Cristina Panti

Are baleen whales exposed to the threat of microplastics? A case study of the Mediterranean fin whale (Balaenoptera physalus)

Baleen whales are potentially exposed to micro-litter ingestion as a result of their filter-feeding activity. However, the impacts of microplastics on baleen whales are largely unknown. In this case study of the Mediterranean fin whale (Balaenoptera physalus), we explore the toxicological effects of microplastics on mysticetes. The study included the following three steps: (1) the collection/count of microplastics in the Pelagos Sanctuary (Mediterranean Sea), (2) the detection of phthalates in surface neustonic/planktonic samples, and (3) the detection of phthalates in stranded fin whales. A total of 56% of the surface neustonic/planktonic samples contained microplastic particles. The highest abundance of microplastics (9.63 items/m(3)) was found in the Portofino MPA (Ligurian Sea). High concentrations of phthalates (DEHP and MEHP) were detected in the neustonic/planktonic samples. The concentrations of MEHP found in the blubber of stranded fin whales suggested that phthalates could serve as a tracer of the intake of microplastics. The results of this study represent the first warning of this emerging threat to baleen whales.

Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies

BackgroundOdontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR) has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers.ResultsTen commonly used housekeeping genes (HKGs) were partially sequenced in the striped dolphin (Stenella coeruleoalba) and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper) which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH) and tyrosine 3-monooxygenase (YWHAZ) always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4) and S18 (RPS18) also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC), phosphoglycerate kinase 1 (PGK1), hypoxanthine ribosyltransferase (HPRT1) and β-2-microglobin (B2M) show variable expression among the studied samples and appear as less suitable reference genes for data normalization.ConclusionIn this work, we have provided essential background information for the selection of control genes in qRT-PCR studies of cetacean skin biopsies, as a molecular technique to investigate ecotoxicological hazard in marine mammals. Of 10 HKGs tested, those encoding for YWHAZ and GAPDH appear as the most reliable control genes for the normalization of qRT-PCR data in the analysis of striped dolphin skin biopsies. Potentially useful reference genes are also those encoding for ribosomal proteins L4 and S18.

Quantitative Real-Time PCR detection of TRPV1-4 gene expression in human leukocytes from healthy and hyposensitive subjects

BackgroundBesides functioning as chemosensors for a broad range of endogenous and synthetic ligands, transient receptor potential vanilloid (TRPV) 1–4 channels have also been related to capsaicin (TRPV1), pain, and thermal stimuli perception, and itching sensation (TRPV1–4). While the expression of the TRPV1–4 genes has been adequately proved in skin, sensory fibres and keratinocytes, less is known about TRPV3 and TRPV4 expression in human blood cells.ResultsTo study the gene expression of TRPV1–4 genes in human leukocytes, a quantitative Real-Time PCR (qRT-PCR) method, based on the calculation of their relative expression, has been developed and validated. The four commonly used house-keeping genes (HKGs), β-Actin (Act-B), glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxanthine ribosyltransferase (HPRT1), and cyclophilin B (hCyPB), were tested for the stability of their expression in several human leukocyte samples, and used in the normalization procedure to determine the mRNA levels of the TRPV 1–4 genes in 30 healthy subjects. cDNAs belonging to all the TRPV1–4 genes were detected in leukocytes but the genes appear to be expressed at different levels. Our analysis did not show significant sex differences in TRPV1–4 cDNA levels in the 30 healthy subjects. The same qRT-PCR assay was used to compare TRPV1–4 expression between healthy controls and patients hyposensitive to capsaicin, pain and thermal stimuli: an almost doubled up-regulation of the TRPV1 gene was found in the pathological subjects.ConclusionThe qRT-PCR assay developed and tested in this study allowed us to determine the relative expression of TRPV1–4 genes in human leukocytes: TRPV3 is the least expressed gene of this pool, followed by TRPV4, TRPV1 and TRPV2. The comparison of TRPV1–4 gene expression between two groups of healthy and hyposensitive subjects highlighted the evident up-regulation of TRPV1, which was almost doubly expressed (1.9× normalized fold induction) in the latter group. All the four house-keeping genes tested in this work (Act-B, GAPDH, hCyPB, HPRT1) were classified as optimal controls and showed a constant expression in human leukocytes samples. We recommend the use of these genes in similar qRT-PCR studies on human blood cells.

Fin whales and microplastics: The Mediterranean Sea and the Sea of Cortez scenarios

The impact that microplastics have on baleen whales is a question that remains largely unexplored. This study examined the interaction between free-ranging fin whales (Balaenoptera physalus) and microplastics by comparing populations living in two semi-enclosed basins, the Mediterranean Sea and the Sea of Cortez (Gulf of California, Mexico). The results indicate that a considerable abundance of microplastics and plastic additives exists in the neustonic samples from Pelagos Sanctuary of the Mediterranean Sea, and that pelagic areas containing high densities of microplastics overlap with whale feeding grounds, suggesting that whales are exposed to microplastics during foraging; this was confirmed by the observation of a temporal increase in toxicological stress in whales. Given the abundance of microplastics in the Mediterranean environment, along with the high concentrations of Persistent Bioaccumulative and Toxic (PBT) chemicals, plastic additives and biomarker responses detected in the biopsies of Mediterranean whales as compared to those in whales inhabiting the Sea of Cortez, we believe that exposure to microplastics because of direct ingestion and consumption of contaminated prey poses a major threat to the health of fin whales in the Mediterranean Sea.

Plastic litter in the sea

On June 2013 a workshop at the University of Siena (Italy) was organized to review current knowledge and to clarify what is known, and what remains to be investigated, concerning plastic litter in the sea. The content of the workshop was designed to contribute further to the European Marine Strategy Framework Directive (MSFD) following an inaugural workshop in 2012. Here we report a number of statements relevant to policymakers and scientists that was overwhelming agreement from the participants. Many might view this as already providing sufficient grounds for policy action. At the very least, this early warning of the problems that lie ahead should be taken seriously, and serve as a stimulus for further research.

Selection of reliable reference genes for qRT-PCR studies on cetacean fibroblast cultures exposed to OCs, PBDEs, and 17β-estradiol

Quantitative real-time PCR (qRT-PCR) represents an effective molecular technique for the detection of mRNA expression in biological samples. Its sensitivity allows the quantification of slight changes in the regulation of gene transcription but is strictly dependent upon the method followed during the normalization procedure. Relative quantification determines changes in the steady-state mRNA levels of genes across multiple samples and it is assessed by comparison with the levels of one or more internal control RNA. In this context, the choice of constitutively expressed control genes, whose transcription is not affected by the contaminants, appears to be fundamental for the reliability of this technique. During this study, fibroblast cell cultures originated from integumentum biopsies, sampled in the cetacean species Stenella coeruleoalba, have been exposed for 6h to increasing concentrations of different mixtures of compounds with endocrine disruptor capacities (EDCs): organochlorines (OCs), polybrominated diphenyl ethers (PBDEs), and 17beta-estradiol. Ten common housekeeping genes have been tested for the expression of their transcripts in exposed cell cultures using qRT-PCR assays and raw data were analyzed with the two Excel applets geNorm and NormFinder. The genes encoding for SDHA, GAPDH and YWHAZ appear to be the most reliable controls, respectively, for the OC, PBDE and 17beta-estradiol treatments. These results clearly show that the transcription of even widely diffused control genes can be regulated by different treatments and underlie the importance of a careful selection of the optimal housekeeping genes in toxicological studies.

Ecotoxicological diagnosis of striped dolphin (Stenella coeruleoalba) from the Mediterranean basin by skin biopsy and gene expression approach

Mediterranean cetacean odontocetes are exposed to environmental stress, in particular to persistent organic pollutants, polycyclic aromatic hydrocarbons and trace elements. In the present study, the response of “gene-expression biomarkers” was evaluated in Mediterranean striped dolphin (Stenella coeruleoalba) skin biopsies collected in three sampling areas: Pelagos sanctuary (Ligurian sea), Ionian sea, and Strait of Gibraltar. The mRNA levels of five putative biomarker genes (aryl hydrocarbon receptor, E2F-1 transcription factor, cytochrome P450 1A, estrogen receptor 1, and heat shock protein 70) were measured for the first time by quantitative real-time PCR in cetacean skin biopsies. The different responses of most of the genes reflected contamination levels in the three sampling areas. Pelagos sanctuary dolphins appeared to be the most exposed to toxicological stress, having the highest up-regulation of CYP1A and AHR. Moreover, a cluster analysis distinguished the populations on the basis of the gene expression biomarker used in our study, showing different pattern between Mediterranean sea and Strait of Gibraltar. Our results suggest that this molecular approach applied to non-destructive biopsy material is a powerful diagnostic tool for evaluating ecotoxicological impact on cetacean populations.

The Pelagos Sanctuary for Mediterranean marine mammals: Marine Protected Area (MPA) or marine polluted area? The case study of the striped dolphin (Stenella coeruleoalba)

The concurrence of man-made pressures on cetaceans in the Mediterranean Sea is potentially affecting population stability and marine biodiversity. This needs to be proven for the only pelagic marine protected area in the Mediterranean Sea: the Pelagos Sanctuary for Mediterranean Marine Mammals. Here we applied a multidisciplinary tool, using diagnostic markers elaborated in a statistical model to rank toxicological stress in Mediterranean cetaceans. As a case study we analyzed persistent, bioaccumulative and toxic chemicals combined with a wide range of diagnostic markers of exposure to anthropogenic contaminants and genetic variation as marker of genetic erosion in striped dolphin (Stenella coeruleoalba) skin biopsies. Finally, a statistical model was applied to obtain a complete toxicological profile of the striped dolphin in the Pelagos Sanctuary and other Mediterranean areas (Ionian Sea and Strait of Gibraltar). Here we provide the first complete evidence of the toxicological stress in cetaceans living in Pelagos Sanctuary.

Plastic Debris Occurrence, Convergence Areas and Fin Whales Feeding Ground in the Mediterranean Marine Protected Area Pelagos Sanctuary: A Modeling Approach

The Mediterranean Sea is greatly affected by marine litter. In this area, research on the impact of plastic debris (including microplastics) on biota, particularly large filter-feeding species such as the fin whale (Balaenoptera physalus), is still in its infancy. We investigated the possible overlap between microplastic, mesoplastic and macrolitter accumulation areas and the fin whale feeding grounds in in a pelagic Specially Protected Area of Mediterranean Importance (SPAMI): the Pelagos Sanctuary. Models of ocean circulation and fin whale potential habitat were merged to compare marine litter accumulation with the presence of whales. Additionally, field data on microplastics, mesoplastics and macrolitter abundance and cetacean presence were simultaneously collected. The resulting data were compared, as a multi-layer, with the simulated distribution of plastic concentration and the whale habitat model. These data showed a high occurrence of microplastics (mean: 0.082 items/m2, STD ± 0.079 items/m2) spatial distribution agreed with our modelling results. Areas with high microplastic density significantly overlapped with areas of high macroplastic density. The most abundant polymer detected in all the sampling sites was polyethylene (PE), suggesting fragmentation of larger packaging items as the primary source. To our knowledge, this is the first study in the Pelagos Sanctuary in which the simulated microplastic distribution has been confirmed by field observations. The overlap between the fin whale feeding habitat and the microplastic hot spots is an important contribution for risk assessment of fin whale exposure to microplastics.

Occurrence, relative abundance and spatial distribution of microplastics and zooplankton NW of Sardinia in the Pelagos Sanctuary Protected Area, Mediterranean Sea

Environmental context Plastic materials are accumulating in the marine environment where they can now be found in the remotest areas of the worlds oceans. Microplastics (plastic particles ≤5mm), abundant in marine surface waters, are also found in neritic and pelagic waters of Mediterranean marine protected areas. Microplastics can accumulate along marine food chains, having noxious effects on marine organisms at different trophic levels and creating a serious threat to marine ecosystems. Abstract Floating plastic debris tends to fragment into smaller pieces, termed microplastics, which may increase the likelihood of ingestion of plastics by marine organisms entering the food web. This study analyses the amount and spatial distribution of microplastics and zooplankton in an area near Asinara National Park (NW Sardinia) and overlapping the Pelagos Sanctuary (Mediterranean Sea). Analysis showed microplastics in 81% of the 27 samples analysed, with a mean value of 0.17±0.32 items m–3. From geographic information system processing of the data, microplastics appeared more abundant (by a factor of four) in the pelagic than in the neritic environment, and showed a size range of the same order as major zooplanktonic taxa determined in the area. These findings suggest a potential risk of mesozooplankton and species preying on plankton mistaking microplastics for food. Further functional and toxicological studies are therefore necessary to assess the hazard associated with microplastics in the marine food web.