Playing "hide and seek" with the Mediterranean monk seal: a citizen science dataset reveals its distribution from molecular traces (eDNA).

Animal conservation relies on assessing the distribution and habitat use of species, but for endangered/elusive animals this can prove difficult. The Monk Seal, Monachus monachus, is one of the world's most endangered species of pinniped, and the only one endemic to the Mediterranean Sea. During recent decades, direct observations have been few and scattered, making it difficult to determine its distribution away from the Aegean Sea (core distribution area of the post-decline relict population). This study relies on environmental DNA (eDNA) analysis to detect the presence of the Monk Seal in 135 samples collected in 120 locations of the central/western Mediterranean Sea, spanning about 1500 km longitudinally and 1000 km latitudinally. A recently described species-specific qPCR assay was used on marine-water samples, mostly collected during 2021 by a Citizen Science (CS) project. Positive detections occurred throughout the longitudinal range, including the westernmost surveyed area (Balearic archipelago). The distribution of the positive detections indicated six "hotspots", mostly overlapping with historical Monk Seal sites, suggesting that habitat-specific characteristics play a fundamental role. We applied single-season occupancy models to correct for detection probability and to assess the importance of site-specific characteristics. The distance from small islets and protected (or access-restricted) areas was correlated negatively with the detection probability. This novel molecular approach, applied here for the first time in an extensive CS study, proved its potential as a tool for monitoring the distribution of this endangered/elusive species.

Polymer composition analysis of plastic debris ingested by loggerhead turtles (Caretta caretta) in Southern Tyrrhenian Sea through ATR-FTIR spectroscopy.

The ingestion of anthropogenic plastic debris by marine wildlife is widespread in the Mediterranean Sea. The endangered status (in the IUCN Red List) of Loggerhead turtle (Caretta caretta, Linnaeus, 1758) is a consequence of its vulnerability. In this study, macro-/meso-plastics (5-170 mm) collected from faeces of twelve loggerhead turtles rescued (live) in the Aeolian Archipelago (Southern Tyrrhenian Sea, Italy) were analyzed by size, weight, shape, color and polymer type through Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy (ATR-FTIR). The defecation rate during hospitalization (7-14 days) varied among turtles (from 0.08 to 0.58). The mean number of plastic expulsions (2.7 ± 1.8 items for turtle) was higher during the 5th day of hospitalization (Kruskal-Wallis test, P = 0.01). However, the mean number of plastic-like items defecated during the common days of hospitalization did not vary among turtles (Kruskal-Wallis test, P > 0.05). All turtles were found to have ingested plastic. A total of 114 debris items were recovered from their faeces, 113 of which were identified as plastic. Their color was mostly white-transparent (64.9%) and light (19.3%). Shape was mainly fragments (52.6%), sheets (38.6%), followed by nylon, net-fragments, elastic plastic, foamed plastic and industrial granules (8.8%). Meso-plastics (5-25 mm) represented 72% of the total number of debris and were found more frequently in turtle with Curved Carapace Length (CCL) ≤ 60 cm (CCL = 30-60 cm, n = 5) than those with CCL >60 cm (CCL = 60-71 cm, n = 7). Plastic items were composed mainly of polyethylene (48.2%) and polypropylene (34.2%). Polypropylene (R2 = 0.95, P < 0.001) and polyisoprene (R2 = 0.45, P = 0.017) were more common in meso-plastics while polyethylene (R2 = 0.44, P < 0.01) in macro-plastics. Finally, high-density polyethylene, polyvinyl chloride, polyamide and polyurethane were also found in some turtles. This study reveals high spreads of plastic contamination in faeces of both turtles with CCL ≤60 cm and CCL >60 cm, particularly vulnerable to the increasing quantity of floating plastic into their foraging sites highlighting the need of further research to associate debris ingestion with turtle diet and their size.

Phthalate esters (PAEs) concentration pattern reflects dietary habitats (δ13C) in blood of Mediterranean loggerhead turtles (Caretta caretta).

Phthalic acid esters (PAEs) are classified as endocrine disruptors, but it remains unclear if they can enter the marine food-web and result in severe health effects for organisms. Loggerhead turtles (Caretta caretta) can be chronically exposed to PAEs by ingesting plastic debris, but no information is available about PAEs levels in blood, and how these concentrations are related to diet during different life stages. This paper investigated, for the first time, six PAEs in blood of 18 wild-caught Mediterranean loggerhead turtles throughout solid-phase extraction coupled with gas chromatography-ion trap/mass spectrometry. Stable isotope analyses of carbon and nitrogen were also performed to assess the resource use pattern of loggerhead turtles. DEHP (12-63 ng mL-1) and DBP (6-57 ng mL-1) were the most frequently represented PAEs, followed by DiBP, DMP, DEP and DOP. The total PAEs concentration was highest in three turtles (124-260 ng mL-1) whereas three other turtles had concentrations below the detection limit. PAEs were clustered in three groups according to concentration in all samples: DEHP in the first group, DBP, DEP, and DiBP in the second group, and DOP and DMP in the third group. The total phthalates concentration did not differ between large-sized (96.3 ± 86.0 ng mL-1) and small-sized (67.1 ± 34.2 ng mL-1) turtles (p < 0.001). However, DMP and DEP were found only in large-sized turtles and DiBP and DBP had higher concentrations in large-sized turtles. On the other hand, DEHP and DOP were found in both small- and large-sized turtles with similar concentrations, i.e. ~ 21.0/32.0 ng mL-1 and ~ 7.1/9.9 ng mL-1, respectively. Winsored robust models indicated that δ13C is a good predictor for DBP and DiBP concentrations (significant Akaike Information criterion weight, AICwt). Our results indicate that blood is a good matrix to evaluate acute exposure to PAEs in marine turtles. Moreover, this approach is here suggested as a useful tool to explain the internal dose of PAEs in term of dietary habits (δ13C), suggesting that all marine species at high trophic levels may be particularly exposed to PAEs, despite their different dietary habitats and levels of exposure.

Fast and Reliable Determination of Phthalic Acid Esters in the Blood of Marine Turtles by Means of Solid Phase Extraction Coupled with Gas Chromatography-Ion Trap/Mass Spectrometry.

The presence of phthalic acid esters (PAEs) in marine environments is an important issue. These chemicals are able to affect marine organisms, particularly marine turtles, and to act as endocrine disrupters. In this paper, for the first time, a simple and reproducible analytical method based on solid-phase extraction (SPE) coupled with gas chromatography-ion trap/mass spectrometry (GC-IT/MS) was developed for the extraction of phthalates from the blood of marine turtles. The extraction was obtained by using C18 phthalates-free as the stationary phase. In order to individuate the best working conditions for the extraction, the adsorption isotherms and breakthrough curves were studied. The overall analytical methodology was validated in terms of limit of detection (LOD, 0.08-0.6 ng mL-1), limit of quantification (LOQ, 0.4-0.8 ng mL-1), and correlation coefficients (>0.9933). By using this procedure, percentage recoveries ranging from 89 to 103% were achieved. The precision parameters (intra-day and inter-day) were studied, and the obtained values were smaller than 12.5%. These data confirm the goodness of the proposed analytical methodology, which is applied to real samples.

Behaviour and vocalizations of two sperm whales (Physeter macrocephalus) entangled in illegal driftnets in the Mediterranean Sea.

Illegal driftnetting causes each year several entanglements and deaths of sperm whales in different Mediterranean areas, primarily in the Tyrrhenian Sea. In summer 2020, during the June-July fishing season, two sperm whales were found entangled in illegal driftnets in the Aeolian Archipelago waters, Southern Italy. These two rare events were an exceptional chance to collect behavioural and acoustics data about entangled sperm whales. We analysed 1132 one-minute sets of breathing/behavioural data and 1575 minutes of acoustic recording, when the whales were found entangled, during the rescue operation, immediately after release, and in the days thereafter. The first whale was generally quiet showing a general status of debilitation/weakness, numerous skin lesions, and low breathing rate (0.31 (0.60)); it collaborated during rescue operations. On the contrary, the second whale showed a high level of agitation with a high breathing rate (1.48 (1.31)) during both the entanglement period and the net cutting operations, vigorously moving its fluke and pectoral fins, opening its mouth, sideway rolling or side fluking and frequently defecating. Acoustically, the first whale produced mainly single clicks in all phases except for two series of creaks during rescuing operations while the second whale produced a wide range of vocalizations (single clicks, likely either slow clicks or regular clicks, creaks, and codas). Our observations indicate that acoustics, respiratory and behavioural parameters may be useful to monitor the physical/physiological status of sperm whales during disentanglement operations.

Movement patterns of large juvenile loggerhead turtles in the Mediterranean Sea: Ontogenetic space use in a small ocean basin.

Mechanisms that determine how, where, and when ontogenetic habitat shifts occur are mostly unknown in wild populations. Differences in size and environmental characteristics of ontogenetic habitats can lead to differences in movement patterns, behavior, habitat use, and spatial distributions across individuals of the same species. Knowledge of juvenile loggerhead turtles' dispersal, movements, and habitat use is largely unknown, especially in the Mediterranean Sea. Satellite relay data loggers were used to monitor movements, diving behavior, and water temperature of eleven large juvenile loggerhead turtles (Caretta caretta) deliberately caught in an oceanic habitat in the Mediterranean Sea. Hidden Markov models were used over 4,430 spatial locations to quantify the different activities performed by each individual: transit, low-, and high-intensity diving. Model results were then analyzed in relation to water temperature, bathymetry, and distance to the coast. The hidden Markov model differentiated between bouts of area-restricted search as low- and high-intensity diving, and transit movements. The turtles foraged in deep oceanic waters within 60 km from the coast as well as above 140 km from the coast. They used an average area of 194,802 km2, where most individuals used the deepest part of the Southern Tyrrhenian Sea with the highest seamounts, while only two switched to neritic foraging showing plasticity in foraging strategies among turtles of similar age classes. The foraging distribution of large juvenile loggerhead turtles, including some which were of the minimum size of adults, in the Tyrrhenian Sea is mainly concentrated in a relatively small oceanic area with predictable mesoscale oceanographic features, despite the proximity of suitable neritic foraging habitats. Our study highlights the importance of collecting high-resolution data about species distribution and behavior across different spatio-temporal scales and life stages for implementing conservation and dynamic ocean management actions.

Antibiotic Resistance of Gram-Negative Bacteria from Wild Captured Loggerhead Sea Turtles.

Sea turtles have been proposed as health indicators of marine habitats and carriers of antibiotic-resistant bacterial strains, for their longevity and migratory lifestyle. Up to now, a few studies evaluated the antibacterial resistant flora of Mediterranean loggerhead sea turtles (Caretta caretta) and most of them were carried out on stranded or recovered animals. In this study, the isolation and the antibiotic resistance profile of 90 Gram negative bacteria from cloacal swabs of 33 Mediterranean wild captured loggerhead sea turtles are described. Among sea turtles found in their foraging sites, 23 were in good health and 10 needed recovery for different health problems (hereafter named weak). Isolated cloacal bacteria belonged mainly to Enterobacteriaceae (59%), Shewanellaceae (31%) and Vibrionaceae families (5%). Although slight differences in the bacterial composition, healthy and weak sea turtles shared antibiotic-resistant strains. In total, 74 strains were endowed with one or multi resistance (up to five different drugs) phenotypes, mainly towards ampicillin (~70%) or sulfamethoxazole/trimethoprim (more than 30%). Hence, our results confirmed the presence of antibiotic-resistant strains also in healthy marine animals and the role of the loggerhead sea turtles in spreading antibiotic-resistant bacteria.

Skin marks in bottlenose dolphins (Tursiops truncatus) interacting with artisanal fishery in the central Mediterranean Sea.

Skin marks occur frequently in many cetacean species across the globe revealing a broad spectrum of causes, including social interactions, infectious diseases and injuries produced by anthropogenic factors. The current study used photo-id data from 2005-2014 to estimate the skin mark pattern on resident bottlenose dolphins (Tursiops truncatus) from the Aeolian Archipelago (Italy). Thirteen skin mark types were identified and their origin, prevalence and permanence time were examined. The pattern of skin marks was assessed for the abundance, richness, distribution and severity in six body regions and compared among age classes, sex and degree of dolphins' interaction with trammel nets (DIN). Our results showed higher prevalence, abundance, richness and distribution of skin marks in adults than in the younger age classes, with the exception of black marks and white ring lesions. The prevalence and abundance of skin marks were higher in males than females, with the exception of scratches and white patches. Moreover, gunshot wounds, mutilations and irregular dorsal fin edges were found only on adult males. Since males showed higher DIN than females and, in dolphins with higher DIN, skin marks were more abundant and frequently distributed in different body regions, the skin mark pattern in regard to DIN seems to be sex-related. The more severe marks were observed on adults, males and dolphins with higher DIN, namely skin disorder, tooth rake marks, small shallow indentations, deep indentations and mutilations. On the contrary, the severity of scratches, white patches and dark ring lesions was higher in females than males, but not significantly related to DIN and age of the individuals. Our results showed that photo-id data provide an efficient and cost-effective approach to document the occurrence of skin marks in free-ranging bottlenose dolphin populations, a critical step toward understanding the cause and supporting the conservation strategies.

Complex Social Structure of an Endangered Population of Bottlenose Dolphins (Tursiops truncatus) in the Aeolian Archipelago (Italy).

We investigated social structure and association patterns for a small population of Mediterranean bottlenose dolphins, Tursiops truncatus, inhabiting the Aeolian Archipelago (southern Italy). Specifically we evaluate the role of sex and age composition, residency patterns and interaction with trammel nets on this social organization. Association data for 23 regularly sighted individuals were obtained from summer photoidentification surveys collected from 2005-2012. Using a combined cluster and social network analysis approach, we found associations between dolphins were hierarchically structured, where two mixed-sex social units were subdivided into smaller temporarily dynamic groups. We found non-random and long-term preferred associations in the population; however, the degree of social cohesion, residence pattern and interaction with trammel nets differed considerably between the two social units. Six of eight females occurred in the more resident social unit-1; in addition, social unit-1 individuals had significantly stronger associations, higher preferred associates, lived in larger groups and occurred less frequently with trammel nets. Nine of eleven males were clustered in social unit-2 and five of these males, interacting with trammel nets, formed small groups and preferred associations. We propose that female and male groups associate in the study area during the breeding season and that some males choose to interact with reproductive females forming a distinct but interrelated social unit. Other males may be associating in a larger fission-fusion network, which consists of dolphins that appear to temporarily join the network from the coastal population. We cannot exclude that some males specialized in trammel net foraging, suggesting that this foraging technique may favor a solitary lifestyle. Large group sizes and high degree of social cohesion for females could be an indication of greater protection and more efficiency in detecting, deterring or repelling anthropogenic pressures. Most likely dolphins' social organization depends on a combination of socio-ecological, demographic and anthropogenic factors.

Genome-wide expression profile of sporadic gastric cancers with microsatellite instability.

Gastric cancers with mismatch repair (MMR) inactivation are characterised by microsatellite instability (MSI). In this study, the transcriptional profile of 38 gastric cancers with and without MSI was analysed. Unsupervised analysis showed that the immune and apoptotic gene networks efficiently discriminated these two cancer types. Hierarchical clustering analysis revealed numerous gene expression changes associated with the MSI phenotype. Amongst these, the p53-responsive genes maspin and 14-3-3 sigma were significantly more expressed in tumours with than without MSI. A tight immunosurveillance coupled with a functional p53 gene response is consistent with the better prognosis of MSI cancers. Frequent silencing of MLH1 and downregulation of MMR target genes, such as MRE11 and MBD4, characterised MSI tumours. The downregulation of SMUG1 was also a typical feature of these tumours. The DNA repair gene expression profile of gastric cancer with MSI is of relevance for therapy response.

Water-related diseases outbreaks reported in Italy.

Water related disease outbreak (WRDO) statistics in Italy from 1998 to 2005 have been discussed in this paper. The true incidence of WRDO is not reflected in the National Surveillance System (NSS), although this study has provided information on pathogens associated to different water sources, incidence in Regions and inadequacy of regulations. 192 outbreaks and 2546 cases of WRD were reported to the NSS, an average of 318 cases per year. Cases were associated to shellfish (58.79%), drinking water (39.94%) and agricultural products (1.25%). WRDs have been detected in 76% of Regions: central and southern Regions showed lower percentage of cases (35.4%) due to under-reporting. Most of WRD cases in the North were related to drinking water; WRDs in marine coastal Regions were mostly related to shellfish. 49% of Districts (Province) notified WRDs, including only 101 Municipalities. Pathogenic microorganisms were identified in a few cases from clinical investigations. They included enteric viruses, Norwalk viruses, Salmonella, Shigella, Giardia and Campylobacter. There is the need to improve the existing NSS in relation to WRDs. An adequate WRDs Surveillance System should be based on connection between health and environmental authorities, priority pathogens and critical areas identification, response capability and contingency plans.

A human cell-based assay to evaluate the effects of alterations in the MLH1 mismatch repair gene.

We describe a new approach to investigate alterations in the human MLH1 mismatch repair (MMR) gene. This is based on complementation of the phenotype of a MLH1-defective subclone of the ovarian carcinoma A2780 cells by transfection of vectors encoding altered MLH1 proteins. Measurements of resistance (tolerance) to methylating agents, mutation rate at HPRT, microsatellite instability (MSI), and steady-state levels of DNA 8-oxoguanine were used to define the MMR status of transfected clones. The approach was validated by transfecting cDNA of wild-type (WT) MLH1, cDNAs bearing two previously identified polymorphisms (I219V and I219L) and two with confirmed hereditary nonpolyposis colorectal cancer (HNPCC) syndrome mutations (G224D and G67R). A low-level expression of two MLH1 polymorphisms partially reversed methylation tolerance and the mutator phenotype, including MSI. Higher levels of I219V resulted in full restoration of these properties to WT. Increased expression of I129L did not fully complement the MLH1 defect, because there was a simultaneous escalation in the level of oxidative DNA damage. The findings confirmed the important relationship between deficient MMR and increased levels of oxidative DNA damage. Mutations from Italian HNPCC families (G224D, G67R, N635S, and K618A) were all ineffective at reversing the phenotype of the MLH1-defective A2780 cells. One (K618A) was identified as a low penetrance mutation based on clinical and genetic observations.

The oxidized deoxynucleoside triphosphate pool is a significant contributor to genetic instability in mismatch repair-deficient cells.

Oxidation is a common form of DNA damage to which purines are particularly susceptible. We previously reported that oxidized dGTP is potentially an important source of DNA 8-oxodGMP in mammalian cells and that the incorporated lesions are removed by DNA mismatch repair (MMR). MMR deficiency is associated with a mutator phenotype and widespread microsatellite instability (MSI). Here, we identify oxidized deoxynucleoside triphosphates (dNTPs) as an important cofactor in this genetic instability. The high spontaneous hprt mutation rate of MMR-defective msh2(-/-) mouse embryonic fibroblasts was attenuated by expression of the hMTH1 protein, which degrades oxidized purine dNTPs. A high level of hMTH1 abolished their mutator phenotype and restored the hprt mutation rate to normal. Molecular analysis of hprt mutants showed that the presence of hMTH1 reduced the incidence of mutations in all classes, including frameshifts, and also implicated incorporated 2-oxodAMP in the mutator phenotype. In hMSH6-deficient DLD-1 human colorectal carcinoma cells, overexpression of hMTH1 markedly attenuated the spontaneous mutation rate and reduced MSI. It also reduced the incidence of -G and -A frameshifts in the hMLH1-defective DU145 human prostatic cancer cell line. Our findings indicate that incorporation of oxidized purines from the dNTP pool may contribute significantly to the extreme genetic instability of MMR-defective human tumors.