Identification of target genes for RNAi-mediated control of the Twospotted Spider Mite

RNA interference (RNAi) is being developed for the management of pests that destroy crops. The twospotted Spider Mite (TSSM), Tetranychus urticae is a worldwide pest due to its unique physiological and behavioral characteristics including extraordinary ability to detoxify a wide range of pesticides and feed on many host plants. In this study, we conducted experiments to identify target genes that could be used for the development of RNAi-based methods to control TSSM. Leaf disc feeding assays revealed that knockdown in the expression genes coding for proteins involved in the biosynthesis and action of juvenile hormone (JH) and action of ecdysteroids [Methoprene-tolerant (Met), retinoid X receptor β, farnesoic acid O-methyltransferase, and CREB-binding protein] caused 35–56% mortality. Transgenic tobacco plants expressing hairpin dsRNA targeting Met gene were generated and tested. About 48% mortality was observed in TSSM raised on transgenic tobacco plants expressing dsMet. These studies not only broaden our knowledge on understanding hormone action in TSSM but also identified target genes that could be used in RNAi-mediated control of TSSM.

RNA interference (RNAi), a post-transcriptional gene silencing mechanism that helped in studies aimed at elucidating the function of genes. In TSSM two RNAi methods have been demonstrated thus far to deliver the double-stranded RNA (dsRNA): an injection into adult females and eggs, and a leaf-disc feeding assay 14,15 . Injecting Distal-less dsRNA into TSSM female adults showed that Distal-less phenotype is maternally inherited 14 . The feeding dsRNA method was shown to be a viable method inducing mortality in TSSM after feeding leaf discs treated with dsRNA targeting four known lethal genes 15 , demonstrating the possibility of leaf disc-mediated delivery of dsRNA. Recently. Suzuki and his colleagues compared five different methods to deliver dsRNA to TSSM: leaves floating on a dsRNA solution, dsRNA-expressing plants, an artificial diet supplemented with dsRNA, or dsRNA-coated leaves, and mite soaking in dsRNA solution 16 .
The goal of the current study is to identify genes involved in hormone biosynthesis and action and explore their use as RNAi targets in the TSSM. The bean-leaf disc assay was performed to investigate the feeding RNAi effect of eight candidate genes. The transgenic tobacco plants expressing one of the genes, Met, were produced and tested. We also tested JH analogs to determine their effect on TSSM. This information helps in understanding TSSM physiology and also lays a foundation for the development of RNAi-based methods to control this pest.

Results and Discussion
Identification of target genes for RNAi-based control of TSSM. From the T. urticae genome website (http://bioinformatics.psb.ugent.be/orcae/overview/Tetur), we retrieved sequences of eight genes, including TuMet (tetur18g03530), TuSRC (tetur41g00280), and FaMet (tuter13g03250), TuEcR (tetur01g15140), TuRXR1 (tetur31g01930), TuRXR2 (tetur01g09240), TuRXR β (tetur01g09220), and TuCBP (tuter07g03920) coding for proteins involved in biosynthesis and action of JH and action of ecdysteroids (Table 1). Gene-specific primers based on these sequences and DNA isolated from TSSM were used to amplify the 300-500 bp fragment of each gene ( Table 2). The PCR products were then used to synthesize dsRNAs, and the dsRNAs were screened in bean leaf disc assay. The bean leaf disc assay developed by Kwon et al. 15 was modified and used to screen candidate genes ( Fig. 1). Nuclease-free water and dsRNA targeting a fragment of the gene coding for green fluorescent protein (GFP) dsRNA were used as negative controls to check the undesirable effects of treatment and dsRNA on TSSM. Approximately, 10% and 20-22% mortality was observed in control TSSM treated with nuclease-free water and GFP dsRNA, respectively. Among the dsRNAs tested, dsTuMet, dsTuCBP, dsTuRXR β, and dsTuFaMet caused significantly higher mortality compared to the mortality in the control treatment (Fig. 2). Most of the mortality caused by knockdown of TuMet, TuCBP, TuRXR β, and FaMet occurred during the last molting stage (Fig. 2). Knockdown efficiency was determined in TuMet fed deutonymphs, prior to the last molt stage. This stage was selected because most of the TSSM died during the last molting stage (Fig. 3A,B). Feeding dsMet caused about 50% reduction in Met mRNA levels (Fig. 3A). These data suggest that Met, CBP, RXR β, and FaMet are required for successful completion of development and molting in TSSM and these genes could serve as target sites for development of RNAi-based methods to control this pest.
Sequencing of the TSSM genome and further studies provided important insights into biosynthesis and action of two major hormones, ecdysteroids and juvenile hormones. PonA and MF have been proposed as the major ecdysteroid and JH, respectively. However, not much is known about the action of these hormones especially about the receptors that transduce hormone signals. The methoprene-tolerant has been identified as a JH receptor in insects. It is possible that spider mites may use Met as an MF receptor since chemical structures of JH and MF are nearly identical, except for the absence of an epoxide group 17 . Several recent studies suggested that crustaceans which mainly synthesize the sesquiterpenoid, methyl farnesoate use Met as a receptor for MF 18,19 . Taken together our data and the previous reports suggest that these mites may use Met as an MF receptor. However, determination on whether or not Met is an MF receptor requires further studies. Interestingly, Silencing of TuFaMet, an enzyme involved in MF biosynthesis, also induced mortality of mites confirming previous findings on the role of MF as a major JH in these mites. In insects, homologs of steroid receptor co-activator (SRC) play essential roles in both JH and 20E action 7 . Silencing of SRC homolog in TSSM did not cause significant mortality suggesting that SRC homolog we tested may not be an essential gene in TSSM development. However, further studies are required to determine if there are other homologs of SRC or some other co-activators involved in hormone action in TSSM. Cyclic AMP response binding protein interacts with multiple transcription factors and other proteins that regulate many developmental processes in insects and other animals. Interestingly, knockdown of the CBP gene caused significant mortality in TSSM suggesting that some of the functions of CBP recently shown in insects may have been conserved in TSSM 20,21 . RXR homolog in insects, ultraspiracle, is an essential partner for EcR in transduction of 20-hydroxyecdysone signals in insects. Knockdown of TSSM RXR β also caused significant mortality (Fig. 2). Knockdown of RXR β but not RXR1 and RXR2 caused significant mortality in TSSM suggesting that RXR β is essential for TSSM development. Differences in the effect of RXR β, RXR1 and RXR2 knockdown on TSSM development is intriguing. In insects, USP and RXR isoforms are produced by alternative splicing. The three RXR β and the other two RXRs seem to be coded by different genes in TSSM. Also, RXR1 and RXR2 are typical nuclear receptors containing canonical DNA-binding and ligand-binding domains. Whereas RXR β protein contains a ligand binding domain but not zinc finger-containing DNA binding domain. Structure of RXR1 and RXR2 proteins suggest that they may have redundant function hence, silencing of each of these genes did not show a significant effect on development. Further work on the structure of RXR genes and isoform-specific effects will help to explain the action of RXRs in TSSM. Whether RXR β is required for PonA action or some other function in TSSM requires further studies as well. Lack of mortality after EcR knockdown is also intriguing and requires further investigation. Taken together, the data included here identified Met, CBP, RXR β, and FaMet as potential targets for the development of RNAi-based methods for controlling TSSM.  Table 2. Sequences of the primers used in this study. T7 promoter sequence TAATACGACTCACTATAGGG was added at the 5′ end of each dsRNA primer.  (Fig. 4A). These plants were evaluated in TSSM bioassays. About 48% mortality was detected in the TSSM growing on TuMet transgenic plants compared to 10% mortality detected in the TSSM on control plants expressing only vector (Fig. 4B). These data confirm results obtained in leaf disc bioassay and demonstrate the feasibility of delivering dsRNA to TSSM through expression in plants.
Poor RNAi efficiency was observed in several feeding assays methods tested in TSSM so far. The methodology/ delivery of dsRNA, physiology of TSSM with the highly effective excretory system, and frequent molting during development may have contributed to a decrease in the effectiveness of RNAi in TSSM. Delivering dsRNA to target organisms is one of the challenges associated with developing RNAi-based pest control methods. Some of the approaches that are being developed are expression and delivery through transgenic plants, spraying, soil application or trunk injection of dsRNA synthesized in vitro or in microorganisms [22][23][24] . Several previous studies demonstrated successful delivery of dsRNAs to target insects after their expression in plants 2,20,22,[24][25][26][27] . When economical and acceptable to the public, this may be the desirable method of delivery of dsRNA to TSSM. Other methods including spray or soil application of dsRNA synthesized in vitro or in microorganisms should also be explored for TSSM control. The mortality in TSSM caused by feeding dsRNA targeting genes coding for proteins involved in JH and Ecdysone biosynthesis and action. The dsRNA targeting eight candidate genes coding for proteins involved in JH biosynthesis and action and ecdysteroid action were fed to TSSM. The mortality of TSSM was recorded on the 9 th day after application of dsRNA. dsGFP and water were used as controls. Juvenile hormone analogs for TSSM control. Since knockdown of genes involved in JH biosynthesis and action caused mortality in TSSM, we evaluated JH analogs (JHA) for their potential to control TSSM. Insect growth regulators, especially JHAs, have been widely used to control mosquitoes, scales, and mealybugs 28,29 . Here, we tested kinoprene, pyriproxyfen, and hydroprene to see whether they cause mortality in TSSM. Treatment with kinoprene caused significant mortality (88.5%) and blocked development. In contrast, treatment with different concentrations of pyriproxyfen and hydroprene was not effective in blocking development or causing mortality of TSSM (Table 3). In the case of kinoprene treatment, mites mostly died during early stages and did not reach the first molt. Insect growth regulators are widely used for controlling pests all over the world. Very few studies have been performed to determine the effect of JHA on the TSSM. Only one study showed the effectiveness of JHA on TSSM, CGA 29′170 and ZR-777 (kinoprene) 30 . Our results confirm these previous studies where 0.15% ZR-777 caused above 60% mortality on day two and 80% mortality on day four after treatment 30 . The mortality observed in our experiment was around 60~80% on day two after treatment which confirms the previous report. Previous studies identified methoprene as the most effective JHA for Aedes aegypti 31 and hydroprene as the most effective one for Tribolium castaneum 26 ; significant differences in the effectiveness of three JHAs against TSSM is interesting. Taken together these data suggest that JHA work differently among arthropods. Whether or not the differences in JHA activity is due to the differences in Met protein sequence coded by genomes of these arthropods remains to be investigated. The data included in the paper not only broaden our knowledge of hormone action in TSSM but also identified target genes for use in RNAi-based control of TSSM.  Total RNA extraction, PCR, dsRNA synthesis and Quantitative real-time PCR (qRT-PCR). Total RNA was isolated from deutonymphs using the TRI reagent (Molecular Research Center Inc., Cincinnati, OH). DNase I (Ambion Inc., Austin, TX) treated RNA was converted cDNA using M-MLV reverse transcriptase (Clontech Laboratories). The cDNA and the primers designed based on target gene sequences (Table 2) were used to amplify fragments of target genes. The PCR products were purified using the QIAquick PCR purification kit (Qiagen Inc, Valencia, CA) and used as templates to synthesize dsRNA using the Ambion MEGAscript transcription kit (Ambion, Austin, TX). Double-stranded RNA was purified by phenol/chloroform extraction followed by ethanol precipitation. The quality of dsRNAs was checked by running them on agarose gels. The concentration of dsRNAs was measured using NanoDrop1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA).
The qRT-PCR was performed in Applied Biosystems StepOnePlus ™ Real-Time PCR System (Life Technologies ™ , Carlsbad, CA) using FastStart SYBR Green Master (Roche Diagnostics, Indianapolis, IN).
Relative mRNA levels were determined in triplicate biological samples using ribosomal protein 49 (RP49) mRNA levels for normalization.
Bean leaf disc assay. The feeding chamber and dsRNA-permeated leaf disc were used as described in a previous study 15 with some modifications to the shape of the chamber and quantity of dsRNA used. The 200 μl of dsRNA (300 ng/μl concentration) was placed underneath the leaf on the first day, followed by adding 50 μl of dsRNA on each day up to nine days. Since the amount dsRNA nearly reached a peak level within the leaf disc by after 24 hr after application 15 , mites were placed on the leaf disc at 24 hr after the application. Twenty-to-thirty larvae were placed on the dsRNA-permeated leaf disc. These chambers were maintained at 23 ± 1 °C, 55 ± 5% relative humidity (RH) at 16:8 (L:D) photoperiod. The green fluorescent protein (GFP) dsRNA was used as a control. When the mites reached adulthood (eight to nine days later), the remaining mites on each leaf disc were counted. The bean leaf disc assay was repeated three times. To determine knockdown efficiency, qRT-PCR was performed using the gene-specific primers ( Table 2). A pool of multiple mites were used for each biological replicate and three biological replicates were included in each treatment.
Transgenic plant production and assay. The transgenic tobacco plants expressing dsRNA targeting TuMet were produced following methods described previously 32 . Tobacco strain (Nicotiana tabacum L. cv. Samsun NN) was used in these experiments. Tobacco plant transformation was done as described previously by Sahoo et al. 32 . Tobacco leaf, cut into to 4 × 4 cm squares, were placed above the wet cotton in the petri dish. Twenty to thirty larvae were placed on the tobacco leaf, and mortality was checked on the seventh day after initiation of feeding. Each experiment was repeated three times. Statistical analysis. Experimental data were analyzed by Student's t-test to compare the difference between the control group and treatment group.

Availability of Materials and Data
The materials used in the experiments and data reported are available upon request.