Fig 1.
Influence of different digestive enzyme inhibitors on survival of S. exigua larvae.
L3 larvae were treated with diet soaked in different concentrations of inhibitors for 5 days. Treated larvae were then reared with untreated diet for 3 days. Survival rates were determined at 8 days after treatment. Each treatment was replicated three times. For each replication, 10 larvae were used. ʻCONʼ represents solvent treatment without inhibitor. Inhibitors include chymostatin specific to α-, β-, γ-, δ- chymotrypsin, papain, cathepsin A, B and D; TPCK (tosyl phenylalanyl chloromethyl ketone) specific to chymotrypsin, cerastocytin, papain, ficin, but not trypsin; TLCK (tosyl-L-lysyl-chloromethane hydrochloride) specific to trypsin, cerastocytin, but not chymotrypsin; cathepsin III inhibitor (CATH) specific to cathepsin, and inhibitor mixture of the four inhibitors at equal mass. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).
Table 1.
Putative chymotrypsin (CHY) genes of S. exigua obtained from Spodobase (http://bioweb.ensam.inra.fr/spodobase/), open reading frame (ORF) sizes, and annotation to GenBank using Blast P.
Fig 2.
Expression analysis of 7 chymotrypsin (CHY) genes selected from S. exigua.
(A) Phylogenetic tree analysis with other insect CHYs. Sequence alignment was performed with Clustal W program. The tree was constructed with MEGA 6.0. Each node contains bootstrap value after 1,000 replications. GenBank accession numbers of CHY genes other than S. exigua CHY (SeCHY) genes are denoted in S2 Table. RT-PCR analysis of SeCHYs in different developmental stages (B) and tissues (C). RL32 was used to validate cDNA integrity. Different developmental stages included larval instars (ʻL1-L5ʼ), pupa (ʻPʼ), and adult (ʻAʼ). Different tissues included hemocyte (ʻHCʼ), fat body (ʻFBʼ), midgut (ʻGUTʼ), and epidermis (ʻEPDʼ).
Fig 3.
Screening of 7 different chymotrypsin (SeCHY) genes of S. exigua with respect to RNA interference (RNAi) efficiency.
All dsRNAs were constructed at ~ 300 bp and injected to each L4 larva at 6 μg. Each treatment was replicated with three different insects. Control dsRNA (ʻdsCONʼ) used EGFP gene. At 48 h post injection, each SeCHY mRNA level was measured by RT-qPCR. mRNA levels were normalized by RL32 gene expression. Relative mRNA levels were calculated based on mRNA level of dsCON treatment in each SeCHY. Asterisk (*) indicates significant difference compared to control level at Type I error = 0.05 (Student’s t test).
Fig 4.
Protein structure and expression profile of SeCHY2.
(A) Prediction of signature motifs of chymotrypsin. Three thin arrows indicate catalytic triad. Six thick arrows indicate conserved cysteine residues. Asterisk indicates the cleavage site of signal peptide. For sequence alignment, Clustal W program of MegAlign (DNASTAR, Version 7.0) was used. GenBank accession numbers of well-known chymotrypsin amino acid are denoted in S1 Table. (B) Expression in digestive tract: foregut (ʻFGʼ), midgut (ʻMGʼ), and hindgut (ʻHGʼ). MG was further separated into three parts: anterior (ʻAʼ), middle (ʻMʼ), and posterior (ʻPʼ). RL32 was used to validate cDNA integrity.
Fig 5.
Insecticidal activity of dsRNA specific to SeCHY2 (dsSeCHY2) against different larval instars of S. exigua.
(A) Effect of dsSeCHY2 on different larval instars by injection or feeding dsRNA. Control dsRNA (‘dsCON’) used EGFP gene. For injection, 3 μg of dsRNA was injected into each larvae. For feeding, 10 μg of dsRNA was fed to each larva. Mortality was determined at 5 days after treatment (ʻ5 DATʼ). (B) Dose-mortality in dsRNA treatment. (C) Time-mortality in dsRNA treatment. Each treatment was replicated three times. For each replication, 10 larvae were used. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).
Fig 6.
Effect of dsRNA specific to SeCHY2 (‘dsSeCHY2’) on enzyme activity of S. exigua chymotrypsin in the midgut lumen.
(A) dsRNA (6 μg/larvae) was injected to 3rd instar. (B) dsRNA (20 μg/ larvae) was fed to 3rd instar. At different periods, gut lumen was collected and used to visualize (upper panel) or quantify (lower panel) CHY enzyme activity using a fluorescent substrate. Control dsRNA (‘dsCON’) was EGFP gene. For each replication, 10 larvae were used. Asterisk (*) indicates significant difference with control level at Type I error = 0.05 (LSD test).
Fig 7.
Immunosuppressive activity of dsRNA specific to SeCHY2 against fifth instar larvae of S. exigua.
(A) Nodulation assay. Heat-killed X. hominickii (5 × 105 cells/larvae) were injected to larva at 48 h after dsSeCHY2 injection. At 8 h post X. hominickii injection, hemocyte nodules were counted. For each treatment, 5 larvae were used. (B) Enhancement of B. thuringiensis (Bt) pathogenicity by injection of dsSeCHY2. Bt was treated with 105 ppm by diet-dipping feeding method. Each treatment was replicated three times. For each replication, 10 larvae were used. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).
Fig 8.
Degradation of dsRNA in hemolymph or midgut juice of S. exigua.
Hemolymph (ʻHLʼ) was collected from fifth instar (ʻL5ʼ). Gut juice was collected from the midgut by collecting supernatant of gut content followed by centrifugation at 10,000 × g for 5 min at 4°C. (A) Effect of hemolymph on dsRNA specific to SeCHY2 after incubating 2 μg dsRNA with 10 μl HL sample with different incubation periods. (B) Effect of gut juice on dsRNA specific to SeCHY2 after incubating 2 μg dsRNA with 10 μL gut juice sample. RNase inhibitor (1 μL, 20 ng) was included in the incubation. After incubation, all RNA samples were subjected to 1% agarose gel electrophoresis and visualized with EcoDye DNA staining solution (SolGent, Daejeon, Korea).
Fig 9.
Construction of recombinant E. coli expressing dsRNA specific to SeCHY2 (dsSeCHY2).
(A) Location of dsSeCHY2 in cDNA of SeCHY2. (B) Directional cloning of dsSeCHY2 into L4440 vector using two restriction sites of HindIII and SpeI. The cloning site was located between two opposite T7 promoters. Recombinant vector was screened after transformation into E. coli in the presence of ampicillin antibiotics. LacZ promoter (ʻlacZʼ) was then induced by IPTG to express T7 RNA polymerase (ʻT7 RPʼ) which recognized two T7 promoters and transcribed dsSeCHY2 in both directions. (C) Resulting dsSeCHY2 and its resistance to RNase A treatment. ʻWildʼ and ʻRCʼ represent non-recombinant and recombinant HT115 bacteria, respectively. ʻMʼ represents DNA marker.
Fig 10.
Variation in insecticidal efficacy of E. coli HT115-expressing dsRNA specific to SeCHY2 (dsSecHY2) after different pretreatments.
Pretreatments used no pretreatment (ʻLiveʼ), heat treatment at 95°C for 10 min (ʻheat-killedʼ), or sonication (ʻSonicatedʼ) using ultrasonication to breakdown bacterial cell membrane. Control (ʻCONʼ) used non-recombinant HT115 bacteria for feeding assay. All treatments were used for oral administration of bacteria by loading 107 cells to diet. After feeding all bacteria, larvae were fed with fresh diet for growth. Fourth instar were used as test larvae. (A) Larvae treated with E. coli HT115 expressing dsSeCHY2 after different pretreatments. (B) Influence of bacterial treatment on different larval developmental parameters. Each treatment was replicated three times. For each replication, 10 larvae were used. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).
Fig 11.
Insecticidal activity of E. coli HT115 expressing dsRNA specific to SeCHY2 against different larval instars (ʻL1-L4ʼ) of S. exigua.
Sonication-pretreated bacteria were used for oral feeding. Recombinant bacteria were loaded to each diet at a dose of 107 cells. (A) Variation in insecticidal efficiencies of recombinant bacteria depending on developmental stages. Each instar treatment was replicated three times with 10 larvae per replication. (B) Variation in RNAi efficiency of bacteria treatment among developmental stages. In each instar treatment, three individuals were randomly selected and used to measure mRNA levels using RT-qPCR. Each individual was an experimental unit. Relative mRNA was calculated by SeCHY2 level of treatment (ʻTRTʼ) divided by that of control (ʻCONʼ). TRT represents feeding treatment of recombinant bacteria, while CON represents feeding treatment of non-recombinant bacteria. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).