Seasonal variability in microbial methanol utilisation in coastal waters of the western English Channel

Methanol is ubiquitous in seawater and the most abundant oxygenated volatile organic compound (OVOC) in the atmosphere where it influences oxidising capacity and ozone formation. Marine methylotrophic bacteria utilise methanol in seawater both as an energy and/or growth substrate. This work represents the first fully resolved seasonal study of marine microbial methanol uptake dynamics. Rates of microbial methanol dissimilation in coastal surface waters of the UK varied between 0.7 – 11.2 nmol l-1 h-1 and reached a maximum in February. Rates of microbial methanol assimilation varied between 0.04 – 2.64 x 10-2 nmol l-1 h-1 and reached a maximum in August. Temporal variability in microbial methanol uptake rates shows that methanol assimilation and dissimilation display opposing seasonal cycles, although overall <1% of methanol was assimilated. Correlative approaches with 16S rRNA pyrosequencing data suggested that bacteria of the SAR11 clade and Rhodobacterales could be significantly influencing rates of methanol dissimilation and assimilation, respectively, at station L4 in the western English Channel

Seasonal variability in microbial methanol utilisation in coastal waters of the western English Channel

Methanol is ubiquitous in seawater and the most abundant oxygenated volatile organic compound (OVOC) in the atmosphere where it influences oxidising capacity and ozone formation. Marine methylotrophic bacteria utilise methanol in seawater both as an energy and/or growth substrate. This work represents the first fully resolved seasonal study of marine microbial methanol uptake dynamics. Rates of microbial methanol dissimilation in coastal surface waters of the UK varied between 0.7 – 11.2 nmol l-1 h-1 and reached a maximum in February. Rates of microbial methanol assimilation varied between 0.04 – 2.64 x 10-2 nmol l-1 h-1 and reached a maximum in August. Temporal variability in microbial methanol uptake rates shows that methanol assimilation and dissimilation display opposing seasonal cycles, although overall <1% of methanol was assimilated. Correlative approaches with 16S rRNA pyrosequencing data suggested that bacteria of the SAR11 clade and Rhodobacterales could be significantly influencing rates of methanol dissimilation and assimilation, respectively, at station L4 in the western English Channel

Biostratigraphic Evidence Relating to the Age-Old Question of Hannibal's Invasion of Italy, II: Chemical Biomarkers and Microbial Signatures

As discussed in Part I, a large accumulation of mammalian faeces at the mire site in the upper Guil Valley near Mt. Viso, dated to 2168cal ¹⁴C yr., provides the first evidence of the passage of substantial but indeterminate numbers of mammals within the time frame of the Punic invasion of Italia. Specialized organic biomarkers bound up in a highly convoluted and bioturbated bed constitute an unusual anomaly in a histosol comprised of fibric and hemist horizons that are usually expected to display horizontal bedding. The presence of deoxycholic acid and ethylcoprostanol derived from faecal matter, coupled with high relative numbers of Clostridia 16S rRNA genes, suggests a substantial accumulation of mammalian faeces at the site over 2000years ago. The results reported here constitute the first chemical and biological evidence of the passage of large numbers of mammals, possibly indicating the route of the Hannibalic army at this time. Combined with the geological analysis reported in Part I, these data provide a background supporting the need for further historical archaeological exploration in this area.

Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: A duplex approach with DNA binding dye chemistry

Schistosomiasis is a chronically debilitating helminth infection with a significant socio-economic and public health impact. Accurate diagnostics play a pivotal role in achieving current schistosomiasis control and elimination goals. However, many of the current diagnostic procedures, which rely on detection of schistosome eggs, have major limitations including lack of accuracy and the inability to detect pre-patent infections. DNA-based detection methods provide a viable alternative to the current tests commonly used for schistosomiasis diagnosis. Here we describe the optimisation of a novel droplet digital PCR (ddPCR) duplex assay for the diagnosis of Schistosoma japonicum infection which provides improved detection sensitivity and specificity. The assay involves the amplification of two specific and abundant target gene sequences in S. japonicum; a retrotransposon (SjR2) and a portion of a mitochondrial gene (nad1). The assay detected target sequences in different sources of schistosome DNA isolated from adult worms, schistosomules and eggs, and exhibits a high level of specificity, thereby representing an ideal tool for the detection of low levels of parasite DNA in different clinical samples including parasite cell free DNA in the host circulation and other bodily fluids. Moreover, being quantitative, the assay can be used to determine parasite infection intensity and, could provide an important tool for the detection of low intensity infections in low prevalence schistosomiasis-endemic areas.

Taxonomic and Functional Diversity of Soil and Hypolithic Microbial Communities in Miers Valley, McMurdo Dry Valleys, Antarctica

The McMurdo Dry Valleys of Antarctica are an extreme polar desert. Mineral soils support subsurface microbial communities and translucent rocks support development of hypolithic communities on ventral surfaces in soil contact. Despite significant research attention, relatively little is known about taxonomic and functional diversity or their inter-relationships. Here we report a combined diversity and functional interrogation for soil and hypoliths of the Miers Valley in the McMurdo Dry Valleys of Antarctica. The study employed 16S rRNA fingerprinting and high throughput sequencing combined with the GeoChip functional microarray. The soil community was revealed as a highly diverse reservoir of bacterial diversity dominated by actinobacteria. Hypolithic communities were less diverse and dominated by cyanobacteria. Major differences in putative functionality were that soil communities displayed greater diversity in stress tolerance and recalcitrant substrate utilization pathways, whilst hypolithic communities supported greater diversity of nutrient limitation adaptation pathways. A relatively high level of functional redundancy in both soil and hypoliths may indicate adaptation of these communities to fluctuating environmental conditions.

Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189 +/- 58 operational taxonomic units (OTUs) but dropped to 27 +/- 12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.

Development of a simple and fast “DNA extraction kit” for sea food identification and marine species

Seafood products fraud, the misrepresentation of them, have been discovered all around the world in different forms as false labeling, species substitution, short-weighting or over glazing in order to hide the correct identity, origin or weight of the seafood products. Due to the value of seafood products such as canned tuna, swordfish or grouper, these species are the subject of the commercial fraud is mainly there placement of valuable species with other little or no value species. A similar situation occurs with the shelled shrimp or shellfish that are reduced into pieces for the commercialization. Food fraud by species substitution is an emerging risk given the increasingly global food supply chain and the potential food safety issues. Economic food fraud is committed when food is deliberately placed on the market, for financial gain deceiving consumers (Woolfe, M. & Primrose, S. 2004). As a result of the increased demand and the globalization of the seafood supply, more fish species are encountered in the market. In this scenary, it becomes essential to unequivocally identify the species. The traditional taxonomy, based primarily on identification keys of species, has shown a number of limitations in the use of the distinctive features in many animal taxa, amplified when fish, crustacean or shellfish are commercially transformed. Many fish species show a similar texture, thus the certification of fish products is particularly important when fishes have undergone procedures which affect the overall anatomical structure, such as heading, slicing or filleting (Marko et al., 2004). The absence of morphological traits, a main characteristic usually used to identify animal species, represents a challenge and molecular identification methods are required. Among them, DNA-based methods are more frequently employed for food authentication (Lockley & Bardsley, 2000). In addition to food authentication and traceability, studies of taxonomy, population and conservation genetics as well as analysis of dietary habits and prey selection, also rely on genetic analyses including the DNA barcoding technology (Arroyave & Stiassny, 2014; Galimberti et al., 2013; Mafra, Ferreira, & Oliveira, 2008; Nicolé et al., 2012; Rasmussen & Morrissey, 2008), consisting in PCR amplification and sequencing of a COI mitochondrial gene specific region. The system proposed by P. Hebert et al. (2003) locates inside the mitochondrial COI gene (cytochrome oxidase subunit I) the bioidentification system useful in taxonomic identification of species (Lo Brutto et al., 2007). The COI region, used for genetic identification - DNA barcode - is short enough to allow, with the current technology, to decode sequence (the pairs of nucleotide bases) in a single step. Despite, this region only represents a tiny fraction of the mitochondrial DNA content in each cell, the COI region has sufficient variability to distinguish the majority of species among them (Biondo et al. 2016). This technique has been already employed to address the demand of assessing the actual identity and/or provenance of marketed products, as well as to unmask mislabelling and fraudulent substitutions, difficult to detect especially in manufactured seafood (Barbuto et al., 2010; Galimberti et al., 2013; Filonzi, Chiesa, Vaghi, & Nonnis Marzano, 2010). Nowadays,the research concerns the use of genetic markers to identify not only the species and/or varieties of fish, but also to identify molecular characters able to trace the origin and to provide an effective control tool forproducers and consumers as a supply chain in agreementwith local regulations.

A new species of gall midge (Diptera: Cecidomyiidae) feeding on ornamental Cordyline fruticosa(Asparagaceae) in Australia

Larvae of an undescribed gall midge were found feeding on leaves and stems within leaf sheaths and between leaf blades of potted plants of Cordyline fruticosa (Asparagaceae) in a production nursery in Queensland. The following varieties of the host plant were infested: Apple Blossom', Glauca', Kilauea', Negra', Pink Diamond, 'Purple Prince' and Willy's Gold'. The new species, Dasineura cordylineaeKolesik sp. nov., is described and its cytochrome oxidase unit I mitochondrial gene segment is sequenced. The new species is the first known gall midge feeding on a plant species of the genus Cordyline. Orange larvae induce oval shallow swellings on the leaf and stem tissue, which becomes necrotised during the later stage of larval feeding. Necrotic areas remain visible to the end of leaves' lives and decrease the market value of the plants. In the production nursery investigated, the lesions caused by the gall midge provided an entry for a fungal infection by Fusarium sp. inflicting further injury to plants. Larvae of the new species were preyed on by larvae of Gaurax sp. (Diptera: Chloropidae). This is the first worldwide record of Chloropidae preying on Cecidomyiidae.

Biogeographical distribution of Rimicaris exoculata resident gut epibiont communities along the Mid-Atlantic Ridge hydrothermal vent sites

Rimicaris exoculata is a deep-sea hydrothermal vent shrimp which enlarged gill chamber houses a complex trophic epibiotic community. Its gut harbours an autochthonous and distinct microbial community. This species dominates hydrothermal ecosystems megafauna along the Mid-Atlantic Ridge, regardless of contrasted geochemical conditions prevailing in them. Here, the resident gut epibiont community at four contrasted hydrothermal vent sites (Rainbow/TAG/Logatchev/Ashadze) was analysed and compiled with previous data to evaluate the possible influence of site location, using 16S rRNA surveys and microscopic observations (TEM, SEM and FISH analyses). Filamentous epibionts inserted between the epithelial cells microvilli were observed on all examined samples. Results confirmed resident gut community affiliation to Deferribacteres, Mollicutes, Epsilonproteobacteria and to a lesser extent Gammaproteobacteria lineages. Still a single Deferribacteres phylotype was retrieved at all sites. Four Mollicutes-related OTUs were distinguished, one being only identified on Rainbow specimens. The topology of ribotypes median-joining networks illustrated a community diversification possibly following demographic expansions, suggesting a more ancient evolutionary history and/or a larger effective population size at Rainbow. Finally, the gill chamber community distribution was also analysed through ribotypes networks based on sequences from R. exoculata collected at Rainbow/Snake Pit/TAG/Logatchev/Ashadze sites. Results allow refining hypotheses on the epibiont role and transmission pathways.

Indole acetic acid and ACC deaminase fromendophytic bacteria improves the growth of Solanum lycopersicum

Background: Endophytic bacteria are ubiquitous in all plant species contributing in host plant\'s nutrient uptake and helping the host to improve its growth. Moringa peregrina which is a medicinal plant, growing in arid region of Arabia, was assessed for the presence of endophytic bacterial strains. Results: PCR amplification and sequencing of 16S rRNA of bacterial endophytes revealed the 5 endophytic bacteria, in which 2 strains were from Sphingomonas sp.; 2 strains from Bacillus sp. and 1 from Methylobacterium genus. Among the endophytic bacterial strains, a strain of Bacillus subtilis LK14 has shown significant prospects in phosphate solubilization (clearing zone of 56.71 mm after 5 d), ACC deaminase (448.3 ± 2.91 nM α-ketobutyrate mg-1 h-1) and acid phosphatase activity (8.4 ± 1.2 nM mg-1 min-1). The endophytic bacteria were also assessed for their potential to produce indole-3-acetic acid (IAA). Among isolated strains, the initial spectrophotometry analysis showed significantly higher IAA production by Bacillus subtilis LK14. The diurnal production of IAA was quantified using multiple reactions monitoring method in UPLC/MS–MS. The analysis showed that LK14 produced the highest (8.7 μM) IAA on 14th d of growth. Looking at LK14 potentials, it was applied to Solanum lycopersicum , where it significantly increased the shoot and root biomass and chlorophyll (a and b) contents as compared to control plants. Conclusion: The study concludes that using endophytic bacterial strains can be bio-prospective for plant growth promotion, which might be an ideal strategy for improving growth of crops in marginal lands.

Molecular identification and localization of filamentous symbiotic bacteria associated with the hydrothermal vent annelid Alvinella pompejana

Alvinella pompejana is a polychaetous annelid that inhabits high temperature environments associated with active deep-sea hydrothermal vents along the East Pacific Rise. A unique and diverse epibiotic microflora with a prominent filamentous morphotype is found associated with the worm's dorsal integument. A previous study established the taxonomic positions of two epsilon proteobacterial phylotypes, 13B and 5A, which dominated a clone library of 16S rRNA genes amplified by PCR from the epibiotic microbial community of an A. pompejana specimen. In the present study deoxyoligonucleotide PCR primers specific for phylotypes 13B and 5A were used to demonstrate that these phylotypes are regular features of the bacterial community associated with A. pompejana. Assaying of other surfaces around colonies of A. pompejana revealed that phylotypes 13B and 5A are not restricted to A. pompejana. Phylotype 13B occurs on the exterior surfaces of other invertebrate genera and rock surfaces, and phylotype 5A occurs on a congener, Alvinella caudata. The 13B and 5A phylotypes were identified and localized on A. pompejana by in situ hybridization, demonstrating that these two phylotypes are, in fact, the prominent filamentous bacteria on the dorsal integument of A. pompejana. These findings indicate that the filamentous bacterial symbionts of A. pompejana are epsilon Proteobacteria which do not have an obligate requirement for A. pompejana.

Chronic Polyaromatic Hydrocarbon (PAH) Contamination Is a Marginal Driver for Community Diversity and Prokaryotic Predicted Functioning in Coastal Sediments

Benthic microorganisms are key players in the recycling of organic matter and recalcitrant compounds such as polyaromatic hydrocarbons (PAHs) in coastal sediments. Despite their ecological importance, the response of microbial communities to chronic PAH pollution, one of the major threats to coastal ecosystems, has received very little attention. In one of the largest surveys performed so far on coastal sediments, the diversity and composition of microbial communities inhabiting both chronically contaminated and non-contaminated coastal sediments were investigated using high-throughput sequencing on the 18S and 16S rRNA genes. Prokaryotic alpha-diversity showed significant association with salinity, temperature, and organic carbon content. The effect of particle size distribution was strong on eukaryotic diversity. Similarly to alpha-diversity, beta-diversity patterns were strongly influenced by the environmental filter, while PAHs had no influence on the prokaryotic community structure and a weak impact on the eukaryotic community structure at the continental scale. However, at the regional scale, PAHs became the main driver shaping the structure of bacterial and eukaryotic communities. These patterns were not found for PICRUSt predicted prokaryotic functions, thus indicating some degree of functional redundancy. Eukaryotes presented a greater potential for their use as PAH contamination biomarkers, owing to their stronger response at both regional and continental scales.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem- resistant Pseudomonas aeruginosa isolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosa isolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXYOprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Response of Core Microbial Consortia to Chronic Hydrocarbon Contaminations in Coastal Sediment Habitats

Traditionally, microbial surveys investigating the effect of chronic anthropogenic pressure such as polyaromatic hydrocarbons (PAHs) contaminations consider just the alpha and beta diversity and ignore the interactions among the different taxa forming the microbial community. Here, we investigated the ecological relationships between the three domains of life (i.e., Bacteria, Archaea, and Eukarya) using 454 pyrosequencing on the 16S rRNA and 18S rRNA genes from chronically impacted and pristine sediments, along the coasts of the Mediterranean Sea (Gulf of Lion, Vermillion coast, Corsica, Bizerte lagoon and Lebanon) and the French Atlantic Ocean (Bay of Biscay and English Channel). Our approach provided a robust ecological framework for the partition of the taxa abundance distribution into 859 core Operational taxonomic units (OTUs) and 6629 satellite OTUs. OTUs forming the core microbial community showed the highest sensitivity to changes in environmental and contaminant variations, with salinity, latitude, temperature, particle size distribution, total organic carbon (TOC) and PAH concentrations as main drivers of community assembly. The core communities were dominated by Gammaproteobacteria and Deltaproteobacteria for Bacteria, by Thaumarchaeota, Bathyarchaeota and Thermoplasmata for Archaea and Metazoa and Dinoflagellata for Eukarya. In order to find associations among microorganisms, we generated a co-occurrence network in which PAHs were found to impact significantly the potential predator – prey relationship in one microbial consortium composed of ciliates and Actinobacteria. Comparison of network topological properties between contaminated and non-contaminated samples showed substantial differences in the network structure and indicated a higher vulnerability to environmental perturbations in the contaminated sediments.

In two waters: contemporary evolution of lagoonal and marine white seabream (Diplodus sargus) populations

Brackish water ecosystems are often exposed to wide variations in environmental variables, including temperature and salinity, which may cause strong selective pressures on organisms modifying the genetic patterns of species. The aim of this work was to test whether there is a ‘divergence-with-gene flow’ in coastal lagoon populations of white seabream (Diplodus sargus) (Ria Formosa, S Portugal and Mar Menor, SE Spain) respect to four marine populations, by using partial sequences of cyt b mitochondrial gene and information from nine microsatellite loci. Genetic diversity was highest in both coastal lagoons (Mar Menor and Ria Formosa) considering mitochondrial and nuclear markers. Although some of FST population pairwise comparisons were not significant, analyses of molecular variance (AMOVAs) detected differences between groups (coastal lagoon and marine) close to significance. Also, only two haplotypes (Cytb-17 and Cytb-18) were detected in both coastal lagoon sampling sites and these localities (Mar Menor and Ria Formosa) showed the highest number of singletons, some of them with a high number of mutations, as has been already described for other Mar Menor populations (Pomatochistus marmoratus and Holothuria polii). Also, several tests detected significant positive and balancing selection considering mtDNA and microsatellite data. These data support the hypothesis of selection as one of the drivers of the genetic differences found between coastal lagoon and marine populations. The life strategy adopted by Diplodus sargus in coastal lagoons allows it to decrease its mortality rate and improve the heritability of its genes. Also, the increase time spent in coastal lagoons with different temperatures and salinities favours the fitness selection and the maintenance of exclusive haplotypes and genotypes in coastal lagoon inhabitants favouring the ‘divergence-with-gene-flow’.