Dafachronic acid promotes larval development in Haemonchus contortus by modulating dauer signalling and lipid metabolism

Here, we discovered an endogenous dafachronic acid (DA) in the socioeconomically important parasitic nematode Haemonchus contortus. We demonstrate that DA promotes larval exsheathment and development in this nematode via a relatively conserved nuclear hormone receptor (DAF-12). This stimulatory effect is dose- and time-dependent, and relates to a modulation of dauer-like signalling, and glycerolipid and glycerophospholipid metabolism, likely via a negative feedback loop. Specific chemical inhibition of DAF-9 (cytochrome P450) was shown to significantly reduce the amount of endogenous DA in H. contortus; compromise both larval exsheathment and development in vitro; and modulate lipid metabolism. Taken together, this evidence shows that DA plays a key functional role in the developmental transition from the free-living to the parasitic stage of H. contortus by modulating the dauer-like signalling pathway and lipid metabolism. Understanding the intricacies of the DA-DAF-12 system and associated networks in H. contortus and related parasitic nematodes could pave the way to new, nematode-specific treatments.


Author summary
In the present study, using an integrative multi-omics approach, we show that dafachronic acid (DA) plays a critical functional role in the developmental transition in larvae of the parasitic nematode Haemonchus contortus (barber's pole worm) by modulating the dauer-like signalling pathway and lipid metabolism. The DA-DAF-12 signalling module in H. contortus provides a paradigm to explore its developmental and reproductive biology at the molecular level, to study physiochemical cross-talk between the parasite and its hosts, and to discover novel anthelmintic targets.
The DA-DAF-12 system is not unique to C. elegans. It has been shown to be functional in the free-living nematode Pristionchus pacificus [12] and in the parasitic nematodes of Ancylostoma ceylanicum (clade V) and Strongyloides stercoralis (clade IV) [13][14][15][16]. Using informatic approaches, components of this system have been identified in parasitic nematodes representing different evolutionary clades, including Trichinella spiralis, Trichuris trichiura (clade I); Brugia malayi and Loa loa (clade III), [17]; and, recently, DA was discovered in Ascaris suum and Toxocara canis (ascaridoids; clade III) [18]. Published information indicates that this endocrine system (controlling dauer formation, or developmental arrest) is relatively conserved for members of the phylum Nematoda [19][20][21], raising interest in the proposition that DAF-12 and/or associated molecules might represent suitable targets for new anthelmintics [13,[22][23][24]. This aspect is particularly important, given the nature and extent of anthelmintic resistance in socioeconomically important parasitic nematodes of animals, and the adverse impact that it has on the agricultural and associated industries through reduced animal productivity [25]. However, surprisingly, as yet there has been no detailed structural or functional investigation of DA-DAF-12 and associated signalling pathways in economically significant nematodes of livestock animals.
The barber's pole worm, Haemonchus contortus (order Strongylida), is particularly well-suited for molecular explorations [26]. It is arguably the most pathogenic nematode of ruminants, and the disease that it causes (haemonchosis) has a major, adverse impact on animal health and production worldwide [27,28]. This worm has a short life-cycle (~28-30 days), has major reproductive potential and, thus, can be readily produced in large numbers in experimental sheep, allowing detailed in vitro studies. The worm develops from the egg to the adult stage through four larval stages, with a dauer-like developmental arrest at the third stage (L3) in the environment, and a possible developmental arrest (hypobiosis) at the fourth stage (L4) within the host animal [29][30][31]. Recently, we established an efficient in vitro-culture system for larval stages of this parasitic nematode [32], which facilitates in-depth studies of developmental processes and mechanisms [33][34][35], underpinned by extensive genomic resources [36][37][38] and enabled by a ready accessibility to transcriptomic, proteomic, lipidomic and informatic technologies [33,35,39,40]. Using these resources and technologies, in the present study, we elucidate the functionality of DA-DAF-12 system and explore how it modulates associated pathways in this highly significant parasitic nematode-H. contortus.

Transcriptional changes link to the dauer-signalling pathway during larval transition, and the identification and quantitation of endogenous Δ7-DA
Haemonchus contortus undergoes a morphological transition from an infective L3, via the exsheathed L3 (called xL3), to the parasitic L4 stage [29], which can be carried out in vitro [32]. Here, we investigated, the transcription of genes inferred to be linked to dauer-signalling [34] during this transition in vitro, and then identified and quantitated Δ7-DA in respective larval stages of the nematode.
Based on prior knowledge for C. elegans [9], these transcriptional alterations ( Fig 1B) suggested that DAs are integral to this developmental transition in H. contortus, because the biosynthesis of DAs likely represents the outcome of the dauer signalling pathway (Fig 1A) [34]. Therefore, we investigated H. contortus L3s for the presence of DA. Endogenous Δ7-DA (retention time: 4.2 min; mass error:~0.5 part per million) was unequivocally identified in L3s (Fig 1C), and then quantified in all larval stages studied here (Fig 1D). The abundance of Δ7-DA increased substantially from L3 to xL3 (24 h following exsheathment) and then decreased gradually in the ensuing 6 days of in vitro-culture ( Fig 1D).

Synthetic, exogenous (25S)-Δ7-DA activates Hc-DAF-12
To examine whether (25S)-Δ7-DA might bind to the ligand-binding domain (LBD) of Hc-DAF-12, we compared the structural model of this predicted LBD with that of Ac-DAF-12 from Ancylostoma caninum (a canine hookworm which is a related strongylid nematode) (cf. [13]). Using three independent algorithms, we showed high structural similarity, achieving a root-mean-square deviation (RMSD) of 1.05, a structural distance measure (SDM) of 20.89 and a Q-score of 0.88 (Fig 2A), suggesting that Hc-DAF-12 and Ac-DAF-12 have a similar binding affinity and ability to activate DAF-12. This proposal was confirmed by showing that, in a luciferase reporter assay, (25S)-Δ7-DA at 50 nM to 1 μM activated Hc-DAF-12 with an EC 50 of 12.54 nM, which is similar to that of Ac-DAF-12 (12.80 nM) ( Fig 2B).

Analysis of differential transcription, protein expression and lipid abundance
Using individual transcriptomic, proteomic and lipidomic data sets produced (S2, S3 and S4 Tables), we studied molecular changes in H. contortus xL3s and xL3s exposed to (25S)-Δ7-DA for 24 h. Extensive changes in mRNA transcription, protein expression and lipid abundance were recorded (S2, S3 and S4 Tables). Specifically, significantly higher levels of 1,055 mRNAs, 101 proteins and 180 lipids, and significantly lower levels of 1,029 mRNAs, 46 proteins and 109 lipids were detected in xL3s (at 24 h) compared with L3s immediately following exsheathment (Fig 3A-3C; S2, S3 and S4 Tables). More differences were seen in xL3s exposed to (25S)-Δ7-DA at 24 h, including significantly increased levels of some mRNAs (n = 1,378), proteins (n = 263) and lipids (n = 177) and significantly decreased levels of other mRNAs (n = 1,362), proteins (n = 126) and lipids (n = 109) (S2, S3 and S4 Tables). Most significant molecular changes detected in xL3s (at 24 h) were identified in (25S)-Δ7-DA-treated xL3s (at 24 h); these changes were inferred to be associated with biological processes including environmental information processing (principally signal transduction), genetic information processing (including folding, sorting and degradation) and lipid metabolism (including fatty acid degradation and steroid hormone biosynthesis) ( Table).

Inhibition of endogenous Δ7-DA by dafadine A compromises larval exsheathment and development, and alters lipid abundance
As a previous study [41] has shown that dafadine A can specifically inhibit the biosynthesis of DAs in C. elegans, we elected to test the effect of this inhibitor on Δ7-DA biosynthesis, larval exsheathment and development of H. contortus. Treatment with dafadine A (100 μM) slowed larval development from the L3 to the L4 stage and significantly reduced the level of endogenous Δ7-DA in dafadine A-treated worms, compared with untreated and (25S)-Δ7-DA-treated worms (Fig 6A and 6B). When L3s were exposed to 100 μM of dafadine A in vitro, exsheathment was significantly inhibited (P < 0.001) compared with unexposed L3 controls (Fig 6C). Similarly, when xL3s were exposed to 100 μM of dafadine A, larval development decreased significantly (P < 0.001) (Fig 6D). The inhibitory effects of dafadine A on the production of 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).

Discussion
This study identified, for the first time, DA in the strongylid nematode H. contortus, and showed that this hormone promotes larval exsheathment and development via a relatively conserved nuclear hormone receptor, DAF-12. In H. contortus, the DA-DAF-12 complex modulates the dauer-like signalling pathway, via a negative feedback circuit, and affects molecular alterations linked to pharynx development, body morphogenesis, cellular growth, lipid signalling and metabolism.
It has been reported that the binding of DA to DAF-12 in parasitic nematodes is similar to that of bile acids to the farnesoid X receptor in mammals, suggesting that a bile acid-like signalling pathway exists in parasitic nematodes [15]. Interestingly, a common hormone-theme has been proposed for physicochemical communications between parasite and host animal [43][44][45]. A good example of this is that prolactin evokes the transmammary transmission of larvae of the ascaridoid nematode T. canis in mice [46]. It is readily possible that the DA-DAF- Dafachronic acid promotes larval development in Haemonchus contortus 12 module in the latter nematode plays a role in regulating or signalling larval activation and transmission, but this involvement needs to be verified molecularly. Clearly, understanding the functionality of the DA-DAF-12 module in parasitic nematodes could provide a paradigm for exploring cross-talk between parasite and host, particularly for worms which can enter into or exit from hypobiosis (arrested development) in their host, such as some members of the families Trichostrongylidae and Ascarididae [47,48].
In H. contortus, the upregulated transcription of particular dauer signalling genes during the developmental transition from the L3 (dauer-like) stage to the L4 stage indicates an active DA biosynthesis in xL3s, confirmed by measuring an increase in the level of Δ7-DA following L3 exsheathment. These alterations are similar to the transcriptional changes and hormone signal amplification seen in C. elegans during its development to the reproductively-active adult stage [6,11]. By contrast, a decreased level of Δ7-DA during the ensuing larval development indicates a reduction of its biosynthesis, which is supported by the observation of a pronounced downregulation of transcription of particular genes linked to dauer-like signalling. The dynamics of these changes in DA and transcription levels suggest that the endogenous synthesis of Δ7-DA is relatively tightly modulated or controlled via an, as yet, uncharacterised feedback circuit. A similar negative feedback mechanism exists in C. elegans, and operates via the let-7 family of microRNAs [5,6]. As let-7 homologues have not yet been identified or characterised in H. contortus, further work is required to establish how this feedback mechanism works in this parasitic nematode. Interestingly, Δ4-DA was not detected in H. contortus, which might be due to its absence or undetectable levels in the larval stages studied. However, both Δ4-DA and Δ7-DA have been detected in both A. suum and T. canis at differing levels [18], suggesting a functional distinctiveness of the two isomers in their involvement in selected biological processes in the latter two nematodes.
Different "signal intensity thresholds" of DA might be required for larval activation versus development; we found that 100 μM of (25S)-Δ7-DA did not induce exsheathment (although there is a possibility that DA does not penetrate the L3 sheath), but did significantly stimulate larval development following exsheathment. The specific inhibition of DAF-9 (cytochrome P450) with dafadine A resulted in a significant reduction of both endogenous Δ7-DA levels and larval exsheathment/development, which could be partially reversed through the supplementation of an excess (1.25 μM) of exogenous (25S)-Δ7-DA. These findings are distinct from those described for Ancylostoma caninum in that (25S)-Δ7-DA can directly activate infective larvae (L3s) of A. caninum and can induce post-parasitic larvae of S. stercoralis to develop to free-living stages [13,16]. The distinct responses to (25S)-Δ7-DA among H. contortus (clade V), A. caninum (clade V) and S. stercoralis (clade IV) might relate to evolutionary divergences in DA-associated signalling pathways within the phylum Nematoda [14,49]. This proposal warrants future evaluation. Exogenous (25S)-Δ7-DA-induced changes in mRNA, protein and lipid profiles in xL3s of H. contortus appear to link to phenotypic distinctiveness (development) and lipid metabolism (i.e. fatty acid degradation, and glycero-and glycerophospho-lipid biosynthesis) via the DA-DAF-12 module. To test the functionality of the DA-DAF-12 module, we blocked the biosynthesis of Δ7-DA using a specific inhibitor (i.e. dafadine A) of DAF-9 (cytochrome P450) [41], which resulted in a reduction of the endogenous Δ7-DA level, and, consequently, inhibited larval development. Similar results were achieved when the cytochrome P450s of Nippostrongylus brasiliensis and S. stercoralis were targeted with a less specific inhibitor, ketoconazole [16,50]. The significant reduction of PC(15:0_20:4, 16:0_17:0), LPC(15:0) and PI(15:0_20:4) levels in dafadine A-treated H. contortus larvae could be reversed by supplementation with (25S)-Δ7-DA, indicating a direct or indirect role for DA-DAF-12 signalling in the metabolism of selected glycerolipids and glycerophospholipids [51]. In addition, the increases in DG (15:0_18:1) and TG(15:0_10:0_18:2) levels in dafadine A-treated worms suggest a role for DAF-9 in glycerolipid metabolism. Interestingly, all of these lipid species are odd-chain fatty acids, which contrasts the situation in C. elegans, in which only small amounts of straight, oddchain fatty acids (likely originating from the worm's food source-Escherichia coli) accumulate in lipids [52]. Surprisingly little is known about the origin and functional roles of these oddchain lipid species in developmental processes of nematodes. Nonetheless, based on the present findings, we propose a dual role for the DA-DAF-12 module in promoting the metabolism of key glycerophospholipids and inhibiting the degradation of some lipids (possibly promoting fat accumulation), which functions via a negative feedback to DAF-9 (see Fig 7G), but, clearly, this hypothesis requires rigorous testing.
Taken together, the findings of the present study provide evidence for a signalling cascade in H. contortus, in which host signals (e.g., CO 2 , pH, insulin and/or metabolites of bile acids) bind to chemoreceptors, which trigger signal transduction from chemosensory neurons to endocrine cells and then hypodermal cells through the interconnected cGMP, TGF-β and IGF-1 pathways. The transduced signal promotes the metabolism of steroids and the biosynthesis of DA, the latter of which activates the nuclear hormone receptor DAF-12, leading to gene transcription and protein expression associated with body morphogenesis and pharynx development as well as lipid metabolism. A high level of DA would modulate phosphatidylinositol signalling that activates PI3K-AKT signalling [53], resulting in phosphorylation-dependent cytoplasmic sequestration of the transcription factors DAF-16/FOXO [9,54]. The activation of DAF-16/FOXO antagonises the upstream cGMP, TGF-β and IGF-1 signalling [53,55], downregulating DA biosynthesis in a feedback circuit, resulting in a reduced lipid metabolism, and, consequently, in fat accumulation (Fig 7G). Understanding the biosynthesis of DAs and nuclear-hormone signal transduction (e.g., via DA-DAF-12) should provide valuable insights into the developmental biology and adaptation of parasitic nematodes to host animals. Experimental evidence [13,23,24] has already shown that S. stercoralis hyperinfection can be prevented by treatment with (25S)-Δ7-DA. Although (25S)-Δ7-DA might regulate developmental processes in H. contortus (order Strongylida) differently from those in Strongyloides [12,13,16], the potential of DAF-9 and DAF-12 as novel intervention targets (cf. [13,22]) should be explored further. Clearly, major success achieved in a recent study [56] opens the door to assessing the functional essentiality of these steroid hormone signalling components in Strongyloides species by RNA interference.
In conclusion, current findings for H. contortus indicate that the hormonal signal complex DA-DAF-12 modulates the dauer-like signalling pathway through a feedback loop, and regulates biological processes associated with cellular growth and lipid metabolism via a conserved DA-DAF-12 signalling module during developmental transition. This module provides a paradigm to investigate aspects of the developmental and possibly reproductive biology of H. contortus and related nematodes, to explore physiochemical cross-talk between parasite and host, and to discover novel intervention strategies against parasitic diseases.

H. contortus stages
A monospecific infection of H. contortus was maintained in sheep under well-controlled experimental conditions [57]; three distinct larval stages of this nematode were produced in vitro using established methods [32]. In brief, third-stage larvae (L3s) were collected from coproculture, purified and maintained at 10˚C in a refrigerated incubator; exsheathed L3s (xL3s) were produced using a well-established hypochlorite-treatment method [32]; and xL3s were cultured (300 per well of 96-well culture plates), under standardised conditions, in Luria Bertani medium (LB) supplemented with Antibiotic-Antimycotic (cat no. 15240-062, Gibco) (LB � ) at 38˚C, 10% v/v CO 2 to yield fourth-stage larvae (L4s) of H. contortus.

Detection of DA in the worm
Endogenous DA was identified by liquid chromatography-mass spectrometric (LC-MS) analysis of lipids extracted from three distinct developmental stages of H. contortus. Lipids were extracted from four replicates (each 1 mg dry weight) of each L3s, xL3s and L4s using an established method [34]. Each replicate was suspended in ice-cold methanol (40%), homogenised using zirconium oxide beads (ZROB05, Next Advance, USA) and extracted with chloroform: methanol (2:1) by centrifugation at 10,000 ×g for 15 min, dried and resuspended in methanol (100%), then subjected to LC-MS analysis in an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 UHPLC using a C30 column (

Structure modelling and DAF-12 reporter assay
The structure of the LBD of DAF-12 of H. contortus (Hc-DAF-12) (using the inferred amino acid sequence: GenBank accession no. MK_256962) was modelled using the program I-TASSER [58] and compared with that of A. caninum (Ac-DAF-12) [15] using UCSF Chimera v.1.12 [59]. Structural similarities between query and template sequences were established using sequence length, overall RMSD, SDM and Q-score.

Assaying the effect of (25S)-Δ7-DA on larval exsheathment and development
First, L3s (300 worms per well; four replicates) were exsheathed by incubating them at 38˚C and 10% v/v CO 2 for 48 h in physiological saline in the presence or absence of 10 μM of (25S)-Δ7-DA. The number and percentage of exsheathed L3s (xL3s) were assessed every 12 h. Second, xL3s (300 worms per well; four replicates) were cultured to L4s at 38˚C, 10% v/v CO 2 in LB � in the absence or presence of (25S)-Δ7-DA (10 μM to 10 � 2 −17 μM). The numbers of xL3s and L4s in culture were calculated every 24 h, and the proportion of L4s was calculated at each time point. Statistical analyses (student's t-test, Spearman's rank correlation and non-linear regression) were performed using Prism 7 (GraphPad, La Jolla, USA).

Transcriptomic, proteomic and lipidomic analyses
The transcriptomes, proteomes and lipidomes were produced from H. contortus xL3s, which had been exsheathed using the established hypochlorite-treatment method [32] and then incubated in LB � (38˚C, 10% v/v CO 2 for 24 h) in the presence or absence of 1.25 μM (25S)-Δ7-DA. For each treatment, four replicates of 30,000 xL3s each were incubated at 38˚C, 10% v/v CO 2 for 24 h.
For transcriptomic analysis, total RNA was extracted from each of the replicates of xL3s, processed and sequenced as described previously [33]. In brief, strand-specific mRNA libraries were constructed using the TruSeq RNA Library Prep Kit (Illumina) and sequenced on the BGISEQ-500 platform. Raw reads were processed and mapped to predicted genes of H. contortus (BioProject: PRJEB506) using Bowtie v.2.1.0 [60] within the software package RSEM v.1.2.11 [61].
For proteomic analysis, proteins were isolated from the replicates as described previously [33]. In brief, protein (50 μg) samples were reduced with Tris(2-carboxyethyl)phosphine (TCEP), alkylated with iodoacetamide and digested with Lys-C/trypsin Mix (cat no. V5072; Promega, USA). The digested samples were acidified with 1.0% (v/v) formic acid and purified using Oasis HLB cartridges (cat no. 186000383; Waters, USA) and then subjected to LC-MS/ MS analysis using a QExactive plus Orbitrap mass spectrometer (Thermo Fisher Scientific, USA) with a nanoESI interface in conjunction with an Ultimate 3000 RSLC nanoHPLC (Dionex Ultimate 3000). Mass spectrometry data were analysed using MaxQuant [62] to identify and quantify peptides.
For lipidomic analysis, lipids were extracted from the replicates and analysed by LC-MS/MS using an Orbitrap Fusion Lumos mass spectrometer [40]. The mRNAs, proteins and lipids quantified were subjected to principal component and hierarchical cluster analyses using the Perseus computational platform [63,64]. Differential transcription was explored using the limma, glimma and edgeR packages [65]; a fold-change (FC) of � 2 and a false discovery rate (FDR) of � 0.01 defined a significant difference, unless otherwise stated (FC � 2 and P � 0.01). Differential protein expression analysis was conducted using the program Perseus v.1.6.1.1, employing an FC of � 1.5 and an FDR � 0.05 as thresholds. For lipids, an FC of � 1.5 and a P value of � 0.01 were used as cut-offs. Differentially transcribed mRNAs and expressed proteins were assigned to Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) terms using BlastKOALA [66], and KEGG annotations were analysed and displayed using FuncTree 2 [67].

Accession numbers
Nucleic acid sequence data from this study are available via the National Center for Biotechnology Information (NCBI) sequence reads archive (SRA) under accession numbers SUB3797117 and SUB5228712. The proteomic data obtained by mass spectrometry have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository and are linked to the dataset identifier PXD012878.
Supporting information S1 Table. Transcription