Preventing illegal seafood trade using machine-learning assisted microbiome analysis.

BACKGROUND: Seafood is increasingly traded worldwide, but its supply chain is particularly prone to frauds. To increase consumer confidence, prevent illegal trade, and provide independent validation for eco-labelling, accurate tools for seafood traceability are needed. Here we show that the use of microbiome profiling (MP) coupled with machine learning (ML) allows precise tracing the origin of Manila clams harvested in areas separated by small geographic distances. The study was designed to represent a real-world scenario. Clams were collected in different seasons across the most important production area in Europe (lagoons along the northern Adriatic coast) to cover the known seasonal variation in microbiome composition for the species. DNA extracted from samples underwent the same depuration process as commercial products (i.e. at least 12 h in open flow systems). RESULTS: Machine learning-based analysis of microbiome profiles was carried out using two completely independent sets of data (collected at the same locations but in different years), one for training the algorithm, and the other for testing its accuracy and assessing the temporal stability signal. Briefly, gills (GI) and digestive gland (DG) of clams were collected in summer and winter over two different years (i.e. from 2018 to 2020) in one banned area and four farming sites. 16S DNA metabarcoding was performed on clam tissues and the obtained amplicon sequence variants (ASVs) table was used as input for ML MP. The best-predicting performances were obtained using the combined information of GI and DG (consensus analysis), showing a Cohen K-score > 0.95 when the target was the classification of samples collected from the banned area and those harvested at farming sites. Classification of the four different farming areas showed slightly lower accuracy with a 0.76 score. CONCLUSIONS: We show here that MP coupled with ML is an effective tool to trace the origin of shellfish products. The tool is extremely robust against seasonal and inter-annual variability, as well as product depuration, and is ready for implementation in routine assessment to prevent the trade of illegally harvested or mislabeled shellfish.

Depuration processes affect the Vibrio community in the microbiota of the Manila clam, Ruditapes philippinarum.

As filter-feeders, bivalve molluscs accumulate Vibrio into edible tissues. Consequently, an accurate assessment of depuration procedures and the characterization of the persistent Vibrio community in depurated shellfish represent a key issue to guarantee food safety in shellfish products. The present study investigated changes in the natural Vibrio community composition of the Ruditapes philippinarum microbiota with specific focus on human pathogenic species. For this purpose, the study proposed a MLSA-NGS approach (rRNA 16S, recA and pyrH) for the detection and identification of Vibrio species. Clam microbiota were analysed before and after depuration procedures performed in four depuration plants, using culture-dependent and independent approaches. Microbiological counts and NGS data revealed differences in terms of both contamination load and Vibrio community between depuration plants. The novel MLSA-NGS approach allowed for a clear definition of the Vibrio species specific to each depuration plant. Specifically, depurated clam microbiota showed presence of human pathogenic species. Ozone treatments and the density of clams in the depuration tank probably influenced the level of contamination and the Vibrio community composition. The composition of Vibrio community specific to each plant should be carefully evaluated during the risk assessment to guarantee a food-safe shellfish-product for the consumer.

Multidisciplinary long-term survey of Manila clam grown in farming sites subjected to different environmental conditions.

In recent years recurrent bivalve mass mortalities considerably increased around the world, causing the collapse of natural and farmed populations. Venice Lagoon has historically represented one of the major production areas of the Manila clam Ruditapes philippinarum in Europe. However, in the last 20 years a 75 % decrease in the annual production has been experienced. While climate change and anthropogenic interventions may have played a key role in natural and farmed stocks reductions, no studies investigated at multiple levels the environmental stressors affecting farmed Manila clam to date. In this work we carried out a long-term monitoring campaign on Manila clam reared in four farming sites located at different distances from the southern Venice Lagoon inlet, integrating (meta)genomic approaches (i.e. RNA-seq; microbiota characterization), biometric measurements and chemical-physical parameters. Our study allowed to characterize the molecular mechanisms adopted by this species to cope with the different environmental conditions characterizing farming sites and to propose hypotheses to explain mortality events observed in recent years. Among the most important findings, the disruption of clam's immune response, the spread of Vibrio spp., and the up-regulation of molecular pathways involved in xenobiotic metabolism suggested major environmental stressors affecting clams farmed in sites placed close to Chioggia's inlet, where highest mortality was also observed. Overall, our study provides knowledge-based tools for managing Manila clam farming on-growing areas. In addition, the collected data is a snapshot of the time immediately before the commissioning of MoSE, a system of mobile barriers aimed at protecting Venice from high tides, and will represent a baseline for future studies on the effects of MoSE on clams farming and more in general on the ecology of the Venice Lagoon.

Fast and Green Method to Control Frauds of Geographical Origin in Traded Cuttlefish Using a Portable Infrared Reflective Instrument.

An appropriate seafood origin identification is essential for labelling regulation but also economic and ecological issues. Near infrared (NIRS) reflectance spectroscopy was employed to assess the origins of cuttlefish caught from five fishing FAO areas (Adriatic Sea, northeastern and eastern central Atlantic Oceans, and eastern Indian and western central Pacific Oceans). A total of 727 cuttlefishes of the family Sepiidae (Sepia officinalis and Sepiella inermis) were collected with a portable spectrophotometer (902-1680 nm) in a wholesale fish plant. NIR spectra were treated with standard normal variate, detrending, smoothing, and second derivative before performing chemometric approaches. The random forest feature selection procedure was executed to select the most significative wavelengths. The geographical origin classification models were constructed on the most informative bands, applying support vector machine (SVM) and K nearest neighbors algorithms (KNN). The SVM showed the best performance of geographical classification through the hold-out validation according to the overall accuracy (0.92), balanced accuracy (from 0.83 to 1.00), sensitivity (from 0.67 to 1.00), and specificity (from 0.88 to 1.00). Thus, being one of the first studies on cuttlefish traceability using NIRS, the results suggest that this represents a rapid, green, and non-destructive method to support on-site, practical inspection to authenticate geographical origin and to contrast fraudulent activities of cuttlefish mislabeled as local.

The new PFAS C6O4 and its effects on marine invertebrates: First evidence of transcriptional and microbiota changes in the Manila clam Ruditapes philippinarum.

There is growing concern for the wide use ofperfluorooctanoic acid (PFOA) because of its toxic effects on the environment and on human health. A new compound - the so called C6O4 (perfluoro ([5-methoxy-1,3-dioxolan-4-yl]oxy) acetic acid) - was recently introduced as one of the alternative to traditional PFOA, however this was done without any scientific evidence of the effects of C6O4 when dispersed into the environment. Recently, the Regional Agency for the Protection of the Environment of Veneto (Italy) detected high levels of C6O4 in groundwater and in the Po river, increasing the alarm for the potential effects of this chemical into the natural environment. The present study investigates for the first time the effects of C6O4 on the Manila clam Ruditapes philippinarum exposed to environmental realistic concentrations of C6O4 (0.1 µg/L and 1 µg/L) for 7 and 21 days. Furthermore, in order to better understand if C6O4 is a valid and less hazardous alternative to its substitute, microbial and transcriptomic alterations were also investigated in clams exposed to 1 µg/L ofPFOA. Results indicate that C6O4 may cause significant perturbations to the digestive gland microbiota, likely determining the impairment of host physiological homeostasis. Despite chemical analyses suggest a 5 times lower accumulation potential of C604 as compared to PFOA in clam soft tissues, transcriptional analyses reveal several alterations of gene expression profile. A large part of the altered pathways, including immune response, apoptosis regulation, nervous system development, lipid metabolism and cell membrane is the same in C6O4 and PFOA exposed clams. In addition, clams exposed to C6O4 showed dose-dependent responses as well as possible narcotic or neurotoxic effects and reduced activation of genes involved in xenobiotic metabolism. Overall, the present study suggests that the potential risks for marine organism following environmental contamination are not reduced by replacing PFOA with C6O4. In addition, the detection of both C6O4 and PFOA into tissues of clams inhabiting the Lagoon of Venice - where there are no point sources of either compounds - recommends a similar capacity to spread throughout the environment. These results prompt the urgent need to re-evaluate the use of C6O4 as it may represent not only an environmental hazard but also a potential risk for human health.

Long-lasting effects of chronic exposure to chemical pollution on the hologenome of the Manila clam.

Chronic exposure to pollutants affects natural populations, creating specific molecular and biochemical signatures. In the present study, we tested the hypothesis that chronic exposure to pollutants might have substantial effects on the Manila clam hologenome long after removal from contaminated sites. To reach this goal, a highly integrative approach was implemented, combining transcriptome, genetic and microbiota analyses with the evaluation of biochemical and histological profiles of the edible Manila clam Ruditapes philippinarum, as it was transplanted for 6 months from the polluted area of Porto Marghera (PM) to the clean area of Chioggia (Venice lagoon, Italy). One month post-transplantation, PM clams showed several modifications to its resident microbiota, including an overrepresentation of the opportunistic pathogen Arcobacter spp. This may be related to the upregulation of several immune genes in the PM clams, potentially representing a host response to the increased abundance of deleterious bacteria. Six months after transplantation, PM clams demonstrated a lower ability to respond to environmental/physiological stressors related to the summer season, and the hepatopancreas-associated microbiota still showed different compositions among PM and CH clams. This study confirms that different stressors have predictable effects in clams at different biological levels and demonstrates that chronic exposure to pollutants leads to long-lasting effects on the animal hologenome. In addition, no genetic differentiation between samples from the two areas was detected, confirming that PM and CH clams belong to a single population. Overall, the obtained responses were largely reversible and potentially related to phenotypic plasticity rather than genetic adaptation. The results here presented will be functional for the assessment of the environmental risk imposed by chemicals on an economically important bivalve species.

Temporal occurrence of Cryptosporidium in the Manila clam Ruditapes philippinarum in northern Adriatic Italian lagoons.

In order to evaluate the temporal occurrence of Cryptosporidium oocysts in Ruditapes philippinarum clams bred along the northeastern Italian Adriatic coast and molecularly characterize the isolates, 2,160 specimens (180 clams per month) were collected from three clam farms from January to December 2004. Two farms (sites A and B) were located in Venice (Chioggia, Veneto region) and one (site C) in the Marano Lagoons (Friuli Venezia Giulia region). Clams from 36 pools (i.e., one pool of 60 clams per month per site) were subjected to a high-sensitivity seminested PCR assay specific for a 360-bp diagnostic region internal to the Cryptosporidium spp. outer wall protein gene. Positive amplicons were sequenced and analyzed. Cryptosporidium DNA was found in clams from seven pools (sites A and B) during 1 month of sampling at site A and 6 months of sampling at site B, with Cryptosporidium hominis and Cryptosporidium parvum being detected. The expected infection rate of the clams was 0.36%. Site B showed a significantly higher expected infection rate (1.15%) than did the other sites (A = 0.14% and C = 0%). Given its high sensitivity and specificity, this seminested PCR assay can be considered a reliable tool for detecting and distinguishing species within the Cryptosporidium genus. The seasonal pattern of contamination and the related public health risks are of particular concern.

Can ecological history influence response to pollutants? Transcriptomic analysis of Manila clam collected in different Venice lagoon areas and exposed to heavy metal.

Chronic exposure to environmental pollutants can exert strong selective pressures on natural populations, favoring the transmission over generations of traits that enable individuals to survive and thrive in highly impacted environments. The lagoon of Venice is an ecosystem subject to heavy anthropogenic impact, mainly due to the industrial activities of Porto Marghera (PM), which led to a severe chemical contamination of soil, groundwater, and sediments. Gene expression analysis on wild Manila clams collected in different Venice lagoon areas enabled to identify differences in gene expression profiles between clams collected in PM and those sampled in clean areas, and the definition of molecular signatures of chemical stress. However, it remains largely unexplored to which extent modifications of gene expression patterns persists after removing the source of contamination. It is also relatively unknown whether chronic exposure to xenobiotics affects the response to other chemical pollutants. To start exploring such issues, in the present study a common-garden experiment was coupled with transcriptomic analysis, to compare gene expression profiles of PM clams with those of clams collected in the less impacted area of Chioggia (CH) during a period under the same control conditions. Part of the two experimental groups were also exposed to copper for seven days to assess whether different "ecological history" does influence response to such pollutant. The results obtained suggest that the chronic exposure to chemical pollution generated a response at the transcriptional level that persists after removal for the contaminated site. These transcriptional changes are centered on key biological processes, such as defense against either oxidative stress or tissue/protein damage, and detoxification, suggesting an adaptive strategy for surviving in the deeply impacted environment of Porto Marghera. On the other hand, CH clams appeared to respond more effectively to copper exposure than PM animals, proposing that chronic exposure to chemical toxicants either lowers the sensitivity to additional toxicants or blunts the capacity to respond to novel chemical challenges in PM clams.