Fig 1.
First, different proglottids (scolex-neck-immature proglottids, mature proglottids, and gravid proglottids) were prepared for RNA extraction, cDNA library construction, and nanopore sequencing. The differentially expressed genes (DEGs) were subsequently screened and subjected to bioinformatics analysis, and the omics sequencing results were subsequently validated using qPCR. Finally, the pyruvate kinase gene family was selected for in-depth analysis, including analysis of gene family composition, evolutionary patterns, and temporal expression profiles. The molecular characteristics and functional mechanisms of representative family members were further characterized. Flow chart was created in BioRender. candy, c. (2025) https://BioRender.com/j04v286.
Table 1.
Statistics of full-length sequence data.
Fig 2.
GO and KEGG analysis of coexpressed differentially expressed genes among different segments of S. mansoni.
A. GO subcategories of coexpressed DEGs across three proglottids. B. Top 10 enriched terms of coexpressed DEGs in three proglottids across the BP, CC, and MF categories. C. KEGG classification diagram of coexpressed DEGs across three proglottids. D. Top 20 significantly enriched KEGG pathways of coexpressed DEGs across three proglottids.
Fig 3.
Analysis of the glycolysis pathway in SNIPs and MPs.
A. Fifteen DEGs involved in the glycolytic pathway were identified in the SNIPs and MPs. The numbers in boxes represent genes. Red symbols represent genes upregulated in MPs. Yellow symbols represent genes with mixed regulation. B. Gene set enrichment analysis of the glycolysis pathway in the SNIPs and MPs. A positive ES indicates significant enrichment of this pathway in MPs. C. Heatmap of 15 DEGs in the glycolysis pathway.
Fig 4.
Protein–protein interaction network analysis of metabolism-related DEGs.
A. PPI network of coexpressed DEGs across three proglottids. The blue nodes represent DEGs. B. PPI network of DEGs between SNIPs and MPs. C. PPI network of DEGs between MPs and GPs. D. PPI network of DEGs between SNIPs and GPs. Red and green nodes represent upregulated and downregulated genes, respectively.
Fig 5.
A and B represent the upregulated genes in SNIPs and MPs, respectively. C and D represent the upregulated genes in MPs and GPs, respectively. E and F represent upregulated genes in SNIPs and GPs, respectively. GAPDH was used for normalization. The results are presented as the means±SEMs (standard means of error) of the samples (n = 3).
Table 2.
Annotation characteristics of SmPKs.
Fig 6.
Analysis of the gene structure and expression patterns of SmPKs.
A. Scaffold-level localization: the green columns represent different scaffolds of S. erinaceieuropaei (GCA_902702965.1). B. Analysis of conserved domains: Red and blue rectangles represent conserved domains of pyruvate kinase and its subfamily, respectively. C. Intron‒exon structure: the red rounded rectangles represent exons; the black lines connecting two exons represent introns. D. Collinearity analysis of SmPKs: the grey shading indicates the gene density. E and F Collinearity analysis of the PKs among medically important cestodes. The grey shading indicates syntenic regions of S. mansoni with other medically important cestodes in non-PK gene families. (E) Collinearity analysis between S. mansoni and Echinococcus granulosus, Hydatigera taeniaeformis, and Taenia asiatica. (F) Collinearity analysis between S. mansoni and Dibothriocephalus latus and Mesocestoides corti. G. PK gene expression of S. mansoni at different stages determined via qRT‒PCR. SNIP: scolex-neck-immature proglottid; MP: mature proglottid; GP: gravid proglottid; Plero.Head: plerocercoid head; Plero.Body: plerocercoid body. GAPDH was used as an internal reference gene. The expression level was measured with the 2-∆∆CT method. The data were averaged from three repeats, and the error bars represent the SDs (n = 3).
Fig 7.
Phylogenetic analysis of pyruvate kinases in medically important cestodes and trematodes via the maximum likelihood method.
Nematodes were selected as the outgroup, and the values on the branches represent the bootstrap values.
Fig 8.
Molecular characterization of SmPK1.
A, B Transcription patterns of the PK gene in various developmental stages of S. mansoni, including eggs, plerocercoids, and adults. (A) Conventional RT‒PCR. (B) Real-time RT‒PCR. The housekeeping gene (GAPDH) was used as an internal reference. H2O was used as a negative control. plero.H.: plerocercoid head; plero.B.: plerocercoid body; SNIP: scolex-neck-immature proglottid; MP: mature proglottid; GP: gravid proglottid; Egg: eggs in the gravid proglottid uterus. C. Temperature gradient-induced recombinant protein expression. M: prestained protein marker; 1: uninduced control; 2: induced at 16 °C; 3: induced at 20 °C; 4: induced at 25 °C; 5: induced at 30 °C. D. IPTG concentration gradient-induced recombinant protein expression. M: prestained protein marker; 1: 0 mM IPTG; 2: 0.05 mM IPTG; 3: 0.1 mM IPTG; 4: 0.2 mM IPTG; 5: 0.4 mM IPTG; 6: 0.5 mM IPTG. E. Time course induction of recombinant protein expression. M: prestained protein marker; 1: uninduced control; 2: 11 h induction; 3: 12 h induction; 4: 13 h induction; 5: 14 h induction; 6: 15 h induction; 7: 16 h induction. F. Solubility analysis and purification of rSmPK1. M: Prestained protein marker; 1: uninduced bacterial cultures; 2: the lysate of the induced recombinant bacteria harbouring pQE-80 L-rSmPK1 after ultrasonication; 3: the protein in the supernatant; 4: the protein in the precipitate; 5: rSmPK1 purified by the Ni-NTA-Sefinose column. G. Determination of the optimal antigen coating concentration. H. Anti-rSmPK immunoserum potency assay. Green, orange, pink, blue, and purple represent serum dilutions of 1:10², 1:10³, 1:10⁴, 1:10⁵, and 1:10⁶, respectively. rSmPK1 antigenicity analysis. I. M: prestained protein marker; 1: rSmPK1 + anti-rSmPK1 serum. J. M: prestained protein marker; 1: rSmPK1 + infected mouse serum; 2: rSmPK1 + normal mouse serum.
Fig 9.
Immunofluorescence analysis of PK1 in various life cycle stages of Spirometra mansoni.
Head: head of the plerocercoid; Body: body of the plerocercoid; IMP: immature proglottid; MP: mature proglottid; GP: gravid proglottid; Eggs: eggs in the uterus of the mature proglottid. A. Normal serum; B. Infected serum; C. Anti-rSmPK serum. GPR, scale of head, body, IMP, MP and GP: 500 µm; uterus: 200 µm; eggs: 100 µm.
Fig 10.
Enzymatic activity analysis of rSmPK1 and evaluation of the metabolic inhibition effect of tannic acid on S. mansoni.
rSmPK1 was incubated with 2.5 mm PEP and 1.25 mm ADP for 10 min under various conditions. The optimal catalytic conditions for rSmPK1 were assessed with various rSmPK1 concentrations (2–24 ng/μL), temperatures (20–70 °C) and buffer solutions with different pH values (2–10). A. The optimum catalytic concentration of rSmPK1 is 20 ng/μl. B. The optimum catalytic temperature of rSmPK1 is 37 °C. C. The optimum catalytic pH of rSmPK1 is 8.0. D. Effects of different metal ions on rSmPK1 activity. K+ and Mg2+ clearly increase rSmPK1 activity. E. Inhibitory effects of tannic acid on rSmPK1 enzymatic activity. F. Standard curve of sodium pyruvate. pH 8.0 and 37 °C: Michaelis‒Menten curves and Lineweaver‒Burk plots of G. PEP and H. ADP. I. Negative control. J. Experimental group. K. Effect of tannic acid on the EA of S. mansoni. L. Effect of tannic acid on the PA content of S. mansoni. M. Effect of tannic acid on the TG content of S. mansoni. N. Effect of tannic acid on the TS content of S. mansoni. EA: enzyme activity; PA: pyruvic acid; TG: triglyceride; TS: total sugar; Statistical analysis was performed using SPSS 26 software. Statistical significance was determined by one-way analysis of variance (ANOVA), with data derived from three technical replicates. *p-value < 0.05 was considered to indicate statistical significance. The error bars represent the means ± standard deviations.