Adult worms of H. contortus (ZJ strain) were collected from a slaughterhouse in Jiaxing, Zhejiang, China. Female adult worms were dissected to collect eggs under a dissecting microscope (Motic, China). Eggs were cultured at 28°C for 7 days to obtain the L3s. Three helminth-free lambs (Hu sheep, 6 months old) were experimentally infected with ∼8000 L3s of H. contortus as described previously (Zhang et al., 2018). Faecal examination was performed to confirm the establishment of infection. Eggs of H. contortus were collected by flotation using saturated NaCl solution (Cox and Todd, 1962). The first-stage larvae (L1s), second-stage larvae (L2s) and L3s of H. contortus were obtained by incubating faecal samples at 28°C for 1, 3 and 7 days, respectively. L4s and adult worms were collected from the abomasum of euthanised lambs at 9 and 45 days after infection, washed in phosphate-buffered saline (PBS, pH7.4) and temporarily maintained in DMEM (Thermo Fisher Scientific, United States) with 10% FBS (Biological Industries, Israel) at 37°C (Shi et al., 2021).

The free-living nematode C. elegans N2 strain was maintained on nematode growth medium (NGM) agar plates at 20°C (Stiernagle, 2006). NGM agar plates were supplied with Escherichia coli OP50 or HT115 mutant bacteria as a food source.

Genomic DNA (gDNA) was extracted from snap-frozen adult worms of H. contortus using a small-scale DNA extraction kit (Takara Bio, Japan) according to the supplier’s instructions. Total RNA was extracted from eggs, L1s, L2s, L3s, L4s and adult worms of H. contortus using Trizol reagent (Invitrogen, United States). The first strand cDNA was synthesised from the total RNA using a first-strand cDNA synthesis kit (Toyobo, Japan). Extracted gDNA and synthesised cDNA were stored at −80°C until use.

The sequence of C. elegans ttr-31 (WormBase ID: WBGene00010225) was used to identify the gene homologue of H. contortus (designated as Hc-ttr-31) from the Sanger Helminths Database (https://www.sanger.ac.uk/resources/downloads/helminths/haemonchus-contortus.html). Based on the available transcript of Hc-ttr-31, primers (Supplementary Table S1) were designed using Primer Premier 5 (PREMIER Biosoft International, United States) to amplify the coding region of this gene. The amplification reaction mix contained 2.5 μL 10 × PCR buffer, 0.5 μL LA Taq (Takara Bio), 2 μL of 2.5 mM dNTPs, 2.0 μL of primer pair (10 μM), 1 μL gDNA of H. contortus and 17 μL of molecular grade water. The thermocycle program comprised of 94°C for 15 s, 62°C for 30 s and 72°C for 30 s. PCR amplification was also performed using cDNA as a template to obtain the transcript of Hc-ttr-31. PCR products were purified, ligated to PMD-19T (Takara Bio) and sequenced in both directions. Based on the obtained nucleotide acid sequence, exons and introns of Hc-ttr-31 were predicted by the “GT-AG” rule using DNASTAR (Version 7.1.0) (Breathnach and Chambon, 1981), with evidence from the obtained transcript sequences. Compara Gene Tree analysis and reciprocal blastp searching were performed based on resources available at WormBase ParaSite (https://parasite.wormbase.org/index.html). Functional domain architecture of the inferred proteins was predicted based on the InterPro database (Mitchell et al., 2018).

Transcriptions of Hc-ttr-31 in all developmental stages (egg, L1, L2, L3, L4 and adult) of H. contortus were determined using Real-time SYBR Green Mix reagent (Toyobo) and a CFX96 Real-time PCR System (Bio-Rad, United States). qRT-PCR was also performed to assess the transcriptional level of ttr-31 in different developmental stages of C. elegans. The thermocycle program was 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 60°C for 15 s and 72°C for 15 s. Actin (act-1) and β-tubulin were used as the internal controls for C. elegans and H. contortus, respectively. Transcriptional levels of key signalling components (i.e., ced-1, ced-4, ced-6, ced-7, ced-9, egl-1, ina-1, nrf-5, par-1, and ttr-52) involved in apoptosis and phagocytosis of apoptotic cells (Reddien and Horvitz, 2004; Wang et al., 2010; Palmisano and Meléndez, 2019) were also assessed. Primer sets used in this section can be found in Supplementary Table S1. Three replicates were included and performed independently. Transcriptional changes of genes were analysed using a 2^-∆∆Ct method and presented as mean ± standard error of mean (SEM). Statistical analysis was conducted using a one-way ANOVA (p < 0.05).

Hc-ttr-31 was subcloned into pET30a vectors for prokaryotic expression of the recombinant protein rHc-TTR-31. In brief, the pET30a-Hc-trr-31 plasmids were transformed into E. coli BL 21 (DE3), which was induced by 0.5 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) at 37°C for 8 h. Bacteria were harvested by a centrifugation at 8,000 × g at 4°C for 5 min, suspended in 50 mM potassium phosphate (pH7.4), and lysed by sonication. The supernatant was separated by a centrifugation at 12,000 × g for 10 min at 4°C, filtered with a 0.22 μm filter (Merck Millipore, United States), then incubated with Ni-NTA resin column (Thermo Fisher Scientific) for 30 min. After washing with 20 mM imidazole, recombinant protein rHc-TTR-31 was eluted with 250 mM imidazole from the column. Purified protein was assessed by SDS-PAGE and a Bradford kit (Fdbio Science, China) using bovine serum albumin (BSA) as standard.

A New Zealand rabbit was immunised with the rHc-TTR-31 to generate polyclonal antibodies against the recombinant protein (Shi et al., 2021). In brief, the rabbit was subcutaneously injected with the rHc-TTR-31 and Freund’s Complete Adjuvant (Sigma-Aldrich, United States). Two boosting shots were conducted with the rHc-TTR-31 and Incomplete Fraud’s Adjuvant in a 2-week interval. Antiserum was collected 10 days after the final immunisation. Western blotting was performed using the anti-6 × His tag polyclonal antibodies (1:2,000; Proteintech, United States) and the generated anti-rHc-TTR-31 polyclonal antibodies to determine the specificity of antibodies.

Immunolocalisation of target protein was performed as described previously (Riou et al., 2005; Shi et al., 2021). Briefly, L3s of H. contortus were exsheathed by incubation with 0.15% sodium hypochlorite for 30 min (Ding et al., 2017); the exsheathed L3s (xL3s), L4s and adults were washed in PBS (pH7.4), fixed in 4% paraformaldehyde (Rothwell and Sangster, 1993) overnight, and then dehydrated. L4s and adults were further embedded in paraffin, sectioned at 5 μm in thickness, dried at 60°C overnight and deparaffinized with xylene and dehydrated as described previously (Qu et al., 2014). Sliced L4s and adults as well as xL3s of H. contortus were incubted in 1% BSA for 2 h, washed twice in PBS, and then incubated with 1:500 diluted anti-Hc-TTR-31 polyclonal antibodies at 4°C overnight. After washing, slides were incubated with goat anti-rabbit IgG (Invitrogen) at 1:1,000 dilution at 37°C for 1 h. Pre-immunisation serum from the same animal was used as a negative control. Fluorescence was detected with a Zeiss LSM710 laser confocal microscope (Zeiss Microscopy, Germany).

To confirm the spatial distribution of Hc-TTR-31, transgenesis of Hc-ttr-31 was conducted in C. elegans (Huang et al., 2021). Briefly, the promoter sequence of Ce-ttr-31 was amplified, ligated to the PMD-18T vector and subcloned into pPD95.67 plasmids (Ce-ttr-31p::gfp), with the Hc-ttr-31 coding sequence inserted into the multiple cloning site (Ce-ttr-31p::Hc-ttr-31::gfp). Transgenic expression of Hc-ttr-31 in C. elegans N2 strain was performed by micro-injection with the Ce-ttr-31p::Hc-ttr-31::gfp (Mello et al., 1991). A Ce-ttr-31p::Ce-ttr-31::gfp was used as a reference control. Considering the homology of Hc-ttr-31 and Ce-ttr-31, transgenesis of Hc-ttr-31 should result in an over-expression of TTR-31 in the treated C. elegans. To explore the effects of exogenous Hc-TTR-31 in C. elegans, transgenic worms were mounted on 2% agar pads containing 1% sodium azide (Solarbio, China) and imaged using a Zeiss LSM710 laser confocal microscope (Zeiss Microscopy).

To explore the function of Hc-ttr-31 in H. contortus, Hc-ttr-31 RNAi-mediated gene knockdown of its orthologue Ce-ttr-31 was performed in C. elegans, using a method described previously (Kwon et al., 2010; Yan et al., 2014). In brief, Hc-ttr-31 was cloned into the plasmid L4440, which was then transformed into E. coli HT115 (DE3), with Ce-ttr-31 and empty L4440 vectors used as positive and negative controls, respectively. NGM plates containing 0.1 mM IPTG were seeded with the transformed HT115 and placed at room temperature for 5 days prior to RNAi assay. Worms of C. elegans N2 strain were synchronized and laid on standard NGM plates supplemented with OP50 (Kamath and Ahringer, 2003). The worms were washed off the plates after 30 h (i.e., L4 stage) and then centrifuged at 1,000 ×g for 5 min. From which, 20 worms were inoculated onto NGM plates with the transformed HT115, and incubated at 25°C for 3 days. Following transcriptional examinations of Hc-ttr-31 and Ce-ttr-31 by qRT-PCR, phenotypic changes of worms were assessed in aspects of brood size, pumping rate, body length and body width (see Wong et al., 1995; Mörck and Pilon, 2006; Papaevgeniou et al., 2019). Three replicates were performed for the RNAi and phenotypic assays. For the analysis of facultative vivipary phenotype, worms were checked using a dissecting microscope (Motic) and the ratio of bagging worms was calculated.

To check whether Hc-ttr-31 plays a role in the apoptosis, the number of apoptotic germ cells in the RNAi-treated worms was determined using a method described elsewhere (Kelly et al., 2000) with some modifications according to manufacturer’s instruction. Briefly, ∼ 100 synchronised adult worms were subjected to RNAi treatment for 36 h and then washed in M9 buffer for 5 times; Worms were suspended in 0.5 ml M9 buffer and stained with acridine orange (20 μg/ml; Solarbio) for 1.0 h; Stained worms were then transferred onto NGM plates seeded with OP50 for 1.5 h for recovery. Recovered worms were kept in 1.0% sodium azide solution at the centre of slide and examined under a Zeiss LSM710 laser confocal microscope (Zeiss Microscopy).

Production of general ROS in RNAi-treated worms was determined using 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime, China) based on the method reported elsewhere (Zhu et al., 2010; Guo et al., 2016). In brief, about 5,000 treated/untreated worms were sonicated in PBS, and the supernatant was collected after centrifugation at 15,000 × g for 45 min. Bradford protein assay kit (Fdbio Science) was performed to determine protein concentration. The lysates were incubated with 300 μM DCFH-DA in black-walled microtiter plate (Corning Incorporated, United States) at 37°C for 3 h. Rosup was used as a positive control in the production of reactive oxygen. Fluorescence intensity was measured using a Synergy H1 hybrid multimode microplate reader (BioTek, United States).

At least three technical replicates were included in each assay and each experiment was repeated three times. Data are presented as mean ± standard error of mean (SEM). One-way ANOVA with Dunnett post-hoc test was performed using Excel 2016 (Microsoft, United States) and GraphPad Prism 5 (GraphPad Software, United States). p < 0.05 was considered statistically significant.

Hc-ttr-31 comprised three exons, encoding a transcript of 432 nt in length (GenBank accession number MW013314; Figure 1A), representing a predicted gene locus (hcontortus_chr2_Celeg_TT_arrow_pilon: 30031855–30032608) in the recently updated chromosome-level genome assembly of H. contortus (MHCO3ISE; WBPS15). The cDNA-confirmed gene model of Hc-ttr-31 was similar to that of Ce-ttr-31 in the free-living model organism C. elegans (Figure 1A), and was predicted a 1-to-1 orthologue of Ce-ttr-31. The inferred protein Hc-TTR-31 was 143 aa in length, contained a signal peptide (SignalP-noTM) and a transthyretin-related family domain, showing a 65% similarity to the amino acid sequence deduced from Ce-ttr-31 (Figure 1B).

Different transcriptional levels of Hc-ttr-31 were detected among the developmental stages of H. contortus. Specifically, Hc-ttr-31 was highly transcribed in the egg and larval (L1, L2, L3 and L4) stages of H. contortus, with the highest level detected in the L1 stage and the lowest in the female adult stage (Figure 1C), showing a decreasing transcriptional pattern of Hc-ttr-31 during the development of this parasitic nematode. A similar developmental transcription of Ce-ttr-31 was also found in C. elegans (Figure 1D). Differently, the highest transcriptional level of Ce-ttr-31 was detected in the egg of the free-living nematode (Figure 1D).

Recombinant Hc-TTR-31 (rHc-TTR-31) was successfully expressed in E. coli BL21 (DE3) and was recognised by the anti-6×His tag polyclonal antibodies (Supplementary Figure S1). Using the recombinant protein, polyclonal antibodies against rHc-TTR-31 were generated and used to detect the native Hc-TTR-31 from the crude protein extract of H. contortus (Supplementary Figure S1A), facilitating immunolocalization of native Hc-TTR-31 in the L3s, L4s, and adults of this parasite (Figure 2 and Supplementary Figure S2). Specifically, predominant cephalic, cuticular/muscular and intestinal distributions of Hc-TTR-31 were observed in the xL3s of H. contortus, with punctate expression detected in the head and tail regions (Figure 2A). Muscular and intestinal distributions of Hc-TTR-31 were also found in L4s (Figure 2B) and adults (Figure 2C) of this parasitic nematode. Differently, the fluorescent intensities observed in the L4s and adults were lower than that in the xL3s of H. contortus (Figures 2B,C), which was in accordance with the decreasing transcriptional pattern of Hc-ttr-31 during the development of this parasite.

Driven by the promoter of Ce-ttr-31 (Ce-ttr-31p), GFP was expressed in the intestine, muscle, and neurons in the pharynx and tail regions of C. elegans (Figure 3), suggesting functional activities of the promoter in these tissues. Therefore, by microinjection of the Ce-ttr-31p::Ce-ttr-31::gfp and Ce-ttr-31p::Hc-ttr-31::gfp plasmids, GFP-fused proteins were expressed in the transgenic worms. Specifically, the Ce-TTR-31-GFP fusion protein was observed in the pharyngeal neurons, muscle, and the U-shape gonad arms, but not detected in the intestine of adult worms (Figure 4A). A similar protein distribution was also found for the Hc-TTR-31-GFP fusion protein in the transgenic C. elegans, including pharyngeal neurons, gonad and musculature (Figures 4B,C).

Microinjection of Ce-ttr-31p::Ce-ttr-31::gfp/Ce-ttr-31p::Hc-ttr-31::gfp in C. elegans N2 strain resulted in expression of Ce-TTR-31 and expression of both Ce-TTR-31 and Hc-TTR-31 in the transgenic worms. Compared with the non-transgenic C. elegans N2 strain, expression of Ce-TTR-31 prolonged the lifespan of transgenic worms (Figure 4D), but did not show any significant (p > 0.05) effect on brood size (Figure 4E) or pumping rate (Figure 4F) of the transgenic worms. Expression of both Ce-TTR-31 and Hc-TTR-31 in C. elegans N2 strain showed similar effects to the expression of Ce-TTR-31 (Figures 4D–F).

To further explore the functional roles of Hc-ttr-31, gene knockdown was successfully conducted by feeding worms with HT115 bacteria that could express silencing RNAs targeting Ce-ttr-31 or Hc-ttr-31. Specifically, compared with negative control, RNAi of Ce-ttr-31 significantly reduced (>50%; p < 0.001) the transcriptional level of this gene in the treated worms (Figure 5A), shortened the lifespan of treated worms (Figure 5B), decreased the number of progeny (p < 0.001), and inhibited the pumping rate (p < 0.001) and growth (body length and width; p < 0.05) of the treated worms (Figures 5C–F). Hc-ttr-31 RNAi mediated gene knock down of Ce-ttr-31 also achieved effective gene silencing (>50%; p < 0.001) of Ce-ttr-31 (Figure 5A), and resulted in similar effects on treated worms, such as shortened lifespan, and reduced brood size, pumping rate, body length and width (Figures 5B–F), implying functional conservation of ttr-31 between C. elegans and H. contortus.

Notably, compared with the untreated C. elegans, effective knockdown of Ce-ttr-31 and Hc-ttr-31-mediated knockdown of Ce-ttr-31 led to significantly increased (8 and 5 fold; p < 0.001) facultative vivipary phenotype (i.e., hatching in vivo or “bagging”; Chen and Caswell-Chen, 2004) in treated worms (Figure 5G), suggesting involvement of TTR-31 in the post-embryonic development and reproduction processes.

By staining with acridine orange, apoptotic cells were observed (brighter than the normal ones due to pyknosis) in the gonad arms of the RNAi-treated C. elegans (Figure 6). Specifically, compared with negative control (Figure 6A), there was an increase (4.28 and 3.77 folds) of apoptotic germ cells in the gonad arms of Ce-ttr-31 and Hc-ttr-31 RNAi-treated worms (Figures 6B,C). In addition to the increased number of apoptotic germ cells, lower transcriptional level of Ce-ttr-31 and Hc-ttr-31 was linked to a significant increase of general ROS (p < 0.01) and transcriptional level of ced-4 (cell death abnormal 4, encodes a key component in the apoptosis activation pathway) (p < 0.001) in RNAi-treated worms (Figure 7A), suggesting activated apoptosis and involvement of ttr-31 in apoptosis.

As the homologous gene ttr-52 (encodes an extracellular phosphatidylserine-binding protein on the apoptotic cell) was reported to be required for cell corpse engulfment (Wang et al., 2010), the function of ttr-31 in apoptosis or clearance of cell corpse was determined by exploring the transcriptional alterations of signalling components involved in phagocytosis of apoptotic cells (Figures 7B–D). Specifically, compared with untreated worms, significant lower transcriptions were detected for ina-1 (encodes an engulfment receptor integrin-1 that functions upstream of CED-2/CED-5/CED-10/CED-12 apoptotic cell removal signalling pathway) (p < 0.001) (Figure 7C), and nrf-5 (encodes a secreted lipid-binding protein) (p < 0.01), ttr-52 (p < 0.001) and ced-6 (encodes CED-6, a key factor in CED-1/CED-6/CED-7 cell corpse engulfment pathway) (p < 0.001) in both Ce-ttr-31 and Hc-ttr-31 RNAi-treated C. elegans (Figure 7D). Differently, a significant higher transcription of ced-7 (encodes an ABC transporter that transfers phosphatidylserine from apoptotic cells to engulfing cells) (p < 0.05) was found in the treated worms (Figure 7C). Transcriptional changes of egl-1 (egg-laying defective-1), ced-9 (an orthologue of bcl-2), psr-1 (encodes a phosphatidylserine-recognizing receptor) and ced-1 (encodes a single-pass transmembrane protein that acts in engulfing cells to promote removal of apoptotic cells) were not consistent between Ce-ttr-31 and Hc-ttr-31 RNAi-treated worms (Figures 7B–D). In summary, ttr-31 is likely to regulate and recognise the apoptotic cells.