Can synergistic effects of marine heatwaves and Vibrio proliferation act as potential triggers of widespread demosponge disease? A case study in the Mediterranean Sea

Can synergistic effects of marine heatwaves and Vibrio proliferation act as potential triggers of widespread demosponge disease? 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A case study in the Mediterranean Sea Loredana Stabili, Elisa Quarta, Francesca Necci, Andrea Toso, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8987753/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Marine heatwaves associated with climate change are increasingly creating favourable conditions for the proliferation of pathogenic bacteria, leading to disease outbreaks and mass mortality events particularly in sessile suspension-feeding invertebrates, such as sponges. In summer 2024, a disease outbreak affecting two demosponge species, Petrosia ficiformis and Agelas oroides , was documented along the Northern Ionian coast of the Mediterranean Sea (Italy). Diseased sponges exhibited extensive surface necrosis or multiple lesions distributed across the body. Necrotic areas appeared whitish and were frequently coated with a thin mucous layer composed of bacterial aggregates. Microbiological culture analyses revealed elevated bacterial densities on sponge surfaces, with total culturable bacteria reaching 4.3 ± 0.2 × 10⁵ CFU/g and culturable vibrios 7.0 ± 0.4 × 10⁴ CFU/g in P. ficiformis , and 3.6 ± 0.3 × 10⁵ CFU/g and 6.6 ± 0.2 × 10⁴ CFU/g, respectively, in A. oroides . All Vibrio isolates obtained from the conspicuous whitish lesions on diseased sponges were identified as Vibrio alginolyticus , based on concordant phylogenetic, morphological, cultural, and biochemical analyses. Vibrio alginolyticus is a well-established pathogen of numerous aquatic organisms, suggesting that marine heatwaves may enhance Vibrio abundance and increase infection frequency during summer periods. Nevertheless, the specific factors initiating the epidemic in the examined sponge populations remain unresolved. We hypothesize that disease onset involves a synergistic mechanism in which environmental stressors, such as elevated temperature, increased nutrient availability, and reduced water circulation, promote the transition of sponge-associated bacteria, particularly V. alginolyticus , toward virulence. Under these conditions, host physiological defences may be compromised, allowing uncontrolled bacterial proliferation. Overall, our findings indicate that the interaction between thermal stress and pathogenic vibrios represents a plausible trigger for sponge disease outbreaks. Further investigations are required to elucidate the etiological pathways involved and to identify the mechanisms underlying sponge recovery following epidemic events that result in mass mortality. A substantial decline in key sponge species may have serious consequences for ecosystem functioning, given their critical role as filter feeders in marine bioremediation and habitat health. Iinjured sponge Epidemic disease Vibrio alginolyticus Climate change Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Human activities are exerting growing pressure on marine ecosystems, leading to significant environmental degradation. Among the most critical outcomes of this anthropogenic impact are rising pollution levels and increasing global temperatures [ 1 , 2 ]. These factors not only threaten marine biodiversity but also create conditions favoriting the proliferation of non-indigenous, thermophilic species and harmful microorganisms, some of which may be invasive or even pathogenic [ 3 , 4 ]. In particular, several above mentioned factors can promote disease outbreaks in marine animals, leading to mass mortality events [ 5 ]. This issue is particularly critical in the Mediterranean Sea, a region strongly influenced by both global and local environmental changes. Over recent decades, multiple mass mortality events affecting sessile benthic invertebrates, such as sponges, anthozoans, bivalves, and ascidians, have been documented throughout the Mediterranean basin [ 6 – 17 ]. In this scenario, there is broad consensus that global warming contributes to the emergence of these severe ecological crises in the Mediterranean Sea [ 18 ]. Elevated seawater temperatures increase the metabolic and respiratory demands of organisms while simultaneously reducing the availability of nutrients and food resources due to “thermal stratification”. Two of the most significant mass-mortality events in the north-western Mediterranean, both in terms of spatial extent (approximately 1,000 km of coastline) and biodiversity impact (around 30 macro-benthic species affected), occurred during the summers of 1999 and 2003. These events coincided with pronounced positive thermal anomalies, characterized by sea temperatures 3–4°C above average and prolonged stability of the water column in late summer [ 18 – 22 ]. Globally, the mean temperature for the decade 2014–2023 was 1.20 ± 0.12°C higher than the 1850–1900 baseline [ 22 ]. This warming trend intensified further in the summer of 2024, when temperatures exceeded those recorded in 2023. Notably, July of both 2023 and 2024 was marked by severe heatwaves across multiple regions of the Northern Hemisphere [ 22 ]. As a consequence, many marine ecosystems are in a poor to bad state [ 23 – 25 ]. During prolonged and unusually warm summers, benthic suspension feeders experience prolonged energetic constraints, determining a compromised stressed physiological state that often culminates in mortality during late summer or early autumn [ 26 , 27 ]. For filter-feeding invertebrates in particular, elevated seawater temperatures can disrupt key physiological functions - such as filtration efficiency, nutrient assimilation, and symbiotic interactions with microorganisms [ 28 ]. These impairments can weaken the organisms’ capacity to cope with environmental fluctuations, thereby increasing their susceptibility to disease and predation [ 29 , 30 ]. In this context, opportunistic microbial infections are suspected contributors, although their exact role remains unclear. A consistent pattern emerging from studies over the past decades indicates that climate change is likely to increase the frequency, severity, and geographic range of disease outbreaks in both terrestrial and marine systems [ 31 , 32 ]. Pathogens and the diseases they cause constitute a significant constraint on ecosystems, with broad social, economic, and environmental consequences. Changing environmental parameters, such as rising temperatures, often increase host susceptibility to infection. Sessile marine invertebrates, constantly threatened by a rich mix of microorganisms present in the surrounding waters, are vulnerable to pathogen attack [ 33 ]. In particular, because Vibrio species thrive at relatively high temperatures, outbreaks in marine animals are expected to become increasingly frequent due to global warming. For example, in recent decades, Vibrio have been implicated in the "bleaching" of subtropical and tropical corals worldwide [ 34 ]. Like other sessile benthic groups, sponges despite their capacity for tissue regeneration, have experienced recurrent mass-mortality events globally [ 8 , 35 , 40 ]. Evidence from long-term monitoring in the Mediterranean Sea suggests that the frequency of these sponge mortality episodes has increased markedly over the last twenty years [ 14 , 15 , 41 , 42 ]. These disease events affect numerous sponge species and occur across wide geographic regions, with impacted sponges exhibiting diverse physiological symptoms. In sponges, as in other marine invertebrates, several environmental drivers, most notably ocean warming and acidification, have been implicated in both the onset and subsequent spread of disease outbreaks [ 10 , 11 , 43 – 48 ]. Persistent thermal stress may also cause tissue necrosis and bleaching in species that rely on photosynthetic symbionts, ultimately leading to death [ 29 ]. Several studies have linked sponge mass-mortality events to marine heatwaves, during which anomalously high temperatures persist for extended periods [ 4 , 30 , 31 ]. Although most studies attribute sponge diseases to microbiome dysbiosis, only a few of them have successfully isolated specific pathogenic agents [ 14 , 15 , 35 , 48 ]. In the Mediterranean Sea, the best-documented disease episodes are referred to the commercially important sponges [ 11 , 12 , 36 , 45 , 49 ] and were likely due to bacteria capable of degrading spongin fibres [ 10 , 36 ]. For other species, Corriero et al. [ 50 ] reported a similar pattern of fibre deterioration across multiple keratose sponges, including Spongia, Sarcotragus , and Ircinia , suggesting a shared bacterial etiology. Maldonado et al. [ 14 ] described a recurrent disease in Ircinia spp. that re-emerges annually, typically after the hottest months. This condition was attributed to an external bacterium, presumably a Vibrio sp., that invades and colonizes the sponge tissues. Likewise, Stabili et al. [ 36 ] documented a disease event in Ircinia variabilis (Schmidt, 1862) observed in September 2009 along the southern Adriatic and Ionian Seas (Apulian coast). The injured sponges displayed extensive surface necrosis and, in severe cases, fragmentation of the body into multiple portions. The necrotic zones appeared whitish and were frequently coated with a thin mucous coat associated with Vibrio proliferation. Several evidence indicate that Vibrio -related diseases are increasing worldwide in response to ocean warming [ 51 ]. Additional studies have linked Vibrio infections to rising mass-mortality events in coastal marine organisms [ 52 – 54 ]. In the present study, we report a recently observed disease outbreak affecting populations of the sponge species Petrosia ficiformis (Poiret, 1789) and Agelas oroides (Schmidt, 1864) along the Northern Ionian Sea (Mediterranean Sea, Italy) in the summer 2024. We document the extent of the outbreak and assess its possible association with Vibrio infection, regional marine heatwaves, and the exceptionally high seawater temperatures recorded during this period. 2. Materials and Methods 2.1 Study sites and studied material During the summer of 2024, episodes of tissue necrosis with whitish areas in the Demospongiae Petrosia ficiformis and Agelas oroides were reported along the Apulian coast (Ionian Sea, Italy, southeastern Mediterranean). Several areas of the Apulian coast were examined to assess the extent of the phenomenon. Specifically, this event was observed in the areas of Otranto (Adriatic Sea), Porto Badisco (Ionian Sea), Santa Maria di Leuca (Ionian Sea), and Santa Caterina (Ionian Sea). For this reason, a field survey was conducted in September 2024, at Santa Maria al Bagno (40.1297° N; 17.9980° E) (Fig. 1 ). The site consists mainly of rocky bottoms alternating with sandy patches, where the substrate includes both exposed surfaces and shaded crevices, creating favourable conditions for benthic assemblages dominated by photophilic algal communities and sponges. Sampling was conducted by scuba diving between 4 and 6 m depth. A linear transect (50 m long and 1 m wide) was placed parallel to the shoreline, and every visible specimen of P. ficiformis and A. oroides was photographed using an Olympus TG-6 camera. The photographs were subsequently examined to assess the frequency of damaged versus healthy specimens. Additionally, small tissue samples (approximately 1 cm³) were collected from affected individuals (n = 15 for P. ficiformis and n = 7 for A. oroides ) for microbiological screening to detect possible pathogenic bacteria. Additionally, seawater and sediment samples were collected from the areas surrounding the diseased sponges, with the sediments exhibiting a whitish biofilm similar to that observed on the tissues of affected sponges. 2.2 Marine Heat Waves calculation In addition, subsurface Marine Heat Waves (MHWs) were detected and quantified following Hobday et al. [ 56 ], which defines them as discrete, prolonged events during which sea temperature exceeds the seasonally varying 90th percentile for at least five consecutive days. We used daily Sea water temperature data from the Mediterranean Sea Physics Reanalysis (Copernicus Marine Service) [ 57 ]. The period 1994–2010 was used to derive the climatological baseline and the seasonally varying 90th percentile thresholds, while MHWs were identified for the period 2011–2024. Temperature values were extracted at the closest depth in the model for 5 m (5.46 m), from the pixel, with spatial resolution 0.042° × 0.042° (~ 4.5 km), corresponding to the coordinates of the site Santa Maria al Bagno. Based on the starting date of each event, MHWs were classified into: Cold-season events (initiated in autumn or winter: October - March), and Warm-season events (initiated in spring or summer: April - September). 2.3 Enumeration, isolation and phylogenetic analysis of vibrios In the laboratory, fragments from damaged P. ficiformis and A. oroides specimens were gently rinsed with sterile, 0.2 µm filtered seawater to remove the bacteria settled on the surfaces. The fragments were then suspended in sterile seawater and subjected to three sonication cycles (Branson Sonifier 2200; 60 W, 47 kHz; 1 min per cycle in an ice bath) to optimize detachment of surface-associated bacteria. Sonication was interrupted for 30 s between cycles, during which samples were shaken manually. To quantify surface-associated bacteria, including Vibrio spp., 1 or 5 mL of each sonicated sample and appropriate decimal dilutions were plated in parallel on Marine Agar 2216 or thiosulfate–citrate–bile salts–sucrose (TCBS) agar (Becton Dickinson and Company). Culturable colonies were enumerated using the CFU method following incubation at 22°C for 7 days (Marine Agar) and at 30°C for 48 h (TCBS). Additionally, fragments from injured tissues exhibiting extensive whitish lesions, were rinsed in sterile, 0.2 µm filtered seawater to remove surface bacteria, then placed directly onto TCBS agar and incubated at 30°C for 48 h. All distinct colonies were isolated and streaked onto Marine Agar to obtain pure cultures. Phenotypic identification of isolates followed the procedures reported by Stabili et al. [ 36 ]. The assays included: Gram staining, cell morphology, motility, growth on TCBS agar, oxidase, catalase, and urease activities, H 2 S production, sensitivity to vibriostatic agent O/129, oxidative/fermentative metabolism, and amino acid decarboxylase reactions. Growth tolerance was tested at 0, 3, and 8% NaCl and at 4°C, 30°C, and 35°C. Additional tests included indole production, gelatinase activity, Voges–Proskauer reaction, citrate utilization, casein and aesculin hydrolysis, and acid production from inositol, arabinose, and salicin. Carbon-source utilization (L-arabinose, N-acetylglucosamine, D-mannose, D-cellobiose, D-glucose, galactose, D-trehalose, D-melibiose, lactose, D-mannitol, sorbitol, amygdalin, D-fructose, glycerol, glycogen, maltose, and sucrose) was evaluated in the presence of added NaCl. Nitrate reduction was also assessed. 2.4 16S rDNA gene sequence analysis of bacterial isolates Bacterial isolates were grown in 100 mL nutrient broth (Beckton Dickinson and Company) containing 3% NaCl with shaking at 30°C to an optical density of 0.8 at 550 nm. High-molecular-weight genomic DNA from the different Vibrio bacterial isolates was obtained using the NucleoSpin Microbial DNA KIT (Macherey-Nagel), and the extracted DNA was stored at 4°C before PCR and sequencing experiments. The extracts were analyzed both quantitatively and qualitatively using agarose gel electrophoresis (1%, 75 V) and spectrophotometric measurements (NanoDrop). The 16S rRNA gene was amplified using AmpliTaq (Thermo Fisher Scientific) and the primer pairs COM1F/COM2F, 785F/1488R, and 20-43F/683R, as previously reported [ 57 ]. The thermal cycling conditions were as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles (denaturation at 95°C for 45 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min), with a final extension at 72°C for 5 min. The quality and quantity of each amplicon were assessed by agarose gel electrophoresis and spectrophotometric measurements, as previously reported. Amplicons were sequenced using the Sanger method (Eurofins Genomics, Ebersberg, Germany). For each isolate, the three resulting amplicons were assembled as previously described [ 58 ]. The obtained sequences were used for taxonomic identification against the rRNA/ITS database via NCBI BLAST. Multiple sequence alignment was performed using the MAFFT tool [ 59 ], and phylogenetic analysis was conducted with IQ-TREE [ 60 ] to construct a phylogenetic tree. The parameters of the tools used, and the reference sequences were as previously reported [ 42 ]. 2.4 Statistical analysis Analysis of variance (ANOVA) was performed using STATISTICA 10.0 software to evaluate differences in bacterial counts between diseased sponges, sediment, and water samples. Significance was set at a critical level of 95% ( p < 0.05). 3. Results 3.1 Impact of the disease on Biota During the summer of 2024, episodes of tissue necrosis were observed in different benthic organisms, such as sponges Petrosia ficiformis, Agelas oroides, Sarcotragus spinosulus (Schmidt, 1862), but also the green algae such as Codium bursa (Linnaeus - C. Agardh 1817) (Fig. 2 ). The coastal zone survey recorded a density of 0.74 ind/m² for P. ficiformis and 0.14 ind/m² for A. oroides . Most sponges exhibited a whitish biofilm, and a necrotic tissue was observed in both species. Moreover, only one individual of P. ficiformis was found completely dead (Fig. 3 ). 3.2 Marine heat waves in the period 2011–2024 Between 2011 and 2024, a total of 68 marine heatwave (MHW) events were identified at 5 m depth (Table S1 ). Event duration ranged from 5 to 119 days, with a marked variability in both temporal persistence and thermal structure. Mean intensity values (above the threshold) varied between 0.84 and 3.42°C, while maximum intensity (above the threshold) reached up to 4.92°C. The seasonal distribution of marine heatwaves out of the 68 total events is: 34 MHWs were classified as cold-season events, and 34 MHWs as warm-season events, indicating an overall balanced seasonal frequency. Despite the similar number of events, the thermal characteristics differed markedly between seasons. Warm-season MHWs generally exhibited higher intensity and cumulative impact compared to cold-season events. The most intense summer-origin MHW occurred in 2019, lasting 119 days, with a cumulative intensity of 242.88°C·days, representing the most powerful event of the entire time series. In contrast, cold-season MHWs tended to exhibit lower mean intensities; however, several events were characterized by exceptionally long durations, resulting in high cumulative thermal stress. The most intense cold-season event occurred between December 2013 and April 2014, lasting 110 days and reaching a cumulative intensity of 170.47°C·days. Similarly, prolonged winter events were observed in 2022–2023 and 2023–2024, both of which exceeded 90 days. From a temporal perspective, the dataset reveals an increase in the frequency, duration, and cumulative intensity of MHWs after 2015, with particularly pronounced events occurring after 2018. The period 2019–2024 is characterized by recurrent long-lasting and high-intensity events, affecting both warm and cold seasons. In 2024, marine heatwaves at 5 m depth occurred during both the cold and warm seasons, with a prolonged winter-spring event, characterised by long duration and high cumulative intensity, followed by an exceptionally intense and persistent summer heatwave, together indicating sustained thermal stress throughout the entire year (Fig. 4 ). The event, which occurred between July and September 2024, represents one of the most severe marine heatwaves recorded at 5 m depth in the study period. This event lasted 66 days, making it one of the longest warm-season MHWs in the dataset. It was characterized by a high mean relative intensity (2.53°C) and a maximum relative intensity of 4.11°C, indicating the presence of strong thermal anomalies above the climatological threshold. In terms of absolute temperature, this event reached an absolute mean intensity of 27.90°C and an absolute maximum intensity of 29.18°C, values among the highest observed across the entire 2011–2024 time series. The prolonged exposure to such elevated absolute temperatures resulted in a very high cumulative intensity (166.72°C·days) and an absolute cumulative intensity of 1841.53°C·days, ranking this event among the most energetically impactful marine heatwaves recorded, second only to the extreme events of 2019 and 2015. 3.3 Enumeration, isolation and phylogenetic analysis of vibrios The results of cultural analysis by using in parallel Marine Agar 2216 and TCBS Agar demonstrated that the abundance of surface bacteria was significantly ( p < 0.001) higher in injured specimens of P. ficiformis (on average 4.3 ± 0.2 x 10 5 CFU/g. for total surface culturable bacteria and 7 ± 0.4 x 10 4 CFU/g for culturable vibrios) and A. oroides (on average 3.6 ± 0.3 x 10 5 CFU/g for total surface culturable bacteria and 6.6 ± 0.2 x 10 4 CFU/g for culturable vibrios) than in the surrounding sediment (on average 2.4 ± 0.3 x 10 5 CFU/g. for total surface culturable bacteria and 3.2 ± 0.2 x 10 4 CFU/g for culturable vibrios) and seawater (on average 6.5 ± 0.4 x 10 2 CFU/mL and 81 ± 0.4 CFU/mL for total surface culturable bacteria and culturable vibrios respectively) (Fig. 5 ). Additionally, Gram-negative, oxidase- and catalase-positive bacteria sensitive to O/129 were isolated from diseased tissue samples, as well as from sediment and seawater samples. These bacteria produced exclusively yellow, sucrose-fermenting colonies on TCBS agar, consistent with those expected for members of the genus Vibrio in the case of sponges and sediments, and predominantly yellow colonies in the case of seawater. Moreover, although pieces of healthy specimens as well as pieces of the damaged sponges of both species, were directly placed on TCBS agar plates, vibrios grew only from the wide whitish areas on the surface of the diseased sponges. From a macroscopic point of view after incubation of the pieces of sponges on TCBS for 48 hour we observed the growth of a yellow coating on the plates incubated due to the growth of sucrose-fermenting vibrios (Fig. 6 ). On TCBS plates, the isolates formed round, smooth, edge tidy, convex and translucent yellow colonies. All these isolated vibrios were identified on the basis of both genotypic and phenotypic analysis. The identity of the isolates was investigated through 16S rRNA–based taxonomic classification and confirmed by phylogenetic analysis. Specifically, BLAST searches against the rRNA/ITS database revealed that all isolates were phylogenetically related to Vibrio alginolyticus strains ATCC 17749 and NBRC 15630, showing sequence identities greater than 99% (Table 1 ). Table 1 Taxonomic identification of the isolates based on 16S rRNA sequences and phylogenetic analysis. AGE = isolate from Agelas oroides ; PET = isolate from Petrosia ficiformis; H 2 O = isolate from seawater; SED = isolate from sediment. Isolate ID Accession number Description Query cover (%) Identity (%) Accession Reference AGE1 PX623002 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 99% 99.72% 99.65% NR_118258.1 NR_113781.1 This study AGE2 PX623003 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 99% 99.72% 99.58% NR_118258.1 NR_113781.1 This study PET1 PX623004 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 100% 99.58% 99.58% NR_118258.1 NR_113781.1 This study PET2 PX623005 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 99% 99.79% 99.72% NR_118258.1 NR_113781.1 This study PET3 PX623006 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 99% 99.65% 99.65% NR_118258.1 NR_113781.1 This study PET4 PX623007 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 99% 99.23% 99.44% NR_118258.1 NR_113781.1 This study PET5 PX623008 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 99% 99.56% 99.56% NR_117895.1 NR_122060.1 This study PET6 PX623009 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 100% 99.72% 99.65% NR_118258.1 NR_113781.1 This study PET7 PX623010 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 99% 99.51% 99.44% NR_118258.1 NR_113781.1 This study PET8 PX623011 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 99% 99.09% 99.58% NR_118258.1 NR_122050.1 This study H 2 O1 PX623012 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 99% 99.58% 99.51% NR_118258.1 NR_113781.1 This study H 2 O2 PX623013 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 99% 99.72% 99.65% NR_118258.1 NR_113781.1 This study SED1 PX623014 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 100% 99.02% 98.95% NR_118258.1 NR_113781.1 This study SED2 PX623015 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 99% 99% 99.72% 99.58% NR_118258.1 NR_113781.1 This study MZ900920.1 V. alginolyticus ATCC 17749 V. alginolyticus NBRC 15630 100% 100% 98.91% 99.35% NR_122050.1 NR_118258.1 Dinçtürk et al. [ 42 ] Interestingly, one of the previously identified isolates ( Vibrio sp. MZ900920.1), associated with mass sponge mortality [ 42 ], was also found to be phylogenetically close to V. alginolyticus ATCC 17749 and V. alginolyticus NBRC 15630 based on rRNA/ITS database comparison (Table 1 ). To confirm the taxonomic identification, a phylogenetic approach was applied by comparing the sequences of the isolates with those previously reported in the literature [ 42 ], as well as with reference sequences of V. alginolyticus ATCC 17749 (NR_118258.1) and V. alginolyticus NBRC 15630 (NR_113781.1) retrieved from the rRNA/ITS database. Figure 7 shows the phylogenetic tree, which illustrates that the sequences of the isolates cluster near the 16S rRNA sequences of bacteria belonging to the Vibrio harveyi group ( V. alginolyticus, V. azureus, V. campbellii, V. harveyi, V. mytili, V. natriegens, V. parahaemolyticus , and V. rotiferianus ) [ 61 ], confirming that the isolates belong to the Harveyi clade. The sequences obtained from the isolates in this study are highly similar to one another and form a subclade that also includes Vibrio sp. MZ900920.1 and V. alginolyticus ATCC 17749 (NR_118258.1). Notably, Vibrio sp. MZ900920.1 shows the greatest similarity to the sequence of the PET4S isolate. Vibrio neocaledonicus , a species previously reported as pathogenic in Patinopecten yessoensis [ 62 ], is closely related to this subclade. In conclusion, all the isolates from the damaged sponges, as well as from the sediment and water samples, belonged to the Group Harvey. The latter includes bacteria known to be pathogenic to fish and marine invertebrates. The assignment of the bacterial isolates to the V. alginolyticus was consistent with the phylogenetic analysis (Fig. 7 ) and the results of morphological, cultural and biochemical tests (Table 2 ) according to the schemes proposed by Alsina and Blanch [ 63 ] and Kaysner et al. [ 64 ] and the species description by Gomez-Gil et al. [ 65 ]. Table 2 The results of analysis of Vibrio alginolyticus morphological and physiological properties. Characteristics/test Isolated bacteria Alsina & Blanch [ 63 ] Kaysner et al. [ 64 ] Gram reaction - - - Cell morphology r r nd 0/129 sensitivity 10 µg + + R 150 µg + (-) S Oxidation/Fermentation (O/F) F F nd Motility + nd nd H₂S production - nd nd Urease - (-) - Decarboxylase Arginine - - - Lysine + + + Ornithine - v + Reduction Nitrates to nitrites + + nd Nitrates to nitrogen + + nd Growth in % NaCl 0 - - - 3 + + + 8 + + + Oxidase + + + Catalase + nd nd Voges Proskauer + + + Indole - + nd Gelatinase + + + Citrate + + nd Hydrolysis Casein - nd nd Aesculin - (-) nd Growth at °C 4 - - - 30 + + + 35 + + + Acid from Inositol - - nd Arabinose - - nd Salicin - (-) nd Carbon sources L-Arabinose - - - Sucrose + (+) + Sorbitol - - nd D-Mannitol + + + N-Acetylglucosamine + nd nd Amygdalin + - nd D-Mannose + nd + D-Cellobiose - nd - D-Fructose + nd nd D-Glucose + + nd Glycerol + nd nd Glycogen + nd nd Galactose - nd nd D-Trehalose + nd nd Maltose + nd nd D-Melibiose - - nd Lactose - - - Identification V. alginolyticus V. alginolyticus V. alginolyticus F = Fermentative; V = Variable; r = Short rod with curve shaped; nd = No Data; + = Positive Reaction; - = Negative Reaction; (-) = Negative for 25–11%; S = Susceptible; R = Resistant. 4. Discussion Sponges (phylum Porifera) are sessile invertebrates regarded as the earliest-diverging lineage of multicellular animals. Owing to their highly efficient filter-feeding systems, sponges play significant ecological and biotechnological roles in nutrient cycling within marine ecosystems. In this study, we document a sponge disease event that occurred along the Italian Apulian coastline (Mediterranean Sea, Italy) during the summer of 2024. Our findings, showing microbial overgrowth in the P. ficiformis and A. oroides population and necrotic tissue in most of the specimens, indicate a substantial sublethal impact. The phenomenon was observed in a spatially patchy manner at five distinct sites along a coastal stretch of approximately 100 km. For P. ficiformis , this pattern is consistent with observations by Cerrano et al. [ 9 ], who emphasized that climate-driven stress events in the Mediterranean, often result not only in acute mortality but also in widespread sublethal effects, potentially impairing long-term survival, ecological functioning, and resilience of sponge assemblages; however, unlike their interpretation, which did not associate tissue damage with bacterial involvement, our results indicate that microbial proliferation may accompany stress-related tissue degradation. During the summer of 2022, Núñez-Pons et al. [ 66 ] reported widespread thermal stress in P. ficiformis populations following a marine heatwave in the Gulf of Naples (Tyrrhenian Sea, Italy). Affected sponges exhibited depigmentation and altered tissue consistency, often progressing to necrosis and mortality. By comparing the microbiomes of healthy and diseased individuals, the authors identified a profound restructuring of their microbial communities, a dysbiotic state characterized by an influx of rare taxa, (e.g. Rhodobacteraceae, Flavobacteriaceae) and the introduction of potentially pathogenic, opportunist groups, including Vibrio spp. While previous investigations broadly described this as “disease-associated dysbiosis,” the present study advances this understanding by isolating and identifying as a specific, well-defined pathogen: Vibrio alginolyticus . This bacterium was found to dominate during the critical necrotic phase preceding sponge death. Notably, this epidemiological response was consistent across the two taxonomically distinct sponge species examined, suggesting that Vibrio proliferations thrive during stress-induced tissue degradation. These findings support the hypothesis that opportunistic bacteria play a decisive role in sublethal impairment and subsequent mortality under extreme environmental stress. These disease events occurred in correspondence with one of the most intense and persistent marine heatwaves recorded in the study area. The summer 2024 event was characterized by exceptionally high maximum absolute temperatures (29.18°C), prolonged duration, and elevated cumulative thermal stress, following an already long-lasting winter-spring heatwave. This sustained thermal pressure likely exceeded the physiological tolerance thresholds of several benthic organisms, including sponges, impacting the marine biodiversity [ 67 ], and driving mass mortality events [ 31 , 41 ]. A variety of factors and conditions have been tentatively linked to mass mortality in sponges, but the agents/mechanisms causing mortality to have rarely been elucidated. Local accumulation of pollutants, the introduction of alien species, ecological imbalances caused by overexploitation of commercial benthic organisms, environmentally induced dysbiosis of the symbiotic microbiome, infection by water column pathogens, and anomalous climate patterns leading to heat waves, among other factors, have been suggested as general scenarios associated with several cases of mass mortality of sponges [ 35 , 36 , 40 , 68 – 70 ]. The latter two (pathogens and water warming) are perceived to be particularly important drivers of sponge mortality, and they can be related to each other through the hypothesis that increasing values of sea surface temperature in turn increase microbial pathogenicity or sponge susceptibility or both [ 46 , 71 ]. Interestingly, in all the damaged sponges of both species P. ficiformis and A. oroides bacteria belonging to the species Vibrio alginolyticus were mainly isolated. Vibrio alginolyticus is a naturally occurring marine bacterium, recognized as an emerging pathogen in humans and animals and the second most common cause of vibriosis. The transmission of V. alginolyticus is a function of its abundance in the environment. Like other Vibrio species, V. alginolyticus is considered a conditionally rare taxon in marine waters, with populations capable of forming large, short-lived blooms under specific environmental conditions. Previous research has established the importance of temperature as one of the main determinants of Vibrio geographical and temporal distribution. Therefore, the high temperatures recorded in the summer of 2024 favoured the development of V. alginolyticus , which likely prevailed over the other Vibrio species present. Indeed, among vibrios, only V. alginolyticus grew around the whitish areas of damaged sponges. Sponges are known to host stable, taxonomically diverse, and structurally complex microbial consortia. These symbiotic microorganisms substantially contribute to host physiology, including growth, secondary metabolite production, chemical defence, and responses to biotic and abiotic stressors. However, changes in microbial composition cause disease in the host sponge. For this reason, the prevalence of V. alginolyticus among the bacterial isolates from damaged sponges suggests a crucial role for this microorganism in the observed event. Under such scenario, the genus of Vibrio bacteria emerges as a putative candidate for at least some cases of sponge mortality, a possibility that, at present, remains poorly investigated in this animal group. Vibrio consists of thermo-dependent bacteria that are often pathogenic or facultative pathogenic to a wide variety of aquatic organisms (corals, bivalves, fish, etc.), and whose niche expansion is being facilitated by global ocean warming [ 42 , 72 ]. While microbial agents, in general, have putatively been blamed as responsible for several episodes of sponge disease [ 14 , 15 , 36 , 45 , 46 , 71 ], very few studies have been able to unequivocally identify the specific pathogens. Here is a rewritten version with enhanced originality, clarity, and polished scientific English while preserving the original meaning: This situation is unsurprising considering the high structural and functional complexity of the sponge microbiome [ 42 , 73 , 74 ]. In the limited number of studies where a microbial pathogen has been conclusively identified [ 71 , 75 – 77 ], the causative agents included Pseudoalteromonas agarivorans (originally misidentified as Sulfitobacter pontiacus ), which has been associated with both sponge white patch (SWP) and sponge boring necrosis (SBN), as well as Hormoscilla sp., implicated in mangrove sponge disease (MSD). To date, the involvement of Vibrio spp. in sponge diseases has been only proposed for Ircinia fasciculata [ 14 , 15 ] and experimentally demonstrated for the closely related species Ircinia variabilis by Stabili et al. [ 36 ], who successfully isolated Vibrio rotiferianus following agar inoculation with tissue from diseased specimens. More recently, Dinçtürk et al. [ 42 ] documented a mass mortality event affecting wild sponge populations in the Aegean Sea (Turkey, Eastern Mediterranean), which impacted the keratose demosponge Sarcotragus foetidus in September 2021. The authors identified, through 16S rRNA, the colonies isolated from the diseased sponges. Three of them resulted putatively pathogenic ( V. fortis , V. owensii , V. gigantis ). Importantly, those vibrios were isolated from only tissues of diseased sponges. In contrast, healthy individuals sampled in both summer and winter led to no Vibrio growth in laboratory cultures. In addition, they reported that a progressive increase in temperature from 1970 to 2021 was recorded with values above 24°C from May to September 2021, reaching an absolute historical maximum of 28.9°C in August 2021. Interestingly the three vibrio species V. fortis , V. owensii , V. gigantis belonged to the Vibrio harveyi group as well as Vibrio rotiferianus that was isolated by Stabili et al. [ 36 ] in the case of diseased I. variabilis . Interestingly, one of the bacterial isolates identified by Dinçtürk et al. [ 42 ], Vibrio sp. MZ900920.1, is taxonomically close to the Vibrio alginolyticus species (Table 1 ). In the present study, 16S rRNA gene sequence analysis of the 14 isolates revealed that all were taxonomically related to V. alginolyticus (Table 1 ) and, consequently, to the Vibrio harveyi group. Phylogenetic analysis further showed that the isolates clustered closely with the reference strain V. alginolyticus ATCC 17749 (NR_118258.1) and with Vibrio sp. MZ900920.1, previously reported by Dinçtürk et al. [ 42 ] in an environmental context characterized by elevated temperatures and mass sponge mortality events, as described before. V. alginolyticus employs several key virulence factors, including the metalloprotease (Asp), serine protease (Pep), immunelike protein (IlpA), outer membrane protein (OmpU), type VI secretion system (T6SS) [ 78 ], hemolysins, and the quorum-sensing (QS) master regulator LuxR. Together, these components facilitate host tissue degradation, immune evasion, and QS-dependent pathogenic behaviors such as motility and biofilm formation [ 79 ]. Several of these virulence factors are transcriptionally upregulated by the alternative sigma factor RpoE, which directly binds to the luxR promoter, thereby enhancing LuxR expression and activating the downstream QS cascade [ 79 ]. Notably, LuxR binding motifs have been shown to vary with temperature [ 80 ]. Moreover, the expression of these virulence factors is strongly temperature dependent [ 79 ]. For instance, hemolysin production and protease secretion are significantly suppressed at lower temperatures [ 81 ]. These findings support the hypothesis that the elevated temperatures observed during the sampling events in this study may enhance the expression of virulence factors in V. alginolitycus as well as promote the proliferation and dissemination of diverse Vibrio spp., consistent with previous reports. Therefore, in agreement with Dinçtürk et al. [ 42 ], based on the results here obtained, we can hypothesize that, as in the case of the Aegean Sea, also in our case the high seawater temperatures maintained for several months in 2024 promoted the proliferation of pathogenic Vibrio species (thermo-dependent bacteria), triggering or exacerbating the course of sponge disease in P. ficiformis and A. oroides . Therefore, vibriosis emerges as one of the putative mechanisms through which global warming of waters in the Mediterranean Sea translates into sponge mortality. This is in agreement with the Marine Climate Change Impacts Partnership (MCCIP) Annual Report 2010–2011, which provides an updated assessment of how climate change is also affecting the seas of the United Kingdom, considering, for the first time, the potential future increase of marine vibrios as an emerging issue. In recent years, several countries in Northwestern Europe have seen an unprecedented increase in the number of infections associated with warm-water Vibrio species, including in humans. For example, during the hot summer of 2006, wound infections related to contact with Baltic and North Sea waters were reported in Germany ( V. vulnificus [ 82 ]), southeastern Sweden ( V. cholerae non-O1/O139 [ 83 ]), the Netherlands ( V. alginolyticus [ 84 ]), and Denmark ( V. alginolyticus and V. parahaemolyticus [ 85 ]). These events have raised growing concern about the potential contribution of climate change to the abundance of Vibrio species in coastal seas. However, despite the wealth of indirect evidence, it remains unclear whether vibrios, known to be temperature-dependent, are increasing within the complex, ecologically regulated bacterial communities of coastal marine waters. This is primarily due to the lack of historical data. Therefore, this study represents a further contribution to evaluating a possible link between the presence of Vibrio and seawater surface temperature, and the increase in diseases affecting humans and marine organisms. Furthermore, beyond a reliable identification of the causative agents responsible for pathogenic events, another aspect requiring further investigation is the ability of sponges to defend themselves from microbial infections. Therefore, further studies will be conducted to evaluate the presence of antimicrobial compounds in the studied species and their efficacy under conditions of sponge weakness related to global change. This is particularly important since, at present, we can suggest, as already hypothesized by Vacelet et al. [ 45 , 46 ], the involvement of a synergistic mechanism in the disease and that presumably under stressful physiological conditions (high temperature, high nutrients and reduced water flow) sponge pathogens, in our case V. alginolyticus , could activate virulence mechanisms and sponges could not be able to control the proliferation of these bacteria. This hypothesis aligns with the findings of Núñez-Pons et al. [ 66 ], who suggested that microbial community stability confers protection to certain host phenotypes, thereby bolstering population-level resilience. Therefore, we suggest that the heightened intensity of the 2024 MHW coupled with the prolonged disruption of microbial stability, likely facilitated the extensive invasion of Vibrio alginolyticus . Furthermore, our results corroborate the classification of P. ficiformis as a sentinel species or, at least, one highly vulnerable to MHWs in the Mediterranean Sea. However, we also demonstrate that this susceptibility extends to other taxa, such as A. oroides . Ultimately, marine heatwaves represent a pervasive threat to the diverse sponge species that underpin the structure of shallow Mediterranean benthic communities. Interestingly, in the Mediterranean Sea, increasing global temperatures not only pose a significant threat to marine biodiversity but also promote environmental conditions conducive to the proliferation and successful establishment of non-indigenous, thermophilic species. [ 86 – 88 ]. Moreover, during the summer of 2024 in the Apulian seas (Mediterranean) mortality and disease events did not only affect the here considered sponges but outbreaks were recorded for mussels belonging to the species Mytilus galloprovincialis [ 89 ] but also for seaweeds of the species Chatemorpha linum grown in an integrated multitrophic aquaculture system in the Gulf of Taranto (personal observation). In the study by Calabrese et al. [ 89 ], the authors concluded that, in addition to high temperatures (maximum 31.01°C), there was a combination of factors triggered by the prolonged heat wave, such as the involvement of excessive algal blooms, including toxic species, and/or the decay of surface organisms due to infections, which also affected the deeper layers and, consequently, mussel growth was prohibitive even at depth. It is therefore clear that the consequences of climate change led to social, economic, and environmental impacts on a global scale [ 90 – 92 ]. A consistent trend observed in recent decades is that, due to climate change, disease outbreaks are expected to occur more frequently, with greater intensity, and with a wider spread both on land and in water [ 33 ]. In this framework investigating diseases affecting marine sponges is essential to gain a comprehensive understanding of benthic ecosystem ecology and dynamics as well as sponge biodiversity, both of which support the sustainable functioning of ocean environments. The loss of a sponge population can trigger unpredictable ecological consequences for surrounding habitats and associated species, given the pivotal biological and ecological roles these organisms play. Marine sponges indeed serve as refuge for numerous small invertebrates [ 55 ] and are key regulators of benthic–pelagic coupling across diverse marine habitats. Moreover, maintaining healthy and stable populations of commercially valuable sponge species is essential for the viability of the sponge industry. Investigating diseases affecting marine sponges is essential to gain a comprehensive understanding of benthic ecosystem ecology and dynamics as well as sponge biodiversity, both of which support the sustainable functioning of ocean environments. The loss of a sponge population can trigger unpredictable ecological consequences for surrounding habitats and associated species, given the pivotal biological and ecological roles these organisms play. Marine sponges indeed serve as refuge for numerous small invertebrates [ 55 ] and are key regulators of benthic–pelagic coupling across diverse marine habitats. Moreover, maintaining healthy and stable populations of commercially valuable sponge species is essential for the viability of the sponge industry. 5. Conclusion Marine heatwaves (MHWs) are increasing in frequency, duration, and intensity, with profound consequences for marine ecosystems worldwide. This phenomenon was evident during the summer of 2024 along the Italian Apulian coastline where extensive mass-mortality events of benthic organisms were documented. Among the most affected were the sponges Petrosia ficiformis and Agelas oroides , which exhibited clear signs of disease. Bacteriological analyses of diseased individuals revealed the presence of Vibrio alginolyticus . The main strengths of this study include: (a) sampling conducted precisely at the time when the condition of maximum virulence became macroscopically evident, namely during the appearance of the whitish mucilaginous plume that smothered the sponge; (b) observation of the phenomenon in two co-occurring sponge species belonging to the same class but different orders, examined across multiple locations within the same habitat; and (c) documentation that all samples collected from the whitish biofilm consistently exhibited a high density of Vibrio alginolyticus , identifying this bacterium as the primary driver of the necrotic process. The anomalously high seawater temperatures that persisted for several months in 2024 likely facilitated the proliferation of this thermophilic pathogen within sponge tissues, initiating or exacerbating disease progression. These observations suggest that vibriosis represents a plausible mechanistic pathway linking Mediterranean warming to sponge mortality. If marine heatwaves continue to recur in the coming summers, repeated disease outbreaks could ultimately decimate local populations of these sponge species, with cascading impacts on habitat structure, biogeochemical cycling, and overall ecosystem functioning. Given these ecological implications, future research should explicitly investigate the role of heat-stress and Vibrio infections as key drivers of sponge disease dynamics under ongoing climate change. Declarations Declaration of Competing Interest The authors declare no conflict of interest. Author Contribution Investigation : L.S., M.C., A.T., F.N., E.Q., P.A. Formal analysis : L.S., M.C., A.T., F.N., E.Q. Visualization : L.S., M.C., A.T., F.N., E.Q. Resources : L.S., M.C., A.T., F.N., E.Q. Methodology : L.S., M.C., A.T., F.N., E.Q. Conceptualization : L.S., S.P., P.A. Writing - Review & Editing : L.S., M.C., A.T., F.N., E.Q. Writing - Original Draft : L.S., M.C., A.T., F.N., E.Q. Supervision : L.S., S.P., P.A. Project administration : L.S., S.P. Funding acquisition : L.S., S.P. Acknowledgement This study was mainly supported by the “National Biodiversity Future Center (NBFC)”, funded by European Union Next Generation EU (PNRR) (Project code CN_00000033, Concession Decree No. 1034 of 14 June 2022 adopted by the Italian Ministry of University and Research, CUP: D33C22000960007, Spoke 2 Activity 3 Task 3.2 CUP B83C22002930006. Data Availability The 16s rRNA gene sequences obtained in this study are available under the following accession numbers: PX623002, PX623003, PX623004, PX623005, PX623006, PX623007, PX623008, PX623009, PX623010, PX623011, PX623012, PX623013, PX623014, PX623015. References Ford HV, Jones NH, Davies AJ, Godley BJ, Jambeck JR, Napper IE, Suckling CC, Williams GJ, Woodall LC, Koldewey HJ (2022) The Fundamental Links between Climate Change and Marine Plastic Pollution. 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Jones J, Bacteriological Analytical Manual (BAM) Chap. 9: Vibrio May pag.6 Gomez-Gil B, Tron-Mayén L, Roque A, Turnbull JF, Inglis V, Guerra-Flores AL (1998) Species of Vibrio isolated from hepatopancreas, haemolymph and digestive tract of a population of healthy juvenile Penaeus vannamei. Aquaculture 163(1–2):1–9 Núñez-Pons L, Cusano LM, Chiarore A, Mirasole A, Teixidó N, Efremova J, Mazzella V (2025) Too hot for my bugs: mediterranean heatwave disrupts associated microbiomes in the sponge Petrosia ficiformis . Environmental Microbiome Wernberg T, Thomsen MS, Burrows MT et al (2025) Marine heatwaves as hot spots of climate change and impacts on biodiversity and ecosystem services. Nat Rev Biodivers 1:461–479. https://doi.org/10.1038/s44358-025-00058-5 Fortunato HFDM, Silva AGD, Teixeira RPA, Carvalho BC, Fleury BG (2022) Abnormal average increase in sea surface temperature may promote the first documented mortality event of a marine sponge in the Southeastern Brazil. Biota Neotrop 22(3):e20211298 Luter HM, Webster NS (2017) Sponge disease and climate change. In Climate change, ocean acidification and sponges: impacts across multiple levels of organization. Cham: Springer International Publishing. ;411–428 Botté ES, Bennett H, Engelberts JP, Thomas T, Bell JJ, Webster NS, Luter HM (2023) Future ocean conditions induce necrosis, microbial dysbiosis and nutrient cycling imbalance in the reef sponge Stylissa flabelliformis . ISME Commun 3(1):53 Sweet M, Bulling M, Cerrano C A novel sponge disease caused by a consortium of micro-organisms, Coral Reefs, Published online 20 March 2015, 10.1007/s00338-015-1284-0 Frydenborg BR, Krediet CJ, Teplitski M, Ritchie KB (2014) Temperature-dependent inhibition of opportunistic vibrio pathogens by native coral commensal bacteria. Microb Ecol 67:392–401. 10.1007/s00248-013-0334-9 Schmitt S, Wehrl M, Bayer K, Siegl A, Hentschel U (2007) Marine sponges as models for commensal microbe-host interactions. Symbiosis 44:43–50 Webster NS, Taylor MW (2012) Marine sponges and their microbial symbionts: love and other relationships. Environ Microbiol 14:335–346. 10.1111/j.1462-2920.2011.02460.x Rützler K (1988) Mangrove sponge disease induced by cyanobacterial symbionts: failure of a primitive immune system? Dis Aquat Org 5:143–149. 10.3354/dao005143 Webster NS, Negri AP, Webb RI, Hill RT (2002) A spongin-boring α-proteobacterium is the etiological agent of disease in the great barrier reef sponge Rhopaloeides odorabile . Mar Ecol Prog Ser 232:305–309. 10.3354/meps232305 Choudhury JD, Pramanik A, Webster NS, Llewellyn LE, Gachhui R, Mukherjee J (2015) The pathogen of the great barrier reef sponge Rhopaloeides odorabile is a new strain of Pseudoalteromonas agarivorans containing abundant and diverse virulence-related genes. Mar Biotechnol 17:463–478. 10.1007/s10126-015-9627-y Attar N (2015) MIXing up T6SS effectors. Nat Rev Microbiol 13:600. https://doi.org/10.1038/nrmicro3560 Gu D, Guo M, Yang M, Zhang Y, Zhou X, Wang Q (2016) A σE-mediated temperature gauge controls a switch from LuxR-mediated virulence gene expression to thermal stress adaptation in Vibrio alginolyticus . PLoS Pathog 12(6):e1005645 Cai J, Hao Y, Xu R, Zhang Y, Ma Y, Zhang Y, Wang Q (2022) Differential binding of LuxR in response to temperature gauges switches virulence gene expression in Vibrio alginolyticus . Microbiol Res 263:127114 Huang Z, Li Y, Yu K, Ma L, Pang B, Qin Q, Kan B (2024) Genome-wide expanding of genetic evolution and potential pathogenicity in Vibrio alginolyticus . Emerg Microbes Infections 13(1):2350164 Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, Olsen GJ (2008) Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appl Environ Microbiol 74:2461–2470. 10.1128/AEM.02272-07 Andersson Y, Ekdahl K (2006) Wound infections due to Vibrio cholerae in Sweden after swimming in the Baltic Sea, summer 2006. Euro Surveill. ;11:E060803.2. 10.2807/esw.11.31.03013-en Schets FM, Van den Berg HHJL, Demeulmeester AA, Van Dijk E, Rutjes SA, Van Hooijdonk HJP et al (2006) Vibrio alginolyticus infections in the Netherlands after swimming in the North Sea. Euro Surveill 11:E0611093. 10.2807/esw.11.45.03077-en Andersen PH (2006) Infections with seawater bacteria. EPI-NEWS 1:26–32 Toso A, Mammone M, Rossi S, Piraino S, Giangrande A (2024) Effect of temperature and body size on anterior and posterior regeneration in Hermodice carunculata (Polychaeta, Amphinomidae). Mar Biol 171:152. https://doi.org/10.1007/s00227-024-04468-5 Furfaro G, Fumarola LM, Toso A, Toso Y, Trainito E, Bariche M, Piraino S (2025) A Mediterranean melting pot: native and non-indigenous sea slugs (Gastropoda, Heterobranchia) from Lebanese waters. BioInvasions Records, ;14(1): 197–221. https://doi.org/10.3391/bir . 2025.14.1.16 Delcour N, Garzia M, Oliver PG, Berrilli E, Toso A, Bariche M, Albano PG, Mariottini P, Salvi D (2025) High genetic diversity and lack of structure underlie the invasion history of the non-indigenous oyster Dendostrea cf. crenulifera (Mollusca, Ostreida, Ostreidae) spreading in the eastern Mediterranean Sea. NeoBiota 101:277–302. https://doi.org/10.3897/neobiota.101.154917 Calabrese C, Arduini D, Portacci G, Quarta E, Giangrande A, Acquaviva MI, Biandolino F, Giandomenico S, Prato E, Stabili L (2025) Farming strategy under climate change: Growth performances and quality of Mytilus galloprovincialis in an Integrated Multi-Trophic Aquaculture system (North-West Mediterranean Sea). Mar Pollut Bull 220:118377. https://doi.org/10.1016/j.marpolbul.2025.118377 Arduini D, Portacci G, Giangrande A, Acquaviva MI, Borghese J, Calabrese C, Giandomenico S, Quarta E, Stabili L (2023) Growth Performance of Mytilus galloprovincialis Lamarck, 1819 under an Innovative Integrated Multi-Trophic Aquaculture System (IMTA) in the Mar Grande of Taranto (Mediterranean Sea, Italy). Water 15(10):1922. https://doi.org/10.3390/w15101922 Charles M, Bernard I, Villalba A, Oden E, Burioli EAV, Allain G, Trancart S, Bouchart V, Houssin M (2020) High mortality of mussels in northern Brittany–Evaluation of the involvement of pathogens, pathological conditions and pollutants. J Invertebr Pathol 170:107308 Villasante S, Rodríguez-González D, Antelo M, Rivero-Rodríguez S, Lebrancón-Nieto J (2013) Why are prices in wild catch and aquaculture industries so different? Ambio 42:937–950 Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx Supplementary Tables Table S1. Summary of detected marine heatwave events at a depth of 5 m, in the time period 2011-2024 in Santa Maria al Bagno. Including the following variables: Event N°, progressive event identifier; Date start, onset date of the marine heatwave; Date peak, date of maximum intensity; Date end, termination date of the event; Duration, event length in days; Intensity mean, average intensity over the event; Intensity max, maximum intensity reached; Intensity var, variance of intensity; Intensity cumulative, cumulative intensity over the event; Intensity mean (abs), mean absolute temperature anomaly; Intensity max (abs), maximum absolute temperature anomaly; Intensity var (abs), variance of absolute temperature anomaly; Intensity cum. (abs), cumulative absolute temperature anomaly. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8987753","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":601439885,"identity":"125c18c3-0bb1-44c7-af15-a59559dacdbe","order_by":0,"name":"Loredana Stabili","email":"data:image/png;base64,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","orcid":"","institution":"National Research Council","correspondingAuthor":true,"prefix":"","firstName":"Loredana","middleName":"","lastName":"Stabili","suffix":""},{"id":601439886,"identity":"95ccf09b-3eee-45d6-aa97-f8e2a397e652","order_by":1,"name":"Elisa Quarta","email":"","orcid":"","institution":"University of Salento","correspondingAuthor":false,"prefix":"","firstName":"Elisa","middleName":"","lastName":"Quarta","suffix":""},{"id":601439887,"identity":"37c066a1-c950-441d-a9fe-c4cb5d15ff2d","order_by":2,"name":"Francesca Necci","email":"","orcid":"","institution":"University of Salento","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Necci","suffix":""},{"id":601439888,"identity":"4468451f-b488-44e7-8c04-98d874a1eabf","order_by":3,"name":"Andrea Toso","email":"","orcid":"","institution":"University of Salento","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Toso","suffix":""},{"id":601439889,"identity":"7dc5a388-dc1e-4af4-a658-fd1a66a26584","order_by":4,"name":"Stefano Piraino","email":"","orcid":"","institution":"University of Salento","correspondingAuthor":false,"prefix":"","firstName":"Stefano","middleName":"","lastName":"Piraino","suffix":""},{"id":601439890,"identity":"c85537a6-9277-4a90-bad0-43409cc67128","order_by":5,"name":"Pietro Alifano","email":"","orcid":"","institution":"University of Salento","correspondingAuthor":false,"prefix":"","firstName":"Pietro","middleName":"","lastName":"Alifano","suffix":""},{"id":601439891,"identity":"17215360-a6f4-4694-8adb-f96a3df868ee","order_by":6,"name":"Matteo Calcagnile","email":"","orcid":"","institution":"University of Salento","correspondingAuthor":false,"prefix":"","firstName":"Matteo","middleName":"","lastName":"Calcagnile","suffix":""}],"badges":[],"createdAt":"2026-02-27 11:54:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8987753/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8987753/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104207771,"identity":"b21caf85-3f6f-42bd-a31c-3844071f8d68","added_by":"auto","created_at":"2026-03-09 07:11:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":327370,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing the study area, with red dots indicating locations where mortality events were observed and the black star marking the sampling site at S. Maria al Bagno.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8987753/v1/c7de63fa9295ad6269889667.jpg"},{"id":104207499,"identity":"61ecd4e4-1deb-4427-9454-6228c614c1e3","added_by":"auto","created_at":"2026-03-09 07:09:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6479415,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative benthic organisms observed at the study site: (A) \u003cem\u003eAgelas oroides\u003c/em\u003e; (B) \u003cem\u003eCodium bursa\u003c/em\u003e; (C) \u003cem\u003eSarcotragus spinosulus\u003c/em\u003e covered by a whitish biofilm.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8987753/v1/6d2be13b27588e7222451c4b.png"},{"id":104207569,"identity":"19661efb-03df-4a3d-82be-a811f27a9c1f","added_by":"auto","created_at":"2026-03-09 07:10:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3779724,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent conditions of \u003cem\u003ePetrosia ficiformis\u003c/em\u003e observed at Santa Maria al Bagno (from A to E). (A) Healthy sponge; (B), (C), (D), (E) sponges showing with different degree of necrosis and whitish biofilm; (F) Whitish biofilm observed over the sediment.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8987753/v1/9469e984a8a940cc53fb728f.png"},{"id":104207461,"identity":"714314a1-b34d-49d1-97b3-964bf0b758c0","added_by":"auto","created_at":"2026-03-09 07:09:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":255544,"visible":true,"origin":"","legend":"\u003cp\u003eSeasonal evolution of seawater temperature at ~ 5.5 m depth during 2024, showing the climatological mean (dashed line), the marine heatwave threshold (red line), and the observed temperature (black line), and marine heatwave periods. Shaded areas indicate winter/autumn and summer/spring heatwaves.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8987753/v1/7c48a694370c2e8a43029815.png"},{"id":104207660,"identity":"c739135b-f619-429c-9da3-308008f2f0e5","added_by":"auto","created_at":"2026-03-09 07:10:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":105357,"visible":true,"origin":"","legend":"\u003cp\u003eCultivable vibrios grew after incubation in Petri dishes containing TCBS agar, specifically: \u003cem\u003ePetrosia ficiformis\u003c/em\u003e (A), \u003cem\u003eAgelas oroides\u003c/em\u003e (B), Sediment in direct contact with diseased sponges (C), Water sampled near diseased sponges (D).\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8987753/v1/f61bc18f2b13779e26a61350.jpg"},{"id":104207816,"identity":"2a9d84d5-820d-445c-957d-ff0f96ce788c","added_by":"auto","created_at":"2026-03-09 07:11:39","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":137559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eVibrios\u003c/em\u003e grew from the large whitish areas on the surface of diseased sponges after incubation in Petri dishes containing TCBS agar, specifically: \u003cem\u003ePetrosia ficiformis\u003c/em\u003e (A) and \u003cem\u003eAgelas oroides\u003c/em\u003e(B). Note the growth of yellow coating on the incubated dishes due to the growth of sucrose-fermenting vibrios.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8987753/v1/a6dc7eea81bb4c339d282d1a.jpg"},{"id":104207566,"identity":"93183ce5-a2c2-432a-9160-fe313d2af025","added_by":"auto","created_at":"2026-03-09 07:10:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1339740,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on 16S rRNA sequences showing that the isolates cluster near species belonging to the Vibrio harveyi group (\u003cem\u003eV. alginolyticus, V. azureus, V. campbellii, V. harveyi, V. mytili, V. natriegens, V. parahaemolyticus, and V. rotiferianus\u003c/em\u003e) confirming that the isolates belong to the Harveyi clade.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-8987753/v1/64073ef6a70bcef831acb9dd.png"},{"id":105516174,"identity":"f22a2488-45ec-49e8-b1d3-25f95840fa42","added_by":"auto","created_at":"2026-03-27 00:09:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15964885,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8987753/v1/4dd44e60-8b7a-4b2b-b3db-fcc550319690.pdf"},{"id":104207589,"identity":"8dd3ce8e-3d6f-4958-808b-b76338f000e1","added_by":"auto","created_at":"2026-03-09 07:10:37","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17545,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Tables\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1. \u003c/strong\u003eSummary of detected marine heatwave events at a depth of 5 m, in the time period 2011-2024 in Santa Maria al Bagno. Including the following variables: Event N°, progressive event identifier; Date start, onset date of the marine heatwave; Date peak, date of maximum intensity; Date end, termination date of the event; Duration, event length in days; Intensity mean, average intensity over the event; Intensity max, maximum intensity reached; Intensity var, variance of intensity; Intensity cumulative, cumulative intensity over the event; Intensity mean (abs), mean absolute temperature anomaly; Intensity max (abs), maximum absolute temperature anomaly; Intensity var (abs), variance of absolute temperature anomaly; Intensity cum. (abs), cumulative absolute temperature anomaly. \"\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8987753/v1/72c846f955fee889f823fc87.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Can synergistic effects of marine heatwaves and Vibrio proliferation act as potential triggers of widespread demosponge disease? A case study in the Mediterranean Sea","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHuman activities are exerting growing pressure on marine ecosystems, leading to significant environmental degradation. Among the most critical outcomes of this anthropogenic impact are rising pollution levels and increasing global temperatures [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These factors not only threaten marine biodiversity but also create conditions favoriting the proliferation of non-indigenous, thermophilic species and harmful microorganisms, some of which may be invasive or even pathogenic [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn particular, several above mentioned factors can promote disease outbreaks in marine animals, leading to mass mortality events [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This issue is particularly critical in the Mediterranean Sea, a region strongly influenced by both global and local environmental changes. Over recent decades, multiple mass mortality events affecting sessile benthic invertebrates, such as sponges, anthozoans, bivalves, and ascidians, have been documented throughout the Mediterranean basin [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this scenario, there is broad consensus that global warming contributes to the emergence of these severe ecological crises in the Mediterranean Sea [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Elevated seawater temperatures increase the metabolic and respiratory demands of organisms while simultaneously reducing the availability of nutrients and food resources due to \u0026ldquo;thermal stratification\u0026rdquo;. Two of the most significant mass-mortality events in the north-western Mediterranean, both in terms of spatial extent (approximately 1,000 km of coastline) and biodiversity impact (around 30 macro-benthic species affected), occurred during the summers of 1999 and 2003. These events coincided with pronounced positive thermal anomalies, characterized by sea temperatures 3\u0026ndash;4\u0026deg;C above average and prolonged stability of the water column in late summer [\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Globally, the mean temperature for the decade 2014\u0026ndash;2023 was 1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u0026deg;C higher than the 1850\u0026ndash;1900 baseline [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis warming trend intensified further in the summer of 2024, when temperatures exceeded those recorded in 2023. Notably, July of both 2023 and 2024 was marked by severe heatwaves across multiple regions of the Northern Hemisphere [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs a consequence, many marine ecosystems are in a poor to bad state [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. During prolonged and unusually warm summers, benthic suspension feeders experience prolonged energetic constraints, determining a compromised stressed physiological state that often culminates in mortality during late summer or early autumn [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For filter-feeding invertebrates in particular, elevated seawater temperatures can disrupt key physiological functions - such as filtration efficiency, nutrient assimilation, and symbiotic interactions with microorganisms [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These impairments can weaken the organisms\u0026rsquo; capacity to cope with environmental fluctuations, thereby increasing their susceptibility to disease and predation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, opportunistic microbial infections are suspected contributors, although their exact role remains unclear. A consistent pattern emerging from studies over the past decades indicates that climate change is likely to increase the frequency, severity, and geographic range of disease outbreaks in both terrestrial and marine systems [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Pathogens and the diseases they cause constitute a significant constraint on ecosystems, with broad social, economic, and environmental consequences. Changing environmental parameters, such as rising temperatures, often increase host susceptibility to infection. Sessile marine invertebrates, constantly threatened by a rich mix of microorganisms present in the surrounding waters, are vulnerable to pathogen attack [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In particular, because \u003cem\u003eVibrio\u003c/em\u003e species thrive at relatively high temperatures, outbreaks in marine animals are expected to become increasingly frequent due to global warming. For example, in recent decades, \u003cem\u003eVibrio\u003c/em\u003e have been implicated in the \"bleaching\" of subtropical and tropical corals worldwide [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Like other sessile benthic groups, sponges despite their capacity for tissue regeneration, have experienced recurrent mass-mortality events globally [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Evidence from long-term monitoring in the Mediterranean Sea suggests that the frequency of these sponge mortality episodes has increased markedly over the last twenty years [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese disease events affect numerous sponge species and occur across wide geographic regions, with impacted sponges exhibiting diverse physiological symptoms. In sponges, as in other marine invertebrates, several environmental drivers, most notably ocean warming and acidification, have been implicated in both the onset and subsequent spread of disease outbreaks [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44 CR45 CR46 CR47\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Persistent thermal stress may also cause tissue necrosis and bleaching in species that rely on photosynthetic symbionts, ultimately leading to death [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Several studies have linked sponge mass-mortality events to marine heatwaves, during which anomalously high temperatures persist for extended periods [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Although most studies attribute sponge diseases to microbiome dysbiosis, only a few of them have successfully isolated specific pathogenic agents [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the Mediterranean Sea, the best-documented disease episodes are referred to the commercially important sponges [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and were likely due to bacteria capable of degrading spongin fibres [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. For other species, Corriero et al. [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] reported a similar pattern of fibre deterioration across multiple keratose sponges, including \u003cem\u003eSpongia, Sarcotragus\u003c/em\u003e, and \u003cem\u003eIrcinia\u003c/em\u003e, suggesting a shared bacterial etiology.\u003c/p\u003e \u003cp\u003eMaldonado et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] described a recurrent disease in \u003cem\u003eIrcinia\u003c/em\u003e spp. that re-emerges annually, typically after the hottest months. This condition was attributed to an external bacterium, presumably a \u003cem\u003eVibrio\u003c/em\u003e sp., that invades and colonizes the sponge tissues. Likewise, Stabili et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] documented a disease event in \u003cem\u003eIrcinia variabilis\u003c/em\u003e (Schmidt, 1862) observed in September 2009 along the southern Adriatic and Ionian Seas (Apulian coast). The injured sponges displayed extensive surface necrosis and, in severe cases, fragmentation of the body into multiple portions. The necrotic zones appeared whitish and were frequently coated with a thin mucous coat associated with \u003cem\u003eVibrio\u003c/em\u003e proliferation.\u003c/p\u003e \u003cp\u003eSeveral evidence indicate that \u003cem\u003eVibrio\u003c/em\u003e-related diseases are increasing worldwide in response to ocean warming [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Additional studies have linked \u003cem\u003eVibrio\u003c/em\u003e infections to rising mass-mortality events in coastal marine organisms [\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, we report a recently observed disease outbreak affecting populations of the sponge species \u003cem\u003ePetrosia ficiformis\u003c/em\u003e (Poiret, 1789) and \u003cem\u003eAgelas oroides\u003c/em\u003e (Schmidt, 1864) along the Northern Ionian Sea (Mediterranean Sea, Italy) in the summer 2024. We document the extent of the outbreak and assess its possible association with \u003cem\u003eVibrio\u003c/em\u003e infection, regional marine heatwaves, and the exceptionally high seawater temperatures recorded during this period.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study sites and studied material\u003c/h2\u003e \u003cp\u003eDuring the summer of 2024, episodes of tissue necrosis with whitish areas in the Demospongiae \u003cem\u003ePetrosia ficiformis\u003c/em\u003e and \u003cem\u003eAgelas oroides\u003c/em\u003e were reported along the Apulian coast (Ionian Sea, Italy, southeastern Mediterranean). Several areas of the Apulian coast were examined to assess the extent of the phenomenon. Specifically, this event was observed in the areas of Otranto (Adriatic Sea), Porto Badisco (Ionian Sea), Santa Maria di Leuca (Ionian Sea), and Santa Caterina (Ionian Sea). For this reason, a field survey was conducted in September 2024, at Santa Maria al Bagno (40.1297\u0026deg; N; 17.9980\u0026deg; E) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The site consists mainly of rocky bottoms alternating with sandy patches, where the substrate includes both exposed surfaces and shaded crevices, creating favourable conditions for benthic assemblages dominated by photophilic algal communities and sponges.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSampling was conducted by scuba diving between 4 and 6 m depth. A linear transect (50 m long and 1 m wide) was placed parallel to the shoreline, and every visible specimen of \u003cem\u003eP. ficiformis\u003c/em\u003e and \u003cem\u003eA. oroides\u003c/em\u003e was photographed using an Olympus TG-6 camera. The photographs were subsequently examined to assess the frequency of damaged versus healthy specimens. Additionally, small tissue samples (approximately 1 cm\u0026sup3;) were collected from affected individuals (n\u0026thinsp;=\u0026thinsp;15 for \u003cem\u003eP. ficiformis\u003c/em\u003e and n\u0026thinsp;=\u0026thinsp;7 for \u003cem\u003eA. oroides\u003c/em\u003e) for microbiological screening to detect possible pathogenic bacteria. Additionally, seawater and sediment samples were collected from the areas surrounding the diseased sponges, with the sediments exhibiting a whitish biofilm similar to that observed on the tissues of affected sponges.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Marine Heat Waves calculation\u003c/h2\u003e \u003cp\u003eIn addition, subsurface Marine Heat Waves (MHWs) were detected and quantified following Hobday et al. [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], which defines them as discrete, prolonged events during which sea temperature exceeds the seasonally varying 90th percentile for at least five consecutive days. We used daily Sea water temperature data from the Mediterranean Sea Physics Reanalysis (Copernicus Marine Service) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The period 1994\u0026ndash;2010 was used to derive the climatological baseline and the seasonally varying 90th percentile thresholds, while MHWs were identified for the period 2011\u0026ndash;2024. Temperature values were extracted at the closest depth in the model for 5 m (5.46 m), from the pixel, with spatial resolution 0.042\u0026deg; \u0026times; 0.042\u0026deg; (~\u0026thinsp;4.5 km), corresponding to the coordinates of the site Santa Maria al Bagno. Based on the starting date of each event, MHWs were classified into:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eCold-season events (initiated in autumn or winter: October - March), and\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eWarm-season events (initiated in spring or summer: April - September).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Enumeration, isolation and phylogenetic analysis of vibrios\u003c/h2\u003e \u003cp\u003eIn the laboratory, fragments from damaged \u003cem\u003eP. ficiformis\u003c/em\u003e and \u003cem\u003eA. oroides\u003c/em\u003e specimens were gently rinsed with sterile, 0.2 \u0026micro;m filtered seawater to remove the bacteria settled on the surfaces. The fragments were then suspended in sterile seawater and subjected to three sonication cycles (Branson Sonifier 2200; 60 W, 47 kHz; 1 min per cycle in an ice bath) to optimize detachment of surface-associated bacteria. Sonication was interrupted for 30 s between cycles, during which samples were shaken manually. To quantify surface-associated bacteria, including \u003cem\u003eVibrio\u003c/em\u003e spp., 1 or 5 mL of each sonicated sample and appropriate decimal dilutions were plated in parallel on Marine Agar 2216 or thiosulfate\u0026ndash;citrate\u0026ndash;bile salts\u0026ndash;sucrose (TCBS) agar (Becton Dickinson and Company). Culturable colonies were enumerated using the CFU method following incubation at 22\u0026deg;C for 7 days (Marine Agar) and at 30\u0026deg;C for 48 h (TCBS).\u003c/p\u003e \u003cp\u003eAdditionally, fragments from injured tissues exhibiting extensive whitish lesions, were rinsed in sterile, 0.2 \u0026micro;m filtered seawater to remove surface bacteria, then placed directly onto TCBS agar and incubated at 30\u0026deg;C for 48 h. All distinct colonies were isolated and streaked onto Marine Agar to obtain pure cultures.\u003c/p\u003e \u003cp\u003ePhenotypic identification of isolates followed the procedures reported by Stabili et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The assays included: Gram staining, cell morphology, motility, growth on TCBS agar, oxidase, catalase, and urease activities, H\u003csub\u003e2\u003c/sub\u003eS production, sensitivity to vibriostatic agent O/129, oxidative/fermentative metabolism, and amino acid decarboxylase reactions. Growth tolerance was tested at 0, 3, and 8% NaCl and at 4\u0026deg;C, 30\u0026deg;C, and 35\u0026deg;C. Additional tests included indole production, gelatinase activity, Voges\u0026ndash;Proskauer reaction, citrate utilization, casein and aesculin hydrolysis, and acid production from inositol, arabinose, and salicin. Carbon-source utilization (L-arabinose, N-acetylglucosamine, D-mannose, D-cellobiose, D-glucose, galactose, D-trehalose, D-melibiose, lactose, D-mannitol, sorbitol, amygdalin, D-fructose, glycerol, glycogen, maltose, and sucrose) was evaluated in the presence of added NaCl. Nitrate reduction was also assessed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 16S rDNA gene sequence analysis of bacterial isolates\u003c/h2\u003e \u003cp\u003eBacterial isolates were grown in 100 mL nutrient broth (Beckton Dickinson and Company) containing 3% NaCl with shaking at 30\u0026deg;C to an optical density of 0.8 at 550 nm. High-molecular-weight genomic DNA from the different \u003cem\u003eVibrio\u003c/em\u003e bacterial isolates was obtained using the NucleoSpin Microbial DNA KIT (Macherey-Nagel), and the extracted DNA was stored at 4\u0026deg;C before PCR and sequencing experiments. The extracts were analyzed both quantitatively and qualitatively using agarose gel electrophoresis (1%, 75 V) and spectrophotometric measurements (NanoDrop).\u003c/p\u003e \u003cp\u003eThe 16S rRNA gene was amplified using AmpliTaq (Thermo Fisher Scientific) and the primer pairs COM1F/COM2F, 785F/1488R, and 20-43F/683R, as previously reported [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The thermal cycling conditions were as follows: initial denaturation at 95\u0026deg;C for 5 min, followed by 30 cycles (denaturation at 95\u0026deg;C for 45 s, annealing at 55\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 2 min), with a final extension at 72\u0026deg;C for 5 min. The quality and quantity of each amplicon were assessed by agarose gel electrophoresis and spectrophotometric measurements, as previously reported. Amplicons were sequenced using the Sanger method (Eurofins Genomics, Ebersberg, Germany). For each isolate, the three resulting amplicons were assembled as previously described [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The obtained sequences were used for taxonomic identification against the rRNA/ITS database via NCBI BLAST. Multiple sequence alignment was performed using the MAFFT tool [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], and phylogenetic analysis was conducted with IQ-TREE [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] to construct a phylogenetic tree. The parameters of the tools used, and the reference sequences were as previously reported [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Statistical analysis\u003c/h2\u003e \u003cp\u003eAnalysis of variance (ANOVA) was performed using STATISTICA 10.0 software to evaluate differences in bacterial counts between diseased sponges, sediment, and water samples. Significance was set at a critical level of 95% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Impact of the disease on Biota\u003c/h2\u003e \u003cp\u003eDuring the summer of 2024, episodes of tissue necrosis were observed in different benthic organisms, such as sponges \u003cem\u003ePetrosia ficiformis, Agelas oroides, Sarcotragus spinosulus\u003c/em\u003e (Schmidt, 1862), but also the green algae such as \u003cem\u003eCodium bursa\u003c/em\u003e (Linnaeus - C. Agardh 1817) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The coastal zone survey recorded a density of 0.74 ind/m\u0026sup2; for \u003cem\u003eP. ficiformis\u003c/em\u003e and 0.14 ind/m\u0026sup2; for \u003cem\u003eA. oroides\u003c/em\u003e. Most sponges exhibited a whitish biofilm, and a necrotic tissue was observed in both species. Moreover, only one individual of \u003cem\u003eP. ficiformis\u003c/em\u003e was found completely dead (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Marine heat waves in the period 2011\u0026ndash;2024\u003c/h2\u003e \u003cp\u003eBetween 2011 and 2024, a total of 68 marine heatwave (MHW) events were identified at 5 m depth (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Event duration ranged from 5 to 119 days, with a marked variability in both temporal persistence and thermal structure. Mean intensity values (above the threshold) varied between 0.84 and 3.42\u0026deg;C, while maximum intensity (above the threshold) reached up to 4.92\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe seasonal distribution of marine heatwaves out of the 68 total events is: 34 MHWs were classified as cold-season events, and 34 MHWs as warm-season events, indicating an overall balanced seasonal frequency.\u003c/p\u003e \u003cp\u003eDespite the similar number of events, the thermal characteristics differed markedly between seasons. Warm-season MHWs generally exhibited higher intensity and cumulative impact compared to cold-season events. The most intense summer-origin MHW occurred in 2019, lasting 119 days, with a cumulative intensity of 242.88\u0026deg;C\u0026middot;days, representing the most powerful event of the entire time series. In contrast, cold-season MHWs tended to exhibit lower mean intensities; however, several events were characterized by exceptionally long durations, resulting in high cumulative thermal stress. The most intense cold-season event occurred between December 2013 and April 2014, lasting 110 days and reaching a cumulative intensity of 170.47\u0026deg;C\u0026middot;days. Similarly, prolonged winter events were observed in 2022\u0026ndash;2023 and 2023\u0026ndash;2024, both of which exceeded 90 days.\u003c/p\u003e \u003cp\u003eFrom a temporal perspective, the dataset reveals an increase in the frequency, duration, and cumulative intensity of MHWs after 2015, with particularly pronounced events occurring after 2018. The period 2019\u0026ndash;2024 is characterized by recurrent long-lasting and high-intensity events, affecting both warm and cold seasons. In 2024, marine heatwaves at 5 m depth occurred during both the cold and warm seasons, with a prolonged winter-spring event, characterised by long duration and high cumulative intensity, followed by an exceptionally intense and persistent summer heatwave, together indicating sustained thermal stress throughout the entire year (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe event, which occurred between July and September 2024, represents one of the most severe marine heatwaves recorded at 5 m depth in the study period. This event lasted 66 days, making it one of the longest warm-season MHWs in the dataset. It was characterized by a high mean relative intensity (2.53\u0026deg;C) and a maximum relative intensity of 4.11\u0026deg;C, indicating the presence of strong thermal anomalies above the climatological threshold.\u003c/p\u003e \u003cp\u003eIn terms of absolute temperature, this event reached an absolute mean intensity of 27.90\u0026deg;C and an absolute maximum intensity of 29.18\u0026deg;C, values among the highest observed across the entire 2011\u0026ndash;2024 time series. The prolonged exposure to such elevated absolute temperatures resulted in a very high cumulative intensity (166.72\u0026deg;C\u0026middot;days) and an absolute cumulative intensity of 1841.53\u0026deg;C\u0026middot;days, ranking this event among the most energetically impactful marine heatwaves recorded, second only to the extreme events of 2019 and 2015.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Enumeration, isolation and phylogenetic analysis of vibrios\u003c/h2\u003e \u003cp\u003eThe results of cultural analysis by using in parallel Marine Agar 2216 and TCBS Agar demonstrated that the abundance of surface bacteria was significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) higher in injured specimens of \u003cem\u003eP. ficiformis\u003c/em\u003e (on average 4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 x 10\u003csup\u003e5\u003c/sup\u003e CFU/g. for total surface culturable bacteria and 7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 x 10\u003csup\u003e4\u003c/sup\u003e CFU/g for culturable vibrios) and \u003cem\u003eA. oroides\u003c/em\u003e (on average 3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 x 10\u003csup\u003e5\u003c/sup\u003e CFU/g for total surface culturable bacteria and 6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 x 10\u003csup\u003e4\u003c/sup\u003e CFU/g for culturable vibrios) than in the surrounding sediment (on average 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 x 10\u003csup\u003e5\u003c/sup\u003e CFU/g. for total surface culturable bacteria and 3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 x 10\u003csup\u003e4\u003c/sup\u003e CFU/g for culturable vibrios) and seawater (on average 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 x 10\u003csup\u003e2\u003c/sup\u003e CFU/mL and 81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 CFU/mL for total surface culturable bacteria and culturable vibrios respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, Gram-negative, oxidase- and catalase-positive bacteria sensitive to O/129 were isolated from diseased tissue samples, as well as from sediment and seawater samples. These bacteria produced exclusively yellow, sucrose-fermenting colonies on TCBS agar, consistent with those expected for members of the genus \u003cem\u003eVibrio\u003c/em\u003e in the case of sponges and sediments, and predominantly yellow colonies in the case of seawater. Moreover, although pieces of healthy specimens as well as pieces of the damaged sponges of both species, were directly placed on TCBS agar plates, vibrios grew only from the wide whitish areas on the surface of the diseased sponges. From a macroscopic point of view after incubation of the pieces of sponges on TCBS for 48 hour we observed the growth of a yellow coating on the plates incubated due to the growth of sucrose-fermenting vibrios (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). On TCBS plates, the isolates formed round, smooth, edge tidy, convex and translucent yellow colonies. All these isolated vibrios were identified on the basis of both genotypic and phenotypic analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe identity of the isolates was investigated through 16S rRNA\u0026ndash;based taxonomic classification and confirmed by phylogenetic analysis. Specifically, BLAST searches against the rRNA/ITS database revealed that all isolates were phylogenetically related to \u003cem\u003eVibrio alginolyticus\u003c/em\u003e strains ATCC 17749 and NBRC 15630, showing sequence identities greater than 99% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTaxonomic identification of the isolates based on 16S rRNA sequences and phylogenetic analysis. AGE\u0026thinsp;=\u0026thinsp;isolate from \u003cem\u003eAgelas oroides\u003c/em\u003e; PET\u0026thinsp;=\u0026thinsp;isolate from \u003cem\u003ePetrosia ficiformis;\u003c/em\u003e H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;=\u0026thinsp;isolate from seawater; SED\u0026thinsp;=\u0026thinsp;isolate from sediment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAccession number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQuery cover (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIdentity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAGE1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.72%\u003c/p\u003e \u003cp\u003e99.65%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003cp\u003eNR_113781.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAGE2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.72%\u003c/p\u003e \u003cp\u003e99.58%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003cp\u003eNR_113781.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePET1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.58%\u003c/p\u003e \u003cp\u003e99.58%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003cp\u003eNR_113781.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePET2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.79%\u003c/p\u003e \u003cp\u003e99.72%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003cp\u003eNR_113781.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePET3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.65%\u003c/p\u003e \u003cp\u003e99.65%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003cp\u003eNR_113781.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePET4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.23%\u003c/p\u003e \u003cp\u003e99.44%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003cp\u003eNR_113781.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePET5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.56%\u003c/p\u003e \u003cp\u003e99.56%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_117895.1\u003c/p\u003e \u003cp\u003eNR_122060.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePET6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.72%\u003c/p\u003e \u003cp\u003e99.65%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003cp\u003eNR_113781.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePET7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.51%\u003c/p\u003e \u003cp\u003e99.44%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003cp\u003eNR_113781.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePET8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.09%\u003c/p\u003e \u003cp\u003e99.58%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003cp\u003eNR_122050.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.58%\u003c/p\u003e \u003cp\u003e99.51%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003cp\u003eNR_113781.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.72%\u003c/p\u003e \u003cp\u003e99.65%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003cp\u003eNR_113781.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSED1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.02%\u003c/p\u003e \u003cp\u003e98.95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003cp\u003eNR_113781.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSED2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePX623015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.72%\u003c/p\u003e \u003cp\u003e99.58%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003cp\u003eNR_113781.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMZ900920.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749\u003c/p\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e98.91%\u003c/p\u003e \u003cp\u003e99.35%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNR_122050.1\u003c/p\u003e \u003cp\u003eNR_118258.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDin\u0026ccedil;t\u0026uuml;rk et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eInterestingly, one of the previously identified isolates (\u003cem\u003eVibrio\u003c/em\u003e sp. MZ900920.1), associated with mass sponge mortality [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], was also found to be phylogenetically close to \u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749 and \u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630 based on rRNA/ITS database comparison (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo confirm the taxonomic identification, a phylogenetic approach was applied by comparing the sequences of the isolates with those previously reported in the literature [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], as well as with reference sequences of \u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749 (NR_118258.1) and \u003cem\u003eV. alginolyticus\u003c/em\u003e NBRC 15630 (NR_113781.1) retrieved from the rRNA/ITS database.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the phylogenetic tree, which illustrates that the sequences of the isolates cluster near the 16S rRNA sequences of bacteria belonging to the \u003cem\u003eVibrio harveyi\u003c/em\u003e group (\u003cem\u003eV. alginolyticus, V. azureus, V. campbellii, V. harveyi, V. mytili, V. natriegens, V. parahaemolyticus\u003c/em\u003e, and \u003cem\u003eV. rotiferianus\u003c/em\u003e) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], confirming that the isolates belong to the \u003cem\u003eHarveyi\u003c/em\u003e clade. The sequences obtained from the isolates in this study are highly similar to one another and form a subclade that also includes \u003cem\u003eVibrio\u003c/em\u003e sp. MZ900920.1 and \u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749 (NR_118258.1). Notably, \u003cem\u003eVibrio\u003c/em\u003e sp. MZ900920.1 shows the greatest similarity to the sequence of the PET4S isolate. \u003cem\u003eVibrio neocaledonicus\u003c/em\u003e, a species previously reported as pathogenic in \u003cem\u003ePatinopecten yessoensis\u003c/em\u003e [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], is closely related to this subclade.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, all the isolates from the damaged sponges, as well as from the sediment and water samples, belonged to the Group Harvey. The latter includes bacteria known to be pathogenic to fish and marine invertebrates. The assignment of the bacterial isolates to the \u003cem\u003eV. alginolyticus\u003c/em\u003e was consistent with the phylogenetic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) and the results of morphological, cultural and biochemical tests (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) according to the schemes proposed by Alsina and Blanch [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] and Kaysner et al. [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] and the species description by Gomez-Gil et al. [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe results of analysis of \u003cem\u003eVibrio alginolyticus\u003c/em\u003e morphological and physiological properties.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics/test\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eIsolated bacteria\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAlsina \u0026amp; Blanch\u003c/em\u003e [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eKaysner et al.\u003c/em\u003e [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram reaction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell morphology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0/129 sensitivity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxidation/Fermentation (O/F)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMotility\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH₂S production\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDecarboxylase\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArginine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLysine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrnithine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ev\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eReduction\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrates to nitrites\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrates to nitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGrowth in % NaCl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxidase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCatalase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVoges Proskauer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGelatinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCitrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHydrolysis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCasein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAesculin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGrowth at \u0026deg;C\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAcid from\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInositol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArabinose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCarbon sources\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Arabinose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSorbitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-Mannitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-Acetylglucosamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmygdalin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-Mannose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-Cellobiose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-Fructose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-Glucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlycerol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlycogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGalactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-Trehalose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaltose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-Melibiose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIdentification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eF\u0026thinsp;=\u0026thinsp;Fermentative; V\u0026thinsp;=\u0026thinsp;Variable; r\u0026thinsp;=\u0026thinsp;Short rod with curve shaped; nd\u0026thinsp;=\u0026thinsp;No Data; + = Positive Reaction; - = Negative Reaction; (-) = Negative for 25\u0026ndash;11%; S\u0026thinsp;=\u0026thinsp;Susceptible; R\u0026thinsp;=\u0026thinsp;Resistant.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eSponges (phylum Porifera) are sessile invertebrates regarded as the earliest-diverging lineage of multicellular animals. Owing to their highly efficient filter-feeding systems, sponges play significant ecological and biotechnological roles in nutrient cycling within marine ecosystems. In this study, we document a sponge disease event that occurred along the Italian Apulian coastline (Mediterranean Sea, Italy) during the summer of 2024. Our findings, showing microbial overgrowth in the \u003cem\u003eP. ficiformis\u003c/em\u003e and \u003cem\u003eA. oroides\u003c/em\u003e population and necrotic tissue in most of the specimens, indicate a substantial sublethal impact. The phenomenon was observed in a spatially patchy manner at five distinct sites along a coastal stretch of approximately 100 km. For \u003cem\u003eP. ficiformis\u003c/em\u003e, this pattern is consistent with observations by Cerrano et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], who emphasized that climate-driven stress events in the Mediterranean, often result not only in acute mortality but also in widespread sublethal effects, potentially impairing long-term survival, ecological functioning, and resilience of sponge assemblages; however, unlike their interpretation, which did not associate tissue damage with bacterial involvement, our results indicate that microbial proliferation may accompany stress-related tissue degradation. During the summer of 2022, N\u0026uacute;\u0026ntilde;ez-Pons et al. [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] reported widespread thermal stress in \u003cem\u003eP. ficiformis\u003c/em\u003e populations following a marine heatwave in the Gulf of Naples (Tyrrhenian Sea, Italy). Affected sponges exhibited depigmentation and altered tissue consistency, often progressing to necrosis and mortality. By comparing the microbiomes of healthy and diseased individuals, the authors identified a profound restructuring of their microbial communities, a dysbiotic state characterized by an influx of rare taxa, (e.g. Rhodobacteraceae, Flavobacteriaceae) and the introduction of potentially pathogenic, opportunist groups, including \u003cem\u003eVibrio\u003c/em\u003e spp. While previous investigations broadly described this as \u0026ldquo;disease-associated dysbiosis,\u0026rdquo; the present study advances this understanding by isolating and identifying as a specific, well-defined pathogen: \u003cem\u003eVibrio alginolyticus\u003c/em\u003e. This bacterium was found to dominate during the critical necrotic phase preceding sponge death. Notably, this epidemiological response was consistent across the two taxonomically distinct sponge species examined, suggesting that \u003cem\u003eVibrio\u003c/em\u003e proliferations thrive during stress-induced tissue degradation. These findings support the hypothesis that opportunistic bacteria play a decisive role in sublethal impairment and subsequent mortality under extreme environmental stress. These disease events occurred in correspondence with one of the most intense and persistent marine heatwaves recorded in the study area. The summer 2024 event was characterized by exceptionally high maximum absolute temperatures (29.18\u0026deg;C), prolonged duration, and elevated cumulative thermal stress, following an already long-lasting winter-spring heatwave. This sustained thermal pressure likely exceeded the physiological tolerance thresholds of several benthic organisms, including sponges, impacting the marine biodiversity [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], and driving mass mortality events [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A variety of factors and conditions have been tentatively linked to mass mortality in sponges, but the agents/mechanisms causing mortality to have rarely been elucidated. Local accumulation of pollutants, the introduction of alien species, ecological imbalances caused by overexploitation of commercial benthic organisms, environmentally induced dysbiosis of the symbiotic microbiome, infection by water column pathogens, and anomalous climate patterns leading to heat waves, among other factors, have been suggested as general scenarios associated with several cases of mass mortality of sponges [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The latter two (pathogens and water warming) are perceived to be particularly important drivers of sponge mortality, and they can be related to each other through the hypothesis that increasing values of sea surface temperature in turn increase microbial pathogenicity or sponge susceptibility or both [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Interestingly, in all the damaged sponges of both species \u003cem\u003eP. ficiformis\u003c/em\u003e and \u003cem\u003eA. oroides\u003c/em\u003e bacteria belonging to the species \u003cem\u003eVibrio alginolyticus\u003c/em\u003e were mainly isolated. \u003cem\u003eVibrio alginolyticus\u003c/em\u003e is a naturally occurring marine bacterium, recognized as an emerging pathogen in humans and animals and the second most common cause of vibriosis. The transmission of \u003cem\u003eV. alginolyticus\u003c/em\u003e is a function of its abundance in the environment. Like other \u003cem\u003eVibrio\u003c/em\u003e species, \u003cem\u003eV. alginolyticus\u003c/em\u003e is considered a conditionally rare taxon in marine waters, with populations capable of forming large, short-lived blooms under specific environmental conditions. Previous research has established the importance of temperature as one of the main determinants of \u003cem\u003eVibrio\u003c/em\u003e geographical and temporal distribution. Therefore, the high temperatures recorded in the summer of 2024 favoured the development of \u003cem\u003eV. alginolyticus\u003c/em\u003e, which likely prevailed over the other \u003cem\u003eVibrio\u003c/em\u003e species present. Indeed, among vibrios, only \u003cem\u003eV. alginolyticus\u003c/em\u003e grew around the whitish areas of damaged sponges. Sponges are known to host stable, taxonomically diverse, and structurally complex microbial consortia. These symbiotic microorganisms substantially contribute to host physiology, including growth, secondary metabolite production, chemical defence, and responses to biotic and abiotic stressors. However, changes in microbial composition cause disease in the host sponge. For this reason, the prevalence of \u003cem\u003eV. alginolyticus\u003c/em\u003e among the bacterial isolates from damaged sponges suggests a crucial role for this microorganism in the observed event. Under such scenario, the genus of \u003cem\u003eVibrio\u003c/em\u003e bacteria emerges as a putative candidate for at least some cases of sponge mortality, a possibility that, at present, remains poorly investigated in this animal group. \u003cem\u003eVibrio\u003c/em\u003e consists of thermo-dependent bacteria that are often pathogenic or facultative pathogenic to a wide variety of aquatic organisms (corals, bivalves, fish, etc.), and whose niche expansion is being facilitated by global ocean warming [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. While microbial agents, in general, have putatively been blamed as responsible for several episodes of sponge disease [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e], very few studies have been able to unequivocally identify the specific pathogens. Here is a rewritten version with enhanced originality, clarity, and polished scientific English while preserving the original meaning: This situation is unsurprising considering the high structural and functional complexity of the sponge microbiome [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. In the limited number of studies where a microbial pathogen has been conclusively identified [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan additionalcitationids=\"CR76\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e], the causative agents included \u003cem\u003ePseudoalteromonas agarivorans\u003c/em\u003e (originally misidentified as \u003cem\u003eSulfitobacter pontiacus\u003c/em\u003e), which has been associated with both sponge white patch (SWP) and sponge boring necrosis (SBN), as well as \u003cem\u003eHormoscilla\u003c/em\u003e sp., implicated in mangrove sponge disease (MSD). To date, the involvement of \u003cem\u003eVibrio\u003c/em\u003e spp. in sponge diseases has been only proposed for \u003cem\u003eIrcinia fasciculata\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and experimentally demonstrated for the closely related species \u003cem\u003eIrcinia variabilis\u003c/em\u003e by Stabili et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], who successfully isolated \u003cem\u003eVibrio rotiferianus\u003c/em\u003e following agar inoculation with tissue from diseased specimens. More recently, Din\u0026ccedil;t\u0026uuml;rk et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] documented a mass mortality event affecting wild sponge populations in the Aegean Sea (Turkey, Eastern Mediterranean), which impacted the keratose demosponge \u003cem\u003eSarcotragus foetidus\u003c/em\u003e in September 2021. The authors identified, through 16S rRNA, the colonies isolated from the diseased sponges. Three of them resulted putatively pathogenic (\u003cem\u003eV. fortis\u003c/em\u003e, \u003cem\u003eV. owensii\u003c/em\u003e, \u003cem\u003eV. gigantis\u003c/em\u003e). Importantly, those vibrios were isolated from only tissues of diseased sponges. In contrast, healthy individuals sampled in both summer and winter led to no \u003cem\u003eVibrio\u003c/em\u003e growth in laboratory cultures. In addition, they reported that a progressive increase in temperature from 1970 to 2021 was recorded with values above 24\u0026deg;C from May to September 2021, reaching an absolute historical maximum of 28.9\u0026deg;C in August 2021. Interestingly the three vibrio species \u003cem\u003eV. fortis\u003c/em\u003e, \u003cem\u003eV. owensii\u003c/em\u003e, \u003cem\u003eV. gigantis\u003c/em\u003e belonged to the \u003cem\u003eVibrio harveyi\u003c/em\u003e group as well as \u003cem\u003eVibrio rotiferianus\u003c/em\u003e that was isolated by Stabili et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] in the case of diseased \u003cem\u003eI. variabilis\u003c/em\u003e. Interestingly, one of the bacterial isolates identified by Din\u0026ccedil;t\u0026uuml;rk et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], \u003cem\u003eVibrio\u003c/em\u003e sp. MZ900920.1, is taxonomically close to the \u003cem\u003eVibrio alginolyticus\u003c/em\u003e species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the present study, 16S rRNA gene sequence analysis of the 14 isolates revealed that all were taxonomically related to \u003cem\u003eV. alginolyticus\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and, consequently, to the \u003cem\u003eVibrio harveyi\u003c/em\u003e group. Phylogenetic analysis further showed that the isolates clustered closely with the reference strain \u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749 (NR_118258.1) and with \u003cem\u003eVibrio\u003c/em\u003e sp. MZ900920.1, previously reported by Din\u0026ccedil;t\u0026uuml;rk et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] in an environmental context characterized by elevated temperatures and mass sponge mortality events, as described before. \u003cem\u003eV. alginolyticus\u003c/em\u003e employs several key virulence factors, including the metalloprotease (Asp), serine protease (Pep), immunelike protein (IlpA), outer membrane protein (OmpU), type VI secretion system (T6SS) [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e], hemolysins, and the quorum-sensing (QS) master regulator LuxR. Together, these components facilitate host tissue degradation, immune evasion, and QS-dependent pathogenic behaviors such as motility and biofilm formation [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Several of these virulence factors are transcriptionally upregulated by the alternative sigma factor RpoE, which directly binds to the \u003cem\u003eluxR\u003c/em\u003e promoter, thereby enhancing LuxR expression and activating the downstream QS cascade [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Notably, LuxR binding motifs have been shown to vary with temperature [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Moreover, the expression of these virulence factors is strongly temperature dependent [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. For instance, hemolysin production and protease secretion are significantly suppressed at lower temperatures [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. These findings support the hypothesis that the elevated temperatures observed during the sampling events in this study may enhance the expression of virulence factors in \u003cem\u003eV. alginolitycus\u003c/em\u003e as well as promote the proliferation and dissemination of diverse \u003cem\u003eVibrio\u003c/em\u003e spp., consistent with previous reports.\u003c/p\u003e \u003cp\u003eTherefore, in agreement with Din\u0026ccedil;t\u0026uuml;rk et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], based on the results here obtained, we can hypothesize that, as in the case of the Aegean Sea, also in our case the high seawater temperatures maintained for several months in 2024 promoted the proliferation of pathogenic \u003cem\u003eVibrio\u003c/em\u003e species (thermo-dependent bacteria), triggering or exacerbating the course of sponge disease in \u003cem\u003eP. ficiformis\u003c/em\u003e and \u003cem\u003eA. oroides\u003c/em\u003e. Therefore, vibriosis emerges as one of the putative mechanisms through which global warming of waters in the Mediterranean Sea translates into sponge mortality. This is in agreement with the Marine Climate Change Impacts Partnership (MCCIP) Annual Report 2010\u0026ndash;2011, which provides an updated assessment of how climate change is also affecting the seas of the United Kingdom, considering, for the first time, the potential future increase of marine vibrios as an emerging issue. In recent years, several countries in Northwestern Europe have seen an unprecedented increase in the number of infections associated with warm-water \u003cem\u003eVibrio\u003c/em\u003e species, including in humans. For example, during the hot summer of 2006, wound infections related to contact with Baltic and North Sea waters were reported in Germany (\u003cem\u003eV. vulnificus\u003c/em\u003e [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]), southeastern Sweden (\u003cem\u003eV. cholerae\u003c/em\u003e non-O1/O139 [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]), the Netherlands (\u003cem\u003eV. alginolyticus\u003c/em\u003e [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]), and Denmark (\u003cem\u003eV. alginolyticus\u003c/em\u003e and \u003cem\u003eV. parahaemolyticus\u003c/em\u003e [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]). These events have raised growing concern about the potential contribution of climate change to the abundance of \u003cem\u003eVibrio\u003c/em\u003e species in coastal seas. However, despite the wealth of indirect evidence, it remains unclear whether vibrios, known to be temperature-dependent, are increasing within the complex, ecologically regulated bacterial communities of coastal marine waters. This is primarily due to the lack of historical data.\u003c/p\u003e \u003cp\u003eTherefore, this study represents a further contribution to evaluating a possible link between the presence of \u003cem\u003eVibrio\u003c/em\u003e and seawater surface temperature, and the increase in diseases affecting humans and marine organisms. Furthermore, beyond a reliable identification of the causative agents responsible for pathogenic events, another aspect requiring further investigation is the ability of sponges to defend themselves from microbial infections. Therefore, further studies will be conducted to evaluate the presence of antimicrobial compounds in the studied species and their efficacy under conditions of sponge weakness related to global change. This is particularly important since, at present, we can suggest, as already hypothesized by Vacelet et al. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], the involvement of a synergistic mechanism in the disease and that presumably under stressful physiological conditions (high temperature, high nutrients and reduced water flow) sponge pathogens, in our case \u003cem\u003eV. alginolyticus\u003c/em\u003e, could activate virulence mechanisms and sponges could not be able to control the proliferation of these bacteria. This hypothesis aligns with the findings of N\u0026uacute;\u0026ntilde;ez-Pons et al. [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], who suggested that microbial community stability confers protection to certain host phenotypes, thereby bolstering population-level resilience. Therefore, we suggest that the heightened intensity of the 2024 MHW coupled with the prolonged disruption of microbial stability, likely facilitated the extensive invasion of \u003cem\u003eVibrio alginolyticus\u003c/em\u003e. Furthermore, our results corroborate the classification of \u003cem\u003eP. ficiformis\u003c/em\u003e as a sentinel species or, at least, one highly vulnerable to MHWs in the Mediterranean Sea. However, we also demonstrate that this susceptibility extends to other taxa, such as \u003cem\u003eA. oroides\u003c/em\u003e. Ultimately, marine heatwaves represent a pervasive threat to the diverse sponge species that underpin the structure of shallow Mediterranean benthic communities.\u003c/p\u003e \u003cp\u003eInterestingly, in the Mediterranean Sea, increasing global temperatures not only pose a significant threat to marine biodiversity but also promote environmental conditions conducive to the proliferation and successful establishment of non-indigenous, thermophilic species. [\u003cspan additionalcitationids=\"CR87\" citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. Moreover, during the summer of 2024 in the Apulian seas (Mediterranean) mortality and disease events did not only affect the here considered sponges but outbreaks were recorded for mussels belonging to the species \u003cem\u003eMytilus galloprovincialis\u003c/em\u003e [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e] but also for seaweeds of the species \u003cem\u003eChatemorpha linum\u003c/em\u003e grown in an integrated multitrophic aquaculture system in the Gulf of Taranto (personal observation). In the study by Calabrese et al. [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e], the authors concluded that, in addition to high temperatures (maximum 31.01\u0026deg;C), there was a combination of factors triggered by the prolonged heat wave, such as the involvement of excessive algal blooms, including toxic species, and/or the decay of surface organisms due to infections, which also affected the deeper layers and, consequently, mussel growth was prohibitive even at depth. It is therefore clear that the consequences of climate change led to social, economic, and environmental impacts on a global scale [\u003cspan additionalcitationids=\"CR91\" citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. A consistent trend observed in recent decades is that, due to climate change, disease outbreaks are expected to occur more frequently, with greater intensity, and with a wider spread both on land and in water [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this framework investigating diseases affecting marine sponges is essential to gain a comprehensive understanding of benthic ecosystem ecology and dynamics as well as sponge biodiversity, both of which support the sustainable functioning of ocean environments. The loss of a sponge population can trigger unpredictable ecological consequences for surrounding habitats and associated species, given the pivotal biological and ecological roles these organisms play. Marine sponges indeed serve as refuge for numerous small invertebrates [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] and are key regulators of benthic\u0026ndash;pelagic coupling across diverse marine habitats. Moreover, maintaining healthy and stable populations of commercially valuable sponge species is essential for the viability of the sponge industry.\u003c/p\u003e \u003cp\u003eInvestigating diseases affecting marine sponges is essential to gain a comprehensive understanding of benthic ecosystem ecology and dynamics as well as sponge biodiversity, both of which support the sustainable functioning of ocean environments. The loss of a sponge population can trigger unpredictable ecological consequences for surrounding habitats and associated species, given the pivotal biological and ecological roles these organisms play. Marine sponges indeed serve as refuge for numerous small invertebrates [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] and are key regulators of benthic\u0026ndash;pelagic coupling across diverse marine habitats. Moreover, maintaining healthy and stable populations of commercially valuable sponge species is essential for the viability of the sponge industry.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eMarine heatwaves (MHWs) are increasing in frequency, duration, and intensity, with profound consequences for marine ecosystems worldwide. This phenomenon was evident during the summer of 2024 along the Italian Apulian coastline where extensive mass-mortality events of benthic organisms were documented. Among the most affected were the sponges \u003cem\u003ePetrosia ficiformis\u003c/em\u003e and \u003cem\u003eAgelas oroides\u003c/em\u003e, which exhibited clear signs of disease. Bacteriological analyses of diseased individuals revealed the presence of \u003cem\u003eVibrio alginolyticus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe main strengths of this study include:\u003c/p\u003e \u003cp\u003e(a) sampling conducted precisely at the time when the condition of maximum virulence became macroscopically evident, namely during the appearance of the whitish mucilaginous plume that smothered the sponge;\u003c/p\u003e \u003cp\u003e(b) observation of the phenomenon in two co-occurring sponge species belonging to the same class but different orders, examined across multiple locations within the same habitat; and\u003c/p\u003e \u003cp\u003e(c) documentation that all samples collected from the whitish biofilm consistently exhibited a high density of \u003cem\u003eVibrio alginolyticus\u003c/em\u003e, identifying this bacterium as the primary driver of the necrotic process.\u003c/p\u003e \u003cp\u003eThe anomalously high seawater temperatures that persisted for several months in 2024 likely facilitated the proliferation of this thermophilic pathogen within sponge tissues, initiating or exacerbating disease progression. These observations suggest that vibriosis represents a plausible mechanistic pathway linking Mediterranean warming to sponge mortality. If marine heatwaves continue to recur in the coming summers, repeated disease outbreaks could ultimately decimate local populations of these sponge species, with cascading impacts on habitat structure, biogeochemical cycling, and overall ecosystem functioning. Given these ecological implications, future research should explicitly investigate the role of heat-stress and \u003cem\u003eVibrio\u003c/em\u003e infections as key drivers of sponge disease dynamics under ongoing climate change.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eInvestigation : L.S., M.C., A.T., F.N., E.Q., P.A. Formal analysis : L.S., M.C., A.T., F.N., E.Q. Visualization : L.S., M.C., A.T., F.N., E.Q. Resources : L.S., M.C., A.T., F.N., E.Q. Methodology : L.S., M.C., A.T., F.N., E.Q. Conceptualization : L.S., S.P., P.A. Writing - Review \u0026amp; Editing : L.S., M.C., A.T., F.N., E.Q. Writing - Original Draft : L.S., M.C., A.T., F.N., E.Q. Supervision : L.S., S.P., P.A. Project administration : L.S., S.P. Funding acquisition : L.S., S.P.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis study was mainly supported by the \u0026ldquo;National Biodiversity Future Center (NBFC)\u0026rdquo;, funded by European Union Next Generation EU (PNRR) (Project code CN_00000033, Concession Decree No. 1034 of 14 June 2022 adopted by the Italian Ministry of University and Research, CUP: D33C22000960007, Spoke 2 Activity 3 Task 3.2 CUP B83C22002930006.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe 16s rRNA gene sequences obtained in this study are available under the following accession numbers: PX623002, PX623003, PX623004, PX623005, PX623006, PX623007, PX623008, PX623009, PX623010, PX623011, PX623012, PX623013, PX623014, PX623015.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFord HV, Jones NH, Davies AJ, Godley BJ, Jambeck JR, Napper IE, Suckling CC, Williams GJ, Woodall LC, Koldewey HJ (2022) The Fundamental Links between Climate Change and Marine Plastic Pollution. 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Ambio 42:937\u0026ndash;950\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Iinjured sponge, Epidemic disease, Vibrio alginolyticus, Climate change","lastPublishedDoi":"10.21203/rs.3.rs-8987753/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8987753/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMarine heatwaves associated with climate change are increasingly creating favourable conditions for the proliferation of pathogenic bacteria, leading to disease outbreaks and mass mortality events particularly in sessile suspension-feeding invertebrates, such as sponges. In summer 2024, a disease outbreak affecting two demosponge species, \u003cem\u003ePetrosia ficiformis\u003c/em\u003e and \u003cem\u003eAgelas oroides\u003c/em\u003e, was documented along the Northern Ionian coast of the Mediterranean Sea (Italy). Diseased sponges exhibited extensive surface necrosis or multiple lesions distributed across the body. Necrotic areas appeared whitish and were frequently coated with a thin mucous layer composed of bacterial aggregates. Microbiological culture analyses revealed elevated bacterial densities on sponge surfaces, with total culturable bacteria reaching 4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026times; 10⁵ CFU/g and culturable vibrios 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 \u0026times; 10⁴ CFU/g in \u003cem\u003eP. ficiformis\u003c/em\u003e, and 3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u0026times; 10⁵ CFU/g and 6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026times; 10⁴ CFU/g, respectively, in \u003cem\u003eA. oroides\u003c/em\u003e. All \u003cem\u003eVibrio\u003c/em\u003e isolates obtained from the conspicuous whitish lesions on diseased sponges were identified as \u003cem\u003eVibrio alginolyticus\u003c/em\u003e, based on concordant phylogenetic, morphological, cultural, and biochemical analyses.\u003c/p\u003e \u003cp\u003e \u003cem\u003eVibrio alginolyticus\u003c/em\u003e is a well-established pathogen of numerous aquatic organisms, suggesting that marine heatwaves may enhance \u003cem\u003eVibrio\u003c/em\u003e abundance and increase infection frequency during summer periods. Nevertheless, the specific factors initiating the epidemic in the examined sponge populations remain unresolved. We hypothesize that disease onset involves a synergistic mechanism in which environmental stressors, such as elevated temperature, increased nutrient availability, and reduced water circulation, promote the transition of sponge-associated bacteria, particularly \u003cem\u003eV. alginolyticus\u003c/em\u003e, toward virulence. Under these conditions, host physiological defences may be compromised, allowing uncontrolled bacterial proliferation. Overall, our findings indicate that the interaction between thermal stress and pathogenic vibrios represents a plausible trigger for sponge disease outbreaks. Further investigations are required to elucidate the etiological pathways involved and to identify the mechanisms underlying sponge recovery following epidemic events that result in mass mortality. A substantial decline in key sponge species may have serious consequences for ecosystem functioning, given their critical role as filter feeders in marine bioremediation and habitat health.\u003c/p\u003e","manuscriptTitle":"Can synergistic effects of marine heatwaves and Vibrio proliferation act as potential triggers of widespread demosponge disease? A case study in the Mediterranean Sea","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-09 07:08:10","doi":"10.21203/rs.3.rs-8987753/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9a4d4956-9649-45af-ae1b-b2f8009fd119","owner":[],"postedDate":"March 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-27T00:09:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-09 07:08:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8987753","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8987753","identity":"rs-8987753","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}