Knockdown of the Trehalose-6-Phosphate Synthase Gene Using RNA Interference Inhibits Synthesis of Trehalose and Increases Lethality Rate in Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Psyllidae)

Simple Summary In this study, we cloned and characterized a trehalose-6-phosphate synthase (TPS) gene from D. citri (DcTPS) for the first time. Meanwhile, we used RNA interference (RNAi) technology to efficiently disrupt DcTPS gene function in order to elucidate its role in the growth and development of D. citri. Our results suggest that dsRNA-mediated gene-specific silencing resulted in a strong reduction in relative expression of DcTPS and survival rate of nymphs, as well as an increase in malformation. This work was undertaken to establish a foundation for further research on the functions of D. citri trehalose-6-phosphate synthase. This will provide a new target for the control of D. citri in the field. Abstract Diaphorina citri Kuwayama is the vector of citrus “huanglongbing”, a citrus disease which poses a significant threat to the global citrus industry. Trehalose-6-phosphate synthase (TPS) plays an important role in the regulation of trehalose levels of insects, while its functions in D. citri are unclear. In this study, full-length cDNA sequences of the TPS gene from D. citri (DcTPS) were cloned and its expression patterns at various developmental stages were investigated. The results indicated that DcTPS mRNA was expressed at each developmental stage and the highest DcTPS expression was found in the fifth-instar nymphs of D. citri. Additionally, mortality and deformity of D. citri were observed after 24 and 48 h by feeding with three different dsRNA concentrations (20, 100 and 500 ng/μL). The results indicated that DcTPS expression was declined, and mortality and malformation in nymphs were increased via feeding with dsDcTPS. Moreover, the enzyme and trehalose content were decreased, while the content of glucose was significantly higher than that of untreated (control) individuals. This suggests that DcTPS might be vital for the growth and development of D. citri and further studies of the genes should be related to molting and metabolism for controlling D. citri.


Introduction
The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a notorious pest that causes huge economic loss to the citrus industry all around the world [1][2][3]. The psyllids can excrete copious amounts of honeydew, leading to bituminous coal sickness [4]. In addition, D. citri

D. citri Rearing and Sample Collection
In this study, healthy adults of D. citri were collected from Murraya exotica plants in Donghu Park, Quanzhou city, Fujian Province. Then, D. citri was maintained in our laboratory for over 3 years and reared continuously in Murraya exotica in insect rearing cages (60 × 60 × 90 cm 3 ). In the meantime, it was not disturbed by any insecticide. The temperature-controlled growth rooms, maintained at the laboratory building of School of Life Science in Nanchang University, were set at 27 ± 1 • C and RH (relative humidity) 70 ± 5%, with a photoperiod of 14:10 (L:D).
Following the methodology of Yu et al (2020), 100 post-mating D. citri females were collected and released onto fresh Murraya exotica plants placed in an insect rearing cage for obtaining D. citri individuals of the same growth and development stage. D. citri nymphs were classified into different stages based on their morphological features, and we continuously collected the nymphs by using a brush until the adults appeared [36]. Seven stages of D. citri were used for analysis of the different developmental stages' expression levels of DcTPS gene including egg, first-, second-, third-, fourth-, fifth-instar nymphs and adults. Thirty individuals of D. citri were used for each sample collection. All stages were performed with three replicates.

RNA Isolation and DcTPS cDNA Synthesis
Firstly, total RNA was extracted from each sample using the Eastep ® Super total RNA Extraction Kit (Shanghai Promega Trading Co., Ltd., Shanghai, China). Each tube of collected sample was homogenized in an ice bath with 300 µL of lysis solution. Then, 300 µL RNA diluent was added into the tube and mixed well. The sample was heated at 70 • C for 5 min and centrifuged at 4 • C, 14,000× g, for 5 min. Then, 500 µL of supernatant was transferred to another new tube, and 250 µL of absolute ethyl alcohol was added and mixed well. The mixture was transferred to a new centrifugal column installed on a collecting pipe and centrifuged at 4 • C, 14,000× g, for 1 min. At the same time, the filtrate was discarded, and 600 µL RNA lotion was added and centrifuged at 4 • C, 14,000× g, for 1 min. Fifty µL of the prepared DNase 1 incubation solution was added to the adsorption film center and incubated at room temperature for 15 min. Then, 600 µL RNA lotion was added and centrifuged at 4 • C, 14,000× g, for 45 s, repeating the process twice. Meanwhile, the filtrate was discarded. The centrifugal column was installed on the collecting pipe and centrifuged at 4 • C, 14,000× g, for 2 min. Next, the centrifugal column was anew installed on an elution tube and 100 µL nuclease-free water was added. Solutions were kept at room temperature for 2 min and centrifuged at 4 • C, 14,000× g, for 1 min. The RNAs were stored at −80 • C. Simultaneously, the concentration and purity of RNA were assayed by NanoPhotometer N60 Touch (IMPLEN GMBH, Munich, Germany) at absorbance ratios of A260/230 and A260/280. The integrity of the total RNA was verified via 1% agarose gel electrophoresis. In accordance with the manufacturer's instructions, total RNA was reverse-transcribed using the PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China). In other words, 1.0 µL of random 6 mers, 1.0 µL of dNTP mixture and 8 µL of total RNA were mixed to reach 10 µL in the tube, which was then incubated at 65 • C for 5 min to improve reverse transcription efficiency. Then, 4.0 µL of 5×PrimeScript II Buffer, 0.5 µL of RNase Inhibitor and 1.0 µL Primer Script II RTase and RNase-free water was added to reach 20 µL. Finally, the mixture was incubated at 45 • C for 50 min and then incubated at 70 • C for 15 min. The cDNA was stored at −20 • C for subsequent experiments.

Molecular Cloning
Fragments of the putative DcTPS gene were procured from the transcriptome database for D. citri. The veracity of the sequences was established by polymerase chain reaction (PCR) using the primers in Table 1. Full-length cDNA was obtained by 5'-and 3'-RACE using SMARTer ® RACE5'/3' kit (Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China) with the specific primers listed in Table 1. We subsequently recovered and purified the PCR product. The purified DNA was ligated onto the PGEM-Teasy Vector (Shanghai Promega Trading Co., Ltd., Shanghai, China) and the dideoxynucleotide method was used for sequencing (Sangon Biotech, Shanghai, China).

Bioinformatic and Phylogenetic Analyses
The cDNA sequence of DcTPS was translated with the Translate tool (http://www.expasy.org/ translate/). Amino acid sequences were deduced using ExPASy (http://web.expasy.org/translate). The molecular weight (MW) and isoelectric point (pI) of the deduced amino acid sequences were predicted by Compute pI/Mw (http://web.expasy.org/compute_pi/). The N-linked glycosylation sites were analyzed using NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/). Sequence comparisons were performed using DNAMAN. Additionally, a phylogenetic tree was constructed using a total of 20 insect TPS protein sequences obtained from NCBI via MEGA7.0 software and Clustal X 1.83 by the maximum likelihood method. Bootstrap values were calculated based on 1000 replicates [37].

Expression of DcTPS Gene
The cDNA templates derived from different developmental stages of D. citri were used for temporal expression tests. Primers were designed for quantitative real time (RT-qPCR) by Prime 5.0 and are listed in Table 1. Expression of the target gene was measured by RT-qPCR and normalized with two stable reference genes, β-Actin (GenBank: DQ675553) and α-tubulin gene (GenBank: DQ675550) [3]. Each PCR reaction was mixed with 10 µL TB green, 7.8 µL ddH 2 O, 1.0 µL cDNA, 0.4 µL Rox dye and 0.4 µL of each primer. The thermal cycling profile consisted of an initial denaturation at 95 • C for 5 min and 40 cycles at 95 • C for 10 s and 60 • C for 20 s. The reactions were performed with the StepOnePlus TM Real-Time PCR Instrument (Thermo Fisher Scientific, Singapore). The relative expression level was calculated using the 2 −∆∆Ct method [38].

Preparation of dsRNA and Feeding
Firstly, dsRNA fragments targeting DcTPS (447 bp) and GFP (GenBank: LN515608) (415 bp) were prepared by using TranscriptAid T7 High Yield Transcription (Thermo Scientific, Lithuania). Secondly, the dsRNA was purified in accordance with instructions for the use of the GeneJET RNA Purification Kit (Thermo scientific, Lithuania). Thirdly, based on the results of a previous study, some adjustments were made to our rearing procedure in the dsRNA ingestion experiment [11]. Briefly, glass cylinders (12.0 cm in length and 3.0 cm in diameter) were used as a feeding chamber. The dsRNA was delivered by an artificial diet placed between two layers of stretched Parafilm. The artificial diet consisted of 20% (w:v) sucrose mixed with dsRNA DcTPS at a final concentration of 20, 100 and 500 ng/µL. Meanwhile, dsGFP as a control group was fed in the same way. Thirty newly emerged fifth-instar nymphs of D. citri were used for each treatment. Meanwhile, there were three biological replicates in each treatment. The number that survived and molted were calculated 24 and 48 h after dsRNA feeding. At the same time, individuals that were clearly of abnormal phenotype were photographed using a Leica Microsystems Ltd. (Leica, Singapore) digital camera. Subsequently, the D. citri individuals (at least 10 individuals) surviving different concentrations of dsRNA treatment at 24 h and 48 h were kept at −80 • C and used for RNA extraction, analysis of relative expression levels and assays of enzyme activity, trehalose content and glucose content. The sample collections of each concentration and each duration were performed with three replicates.

Quantitative Detection of Trehalose-6-Phosphate Synthase Content in D. citri
The content of trehalose-6-phosphate synthase in D. citri was measured using a modified protocol based on a previous report [20]. The content of DcTPS in D. citri was quantified by the insect trehalose-6-phosphate synthase ELISA Assay Kit (Jonln, Shanghai, China). Briefly, each sample containing three surviving individuals of D. citri was homogenized in 300 µL phosphate buffer saline (PBS, pH 7.0) and centrifuged at 4 • C, 5000× g, for 10 min. Then, 100 µL of HRP-conjugate reagent was added to 50 µL of supernatant and incubated at 37 • C for 60 min. Liquid was aspirated from each well and all wells were washed with 350 µL wash solution, repeating the process four times for a total of five washes. Then, 50 µL of chromogen solution A and 50 µL of chromogen solution B were added to each well with gentle mixing and incubated for 15 min at 37 • C under protection from light. Then, 50 µL of stop solution was added into each well. The absorbance was measured at 450 nm. The content of trehalose-6-phosphate synthase in the sample solution was calculated based on a standard curve. Three technical replicates were required for each sample measurement.

Measurements of Trehalose and Glucose Content
Five surviving individuals of D. citri were collected for the trehalose and glucose content assays. Following the assay procedure specified in the Insect Trehalose ELISA Kit and Insect Glucose ELISA Kit (Xinquan, China), samples were homogenized in an ice bath with 100 µL of extraction solution and centrifuged at 4 • C, 3000× g, for 10 min. Then, 40 µL of sample diluent was added to 10 µL of testing sample in each well. Then, 100 µL of HRP-conjugate reagent was added and the well was incubated at 37 • C for 1 h. Then, 50 µL of chromogen solutions A and B were mixed in after the wash followed by incubation for 15 min at 37 • C. The concentrations of trehalose and glucose were measured photometrically at 450 nm. The trehalose and glucose content in the sample solution were calculated based on a standard curve.

Statistical Analysis
The data were summarized as the mean ± SE (standard error) for all data sets. The data were then subjected to a one-way analysis of variance (ANOVA) using SPSS 26.0. Differences among means were tested using a Student-Newman-Keuls (S-N-K) test for multiple comparisons. All experiments were performed with three biological replicates. Each biological replicate was performed with three technical repetitions. Differences were considered statistically significant at the 5% level (p < 0.05).

Sequence Analysis of DcTPS cDNA
The full-length cDNA sequence of DcTPS was cloned and deposited in the GenBank database (MT675285). DcTPS cDNA is made up of 2162 nucleotides with an open reading frame (ORF) of 1785 nucleotides (Figure 1), which encodes a protein of 594 amino acids with a predicted molecular mass of 67.057 kDa and a theoretical isoelectric point (pI) of 4.82. DcTPS has two glycosylation sites. The BLAST analysis revealed that DcTPS shares 76% similarity identity with other insects' TPS genes. Multiple sequence alignments showed that two signatures (HDYHL and DGMNLV) unique to TPS were well conserved in DcTPS (Figure 2). The phylogenetic tree showed that the DcTPS deduced amino acid sequence was more closely related to TPS from Hemiptera (Acyrthosiphon pisum and Diuraphis noxia) than its counterparts from Diptera, Lepidoptera, Orthoptera and Coleoptera ( Figure 3).

Sequence Analysis of DcTPS cDNA
The full-length cDNA sequence of DcTPS was cloned and deposited in the GenBank database (MT675285). DcTPS cDNA is made up of 2162 nucleotides with an open reading frame (ORF) of 1785 nucleotides (Figure 1), which encodes a protein of 594 amino acids with a predicted molecular mass of 67.057 kDa and a theoretical isoelectric point (pI) of 4.82. DcTPS has two glycosylation sites. The BLAST analysis revealed that DcTPS shares 76% similarity identity with other insects' TPS genes. Multiple sequence alignments showed that two signatures (HDYHL and DGMNLV) unique to TPS were well conserved in DcTPS (Figure 2). The phylogenetic tree showed that the DcTPS deduced amino acid sequence was more closely related to TPS from Hemiptera (Acyrthosiphon pisum and Diuraphis noxia) than its counterparts from Diptera, Lepidoptera, Orthoptera and Coleoptera ( Figure  3).

Developmental Stage-Specific Expression Pattern of DcTPS
The relative expression levels of DcTPS at various stages were determined by RT-qPCR. The results suggested that DcTPS is continuously expressed at all developmental stages (F6,14 = 173.482, p = 0.0001). The expression of DcTPS increased steadily from the egg stage and reached a maximum in fifth-instar nymphs (Figure 4). The expression of DcTPS in adults declined slightly ( Figure 4). As shown by the data, the expression level of DcTPS in the fifth-instar nymphs was 19.13 times higher than in the eggs. At the same time, the expression level in adult D. citri was 8.93 times higher than in the eggs. The different temporal expression patterns evident from the data suggest distinct physiological roles of DcTPS.

Developmental Stage-Specific Expression Pattern of DcTPS
The relative expression levels of DcTPS at various stages were determined by RT-qPCR. The results suggested that DcTPS is continuously expressed at all developmental stages (F 6,14 = 173.482, p = 0.0001). The expression of DcTPS increased steadily from the egg stage and reached a maximum in fifth-instar nymphs (Figure 4). The expression of DcTPS in adults declined slightly ( Figure 4). As shown by the data, the expression level of DcTPS in the fifth-instar nymphs was 19.13 times higher than in the eggs. At the same time, the expression level in adult D. citri was 8.93 times higher than in the eggs. The different temporal expression patterns evident from the data suggest distinct physiological roles of DcTPS.

Phenotype and Survival Rate Analysis after Feeding with dsRNA
With the successful silencing of the DcTPS gene, D. citri subjected to RNAi exhibited abnormal phenotypes after feeding with dsDcTPS ( Figure 5B,C). The data on survival rates of nymphs at 24 and 48 h showed that there were significant differences (p < 0.05) between control and 20, 100 and 500 ng/μL, with average survival rates of 97, 61, 51, 43%, respectively ( Figure 6A

Phenotype and Survival Rate Analysis after Feeding with dsRNA
With the successful silencing of the DcTPS gene, D. citri subjected to RNAi exhibited abnormal phenotypes after feeding with dsDcTPS ( Figure 5B,C). The data on survival rates of nymphs at 24 and 48 h showed that there were significant differences (p < 0.05) between control and 20, 100 and 500 ng/µL, with average survival rates of 97, 61, 51, 43%, respectively ( Figure 6A

Phenotype and Survival Rate Analysis after Feeding with dsRNA
With the successful silencing of the DcTPS gene, D. citri subjected to RNAi exhibited abnormal phenotypes after feeding with dsDcTPS ( Figure 5B,C). The data on survival rates of nymphs at 24 and 48 h showed that there were significant differences (p < 0.05) between control and 20, 100 and 500 ng/μL, with average survival rates of 97, 61, 51, 43%, respectively ( Figure 6A   Furthermore, after continuous feeding on the dsDcTPS-containing diet, the average malformation rate increased to 7, 12 and 26% on treatments after 48 h (F3,8 = 36.970, p = 0.0001) separately. This was significantly higher than malformation rates in the dsGFP group ( Figure 6B).
To demonstrate whether target mRNA was suppressed though feeding on the dsDcTPS diet, the relative expression levels of the DcTPS gene were compared between the dsGFP and dsDcTPS (20, 100, and 500 ng/μL) treatments at 24 h (F3,8 = 343.308, p = 0.0001) and 48 h (F3,8 = 24.442, p = 0.0001). The results indicated that the relative expression level of DcTPS in D. citri decreased with increasing rates of applied dsDcTPS ( Figure 6C). The most serious inhibition level (∼85%) was observed at 500 ng/μL after 48 h ( Figure 6C).
Furthermore, compared to the dsGFP group ( Figure 5A,D), three distinct phenotypic characteristics were evident in D. citri after silencing of the DcTPS gene. Firstly, fifth-instar nymphs Furthermore, after continuous feeding on the dsDcTPS-containing diet, the average malformation rate increased to 7, 12 and 26% on treatments after 48 h (F 3,8 = 36.970, p = 0.0001) separately. This was significantly higher than malformation rates in the dsGFP group ( Figure 6B).
To demonstrate whether target mRNA was suppressed though feeding on the dsDcTPS diet, the relative expression levels of the DcTPS gene were compared between the dsGFP and dsDcTPS (20, 100, and 500 ng/µL) treatments at 24 h (F 3,8 = 343.308, p = 0.0001) and 48 h (F 3,8 = 24.442, p = 0.0001). The results indicated that the relative expression level of DcTPS in D. citri decreased with increasing rates of applied dsDcTPS ( Figure 6C). The most serious inhibition level (∼85%) was observed at 500 ng/µL after 48 h ( Figure 6C).
Furthermore, compared to the dsGFP group ( Figure 5A,D), three distinct phenotypic characteristics were evident in D. citri after silencing of the DcTPS gene. Firstly, fifth-instar nymphs were not able to complete a normal molt before death ( Figure 5E). Secondly, some of the emerged adults fed with dsDcTPS showed "misshapen wings" (Figure 5B,F). Thirdly, partially deformed adults could not get rid of the cuticle from the nymphal stage ( Figure 5C). Individuals from the dsGFP control group did not exhibit prominent variation in phenotype. were not able to complete a normal molt before death ( Figure 5E). Secondly, some of the emerged adults fed with dsDcTPS showed "misshapen wings" (Figure 5B,F). Thirdly, partially deformed adults could not get rid of the cuticle from the nymphal stage ( Figure 5C). Individuals from the dsGFP control group did not exhibit prominent variation in phenotype.

Discussion
In this study, a cDNA sequence encoding TPS from D. citri was cloned and characterized for the first time. Sequence analysis showed that there are HDYHL and DGMNLV motifs in the putative DcTPS amino acid sequence. This result is consistent with previous studies [20,34]. Furthermore, multiple sequence alignment and the phylogenetic tree demonstrate higher identity and a closer evolutionary relationship of DcTPS with TPS from other Hemipteran insects such as A. pisum, D. noxia and N. lugens. These results are in accordance with the fact that TPS genes of similar species might have a closer evolutionary relationship and therefore could be clustered together [3,21,34].
The expression of the DcTPS gene in multiple developmental stages has been reported in S. exigua [34], N. lugens [21], L. decemlineata [39], H. armigera [40] and M. domestica [41]. The result indicates that it might have a unique role in insect growth and development [20]. Like other insects, the expression level of the DcTPS gene was found to be stage specific. Its expression was highest before molting, which has been verified in many insect species [20,23,41]. Therefore, DcTPS may be related to molting, and this is possibly related to the requirement for trehalose during metamorphosis. Trehalose is a stored energy source for flight and can be hydrolyzed by cleavage of the glycosidic linkage to release two molecules of glucose and release of substantial energy to meet demand [26,42]. As a trehalose synthase, energy requirements of adults for flight should result in higher expression of DcTPS at this stage [41]. On the other hand, higher expression in adults may be also related to energy requirements for mating and spawning [26].
Many previous research reports have demonstrated the feasibility of RNAi through feeding [8,11,21,30]. Our results have shown that feeding-based RNAi of the DcTPS gene can specifically restrain the expression of the DcTPS gene and significantly affects nymphal growth and development in D. citri, leading to increased mortality and deformity. Consistent with these studies, RNAi of BmTPS and HvTPS decreased the larval survival rate and caused mortality or deformed phenotypes in B. minax [20] and H. vitessoides [23]. Many adults of D. citri did not molt normally and presented with misshapen wings after molting as a result of feeding on dsDcTPS. There might be many explanations for the molting abnormalities. Firstly, the regulation of chitin biosynthesis is related to the concentration of trehalose substrate and the expression of main genes in the pathway of chitin biosynthesis [21]. As a result of limited energy supply, chitin synthesis is affected when insects undergo metamorphosis [20,24,30,43]. Secondly, paucity of trehalose would weaken protection against abiotic stresses [23,29,32]. Thirdly, insect metamorphosis and wing formation are regulated by the TPS gene [21]. Meanwhile, lack of trehalose might weaken energy metabolism in healthy individuals of D. citri [21,41]. Therefore, there are some negative effects on the growth of nymphs and adults which cause even higher mortality and malformation in D. citri following the knockdown of the DcTPS gene. These results suggest that RNAi technology could be used for in-field control of D. citri [8].
Knockdown of the DcTPS gene significantly decreased DcTPS and trehalose content in D. citri. TPS is involved in trehalose synthesis as an important enzyme [26]. Silencing of the DcTPS gene could further weaken trehalose synthesis and lessen the trehalose content in D. citri. Consequently, a sharp drop in trehalose content could make nymphs more susceptible to stress conditions [41]. Trehalose is not only an energy store but also plays a crucial role in combating the negative effects of stress [26]. Moreover, the content of glucose increased in D. citri after RNAi. The reason behind this result may be that less reactants are involved in the trehalose synthesis chain so that glucose is continually accumulated. Our results are also in keeping with previous research showing that there is a negative correlation between trehalose and glucose content in insects [34].

Conclusions
Full-length cDNA of the DcTPS gene in D. citri was cloned for the first time. Developmental stage expression analysis showed that DcTPS expression was the highest at the fifth-instar nymph stage. In addition, there is a sharp drop in the expression of DcTPS and the number of surviving D. citri individuals attributed to dsRNA-mediated gene-specific silencing. There is also clear evidence of enhanced malformation. The significant change in trehalose and glucose concentration after RNAi suggests that DcTPS is related to trehalose metabolism of D. citri. These results establish a foundation for future studies on the physiological function of the DcTPS gene and provide a potential pest control target for management of D. citri. In our next study, we will design some novel biological insecticides which are directly targeted at D. citri trehalose metabolism and combined with RNAi to control D. citri in the field.