Molecular typing and genome sequencing allow the identification of persistent Listeria monocytogenes strains and the tracking of the contamination source in food environments.

The presence of Listeria monocytogenes (Lm) in the food processing environment (facilities and products) is a challenging problem in food safety management. Lm is one of the main causes of mortality in foodborne infections, and the trend is continuously increasing. In this study, a collection of 323 Lm strain isolates recovered from food matrices and food industry environments (surfaces and equipment) over four years from 80 food processing facilities was screened using a restriction site-associated tag sequencing (2b-RAD) typing approach developed for Lm. Thirty-six different restriction site-associated DNA (RAD) types (RTs) were identified, most of which correspond to lineage II. RT1, the most represented genotype in our collection and already reported as one of the most prevalent genotypes in the food environment, was significantly associated with meat processing facilities. The sequencing of the genomes of strains belonging to the same RT and isolated in the same facility in different years revealed several clusters of persistence. The definition of the persistent strains (PSs) allowed the identification of the potential source of contamination in the incoming raw meat that is introduced in the facility to be processed. The slaughterhouses, which, according to the European Union (EU) regulation, are not inspected for the presence of Lm could be hotspots for the persistence of Lm PSs.

Effects of environmental concentrations of the fragrance amyl salicylate on the mediterranean mussel Mytilus galloprovincialis.

Amyl salicylate (AS) is a fragrance massively used as a personal care product and following the discharged in wastewaters may end up in the aquatic environment representing a potential threat for the ecosystem and living organisms. AS was recently detected in water of the Venice Lagoon, a vulnerable area continuously subjected to the income of anthropogenic chemicals. The lagoon is a relevant area for mollusc farming, including the Mediterranean mussels (Mytilus galloprovincialis) having an important economic and ecological role. Despite high levels of AS occurred in water of the Lagoon of Venice, no studies investigated the possible consequences of AS exposures on species inhabiting this ecosystem to date. For the first time, we applied a multidisciplinary approach to investigate the potential effects of the fragrance AS on Mediterranean mussels. To reach such a goal, bioaccumulation, cellular, biochemical, and molecular analyses (RNA-seq and microbiota characterization) were measured in mussels treated for 7 and 14 days with different AS Venice lagoon environmental levels (0.1 and 0.5 µg L-1). Despite chemical investigations suggested low AS bioaccumulation capability, cellular and molecular analyses highlighted the disruption of several key cellular processes after the prolonged exposures to the high AS concentration. Among them, potential immunotoxicity and changes in transcriptional regulation of pathways involved in energy metabolism, stress response, apoptosis and cell death regulations have been observed. Conversely, exposure to the low AS concentration demonstrated weak transcriptional changes and transient increased representation of opportunistic pathogens, as Arcobacter genus and Vibrio aestuarianus. Summarizing, this study provides the first overview on the effects of AS on one of the most widely farmed mollusk species.

Transcriptome analysis reveals a complex response to the RGNNV/SJNNV reassortant Nervous Necrosis Virus strain in sea bream larvae.

The gilthead sea bream (Sparus aurata) is a marine fish of great importance for Mediterranean aquaculture. This species has long been considered resistant to Nervous Necrosis Virus (NNV), an RNA virus that causes massive mortalities in several farmed fish animals. However, the recent appearance of RGNNV/SJNNV reassortant strains started to pose a serious threat to sea bream hatcheries, as it is able to infect larvae and juveniles of this species. While host response to NNV has been extensively studied in adult fish, little attention has been devoted to early life history stages, which are generally the most sensitive ones. Here we report for the first time a time-course RNA-seq analysis on 21-day old fish gilthead sea bream larvae experimentally infected with a RGNNV/SJNNV strain. NNV-infected and mock-infected samples were collected at four time points (6 h, 12 h, 24 h, and 48 h post infection). Four biological replicates, each consisting of five pooled larvae, were analysed for each time point and group. A large set of genes were found to be significantly regulated, especially at early time points (6 h and 12 h), with several heat shock protein encoding transcripts being up-regulated (e.g. hspa5, dnaj4, hspa9, hsc70), while many immune genes were down-regulated (e.g. myd88 and irf5 at T06, pik3r1, stat3, jak1, il12b and il6st at T12). A gene set enrichment analysis (GSEA) identified several altered pathways/processes. For instance, the formation of peroxisomes, which are important anti-viral components as well as essential for nervous system homeostasis, and the autophagy pathway were down-regulated at 6 h and 24 h post infection (hpi). Finally, two custom "reactomes" (i.e. significant gene sets observed in other studies) were defined and used. The first reactome integrated the transcriptomic response to NNV in different fish species, while the second one included all genes found to be stimulated either by interferon (IFN) or by IFN and Chikungunya virus in zebrafish. Genes in both reactomes showed predominant up-regulation at 6hpi and 12hpi and a general down-regulation at 24hpi. Such evidence suggest a certain degree of similarity between the response of sea bream and that of other fish species to NNV, while the observed down-regulation of IFN- and viral-stimulated pathways argues for a possible interference of NNV against the host response.

The effects of glyphosate and AMPA on the mediterranean mussel Mytilus galloprovincialis and its microbiota.

Glyphosate, the most widely used herbicide worldwide, targets the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme in the shikimate pathway found in plants and some microorganisms. While the potential for glyphosate to induce a broad range of biological effects in exposed organisms has been demonstrated, the global molecular mechanisms of toxicity and potential effects in bacterial symbionts remain unclear, in particular for ecologically important marine species such as bivalve molluscs. Here, the effects of glyphosate (GLY), its degradation product aminomethylphosphonic acid (AMPA), and a mixture of both (MIX) on the mussel M. galloprovincialis were assessed in a controlled experiment. For the first time, next generation sequencing (RNA-seq and 16S rRNA amplicon sequencing) was used to evaluate such effects at the molecular level in both the host and its respective microbiota. The results suggest that the variable capacity of bacterial species to proliferate in the presence of these compounds and the impairment of host physiological homeostasis due to AMPA and GLY toxicity may cause significant perturbations to the digestive gland microbiota, as well as elicit the spread of potential opportunistic pathogens such as Vibrio spp.. The consequent host-immune system activation identified at the molecular and cellular level could be aimed at controlling changes occurring in the composition of symbiotic microbial communities. Overall, our data raise further concerns about the potential adverse effects of glyphosate and AMPA in marine species, suggesting that both the effects of direct toxicity and the ensuing changes occurring in the host-microbial community must be taken into consideration to determine the overall ecotoxicological hazard of these compounds.

Habitat segregation and cryptic adaptation of species of Periophthalmus (Gobioidei: Gobiidae).

A study was conducted on the habitat distribution of four sympatric species of Periophthalmus (the silver-lined mudskipper Periophthalmus argentilineatus, the slender mudskipper Periophthalmus gracilis, the kalolo mudskipper Periophthalmus kalolo and the Malacca mudskipper Periophthalmus malaccensis) from northern Sulawesi. Molecular phylogenetic reconstructions based on one mtDNA marker (16S) were used to validate the morphological taxa, identifying five molecular clades. Periophthalmus argentilineatus includes two molecular species, which are named Periophthalmus argentilineatus clades F and K. Multivariate direct gradient analysis show that these species form three distinct ecological guilds, with the two molecular species occurring in different guilds. Periophthalmus clade F is ecologically eurytypic; Periophthalmus clade K and P. kalolo are prevalent in ecosystems isolated by strong oceanic currents and at shorter distances from the sea; P. gracilis plus P. malaccensis are prevalent in ecosystems connected by shallow coastal waters, in vegetated habitats at larger distances from the sea. This indicates for the first time that mudskipper species exhibit a range of adaptations to semiterrestrialism not only within genera, but even within morphospecies, delineating a much more complex adaptive scenario than previously assumed.

Phylogeography and demographic history of two widespread Indo-Pacific mudskippers (Gobiidae: Periophthalmus).

This study provides a first description of the phylogeographic patterns and evolutionary history of two species of the mudskipper genus Periophthalmus. These amphibious gobies are distributed throughout the whole Indo-Pacific region and Atlantic coast of Africa, in peritidal habitats of soft-bottom coastal ecosystems. Three sequence datasets of two widely distributed species, Periophthalmus argentilineatus and P. kalolo, were obtained by amplifying and sequencing two mtDNA markers (D-loop and 16S rDNA) and the nDNA rag1 region. The three datasets were then used to perform phylogeographic, demographic and population genetic analyses. Our results indicate that tectonic events and past climatic oscillations strongly contributed to shape present genetic differentiation, phylogeographic and demographic patterns. We found support for the monophyly of P. kalolo, and only shallow genetic differentiation between East-African and Indo-Malayan populations of this species. However, our collections of the morphospecies P. argentilineatus include three molecularly distinct lineages, one of them more closely related to P. kalolo. The presence of Miocenic timings for the most recent common ancestors of some of these morphologically similar clades, suggests the presence of strong stabilising selection in mudskippers' habitats. At population level, demographic analyses and palaeoecological records of mangrove ecosystems suggest that Pleistocene bottlenecks and expansion plus secondary contact events of the studied species were associated with recurrent sea transgressions during interglacials, and sea regressions or stable regimes during glacials, respectively.