Browsing by Author "Vejsnaes, Flemming"
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- 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.
- Identification of botanical origin of bee-collected mixed pollen samples: a comparison between palynological and DNA metabarcoding methodsPublication . Quaresma, Andreia; Steen, Jozef van der; Amaral, Joana S.; Biron, David G.; Brodschneider, Robert; Brusbardis, Valters; Carreck, Norman L.; Formato, Giovanni; Gratzer, Kristina; Hatjina, Fani; Kilpinem, Ole; Pietropaoli, Marco; Rufino, José; Vejsnaes, Flemming; Pinto, M. AliceIdentification of botanical origin of mixed pollen samples has several applications, including unraveling plant-pollinator interactions, determining botanical origin of honey, monitoring allergy-related airborne pollen sources, or even monitoring pesticide use in crops. These applications have typically been addressed 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 classical palynology. 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 two approaches on 61 bee-collected pollen samples from eight European countries. The samples were homogenized and split into two sub-samples. One set of 61 sub-samples was analyzed 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. Comparisons of the relative abundances at the family level show no significant differences (P ≥ 0.1057, Wilcoxon signed-rank test) and high correlation values (0.2736 ≤ r ≤ 1.000, 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 = 0.5149). These results suggest that ITS2 metabarcoding offers a reliable alternative to classical palynology and this 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.
- 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.