Table 1.
Sequences of TGF β type II receptors used for phylogenetic and alignment analysis.
Fig 1.
Alignments for Haemonchus contortus Hc-TGFBR2 with other homologues of TGF-β type II receptors.
Transmembrane domain is marked in blue, β strands are in red, α helices are marked in green and DFG motif is marked in yellow, the characteristic cluster (CXCX4C) of TGF-β type II receptors is boxed in red, the protein kinase domain is shown between the sequences marked with the two red stars below. In the protein kinase domain, the activation loop is boxed in blue, the conserved residues involved in forming the hydrophobic pocket are shown as red dots below the sequences and the conserved residues involved in forming water-bridged hydrogen bond are shown as red triangles below the sequences.
Fig 2.
Phylogenetic relationship of Haemonchus contortus Hc-TGFBR2 with the TGF-β type II receptors of eight nematodes and six metazoan, whose sequences are listed in Table 1.
A TGF-β type I receptor of Caenorhabditis elegans (GenBank accession no. CCD62175.1) was set as an outgroup. The Jones-Taylor-Thornton (JTT) model was used employing the MEGA program v.6.0. Nodal support values were shown above or below the branches.
Fig 3.
Gene structures of TGF-β type II receptor homologues from Haemonchus contortus (Hc-tgfbr2) and Caenorhabditis elegans (Ce-daf-4a, Ce-daf-4b, Ce-daf-4c, Ce-daf-4d and Ce-daf-4e).
Black boxes represent exons and the numbers above displayed the lengths of exons. Lines between the exons represent introns, and the numbers above indicate the lengths of introns.
Fig 4.
Transcriptional levels of Hc-tgfbr2 gene in eight developmental stages of Haemonchus contortus.
The relative abundance of Hc-tgfbr2 was assessed in eight developmental stages of H. contortus, including eggs (Egg), first-stage larvae (L1), second-stage larvae (L2), third-stage larvae (L3), females of fourth-stage larvae (L4F), males of fourth-stage larvae (L4M), adult females (AdF) and adult males (AdM). The relative quantities (compared with egg, egg = 1) are shown as mean values (± standard error of the mean, SEM). The significant differences between stages were indicated by different letters while one same letter present between stages indicated no difference. The different lowercase letter (a, b) means P < 0.05 while the same lowercase letters mean P > 0.05; the different capital letter (A, B) means P < 0.01 while the same capital letter indicates P > 0.01.
Fig 5.
The localization of Hc-TGFBR2 in Haemonchus contortus adults by immunohistochemistry.
(A-H) the localization of Hc-TGFBR2 in H. contortus adult females, (I-P) the localization of Hc-TGFBR2 in H. contortus adult males. The ‘positive’ serum was the serum from the final bleed after the last immunisation (containing the antibody against recombinant Hc-TGFBR2); the ‘negative’ serum was the serum from the pre-bleed before the first immunization with recombinant Hc-TGFBR2 (exposure time is 2500 ms). a: intestine; b: eggs within the uterus; c: ovaries; d: testes, scale-bar: 50 μm.
Fig 6.
Effects of Hc-tgfbr2 dsRNA-treatment on Haemonchus contortus development.
(A) Hc-tgfbr2 transcription in H. contortus following RNAi assessed by RT-PCR. (B) Larvae at the xL3 stage. The inset shows the enlarged view of the buccal region. (C) Larvae at the L4 stage. The inset shows the well-developed and functional mouth (buccal cavity) (D) The number of L4s that developed in vitro for another 7 days after RNAi. Scale-bar: 25 μm; * means P < 0.05; ** means P < 0.01 and *** means P < 0.001.