Species-specific responses of dew fly larvae to mycotoxins

Abstract   The larval stages of saprophagous insects and filamentous fungi have been demonstrated to be serious competitors on decaying organic matter. When filamentous fungi appear to be competitively superior, fungal mycotoxins have frequently been suggested to constitute chemical weapons, causing high mortality among insect larvae. In this study, we tested whether typical fungal secondary compounds can indeed be considered as the underlying mechanism of interference competition between filamentous fungi and various saprophagous Drosophila species. In contrast to our expectation, we found no grand mycotoxin-specific effects, but insect survival appeared to be generally determined by complex interaction between toxin identity, toxin concentration and insect species. Three out of five drosophilids seemed to be equally affected by the mycotoxins used in this study, whereas two species showed toxin-specific changes in survival. Only two (Kojic acid and Ochratoxin A) out of seven mycotoxins caused insect-specific responses. Moreover, we discovered correlations between survival in toxin-free and spoiled substrates, which may indicate an interrelationship between intra-specific competitive ability and resistance to mycotoxins. We discuss the significance of mycotoxins as underlying mechanisms driving competitive insect–fungus interactions.     

Fungal chemical defence alters density‐dependent foraging behaviour and success in a fungivorous soil arthropod

1. Aggregative behaviour in fungivorous soil arthropods is widespread; its adaptive value, however, is largely unknown. In this study, the spatial foraging behaviour of a collembolan, Folsomia candida, and the fitness consequences of feeding at different densities on the filamentous fungus Aspergillus nidulans were investigated. The effect of two fungal strains were compared; a wild‐type (wt) and a transgenic strain that lacks the ability to express the global secondary metabolite regulator LaeA (ΔlaeA). 2. In laboratory foraging tests, F. candida exhibited aggregated distributions of individuals across four distinct fungal colonies that were arranged in short distances from each other. By quantifying the extent of the feeding damage at each single colony, a more evenly distributed feeding activity was found among wt colonies than among chemical‐deficient colonies. 3. In a fitness experiment, where collembolans at different densities were restricted to feed on single A. nidulans colonies, mean growth rate of F. candida was positively related to density on the wt A. nidulans strain, but negatively related to density on the chemical‐deficient strain. 4. Depending on the fungus' ability to express secondary chemicals and availability of fungal food sources, F. candida may employ different foraging strategies: (i) avoidance of prolonged feeding on single colonies in a rich habitat (travel costs low), and (ii) intensified group feeding on single colonies in a resource‐limited habitat (travel costs high). It was hypothesised that flexibility in fungivore foraging behaviour (clumping vs. spreading feeding activity) is adaptive because it allows avoidance/overcoming induced fungal chemical defence.     

Heterologous expression system in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> for fungal biosynthetic gene clusters of secondary metabolites

Fungal secondary metabolites have been considered promising resources in the search for novel bioactive compounds. Given the high potential of fungi as genetic resources, it is essential to find an efficient way to link biosynthetic genes to the product in a heterologous system, because many genes for the secondary metabolite in the original strain are silent under standard laboratory conditions. In a previous study, we constructed a heterologous expression system for a biosynthetic gene cluster using Aspergillus oryzae as the host. To make the host more versatile for the expression of secondary metabolism genes, the expression levels of a global regulator, laeA, were increased by placing the A. oryzae laeA gene under the control of the constitutive active pgk promoter. In the A. oryzae overexpressing laeA, two clusters of heterologous biosynthetic genes [the monacolin K (MK) gene cluster from Monascus pilosus and the terrequinone A (TQ) gene cluster from Aspergillus nidulans] were successfully overexpressed, resulting in the production of the corresponding metabolite, MK or TQ. The successful production of secondary metabolites belonging to different structural groups, namely MK as a polyketide and TQ as a hybrid of amino acid and isoprenoid, indicated that the laeA-enriched A. oryzae was a versatile host for the heterologous expression of the biosynthetic gene cluster.     

Induced Fungal Resistance to Insect Grazing: Reciprocal Fitness Consequences and Fungal Gene Expression in the Drosophila-Aspergillus Model System

Background

Fungi are key dietary resources for many animals. Fungi, in consequence, have evolved sophisticated physical and chemical defences for repelling and impairing fungivores. Expression of such defences may entail costs, requiring diversion of energy and nutrients away from fungal growth and reproduction. Inducible resistance that is mounted after attack by fungivores may allow fungi to circumvent the potential costs of defence when not needed. However, no information exists on whether fungi display inducible resistance. We combined organism and fungal gene expression approaches to investigate whether fungivory induces resistance in fungi.

Methodology/Principal Findings

Here we show that grazing by larval fruit flies, Drosophila melanogaster, induces resistance in the filamentous mould, Aspergillus nidulans, to subsequent feeding by larvae of the same insect. Larval grazing triggered the expression of various putative fungal resistance genes, including the secondary metabolite master regulator gene laeA. Compared to the severe pathological effects of wild type A. nidulans, which led to 100% insect mortality, larval feeding on a laeA loss-of-function mutant resulted in normal insect development. Whereas the wild type fungus recovered from larval grazing, larvae eradicated the chemically deficient mutant. In contrast, mutualistic dietary yeast, Saccharomyces cerevisiae, reached higher population densities when exposed to Drosophila larval feeding.

Conclusions/Significance

Our study presents novel evidence that insect grazing is capable of inducing resistance to further grazing in a filamentous fungus. This phenotypic shift in resistance to fungivory is accompanied by changes in the expression of genes involved in signal transduction, epigenetic regulation and secondary metabolite biosynthesis pathways. Depending on reciprocal insect-fungus fitness consequences, fungi may be selected for inducible resistance to maintain high fitness in fungivore-rich habitats. Induced fungal defence responses thus need to be included if we wish to have a complete conception of animal-fungus co-evolution, fungal gene regulation, and multitrophic interactions.     

Fungal Secondary Metabolites and Their Fundamental Roles in Human Mycoses

Understanding which fungal factors allow colonization and infection of a human host is critical to lowering the incidence of human mycoses and related mortalities. In the pathogen Aspergillus fumigatus, secondary metabolites, small bioactive molecules produced by many opportunistic fungal pathogens, have important roles in suppressing and providing protection from host defenses. Deletion of LaeA, a global regulator of secondary metabolism in fungi, significantly decreases A. fumigatus virulence, in part owing to loss of gliotoxin and hydrophobin production. In addition to gliotoxin, dihydroxynaphthalene (DHN) melanin and siderophores are other A. fumigatus virulence factors; all three metabolites are derived from hallmark secondary metabolite gene clusters. Many of the gene clusters producing toxin metabolites have yet to be deciphered, and the study of secondary metabolites and their role in the virulence of human pathogens is a nascent field.     

LaeA regulation of secondary metabolism modulates virulence in Penicillium expansum and is mediated by sucrose

Penicillium expansum, the causal agent of blue mould rot, is a critical health concern because of the production of the mycotoxin patulin in colonized apple fruit tissue. Although patulin is produced by many Penicillium species, the factor(s) activating its biosynthesis are not clear. Sucrose, a key sugar component of apple fruit, was found to modulate patulin accumulation in a dose‐responsive pattern. An increase in sucrose culture amendment from 15 to 175 mm decreased both patulin accumulation and expression of the global regulator laeA by 175‐ and five‐fold, respectively, whilst increasing expression of the carbon catabolite repressor creA. LaeA was found to regulate several secondary metabolite genes, including the patulin gene cluster and concomitant patulin synthesis in vitro. Virulence studies of ΔlaeA mutants of two geographically distant P. expansum isolates (Pe‐21 from Israel and Pe‐T01 from China) showed differential reduction in disease severity in freshly harvested fruit, ranging from no reduction for Ch‐Pe‐T01 strains to 15%–25% reduction for both strains in mature fruit, with the ΔlaeA strains of Is‐Pe‐21 always showing a greater loss in virulence. The results suggest the importance of abiotic factors in LaeA regulation of patulin and other secondary metabolites that contribute to pathogenicity.     

Experimental evolution of resistance against a competing fungus in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Competition between microorganisms and arthropods has been shown to be an important ecological interaction determining animal development and spatial distribution patterns in saprophagous communities. In fruit-inhabiting Drosophila, variation in insect developmental success is not only determined by species-specific effects of various noxious filamentous fungi but, as suggested by an earlier study, also by additive genetic variation in the ability to successfully withstand the negative impact of the fungi. If this variation represents a direct adaptive response to the degree to which insect breeding substrates are infested with harmful fungi, genetic variation for successful development in the presence of fungi could be maintained by variation in infestation of resource patches with fungi. We selected for the ability to resist the negative influence of mould by maintaining replicated Drosophila melanogaster populations on substrates infested with Aspergillus nidulans. After five cycles of exposure to the fungus during the larval stage, the selected populations were compared with unselected control populations regarding adult survival and reproduction to reveal an evolved resistance against the fungal competitor. On fungus-infested larval feeding substrates, emerged adults from mould-selected populations had higher survival rates and higher early fecundity than the control populations. In the unselected populations, females had higher mortality rates than males, and a high proportion of both females and males appeared to be unable to lay eggs or fertilise eggs, respectively. When larvae developed on non-infested food we found indications of a loss of resistance to abiotic and starvation stress in the adult stage in flies from the selected populations. This suggests that there are costs associated with an increase in resistance against the microbial competitor. We discuss the underlying mechanisms that might have selected for increased resistance against harmful fungi.     

Genetic Variation and the Role of Insect Life History Traits in the Ability of Drosophila Larvae to Develop in the Presence of a Competing Filamentous Fungus

Competitive interactions between organisms from distantly related phylogenetical branches have been suggested as being one of the most pervasive forms of interspecific competition. However, so-called inter-kingdom competition has rarely been the focus of ecological and evolutionary studies. Thus, a relatively novel hypothesis has been proposed on the basis that saprophagous insects might intensively compete with filamentous fungi for ephemeral resources (e.g. decaying plant tissue). Consideration that life history traits (e.g. developmental time) are adaptive in determining developmental success in the presence of con- or hetero-specifics competitors implies that these traits have been progressively established by natural selection. Because a similar scenario may apply to antagonistic interactions between saprophagous insects and filamentous fungi, one can expect the existence of heritable variation in developmental success when insect larvae are forced to grow in the presence of noxious mould. Therefore, this study aimed at discovering whether a local population of Drosophila melanogaster indeed harbours genetic variation in developmental success in the presence of the mould Aspergillus niger. By using the isofemale line technique, single larvae forced to feed on fungal infected or uninfected substrate were analysed for variation in survival probability to the adult stage, developmental time and body size of emerged adults. I found genetic variation in survival probability in fungal infected substrates but not in uninfected larval food sources. Mean developmental time and body size varied significantly among isofemale lines in both types of larval environment. Survival was negatively correlated with developmental time on fungal infected substrate, but variation in developmental time on fungal-free substrates was not correlated with survival on fungal infected food patches. Within-trait correlation between fungal infected and uninfected substrates was surprisingly weak, and developmental time was not correlated with body size. The results of this study demonstrate (a) the existence of genetic variation for larval developmental success in the presence of A. niger in a Drosophila population, and (b) heritability of important insect life history traits differed as a function of the larval environment (fungal infected or uninfected feeding substrate). I discuss models that might explain heritability differences and the evolutionary consequences of these results.     

The global regulator LaeA controls production of citric acid and endoglucanases in <Emphasis Type="Italic">Aspergillus carbonarius</Emphasis>

The global regulatory protein LaeA is known for regulating the production of many kinds of secondary metabolites in Aspergillus species, as well as sexual and asexual reproduction, and morphology. In Aspergillus carbonarius, it has been shown that LaeA regulates production of ochratoxin. We have investigated the regulatory effect of LaeA on production of citric acid and cellulolytic enzymes in A. carbonarius. Two types of A. carbonarius strains, having laeA knocked out or overexpressed, were constructed and tested in fermentation. The knockout of laeA significantly decreased the production of citric acid and endoglucanases, but did not reduce the production of beta-glucosidases or xylanases. The citric acid accumulation was reduced with 74–96 % compared to the wild type. The endoglucanase activity was reduced with 51–78 %. Overexpression of LaeA seemed not to have an effect on citric acid production or on cellulose or xylanase activity.     

Heterochromatic marks are associated with the repression of secondary metabolism clusters in Aspergillus nidulans

Fungal secondary metabolites are important bioactive compounds but the conditions leading to expression of most of the putative secondary metabolism (SM) genes predicted by fungal genomics are unknown. Here we describe a novel mechanism involved in SM‐gene regulation based on the finding that, in Aspergillus nidulans, mutants lacking components involved in heterochromatin formation show de‐repression of genes involved in biosynthesis of sterigmatocystin (ST), penicillin and terrequinone A. During the active growth phase, the silent ST gene cluster is marked by histone H3 lysine 9 trimethylation and contains high levels of the heterochromatin protein‐1 (HepA). Upon growth arrest and activation of SM, HepA and trimethylated H3K9 levels decrease concomitantly with increasing levels of acetylated histone H3. SM‐specific chromatin modifications are restricted to genes located inside the ST cluster, and constitutive heterochromatic marks persist at loci immediately outside the cluster. LaeA, a global activator of SM clusters in fungi, counteracts the establishment of heterochromatic marks. Thus, one level of regulation of the A. nidulans ST cluster employs epigenetic control by H3K9 methylation and HepA binding to establish a repressive chromatin structure and LaeA is involved in reversal of this heterochromatic signature inside the cluster, but not in that of flanking genes.     

Key role of LaeA and velvet complex proteins on expression of β-lactam and PR-toxin genes in <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>: cross-talk regulation of secondary metabolite pathways

Penicillium chrysogenum is an excellent model fungus to study the molecular mechanisms of control of expression of secondary metabolite genes. A key global regulator of the biosynthesis of secondary metabolites is the LaeA protein that interacts with other components of the velvet complex (VelA, VelB, VelC, VosA). These components interact with LaeA and regulate expression of penicillin and PR-toxin biosynthetic genes in P. chrysogenum. Both LaeA and VelA are positive regulators of the penicillin and PR-toxin biosynthesis, whereas VelB acts as antagonist of the effect of LaeA and VelA. Silencing or deletion of the laeA gene has a strong negative effect on penicillin biosynthesis and overexpression of laeA increases penicillin production. Expression of the laeA gene is enhanced by the P. chrysogenum autoinducers 1,3 diaminopropane and spermidine. The PR-toxin gene cluster is very poorly expressed in P. chrysogenum under penicillin-production conditions (i.e. it is a near-silent gene cluster). Interestingly, the downregulation of expression of the PR-toxin gene cluster in the high producing strain P. chrysogenum DS17690 was associated with mutations in both the laeA and velA genes. Analysis of the laeA and velA encoding genes in this high penicillin producing strain revealed that both laeA and velA acquired important mutations during the strain improvement programs thus altering the ratio of different secondary metabolites (e.g. pigments, PR-toxin) synthesized in the high penicillin producing mutants when compared to the parental wild type strain. Cross-talk of different secondary metabolite pathways has also been found in various Penicillium spp.: P. chrysogenum mutants lacking the penicillin gene cluster produce increasing amounts of PR-toxin, and mutants of P. roqueforti silenced in the PR-toxin genes produce large amounts of mycophenolic acid. The LaeA-velvet complex mediated regulation and the pathway cross-talk phenomenon has great relevance for improving the production of novel secondary metabolites, particularly of those secondary metabolites which are produced in trace amounts encoded by silent or near-silent gene clusters.     

Host–parasitoid interaction as affected by interkingdom competition

Although still underrepresented in ecological research, competitive interactions between distantly related organisms (so-called “interkingdom competition”) are expected to be widespread in various ecosystems, with yet unknown consequences for, e.g. trophic interactions. In the model host–parasitoid system Drosophila melanogaster–Asobara tabida, toxic filamentous fungi have been shown to be serious competitors that critically affect the density-dependent survival of host Drosophila larvae. This study investigates the extent to which the competing mould Aspergillus niger affects key properties of the well-studied Drosophila–parasitoid system and how the host–parasitoid interaction influences the microbial competitor. In contrast to slightly positive density-dependent host mortality under mould-free conditions, competing A. niger mediated a strong Allee effect for parasitised larvae, i.e. mortality decreased with increasing larval density. It was found that the common toxic fungal metabolite kojic acid is not responsible for higher death rates in parasitised larvae. Single parasitised Drosophila larvae were less harmful to fungal reproduction than unparasitised larvae, but this effect vanished with an increase in larval density. As predicted from the negative effect of fungi on host survival and thus on parasitoid fitness at low larval densities, A. tabida females spent less time foraging in fungus-infested patches. Interestingly, even though high host larval densities increased host survival, parasitoids still reduced their search efforts in fungus-infested patches, indicating a benefit for host larvae from feeding in the presence of noxious mould. Thus, this experimental study provides evidence of the potentially important role of interkingdom competition in determining trophic interactions in saprophagous animal communities and the dynamics of both host–parasitoid and microbial populations.     

Fungus-Specific Sirtuin HstD Coordinates Secondary Metabolism and Development through Control of LaeA

The sirtuins are members of the NAD⁺-dependent histone deacetylase family that contribute to various cellular functions that affect aging, disease, and cancer development in metazoans. However, the physiological roles of the fungus-specific sirtuin family are still poorly understood. Here, we determined a novel function of the fungus-specific sirtuin HstD/Aspergillus oryzae Hst4 (AoHst4), which is a homolog of Hst4 in A. oryzae yeast. The deletion of all histone deacetylases in A. oryzae demonstrated that the fungus-specific sirtuin HstD/AoHst4 is required for the coordination of fungal development and secondary metabolite production. We also show that the expression of the laeA gene, which is the most studied fungus-specific coordinator for the regulation of secondary metabolism and fungal development, was induced in a ΔhstD strain. Genetic interaction analysis of hstD/Aohst4 and laeA clearly indicated that HstD/AoHst4 works upstream of LaeA to coordinate secondary metabolism and fungal development. The hstD/Aohst4 and laeA genes are fungus specific but conserved in the vast family of filamentous fungi. Thus, we conclude that the fungus-specific sirtuin HstD/AoHst4 coordinates fungal development and secondary metabolism via the regulation of LaeA in filamentous fungi.     

Saprophagous insect larvae,Drosophila melanogaster,profit from increased species richness in beneficial microbes

Female fruit flies, Drosophila melanogaster, lay their eggs on decaying plant material. Foraging fly larvae strongly depend on the availability of dietary microbes, such as yeasts, to reach the adult stage. In contrast, strong interference competition with filamentous fungi can cause high mortality among Drosophila larvae. Given that many insects are known for employing beneficial microbes to combat antagonistic ones, we hypothesized that fly larvae engaged in competition with the noxious mould Aspergillus nidulans benefit from the presence of dietary yeast species, especially when they are associated with increasingly species rich yeast communities (ranging from one to six yeast species per community). On a nutrient‐limited fruit substrate infested with A. nidulans, both larval survival and development time were positively affected by more diverse yeast communities. On a mould‐free fruit substrate, merely larval development but not survival was found to be affected by increasing species richness of dietary yeasts. Not only yeast diversity had an effect on D. melanogaster life‐history traits, but also the identity of the yeast combinations. These findings demonstrate the importance of the structure and diversity of microbial communities in mutualistic animal–microbe interactions.     

Aspergillus oryzae laeA Regulates Kojic Acid Synthesis Genes

Kojic acid synthesis genes regulation was investigated in Aspergillus oryzae. Our results indicate that kojic acid production was lost in the laeA disruption strain, but was recovered in the LaeA complement strain. Real-time PCR also confirmed that expression of kojic acid biosynthesis genes decreased in the laeA disruption strain, indicating that these genes are under the control of LaeA.     

Lack of Host Specialization in Aspergillus flavus

Aspergillus spp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans for their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains of A. flavus did not affect nonwounded bean leaves, corn kernels, or insects at 22°C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton bolls, is produced by most A. flavus isolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. All A. flavus strains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast to A. flavus, strains of A. nidulans and A. fumigatus could not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate that A. flavus has parasitic attributes that are lacking in A. fumigatus and A. nidulans but that individual strains of A. flavus are not specialized to particular hosts.