Figure 1.
Phylogenetic tree of the protein sequences of LKR/SDH genes.
The LKR/SDH amino acid sequences used were downloaded from GenBank and aligned in MEGA4 with CLUSTALW and the plylogenetic tree made by MEGA version 4.0 based on the neighbor-joining method. Values with 1,000 replications. The sequences used were A. gambiae (XP314728), Aedes aegypti (XP001649464), D. melanogaster (AAF52559), H. sapiens (CAA07619), M. musculus (CAA12114), Equus caballus (XP001502225), Arabidopsis thaliana (AAB53975), Zea mays (AAG21985), Saccharomyces cerevisiae LYS9 (CAA96331) (LYS9-L-glutamate forming), Cryptococcus neoformans (LYS9-L-glutamate forming) (AAW40810), S. cerevisiae LYS1 (CAA86194), (LYS1-L-lysine forming), and Aspergillus fumigatus LYS1P (XP754450) (LYS1-L-lysine forming).
Figure 2.
Transcription analysis of the LKR/SDH mRNA.
The RT-PCR results shown are representative of 3 independent experiments with the same protocol. A, Stage-specific expression profiles of LKR/SDH shown by RT-PCR analyses. Lane 1, eggs; lane 2, unfed larvae; lane 3, fed larvae; lane 4, unfed nymphs; lane 5, fed nymphs; lane 6, unfed adult females, lane 7, engorged females of H. longicornis. RT-PCR was performed using LKR/SDH-specific primers and amplified 487-bp fragments. A 540 bp fragment of the constitutively expressed H. longicornis β-actin was amplified as an internal control. B, RT-PCR analysis of LKR/SDH mRNA expression in different tissues during blood feeding, Mg, midguts; Sg, salivary glands; Ov, ovaries; Fb, fat bodies; Sy, synganglions; Hg & Mt, hindguts with malphighian tubule. UF, unfed adult females; d1, females fed 1 day; d2, females fed 2 days; d3, females fed 3 days; d4, females fed 4 days, d5, females fed 5 days; Eg, engorged females.
Figure 3.
Expression of the recombinant protein.
Recombinant proteins containing the LKR domain, SDH domain and full length LKR/SDH were resolved by 12% SDS-PAGE. A, lane 1, protein extracts from induced E. coli carrying pGEX-4T-3/LKR; lane 2, purified GST/LKR fusion protein; B, lane 1, lysate of E. coli without pGEX-4T-3; lane 2, protein extracts from induced E. coli carrying pGEX-4T-3/SDH; lane 3, purified GST/SDH fusion protein. C, lane 1, purified recombinant His-LKR/SDH protein; M, molecular weight marker.
Figure 4.
A. Native and recombinant proteins were analyzed for LKR and SDH activities under conditions of excess concentrations of all LKR substrates or all SDH substrates, respectively. Data are presented as units/mg protein. Mean±S.D (n = 3). B. Separation of LKR and SDH activities by native-PAGE gel electrophoresis. Samples of partially purified midgut and ovaries, and recombinant His-LKR/SDH were separated by native PAGE and subsequently stained for SDH and LKR activity. In the control gel, samples incubated without L-lysine and saccharopine are labeled “Lys-” and “Sacch-”. In the LKR and SDH activity staining gel, the samples are labeled “Lys+” and “Sacch+”. Lane 1, midgut; lane 2, ovary; lane 3, His-LKR/SDH. C. Optimum pH of LKR/SDH native protein.
Figure 5.
Immunoblotting analysis of native LKR/SDH.
The partially purified LKR/SDH enzyme from midgut, ovary and egg lysates of partially fed adults, and whole body lysates of larvae, nymphs, unfed adult, and fed adult H. longicorns were used as antigens. A. Lane M, protein marker; lane 1, egg lysate; lane 2, larva lysate; lane 3, nymph lysate; lane 4, unfed tick lysate; lane 5, engorged tick lysate. B. Lane M, protein marker; lane 1, adult midgut; lane 2, adult ovary.
Figure 6.
Localization of LKR/SDH in ovary and midgut cells by IFAT.
Slides of ovary or midgut tissue from adults 4 days post-engorgement were used with mouse anti-SDH serum as primary antibodies. The mouse anti-IgG conjugated with Alexa 488 was used as a secondary antibody for ovarian tissues and mouse anti-IgG conjugated with Alexa 594 for midgut tissues. DGC, digestive cell; Oc, oocyte; L, midgut lumen. Magnification, ×20.
Figure 7.
Histochemical detection of SDH activity in sectioned midgut and ovary.
A. In the SDH activity staining tissue, slides stained with saccharopine are labeled with “Sacch+” and control slides not incubated with saccharopine labeled with “Sacch-”. Positive reaction shows SDH specific staining as brown dots on the oocytes and digestive cells of the midgut, whereas negative reaction incubated without saccharopine shows no specific staining. Mg, midgut; DGC, digestive cell; L, lumen; Ov, Ovary; Oc, oocyte. B. IFAT; The mouse anti-IgG conjugated with Alexa 594 was used as a secondary antibody. DGC, digestive cell; L, lumen; C. Mitochondria visualization by MitoTracker probe. MitoTracker probe, midgut tissues stained with 50 nM of Mitotracker Red CMXRos. DGC, digestive cell; L, midgut lumen. Magnification, ×20.
Figure 8.
Expression profiles in tick starvation and after injections of lysine and saccharopine.
Total RNA was pooled from the same concentration of RNA samples from starved ticks. The RT-PCR results shown are representative of 3 independent experiments with the same protocol. A. m3, females starved for 3 months; m6, females starved for 6 months; m10, females starved for 10 months. B. Effect of injections of synthetic L-lysine and saccharopine on LKR/SDH gene expression. Ticks starved for 1 month were injected with synthetic L-lysine (50 mM solution of L-lysine 0.5 µl/tick), L- Saccharopine (20 mM solution L-saccharopine 0.5 µl/tick) and PBS (0.5 µl/tick), respectively. Mg; midguts; Sg, salivary glands; Ov, ovaries; Fb, fat bodies. The data are expressed as the ratio of the density of LKR/SDH to the density of actin gene products from the same template. The RT-PCR results shown are representative of 3 to 4 independent experiments with the same protocol.
Figure 9.
Effect of dsRNA treatment on LKR and SDH gene disruption.
A. Reverse transcription PCR analysis. B. Western blotting analysis. LKR-, SDH- and Luc- dsRNAs and PBS were injected into the each group of female H. longicornis adults. The injected ticks (15 individuals for each group) were infested on rabbits, and ticks recovered after 4 days of feeding. Lane 1, LKR dsRNA-injected ticks; lane 2, SDH dsRNA-injected ticks; lane 3, PBS-injected ticks; lane 4, firefly gene of Luciferase (Luc) dsRNA-injected ticks.
Table 1.
Effect of dsRNA treatment on tick oviposition.
Figure 10.
Morphological changes in LKR silenced ticks.
The macrophotographs of dsLKR RNA-injected tick (above) and PBS-injected tick (below). A, Ventral aspect of dsLKR RNA-injected ticks 7 days after engorgement, Rs, rectal sac (arrow). B, Magnification of A and rectal sac of dsLKR RNA-injected ticks show significant enlargement due to filling with fluid; C and D, Dorsal and ventral aspects of dsLKR RNA-injected tick 14 days after engorgement,show edema-like condition and hernia-like protrusion of Gene's organ. Mg, midgut; Mt, Malpighian tubule, Rs, rectal sac; Go, Gene's organ; E, Ventral aspect of dsLKR RNA-injected tick 25 days after engorgement, Rs, rectal sac changed color from white to red (arrow); F to J; PBS-injected tick. F, Ventral aspect of PBS-injected tick 7 day after engorgement; G, Magnification of F and rectal sac; H, Dorsal aspect 14 days after engorgement; I, Ventral aspect 14 days after engorgement; J, tick after egg oviposition 25 days after engorgement.
Figure 11.
Microphotographs of dsRNA-injected tick and macro photograph of pathological organs.
A. Ovaries of PBS (normal), LKR and SDH dsRNA-injected ticks showing pathological phenotypes. B. Macrophotograph of pathological organs. Panel a, Normal midgut of PBS-injected ticks on day 25 after engorgement, Mg, midgut; panel b, Destroyed structure and appearance of liquid in the midgut of LKR dsRNA-injected tick on day 25 after engorgement, Mg, midgut; panel c, Magnification of Figure 11E, rectal sac of LKR dsRNA-injected tick on day 25 after engorgement shows significant enlargement due to filling with fluid-like redden liquid, Rs, rectal sac; panel d, Hernia-like protrusion of Gene's organ, LKR dsRNA-injected tick on day 14 after engorgement, Go; Gene's organ.