Tiziana Cubeddu

Non-tuberculous mycobacteria are recognized as pathogens affecting aquatic animals and humans. Although Mycobacterium marinum is traditionally considered the principal etiological agent of fish mycobacteriosis and fish-tank granuloma in humans, the contribution of Mycobacterium chelonae in fish as well as in humans remains underestimated, particularly in Ziehl–Neelsen (ZN)-negative lesions. In this study, granulomatous lesions of marine fish species and human skin biopsies were investigated using histopathology and molecular diagnostics. Following histological evaluation, all samples were analysed by PCR amplification and sequencing of the hsp65 gene to identify the causative agent. Fish and human tissues showed similar chronic granulomatous lesions, with all cases negative for acid-fast bacilli by ZN staining. Molecular analysis consistently identified M. chelonae in both marine fish organs and human skin lesions exhibiting clinicopathological features traditionally attributed to M. marinum. Phylogenetic analysis revealed high genetic similarity between fish- and human-derived sequences, suggesting shared environmental reservoirs rather than host-specific lineages. These findings identify M. chelonae as an underrecognized cause of granulomatous disease in fish and humans and demonstrate the limitations of histochemical methods in these infections. Incorporating molecular diagnostics into routine investigations of granulomatous disease supports more accurate diagnosis across veterinary and human medicine and strengthens One Health surveillance of aquatic non-tuberculous mycobacterial infections.

Innovation in the field of bioinspired therapeutic anticoagulants, as an alternative to heparin and its derivatives, is increasingly focused toward the discovery of new molecules from natural sources. Inspired by the compelling observation that marine organisms possess a wide array of acidic polysaccharides (APs) within their extracellular matrix (ECM) with promising anticoagulant potential, this study investigates AP topographic distribution, content, and bioactivity in the body of seven phylogenetically distant Mediterranean sponge species. Tissue architecture was evidenced by Alcian Blue histological staining whereas biochemical analyses were carried out on APs purified from papain-digested tissue extracts using anion-exchange chromatography. Four polysaccharide fractions were obtained and assessed for hexuronic acid content. The two most abundant ones, up to 98% of the total, were characterized by electrophoretic analyses, and assessed in vitro for inhibitory activity on blood coagulation. Histology evidenced a heterogeneous distribution of APs within the sponge’s ECM, along with marked interspecific variability in both concentration and electrophoretic profiles. Neither Tethya aurantium nor Crambe crambe showed any significant in vitro effects on coagulation, whereas the other species exhibited a strong inhibitory effect on both activated partial thromboplastin time (aPTT) and thrombin time (TT), comparable to standard heparin (Hep). Concerning aPTT, both I. retidermata and H. communis reached the endpoint of 300 s at 5 µgUA/mL, whereas S. spinosulus, O. lobularis, A. aerophoba reached it at 10 µgUA/mL. With reference to TT, A. aerophoba, I. retidermata, S. spinosulus and H. communis had comparable effects on coagulation time with respect to Hep (endpoint at 5 µgUA/mL), whereas O. lobularis was less effective (endpoint at 100 µgUA/mL). These findings show that Porifera, one of the most basal Metazoa, have an ECM rich in APs with anticoagulant properties towards both intrinsic and common pathways of coagulation, consistent with known inhibitory mechanisms reported for certain marine sulfated polysaccharides derived from other invertebrate taxa. These molecules, obtainable through sustainable blue technology, represent compelling candidates for bioinspired next-generation anticoagulant therapeutics, with broader applications in regenerative medicine and tissue engineering.

Faunistic investigations on freshwater bodies of the Bangkok Metropolitan Area resulted in the discovery of a freshwater sponge population fragmented in small lentic microhabitats at the Suan Luang Rama IX Park. Sponges living in the lotus and waterlily garden and tropical greenhouse were photographed in situ, and some representative, small samples were collected and stored at the Division of Biology (Porifera Collection), Rajamangala University of Technology Krungthep, Bangkok. The taxonomic integrative analyses focused on diagnostic morphotraits, barcoding, and biogeography. The sponge population was ascribed to Radiospongilla cf. cerebellata (Porifera: Demospongiae: Spongillida: Spongillidae) and is a new record for the Bangkok area and the entire Thai inland water. Comparative integrative analyses vs spongillid taxa worldwide resulted in the discovery of new gemmular morphotraits for the genus. Although the Thai Radiospongilla cf. cerebellata diverges from the Indian type of Radiospongilla cerebellata at the level of gemmular theca architecture, molecular analyses highlighted the matching of the former with the Radiospongilla cluster. A possible allochthonous origin of the Bangkok Radiospongillas population is hypothesised. This investigation on botanical gardens aquatic fauna and a review of appropriate literature highlights that sponges are able to inhabit very confined and fragmented, shallow water bodies, i.e., scattered terracotta pots, plastic containers, and small ponds with aquatic plants also in megalopolis gardens.

Meagre (Argyrosomus regius) is one of the fast-growing species considered for sustainable aquaculture development along the Mediterranean and Eastern Atlantic coasts. The emergence of Systemic Granulomatosis (SG), a disease marked by multiple granulomas in various tissues, poses a significant challenge in meagre aquaculture. In the current study, we investigate the association of Mycobacterium spp. and SG in offshore aquaculture facilities in Sardinia, Italy. A total of 34 adult seemingly healthy meagre were arbitrarily collected and analyzed, combining histological, microbiological, molecular, metagenomics, and in situ techniques to investigate the presence of pathogens. Ziehl–Neelsen (ZN), periodic acid–schiff (PAS), and Giemsa stains were performed for the detection of acid-fast bacteria, common parasites, and fungi within granulomas, respectively. Granulomas were detected in 91% (31/34) of fish. The affected organs were kidney (88%), liver (47%), heart (41%), intestine (17.6%), and brain (5%). Acid-fast staining, along with Mycobacterium spp. specific quantitative PCR (qPCR), in situ hybridization (ISH) assay, and microbiological analyses showed negative results for the detection of Mycobacterium spp. and other bacteria implicated in granuloma formation. However, PCR amplification and sequencing of the 65-kDa heat shock protein gene revealed the presence of M. chelonae in 13% of both formalin-fixed and frozen liver tissues. Bacterial isolation failed to detect nontuberculous mycobacteria (NTM) and other bacteria typically associated with granulomas. Consistently, the use of an M. chelonae-specific probe in ISH failed to identify this bacterial species in granulomas. Collectively, results do not support the role of M. chelonae in the development of granulomas and suggest rejecting the hypothesis of a potential link between NTM and SG.

A new occurrence of the genus Corvospongilla (Porifera: Demospongiae) is recorded from the Southeast Asia freshwater of Khorat Plateau, northeast Thailand. This is the second record of the genus from the Lower Mekong Basin with the discovery the new species Corvospongilla lampaoensis sp. nov. inhabiting a eutrophic lentic habitat (in Lam Pao Reservoir) on manmade structures of fish farming and fishery. Skeleton and gemmules morphotraits, diagnostic for Genus Corvospongilla of the family Spongillidae (Order Spongillida), were examined by Light and Scanning Electron Microscopy differentiating a new species from its twenty congeners known worldwide. In addition, the comparative morpho-analysis highlighted distant sympatric lineages with a notable morphotraits divergence of Corvospongilla lampaoensis sp. nov. vs Corvospongilla siamensis in two western tributaries of the Lower Mekong. The results from this study expand our knowledge of the order Spongillida adaptive radiation in Asia and fill a gap in the geographic range of the genus Corvospongilla in the Oriental-Indomalayan Region.

Thromboembolic conditions are the most common cause of death in developed countries. Anticoagulant therapy is the treatment of choice, and heparinoids and warfarin are the most adopted drugs. Sulphated polysaccharides extracted from marine organisms have been demonstrated to be effective alternatives, blocking thrombus formation by inhibiting some factors involved in the coagulation cascade. In this study, four acidic glycan fractions from the marine sponge Sarcotragus spinosulus were purified by anion-exchange chromatography, and their anticoagulant properties were investigated through APTT and PT assays and compared with both standard glycosaminoglycans and holothurian sulphated polysaccharides. Moreover, their topographic localization was assessed through histological analysis, and their cytocompatibility was tested on a human fibroblast cell line. A positive correlation between the amount of acid glycans and the inhibitory effect towards both the intrinsic and extrinsic coagulation pathways was observed. The most effective anticoagulant activity was shown by a highly charged fraction, which accounted for almost half (about 40%) of the total hexuronate-containing polysaccharides. Its preliminary structural characterization, performed through infrared spectroscopy and nuclear magnetic resonance, suggested that it may consist of a fucosylated chondroitin sulphate, whose unique structure may be responsible for the anticoagulant activity reported herein for the first time.

This study compares the skeletal calcification pattern of batoid Raja asterias with the endochondral ossification model of mammalians Homo sapiens and teleost Xiphias gladius. Skeletal mineralization serves to stiffen the mobile elements for locomotion. Histology, histochemistry, heat deproteination, scanning electron microscopy (SEM)/EDAX analysis, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and Fourier transform infrared spectrometry (FTIR) have been applied in the study. H. sapiens and X. gladius bone specimens showed similar profiles, R. asterias calcified cartilage diverges for higher water release and more amorphous bioapatite. In endochondral ossification, fetal calcified cartilage is progressively replaced by bone matrix, while R. asterias calcified cartilage remains un‐remodeled throughout the life span. Ca²⁺ and PO₄³⁻ concentration in extracellular matrix is suggested to reach the critical salts precipitation point through H₂O recall from extracellular matrix into both chondroblasts or osteoblasts. Cartilage organic phase layout and incomplete mineralization allow interstitial fluids diffusion, chondrocytes survival, and growth in a calcified tissue lacking of a vascular and canalicular system.Highlights: Comparative physico‐chemical characterization (TGA, DTG and DSC) testifies the mass loss due to water release, collagen and carbonate decomposition of the three tested matrices. R. asterias calcified cartilage water content is higher than that of H. sapiens and X. gladius, as shown by the respectively highest dehydration enthalpy values. Lower crystallinity degree of R. asterias calcified cartilage can be related to the higher amount of collagen in amorphous form than in bone matrix. These data can be discussed in terms of the mechanostat theory (Frost, 1966) or by organic/inorganic phase transformation in the course evolution from fin to limbs. Mineral analysis documented different charactersof R. asterias vs H. sapiens and X. gladius calcified matrix.

This paper focuses on the skeletal architecture and morphotraits of the Mediterranean horny sponge Sarcotragus spinosulus (Demospongiae, Keratosa, Dictyoceratida, Irciniidae). This special endoskeletal system consists of a dense, variably complex connective architecture, which extends throughout the entire sponge body and is embedded in an abundant jelly-like extracellular matrix (ECM). To investigate the topographic arrangement and micro-morphotraits of these connective structures in detail and by colour, also during morphogenetic processes, histology techniques using light microscopy are essential. New information is provided on the coordinated morphogenetic processes that characterize the growth and assembly of collagenic prototype structures in the matrix of fibrous skeletal elements and drive skeleton remodelling. Our results also highlight some novelties and some remarkable peculiarities of fibrous, filamentous and fibrillar components at the levels of both composition and structure. The morphofunctional significance of skeletal architecture is suggested in the background of the anatomical complexity of S. spinosulus.