miRNAs as Biomolecular Markers for Food Safety, Quality, and Traceability in Poultry Meat-A Preliminary Study.

In this study, the expression and abundance of two candidate chicken (Gallus gallus; gga) microRNAs (miRNAs, miR), gga-miR-21-5p (miR-21) and gga-miR-126-5p (miR-126), have been analyzed in order to identify biomarkers for the traceability and quality of poultry meat. Two breeds of broiler chickens were tested: the most common Ross308 (fast-growing) and the high-quality Ranger Gold (slow-growing). A preliminary analysis of the two miRNAs expressions was conducted across various tissues (liver, lung, spleen, skeletal muscle, and kidney), and the three tissues (lung, spleen, and muscle) with a higher expression were chosen for further analysis. Using quantitative reverse transcription polymerase chain reaction (RT-qPCR), the expression of miRNAs in the three tissues of a total of thirteen animals was determined. The results indicate that miR-126 could be a promising biomarker for the lung tissue in the Ranger Gold (RG) breed (p < 0.01), thus suggesting a potential applicability for tracing hybrids. RG exhibits a significantly higher miR-126 expression in the lung tissue compared to the Ross308 broilers (R308), an indication of greater respiratory capacity and, consequently, a higher oxidative metabolism of the fast-growing hybrid. During sampling, two R308 broilers presented some anomalies, including airsacculitis, hepatic steatosis, and enlarged spleen. The expression of miR-126 and miR-21 was compared in healthy animals and in those presenting anomalies. Chickens with airsacculitis and hepatic steatosis showed an up-regulation of miR-21 and miR-126 in the most commercially valuable tissue, the skeletal muscle or breast (p < 0.05).

Sorbed environmental contaminants increase the harmful effects of microplastics in adult zebrafish, Danio rerio.

Aquatic animals ingest Microplastics (MPs) which have the potential to affect the uptake and bioavailability of sorbed co-contaminants. However, the effects on living organisms still need to be properly understood. The present study was designed to assess the combined effects of MPs and environmental contaminants on zebrafish (Danio rerio) health and behavior. Adult specimens were fed according to three different protocols: 1) untreated food (Control group); 2) food supplemented with 0.4 mg/L pristine polyethylene-MPs (PE-MPs; 0.1-0.3 mm diameter) (PEv group); 3) food supplemented with 0.4 mg/L PE-MPs previously incubated (PEi group) for 2 months in seawater. Analysis of contaminants in PEi detected trace elements, such as lead and copper. After 15 days of exposure, zebrafish underwent behavioral analysis and were then dissected to sample gills and intestine for histology, and the latter also for microbiome analysis. Occurrence of PEv and PEi in the intestine and contaminants in the fish carcass were analyzed. Both PEv- and PEi-administered fish differed from controls in the assays performed, but PEi produced more harmful effects in most instances. Overall, MPs after environmental exposure revealed higher potential to alter fish health through combined effects (e.g. proportion of microplastics, pollutants and/or microorganisms).

Fin Whale (Balaenoptera physalus) Mortality along the Italian Coast between 1624 and 2021.

The Mediterranean Sea hosts a population of fin whale (Balaenoptera physalus), the only species of Mysticete regularly occurring in the basin. Observed and inferred mortality suggests that the population is likely declining. Accordingly, understanding the causes of mortality and assessing the health status is pivotal to the survival of this endangered population. While such studies are inherently difficult for a highly roaming species with a pelagic distribution, mortality events provide the opportunity to investigate biological and epidemiological traits linked to these events, and evaluate the footprint of human activity, especially when long-term data series exist. We present a comprehensive spatial-temporal overview of fin whale mortality events along the Italian coast encompassing four centuries (1624-2021). Time series analysis was used to highlight structural changes in the evolution of mortality through time, while spatial-temporal patterns in the distribution of mortality events were assessed through emerging hot spot analysis methods. Recent mortality events (1964-2021) were further explored to evaluate, where possible, the primary causes of mortality and to identify anthropogenic threats of conservation concerns. This long-term survey offers the basis for an understanding of the health status of this B. physalus population and provides much-needed information for developing an effective management and conservation plan for the species in the region.

Genome-Wide DNA Methylation Protocol for Epigenetics Studies.

Epigenetic modification are heritable changes in gene expression not encoded by the DNA sequence therefore playing a significant role in a broad range of biological processes and diseases.A key player of the epigenetic modifications is the DNA methylation, a process involving the covalent transfer of a methyl group to the C-5 position of the cytosine ring of DNA forming 5-methylcytosine (5mC), catalyzed by DNA methyltransferases. Altering the structure of the chromatin, DNA methylation has the potential to down-regulate gene expression.The here presented protocol shows a method to obtain DNA samples ready for NGS sequencing for genome-wide methylation analysis.

Plastic ingestion by Atlantic horse mackerel (Trachurus trachurus) from central Mediterranean Sea: A potential cause for endocrine disruption.

Plastics in the oceans can break up into smaller size and shape resembling prey or particles selected for ingestion by marine organisms. Plastic polymers may contain chemical additives and contaminants, including known endocrine disruptors that may be harmful for the marine organisms, in turn posing potential risks to marine ecosystems, biodiversity and food availability. This study assesses the presence of plastics in the contents of the gastrointestinal tract (GIT) of a commercial fish species, the Atlantic horse mackerel, Trachurus trachurus, sampled from two different fishing areas of central Mediterranean Sea. Adverse effect of plastics occurrence on T. Trachurus health were also assessed quantifying the liver expression of vitellogenin (VTG), a biomarker for endocrine disruption. A total of 92 specimens were collected and morphometric indices were analysed. A subgroup was examined for microplastics (MP < 1 mm) and macroplastics (MaP >1 cm) accumulation in the GIT and for VTG expression. Results indicated that specimens from the two locations are different in size and maturity but the ingestion of plastic is widespread, with microplastics (fragments and filaments) abundantly present in nearly all samples while macroplastics were found in the larger specimens, collected in one of the two locations. Spectroscopic analysis revealed that the most abundant polymers in MP fragments were polystyrene, polyethylene and polypropylene, whereas MP filaments were identified mainly as nylon 6, acrylic and polyester. MaP were composed mainly of weathered polyethylene or polypropylene. The expression of VTG was observed in the liver of 60% of all male specimens from both locations. The results of this study represent a first evidence that the ingestion of plastic pollution may alter endocrine system function in adult fish T. Trachurus and warrants further research.

Effects of microplastics on head kidney gene expression and enzymatic biomarkers in adult zebrafish.

Due to massive production, improper use, and disposal of plastics, microplastics have become global environmental pollutants affecting both freshwater and marine ecosystems. Several studies have documented the uptake of microplastics in wild species and the correlated biological effects, such as epithelial damage, inflammation, metabolic alterations, and neurotoxicity. However, the effects of microplastics are not fully understood yet. In this study, adult zebrafish have been exposed for twenty days to two concentrations of a mix of polystyrene and high-density polyethylene microplastics. The biological effects were investigated through the expression levels of a set of selected genes in head kidney samples and two enzymatic biomarkers, acetylcholinesterase and lactate dehydrogenase, in head and body homogenates respectively. The lowest microplastic concentration up-regulated genes involved in xenobiotics catabolic processes (cyp2p8), and adaptive immunity (tcra). Acetylcholinesterase activity was inhibited by the highest microplastics exposure, while a weaker and no significant inhibition was induced by the lowest concentration. No significant effects on lactate dehydrogenase activity were observed. The results presented in this study support the hypothesis that MPs exposure could induce the activation of an immune response and the xenobiotic metabolism, suggesting also that the cytochrome P450 enzyme cyp2p8 and acetylcholinesterase may be sensitive to MPs contamination.

Transcriptome analysis of cadmium exposure in kidney fibroblast cells of the North Atlantic Right Whale (Eubalaena glacialis).

An 8X15k oligonucleotide microarray was developed consisting of 2334 Eubalaena glacialis probes and 2166 Tursiops truncatus probes and used to measure the effects, at transcriptomic level, of cadmium exposure in right whale kidney fibroblast cells. Cells were exposed to three concentrations (1 µM, 0.1 µM, and 0.01 µM) of cadmium chloride (CdCl2) for three exposure times (1, 4, and 24 h). Cells exposed to 1 µM CdCl2 for 4 h and 24 h showed upregulated genes involved in protection from metal toxicity and oxidative stress, protein renaturation, apoptosis inhibition, as well as several regulators of cellular processes. Downregulated genes represented a suite of functions including cell proliferation, transcription regulation, actin polymerization, and stress fiber synthesis. The collection of differentially expressed genes in this study support proposed mechanisms of cadmium-induced apoptosis such as ubiquitin proteasome system disruption, Ca2+ homeostasis interference, mitochondrial membrane potential collapse, reactive oxygen species (ROS) production, and cell cycle arrest. The results also have confirmed the right whale microarray as a reproducible tool in measuring differentiated gene expression that could be a valuable asset for transcriptome analysis of other baleen whales and potential health assessment protocols.

Skin distress associated with xenobiotics exposure: An epigenetic study in the Mediterranean fin whale (Balaenoptera physalus).

The phenotypic plasticity of many organisms is mediated in part by epigenetics, the heritable changes in gene activity that occur without any alterations to DNA sequence. A major mechanism in epigenetics is the DNA methylation (DNAm). Hypo- and hyper-methylation are generalized responses to control gene expression however recent studies have demonstrated that classes of contaminants could mark specific DNAm signatures, that could usefully signal prior environmental exposure. We collected skin and blubber from 6 free-ranging fin whale (Balaenoptera physalus) individuals sampled as a part of a previous published study in the northern Mediterranean Sea. Genomic DNA extracted from the skin of the fin whales and levels of contaminants measured in the blubber of the same individuals were used for DNAm profiling through reduced representation bisulfite sequencing (RRBS). We tested the hypothesis that differences in the methylation patterns could be related to environmental exposure to contaminants and load in the whale tissues. The aims of this study were to determine the DNAm profiles of the methylation contexts (CpGs and non-CpGs) of differently contaminated groups using the RRBS, and to identify potential contaminant exposure related genes. Amount and proportion of methylcytosines in CpG and non-CpG regions (CHH and CHG) was very similar across the 6 samples. The proportion of methylcytosines sites in CpG was n = 32,682, the highest among all the sequence contexts (n = 3216 in CHH; n = 1743 in CHG). The majority of the methylcytosine occurred in the intron regions, followed by exon and promoter regions in CpG, CHH and CHG. Gene Ontology results indicated that DNAm affected genes that take place in cell differentiation and function in cutaneous, vascular and nervous systems. The identification of cellular response pathways allows a better understanding of the organism biological reaction to a specific environmental challenge and the development of sensitive tools based on the predictive responses. Eco-epigenetics analyses have an extraordinary potential to address growing issues on pollution biomonitoring, ecotoxicity assessment, conservation and management planning.

Adverse effects of plastic ingestion on the Mediterranean small-spotted catshark (Scyliorhinus canicula).

Plastics are widely diffused in the oceans and their ingestion by marine organisms is raising concern for potentially adverse effects. The risk of harmful interactions with marine plastic pollution depends on the biology of the species as well as the distribution and abundance of the different plastic types. The aim of this study was to assess the occurrence of plastic ingestion by the small-spotted catshark (Scyliorhinus canicula), one of the most abundant elasmobranchs in the Mediterranean Sea. The expression levels of genes indicative of total immune system function were analyzed to gather preliminary data for further investigation of any potential correlations between plastic presence and immune activation. One hundred catsharks were collected during the Spring 2018 in two geographic locations in the southern region of the central Mediterranean Sea: 1) near Mazara del Vallo, SW Sicily and 2) near Lampedusa island, Italy's southernmost. Standard measurements were recorded for each specimen and its organs and sex was determined. The gastrointestinal tract (GIT) was preserved for plastic detection and identification. Where present, plastics (macro- and micro-) were characterized in terms of size, shape and polymer typology through microscopy and µ-Raman spectroscopy. Spleen from a subset of thirty samples was preserved for RNA extraction, then used to quantify by real time PCR the transcripts of T cell receptor beta (TCRB), T cell receptor delta (TCRD) and IgM genes. The results indicated that ingestion of plastic is widespread, with microplastics (MP, from 1 µm to <1 mm) abundantly present in nearly all samples and macroplasticplastic (MaP, > 1 cm) in approximately 18% of the specimens collected. A significant increase in the expression of TCRB, TCRD and IgM was observed in the spleen of MaP + specimens from Mazara del Vallo waters, in parallel with 67% increase in liver weight. While the presence of MP alone is not enough to induce a strong activation of the immunity, some type of plastics falling into the MaP category may be more toxic than others and crucial in the activation of the immune response. The results of this study represent a first evidence that plastic pollution represents an emerging threat to S. canicula, the Mediterranean food web and human consumers.

Microplastics induce transcriptional changes, immune response and behavioral alterations in adult zebrafish.

Microplastics have become pervasive environmental pollutants in both freshwater and marine ecosystems. The presence of microplastics have been recorded in the tissues of many wild fish species, and laboratory studies have demonstrated that microplastics can exert adverse health effects. To further investigate the biological mechanisms underlying microplastics toxicity we applied an integrated approach, analyzing the effects of microplastics at transcriptomic, histological and behavioral level. Adult zebrafish have been exposed to two concentrations of high-density polyethylene and polystyrene microplastics for twenty days. Transcriptomic results indicate alterations in the expression of immune system genes and the down-regulation of genes correlated with epithelium integrity and lipid metabolism. The transcriptomic findings are supported by tissue alterations and higher occurrence of neutrophils observed in gills and intestinal epithelium. Even the daily rhythm of activity of zebrafish appears to be affected, although the regular pattern of activity is recovered over time. Considering the transcriptomic and histological findings reported, we hypothesize that the effects on mucosal epithelium integrity and immune response could potentially reduce the organism defense against pathogens, and lead to a different utilization of energy stores.

Isolation and phenotyping of potential stem cells from the umbilical cord of the bottlenose dolphin(Tursiops truncatus).

We have successfully isolated cells with stem-like properties from bottlenose dolphin (Tursiops truncatus) umbilical cord. Our results show that this cetacean species has embryonic fetal and adult stem cells as do humans and other studied mammals. This accomplishment allows to eventually investigate whether dolphins, due to their unique adaptations to aquatic environments, have special stem cell lineages or distinctive mechanisms of cell programming. Further characterization of their potency to differentiate into multiple cell lineages would fulfill numerous applicative purposes. We characterized, developed and refined a new protocol for obtaining potential stem cells from umbilical cord tissues of the bottlenose dolphin. Tissue samples were taken from umbilical cords of successful deliveries immediately after placenta ejection and collection from the water. Umbilical cord samples (2-3 cm3) were excised and subjected to enzymatic digestion and mechanical dissociation. Viable cells from specimens resident in the Oceanografic Valencia were cultured and subsequently isolated and tested for pluripotent characteristics (cell morphology, phenotype and expression of surface markers). Cell viability was confirmed also after freezing/thawing. The established protocol is suitable for collection/isolation/culture of dolphin potential mesenchymal stem cells from dolphin umbilical cord, which can be deposited in cell banks for future research needs.

On the revolution of cetacean evolution.

The order of Cetacea with 88 species including Odontoceti (or toothed whales) and Mysticeti (or baleen whales) is the most specialized and diversified group of mammals. The blue whale with a maximum recorded length of 29.9 m for 173 t of weight is the largest animal known to have ever existed, and any dolphin's brain is most powerful and complex than any other brain in the animal kingdom, second only to primate's. Nevertheless, Cetacea are mammals that re-entered the oceans only a little over 50 million years ago, a relatively short time on the evolutionary scale. During this time cetaceans and humans have developed marked morphological and behavioral differences, yet their genomes show a high level of similarity. This present review is focused on the description and significance of newly accessible cetacean genome tools and information, and their relevance in the study of the evolution of successful phenotypic adaptations associated to mammal's marine existence, and their applicability to the unresolved disease mechanisms in humans.

The chronicles of the contaminated Mediterranean seas: a story told by the cetaceans' skin genes.

Wild animals in their natural environment could provide a big source of information, but sampling can be very challenging, above all for protected species, like marine mammals. Nevertheless, significant data can be obtained sampling stranded animals right after their death, taking into account proper sampling time and methodology. RNA samples from the skin of 12 individuals including the species Stenella coeruleoalba, Tursiops truncatus, and Grampus griseus were used to test 4 potential gene markers of anthropogenic contaminants exposure. The individuals were sampled in 3 geographic areas: the Adriatic, Ionian and Tyrrhenian seas. Three out of the 4 genes tested showed higher expression in the samples collected from the Adriatic Sea. Minute skin samples tell the story of the specific geographic location where the marine mammal spent its life, thanks to the different impact on gene expression exerted by different contamination levels.

Transcriptomic analysis of bottlenose dolphin (Tursiops truncatus) skin biopsies to assess the effects of emerging contaminants.

Chemicals discovered in water at levels that may be significantly different than expected are referred to as contaminants of emerging concern (CECs) because the risk to environmental health posed by their occurrence/frequency is still unknown. The worldwide distributed compounds perfluorooctanoic acid (PFOA) and bisphenol A (BPA) may fall into this category due to effects on endocrine receptors. We applied an ex vivo assay using small slices of bioptic skin from the bottlenose dolphin, Tursiops truncatus, cultured and treated for 24 h with different PFOA or BPA concentrations to analyze global gene expression. RNA was labeled and hybridized to a species-specific oligomicroarray. The skin transcriptome held information on the contaminant exposure, potentially predictive about long-term effects on health, being the genes affected involved in immunity modulation, response to stress, lipid homeostasis, and development. The transcriptomic signature of dolphin skin could be therefore relevant as classifier for a specific contaminant.

Convergent evolution of the genomes of marine mammals.

Marine mammals from different mammalian orders share several phenotypic traits adapted to the aquatic environment and therefore represent a classic example of convergent evolution. To investigate convergent evolution at the genomic level, we sequenced and performed de novo assembly of the genomes of three species of marine mammals (the killer whale, walrus and manatee) from three mammalian orders that share independently evolved phenotypic adaptations to a marine existence. Our comparative genomic analyses found that convergent amino acid substitutions were widespread throughout the genome and that a subset of these substitutions were in genes evolving under positive selection and putatively associated with a marine phenotype. However, we found higher levels of convergent amino acid substitutions in a control set of terrestrial sister taxa to the marine mammals. Our results suggest that, whereas convergent molecular evolution is relatively common, adaptive molecular convergence linked to phenotypic convergence is comparatively rare.

Microarray applications to understand the impact of exposure to environmental contaminants in wild dolphins (Tursiops truncatus).

It is increasingly common to monitor the marine environment and establish geographic trends of environmental contamination by measuring contaminant levels in animals from higher trophic levels. The health of an ecosystem is largely reflected in the health of its inhabitants. As an apex predator, the common bottlenose dolphin (Tursiops truncatus) can reflect the health of near shore marine ecosystems, and reflect coastal threats that pose risk to human health, such as legacy contaminants or marine toxins, e.g. polychlorinated biphenyls (PCBs) and brevetoxins. Major advances in the understanding of dolphin biology and the unique adaptations of these animals in response to the marine environment are being made as a result of the development of cell-lines for use in in vitro experiments, the production of monoclonal antibodies to recognize dolphin proteins, the development of dolphin DNA microarrays to measure global gene expression and the sequencing of the dolphin genome. These advances may play a central role in understanding the complex and specialized biology of the dolphin with regard to how this species responds to an array of environmental insults. This work presents the creation, characterization and application of a new molecular tool to better understand the complex and unique biology of the common bottlenose dolphin and its response to environmental stress and infection. A dolphin oligo microarray representing 24,418 unigene sequences was developed and used to analyze blood samples collected from 69 dolphins during capture-release health assessments at five geographic locations (Beaufort, NC, Sarasota Bay, FL, Saint Joseph Bay, FL, Sapelo Island, GA and Brunswick, GA). The microarray was validated and tested for its ability to: 1) distinguish male from female dolphins; 2) differentiate dolphins inhabiting different geographic locations (Atlantic coasts vs the Gulf of Mexico); and 3) study in detail dolphins resident in one site, the Georgia coast, known to be heavily contaminated by Aroclor 1268, an uncommon polychlorinated (PCB) mixture. The microarray was able to distinguish dolphins by sex, geographic location, and corroborate previously published health irregularities for the Georgia dolphins. Genes involved in xenobiotic metabolism, development/differentiation and oncogenic pathways were found to be differentially expressed in GA dolphins. The report bridges the advancements in dolphin genome sequencing to the first step towards providing a cost-effective means to screen for indicators of chemical toxin exposure as well as disease status in top level predators.

Machine learning approaches to investigate the impact of PCBs on the transcriptome of the common bottlenose dolphin (Tursiops truncatus).

As top-level predators, common bottlenose dolphins (Tursiops truncatus) are particularly sensitive to chemical and biological contaminants that accumulate and biomagnify in the marine food chain. This work investigates the potential use of microarray technology and gene expression profile analysis to screen common bottlenose dolphins for exposure to environmental contaminants through the immunological and/or endocrine perturbations associated with these agents. A dolphin microarray representing 24,418 unigene sequences was used to analyze blood samples collected from 47 dolphins during capture-release health assessments from five different US coastal locations (Beaufort, NC, Sarasota Bay, FL, Saint Joseph Bay, FL, Sapelo Island, GA and Brunswick, GA). Organohalogen contaminants including pesticides, polychlorinated biphenyl congeners (PCBs) and polybrominated diphenyl ether congeners were determined in blubber biopsy samples from the same animals. A subset of samples (n = 10, males; n = 8, females) with the highest and the lowest measured values of PCBs in their blubber was used as strata to determine the differential gene expression of the exposure extremes through machine learning classification algorithms. A set of genes associated primarily with nuclear and DNA stability, cell division and apoptosis regulation, intra- and extra-cellular traffic, and immune response activation was selected by the algorithm for identifying the two exposure extremes. In order to test the hypothesis that these gene expression patterns reflect PCB exposure, we next investigated the blood transcriptomes of the remaining dolphin samples using machine-learning approaches, including K-nn and Support Vector Machines classifiers. Using the derived gene sets, the algorithms worked very well (100% success rate) at classifying dolphins according to the contaminant load accumulated in their blubber. These results suggest that gene expression profile analysis may provide a valuable means to screen for indicators of chemical exposure.

Quantitative methods to characterize morphological properties of cell lines.

Descriptive terms are often used to characterize cells in culture, but the use of nonquantitative and poorly defined terms can lead to ambiguities when comparing data from different laboratories. Although recently there has been a good deal of interest in unambiguous identification of cell lines via their genetic markers, it is also critical to have definitive, quantitative metrics to describe cell phenotypic characteristics. Quantitative metrics of cell phenotype will aid the comparison of data from experiments performed at different times and in different laboratories where influences such as the age of the population and differences in culture conditions or protocols can potentially affect cellular metabolic state and gene expression in the absence of changes in the genetic profile. Here, we present examples of robust methodologies for quantitatively assessing characteristics of cell morphology and cell-cell interactions, and of growth rates of cells within the population. We performed these analyses with endothelial cell lines derived from dolphin, bovine and human, and with a mouse fibroblast cell line. These metrics quantify some characteristics of these cells lines that clearly distinguish them from one another, and provide quantitative information on phenotypic changes in one of the cell lines over large number of passages.

Health status, infection and disease in California sea lions (Zalophus californianus) studied using a canine microarray platform and machine-learning approaches.

Conservation biologists face many challenges in assessing health, immune status and infectious diseases in protected species. These challenges include unpredictable sample populations, diverse genetic and environmental backgrounds of the animals, as well as the practical, legal and ethical issues involved in experimentation. The use of whole genome scale transcriptomics with animal samples obtained in a minimally invasive manner is an approach that shows promise for health assessment. In this study we assessed the utility of a microarray to identify changes in gene expression predictive of health status by interrogating blood samples from California sea lions (Zalophus californianus) in rehabilitation. A custom microarray was developed from the commercially available dog microarray (Canis familiaris) by selecting probes that demonstrated reliable cross-hybridization with RNA in sea lion blood. This custom microarray was used for the analysis of RNA from 73 sea lion blood samples, from animals with a broad spectrum of health changes. Both traditional classifying techniques and newer artificial neural network approaches correctly classified sea lions with respect to health status, primarily distinguishing between leptospirosis infection and domoic acid exposure. Real time PCR validation for a small set of genes, followed by sequencing, showed good correlation with array results and high identity (96-98%) between the dog and sea lion sequences. This approach to health status classification shows promise for disease identification in a clinical setting, and assessment of health status of wildlife.