Transcriptional responses to in vitro macrocyclic lactone exposure in Toxocara canis larvae using RNA-seq

Abstract

Toxocara canis, the causative agent of zoonotic toxocariasis in humans, is a parasitic roundworm of canids with a complex lifecycle. While macrocyclic lactones (MLs) are successful at treating adult T. canis infections when used at FDA-approved doses in dogs, they fail to kill somatic third-stage larvae. In this study, we profiled the transcriptome of third-stage larvae derived from larvated eggs and treated in vitro with 10 μM of the MLs - ivermectin and moxidectin with Illumina sequencing. We analyzed transcriptional changes in comparison with untreated control larvae. In ivermectin-treated larvae, we identified 608 differentially expressed genes (DEGs), of which 453 were upregulated and 155 were downregulated. In moxidectin-treated larvae, we identified 1,413 DEGs, of which 902 were upregulated and 511 were downregulated. Notably, many DEGs were involved in critical biological processes and pathways including transcriptional regulation, energy metabolism, neuronal structure and function, physiological processes such as reproduction, excretory/secretory molecule production, host-parasite response mechanisms, and parasite elimination. We also assessed the expression of known ML targets and transporters, including glutamate-gated chloride channels (GluCls), and ATP-binding cassette (ABC) transporters, subfamily B, with a particular focus on P-glycoproteins (P-gps). We present gene names for previously uncharacterized T. canis GluCl genes using phylogenetic analysis of nematode orthologs to provide uniform gene nomenclature. Our study revealed that the expression of Tca-glc-3 and six ABCB genes, particularly four P-gps, were significantly altered in response to ML treatment. Compared to controls, Tca-glc-3, Tca-Pgp-11.2, and Tca-Pgp-13.2 were downregulated in ivermectin-treated larvae, while Tca-abcb1, Tca-abcb7, Tca-Pgp-11.2, and Tca-Pgp-13.2 were downregulated in moxidectin-treated larvae. Conversely, Tca-abcb9.1 and Tca-Pgp-11.3 were upregulated in moxidectin-treated larvae. These findings suggest that MLs broadly impact transcriptional regulation in T. canis larvae.

Keywords: ATP-Binding Cassette Transporters; Dogs; RNA-Seq; Toxocara canis; Toxocariasis; glutamate-gated chloride channels; ivermectin; mRNA; moxidectin; transcriptome.

Figure 1.

Brief graphical depiction of the experimental methods for the transcriptomic study of Toxocara canis third-stage larvae exposed to ivermectin, moxidectin, or RPMI-1640 (controls). Tins figure was created using Biorender.com.

Figure 2.

(A) Principal Component Analysis (PCA) of Toxocara canis third-stage larvae (L3s) exposed to RPMI-1640 medium (controls, blue circle), ivermectin (yellow square),or moxidectin (red diamond). Each point represents a pool of ~500 L3s (one biological replicate); (B-C) Volcano plots of differentially expressed genes in (B) ivermectin- or (C) moxidectin-treated larvae compared to untreated controls. Significance thresholds were set at log2FC > 1 (two-fold change) for upregulated genes, and log2FC < −1 for downregulated genes and adjusted p-value < 0.05. upregulated genes are shown in red, downregulated genes in blue, and non-significant genes in gray. A total of 18,596 genes were analyzed, with 454 significantly upregulated and 155 significantly downregulated in ivermectin-treatcd L3s and 902 significantly upregulated and 511 significantly downregulated in moxidectin-treated L3s; (D-E) Venn diagrams dipicting the overlap of shared upregulated (D) and downregulated (E) genes in larvae treated with ivermectin and moxidectin The upregulated genes show an overlap of 240 shared genes, while the downregulated genes show an overlap of 83 shared genes, (F) Heatmap of normalized expression (vst) values for all 18,596 genes annotated in the T. canis genome for control, ivermectin-, and moxidectin- treated L3s, Color intensity represents normalized expression values, with the scale ranging from black (lowest expression) to red (highest expression).

Figure 3.

(A&C) Gene Ontology (GO) bubble plots of genes (A) upregulatcd and (C) downregulated in Toxocara canis third-stage larvae (L3s) treated with ivermectin. The size of the bubble represents the number of genes associated with each GO term, and the color intensity of the bubble indicates the significance (adjusted p-value) of enrichment. GO terms are grouped by biological process (BP, blue), molecular function (MF, red), and cellular component (CC, yellow) categories; (B&D) Lollipop plots representing pathway enrichment analysis for the top 10 most significantly enriched pathways for genes (B) upregulated and (C) downregulated in T. canis ivermectin-treated L3s. The length and color of the lollipops corresponds to fold enrichment, with blue indicating lower fold enrichment and red indicating higher fold enrichment. The size of the dots represents the number of genes associated with each pathway.

Figure 4.

(A&C) Gene Ontology (GO) bubble plots of genes (A) upregulated and (C) downregulated in Toxocara canis third-stage larvae (L3s) treated with moxidectin. The size of the bubble represents the number of genes associated with each GO term, and the color intensity of the bubble indicates the significance (adjusted p-value) of enrichment. GO terms are grouped by biological process (BP, blue), molecular function (MF, red), and cellular component (CC, yellow) categories; (B&D) Lollipop plots representing pathway enrichment analysis for the top 10 most significantly enriched pathways for genes (B) upregulated and (C) downregulated in T. canis moxidectin-treated L3s. The length and color of the lollipops corresponds to fold enrichment, with blue indicating lower fold enrichment and red indicating higher fold enrichment. The size of the dots represents the number of genes associated with each pathway.

Figure 5.

(A&C) Gene Ontology (GO) bubble plots of the overlapping (A) upregulated and (C) downregulated shared genes between ivermectin- and moxidectin-treated Taxocara canis third-stage larvae (L3s). The size of the bubble represents the number of genes associated with each GO term, and the color intensity of the bubble indicates the significance (adjusted p-valuc) of enrichment. GO terms an: grouped by biological process (BP,, blue),, molecular function (MF, red), and cellular component (CC, yellow) categories; (B&D) Lollipop plots representing pathway enrichment analysis for the top 10 most significantly enriched pathways for the overlapping (B) upregulated and (C) downregulated shared genes between ivermectin- and moxidectin-trcatcd T. canis L3s The length and color of the lollipops corresponds to fold enrichment, with blue indicating lower fold enrichment and red indicating higher fold enrichment. The size of the dots represents the number of genes associated with each pathway.

Figure 6.

A maximum-likelihood phylogenetic tree Of Toxocara canis glutamate-gated chloride channels (GluCls) and related nematode sequences obtained from GenBank. The tree was constructed using PhyML with the SMS to determine the best fitting model and visualized using iTOL and ggtree. Branch support values were calculated from 1000 bootstrap replicates, with values > 75 shown. The tree is rooted using a GluCl sequence from Apfysia californica (California sea hare). The tree revealed six distinct GluCl cladcs (avr-14, glc-5, glc-3, glc-2, glc-4, and glc-6), as well as distinct clades for nematode ligand-gated channels (Igc).

Figure 7.

(A) Hcatmap of normalised expression (vst) values for all 27 Toxocara canis genes of interest, including six glutamate-gated chloride channels (GluCls). 19 ATP-Binding Cassette Transporters, subfamily 13 (13 of which are P-glycoproteins (Pgps)), and one ligand-gated channel (Igc), for control, ivermectin-, and moxidectin-treated third-stage larvae (L3s). Color intensity represents normalized expression values, with the scale ranging from black (lowest expression) to red (highest expression), (B) Boxplots of normalized expression (vst) values for the 27 T. canis genes of interest. The y-axis represents normalized expression. Statistical significance between treatment groups was assessed using Tukey’s multiple comparison. Control L3s arc represented as blue, ivermectin-treated L3s as yellow, and moxidectin-treated L3s as red shaded boxes.