Coevolution of parasitic fungi and insect hosts

Accepted Manuscript Title: Coevolution of parasitic fungi and insect hosts Author: Gerrit Joop Andreas Vilcinskas PII: DOI: Reference:

S0944-2006(16)30055-1 http://dx.doi.org/doi:10.1016/j.zool.2016.06.005 ZOOL 25522

To appear in: Received date: Revised date: Accepted date:

14-11-2015 26-2-2016 15-6-2016

Please cite this article as: Joop, Gerrit, Vilcinskas, Andreas, Coevolution of parasitic fungi and insect hosts.Zoology http://dx.doi.org/10.1016/j.zool.2016.06.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REVIEW

Coevolution of parasitic fungi and insect hosts Gerrit Joopa,*, Andreas Vilcinskasa, b

a

Institute for Insect Biotechnology, Justus Liebig University of Giessen, Heinrich-Buff-Ring

26-32, D-35392 Giessen, Germany b

Department of Bioresources, Fraunhofer Institute for Molecular Biology and Applied

Ecology, Winchesterstrasse 2, D-35394 Giessen, Germany

*

Corresponding author.

E-mail address: [email protected] (G. Joop).



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Graphical abstract

Initially resistant host: Tribolium castaneum

Initially susceptible host: Galleria mellonella

Overlapping generations of T.  castaneum: larvae, pupae and  adults live together.

The original barrier: • Resistant due to external secretion of  quinones (MBQ & EBC) by adult beetles,  which coat the cuticle and disperse into the  flour environment. • Causing oxidative stress in the fungus,  resulting in growth inhibition.

The new virulence factor: • Fungal gain in resistance towards external  secretion. • Increased metabolic activity in coevolved  fungi. • Differences in gene expression profile upon  MBQ exposure in ancestral vs. coevolved  fungi: most important GO term is ‚pathogenesis‘,  including several genes relevant in  resistance to oxidative stress.

(Rafaluk et al., unpublished data; Pedrini et al., 2015;  Leal, 2015)

Larvae of G. mellonella,  with fungal infection  (black spots).

loss

Host resistance

gain

A snapshot of  host–parasite coevolution contrasting evolutionary  outcome in two  model host systems

gain

Parasite  virulence

The new barrier: • Melanic larvae are more resistant towards  fungal infection. • During coevolution larvae specifically  upregulate defence systems in the cuticle  and epidermis of the integument (IMPI, PO)  upon fungal exposure. • Stress management factors also become  upregulated. (Dubovskiy et al., 2013 a & b)

The original virulence factor: 2.  Germination upon host  contact

loss 1. Conidia  spores

Parasitic fungi: Beauveria bassiana &  Metarhizium anisopliae

6. Typical  ‘white bloom’  when growing  out of host,  production  of  new conidia

In insect host: asexually reproducing  Beauveria bassiana anamorph

3. Formation of germ  tube, starting  enzymatic secretion  to dissolve the  insect’s cuticle 4. Hyphal growth into host,  production  of  beauvericin to  weaken host’s  immune system

5. After host death  production of  oosporein, an  antibiotic, to  prevent being  outcompeted  by  bacteria

(Nikoh & Fukatsu, 2000)

Insect haemolymph with hyphae of M. anisopliae and apoptotic haemocytes.



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Highlights 

Coevolution between virulence-associated proteinases and immunity-related proteinase inhibitors plays a major role in the arms race between parasitic fungi and their insect hosts.



Snapshot of host–parasite coevolution: experimental coevolution as a powerful tool.



Comparative analysis of experimental host–parasite coevolution in two model insects.

 



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Abstract Parasitic fungi and their insect hosts provide an intriguing model system for dissecting the complex co-evolutionary processes, which result in Red Queen dynamics. To explore the genetic basis behind host–parasite coevolution we chose two parasitic fungi (Beauveria bassiana and Metarhizium anisopliae, representing the most important entomopathogenic fungi used in the biological control of pest or vector insects) and two established insect model hosts (the greater wax moth Galleria mellonella and the red flour beetle Tribolium castaneum) for which sequenced genomes or comprehensive transcriptomes are available. Focusing on these model organisms, we review the knowledge about the interactions between fungal molecules operating as virulence factors and insect host-derived defense molecules mediating antifungal immunity. Particularly the study of the intimate interactions between fungal proteinases and corresponding host-derived proteinase inhibitors elucidated novel coevolutionary mechanisms such as functional shifts or diversification of involved effector molecules. Complementarily, we compared the outcome of coevolution experiments using the parasitic fungus B. bassiana and two different insect hosts which were initially either susceptible (Galleria mellonella) or resistant (Tribolium castaneum). Taking a snapshot of host–parasite coevolution, we show that parasitic fungi can overcome host barriers such as external antimicrobial secretions just as hosts can build new barriers, both within a relatively short time of coevolution. Keywords: External immune defense; Host–parasite coevolution; Immunity; Tribolium castaneum; Galleria mellonella



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1. Introduction Host–parasite coevolution is a special form of coevolution involving reciprocal adaptive genetic changes in two antagonists, i.e. the particular host and parasite species, based on the selective pressures each partner confers on the other (Woolhouse et al., 2002; also see Milutinovic et al., 2016, current issue). The Red Queen hypothesis proposing continuous adaptation merely to maintain parity with other evolving species (Van Valen, 1973) is clearly demonstrated by the arms race between hosts and parasites (for a description of different modes within the Red Queen, compare Brockhurst et al., 2014). The inspiration for this hypothesis is the following quote: “Now, here, you see, it takes all the running you can do, to keep in the same place” (Caroll, 1871). However, the runners during coevolution do not keep pace – instead, one or the other may always be ahead. This involves adaptation and counter-adaptation, and it is most interesting to understand how the evolution of resistance in the host is matched by the evolution of virulence in the parasite, and vice versa. Insects and other invertebrates represent excellent model hosts because of their short generation times, allowing us to consider evolutionary strategies against a range of pathogens and parasites, including helminths, protozoa, bacteria and even viruses. For simplicity in the following we will refer to all these organisms as parasites. One well-known study system for host–parasite coevolution, and in support of the Red Queen hypothesis, is the aquatic invertebrate Daphnia spp. and its parasites (reviewed in Ebert, 2008). In this same system Decaestecker and colleagues (2007) took advantage of the natural preservation of spatiotemporal interactions in different layers of lake sediment. This natural preservation is not possible in most systems, so researchers have turned to models that can be reared and tested in experimental settings. Experimental host–parasite coevolution is a powerful tool to investigate the evolution of host defense mechanisms and resistance as well as parasite virulence factors (e.g., Schulte et al., 2010; Masri et al., 2015; see Brockhurst and Koskella, 2013 for a review). Samples taken after different time points provide insight into evolutionary adaptations and counter-adaptations and such experiments have been carried out



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using model hosts such as Caenorhabditis elegans (Schulte et al., 2010; Masri et al., 2015), Daphnia spp. (Carius et al., 2001) and Tribolium castaneum (Wegner et al., 2008). Most of these experiments have considered bacterial parasites (but see Wegner et al., 2008), whereas fungi are neglected parasites of insects despite their importance (Madelin, 1966). Furthermore, as resistance against pesticides is spreading through populations of insect pests and vectors (Metcalf, 1989; Hemingway and Ranson, 2000), the search for alternative biological pest control agents is becoming more important. One such approach is indeed the application of entomopathogenic fungi (Ferron, 1978), which are natural parasites of insects (Nikoh and Fukatsu, 2000) that share several million years of coevolution with their hosts, making them ecologically important natural regulators of insect populations in almost all ecosystems (OrtizUrquiza et al., 2015). We here review coevolution experiments using the entomopathogenic fungus Beauviera bassiana as parasite (Dubovskiy et al. 2013 a, b; Rafaluk et al., unpublished data), which has been established as a “model parasite” for the screening of genes involved in virulence (ValeroJimenez et al., 2016). Coevolutionary outcome differed substantially depending on whether a susceptible host (the greater wax moth, Galleria mellonella) or a resistant host (the red flour beetle, Tribolium castaneum) was used. The susceptible host became resistant during the course of the experiment due to increased host resistance, whereas the resistant host became more susceptible because the parasite became more virulent. Both approaches yield important insight into the nature of adaptation and counter-adaptation and increase our understanding of the evolution of virulence and resistance.

2. Red Queen dynamics between fungal parasites and their insect hosts In order to explore the genetic basis of host–parasite coevolution we selected two parasitic fungi and two model host insects for which either completely sequenced genomes or comprehensive transcriptomic data are available. Beauviera bassiana and Metarhizium anisopliae are the most



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important parasitic fungi used as biological control agents against vector and pest insects. M. anisopliae was the first entomopathogenic fungus for which a whole sequenced genome became available (Gao et al., 2011; Staats et al., 2014). The genomic sequence of B. bassiana revealed similar adaptations to the parasitic life style as that of M. anisopliae (Xiao et al., 2012). Both fungi can kill a wide range of insects but occur in a variety of strains, which exhibit pronounced virulence against particular insect hosts. These fungi also share the ability to infect their insect hosts directly through the cuticle (Vilcinskas and Götz, 1999; Gillespie et al., 2000). For our comparative analysis of host–parasite coevolution between parasitic fungi and their host insects we selected the greater wax moth G. mellonella and the red flour beetle T. castaneum. The latter is a pest of stored products that occurs worldwide and has emerged as a model in developmental, molecular and evolutionary biology. It has therefore been selected as the first beetle for which a complete genome has been sequenced (Tribolium Genome Sequencing Consortium, 2008). Its immune gene repertoire has been analyzed by different methodological approaches (Zou et al., 2007; Altincicek et al., 2008). And it became a valuable model host for testing hypotheses addressing interactions of insects with pathogens and transgenerational immune priming (Roth et al., 2010; Knorr et al., 2015; Milutinovic et al., 2016, current issue). G. mellonella represents a classic model host for entomopathogenic fungi (Vilcinskas and Götz, 1999) and has recently also emerged as a model host for human pathogens, with its repertoire of immunity-related defense molecules being intensively studied (Vilcinskas, 2011a, b). Its value as a model host has been remarkably expanded since a comprehensive transcriptomic database enriched for immunity-related genes (Vogel et al., 2011) and microRNAs has become available (Mukherjee and Vilcinskas, 2014). Regarding the reconstruction of the Red Queen dynamics between parasitic fungi and their insect hosts the selected model organisms provide the advantage that their interacting molecules have been functionally characterized (Fig. 1). The innate immune system of G. mellonella relies on a broad spectrum of antimicrobial peptides (AMPs) (Vilcinskas, 2011a) among which, e.g.,



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the defensin-like peptide gallerimycin displays exclusive activity against filamentous fungi, including M. anisopliae (Schuhmann et al., 2003; Langen et al., 2006). In contrast, the three defensins identified in T. castaneum display only activity against Gram-positve bacteria (Tonk et al., 2015). Instead, this beetle was shown to possess thaumatin-like peptides, which were previously only known from plants. A recombinantly expressed T. castaneum thaumatin was shown to inhibit germination of B. bassiana conidia (Knorr et al., 2009). These studies imply that insects have independently acquired AMPs mediating immunity against fungi (Mylonakis et al., 2016). As a counterstrategy against the presence of inducible antifungal peptides in insects these parasitic fungi have independently evolved the ability to suppress innate immune responses in the infected host (Vilcinskas and Matha, 1997b; Vilcinskas et al., 1997a, b). Aside from the suppression of the AMP synthesis in the infected host, entomopathogenic fungi produce virulence-associated proteinases capable of both digesting host defense molecules including AMPs and suppressing its cellular immune responses (Griesch and Vilcinskas, 1998). The countermeasures of insects against fungal infection encompass the co-option of inducible peptidic inhibitors for antifungal immune responses to neutralize fungal proteases operating as virulence factors (Vilcinskas and Wedde, 1997). The simultaneous synthesis of AMPs along with peptidic inhibitors against fungal proteinases during immune responses results in their synergistic activity against fungi. The intimate interaction between virulence-associated proteinases of parasitic fungi and immunity-related proteinase inhibitors of G. mellonella provides the possibility to study host–parasite coevolution directly at the frontline. We will address this topic in more detail in Section 3. Another interesting fact is that B. bassiana and M. anisopliae are known to produce secondary metabolites, e.g. cyclic peptides such as beauverolides, cyclosporines or destruxins which have been implicated in contributing to the suppression of immune response in the infected host (Vilcinskas et al., 1999). For example, destruxins produced by M. anisopliae have been shown to induce apoptosis in immune competent hemocytes of G. mellonella (Vilcinskas et al.,



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1997b). Cyclosporin A, which is produced by B. bassiana and other entomopathogenic fungi, inhibits P-glycoprotein which mediates detoxification by removing a variety of toxic compounds from cells. Inhibition of this membrane-associated ATP-dependent efflux pump in an infected insect hosts promotes its poisoning by toxic compounds taken up, e.g., with its plant diet (Podsiadlowski et al., 1998). Despite the high toxicity of cyclosporin A for dipterans, G. mellonella was found to be highly resistant even to high concentrations which were either injected or orally administered. The resistance of G. mellonella against this fungal toxin has been attributed to its lipophorins, which mediate binding and storage of the compound in the fat body in a physiologically inactive form (Vilcinskas et al., 1997c). In contrast to the lepidopteran G. mellonella, the antifungal defense of the beetle T. casteaneum against fungi encompasses phenoloxidase activity (Yokoy et al., 2015), antifungal peptides (Altinciek et al., 2008) and secreted quinones (Li et al., 2013, Pedrini et al., 2015). The latter are produced by stink glands and mediate the sanitation of the microhabitat (Joop et al., 2014).

3. Coevolution of fungal proteinases as virulence factors and of insect proteinase inhibitors as host barriers All parasites including fungi produce proteinases to prepare the infected host as a source of nutrients and to degrade host defense molecules. Fungal parasites of insects may even need proteinases to initiate infection if their primary route of ingress is the direct penetration of the proteinaceous cuticle, which provides a robust physical barrier against most microbes. Proteinases that enable fungi such as B. bassiana and M. anisopliae to penetrate the insect cuticle and damage host immune responses have been recognized as virulence factors (Griesch and Vilcinskas, 1998; Santi et al., 2010). Insects are not defenseless against such invasive fungal proteinases. For example, innate immune responses in G. mellonella include three serine proteinase inhibitors (6.3–9.2 Da) that are induced and secreted into the hemolymph along with antimicrobial peptides, thus inhibiting the serine proteinases produced by B. bassiana and M.



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anisopliae. All three serine proteinase inhibitors inhibit the trypsin-like proteinase (Pr2) produced by M. anisopliae, and one is also active against the chymoelastase produced by the same species (Vilcinskas and Wedde, 1997; Fröbius et al., 2000; Vilcinskas, 2011b). Theory suggests that only proteinases that are not or not completely inactivated by proteinase inhibitors present in the host cuticle, tissues or hemolymph can operate as virulence factors (Vilcinskas and Götz, 1999; Vilcinskas, 2010), as parasites would waste resources if they synthesized proteinases susceptible to host proteinase inhibitors. We therefore hypothesized that fungal proteinases lacking corresponding host proteinase inhibitors would be subject to positive selection, because these enzymes would fulfill their pathogenesis-related functions even when produced in relatively small amounts (Vilcinskas, 2010). Indeed, M. anisopliae synthesizes a thermolysin-like metalloproteinase with precisely these properties (St Leger et al., 1994). Metalloproteinases of the M4 family (the prototype is thermolysin from Bacillus thermoproteolyticus) have been recognized as active virulence factors in most human parasites because vertebrates largely failed to evolve corresponding inhibitors during host–parasite coevolution (Adekoya and Sylte, 2009). Insects produce tissue inhibitors of matrix metalloproteinases (TIMPs) but generally they do not produce specific inhibitors of microbial metalloproteinases (Vilcinskas and Wedde, 2002). However, it is also predicted that the coevolution between parasites and hosts will result in positive selection for proteinase inhibitors that neutralize virulence-associated microbial enzymes (Vilcinskas, 2010). The first peptide-based and specific inhibitor of microbial metalloproteinases was thus discovered in the hemolymph of immune-challenged G. mellonella larvae (Wedde et al., 1998) and this finding provided a unique opportunity and starting point to test for the existence of reciprocal adaptations.

3.1. The insect metalloproteinase inhibitor



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The insect metalloproteinase inhibitor (IMPI) is an immunity-related G. mellonella peptide with a molecular mass of 8364 Da, which is stable against heat and acids due to the presence of five intramolecular disulfide bonds. The corresponding cDNA was identified by injecting G. mellonella larvae with bacterial lipopolysaccharide and screening for induced genes, using suppression subtractive hybridization (Seitz et al., 2003). The IMPI open reading frame gene comprises 510 nucleotides, corresponding to 170 amino acids. The amino acid sequence is not homologous to any other known peptide or protein (Wedde et al., 1998). However, searching the Pfam database (Finn et al., 2014) for conserved protein domains revealed significant similarity to a trypsin inhibitor-like cysteine-rich domain (Clermont et al., 2004) and IMPI was therefore assigned to the protease inhibitor family I8 (Rawlings et al., 2004). The data summarized above inspired us to postulate that IMPI is the outcome of the coevolution between G. mellonella and its fungal pathogens. To test this hypothesis, we produced recombinant IMPI in Escherichia coli and co-crystalized the peptide with thermolysin from B. thermoproteolyticus in order to determine its three-dimensional structure (Arolas et al., 2011). A complete diffraction dataset was collected at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The 3D-structure supports that this immunity-related defense molecule originates from serine proteinase inhibitors belonging to the I8 family. Strikingly, the IMPI represents the first reported example of a peptidic inhibitor whose reactive site loop has been adapted during host–parasite coevolution to specifically inhibit another class of proteinases, and this unique evolutionary origin may explain why the IMPI displays an unprecedented mechanism of metalloproteinase inhibition (Arolas et al., 2011). Because pathogens and parasites tend to have shorter generation times than their hosts, their capacity to generate new virulence factors through random mutation and selection is high. Accordingly, they have evolved enzymes which enable them to degrade host proteins such as collagen, which are normally resistant against proteolytic digestion. Also, M. anisopliae produces a number of metalloproteinase isoforms that differ in their capacity to degrade denaturated collagen (Qazi and Khachatourians, 2007). We screened the recently published M.



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anisopliae genome (Gao et al., 2011) as well as the available sequences of other entomopathogenic fungi (Hu et al., 2014; Pattemore et al., 2014) and found a number of distinct genes encoding thermolysin-like metalloproteinases (Fig. 2). In order to test our hypothesis that the diversification of parasite proteinases promotes host selection for mechanisms allowing the reciprocal diversification of host-derived proteinase inhibitors, we analyzed the G. mellonella immunity-related transcriptome with a special focus on IMPI on the GS FLX platform (454 Life Sciences/Roche, Branford, CT, USA) combined with traditional Sanger sequencing to obtain a comprehensive transcriptome dataset (Vogel et al., 2011). Sequence diversity was maximized by pooling RNA extracted from different developmental stages, larval tissues (including hemocytes), and immune-challenged larvae. We identified three immunity-inducible transcripts encoding the previously discovered inhibitors of serine proteinases produced by M. anisopliae (Fröbius et al., 2000) as well as eight genes encoding putative homologs of IMPI, with signatures suggesting recent gene duplication events (Vogel et al., 2011). Domain duplication and shuffling by recombination are the most important forces driving the evolution of antimicrobial peptides and immunity-related proteinase inhibitors (Vogel et al., 2005; Vilcinskas, 2013). Accordingly, we found two IMPI peptide sequences on either side of a furin cleavage site. Both peptides, which appear to result from gene duplication and divergent evolution, were expressed as individual recombinant proteins and tested for their specificity, revealing that they were active against different metalloproteinases. IMPIα encoded by the N-terminal part of the IMPI sequence is active exclusively against microbial metalloproteinases whereas IMPIβ is not active against the virulence-associated metalloproteinases we tested, but shows moderate activity against matrix metalloproteinases (Wedde et al., 2007). Detailed analysis of the immunity-related G. mellonella transcriptome (Vogel et al., 2011) provides evidence for the recent diversification of the IMPI gene (Fig. 3). The diversification of both the thermolysin-like metalloproteinases in M. anisopliae (Qazi and Khachatourians, 2007) and IMPI genes in G. mellonella (Fig. 3)



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may therefore represent an intriguing example of reciprocal adaptations and counteradaptations between parasites and their hosts.

4. Experimental coevolution with an initially susceptible host We have successfully established G. mellonella infected with the entomopathogenic fungi B. bassiana and M. anisopliae as a model system for investigating host–parasite coevolution, including the molecular level, focusing on the evolutionary dynamics between parasiteassociated proteinases operating as virulence factors and host proteinase inhibitors involved in antifungal defense (Vilcinskas and Götz, 1999; Vilcinskas, 2010; Dubovskiy et al., 2013a,b). G. mellonella is susceptible to entomopathogenic fungi, and infection causes black spots to appear on the cuticle which represent the local activation of phenoloxidases to counteract fungal proteases resulting in the formation of melanin (Vilcinskas, 2010). Dubovskiy et al. (2013a) found that a naturally melanic morph of G. mellonella is less susceptible to infection by B. bassiana than a non-melanic morph. Although conidial adhesion and germination occurred in the same manner on both morphs, the mechanical and chemical defense of the cuticle was stronger in the melanic morph. Specifically, the cuticle was thicker, therefore taking longer to penetrate, and phenoloxidase activity as a first line of immune defense (Gillespie et al., 2000; Schmid-Hempel, 2011) was induced more strongly. In contrast, the hemolymph phenoloxidase activity was always higher in the non-melanic larvae, regardless of the infection status. Differences were also observed in immunity-related and stress-related gene expression. Basal expression in uninfected larvae was always higher in the melanic morph, with the exception of IMPI, which was expressed at a lower level. Following topical infection with the fungi, most of the immunity and stress genes were only marginally upregulated in the melanic morphs, whereas the non-melanic larvae experienced a strong induction of gene expression 24–48 h post-infection. Ultimately, the gene expression profiles in both morphs were the same, given that the low level of induction in the melanic morphs compensated for the



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higher constitutive expression level (Dubovskiy et al., 2013a). In agreement with the hypothesis that elevated immune defense comes at a cost (Schmid-Hempel, 2011), the melanic larvae gave rise to smaller pupae than the non-melanic larvae, potentially reflecting the shorter larval development period, and the oviposition rate was reduced in melanic individuals (Dubovskiy et al., 2013a). To determine whether the insects could evolve to acquire resistance to the fungi, experimental coevolution was carried out using G. mellonella and B. bassiana (Dubovskiy et al., 2013b). A melanic G. mellonella population was selected over 25 generations for increased resistance towards B. bassiana. The selected cohort displayed significantly enhanced resistance towards B. bassiana but not against M. anisopliae. These specific experimentally selected adaptations occurred rather rapidly and therefore might not be mediated by changes in the germline DNA sequence but imply the involvement of epigenetic mechanisms (Vilcinskas, 2016, current issue). Just as in the comparison of melanic and non-melanic morphs (Dubovskiy et al., 2013a), we found that phenoloxidase activity 24 h post-infection was higher in the cuticles of the exposed cohort compared to the controls. Hemolymph phenoloxidase activity was only induced in the previously non-exposed cohort upon exposure to M. anisopliae, whereas B. bassiana did not elicit a significant response. The immunity and stress-related genes discussed above were expressed at lower levels in the exposed cohort than in the unselected control cohort. In terms of the cost of immunity, melanism did not reduce the pupal biomass, but the larvae from the exposed cohort took longer to develop than those from the unselected cohort. Melanism in G. mellonela offers sufficient protection against fungal infection, but it comes at a cost. This defense mechanism is found naturally (Dubovskiy et al., 2013a) or can be the outcome of experimental coevolution (Dubovskiy et al., 2013b), indicating that natural melanism in this species may have evolved as a heritable response to fungal infection. Experimentally, resistance against B. bassiana did not offer simultaneous resistance against M. anisopliae, showing that the response is species-dependent rather than generic. This suggests the insect possesses a recognition apparatus that distinguishes among different species of fungal



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pathogens (Altincicek et al., 2009). Following parasite recognition, the host responds by activating its defense systems. Here in particular, the elevated expression of IMPI following the detection of B. bassiana is interesting because IMPI may inhibit the fungal metalloproteases produced during the course of infection (Vilcinskas, 2011b). The latter would indicate that the coevolution of fungal proteinases and insect proteinase inhibitors is a major component of the dynamics between the virulence of fungal parasites and insect host resistance.

5. Experimental coevolution with an initially resistant host We also established an experimental system for the analysis of host–parasite coevolution in T. castaneum and B. bassiana (Rafaluk et al., unpublished data), the former representing a resistant host in contrast to G. mellonella. The system was established to better understand by which means this host has evolved resistance to most parasitic fungi (Akbar et al., 2004; Khashaveh et al., 2011, Golshan et al., 2014). T. castaneum adults, in addition to the standard insect immune defenses, secrete antimicrobial substances that spread from the cuticle into the environment (Tschinkel, 1975; Unruh et al., 1998; Yezerski et al., 2007; Li et al., 2013). This provides communal protection, i.e. larvae in an environment previously conditioned by adult secretions are better protected against a natural parasite than larvae in an unconditioned environment (Joop et al., 2014). However, the adult secretion is toxic to the larvae (Omaye et al., 1981), so protection against pathogens comes at the cost of increased larval mortality in the presence of the secretion alone, depending on the environmental concentration (Joop et al., 2014). Quinones represent a major component of the secretion (Villaverde et al., 2007; Li et al., 2013), and these redox-active molecules are known to generate reactive oxygen species (ROS) that induce oxidative stress by oxidizing macromolecules such as lipids, proteins and nucleic acids, the latter associated with aging and carcinogenesis (Bolton et al., 2000). Li et al. (2013), by silencing candidate genes linked to quinone production, could show that this external defensive secretion provides an effective barrier against parasitic fungi, as beetles after RNAi were unable to inhibit fungal growth.



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Strategies used by parasitic fungi to gain virulence against T. castaneum have been investigated using a gene knockout approach, focusing on the defensive barrier provided by the redox-active quinones. Pedrini et al. (2015) identified the B. bassiana gene BbbqrA0, which encodes a benzoquinone reductase expressed in response to benzoquinone exposure. They created a disruption mutant (ΔBbbqrA) and an overexpression strain (Bb::bqrAΟ), both of which were tested in the presence of benzoquinone alone and in the presence of the complete beetle extract. They found that the ΔBbbqrA mutant was more susceptible than the wild-type fungus, whereas Bb::bqrAΟ was less susceptible, indicating that the benzoquinone reductase may promote fungal virulence towards T. castaneum. Benzoquinones delayed germination in all three strains, but the Bb::bqrAΟ strain rapidly compensated for the delay and the wild-type strain did so at a slower rate. To determine whether benzoquinone susceptibility is contra-directionally correlated with increased virulence, a T. castaneum survival test was conducted against all three B. bassiana strains (Pedrini et al. 2015). The mortality rate of beetles infected with Bb::bqrAΟ was greater than 40%, whereas those exposed to the wild-type strain experienced ~20% mortality and those exposed to ΔBbbqrA experienced only 7% mortality. These experiments confirmed the importance of the external secretion as a key defense strategy against fungal infection. To determine whether the upregulation of benzoquinone reductase is also a route the fungus might take naturally, we conducted a coevolution experiment in which we added B. bassiana to the flour environment and used T. castaneum with partially overlapping generations to ensure a continuous supply of benzoquinones (Rafaluk et al., unpublished data). The experiment comprised 13 host transfers. Throughout the course of coevolution we sampled coevolved B. bassiana from the experimental environment. These fungal samples were tested for susceptibility towards natural beetle secretions as well as a methyl-1,4-benzoquinone (MBQ) standard. Inhibition tests showed that coevolved strains were less susceptible to the secretion and to pure MBQ, as shown for the overexpression strains described above (Pedrini et al., 2015). Rafaluk et al. (unpublished data) also compared the metabolic activity of the ancestral



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and coevolved strains of B. bassiana. The coevolved isolates had a greater metabolic activity than the ancestral strain in the presence or absence of MBQ and also compensated more rapidly for the abovementioned MBQ-dependent delay in germination. To determine whether this also increased the virulence of the fungi, a survival assay was carried out in which a T. castaneum control line was exposed to the ancestral and coevolved strains of B. bassiana. Exposure to the ancestral B. bassiana strains caused ~7% mortality, whereas exposure to the coevolved strains caused up to 45% mortality, confirming the increase in virulence due to coevolution. This increase in virulence was confirmed in a comparable coevolution experiment involving T. castaneum and a microsporidian parasite (Rafaluk et al., 2015), which are close relatives of the true fungi. Here the increase in mortality was so rapid that the host became extinct within five transfers. Our results confirmed that secretion is the major host barrier protecting T. castaneum against fungal infection but that this defense mechanism can be overcome by B. bassiana during relatively short periods of coevolution. The genetic basis of this decreasing susceptibility and hence increasing virulence was addressed by using Illumina HiSeq2000 technology (Illumina, San Diego, CA, USA) to compare the transcriptomes of one ancestral and two coevolved fungal isolates (Rafaluk et al., unpublished data). The influence of MBQ on gene expression was investigated by comparing the gene expression profiles of the different fungal strains in the presence and absence of MBQ. This revealed differences between the ancestral and coevolved transcripts. The coevolved strains induced a large set of genes in one cluster in the presence of MBQ, indicating its relevance in the context of MBQ susceptibility. Gene Ontology (GO) enrichment analysis in this cluster using the whole transcriptome as a background showed that pathogenesis was the most significantly upregulated GO term. Most of the significantly upregulated genes in this group are involved in oxidative stress resistance, resolving to the same functional group as suggested by Pedrini et al. (2015). However, this was only the case for 2 out of 17 coevolved strains, so the non-tested strains may have followed alternative routes towards a loss in MBQ susceptibility and an increase in virulence towards T. castaneum.



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Our work confirms that T. castaneum secretions (benzoquinones and the oxidative stress they induce) provide an efficient barrier against fungal infection. We also show that the increase in virulence can be acquired by greater oxidative stress resistance in the parasite which can be achieved via several routes. Evolution will depend on additional selective forces, so differences may reflect the diverse host and parasite strains used in different experiments, and the results may not reflect what is happening under natural conditions given that B. bassiana has a broad host range. Therefore, the easiest evolutionary path for the fungus may be to infect another species with lower barriers, thus avoiding the inevitable trade-off between overcoming a challenging host and infecting many others with comparative ease.

6. Conclusions Our comparative analysis of coevolution experiments with two distinct parasitic fungi and two model host insects enabled us to elucidate opposing dynamics that can result from different baseline situations. In the G. mellonella–B. bassiana system, the fungus was initially ahead but the insect host was able to close the gap, whereas in the T. castaneum–B. bassiana system, the beetle was initially ahead, but the fungus was able to draw level or even outpace the insect host. We can only speculate what we would have seen if these experiments had been run for a longer period, with regular sampling, but presumably the race would have continued, with host and parasite taking turns to lead. Taking a snapshot, i.e. stopping coevolution at a defined point, provides an opportunity to identify host barriers and parasite virulence factors at a given moment, and to compare these to the situation at the start of the experiment (also see Leal, 2015). Many factors at the baseline situation such as the population size or non-detected infections with other pathogens can influence the outcome of host–parasite coevolution (Papkou et al., 2016; Schulte, 2016, current issue). Tolerance may also be of importance (Kutzer and Armitage, 2016, current issue). Understanding host defenses, parasite virulence factors and their evolution will help us to fight diseases, develop new drugs and mycoinsecticides, and avoid the emergence of resistant populations of parasites, pathogens and



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pests by, e.g., more carefully choosing pesticides and adjusting how we apply these. Even our basic results may have useful applications; e.g., selecting B. bassiana strains that tolerate oxidative stress could provide broader strategies for pest control, and these findings could also be transferred to other entomopathogenic fungi (Ortiz-Urquiza et al., 2015).

Acknowledgements Both authors are grateful for funding within the host–parasite coevolution priority program of the German Science foundation (SPP 1399) with grants to G.J. (JO963/1-1) and A.V. (VI 219/3-1). G.J. also kindly acknowledges funding by the Volkswagen Foundation, and currently both authors are funded by the Hessen State Ministry of Higher Education, Research and the Arts (HMWK) via the “LOEWE Center for Insect Biotechnology and Bioresources”. The authors thank Dr. Richard M. Twyman for editing the manuscript, Mark Salzig, André Billion and Daniel Amsel for providing sequence alignments, Tilottama Biswas and Thorben Grau for providing the Tribolium picture and two anonymous referees for comments on an earlier version of this text.



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Figure legends Fig. 1. Depicting the arms race of parasitic fungi with their insect hosts on the molecular level. As discussed in the text, the number of arrows represents the current snapshot of coevolution, with Tribolium castaneum currently being resistant and Galleria mellonella being susceptible to parasitic fungi. Abbreviations: MBQ, methyl-para-benzoequinone; EBQ, ethyl-parabenzoequinone. Fig. 2. Alignment of the protein sequence of seven M4-metalloprotease sequences. Multiple sequence alignment was perfomed with ClustalW2.0.12. and drawn with Jalview with the Clustal X default colour scheme (Gao et al., 2011; Hu et al., 2014; Pattemore et al., 2014). Fig. 3. Sequence alignment of genes encoding the IMPIα homologs identified in the transcriptome of G. mellonella (Vogel et al., 2011). Identical amino acid residues are labelled with yellow and similar residues with blue.



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Figure 1

Insect host: Tribolium castaneum Red flour beetle

Entomopathogenic fungi: Beauveria bassiana & Metharizium anisopliae

Insect host: Galleria mellonella Greater wax moth

Humoral factors mediating resistance  to  fungal infection:

Fungal factors determining their virulence  towards insect hosts:

Humoral factors mediating resistance  to  fungal infection:

Antifungal proteins: thaumatin Altincicek et al. 2008 matrix metalloproteinase             Knorr et al. 2009 ribosomal proteins                          Lord et al. 2010

lipases directed towards the insect cuticle:           p450 cytochromes                       Pedrini et al. 2010 ‐> degradation products serving as nutrients  for fugal growth

Antifungal proteins: lysozyme                              Vilcinskas & Matha 1997a cecropins gallerimycin Schuhmann et al. 2003

Phenoloxidase activity                  Yokoi et al. 2015   

Exogenous proteinases:    Griesch & Vilcinskas 1998 metalloproteinase subtilisin‐like proteinase                 Sun & Liu 2006 benzoquinone‐oxidoreductase Pedrini et al. 2015

Protease inhibitors: metalloproteinase inhibitor       Wedde et al. 1998 serine proteinase inhibitors      Fröbius et al. 2000

External secretion mediating resistance  to  fungal infection: Benzoquinones (MBQ & EBQ)          Li et al. 2013

Fungal toxins: destruxins beauverolides cyclosporins cytochalasins

Detoxification proteins: lipophorin Vilcinskas et al. 1997b Vilcinskas et al. 1999 Vilcinskas et al. 1999

Vilcinskas et al 1997c

Cellular immune reactions mediating  resistance  to fungal infection: Phagocytosis                            Vilcinskas et al. 1997a morphology & cytosceleton of  plasmatocytes Vilcinskas et al. 1997b Multicellular encapsulation

host –parasite coevolution specific response to a known target (group) general response and/or specific target unknown Arrows only point towards a target or into the direction of action but do NOT depict the order of successive events as this is unknown up to date.



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Figure 2

Metarhizium album ARSEF 1941 Metarhizium acridum CQMa 102 Metarhizium majus ARSEF 297 Metarhizium guizhouense ARSEF 977 Metarhizium anisopliae Metarhizium anisopliae BRIP 53293 Metarhizium robertsii ARSEF 23

consensus

Metarhizium album ARSEF 1941 Metarhizium acridum CQMa 102 Metarhizium majus ARSEF 297 Metarhizium guizhouense ARSEF 977 Metarhizium anisopliae Metarhizium anisopliae BRIP 53293 Metarhizium robertsii ARSEF 23 consensus Metarhizium album ARSEF 1941 Metarhizium acridum CQMa 102 Metarhizium majus ARSEF 297 Metarhizium guizhouense ARSEF 977 Metarhizium anisopliae Metarhizium anisopliae BRIP 53293 Metarhizium robertsii ARSEF 23

consensus

Metarhizium album ARSEF 1941 Metarhizium acridum CQMa 102 Metarhizium majus ARSEF 297 Metarhizium guizhouense ARSEF 977 Metarhizium anisopliae Metarhizium anisopliae BRIP 53293 Metarhizium robertsii ARSEF 23

consensus

Metarhizium album ARSEF 1941 Metarhizium acridum CQMa 102 Metarhizium majus ARSEF 297 Metarhizium guizhouense ARSEF 977 Metarhizium anisopliae Metarhizium anisopliae BRIP 53293 Metarhizium robertsii ARSEF 23

consensus

Metarhizium album ARSEF 1941 Metarhizium acridum CQMa 102 Metarhizium majus ARSEF 297 Metarhizium guizhouense ARSEF 977 Metarhizium anisopliae Metarhizium anisopliae BRIP 53293 Metarhizium robertsii ARSEF 23

consensus



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Figure 3

 



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