The Arrangement of the Peripheral Olfactory System of Pleuragramma antarcticum: A Well-Exploited Small Sensor, an Aided Water Flow, and a Prominent Effort in Primary Signal Elaboration.

The olfactory system is constituted in a consistent way across vertebrates. Nasal structures allow water/air to enter an olfactory cavity, conveying the odorants to a sensory surface. There, the olfactory neurons form, with their axons, a sensory nerve projecting to the telencephalic zone-named the olfactory bulb. This organization comes with many different arrangements, whose meaning is still a matter of debate. A morphological description of the olfactory system of many teleost species is present in the literature; nevertheless, morphological investigations rarely provide a quantitative approach that would help to provide a deeper understanding of the structures where sensory and elaborating events happen. In this study, the peripheral olfactory system of the Antarctic silverfish, which is a keystone species in coastal Antarctica ecosystems, has also been described, employing some quantitative methods. The olfactory chamber of this species is connected to accessory nasal sacs, which probably aid water movements in the chamber; thus, the head of the Antarctic silverfish is specialized to assure that the olfactory organ keeps in contact with a large volume of water-even when the fish is not actively swimming. Each olfactory organ, shaped like an asymmetric rosette, has, in adult fish, a sensory surface area of about 25 mm2, while each olfactory bulb contains about 100,000 neurons. The sensory surface area and the number of neurons in the primary olfactory brain region show that this fish invests energy in the detection and elaboration of olfactory signals and allow comparisons among different species. The mouse, for example-which is considered a macrosmatic vertebrate-has a sensory surface area of the same order of magnitude as that of the Antarctic silverfish, but ten times more neurons in the olfactory bulb. Catsharks, on the other hand, have a sensory surface area that is two orders of magnitude higher than that of the Antarctic silverfish, while the number of neurons has the same order of magnitude. The Antarctic silverfish is therefore likely to rely considerably on olfaction.

Quantification of neurons in the olfactory bulb of the catsharks Scyliorhinus canicula (Linnaeus, 1758) and Galeus melastomus (Rafinesque, 1810).

In vertebrates, the olfactory bulb (OB) is the zone of the brain devoted to receiving the olfactory stimuli. The size of the OB relative to the size of the brain has been positively correlated to a good olfactory capability but, recently, this correlation was questioned after new investigation techniques were developed. Among them, the isotropic fractionator allows to estimate the number of neurons and non-neurons in a given portion of nervous tissue. To date, this technique has been applied in a number of species; in particular the OB was separately analyzed in numerous mammals and in a single crocodile species. Thus, a quantitative description of the OB's cells is available for a small portion of vertebrates. Main aim of this work was to apply isotropic fractionator to investigate the olfactory capability of elasmobranch fishes, whose traditional concept of outstanding olfaction has recently been scaled down by anatomical and physiological studies. For this purpose, the OB of two elasmobranch species, Galeus melastomus and Scyliorhinus canicula, was studied leading to the determination of the number of neurons vs non-neurons in the OB of the specimens. In addition, the obtained cell quantification was related to the olfactory epithelium surface area to obtain a new parameter that encapsulates both information on the peripheral olfactory organ and the OB. The analyzed species resulted in an overall similar quantitative organization of the peripheral olfactory system; slight differences were detected possibly reflecting different environment preference and feeding strategy. Moreover, the non-neurons/neurons ratio of these species, compared to those available in the literature, seems to place elasmobranch fishes among the vertebrate species in which olfaction plays an important role.

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.

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.

Simulated microgravity induces nuclear translocation of Bax and BCL-2 in glial cultured C6 cells.

Alterations in the control of apoptotic processes were observed in cells during space flight or under simulated microgravity, the latter obtained with the 3D-Random Positioning Machine (3D-RPM). Usually the proteins Bax and Bcl-2, act as pro- or anti-apoptotic regulators. Here we investigated the effects of simulated microgravity obtained by the 3D-RPM on cell viability, localization and expression of Bax and Bcl-2 in cultures of glial cancerous cells. We observed for the first time a transient cytoplasmic/nuclear translocation of Bax and Bcl-2 triggered by changing gravity vector. Bax translocates into the nucleus after 1 h, is present simultaneously in the cytoplasm after 6 h and comes back to the cytoplasm after 24 h. Bcl-2 translocate into the nucleus only after 6 h and comes back to the cytoplasm after 24 h. Physiological meaning, on the regulation of apoptotic event and possible applicative outcomes of such finding are discussed.

Secondary Folds Contribute Significantly to the Total Surface Area in the Olfactory Organ of Chondrichthyes.

The olfactory organ of Chondrichthyes is characterized by a central support with several lamellae covered by a sensory olfactory epithelium. Although secondary folds are present on the lamellae in all the chondrichthyan species analyzed to date, their shape and size have not been described. We here analyze the olfactory organ of 13 elasmobranch and 1 holocephalan species, describe the shape of the secondary folds and evaluate how they contribute to the epithelial surface area. The secondary folds vary in shape and size, and they should always be considered when comparing the epithelial surface area among species; in fact, the increase of the area, due to the presence of the secondary folds, spans from 70 to 495% in the considered species. Because of the complexity of the shapes, we approach the description of the secondary folds by analyzing histological sections of the olfactory lamellae to obtain illustrative silhouettes. We introduce two indexes in order to describe a 2D-sectioned shape of the secondary folds. Considering the different numerical parameters which describe the morphology of the olfactory organ (secondary folds included), a principal component analysis elucidates the possible ecological role and phylogenetic relationship of the chondrichthyan olfactory organ.

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.

The earthworm Dendrobaena veneta (Annelida): A new experimental-organism for photobiomodulation and wound healing.

Photobiomodulation (PBM) is a manipulation of cellular behavior using non-ablative low intensity light sources. This manipulation triggers a cascade of metabolic effects and physiological changes resulting in improved tissue repair, of benefit in the treatment of tissue injury, degenerative or autoimmune diseases. PBM has witnessed an exponential increase in both clinical instrument technology and applications. It is therefore of benefit to find reliable experimental models to test the burgeoning laser technology for medical applications. In our work, we proposed the earthworm Dendrobaena veneta for the study of non-ablative laser-light effects on wound healing. In our preliminary work, D. veneta has been shown to be positively affected by PBM. New tests using D. veneta were set up to evaluate the effectiveness of a chosen 808 nm-64 J/cm2-1W-CW laser therapy using the AB2799 hand-piece with flat-top bean profile, on the wound healing process of the earthworm. Effective outcome was assimilated through examining the macroscopic, histological, and molecular changes on the irradiated posterior-segment of excised-earthworms with respect to controls. Three successive treatments, one every 24 hours, were concluded as sufficient to promote the wound healing, by effects on muscular and blood vessel contraction, decrement of bacteria load, reduction of inflammatory processes and tissue degeneration. D. veneta was demonstrated to be a reliable experimental organism that meets well the 3Rs principles and the National Science Foundation statement. Through their genetic and evolutionary peculiarity, comparable to those of scientifically accredited models, D. veneta allows the effect of laser therapies by multidisciplinary methods, at various degree of complexity and costs to be investigated.

Production, Characterization and Biocompatibility Evaluation of Collagen Membranes Derived from Marine Sponge Chondrosia reniformis Nardo, 1847.

Collagen is involved in the formation of complex fibrillar networks, providing the structural integrity of tissues. Its low immunogenicity and mechanical properties make this molecule a biomaterial that is extremely suitable for tissue engineering and regenerative medicine (TERM) strategies in human health issues. Here, for the first time, we performed a thorough screening of four different methods to obtain sponge collagenous fibrillar suspensions (FSs) from C. reniformis demosponge, which were then chemically, physically, and biologically characterized, in terms of protein, collagen, and glycosaminoglycans content, viscous properties, biocompatibility, and antioxidant activity. These four FSs were then tested for their capability to generate crosslinked or not thin sponge collagenous membranes (SCMs) that are suitable for TERM purposes. Two types of FSs, of the four tested, were able to generate SCMs, either from crosslinking or not, and showed good mechanical properties, enzymatic degradation resistance, water binding capacity, antioxidant activity, and biocompatibility on both fibroblast and keratinocyte cell cultures. Finally, our results demonstrate that it is possible to adapt the extraction procedure in order to alternatively improve the mechanical properties or the antioxidant performances of the derived biomaterial, depending on the application requirements, thanks to the versatility of C. reniformis extracellular matrix extracts.

Permethrin drastically affects the developmental cycle of the non-target slime mould Dictyostelium discoideum.

The use of pyrethroids has increased throughout the world over the past few decades, as organophosphate, carbamate and organochlorine insecticides are being phased out. Permethrin is widely used in the USA for crops treatment, at concentrations around 750 × 103 µg/L. In our study 3.6 µg/L permethrin decreases the fission-rate and the fruiting bodies formation of slime mould Dictyostelium discoideum. Whereas 3.6 × 104 µg/L kills the 100% of amoebae, showing a 24 h-LC50 = 96.6 µg/L. This concentration induces an increase in the pseudocholinesterase activity as well as in both butyrylcholinesterase and heat-shock-protein 70 presence. Our results highlight the high sensitivity of Dictyostelium to permethrin, at concentration of about 105 lesser than what used for agricultural pest control. If we match our results on 6 days of exposure, with the permethrin relatively slow permanence (30 days) in the aerobic soil, as well as the higher effect of permethrin than organophosphate, carbamate and organochlorine pesticides on D. discoideum, the damage on the dictyostelids community, by use of permethrin, is clear. Our data suggest that, if the sustainable agriculture implementation is a topic of the modern "industrial" farming, the permethrin cannot represent a reliable alternative to organochlorine, organophosphate or carbamate pesticides, in implementing Integrated Pest Management programmes.

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.

Clarification of the Terminology of the Olfactory Lamellae in Chondrichthyes.

Several papers regard the anatomy of the peripheral olfactory organ, the olfactory rosette, in the class of Chondrichtyes. The complex shape of this organ and the differences among species give clues to functional, evolutionary, and ecological observations; data on a larger number of species are needed in order to have a more complete insight. The rosette is made up of a central support and of numerous lamellae, which are lined by the sensory epithelium. The size, shape and number of these lamellae, which are highly variable among species, are noteworthy because they affect the sensory surface area, the water dynamic within the olfactory chamber, and the organization of the olfactory bulb. In the literature about Chondrichthyes, the definition of olfactory lamella is confused, because authors use the same words for different structures. The average number of lamellae is distinctive for each species, and the meaning of this difference is not completely understood and, in this frame, a not unambiguous definition of lamella leads to difficulties in comparing data from different publications and analyzing them together. We aim to give here an unambiguous definition of olfactory lamella, that should be each single fold of tissue extending from the raphe, and of lamellar number of a given species, that should be the average number of lamellae in one olfactory organ. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:2039-2045, 2017. © 2017 Wiley Periodicals, Inc.

Gross anatomy and histology of the olfactory rosette of the shark Heptranchias perlo.

Sharks belonging to the family Hexanchidae have six or seven gill slits, unlike all other elasmobranchs, which have five gill slits. Their olfactory organs have a round shape, which is common for holocephalans, but not for elasmobranchs. Thus, the shape of the olfactory organ represents a further, less striking, peculiarity of this family among elasmobranchs. Despite that, the microscopic anatomy and histology of the olfactory organ have not yet been studied in any species of this family. Here, an anatomical and histological description of the olfactory organ of the sharpnose sevengill shark Heptranchias perlo is given. The organ is a rosette, with a central raphe and 31-34 primary lamellae, which bear secondary lamellae with a more or less branched shape. The elastic connective capsule which envelops the olfactory rosette possibly changes its shape along with water influx. In the olfactory epithelium, the supporting cells also have a secretory function, while no specialized mucous cells are visible; regarding this feature the olfactory epithelium of H. perlo differs from that of other chondrichthyan species. The immunohistochemical investigation of the sensory epithelium shows the absence of immunoreactivity for Gαolf in receptor neurons, which confirms previous observations in Chondrichthyes.

The Effect of Photobiomodulation on the Sea Urchin Paracentrotus lividus (Echinodermata) Using Higher-Fluence on Fertilization, Embryogenesis, and Larval Development: An In Vitro Study.

OBJECTIVE: The aim of this study was to investigate the photobiomodulation (PBM) effect of the 808 nm diode laser irradiation on spermatozoa, eggs, fertilized eggs, embryos, and larvae of Paracentrotus lividus, using two different power settings. BACKGROUND DATA: Studies have shown the possible use of PBM in artificial insemination. These have shown the potential effect of low-power laser irradiation on spermatozoa, while there are few studies on the effect of laser photonic energy on oocytes and almost no reports on the influence of lasers in embryogenesis. METHODS: P. lividus gametes, zygotes, embryos, and larvae were irradiated using the 808 nm diode laser (fluence 64 J/cm2 using 1 W or 192 J/cm2 with 3 W) with a flat-top hand-piece delivery, compared to a control without laser irradiation (0 J/cm2-0 W). The fertilization rate and the early developmental stages were investigated. RESULTS: The fertilization ability was not affected by the sperm/egg irradiation. At the gastrula stage, no significant differences were observed compared with the control samples. In the late pluteus stage, there were no differences in the developmental percentage observed between the control and the treated samples (1 W), with the exception of larvae from gastrulae and larvae, which were irradiated at 3 W. CONCLUSIONS: This study has demonstrated that both the 64 J/cm2-1 W and the 192 J/cm2-3 W do not induce morphological damage on the irradiated P. lividus gametes whose zygotes generate normal embryos and larvae. Our data therefore support the assumption to use higher fluence in preliminary studies on in vitro fertilization.

Photobiomodulation by Infrared Diode Laser: Effects on Intracellular Calcium Concentration and Nitric Oxide Production of Paramecium.

In Paramecium, cilia beating is correlated to intracellular calcium concentration ([Ca2+ ]i) and nitric oxide (NO) synthesis. Recent findings affirm that photobiomodulation (PBM) can transiently increase the [Ca2+ ]i in mammalian cells. In this study, we investigated the effect of both 808 and 980 nm diode laser irradiated with flat-top hand-piece on [Ca2+ ]i and NO production of Paramecium primaurelia, to provide basic information for the development of new therapeutic approaches. In the experiments, the laser power in CW varied (0.1; 0.5; 1; and 1.5 W) to generate the following respective fluences: 6.4; 32; 64; and 96 J cm-2 . The 6.4 J cm-2 did not induce PBM if irradiated by both 808 and 980 nm diode laser. Conversely, the 32 J cm-2 fluence had no effect on Paramecium cells if irradiated by the 808 nm laser, while if irradiated by the 980 nm laser induced increment in swimming speed (suggesting an effect on the [Ca2+ ]i, NO production, similar to the 64 J cm-2 with the 808 nm wavelength). The more evident discordance occurred with the 96 J cm-2 fluence, which had the more efficient effect on PBM among the parameters if irradiated with the 808 nm laser and killed the Paramecium cells if irradiated by the 980 nm laser. Lastly, the 980 nm and 64 or 96 J cm-2 were the only parameters to induce a release of stored calcium.

Aquaporin in Chondrosia reniformis Nardo, 1847 and Its Possible Role in the Interaction Between Cells and Engulfed Siliceous Particles.

The sponge Chondrosia reniformis selectively engulfs siliceous particles that, when in crystalline form, become quickly dissolved in its ectosome. The molecular mechanism, identity, and physiological significance of the cells involved in this process are not completely understood. In the present study, we applied light and electronic microscopic techniques to show how the quartz particles in C. reniformis are enveloped through collagen fibers and host cells near the surface of these organisms. As various aquaporins from bacteria, animals, and plants bidirectionally conduct metalloids-including silicon ions--through the cell membrane, the presence and potential involvement of aquaporins in quartz dissolution in C. reniformis have been investigated. An aquaporin-like transcript (CrAQP) was isolated according to the transcriptome sequencing results in C. reniformis The full-length CrAQP cDNA is 907 nucleotides long, with a 795-base pair (bp), open reading frame encoding a protein of 265 amino acids, a 29-bp, 5'-non-coding region, and a 83-bp, 3'-untranslated region. The Bayesian phylogenetic inference suggests that CrAqp is closely related to the Aqp8L grade, which is also implicated in H2O2 transport. Quantification of CrAQP mRNA through qPCR indicated that the transcript level was higher in the ectosome than in the choanosome. Immunofluorescence of a mammalian AQP8 in C. reniformis showed positivity in some cells near the quartz particles, a finding that may support the initial hypothesis of the potential involvement of CrAQP in quartz erosion. However, the features of the primary structure of this protein offer a new viewpoint about the functional role of these molecules in this process: that CrAQP may be involved in the permeation of H2O2 released during silica erosion.

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.

First Description of a Palatal Organ in Chimaera monstrosa (Chondrichthyes, Holocephali).

Chimaeroid fishes are the only extant Holocephali, a subclass of Chondrichthyes. We describe for the first time a well evidenced structure localized in the palate of the chimaeroid Chimaera monstrosa, here named a palatal organ (PO). Attention has been paid to the holocephalan head morphology, but there has been no mention of this particular organ in the literature. The PO is a soft-tissue mass located within a slight hollow in between the two vomerine toothplates, and it protrudes into the oral cavity, resembling the mammalian incisive papilla. It is characterized by dense connective tissue with abundant collagen and elastic fibers and no muscular tissue. The robust innervation but low density of taste buds suggest a role in gustation for the PO, but primary utility in general mechanical sensitivity likely implicated in food sorting. The presence of numerous multicellular serous glands in the anterior/dorsal part of the PO is quite surprising because, in gnathostome fish, the presence of multicellular glands within the mouth has been reported in only the rare case of teeth-associated venom glands. Hypothesized roles for these glands could include food lubrication, digestion and defense against pathogens. In the literature, the presence of a PO has been demonstrated in many published images of chimaeroid fishes, but has gone unnoticed. This trait could represent a peculiar characteristic of all or a subset of holocephalans.

Molecular Cloning, Characterization, and Expression Analysis of a Prolyl 4-Hydroxylase from the Marine Sponge Chondrosia reniformis.

Prolyl 4-hydroxylase (P4H) catalyzes the hydroxylation of proline residues in collagen. P4H has two functional subunits, α and ß. Here, we report the cDNA cloning, characterization, and expression analysis of the α and ß subunits of the P4H derived from the marine sponge Chondrosia reniformis. The amino acid sequence of the α subunit is 533 residues long with an M r of 59.14 kDa, while the ß subunit counts 526 residues with an M r of 58.75 kDa. Phylogenetic analyses showed that αP4H and ßP4H are more related to the mammalian sequences than to known invertebrate P4Hs. Western blot analysis of sponge lysate protein cross-linking revealed a band of 240 kDa corresponding to an α2ß2 tetramer structure. This result suggests that P4H from marine sponges shares the same quaternary structure with vertebrate homologous enzymes. Gene expression analyses showed that αP4H transcript is higher in the choanosome than in the ectosome, while the study of factors affecting its expression in sponge fragmorphs revealed that soluble silicates had no effect on the αP4H levels, whereas ascorbic acid strongly upregulated the αP4H mRNA. Finally, treatment with two different tumor necrosis factor (TNF)-alpha inhibitors determined a significant downregulation of αP4H gene expression in fragmorphs demonstrating, for the first time in Porifera, a positive involvement of TNF in sponge matrix biosynthesis. The molecular characterization of P4H genes involved in collagen hydroxylation, including the mechanisms that regulate their expression, is a key step for future recombinant sponge collagen production and may be pivotal to understand pathological mechanisms related to extracellular matrix deposition in higher organisms.