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 life cycle. 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. 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 1413 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, body 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 and transporter genes using phylogenetic analysis of nematode orthologs to provide uniform gene nomenclature. Our study revealed that the expression of two GluCls and eight ABCB genes, particularly five P-gps were significantly altered in response to ML treatment. Compared to controls, Tca-glc-3, Tca-avr-14, Tca-haf-10, and Tca-Pgp-13.2 were downregulated in ivermectin-treated larvae, while Tca-abcb7, Tca-Pgp-11.2, and Tca-Pgp-2 were downregulated in moxidectin-treated larvae. Conversely, Tca-haf-9, Tca-Pgp-11.3, and Tca-Pgp-16.3 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; Glutamate-gated chloride channels; Ivermectin; Moxidectin; RNA-Seq; Toxocara canis; Toxocariasis; Transcriptome; mRNA.

Graphical abstract

Fig. 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). This figure was created using Biorender.com.

Fig. 2

(A) Principal Component Analysis (PCA) of Toxocara canis third-stage larvae (L3s) exposed to RPMI-1640 medium (controls; blue circles), ivermectin (yellow squares), or moxidectin (red diamonds). Each point represents a pool of ∼500 - 1000 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-treated L3s and 902 significantly upregulated and 511 significantly downregulated in moxidectin-treated L3s; (D–E) Venn diagrams depicting 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).

Fig. 3

(A&C) Gene Ontology (GO) bubble plots of genes (A) upregulated 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 correspond 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.

Fig. 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 correspond 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.

Fig. 5

(A&C) Gene Ontology (GO) bubble plots of the overlapping (A) upregulated and (C) downregulated shared genes between ivermectin- and moxidectin-treated Toxocara 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-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 the overlapping (B) upregulated and (C) downregulated shared genes between ivermectin- and moxidectin-treated T. canis L3s. The length and color of the lollipops correspond 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.

Fig. 6

A maximum-likelihood phylogenetic tree of Toxocara canis glutamate-gated chloride channels (GluCls) (A) and half ATP-binding cassette (haf) transporters (B) with related nematode sequences obtained from GenBank. Both trees were 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 GluCl tree (A) is rooted using a GluCl sequence from Aplysia californica (California sea hare) and the haf tree (B) was rooted using a P-gp sequence from Canis lupus familiaris (dog).

Fig. 7

(A) Heatmap of normalized expression (vst) values for all 25 Toxocara canis genes of interest, including seven glutamate-gated chloride channels (GluCls), and 19 ATP-Binding Cassette Transporters, subfamily B (13 of which are P-glycoproteins (Pgps)), 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 25 T. canis genes of interest. The y-axis represents normalized expression. Statistical significance between treatment groups was assessed using the Kruskal-Wallis test followed by Dunn's post-hoc comparisons. Control L3s are represented as blue, ivermectin-treated L3s as yellow, and moxidectin-treated L3s as red shaded boxes.

Fig. 8

qPCR validation of RNA-Seq results. Fold change in expression of 15 ML-associated genes of interest following ivermectin (yellow) or moxidectin (red) treatment of larvae hatched in vitro compared to control larvae (blue). Fold change was calculated using the Pfaffl method using T. canis 18S rRNA as the reference gene. Statistical significance was assessed using the Kruskal–Wallis test followed by Dunn's post-hoc comparisons.