Natural variation in the C. elegans autophagy response and clearance of intracellular microsporidia

Natural genetic variation can determine the outcome of an infection, and can reflect the co-evolutionary battle between hosts and pathogens. We previously found that a natural variant of the nematode C. elegans from Hawaii (HW) has increased resistance against natural microsporidian pathogens in the Nematocida genus compared to the standard laboratory strain of N2. Strikingly, HW animals can clear infection, while N2 animals cannot. HW animals can clear infection only at the first larval stage of life, which is when Nematocida imposes selective pressure on this host. Here we investigate how this clearance ability relates to xenophagy, which is the process of autophagy used for degrading intracellular microbes. We find there is much better targeting of autophagy machinery to parasites under conditions where they are cleared. In particular, ubiquitin targeting to Nematocida cells correlates very well with their subsequent clearance in terms of timing, host strain and age, as well as Nematocida species. Furthermore, clearance correlates with targeting of the LGG-2/LC3 autophagy protein to parasite cells, with HW animals having much more efficient targeting of LGG-2 to parasite cells than N2 animals. Surprisingly, however, we found that HW lgg-2 mutants still can clear Nematocida infection, although they have increased pathogen load early in infection. These findings indicate that lgg-2 regulates intracellular colonization of microsporidia inside intestinal cells, and a non-lgg-2-mediated process controls clearance. Thus, natural variation in xenophagy protein targeting correlates with, but is not required for clearance in this natural infection model.


Introduction
! 3! Natural genetic variation underlies differences in susceptibility to infection and inflammation among individuals. 1 Genome-wide association studies in humans have revealed genetic variation in innate immune genes that predispose individuals to increased risk of infection and autoimmune disease. For example, polymorphisms in autophagy genes Nod2, ATG16L, and IRGM are associated with increased risk for Crohn's disease, which is an inflammatory bowel disease characterized by a dysregulated gut microbiome. 2,3 The discovery that natural variation in autophagy genes can lead to differences in inflammation is part of a large body of evidence indicating a close connection between inflammation, intestinal immunity, and autophagy. 4,5 Autophagy is a broadly conserved cellular process that was originally studied for its role in degrading and recycling bulk intracellular material, and subsequently has been shown to play a role in immunity. 6 When autophagy is used to degrade intracellular pathogens it is called xenophagy. 7-10 Xenophagy often begins with localization of host ubiquitin to intracellular microbes, followed by localization of autophagy proteins, formation of an autophagosome to enclose the microbe, and then fusion with a lysosome to degrade microbial cargo. Initial studies of the role for autophagy in immunity focused on xenophagy, although it is now appreciated there are 'non-canonical' forms of autophagy that also promote anti-microbial defense. 11 For example, autophagy can regulate secretion of anti-microbials outside the cell during stress. 12 Given that natural variation in autophagy underlies human risk of intestinal inflammatory disease, studying how autophagy responses vary among individuals in a species and how these responses contribute to immunity could provide insights into human disease.
The nematode Caenorhabditis elegans provides an attractive system for study of natural variation in innate immunity in the intestine and the role of autophagy. 13 There are many natural isolates of C. elegans, and various natural pathogens associated with these strains. 14 The most common cause of infection for C. elegans in the wild is microsporidia, which comprise a phylum of >1400 species of obligate intracellular microbes related to fungi. 15,16 Microsporidia invade host cells using a polar tube that 'fires' to deliver a parasite cell called a sporoplasm directly into the host cell. The first microsporidian species shown to infect C. elegans was isolated from wildcaught nematodes near Paris, France and we named it Nematocida parisii strain ERTm1 (nematode-killer from Paris), because it causes a lethal intestinal infection. 17 In the early stages of infection, N. parisii grows in direct contact with the host cytoplasm, and RNAseq analysis identified several predicted host ubiquitin ligase components induced by N. parisii infection. 18,19 Because ubiquitin ligases can act upstream of autophagy by targeting ubiquitin to microbes exposed to the cytoplasm, we explored the role of autophagy/xenophagy in resistance to N.
parisii. C. elegans has conserved autophagy machinery, and other studies have demonstrated a role for autophagy in response to infection with extracellular, clinically relevant pathogens. 13,[20][21][22][23] However, bona fide xenophagy (i.e. autophagy targeting to microbes that mediates subsequent clearance of pathogen) has not yet been demonstrated in C. elegans. In RNAi knock-down studies we found that autophagy is necessary and sufficient to regulate N. parisii load in the standard N2 laboratory strain of C. elegans, but only to a modest extent. 19 We did find a low level of autophagy targeting to parasites cells, but in subsequent studies we have shown that these animals are not able to clear infection. 19 consequences for the host. Quantitative genetic analysis indicated that the HW genomic regions that confer increased immunity contain genes predicted to encode ubiquitin ligase adaptor proteins. 24 Therefore, we hypothesized that HW C. elegans may be better than N2 at targeting Nematocida cells with ubiquitin to direct autophagy machinery for clearance of these parasites via xenophagy. Indeed, because C. elegans lacks professional immune cells, and its intestinal epithelial cells are non-renewable, xenophagy provides an attractive candidate for a process that could explain how C. elegans can clear microsporidia infection from their intestinal cells. 26,27 Here we show natural variation in both the host and pathogen side in the C. elegans autophagy response to microsporidia, with a striking correlation between autophagy targeting to Nematocida cells and the ability to clear these pathogens from host intestinal cells. We find that HW animals have much higher levels of ubiquitin targeting to Nematocida cells compared to N2 animals. Furthermore, this targeting happens at the L1 stage, when HW have the ability clear infection, but not at the L4 stage, when HW cannot clear infection. We also find that ubiquitin targeting is more efficient to cells of a Nematocida species that is cleared, compared to two Nematocida species that are not cleared. In addition, we find much better targeting of the autophagy protein LGG-2/LC3 to Nematocida cells in HW animals compared to N2 animals.
Surprisingly however, we find that LGG-2 is not required for clearance in HW animals, although it does affect pathogen load early during infection. Thus, lgg-2 has an early role in regulating establishment of Nematocida inside intestinal cells, but is not important for clearing them via xenophagy. Altogether our findings show natural variation in xenophagic targeting to ! 6! microsporidia that are cleared from the C. elegans intestine, but the host appears to have separate, non-lgg-2 related mechanisms for clearing this co-evolved pathogen.

Nematocida infection assays and imaging
Infection experiments were performed with N. ironsii strain ERTm5, N. parisii strain ERTm1, and N. ausubeli strain ERTm2. 25, 31 Spores were prepared and quantified as previously described. 32 Host animals were either continuously exposed to Nematocida spores or pulse-inoculated with ! 7! spores for 3 hours as described in the figure legends. Infections of L1 stage animals or L4 stage animals were initiated as described previously. 24  Nematocida infections, fixed and stained animals were mounted on 5% agarose pads and imaged using a ×40 oil immersion objective on a Zeiss LSM700 confocal microscope run by ZEN2010 software. At least 10 infected transgenic animals were imaged per sample and Nematocida cells were counted as having transgenic proteins localized around them or not.

Measurements of transgenic protein levels
For Western blot measurements of protein levels, animals were washed off NGM plates with M9 buffer, and then washed once with PBS with 0.1% Tween 20 to remove bacteria. Samples were then resuspended in sample buffer with 1% SDS and 50 mM DTT and boiled at 95°C for 10 minutes. Lysates were then run on a 4-20% gradient SDS-PAGE gel (Bio-Rad) and transferred to PVDF membrane (Bio-Rad). The blots were stained with Ponseau S stain (Sigma-Aldrich) and imaged for total protein before staining with an anti-GFP antibody made in rabbits (a gift from Arshad Desai and Karen Oegema labs at UC San Diego) diluted at 1:5000 overnight at 4°C, then staining with an antirabbit HRP antibody at 1:10000 for 45 minutes at room temperature. The blots were treated with ECL reagent (Amersham GE Healthcare Life Sciences) and imaged on a Bio-Rad ChemiDoc. For measurements of GFP protein levels in live animals, uninfected and N. ironsii infected animals were collected 15 hpi and measured on the COPAS Biosorter (Union Biometrica) for size (TOF) and GFP (green). More than 1000 animals were measured per sample. Animals expressing transgenic GFP were distinguished from nonexpressing animals by measuring GFP levels in wild type N2 animals and only analyzing GFP levels in animals above this threshold.

CRISPR-Cas9 generation of lgg-2 deletion alleles
Using the CRISPR design tool (http://crispr.mit.edu), DNA sequences with high scores were chosen in regions upstream from the start and downstream from the stop codon of lgg-2 to create crRNAs (synthesized by Integrated DNA Technologies). crRNAs were annealed to the tracrRNA, and sgRNA products were co-injected with Cas9 protein and dpy-10 sgRNA marker. 33 Dumpy F1 animals were screened for lgg-2 deletions using PCR analysis. Isolated homozygous lines were confirmed and characterized by DNA sequence analysis and backcrossed three times to the original strain, CB4856. The jy44 allele contains a 1376-nucleotide deletion, which removes the entire lgg-2 gene and an additional 132 nucleotides upstream and 146 nucleotides downstream of the gene. Similarly, the whole lgg-2 gene together with 138 nucleotides upstream and 146 nucleotides downstream (1382 nucleotides in total) are substituted for a single nucleotide insertion in jy45 allele.

Feeding rate measurements
Fluorescent beads (Fluoresbrite TM Polychromatic Red 0.5 µm Microspheres, Polysciences, Inc.) were added in 1:50 ratio to the mixes of starved L1 animals, OP50 food source and N. ironsii spores in M9 buffer, which were prepared following the protocol used in the clearance experiments. After 30 minutes incubation at 25°C, animals were fixed in 4% paraformaldehyde and imaged using a Zeiss AxioImager. Fluorescence levels were analyzed using the FIJI program. Mean fluorescence per animal was calculated for 50 animals per strain, per replicate, in three separate experiments. Background fluorescence levels were subtracted from obtained results.

C. elegans than the N2 strain
To investigate the hypothesis that HW C. elegans clear N. ironsii infections through improved targeting of ubiquitin to parasite cells followed by autophagic clearance, we examined the localization of ubiquitin to parasite cells in the HW host compared to the N2 host strain, which does not clear infections. First, we generated transgenic N2 and HW strains that express GFPtagged ubiquitin under the control of an intestinal-specific promoter (GFP::UBQ-2). Ubiquitin is broadly conserved in eukaryotes, 34 and UBQ-2 encodes a canonical ubiquitin protein fused to the L40 ribosomal large subunit protein. We pulse-infected these GFP::UBQ-2-expressing strains at the first larval (L1) stage by feeding animals with N. ironsii spores for three hours, then removed them from spores and fixed them at different hours post-inoculation (hpi). We performed FISH staining on these fixed animals using a probe that targets Nematocida rRNA to label parasite cells and then determined the percentage of those cells that also have GFP::UBQ-2 localization ( Figure 1A). With this method, we found that a much higher percentage of parasite cells had ubiquitin localization to parasite cells in HW animals than in N2 animals. For example, at 15 hpi 84% out of 19 parasite cells analyzed in 10 separate animals ! 10! had GFP::UBQ-2 targeting in HW animals ( Figure 1B), and every animal had at least one parasite cell with GFP::UBQ-2 localization. In contrast, only 11% of parasite cells were targeted with GFP::UBQ-2 in N2 animals at 15 hpi ( Figure 1B).
Next we analyzed how ubiquitin targeting to parasites correlates with their clearance. In previous clearance experiments at 25°C we had found parasite load much lower in HW animals at 20 hpi compared to 3 hpi, but did not measure when clearance began between those two timepoints. 24 Here we analyzed clearance at several timepoints after 3 hpi and saw that clearance begins around 10 hpi at 25°C ( Figure S1A). 25°C is a slightly stressful condition for C. elegans, and autophagy is more active at this temperature. 35 We have also found that there are more proteins aggregates at this temperature, which can make localization experiments challenging, 19 so we have performed our autophagy localization experiments at 20°C (this paper and 19 ). Therefore, we also analyzed clearance at 20°C to ensure that clearance is not just a high-temperature phenomenon, and to better compare the kinetics of clearance and ubiquitin targeting. We found that parasite clearance also occurs at 20°C (Figure S1B), indicating it is not specific to higher temperature. Furthermore, we were able to compare the kinetics of clearance with ubiquitin targeting at the same temperature, and found that the peak of ubiquitin targeting correlates with the start of clearance (compare Figure 1B and Figure S1B). These findings are consistent with ubiquitin targeting driving parasite clearance.
One potential explanation for increased targeting of ubiquitin to parasite cells in HW animals is increased expression from the GFP::UBQ-2 transgene in HW animals. To investigate this possibility, we compared protein levels of GFP::UBQ-2 with two methods. First, we performed Western blots with anti-GFP antibodies on lysates from transgenic HW and N2 animals to compare levels of GFP::UBQ-2 across populations of animals. Here we found that HW animals ! 11! do not have increased levels of GFP::UBQ-2 compared to N2 animals ( Figure S2). Second, we analyzed GFP::UBQ-2 fluorescence on a per-animal level using a COPAS Biosort 'worm sorter'.
While there was animal-to-animal variability in fluorescence levels of GFP-ubiquitin, the overall levels were not higher in HW compared to N2 animals ( Figure 1C). Therefore, the increased targeting of GFP-ubiquitin to N. ironsii cells in HW compared to N2 does not appear to be simply due to increased GFP::UBQ-2 levels in HW animals.

Localization of ubiquitin to parasite cells correlates with their clearance in terms of host age and parasite species
In previous work, we showed that HW animals can clear infection by N. ironsii only during the first larval stage (L1), and this ability is lost in later larval stages. 24 To characterize genetic variation in clearance on the parasite side, we infected HW at the L1 and L4 stages with three different Nematocida species. We observed variation in clearance of these Nematocida species by HW animals, where N. ironsii was cleared by L1, but not L4 animals while N. parisii and N. ausubeli were cleared poorly by both L1 and L4 animals (Figure 2A, B). Therefore, the clearance of microsporidia by HW animals is specific to N. ironsii infections at the L1 stage.
To determine how these patterns of clearance related to the association of host ubiquitin with parasite cells, we examined the frequency of ubiquitin localization to Nematocida cells in L1 HW animals over time. Here we observed a high frequency of ubiquitin localization around N. ironsii cells but not N. parisii or N. ausubeli cells ( Figure 2C). Furthermore, the high frequency of ubiquitin localization to N. ironsii cells was observed in HW animals infected at the L1 stage but not in HW animals infected at the L4 stage or N2 animals at either stage ( Figure 2D). Thus, the frequency of host ubiquitin localization around microsporidia cells in C. elegans is highly correlated with clearance of infection.

Localization of LGG-2/LC3 but not LGG-1/GABARAP correlates with parasite clearance
Xenophagic clearance of intracellular pathogens involves targeting of autophagic machinery after ubiquitin targeting to pathogen cells. One of the most well-studied autophagic events is activation and conjugation of proteins in the LC3/GABARAP family to the developing autophagosome, which are required for fusion with the lysosome. 36 Therefore, we examined whether LGG-1 (homolog of GABARAP in mammals) or LGG-2 (homolog to LC3 in mammals), localized more frequently to parasite cells in HW worms compared to N2 worms. To analyze localization, we first generated N2 and HW transgenic animals with lgg-1p::GFP::LGG-1 or lgg-2::GFP::LGG-2 transgenes. Then we performed experiments similar to the ubiquitin studies in Figure 1. We pulse-infected these animals for 3 hours, fixed them at various timepoints after inoculation, stained with FISH to label N. ironsii cells and then quantified parasite localization with the GFP fusions ( Figure 3A). Here we found that there was a gradual increase in GFP::LGG-1 targeting to N. ironsii cells over time in both N2 and HW animals, with no obvious difference between the two strains ( Figure 3B). In contrast, we found that there was a much greater percentage of parasite cells targeted with GFP::LGG-2 in HW animals, compared with N2 animals ( Figure 3B). This increased localization of LGG-2 was comparable to the increased ubiquitin targeting seen in HW compared to N2 ( Figure 1B).
To eliminate the possibility that the increased targeting of LGG-2 in HW animals was simply due to increased levels of GFP::LGG-2 expression, we performed Western blot analysis ( Figure S3) and COPAS Biosort 'worm sorter' analysis of GFP::LGG-2 (and GFP::LGG-1)( Figure 3C).
Similar to our analysis of ubiquitin levels, we did not see an increased level of GFP::LGG-2 (or GFP::LGG-1) expression in HW compared to N2 animals. Additionally, we note that N. ironsii infection increased the levels of transgenic GFP::LGG-1 proteins in both N2 and HW strains ! 13! ( Figure 3C). Infection with N. parisii does not lead to differences in mRNA expression of lgg-1 (or ubq-2 or lgg-2) based on RNAseq analysis, which suggests that the autophagy protein LGG-1 may be differentially regulated during microsporidia infection post-transcriptionally.
LGG-2/LC3 is not required for clearance, but does affect intracellular colonization of N.

ironsii inside intestinal cells
The increased localization of LGG-2 to parasite cells in HW, and the ability of this strain to clear infection suggested that LGG-2 was important for the clearance ability in this strain. Therefore, we examined a functional role for LGG-2 in defense against N. ironsii. To do this we used CRISPR/Cas9 editing to generate a complete deletion of the lgg-2 locus in the HW strain background. We isolated and characterized two independent alleles (called HW lgg-2(jy44) and HW lgg-2(jy45)) in which the entire lgg-2 locus is deleted and should produce no LGG-2 protein ( Figure 4A). We backcrossed these strains and then analyzed their ability to clear N. ironsii. In several independent experiments, we saw that HW lgg-2 mutants were still able to clear N.
ironsii between 3 hpi and 20 hpi ( Figure 4B, Figure S2A). Therefore, despite the striking increase in localization of LGG-2 to N. ironsii cells in HW animals compared to N2 animals, LGG-2 is not required for clearance of N. ironsii under these conditions. We did notice however, that HW lgg-2 mutants had a higher initial dose of parasite at 3 hpi compared to wild-type HW animals. In particular, we saw that HW lgg-2 mutants had a higher percentage of animals infected with N. ironsii compared to wild-type animals ( Figure 4B, S4A).
Furthermore, when we analyzed just the infected animals, we found that HW lgg-2 animals had higher number of sporoplasms per animal ( Figure 4C, S4B). One potentially trivial explanation for this difference could be that HW lgg-2 mutants have greater exposure to the pathogen because it accumulates to a higher level within the intestinal lumen, either through increased ! 14! feeding or decreased defecation. To examine this possibility we used a fluorescent beadfeeding assay and measured accumulation of beads inside the intestinal lumen in HW and HW lgg-2 mutants. Here we found that there was no increase in the accumulation of beads in HW lgg-2 mutants, indicating that their increased parasite load at 3hpi is likely not due to increased exposure to parasites ( Figure 4D; Figure S4C). Therefore, lgg-2 may be acting at a very early stage to regulate pathogen load of the natural intracellular parasite N. ironsii in HW animals.
Although clearance still occurred in HW lgg-2 mutants these animals had slightly less efficient clearance between 3 hpi and 20 hpi compared to HW wild-type animals. To determine whether this was due to the difference in initial dose at 3 hpi between HW and HW lgg-2 mutants, we infected HW animals with a higher spore dose than HW lgg-2 animals such that both strains had roughly equivalent initial sporoplasm number at 3 hpi ( Figure S5). Under this condition, we found that clearance from 3 hpi to 20 hpi was similar in HW and HW lgg-2 mutants, indicating that lgg-2 is not required for clearance of N. ironsii, but is required for regulating the initial colonization of this natural parasite inside host intestinal cells.

Discussion
These studies demonstrate natural variation in C. elegans targeting of autophagy machinery to natural Nematocida pathogens, and show that targeting correlates very well with clearance of these pathogens ( Figure 5). In particular, we found that HW animals, which can clear N. ironsii infection, have increased ubiquitin targeting to parasite cells compared to N2 animals, which cannot clear infection. Furthermore, this efficient targeting only occurs as the L1 stage when HW animals can clear infection, but not at the L4 stage when HW cannot clear infection. In addition, there is a correlation between clearance and localization of the autophagy protein LGG-2 to N. ! 15! ironsii cells in HW animals. Surprisingly however, we found that deletion of lgg-2 in HW animals did not substantially alter the ability of this host to clear the parasite. In mammals there are six proteins in the LC3/GABARAP family involved in autophagy, three in the LC3 family and three in the GABARAP family, with redundancy among them. 36 C. elegans just has two proteins in this family, LGG-2 (homolog of LC3) and LGG-1 (homolog of GABARAP), and they do have redundant function in other contexts. 30, 37 Thus, it is possible that LGG-1 (GABARAP ortholog) is able to compensate for LGG-2 in clearance of microsporidia in HW C. elegans. Unfortunately, it is difficult to test this model, because LGG-1 is an essential gene, and HW worms are resistant to RNAi. 38 However, it seems somewhat unlikely that LGG-1 could substitute for LGG-2, given that there is extensive LGG-1 localization to parasite cells in N2 animals (at the equivalent level to HW animals, see Figure 3) and in this context LGG-1 is not sufficient for clearance.
How are Nematocida pathogens cleared from HW animals? Our findings do not rule out LGG-2 mediated xenophagy as playing a role in clearance, but they show it is not required. It is possible that LGG-2 does mediate clearance through xenophagy, and this process is redundant with another form of clearance ( Figure 5). Given that C. elegans and Nematocida species appear to have a long co-evolutionary relationship, 15 it is possible that C. elegans evolved additional forms of clearance as part of an arms race between this host and its natural pathogen. Studies in other systems have described pathogen virulence factors that suppress host autophagy, for example through removing LC3 conjugation from the autophagosomal membrane. 9 Perhaps because another pathogen suppressed LGG-2-mediated autophagy in its evolutionary past, C. elegans evolved a separate pathway for clearance that functions here in the absence of LGG-2 to clear N. ironsii. Regardless of the reason, it is clear that HW animals have a pathway separate from LGG-2 that mediates clearance of N. ironsii. Given the extremely close correlation between ubiquitin targeting and Nematocida clearance (Figure 1-3), perhaps ! 16! ubiquitin is functionally relevant for this pathway, and serves to recruit another type of degradative machinery to the parasite in parallel to LGG-2. Unfortunately, because ubiquitin is an essential gene, it is difficult to test a functional role here. It is also possible that the ubiquitin targeting we see is not functionally relevant but is simply correlative with clearance. Indeed, there are several other studies where targeting of autophagic machinery to microbes is not indicative of a functional role in clearance. 10,39 It is difficult to thoroughly test a role for autophagy in the clearance of Nematocida from HW animals, because many autophagy proteins are essential, and as mentioned above, RNAi knock-down in HW is relatively inefficient.
Nonetheless, our findings suggest C. elegans evolved additional, non-xenophagic strategies for clearance of natural pathogens.
Interestingly, while lgg-2 is not required for clearance, we found that HW animals defective in lgg-2 do have more microsporidia sporoplasms inside intestinal cells compared to wild-type animals. These results indicate that lgg-2 has a role in controlling early Nematocida colonization inside of intestinal cells, after the spores are ingested into the intestinal lumen. One explanation for these results is that lgg-2 has a role in regulating the viability of microsporidia spores in the extracellular lumenal space, or in regulating their ability to fire the polar tube for invasion of intestinal cells. If so, this effect would be consistent with lgg-2 acting through secretion of antimicrobial compounds, as has been shown for regulation of lysozyme secretion by the autophagy pathway in the mammalian intestine. 12 Another possibility is that lgg-2 regulates the intracellular establishment of sporoplasms after they have been delivered into the cytoplasm by the polar tube. Nothing is known about whether the host cells control this stage of microsporidia infection, as polar tube firing is described as a mechanical rupture that 'forces' a parasite cell into the host cell. 16 However, it is exciting to consider that host cells may regulate this step.

17!
Our observations that lgg-2 control sporoplasm levels at 3 hpi are arguably the earliest stage at which a host factor has been shown to control microsporidia load in any system. Studies of microsporidia species that infect mammalian cells have implicated host glycosaminoglycans 40 and a transferrin receptor protein 41 in regulation of early steps, but the exact stage and mechanism by which they act is unknown. It will be interesting to further explore this early role for lgg-2. However, as polar tube firing has not been visualized in a live host it is difficult to determine whether lgg-2 regulates polar tube firing or a later step in this live C. elegans/Nematocida system. Analysis in a tissue culture system might aid in distinguishing among these possibilities, although no such system exists for C. elegans. Thus, a role for the autophagy protein lgg-2 in this very early step of sporoplasm colonization is intriguing but challenging to pursue further with existing tools. Nonetheless, the observations here paired with further analysis could shed light on the poorly understood question of how hosts control microsporidia infection.        and lgg-2(jy45) mutants show similar feeding rate. Feeding assay was performed in triplicate, 50 animals were analyzed per strain per experiment. (B, C) A student's t-test was used to calculate p values; p < 0.001 is indicated with four asterisks; ns indicates non-significant difference (p > 0.05). (A-C) All experiments were performed at 25°C.

Figure S5. Ability to clear infection is similar in wild-type HW animals and lgg-2 deletion mutants if initial infection rate is similar.
(A, B) N. ironsii clearance in HW wild-type (green line) and lgg-2(jy44) animals (orange line) is similar if HW wt animals are infected with a higher dose of microsporidia spores than HW lgg-2(jy44) mutants, to achieve similar initial intestinal colonization in both strains. (A) N. ironsii clearance in HW wild-type animals (green line) and lgg-2(jy44) mutants (orange line). 100 animals were analyzed per strain at 3 hpi and 20 hpi (x-axis). (B) Box-and-whiskers plot shows