Body burdens and distribution of mercury and selenium in bottlenose, striped and Risso's dolphins along the Adriatic coast: A 20-year retrospective.

Top marine predators present high mercury concentrations in their tissues due to biomagnification in the marine food chain. This study reports mercury (Hg) and selenium (Se) status, and the Hg:Se molar ratio assessment in bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's dolphins (Grampus griseus). Total Hg and Se concentrations were determined in muscle, liver, kidney, lung, spleen, adipose tissue and skin collected from 186 specimens stranded in the Croatian part of Adriatic Sea from 1995 to 2014. Total Hg concentrations in tissue samples ranged from 0.001 in the spleen to 2238 mg/kg wet weight in liver. Se concentrations in dolphin samples ranged from 0.010 to 2916 mg/kg ww. Minimum Se concentration was found in muscle and maximum Se concentration were found in liver of bottlenose dolphin. Hg and Se levels in Risso's dolphins showed higher concentrations in all tissues in comparison to bottlenose and striped dolphins. Significant and positive correlations were observed between age and Hg concentrations (P < 0.05). In 66.6 % of Risso's, 15.3 % of bottlenose dolphins and one stranded striped dolphin in this study, the hepatic concentration of Hg exceeded the higher toxic thresholds (400 mg/kg w.w.) previously defined as evidence of liver damage in marine mammals. The Hg:Se molar ratio in the liver of Risso's dolphin was 0.670. The liver of adult bottlenose dolphins showed expected values (0.870), while the liver of young dolphins had a high ratio (0.750), non-specific for the age group. The Hg:Se molar ratio in the liver of striped dolphins was 0.390, which is lower than the literature values.

Lead and Other Trace Element Levels in Brains of Croatian Large Terrestrial Carnivores: Influence of Biological and Ecological Factors.

Trace element pollution can adversely affect the brains of individuals and thus impact the entire population of apex predators, such as large European carnivores. We assessed exposure to prominent neurotoxicants As, Cd, Hg and Pb by measuring their brain stem levels in brown bears (n = 114), grey wolves (n = 8), Eurasian lynx (n = 3), and golden jackals (n = 2) sampled in 2015-2022 in Croatia. The highest of the non-essential elements was the Pb level in the bears' brains (median, Q1-Q3; 11.1, 7.13-24.1 µg/kg wet mass), with 4% of animals, all subadults, exceeding the established normal bovine levels (100 µg/kg wet mass). Species-specific differences were noted for Ca, Cd, Cu, Fe, Pb and Se brain levels. Female brown bears had higher As brain levels than males. Cubs and yearlings had lower brain Cd, but higher Zn, while subadults had higher Cu than adult bears. Hepatic As, Cd, Cu and Hg levels were shown to be a moderate proxy for estimating brain levels in bears (rS = 0.30-0.69). Multiple associations of As, Cd, Hg and Pb with essential elements pointed to a possible interaction and disturbance of brain Ca, Cu, Fe, Se and Zn homeostasis. Non-essential element levels in the brains of four studied species were lower than reported earlier for terrestrial meso-carnivores and humans. The age and sex of animals were highlighted as essential factors in interpreting brain element levels in ecotoxicological studies of large carnivores.

Microbiota and gut ultrastructure of Anisakis pegreffii isolated from stranded cetaceans in the Adriatic Sea.

BACKGROUND: Inferring the microbiota diversity of helminths enables depiction of evolutionarily established ecological and pathological traits that characterize a particular parasite-host interaction. In turn, these traits could provide valuable information for the development of parasitosis control and mitigation strategy. The parasite Anisakis pegreffii (Nematoda: Anisakidae) realizes the final stage of its life-cycle within gastric chambers of aquatic mammals, causing mild-to-moderate granulomatous gastritis with eosinophilic infiltrate, to severe ulcerative gastritis with mixed inflammatory infiltrate, often associated with bacterial colonies. However, its interaction with the host microbiota remains unknown, and might reveal important aspects of parasite colonization and propagation within the final host. METHODS: MySeq Illumina sequencing was performed for the 16S rRNA gene from microbiota isolated from larvae, and uterus and gut of adult A. pegreffii parasitizing stranded striped dolphins (Stenella coeruleoalba). To assess the potential presence of Brucella ceti within isolated microbiota, Brucella-targeted real-time PCR was undertaken. In addition, TEM of the gastrointestinal tract of the infective third-stage (L3) and transitioning fourth-stage larvae (L4) was performed to characterize the morphological differences and the level of larval feeding activity. RESULTS: In total, 230 distinct operational taxonomic units (OTUs) were identified across all samples (n = 20). The number of shared taxa was lower than the number of taxa found specifically in each parasite stage or organ. The dominant taxon was Mycoplasmataceae (genus Mycoplasma) in the gut and uterus of adult A. pegreffii, whereas Fusobacteriaceae (genus Cetobacterium) was the most abundant in 40% of larvae, alongside Mycoplasmataceae. No B. ceti DNA was detected in any of the microbiota isolates. TEM revealed differences in gut ultrastructure between L3 and L4, reflecting a feeble, most likely passive, level of feeding activity in L3. CONCLUSIONS: Microbiota from L3 was more related to that of the gut rather than the uterus of adult A. pegreffii. Taxa of the larval microbiota showed qualitative and quantitative perturbations, likely reflecting the propagation through different environments during its life-cycle. This suggests an ontogenetic shift in the alpha and beta diversity of microbial communities from uterus-derived towards cetacean-derived microbiota. Although TEM did not reveal active L3 feeding, microbiota of the latter showed similarity to that of an actively feeding adult nematode.

Manganese Concentrations in Tissues and Skin of Three Dolphin Species Stranded in the Croatian Waters of the Adriatic Sea from 1995 to 2013.

Manganese concentrations were determined in muscle, kidney, liver, spleen, lung and fat tissues and skin of three cetacean species, the bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's (Grampus griseus) dolphins, stranded along the Croatian Adriatic coast between 1995 and 2013. Mean ranges determined in tissues were (mg/kg, ww): muscle 0.23-0.27, liver 2.87-4.00, kidney 0.66-1.26, spleen 0.37-0.79, lung 0.18-0.57, skin 0.11-0.97, fat 0.06-0.42. The highest Mn mean levels in tissues were measured in species (mg/kg, ww): T. truncates lung 0.41, skin 0.97, fat 0.42; S. coeruleoalba muscle 0.26; G. griseus kidney 1.26, liver 4.00, spleen 0.64. Significant differences of Mn concentrations in the liver (p = 0.034), spleen (p = 0.037) and skin (p = 0.013) were found among the three dolphin species. Significant differences in Mn levels were found between young and adult T. truncates in kidney (p = 0.042), lung (p = 0.0040) and skin (p = 0.0034).

Whole-Genome Sequence of the First Sequence Type 27 Brucella ceti Strain Isolated from European Waters.

Brucella spp. that cause marine brucellosis are becoming more important, as the disease appears to be more widespread than originally thought. Here, we report a whole and annotated genome sequence of Brucella ceti CRO350, a sequence type 27 strain isolated from a bottlenose dolphin carcass found in the Croatian part of the northern Adriatic Sea.

Evidence of Brucella strain ST27 in bottlenose dolphin (Tursiops truncatus) in Europe.

Marine mammal brucellosis has been known for more than 20 years, but recent work suggests it is more widespread than originally thought. Brucella (B.) pinnipedialis has been isolated from pinnipeds, while B. ceti strains have been associated with cetaceans. Here we report a Brucella strain isolated from multiple lymph nodes of one bottlenose dolphin (Tursiops truncatus) during routine examination of dolphin carcasses found in the Croatian part of the northern Adriatic Sea during the summer of 2015. Classical bacteriological biotyping, PCR-based techniques (single, multiplex, PCR-RFLP) and 16S rRNA DNA sequencing were used to identify Brucella spp. Multiple-locus variable number tandem repeat analysis of 16 loci and multilocus sequence typing of 9 loci were used for genotyping and species determination. The combination of bacteriological, molecular and genotyping techniques identified our strain as ST27, previously identified as a human pathogen. This report provides, to our knowledge, the first evidence of ST27 in the Adriatic Sea in particular and in European waters in general. The zoonotic nature of the strain and its presence in the Adriatic, which is inhabited by bottlenose dolphins, suggest that the strain may pose a significant threat to human health.

Copper Levels in Tissues of Dolphins Tursiops truncatus, Stenella coeruleoalba and Grampus griseus from the Croatian Adriatic Coast.

Copper concentrations were determined in muscle, liver, kidney, spleen and lung tissues of three dolphin species. Dolphins of Tursiops truncatus (young and adult), Stenella coeruleoalba and Grampus griseus were stranded along the Croatian coast. Concentrations in tissues of all three dolphin species were highest in the liver (4.92-16.5 µg/g) followed by kidney (2.85-5.29 µg/g). Similar levels were measured in muscle, spleen and lung in range 1.13-3.67 µg/g. Statistics analysis showed significant differences of Cu concentrations for muscle (p = 0.008), kidney (p = 0.04) and liver (p = 0.02) between the three dolphin species. Also, for all three species significant differences between tissue types of the same species were determined (p < 0.001, all). However, there were no significant differences in Cu levels of the same tissues between males and females within same species. Also, significant differences of body length and weight between three dolphin were found (p < 0.001, both).

Three Anisakis spp. isolated from toothed whales stranded along the eastern Adriatic Sea coast.

Knowledge concerning cetacean ecology in the Mediterranean is limited but important for sustainable planning and enforcement of appropriate conservation measures. Any information that might help to elucidate their ecology is essential. We explored the population and genetic structures of Anisakis spp. nematodes isolated from four toothed whale species - bottlenose dolphins (Tursiops truncatus), striped dolphins (Stenella coeruleoalba), Risso's dolphins (Grampus griseus) and Cuvier's beaked whales (Ziphius cavirostris) - stranded along the eastern Adriatic Sea coast (1990-2012) to reveal more information on host ecological patterns. Lower parasite prevalence was observed in resident dolphin species compared with occasionally occurring species, as well as in young compared with adult dolphins, indicating different feeding habits related to age. No unequivocal relationship between the biological traits of a host (age, body length, body mass and blubber depth) and Anisakis population parameters was observed. Phylogenetic analysis revealed a new geographical record of Anisakis simplex sensu stricto (1.96%) and Anisakis physeteris (1.31%) in the Adriatic Sea in addition to resident Anisakis pegreffii (96.73%). In an assessment of the Adriatic Sea and oceans worldwide, the genetic structure of Anisakis revealed that A. pegreffii populations do not differ among various final host species but do differ with respect to geographical location in contrast to previously accepted Anisakis panmixia.

Toxic element concentrations in the bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's (Grampus griseus) dolphins stranded in eastern Adriatic Sea.

Concentrations of cadmium (Cd), arsenic (As), mercury (Hg) and lead (Pb) were measured in muscle, liver and kidney of three cetacean species, the bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's (Grampus griseus) dolphins from the Croatian waters of the Adriatic Sea. In all three dolphin species Cd levels decreased in tissues in the order: kidney > liver > muscle, while As and Pb decreased in the order: liver > kidney > muscle for striped and Risso's dolphins, but with the order reversed for liver and kidney in the bottlenose dolphin for Pb. Levels of Hg consistently followed the order: liver > muscle > kidney, with mean concentrations in the liver being 11-34 times higher than in the other tissues. The highest mean concentrations of trace elements were measured in Risso's dolphins at 14.9 µg/g wet weight, for Cd in the kidney, and concentrations in the liver of 2.41, 1,115 and 0.63 µg/g for As, Hg and Pb, respectively. Statistically significant differences between the three dolphin species were determined for Cd, Hg and Pb in liver tissues, for As in muscle and for Cd in kidney. Significant correlations of metals between tissues were determined in all three species. The results presented give an indication of the environmental condition with regard to the content of toxic metals along the eastern coast of the Adriatic Sea.

Repetitive sequences in Eurasian lynx (Lynx lynx L.) mitochondrial DNA control region.

Mitochondrial DNA (mtDNA) control region (CR) of numerous species is known to include up to five different repetitive sequences (RS1-RS5) that are found at various locations, involving motifs of different length and extensive length heteroplasmy. Two repetitive sequences (RS2 and RS3) on opposite sides of mtDNA central conserved region have been described in domestic cat (Felis catus) and some other felid species. However, the presence of repetitive sequence RS3 has not been detected in Eurasian lynx (Lynx lynx) yet. We analyzed mtDNA CR of 35 Eurasian lynx (L. lynx L.) samples to characterize repetitive sequences and to compare them with those found in other felid species. We confirmed the presence of 80 base pairs (bp) repetitive sequence (RS2) at the 5' end of the Eurasian lynx mtDNA CR L strand and for the first time we described RS3 repetitive sequence at its 3' end, consisting of an array of tandem repeats five to ten bp long. We found that felid species share similar RS3 repetitive pattern and fundamental repeat motif TACAC.

Genotyping success of historical Eurasian lynx (Lynx lynx L.) samples.

Historical samples, like tanned hides and trophy skulls, can be extremely important for genetic studies of endangered or elusive species. Selection of a sampling protocol that is likely to provide sufficient amount and quality of DNA with a minimum damage to the original specimen is often critical for a success of the study. We investigated microsatellite genotyping success of DNA isolated from three different types of Eurasian lynx historical samples. We analysed a total of 20 microsatellite loci in 106 historical samples from the endangered Dinaric lynx population, established from re-introduction of three pairs of lynx in 1973 from Slovakian Carpathians. Of the three tested sample types, turbinal bone and septum from the nasal cavity of the trophy skulls had the lowest percentage of samples successfully genotyped for all 20 microsatellite loci. Footpad samples, collected using a cork drill, exhibited better results in polymerase chain reaction amplification and genotyping than samples of footpad epidermis cut with a scalpel. We report simple and efficient sampling protocols, which could be widely applied for future studies utilizing historical samples.

Isolation of Clostridium tertium from a striped dolphin (Stenella coeruleoalba) in the Adriatic Sea.

Bacteriologic examination of an abscess found between blubber and musculature of a striped dolphin (Stenella coeruleoalba) found dead on 30 March 2002 in the Adriatic Sea, Croatia, was performed and an aerotolerant, nontoxogenic Clostridium tertium was isolated in pure culture. National Collections of Industrial Food and Marine Bacteria (NCIMB Ltd., Aberdeen, Scotland, UK) confirmed the results. Sequencing results showed it to be C. tertium with 100% similarity. The strain was named Clostridium tertium strain Zagreb, acceded to the culture collection and assigned the accession number NCIMB 13970. This is the first record of C. tertium in marine mammals.