Fig 1.
Transcriptional changes pertaining to dauer signalling genes, and quantification of dafachronic acids in Haemonchus contortus during developmental transition.
(A) Model of the cyclic guanosine monophosphate (cGMP) (red), DAF-7-related transforming growth factor-β (TGF-β) (orange), DAF-2-related insulin-like growth factor 1 (IGF-1) (green) and steroid hormone signalling (blue) pathways proposed for H. contortus [34]. This model is predicted to play a role in integrating environmental signals to control the biosynthesis of one or more dafachronic acids (DAs), which activate the nuclear hormone receptor DAF-12. The DA-DAF-12 module might serve as a checkpoint for developmental decisions and associate with nutrient metabolism in parasitic nematodes [8,14,17,34]. (B) Transcriptional profiles (Z-score normalised, mapped reads per million) of 61 gene homologues involved in the cGMP (red), TGF-β (orange), IGF-1 (green) and steroid hormone (blue) signalling pathways are displayed for the developmental transition from the dauer-like third larval stage (L3), via exsheathed L3 (xL3), to the parasitic fourth larval stage (L4) of H. contortus in vitro. (C) Using (25S)-Δ4-DA (calculated mass: 413.3061, retention time: 4.0 min) and (25S)-Δ7-DA (calculated mass: 413.3061; retention time: 4.2 min) as references (blue peaks), endogenous Δ7-DA (retention time: 4.2 min; red peak) was detected in H. contortus with mass error estimated at 0.5 part per million (ppm). (D) The relative abundance of endogenous Δ7-DA following larval exsheathment and in the ensuing larval development in vitro is indicated.
Fig 2.
The influence of (25S)-Δ7-DA on larval activation and development.
(A) Comparison of the ligand-binding domain (LBD) of DAF-12 of Haemonchus contortus (Hc-DAF-12) with that of Ac-DAF-12 from Ancylostoma caninum, using the following parameters: sequence length, root-mean-square deviation (RMSD), structural distance measure (SDM) and Q-score. (B) Activation of Hc-DAF-12 and Ac-DAF-12 by (25S)-Δ7-DA) in a luciferase reporter assay. The effects of 10 μM of (25S)-Δ7-DA on (C) larval exsheathment and (D) larval development. The effect of (25S)-Δ7-DA on larval development is both (E) dose- and (F) time- dependent. An error bar indicates a standard deviation (SD; four replicates). Statistical significance is indicated with one or more asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, using Student’s t-test).
Fig 3.
Alterations in mRNA transcription, protein expression or lipid metabolism in Haemonchus contortus following larval exsheathment.
Differential analyses of (A) mRNA, (B) protein or (C) lipid levels between exsheathed L3s (xL3s) at 0 h and xL3s at 24 h. Indicated is a significant up-regulation (red) or down-regulation (blue) of mRNA transcription, protein expression or lipid levels in larvae (xL3s) 24 h after exsheathment. (D) By integrating all results, we showed that molecules (mRNAs encoded by particular genes, proteins and lipids) with significant differential transcription, expression or abundance were specifically associated with fatty acid degradation, glycerolipid metabolism, glycerophospholipid biosynthesis, ether lipid or sphingolipid metabolism and/or steroid hormone biosynthesis. Down-regulated (blue) or up-regulated (red) molecule or pathway indicated; gene and protein designations derived from Caenorhabditis elegans homologues (WormBase).
Fig 4.
Effect of (25S)-Δ7-DA on the transcription of dauer signalling genes following larval exsheathment.
Transcription levels of dauer-like signalling genes in Haemonchus contortus exsheathed third-stage larvae (xL3s; 0 h and 24 h) and (25S)-Δ7-DA-treated xL3s (24 h) following exsheathment are indicated in the heat map. Genes involved in the cyclic guanosine monophosphate (cGMP), transforming growth factor-β (TGF-β) and insulin-like growth factor 1 (IGF-1) and steroid hormone signalling pathways are listed. Colour scales indicate scaled read counts per million in the rows; up-regulation (red) or down-regulation (blue) is indicated. Gene designations relate to the dauer-signalling pathway model for H. contortus [34].
Fig 5.
Transcriptomic, proteomic and lipidomic differences between treated and untreated worms.
Differential analyses of the (A) mRNA, (B) protein and (C) lipid levels between exsheathed the third-stage larvae (xL3s) and (25S)-Δ7-DA-treated xL3s of Haemonchus contortus at 24 h following exsheathment. Molecules that were significantly up-regulated (red) or down-regulated (blue) in (25S)-Δ7-DA-treated xL3s are indicated. (D) These molecular alterations inferred to associate with cellular growth, lipid signalling, fat degradation, pharynx development and body morphogenesis; gene and protein designations derived from Caenorhabditis elegans homologues (WormBase).
Fig 6.
Effects of dafadine A on DA biosynthesis, larval development and lipid metabolism.
(A) Treatment with 100 μM of dafadine A results in a reduced larval development, which is linked to (B) a significantly lower level of endogenous Δ7-DA in dafadine-treated worms. The inhibitory effect of dafadine A and rescuing effect of 1.25 μM of (25S)-Δ7-DA on (C) larval exsheathment and (D) development. An error bar indicates a standard deviation (SD; four replicates). Statistical significance is indicated with one or more asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, using the Student’s t-test).
Fig 7.
Effects of dafadine A (100 μM) on the abundances of glycerolipids and glycerophospholipids.
The abundances of particular (A) diradylglycerol (DG), (B) triacylglycerol (TG), (C and D) phosphatidylcholine (PC), (E) lysophosphatidylcholine (LPC) and (F) phosphatidylinositol (PI) in untreated xL3s; dafadine A-inhibited xL3s; 1.25 μM of (25S)-Δ7-DA-rescued xL3s; and 1.25 μM of (25S)-Δ7-DA-treated xL3s. An error bar indicates a standard deviation (SD; four replicates). Statistical significance is indicated with asterisk (*P < 0.05, **P < 0.01, ***P < 0.001, using the Student’s t-test). (G) A schematic showing the DA-DAF-12 module and its proposed functional roles in regulating fat degradation/accumulation, cell growth and cellular signalling. It is proposed that DA produced by DAF-9 (cytochrome P450) activates the nuclear hormone receptor DAF-12, which promotes the degradation of glycerolipids [e.g., DG(15:0_18:1) and TG(15:0_10:10_18:2)] for the subsequent production of glycerophospholipids [e.g., PC(15:0_20:4), PC(16:0_17:0) and PI(15:0_20:4)], and which negatively regulates DA biosynthesis to reduce lipid degradation for fat accumulation. The solid arrow indicates the production of endogenous DA; a dashed line with an arrow indicates an indirect pathway; and a dashed line with a bar indicates a negative feedback loop.