Identification of the Genes daf-12, sdc-2, and sex-1 Associated with Sexual Differentiation of Haemonchus contortus

1. Introduction

Haemonchosis is a parasitic disease caused by the blood-feeding nematode Haemonchus contortus (H. contortus), which primarily infects small ruminants such as sheep and goats. The economic burden of this parasite is considerable, as it reduces meat and milk production and compromises animal health and welfare [1,2].

Sexual reproduction is essential for population growth in most animals and depends on the generation of two distinct sexes. Sex determination usually occurs at early developmental stages and has profound consequences for development and behavior [3]. In insects, sex is determined by different chromosomal systems (e.g., XY/XX in flies and beetles, ZZ/ZW in butterflies and moths, and XO/XX or Z0/ZZ in some groups), while Hymenoptera use a haplodiploid system in which unfertilized eggs develop into haploid males and fertilized eggs into diploid females [4]. In Drosophila melanogaster, sex determination relies on activation of the sex-lethal gene rather than the presence of a Y chromosome. In mammals such as mice (Mus musculus), the testis-determining gene on the Y chromosome triggers testis formation and systemic hormonal regulation of development and behavior [4,5].

In nematodes, Genetic Sex Determination (GSD) occurs, where sex is defined by the presence or absence of sex chromosomes: females are homogametic XX and males heterogametic XO. Nematode species with chromosomal sex determination typically display a 1:1 sex ratio [6,7]. GSD begins with an initial signal based on the ratio of X chromosomes to autosomes (X:A). This signal initiates a cascade of interacting genes that regulate sexual identity and coordinate the development of male or female traits [8,9]. In addition to sex determination, this cascade also regulates X-chromosome dosage compensation [3].

The free-living nematode Caenorhabditis elegans (C. elegans) has been a fundamental model for studying sex determination. Its pathway is divided into two segments: an upstream branch (from X:A signal to sdc-2) that coordinates sex determination and dosage compensation, and a downstream branch (from her-1 to tra-1) that controls sexual fate [10,11]. Key regulators include xol-1, which promotes male development in XO animals, and sdc-2, which directs female/hermaphrodite fate in XX animals. Downstream, the fem genes (fem-1, fem-2, fem-3) promote tra-1 degradation to enable male development, while active tra-1 specifies female/hermaphrodite identity [3,8,12].

In H. contortus, sexual differentiation also follows an XX/XO system, as demonstrated by microsatellite markers. Males are consistently monomorphic, while females are diploid with expected heterozygosity, and polyandry has been reported, increasing offspring genetic variability [13,14]. Comparative analyses suggest that while some regulators are conserved between C. elegans and H. contortus, others differ. For example, fox-1 and sex-1 orthologs are present but autosomal, and regulators such as xol-1 and sdc-1, sdc-2, sdc-3 appear absent. Most downstream components, including orthologs of her-1 and fem-1/fem-2, are present, though tra-2 and fem-3 were not detected [15,16].

These differences suggest that although H. contortus shares the XX/XO system with C. elegans, its regulatory network is only partially conserved. The aim of this study is to identify and characterize orthologs of key sex-differentiation genes in H. contortus, in order to improve understanding of its reproductive biology and to identify potential molecular targets for novel parasite control strategies.

2. Materials and Methods

2.1. Obtaining H. Contortus L3

The L3 were obtained from the feces of Columbia breed sheep previously infected with a pure strain of H. contortus. This strain has been maintained through successive infections in animals free of other nematodes and was donated by the research group of Dr. Fernando Alba Hurtado from the Multidisciplinary Research Unit of FES-Cuautitlán.

2.2. Sucrose Washing and Recovery of H. Contortus L3

A sucrose solution was prepared by dissolving 20 g of sucrose in 200 mL of pre-heated distilled water. Ten milliliters of this solution were dispensed into four 15-mL conical tubes, and 5 mL of a suspension containing H. contortus L3 were added to each tube, reaching a final volume of 15 mL. The sucrose solution created a density gradient, allowing the larvae to remain suspended.

Tubes were centrifuged at 6,000 × g for 5 min, and the supernatant was decanted. One milliliter of the interphase from each tube was transferred to new 15-mL conical tubes, diluted with 14 mL of distilled water, and centrifuged again under the same conditions. This washing step was repeated three times. Larval pellets obtained after centrifugation were resuspended in 5 mL of 1X PBS and transferred to fresh conical tubes.

To prevent bacterial and fungal growth in the suspension containing H. contortus L3, a 20 µL antibiotic–antimycotic treatment was added, consisting of penicillin (10,000 U/mL), streptomycin (10,000 mg/mL), and amphotericin B (25 µg/mL). The treatment was applied for 24 h under refrigeration at 4 °C.

2.3. Exsheathment and Migration of H. Contortus L3

The conical tube containing H. contortus L3 was centrifuged at 6,000 × g for 5 min, and the supernatant was discarded. Ten milliliters of 1X PBS and 500 µL of sodium hypochlorite were added, followed by centrifugation at 6,000 × g for 5 min and decantation. The larvae were washed three times by adding 10 mL of distilled water, centrifuging under the same conditions, and discarding the supernatant.

A 50-mL conical tube was prepared with 45 mL of 1X PBS supplemented with 90 µL of an antibiotic–antimycotic mixture (penicillin, 10,000 U/mL; streptomycin, 10,000 mg/mL; amphotericin B, 25 µg/mL) and 14.4 µL of ceftriaxone. A mini-Baermann apparatus was assembled by cutting the bottom end of a 15-mL conical tube and placing it inside the 50-mL tube. The open end of the 15-mL tube was covered with a lens-cleaning tissue secured with a rubber band, serving as a filter. The 50-mL tube was then filled with 15 mL of 1X PBS containing antibiotics until the liquid reached the lower edge of the 15-mL tube in contact with the tissue [17].

Finally, the suspension of H. contortus L3 was placed inside the 15-mL tube, allowing the larvae to migrate through the tissue by hydrotaxy while shedding their cuticle. The mini-Baermann apparatus was incubated for 24 h at 37 °C.

2.4. Preservation of H. Contortus L3

The conical tube containing the suspension of H. contortus L3 under antibiotic–antimycotic treatment was removed from the incubator, adjusted to 14 mL with 1X PBS, centrifuged at 6,000 × g for 5 min, and the supernatant discarded. This step was repeated twice. Finally, 3 mL of Trizol (Cat. 15596026, Invitrogen) were added to the L3, the solution was homogenized, and aliquoted into three 1.5-mL microtubes (1 mL each), which were stored at –70 °C until further use.

2.5. Collection of H. Contortus Adult Males and Females

Adult male and female H. contortus were kindly provided by the Helminthology Unit, headed by Dr. María Eugenia López Arellano at CENID-SAI. Parasites were collected directly from the abomasum during necropsies of sheep experimentally infected with H. contortus, which had not been exposed to other pathogens or anthelmintic treatments. These animals belonged to the production unit of CENID-SAI. The collected specimens were washed with 1X PBS and placed in Trizol and stored at –70 °C until use.

2.6. Identification of Genes Associated with Sexual Differentiation in C. elegans

An in-silico search was performed across multiple databases (ScienceDirect, Web of Science, PubMed, Scopus, Wiley, and Bidi-UNAM) using keywords such as “C. elegans,” “sex differentiation,” “sex determination,” and “sex-specific gene expression.” Articles from the last 10 years were prioritized, but earlier studies were included if relevant. Selected studies were evaluated for information on gene function, regulation, and expression related to sexual differentiation.

2.7. Identification of Gene Sequences Related to Sexual Differentiation in the H. contortus Genome

A bioinformatic analysis was performed using C. elegans genes as references. Amino acid sequences were aligned via BLASTp in NCBI, Swiss-Prot, and KEGG databases to identify orthologs in H. contortus. Nucleotide sequences were obtained or inferred from amino acid sequences when unavailable. Genes were selected based on sequence availability and functional relevance. The constitutive gene sod was used as a reference to monitor amplification and normalize results.

2.8. Primer Design for Selected Gene Sequences

Oligonucleotides were designed using the DNA sequences obtained from the conversion of amino acid sequences to nucleotide sequences via the Sequence Manipulation Suite. Complementary strands were generated in the 5’–3’ direction. Oligonucleotide length was optimized to 17–20 base pairs (Table 1). The melting temperature (Tm) was calculated to ensure specific annealing to the template DNA, with an ideal range of 55–65 °C. Terminal complementarity was checked to prevent self- or cross-dimer formation. Designed oligonucleotides were synthesized at the Institute of Biotechnology, UNAM.

2.9. Genomic DNA Extraction

Genomic DNA (gDNA) was extracted from a sample of 1000 exsheathed L3 of H. contortus suspended in 500 µL of 1X PBS. The sample was subjected to lysis by thermal shock in a dry bath at 65 °C for 5 min, followed by mechanical maceration and disruption with a sterile pestle, and immersion in liquid nitrogen for 5 min. This cycle was repeated three times. DNA was then purified using the FavorPrep™ Tissue Genomic DNA Extraction kit (Favorgen®).

gDNAquantification was performed using a Nabi-UV/Vis nanophotometer. RNA integrity was assessed by electrophoresis on 2% agarose gels using 6X TriTack DNA Load-ing Dye (Thermo Scientific®). Gels were documented with the EpiChemi3 Darkroom system and ToupView software.

2.10. RNA Extraction

Total RNA was extracted from a sample of 1000 exsheathed L3 and 10 adult males and females of H. contortus suspended in 500 µL of Trizol. Each sample was subjected to lysis by thermal shock in a dry bath at 65 °C for 5 min, followed by mechanical maceration and disruption with a sterile pestle, and immersion in liquid nitrogen for 5 min. This cycle was repeated three times. RNA was then purified using the Direct-zol™ RNA Miniprep Plus kit (Zymo Research®).

RNA quantification was performed using a Nabi-UV/Vis nanophotometer. RNA integrity was assessed by electrophoresis on 2% agarose gels using 6X TriTack DNA Loading Dye (Thermo Scientific®). Gels were documented with the EpiChemi3 Darkroom system and ToupView software.

2.11. PCR

To confirm the presence of genes associated with sexual differentiation in the H. contortus genome, endpoint PCR was performed using the GoTaq® Green Master Mix 2X kit (Promega). The reaction was carried out with previously extracted gDNA, and primers specifically designed for this analysis.

PCR products were resolved on 2% agarose gels stained with ethidium bromide and compared with a 50 bp molecular marker (Jena Bioscience®). Gels were documented with the EpiChemi3 Darkroom system, and amplicon sizes were estimated using a calibration curve based on marker migration distances.

2.12. RT-PCR

Total RNA previously extracted from L3 and adult males and females was used as a template. RT-PCR reactions were carried out with the GoScript™ Reverse Transcription System kit (Promega®), following the manufacturer’s instructions.

RT-PCR products were resolved on 2% agarose gels stained with ethidium bromide and compared with a 50 bp molecular marker (Jena Bioscience®). Band intensities were quantified using ImageJ, averaged from three replicates, and normalized to the constitutive control (100%).

2.13. Statycal Analysis

Statistical analysis was performed using one-way analysis of variance (ANOVA) on optical densitometry values expressed as relative percentages. Analyses were conducted using GraphPad Prism 9 software, and differences were considered statistically significant at p ≤ 0.05.

2.14. Modeling of the daf-12, fem-1, and sdc-2 Gene Structures

3D models of daf-12, fem-1, and sdc-2 were generated with AlphaFold and validated using SAVES. Stereochemical quality was assessed with PROCHECK Ramachandran plots and ERRAT, yielding Overall Quality Factors comparable to experimentally resolved proteins.

2.15. Phylogenetic Analysis

Phylogenetic trees were constructed using amino acid sequences of DAF-12, FEM-1, and SDC-2 obtained from NCBI and Swiss-Prot (Appendix A). Sequences with high identity and conserved domains were aligned with ClustalW (MEGA 12), and a Neighbor-Joining tree was generated with 1500 Bootstrap replicates.

3. Results

3.1. Determination of the Presence of Genes Related to Sexual Differentiation in the H. contortus Genome

Three amplicons were detected: daf-12 (660 bp), sdc-2 (469 bp), and fem-1 (154 bp) (Figure 1).

3.1.1. daf-12

For daf-12, amplicons of 61 bp were detected, compared with the constitutive sod gene (36 bp). Optical densitometric analysis revealed relative expression levels of 167% in L3, 117% in males, and 101% in females of H. contortus. One-way ANOVA indicated statistically significant differences between L3–males (p ≤ 0.01) and L3–females (p ≤ 0.001) (Figure 2).

3.1.2. fem-1

For fem-1 amplicons of 81 bp were observed in comparison with the constitutive gene sod (36 bp). Optical densitometric analysis revealed relative expression levels of 57% in L3, 140% in males, and 48% in females of H. contortus. Statistical analysis using ANOVA showed significant differences between L3–males (p ≤ 0.001) and males–females (p ≤ 0.0001) (Figure 3).

3.1.3. sdc-2

Amplicons of 74 bp were observed in comparison with the constitutive gene sod (36 bp). Optical densitometric analysis revealed relative expression levels of 72% in L3, 56% in males, and 127% in females of H. contortus. Statistical analysis using ANOVA showed significant differences between L3–females (p ≤ 0.05) and males–females (p ≤ 0.01) (Figure 4).

3.2. Bioinformatic Analysis

3.2.1. 3D Modeling of daf-12

The daf-12 sequence was analyzed in silico and found to encode DAF-12, a protein composed of 686 amino acids (NCBI: QEA03487.1) containing a characteristic nuclear hormone receptor domain. Visual inspection of the 3D model of DAF-12 predicted by AlphaFold revealed a well-defined core composed of α-helices, represented in navy blue, indicating high confidence in the structural prediction for this region (pLDDT > 90). In contrast, orange-colored regions were observed at the termini of the protein, indicating lower confidence (pLDDT < 50) and likely corresponding to flexible or disordered regions (Figure 5a). The 3D model of DAF-12 was further validated using SAVES with the ERRAT and PROCHECK tools. ERRAT generated an overall quality factor of 94.76%, while PROCHECK provided a stereochemical assessment through a Ramachandran plot, which evaluates the orientation of phi (φ) and psi (ψ) angles of amino acids to determine the proper folding and structural stability of the model. The analysis showed that 81.1% of the residues were in the most favored regions of the plot, 9.8% in additionally allowed regions, 3.8% in generously allowed regions, and 5.8% in disallowed regions (Figure 5b).

3.2.2. 3D Modeling of fem-1

The fem-1 sequence was analyzed in silico and found to encode FEM-1, a protein composed of 652 amino acids (NCBI: XGW30565.1) containing an ankyrin repeat domain. Visual inspection of the 3D model of FEM-1 predicted by AlphaFold revealed predominantly α-helices organized in a repetitive and compact pattern, represented in navy blue (pLDDT > 90) and light blue (pLDDT = 90–70), indicating high confidence in the structural prediction for this region. At the protein termini, yellow and orange regions were observed, corresponding to low-confidence predictions (pLDDT < 50) that likely represent flexible segments (Figure 6a). The 3D model of FEM-1 was validated using SAVES with the ERRAT and PROCHECK tools. ERRAT generated an overall quality factor of 76.79%, while PROCHECK provided a stereochemical evaluation through a Ramachandran plot. The analysis showed that 82.4% of residues were in the most favored regions and 17% in additionally allowed regions, with no residues found in generously allowed or disallowed regions (Figure 6b).

3.2.3. 3D Modeling of sdc-2

The sdc-2 sequence was analyzed in silico and found to encode SDC-2, a protein composed of 486 amino acids (NCBI: CDJ84099.1). Visual inspection of the 3D model of SDC-2 predicted by AlphaFold revealed α-helices, some represented in navy blue (pLDDT > 90) and others in light blue (pLDDT = 90–70), indicating high and moderate confidence in the structural prediction. At the protein termini, yellow (pLDDT > 50) and orange (pLDDT < 50) regions were observed, corresponding to low-confidence predictions that are likely to represent flexible regions (Figure 7a). The 3D model of SDC-2 was validated using SAVES with the ERRAT and PROCHECK tools. ERRAT generated an overall quality factor of 86.24%, while PROCHECK provided a stereochemical evaluation through a Ramachandran plot. The analysis showed that 74.8% of residues were located in the most favored regions, 24.5% in additionally allowed regions, 0.5% in generously allowed regions, and 0.2% in disallowed regions (Figure 7b).

3.3. Phylogenetic Analysis

Phylogenetic trees were reconstructed for daf-12, fem-1, and sdc-2 genes using representative protein sequences from invertebrates (insects, arthropods, nematodes, cestodes, trematodes) and vertebrates (reptiles, birds, fish, mammals) available in GenBank and Swiss-Prot. Analyses were performed using the Neighbor-joining method with 1500 bootstrap (BS) replicates.

For daf-12, the tree showed a clear separation among major taxonomic groups, with well-supported clades for insects, arthropods, nematodes, trematodes, cestodes, fish, amphibians, and mammals. H. contortus clustered within the nematode clade together with C. elegans (BS = 100), Oesophagostomum dentatum (BS = 96), and Toxocara canis (BS = 100), indicating conservation of daf-12 among free-living and parasitic nematodes (Figure 8). Lower similarity was observed between nematodes and vertebrates, with BS = 78 for Ovis aries and Capra hircus, the definitive hosts of H. contortus (Figure 8). These results suggest that daf-12 functions in developmental regulation are conserved across taxa.

For fem-1, a similar pattern was observed, with well-defined clades for all major groups. H. contortus grouped with C. elegans (BS = 100), Ancylostoma caninum (BS = 100), and Toxocara canis (BS = 100), confirming conservation of fem-1 in sexual differentiation processes of free-living and parasitic nematodes (Figure 9). In vertebrates, mammalian sequences were strongly supported (BS = 100 for Ovis aries and Capra hircus), suggesting possible roles of fem-1 in the parasite–host interaction (Figure 9).

For sdc-2, the phylogenetic tree also revealed distinct clades for arthropods, nematodes, trematodes, fish, reptiles, birds, and mammals. H. contortus clustered with C. elegans (BS = 92), supporting functional conservation of sdc-2 in developmental regulation, including X-chromosome dosage compensation during sexual differentiation (Figure 10). However, moderate support was obtained for Trichinella spiralis (BS = 56), indicating incomplete conservation of sdc-2 among nematodes. In vertebrates, mammalian sequences showed high support, particularly Ovis aries (BS = 96) (Figure 10).

4. Discussion

Haemonchosis caused by H. contortus is a globally recognized disease due to its pathogenicity in sheep and goats, although it has also been documented in other ruminants such as cattle, deer, antelopes, and camelids. This parasite represents a major challenge to the livestock industry because of the severe economic losses it causes. It is estimated that H. contortus is responsible for global losses exceeding USD 1.3 billion, associated with decreased animal productivity (e.g., weight gain, meat and milk production), management costs, anthelmintic treatments, and mortality [18]. Control currently relies mainly on the use of anthelmintics; however, their intensive and indiscriminate use has resulted in the widespread emergence of resistance among gastrointestinal nematode populations [19]. This situation highlights the urgent need for alternative, sustainable, and specific control strategies that do not rely solely on anthelmintics. A deeper understanding of the biology of H. contortus, particularly essential processes such as development, reproduction, and sexual differentiation, may offer new opportunities for control. Sexual differentiation is of particular interest given the parasite’s life cycle and infective capacity: adult females can produce 1,000–5,000 eggs per day, which are shed into the environment through feces, contaminating pastures and perpetuating infection [20]. Understanding the mechanisms underlying sexual differentiation could therefore provide strategies to interfere with reproduction and persistence of the parasite in animal production systems.

In free-living nematodes such as C. elegans, several genes have been shown to play key roles in sexual differentiation, acting within highly conserved regulatory cascades [21]. However, in H. contortus these genes remain poorly characterized, and their functions in the dynamics of parasitism are largely unknown. Characterizing the presence, expression, and structure of genes associated with sexual differentiation—such as daf-12, fem-1, and sdc-2—provides valuable insights not only into the basic biology of H. contortus, but also into the identification of potential molecular targets for novel antiparasitic therapies or reproductive control strategies.

In C. elegans, daf-12 encodes a nuclear receptor belonging to the steroid hormone receptor family. It plays a central role in the dafachronic acid-mediated signaling pathway, regulating crucial developmental processes such as diapause, reproductive transitions, and longevity [22,23]. Orthologous sequences identified in H. contortus revealed conserved functional domains, particularly the ligand-binding domain (LBD) and the DNA-binding domain (DBD) [24], suggesting functional conservation despite parasitic adaptations. Molecular analyses confirmed the presence and expression of daf-12. Amplicons of different sizes were obtained: 660 bp from genomic DNA and 61 bp from cDNA, differences consistent with intron–exon organization. Relative expression analysis revealed high expression in infective L3 (67%), but markedly lower levels in adult males (17%) and females (1%). This stage-dependent regulation aligns with the infective role of L3 and with observations in C. elegans, where daf-12 controls developmental arrest and latency exit [22,25]. Structural modeling using AlphaFold revealed a compact α-helical core typical of nuclear receptors, validated by SAVES with an Overall Quality Factor of 94.76% and favorable stereochemical parameters. Phylogenetic analysis further confirmed strong clustering of H. contortus with other nematodes, supported by high bootstrap values, while vertebrate orthologs diverged functionally, including homologs such as the vitamin D receptor and bile acid receptor [26,27]. Collectively, these findings highlight daf-12 as a key regulator of development and sexual differentiation in H. contortus and a potential molecular target for novel control strategies.

In C. elegans, fem-1 is essential for male sexual determination. It promotes degradation of the transcription factor TRA-1 via the ubiquitin–proteasome pathway in association with FEM-2 and FEM-3, thereby enabling male development and spermatogenesis [28,29]. The ortholog identified in H. contortus exhibited strong similarity with C. elegans FEM-1, particularly within ankyrin repeat domains, key for protein–protein interactions [30]. PCR confirmed both presence and expression of fem-1, with amplicons of 154 bp from genomic DNA and 81 bp from cDNA, again reflecting intron–exon structure. Expression analysis revealed sex- and stage-dependent patterns: subexpression in L3 (43%), higher expression in adult females (52%), and a relative overexpression in adult males (40%). This expression profile is consistent with a conserved role in male sexual differentiation, as reported in C. elegans. Structural predictions revealed an ankyrin repeat-rich α-helical protein with high-confidence regions, validated by SAVES (Overall Quality Factor 76.79%) and PROCHECK stereochemistry analyses. Phylogenetic analysis placed H. contortus within a well-supported nematode clade alongside C. elegans and other parasites, indicating evolutionary conservation of fem-1 functions. In vertebrates, orthologs were more divergent and associated with broader roles such as apoptosis and neuronal development [31]. Overall, fem-1 appears to play a conserved role in male differentiation and reproductive processes in nematodes, making it a promising target for antiparasitic interventions.

In the case of sdc-2 in C. elegans has dual functions in female sex determination and dosage compensation in XX individuals, initiating assembly of the dosage compensation complex (DCC) and repressing her-1 to promote female development [32,33]. In H. contortus, an ortholog was identified, suggesting partial conservation of dosage compensation and sexual regulation mechanisms. PCR confirmed presence and expression of sdc-2, with amplicons of 469 bp from genomic DNA and 74 bp from cDNA, consistent with intron–exon differences. Expression analysis revealed low levels in L3 (28%) and males (44%), but significant overexpression in females (27%), in agreement with its role in sex determination and X-chromosome regulation. Structural modeling predicted an α-helical protein core with flexible terminal regions, validated by SAVES (Overall Quality Factor 86.24%) and PROCHECK analysis, indicating a reliable structural model. Phylogenetic analysis grouped H. contortus with C. elegans and Trichinella spiralis within a conserved nematode clade, while arthropods, trematodes, and vertebrates formed distinct clades. Notably, vertebrate orthologs showed divergent functions, including roles in transcriptional regulation, angiogenesis, and even tumor invasiveness [34,35]. These results suggest that while sdc-2 retains core regulatory functions in nematodes, its role has diversified in vertebrates.

Together, these findings confirm that daf-12, fem-1, and sdc-2 play crucial and conserved roles in the sexual differentiation and development of H. contortus. Their evolutionary conservation among nematodes, combined with structural and expression analyses, underscores their importance in reproductive regulation. Importantly, these genes represent promising molecular targets for developing novel strategies to disrupt parasite reproduction and control haemonchosis in livestock.

5. Conclusions

This study confirmed the presence and expression of daf-12, fem-1, and sdc-2 in H. contortus, with stage- and sex-specific expression patterns consistent with their described roles in C. elegans. Structural modeling and validation confirmed conserved functional domains, while phylogenetic analyses revealed strong evolutionary conservation among nematodes and divergence from other phyla. These findings underscore the importance of these genes in the development and reproduction of H. contortus and support their potential as molecular targets for novel antiparasitic strategies.

Appendix A.1. Accession Data of the Sequences Used for the Phylogenetic Analysis of DAF-12

Accession number	Species	Protein	Data base
XP 0539946974.1	Anastrepha ludens	Nuclear hormone receptor HR96	NCBI
NP 524493.1	Drosophila melanogaster	Hormone receptor-like in 96	NCBI
XP 037952769.1	Teleopsis dalmanni	Nuclear hormone receptor HR96-like	NCBI
XP 030369834.1	Scaptodrosophila lebanonensis	Nuclear hormone receptor HR96 isoform X1	NCBI
KAH8407185.1	Zaprionus bogoriensis	Nuclear hormone receptor HR96 isoform X1	NCBI
XP 013110462.2	Stomoxys calcitrans	Nuclear hormone receptor HR96	NCBI
AEC03603.1	Musca domestica	Nuclear receptor HR96	NCBI
XP 065371768.1	Calliphora vicina	Nuclear hormone receptor HR96	NCBI
KNC27967.1	Lucilia cuprina	Nuclear hormone receptor HR96	NCBI
XP 001981892.1	Drosophila erecta	Nuclear hormone receptor HR96	NCBI
XP 016036188.1	Drosophila simulans	Nuclear hormone receptor HR96	NCBI
XP 033163627.1	Drosophila mauritiana	Nuclear hormone receptor HR96	NCBI
XP 037570853.1	Dermacentor silvarum	Vitamin D3 receptor	NCBI
XP 037523479.1	Rhipicephalus sanguineus	Vitamin D3 receptor isoform X1	NCBI
XP 037286542.1	Rhipicephalus microplus	Vitamin D3 receptor-like	NCBI
XP 003748267.1	Galendromus occidentalis	Vitamin D3 receptor	NCBI
KAK5968683.1	Trichostrongylus colubriformis	Nuclear receptor subfamily 6 group A member 1	NCBI
XSG12237.1	Toxocara canis	Nuclear hormone receptor DAF-12, partial	NCBI
ALF38191.1	Anisakis simplex	Nuclear hormone receptor, partial	NCBI
KHJ95871.1	Oesophagostomum dentatum	Ligand-binding domain of nuclear hormone receptor, partial	NCBI
NP 001041239.1	Caenorhabditis elegans	Nuclear hormone receptor family member daf-12	NCBI
KJH46651.1	Dictyocaulus viviparus	Zinc finger, C4 type	NCBI
KAK6030423.1	Ostertagia ostertagi	Zinc finger, C4 type	NCBI
QEA03487.1	Haemonchus contortus	Nuclear hormone receptor DAF-12	NCBI
KAG5443350.1	Clonorchis sinensis	Nuclear hormone receptor member daf-12	NCBI
XP 035587464.2	Schistosoma haematobium	Nuclear hormone receptor, partial	NCBI
TPP66369.1	Fasciola gigantica	Nuclear hormone receptor HR96	NCBI
THD27323.1	Fasciola hepatica	Nuclear hormone receptor HR96	NCBI
KAF8568779.1	Paragonimus westermani	Nuclear hormone receptor, partial	NCBI
KAH9283862.1	Echinococcus granulosus	Nuclear hormone receptor family member daf-12	NCBI
KAL5964423.1	Taenia solium	Nuclear hormone receptor family member daf-12	NCBI
CDS25950.1	Hymenolepis microstoma	Nuclear hormone receptor HR96	NCBI
CDS43059.1	Echinococcus multilocularis	Nuclear hormone receptor HR96	NCBI
XP 040281812.1	Bufo bufo	Vitamin D3 receptor isoform X1	NCBI
XP 040196717.1	Rana temporaria	Vitamin D3 receptor isoform X1	NCBI
XP 002935703.1	Xenopus tropicalis	Vitamin D3 receptor	NCBI
XP 044143545.1	Bufo gargarizans	Vitamin D3 receptor	NCBI
XP 033795349.1	Geotrypetes seraphini	Vitamin D3 receptor	NCBI
XP 069509284.1	Ambystoma mexicanum	Vitamin D3 receptor isoform X1	NCBI
XP 072262732.1	Pyxicephalus adspersus	Vitamin D3 receptor	NCBI
XP 030054021.1	Microcaecilia unicolor	Vitamin D3 receptor	NCBI
XP 029450950.1	Rhinatrema bivittatum	Vitamin D3 receptor isoform X1	NCBI
XP 007240874.1	Astyanax mexicanus	Vitamin D3 receptor A	NCBI
XP 026775925.1	Pangasianodon hypophthalmus	Vitamin D3 receptor A	NCBI
NP 570994.1	Danio rerio	Vitamin D3 receptor A	NCBI
XP 028855014.1	Denticeps clupeoides	Vitamin D3 receptor	NCBI
XP 014868079.1	Poecilia mexicana	Vitamin D3 receptor	NCBI
XP 020468294.1	Monopterus albus	Vitamin D3 receptor	NCBI
NP 001121989.1	Oryzias latipes	Vitamin D3 receptor	NCBI
W5PWT0	Ovis aries	Bile acid receptor	Swiss-Prot
Q96RI1	Homo sapiens	Bile acid receptor	Swiss-Prot
Q60641	Mus musculus	Bile acid receptor	Swiss-Prot
Q3SZL0	Bos taurus	Bile acid receptor	Swiss-Prot
A0A8D0VZF6	Sus scrofa	Nuclear receptor subfamily 1 group H member 4	Swiss-Prot
A0A452FGV9	Capra hircus	Nuclear receptor subfamily 1 group H member 4	Swiss-Prot
A0A0G2K6D2	Rattus norvegicus	Nuclear receptor subfamily 1, group H, member 4	Swiss-Prot

Appendix A.2. Accession Data of the Sequences Used for the Phylogenetic Analysis of FEM-1

Accession number	Species	Protein	Data base
NP 611508.1	Drosophila melanogaster	Fem-1, isoform A	NCBI
XP 047115103.1	Schistocerca piceifrons	Protein fem-1 homolog B	NCBI
XP 063885865.1	Scylla paramamosain	Protein fem-1 homolog B-like isoform X1	NCBI
XP 001654238.1	Aedes aegypti	Protein fem-1 homolog B-like isoform X1	NCBI
XP 023933934.1	Bicyclus anynana	Protein fem-1 homolog B-like isoform X1	NCBI
XP 011202197.2	Bactrocera dorsalis	Protein fem-1 homolog B-like isoform X1	NCBI
XP 002138527.2	Drosophila pseudoobscura	Protein fem-1 homolog B-like isoform X1	NCBI
XP 050712882.1	Eriocheir sinensis	Protein fem-1 homolog B-like isoform X1	NCBI
XP 036330667.1	Rhagoletis pomonella	Protein fem-1 homolog B-like isoform X1	NCBI
XP 031341515.1	Photinus pyralis	Protein fem-1 homolog B	NCBI
XP 061507539.1	Anopheles gambiae	Protein fem-1 homolog B	NCBI
XP 022128557.1	Pieris rapae	Protein fem-1 homolog B-like isoform X1	NCBI
XP 019768800.1	Dendroctonus ponderosae	Protein fem-1 homolog B-like isoform X1	NCBI
XP 023012557.1	Leptinotarsa decemlineata	Protein fem-1 homolog B-like isoform X1	NCBI
XP 049701068.1	Helicoverpa armigera	Protein fem-1 homolog B-like isoform X1	NCBI
XP 068896437.1	Tenebrio molitor	Protein fem-1 homolog B-like isoform X1	NCBI
XP 050044960.1	Dermacentor andersoni	Protein fem-1 homolog B	NCBI
XP 037563812.1	Dermacentor silvarum	Protein fem-1 homolog B	NCBI
XP 037511638.1	Rhipicephalus sanguineus	Protein fem-1 homolog B	NCBI
XP 037291813.1	Rhipicephalus microplus	Protein fem-1 homolog B-like	NCBI
XP 022670278.1	Varroa destructor	Protein fem-1 homolog B-like isoform X1	NCBI
XGW30566.1	Haemonchus contortus	Protein fem-1 homolog B	NCBI
KAK5970382.1	Trichostrongylus colubriformis	Sex-determining protein fem-1	NCBI
RCN43014.1	Ancylostoma caninum	Ankyrin repeat protein	NCBI
KJH51017.1	Dictyocaulus viviparus	Ankyrin repeat protein	NCBI
KAK6025858.1	Ostertagia ostertagi	Ankyrin repeat protein	NCBI
KHN80228.1	Toxocara canis	Sex-determining protein fem-1	NCBI
PIO65957.1	Teladorsagia circumcincta	Ankyrin repeat protein	NCBI
MCP9265608.1	Dirofilaria immitis	Protein fem-1	NCBI
NP 500824.1	Caenorhabditis elegans	Sex-determining protein fem-1	NCBI
THD25860.1	Fasciola hepatica	Protein fem-1	NCBI
KAA0198803.1	Fasciolopsis buskii	Fem-1 A	NCBI
TPP64208.1	Fasciola gigantica	Protein fem-1	NCBI
KAG5441328.1	Clonorchis sinensis	Protein fem-1 C	NCBI
TNN08969.1	Schistosoma japonicum	Protein fem-1	NCBI
XP 035585568.1	Schistosoma haematobium	Protein fem-1 C	NCBI
XP 018647049.1	Schistosoma mansoni	Sex-determining protein fem-1	NCBI
KAH9279482.1	Echinococcus granulosus	Protein fem-1 -like protein C	NCBI
CDS43292.1	Echinococcus multilocularis	Sex-determining protein fem-1	NCBI
KAL5961090.1	Taenia solium	Sex-determining protein fem-1	NCBI
CDS26165.1	Hymenolepis microstoma	Sex-determining protein fem-1	NCBI
XP 005722081.1	Pundamilia nyererei	Protein fem-1 homolog C	NCBI
XP 003446110.1	Oreochromis niloticus	Protein fem-1 homolog C	NCBI
XP 006789826.1	Neolamprologus brichardi	Protein fem-1 homolog C	NCBI
XP 015828271.1	Nothobranchius furzeri	Protein fem-1 homolog C	NCBI
XP 013870593.1	Austrofundulus limnaeus	Protein fem-1 homolog C-like	NCBI
XP 064901447.1	Columba livia	Protein fem-1 homolog C isoform X1	NCBI
XP 065717267.1	Patagioenas fasciata	Protein fem-1 homolog C	NCBI
XP 065513711.1	Caloenas nicobarica	Protein fem-1 homolog C	NCBI
NXK13339.1	Herpetotheres cachinnans	FEM1C protein	Swiss-Prot
XP 072716475.1	Ciconia boyciana	Protein fem-1 homolog C	NCBI
NXV77506.1	Atlantisia rogersi	FEM1C protein, partial	NCBI
XP 027827254.2	Ovis aries	Protein fem-1 homolog C	NCBI
XP 017909215.1	Capra hircus	Protein fem-1 homolog C isoform X1	NCBI
P0C6P7	Rattus norvegicus	Protein fem-1 homolog B	Swiss-Prot
A0A2I3SXM4	Pan troglodytes	Fem-1 homolog B	Swiss-Prot
G7MY25_MACMU	Macaca mulatta	FEM1B	Swiss-Prot
I3L6T4	Sus scrofa	Fem-1 homolog B	Swiss-Prot
A0A8C0K439	Canis lupus dingo	Fem-1 homolog B	NCBI
A0A8C9P293_SPEDA	Spermophilus dauricus	Fem-1 homolog B	Swiss-Prot
NP 001039691.1	Bos taurus	Protein fem-1 homolog A	NCBI
Q9Z2G0	Mus musculus	Protein fem-1 homolog B	Swiss-Prot
Q9UK73	Homo sapiens	Protein fem-1 homolog B	Swiss-Prot

Appendix A.3. Accession Data of the Sequences Used for the Phylogenetic Analysis of SDC-2

Accession number	Species	Protein	Data base
XP 050040600.1	Dermacentor andersoni	Syndecan 3	NCBI
XP 037507833.1	Rhipicephalus sanguineus	Syndecan-like	NCBI
XP 037507832.1	Rhipicephalus sanguineus	Syndecan	NCBI
XP 037570033.1	Dermacentor silvarum	Syndecan-like	NCBI
XP 065291937.1	Dermacentor albipictus	Syndecan-like	NCBI
CDJ84099.1	Haemonchus contortus	CBN-SDC-2 protein	NCBI
NP 509924.1	Caenorhabditis elegans	Sex determination and dosage compensation protein sdc-2	NCBI
XP 003379719.1	Trichinella spiralis	Syndecan-2	NCBI
CAH8611371.1	Schistosoma bovis	Syndecan	NCBI
KAH8873224.1	Schistosoma japonicum	Syntenin-2 (Syndecan-binding protein 2)	NCBI
XP 018653325.1	Schistosoma mansoni	Putative syntenin-2 (Syndecan-binding protein 2)	NCBI
CAH8865890.1	Trichobilharzia regenti	Syndecan	NCBI
XP 062265467.1	Platichthys flesus	Syndecan-2	NCBI
XP 036793385.1	Oncorhynchus mykiss	Syndecan-2 isoform X1	NCBI
XP 067257318.1	Chanodichthys erythropterus	Syndecan-2-A-like isoform X1	NCBI
XP 053302077.1	Pleuronectes platessa	Syndecan-2	NCBI
XP 060948873.1	Limanda limanda	Syndecan-2	NCBI
XP 034455044.1	Hippoglossus hippoglossus	Syndecan-2	NCBI
XP 028255454.1	Parambassis ranga	Syndecan-2-A-like	NCBI
XP 037748165.1	Chelonia mydas	Syndecan-2-A-like isoform X1	NCBI
XP 038247599.1	Dermochelys coriacea	Syndecan-2-A-like isoform X2	NCBI
XP 044859986.1	Mauremys mutica	Syndecan-2	NCBI
XP 053874007.1	Malaclemys terrapin pileata	Syndecan-2	NCBI
XP 039382872.1	Mauremys reevessi	Syndecan-2	NCBI
NP 001001462.2	Gallus gallus	Syndecan-2 precursor	NCBI
XP 015711411.1	Coturnix japonica	Syndecan-2	NCBI
XP 021243681.1	Numida meleagris	Syndecan-2	NCBI
NP 001290149.1	Meleagris gallopavo	Syndecan-2 precursor	NCBI
XP 005042472.1	Ficedula albicollis	Syndecan-2	NCBI
XP 042665043.1	Centrocercus urophasianus	Syndecan-2	NCBI
XP 065609569.1	Cyrtonyx montezumae	Syndecan-2	NCBI
XP 014953400.1	Ovis aries	Syndecan-2-A-like isoform X1	NCBI
NP 001029960.2	Bos taurus	Syndecan-2 precursor	NCBI
NP 032330.1	Mus musculus	Syndecan-2 precursor	NCBI
NP 037214.1	Rattus norvegicus	Syndecan-2 precursor	NCBI
NP 001247893.1	Macaca mulatta	Syndecan-2 precursor	NCBI
NP 001302683.1	Sus scrofa	Syndecan-2 precursor	NCBI
NP 002989.2	Homo sapiens	Syndecan-2 precursor	NCBI