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• Nematodes H. bacteriophora and S. feltiae were multiplied on Galleria mellonella larvae. • Honeybees were treated with potentially immuno- modulating substances (plant alkaloid and probiotics) during 8 days of development. • Bee larvae and pupae were collected and infected on Petri dishes with the dose 1, 5, 10, 15 or 20 EPN/larva (Fig. 4). • 48 hours after infection larvae and pupae were scored for mortality. • H. bacteriophora harbouring GFP labeled P. luminescens was used to monitor the infection (Fig. 5). 1 Department of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic 2 Department of Microbiology, Nutrition and Dietetics, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Czech Republic 3 Bee Research Institute; Libcice nad Vltavou, Czech Republic 4 Institute of Animal Physiology and Genetics v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic 5 Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Czech Republic P avel Hyršl 1 , Pavel Dobeš 1 , Libor Vojtek 1 , Jakub Berka 1 , Jana Hurychová 1 , Jaroslav Havlík 2 , Martin Kamler 2,3 , Zuzana Hroncová 2 , Jiří Killer 4 , Jan Tyl 3 , Dalibor Titěra 2,5 Plant alkaloid and probiotics increase resistance of honeybees to nematobacterial infection Our research is supported by research grants from Ministry of Agriculture of Czech Republic (NAZV-KUS QJ1210047), MUNI/C/1406/2014 and by the Program of „Employment of Newly Graduated Doctors of Science for Scientific Excellence“ (CZ.1.07/2.3.00/30.0009) co-financed from European Social Fund and the state budget of the Czech Republic. Bees are used by mankind for several thousand years, but their immune system is still far from being fully understood and moreover we still don’t have clear idea about all immune mechanisms, which mediate bees’ response to the pathogens. These pathogens negatively influence life of the bees and very often even their viability, causing direct impact on agriculture and industry. Detailed knowledge of bee immunity is crucial for successful fight against bee diseases. We successfully used nematode infections in our previous research to study immunity of Drosophila melanogaster and determined several genes involved in the immune reaction against nematobacterial complex. We also previously showed that honeybee larvae are suitable hosts for nematodes and our tripartite model (honeybee, nematodes, bacteria) can be used as standard procedure for testing honeybees' immune response (Fig. 2). Our current aim was to optimize the number of nematode infective juveniles (IJ) needed to set the sublethal dose for honey bee larvae model and subsequently use it to test immune defence of larvae treated with potentially immuno-modulating substances (plant alkaloid and probiotics). B Conclusions • Bee larvae and pupae can be infected with entomopathogenic nematodes that will enable detailed studies of their immune response. • Infection with H. bacteriophora can be visualized using GFP labeled symbiotic bacteria. • We optimized the natural infection for EPN Heterorhabditis bacteriophora and Steinernema feltiae; both species show dose dependence infection (higher EPN dose resulted also in higher amount of invaded parasites). • As an ideal sub-lethal dose was chosen 10 IJ per larva was selected. • Bee larvae are more susceptible to the infection than pupae. • Larvae fed by probiotics and plant alkaloid showed higher resistance against EPN infection which supports our immuno-stimulating hypothesis. BODY BARRIERS HEMOCOEL EXTERNAL SPACE Humoral Immunity Cellular Immunity invasion regurgitation of EPB Barrier Immunity PRODUCTS OF EPB • lectins • toxins • hydrolytic enzymes • antibiotics • pigments FUNCTION IN INFECTION PROCESS • lectin-mediated attachment • disruption of cytoskelet, impaired hemocyte movement • lysis of hemocytes and tissue cells • inhibition of phenoloxidase system • protecting niche from competitive organism hemocytes fat body toxicaemia, septicaemia Entomopathogenic nematodes (EPN, Fig. 1A) Heterorhabditis bacteriophora and Steinernema feltiae are obligate and lethal insect parasites. These EPN are symbiotically associated with entomopathogenic bacteria Photorhabdus luminescens or Xenorhabdus bovienii respectively, creating the highly pathogenic nematobacterial complex that is able to kill the host within 24 to 48 hours. EPN with its bacterial symbionts are able to infect a broad spectrum of insect species including e.g. larvae of flies, moths (Fig. 1B, C) or bees. Symbiotic bacteria help to digest host tissues and provide nutrients for themselves and developing nematodes. Fig. 1 A: Infective juveniles (IJ) of H. bacteriophora with GFP labeled bacteria P. luminescens in the gut. B, C: Drosophila melanogaster and Galleria mellonella larvae infected by nematobacterial complex Heterorhabditis–Photorhabdus. Cadavers have the typical coloration caused by pigments produced by symbiotic bacteria. A A B Entomopathogenic nematodes (EPN) Infection of honeybee larvae A Honeybee immunity GFP as a tool to track the infection Material and method Fig. 2: For successful development within the host, EPN and their symbiotic bacteria must overcome insect defences including both cellular and humoral immune responses. Results Fig. 5: To demonstrate the role of symbiotic bacteria we exchanged the natural symbiont with TT01-GFP expressing strain. The bacteria are localized in the gut of IJ and cause septicemia after release into the insect body. Pictures shows infected bee larvae infected in honeycomb under fluorescence (A), uninfected and infected larvae under day light (B) and fluorescence (C). control S. feltiae H. bacteriophora Fig. 3 C Larvae as well as pupaes were successfully infected by two entomopathogenic nematode species (H. bacteriophora is more pathogenic for larvae). We observed dose dependence in mortality (Fig. 6) and as a sublethal dose were chosen 10 IJ per larvae. Results from Petri dishes confirmed the immuno-stimulating effect of probiotics and plant alkaloid – stronger effect (up to 30 % decrease of mortality compared to control) was observed against S. feltiae but even H. bacteriophora showed better defence response (10 % decrease of mortality compared to control, Fig. 7). 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1IJ 5IJ 10IJ 15IJ 20IJ mortality (%) pupae H. bacteriophora S. feltiae 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1IJ 5IJ 10IJ 15IJ 20IJ mortality (%) larvae H. bacteriophora S. feltiae Fig. 4: Bee larvae and pupae infected by nematobacterial complex Heterorhabditis–Photorhabdus (A, B). Melanized wounds caused by nematodes during invasion are visible in the cuticle (C). B 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% control probiotics plant alkaloid mortality (%) S. feltiae (10 IJ/larva) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% control probiotics plant alkaloid mortality (%) H. bacteriophora (10 IJ/larva) Fig. 6: Mortality of larvae (A) and pupae (B) bee larvae is dependent on dose of IJ used for infection. Bees were infected with nematobacterial complex Heterorhabditis–Photorhabdus and Steinernema-Xenorhabdus. Fig. 7: Immuno-stimulating effect of plant alkaloid and probiotics on bee larvae using 10 IJ of S. feltiae (A) and H. bacteriophora (B) per larva, expressed as % of mortality ± SD; n=20 per group; tested in triplicates. C B B B C A Contact e-mail: [email protected]
