Molecular characterization of cetacean poxviruses along the coast of mainland Portugal.

Cetacean poxvirus (CePV) is the causative agent of tattoo skin disease (TSD) in dolphins, porpoises and whales, a condition characterized by pinhole, ring-like lesions or generalized tattoo-like skin lesions. This study genetically characterized cetacean poxviruses from stranded animals along mainland Portugal. Samples from skin lesions compatible with TSD were obtained from 4 odontocete species (Delphinus delphis, Stenella coeruleoalba, Phocoena phocoena, and Tursiops truncatus) and analyzed using a conventional PCR assay targeting the DNA polymerase gene partially. Among the positive samples (n = 29, 65.9%), a larger DNA polymerase gene fragment was obtained, allowing a robust phylogenetic analysis. Nineteen samples (43.2%) were successfully amplified and sequenced using Sanger sequencing. By combining 11 of these sequences with those from public databases, a maximum likelihood phylogenetic tree was constructed, revealing high heterogeneity within the group. These findings contribute to a better understanding of the genetic diversity, epidemiology, phylogenetics, and evolution of CePV.

Harbor porpoise losing its edge: Genetic time series suggests a rapid population decline in Iberian waters over the last 30 years.

Impact of climate change is expected to be especially noticeable at the edges of a species' distribution, where they meet suboptimal habitat conditions. In Mauritania and Iberia, two genetically differentiated populations of harbor porpoises (Phocoena phocoena) form an ecotype adapted to local upwelling conditions and distinct from other ecotypes further north on the NE Atlantic continental shelf and in the Black Sea. By analyzing the evolution of mitochondrial genetic variation in the Iberian population between two temporal cohorts (1990-2002 vs. 2012-2015), we report a substantial decrease in genetic diversity. Phylogenetic analyses including neighboring populations identified two porpoises in southern Iberia carrying a divergent haplotype closely related to those from the Mauritanian population, yet forming a distinct lineage. This suggests that Iberian porpoises may not be as isolated as previously thought, indicating possible dispersion from Mauritania or an unknown population in between, but none from the northern ecotype. Demo-genetic scenario testing by approximate Bayesian computation showed that the rapid decline in the Iberian mitochondrial diversity was not simply due to the genetic drift of a small population, but models support instead a substantial decline in effective population size, possibly resulting from environmental stochasticity, prey depletion, or acute fishery bycatches. These results illustrate the value of genetics time series to inform demographic trends and emphasize the urgent need for conservation measures to ensure the viability of this small harbor porpoise population in Iberian waters.

Microplastics in Cetaceans Stranded on the Portuguese Coast.

This study characterises microplastics in small cetaceans on the coast of Portugal and assesses the relationship between several biological variables and the amount of detected microplastics. The intestines of 38 stranded dead cetaceans were processed in the laboratory, with digestion methods adapted to the amount of organic matter in each sample. The influence of several biological and health variables (e.g., species, sex, body condition) on the amount of microplastics was tested in all analysed species and particularly in common dolphins, due to the larger number of available samples. Most of the analysed individuals had microplastics in the intestine (92.11%), with harbour porpoises revealing a significantly higher median number of microplastics than common dolphins, probably due to their different diets, use of habitat and feeding strategies. None of the other tested variables significantly influenced the number of microplastics. Moreover, the microplastics found should not be enough to cause physical or chemical sublethal effects, although the correlation between microplastic ingestion and plastic additive bioaccumulation in cetacean tissues requires further investigation. Future monitoring in biota should rely on improved and standardised protocols for microplastic analyses in complex samples to allow for accurate analyses of larger samples and spatio-temporal comparisons.

Assessment of Harbour Porpoise Bycatch along the Portuguese and Galician Coast: Insights from Strandings over Two Decades.

The Iberian harbour porpoise population is small and fisheries bycatch has been described as one of its most important threats. Data on harbour porpoise strandings collected by the Portuguese and Galician stranding networks between 2000 and 2020 are indicative of a recent mortality increase in the western Iberian coast (particularly in northern Portugal). Overall, in Portugal and Galicia, individuals stranded due to confirmed fishery interaction represented 46.98% of all analysed porpoises, and individuals stranded due to probable fishery interaction represented another 10.99% of all analysed porpoises. Considering the Portuguese annual abundance estimates available between 2011 and 2015, it was possible to calculate that an annual average of 207 individuals was removed from the population in Portuguese waters alone, which largely surpasses the potential biological removal (PBR) estimates (22 porpoises, CI: 12-43) for the same period. These results are conservative and bycatch values from strandings are likely underestimated. A structured action plan accounting for new activities at sea is needed to limit the Iberian porpoise population decline. Meanwhile, there is an urgent need for a fishing effort reorganization to directly decrease porpoise mortality.

Harbour Porpoise Abundance in Portugal over a 5-Year Period and Estimates of Potential Distribution.

The Iberian porpoise population is small and under potentially unsustainable removal by fisheries bycatch. Recently, a marine Site of Community Importance (SCI) was legally approved in Portugal, but no measures ensued to promote porpoise conservation. Information about porpoise abundance and distribution is fundamental to guide any future conservation measures. Annual aerial surveys conducted between 2011 and 2015 show a low overall porpoise abundance and density (2254 individuals; 0.090 ind/km2, CV = 21.99%) in the Portuguese coast. The highest annual porpoise estimates were registered in 2013 (3207 individuals, 0.128 ind/km2), followed by a sharp decrease in 2014 (1653 individuals, 0.066 ind/km2). The porpoise density and abundance estimated in 2015 remained lower than the 2013 estimates. A potential distribution analysis of the Iberian porpoise population was performed using ensembles of small models (ESMs) with MaxEnt and showed that the overall habitat suitability is particularly high in the Portuguese northern area. The analysis also suggested a different pattern in porpoise potential distribution across the study period. These results emphasize the importance of further porpoise population assessments to fully understand the spatial and temporal porpoise habitat use in the Iberian Peninsula as well as the urgent need for on-site threat mitigation measures.

Speciation in the deep: genomics and morphology reveal a new species of beaked whale Mesoplodon eueu.

The deep sea has been described as the last major ecological frontier, as much of its biodiversity is yet to be discovered and described. Beaked whales (ziphiids) are among the most visible inhabitants of the deep sea, due to their large size and worldwide distribution, and their taxonomic diversity and much about their natural history remain poorly understood. We combine genomic and morphometric analyses to reveal a new Southern Hemisphere ziphiid species, Ramari's beaked whale, Mesoplodon eueu, whose name is linked to the Indigenous peoples of the lands from which the species holotype and paratypes were recovered. Mitogenome and ddRAD-derived phylogenies demonstrate reciprocally monophyletic divergence between M. eueu and True's beaked whale (M. mirus) from the North Atlantic, with which it was previously subsumed. Morphometric analyses of skulls also distinguish the two species. A time-calibrated mitogenome phylogeny and analysis of two nuclear genomes indicate divergence began circa 2 million years ago (Ma), with geneflow ceasing 0.35-0.55 Ma. This is an example of how deep sea biodiversity can be unravelled through increasing international collaboration and genome sequencing of archival specimens. Our consultation and involvement with Indigenous peoples offers a model for broadening the cultural scope of the scientific naming process.

Floating marine litter and their risks to cetaceans off Portugal.

Floating Dangerous Debris For Cetaceans (DDFC) and cetacean abundances were assessed by shipboard visual surveys off continental Portugal. Plastic litter (other than fishing gear) was the most abundant category, corresponding to 56.53% of the total recorded DDFC. Areas of DDFC and cetacean potential overlap suggest that cetaceans are generally exposed to DDFC at different risk levels within the study area. The highest concentration areas of DDFC and cetaceans did not overlap, whereas lower DDFC concentration areas (70% kernel probability contour) overlapped with 45% of the distribution area obtained for cetaceans. Future marine litter management measures (prevention or mitigation) should be focused on the predicted high DDFC-cetacean interaction risk area in the central region of the study. DDFC are representative of the overall floating litter distribution and future floating litter assessments could be based on DDFC categories alone.

Striped dolphins as trace element biomonitoring tools in oceanic waters: Accounting for health-related variables.

In Europe, monitoring contaminant concentrations and their effects in the marine environment is required under the Marine Strategy Framework Directive (MSFD, 2008/56/EC). The striped dolphin (Stenella coeruleoalba) is the most abundant small cetacean species in Portuguese oceanic waters, representing a potential biomonitoring tool of contaminant levels in offshore waters. Concentrations of nine trace elements were evaluated by ICP-MS in kidney, liver and muscle samples of 31 striped dolphins stranded in the Portuguese continental coast. The mean renal Cd concentration was high (19.3 µg.g-1 wet weight, range 0.1-69.3 µg.g-1 wet weight) comparing to striped dolphins from other locations. Therefore, the present study reports a possibly concerning level of Cd in the oceanic food chain in Portuguese offshore areas. This study also aimed at evaluating potential relationships between trace element concentrations and striped dolphins' biological and health-related variables. Individual length was related with some of the trace element concentrations detected in striped dolphins. Indeed, Cd, Hg and Se bioaccumulated in larger animals, whereas the reverse was observed for Mn and Zn. Striped dolphins with high parasite burdens showed higher levels of Hg, while animals showing gross pathologies presented higher concentrations of Cd and Se. This study reported relationships between trace element concentrations and health-related variables for the first time in striped dolphins and it also provided information on the relative contamination status of Portuguese oceanic waters in comparison to other regions in the world.

Microbiota fingerprints within the oral cavity of cetaceans as indicators for population biomonitoring.

The composition of mammalian microbiota has been related with the host health status. In this study, we assessed the oral microbiome of 3 cetacean species most commonly found stranded in Iberian Atlantic waters (Delphinus delphis, Stenella coeruleoalba and Phocoena phocoena), using 16S rDNA-amplicon metabarcoding. All oral microbiomes were dominated by Proteobacteria, Firmicutes, Bacteroidetes and Fusobacteria bacteria, which were also predominant in the oral cavity of Tursiops truncatus. A Constrained Canonical Analysis (CCA) showed that the major factors shaping the composition of 38 oral microbiomes (p-value < 0.05) were: (i) animal species and (ii) age class, segregating adults and juveniles. The correlation analysis also grouped the microbiomes by animal stranding location and health status. Similar discriminatory patterns were detected using the data from a previous study on Tursiops truncatus, indicating that this correlation approach may facilitate data comparisons between different studies on several cetacean species. This study identified a total of 15 bacterial genera and 27 OTUs discriminating between the observed CCA groups, which can be further explored as microbiota fingerprints to develop (i) specific diagnostic assays for cetacean population conservation and (ii) bio-monitoring approaches to assess the health of marine ecosystems from the Iberian Atlantic basin, using cetaceans as bioindicators.

Mercury, Lead and Cadmium Concentrations in Talpa occidentalis and in Their Digeneans of the Genus Ityogonimus.

INTRODUCTION: Many parasites living in aquatic ecosystems are useful indicators of environmental health. On the other hand, information is scarcer with respect to the use of helminth parasites of vertebrates living in terrestrial ecosystems as monitoring tools for toxic element environmental pollution. The present study evaluates the suitability of the model Talpa occidentalis/Ityogonimus spp. as a bioindicator system for mercury (Hg), lead (Pb) and cadmium (Cd) contamination in agricultural soils from Asturias (Spain). METHODS: Kidney and liver samples collected from T. occidentalis specimens (n = 36) and Ityogonimus spp. samples collected from 14 infected hosts were analyzed by ICP-MS. RESULTS: The highest mean levels of Hg and Pb were found in Ityogonimus individuals (20.9 and 12.4 µg g-1 wet weight, respectively). Considering renal and hepatic concentrations in T. occidentalis, bioaccumulation factors of Ityogonimus for Hg were 83.7 and 58.6, respectively, whereas concerning Pb bioaccumulation factors were 38.2 and 82.9, respectively. No bioaccumulation was detected in Ityogonimus in the case of Cd. CONCLUSIONS: More studies involving digenean parasites of small mammals are needed, especially when biomonitoring environmental toxic element pollution in terrestrial ecosystems. The present results support the above-mentioned model as a suitable biomonitoring system to evaluate environmental Hg and Pb contamination in terrestrial non-urban Iberian habitats. Similar models involving other species (Talpa spp./Ityogonimus spp.) might be used in a much wider geographical range.

Trace elements in loggerhead turtles (Caretta caretta) stranded in mainland Portugal: Bioaccumulation and tissue distribution.

Pollution is among the most significant threats that endanger sea turtles worldwide. Waters off the Portuguese mainland are acknowledged as important feeding grounds for juvenile loggerheads. However, there is no data on trace element concentrations in marine turtles occurring in these waters. We present the first assessment of trace element concentrations in loggerhead turtles (Caretta caretta) occurring off the coast of mainland Portugal. Also, we compare our results with those from other areas and discuss parameters that may affect element concentrations. Trace element concentrations (As, Cd, Cu, Pb, Mn, Hg, Ni, Se, Zn) were determined in kidney, liver and muscle samples from 38 loggerheads stranded between 2011 and 2013. As was the only element with higher concentrations in muscle (14.78 µg g-1 ww) than in liver or kidney. Considering non-essential elements, Cd presented the highest concentrations in kidney (34.67 µg g-1) and liver (5.03 µg g-1). Only a weak positive link was found between renal Cd and turtle size. Inter-elemental correlations were observed in both liver and kidney tissues. Hepatic Hg values (0.30 ± 0.03 µg g-1) were higher than values reported in loggerheads in the Canary Islands but lower than in Mediterranean loggerheads. Cd concentrations in the present study were only exceeded by values found in turtles from the Pacific. Although many endogenous and exogenous parameters related with complex life cycle changes and wide geographic range may influence trace element accumulation, the concentrations of Cd are probably related to the importance of crustaceans in loggerhead diet in the Portuguese coast.

The microbiome of a striped dolphin (Stenella coeruleoalba) stranded in Portugal.

Infectious diseases with epizootic consequences have not been fully studied in marine mammals. Presently, the unprecedented depth of sequencing, made available by high-throughput approaches, allows detailed comparisons of the microbiome in health and disease. This is the first report of the striped dolphin microbiome in different body sites. Samples from one striped female edematous dolphin were acquired from a variety of body niches, including the blowhole, oral cavity, oral mucosa, tongue, stomach, intestines and genital mucosa. Detailed 16S rRNA analysis of over half a million sequences identified 235 OTUs. Beta diversity analyses indicated that microbial communities vary in structure and cluster by sample origin. Pathogenic, Gram-negative, facultative and obligate anaerobic taxa were significantly detected, including Cetobacterium, Fusobacterium and Ureaplasma. Phocoenobacter and Arcobacter dominated the oral-type samples, while Cardiobacteriaceae and Vibrio were associated with the blowhole and Photobacterium were abundant in the gut. We report for the first time the association of Epulopiscium with a marine mammal gut. The striped dolphin microbiota shows variation in structure and diversity according to the organ type. The high dominance of Gram-negative anaerobic pathogens evidences a cetacean microbiome affected by human-related bacteria.

Bioaccumulation of trace element concentrations in common dolphins (Delphinus delphis) from Portugal.

The common dolphin (Delphinus delphis) is one of the most abundant species in Atlantic Iberia, representing a potentially important tool to assess the bioaccumulation of trace elements in the Iberian marine ecosystem. Nine elements (As, Cd, Cu, Hg, Mn, Ni, Pb, Se and Zn) were evaluated in 36 dolphins stranded in continental Portugal. Dolphins had increasing Hg concentrations (16.72µg·g-1 ww, liver) compared with previous studies in Atlantic Iberia, whereas Cd concentrations (2.26µg·g-1 ww, kidney) fell within reported ranges. The concentrations of some trace elements (including Cd and Hg) presented positive relationships with dolphin length, presence of parasites and gross pathologies. Common dolphins may help biomonitoring more offshore Atlantic Iberian areas in future studies, which would otherwise be difficult to assess.

New insight into dolphin morbillivirus phylogeny and epidemiology in the northeast Atlantic: opportunistic study in cetaceans stranded along the Portuguese and Galician coasts.

BACKGROUND: Screening Atlantic cetacean populations for Cetacean Morbillivirus (CeMV) is essential to understand the epidemiology of the disease. In Europe, Portugal and Spain have the highest cetacean stranding rates, mostly due to the vast extension of coastline. Morbillivirus infection has been associated with high morbidity and mortality in cetaceans, especially in outbreaks reported in the Mediterranean Sea. However, scarce information is available regarding this disease in cetaceans from the North-East Atlantic populations. The presence of CeMV genomic RNA was investigated by reverse transcription-quantitative PCR in samples from 279 specimens stranded along the Portuguese and Galician coastlines collected between 2004 and 2015. RESULTS: A total of sixteen animals (n = 16/279, 5.7 %) were positive. The highest prevalence of DMV was registered in striped dolphins (Stenella coeruleoalba) (n = 14/69; 20.3 %), slightly higher in those collected in Galicia (n = 8/33; 24.2 %) than in Portugal (n = 6/36; 16.7 %). CONCLUSIONS: Phylogenetic analysis revealed that, despite the low genetic distances between samples, the high posterior probability (PP) values obtained strongly support the separation of the Portuguese and Galician sequences in an independent branch, separately from samples from the Mediterranean and the Canary Islands. Furthermore, evidence suggests an endemic rather than an epidemic situation in the striped dolphin populations from Portugal and Galicia, since no outbreaks have been detected and positive samples have been detected annually since 2007, indicating that this virus is actively circulating in these populations and reaching prevalence values as high as 24 % among the Galician samples tested.

Spatial distribution of floating marine debris in offshore continental Portuguese waters.

This study presents data on abundance and density of macro-floating marine debris (FMD), including their composition, spatial distribution and potential sources off continental Portugal. FMD were assessed by shipboard visual surveys covering ±252,833 km(2) until the 220 nm limit. The FMD average density was 2.98 items/km(2) and abundance amounted to 752,740 items. Unidentified plastics constitute the major bulk of FMD (density=0.46 items/km(2); abundance=117,390 items), followed by styrofoam, derelict or lost materials from fisheries, paper/cardboard and wood material. The North sector of the area presents higher FMD diversity and abundances, probably as a result of the high number of navigation corridors and fisheries operating in that sector. Most FMD originate from local sources, namely discharges from vessels and derelict material from fisheries. Considering the identifiable items, cables and fishing lines were the only fishing related items among the top ten FMD items in Portuguese offshore waters.

Biological variables and health status affecting inorganic element concentrations in harbour porpoises (Phocoena phocoena) from Portugal (western Iberian Peninsula).

The coastal preferences of harbour porpoise (Phocoena phocoena) intensify their exposure to human activities. The harbour porpoise Iberian population is presently very small and information about the threats it endures is vital for the conservation efforts that are being implemented to avoid local extinction. The present study explored the possible relation between the accumulation of trace elements by porpoises and their sex, body length, nutritional state, presence of parasites and gross pathologies. The concentrations of arsenic (As), cadmium (Cd), copper (Cu), mercury (Hg), manganese (Mn), nickel (Ni), lead (Pb), zinc (Zn) and selenium (Se) were evaluated in 42 porpoises stranded in Portugal between 2005 and 2013. Considering European waters, porpoises stranded in Portugal present the highest Hg concentrations and the lowest Cd concentrations, which may reflect dietary preferences and the geographic availability of these pollutants. While no effect of sex on trace element concentrations was detected, there was a positive relationship between porpoise body length and the concentration of Cd, Hg and Pb. Animals in worse nutritional condition showed higher levels of Zn. Harbour porpoises with high parasite burdens showed lower levels of Zn and As in all analysed tissues and also lower levels of renal Ni, while those showing gross pathologies presented higher Zn and Hg levels. This is the first data on the relationship between trace elements and health-related variables in porpoises from southern European Atlantic waters, providing valuable baseline information about the contamination status of this vulnerable population.

Ingestion of marine litter by loggerhead sea turtles, Caretta caretta, in Portuguese continental waters.

The accumulation of litter in marine and coastal environments is a major threat to marine life. Data on marine litter in the gastrointestinal tract of stranded loggerhead turtles, Caretta caretta, found along the Portuguese continental coast was presented. Out of the 95 analysed loggerheads, litter was present in 56 individuals (59.0%) and most had less than 10 litter items (76.8%) and less than 5 g (dm) (96.8%). Plastic was the main litter category (frequency of occurrence=56.8%), while sheet (45.3%) was the most relevant plastic sub-category. There was no influence of loggerhead stranding season, cause of stranding or size on the amount of litter ingested (mean number and dry mass of litter items per turtle). The high ingested litter occurrence frequency in this study supports the use of the loggerhead turtle as a suitable tool to monitor marine litter trends, as required by the European Marine Strategy Framework Directive.

Ecological variables influencing trace element concentrations in bottlenose dolphins (Tursiops truncatus, Montagu 1821) stranded in continental Portugal.

Both the conservation status of bottlenose dolphins (Tursiops truncatus) (Habitats Directive 92/43/CEE, Annex II) and the Marine Strategy Framework Directive demand for data on their ecology and anthropogenic threats. To evaluate the bottlenose dolphin's toxicological status in continental Portugal, several trace elements (As, Cd, Cu, Hg, Mn, Ni, Pb, Se, Zn) were determined in 25 stranded individuals. The potential effect of sex, body length and stranding location on trace element concentrations was analysed. In the present study, bottlenose dolphins presented high mercury levels, only exceeded by animals from the Mediterranean and Adriatic seas. Only essential elements were influenced by dolphin sex, whereas Cd, Hg and Pb bioaccumulated in larger dolphins, and hepatic Hg and Cd concentrations were higher in the northwest coast of continental Portugal. The location effect may relate to variations in bottlenose diet and trace element availability, according to the proximity to anthropogenic sources in the Atlantic Iberian coast.

Effect of Intestinal Tapeworm Clestobothrium crassiceps on Concentrations of Toxic Elements and Selenium in European Hake Merluccius merluccius from the Gulf of Lion (Northwestern Mediterranean Sea).

The capacity for heavy metal bioaccumulation by some fish parasites has been demonstrated, and their contribution to decreasing metal concentrations in tissues of parasitized fish has been hypothesized. The present study evaluated the effect of the cestode Clestobothrium crassiceps on the accumulation of trace elements in 30 European hake, Merluccius merluccius, in Spain (half of them infested by C. crassiceps). Tissue samples from all M. merluccius and specimens of C. crassiceps from the infected hakes were collected and stored until element analysis by inductively coupled plasma mass spectrometry (ICP-MS). Arsenic, mercury, and selenium were generally present in lower levels in the cestode than in all hake tissues. The mean value of the muscular Se:Hg molar ratio in the infested subsample was higher than that in hakes without cestodes. Values indicate that the edible part of infested hakes presents a lower amount of Cd and Pb in relation to noninfested hakes.