Fungi activate Toll-1 dependent immune evasion to induce cell loss in the host brain

Fungi evolve within the host, ensuring their own nutrition and reproduction, at the expense of host health. They intervene in hosts’ brain function, to alter host behaviour and induce neurodegeneration. In humans, fungal infections are emerging as drivers of neuroinflammation, neurodegenerative diseases and psychiatric disorders. However, how fungi alter the host brain is unknown. Fungi trigger an innate immune response mediated by the Toll-1/TLR receptor, the adaptor MyD88 and the transcription factor Dif/NFκB, that induce the expression of antimicrobial peptides (AMPs). However, in the nervous system, Toll-1/TLR could also drive an alternative pathway involving the adaptor Sarm, which causes cell death instead. Sarm is the universal inhibitor of MyD88 and could drive immune evasion. The entomopathogenic fungus Beauveria bassiana is well-known to activate Toll-1 signalling in innate immunity in Drosophila. In fruit-flies, the adaptor Wek links Toll-1 to Sarm. Thus, here we asked whether B. bassiana could damage the Drosophila brain via Toll-1, Wek and Sarm. We show that exposure to B. bassiana reduced fly lifespan and impaired locomotion. B. bassiana entered the brain and induced the up-regulation of AMPs, as well as wek and sarm, within the brain. Exposure to B. bassiana caused neuronal and glial loss in the adult Drosophila brain. Importantly, RNAi knockdown of Toll-1, wek or sarm concomitantly with infection prevented B. bassiana induced cell loss. By contrast, over-expression of wek or sarm was sufficient to cause dopaminergic neuron loss in the absence of infection. These data show that B. bassiana caused cell loss in the host brain via Toll-1/Wek/Sarm signalling driving immune evasion. We conclude that pathogens can benefit from an innate immunity receptor to damage the host brain. A similar activation of Sarm downstream of TLRs in response to fungal infections could underlie psychiatric and neurodegenerative diseases in humans.


INTRODUCTION
Fungi such as Ophiocordyceps unilateralis and Entomopthora muscae can manipulate the behaviour of their insect hosts, ultimately causing their death (de Bekker, 2019, de Bekker and Das, 2022, Elya et al., 2018).Even only exposure to fungal volatiles is sufficient to reduce lifespan, decrease dopamine levels, induce neurodegeneration and impair locomotion in fruit-flies (Inamdar et al., 2013, Inamdar et al., 2010).This is concerning also for human health.Abundant fungal spores, mould and fungal volatiles are commonly found in indoor damp conditions (Provost et al., 2013, Shi et al., 2014).Fungal spores have been found in brains of Parkinson's and Alzheimer's disease patients, and fungal infections are emerging as drivers of neuroinflammation, neurodegenerative diseases and psychiatric disorders (Chen et al., 2022, Alonso et al., 2014, Pisa et al., 2020, Cannon and Gruenheid, 2022).How fungi and neuroinflammation drive disease in the host brain is unknown (Cannon andGruenheid, 2022, Chen et al., 2022).
Here, we asked whether fungal infections could induce neurodegeneration in the host brain via Toll-Wek-Sarm signalling.We used Beauveria bassiana, an entomopathogenic fungus that induces disease in over 700 arthropod species, can be used for the control of insect pests, and is a well-known activator of Toll signalling in Drosophila (Evans, 1982, Gottar et al., 2006, Inglis et al., 2001, Lemaitre et al., 1997, McCoy, 1990, Samson et al., 1988, Quesada-Moraga et al., 2023).We show that the fungus benefits from immune receptor signaling as it induces immune evasion and cell death in the host brain.

B. bassiana exposure decreased lifespan and impaired locomotion
To ask whether and how fungi could affect the brain, we aimed to mimic natural exposure of flies to spores.We used an infection chamber with a fungal culture at the base and a separate compartment for fly-food affixed to the bottle's side wall, thus preventing fungal growth from limiting food and hydration supply (Figure 1A).Whereas non-infected control flies lived up to 70 days, flies exposed to B. bassiana died within less than 20 days (Figure 1B).By day seven over 50 percent of the flies in the infection chamber had perished (Figure 1B).Next, we tested whether exposure to B. bassiana affected fly behaviour.Negative geotaxis -also known as startle induced negative geotaxis (SING) or the climbing assay -is commonly used to measure locomotor impairment caused by neurodegeneration (Feany andBender, 2000, Riemensperger et al., 2013).Amongst the surviving flies, three days exposure to B. bassiana had no effect on flies' climbing ability, but by seven days post exposure, climbing was significantly impaired (Figure 1C).Thus, from here onwards, we used threeand seven-days' exposures for further tests.
Altogether, these data showed that exposure to B. bassiana compromised locomotion and longevity, which are common indicators of neurodegeneration in flies.

B. bassiana penetrate adult fly brains
In the light of the locomotion impairment, we wondered whether B. bassiana could affect brain function directly, by entering the brain.We exposed adult flies to B. bassiana spores for three days and using the FM4-64 dye, distinctive oval-shaped structures could be seen within the brain optic lobes (Figure 1D).Importantly, here, FM4-64 colocalised with the nuclear dye DAPI, confirming that they were spores (Figure 1D).We used a second dye, calcofluor white (CLW), which stains cell walls of algae, plants and fungi, together with the pan-neuronal marker Elav, and CLW+ signal was found in the optic lobes and central brain after three days of B. bassiana exposure (Figure 1E).Finally, when adult flies were exposed to a GFP transgenic B. bassiana strain (EABb 04/01-Tip GFP5, Figure 1F) (Landa et al., 2013), GFP+ spores were found both within the optic lobes and central brain (Figure 1G).
Together these findings show that B. bassiana spores infiltrated the adult brain.
The blood brain barrier (BBB) shelters the brain from bodily fluids and foreign agents, thus, to enter the brain, B. bassiana would either have to damage the BBB or be carried across by macrophages (Winkler et al., 2021).The dye dextran red is commonly used to test the integrity of the BBB in Drosophila (Bainton et al., 2005).The dye is injected into the flies' thorax, and if the BBB is intact, dextran red outlines the retina's edge, whereas if the integrity of the BBB is compromised, the dye leaks into the retina (Bainton et al., 2005).We found that in non-exposed flies the dye accumulated in the retina's periphery, whereas in flies that had been exposed to B. bassiana for seven days, dextran red had spread within the retina (Figure 1H), meaning that the BBB was damaged.
Altogether, these findings showed that following exposure, B. bassiana penetrated the fruit-fly brain.

Exposure to B. bassiana activated Toll signalling inside the adult brain
B. bassiana could invade the brain through the optic lobes, the head capsule, and the proboscis.Fungi can degrade insects' cuticle, enabling them to penetrate hosts' bodies (Boucias et al., 1988, Holder et al., 2007, Ortiz-Urquiza and Keyhani, 2016, Quesada-Moraga et al., 2020).The fly uses the proboscis for feeding, and as it traverses the brain to join the oesophagus, B. bassiana could enter the brain through the proboscis during feeding.
On the other hand, conceivably, B. bassiana could trigger repulsion or avoidance in the fly.
In C.elegans worms, pathogenic bacteria trigger a behavioural avoidance response, inducing the worms to turn away from the source of pathogen (Pradel et al., 2007, Pujol et al., 2001).Thus, we tested whether flies like or dislike to feed on B. bassiana spores.We offered them either water or sucrose or spores mixed with a blue dye and measured the optical density (OD) of their abdomens after feeding.OD values were higher in flies that fed on spores compared to those that fed on water controls.This showed that flies fed on sucrose -which they are known to like -and similarly fed abundantly on spores (Figure 2A).Thus, ingestion would provide B. bassiana with an effective route into the brain.
Above findings showed B. bassiana inside the brain, that flies were not repelled by B. bassiana and instead fed on it.This was surprising for two reasons.Firstly, it contrasts with the bacterial avoidance response of C.elegans which depends on Tol-1 (Pradel et al., 2007, Pujol et al., 2001).Secondly, in Drosophila, the innate immune response driven by Toll-1 should lead to clearance of the fungus (Imler and Hoffmann, 2001).Thus, we wondered whether perhaps B. bassiana did not lead to the activation of Toll-1 inside the brain.First, we asked whether Toll-1 and sarm might be expressed in the proboscis, which is required for feeding.If they were not, this would explain the lack of avoidance response to B. bassiana.
We had previously shown that both Toll-1 and sarm are expressed in the sub-aesophageal ganglion, where the feeding circuit links to the proboscis (Li et al., 2020).Here, using the in vivo reporter Toll-1>FlyBow we found expression in sensory neurons of the proboscis, including in dendrites of the labellum (Figure 2B).Similarly, the Toll signalling adaptor sarm was also expressed in sensory neurons of the proboscis (Figure 2B).To further verify this, we tested what consequences activating Sarm+ neurons might have on the proboscis extension response (PER), which is required for feeding.Activating Sarm neurons with TrpA1 (using two independent lines) did not prevent feeding, and instead increased the incidence of PER events compared to unstimulated controls (Figure 2C,D).This suggests that sarm neurons are not involved in avoidance of B. bassiana, but instead could be involved in feeding.
Next, we asked whether B. bassiana could trigger Toll signalling in the brain, which would result in the up-regulation of its immunity downstream effectors.We had previously shown that seven of the nine Toll receptors, as well as its adaptors MyD88 and sarm, are normally expressed in the adult fly brain (Li et al., 2020).Single-cell RNAseq analysis of the adult fly brain revealed that the fungal PRR GNBP3 as well as the downstream SPE protease, spz-1 ligand of Toll-1, downstream Toll-1 adaptor MyD88, downstream effector Dif/NF-kB and target antimicrobial peptide genes drs and mtk, are all normally expressed in the adult brain (Scope Fly Atlas) (Davie et al., 2018).Thus, we asked whether exposure to B. bassiana might activate innate immunity in the brain.We used qRT-PCR in dissected adult brains, and found that at seven days post-infection, expression of AMPs drs and mtk mRNA was upregulated (Figure 2E).Intriguingly, the expression of the Toll adaptors wek and sarm was also upregulated (Figure 2E).Similarly, wek expression was also shown to increase upon E. muscae infection in adult flies (Elya et al., 2018), consistent with our findings.
To conclude, our data showed that exposure to B. bassiana induced immune Toll signalling within the adult brain.Moreover, this supported the data shown in (Figure 1) that B. bassiana entered the brain.Intriguingly, the data showed that B. bassiana also triggered an alternative Toll signalling route engaging Wek and Sarm (Figure 2F).Wek can interact with MyD88, but it is not required for innate immunity (Chen et al., 2006), and it links Tolls to Sarm instead (Foldi et al., 2017).Sarm inhibits MyD88 and immune signalling, and instead, Toll signalling via Wek and Sarm induces apoptosis downstream (Foldi et al., 2017).Sarm also induces axonal destruction (Osterloh et al., 2012).Remarkably, the infection-dependent up-regulation of wek and sarm had the potential to drive neurodegeneration in response to Toll signalling (Figure 2F).

Exposure to B. bassiana caused neuronal and glial loss
To ask whether B. bassiana could induce neurodegeneration via Toll-1, Wek and Sarm, we first tested whether exposure to B. bassiana altered cell number in the brain.If Sarm drove neurodegeneration in response to infection, these cells would be lost.Thus, to test if the number of Sarm+ neurons was affected, we used the nuclear reporter histone-YFP to visualize Sarm+ cells in the central brain (sarm NP0257 >hisYFP) and quantified Sarm+ cells automatically.Seven days exposure to B. bassiana decreased the number of Sarm+ cells in the central brain compared to non-exposed controls (Figure 3A,B).To ask whether glial cells might also be affected, glial cells were labelled with pan-glial anti-Repo antibodies, and we found that the number of glial cells in infected flies also decreased (Figure 2C,D).Thus, B. bassiana infection caused loss of Sarm+ neurons and Repo+ glia.
Exposure of fruit-flies to the fungal volatile 1-octen-3-ol caused loss of dopaminergic neurons (Inamdar et al., 2013).Thus, we asked whether exposure to B. bassiana spores could elicit similar effects.First, we tested whether seven-day exposure to B. bassiana spores might affect the expression of Tyrosine Hydroxylase (TH), the enzyme that catalyses the conversion of tyrosine to L-Dopa, which is then decarboxylated to produce dopamine (Daubner et al., 2011).Using qRT-PCR, we found a significant decrease in TH mRNA levels within infected adult brains (Figure 3E), showing that B. bassiana exposure decreased dopamine production.Next, we used anti-TH antibodies to visualise dopaminergic neurons in the adult brain.By seven days post-exposure, the number of PPL1, PPL2, PPM1/2, PPM3 (Figure 3F-I), and PAM (Figure 3I,J,K) dopaminergic neurons had decreased in infected brains.These data show that exposure to B. bassiana caused dopaminergic neuron cell loss within the adult brain.
Altogether, seven days exposure to B. bassiana caused loss of Sarm>his-YFP+ neurons, Repo+ glia and TH+ dopaminergic neurons in the infected adult fly brain.

B. bassiana requires Toll-1 to induce cell loss in the fly brain
Having established that exposure to B. bassiana induced Toll-1 signalling and caused cell loss in the adult brain, we asked whether cell loss depended on Toll-1.Raised levels of antimicrobial peptides of the IMD pathway, as well as drs of the Toll-1 pathway, can induce neurodegeneration in the fly brain (Cao et al., 2013, Kounatidis et al., 2017, Shukla et al., 2019).On the other hand, Toll-1 signalling can induce cell death via the Wek/Sarm/JNK pathway (Foldi et al., 2017).Sarm is the only inhibitor of MyD88, and this function is highly evolutionarily conserved (Belinda et al., 2008, Carty et al., 2006, Peng et al., 2010, Carty and Bowie, 2019).Thus, in MyD88+ cells, Toll-1 could potentially drive signalling via the two alternative MyD88 and Sarm pathways, in co-expressing cells.Thus, to account for both potential Toll-1 signalling routes, we visualised adult MyD88+ cells with MyD88>HisYFP and counted cell number automatically.Seven-day exposure to B. bassiana induced loss of MyD88+ cells in an otherwise wild-type background (Figure 4A,C).
To knock-down Toll-1 specifically in adult flies, we used the temperature-sensitive Gal4 repressor tubulin-Gal80ts (Figure 4B,tubGAL80ts,MyD88>hisYFP).In non-infected controls, adult Toll-1 RNAi knockdown using line UAS-Toll-1RNAi kk/100078 (shown to work effectively in (Li et al., 2020)) caused an increase in MyD88-YFP+ cells within the central brain (Figure 4A-C).This increase in cell number could correspond to the induction of proliferation or an increase in the number of surviving cells.In fact, there is abundant cell death in the developing CNS and Toll-1 is known to induce apoptosis in at least larvae and pupae (Foldi et al., 2017).There, Toll-1 loss of function increased neuronal number, and constitutively active Toll-1 10b decreased neuron number and increased the number of Dcp1+ apoptotic cells (Foldi et al., 2017).This suggests that the increase in cell number caused by Toll-1-RNAi knock-down would be more likely to result from decreased cell death.
Importantly, in infected adult brains, Toll-1 RNAi knockdown prevented the loss of MyD88-YFP+ cells that would have been otherwise caused by B. bassiana exposure (Figure 4A-C).
These data mean that B. bassiana induced cell loss depends on Toll-1 signalling.
Neuronal loss can cause glial loss (Hidalgo et al., 2001), MyD88 is also expressed in some Repo+ glial cells (Li et al., 2020) and Toll-1 is also expressed in glial cells (Davie et al., 2018).Thus, we tested whether adult specific Toll-1 RNAi knockdown in MyD88 cells affected glial cell number, stained with anti-Repo.When Toll-1 was downregulated in noninfected flies, the number of Repo+ glial cells in the brain was not altered compared to controls (Figure 4D,E).However, Toll-1 RNAi knock-down in MyD88+ cells prevented the decrease in glial cell number caused by B. bassiana infection (Figure 4D,E).This meant that B. bassiana induced glial cell loss requires Toll-1 signalling in MyD88+ cells.
We also tested the effect of Toll-1 RNAi knock-down in dopaminergic neurons (DANs).We first tested if Toll-1 is expressed in DANs, by labelling Toll-1 cells with Toll-1> HistoneYFP and dopaminergic neurons with anti-TH antibodies.There was colocalization within multiple DAN clusters, most particularly within the PAL, PPM3, PPL2, and PPL1 clusters (Supplementary Figure 1A).By contrast, very few neurons within the large PAM clusters were Toll-1>hisYFP+ (Supplementary Figure 1A).Following Toll-1 RNAi knockdown in MyD88+ cells in the adult brain, we counted the number of cells in each DAN cluster.In non-infected flies, Toll-1 knockdown did not alter DAN cell number (Figure 4F,G).

B. bassiana benefits from non-immune Wek-Sarm signalling to induce cell loss
Apoptotic Toll-1 signalling requires Wek, which binds Sarm, which induces cell death (Foldi et al., 2017).As we had seen that exposure to B. bassiana induced the upregulation of wek and sarm in the fly brain (Figure 2D), and B. bassiana induced cell loss depended on Toll, we asked whether wek and sarm might also be required for infection-dependent cell loss.
We first asked whether adult specific knock-down of wek might affect the cellular response to B. bassiana infection.In non-infected controls, adult specific knock-down using line UAS-wek MH046534 -RNAi (shown to work effectively in (Foldi et al., 2017, Li et al., 2020)) in MyD88+ cells did not alter MyD88-hisYFP+ cell number (Figure 5A,C).However, when wek was knocked-down with RNAi, this rescued B. bassiana-induced MyD88-HisYFP+ cell loss (Figure 5A,C).In fact, like before, cell number increased further.In non-infected control flies, wek-RNAi knockdown did not alter the number of Repo+ glial cells in the brain (Figure 5D,E).However, no difference was found in Repo+ glial cell number between infected and non-infected brains upon wek-RNAi knock-down (Figure 5D,E).These data means that B. bassiana could not induce MyD88+ and Repo+ cell loss without Wek.
By contrast, wek knock-down in non-infected adult flies caused a significant decrease in the number of DANs of the PPL1, PPL2 and PPM3 clusters (Figure 5F,G and Supplementary Figure 2).This suggested that Wek is required to maintain DAN cell survival or promote neurogenesis or differentiation (e.g.TH expression) in the adult brain.
Importantly, in wek-RNAi knock-down flies, B. bassiana infection failed to induce further neuronal loss (Figure 5F,G).These data are consistent with the known pleiotropic functions of wek, including in the adult brain (Chen et al., 2006, Foldi et al., 2017, Li et al., 2020).They have also revealed that in the absence of Wek, some DANs do not develop normally and only a few DANs remain, but these remaining DANs are not susceptible to further damage by B. bassiana infection.
Next we tested sarm.In the absence of infection, when we downregulated sarm expression with RNAi in adult MyD88 cells (using line UAS-sarm-RNAi JF01681 , shown to work effectively in (Foldi et al., 2017)), there was no effect compared to controls (Figure 6A-C).
However, RNAi knock-down of sarm rescued B. bassiana-induced MyD88>hisYFP+ cell loss (Figure 6A-C).Similarly, sarm RNAi knock-down had no effect on the number of glial cells in the adult brain compared to non-infected controls, but it rescued the Repo+ cell loss caused by B. bassiana infection (Figure 6D,E).And finally, using anti-TH antibodies we showed that sarm RNAi knock-down did not affect PPM3 cell number in non-infected controls, but it rescued the loss of PPM3 DANs caused by B. bassiana infection (Figure 6F,G).Altogether, these data showed that sarm is required for B. bassiana induced loss of MyD88-HisYFP+, Repo+ and TH+ PPM3 cells (Figure 6H).
Altogether, these data show that signalling via Wek and Sarm is required for B. bassiana-induced cell loss in the brain.This suggests that the upregulation of wek and sarm expression in the brain upon B. bassiana infection could lead to neurodegeneration in the host (Figure 2F and Figure 6H).

Increased Toll-1, Wek and Sarm levels could induce cell loss
Finally, we asked whether over-expression of activated Toll-1, wek or sarm could be sufficient to induce cell loss in the absence of infection.Wek has pleiotropic functions downstream of Tolls: it binds Tolls and MyD88 to enable canonical signalling via NFkB/Dorsal/Dif downstream, which in the CNS promotes quiescence and cell survival; it can also bind Sarm, to promote cell death; or function independently of both, to promote neurogenesis in the adult brain from quiescent MyD88+ progenitor cells (Chen et al., 2006, Foldi et al., 2017, Li et al., 2020).Thus, the pleiotropic functions of wek could lead to compound phenotypes.To simplify this, we tested the effect of activated Toll-1 10b , wek or sarm over-expression on PAM neurons, which are differentiated neurons.We used a DAN specific GAL4 driver (THGAL4; R58E02GAL4), visualised PAMs with histone-YFP and counted them automatically.Toll-1 is normally expressed only in a fraction of PAM neurons (Supplementary Figure 1).Over-expressed activated Toll-10 1b in DANs caused a mild decrease in PAM cell number, which however was not statistically significantly different from the control (Figure 7A,B).This suggested that not many PAMs may express wek, sarm or both in the un-infected brain.Importantly, over-expression of either wek (Figure 7A,B

DISCUSSION
We show here that the fungus B. bassiana induces cell loss in the host Drosophila brain via the innate immunity Toll-1 receptor.In innate immunity, Toll-signalling via MyD88 and NFkB/Dif results in the upregulation of anti-microbial peptide gene expression to eliminate the fungus (Imler and Hoffmann, 2001, Lemaitre et al., 1996, Leulier and Lemaitre, 2008).
However, we show that in the brain, B. bassiana can also activate an alternative Toll-1 signalling pathway, involving Wek and Sarm, that causes neurodegeneration instead.Sarm is the highly evolutionarily conserved and only inhibitor of Toll/TLR-dependent innate immunity, from flies to humans (Anthoney et al., 2018, Belinda et al., 2008, Carty and Bowie, 2019, Peng et al., 2010, Yuan et al., 2010).Through its ability to up-regulate Sarm, B. bassiana drives immune evasion, causing neurodegeneration in the host brain.
B. bassiana spores infiltrated the brain, damaged the BBB and upregulated the expression of antifungal peptides drs and mtk in infected brains.B. bassiana might enter the brain by first adhering to and degrading the cuticle (Boucias et al., 1988), in the optic lobes, head capsule and proboscis.Once attached, B. bassiana spores germinate and produce hyphae that degrade the cuticle by secreting various chitinases and proteases (Fan et al., 2007, Fang et al., 2009, Zhang et al., 2008).Spores in the brain and a compromised bloodbrain barrier were also found following E. muscae infection in Drosophila (Elya et al., 2023, Elya et al., 2018).B. bassiana could also trigger a host response facilitating macrophage entry across the BBB and into the brain (see (Winkler et al., 2021)).In many insects, the likelihood of infection is highest at less sclerotized cuticle regions like the mouth (Ortiz-Urquiza and Keyhani, 2016).As the proboscis traverses the brain, dissolving the proboscis cuticle would provide B. bassiana a direct route to the brain.Similarly, fungi also invade the human brain (Cannon and Gruenheid, 2022, Chen et al., 2022, Pisa et al., 2020, Pisa et al., 2015a, Pisa et al., 2015b).The most common infection is by Cryptococcus, which causes meningoencephalitis, and Cryptococcus enters the human brain through the nose, by inhalation, followed by degradation of the BBB (Chen et al., 2022).
We showed that exposure to B. bassiana decreased fly longevity and impaired locomotion, which correlated with widespread cell loss in the brain.B. bassiana induced-cell loss was verified with multiple cell markers: loss of His-YFP labelled Sarm+ and MyD88+ cells, loss of Repo+ glial cells and loss of TH+ DANs.Furthermore, B. bassiana infection reduced the expression of TH, which is responsible for dopamine synthesis, and dopamine is required for locomotion (Riemensperger et al., 2013).Our findings are consistent with reports that fungal volatiles induce neurodegeneration in the Drosophila brain (Inamdar et al., 2013, Inamdar et al., 2010).Evidence of neurodegeneration were decreased longevity, impaired climbing, decreased dopamine levels and dopaminergic neuron loss (Inamdar et al., 2013, Inamdar et al., 2010).These same phenotypes are also measures of neurodegeneration in other contexts, such as Drosophila models of Parkinson's disease (Riemensperger et al., 2013, Cassar et al., 2015, Feany and Bender, 2000).Similarly, our work demonstrates that exposure to B. bassiana reduced longevity, impaired locomotion, caused cell loss -including of DANs -and reduced TH levels.Altogether, exposure to B. bassiana induced a signature characteristic of neurodegeneration.Notably, B. bassiana is also a plant endophyte, providing protection to plants against insects (Behie et al., 2012, Behie et al., 2015, Branine et al., 2019).
By reducing the climbing ability of flies, B. bassiana may facilitate greater spore adherence and germination on the fly cuticle, ultimately leading to the insect's demise.
Absence of Wek in PAMs would prevent Toll-1 from driving apoptosis, even in the presence of Sarm, as Tolls cannot bind Sarm without Wek (Foldi et al., 2017).Instead, overexpression of wek or sarm was sufficient to induce PAM cell loss.On the other hand, some DANs (PPL1, PPL2 and PPM3) were lost with wek knock-down, meaning that wek is required for their cell survival, neurogenesis or differentiation.This is consistent with the fact that Toll-2 promotes adult neurogenesis via Wek (Li et al., 2020).It is currently unknown if wek can directly regulate neuronal differentiation, but it is not unlikely, as Wek encodes a zinc-finger transcription factor (Chen et al., 2006).Either way, the data further support the notion that Wek has pleiotropic functions that depend on cellular context (Foldi et al., 2017, Chen et al., 2006).
The data also suggest that signalling by Toll-1/Wek/Sarm may not be enough to induce widespread neurodegeneration after infection.One possibility is that other Tolls might also induce cell death when Wek and Sarm levels rise.In fact, Spz-1 can bind at least Toll-7 in flies (Chowdhury et al., 2019), and Toll-5 in mosquito (Saucereau et al., 2022).
Alternatively, anti-microbial peptides induced by Toll-1 via MyD88/NFkB/Dif signalling could also drive neurodegeneration non-autonomously.In fact, anti-microbial peptides regulated by NFkB Rel downstream of the Imd pathway can induce neurodegeneration (Kounatidis et al., 2017), and over-expression of both drs and mtk can induce DAN cell loss (Shukla et al., 2019).In C. elegans, TIR-1/Sarm promotes the expression of anti-microbial peptides in the epidermis, which bind a receptor in neurons to cause dendrite degeneration (E et al., 2018).Thus, B. bassiana's ability to induce neurodegeneration in the host may involve a wider molecular machinery than explored here.
Our data suggest that the activation of Sarm signalling by B. bassiana is one of a range of adaptive interactions with its hosts.For example, in mosquitos, B. bassiana can reduce the host immune response by exporting a microRNA that downregulates the levels of Spz4, a ligand of Toll (Cui et al., 2019).In flies, B. bassiana can bypass the host pattern recognition receptor GNBP3 by releasing virulence factors that promote host immune evasion and fungal growth within the host (Eley et al., 2007, Molnár et al., 2010, Pedrini, 2022).In return, flies can detect the virulence factors too, to activate innate immunity bypassing detection of the fungal cell wall through GNBP3.In fact, the virulence factor PR1 gets proteolytically cleaved by Persephone, and activates the Toll-signalling cascade independently of GNBP3 (Gottar et al., 2006).Similarly, in mammals, Toll-Like Receptor-4 (TLR-4) activates the immune response against C. albicans, while C. albicans activates TLR-2, which leads to production of anti-inflammatory chemokines that help C. albicans to evade the host immune system (Netea et al., 2004, Netea et al., 2002, Netea et al., 2007).In this context, our data show that B. bassiana can induce the Sarm pathway downstream of Toll-1 harming the host.What controls the up-regulation of wek and sarm after infection, enabling degenerative signalling, is unknown, but it could be Toll signalling itself, activated by B. bassiana.In fact, signalling downstream of Toll-6 and -7 upregulates the expression of various Toll downstream effectors, including NFkB homologues dorsal and dif, and their inhibitor cactus (McIlroy et al., 2013).Importantly, the functions of Sarm in inhibiting canonical Toll/TLR signalling and in inducing neuronal apoptosis, axon destruction and neurodegeneration are all highly evolutionarily conserved (Anthoney et al., 2018, Belinda et al., 2008, Carty and Bowie, 2019, Sarkar et al., 2023).In flies and mammals, Sarm induces apoptosis via JNK signalling and in C. elegans, via MAPK/p38 signalling (Carty et al., 2006, Essuman et al., 2017, Izadifar et al., 2021, Mukherjee et al., 2015, Osterloh et al., 2012, Peng et al., 2010, Veriepe et al., 2015, Yuan et al., 2010, Foldi et al., 2017).Moreover, Sarm has a TIR domain that has catalytic NADase activity, which drives neurite destruction as well as cell death (Essuman et al., 2017, Osterloh et al., 2012, Summers et al., 2016).Most intriguingly, similarly to Sarm, some prokaryotic proteins also bear a TIR domain with NADase catalytic function, and they function in immune evasion (Sarkar et al., 2023).
To conclude, our data show a novel tactic in the evolutionary arms race between the host Drosophila and the fungus B. bassiana taking place in the brain.B. bassiana is detected in the Drosophila brain, that activates Toll-1 dependent innate immunity signalling against the fungus to protect the brain.In turn, B. bassiana benefits from the concomitant activation of the Toll-1 immune-evasion Sarm pathway driving neurodegeneration in the host brain.Importantly, human neurodegenerative and psychiatric diseases have been linked to fungal infections and neuro-inflammation (Chen et al., 2022, Alonso et al., 2014, Pisa et al., 2020, Cannon and Gruenheid, 2022).There is also evidence linking altered Sarm function to neurodegenerative diseases (Bloom et al., 2022, Miao et al., 2024, Veriepe et al., 2015, Murata et al., 2023).For example, constitutively active Sarm1 variants are enriched in ALS patients (Bloom et al., 2022).It will be important to find out whether a similar activation of Sarm downstream of TLRs in response to fungal infections is responsible for inducing psychiatric and neurodegenerative diseases in humans.

MATERIALS AND METHODS
Drosophila genetics: Please see Table 2.1 for the list of the stocks used.Conditional overexpression and knock-down in adult flies was carried out using ubiquitously expressed tub-GAL80 ts .GAL80 ts is a temperature sensitive GAL4 repressor, that prevents GAL4 expression at 18°C and enables it at 30°C.The temperature regimes we used are indicated in the figures and were set to enable GAL4 adult-onset and B. bassiana growth together.

Spores:
To isolate B. bassiana spores, 10 ml of distilled water was poured on the B. bassiana culture in the petri plate and culture was scraped with a spreader.The solution was centrifuged at 4,000 rpm for 15mins, the supernatant was removed, the pellet was dissolved in 2-5ml of water and spun again for 1-2mins.The pellet was dissolved in 1ml distilled water and labelled as the principal solution.The concentration of the principal solution was calculated by counting spores using a haemocytometer.10µl of principal solution was diluted in 90µl of water and marked as dilution X10 and spores were counted again by using the haemocytometer.This step was repeated until a spore concentration of 3.7 x 10 9 spores/ml was achieved.
Infection chamber: To enable natural infection whilst avoiding damage to the body that would induce an injury-response, a natural infection chamber was devised.An infection chamber consists of a glass bottle with a carpet of B. bassiana growing on sabouraud dextrose agar (SDA) medium at the bottom, and a cut 50ml falcon tube bearing standard flyfood stuck with double-sided sticky tape to the bottle side wall.To prepare SDA media, 16.25g of 4% SDA was added to 250 ml of distilled water and autoclaved.Next, 10ml SDA medium was transferred into autoclaved glass bottles and once it had set, 1ml of 3.7 x 10 9 spores/ml B. bassiana solution was poured into each bottle onto the medium.After plugging the bottles, they were transferred to 25°C incubator for 7-10 days.Next, the falcon tube containing fly-food was inserted into the bottle, and then the flies.In this way, flies can freely access fly-food devoid of B. bassiana, for feeding and hydration, whilst being naturally exposed to spores.

Staining of B. bassiana spores
Adult Drosophila brains were dissected and fixed in 4% paraformaldehyde (PFA) in Phosphate buffer saline (PBS) at room temperature for 20 minutes.Following washes in 0.5% Triton in PBS, brains were incubated in FM4-64 dye at 1:200 dilution, or calcofluor white (CLW) stain (50mg/ml) 1:1000 dilution for 1h at room temperature.The brains were washed and mounted in DAPI containing Vectashield, prior to imaging.

Drosophila behaviour tests
Longevity was measured as described in (Piper and Partridge, 2016), using F1 wild-type Oregon/CantonS flies raised on standard cornmeal food.They were placed in an infection chamber, the fly food was replaced every four days in both control and experimental setups, and the number of dead flies and censor events were manually recorded after every transfer.
Both infected flies and non-infected controls were kept at 25°C.Dead flies were scored as 1 and flies that got stuck to the food or escaped were scored as 0. Data were analysed using GraphPad prism and Log rank (Mantel-Cox test) to analyse the data and generate a Kaplan-Meier survival curve.The experiment was conducted with n=104 flies for each condition, from two biological replicates of n=58 and n=46 flies each.

Climbing assay.
Startled induced negative geotaxis assay was carried out as described in (Barone and Bohmann, 2013).The test was carried out in a humidity and temperature controlled lab (25°C).The infected flies were flipped 2-3 times to remove excess spores attached to their bodies before being transferred to climbing assay vials.The vials with flies inside were tapped, and the flies were filmed for 10 seconds, followed by a 30-second rest period, and the number of flies climbing above the 2cm mark were counted.This process was repeated 15 times for each cohort.Three cohorts of 7-10 flies each were used per genotype, and the entire process was repeated a second time, using new flies.

Proboscis extension response (PER) assay.
The PER assay was adapted from (Smith and Burden, 2014).Flies were starved in a 25°C incubator for 24 hours in vials containing agar before performing the PER assay.After 24hrs, the flies were transferred to a 20µl pipette tip using a fly aspirator.Using a sharp razor blade, the tip was cut, and the fly was then carefully placed in position with the help of a wick made of Kimtech wipe.The 20µl tip with the fly head emerging was fixed on a flat surface and a camera was positioned around it.To verify that the immobilized flies in the 20µl tip were fit for the PER assay, they were first given water and 100 mM sucrose solution using tissue paper wicks and the response was recorded for 60 seconds.The flies had been hydrated but starved before the experiment, so any flies that responded to water or did not respond to the 100 mM sucrose solution were discarded.The TrpA1 experiment was conducted inside an environmental chamber at 30°C.After the flies had been inside the chamber for 2-3 minutes, they were recorded for 60 seconds.The experiment was conducted using 10 flies per genotype, and the process was repeated three times using new biological replicates of the crosses.

Feeding assay.
The feeding assay was adapted from (Cheriyamkunnel et al., 2021).To prepare a fly cage, an empty food vial was used, and was cut at 4mm from its base using a heated knife.The base was then sterilized with 70% ethanol.Solutions mixed with 4% brilliant blue dye were transferred to the base and placed in an egg-laying chamber.For the feeding assay, four solutions were prepared, with each egg laying chamber containing only one solution: Negative control 1: distilled H2O; Negative control 2: distilled H2O (950ul) + 50ul 4% brilliant blue dye (95%/5% v/v); Positive control: 100mM Sucrose solution (950ul) + 50ul 4% brilliant blue dye (95%/5% v/v); Test: 7.0025 x 10 7 spores/ml (950ul) + 50ul 4% brilliant blue dye (95%/5% v/v).Equal number of wildtype Oregon male and female flies were used (n=10 males and 10 females) and 4 biological replicates were performed, flies were anesthetized on ice and transferred to the 4-feeding assay setups containing the above solutions.The setups were then placed at 25°C incubator for 24hrs.Next, the flies were collected by anesthetising the flies with CO2 and transferred to the 1ml Eppendorf tube.The Eppendorf containing flies were dipped in liquid nitrogen for 5 secs, placed in the 50ml Falcon tube, and dropped from a height to separate the flies' heads from their abdomens.The abdomen of the flies was collected using forceps and transferred to an Eppendorf containing 50ul of distilled water.Using a pestle abdomen was homogenised in the Eppendorf and 950ul of dH2O was added to it.The Eppendorf was then vortexed and centrifuged for 5 mins at 10,000rpm.The supernatant was transferred into the new Eppendorf and kept on ice until analysis.The homogenised solution was transferred to 1ml cuvette, and the absorbance was recorded using spectrophotometer, at 630nm and 750nm.The corrected absorbance was calculated by subtracting the mean of 3 readings at 750nm (background) from the mean of 3 readings at 630nm (peak of blue dye).

Injecting flies with Dextran red fluorescent dye.
This protocol was adapted from (Bainton et al., 2005).A glass pulled capillary needle was inserted into a glass pasteur pipette and sealed with parafilm at the point of insertion, and needle was loaded with 0.1µl of 25 mg/µl fluorescent Dextran red dye.The needle was carefully inserted into the thorax beneath the wing of anaesthetised flies.The flies were then transferred to a vial containing fly food and placed in a 25°C incubator for 24hrs to recover.
After the 24hrs recovery period, the flies were fixed onto a glass slide using glue, and their retinas were imaged using Leica SP8 confocal fluorescent microscope, n=10 flies.

Quantitative Reverse Transcription (qRT)-PCR.
qRT-PCR was carried out from 20 adult brains per sample, dissected from wild type Oregon non-infected and infected flies.The dissected adult brains were cleaned removing all fat body and then immediately transferred into the Eppendorf containing TRI reagent (Ambion #AM9738).RNA extraction was carried out according to the TRI reagent protocol using isopropanol.Extracted RNA were treated with DNase to get rid of genomic DNA contamination using DNA-free kit (Ambion #AM1906).200ng of RNA was reverse transcribed into cDNA by using random primer by following the manuscript of GoScript TM Reverse Transcription System (Promega #A50001) (Table 2.1).A PCR was performed to check the contamination in the RNA and Reverse Transcribed (RT) cDNA (Table 2.2).This is followed by a qRT-PCR.All qRT-PCR experiments were done in triplicate (i.e., 3 well condition).A DNA binding dye SYBR was used to observe the dynamics of the PCR (SensiFast TM SYBR®) and used ABI Prism 7000SDS machine for the qPCR (Table 2.3).
Following qRT-PCR, quantification were performed using the CT value generated by the qPCR machine, as described in (Rao et al., 2013).For the internal control GAPDH

Immunostainings.
Adult fly brains were dissected and fixed following standard methods, as in (Li et al., 2020).
Antibodies used, their source and their working dilutions are given in Table 3.

Microscopy and imaging
B. bassiana spores in the adult brain stained with FM4-64 dye, Calcofluor white stain, and transgenic B. bassiana-GFP spores were imaged with Leica SP8 confocal microscope with a 20X oil immersion lens, at a resolution of 1024x1024 pixels, zoom 1 and scanning speed of 400 Hz, and 0.96 µm Z step.High magnification images of B. bassiana spores in adult brain were obtained using a 63X oil immersion lens at 1024x1024, zoom 2.0, speed of 400Hz, line average of 4 and Z step 0.96 µm.
Diffusion of Dextran Red dye in the retina of adult flies were imaged using Leica SP8 confocal microscope with a 10X lens, at a resolution of 1024x1024 pixels.The zoom was set to 0.8 and scanning speed was 400Hz.
Brains for cell counting were imaged as follows: Anti-Repo and SarmNP0257> hisYFP were imaged using a Zeiss LSM 710 confocal microscope with a 25x oil immersion lens, at a resolution of 1024x1024 pixels, zoom 0.6 or 1, speed of 7 frames per second (fps), line average of 1 and 0.96 µm step.MyD88>hisYFP samples were imaged using a Leica SP8 confocal microscope with a 20X oil immersion lens, at 1024x1024, zoom 1, speed 400 Hz and Z step 0.96 µm.
Anti-TH+ stained Toll->HisYFP adult brains were imaged with the Leica SP8 confocal microscope with a 20X oil immersion lens, at 1024x1024 pixels, zoom 1.4, speed 400 Hz and Z step 0.96 µm.For anti-TH in wild-type brains, the Zeiss LSM 710 confocal microscope was used, with a 25x oil immersion lens, 1024x1024 at 8 fps, line average of 1 and Z step 0.96 µm.For MyD88>hisYFP anti-TH samples, adult brains were scanned with the Leica SP8 confocal microscope with a 20X oil immersion lens, at 1024x1024, zoom 1.4, speed 400 Hz and Z step 0.96 µm.

Quantification of cell number using DeadEasy
Cells were counted automatically using DeadEasy software, developed as ImageJ plug-ins, as reported in (Li et al., 2020).Repo+ cells in the adult brain were counted automatically with the DeadEasy Glia Adult ; Sarm>HisYFP+ and MyD88>HisYFP+ cells were quantified with DeadEasy adult central brain; and THGAL4 R58E02GAL4>HisYFP+ cells were counted with DeadEasy Kenyon Cells.

Statistical analysis
Data were collected using Excel (Microsoft) and analysed using GraphPad Prism®.
Longevity assay data were analysed using Log Rank (Mantel-Cox test).Categorical proboscis extension response data were analysed using Chi-square test.Cell number counting data were numerical and continuous.Datasets with four or more groups were first tested for normality.For sample groups with two sample types, unpaired Student t-tests were conducted; and Mann-Whitney U tests if not normally distributed.For comparisons involving infection vs, not-infected, and two genotypes, Two-Way ANOVA followed by Turkey's multiple comparison tests were used, using 95% confidence.
) or sarm (Figure 7C,D) was sufficient to cause a significant reduction in the number of PAM neurons in the absence of infection.This shows that increased Wek and Sarm levels could induce PAM cell loss.Altogether, these data support the notion that Toll-1 signalling via Wek and Sarm can induce cell loss in the brain.They indicate that the upregulation of wek and sarm caused by B. bassiana infection leads to cell loss in the host brain.
. Importantly, knocking down either Toll-1, sarm or wek rescued the cell loss caused by B. bassiana infection, and over-expression of wek or sarm was sufficient to induce cell loss in the absence of infection.Together, these data demonstrate that B. bassiana-induced cell loss requires Toll signalling via Wek and Sarm.Importantly, we showed that although Toll-1 expression varies among different DAN clusters, Toll-1 knock-down rescued B. bassiana-induced loss of cells expressing Toll-1.The variable effects in distinct DANs also suggest that different DAN clusters may express different combinations of Tolls, downstream adaptors and effectors driving distinct outcomes.

(
housekeeping\reference gene) was used.The expression of the target gene (ΔCT) was normalised in infected and non-infected flies by subtracting the CT value of the GAPDH (reference gene) from the CT value of the target gene.ΔΔCT was calculated by subtracting the ΔCT (Target gene) of the infected flies from the ΔCT (Target gene) of the non-infected flies.The relative gene expression of the target gene was then calculated in infected and non-infected flies and expressed as fold change (2 -ΔΔCT ).The statistical analysis was conducted on the ΔCT values.3 biological replicates were used each biological replicate consist of 20 flies.ΔCT = CT (a target gene) -CT (a reference gene\GAPDH); ΔΔCT = ΔCT (non-infected sample) -ΔCT (infected sample); Fold change = 2 -ΔΔCT

Figure 1 .
Figure 1.B. bassiana spores were detected within the fly brain.(A) Infection chamber.
Figure 5B.bassiana induced cell loss requires Wek, but Wek has pleiotropic

Figure 6 BFigure 5
Figure 6 B. bassiana induced cell loss requires sarm (A) Seven-day exposure to the B. bassiana caused loss of MyD88>HisYFP+ cells, and this was rescued with adult-specific sarm-RNAi JF01681 knockdown, quantification in (C).(B)