My research is directed to the understanding of the molecular mechanisms related to sensory perception, synaptic plasticity underlying learning and memory and complex behaviour in the honeybee Apis mellifera. My main focuses are the neurotransmitter systems of the central nervous system (CNS) and the genetic basis of hygienic behaviour.

A particular emphasis is put on the development of molecular tools to interfere specifically with gene expression. We are using the technique of RNA interference (RNAi) to inhibit gene expression and attempts are made to transfect in vivo brain neurons of adult honeybees with functional expression vectors.

 

Hygienic Behaviour

This project, in collaboration with Dr. Tanja Gempe and Prof. Martin Beye (Heinrich-Heine Universität, Düsseldorf), is part of a cooperative project aiming towards the development of diagnostic tools for the selection of pest resistant honey bees. Since a few years, the number of bee colonies decreases continuously in Europe. One reason for this is pests affecting honeybee brood (e.g. the mite Varroa destructor, the fungus Ascosphaera apis). Some honeybees present a hygienic behaviour, meaning that they are able to detect and discard infested brood. This behaviour has a strong genetic component in the honeybee. The genetic differences of behaviour can most directly be described as relative changes of gene products in the nervous system. For this reason, we compare the full transcriptome profiles of honey bee brains simultaneously by applying DNA array.

 

Neurotransmitter systems

The CNS is composed of several neurotransmitter systems. The knowledge about their identity and their organisation is limited. One main research orientation consists in the allocation of neurotransmitter system to specific neuropiles or group of neurons and to study their function in behaviour.

The glutamatergic neurotransmission retains most of our attention. We characterised the NMDA glutamate receptor in the honeybee brain and we are studying its role in learning and memory by using RNAi. Our first analysis showed that this receptor is playing an important role in the formation of specific memory phases in appetitive conditioning. We are now analysing the spatiotemporal aspects of the receptor’s activation in learning and memory. These findings also raise interesting questions about the molecular correlates of the different memory phases.

Another project, in collaboration with Catherine Armengaud (Paul Sabatier University, France) focus on inhibitory glutamate receptor channels that are expressed only in invertebrates and are for this reason the target of pesticides. Since little is known about their physiological role, we are applying a multidisciplinary approach to study their function by combining RNAi, pharmacology, electrophysiology and behaviour.

 

Expression systems

Our RNAi protocol is an efficient technique to knock down gene expression, however it has some limitations. For example, it allows only inhibitions of moderate amplitude and the spatial resolution of the manipulation is not precisely controlled. For this reason efforts are invested to develop in vivo expression tools allowing the transfer of expression vectors in neurons. We concentrate on in vivo electroporation and viral-based expression vectors.

We want to use this technologies to both over-express a protein of interest and to inhibit gene expression by induction of RNAi. In addition, these tools will permit us to design strategies allowing the transfection of selected regions of the brain.

 

I accomplished my Ph.D. at the Free University of Brussels (Applied Genetics - Prof. Alex Bollen) in molecular biology, in immunobiology and parasitology. The aim was to characterize Ixodes ricinus tick factors that modulate the host’s defences and that could enable the transmission of pathogens (such as Borrelia burgdorferi).

Since I joined the institute for Neurobiology at the Free University of Berlin (Prof. Randolf Menzel), I studied PKA activation in the honeybee brain. I also contributed to the annotation process of the honeybee genome by focusing on the CREB and PKA gene families. I also collaborated on a project investigating the achromatic visual system based on one of the photoreceptor types, the long wavelength receptor.

Subtractive and ‘full length’ cDNA libraries. Recombinant proteins expression, in vitro activity tests and DNA vaccination. In situ hybridisation and immunohistochemistry. Western blot, ELISA and real-time PCR. Induction of RNAi and in vivo electroporation. Viral-based expression system. Behavioural tests.

[1] Müßig L., Richlitzki A., Rößler R., Eisenhardt D., Menzel R., Leboulle G.: Acute Disruption of the NMDA Receptor Subunit NR1 in the Honeybee Brain Selectively Impairs Memory Formation (2010) J. Neuroscience 30(23): 7817-7825

[2] Niggebrugge C., Leboulle G., Menzel R., Komischke B., Hempel de Ibarra, N.: Fast learning but coarse discrimination of colours in restrained honeybees (2009) J. Exp. Biol. 212(9): 1344-1350.

[3] Daix V., Praet N., Georgin J.-P., Chiappino I., Gillet L., Defays K., Decrem Y., Leboulle G., Godfroid E., Bollen A., Pastoret P.-P., Gern L., Sharp P. M., Vanderplasschen A. Ixodes ticks encode a new family of anti-complement proteins” (2007) – Insect Mol. Biol. – 16(2): 155-166.

[4] Niggebruegge C., Hempel de Ibarra N., Komischke B., Leboulle G., Menzel R.: Classical conditioning of coloured stimuli in the honeybee, Apis mellifera (2006) Perception 35: 138-139   Supplement: S

[5] Bee Genome Sequencing Consortium, (2006) Insights into social insects from the genome of the honeybee, Apis mellifera. Nature  443  (7114): 931-949. Dorothea Eisenhardt and Gérard Leboulle are co-authors of this publication.

[6] Eisenhardt, D., Kühn,C.; Leboulle, G. (2006) The PKA-CREB system encoded by the honeybee genome. Insect Molecular Biology 15(5): 551-561.

[7] Zannat M.T., Locatelli F., Rybak J., Menzel R., Leboulle G. Identification and localisation of the NR1 sub-unit homologue of the NMDA glutamate receptor in the honeybee brain (2006). Neurosci Lett. 398: 274-279.

[8] Menzel R., Leboulle G., Eisenhardt D. Small brains, bright minds (2006). Cell 124(2): 237-9.

[9] Leboulle G., Müller U. Synergistic activation of insect cAMP-dependent protein kinase A (type II) by cyclicAMP and cyclicGMP (2004). FEBS letters 576: 216-220.

[10] Leboulle G., Crippa M., Decrem Y., Mejri N., Brossard M., Bollen A. and Godfroid E.: Characterization of a novel salivary immunosuppressive protein from Ixodes ricinus ticks (2002). J. Biol. Chem.  277: 10083-10089.

[11] Leboulle G., Rochez C., Louahed J., Rutti B., Brossard M., Bollen A., and Godfroid E.: Isolation of Ixodes ricinus salivary gland mRNA encoding factors induced during blood feeding (2002). Am. J. Trop. Med. & Hyg. 66(3): 225-233.