Conservation of Nematocida microsporidia gene expression and host response in Caenorhabditis nematodes

Yin Chen Wan; Emily R Troemel; Aaron W Reinke

doi:10.1371/journal.pone.0279103

. 2022 Dec 19;17(12):e0279103. doi: 10.1371/journal.pone.0279103

Conservation of Nematocida microsporidia gene expression and host response in Caenorhabditis nematodes

Yin Chen Wan ¹, Emily R Troemel ², Aaron W Reinke ^1,^*

Editor: Nicolas Corradi³

PMCID: PMC9762603 PMID: 36534656

Abstract

Microsporidia are obligate intracellular parasites that are known to infect most types of animals. Many species of microsporidia can infect multiple related hosts, but it is not known if microsporidia express different genes depending upon which host species is infected or if the host response to infection is specific to each microsporidia species. To address these questions, we took advantage of two species of Nematocida microsporidia, N. parisii and N. ausubeli, that infect two species of Caenorhabditis nematodes, C. elegans and C. briggsae. We performed RNA-seq at several time points for each host infected with either microsporidia species. We observed that Nematocida transcription was largely independent of its host. We also observed that the host transcriptional response was similar when infected with either microsporidia species. Finally, we analyzed if the host response to microsporidia infection was conserved across host species. We observed that although many of the genes upregulated in response to infection are not direct orthologs, the same expanded gene families are upregulated in both Caenorhabditis hosts. Together our results describe the transcriptional interactions of Nematocida infection in Caenorhabditis hosts and demonstrate that these responses are evolutionarily conserved.

Introduction

Microsporidia are obligate eukaryotic intracellular pathogens [1]. This fungal-related phylum contains over 1400 described species that infect a wide range of animal hosts including invertebrates, vertebrates, and protists [2, 3]. Although as a phylum microsporidia infect a wide range of hosts, most species only infect one or several closely related hosts [4, 5]. Throughout evolution, microsporidia have lost many metabolic and biosynthesis genes that are present in other eukaryotes [6]. These adaptations to survive within their hosts have resulted in microsporidia having the smallest known eukaryotic genomes [7]. These features of microsporidia being able to specifically infect most types of animals with a limited coding capacity have made microsporidia a powerful model for understanding the evolution of intracellular parasites [8–11].

The model nematode Caenorhabditis elegans has become an important system for studying microsporidia infections [12]. Nematocida parisii and Nematocida ausubeli (referred to as Nematocida sp. 1 in some publications) are the two microsporidia species most commonly observed to infect Caenorhabditis elegans [13, 14]. These two species of Nematocida are also commonly found to infect Caenorhabditis briggsae, a related species which has been used as a model organism to facilitate comparative analyses to C. elegans [15, 16]. These two nematode species live in overlapping geographical locations [17, 18]. Both of these species are hermaphrodites, but they have some differences in development and response to temperature [19, 20]. N. parisii was first found infecting C. elegans outside of Paris and N. ausubeli was originally found infecting C. briggsae in India. Since then, both microsporidia species have been found infecting both hosts in Europe [13, 21].

The infection cycle of N. parisii and N. ausubeli in Caenorhabditis hosts begins with ingestion of the microsporidia spores, an environmentally resistant and dormant stage of the pathogen [22, 23]. In the intestine, the sporoplasm in the spore is ejected through the polar tube and deposited into the intestinal cells. This is followed by replication into meronts and differentiation into new spores, which then exit intestinal cells into the intestinal lumen. The spores are defecated by the nematodes, and the infection cycle repeats when they are ingested by another host [22, 24, 25].

There are some similarities in the infections caused by these two Nematocida species; they both exclusively infect the intestine, they infect C. elegans and C. briggsae, they cause intestinal cells to fuse, and they have similar life cycles [13, 14, 26]. Both species also use similar types of secreted and membrane bound proteins to interface with host proteins [21]. Although the divergence time between N. parisii and N. ausubeli is unknown, the two Nematocida species share 68.3% amino acid identity [24, 25]. These two species also have distinct growth and phenotypic characteristics during infection in C. elegans. For example, N. ausubeli displays faster growth and increased impairment of host fitness [26].

In response to microsporidia infection, hosts often display large transcriptional changes. The gene expression of C. elegans in response to N. parisii infection is reported to be distinct from responses to bacteria, but similar to nodavirus infection [27]. Although the response to N. parisii infection also shares some similarities to the response against fungi such as Drechmeria coniospora and Harposporium, the genes upregulated during N. parisii infection are still mostly specific to either microsporidia or viral infection [27]. The unique transcriptional response against Nematocida has been termed the intracellular pathogen response (IPR) [28]. Among upregulated IPR genes, many contain F-box, FTH, and MATH domains that are implicated in substrate recognition during ubiquitin-mediated degradation [27, 29]. The IPR also includes upregulation of several Caenorhabditis specific families that are still poorly understood, such as the PALS family [30]. Mutants that cause the IPR to be upregulated are resistant to infection and can clear N. parisii infections [31–33].

Studies looking at infection of nematodes by different bacterial and fungal pathogens showed both significant overlap between pathogen infections, as well as species-specific responses [34–36]. In addition, recognition of two species of oomycete displayed a shared transcriptional response [37]. Similarities of responses between different host species have also been observed. For example, the response to nodavirus infection is conserved between C. elegans and C. briggsae [38]. An intergenerational transcriptional response to Pseudomonas vranovensis was conserved between some, but not all species of Caenorhabditis [39]. A study looking at four different species of bacteria infecting the nematode Pristionchus pacificus also showed both similarities and differences between the transcriptional responses in the two hosts [40]. For example, while genes in the FOXO/DAF-16, TGF-beta and p38 MAP kinase pathways are suggested to be conserved between C. elegans and P. pacificus in response to bacterial infection, host-specific transcriptional responses are also observed, such as the enrichment of lipid metabolism related domains in the response of P. pacificus to different bacteria [40]. Although conservation of responses between pathogen and hosts in C. elegans is observed, different strains of bacteria can elicit different responses, and different strains of C. elegans can have distinct responses [36, 41].

To determine the extent that microsporidia gene expression is influenced by its host and how conserved the host response is to microsporidia infection, we generated and analysed transcriptional data of N. parisii and N. ausubeli infecting C. elegans and C. briggsae. For the hosts, we used C. elegans and C. briggsae, which diverged ~18 million years ago [42]. About 60% of the genes from these species are orthologous and the median percent identity between these proteins is 80%, which is approximately the same extent of divergence between humans and mice [15]. We chose these two host models because both are infected by N. parisii and N. ausubeli efficiently in a laboratory setting, which recapitulates the natural interactions that exist in the wild [13, 14]. We observe similar transcriptional patterns of each Nematocida species in the two species of hosts with only a small set of differentially regulated genes. The host response to either of the two microsporidia species was also similar. This transcriptional response is conserved across C. elegans and C. briggsae. Altogether, our results suggest that different Nematocida species do not have distinct expression programs depending on the host, and transcriptional responses of Caenorhabditis hosts to Nematocida infection are conserved.

Materials and methods

Infection of nematodes with microsporidia

C. elegans strain N2 and C. briggsae strain AF16 were maintained at 21°C on 10-cm nematode growth medium (NGM) plates seeded with Escherichia coli OP50-1 for at least three generations without starvation. Three plates each of mixed stage populations of animals were washed with M9 and embryos extracted by treating with sodium hypochlorite/1 M NaOH for 2.5 minutes. Embryos were hatched by incubating for 18 hours at 21°C. ~30,000 L1s of each species were placed on a 10-cm plates along with 1 ml M9 and 10 μl of 10X OP50-1. Three plates of each species were prepared. Animals were infected with either 20 million N. parisii (ERTm1) or N. ausubeli (ERTm2) spores. These spores were prepared as described previously [22]. Uninfected plates were used as the control. Plates were dried for 1 hour in a clean cabinet and incubated an additional 1.5 hours at 21°C. Animals were then removed from plates using two 5 ml washes of M9. After washing samples twice with 10 ml M9, all but 1 ml of M9 was removed from the samples. An additional 3 ml of M9 and 1 ml of 10X OP50-1 was added to each sample, and 1 ml of this solution was plated onto 4 10-cm plates per sample, resulting in ~6,000 animals per plate. At either 10, 20, or 28 hours post infection, plates were harvested by washing off worms 3 times with 725 μl M9. Samples were then washed 3 times with 1 ml M9/0.1% Tween-20.

To determine the extent of infection, ~1,000 animals from each sample were removed, fixed, and stained using an N. parisii 18S rRNA fluorescent in situ hybridization probe as previously described [21]. Vectashield containing DAPI (Vector Labs) was added and samples imaged using an Zeiss Axioimager 2.

RNA extraction and sequencing

After the last wash, 1 ml of TRIzol was added to each sample and RNA was extracted similar to as described previously [27]. mRNA libraries were prepared by the UCSD IGM Genomics Center using Illumina Stranded mRNA Prep kit and sequenced on a single lane of an Illumina HiSeq 4000, using 100 bp paired-end reads.

Microsporidia gene expression analysis

The respective N. parisii and N. ausubeli genome annotation files were converted into.gtf format using Gffread v0.12.3. Reads from each sample were mapped to either Nematocida parisii strain ERTm1 (Genbank: GCF_000250985.1) or Nematocida ausubeli strain ERTm2 (Genbank: GCA_000250695.1) using TopHat v2.1.2, which also calculates the overall read mapping rate [43]. Using FastQC, quality control was performed on the Tophat aligned files to ensure that they are of good quality (quality scores across all bases >30) and that the adapters were properly removed from the sequencing datasets prior to downstream analysis [44]. Transcriptome assemblies were then generated using Cufflinks v2.2.1 [45] and Cuffmerge in the Cufflinks package. Differentially expressed genes were determined using Cuffdiff v2.2.1 [46] and were visualised using the R package cummeRbund v2.36.0 [47].

The Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values of N. parisii and N. ausubeli transcripts in each Caenorhabditis host were calculated by Cuffdiff v2.2.1 [46]. The linear correlation between gene expression in each host was determined using the R packages ggplot2 v3.3.5 and ggpubr v0.4.0; the ratio of FPKM values between the two hosts was calculated by dividing the microsporidia’s FPKM values in C. elegans by that in C. briggsae. Hierarchical clustering of log10-transformed FPKM+1 values was done using the hclust function from the R package stats v4.1.1; heatmaps were generated by ggplot2 v3.3.5 in R.

Caenorhabditis RNA-seq analysis

The paired end reads of each 10 hours post infection sample were submitted to Alaska v1.7.2 (http://alaska.caltech.edu), an online automatic C.elegans RNA-seq analysis pipeline. During the pipeline, briefly, Bowtie2 [48], Samtools [49], RSeQC [50], FastQC [44], and MultiQC [51] were used for quality control of the input files. Then, Kallisto [52] was used for read alignment and quantification, followed by differential analysis using Sleuth [53]. For C. elegans samples, the reads were aligned to the N2 reference of the WS268 release (accession: PRJNA13758); the reads from C. briggsae samples were aligned to reference genome of the WS268 release (accession: PRJNA10731). The 20 and 28 hours samples were not submitted for analysis due to the lack of replicates at these timepoints.

Log2 fold-change values of duplicated genes in each of the eight samples were averaged using the R package dplyr v1.0.8. Genes with FDR-adjusted p-value of <0.05 were regarded as significant. Differentially upregulated genes were defined as those with an FDR-adjusted p-value <0.05 and log2 fold change ≥0 (infected vs. control); differentially downregulated genes were defined as those with an FDR-adjusted p-value <0.05 and log2 fold change ≤0 (infected vs. control).

Principal component analysis

The normalised abundance measurements in C. elegans and C. briggsae generated by Alaska, were read by the readRDS() function of the R package base v4.1.1. Normalized counts for each gene in each host species were generated via sleuth_to_matrix() with the “obs_norm” data and “tpm” units. PCA of gene expression was performed using samples from each species via the R package pcaexplorer v2.20.2 [54].

Determination of gene expression overlap

Genes with an FDR<0.05 were used to compare expression overlap. Values of duplicated genes in each sample were averaged using the R package dplyr v1.0.8. Overlap between samples was determined using the R package gplots v3.1.1. The p-values were calculated using the Fisher exact test. Statistical enrichment tests of shared and non-shared genes were performed using PANTHER on pantherdb.org [55]. Each input list was statistically tested for enrichment against the annotation sets: “GO biological process complete”, “GO cellular component complete”, “GO molecular function complete”, “PANTHER GO-Slim Biological Process”, “PANTHER GO-Slim Cellular Component”, “PANTHER GO-Slim Molecular Function”, “PANTHER Pathways”, “Reactome pathways” and “PANTHER Protein Class”. Results from these tests with p-value <0.05 are in S4 Table.

To determine overlap in our samples compared to differentially regulated genes from Bakowski et al., we first used Alaska to process the read files of C. elegans infected by N. parisii samples at 8, 16, 30, 40 and 64 hours. To compare our samples’ expression with data from Bakowski et al., genes expressed in at least four of those samples were used for hierarchical clustering by hclust function in the R package stats v4.1.1. The dendrogram in the was produced using the R package ggdendrogram v0.1.23; the heatmap was generated by ggplot2 v3.3.5. Overlap between our samples, the Bakowski et al., (2014) samples, and the Chen et al. (2017) samples containing differentially regulated genes for C. elegans N2 infected by Orsay virus or N. parisii, and C. briggsae infected by Santeuil virus were calculated as described above.

Domain enrichment analysis

The type of gene classes and domains investigated for the enrichment analyses are listed in S6 Table. Among these, we actively chose to look at F-box, MATH/BATH, PALS, DUF684, DUF713, C-type lectins, and skr domains containing genes, implicated from differentially regulated genes of N. parisii infected C. elegans in previous publications [27, 38]. We also examined additional types of genes and domains enriched in our datasets using DAVID Bioinformatics Resources (2021) [56, 57] for C. elegans and the WormBase simple Ggne queries tool for C. briggsae. A list of gene names from respective gene classes were downloaded separately from Wormbase (http://wormbase.org/) and the corresponding protein-coding sequences were extracted from Wormbase ParaSite (https://parasite.wormbase.org/). Alignments of other domains or classes were downloaded directly from Pfam (http://pfam.xfam.org/) separately. After deleting duplicated sequences, protein sequences of these classes of genes were converted to Stockholm format using Clustal Omega Multiple Sequence Alignment tool (https://www.ebi.ac.uk/Tools/msa/clustalo/). Hmmbuild of HMMER v3.3.2 [58] was used to build respective profile HMMs, which was then searched against C. elegans (accession: PRJNA13758) proteome of WS268 release using hmmsearch. Output genes with E-value <1e-5 were used as the gene list for the enrichment analyses. In the output genes, MATH and BATH genes were combined into one list; fbxa, fbxb and fbxc genes were merged into the gene list for F-box while genes with CUB and CUB-like domains were categorised together (S6 Table). Using the R package gplots v3.1.1, the number of genes overlapping between the lists and samples were computed, then plotted using R package ggplot2 v3.3.5. Genes that do not fall into any of these gene classes or have any of these domains were categorized as “other”.

Determination of orthologs between C. elegans and C. briggsae

Orthologous genes were determined between C. elegans (accession: PRJNA13758) and C. briggsae (accession: PRJNA13758) proteomes of the WS268 release using Orthofinder v2.5.2 with the default settings [59].

Single copy orthologs analysis

Genes that are single copy orthologs were extracted, based on the single copy orthologs list computed by Orthofinder. Single copy orthologs expressed in at least one out of the four samples were used for hierarchical clustering via the function hclust() from the R package stats v4.1.1, then plotted using R packages ggdendrogram and ggplot2 v3.3.5. The linear correlation of each single copy ortholog expression between the two host was calculated using ggpubr v0.4.0. The scatterplots and volcano plots were generated by ggplot2 v3.3.5.

Comparison of gene expression in expanded gene families

The C. elegans and C. briggsae genes in PALS and DUF713 families were obtained from the output of HMMER v3.3.2 [58] using a E-value threshold of <1e-5. The protein sequences of the genes were obtained from Wormbase ParaSite (https://parasite.wormbase.org/) and aligned by M-Coffee using default settings [60, 61]. A phylogenetic tree for each family was generated using MrBayes [62, 63] with the following settings: lset nst = 1, rates = invgamma, nruns = 2, stoprule = YES, stopval = 0.05 and mcmcdiagn = YES. A dendrogram of the phylogenetic tree of each gene family was created using the function ReadDendrogram from the R package DECIPHER v2.22.0. For each gene family, the orthologs’ log2 fold change values across the C. elegans and C. briggsae samples were plotted as a heatmap using ggplot2 v3.3.5.

Results

Gene expression of N. parisii and N. ausubeli is similar in C. elegans and C. briggsae

To understand how gene expression of related microsporidia and hosts species is conserved, we infected Caenorhabditis nematodes with Nematocida microsporidia (Fig 1A). We designed our infection experiment to compare transcriptional differences between related microsporidia and host species (Fig 1B). Unlike previous transcriptional profiling experiments of Nematocida infection, which were done as continuous infections at 25°C [27, 38], we infected the worms at 21°C for a short period of time to synchronize the infections. We pulse-infected C. elegans and C. briggsae with either N. parisii, N. ausubeli, or a mock treatment for 2.5 hours, washed to remove spores from outside the worms, and then replated the animals for a total of 10, 20, or 28 hours of infection at 21°C. We chose these three timepoints because at 21°C, Nematocida is in the sporoplasm stage at 10 hour post infection, and in the meront stage at 20 and 28 hours post infection, which allowed us to compare gene expression levels at these growth stages [26, 27] (Fig 2A). Each condition was done once, except for the 10-hour time point which was performed in duplicate (Fig 1A). Samples were stained using a probe specific to the Nematocida 18S rRNA and we observed that greater than 75% of each population was infected (Fig 2A and 2B). Compared to N. parisii-infected animals, we observed more parasite in N. ausubeli-infected hosts at 28 hours, consistent with a previous report that N. ausubeli grows faster than N. parisii [26]. RNA from infected and uninfected animals was extracted and sequenced. To determine the expression of microsporidia genes, we mapped reads of N. parisii and N. ausubeli to their respective genomes (S1 Table). At 10 hours post infection, the overall percentage of reads from either N. parisii or N. ausubeli were <1%, which increased to ~3–5% at 28 hours post infection (Fig 2C).

Fig 1 — (A) Schematic diagram of the RNA-seq experiment. C. *elegans* and C. *briggsae* were either not infected or infected with N. *parisii* or N. *ausubeli* separately at the L1 stage for 2.5 hours. Next, animals were washed to remove any remaining microsporidia spores and replated. Worms were harvested for RNA-sequencing at 10, 20 and 28 hours after infection. (B) The three comparisons of transcriptional responses made in this study are represented. Comparison 1 is microsporidia gene expression between hosts, comparison 2 is the response of each host species to different microsporidia species, and comparison 3 is the conserved response between different hosts to each microsporidia species. Figure was created with BioRender (www.biorender.com).

Fig 2 — (A) Pulse-infected C. *elegans* or C. *briggsae* were fixed and then stained with DAPI to detect nuclei and a FISH probe specific to *Nematocida* 18S rRNA to detect either N. *parisii* or N. *ausubeli* at 10, 20 and 28 hours after infection. Representative images of infection are shown. Scale bars are 23 μm. (B) Percentage of infected animals at different timepoints. Between 39 to 220 animals were counted for each sample. (C) Overall read mapping rate of *Nematocida* in each host at different time points, calculated by TopHat during alignment to the respective reference genomes. R1 or R2 indicates the replicate samples at the 10-hour timepoint.

To define the transcriptional patterns of N. parisii and N. ausubeli during infection, we analysed RNA-seq results of each microsporidia species between C. elegans and C. briggsae at 10, 20, and 28 hours post infection. First, we used principal component analysis to compare microsporidia gene expression in each of these samples. We observed that at the 10-hour time point, there is a larger difference between expression in the two hosts, and at the later time points, expression in the two hosts is similar (Fig 3A and 3B). The 20- and 28-hour time points in N. parisii cluster closely together, but there is a larger difference between these time points in N. ausubeli, likely due to the accelerated growth of this species [26]. Moreover, we observed the expression of genes in each microsporidia species to be similar between the two Caenorhabditis hosts across the timepoints (Fig 3C and 3D, S1A and S1B Fig). Strong correlation of Nematocida gene expression between C. elegans and C. briggsae was also observed, with similar levels of correlation of each microsporidia species in either host than between replicates in the same host (Fig 3E and 3F, S1C and S1D Fig). These similarities between expression in different host species also did not change across time points as the parasite had gone through larger amounts of replication. At 10 hours post infection, over 45% of Nematocida genes are within 2-fold of each other, and over 60% are within four-fold of each other. As the time post infection increased to 28 hours, over 75% of Nematocida genes are within two-fold of each other, and more than 89% are within four-fold of each other. The similarity is even more pronounced in highly expressed genes (genes with greater than 100 FPKM in either host) at the 20- and 28-hour time point where 95–99% are within four-fold of each other.

Fig 3 — Principal component analysis of (A) N. *parisii* and (B) N. *ausubeli* in C. *elegans* and C. *briggsae* at 10, 20 and 28 hours. Circles represent confidence ellipses around each strain at 95% confidence interval. (C-D) Heatmap of transcriptional profiles of genes expressed in N. *parisii* (C) and N. *ausubeli* (D). Rows represent gene clustered hierarchically. Scale is of differential regulation of infected compared to uninfected samples. (E-F) Scatterplot of log10 FPKM values in N. *parisii* (E) and N. *ausubeli* (F) when infecting C. *briggsae* and C. *elegans*. Each point represents a microsporidian gene. Pearson correlation value and p-value are indicated on the top left of each plot. The ratio of FPKM values for each gene between C. *elegans* and C. *briggsae* is demonstrated by the colour of the points, which is described in the legend at the bottom.

To determine genes that are significantly differentially regulated depending upon the host species infected, we compared the 10-hour time points of each Nematocida species between the two hosts at 10 hours post infection (Fig 4A and 4B). We identified 34 differentially regulated genes in N. parisii and 11 in N. ausubeli (alpha< 0.05) (S1 Table). Most of these differentially regulated genes had higher expression in C. elegans than C. briggsae (31/34 in N. parisii and 7/11 in N. ausubeli). Notably, differentially regulated genes were significantly enriched for ribosomal protein genes in both N. parisii (17/34, Fisher’s exact test p-value = 2.2x10^-18) and N. ausubeli (6/11, Fisher’s exact test p-value = 1.2x10^-8). Taken together, our results indicate that the transcriptional programs of N. parisii and N. ausubeli are largely independent of which Caenorhabditis hosts they infect.

Fig 4 — (A-B) Line plots of the FPKM values with confidence intervals, calculated for the differentially regulated genes (FDR<0.05) in N. *parisii* (A) and N. *ausubeli* (B) at 10 hours post infection. * indicates ribosomal protein-encoding genes. The aggregate sample value of the two replicates is noted as a black dot within the confidence interval, while the value for each replicate is shown as a dot of matching colour along the confidence interval. Error bars of each gene show the variability of the distribution of FPKM values.

Shared and unique transcriptional responses of each Caenorhabditis host to N. parisii and N. ausubeli infection

Different microsporidia species may induce similar or distinct transcriptional responses in the same host. To address this question, we compared how either C. elegans or C. briggsae responded to infection with either N. parisii or N. ausubeli at the 10-hour timepoint for which we had replicate data. We first mapped the reads from each sample to the corresponding host genome (S2 Table). In C. elegans, we identified 875 genes that are significantly differentially regulated in N. parisii infected samples and 735 genes that are significantly differentially regulated in N. ausubeli infected samples (Fig 5A and 5B and S3 Table). For C. briggsae, 1091 genes are significant in N. parisii infected samples while 449 were significant in N. ausubeli infected samples (Fig 5C and 5D and S3 Table). There are more downregulated than upregulated genes in each sample. Next, we directly compared shared genes that are upregulated in each of the Caenorhabditis hosts when infected by N. parisii or N. ausubeli. We observed significant overlap in the transcriptional response to these infections with 222 upregulated genes shared in C. elegans and 117 upregulated genes shared in C. briggsae.

Fig 5 — (A-D) Venn diagrams of genes in C. *elegans* (A-B) or C. *briggsae* (C-D) that are upregulated (A and C) or downregulated (B and D) when infected by N. *parisii* or N. *ausubeli*. npa (N. *parisii)* and nau (N. *ausubeli*).

We next compared both the overlapping and unique genes between samples to determine if they are enriched in known biological functions. Statistical enrichment tests indicate that only broad categories were enriched in the unique or shared genes between samples. We observed that the shared upregulated genes in N. parisii and N. ausubeli infected C. elegans are enriched for GO Biological Process associated with metabolism; as well as GO Molecular Process associated with catalytic activity (FDR<0.05, S4 Table). We did not observe any enrichment in the shared upregulated genes in C. briggsae, however the 267 shared downregulated C. briggsae genes showed enrichment for small molecule binding and structural constituent of cuticle (S4 Table). Of the non-shared genes, only the 415 downregulated genes in C. briggsae infected with N. parisii showed enrichment for GO Molecular Process associated with structural constituent of cuticle and protein binding (S4 Table).

We then compared our RNA-seq data to previously published studies of the transcriptional response to N. parisii infection (S5 Table). Bakowski et al. performed RNA-seq at multiple time points of a germline-deficient mutant of C. elegans continuously infected with spores at the L3/L4 stage at 25°C. We compared our 10-hour time point to the five time points in the Bakowski et al. study and observed strong overlap especially at the 8-, 16-, and 30-hour time points (S2A Fig). We observed significant overlap for all the time points with both our N. parisii and N. ausubeli 10-hour post infection samples (S2B and S2C Fig). We also compared our data to Chen et al. where C. elegans was infected with N. parisii at the L3 stage at 20°C. We observed significant overlap with both our 10-hour N. parisii and N. ausubeli data (S2D and S2E Fig). Chen et al. also measured Orsay virus infected C. elegans and Santeuil virus infected C. briggsae and we saw significant similarities to these as well (S2D and S2E Fig). Together our analysis demonstrates that despite the experimental differences between studies, a largely similar upregulated host response is consistently observed.

Evolutionary conserved host response to Nematocida infection

To investigate the evolutionary conserved response of Caenorhabditis to Nematocida infection, we studied the expression levels of C. elegans and C. briggsae orthologs. We identified orthologs using Orthofinder [59]. Additionally, we determined the subset of orthologs that only had a single copy present in each nematode species. Of the orthogroups shared between C. elegans and C. briggsae, about 76% are single-copy orthologs (10826/14183). We first clustered the expression of these one-to-one orthologs, which shows a smaller cluster of upregulated genes and a larger cluster of downregulated genes (Fig 6A). When we compared the host response to infection between the two host species, we observed a modest but significant corelation of response to infection between C. elegans and C. briggsae (Fig 6B). We then compared the statistical significance to the fold change of differentially expressed genes from the different infection conditions (Fig 6C and 6D) and investigated which of these differentially expressed genes were one-to-one orthologs (Fig 6E and 6F). We observed that only 8–17% of the differentially upregulated genes were one-to-one orthologs. Furthermore, of the most highly expressed genes (greater than four-fold), only 0–2% were one-to-one orthologs. In contrast to less than 20% of significantly upregulated genes not having one-to-one orthologs, between 29–42% of significantly downregulated genes did.

Fig 6 — (A) Heatmap of transcriptional profiles of single-copy orthologs in C. *elegans* and C. *briggsae* after infection by N. *parisii* or N. *ausubeli*. Rows represent genes clustered hierarchically. Scale is of differential regulation of infected compared to uninfected samples. **(B)** Scatterplot of log2 fold change values of single-copy orthologs expressed in C. *elegans* and C. *briggsae*. Each point represents a single-copy ortholog between the two host species. Pearson correlation values and p-values are indicated on top left of each plot. The ratio of log2 fold change values for each single copy ortholog between C. *elegans* and C. *briggsae* is demonstrated by the colour of the points. (C-D) Volcano plots of genes expressed in *Nematocida* infected C. *elegans* (C) and C. *briggsae* (D). Upregulated genes are represented with orange points; downregulated genes are represented with pink points. The top 5 upregulated and downregulated genes are labelled. (E-F) Volcano plots of genes expressed in *Nematocida* infected C. *elegans* (E) and C. *briggsae* (F). Single-copy orthologs are represented with red points and non-single copy orthologs represented with grey points. npa (N. *parisii)* and nau (N. *ausubeli*).

As much of the upregulated response is in the non-single copy orthologs, we analysed these genes for commonalties between samples. We performed domain enrichment analysis to determine the types of proteins induced in the non-single copy orthologs by microsporidia infection. Previous transcriptional analysis of C. elegans infected with either N. parisii or Orsay virus has identified serval types of domains enriched in expression, such as F-box, MATH/BATH, PALS, DUF713, DUF684 and C-type lectins [27, 38]. In addition to these domains, we also included other types of domains that are enriched in the significantly upregulated and downregulated non-single copy orthologs, for a total of thirteen domains that we analysed (S6 Table). From this analysis, we found additional domains and genes enriched with greater than three domain-containing proteins in any of our samples. These identified domains are chitinase-like (chil) proteins, CUB and CUB-like domains, cytochrome P450, glucosyltransferase family 92, nematode cuticle collagen N-terminal domain, and UDP-glucuronosyltransferase.

The proportion of each type of domain or gene family in the upregulated and downregulated non-single copy orthologs was determined (Fig 7A and 7B). For C. elegans infected with N. parisii, we observed that ~29% of the significantly expressed non-single copy orthologs contained at least one of these thirteen domains, with genes containing PALS domains being the most common. For genes upregulated after infection with N. ausubeli, we observed similar trends with ~35% containing one of these domains. Overall, our results suggest that expanded gene families, especially of IPR, are upregulated during Nematocida infection in Caenorhabditis species [38, 42]. The downregulated genes showed similar domain patterns for both microsporidia species, with cuticle collagen and glycosyltransferase being the two most common domains. Similar patterns regarding types of domains in regulated genes induced by the two microsporidia species was also observed in C. briggsae (Fig 7B). Interestingly, cytochrome P450 genes, implicated to be involved in detoxification, are upregulated in both hosts, suggesting that the upregulated response to Nematocida overlaps with the metabolism and elimination of potentially toxic xenobiotics [64]. Moreover, we observe the upregulation of a few chil genes in C. elegans and C. briggsae, previously shown to be upregulated in response to epidermally-infecting oomycetes [37, 65].

As some of the same expanded gene families are upregulated in both C. elegans and C. briggsae, we sought to determine the relationships between expression of these families in response to infection. We first constructed gene trees of PALS and DUF713 families (S3 and S4 Figs). Next, we compared the expression levels of these orthologs in our 10-hour samples. This analysis shows that some phylogenetically related PALS and DUF713 genes are upregulated in both C. elegans and C. briggsae in response to Nematocida infection (Fig 8A and 8B).

Fig 8 — (A-B) Heatmap of transcriptional profiles of differentially regulated genes (FDR<0.05) in the PALS (A) and DUF713 (B) gene families. Scale is of differential regulation of infected compared to uninfected samples. npa (N. *parisii)* and nau (N. *ausubeli*).

Discussion

To understand the conservation of transcriptional responses during microsporidia infection, we took the approach of comparing interactions between related microsporidia and host species. We found that responses between Caenorhabditis and Nematocida species are largely conserved. However, there are several limitations to our study. Although we monitored infection at multiple time points, we only performed duplicates at 10 hours post infection. Although a small number of replicates limits the ability to detect differentially regulated genes [66], we believe that our conclusions are not simply a consequence of the lack of statistical power for the following reasons. Firstly, we saw a large overlap in significantly regulated genes compared to previously published transcriptional datasets of N. parisii infected C. elegans [13, 14]. The conclusions we reach in our study also agree with those from another transcriptional response comparison from our group that generated RNA-seq data in triplicate of C. elegans infected at a latter timepoint with four different species of microsporidia, including N. parisii and N. ausubeli [67]. Secondly, previous statistical power calculations for RNA-seq analysis over a range of fold-changes and number of replicates suggests that a small number of replicates is sufficient to capture highly-expressed genes, such as the highly expressed IPR genes observed in our study [66, 68, 69]. Finally, previously published transcriptional datasets from Bakowski et al., (2014) and Chen et al., (2017), which we compared our dataset to, were also performed in duplicates or triplicates. Another limitation is that our study only analysed two pairs of hosts and microsporidia species. Additionally, differences in transcriptional responses between species could be dependent upon particular host and microsporidia strains or environmental conditions.

Our results suggest that Nematocida microsporidia species mostly do not sense and respond differently depending upon their host environment. The main difference we observed in Nematocida gene expression between the two hosts was an upregulation of both small and large ribosomal subunit genes in C. elegans at 10 hours post infection. We also observed about three-fold more ribosomal genes being differentially regulated in N. parisii than N. ausubeli. After invasion both species undergo a lag period before starting replication. This lag period is slightly shorter in N. ausubeli and the larger increase of ribosomal genes in N. parisii is potentially related to these differences in lag time [26]. The lack of large differences in the host response also suggests that these Nematocida microsporidia are likely not differentially regulating host genes for their own benefit.

How hosts sense microsporidia infection is not fully understood. In mammals, several toll-like receptors are necessary for immune activation [70], but the proteins that C. elegans uses to sense and respond to microsporidia infection are mostly unknown. Several negative regulators of the IPR are known, including PALS-22, LIN-35, and PNP-1, but none of these are known to be necessary for the response to infection [28, 31–33]. Recently, a basic-region leucine-zipper transcription factor, ZIP-1, was found to positively regulate a subset of IPR genes [71]. A viral RNA receptor, DRH-1, is necessary for activation of the IPR by the Orsay virus, but not for microsporidia infection [72]. Whether there is an equivalent protein that is specifically involved in detecting microsporidia infection in C. elegans is unknown. Although a previous study showed that three IPR genes were induced to a lesser extent by N. ausubeli than N. parisii [13], by comparing the full transcriptional profile we see a largely similar set of genes being induced. As these different microsporidia species induce a similar transcriptional response, this suggests that some common feature, such as invasion, is detected by the host.

Nematodes, insects, and vertebrates all have strong transcriptional responses to infection by microsporidia, though the responses appear to be quite diverse [73, 74]. For example, antimicrobial peptides are observed to be upregulated in silkworms and cytokines are induced in infected human cells [75, 76]. The responses seen in these other animals are quite different than what is observed in Caenorhabditis. One reason for this is that many IPR genes are part of large gene families, such as PALS [30], that are not conserved outside of Caenorhabditis. Little is known about how a host responds to different species of microsporidia. A study examining two species of microsporidia that infect the same mosquito found that a horizontal transmitted species elicited a strong transcriptional immune response, but this response was not enriched during infection with a vertically transmitted species [77]. Further studies will be necessary to know if the similar immune responses we observe to infection by two horizontally transmitted microsporidia species are common in other hosts infected by different microsporidia.

Microsporidia that infect free-living terrestrial nematodes appear to be common, and there are other genera and families of nematodes that can be cultured in the laboratory and infected with microsporidia. Additionally, there are other genera of microsporidia that have been shown to infect C. elegans and other nematodes. Some nematode-infecting microsporidia also infect multiple genera of nematodes, which could facilitate cross-species host expression [13]. These types of broader comparisons would allow for a fuller view of how animals evolve transcriptional responses to microsporidia infection.

Supporting information

S1 Fig. Transcriptional response of Nematocida species in Caenorhabditis hosts.

(A-B) Scatterplot of N. parisii (A) and N. ausubeli (B) log10 FPKM values between C. briggsae and C. elegans replicates at 10 hours. Pearson correlation values and p-values are indicated on the top left of each plot. The ratio of FPKM values for each gene between the replicates is demonstrated by the colour of the points. (C-D) Gene expression pattern of N. parisii (C) and N. ausubeli (D) in C. elegans and C. briggsae.

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S2 Fig. Data from this study has high overlap with previously published intracellular infection samples.

(A) Heatmap of transcriptional profiles of differentially regulated genes (FDR<0.05) across five samples from Bakowski et al. and our 10hr N. parisii and N. ausubeli infected C. elegans samples. Only genes expressed in at least four out of the eight total samples are included. (B-C) Statistical overlap of significant genes between respective N. parisii or N. ausubeli infected C. elegans samples and Bakowski et al. samples across five timepoints. (D-E) Statistical overlap of significant genes between N. parisii (D) or N. ausubeli (E) infected C. elegans samples and Chen et al. Orsay virus infected C. elegans sample at 12 hours. (F) Statistical overlap of significant genes between respective N. parisii or N. ausubeli infected C. briggsae sample and Chen et al. Santeuil virus infected C. briggsae sample at 12 hours. The p-value of each comparison is indicated on top of each Venn diagram. npa (N. parisii) and nau (N. ausubeli).

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S3 Fig. Phylogram of C. elegans and C. briggsae PALS family genes.

Genes with the same name that end with different letters indicate protein isoforms. Node values indicate posterior probabilities for each split as a percentage. The scale bar indicates average branch length measured in expected substitutions per site.

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S4 Fig. Phylogram of C. elegans and C. briggsae DUF713 family genes.

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S5 Fig

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S1 Table. FPKM values of gene expression and differentially expressed microsporidia genes.

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S2 Table. Normalised RNA-seq counts of C. elegans and C. briggsae for the transcriptional analysis generated by Alaska.

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S3 Table. Differentially expressed genes of C. elegans and C. briggsae exposed to the two species of microsporidia.

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S4 Table. PANTHER GO enrichment analyses.

Sheet S1 contains results of the statistical enrichment tests of the 222 significantly upregulated genes shared between N. parisii and N. ausubeli infected C. elegans. Sheet S2 contains results of the statistical enrichment tests of the 267 significantly downregulated genes shared between N. parisii and N. ausubeli infected C. briggsae. Sheet S3 contains results of the statistical enrichment tests of the 415 significantly downregulated genes specifically in N. parisii infected C. briggsae.

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S5 Table. List of gene set overlaps with previously published infection samples.

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S6 Table. Gene classes and domains used for enrichment analyses.

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Acknowledgments

We thank Hala Tamim El Jarkass, Meng A. Xiao, and Winnie Zhao for providing helpful comments on the manuscript. We thank Winnie Zhao for technical help for the comparisons to the Bakowski data. Some strains were provided by the CGC and we thank WormBase.

Data Availability

Data availability All samples were deposited under NCBI BioProject PRJNA841614 and the sequence reads for all samples were submitted to the NCBI Sequence Read Archive. All other relevant data are within the paper and its Supporting information files.

Funding Statement

Canadian Institutes of Health Research grant no. 400784 (to A.R.) Alfred P. Sloan Research Fellowship FG2019-12040 (to A.R.) NIH R01’s AG052622 and GM114139 to E.T. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0279103.r001

Decision Letter 0

Nicolas Corradi

19 Sep 2022

PONE-D-22-23486Conservation of Nematocida microsporidia gene expression and host response in Caenorhabditis nematodesPLOS ONE

Dear Dr. Reinke,

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: In this manuscript, the authors used RNAseq data to investigate if distinct microsporidia species from the genus Nematocida (N. parisii and N. ausubeli) express different genes when infecting different nematode hosts from the genus Caenorhabditis (C. elegans and C. briggsae). They also investigated if these nematodes hosts differ in their response to the Nematocida spp. infections. From their findings, the authors conclude that neither the pathogens nor the hosts express different genes depending on which species is infecting or being infected, respectively.

Overall, I found the manuscript to be easy to read and did not detect any major flaw (my comments are described below). I believe that the manuscript will be suitable for publication in PLOS one pending minor revisions.

# 1. The authors spent a lot of effort investigating potential expression differences between two species of Nematocida infecting two species of Caenorhabditis and the overall take home message of their manuscript is that they found no major difference. However, one of the main questions that came to my mind while reading this manuscript is: Are those species really that different? Because if not, then one should not expect any major difference in gene expression levels.

In the introduction (lines 46-61) the authors mention that N. ausubeli grows faster than the other microsporidian species. Likewise, the authors state that the two nematodes live in distinct but overlapping locales but that doesn’t tell us much about their biological differences, if any. What else makes these species distinct? I am neither a Caenorhabditis nor a Nematocida expert and, overall, I felt that I was missing some crucial information to properly appreciate the authors findings while going through the manuscript. Although the authors somewhat address this question later on at the start of their discussion (on page 5, lines 249-254) by talking about amino acid differences (are those significant?), this should be clearly addressed in the introduction.

In the same trail of thought, were the nematode host species C. elegans and C. briggsae selected because they showed distinct responses to other fungal pathogens? The authors succinctly mention nematode responses to fungal infections in their introduction and instead focus on their response to bacterial infections (lines 75-88). However, microsporidia are fungi (or at least closely related) and expanding on nematode responses to fungal infections would be more informative than using bacteria as a frame of reference.

Other minor comments:

# 2. I’m assuming that the 10, 20 and 28h post-infection time points for RNAseq are relevant to the microsporidia biology but it would be informative to have a brief statement explaining why these time points were selected. Was it based on the infection progression as observed in the FISH results from Fig 2A?

# 3. Page 4 lines. 175-178. 'Using statistical enrichment tests, we observed that the shared upregulated genes in N. parisii and N. ausubeli infected C. elegans are enriched for GO Biological Process associated with metabolism; as well as GO Molecular Process associated with catalytic activity (FDR<0.05, Supplementary table S4)'.

This statement in uninformative. Metabolism and catalytic activity are incredibly broad categories. Growing organisms have metabolic/catabolic activity. That is a given. Is there any gene ontology term other than these vague categories that stands out?

# 4. Page 7. RNA sequencing. Which library kit was used to prepare the sequencing libraries? The authors state that mRNA libraries were generated so a rRNA depletion step was likely not required but how were those mRNA libraries generated? Different kits can have different biases and this is also important to know which adapters might be present in the sequencing datasets.

# 5. Page 7. Microsporidia gene expression analysis. Were the adapters properly removed from the sequencing datasets prior to the analyses? The authors used two different methods to analyze RNAseq data, one for the microsporidia datasets and one for the nematode datasets (using the WormBase Alaska pipeline). The latter mention some sort of quality control (QC) but the first one does not mention any such QC step.

# 6. Page 7 lines 366-367: 'The paired end reads of each sample 10 hours post infection sample were submitted to Alaska v1.7.2 (http://alaska.caltech.edu)'.

What about the 20 and 28 PI samples? Were they also analyzed with the Alaska pipeline from WormBase? Did the authors mean: The paired end reads of each sample were submitted to Alaska v1.7.2 (http://alaska.caltech.edu)? The authors should also explicitly state what Alaska is. I had to reread that sentence twice as I found it confusing.

# 7. Page 7 methods and page 12 Fig 2C. From which RNAseq analysis method were the mapping metrics in Fig 2C derived; Alaska, the microsporidia one, both? A subtraction would not do here if contamination from a third party was present. Also, while I understand using the WormBase pipeline to analyze the Caenorhabditis data, this pipeline uses completely different tools than the ones used to analyze microsporidia data. Did the authors check for congruency between the two approaches?

# 8. Page 8. Lines 420 - 424. 'From this analysis, we found additional domains and genes enriched with greater than three domain-containing proteins in any of our samples. These identified domains are chitinase-like (chil) proteins, CUB and CUB-like domains, cytochrome P450, glucosyltransferase family, nematode cuticle collagen N terminal domain, and UDP-glucuronosyltransferase'.

These sentences read like results rather than methods. Also, P450 cytochromes are involved in the detoxification of xenobiotics in insects. If those were enriched/overexpressed, something important might be going on here. Microsporidia spores are also made of chitin so chitinases appear highly relevant here.

# 9. On page 1, lines 50-51. 'These two species of Nematocida are also commonly found to infect Caenorhabditis briggsae, which has been developed as a comparative species to C. elegans (Stein et al., 2003)'.

Weird phrasing: species do not arise to be compared to something and Caenorhabditis briggsae was not developed/created in lab (or was it?). I’m assuming that the authors meant that this species was used as an alternate model to facilitate comparative analyses or something like that…

# 10. On page 2, lines 83-85 'A study looking at different species of bacterial infection in the nematode Pristionchus pacificus also showed both similarities and differences between the transcriptional responses in the two hosts (Sinha et al., 2012)'.

'showed both similarities and differences' This is vague. Minor/Major differences? A few but crucial ones?

# Figures 3 and 4 legends. Replace Microsporidia by Nematocida in the titles. These results are not representative of the whole group, and they would likely differ in non-Caenorhabditis hosts.

# 12. Formatting: References are not formatted according to PLOS guidelines.

Reviewer #2: The manuscript by Wan, Troemel and Reinke characterizes the microsporidian and host transcriptomes of two Nematocida spp. infecting two Caenorhabditis spp. The central questions posed by the authors are: 1. whether the parasites change their gene expression profiles depending on the host infected, and 2. whether the host gene expression profiles differ when infected by either Nematocida spp. The conclusion is that parasite gene expression profiles are very similar, no matter what host is being used, and that the same could be said for the host as well.

I think it is an interesting paper, well written and with very nice illustrations, which is worth of publication.

I have three questions to the authors that may (or may not) be explored in order to build an improved version of the manuscript.

1) Hosts were experimentally infected and RNA was extracted at 10, 20 and 28 hours post infection, with one replicate only for the 10 hpi timepoint. On line 257 of the discussion the authors recognize that the lack of replications could have limited their ability to detect differentially expressed genes. It would be interesting to know the extent by which their conclusions have been biased by the lack of replicates. Is it possible to estimate that in any way (e.g. by generating pseudo replicates)? Alternatively, would it be possible to provide at least one good argument to the reader giving support for their claims, i.e., that the conclusions are not simply a consequence of the lack of statistical power?

2) Another aspect that needs clarification is the statement that expression profiles are “evolutionarily conserved”. I am not sure whether you could say that for such closely related species, but the reader should at least know what evolutionary scale is actually being assessed in the study. Somewhere it should be mentioned to what degree both microsporidia and both hosts genetically diverge from each other (for any standard marker such as rRNA or other phylogenetic marker gene).

3) I am wondering whether it wouldn’t be possible, with the data at hand, to test other hypotheses. It would be interesting to know, for instance, what are the differences in expression 1. between parasites independently of the host, or 2. between hosts independently of the parasite. The first is going to be an unavoidable curiosity of the manuscript reader, considering that N. parisii and N. ausubeli differ in their infection phenotypes (lines 60-61).

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Reviewer #2: No

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PLoS One. 2022 Dec 19;17(12):e0279103. doi: 10.1371/journal.pone.0279103.r002

Author response to Decision Letter 0

29 Nov 2022

We thank the reviewers for their comments. We have provided a revised manuscript (where all changes are tracked) to address the reviewers’ concerns. Points raised by the reviewers have been incorporated into the manuscript and we have provided a point-by-point response to each of the reviewer’s comments:

We thank the reviewer for their kind comments.

To address this comment, we have added additional information in the following paragraphs to the introduction:

Lines 62-69:

There are some similarities in the infections caused by these two Nematocida species; they both exclusively infect the intestine, they infect C. elegans and C. briggsae, they cause intestinal cells to fuse, and have similar life cycles (Balla et al., 2016; Zhang et al., 2016; Wadi et al., 2022). Both species also use similar types of secreted and membrane bound proteins to interface with host proteins (Reinke et al., 2017). Although the divergence time between N. parisii and N. ausubeli is unknown, the two Nematocida species share 68.3% amino acid identity (Cuomo et al., 2012; Luallen et al., 2016). These two species also have distinct growth and phenotypic characteristics during infection in C. elegans; specifically N. ausubeli displays faster growth and increased impairment of host fitness (Balla et al., 2016).

Lines 107-110:

For the hosts, we used C. elegans and C. briggsae, which diverged ~18 million years ago (Cutter, 2008). About 60% of the genes from these species are orthologous and the median percent identity between these proteins is 80%, which is approximately the same extent of divergence between humans and mice (Stein et al., 2003).

As described above, we have modified the introduction to better clarify the differences and similarities between the Caenorhabditis and Nematocida species in lines 44-69 and 106-111.

C. elegans and C. briggsae were chosen because these two host species are infected efficiently by N. parisii and N. ausubeli under laboratory setting, similar to their natural interactions in the wild. Thus these host-parasite pairs are an ideal set of species to study transcriptional responses to microsporidia in related species. We have added additional information to clarify why we choose these species in lines 44-69 and 106-111.

Although microsporidia are early-diverging fungi, Caenorhabditis nematodes have distinct responses to Nematocida compared to other fungi. Transcriptomic response of different nematodes to pathogenic bacteria is more studied, therefore we included bacteria as a frame of reference. For clarity, we have differentiated the response to different pathogens by integrating the following text into the introduction:

Lines 80-87

In response to microsporidia infection, hosts often display large transcriptional changes. The gene expression of C. elegans in response to N. parisii infection is reported to be distinct from responses to bacteria, but similar to nodavirus infection (Bakowski et al., 2014). Although the response to N. parisii infection also shared some similarities to the response against fungi such as Drechmeria coniospora and Harposporium, the genes upregulated during N. parisii infection are still mostly specific to either microsporidia or viral infection (Bakowski et al., 2014).

Other minor comments:

Yes, to address this, we have added the following text to the results:

Line 285-288

We chose these three timepoints because at 21°C, Nematocida is in the sporoplasm stage at 10 hour post infection, and in the meront stage at 20 and 28 hours post infection, which allowed us to compare gene expression levels at these growth stages (Bakowski et al., 2014; Balla et al., 2016) (Fig 2A).

We agree with the reviewers that these are vague categories, but they are the ones that are enriched. We have modified the text in the relevant paragraph to clarify that only these broad categories were enriched.

Line 351-352

“Statistical enrichment tests indicate that only broad categories were enriched in the unique or shared genes between samples.”

Libraries were prepared using a polyA capture library and a rRNA depletion step was not performed. We have added the following to the methods:

Lines 148-150

mRNA libraries were prepared by the UCSD IGM Genomics Center using Illumina Stranded mRNA Prep kit and sequenced on a single lane of an Illumina HiSeq 4000, using 100 bp paired-end reads.

We used FastQC to perform the quality control on the microsporidia unaligned .fastq and Tophat-aligned .bam datasets. The adapters in the .fastq files were reported as overrepresented sequences by FastQC, but not in the .bam datasets, which indicates that they have been removed after alignment. We have added the following text in the methods section:

Line 157-159

“Using FastQC, quality control was performed on the Tophat aligned files to ensure that they are of good quality (Quality scores across all bases >30) and that the adapters were properly removed from the sequencing datasets prior to downstream analysis (Andrews, Simon, 2015).”

# 6. Page 7 lines 366-367: 'The paired end reads of each sample 10 hours post infection sample were submitted to Alaska v1.7.2 (http://alaska.caltech.edu)'.

We have added the following text into the methods section to state what Alaska is, and explain why the 20 and 28 PI samples are not analysed with the Alaska pipeline:

Line 174-175:

“The paired end reads of each sample 10 hours post infection sample were submitted to Alaska v1.7.2 (http://alaska.caltech.edu), an online automatic C. elegans RNA-seq analysis pipeline.”

Line 181-182:

“The 20 and 28 hours samples were not submitted for analysis due to the lack of replicates at these timepoints.”

We have added the following text to explain that the overall read mapping rate in Fig 2C and the description in page 7 methods are from the RNAseq analysis of microsporidia.

Line 154-157

“Reads from each sample were mapped to either Nematocida parisii strain ERTm1 (Genbank: GCF_000250985.1) or Nematocida ausubeli strain ERTm2 (Genbank: GCA_000250695.1) using TopHat v2.1.2, which also calculates the overall read mapping rate (Trapnell, Pachter and Salzberg, 2009).”

Figure legend of Figure 2C

“(C) Overall read mapping rate of Nematocida in each host at different time points, calculated by TopHat during alignment to the respective reference genomes.”

For the final point, Cufflinks had also been used previously for RNA-seq analysis of Nematocida (Cuomo et al., 2012). Since we did not directly compare the RNA-seq expression between the hosts and the microsporidian pathogens, we did not check for congruency between Alaska and Cufflinks.

To address the first point, we moved those sentences from the methods into the results:

Line 404-408

“From this analysis, we found additional domains and genes enriched with greater than three domain-containing proteins in any of our samples. These identified domains are chitinase-like (chil) proteins, CUB and CUB-like domains, cytochrome P450, glucosyltransferase family 92, nematode cuticle collagen N-terminal domain, and UDP-glucuronosyltransferase.”

We also added more text in the results elaborating the potential implication of enriched P450 cytochromes and chil genes:

Line 420-424

“Interestingly, cytochrome P450 genes, implicated to be involved in detoxification, are upregulated in both hosts, suggesting that the upregulated response to Nematocida overlaps with the metabolism and elimination of potentially toxic xenobiotics (Larigot et al., 2022). Moreover, we observe the upregulation of a few chil genes in C. elegans and C. briggsae, previously shown to be upregulated in response to epidermally infecting oomycetes (Osman et al., 2018; Grover et al., 2021).

We have modified the relevant text in the introduction:

Line 47-50:

“These two species of Nematocida are also commonly found to infect Caenorhabditis briggsae, a related species which has been used as a model organism to facilitate comparative analyses to C. elegans (Stein et al., 2003; Uyar et al., 2012).”

'showed both similarities and differences' This is vague. Minor/Major differences? A few but crucial ones?

To address this, we have described some major similarities and differences between the transcriptional responses of Pristionchus pacificus and C. elegans in the introduction:

Line 91-100

“A study looking at four different species of bacteria infecting the nematode Pristionchus pacificus also showed both similarities and differences between the transcriptional responses in the two hosts (Sinha et al., 2012). For example, while genes in the FOXO/DAF-16, TGF-beta and p38 MAP kinase pathways are suggested to be conserved between C. elegans and P. pacificus in response to bacterial infection, host-specific transcriptional responses are also observed, such as the enrichment of lipid metabolism related domains in the response of P. pacificus to different bacteria (Sinha et al., 2012).”

# 11. Figures 3 and 4 legends. Replace Microsporidia by Nematocida in the titles. These results are not representative of the whole group, and they would likely differ in non-Caenorhabditis hosts.

We have replaced “Microsporidia” with “Nematocida” in the legend titles of Figure 3 and 4.

# 12. Formatting: References are not formatted according to PLOS guidelines.

The references have now been formatted according to PLOS guidelines.

I think it is an interesting paper, well written and with very nice illustrations, which is worth of publication.

We thank the reviewer for their kind comments.

I have three questions to the authors that may (or may not) be explored in order to build an improved version of the manuscript.

To address the possible bias by the lack of replicates, we provide the following arguments in the discussion:

Line 441-453

“Although a small number of replicates limits the ability to detect differentially regulated genes (Conesa et al., 2016), we believe that our conclusions are not simply a consequence of the lack of statistical power for the following reasons. Firstly, we saw a large overlap in significantly regulated genes compared to previously published transcriptional datasets of N. parisii infected C. elegans (Bakowski et al., 2014; Chen et al., 2017). The conclusions we reach in our study also agree with those from another transcriptional response comparison from our group that generated RNA-seq data in triplicate of C. elegans infected at a latter timepoint with four different species of microsporidia, including N. parisii and N. ausubeli (Mok et al., 2022). Secondly, previous statistical power calculations for RNA-seq analysis over a range of fold-changes and number of replicates suggests that a small number of replicates is sufficient to capture highly-expressed genes, such as the highly expressed IPR genes in our study (Conesa et al., 2016; Schurch et al., 2016; Lamarre et al., 2018). Finally, previously published transcriptional datasets from Bakowski et al., (2014) and Chen et al., (2017), which we compared our dataset to, were also performed in duplicates or triplicates.”

We have addressed these points above in the responses to reviewer #1.

As microsporidia only replicate inside of a host, it is not possible to measure the proliferative stage of infection independently. These parasites do exist outside of the host as dormant spores, but we did not measure mRNA from the spores. To clarify the life cycle of microsporidia infection more clearly, we have added the following text in the introduction:

Line 56-62

The infection cycle of N. parisii and N. ausubeli in Caenorhabditis hosts begins with ingestion of the microsporidia spores, an environmentally resistant and dormant stage of the pathogen (Troemel et al., 2008; Vávra and Ronny Larsson, 2014). In the intestine, the sporoplasm in the spore is ejected through the polar tube and deposited into the intestinal cells. This is followed by replication into meronts and differentiation into new spores, which then exit intestinal cells into the intestinal lumen. The spores are defecated by the nematodes, and the infection cycle repeats when they are ingested by another host (Troemel et al., 2008; Cuomo et al., 2012; Luallen et al., 2016).

Although our data could be used to compare differences in expression between C. elegans and C. briggsae independent of infection, this was not a goal of our study. Additionally, a previous study (Grün et al., 2014) compared expression changes between C. elegans and C. briggsae at 7 time points. We have now added this reference to line 52.

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PLoS One. doi: 10.1371/journal.pone.0279103.r003

Decision Letter 1

Nicolas Corradi

1 Dec 2022

Conservation of Nematocida microsporidia gene expression and host response in Caenorhabditis nematodes

PONE-D-22-23486R1

Dear Dr. Reinke,

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PLoS One. doi: 10.1371/journal.pone.0279103.r004

Acceptance letter

Nicolas Corradi

6 Dec 2022

PONE-D-22-23486R1

Conservation of Nematocida microsporidia gene expression and host response in Caenorhabditis nematodes

Dear Dr. Reinke:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Transcriptional response of Nematocida species in Caenorhabditis hosts.

(TIF)

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S2 Fig. Data from this study has high overlap with previously published intracellular infection samples.

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S3 Fig. Phylogram of C. elegans and C. briggsae PALS family genes.

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S4 Fig. Phylogram of C. elegans and C. briggsae DUF713 family genes.

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S5 Fig

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S1 Table. FPKM values of gene expression and differentially expressed microsporidia genes.

(XLSX)

Click here for additional data file.^{(416.9KB, xlsx)}

S2 Table. Normalised RNA-seq counts of C. elegans and C. briggsae for the transcriptional analysis generated by Alaska.

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S3 Table. Differentially expressed genes of C. elegans and C. briggsae exposed to the two species of microsporidia.

(XLSX)

Click here for additional data file.^{(15.4MB, xlsx)}

S4 Table. PANTHER GO enrichment analyses.

(XLSX)

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S5 Table. List of gene set overlaps with previously published infection samples.

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S6 Table. Gene classes and domains used for enrichment analyses.

(DOCX)

Click here for additional data file.^{(13.8KB, docx)}

Attachment

Submitted filename: plosone_response_letter_final.pdf

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Data Availability Statement

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PERMALINK

Conservation of Nematocida microsporidia gene expression and host response in Caenorhabditis nematodes

Yin Chen Wan

Emily R Troemel

Aaron W Reinke

Roles

Abstract

Introduction

Materials and methods

Infection of nematodes with microsporidia

RNA extraction and sequencing

Microsporidia gene expression analysis

Caenorhabditis RNA-seq analysis

Principal component analysis

Determination of gene expression overlap

Domain enrichment analysis

Determination of orthologs between C. elegans and C. briggsae

Single copy orthologs analysis

Comparison of gene expression in expanded gene families

Results

Gene expression of N. parisii and N. ausubeli is similar in C. elegans and C. briggsae

Fig 1. Overview of study design.

Fig 2. Nematocida infection of Caenorhabditis hosts.

Fig 3. Nematocida gene expression in different host species.

Fig 4. Differentially regulated genes of Nematocida.

Shared and unique transcriptional responses of each Caenorhabditis host to N. parisii and N. ausubeli infection

Fig 5. Transcriptional responses of Caenorhabditis hosts to Nematocida infection.

Evolutionary conserved host response to Nematocida infection

Fig 6. Transcriptional responses of single copy orthologs in Caenorhabditis hosts to Nematocida infection.

Fig 7. Transcriptional responses of non-single copy orthologs in Caenorhabditis hosts to Nematocida infection.

Fig 8. Conserved response of Caenorhabditis hosts to Nematocida infection.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Nicolas Corradi

Roles

Author response to Decision Letter 0

Decision Letter 1

Nicolas Corradi

Roles

Acceptance letter

Nicolas Corradi

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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