Fig 1.
Sequence features and expression pattern of 12 far genes in N. brasiliensis.
(A) Location of far genes on the chromosome of N. brasiliensis. Detailed location information was shown in S1 Table. (B) Sequence identity analysis of 12 FAR proteins in N. brasiliensis. (C) Maximum likelihood protein phylogenetic tree of 12 FAR proteins from N. brasiliensis. Bootstrap values were shown in the nodes. The scale bar represents the number of amino acid substitutions per site. (D) Expression pattern of far genes across developmental stages of N. brasiliensis using RNA-seq data. Detailed expression values were shown in S2 Table. (E) Expression pattern of the Nb-far-1 gene across developmental stages of N. brasiliensis by qPCR (n = 3).
Fig 2.
Analysis of the binding activities of Nb-FAR-1 protein with different fatty acids and retinol.
(A) Prokaryotic expression and purification of recombinant Nb-FAR-1 protein. (B) Change in relative fluorescence intensity of DAUDA (10 μM) in the presence of increasing concentrations of Nb-FAR-1 protein. The best fit curve was used to determine the equilibrium dissociation constant (Kd) for the DAUDA: Nb-FAR-1 interaction. (C) Similar analysis of binding affinity and Kd calculation for the retinol: Nb-FAR-1 interaction. (D) Competition binding effect of a 5-fold excess of unlabeled fatty acids with different carbon chain lengths and number of double bonds on the fluorescence intensity of DAUDA-Nb-FAR-1 complex (measured at 380~700 nm). * p <0.0001 means difference in mean fluorescence intensity compared to the DAUDA-Nb-FAR-1 control group (n = 3). C16:0, palmitic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C20:4, arachidonic acid; C20:5, eicosapentaenoic acid.
Table 1.
The compositions of fatty acids in N. brasiliensis and rat intestine parasitized with adult worms (%, n = 4).
Fig 3.
Identification of lentivirus mediated RNAi effects on Nb-far-1 expression.
(A) Identification of RNAi effects of four siRNAs on L3s by qPCR analysis and alignment of the nucleotide sequences of siRNA-Nb-far-1-651 and siRNA-Nb-far-1-310 with Nb-far-1 gene. (B) Lentiviral packaging plasmids were transfected and expressed in 293T cells. (C) PCR identification of shRNAmir-Nb-far-1-651 and shRNAmir-Nb-far-1-310 in the lentivirus from the supernatant of 293T cells. (D) PCR analysis of total DNA to identify shRNAmir-Nb-far-1-651 and shRNAmir-Nb-far-1-310 integrated into F0 of L3s. (E-F) Nb-far-1 gene transcription in F0 of L3s treated with lentivirus LV-Nb-far-1-651 and LV-Nb-far-1-310 at MOI of 200 and 400 by qPCR analysis. (G) Nb-far-1 gene transcription in F4 of L3s after lentivirus treatment by qPCR analysis. WT: wild type; NC: non-specific control siRNA; siRNA-Nb-far-1-651 and siRNA-Nb-far-1-310: the positions of the siRNAs on the Nb-far-1 gene. Data were mean ± SD of three independent experiments (n = 3). *p < 0.05, **p < 0.005.
Fig 4.
Oil Red O staining of lipid droplets in L3 N. brasiliensis with LV mediated Nb-far-1 RNAi.
Fig 5.
Effects of lentivirus-mediated siRNA of N. brasiliensis Nb-far-1 gene on female egg-shedding and larval development after treatment with lentivirus LV-Nb-far-1-310.
(A) Effects of Nb-far-1 RNAi on the egg-shedding curve of adult female worms (n = 3). (B) Effects of Nb-far-1 RNAi on the morphology of fecal eggs. Arrow indicates the increased space between blastomere and egg shell. (C) Effects of Nb-far-1 RNAi on changes in egg hatchability (n = 3). (D) Expression pattern of sperm-related genes in N. brasiliensis with Nb-far-1 RNAi and across developmental stages with RNA-seq data, respectively. (E-F) Effects of Nb-far-1 RNAi on the morphological changes of L1s and L2s. (G) Effects of Nb-far-1 RNAi on the changes of larval development from L1s to L3s (n = 3). (H) Effects of Nb-far-1 RNAi on the changes of larval development from L3s to adults (n = 3). **p < 0.01.
Fig 6.
Interference with Nb-far-1 gene expression impeded cuticle formation in N. brasiliensis after treatment with lentivirus LV-Nb-far-1-310.
(A) SEM and TEM observation of the morphological changes in adult (F1 generation) N. brasiliensis. L: lip, LP: labial papilla, R: ridge, EC: epicuticle, C: cortical, M: medial, F: fiber, BM: basal membrane, MB: muscle bundle, MBG: muscle bundle gap. (B) Expression pattern of collagen in N. brasiliensis with Nb-far-1 RNAi and across developmental stages with RNA-seq data, respectively.