In pursuit of key enzymes

Toxigenomics: How honey bees detoxify plant protection products

Honey bees have metabolic mechanisms that can effectively break down certain insecticides quickly, allowing the applied dose to remain toxic to the target pest insects, yet not to bees. Due to this, it is possible to use certain insecticides on flowering crops which are attractive to bees. Bayer researchers are currently investigating which enzymes are involved in this metabolically driven detoxification process. Armed with this knowledge, they hope to become even more targeted in their development of plant protection products that are effective against pests while remaining low-toxic for honey bees.

Toxigenomics: How honey bees detoxify plant protection products


// Honey bees have metabolic mechanisms that can effectively break down certain insecticides.

// Bayer researchers have now discovered how enzymes in bees break down certain insecticides particularly quickly.

// This knowledge can be used to facilitate the development of new, bee-friendly insecticides.

Teamwork at the lab

Teamwork in the lab: Denise Steinbach, Dr Ralf Nauen, Harald Köhler and Marion Zaworra (from left) not only work with bees; they also analyze how some pests can become resistant to crop protection products.

The molecular mechanisms and tools which honey bees use to break down certain plant protection products lead to some remarkable differences. While a certain amount of one insecticide can kill a honey bee, another may have no effect on their health. “Honey bees seem to have very effective metabolic processes for coping with certain insecticides,” explains Dr Ralf Nauen, Head of the Bee Toxicogenomics project for insect research at Bayer. Dr Nauen and his team intend to document which metabolic processes are involved in bee detoxification mechanisms.

In order to achieve this, the researchers expose bees to different kinds of insecticides. “We’ve tested several products containing insecticides that are used against pests such as aphids and leaf-eating beetles. While certain compounds such as thiacloprid had no impact on bee health at a certain dose, others from the same chemical class were acutely toxic to the bees at comparable exposure rates,” explains Dr Nauen. Even though some of the molecules are only slightly different in their structure and react with the same receptors in an insect’s body. “The findings also show that you can’t make generalizations about the effects of insecticides of a chemical class such as neonicotinoids,” says Dr Nauen. He and his team wanted to get to the bottom of why that is – and indeed there are natural resistance or self-defense mechanisms involved to protect honey bees from active ingredients which have, as a consequence, a low acute toxicity to bees, such as thiacloprid.

intra-abdominal microinjection apparatus administers an antisense RNA to an anesthetized honey bee

Precision work
The intra-abdominal microinjection apparatus administers an antisense RNA to an anesthetized honey bee.

The researchers therefore analyzed the honey bee genome which was published in 2006. They examined almost 10,000 genes in an effort to find those involved in detoxification of certain insecticides. They cloned and expressed many of them to investigate their detoxification capacity in insect cell lines. However, that is not all: “In order to determine the function of a gene, we have to shut it down,” explains Dr Cristina Manjon, a postdoctoral researcher in Dr Nauen’s laboratory. To do this, you anesthetize a honey bee and carefully administer an “antisense RNA”. This type of Ribonucleic Acid (RNA) targets an intermediate product of a particular gene called messenger RNA, by binding to it and blocking it.

The gene can then no longer perform its normal function. The bees which have this silenced gene are then exposed to an insecticide they would normally resist. A control group of untreated bees is also exposed to the same substance. “We then examine how the bees react to the practically non-toxic insecticide – whether it affects their well-being or triggers symptoms of poisoning,” she explains.

researchers applied certain insecticides under controlled conditions

The researchers applied certain insecticides under controlled conditions, to compare untreated insects with ones containing an inactive copy of a particular gene.

The gene encodes a particular enzyme, a protein molecule catalyzing specific metabolic reactions. This protein molecule helps the honey bee to break down the insecticide within minutes – so rapidly that there is not time for the insecticide to take effect.

Dr Manjon was even able to identify where the enzyme acts: “Surprisingly, several insecticides are not broken down in the midgut as in most insects. Instead, they are metabolized and broken down in the nerve cells of the brain and the Malpighian tubules – the insects’ kidneys.”

These findings from Dr Nauen’s lab could revolutionize the development of plant protection products: “If we learn and understand more about the detoxification mechanisms and about which enzymes and mechanisms are involved, we can develop more targeted plant protection products,” explains Dr Nauen. The researchers could then test and select active ingredients that are practically non-toxic for honey bees much more easily and earlier in the development process. This would make the development of bee-friendly plant protection products a great deal more efficient.

The scientists are also researching other molecular mechanisms and tools that honey bees use to break down plant protection products. They hope to find out, for example, whether such enzymes can also reduce the effects of certain insecticides. As the researchers want to avoid that their study ‘goes into hibernation’ alongside the honey bees that they are studying, they conduct tests using another insect during the fall and winter: “We transfer the bee genes of interest to fruit flies, which then also produce the enzyme. In this way, we can carry out tests all year round,” explains Dr Manjon. She and the rest of the team are also looking for other enzymes that can quickly break down insecticides. Dr Nauen notes, “We want to learn about all natural resistance and the metabolic defense mechanisms in honey bees. The more we know about what distinguishes them from other insects, the better we can protect them in agriculture.”


Way forward

Plant protection products must also be safe for bee species other than the honey bee. Dr Nauen’s team is, therefore, working with scientists at Rothamsted Research in the UK. They are investigating the same biochemical and genetic mechanisms in two other important bee pollinator species: the red mason bee, a solitary wild bee species, and the buff-tailed bumble bee often used for pollination in greenhouses. The team also has plans to research other types of bee in the future, such as stingless bees, which play an important role for pollination in Brazil.

scientists at Rothamsted Research in the UK

Chris Bass, Head of the Bee Toxicogenomics project at Rothamsted Research in the UK

Spotlight on wild bees

Chris Bass is Head of the Bee Toxicogenomics project at Rothamsted Research in the UK. Since June 2014, he and his team have been studying the resistance mechanisms of two wild bee species. Specifically, they are looking at how insecticides such as neonicotinoids affect them.

What is your focus in the Bee Toxicogenomics project?

The primary focus of the group at Rothamsted is to expand on the work of Bayer on honey bees by exploring the molecular basis of insecticide selectivity in additional bee species, principally the social bumble bee Bombus terrestris and the solitary bee Osmia bicornis. Our specific objectives are to generate new genomic and transcriptomic (which means the complete set of messenger RNA transcripts produced by the genome) information and to compare the toxicological profile of these wild bees with that of honey bees. The final aim is to identify and characterize the gene(s) involved in bee detoxification of insecticides.

How do your research results differ from the results with honey bees?

The results have revealed interesting similarities, but we have also observed some differences: It is clear that, as shown for honey bees, profound differences exist in the sensitivity of these bee species to different insecticides within the same class. This finding suggests an understanding of the molecular basis of this observed selectivity could be harnessed to develop pest-specific insecticides that are not harmful to these bee species. We also find similarities across the bee species in the type of specific detoxification enzyme family that is metabolizing the less toxic compounds, but there are differences at the level of the specific genes involved. We are excited with the results generated to date. In the case of Osmia bicornis, the speed with which the results have been obtained is a reflection of the powerful next-generation sequencing technologies that have become available over the last few years. These technologies allow unprecedented power to rapidly characterize whole insect transcriptomes, produce draft genomes in non-model organisms that are typically not well-studied, and identify whole super-families of genes involved in insect detoxification.

What are the next steps for the project?

A huge amount of data and novel information has been generated so far. Our priority is to see this initial data published as swiftly as possible. Beyond this, we will start to explore the molecular basis of selectivity observed in other insecticide classes. We also want to focus on translation of the knowledge generated into rapid and user-friendly test methods that can be used as a future screening tool to develop next-generation, bee-friendly insecticides.

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