Transcriptional profiles in Strongyloides stercoralis males reveal deviations from the Caenorhabditis sex determination model

Abstract

The human and canine parasitic nematode Strongyloides stercoralis utilizes an XX/XO sex determination system, with parasitic females reproducing by mitotic parthenogenesis and free-living males and females reproducing sexually. However, the genes controlling S. stercoralis sex determination and male development are unknown. We observed precocious development of rhabditiform males in permissive hosts treated with corticosteroids, suggesting that steroid hormones can regulate male development. To examine differences in transcript abundance between free-living adult males and other developmental stages, we utilized RNA-Seq. We found two clusters of S. stercoralis-specific genes encoding predicted transmembrane proteins that are only expressed in free-living males. We additionally identified homologs of several genes important for sex determination in Caenorhabditis species, including mab-3, tra-1, fem-2, and sex-1, which may have similar functions. However, we identified three paralogs of gld-1; Ss-qki-1 transcripts were highly abundant in adult males, while Ss-qki-2 and Ss-qki-3 transcripts were highly abundant in adult females. We also identified paralogs of pumilio domain-containing proteins with sex-specific transcripts. Intriguingly, her-1 appears to have been lost in several parasite lineages, and we were unable to identify homologs of tra-2 outside of Caenorhabditis species. Together, our data suggest that different mechanisms control male development in S. stercoralis and Caenorhabditis species.

Figure 1

Components of the Caenorhabditis sex determination pathway are present in S. stercoralis, and key transcription factors likely have conserved function. (A) Genes that compose the “core” sex determination pathway in Caenorhabditis species, along with accessory genes that play a role in somatic (white boxes) and germline (gray boxes) sex determination, are diagrammed, with feminizing factors in red and masculinizing factors in blue. Homologs in S. stercoralis (black circle), homologs encoding a similar type of protein (gray circle), and genes for which no homolog was identified in the S. stercoralis genome (white circle), are indicated. Adapted from. (B–H) Transcript abundances of the S. stercoralis homologs are represented using TMM-normalized counts per million (CPM) for the following developmental stages: free-living adult males (FL Male), free-living gravid adult females (FL Female), post-free-living first-stage larvae (PFL L1), developmentally arrested infectious third-stage larvae (iL3), L3 activated inside a permissive host (L3 +), parasitic gravid adult females (P Female), heterogonically-developing post-parasitic L1 (PP L1), and heterogonically-developing post-parasitic L3 enriched for females (PP L3). Graphs were constructed using GraphPad Prism v.9.0.0; bars indicate means (horizontal) and 95% confidence intervals (vertical) for each of the three biological replicates represented as individual data points. Asterisks (*) indicate a significant (fold change > 2.0; FDR < 0.05) difference between FL Male and FL Female.

Figure 2

S. stercoralis developmental stages have distinct transcript expression profiles. (A) S. stercoralis developmental stages examined by RNA-Seq are well-separated, and the three biological replicates group closely, by multidimensional scaling (MDS) analysis. (B) Numerous transcripts are significantly (log₂ fold change > 1.0; FDR < 0.05) up-regulated (red) and down-regulated (blue) in free-living adult males, in comparison to free-living adult females, by mean-difference (MD) analysis. A subset of transcripts are significantly up-regulated in free-living adult males to a greater extent than transcripts are down-regulated. Plots were constructed using EdgeR v.3.32.0.

Figure 3

Transcripts encoding S. stercoralis nuclear hormone receptors and cytochrome P450s are differentially regulated between males and females. (A) Homologs of SEX-1, NHR-23, and ECR-1 in S. stercoralis (arrows) and other clade I, III, IV, and V nematodes are identified in a neighbor-joining phylogenetic tree with 100 iterations of bootstrapping constructed with Geneious v.11.1.5. Gene names and accession numbers are listed after the species names. (B-H) Transcript abundances of the S. stercoralis homologs encoding relevant nuclear hormone receptors and cytochrome P450s are represented using TMM-normalized counts per million (CPM) for the following developmental stages: free-living adult males (FL Male), free-living gravid adult females (FL Female), post-free-living first-stage larvae (PFL L1), developmentally arrested infectious third-stage larvae (iL3), L3 activated inside a permissive host (L3 +), parasitic gravid adult females (P Female), heterogonically-developing post-parasitic L1 (PP L1), and heterogonically-developing post-parasitic L3 enriched for females (PP L3). Graphs were constructed using GraphPad Prism v.9.0.0; bars indicate means (horizontal) and 95% confidence intervals (vertical) for each of the three biological replicates represented as individual data points. Asterisks (*) indicate a significant (fold change > 2.0; FDR < 0.05) difference between FL Male and FL Female.

Figure 4

The S. stercoralis genome encodes three GLD-1 paralogs with transcripts that are sex specific. (A) Homologs of GLD-1 and ASD-2 in S. stercoralis (arrows) and other III, IV, and V nematodes are identified in a neighbor-joining phylogenetic tree with 100 iterations of bootstrapping constructed with Geneious v.11.1.5. Gene names and accession numbers are listed after the species names. (B-D) Transcript abundances of the three S. stercoralis paralogs are represented using TMM-normalized counts per million (CPM) for the following developmental stages: free-living adult males (FL Male), free-living gravid adult females (FL Female), post-free-living first-stage larvae (PFL L1), developmentally arrested infectious third-stage larvae (iL3), L3 activated inside a permissive host (L3 +), parasitic gravid adult females (P Female), heterogonically-developing post-parasitic L1 (PP L1), and heterogonically-developing post-parasitic L3 enriched for females (PP L3). Graphs were constructed using GraphPad Prism v.9.0.0; bars indicate means (horizontal) and 95% confidence intervals (vertical) for each of the three biological replicates represented as individual data points. Asterisks (*) indicate a significant (fold change > 2.0; FDR < 0.05) difference between FL Male and FL Female.

Figure 5

Genes encoding pumilio family proteins appear to have been duplicated in Strongyloides species. (A) Homologs of pumilio family proteins in S. stercoralis (arrows) and other clade III, IV, and V nematodes are identified in a neighbor-joining phylogenetic tree with 100 iterations of bootstrapping constructed with Geneious v.11.1.5. The genes encoding Ss-PUM-1, Ss-PUM-2, and Ss-PUM-3 appear to have resulted from a duplication in an ancestor of Strongyloides species. Gene names and accession numbers are listed after the species names. (B-F) Transcript abundances of the S. stercoralis homologs are represented using TMM-normalized counts per million (CPM) for the following developmental stages: free-living adult males (FL Male), free-living gravid adult females (FL Female), post-free-living first-stage larvae (PFL L1), developmentally arrested infectious third-stage larvae (iL3), L3 activated inside a permissive host (L3 +), parasitic gravid adult females (P Female), heterogonically-developing post-parasitic L1 (PP L1), and heterogonically-developing post-parasitic L3 enriched for females (PP L3). Graphs were constructed using GraphPad Prism v.9.0.0; bars indicate means (horizontal) and 95% confidence intervals (vertical) for each of the three biological replicates represented as individual data points. Asterisks (*) indicate a significant (fold change > 2.0; FDR < 0.05) difference between FL Male and FL Female.