Conserved microRNAs miR-8-3p and miR-2a-3 targeting chitin biosynthesis to regulate the molting process of Sogatella furcifera (Horváth)(Hemiptera: Delphacidae).

Molting is a key solution to growth restriction in insects. The periodic synthesis and degradation of chitin, one of the major components of the insect epidermis, is necessary for insect growth. MicroRNA (miRNA) have been implicated in molting regulation, yet their involvement in the interplay interaction between the chitin synthesis pathway and 20-hydroxyecdysone signaling remains poorly understood. In this study, soluble trehalase (Tre1) and phosphoacetylglucosamine mutase (PAGM) were identified as targets of conserved miR-8-3p and miR-2a-3, respectively. The expression profiles of miR-8-3p-SfTre1 and miR-2a-3-SfPAGM exhibited an opposite pattern during the different developmental stages, indicating a negative regulatory relationship between them. This relationship was confirmed by an in vitro dual-luciferase reporter system. Overexpression of miR-8-3p and miR-2a-3 by injection of mimics inhibited the expression of their respective target genes and increased mortality, leading to death in the pre-molting, and molting death phenomena. They also caused a decrease in chitin content and expression levels of key genes in the chitin synthesis pathway (SfTre1, SfTre2, SfHK, SfG6PI, SfGFAT, SfGNA, SfPAGM, SfUAP, SfCHS1, SfCHS1a, and SfCHS1b). Conversely, the injection of miRNA inhibitors resulted in the upregulation of the expression levels of these genes. Following 20E treatment, the expression levels of miR-8-3p and miR-2a-3 decreased significantly, while their corresponding target genes increased significantly. These results indicate that miR-8-3p and miR-2a-3 play a regulatory role in the molting of Sogatella furcifera by targeting SfTre1 and SfPAGM, respectively. These findings provide new potential targets for the development of subsequent new control strategies.

Wing expansion functional analysis of ion transport peptide gene in Sogatella furcifera (Horváth) (Hemiptera: Delphacidae).

Ion transport peptide (ITP), a superfamily of arthropod neuropeptides, serves a crucial role in regulating various physiological processes such as diuresis, ecdysis behavior, and wing expansion. However, the molecular characteristics, expression profile, and role of ITP in Sogatella furcifera are poorly understood. To elucidate the characteristics and biological function of ITP in S. furcifera, we employed reverse transcription-polymerase chain reaction (RT-PCR) and RNA interference (RNAi) methods. The identified SfITP gene encodes 117 amino acids. The expression of SfITP gradually increased followed the formation of 3-day-old of 5th instar nymph, peaking initially at 40 min after eclosion, and reaching another peak 24 h after eclosion, with particularly high expression levels in thorax and wing tissues. Notably, SfITP RNAi in 3rd instar nymphs of S. furcifera significantly inhibited the transcript levels of SfITP, resulting in 55% mortality and 78% wing deformity. These findings suggests that SfITP is involved in the regulation of wing expansion in S. furcifera, providing insights into the regulation of insect wing expansion and contributing to the molecular understanding of this process.

Buprofezin affects the molting process by regulating nuclear receptors SfHR3 and SfHR4 in Sogatella furcifera.

Nuclear receptors play a crucial role in various signaling and metabolic pathways, such as insect molting and development. Buprofezin (2-tert-butylimino-3-isopropyl-5-phenyl-perhydro-1, 3, 5-thiadiazin-4-one), a chitin synthesis inhibitor, causes molting deformities and slow death in insects by inhibiting chitin synthesis and interfering with their metabolism. This study investigated whether buprofezin affects insect ecdysteroid signaling pathway. The treatment of buprofezin significantly suppressed the transcription levels of SfHR3 and SfHR4, two nuclear receptor genes, in third-instar nymphs of Sogatella furcifera. Meanwhile, the transcription levels of SfHR3 and SfHR4 in first-day fifth-instar nymphs were induced at 12 h after 20E treatment. In addition, the silencing of SfHR3 and SfHR4 genes in first-day fifth-instar nymphs caused severe developmental delay and molting failure, resulting in a significant reduction of survival rates at 7.36% and 2.99% on the eighth day, respectively. Further analysis showed that the silencing SfHR3 and SfHR4 significantly inhibited the transcription levels of chitin synthesis and degradation-related genes. These results indicate that buprofezin can inhibits chitin synthesis and degradation by suppressing the signal transduction of 20E through SfHR3 and SfHR4, leading to molting failure and death. This study not only expands our understanding of the molecular mechanism of buprofezin in pest control but also lays a foundation for developing new control strategies of RNAi by targeting SfHR3 and SfHR4.

Knockdown of Two Trehalase Genes by RNA Interference Is Lethal to the White-Backed Planthopper Sogatella furcifera (Horváth) (Hemiptera: Delphacidae).

Trehalase (Tre) is a crucial enzyme involved in trehalose metabolism, and it plays pivotal roles in insect development and metamorphosis. However, the biological function of Tre genes in Sogatella furcifera remains unclear. In the present study, two Tre genes-SfTre1 and SfTre2-were cloned and identified based on the S. furcifera transcriptome data. Bioinformatic analysis revealed that the full-length complementary DNA of SfTre1 and SfTre2 genes were 3700 and 2757 bp long, with 1728- and 1902-bp open reading frame encoding 575 and 633 amino acid residues, respectively. Expression analysis indicated that SfTre1 and SfTre2 were expressed at all developmental stages, with the highest expression in day two adults. Furthermore, the highest expression levels of SfTre1 and SfTre2 were observed in the ovary; enriched expression was also noted in head tissues. The knockdown of SfTre1 and SfTre2 via injecting double-stranded RNAs decreased the transcription levels of the corresponding mRNAs and led to various malformed phenotypes and high lethality rates. The results of our present study indicate that SfTre1 and SfTre2 play crucial roles in S. furcifera growth and development, which can provide referable information for Tre genes as a potential target for planthopper control.

SfDicer2 RNA Interference Inhibits Molting and Wing Expansion in Sogatella furcifera.

Endoribonuclease 2 (Dicer2) is a key nicking endonuclease involved in the small interfering RNA biosynthesis, and it plays important roles in gene regulation and antiviral immunity. The Dicer2 sequence was obtained using the transcriptomic and genomic information of Sogatella furcifera (Horváth), and the spatiotemporal characteristics and functions of molting and wing expansion regulation were studied using real-time quantitative polymerase chain reaction and RNA interference (RNAi) technology. The expression of SfDicer2 fluctuated during the nymphal stage of S. furcifera. Its expression decreased significantly over the course of molting. SfDicer2 exhibited the highest transcript level in the nymphal stage and adult fat body. After SfDicer2 was silenced, the total mortality rate was 42.69%; 18.32% of the insects died because of their inability to molt. Compared with the effects of dsGFP or water, 44.38% of the insects subjected to the silencing of SfDicer2 exhibited wing deformities after successful eclosion. After SfDicer2 RNAi, the expression of chitinase, chitin deacetylase, trehalase, chitin synthase 1, and wing expansion-related genes was significantly inhibited. These findings indicate that SfDicer2 controls molting by affecting genes associated with chitin synthesis and degradation and regulates wing expansion by altering the expression of wing expansion-related genes in S. furcifera.

Molecular Characterization of UDP-N-Acetylglucosamine Pyrophosphorylase and Its Role in the Growth and Development of the White-Backed Planthopper Sogatella furcifera (Hemiptera: Delphacidae).

UDP-N-acetylglucosamine pyrophosphorylase (UAP) is a key enzyme in the chitin biosynthesis pathway of insects. Here, we described the gene SfUAP in the white-backed planthopper Sogatella furcifera (Horváth) with an open reading frame of 1470 bp. Quantitative real-time polymerase chain reaction (qPCR) suggested that SfUAP exhibits a different developmental expression pattern and a higher expression after molting. The highest expression of SfUAP was observed in the integument tissues of adults, whereas head tissues showed negligible expression. RNAi-based gene silencing decreased the mRNA transcript levels in S. furcifera nymphs injected with double-stranded RNA of SfUAP. Finally, SfUAP silencing led to 84% mortality and malformed phenotypes in nymphs. Thus, our results can help better understand the role of SfUAP in S. furcifera.

Identification and profiling of Sogatella furcifera microRNAs and their potential roles in regulating the developmental transitions of nymph-adult.

Sogatella furcifera is one of the most serious insect pests that affect rice in Asia. One class of small RNAs (sRNAs; ~22 nt long) is miRNAs, which participate in various biological processes by regulating the expression of target genes in a spatiotemporal manner. However, the role of miRNAs in nymph-to-adult transition in S. furcifera remains unknown. In this study, we sequenced sRNA libraries of S. furcifera prepared from individuals at three different developmental stages (pre-moult, moulting and early adult). A total of 253 miRNAs (134 known and 119 novel) were identified, of which 12 were differentially expressed during the nymph-to-adult developmental transition. Moreover, Real time quantitative PCR (RT-qPCR) analysis revealed that all 12 miRNAs were differentially expressed among five different nymph tissues and 14 different developmental stages (first to fifth instar nymphs and 1-day-old adults). Injection of miR-2a-2 mimic/antagomir and miR-305-5p-1 mimic/antagomir into 1-day-old fifth instar nymphs significantly increased the mortality rate. In addition, a defective moulting phenotype was observed in nymphs injected with miR-2a-2 and miR-305-5p-1, suggesting that these miRNAs are involved in S. furcifera nymph-adult transition. In conclusion, these results reveal the function of critical miRNAs in S. furcifera nymph-adult transition, and also provide novel potential targets of insecticides for the long-term sustainable management of S. furcifera.

Silencing of Decapentaplegic (Dpp) gene inhibited the wing expansion in the white-backed planthopper, Sogatella furcifera (Horváth) (Hemiptera: Delphacidae).

The Decapentaplegic gene controls wing patterning and spreading by regulating downstream genes in many insect species. However, the molecular characteristics, expression, and biological function of Dpp in Sogatella furcifera remain poorly understood. In this study, we cloned the Dpp gene from S. furcifera and examined its expression levels in different development stages, wing typed adults, and tissues. Then, the function of SfDpp gene was analyzed using an RNA interference (RNAi)-based approach. The results showed that the full-length complementary DNA  of the SfDpp gene consists of 1034 bp and contains a 954-bp open reading frame encoding 317 amino acids. SfDpp has a transforming growth factor-ß (TGF-ß) propeptide superfamily domain and a TGF-ß superfamily domain, typical of members of the TGF-ß superfamily. Quantitative real-time polymerase chain reaction showed that the expression of SfDpp reached its highest expression level 40 min after eclosion. RNAi-based gene silencing inhibited transcript levels of the corresponding messenger RNA in S. furcifera nymphs injected with double-stranded RNA of SfDpp and resulted in death of 29.17% and 26.67% of 4th and 5th instar nymphs, respectively. The wing deformity rate of the adults was 74.12% and 3.41% after SfDpp gene silencing in 4th and 5th instar nymphs, respectively. Examining wing development-associated genes showed that two target genes of Dpp (Vestigial and Spalt) were both dramatically downregulated after SfDpp was silenced. Our results demonstrate that downregulated SfDpp in early development causes wing expansion failure in S. furcifera. Thus, Dpp may be a target gene for restricting the migration of rice-damaging planthoppers.

Juvenile Hormone Synthesis Pathway Gene SfIPPI Regulates Sogatella furcifera Reproduction.

The juvenile hormone (JH) is crucial for insect reproduction, and isopentenyl pyrophosphate isomerase (IPPI) is a key enzyme in the JH synthesis pathway. However, few studies have investigated how IPPI regulates insect reproduction. This study identifies and characterizes the IPPI gene (SfIPPI) from the important agricultural pest Sogatella furcifera. A phylogenetic analysis reveals a high homology of SfIPPI with the IPPI amino acid sequences of Laodelphax striatellus and Nilaparvata lugens (Stål). Furthermore, SfIPPI is expressed at various developmental stages and in various tissues of S. furcifera, and is significantly higher on the 5th day of adult emergence and in integument tissue, while lower levels are found on the 3rd day of adult emergence and in fat body and gut tissue. After silencing SfIPPI using RNA interference, the ovarian development is significantly inhibited and the fecundity is significantly reduced when compared with the control group. Additionally, SfIPPI silencing significantly decreases the expression levels of downstream JH signal transduction pathway genes (SfJHAMT, SfFAMeT, and SfKr-h1) and SfVg. Our findings are helpful in elucidating the molecular mechanism underlying the regulation of insect reproduction through genes in the JH synthesis pathway, and they provide a theoretical basis for the development of pest control treatments targeting SfIPPI.

Identification and RNAi-Based Functional Analysis of Four Chitin Deacetylase Genes in Sogatella furcifera (Hemiptera: Delphacidae).

Chitin deacetylases (CDAs) are chitin-degrading enzymes that play a key role in insect molting. In this study, we identified and characterized four full-length cDNAs of CDAs from Sogatella furcifera (Horváth). Developmental expression showed that SfCDA1 and SfCDA2 were expressed at all nymph developmental stages, SfCDA3 and SfCDA4 were mainly expressed in the third-instar to fifth-instar nymph stages, whereas tissue-specific analyses indicated that four CDA genes were mainly high expressed in the integument and head during the fifth-instar nymph. RNA interference (RNAi) results revealed that SfCDA1, SfCDA2, and SfCDA4 are associated with molting defect and high mortality with nymph-adult molting. Furthermore, transcripts of chitin synthase 1 variants (SfCHS1, SfCHS1a, and SfCHS1b) were significantly downregulated and causing significant changes in the expression levels of trehalases (TRE1 and TRE2) in the SfCDA1, SfCDA2, and SfCDA4 dsRNA treatment groups. By contrast, no significant phenotypic characteristics were observed after dsSfCDA3 injection. Taken together, our results suggest that SfCDA1, SfCDA2, and SfCDA4 play a vital role in nymph-adult transition, and these genes could regulate chitin biosynthesis expression levels.

Role of SfJHAMT and SfFAMeT in the reproductive regulation of Sogatella furcifera and its expression under insecticide stress.

The isoprene branching pathway is a unique downstream synthesis pathway of juvenile hormone (JH) in arthropods, which plays an important role in the growth, development, and reproduction of insects. Juvenile hormone acid O-methyltransferase (JHAMT) and farnesoic acid O-methyltransferase (FAMeT) are two key proteins that are regulated in the isoprene branching pathway. Based on the available transcriptomic and genomic data of Sogatella furcifera, full-length cDNAs of SfJHAMT and SfFAMeT were identified. In vitro injection of dsRNA targeted to silence SfJHAMT and SfFAMeT inhibited the fecundity, ovarian development, and transcription levels of SfKr-h1 and SfVg significantly. Of note, The transcription levels of SfJHAMT and SfFAMeT are regulated mutually; i.e., silencing of SfJHAMT causes an increase in the SfFAMeT transcription level and vice versa, and the negative effect of simultaneous silencing on reproduction is greater. The results revealed a coordinated effect of SfJHAMT and SfFAMeT on the reproductive capabilities of S. furcifera. Furthermore, a JH analog (methoprene) partially rescued the negative effect of simultaneous silencing by SfJHAMT and SfFAMeT on reproduction. In addition, the expression profile analysis after insecticide stress showed that triazophos (LC25) can induce the transcription of SfMet and SfKr-h1 to promote JH signal transduction, which affects the transcription of SfVg and ultimately promotes the reproduction of S. furcifera. The results of the present study lay a foundation to further explain the isoprene branch pathway function in insect reproduction and can open up new avenues for sustainable pest control while expanding the current understanding of molecular mechanisms through which insecticides stimulate reproduction and lead to pest resurgence.

Effects of Sublethal Concentrations of Insecticides on the Fecundity of Sogatella furcifera (Hemiptera: Delphacidae) via the Regulation of Vitellogenin and Its Receptor.

White-backed planthopper (Sogatella furcifera, Hemiptera: Delphacidae) is an important migratory pest of rice. It causes severe economic losses by reducing crop production. Vg and VgR are important proteins that help in the successful reproduction of insects and have been studied in many insects. To understand the molecular mechanisms underlying the effects of insecticides on white-backed planthopper reproduction, we studied the expression profiles of SfVg, SfVg-like, and SfVgR in white-backed planthopper exposed to insecticides. SfVg and SfVgR silencing inhibited the ovarian development, number of eggs laid by, and hatching rate of white-backed planthopper. Thiamethoxam LC10 significantly inhibited SfVg-like and SfVgR expression. In contrast, triazophos LC25 significantly promoted SfVg, SfVg-like, and SfVgR expression and increased vitellogenin content in white-backed planthopper. These results demonstrate that insecticides can regulate the reproduction of white-backed planthopper by altering the expression of SfVg and SfVgR, thereby affecting the population density of white-backed planthopper. These findings build a foundation for improving our understanding of the molecular mechanisms underlying the effects of insecticides on the reproduction and resurgence of pests.

Effects of abiotic stress on the expression of Hsp70 genes in Sogatella furcifera (Horváth).

Sogatella furcifera (Horváth), a prominent rice pest in Asia, is a typical R-strategic and highly adaptable insect. Heat shock proteins (Hsps) are highly conserved molecular chaperones regulating responses to various abiotic stresses; however, limited information is available regarding their role in responding to abiotic stress in S. furcifera. This study aimed to investigate the effect of abiotic stresses on the expression of Hsp70 genes in the S. furcifera. Five Hsp70 genes were isolated from S. furcifera, and the expression patterns at different developmental stages and temperatures, upon treatment with different insecticides and ultraviolet A (UV-A) stress, were analyzed. Hsp70 genes were expressed at different developmental stages. Hsp70-2, Hsp70-5, and Hsp70-6 were significantly upregulated upon heat shock at 40 °C for 30 min. Hsp70-3 and Hsp70-4 were significantly upregulated upon heat shock at 30 °C for 30 min. Under UV-A stress, Hsp70-3, Hsp70-4, Hsp70-5, and Hsp70-6 were significantly upregulated. Conversely, Hsp70-2 was significantly downregulated under UV-A stress. The five Hsp70 genes were significantly downregulated in 3rd-instar nymphs on exposure to thiamethoxam, buprofezin, and avermectin at LC10 and LC25 concentrations. Hence, Hsp70 genes significantly contribute to the tolerance of S. furcifera to temperature and UV-A stress; however, they are not involved in the response to insecticides.

Effects of Insecticide Stress on Expression of NlABCG Transporter Gene in the Brown Planthopper, Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens, is an important pest of rice that severely affects production. Insecticides are an important means of controlling BPH, but their long-term use has led to resistance. To provide insight into BPH responses to insecticide stress, we determined the expression levels of BPH ABCG transporter genes under treatment with thiamethoxam, abamectin, and cyantraniliprole at LC10, LC25, LC50, and LC90. We cloned 13 BPH ABCG transporters, named NlABCG1 to NlABCG13. Conservative domain analysis showed that all 13 transporters have one nucleotide binding domain and one transmembrane domain, typical of semi-molecular transporters. Real-time quantitative PCR showed that thiamethoxam, abamectin, and cyantraniliprole stress increased the expression of some NlABCG transporters gene in BPH. However, after treatment with thiamethoxam at LC25 and abamectin at LC10, there was no significant upregulation of NlABCG. These results indicate that the expression of NlABCG varies in response to stress from different insecticides. These findings provide baseline information for further understanding of the molecular mechanisms of insecticide resistance in BPH.

Molecular cloning, expression, and functional analysis of the chitin synthase 1 gene and its two alternative splicing variants in the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae).

Chitin synthase is responsible for chitin synthesis in the cuticles and cuticular linings of other tissues in insects. We cloned two alternative splicing variants of the chitin synthase 1 gene (SfCHS1) from the white-backed planthopper, Sogatella furcifera. The full-length cDNA of the two variants (SfCHS1a and SfCHS1b) consists of 6408 bp, contains a 4719-bp open reading frame encoding 1572 amino acids, and has 5' and 3' non-coding regions of 283 and 1406 bp, respectively. The two splicing variants occur at the same position in the cDNA sequence between base pairs 4115 and 4291, and consist of 177 nucleotides that encode 59 amino acids but show 74.6% identity at the amino acid level. Analysis in different developmental stages showed that expression of SfCHS1 and SfCHS1a were highest just after molting, whereas SfCHS1b reached its highest expression level 2 days after molting. Further, SfCHS1 and SfCHS1a were mainly expressed in the integument, whereas SfCHS1b was predominately expressed in the gut and fat body. RNAi-based gene silencing inhibited transcript levels of the corresponding mRNAs in S. furcifera nymphs injected with double-stranded RNA of SfCHS1, SfCHS1a, and SfCHS1b, resulted in malformed phenotypes, and killed most of the treated nymphs. Our results indicate that SfCHS1 may be a potential target gene for RNAi-based S. furcifera control.

Comparative transcriptome analysis of Sogatella furcifera (Horváth) exposed to different insecticides.

White-backed planthopper, Sogatella furcifera (Horváth) (Hemiptera: Delphacidae), one of the main agricultural insect pests in China, is resistant to a wide variety of insecticides. We used transcriptome analysis to compare the expression patterns of resistance- and stress-response genes in S. furcifera subjected to imidacloprid, deltamethrin, and triazophos stress, to determine the molecular mechanisms of resistance to these insecticides. A comparative analysis of gene expression under imidacloprid, deltamethrin, and triazophos stress revealed 1,123, 841, and 316 upregulated unigenes, respectively, compared to the control. These upregulated genes included seven P450s (two CYP2 clade, three CYP3 clade, and two CYP4 clade), one GST, one ABC transporter (ABCF), and seven Hsps (one 90 and six Hsp70s) under imidacloprid stress; one P450 (CYP3 clade), two ABC transporters (one ABCF and one ABCD), and one Hsp (Hsp90) under deltamethrin stress; one P450 (CYP3 clade) and one ABC transporter (ABCF) under triazophos stress. In addition, 80 genes were commonly upregulated in response to the three insecticide treatments, including laminin, larval cuticle protein, and fasciclin, which are associated with epidermal formation. These results provide a valuable resource for the molecular characterisation of insecticide action in S. furcifera, especially the molecular characteristics of insecticide cross resistance.

Protective and Detoxifying Enzyme Activity and ABCG Subfamily Gene Expression in Sogatella furcifera Under Insecticide Stress.

Sogatella furcifera, an important migratory pest of rice, has substantial detrimental effects on rice production. To clarify the mechanism whereby S. furcifera responds to insecticide stress, we measured the activity of its protective [superoxide dismutase (SOD); peroxidase (POD); catalase (CAT)] and detoxifying [carboxylesterase (CarE); glutathione S-transferase (GST); mixed-function oxidase (MFO)] enzymes and the expression levels of its ATP-binding cassette subfamily G (ABCG) transporter genes in response to sublethal concentrations (LC10 and LC25) of the insecticides thiamethoxam, buprofezin, and abamectin. On the bases of the transcriptome data and the ABCG genes of Laodelphax striatellus, we obtained 14 full-length ABCG sequences for S. furcifera. RT-qPCR results showed that 13, 12, and 9 sfABCG genes were upregulated in the presence of thiamethoxam, buprofezin, and abamectin, respectively, at LC10. Moreover, 13 and 7 sfABCG genes were upregulated following treatment with thiamethoxam and abamectin, respectively, at LC25. Enzyme activity assays showed that although thiamethoxam, buprofezin, and abamectin induced GST, CarE, CAT, POD, and SOD activity, they did so at different concentrations and exposure times. The activity of MFO was generally inhibited with prolonged exposure to the three insecticides, with the inhibitory effect being most significant at 72 h. These results indicate that S. furcifera differs in its response to different types or concentrations of insecticides. Taken together, our results lay the foundations for gaining a deeper understanding of the mechanisms underlying the adaptation of S. furcifera to different types of insecticides, which would be of considerable significance for the development of effective pest management strategies.