The emergence of the final apiculturist citizen science guidelines

The INSIGNIA study has been running now for two years and we are entering the final stage. In these two years, we have gone through all stages of organizing an apiculturist citizen science study. We developed the passive sampler APIStrip, the efficient matrix to capture pesticides, and all the pitfalls that came on the way like the “not-to-be -misinterpretable” label, best practice for storage shipping, sampling schemes etc. Now it is time to bring all of this information together in the “Guideline for apiculturist citizen science for applying honeybee colonies for bio-monitoring of the environment  – subject pesticides”.  Given the Covid-19 restrictions,  physical meetings to discuss all of the stages, best wording, most logical set-up, best flow-charts, and whatever it takes to write the best guidelines, would be great but are sadly impossible. Therefore we have started a scheme of virtual Teams-meetings with a strict agenda to go through all of these aspects. We started in December 2020 and will be finished in March 2021. There will be as many Teams-meetings as it takes to get the job done in time.

Jozef van der Steen

coordinator Insignia


Dear participant of the Insignia study 2020

In my virtual meetings with the beekeeper citizen scientists, I am asked sometimes: “what is in it for me?” A fair question. Let me explain what is in it for you… You will sample pollen and APIStrips from two colonies on ten occasions. Pollen and APIStrips will be analyzed at the apiary level; pollen for botanical identity and the APIStrips for pesticide residues in the hive. These results will be shared with you and will give you the information about what plants your bees have collected pollen from, and what pesticides your bees have brought along with the pollen and nectar. The latter is qualitative, meaning yes/ no pesticide present in the APIStrip. We detect different amounts but what this means for the actual exposure of your bees in the hive can’t be told with these data. This requires another study set-up which hopefully can be done in the future. Now the reward in monetary terms. Each APIStrip with its analysis costs about €200 each, and each pollen analysis also costs about €200. So together this is €400, with transport, administration, quality control, statistics and reports not included. A very cautious estimate of the costs per beekeeper for Insignia will be 10 sampling x € 400 plus extra costs = €4,500. 60% of this is paid by the European Union = € 2,700, the remainder €1,800 is paid by your national coordinator’s institute and you will have this information about your bees for free.

Additionally and not directly for your bees, the value is that we will use the data collected for:
1. Protocol development to apply honey bee colonies for environmental monitoring;
2. Provide information about the level of environmental (pesticide) pollution in Europe;
3. Provide information about food availability and diversity for honey bees and other pollinators at a landscape level and;
4. Model building to assess pesticide exposure risk for insect pollinators.

So your contribution is very valuable, greatly appreciated, highly important, indispensable to the project, and fundamental for applied science.

Best regards

Sjef van der Steen (Insignia coordinator)

Insignia bumblebee colony sampling round 1

Today, the first APIStrips were removed after a 14-days exposure period. Curious to see if the APIStrips absorbed pesticides in detectable amounts. Also, the first pollen was taken from the pollen pots. Relying on the purple pollen color, I think the bees foraged among others on the horse chestnut (Aesculus spp.) and/or red deadnettle (Lamium purpureum). Alice and her team in Bragança will tell after the metabarcoding analysis. The APIStrips and pollen are labeled and stored in the freezer until shipping to the labs.

Sjef van der Steen

APIStrip in a bumblebee colony

Next to the honeybees, the bumblebees are part of the Insignia study. This year we are testing the pesticides, collected by bumblebees in the field along with pollen and nectar, with the APIStrip. I will work this year with two colonies at the Sinderhoeve, the experimental station of Wageningen University, Environmental Science in Heelsum. Bumblebees are blind for the longer wavelength light spectrum, therefore, manipulation of the colony is done in red light. This keeps them rather calm. Bumblebees do not have beelanes, actually, compared to honeybees their brood nest is a mess. But, as honeybees, they are very curious. To prevent disturbance of the colony during changing the APIStrip, I inserted the strip via the top- grid near the entrance of the bumblebee box positioned in the very proximity of the brood cells. On the reddish photo you see them crawling over the APIStrip (with the black tyrap). Box entering and box- leaving bumblebees will be in contact with the strip as will the in-box bees. Time will learn whether the APIStrip will be incorporated in the brood nest or left apart in the 2-weeks exposure period. In two weeks the first APIStrips will go out and the second will go in, simultaneously to the honeybee colony sampling. Therefore, two photos of reddish. The APIStrip is prevented from falling into the box by a tyrap. Let’s follow how it will go in the next six weeks.


Sjef van der Steen

Colleagues meet at the Insignia plenary meeting in Almeria

Meetings as the annual plenary meeting are about more than data exchange and scientific discussion; they are also about meeting colleagues, social interaction, interests in colleague’s family life and relaxation in eating and drinking together. The early birds that arrived already on Sunday 12th January were invited for a tapas experience in Almeria city and both meeting evenings were great experiences with Spanish fish meals, regional culinary highlights, Spanish wine, and Portuguese port. In the coffee- and lunch breaks there was time for chats in the sun. This socializing, chatting and having a good time is the basis for mutual respect which, together with full commitment to the well-being of the honeybee and the interests in the environment and its interaction with the bees, form the fundament for a good functioning team; and a good functioning team we are!

Sjef van der Steen


The plenary meeting in Almeria, group at work

At the University of Almeria, Amadeo Fernandez-Alba and his team hosted 24 scientists from 10 countries for the plenary Insignia meeting on 14th and 15th January 2020. In a great ambiance, the results of the 2019 study were presented, discussed and comprised into the 2020 protocol, to be tested in 9 countries in 81 apiaries. The results presented clearly showed a difference between the four (4) pesticide residue matrices, the two biological matrices: trapped pollen, beebread, and the two non-biological non-invasive passive sampling matrices: Beehold tube and the in-hive APIStrip. The latter proved to have the best binding capacities for the number of pesticides. Trapped pollen proved to be a very poor matrix for this purpose. Beebread and the Beehold tube functioned in between. For pollen diversity, three matrices were tested: trapped pollen, beebread, and the Beehold tube. Comparison between classic microscope palynology and ITS2 pollen metabarcoding revealed that ITS2 pollen metabarcoding results are comparable outcomes on family level. For pollen diversity, the three (3) matrices gave similar results. Based on the 2019 results it was decided to apply in the 2020 study the best pesticide residue matrix APIStrip and the most practical pollen matrix: trapped pollen for ITS2 metabarcoding. Insignia is about protocol development for citizen science study. The interaction between scientist and apiculturist has been studied in a sociological study. Non surprisingly it can be concluded that citizen scientist involvement is based on personal interests like participating in a scientific study or interest in the environment primarily on mutual respect between the scientist and beekeeper and mutual concern for the well-being of the honeybee.

All the aspects, from the interaction between scientist and beekeeper and instruction workshops to practical issues as best exposure location, matrix storage, and transport were point by point discussed and agreed. This will result in an updated instruction/ picture manual for all participating beekeepers.

Sjef van der Steen


Follow your scientist – The INSIGNIA project

Last week note nr 2. “Follow your scientist – the INSIGNIA project”  by Flemming Vejsnæs was sent out to the stakeholders. it draws the attention to our social media and website to inform and to enhance the cooperation and commitment of the stakeholders to the INSIGNIA project. Soon similar notes will be sent to the beekeeper’s journals in the INSIGNIA countries. More notes will follow

Flemming Vejsnæs,  Sjef van der Steen 


Our social media
our website

Sample analysis – Greek Lab BPI

After samples’ arrival, they are all coded and then analysed or stored in the freezer till the day of analysis. For their preparation, extraction and clean-up steps are taking place, to separate the analytes from interfering compounds and obtain the optimum results. For the quantification of the pesticide residues analysed, both liquid and gas chromatography combined with tandem mass spectrometry is used. The last stage of this procedure is the data analysis and the evaluation of the results.

Lab material and equipment used by the lab staff (us), are presented in the following pictures. In particular, in the picture on the left, 50mL falcons containing salts for the clean-up step of the samples and 15mL falcons containing samples after the clean-up step are shown in the back and front part of the picture, respectively. Vials to be filled and vials ready for injection in LC-MS/MS and GC-MS/MS are also presented in the following pictures.

Effrosyni Zafeiraki

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