Silencing of vitellogenin gene contributes to the promise of controlling red palm weevil, Rhynchophorus ferrugineus (Olivier)

Red palm weevil [Rhynchophorus ferrugineus (Olivier)], is native to South Asia and expanding its distribution range globally. Recent invasions of red palm weevil around the world, including Saudi Arabia, has become a global constraint for the production of palm species. Although, several control measures have been tested, none of them seemed successful against this invasive species. Therefore, we focused on silencing the reproduction control gene vitellogenin (Vg) based on RNA interference (RNAi) strategy for its possible application in the management of R. ferrugineus. The Vg is a major yolk protein precursor critical for oogenesis. To do this, fat body transcriptome of R. ferrugineus female adults was sequenced, which provided partial Vg gene transcript (FPKM 5731.60). A complete RfVg gene transcript of 5504 bp encoding 1787 amino acids was then sequenced using RCAE-PCR strategy and characterized. Phylogenetic analysis suggested that RfVg has closer ancestry to the coleopteran insects. The RfVg-based RNAi significantly suppressed the expressions of Vg gene. The 15, 20 and 25 days post-injection periods suppressed Vg expressions by 95, 96.6 and 99%, respectively. The suppressed Vg expressions resulted in the dramatic failure of Vg protein expression, which caused atrophied ovaries or no oogenesis and ultimately eggs were not hatched. These results suggest that knockdown of Vg gene involved in R. ferrugineus reproduction is a promising target for RNAi-based management of R. ferrugineus.


Results
Full-length sequencing of RfVg and structural analysis. The complete RfVg gene transcript was of 5504 bp, which encoded 1787 residues mature protein representing all conserved structures typical of insects' Vgs. In the Vg protein analysis, first twenty amino acid residues were predicted as signal peptide [analyzed with the SignalP program (www. cbs. dtu. dk/ servi ces/ Signa lP/)]. The deduced R. ferrugineus Vg protein contained five putative cleavage recognition sites, i.e., RRSR (361-364), RSRR (362-365), REGR (593-596), RLAR (666-669) and RPQR (1679-1682). In addition, all conserved motifs such as DGXR and GL/ICG, which are usually found at C-terminus region of Vgs also existed in the Vg of R. ferrugineus. The sequence of DGXR motif was DGKR (amino acids position 1,622-1,625), while of GL/ICG motif was GLCG (amino acids position 1,641-1,644). The RfVg contained 27 cysteine residues, of which seven were located at the C-terminus. As predicted by the NetNGlyc 1.0 program (www. cbs. dtu. dk/ servi ces/ NetNG lyc/), RfVg contained 10 putative glycosylation sites (NXT/S). Moreover, RfVg contained 149 putative phosphorylation sites, including 90 serine (S), 26 threonine (T) and 33 tyrosine (Y). Moreover, three conserved domains were identified in the RfVg by using NCBI conserved domain database (CDD) search (www. ncbi. nlm. nih. gov/ Struc ture/ cdd/ wrpsb. cgi). Among these conserved domains, one was Vg_N spanning from amino acids 21 to 735. The second domain was unknown function 1943 (DUF1943) spanning from amino acid residues 769 to 1059. The third was Von Willebrand factor domain (VWD) located at the C-terminus and spanned from amino acid residues 1467 to 1657 (Fig. S1). To elucidate the evolutionary relationship of RfVg, a neighbor joining phylogenetic tree was generated based on 99 insect and non-insect Vgs sequences present in the NCBI database (Fig. 1). The sequence of RfVg was grouped with other coleopteran Vgs as expected. The evolutionary relationship based on the current phylogenetic tree analysis showed that Vgs from insects are closer to the Vg of nematode and arachnids as compared to Vg of vertebrate and crustacean.
Expression pattern and developmental traits of RfVg gene. The RT-PCR was conducted to determine the sex, tissue, and stage-specific expression and probe the temporal profiles of RfVg gene transcription. The RT-PCR studies were performed by amplifying RfVg-specific region by using gene-specific primers (RfVgRTF2, RfVgRTR2) ( Table S1). The RfVg gene was exclusively expressed in the female fat body cells as demonstrated by a single band, whereas no expression was observed for other tissue ( Fig. 2A). The expression of tubulin gene -an internal control-in all tissues confirms the quality of the cDNAs used in these studies. To analyze the temporal expression profile of RfVg gene, total RNAs were extracted from the fat body of adult R. ferrugineus females up to three weeks. The RT-PCR was conducted to determine the developmental profile of RfVg gene transcription. The expression of RfVg gene was detected from the first day of R. ferrugineus female adults, which was still present inside the cocoons but with light bands. The expression of RfVg gene and intensity of the bands gradually increased after the emergence of adult females from the cocoons. The expression level of RfVg gene remained almost the same from day 10 to 21 (Fig. 2B). Biological studies to assess the impact of RfVg gene silencing in knockdown phenotypes. The phenotypic reflection of Vg gene knockdown in adult females was evaluated by analyzing pre-oviposition period   www.nature.com/scientificreports/ to assess a possible delay in egg laying, oviposition period, post-oviposition period, total number of eggs laid per female, eggs hatchability percentage, and female lifespan ( Fig. 5A-F) in addition to ovarian development. The RfVg knockdown greatly influenced pre-oviposition period in dsRNA-treated females as compared to control groups. The data represented that pre-oviposition periods were significantly different and delayed in dsRNAtreated females (df = 2, F = 14.9, P ˂ 0.0001) in RfVg-dsRNA-treated females as compared to the females in NFW and NI groups (Fig. 5A). Likewise, a significant difference (df = 2, F = 6.08, P ˂ 0.007) was recorded in the postoviposition period of RNAi-treated females and those in NFW and NI groups (Fig. 5C). A significant difference was also noted in total number of eggs laid per female (df = 2, F = 13.3, P ˂ 0.0001) where RfVg-dsRNA-injected group laid lesser number of eggs as compared to NFW and NI groups (Fig. 5D). Moreover, no eggs hatchability (or 0%) was observed in RfVg-dsRNA-injected group, whereas NFW and NI groups recorded 75 and 79% eggs hatchability, respectively (Fig. 5E). However, non-significant differences were recorded for oviposition period (df = 2, F = 1.2, P = 0.3) (Fig. 5B) and adult female lifespan (df = 2, F = 1.1, P = 0.3) (Fig. 5F).

Silencing of
The investigations on ovarian development in RfVg-dsRNA-injected females also revealed remarkable phenotypic repercussions. The ovaries were decreased with lesser yolk compared the ovaries from NFW and NI groups where oocytes were well-developed and ovaries were larger in size (Fig. 6A). Moreover, a drastic decrease in the egg size was observed in RNAi-treated females as compared to the control groups (Fig. 6B).
The length (df = 2, F = 170.4, P ˂ 0.0001) and width (df = 2, F = 73.7, P ˂ 0.0001) of eggs significantly differed among treatment groups (Fig. 7). Overall, low egg production, delayed oviposition periods, smaller-sized eggs with no hatchability were recorded for RfVg-dsRNA-injected females suggesting that RNAi-targeting Vg gene has the ability to inhibit reproduction in R. ferrugineus. To test the statistical significance among different biological parameters, one-way analysis of variance was performed at (α = 0.05). Different letters above the bars show significant difference among groups. www.nature.com/scientificreports/

Discussion
Red palm weevil [Rhynchophorus ferrugineus (Olivier)] has become a noxious pest of palm trees around the world. It has gained significant importance due to its global invasion and associated economic costs. Recent molecular studies have revealed that Vg structures and functions seemed conserved across diverse insect taxa, although some deviations exist in post-translational processing/number of cleavage sites, number of Vg genes, and in the hormonal system regulating these genes 6,44,45 . The Vgs proteins have been sequenced and characterized from several insect species representing different orders, including dictyoptera 14,15,25,46 , hemiptera 27 , lepidoptera 16 , hymenoptera 47,48 , diptera 49 , and coleoptera 50-52 due to their prime importance in insect reproduction. However, there is no information available on molecular mechanism of R. ferrugineus reproduction. The present study, therefore, focused on molecular characterization, expression profiling, and silencing of the reproduction control gene Vg in R. ferrugineus.
The RfVg gene transcript comprised of 5,504 bp nucleotides that encoded deduced protein of 1,787 amino acids. The molecular weight of RfVg protein was 210 kDa, which is almost similar to other insects' Vgs, including coleopterans 50,51,53 . Like other coleopterans, conserved domains were present in RfVg (Fig. S1). Conserved domains play critical role in organisms' physiology 54,55 . The RfVg protein have 5 post-translational cleavage sites without polyserine clusters, which shows robust structural similarity with known Vgs of coleopteran [50][51][52] and some other insects including cotton leafworm (Spodoptera litura) 16 , parasitoid wasp (Encarsia formosa) 56 , and fire ant (Solenopsis invicta) 57 . The precursor Vg in most of the insects are post-translationally modified and   www.nature.com/scientificreports/ proteolytically cleaved at a consent cleavage site (RXXR) into subunits by dibasic endoproteases 58 . Moreover, presence of 149 phosphorylation sites (S, T and Y) in RfVg protein sequence (Fig. S1) indicated that RfVg is highly phosphorylated similar to several other insects 50,51,59 . In addition, the presence of GL/ICG motif and cysteine residues at C-terminus are essential for oligomerization 60,61 . Furthermore, this study first time reports the data, which support potential use of RfVg gene silencing as a tool for the management of R. ferrugineus. The gene silencing approach has been successfully demonstrated against several specific genes in targeted insects, including Vg using different delivery methods, i.e., injection, feeding, and drops [30][31][32][33][34][35][36][37][38][39]62,63 .
The results of RfVg-based RNAi revealed a drastic decrease in the expressions of Vg gene in RfVg-dsRNA group as compared to females in NFW and NI groups (Fig. 3). Almost 95.3% suppression was noted in the expressions of Vg mRNA for RfVg-dsRNA injected group 15 days after injection, whereas 96.6% and 99.4% suppression was recorded on 20 and 25 days after injection, respectively. These findings clearly demonstrate that expressions of RfVg gene in R. ferrugineus females was strongly affected by RfVg-dsRNA treatment. The drastic reduction in Vg mRNA level also confirm the sensitivity of R. ferrugineus to RNAi. Generally, literature indicates that coleopteran species are more sensitive to RNAi than other insect groups as shown in western corn rootworm (Diabrotica virgifera) 37 , red flour beetle (Tribolium castaneum) 64 , Colorado potato beetle (Leptinotarsa decemlineata) 65 , and cotton boll weevil (Anthonomus grandis) 62 . For example, Vg gene expressions in cotton boll weevil were reduced by 97% just within 72 h after injection 62 . The persistence of RfVg-dsRNA was examined on 15, 20, and 25 days after injection in the current study in addition to reproduction performance and a drastic reduction in RfVg expression (99%) was observed within 25 days after injection. Likewise, results of western corn rootworm neonates fed on V-ATPase-A gene based transgenic plants indicated the silencing of a particular gene, which lead to death of treated neonates 37 . Likewise, feeding of Actin and Copβ gene based dsRNA expressed bacteria to Colorado potato beetle reduced growth and caused mortality 65 . In contrast, RNAi efficiency is less supported in other insect groups, i.e., lepidopteran insect species such as light brown apple moth (Epiphyas postvittana) where third instar larvae were fed with carboxylesterase-based dsRNA through droplets and < 50% mRNA silencing was achieved 66 . The major factor between coleopteran and lepidoptera RNAi efficiency includes the uptake of dsRNA and its processing to siRNA. The cells and tissues in coleopteron insects uptake dsRNA quickly and process it into siRNA faster than lepidopteran species 41 . Similarly, RNAi indicated 50 and 40% efficiency against mid-gut protein tsetse EP and nitroporin-2 in Glossina morsitans morsitans and Rhodnius prolixus, respectively 67,68 .
Additionally, a striking reduction in the expressions of RfVg gene resulted in dramatic failure of Vg protein expression in the RfVg-dsRNA-injected group as compared to NFW and NI (Fig. 4). Silencing of Vg gene not only caused failure of Vg protein expression, but also affected the transport of other associated nutrients 63,69 .
Furthermore, present study also confirmed the consequences of RfVg-dsRNA on reproductive performance of R. ferrugineus females, where several parameters were observed, including pre-oviposition and oviposition periods, fecundity, egg hatchability, post-oviposition period, and female lifespan. No egg hatchability was recorded for RfVg-dsRNA-injected group, 75.2 and 78.6% egg hatchability was found in NFW-treated and NI groups, respectively (Fig. 5E). No egg hatchability was the result of significant reduction in expression level of RfVg gene, which caused inadequate production of vitellogenin protein to confirm normal egg size and hatchability. Moreover, this study revealed a significant difference in number of eggs laid per female among the treatment groups (Fig. 5D) in addition to no egg hatchability. Likewise, eggs' viability was dramatically decreased (˂1%) in Vg dsRNA-injected females of cotton boll weevil and eggs were unable to hatch. However, injection of Vg-dsRNA had no effect on fecundity of this pest 62 . Moreover, in bedbug (Cimex lectularius) Vg-dsRNA injection radically suppressed egg production compared to control group and egg production was entirely ceased two weeks after Vg-based dsRNA injection 63 . Additionally, results of Vg dsRNA injection also decreased the eggs production in lubber grasshopper (Romalea microptera) 69 .
The pre-oviposition periods were delayed in dsRNA-treated females. The effect of Vg knockdown on preoviposition period has been reported earlier 63 ; however, no effect of RNAi on pre-oviposition period of cotton boll weevil has also been reported 62 . Besides, present study revealed that ovaries were rigorously deformed with short and unorganized eggs and ovary size (Fig. 6A,B). The size of eggs was significantly decreased in RfVg-dsRNA-treated group as compared to NFW and NI groups (Fig. 7). Similarly, eggs development in cotton boll weevil was severely affected in Vg dsRNA-injected group 62 . Moreover, results of RNAi injection revealed shrunken ovaries in bedbug with no developed oocytes in Vg dsRNA-treated female as compared to control where ovaries were normal in size with mature oocytes 63 . Likewise, ovaries were rigorously deformed in rice moth having small ovarioles and disorganized egg sizes in Vg dsRNA-treated females relative to un-injected females 31 .
The present findings along with previous reports conclusively demonstrate a high potential of Vg-based RNAi technology for pest management. Silencing of RfVg gene provides evidence that RNAi technology could be a smart alternative to traditional management methods for coleopteran pests, particularly for R. ferrugineus. Undoubtedly, the success and effectiveness of RNAi varies with species, selection of target genes, and the mode of dsRNA delivery 40,41,70 . The present study has provided evidence that Vg gene is the best target for RNAi-based management of R. ferrugineus. Choosing a suitable tactic to deliver the dsRNA successfully after selection and identifying the target gene is a main challenge in RNAi-based plant protection method. Although microinjection is a suitable approach for functional genomic studies, this strategy is not appropriate to manage the pest in the field. However, numerous developments on dsRNA delivery made this technique more efficient in the field. For example, delivering dsRNA to insect pests through transgenic plant has been tried 37 . Moreover, successful feeding of insects pest via bacterially expressed dsRNA 65 and application of dsRNA through nanoparticles 71 have also been practiced. The silencing of RfVg gene by RNAi may have the potential to stop the reproduction of R. ferrugineus and RfVg could be an auspicious target candidate gene for developing an alternative pest management strategy for the pest at molecular level. Therefore, the future research should be focused on the delivery of RfVg-dsRNA for the management of R. ferrugineus in the field. This study supports the potential use of emerging www.nature.com/scientificreports/ RNAi technology for pest control and might provide an alternative to the conventional methods being used for the management of R. ferrugineus.

Materials and methods
Rearing of the red palm weevil. Red palm weevils were originally collected from infested date palm trees in Dirab, Kingdom of Saudi Arabia (24.4164°N, 46.5765°E). The adults were provided a piece of cotton saturated with 10% sugar solution 72 and kept in plastic box (L: 17 cm; W: 11 cm; H: 7 cm). The laid eggs were collected with the help of forceps and shifted to wet filter papers placed in small plastic cup (d: 6 cm; h: 2.5 cm). The larvae were fed with artificial diet (250 g/5 larvae) for further development in the plastic box (L: 17 cm; W: 11 cm; H: 7 cm). Finally, the last instar larvae were shifted into a sugarcane set (10 cm) for pupation in plastic boxes (L: 17 cm; W: 11 cm; H: 7 cm). The R. ferrugineus culture was maintained in the growth chamber at 25 ± 1 °C, 70 ± 5% relative humidity 72 .
PCR amplification and sequencing to obtain full length RfVg and phylogenetic analysis. The partial sequence of RfVg gene transcript was obtained through the next-generation sequencing (NGS) of R. ferrugineus fat body tissues from Beijing Genomics Institute (BGI), China. The gene-specific primers (RfVgF1), which were designed based on the partial RfVg sequence and the adopter primer 1 (AP1) (Clontech) ( Table S1) were used for 3ʹ RACE-PCR in order to get the full-length sequence of RfVg gene. The ds cDNA library was subjected to PCR by using the Gene Amp PCR system 9700 thermo cycler (Applied Biosystems, USA). The PCR conditions were; initial denaturation at 94 ºC for 1 min followed by 35 cycles of denaturation at 94 ºC for 30 s, annealing at 68 ºC for 3 min, and a final extension of 68 ºC for 5 min. The amplified PCR products were purified by using illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Sciences, USA). The purified PCR products were sequenced from BGI, China. The obtained sequences were analyzed and checked for homology with other insects Vg sequences by using basic local alignment search tool (BLAST) of National Center for Biotechnology Information (NCBI). Finally, RfVg sequence was submitted to the NCBI GenBank database (accession number ALN38803) after confirmation. The RfVg sequence was aligned to other known insects Vgs sequences available in the NCBI database using the clustalW program 73 . Phylogenetic tree was constructed using neighbor-joining method on MEGA version 6 74 .

Expression pattern and developmental traits of RfVg gene.
To investigate the tissue, genderspecific expression, and developmental profile of RfVg gene transcription, total RNAs were extracted from the female fat body, ovary, mid-gut, muscle, male fat body, and female pupa. Total RNA was extracted from fat body of adult R. ferrugineus females up to three weeks (for one week when the female adults were inside the cocoons and two weeks after the eclosion) by using Tri-RNA reagent (Favorgen Biotech Corp, Taiwan) to analyze the developmental expression profile of RfVg gene.The RNA samples were treated with DNase I (Invitrogen, USA) to remove DNA contamination. A 2-μg of total RNA from each sample was reverse transcribed to cDNA using ReverTra Ace cDNA synthesis kit (Toyobo Co. Ltd, Japan). The reverse transcription polymerase chain reaction (RT-PCR) was performed by using gene-specific primers (RfVgRTF1, RfVgRTR1, RfVgRTF2, RfVgRTR2) and tubulin was used as internal control (TubulinRfer-F, TubulinRfer-R) ( Table S1). The cDNA was subjected to RT-PCR by using Gene Amp PCR system 9700 thermo cycler (Applied Biosystem, USA). The following thermal programs were applied; 94 ºC for 1 min for denaturation followed by 30 cycles of 94 ºC for 30 s, 68 ºC for 30 s, and 72 ºC for 1 min. The PCR amplified products were run on 2% agarose gel, stained with ethidium bromide, and visually confirmed under ultra violet (UV) light by using gel documentation BioDocAnalyze system (Biometra, Germany). The experiment consisted of three biological treatment groups, which were RfVg-dsRNA-injected, nuclease free water (NFW)-injected, and no injection (NI) 31,39,[76][77][78] . A total 2-μg (10-μl) of RfVg-dsRNA was injected dorsally in the second abdominal segment of each 10-12 days-old female pupae by using 0.5 ml BD Micro-Fine tm Plus syringe (Becton, Dick-inson Co, USA), whereas 10-μl of NFW was injected as control. The pupae used for injection were taken from the cocoons, which were placed again in the cocoon after injection for further development and kept in growth chamber at 25 ± 1 °C, 70 ± 5% relative humidity and photoperiod of 12:12 (L:D) 5 . When pupae converted to adults, they remained inside the cocoon for almost one week before eclosion. The female pupae were selected on the base of snout as R. ferrugineus female has smooth narrow long snout, while male has shorter and wider snout with some tuft hairs. Furthermore, females were also confirmed after adult eclosion. The newly emerged females (from cocoon) were shifted to a separate box having a piece of cotton saturated with 10% sugar solution. The females were harvested on three post-injection periods (i.e., 15, 20, and 25 days) for the collection of fat body (for cDNA synthesis to analyze Vg transcript levels), hemolymph (for Vg protein expression analysis) and ovaries. The RNAi experiments were validated using quantitative real time polymerase chain reaction (qRT-PCR), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and by observing phenotypic effects of RNAi on ovarian development. The cDNA and hemolymph samples were prepared for all biological groups. Total RNAs were extracted from the fat body by using Tri-RNA reagent (Favorgen Biotech Corp, Taiwan) to make cDNA. The RNA samples were treated with DNase I (Invitrogen, USA) to remove the DNA contamination. A 2-μg of total RNA from each sample was reverse transcribed to cDNA using ReverTra www.nature.com/scientificreports/ Ace cDNA synthesis kit (Toyobo Co. Ltd, Japan). Hemolymph was collected with the help of micropipette after the snout of weevil was amputated using a fine scissors and diluted to 1:50 with the sample buffer.

Validation of RfVg gene silencing through qRT-PCR. A qRT (quantitative real time)-PCR, analysis
was performed by using the RfVg-gene-specific primers (RfVgRTF3 and RfVgRTR3) to corroborate the impact of RfVg-based RNAi on Vg gene expression (Table S1). Expression levels of the RfVg gene were normalized by quantifying the expression levels of tubulin (a housekeeping gene) using TubulinRfer-F and TubulinRfer-R primers (Table S1). The qRT-PCR trials were designed based on three biological groups, which were RfVg-dsRNA, NFW, and NI. All groups contained three replicates, each replicate had a single animal, while there were three technical replicates. The qRT-PCR was accomplished using CFX-96 Touch™ Real-Time PCR Detection System (BioRad, USA), while reactions (each contained a volume of 20-μl) were performed using SsoAdvanced™ Universal SYBR ® Green Supermix (BioRad, USA). The following qRT-PCR conditions were applied for amplifying the cDNA; 95 ºC for 30 s, 40 cycles of 95 ºC for 15 s, 60ºC for 60 s, followed by melting curve analysis at 65-95 ºC with an increment of 0.5 ºC every 5 s. The 2 −∆∆CT method was used to analyze the relative expression levels of RfVg gene by normalizing them to tubulin and control (NI) group.
Validation of RfVg gene silencing through SDS-PAGE. The Vg protein expression levels in dsRNAinjected females were analyzed through SDS-PAGE as reported previously 15,27 to affirm the efficiency of RNAi. Protein analyses were conducted using samples of hemolymph and egg extracts (each 10 μl) run on 8% polyacrylamide gels. Three post-injection periods, i.e., 15, 20 and 25 days (after injection with dsRNA) with three biological groups (RfVg-dsRNA, NI, and NFW) were analyzed. All groups contained three replicates; each replicate was an individual of R. ferrugineus, while there were three technical replicates. The Vg protein expression levels in RfVg-dsRNA injected-weevils were compared with the NI and NFW groups. The gels were stained with Coomassie blue and washed with de-staining solution. The protein bands were visualized under the white light and photographed by using gel documentation BioDocAnalyze system (Biometra, Germany).
Biological studies to assess the impact of RfVg gene silencing in knockdown phenotypes. The phenotypic manifestation of RfVg gene silencing in RNAi-treated R. ferrugineus females was assessed based on the biological markers including pre-oviposition periods, oviposition periods, total number of eggs laid per female, eggs hatchability %, post-oviposition periods, and female life span in addition to the ovarian development. To inspect these biological traits, newly emerged adult females (dsRNA-injected) were paired with the normal males, transferred to a separate plastic box (1 kg) having a piece of cotton saturated with 10% sugar solution and kept in growth chamber at 25 ± 1 °C, 70 ± 5% relative humidity 72 . There were again three biological groups, i.e., RfVg dsRNA-injected (dsRNA), NFW-injected, and NI. All groups contained nine replicates and each replicate was an individual pair (normal male + dsRNA-treated female) of R. ferrugineus. All pairs were allowed to mate and lay eggs until females died. The oviposition periods were observed and the number of eggs and hatchability percentage were scored. Completely randomized design (CRD) was used in this experiment.
To investigate the impact of Vg-based RNAi on ovarian development, ovaries from all three groups (RfVg-dsRNA, NFW and NI) were isolated in the phosphate buffered saline (PBS) 20 days post-injection periods, viewed under stereomicroscope (DM 165 C, Leica, Wetzlar, Germany) and photographed using auto-montage software system (Syncroscopy, Cambridge, UK) to measure the ovarian development. Eggs from dsRNA-injected groups were compared with NFW and NI groups, observed under the stereomicroscope and photographed (Leica MZ 125, Helicon focus 6 software, Germany). Furthermore, 15 eggs from each group (NI, NFW, and RfVg-dsRNA injected groups) were measured using Dino-Lite Digital Microscope AM4815ZT (AnMo Electronic Corp, USA) to clarify the impact of RfVg based RNAi on egg size (length and width).
Statistical analysis. The qRT-PCR quantification results were analyzed following 2 −∆∆CT method (Livak and Schmittgen, 2001). One-way analysis of variance was performed at (α = 0.05) by using SAS program ver. 9.2. To analyze the statistical significant differences among three experimental groups (RfVg-dsRNA, NI and NFW) for qRT-PCR data and biological studies 75 . Ethics approval. The work is original. Moreover, no legal permission was required to conduct the experiments.

Data availability
All the data is present in the manuscript.