The role of fungal secondary metabolites in Collembola ― fungi interactions

SUMMARY Soil organisms, in particular fungi and decomposer insects are primary drivers of organic matter recycling and energy fluxes (Swift et al. 1979; Cadish and Giller 1997; Bardgett et al. 2005). Fungi play a crucial role in the cycling of carbon, nitrogen and phosphorus in terrestrial ecosystems functioning while having to deal in the same time with relentless attacks from fungivores. Only few studies, however, investigated the structuring forces of the population dynamics of fungi and the abundant decomposer fungivores, such as Collembola, with whom they continuously interact. This thesis investigated the interactions between fungi and Collembola focussing particularly on the effects of fungal secondary metabolites from different perspectives. Fungal secondary metabolites are believed to be one of the main vectors driving this interaction. Aiming to get specific insights into the nature of the mechanisms driving this interaction I focused on testing three overarching hypothesis: H1. Fungal secondary compounds mediate the Collembola – fungi interaction H2. Collembola have evolved means to detect fungal toxicity H3. Genetic evidence (transcript regulation) can be used to understand the molecular nature of the Collembola – fungi interaction The above three overarching hypothesis have been addressed in three experimental studies, each with several pointed hypothesis. H1. The first experimental study consisted of a feeding choice experiment offering single and mixed fungal diets using labelled fungal species (C3 and C4; 13C and 15N) of different toxicity. Collembola fractionation and carbon/ nitrogen incorporation of fungal species were assessed via stable isotope analysis. Four knock out mutants of Aspergillus nidulans with the sterigmatocystin production blocked at different steps along the biosynthetic pathway were combined in mixed diets with either the high quality fungus Cladosporium cladosporioides or the low quality fungus A. nidulans (wildtype). This study aimed at understanding the impact of fungal secondary metabolites and more specifically sterigmatocystin (ST) on Collembola performance in single and mixed diets and stabile isotope fractionation. It was hypothesised that (i) presence of sterigmatocystin (ST) impairs Collembola performance with increasing fungal toxicity of the A. nidulans strains, (ii) mixed diets will be beneficial to Collemboal fitness due to toxin dilution and (iii) the fractionation of 13C and 15N it is more pronounced in more toxic diets. We found that ST generally but not uniformly diminished springtail SUMMARY v fitness partially supporting the idea that secondary compounds act as shield against fungivory. However, the use of knockout mutants A. nidulans of the ST pathway (S3-S6) led to rather idiosyncratic responses. Although Collembola fitness was not uniformly increased in mixed diets (suggesting a species specific response) the results still support the toxin dilution hypothesis since no correlation between fungal N content and ingestion could be found. Strong and specific responses of the two Collembola species to mixed diets, knock out mutants and toxins suggest the evolution of species specific strategies to cope with the constraints associated with living in different soil layers. The hypothesis suggesting a link between stable isotope fractionation and fungal toxins has been partially supported with the results suggesting that fungal toxin content may be more important than the nutrient content in controlling stable isotope fractionation of 13C and 15N. H2. The second study focused on the olfactory ability of Collembola to perceive fungal toxicity via olfactory/volatile cues. By means of an olfactometer approach this experiment hypothesized that (i) Collembola are able to olfactorily perceive and distinguish fungal species/strains differing in secondary metabolism, (ii) that Collembola are able to sense and respond to fungal grazing by avoiding to forage on grazed fungi and that (iii) grazing by Collembola triggers in secondary metabolite gene expression in one Basidiomycete and one Ascomycete fungal species using a custom made cDNA microarrays (Chapter 3). All investigated Collembola species recognized fungal olfactory cues and directed their movement to fungal patches and moreover towards fungal strains with suppressed secondary metabolites, in particular towards the mutant ΔlaeA with the main part of secondary metabolites silenced. The volatile cues of conspecifically grazed fungi provoked a movement from two of the three Collembola species (H. nitidus and S. furcifera) towards ungrazed fungi. However, the response of S. furcifera was restricted to fungi extensively exposed to grazing (5 days) suggesting that the response varies between Collembola species. Surprisingly, the investigated fungal gene spectrum did not significantly respond to grazing by Collembola. The results support the first and second hypothesis indicating that Collembola are able to olfactorily differentiate fungi of different toxicity, orientate their movement towards more palatable fungi and avoid movement towards fungi previously exposed to grazing. The lack of changes in fungal gene regulation by grazing suggests that refined methods need to be adopted to investigate the genetic response of fungi to grazing. SUMMARY vi H3. The third study investigated the impact of fungal secondary metabolites on transcript regulation of stress related expressed sequence tags (ESTs) of Folsomia candida, the Collembola species used as model species in ecotoxicology. Aspergillus nidulans wildtype (WT; Ascomycota) able to produce secondary metabolites including sterigmatocystin (ST) and a knockout mutant with reduced secondary metabolism (A. nidulans ΔLaeA) were combined with the high quality fungus C. cladosporioides as mixed diets or offered as single diets. I hypothesized that (i) A. nidulans WT triggers more genes associated with stress responses compared to the A. nidulans ΔlaeA strain with suppressed secondary metabolism, (ii) C. cladosporioides causes significantly different transcript regulation than the A. nidulans strains ΔlaeA and WT, and (iii) mixed diets will cause significantly different transcript expression levels than single diets. All three hypotheses are generally supported despite the fact that many functions of the affected ESTs are unknown. The results bring molecular evidence for the existence of a link between fungal secondary metabolites and responses in springtails supporting the hypothesis that fungal secondary metabolites act as a shield against fungivory. Overall, the work conducted in this thesis suggests that fungal secondary metabolites act as a structuring force in Collembola-fungi interactions and population dynamics. Using multiple approaches (food choice, olfactory and genetical) the results brings new insights supporting the hypothesis that fungal secondary metabolites act as a shield against fungivory.