Expression and Role of Vitellogenin Genes in Ovarian Development of Zeugodacus cucurbitae

Simple Summary Vitellogenin (Vg) is the precursor of the yolk protein gene, which is crucial for insect reproduction. In this study, we identified four Vg genes in Zeugodacus cucurbitae (Coquillett). Their molecular characteristics and expression patterns were analyzed. The four genes are mainly expressed in the fat body tissue of adult female melon flies, and their expression is regulated by the juvenile hormone and ecdysone. Nutritional stress significantly down-regulated their expression, indicating that nutrition-dependent vitellogenic development occurs during ovarian development. RNAi-mediated inhibition of the expression of the four genes resulted in significantly delayed ovarian development in Z. cucurbitae. The results indicate that the four genes play an important role in the development of ovaries in Z. cucurbitae. Abstract Vitellogenin (Vg) genes encode the major egg yolk protein precursor in arthropods. In this study, four Vgs were identified in Zeugodacus cucurbitae (Coquillett). Sequence analysis showed that four ZcVgs had the conserved Vg domain. Phylogenetic analysis indicated that four ZcVgs were homologous to the Vgs of Tephritidae insects. The temporal and spatial expression patterns of ZcVgs were analyzed by quantitative real-time polymerase chain reaction (RT-qPCR), and the four ZcVgs showed high expression levels in female adults, especially in the fat body. The expression of ZcVg1 and ZcVg3 was down-regulated by a low dosage (0.5 μg) of 20-hydroxyecdysone (20E), and ZcVg2, ZcVg3, and ZcVg4 were up-regulated by a high dosage (1.0 and 2.0 μg) of 20E. The expression of ZcVg1 and ZcVg2 was up-regulated by 5 μg of juvenile hormone (JH), while all of the ZcVgs were down-regulated by a low and high dosage of JH. Expression of ZcVgs was down-regulated after 24 h of starvation and recovered to normal after nutritional supplementation. After micro-injection of the gene-specific double-stranded RNA, the ZcVgs’ expression was significantly suppressed, and ovarian development was delayed in Z. cucurbitae females. The results indicate that RNA interference of reproduction-related genes is a potential pest control method that works by manipulating female fertility.


Introduction
Vitellogenin (Vg) genes encode the major egg yolk protein precursor in arthropods. In insects, Vg is mainly synthesized in the fat body in a tissue-, sexual-, and stage-specific manner [1]. It is secreted into the hemolymph, transported to the ovary, and absorbed by the receptor in oocytes [2]. The insect Vg protein was first discovered in the cecropia moth, Hyalophora cecropia, as a female-specific protein precursor for vitellin or yolk protein [1]. The Vg protein is a large glycolipophosphoprotein. The molecular weights vary from 150 to 200 kDa for large subunits and 40 to 65 kDa for small subunits belonging to the large lipid transfer protein (LLTP) superfamily [3]. Vg protein sequences are evolutionarily conserved

Insect Rearing
The insect stock was collected from Haikou, Hainan Province, China, in 2016. The adults were reared in an environmentally controlled insectary, with a temperature of 26-27 • C, a relative humidity (RH) of 65-75%, and a photoperiod of 14:10 h (light: dark). As prescribed in previously published work, Z. cucurbitae larvae were reared on an artificial diet consisting of bitter gourd, yeast powder, corn, wheat flour, and sucrose; adults were fed a mixture of sucrose, yeast powder, and honey [27].

RNA Isolation and cDNA Synthesis
Same-aged females were cultured under the same conditions as the stock flies. Five-dayold virgin female adults were collected. The total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA sample's concentration was determined by a NanoDrop One spectrophotometer (Thermo Scientific, Madison, WI, USA), that is, the purity of RNA samples (OD260/280 and OD260/230). The genomic DNA contamination of 1 µg of total RNA was removed using an RQ1 RNase-Free DNase kit (Promega, Madison, WI, USA). The first-strand cDNA was synthesized using the PrimeScript ® RT reagent Kit (TaKaRa, Dalian, China) and stored at −20 • C until use.

Molecular Cloning and Sequence Analysis
The homologous Vgs from Bactrocera dorsalis (AF368053.1, AF368054.1, and ARV91014.1) were used as the query sequences to find the Vgs in Z. cucurbitae with a BLAST search against the genome in the National Center for Biotechnology Information (NCBI) database. Four fragment candidates were downloaded for the full-length confirmation by PCR. The accession number for the melon fly genome was ASM80634v1. The gene-specific primers were designed by Primer Premier 5.0 (PREMIER Biosoft International, Palo Alto, CA, USA) ( Table 1). The PCR amplification was run on a Bio-Rad PCR machine (Bio-Rad, Singapore). PCR was conducted with 25 µL of a mixture consisting of 12.5 µL of 2× Taq PCR Master Mix (Biomed, Beijing, China), 9.5 µL of RNase-free water, 1.0 µL of the cDNA template (described above), and 1.0 µL each of forward and reverse primers (10 µM). PCR amplification was performed using the following procedure: initial denaturation at 95 • C for 3 min, followed by 35 cycles at 95 • C for 30 s, 55 • C for 30 s, 72 • C for 1 min, and a final extension at 72 • C for 10 min. The product was detected by 1% agarose gel electrophoresis. A QIAquick PCR Purification Kit (QIAGEN, Dusseldorf, Germany) was used to purify the positive products. The target product was connected to a pGEM-T by pGEM ® -T Easy Vector (Promega, Madison, WI, USA) and transformed into E. coli Trans 5α (TSINGKE, Beijing, China). Finally, positive clones were obtained through blue-white spot screening and then sequenced. Table 1. Primers used in this study.  The conserved domains of ZcVgs were analyzed by the SMART online website (http: //smart.embl-heidelberg.de, accessed on 16 August 2021) [28]. For the alignment and phylogenetic tree construction, the homologs of Vg in other insects were derived from the NCBI database by BLASTp. Multiple sequence alignment was performed using Clustal Omega and visualized with Jalview 2.10 software [29]. The unrooted phylogenetic tree was constructed by the neighbor-joining method with 1000 bootstrap replicates using MEGA7 software [30].

Spatio-Temporal Expression Revealed by RT-qPCR
Virgin female adults were reared separately and collected to analyze the expression pattern of ZcVgs on each day from 0 to 9 d, with four biological replicates. Total RNA and cDNA preparation was performed as described above. Ribosomal protein subunit 3 (Rps3) and ribosomal protein L13 (Rpl13) were selected as the internal reference genes to explore the expression patterns of ZcVgs at different developmental stages [31]. The tissue samples from the midgut, fat body, Malpighian tubules, and ovary were dissected from 5 d old virgin female adults to determine the expression pattern of ZcVgs. Four biological replicates were prepared, and each sample was dissected from 20 individuals. α-Tubulin (α-Tub) and β-tubulin 1 (β-Tub1) were selected as the internal reference genes to analyze the expression in various tissues of female adults [31]. The primers for the RT-qPCR were designed by Primer Premier 5.0 (Table 1), followed by the amplification efficiency determination. After the primers were synthesized, the standard curve was calculated to evaluate the amplification efficiency. Efficiencies in the range of 90-110% were acceptable. The qPCR reaction was run on a CFX384 Optics Module (Bio-Rad, Singapore) using a NovoStart SYBR qPCR SuperMix Plus Kit (Novoprotein, Shanghai, China). RT-qPCR was conducted with 10 µL of a mixture consisting of 5 µL of 2× qPCR mixture, 3.5 µL of RNase-free water, 0.5 µL of cDNA, and 0.5 µL each of forward and reverse primers (10 µM). The reaction program was 95 • C for 2 min, followed by 40 cycles at 95 • C for 30 s, 60 • C for 30 s, and a final at 60 • C for 1 min, 95 • C for 10 min. Melting curve analysis at 60-95 • C was performed to ensure the specificity of each primer. The gene expression was analyzed using qBase plus software [32].

Hormone-Induced Expression
First, 20E (TCI, Shanghai, China) was diluted in ethanol (Chuandong, Chongqing, China) to produce a stock solution with a concentration of 20 mg/mL. Then, the stock solution was diluted in ethanol to 0.5, 1.0, and 2.0 µg/µL as the work solutions. More than 300 female flies that had emerged on the same day were collected. After that, 1 µL of each solution was dripped onto the pronotum of each female adult on day 4, that is, 0.5, 1.0, and 2.0 µg/fly [33]. After treatment, all females were reared and collected for total RNA isolation after 12, 24, and 48 h. Females with an equal amount of ethanol dripped onto them served as the control group.
The JH analog Methoprene (Sigma, St. Louis, MO, USA) was dissolved in acetone (Chuandong, Chongqing, China) to produce a stock solution with a concentration of 20 mg/mL. The stock solution was then diluted in acetone to 2.5, 5.0, and 10.0 µg/µL. Then, 1 µL of each solution was dripped onto the pronotum of each female adult on day 4, that is, 2.5, 5.0, and 10.0 µg/fly. After treatment, all females were reared and collected for total RNA isolation after 12, 24 and 48 h. The females treated with an equal amount of acetone served as the control group. The Rps3 and actin 3 (Act3) were preliminarily evaluated as the internal reference genes to explore ZcVgs' expression induced by 20E; Rps3 and α-Tub were preliminarily evaluated as the internal reference genes to explore the expression of ZcVgs induced by Methoprene. The expression of ZcVgs was detected by RT-qPCR.

Nutritional Stress
A total of 150 4 d old virgin females were reared and divided into three groups. Fifty females in group I were normally raised with sufficient water and nutrition. The females in group II were provided water only. After 12, 24 and 48 h, the females in groups I and II were collected for total RNA isolation. The females in group III were starved for 24 h and then provided with an adult diet for supplementary nutrition for another 24 h. The females in groups III were collected for total RNA isolation. Rps3 and RPl13 were selected as the internal reference genes to explore ZcVgs' expression patterns under nutritional stress. The expression of ZcVgs was detected by RT-qPCR.

RNA Interference and Functional Analysis
Gene-specific primers of four ZcVgs were designed for dsRNA synthesis using the Primer Premier 5.0. After amplifying the target fragments of 577, 533, 489, and 571 bp in ORFs, the dsRNAs were synthesized and purified using a Transcript Aid T7 High Yield Transcription Kit (Thermo Scientific, Vilnius, Lithuania) according to the manufacturer's instructions. The concentration of dsRNA was tested with a NanoDrop One spectrophotometer. The integrity of the dsRNA was detected by 1% agarose gel electrophoresis. More than 500 female flies that had emerged on the same day were collected. Based on the spatiotemporal expression pattern of melon fly ZcVgs, the 3 d old Z. cucurbitae females were injected with 2 µg (50 nL) of dsRNA into the last ventral segment using a micromanipulator M3301R (World Precision Instruments, Sarasota, FL, USA). The females who delivered dsGFP fragment (378 bp) were set as the control group. Four biological replicates were conducted. Total RNA and cDNA preparation was performed as described above. Rps3 and α-Tub were selected as reference genes to normalize the expression of ZcVgs on day 5 after RNAi [31]. Ovaries were dissected to determine the size on day 5. Thirty samples were dissected in each treatment, and the ovarian area (mm 2 ) was measured using a Digital MicroscopeVHX-5000 (KEYENCE, Itasca, IL, USA).

Statistical Analysis
Gene expression was analyzed using the qBase plus software [32]. The significant differences among spatio-temporal expression and gene expression induced by the hormones were analyzed by the one-way analysis of variance with the least significant difference method (p < 0.05). The gene-silencing efficiency and phenotype (ovarian size) were analyzed by an independent sample t-test (p < 0.05). All of the analyses were performed by SPSS Statistics 22.0 (IBM, Chicago, IL, USA).

Sequence Analysis and Phylogenetic Analysis
The full-length sequence confirmation showed that ZcVg1, ZcVg2, ZcVg3, and ZcVg4 had open reading frames (ORFs) of 1308, 1275, 1290, and 1296 bp, respectively, encoding 435, 424, 429, and 427 amino acids, respectively. The sequence similarity of these four ZcVgs ranged from 53.90% to 92.81%. The sequence similarity of the four ZcVgs compared to other Dipteran Vgs ranged from 47.30% to 97.45%, while the sequence similarity of Vgs between Dipteran and B. mori Vgs ranged from 2.02% to 2.97% (Table S1). Sequences of the four genes (ZcVg1, ZcVg2, ZcVg3, and ZcVg4) were deposited in the NCBI GenBank database with the accession numbers OL546555, OL546556, OL546557, and OL546558, respectively. Sequence analysis revealed that all four ZcVgs possessed conserved domains with the LPD_N. They had putative cleavage sites ( R / K XX R / K ) and signal peptides. In addition, serine residues (the potential phosphorylation sites) and asparagines (NELV, NGPA, NVIE, and NERN-the glycosylation sites) were identified ( Figure 1). The conserved lipase domains of ZcVg1, ZcVg2, ZcVg3, and ZcVg4 existed in 107-407, 91-399, 96-403, and 101-405 amino acids, respectively. The phylogenetic tree showed that ZcVg3 and ZcVg4 are most closely related to the Vgs of Z. tau, but ZcVg1 and ZcVg2 are most closely related to the Vgs of B. dorsalis ( Figure 2).
Insects 2022, 13, x FOR PEER REVIEW 6 of 15 database with the accession numbers OL546555, OL546556, OL546557, and OL546558, respectively. Sequence analysis revealed that all four ZcVgs possessed conserved domains with the LPD_N. They had putative cleavage sites ( R /KXX R /K) and signal peptides. In addition, serine residues (the potential phosphorylation sites) and asparagines (NELV, NGPA, NVIE, and NERN-the glycosylation sites) were identified ( Figure 1). The conserved lipase domains of ZcVg1, ZcVg2, ZcVg3, and ZcVg4 existed in 107-407, 91-399, 96-403, and 101-405 amino acids, respectively. The phylogenetic tree showed that ZcVg3 and ZcVg4 are most closely related to the Vgs of Z. tau, but ZcVg1 and ZcVg2 are most closely related to the Vgs of B. dorsalis (Figure 2). The signal peptide is underlined by a black line. The height of the yellow/brown bars below the alignments indicates the degree of similarity of the amino acid above.
Putative cleavage sites ( R / K XX R / K ) Putative asparagine-linked glycosylation sites Serine residues Signal peptide

Spatio-Temporal Expression of ZcVgs
The expression patterns of ZcVgs in different developmental stages showed that all of the genes were highly expressed from 5 to 9 d (Figure 3). In particular, ZcVg1 and ZcVg2 were significantly highly expressed in females from 5 to 9 d, with the highest expression at 6 d. ZcVg3 and ZcVg4 were significantly highly expressed in females from 5 to 9 d, with the highest expression at 8 d.

Spatio-Temporal Expression of ZcVgs
The expression patterns of ZcVgs in different developmental stages showed that all of the genes were highly expressed from 5 to 9 d ( Figure 3). In particular, ZcVg1 and ZcVg2 were significantly highly expressed in females from 5 to 9 d, with the highest expression at 6 d. ZcVg3 and ZcVg4 were significantly highly expressed in females from 5 to 9 d, with the highest expression at 8 d. The expression patterns of ZcVgs in different tissues of female adults were analyzed. The results showed that the expression levels of ZcVg1, ZcVg2, ZcVg3, and ZcVg4 in the fat body were significantly higher than those in other tissues (Figure 4). Therefore, the elevated transcript levels in the fat body are consistent with Vg biosynthesis in this tissue. The expression patterns of ZcVgs in different tissues of female adults were analyzed. The results showed that the expression levels of ZcVg1, ZcVg2, ZcVg3, and ZcVg4 in the fat body were significantly higher than those in other tissues ( Figure 4). Therefore, the elevated transcript levels in the fat body are consistent with Vg biosynthesis in this tissue. In addition, these genes are also expressed in the ovary but with low expression levels. The expression patterns of ZcVgs in different tissues of female adults were analyzed. The results showed that the expression levels of ZcVg1, ZcVg2, ZcVg3, and ZcVg4 in the fat body were significantly higher than those in other tissues ( Figure 4). Therefore, the elevated transcript levels in the fat body are consistent with Vg biosynthesis in this tissue. In addition, these genes are also expressed in the ovary but with low expression levels.

Effects of Hormone Induction on ZcVgs' Expression
Four ZcVgs were induced by the exogenous hormone 20E with a different pattern ( Figure 5). After 12 h, the expression of ZcVg2, ZcVg3, and ZcVg4 in the 1 µg treatment group was up-regulated, and the expression of ZcVg4 in the 2 µg treatment group was up-regulated. After 24 h, the expression of ZcVg3 in the 0.5 µg treatment group was down-regulated. After 48 h, the expression of ZcVg1 in the 0.5 µg treatment group was down-regulated.
Four ZcVgs were induced by the exogenous JH analogue Methoprene ( Figure 6). After 12 h, the expression of ZcVg1 and ZcVg2 in the 5 µg treatment group was up-regulated, and the expression of ZcVg3 and ZcVg4 in the 2.5 µg and 10 µg treatment groups was down-regulated. After 24 h, the expression of ZcVg1 and ZcVg2 in the 2.5 µg treatment group was down-regulated, and the expression of ZcVg2 in the 10 µg treatment group was down-regulated. After 48 h, the expression of ZcVg1 in the 2.5 µg treatment group was down-regulated.

Effects of Starvation on ZcVgs' Expression
The results showed that the expression levels of four ZcVgs were down-regulated after 12, 24, and 48 h of starvation (Figure 7). After 12 h of starvation, the expression of ZcVg1 was relatively less affected. Feeding after starvation can significantly induce Vg gene expression and return it to a normal level, or even a higher level than normal, such as in ZcVg3.

Effects of Hormone Induction on ZcVgs' Expression
Four ZcVgs were induced by the exogenous hormone 20E with a different pattern ( Figure 5). After 12 h, the expression of ZcVg2, ZcVg3, and ZcVg4 in the 1 μg treatment group was up-regulated, and the expression of ZcVg4 in the 2 μg treatment group was up-regulated. After 24 h, the expression of ZcVg3 in the 0.5 μg treatment group was downregulated. After 48 h, the expression of ZcVg1 in the 0.5 μg treatment group was downregulated.

Effects of Starvation on ZcVgs' Expression
The results showed that the expression levels of four ZcVgs were down-regulated after 12, 24, and 48 h of starvation (Figure 7). After 12 h of starvation, the expression of ZcVg1 was relatively less affected. Feeding after starvation can significantly induce Vg gene expression and return it to a normal level, or even a higher level than normal, such as in ZcVg3.

Effects of RNAi-Mediated Knockdown on Ovarian Development
The results of RNAi showed that ZcVg1, ZcVg2, ZcVg3, and ZcVg4 were significantly down-regulated by 54.9%, 58.0%, 75.1%, and 82.1%, respectively ( Figure 8A-D). The ovarian area of the dsVgs treatment group was significantly smaller than that of the dsGFP control group, and ovarian development was significantly delayed compared to the control group ( Figure 8E,F).

Effects of Starvation on ZcVgs' Expression
The results showed that the expression levels of four ZcVgs were down-regulated after 12, 24, and 48 h of starvation (Figure 7). After 12 h of starvation, the expression of ZcVg1 was relatively less affected. Feeding after starvation can significantly induce Vg gene expression and return it to a normal level, or even a higher level than normal, such as in ZcVg3.

Effects of RNAi-Mediated Knockdown on Ovarian Development
The results of RNAi showed that ZcVg1, ZcVg2, ZcVg3, and ZcVg4 were significantly down-regulated by 54.9%, 58.0%, 75.1%, and 82.1%, respectively ( Figure 8A-D). The ovarian area of the dsVgs treatment group was significantly smaller than that of the dsGFP control group, and ovarian development was significantly delayed compared to the control group ( Figure 8E,F).

Discussion
The Vg hemolymph proteins are the precursors of yolk protein in the egg. The conserved Vgs have been studied extensively in various animals, both vertebrates and invertebrates, including insects [4]. In this study, we identified four Vgs in Z. cucurbitae based on the genome: ZcVg1, ZcVg2, ZcVg3, and ZcVg4. Multiple Vgs work together to

Discussion
The Vg hemolymph proteins are the precursors of yolk protein in the egg. The conserved Vgs have been studied extensively in various animals, both vertebrates and invertebrates, including insects [4]. In this study, we identified four Vgs in Z. cucurbitae based on the genome: ZcVg1, ZcVg2, ZcVg3, and ZcVg4. Multiple Vgs work together to ensure effective Vg production required for ovarian development. Variations in the Vg gene number in insects may reflect evolutionary selection and strategies to adapt to the environment [2]. Sequence analysis of Vgs revealed that the four ZcVgs possess conserved domains in Vgs and contain putative cleavage sites ( R / K XX R / K ), serine residues, and putative asparagine-linked glycosylation sites. These polyserine bundles are considered to be substrates for kinases. The negatively charged phosphoserine may affect protein solubility or chelate essential metal ions required for yolk production [34]. N-glycans may have a significant role in keeping this large, hydrophobic protein in the hemolymph to improve its transport to the ovary [5]. Vg, composed of the LPD_N for lipid binding, DUF1943, and vWFD, is highly conserved in most insect species except for aphids [35]. In addition, the length of the primary protein structure of the four ZcVgs is much smaller than that of most other order insects [36]. These much smaller Vgs are associated with the mammalian triglyceride lipase gene family [4]. Phylogenetic analysis indicated that ZcVgs are most closely related to the Vgs of other Tephritidae, such as B. dorsalis and Z. tau.
In this study, ZcVg1, ZcVg2, ZcVg3, and ZcVg4 showed similar expression patterns, with the highest expression in the fat body. This expression pattern was similar to that of B. dorsalis, and Vg was synthesized in the fat body [37]. The VgR takes Vg up from the hemolymph and transports it into developing oocytes [4]. Therefore, based on the expression pattern of Vgs in Z. cucurbitae, the specific expression pattern of Vg indicates its function in synthesizing nutrition in the fat body. In contrast, low expression of Vg was observed in the male fat body and testis of B. dorsalis [37]. Analysis of the expression levels of ZcVgs in 0 to 9 d old female adults in Z. cucurbitae revealed that the mRNA level of ZcVgs was very high in 5 to 9 d old adults during the vitellogenic stage. Therefore, the expression of ZcVg1, ZcVg2, ZcVg3, and ZcVg4 is correlated with the ovarian development of Z. cucurbitae. There are similar expression patterns in other insects, such as Bemisia tabaci [38] and Actias selene [39]. However, the up-regulation of Vg expression in some insects is observed earlier, with up-regulation beginning during the pupal stage. For instance, Vg expression is first detected during the late pupal stage in Spodoptera litura [40].
The expression of Vgs in insects is regulated by the ecdysone and JH, while the transcriptional mechanisms are different [1,41]. For instance, the transcription of the Vg gene in Heliothis virescens is only regulated by JH [1], while in T. castaneum, both JH and 20E are required for Vg gene expression [42]. After 20E induction, the expression of ZcVg1 and ZcVg3 was down-regulated by a low dosage, and the expression of ZcVg2, ZcVg3, and ZcVg4 was up-regulated by a high dosage. Only the expression of ZcVg1 and ZcVg2 was up-regulated by 5 µg of JH, while all of the ZcVgs genes were down-regulated by a low and high dosage of JH. The following inferences are possible. The pathways activated by exogenous 20E after induction of a low dose and high dose were different in regulating Vg synthesis. A low dose and high dose of exogenous JH induced the same activation of the Vg synthesis pathway, but 5 µg of exogenous JH inducing the activation of the Vg synthesis pathway differs with a low dose or high dose. The pathway activated by low-dose exogenous 20E to regulate Vg synthesis has the same function as the pathway activated by low-dose and high-dose exogenous JH to promote Vg expression. The activation of the Vg synthesis pathway induced by exogenous 20E at a high dose was the same as that induced by exogenous JH at 5 µg, which inhibited Vg expression. The results of this study indicate that ZcVgs are regulated by the ecdysis hormone and JH, and the regulatory system is relatively complex. However, in the female American cockroach (Periplaneta americana), the Vg gene is activated by JH III and repressed by 20E via cis-regulatory elements in a dose-dependent manner [43]. JH III at doses of 0.1, 1, and 10 µg induced Vg synthesis in a dose-dependent manner, with doses as low as 0.1 µg being sufficient to significantly induce Vg synthesis in Blattella germanica [44]. The transcription of Vg1 and Vg2 in P. americana is induced by JH and inhibited by 20E in a dose-dependent manner [1], while both JH and 20E induce the Vg expression in T. castaneum [42]. Interestingly, in Lymantria dispar, JH induced or even down-regulated the expression of the Vg gene [1]. Both 20E and JH are extremely important for ovarian development by regulating the Vg expression. In Nilaparvata lugens, gene silencing using RNAi demonstrates the importance of JH signaling in ovarian development [45].
Nutrients play a pivotal role in insect vitellogenesis [2]. In this study, during ovarian development, the expression of ZcVgs was down-regulated after 24 h of starvation in melon flies, and the expression of ZcVgs could return to normal levels after nutritional supplementation. These results are similar to the effect of nutrition on the expression of Vg in T. castaneum, in which the starvation of female T. castaneum caused Vg synthesis to be blocked but did not cause oocyte development to enter the quiescent phase. Vg synthesis continued and oocyte development shifted from the quiescent phase to the mature phase after nutritional supplementation [17].
Gene silencing using RNAi is an effective method to explore gene function [46], and its use has proven successful in Diptera [47]. In this study, gene-specific dsRNA suppressed the ZcVgs' transcription and delayed ovarian development. These results indicate that ZcVgs play an important role in the development of the ovary in Z. cucurbitae. Interference with the expression of these four genes had a negative effect on ovarian development. This suggests that the expression of ZcVg1, ZcVg2, ZcVg3, and ZcVg4 is required for ovarian development. It also demonstrates that multiple Vgs work together to ensure effective production of the Vg required for ovarian development. In addition, RNAi-mediated inhibition of ZcVgs showed an off-target effect as the conserved domain in sequences. It was speculated that the expression of one ZcVg was affected by the other ZcVgs. This off-target silencing of Vgs was also found in Haemaphysalis longicornis; after silencing each Vg, the expression of other Vgs was also significantly reduced, and the body weight and egg weight of ticks were significantly reduced [48]. In any case, the role of ZcVgs in ovarian development was validated by the RNAi work, showing potential targets for pest control. Since these four Vg sequences have a high similarity, this conserved sequence may be an excellent fragment target for pest control based on RNA interference. Previous studies have shown that the successful reproduction of insects depends on the production and deposition of Vg and the uptake of Vgs by VgR in developing oocytes [49]. The silencing of Vgs also leads to decreased reproduction in other insects and abnormal ovarian development. For example, suppressing C. lectularius Vg caused ovarian tissue atrophy and reduced egg production [20].

Conclusions
In this study, four ZcVgs were identified and cloned. Their molecular characteristics and expression patterns were analyzed. ZcVgs are mainly expressed in the fat body tissue of adult female melon flies. In addition, the expression of ZcVgs is regulated by JH and 20E in a dose-dependent manner. Nutritional stress significantly down-regulated the ZcVgs' expression, indicating that nutrition-dependent vitellogenic development occurs during ovarian development. RNAi-mediated inhibition of the ZcVgs' expression resulted in significantly delayed ovarian development. These results indicate that ZcVgs play an important role in the ovarian development of Z. cucurbitae. The results demonstrate that Vgs are potential candidates for a pest control method that works by suppressing their expression using RNAi to manipulate ovarian development.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/insects13050452/s1, Table S1. Identities of amino acid sequences of ZcVgs compared to other insect Vgs.