The plants and insects
The colony of B. tabaci Mediterranean (MED, Q biotype) was maintained on cucumber plants (Cucumis sativus L.cv. Bojie-107) in cages (60 cm×60 cm×80 cm) in the greenhouse at 28±1℃, L:D=16 h:8 h and 75±1% relative humidity. The genetic purity of B. tabaci Q biotype cultures was monitored every 3 generations using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) technique combined with the sequencing of mtCO1 gene . To obtain CCYV-infected plant cultures, cucumber plants at 2 true-leaf stage were inoculated with Agrobacterium tumefaciens-mediated CCYV clones . Plants of cucumber were kept under above-mentioned conditions.
Quantification of CCYV in cucumber plants and whiteflies
Whiteflies of the same colony were fed on CCYV-infected cucumber plants in a clip cage for 48 h, and were collected in group of 10 individuals of each stage of 1st to 4th instar nymphs and adults for RNA extraction. Total RNA of whiteflies or infected cucumber plants (100 mg) was extracted using TRIzol® Reagent (Invitrogen Carlsbad, CA, USA) following the manufacturer’s instructions. RNA concentration and purity were measured in a NanoDropTM spectrophotomer (Thermo Scientiﬁc Wilmington, DE, USA) and stored at -80℃ for subsequent analysis. Total RNA (1 μg) from each sample was reverse transcribed to generate the first-strand cDNA using the PrimeScript® RT reagent Kit (Takara, Dalian, China).
Primers were designed based on coding sequences of CCYV coat protein (CP) by using primer premier 5 software and the nucleotide sequence in GenBank (Accession No: HM581658.1). The primers used are shown in Table 1. Subsequent primer-blast searches showed that they had a high specificity towards CCYV. PCR products were connected with pMD18-T vector to construct standard recombinant plasmid. Six gradients (3.40×103-3.40×108 copies/μL) of standard recombinant plasmids were set up as a template for real-time qRT-PCR, with three replicates for each concentration, meanwhile blank control and negative control were set up. Amplification reactions were performed as follows: 94℃ for 2 min, 40 cycles of 94℃ for 15 s, 60℃ for 20 s, 72℃ for 20s. According to the standard curve automatically generated by the instrument, the correlation coefficient R2=0.9984, the amplification efficiency E=95%, and the standard curve equation is Y=-3.3396lgX+27.8480 (Figure S1). Ct value of each sample was detected by qRT-PCR, three replicates were examined, and the absolute quantification of CCYV mRNA molecules in cucumber plants or B. tabaci were calculated.
Table 1 PCR primers for CCYV detection. Note: Primers were designed based on coding sequences of CCYV coat protein (CP) by using primer premier 5 software and the nucleotide sequence in GenBank (Accession No: HM581658.1).
Impacts of CCYV on biology of B. tabaci
A couple of 3-d adults were placed with a clip cage on a leaf of healthy cucumber plant for oviposition. After 24 hours, the adults were transferred to CCYV-infected cucumber plant for another 24 hours, then the adults were removed from plants. Thirty eggs on each leaf were marked under the super-depth microscope (Keycence digital microscope VHX-600E) and other eggs were removed. Three replicates were used for each treatment. Observations were taken every day under the super-depth microscope until all eggs hatched and 1st-instar nymphs were fixed. Locations of the nymphs were marked. The egg hatching rates (P0), nymph survival rates (Pn) , and sex ratio (P) of newly emerging adults were calculated with the following equations: see equations 1, 2, and 3 in the supplementary files.
Where N1 is the number of 1st-instar nymphs; K is a constant of 30; Pn is the survival rates of each instar nymphs (n=1, 2, 3, 4); M is the number of males and F is the number of females. The nymph duration and adult longevity were recorded separately. Sizes of each individual of adults were measured.
In another set of experiments, a couple of newly emerging adults were placed with a clip cage on a leaf of healthy or CCYV-infected cucumber plant. The insects were moved to a new plant every 24 hours. Eggs on all leaves were counted, and dead male adults were replaced with new males until the female adults died. Ovipositional capacity was calculated. For this sets of experiments, 40 female adults were used.
IBM SPSS Statistics 21.0 was used to conduct data analyses. Comparisons in body size, oviposition, nymph duration, adult longevity as well as sex ratio of insects on healthy and CCYV-infected cucumber plants were made using Independent-Samples t-test; one-way ANOVA, LSD test was used to analyze fertility rate, nymph survival rates and the amount of CCYV mRNA molecules among all instar nymphs and adults. Significant differences were tested at the 0.05 or 0.01 level. All data were expressed as Mean±SE of three independent experiments.