Browsing by Author "Keller, Alexander"
Now showing 1 - 8 of 8
Results Per Page
Sort Options
- Honey bee collected pollen for botanical identification via its2 metabarcoding: a comparison of preservation methods for citizen sciencePublication . Quaresma, Andreia; Brodschneider, Robert; Gratzer, Kristina; Gray, Alison; Keller, Alexander; Kilpinen, Ole; Rufino, José; Steen, Jozef van der; Vejsnaes, Flemming; Pinto, M. AliceWhile classical palynology has been the method of choice to assess botanical diversity of bee-collected pollen for multiple purposes, DNA metabarcoding is emerging as a powerful alternative being able to achieve high taxonomic identification accuracy. Moreover,DNA metabarcoding allows analysis of hundreds of samples in a single high-throughput sequencing run, therefore offering unprecedented scale in citizen science projects. Biases in metabarcoding can be introduced at any stage of sample processing and preservation is at the forefront of the pipeline. Hence, it is important to test how sample preservation influences quality and quantitative performance of pollen metabarcoding. While inmetabarcoding studies pollen has typically been preserved at −20°C (FRZ), this is not the best method to be applied by citizen scientists.
- Honey bee collected pollen for botanical identification via its2 metabarcoding: a comparison of preservation methods for citizen sciencePublication . Quaresma, Andreia; Brodschneider, Robert; Gratzer, Kristina; Gray, Alison; Keller, Alexander; Kilpinen, Ole; Rufino, José; Steen, Jozef van der; Vejsnaes, Flemming; Pinto, M. AliceDNA metabarcoding is emerging as a powerful method for botanical identification of bee-collected pollen, allowing analysis of hundreds of samples in a single high-throughput sequencing run, therefore offering unprecedented scale in citizen science projects. Biases in metabarcoding can be introduced at any stage of sample processing and preservation is the first step of the pipeline. Hence, it is important to test whether the pollen preservation method influences metabarcoding performance. While in metabarcoding studies pollen has typically been preserved at −20°C, this is not the best method to be applied by citizen scientists. Here, we compared the freezing method (FRZ) with ethanol (EtOH), silica gel (SG) and room temperature (RT) in 87 pollen samples collected from hives in Austria and Denmark.
- Its2 metabarcoding: a promising approach for identification of botanical origin of bee-collected pollenPublication . Quaresma, Andreia; Van der Steen, Joseph; Amaral, Joana S.; Biron, David G.; Brodschneider, Robert; Brusbardis, Valters; Carreck, Norman L.; Mary, Frances Coffey; Formato, Giovanni; Graaf, Dirk C.; Gratzer, Kristina; Hatjina, Fani; Kilpinem, Ole; Keller, Alexander; Laget, Dries; Pietropaoli, Marco; Rufino, José; Vejsnaes, Flemming; Pinto, M. AliceBee products have long been used in human’s diet and their consumption has increasingly been recognized has beneficial for human’s health. One such product is pollen, which is a particularly interesting food as it contains bioactive compounds and all the essential amino-acids needed by humans. However, the composition of bee-collected pollen depends on the environment where the visited plants grow (e.g.: climatic conditions, soil type) and, above all, on the plant species [1]. Therefore, identification of the botanical origin of bee-collected pollen is important for a fuller characterization of this food product. Until recently, pollen identification has been carried out using light microscopy, a costly approach that often provides low taxonomic resolution. However, with high-throughput sequencing (HTS) becoming increasingly affordable, DNA metabarcoding is emerging as a promising alternative to light microscopy. In addition to be time- and cost-effective for large sample sizes, metabarcoding has the potential to allow identification of pollen mixtures at the species level. However, before it can be widely employed in pollen analysis, the reliability of this molecular tool must be appraised. Herein, we compared the relative abundances obtained by the two approaches on 108 bee-collected pollen samples from 10 European countries. To that end, the 108 samples were first homogenized and split into two identical sub-sets. One sub-set was analysed by palynology experts from the “Institut für Bienenkunde”, Germany, and the other one was subjected to HTS, using ITS2 as the barcode, in the labs of CIMO and CIBIO. Pairwise comparisons of the relative abundances at the family level of the 108 samples show no significant differences (P ≥ 0.1057, Wilcoxon signed-rank test) and high correlation values (0.2736 ≤ r ≤ 0.9842, Pearson’s correlation) between the two approaches. The highest correlation values were observed for Italian samples (0.7245 ≤ r ≤ 0.9842; global r = 0.8958) and the lowest for Greek samples (0.0266 ≤ r ≤ 0.9703; global r = 0.4929). Despite, the few outliers, which can be improved by further optimization of the protocols, these results suggest that ITS2 metabarcoding promises to be a reliable alternative to light microscopy. This molecular approach is now being employed in the European project INSIGNIA (https://www.insignia-bee.eu/), which is developing a standard protocol for using the honey bee as a tool for environmental monitoring.
- Pollen identification by its2 metabarcoding: curation of the sequences retrieved from genbank to build a reference databasePublication . Quaresma, Andreia; Keller, Alexander; Rufino, José; Steen, Jozef van der; Pinto, M. AliceA powerful way of studying the quality of the environment is by examining the pollen collected by honey bees (Apis mellifera) as it contains information on available plant sources, spatial and temporal floral diversity, as well as on chemical contaminants. This entails botanical identification of pollen which has typically been addressed by classical palynology, a costly approach that often provides low taxonomic resolution, is time-consuming, labour intensive, and requires plant taxonomy expertise. However, with high-throughput sequencing becoming increasingly affordable, pollen metabarcoding is gaining momentum, and it is a promising alternative to classical palynology. But one of the main drawbacks of pollen metabarcoding is the lack of good quality reference databases for the barcode of choice. BCdatabaser (Keller et al. 2020) was developed to automatically generate a standardized database for the ITS2 barcode from the primary sequence database GenBank. While using BCdatabaser to construct an ITS2 reference database for identification of bee-collected pollen, we noticed several misidentified sequences retrieved from GenBank, which would impact identification accuracy. There were two types of problems: plant sequences that were assigned to the wrong plant species and fungi sequences that were identified as plants. To overcome these issues, we developed scripts in bash and R to curate an ITS2 reference database for pollen identification purposes. These scripts allowed us to identify the Fungi sequences retrieved from GenBank for subsequent removal from the database, to perform a pairwise alignment of all the sequences using vsearch v2.14.1 (Rognes et al., 2016) and, then to remove all the sequences with low identity percentage using an iteration process in R v4.1.2. The database curation is automated therefore enabling easy update of the ITS2 database to take advantage of the new sequences that are regularly deposited in GenBank.
- Pollen identification by its2 metabarcoding: curation of the sequences retrieved from genbank to build a reference databasePublication . Quaresma, Andreia; Keller, Alexander; Rufino, José; Steen, Jozef van der; Pinto, M. AliceA powerful way of studying the quality of the environment is by examining the pollen collected by honey bees (Apis mellifera) as it contains information on available plant sources, spatial and temporal floral diversity, as well as on chemical contaminants. This entails botanical identification of pollen which has typically been addressed by classical palynology, a costly approach that often provides low taxonomic resolution, is time-consuming, labour intensive, and requires plant taxonomy expertise
- Preservation methods of honey bee-collected pollen are not a source of bias in ITS2 metabarcodingPublication . Quaresma, Andreia; Brodschneider, Robert; Gratzer, Kristina; Gray, Alison; Keller, Alexander; Kilpinen, Ole; Rufino, José; Steen, Jozef van der; Vejsnæs, Flemming; Pinto, M. AlicePollen metabarcoding is emerging as a powerful tool for ecological research and offers unprecedented scale in citizen science projects for environmental monitoring via honey bees. Biases in metabarcoding can be introduced at any stage of sample processing and preservation is at the forefront of the pipeline. While in metabarcoding studies pollen has been preserved at − 20 °C (FRZ), this is not the best method for citizen scientists. Herein, we compared this method with ethanol (EtOH), silica gel (SG) and room temperature (RT) for preservation of pollen collected from hives in Austria and Denmark. After ~ 4 months of storage, DNAs were extracted with a food kit, and their quality and concentration measured. Most DNA extracts exhibited 260/280 absorbance ratios close to the optimal 1.8, with RT samples from Austria performing slightly worse than FRZ and SG samples (P < 0.027). Statistical differences were also detected for DNA concentration, with EtOH samples producing lower yields than RT and FRZ samples in both countries and SG in Austria (P < 0.042). Yet, qualitative and quantitative assessments of floral composition obtained using high-throughput sequencing with the ITS2 barcode gave non-significant effects of preservation methods on richness, relative abundance and Shannon diversity, in both countries. While freezing and ethanol are commonly employed for archiving tissue for molecular applications, desiccation is cheaper and easier to use regarding both storage and transportation. Since SG is less dependent on ambient humidity and less prone to contamination than RT, we recommend SG for preserving pollen for metabarcoding. SG is straightforward for laymen to use and hence robust for widespread application in citizen science studies.
- Semi-automated sequence curation for reliable reference datasets in ITS2 vascular plant DNA (meta-)barcodingPublication . Quaresma, Andreia; Ankenbrand, Markus J.; Garcia, Carlos Ariel Yadró; Rufino, José; Honrado, Mónica; Amaral, Joana S.; Brodschneider, Robert; Brusbardis, Valters; Gratzer, Kristina; Hatjina, Fani; Kilpinen, Ole; Pietropaoli, Marco; Roessink, Ivo; Steen, Jozef van der; Vejsnæs, Flemming; Pinto, M. Alice; Keller, AlexanderOne of the most critical steps for accurate taxonomic identification in DNA (meta)-barcoding is to have an accurate DNA reference sequence dataset for the marker of choice. Therefore, developing such a dataset has been a long-term ambition, especially in the Viridiplantae kingdom. Typically, reference datasets are constructed with sequences downloaded from general public databases, which can carry taxonomic and other relevant errors. Herein, we constructed a curated (i) global dataset, (ii) European crop dataset, and (iii) 27 datasets for the EU countries for the ITS2 barcoding marker of vascular plants. To that end, we first developed a pipeline script that entails (i) an automated curation stage comprising five filters, (ii) manual taxonomic correction for misclassified taxa, and (iii) manual addition of newly sequenced species. The pipeline allows easy updating of the curated datasets. With this approach, 13% of the sequences, corresponding to 7% of species originally imported from GenBank, were discarded. Further, 259 sequences were manually added to the curated global dataset, which now comprises 307,977 sequences of 111,382 plant species.
- Standard methods for pollen researchPublication . Campos, Maria G.; Anjos, Ofélia; Chica, Manuel; Campoy, Pascual; Nozkova, Janka; Almaraz-Abarca, Norma; Barreto, Lidia M.R.C.; Nordi, João Carlos; Estevinho, Leticia M.; Pascoal, Ananias; Paula, Vanessa B.; Choupina, Altino; Dias, L.G.; Tešić, Živoslav L. j.; Mosić, Mirjana D.; Kostić, Aleksandar Ž.; Pešić, Mirjana B.; Milojković-Opsenica, Dušanka M.; Sickel, Wiebke; Ankenbrand, Markus J.; Grimmer, Gudrun; Steffan-Dewenter, Ingolf; Keller, Alexander; Förster, Frank; Tananaki, Chrysoula H.; Liolios, Vasilios; Kanelis, Dimitrios; Rodopoulou, Maria-Anna; Thrasyvoulou, Andreas; Paulo, Luísa; Kast, Christina; Lucchetti, Matteo A.; Glauser, Gaëtan; Lokutova, Olena; Almeida-Muradian, Ligia Bicudo; Szczęsna, Teresa; Carreck, Norman L.“Bee pollen” is pollen collected from flowers by honey bees. It is used by the bees to nourish themselves, mainly by providing royal jelly and brood food, but it is also used for human nutrition. For the latter purpose, it is collected at the hive entrance as pellets that the bees bring to the hive. Bee pollen has diverse bioactivities, and thus has been used as a health food, and even as medication in some countries. In this paper, we provide standard methods for carrying out research on bee pollen. First, we introduce a method for the production and storage of bee pollen which assures quality of the product. Routine methods are then provided for the identification of the pollen’s floral sources, and determination of the more important quality criteria such as water content and content of proteins, carbohydrates, fatty acids, vitamins, alkaloids, phenolic and polyphenolic compounds. Finally, methods are described for the determination of some important bioactivities of bee pollen such as its antioxidant, anti-inflammatory, antimicrobial and antimutagenic properties. Métodos estándar Para la investigación del polen El "polen de abeja" es el polen recogido de las flores por las abejas melíferas. El polen de abeja es utilizado para nutrir a las propias abejas, principalmente para proporcionar jalea real y alimento para las crías, pero también se utiliza para la nutrición humana. Para este último fin, se recoge en la entrada de la colmena en forma de gránulos que las abejas llevan a la colmena. El polen de abeja tiene diversas bioactividades, por lo que se hautilizado como alimento para la salud, e incluso como medicamento en algunos países. En este artículo, proporcionamos métodos estándar para llevar a cabo investigaciones sobre el polen de abeja. En primer lugar, presentamos un método de producción y almacenamiento de polen de abeja que garantiza la calidad del producto. A continuación, se ofrecen métodos de rutina para la identificación de las fuentes florales del polen y la determinación de los criterios de calidad más importantes, como el contenido de agua y de proteínas, carbohidratos, ácidos grasos, vitaminas, alcaloides y compuestos fenólicos y polifenólicos. Por último, se describen métodos para la determinación de algunas bioactividades importantes del polen de abeja, como sus propiedades antioxidantes, antiinflamatorias, antimicrobianas y antimutagénicas.