Arctic marine phytobenthos of northern Baffin Island

Peer reviewed

Arctic marine phytobenthos of northern Baffin Island

Funded by UK Natural Environment Research Council European Commission. Grant Number: 227799 TOTAL Foundation MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland) Scottish Funding Council. Grant Number: HR09011

Applicazione di un veloce metodo di estrazione del DNA per l’identificazione di prodotti ittici marini.

L'identificazione dei prodotti ittici è uno dei temi chiave in materia di sicurezza alimentare. L’errata etichettatura dei prodotti alimentari e la sostituzione di alcuni ingredienti rappresentano questioni emergenti in termini di qualità e sicurezza alimentare e nutrizionale. L'autenticazione e la tracciabilità dei prodotti alimentari, gli studi di tassonomia e di genetica di popolazione, così come l'analisi delle abitudini alimentari degli animali e la selezione delle prede, si basano su analisi genetiche tra cui la metodica molecolare del DNA barcoding, che consiste nell’amplificazione e nel sequenziamento di una specifica regione del gene mitocondriale chiamata COI. Questa tecnica biomolecolare è utilizzata per fronteggiare la richiesta di determinazione specifica e/o la reale provenienza dei prodotti commercializzati, nonché per smascherare errori di etichettatura e sostituzioni fraudolente, difficile da rilevare soprattutto nei prodotti ittici trasformati. Sul mercato sono disponibili differenti kit per l'estrazione del DNA da campioni freschi e conservati; l’impiego dei kit, aumenta drasticamente il costo dei progetti di caratterizzazione e di genotipizzazione dei campioni da analizzare. In questo scenario è stato messo a punto un metodo veloce di estrazione del DNA. Esso non prevede nessuna fase di purificazione per i prodotti ittici freschi e trasformati e si presta a qualsiasi analisi che preveda l’utilizzo della tecnica PCR. Il protocollo consente l'amplificazione efficiente del DNA da qualsiasi scarto industriale proveniente dalla lavorazione del pesce, indipendentemente dal metodo di conservazione del campione. L’applicazione di questo metodo di estrazione del DNA, combinato al successo e alla robustezza della amplificazione PCR (secondo protocollo barcode) ha permesso di ottenere, in tempi brevissimi e con costi minimi, il sequenziamento del DNA.

Fungi associated with foliar diseases of wild and cultivated rice (Oryza spp.) in northern Queensland

During surveys of wild and cultivated rice in northern Queensland in 2014 and 2015, 92 fungal isolates were obtained from plants that were afflicted by foliar diseases, including the rice blast pathogen, Pyricularia oryzae, and the brown spot pathogen, Bipolaris oryzae. Seven species of Curvularia were found, viz. Curvularia aeria, C. alcornii, C. asianensis, C. clavata, C. lunata, C. muehlenbeckiae and an undescribed species. To remove uncertainty about the identity of the host plants from which the fungi were isolated, a DNA barcoding strategy was developed using regions of the chloroplast genome. Pathogenicity tests using wild rice isolates of P. oryzae indicated that many local rice varieties are susceptible to infection.

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.

First record of the coloured righteye flounder, Poecilopsetta colorata (Teleostei: Poecilopsettidae) from the Sakalaves seamounts in the Mozambique Channel

Background: The coloured righteye flounder, Poecilopsetta colorata Günther, 1880 was previously known from the eastern Indian Ocean to the South China Sea and Indonesia. Here, a new record from the western Indian Ocean is reported. Results: The new record is based on a specimen collected on the Sakalaves seamounts at 375 m in depth in the Mozambique Channel during a recent oceanographic survey. Four other teleost fish species including an uncommon ophidiid species, Neobythites somaliaensis Nielsen, 1995 were also collected on the same seamounts. Conclusions: The presence of P. colorata in the Mozambique Channel suggests a broad and Indo-West Pacific wide distribution for this relatively rare deep-sea species. The sequence of the cytochrome oxidase subunit-I for the collected specimen is provided as a genetic reference for further DNA barcoding and systematic studies.

ANALYSIS OF MACROINVERTEBRATE COMMUNITIES IN SEASONAL WETLANDS THROUGH TIME, ACROSS SPACE, AND USING SPECIES TRAITS

Restoration of natural wetlands may be informed by macroinvertebrate community composition. Macroinvertebrate communities of wetlands are influenced by environmental characteristics such as vegetation, soil, hydrology, land use, and isolation. This dissertation explores multiple approaches to the assessment of wetland macroinvertebrate community composition, and demonstrates how these approaches can provide complementary insights into the community ecology of aquatic macroinvertebrates. Specifically, this work focuses on macroinvertebrates of Delmarva Bays, isolated seasonal wetlands found on Maryland’s eastern shore. A comparison of macroinvertebrate community change over a nine years in a restored wetland complex indicated that the macroinvertebrate community of a rehabilitated wetlands more rapidly approximated the community of a reference site than did a newly created wetland. The recovery of a natural macroinvertebrate community in the rehabilitated wetland indicated that wetland rehabilitation should be prioritized over wetland creation and long-term monitoring may be needed to evaluate restoration success. This study also indicated that characteristics of wetland vegetation reflected community composition. The connection between wetland vegetation and macroinvertebrate community composition led to a regional assessment of predaceous diving beetle (Coleoptera: Dytiscidae) community composition in 20 seasonal wetlands, half with and half without sphagnum moss (Sphagnum spp.). Species-level identifications indicated that wetlands with sphagnum support unique and diverse assemblages of beetles. These patterns suggest that sphagnum wetlands provide habitat that supports biodiversity on the Delmarva Peninsula. To compare traits of co-occurring beetles, mandible morphology and temporal and spatial variation were measured between three species of predaceous diving beetles. Based on mandible architecture, all species may consume similarly sized prey, but prey characteristics likely differ in terms of piercing force required for successful capture and consumption. Therefore, different assemblages of aquatic beetles may have different effects on macroinvertebrate community structure. Integrating community-level and species-level data strengthens the association between individual organisms and their ecological role. Effective restoration of imperiled wetlands benefits from this integration, as it informs the management practices that both preserve biodiversity and promote ecosystem services.

Development of a novel molecular tool for the rapid assessment of the biodiversity changes of benthic nematodes assemblages. Mares Conference Marine Ecosystems Helath and conservation

The molecular profiling system was developed using directed terminal-restriction fragment length polymorphism (dT-RFLP) to characterize soil nematode assemblages by relative abundance of feeding guilds and validation by comparison to traditional morphological method. The good performance of these molecular tools applied to soil nematodes assemblages create an opportunity to develop a novel approach for rapid assessment of the biodiversity changes of benthic nematodes assemblages of marine and estuarine sediments. The main aim of this research is to combine morphological and molecular analysis of estuarine nematodes assemblages, to establish a tool for fast assessment of the biodiversity changes within habitat recovery of Zostera noltii seagrass beds; and validate the dT-RFLP as a high-throughput tool to assess the system recovery. It was also proposed to develop a database of sequences related to individuals identified at species level to develop a new taxonomic reference system. A molecular phylogenetic analysis of the estuarine nematodes has being performed. After morphological identification, barcoding of 18S rDNA are being determined for each nematode species and the results have shown a good degree of concordance between traditional morphology-based identification and DNA sequences. The digest strategy developed for soil nematodes is not suitable for marine nematodes. Then five samples were cloned and sequenced and the sequence data was used to design a new dT-RFLP strategy to adapt this tool to marine assemblages. Several solutions were presented by DRAT and tested empirically to select the solution that cuts most efficiently, separating the different clusters. The results of quantitative PCR showed differences in nematode density between two sampling stations according the abundance of the nematode density obtained by the traditional methods. These results suggest that qPCR could be a robust tool for enumeration of nematode abundance, saving time.

Fixing, storage time and DNA extraction applied to dT-RFLP for ecological and molecular studies of marine nematodes assemblages

The application of molecular methods offers an alternative faster than traditional methods based on morphology It is nearly impossible to process all the samples in short period using traditional methods, and the deterioration of marine sediments rapidly occurs The dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows a rapid assessment of biodiversity changes of nematodes assemblages The use of a not suitable fixing, storage time and DNA extraction could be a limitation in molecular analysis like dT-RFLP and real time PCR.Objetives: the best fixative •the level of DNA degradation over the time •the best DNA extraction method for marine nematodes and suitable for dT-RFLP analysis

Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.