The earliest evidence of deep-sea vertebrates.

Vertebrate macroevolution has been punctuated by fundamental habitat transitions from shallow marine origins to terrestrial, freshwater, and aerial environments. Invasion of the deep sea is a less well-known ecological shift because of low fossilization potential and continual loss of abyssal fossil record by ocean floor subduction. Therefore, there has been a lack of convincing evidence of bottom-living vertebrates from pre-Paleogene deep seas. Here, we describe trace fossils from abyssal plain turbidites of the Tethys Ocean, which, combined with nannofossil dating, indicate that fishes have occupied the deep seafloor since at least the Early Cretaceous (Hauterivian-Barremian). These structures are identical to those produced by modern demersal fishes that feed by either scratching the substrate or expose their prey by water flow generated by suction or jetting. The trace fossils suggest activity of at least three fish species exploiting a productive abyssal invertebrate sediment fauna. These observations are consistent with Early Cretaceous vertebrate transition to the deep sea triggered by the availability of new food sources. Our results anticipate the appearance of deep-seafloor fishes in the fossil record by over 80 My while reassessing the mode of vertebrate colonization of the deep sea.

Ancient and Nonuniform Loss of Olfactory Receptor Expression Renders the Shark Nose a De Facto Vomeronasal Organ.

Cartilaginous fishes are renowned for a keen sense of smell, a reputation based on behavioral observations and supported by the presence of large and morphologically complex olfactory organs. At the molecular level, genes belonging to the four families coding for most olfactory chemosensory receptors in other vertebrates have been identified in a chimera and a shark, but it was unknown whether they actually code for olfactory receptors in these species. Here, we describe the evolutionary dynamics of these gene families in cartilaginous fishes using genomes of a chimera, a skate, a sawfish, and eight sharks. The number of putative OR, TAAR, and V1R/ORA receptors is very low and stable, whereas the number of putative V2R/OlfC receptors is higher and much more dynamic. In the catshark Scyliorhinus canicula, we show that many V2R/OlfC receptors are expressed in the olfactory epithelium in the sparsely distributed pattern characteristic for olfactory receptors. In contrast, the other three vertebrate olfactory receptor families are either not expressed (OR) or only represented with a single receptor (V1R/ORA and TAAR). The complete overlap of markers of microvillous olfactory sensory neurons with pan-neuronal marker HuC in the olfactory organ suggests the same cell-type specificity of V2R/OlfC expression as for bony fishes, that is, in microvillous neurons. The relatively low number of olfactory receptors in cartilaginous fishes compared with bony fishes could be the result of an ancient and constant selection in favor of a high olfactory sensitivity at the expense of a high discrimination capability.

Near-Infrared 810 nm Light Affects Porifera Chondrosia reniformis (Nardo, 1847) Regeneration: Molecular Implications and Evolutionary Considerations of Photobiomodulation-Animal Cell Interaction.

Chemotrophic choice as a metabolic source of energy has characterised animal cell evolution. However, light interactions with animal cell photoacceptors that are able to increase energetic metabolism (photo-biomodulation (PBM)) have been previously described. In the present study, we cut three specimens of Chondrosia reniformis into four equal parts (12 fragments), and we irradiated the regenerating edge of six fragments with the previously characterised 810 nm near-infrared light, delivered at 1 W, 60 J/cm2, 1 W/cm2, and 60 J in a continuous-wave mode for 60 s through a flat-top hand-piece with a rounded spot-size area of 1 cm2. Six fragments were irradiated with 0 W for 60 s as the controls. We performed irradiation at the time 0 h and every 24 h for a total of five administrations. We monitored the regeneration process for five days (120 h) in aquaria by examining the macroscopic and histological changes. We analysed the gene expression profile of the inflammatory processes, apoptosis, heat stress, growth factors, and collagen production and determined oxidative stress enzyme activity and the total prokaryotic symbiont content. PBM sped up C. reniformis regeneration when compared to the controls. Particularly, transforming growth factor TGF3 and TGF6 upregulation during the early phase of regeneration and TGF5 upregulation 120 h postinjury in the irradiated samples supports the positive effect of PBM in sponge tissue recovery. Conversely, the expression of TGF4, a sponge fibroblast growth factor homologue, was not affected by irradiation, indicating that multiple, independent pathways regulate the TGF genes. The results are consistent with our previous data on a wide range of organisms and humans, suggesting that PBM interaction with primary and secondary cell targets has been conserved through the evolution of life forms.

Potential Biomedical Applications of Collagen Filaments derived from the Marine Demosponges Ircinia oros (Schmidt, 1864) and Sarcotragus foetidus (Schmidt, 1862).

Collagen filaments derived from the two marine demosponges Ircinia oros and Sarcotragus foetidus were for the first time isolated, biochemically characterised and tested for their potential use in regenerative medicine. SDS-PAGE of isolated filaments revealed a main collagen subunit band of 130 kDa in both of the samples under study. DSC analysis on 2D membranes produced with collagenous sponge filaments showed higher thermal stability than commercial mammalian-derived collagen membranes. Dynamic mechanical and thermal analysis attested that the membranes obtained from filaments of S. foetidus were more resistant and stable at the rising temperature, compared to the ones derived from filaments of I. oros. Moreover, the former has higher stability in saline and in collagenase solutions and evident antioxidant activity. Conversely, their water binding capacity results were lower than that of membranes obtained from I. oros. Adhesion and proliferation tests using L929 fibroblasts and HaCaT keratinocytes resulted in a remarkable biocompatibility of both developed membrane models, and gene expression analysis showed an evident up-regulation of ECM-related genes. Finally, membranes from I. oros significantly increased type I collagen gene expression and its release in the culture medium. The findings here reported strongly suggest the biotechnological potential of these collagenous structures of poriferan origin as scaffolds for wound healing.

In-vivo genetic ablation of metabotropic glutamate receptor type 5 slows down disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease due to motor neuron (MN) loss. The mechanisms causing selective MN death are largely unknown, thus prejudicing successful pharmacological treatments. Major causes of MN damage are effects downstream of the abnormal glutamate (Glu) neurotransmission. Group I metabotropic Glu receptors (mGluR1, mGluR5) actively contribute to the excitotoxicity in ALS and represent druggable molecular targets. We previously demonstrated that halving mGluR1 or mGluR5 expression in the widely studied SOD1G93A mouse model of ALS had a positive impact on disease onset, clinical progression and survival, as well as on cellular and biochemical parameters altered in ALS. Whereas these effects were similar in female and male mGluR1 heterozygous SOD1G93Amice, only male mGluR5 heterozygous SOD1G93A mice showed improved motor skills during disease progression. To further validate the role of Group I mGluRs in ALS, we generated in this study mGluR1 or mGluR5 null mice expressing the SOD1G93A mutation (SOD1G93AGrm1crv4/crv4 or SOD1G93AGrm5-/-, respectively). SOD1G93AGrm1crv4/crv4 mice showed early and progressive motor impairments and died even before SOD1G93A mice, while SOD1G93AGrm5-/- mice exhibited delayed disease onset, longer survival, and ameliorated motor skills than SOD1G93A mice. No difference between female and male SOD1G93AGrm5-/- mice were observed. These effects were associated with enhanced MN preservation and decreased astrocytic and microglial activation. Our results strongly support the assumption that constitutively lowering of mGluR5 expression has a positive impact in mice with ALS by counteracting the abnormal Glu transmission and this could be a potentially effective pharmacological target in ALS.

Insights into the evolution of metazoan regenerative mechanisms: roles of TGF superfamily members in tissue regeneration of the marine sponge Chondrosia reniformis.

Tissue repair is an adaptive and widespread metazoan response. It is characterised by different cellular mechanisms and complex signalling networks that involve numerous growth factors and cytokines. In higher animals, transforming growth factor-ß (TGF-ß) signalling plays a fundamental role in wound healing. In order to evaluate the involvement of TGF superfamily members in lower invertebrate tissue regeneration, sequences for putative TGF ligands and receptors were isolated from the transcriptome of the marine sponge Chondrosia reniformis We identified seven transcripts that coded for TGF superfamily ligands and three for TGF superfamily receptors. Phylogenetically, C. reniformis TGF ligands were not grouped into any TGF superfamily clades and thus presumably evolved independently, whereas the TGF receptors clustered in the Type I receptor group. We performed gene expression profiling of these transcripts in sponge regenerating tissue explants. Data showed that three ligands (TGF1, TGF3 and TGF6) were mainly expressed during early regeneration and seemed to be involved in stem cell maintenance, whereas two others (TGF4 and TGF5) were strongly upregulated during late regeneration and thus were considered pro-differentiating factors. The presence of a strong TGF inhibitor, SB431542, blocked the restoration of the exopinacoderm layer in the sponge explants, confirming the functional involvement of the TGF pathway in tissue regeneration in these early evolved animals.

Extramitochondrial energy production in platelets.

BACKGROUND INFORMATION: Energy demand in human platelets is very high, to carry out their functions. As for most human cells, the aerobic metabolism represents the primary energy source in platelets, even though mitochondria are negligibly represented. Following the hypothesis that other structures could be involved in chemical energy production, in this work, we have investigated the functional expression of an extramitochondrial aerobic metabolism in platelets. RESULTS: Oximetric and luminometric analyses showed that platelets consume large amounts of oxygen and produce ATP in the presence of common respiring substrates, such as pyruvate + malate or succinate, although morphological electron microscopy analysis showed that these contain few mitochondria. However, evaluation of the anaerobic glycolytic metabolism showed that only 13% of consumed glucose was converted to lactate. Interestingly, the highest OXPHOS activity was observed in the presence of NADH, not a readily permeant respiring substrate for mitochondria. Also, oxygen consumption and ATP synthesis fuelled by NADH were not affected by atractyloside, an inhibitor of the adenine nucleotide translocase, suggesting that these processes may not be ascribed to mitochondria. Functional data were confirmed by immunofluorescence microscopy and Western blot analyses, showing a consistent expression of the ß subunit of F1 Fo -ATP synthase and COXII, a subunit of Complex IV, but a low signal of translocase of the inner mitochondrial membrane (a protein not involved in OXPHOS metabolism). Interestingly, the NADH-stimulated oxygen consumption and ATP synthesis increased in the presence of the physiological platelets agonists, thrombin or collagen. CONCLUSIONS: Data suggest that in platelets, aerobic energy production is mainly driven by an extramitochondrial OXPHOS machinery, originated inside the megakaryocyte, and that this metabolism plays a pivotal role in platelet activation. SIGNIFICANCE: This work represents a further example of the existence of an extramitochondrial aerobic metabolism, which can contribute to the cellular energy balance.

First identification of a fatal fungal infection of the marine sponge Chondrosia reniformis by Aspergillus tubingensis.

Sponges are considered promising sources of biomolecules for both pharmaceutical and cosmetic interests as well as for the production of biomaterials suitable for tissue engineering and regenerative medicine. Accordingly, the ability to grow sponges in captivity and in healthy conditions to increase their biomass is a required goal for the development of sponge aquaculture systems. To date, little information is available about the pathogenicity of fungi associated with sponges. In our study, we identified an infection in freshly collected specimens of Chondrosia reniformis (Porifera, Demospongiae) and determined that the fungus Aspergillus tubingensis was the pathogen responsible. This is the first description of a natural infection of C. reniformis by A. tubingensis. Despite raising an inflammatory response by means of an increase in tumour necrosis factor (TNF) mRNA, the infected C. reniformis specimens were not able to control the fungal infection, leading to rotting in 15 d. Characterization of this infection shows that a widely distributed fungus can represent a potential hazard to sponge aquaculture industries and how, especially in stressed or compromised marine environments, this fungus could represent a fatal opportunistic pathogen.

Fipronil (Phenylpyrazole) induces hemato-biochemical, histological and genetic damage at low doses in common carp, Cyprinus carpio (Linnaeus, 1758).

This study was conducted to investigate the toxic effects of fipronil on common carp (Cyprinus carpio). The trial was performed in laboratory of Department of Life Sciences at the Islamia University of Bahawalpur, Punjab, Pakistan. Total of 72 fishes, with apparently no clinical signs were purchased from local fish hatchery near Bahawalpur, Punjab, Pakistan. After 2 weeks of acclimatization fish were divided into six groups in glass aquaria (each having 100 liters of water capacity) and treated with different concentrations of fipronil for 12 days: 0 mg/L-control group (A),0.02 mg/L (B),0.04 mg/L (C),0.06 mg/L (D),0.08 mg/L (E) and 0.10 mg/L (F). Blood samples were taken every 4 days for evaluation of hemato-biochemical parameters. Fish in high doses treated groups show severe abnormalities in clinical-hematological and biochemical parameters. At high dose in group E and F fish show clinical ailments such as convulsions, jerking, faintness, increase in operculum movement, body curvature and breathing difficulty. Erythrocyte count, hemoglobin, hematocrit were decreased significantly (P < 0.05) and mean corpuscular volume, total leukocyte count, neutrophils, monocytes, and lymphocytes were significantly increased. Biochemical parameters including urea, creatinine, cholesterol, triglyceride, glucose were significantly increased but albumin was significantly decreased. Nuclear and cellular abnormalities were also increased in high dose treated groups. Hence fipronil induces clinico-hematological and serum biochemical changes in common carp C. carpio.

Silica-induced fibrosis: an ancient response from the early metazoans.

Exposure to crystalline silica particles causes silicosis, an occupational disease leading to an overproduction of collagen in the lung. The first step of this pathology is characterized by the release of inflammatory mediators. Tumour necrosis factor (TNF) is a pro-inflammatory cytokine directly involved in silica-induced pulmonary fibrosis. The marine demosponge Chondrosia reniformis is able to incorporate silica grains and partially dissolve the crystalline forms apparently without toxic effects. In the present work, C. reniformis tissue explants were treated with fine quartz dust and the expression level of fibrogenic genes was assayed by qPCR, demonstrating an overexpression of a fibrillar and a non-fibrillar collagen and of prolyl-4-hydroxylase enzyme. The deposition of new collagen could also be documented in quartz-treated sponge explants. Furthermore, TNF pro-inflammatory cytokine overexpression and involvement in silica-induced sponge collagen biosynthesis was demonstrated in quartz-treated explants as compared with controls by means of specific TNF inhibitors affecting the fibrogenic gene response. As no documentable detrimental effect was observed in treated explants, we conclude that the C. reniformis unique quartz engulfment and erosion is physiological and beneficial to the animal, leading to new collagen synthesis and strengthening of the body stiffness. Thus, we put forward the hypothesis that an ancient physiological behaviour from the lowest of the Metazoa, persisting through evolution via the same molecular mediators such as TNF, may have become the cause of disease in the specialized tissues of higher animals such as mammals.

Effects of polystyrene microbeads in marine planktonic crustaceans.

Plastic debris accumulates in the marine environment, fragmenting into microplastics (MP), causing concern about their potential toxic effects when ingested by marine organisms. The aim of this study was to verify whether 0.1µm polystyrene beads are likely to trigger lethal and sub-lethal responses in marine planktonic crustaceans. MP build-up, mortality, swimming speed alteration and enzyme activity (cholinesterases, catalase) were investigated in the larval stages of Amphibalanus amphitrite barnacle and of Artemia franciscana brine shrimp exposed to a wide range of MP concentrations (from 0.001 to 10mgL-1) for 24 and 48h. The results show that MP were accumulated in crustaceans, without affecting mortality. Swimming activity was significantly altered in crustaceans exposed to high MP concentrations (> 1mgL-1) after 48h. Enzyme activities were significantly affected in all organisms exposed to all the above MP concentrations, indicating that neurotoxic effects and oxidative stress were induced after MP treatment. These findings provide new insight into sub-lethal MP effects on marine crustaceans.

Histopathological analysis of the olfactory epithelium of zebrafish (Danio rerio) exposed to sublethal doses of urea.

Chronic renal disease is known to alter olfactory function, but the specific changes induced in olfactory organs during this process remain unclear. Of the uraemic toxins generated during renal disease, high levels of urea are known to induce hyposmic conditions. In this study, the effects of environmental exposure to elevated concentrations of urea (7, 13.5 and 20 g L(-1)) on the sensory mucosa of zebrafish in acute toxicity and chronic toxicity tests were described. It was observed that lamellae maintained structural integrity and epithelial thickness was slightly reduced, but only following exposure to the highest concentrations of urea. Pan-neuronal labelling with anti-Hu revealed a negative correlation with levels of urea, leading to investigation of whether distinct neuronal subtypes were equally sensitive. Using densitometric analysis of immunolabelled tissues, numbers of Gα olf-, TRPC2- and TrkA-expressing cells were compared, representing ciliated, microvillous and crypt neurons, respectively. The three neuronal subpopulations responded differently to increasing levels of urea. In particular, crypt cells were more severely affected than the other cell types, and Gα olf-immunoreactivity was found to increase when fish were exposed to low doses of urea. It can be concluded that exposure to moderate levels of urea leads to sensory toxicity directly affecting olfactory organs, in accordance with the functional olfactometric measurements previously reported in the literature.

Review: Morphofunctional and biochemical markers of stress in sea urchin life stages exposed to engineered nanoparticles.

We describe the use of different life stages of the Mediterranean sea urchin Paracentrotus lividus for the assessment of the possible risk posed by nanoparticles (NPs) in the coastal water. A first screening for the presence of NPs in sea water may be obtained by checking their presence inside tissues of organisms taken from the wild. The ability of NPs to pass from gut to the coelomic fluid is demonstrated by accumulation in sea urchin coelomocytes; the toxicity on sperms can be measured by embryotoxicity markers after sperm exposure, whereas the transfer through the food chain can be observed by developmental anomalies in larvae fed with microalgae exposed to NPs. The most used spermiotoxicity and embryotoxicity tests are described, as well as the biochemical and histochemical analyses of cholinesterase (ChE) activities, which are used to verify toxicity parameters such as inflammation, neurotoxicity, and interference in cell-to-cell communication. Morphological markers of toxicity, in particular skeletal anomalies, are described and classified. In addition, NPs may impair viability of the immune cells of adult specimens. Molecular similarity between echinoderm and human immune cells is shown and discussed. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1552-1562, 2016.

Functional Expression of Electron Transport Chain and FoF1-ATP Synthase in Optic Nerve Myelin Sheath.

Our previous studies reported evidence for aerobic ATP synthesis by myelin from both bovine brainstem and rat sciatic nerve. Considering that the optic nerve displays a high oxygen demand, here we evaluated the expression and activity of the five Respiratory Complexes in myelin purified from either bovine or murine optic nerves. Western blot analyses on isolated myelin confirmed the expression of ND4L (subunit of Complex I), COX IV (subunit of Complex IV) and ß subunit of F1Fo-ATP synthase. Moreover, spectrophotometric and in-gel activity assays on isolated myelin, as well as histochemical activity assays on both bovine and murine transversal optic nerve sections showed that the respiratory Complexes are functional in myelin and are organized in a supercomplex. Expression of oxidative phosphorylation proteins was also evaluated on bovine optic nerve sections by confocal and transmission electron microscopy. Having excluded a mitochondrial contamination of isolated myelin and considering the results form in situ analyses, it is proposed that the oxidative phosphorylation machinery is truly resident in optic myelin sheath. Data may shed a new light on the unknown trophic role of myelin sheath. It may be energy supplier for the axon, explaining why in demyelinating diseases and neuropathies, myelin sheath loss is associated with axonal degeneration.

Pharmacological characterization of NMDA-like receptors in the single-celled organism Paramecium primaurelia.

Paramecium primaurelia is a unicellular eukaryote that moves in freshwater by ciliary beating and responds to environmental stimuli by altering motile behaviour. The movements of the cilia are controlled by the electrical changes of the cell membrane: when the intraciliary Ca(2+) concentration associated with plasma membrane depolarization increases, the ciliary beating reverses its direction, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca(2+) influx. Here, we evaluated the effects due to the activation or blockade of N-methyl-d-aspartic acid (NMDA) receptors on swimming behaviour in Paramecium. Paramecia normally swim forward, drawing almost linear tracks. We observed that the simultaneous administration of NMDA and glycine induced a partial ciliary reversal (PaCR) leading to a continuous spiral-like swim. Furthermore, the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, was longer in NMDA+glycine-treated cells. NMDA action required the presence of Ca(2+), as the normal forward swimming was restored when the ion was omitted from the extracellular milieu. The PaCR and the enhancement of CCR duration significantly decreased when the antagonists of the glutamate site D-AP5 or CGS19755, the NMDA channel blocker MK-801 or the glycine site antagonist DCKA was added. The action of NMDA+glycine was also abolished by Zn(2+) or ifenprodil, the GluN2A and the GluN2B NMDA-containing subunit blockers, respectively. Searches of the Paramecium genome database currently available indicate that the NMDA-like receptor with ligand-binding characteristics of an NMDA receptor-like complex, purified from rat brain synaptic membranes and found in some metazoan genomes, is also present in Paramecium. These results provide evidence that functional NMDA receptors similar to those typical of mammalian neuronal cells are present in the single-celled organism Paramecium and thus suggest that the glutamatergic NMDA system is a phylogenetically old behaviour-controlling mechanism.

Effects of urea on the molecules involved in the olfactory signal transduction: a preliminary study on Danio rerio.

Among vertebrates, the physiologically uremic Chondrichthyes are the only class which are not presenting the ciliated olfactory receptor neurons in the olfactory neuroepithelium. The only sequenced genome for this class revealed only three olfactory receptor genes and the immunohistochemical detection of G protein alpha subunit typically coupled to the olfactory receptors (Gα(olf)) failed in different species. Chronic renal disease can represent a cause of olfactory impairment in human. In this context, our present study focused on investigating potential effects of high urea concentration on the olfactory epithelium of vertebrates. Larvae of the teleost fish Danio rerio were exposed to urea in order to assess the effects on the olfactory signal transduction; in particular on both the olfactory receptors and the Gα(olf). The endocytosis of neutral red dye in the olfactory mucosa was detected in control and urea-exposed larvae. The amount of neutral red dye uptake was used as a marker of binding and internalization of the Gα(olf). The neutral red dye endocytosis was not affected by urea exposure, hence suggesting that the presence of the Gα(olf) and their binding to the odorants are not affected by urea treatment, either. The presence and distribution of Gα(olf) were investigated in the olfactory epithelium of control and urea-exposed larvae, using a commercial antibody. The immunoreactivity was increased after urea treatment, suggesting an effect of urea on the expression or degradation of this G protein alpha subunit.

Effects of selected metal oxide nanoparticles on Artemia salina larvae: evaluation of mortality and behavioural and biochemical responses.

The aim was to investigate the toxicity of selected metal oxide nanoparticles (MO-NPs) on the brine shrimp Artemia salina, by evaluating mortality and behavioural and biochemical responses. Larvae were exposed to tin(IV) oxide (stannic oxide (SnO2)), cerium(IV) oxide (CeO2) and iron(II, III) oxide (Fe3O4) NPs for 48 h in seawater, with MO-NP suspensions from 0.01 to 1.0 mg/mL. Mortality and behavioural responses (swimming speed alteration) and enzymatic activities of cholinesterase, glutathione-S-transferase and catalase were evaluated. Although the MO-NPs did not induce any mortality of the larvae, they caused changes in behavioural and biochemical responses. Swimming speed significantly decreased in larvae exposed to CeO2 NPs. Cholinesterase and glutathione-S-transferase activities were significantly inhibited in larvae exposed to SnO2 NPs, whereas cholinesterase activity significantly increased after CeO2 NP and Fe3O4 NP exposure. Catalase activity significantly increased in larvae exposed to Fe3O4 NPs. In conclusion, swimming alteration and cholinesterase activity represent valid endpoints for MO-NP exposure, while glutathione-S-transferase and catalase activities appear to be NP-specific.

Insight on thermal stress response of demosponge Chondrosia reniformis (Nardo, 1847).

Global warming has led to an increase in extreme weather and climate phenomena, including floods and heatwaves. Marine heatwaves have frightening consequences for coastal benthic communities around the world. Each species exhibits a natural range of thermal tolerance and responds to temperature variations through behavioral, physiological, biochemical, and molecular adjustments. Physiological stress leading to disease and mass mortality appears when tolerance thresholds are exceeded. Sessile species are therefore particularly affected by these phenomena. Among these sessile species, marine sponges are important members of coral reef ecosystems. To better understand the sponge thermal stress response, we tested the response of demosponge Chondrosia reniformis (Nardo, 1847) to three different temperatures (8 °C, 24 °C and 30 °C) during two exposure periods of time (4 and 14 h). Histological studies of whole parts of the sponge, biochemical analyses (Defense enzymes) and gene expression levels of some target genes were undertaken in this study. The exposure to cold temperature (8 °C) resulted in inhibition of antioxidant enzymes and less modification in the gene expression level of the heat shock proteins (HSPs). These latter were strongly upregulated after exposure to a temperature of 24 °C for 4 h. However, exposure to 30 °C at both periods of time resulted in indication of HSP, antioxidant enzymes, the gene involved in the apoptosis process (Bcl-2: B-cell lymphoma 2), the gene involved in inflammation (TNF: Tumor Necrosis Factor), as well as the aquaporin gene, involved in H2O2 permeation. Moreover, the normal organization of the whole organism was disrupted by the extension and fusion of choanocyte chambers and alteration of the pinacoderm. Interestingly, exposure to sublethal temperatures may show that this sponge has an adaptation threshold temperature. These insights into the adaptation mechanisms of sponges contribute to better management and conservation of sponges and to the prediction of ecosystem trajectories with future climate change.

New Insights into Geometric Morphometry Applied to Fish Scales for Species Identification.

The possibility of quick and cheap recognition of a fish species from a single dermal scale would be interesting in a wide range of contexts. The methods of geometric morphometry appear to be quite promising, although wide studies comparing different approaches are lacking. We aimed to apply two methods of geometric morphometry, landmark-based and outline-based, on a dataset of scales from five different teleost species: Danio rerio, Dicentrarchus labrax, Mullus surmuletus, Sardina pilchardus, and Sparus aurata. For the landmark-based method the R library "geomorph" was used. Some issues about landmark selection and positioning were addressed and, for the first time on fish scales, an approach with both landmarks and semilandmarks was set up. For the outline-based method the R library "Momocs" was used. Despite the relatively low number of scales analyzed (from 11 to 81 for each species), both methods achieved quite good clustering of all the species. In particular, the landmark-based method used here gave generally higher R2 values in testing species clustering than the outline-based method, but it failed to distinguish between a few couples of species; on the other hand, the outline-based method seemed to catch the differences among all the couples except one. Larger datasets have the potential to achieve better results with outline-based geometric morphometry. This latter method, being free from the problem of recognizing and positioning landmarks, is also the most suitable for being automatized in future applications.

Amphioxus (Branchiostoma lanceolatum) in the North Adriatic Sea: ecological observations and spawning behavior.

The European amphioxus (Branchiostoma lanceolatum) is a member of the chordate subphylum Cephalochordata, and, as such, a key model organism for providing insights into the origin and evolution of vertebrates. Despite its significance and global distribution, detailed characterizations of natural populations of cephalochordates are still very limited. This study investigates the abundance, habitat, and spawning behavior of amphioxus in the North Adriatic Sea. Across 32 sampled sites, adult amphioxus were consistently present, reaching densities exceeding 300 individuals m- 2. DNA barcoding confirmed the species as B. lanceolatum, and environmental analyses revealed an amphioxus preference for slightly gravelly sand with low silt content and a correlation between amphioxus density and the presence of specific macroinvertebrate taxa. Remarkably, the amphioxus population was breeding in early spring and possibly late fall, in contrast to the typical late spring/early summer spawning season described for other populations of European amphioxus. Amphioxus adults kept in captivity maintained the spawning seasonality of their place of origin, suggesting the possibility of extending the overall spawning season of European amphioxus in laboratory settings by exploiting populations from diverse geographic origins. This study thus expands our understanding of B. lanceolatum ecology and reproduction in the Mediterranean Sea, emphasizing the role of the North Adriatic Sea as a substantial reservoir.