Final Report Summary - FARMING IN BEETLES (Mechanisms of Fungiculture in Ambrosia Beetles)
Humans are not the only living beings that culture their own food. Three insect lineages have evolved the ability to do so as well. Sixty million years before the rise of human agriculture, some ants, termites and ambrosia beetles started to farm fungi for food. The animals and their crops live in a symbiosis; while the fungus provides food, it depends on the insects for dispersal to new habitats. Neither the insects nor the fungi are able to live independently of the partner. Fungus farming by insects involves plantation, cultivation and sustainable harvesting of the crops. Workers of ants and termites weed their gardens and apply antibiotics-producing microbes onto them. The mechanisms of fungiculture by ambrosia beetles are hardly known and were the focus of this IEF Marie-Curie project.
The main research objective of this project was to unravel some of the behavioural and chemical mechanisms of fungus farming by ambrosia beetles: Can ambrosia beetles actively influence the species community of their fungus gardens? If so, can beetles defend their gardens against fungal pathogens? How do they promote the growth of their fungal cultivars? And can they even fertilize their gardens? All these questions I tried to tackle in the last two years and although we do not have satisfactory replies on all answers, we made some exciting discoveries. These findings have the potential of completely changing the view on how we see these systems and will open up entirely new avenues of research.
First, we got quite clear indications that the ambrosia beetle Xyleborinus saxesenii can control the fungus species community growing in their gardens. Although final results of our experiment, where we removed beetles from half of the nests to check whether their presence affects fungus community, are not yet available, another experiment delivered quite clear indications. Together with a MSc student we found that adult females enhanced their general activity and especially their grooming frequencies immediately after we injected buffer solution with pathogenic fungus spores in laboratory nests (in comparison to a control group treated with sterile buffer solution). This grooming activity significantly reduced the pathogen-spore loads. This result is remarkable because it shows for the first time that hygienic behaviour is inducible in ambrosia beetles and that they show a social immune defence against pathogens comparable to that of eusocial societies of ants or bees.
What has remained unknown after this first finding is the mechanistic basis behind the disinfection of the pathogen spores. Possibly beetles apply antibiotics-producing microbes that inhibit growth of pathogens. To test this we looked at the growth-inhibition effect (which is an indication for antibiotics) of two symbionts (a yeast and a bacterium that are commonly found with this beetle) against two pathogenic mould fungi as well as two nutritional ambrosia fungi on different growth media in the laboratory. Both symbionts did not suppress the growth of the pathogens, so beetles must have other means to disinfect pathogens.
A potential chemical that might be produced by symbionts in the nest and which we found to selectively suppress growth of pathogens is ethanol. A field study had shown that beetles reproduce best in trees with high ethanol concentrations. This suggested that ambrosia fungi are adapted to substrates that contain ethanol, which is normally regarded mycotoxic. Indeed, we found that all kind of pathogenic fungi in beetle nests showed a strong negative growth response with rising ethanol levels between 0 and 5% in growth media, whereas several ambrosia fungi were almost insensitive to ethanol (growth is constant) or even showed the best growth at 1% ethanol concentration. In the future I plan to screen symbionts for their capabilities to produce ethanol and specifically test these in growth-inhibition assays against pathogens and cultivars.
But can beetles also promote the growth of their nutritional ambrosia fungi? It is likely as these fungi produce nutrient-rich fruiting structures only in the presence of the beetles. Fascinatingly, my assays between larvae, pupae and adults of X. saxesenii also revealed that beetles spread specific symbionts, which induced the fruiting of the ambrosia fungus. Currently, we are characterizing the chemical compounds produced by this symbiont and test them individually with the fungus to find out about the underlying chemistry of this effect. This finding of a third microbial player in the beetle-fungus mutualism is a major breakthrough for research on fungus-farming insects as for all farming insects it has been unknown how the insects induce fruiting in their food fungi.
Finally, we tested whether ambrosia beetles use nitrogen-fixing bacteria to fertilize their crops. Screens of nitrogen-fixing genes in bacterial symbionts of X. saxesenii revealed that several strains may have nitrogen fixing abilities. These studies could not be finished yet, but if this pilot date holds true this would mean that ambrosia beetles as well as humans depend on nitrogen-fixing bacteria as fertilizers in their agriculture.
Even though the IEF Marie-Curie project is now finished, I will continue to work on the research direction and system developed during this project. I received a renowned Emmy Noether Fellowship by the German Science Foundation (DFG) to build my own group at the University of Würzburg, Germany. So all the objectives presented above will be investigated in more detail in the next years by me and my co-workers. I will especially focus research on the two major findings of this IEF project: (i) the importance of social behaviour for fungus farming and (ii) the microbial mechanisms underlying the fruiting of the ambrosia fungi and probably also the defence against pathogens. With the various technical and social skills I learned during this excellent period in the group of Prof. Martin Kaltenpoth at the MPI-CE I feel well equipped to become independent and lead my own research group in Würzburg. For some more information see the new website of my group: available online from June 1st 2017
The main research objective of this project was to unravel some of the behavioural and chemical mechanisms of fungus farming by ambrosia beetles: Can ambrosia beetles actively influence the species community of their fungus gardens? If so, can beetles defend their gardens against fungal pathogens? How do they promote the growth of their fungal cultivars? And can they even fertilize their gardens? All these questions I tried to tackle in the last two years and although we do not have satisfactory replies on all answers, we made some exciting discoveries. These findings have the potential of completely changing the view on how we see these systems and will open up entirely new avenues of research.
First, we got quite clear indications that the ambrosia beetle Xyleborinus saxesenii can control the fungus species community growing in their gardens. Although final results of our experiment, where we removed beetles from half of the nests to check whether their presence affects fungus community, are not yet available, another experiment delivered quite clear indications. Together with a MSc student we found that adult females enhanced their general activity and especially their grooming frequencies immediately after we injected buffer solution with pathogenic fungus spores in laboratory nests (in comparison to a control group treated with sterile buffer solution). This grooming activity significantly reduced the pathogen-spore loads. This result is remarkable because it shows for the first time that hygienic behaviour is inducible in ambrosia beetles and that they show a social immune defence against pathogens comparable to that of eusocial societies of ants or bees.
What has remained unknown after this first finding is the mechanistic basis behind the disinfection of the pathogen spores. Possibly beetles apply antibiotics-producing microbes that inhibit growth of pathogens. To test this we looked at the growth-inhibition effect (which is an indication for antibiotics) of two symbionts (a yeast and a bacterium that are commonly found with this beetle) against two pathogenic mould fungi as well as two nutritional ambrosia fungi on different growth media in the laboratory. Both symbionts did not suppress the growth of the pathogens, so beetles must have other means to disinfect pathogens.
A potential chemical that might be produced by symbionts in the nest and which we found to selectively suppress growth of pathogens is ethanol. A field study had shown that beetles reproduce best in trees with high ethanol concentrations. This suggested that ambrosia fungi are adapted to substrates that contain ethanol, which is normally regarded mycotoxic. Indeed, we found that all kind of pathogenic fungi in beetle nests showed a strong negative growth response with rising ethanol levels between 0 and 5% in growth media, whereas several ambrosia fungi were almost insensitive to ethanol (growth is constant) or even showed the best growth at 1% ethanol concentration. In the future I plan to screen symbionts for their capabilities to produce ethanol and specifically test these in growth-inhibition assays against pathogens and cultivars.
But can beetles also promote the growth of their nutritional ambrosia fungi? It is likely as these fungi produce nutrient-rich fruiting structures only in the presence of the beetles. Fascinatingly, my assays between larvae, pupae and adults of X. saxesenii also revealed that beetles spread specific symbionts, which induced the fruiting of the ambrosia fungus. Currently, we are characterizing the chemical compounds produced by this symbiont and test them individually with the fungus to find out about the underlying chemistry of this effect. This finding of a third microbial player in the beetle-fungus mutualism is a major breakthrough for research on fungus-farming insects as for all farming insects it has been unknown how the insects induce fruiting in their food fungi.
Finally, we tested whether ambrosia beetles use nitrogen-fixing bacteria to fertilize their crops. Screens of nitrogen-fixing genes in bacterial symbionts of X. saxesenii revealed that several strains may have nitrogen fixing abilities. These studies could not be finished yet, but if this pilot date holds true this would mean that ambrosia beetles as well as humans depend on nitrogen-fixing bacteria as fertilizers in their agriculture.
Even though the IEF Marie-Curie project is now finished, I will continue to work on the research direction and system developed during this project. I received a renowned Emmy Noether Fellowship by the German Science Foundation (DFG) to build my own group at the University of Würzburg, Germany. So all the objectives presented above will be investigated in more detail in the next years by me and my co-workers. I will especially focus research on the two major findings of this IEF project: (i) the importance of social behaviour for fungus farming and (ii) the microbial mechanisms underlying the fruiting of the ambrosia fungi and probably also the defence against pathogens. With the various technical and social skills I learned during this excellent period in the group of Prof. Martin Kaltenpoth at the MPI-CE I feel well equipped to become independent and lead my own research group in Würzburg. For some more information see the new website of my group: available online from June 1st 2017