Metabarcoding of pollen

At the IPB (Instituto Politécnico de Bragança in Portugal), the botanical origin of the Insignia pollen is determined molecularly. The botanic origin of pollen is determined by its DNA.

As all living organisms, all plants have DNA, and all plant DNA has an ITS2 (internal transcribed spacer) fragment. This ITS2 fragment is a specific part of the DNA strand and is unique for a plant family/ genus/ species. Of every pollen grain in the homogenised subsamples collected from a pollen trap, beebread and Beehold tube, the Insignia pollen matrices, the ITS2 fragment is isolated and multiplicated to millions of copies by an in vitro reaction called PCR.

In the isolation- and multiplication process an Index molecule is coupled to each ITS2 fragment. This Index is unique for a sample. For example, all pollen collected in a trap in front of hive 1 on a particular sampling date has the same Index and of hive 2, another Index. After coupling the Index, the order of DNA bases (G: guanine, T: thymine, A: adenine and C: cytosine) of each ITS2 DNA fragment is determined by using a technology commonly known as next-generation sequencing (NGS). The process of using a short fragment of a specific gene (in this case ITS2) to identify organisms (in this case plants from pollen) is known as “DNA barcoding”. Because a pollen sample collected in a trap combines pollens originating from many different plant species, then identification by DNA barcoding is called “DNA metabarcoding”. This DNA bases order or barcode is unique for a plant family/genus/species.

To determine the relative abundances of different pollen sources in mixed pollen pellets collected by bees, the number of sequence reads is recorded in the next generation sequencing (NGS) process. Each “read” is a specific sequence of DNA bases of ITS2 DNA fragment obtained in the multiplication process, and the more a specific pollen source (e.g. Brassica napus) is present in the mixed-pollen sample, the more reads of the ITS2 fragment of the specific source will be produced. Consequently, the more reads this specific ITS2 fragments have, the higher the relative abundance.

This process from DNA isolation from the pollen until the sequencing of the DNA bases is time consuming compared to microscopic morphological determination. However, several hundreds of samples (= many thousands of ITS2’s) are analysed simultaneously in one sequence run, which makes it time-saving. The number of samples that can be sequenced simultaneously depends on the number of unique Indices available.

In microscopical determination, several hundreds of pollen grains are examined. In metabarcoding millions of pollen grains are examined. As for microscopical analyses, a reference database of ITS2 sequences is needed for comparison and as for microscopic analysis, the richer the reference database is, the better the determination. The more plant species and the higher the geographical coverage, the richer the reference database. In addition to the molecular determination of the botanic origin of pollen, in the Insignia study, the ITS2 European reference database will be enriched.

The photo of the screen with the coloured lines shows the result of sequencing each colour represents a DNA base. The second photo shows the microcentrifuge tubes with the DNA extracts. The third photo shows the ITS2 fragment multiplicated by PCR. The fourth photo is the IPB team working in Insignia and me.


Sjef van der Steen

INSIGNIA at the ICPPR congress in Bern 23, 24, 25 October 2019

Every 2 years the Bee Protection group of the International Commission for Plant-Pollinator Relationships (ICPPR) organises a congress about the hazard of pesticides to bees. Bee researchers from academia, contract labs, industry, governmental institutions, EFSA, organisations involved in bee protection and conservation and student meet there to present and discuss new developments and draft methods and outcomes in assessing the hazard of pesticides to bees both for first to higher tier studies, current hazard assessments are evaluated and new guidelines are presented. This Bee Protection group is both stakeholders of the Insignia pilot study and a target international body to present Insignia. There was a lot of interest in our study, particularly for the non-biological passive samplers. It took some effort to explain the role of the honeybee colony in this study, being not the major subject but the data collecting tool. Contact is made with similar monitoring studies, working groups and EFSA for cooperation and data exchange. Both cooperation and data exchanges are subject currently discussed with the EC-DG SANTE and the consortium. It has shown the need for data of pesticide residues in the environment and the role of the honeybee-colony-tool fulfill in this hot topic.

Sjef van der Steen
coordinator Insignia


Insignia announced in Belgium

Belgium is one of the countries in which nine apiaries will be selected for the 2020 trials. The Flemish beekeepers were informed about the Insignia project in October 2019. Beekeeping in Flanders is much more developed than the front page of the magazine suggests. It is a giant step from making straw skeps to applying honeybee colonies as a monitoring tool for pesticides. An application of the honeybee, completely non-existing when the straw skeps were developed some centuries ago. The big difference between beekeeping with the skep and the Insignia study is that bees in the skep were killed to harvest the honey and that the Insignia project aims to result in a  non-invasive (= no bees sampled and killed) citizen scientist sampling protocol; a progress in bee- welfare and bee research.

Science with communication

How easy can science be on your own, no protests, no objections, bright ideas and always being right? However that’s not science, that is vanity.

Science can only blossom and flourish in cooperation and respectful communication with each other. That is why the GoTo Telemeetings occupy a prominent place in the Insignia consortium. The course of the pilot study, new ideas, results clarification and evaluations and any issue of whatever importance are discussed in these meetings. First in small groups of specialists in the consortium to be broadened up with more subjects-involved participants in the course of the subject.
Continue reading “Science with communication”

Colony strength assessment

The amounts of substances, detectable in a colony, depends on how much is bio-sampled outside. This is directly linked tot he number of foragers. Therefore part of recordings done in the Insignia project is colony strength assessment with Image J. To do so the number of hive entering bees is counted with beecounters during about one week and in this week all frames with bees are photographed. The number of bees per photograph = one frame side is calculated by recording the surface covered with bees. One cm2 is covered by 1.25 bees (Delaplane, Guzman-Novoa, Steen, 2013).

These recordings are done in Rome by Marco and will be done next year in Denmark by Flemming, by Valters in Latvia and by Ivo and myself in the Netherlands. However want to join, please join.

With the colony strength data and number of hive entering bees, we study the linkage between these two parameters in order to have a simple citizen science tool that the colony strength can be assessed by counting the beelanes between frames of specific sizes. That is why the question of frame sizes and occupied bee lanes is added to the lime survey.

Keith S Delaplane, Jozef van der Steen & Ernesto Guzman-Novoa (2013) Standard methods for estimating strength parameters of Apismellifera colonies, Journal of Apicultural Research, 52:1, 1-12

Sjef van der Steen

The first eight months of the Insignia pilot study

A mail, drawing my attention to an EU bio-monitoring call caught my attention. Wow! This is an ultimate chance to demonstrate the honeybee colony’s bio-sample feature as a tool for environmental monitoring. More colleagues had this idea and we found each other in this shared interest. Obviously, this resulted in the start of a consortium which expanded like a swan glue game, resulting in the final Insignia consortium with experts of all required scientific disciplines. Many telemeetings later, with launching, discussing and evaluating ideas, we had a clear interpretation of the call and routes to achieve this. Our focus should be on developing and testing a scientifically substantiated citizen science protocol and the application of passive samplers to meet both the non-invasive and innovative requirements in the call and applying the common matrices trapped pollen and beebread as benchmarks.

Writing the final submission, waiting for the selection and the selection itself sounds simple but was stressful, hectic and required skills for all of us as there were science, statistics, grammar, flow charts, budgeting, summarizing, planning, financial recordings, (very) frequent communicating and reporting, use of social media, etc. etc..

Now we are eight months ahead and we already do have an impressive palmarès thanks to all particip[ants involvement and energy. Passive samplers were developed, testing schemes were  made, discussed and implemented,  picture/ instruction manual written, test apiaries selected, five samplings done, first analyses done, progress in molecular detection of pollen made, social study conducted, a lime survey running, the first data for evaluation of the Corine database collected, an active social media life and several practical issues addressed, solved or parked.

Still, five samplings to go in year 1 and the plenary discussions in January 2020 on best practices both scientifically, practically and citizen-scientifically. It feels good to  cooperate in the consortium and, I am looking forward to the coming exciting 20 months.

The enigma of sample coding

Communication in itself is simply a matter of sending and receiving a message. This implies on one hand, a clear message. On the other hand, every receiver interprets the message within his/her own reference frame. This makes communication a difficult issue anyway. In Insignia, this shows in interpreting the coding of the Insignia samples. This coding is clearly explained and communicated in the picture manual.   On the other hand, other lay-outs are suggested in the conviction that his/ her interpretation is better. Remember that there is a chain of “receivers” and the coding is six-dimensional: country, apiary, colony, date, material and replicates in time. Miscoding will for sure results in potential misinterpretations and certainly in time and energy loss in sorting out the samples. The solution is simple. Stick to the appointments and don’t make labeling

20919709 – human head silhouette with question mark concept

an enigma. 

The 5th sampling in Denmark by Flemming and Ole and assistance of Valters and Sjef

The best way to learn all the ins and outs of Insignia sampling is simply doing it. Flemming and Ole were so kind as to invite me and Valters to assist in the 5th sampling. Due to mine and Valters agenda, the 5th sampling in Denmark was brought forward to Thursday and Friday 4 and 5 July. It was an interesting learning experience for myself and Valters to prepare package and coding,  to do the pollen collection,  taking out and inserting respectively the old and new APIStrips and Beehold tubes, collecting beebread and emptying the pollen trap, to have an indication of the time and energy spent on the labour before and after the field work. I learned/ was confirmed that coding/ sample preparation requires a consequent and accurate effort, beebread picking takes time in two ways, looking for the best frame and the picking itself, the facility of the APIStrip and Beehold tube change and the every time wonderful view of the diversity of pollen in the trap.  All samples are stored in the freezer.

Just a reminder for sending the samples latest half July as described in the picture manual except alcohol on trapped pollen for PCR (and not for pesticide analyses).

Pollen identification
– trapped pollen for pollen identification are sent to Alice in alcohol 96%,
– Beehold tubes and beebread straws are sent, wrapped together with frozen icepacks in an insulating package of e.g. plastic foil with air bubbles or styrofoam box.

Pesticide residues
– APIStrips, Beehold tubes, trapped pollen and bee bread  are sent to the labs wrapped together with frozen icepacks from the freezer in an insulating package of e.g. plastic foil with air bubbles or styrofoam box.
– The division, where to send the samples for pesticide residue analyses will be announced in time.