very widespread phenomenon in marine environments, yet poorly understood at the molecular mechanistic level. We show that a simple loss-of-function mutation in the light-receptive cryptochrome (lcry) gene, sufficient to alter the worm´s lunar reproductive rhythms in lab conditions, significantly impacts a variety of life-history traits. Indeed, knockouts display dampened rate of body and germline growth, delayed sexual maturation, which expands worms´ overall lifespan, as well as increased adult size and fecundity. Quantitative transcriptomics performed in different developmental stages identifies the endocrine system as significantly de-regulated in lcry knockouts. In particular, our analyses suggest that annelid sexual development and growth are coordinated by a combination of hormonal players regulating such aspects in either arthropods or vertebrates. In sum, our study reveals remarkable changes in life-history traits associated to the loss of a photoreceptor, identifies novel molecular mechanisms synchronizing animal reproduction with the lunar cycle, and unveils unexpected similarities in the endocrine dynamics underlying invertebrate and vertebrate sexual maturation. These aspects result particularly relevant to better understand the consequences of light pollution in the marine ecosystem, and shed light on the evolution of the molecular machinery which orchestrates animal reproduction.

ABSTRACT. Clonal reproductive systems abound among marine species, including important ecosystem engineers such as corals and seagrasses. According to conventional wisdom, under a clonal mode of reproduction, (adaptive) evolutionary change is impossible owing to identical replication of modules (shoots /colonies) via mitosis during clonal expansion. Using full-genome data from the model seagrass Zostera marina (eelgrass), I show that clones evolve, accumulating genetic differences (SNPs) among modules over time. Using clone mates of known divergence date, a calibration of the somatic genetic clock was possible, allowing, for the first time, absolute dating of clone ages. At the same time, findings of within-clone evolution may explain a long-standing ecological riddle, namely why apparently large and old clones are productive and resilient through time although they feature zero genetic diversity, an observation inconsistent with the notion of genetic diversity determining population viability. An ongoing and difficult next step is the detection of asexual selective sweeps among clonal lineages, for which methods from cancer evolutionary genetics are adapted to free-living clones. Findings of within-clone genetic diversity have implications for defining individuality in modular species, and may require that some conservation genetic rules need modification under clonality.

ABSTRACT. Aim Skates (Rajiformes) represent one of the most intriguing cases of high species richness coupled with extremely conservative morphological and ecological traits. Small differences are described within and between species and increasing cases of cryptic speciation have been recorded among sister and non-sister taxa. More interestingly, this condition has been frequently observed in Central Atlantic Ocean (CAO), along the coasts of Western Africa, between Angola and Senegal, where oceanographic heterogeneities such as current systems, may play a key role for ecological speciation in skates. Methods To test the hypothesis that restricted gene flow and genetic divergence within Raja species reflect known climate and bio-oceanographic discontinuities the use of multiple mtDNA markers and its integration with nuDNA microsatellite loci has been successfully applied. We estimated the genetic variation and reconstructed phylogenetic relationships, phylogeographic patterns and genetic connectivity in skate species inhabiting the CAO. Results The widely distributed Raja miraletus species complex counts five deeply distinct lineages three of which co-occur in CAO: the Angolan Raja cf. miraletus, the Senegalese and sympatric R. parva and Raja cf. miraletus. In addition, an ancient, hidden lineage nesting in the clade Raja straeleni/R. clavata/R. maderensis was identified in Angola. Main conclusions The complexity of oceanographic system acting in the CAO (i.e., the Benguela Current region and the intertropical Canary current inflowing from the northeast) may have influenced the diversification of the Angolan and Senegalese taxa, boosting the biodiversity of the area, in an unprecedented anthology of species-specific evolutionary histories.

ABSTRACT. Abstract Aim Nudibranchs are a group of specialized molluscs characterized by an adult stage without the shell and the parallel evolution of unique alternative defensive strategies. They are characterized by a great adaptative radiation having colonised almost all the marine habitats from deep waters to sea surface, where only few specialized Cladobranchia can live. This is the case of the worldwide rafting Fiona pinnata (Eschscholtz, 1831) mainly feeding on the goose barnacles anchored on floating substrates. Even if this nudibranch has been investigated by Trickey et al. (2016), who unveiled a possible species complex, this case was not resolved and their results forgotten, so that F. pinnata species complex is currently accepted as a valid species. Considering that Mediterranean specimens have never been studied, we investigated this unsettled case through an integrative approach, adding samples from all the Italian seas (i.e. Tyrrhenian, Ionian and Adriatic Seas).

Methods Bayesian and Maximum likelihood analyses were performed on four molecular markers (COI, 16S, ITS2, H3) on a broader dataset including unpublished Mediterranean and GenBank sequences from all over the world. Additionally, anatomical analysis of the reproductive system, optical and SEM observations of the buccal apparatus, and morphological larvae development, were performed.

Results Morphological and molecular results confirmed all the Mediterranean individuals belong to a species well separated from the ‘true’ F. pinnata and consequently, we propose reinstating Fiona marina (Forsskål, 1775) as a valid species.

Main conclusion The case of F. pinnata species complex was resolved, and the congeneric F. marina reinstated as valid. These results are particularly intriguing and highlight the integrative taxonomy approach as powerful to unravel cryptic diversity. Moreover, this study confirmed the need to analyse Mediterranean specimens especially when these were used in the original descriptions of the species.

ABSTRACT. Similarly to other marine organisms, fish interacts with microbial communities, establishing either mutualistic or pathogenic relations. The microbial populations associated to fish gut, gills and skin have a strong influence on their host’s physiological functions, i.e. nutrition, immunity, and growth. The structure of the fish-associated bacterial communities could be affected by host genetics, developmental stage, feeding habits, and environmental conditions. The contribution of these factors on the establishment of fish-bacteria interaction is still unclear. The study aimed at exploring the microbiota from specimens of Xyrichtis novacula, Serranus scriba, Parablennius sanguinolentus, Trachinus draco, and Gobis cobitis. These fish species commonly inhabit the shallow hydrothermal site of Levante Bay (Vulcano island), where they play an important ecological role as consumers of benthic species. To the best of our knowledge, for the first time gut and gill samples were collected from three adult individuals for each species and the associated microbial diversity was assessed by using Illumina MiSeq sequencing of bacterial 16S rDNA region V3-V4. A total of 58 and 49 bacterial phyla were detected in fish gut and gills, respectively. Protebacteria, Firmicudes, Bacteriodota and Actinobacteria were the most representative groups (> 10% of the total microbial communities). Archaeal communities were less represented (< 1%). Overall, while the gill microbial communities was quite similar across the different species, the gut microbial communities showed species-specific variability

ABSTRACT. The Moroccan Mediterranean region represents a complex and delicate ecosystem that faces multiple challenges. Among these challenges are intensified fishing activities and changes in climatic and hydrological systems, particularly in the area around the Strait of Gibraltar. These factors pose significant threats to the local populations and the biodiversity of the region. One notable cephalopod species found in this area is the common octopus (Octopus vulgaris), which holds considerable commercial value in global markets, making it an economically important resource for fishing and a crucial player in Morocco's social economy. The primary objective of our current research is to investigate the feeding and trophic behavior of Octopus vulgaris. We conducted our study on cephalopods captured off the north coast of Morocco in the Western Mediterranean Sea between 2017 and 2018. Specifically, we examined the stomach contents of 237 common octopuses obtained from the scientific survey called 'Charif El Idrissi.' Out of these, 220 specimens had identifiable food remnants. To analyze their feeding habits, we employed qualitative and quantitative methods, including numerical frequency indices, frequency of occurrence, weight frequency, vacuity index, and index of relative importance. Our findings revealed that the common octopuses exhibited opportunistic feeding behavior, as they consumed a diverse range of prey items from three main taxa: Mollusca, Crustacea, and Teleostei. The most significant prey species were Liocarcinus, contributing to 61% of the index of relative importance, along with other decapod crustaceans. Among the various prey groups found in the octopuses' stomachs, mollusks (including bivalves and gastropods) were the most frequently consumed, based on the index of occurrence and importance in both weight and number, followed by crustaceans (specifically brachyurans) and teleosts. Furthermore, we observed that the diversity of stomach contents was relatively higher in female octopuses compared to males, as well as in mature specimens compared to immature ones of the Octopus vulgaris species. These findings provide valuable insights into the feeding patterns and ecological dynamics of this cephalopod species in the Moroccan Mediterranean region.

ABSTRACT. Aim Ocean acidification (OA) responses in marine organisms are often not univocal due to non-uniform exposure setups or different ecological histories of the specimens. To this end, we assessed OA response in sea urchins Paracentrotus lividus from sites with different environmental features: the Lagoon of Venice and a pristine coastal area in Adriatic Sea.

Methods Animals were maintained in laboratory at natural conditions (pH 8.04) and end-of-the-century predicted pH (-0.4 units). For six months behavioural and physiological (respiration rate, ammonia excretion) endpoints were investigated monthly. After eight months of exposure, the antioxidant and detoxification capabilities were assessed at biochemical level in gonads and digestive tract, whereas immunosurveillance was investigated in the coelomic fluid. In echinoderms, the larval phase is the most susceptible to environmental stressors. Hence, after 6 months of exposure, embryos were obtained from urchin adults and reared at pH 8.1 and 7.7, assessing larval development after 48 hours.

Results In coastal urchins, reduced pH had a greater effect compared to lagoonal animals, influencing more heavily both behaviour and physiology. As the exposure continued, animals from both sites were able to acclimate. However, slight effects of pH on the antioxidant and immunological responses were highlighted, with differences between origin sites. The coastal site had higher levels of antioxidant and immune-related biomarkers. Regarding transgenerational endpoints, larvae reared at low pH showed developmental delay, higher abnormality rate and were smaller than those at pH 8.1, regardless of the parental pH. However, when parents were kept at low pH, the detrimental effects of pH 7.7 were reduced.

Main conclusion Results suggest adult acclimation and transgenerational beneficial effects in P. lividus after long-term exposure to reduced pH, with higher adaptation plasticity in animals from a more variable environment, such as the lagoon, possibly allowing this population to better cope with future pH conditions.

ABSTRACT. Aim: Bivalves are aquatic filter-feeding molluscs characterized by a highly rich and diverse set of transposable elements (TEs) as well as by high levels of heterozygosity. In this study, we used five high-quality, long-read chromosome-level oyster assemblies and whole-genome resequencing data to investigate the impact of TEs in the emergence of within- and between-individual structural variants (SVs). We hypothesize that transposons can be an overlooked but important source of genetic variability both within and between individuals in bivalves and that they can contribute to population differentiation.

Methods: We combined multiple long-read pipelines and simulations to characterize high-confidence within-individual SVs. We have then estimated the role of TEs in their emergence. To characterize between-individual SVs we used 120 publicly available Crassostrea ariakensis short read resequenced samples encompassing the entire Chinese coasts. This SV set was used in classical population genomics analyses to detect population structure, differentiation, and putative variants under selection.

Results: Between 4% and 14% of oyster genomes exhibit structural variability between homologous chromosomes. Most of these regions are enriched in transposable elements, including both putative TE insertions and deletions, but are depleted of host genes. Similar results were obtained when analyzing SVs between individuals. Population structure based on more than 60,000 high-confidence SVs reflects the geographic origin of samples, reflecting what can be recovered using SNPs. Among these SVs, we identified multiple loci under putative diversifying selection between southern and northern populations.

Main conclusions: Applying state-of-the-art methods and simulations we suggest an improved workflow for within-individual SVs calling. We discovered that transposon can be an important hotspot of genetic variability in bivalves providing raw material for potential local adaptations. Our analyses highlight the importance of considering structural variants in population genomics analyses in bivalves, especially with the rise of the pan-genomic era.

ABSTRACT. Aim Lomanotus vermiformis is a dendronotid nudibranch (Gastropoda, Heterobranchia) with circumtropical distribution. It is exclusively reported on the hydroid Macrorhynchia philippina Kirchenpauer, 1872, where it lives undisturbed due to its body pattern perfectly resembling the shape of the colony. In fact, this nudibranch is adapted to this powerful stinging hydrozoan on which it is supposed to feed and lay its eggs. Originally reported from the Red Sea, the scanty description was followed by a more detailed one based on specimens from Australia, a location far from its type locality. Considering that M. philippina has recently colonized the Mediterranean Levantine basin, the possible co-occurrence of its associated nudibranch was investigated and confirmed for the first time.

Methods Within the framework of the project BLUE TYRE – Local Partnership for Sustainable Marine and Coastal Development (AID 012314/01/6), five branches of M. philippina were collected while scuba diving in Tyre (Lebanon) and sorted in laboratory. Morphological analysis was carried out using light and SEM microscopy on the nudibranch, its gut content, and the hydroid.

Results Six L. vermiformis were identified, confirming the presence of the species in the Mediterranean Sea. An updated redescription of the diagnostic features (buccal apparatus and reproductive system) is provided based on specimens coming from the type locality and Lessepsian in the Mediterranean basin. Furthermore, the trophic relationship between the hydroid and its host was demonstrated through analyses of the nudibranch's radula and gut content, revealing, for the first time the presence of microbasic mastigophore nematocysts that match those observed in the nematothecae of M. philippina.

Main conclusion The present finding emphasizes the importance of monitoring Mediterranean alien structural tridimensional species to early detection of associated non-indigenous fauna. Moreover, new insights are provided on this strong adaptive association, shedding lights on the poorly known biology characterizing mimetic and elusive nudibranch species.

ABSTRACT. Aim The ecological adaptation among individuals of different ages of habitat-forming species, i.e., Gongolaria barbata, needs to be better investigated. Filling this knowledge gap could lead to a better prediction of how climate change will affect these ecosystems and their ecological processes. To pursue this aim, individuals of different ages of G. barbata collected from the Venice Lagoon (Venice, Italy) were analysed for some peculiar structural and functional traits and put in relationship with their growth environment and ecological variables experienced during their development.

Methods In vivo chlorophyll fluorescence emission analysis was performed on adult and young thalli of G. barbata, utilizing a pulse amplitude-modulated fluorometer (Junior-PAM, Walz Gmbh, Effeltrich, Germany). On samples, the expression of the D1 protein of photosystem II, photosynthetic pigments, and antioxidant activity was also determined to study the adaptive response of G. barbata thalli of different ages from the Venice Lagoon.

Results The overall analyses showed increased thermal energy dissipation processes in adults with high pigment concentration and dry matter content; conversely, young individuals exhibited a higher photosynthetic yield, electron transport rate, and an elevated antioxidant level. Furthermore, no difference in maximum photochemical efficiency and Photosystem II D1 protein was found between thalli of different ages.

Main conclusion Our analysis highlighted remarkable differences between thalli of diverse age in G. barbata: the youngers allocate more energy into chemical defences and are focused on growth, whereas adults are more concerned about photoprotection and may have adapted over time, adjusting the photosynthetic rate, and opportunely modulating pigments and antioxidants concentration.

ABSTRACT. Aim Microplastics (MPs) and Nanoplastics (NPs) pollution has become a global environmental problem due to their abundance, poor biodegradability, toxicological properties, and negative effects on aquatic and terrestrial organisms including humans (Anik et al., 2021). In our study, we investigated the cytotoxic effects of MPs and NPs in Mytilus galloprovincialis, a good bioindicator of marine pollution (Curpan et al., 2022).

Methods We detected the effects of polystyrene on the fitness of mussels, analysing gills, digestive glands and byssus apparatus respectively involved in the respiratory, feeding process and adhesion to the substrate. Animals were exposed to polystyrene (5 or 0.1 µm) for 1, 3 and 11 days at environmental concentrations (Vroom et al., 2017). Samples were processed for light microscopy using different specific histological (Haematoxylin-eosin, Mallory’s Trichrome, PAS, Alcian Blu and Picrosirius Red) or Immunohistochemistry staining.

Results Histological analysis demonstrated that MPs and NPs are cytotoxic for mussels interfering with structure of gills tissues. Disorganization on the septum and lamellar structures were observed. Melanin and lipofuscin infiltrations were detected, alteration in numbers distribution of mucus cells after treatments also were observed. Immunocytochemistry investigations by PCNA demonstrated an increasing number of positive cells to the antibody confirming alterations in cell cycle. HSP70 detection indicated marked cellular stress response. MPs and NPs affect the digestive gland structures in the tubules and ducts organization, interfering with the proliferative activity, inducing collagen deposition within the digestive tubules. Alterations in number and organization of mucus cells after short and long-time exposure were observed. Impairment in the byssus glands structures and altered byssal thread production were detected.

Main conclusion Polystyrene induces damage in gills, digestive glands and byssus apparatus of mussels, altering their functions, this could represent a risk for their health, for the food chain and indirectly for human consumers.

ABSTRACT. Phylosymbiosis is the association between the phylogenetic relatedness of hosts and the composition of their microbial communities. This phenomenon is widespread in diverse animal taxa. However, the generality of the existence of such a pattern has been questioned, and there seems to be evidence against its occurrence in small-sized aquatic animals, for which the microbiota composition is mostly shaped by local environmental factors rather than the hosts' phylogenetic relationships.

This study aims to investigate the microbial communities associated with poorly known marine interstitial nemerteans to uncover their microbiota diversity and assess the occurrence of phylosymbiosis. Through a comprehensive approach, specimens from various Central American sites were analyzed using morphology-based taxonomy and molecular techniques targeting the host 18S rRNA gene, while their microbial associations were analyzed by targeting the prokaryotic 16S rRNA gene. Phylogenetic and statistical analyses were conducted to examine the potential effects of host nemertean taxa and sampling locations on the host-associated microbial communities.

The results provide compelling evidence of phylosymbiosis in meiofaunal nemerteans, emphasizing the significant impact of host genetic relatedness on microbiome diversity, even in small-sized animals. Additional research is needed to fully unravel potential symbiotic relationships, as well as the complex mechanisms that govern the relationships between hosts and their microbiota across various organisms and ecological settings. The remarkable diversity of meiofaunal animals, spanning various animal phyla with different lifestyles and inhabiting diverse ecosystems, combined with advancements in multi-omics approaches, offers a promising avenue for a comprehensive understanding of the evolutionary and ecological interactions between hosts and their microbiota throughout the animal tree of life.