Loss-of-function in testis-specific serine/threonine protein kinase (TSSKs) triggers male infertility in an invasive moth

Loss-of-function in testis-specific serine/threonine protein kinase (TSSKs) triggers male infertility in an invasive moth | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Loss-of-function in testis-specific serine/threonine protein kinase (TSSKs) triggers male infertility in an invasive moth Xueqing Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4156281/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Oct, 2024 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Genetic control system at molecular level presents a promising and eco-friendly strategy for the management of pest and insect-transmitted diseases. Although considerable advancements have been achieved in gene drive applications targeting mosquitoes, endeavors to combat agricultural pests have been somewhat restricted. Here, we identified that the testis-specific serine/threonine kinases ( TSSKs ) family is uniquely expressed in the testes of Cydia pomonella , a prominent global invasive species. We further generated male moths with disrupted the expression of TSSKs and those with TSSKs completely knocked out using RNA interference and CRISPR/Cas 9 genetic editing techniques, resulting in significant disruptions in spermiogenesis, decreased sperm motility, and hindered development of fertilized oocytes. Further explorations into the underlying post-transcriptional regulatory mechanisms have revealed the involvement of lnc117962 as a competing endogenous RNA (ceRNA) for miR-3960, thereby regulating TSSKs . Notably, orchard trials have demonstrated that the release of male strains can effectively suppress population growth. Our findings indicate that targeting TSSKs could serve as a feasible avenue for managing C. pomonella populations, offering significant insights and potential strategies for controlling invasive pests through genetic sterile insect technique (gSIT) technology. TSSK Cydia pomonella CRISPR/Cas9 SIT Post-transcriptional regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Arthropods, constituting approximately 1.1 to 1.2 million species, are widely distributed in various habitats, including soil and plant surfaces, and are known to parasitize both humans and animals 1 . Insects, the largest class among arthropods, have been extensively studied with a recorded count exceeding one million species 2 . According to the Food and Agriculture Organization of the United Nations (FAO), insect pests contribute to nearly 40% of annual global crop yield losses, resulting in losses of over $ 70 billion primarily due to invasive species. The challenges posed by insects have heightened in recent years due to factors such as climate change, rapid globalization and urbanization, and the proliferation of invasive species 3,4 . Integrated Pest Management (IPM) is a methodology for pest control that emphasizes technical integration of various strategies to maintain pest populations below economically acceptable thresholds 5 . In light of the imperative for increased food production driven by population growth, the utilization of pesticides remains pivotal within the framework of IPM 6,7,8 . Nevertheless, challenges arose following the initiation of insecticide-based control measures, leading to detrimental consequences such as insect outbreaks, ecological disruption, and the persistence of pesticide residues 9 . The adverse effects were linked to the eradication of natural enemies and the development of insecticide resistance 10 . Hence, there is a critical need to explore sustainable and eco-friendly approaches to pest management in agricultural and forestry practices. The sterile insect technique (SIT) has become a crucial method for producing and releasing sterile males targeting specific species 11,12,13 . Initially developed in the mid-1930s, SIT utilizes radiation to induce dominant lethal mutations, as seen in the successful eradication of Cochliomyia hominivorax in United States 11 . The Okanagan-Kootenay Insect Sterile Release (OK SIR) project is a prominent example of regional management for C. pomonella , covering extensive orchards and neighboring areas urban in British Columbia, Canada 14,15 . Traditional sterilization methods using DNA-damaging agents can reduce the fitness and mating competitiveness of released males 16 . Newer approaches include microbial-mediated of SIT variations like the Wolbachia-based incompatible insect technique (IIT) 17 , genetically similar SIT-like systems such as release of insects carrying dominant lethal genes (RIDL) 18 , and methods targeting female elimination like female-specific RIDL (fsRIDL) 19 and autosomal linked x-chromosome powder machine 17 . While these first-generation gene SIT technologies present a notable advancement, but each approach has its limitations. For instance, IIT requires that infected females cannot be released, posing practical challenges in field settings 20 . The use of tetracycline, an antibiotic in the microbiome, could impact the fitness of RIDL/fsRIDL males 16 . X-chromosome shredders are confined to species with sex chromosome heterogamy, restricting their application to other species 21 . Recently, genetic modification sterile technology strategies (gSIT) have emerged to suppress insect populations 22 . gSIT offers advantages like enhanced individual competitiveness and survival, successfully applied in managing agricultural pests such as fruit flies, silkworms, and diamondback moths 23 . Therefore, the deployment of more efficient gSIT technologies that sterilize males without compromising their fitness significantly is beneficial 24 . To achieve this, identifying molecular targets linked to male sterility is essential for the optimal development of genetic technologies. Numerous studies have been conducted to explore the genetic and molecular processes that govern spermatogenesis to identify potential targets for gene-specific treatments for infertility 25,26 . The testicular-specific serine/threonine protein kinases ( TSSKs ) are exclusively expressed after sperm cell meiosis, and their distinct patterns of expression across developmental stages and in various tissues suggest their involvement in the regulation of spermatogenesis 27,28 . For example, experiments involving the knockdown of TSSK1 in D rosophila melanogaster and Zeugodacus cucurbitae have demonstrated a significant decrease in sperm viability and a detrimental effect on male fertility, resulting in a decrease in the hatching of eggs 29,30 . Similarly, the use of CRISPR/Cas9 technology to knockout the serine protease 2 ( ser2 ) gene in Bombyx mori has resulted in the inability of sperm and eggs to fertilize, ultimately leading to infertility in subsequent generations 23 . Furthermore, the reduction of the tektin gene, which is associated with sperm flagella, has been found to cause a significant decrease in the hatching rate of offspring 29 . These results suggest that genes specifically expressed in testis play a crucial role in male reproduction, although the exact regulatory mechanism remains uncertain. Eukaryotic gene expression has traditionally been assessed through steady-state mRNA levels. However, this fails to consider translational activation variability or the stability of specific mRNA types. Consequently, post-transcriptional regulation plays a pivotal role in the overall coordination of gene expression 31 . It is important to highlight that the maturation of insect sperm involves extensive post-transcriptional regulation, whereby a significant number of transcripts are preserved for translation at a later stage 32 . In recent years, long non-coding RNAs (lncRNAs) have emerged as crucial factors in post-transcriptional regulatio 33,34 . These lncRNAs play a significant role in regulating gene expression through various mechanisms, including chromatin modification, RNA decoying, transcriptional co-activation, ribonucleoprotein complex formation, and microRNA sequestration 35,36,37 . LncRNAs exhibit a multifaceted modus operandi involving various regulatory modalities, encompassing direct inhibition of gene expression by lncRNAs, as well as their role as competitive endogenous RNAs (ceRNAs), sequestering microRNAs (miRNAs) to modulate the expression of messenger RNAs (mRNAs) 34 . Notably, the knockdown of 128 testicle-specific lncRNAs in fruit flies demonstrated that silencing nine of these lncRNAs led to sterility or reduced fertility 38 . The lnc94638 is crucial for spermatogenesis in Z. cucurbitae and significantly affects male fertility 39 . Therefore, further investigation of the post-transcriptional regulatory mechanisms involved in spermatogenesis is necessary to advance the development of male sterile technology. The invasive agricultural pest, C. pomonella , has been listed among the world's 20 most resistant pests to insecticides by the Insecticide resistance action committee (IRAC) 40 . To address resistance problem, the use of SIT as part of area-wide integrated pest management (AW-IPM) has been proposed as an environmentally friendly control technology 12 . Previous studies have shown the effectiveness of SIT in controlling C. pomonella 15 . However, the most commonly used radiation-based sterile technology (rSIT) technology is not aligned with the current trend of green agriculture development due to issues such as cobalt source waste, waste source treatment and radioactive safety hazards 23 . In order to explore the concept of gSIT for pest control, this study focused on investigating genes related to male reproduction. Through genome and testis transcriptome analysis, five TSSKs genes were identified. To understand the functional role of TSSKs , an RNA interference (RNAi) and CRISPR/Cas9-based loss-of-function approach was employed, comparing C. pomonella mutant and wild-type (wt) individuals. The study revealed that TSSKs are highly expressed in the testes of adult males. Surprisingly, disruption of the TSSKs gene in males resulted in sterility, reduced sperm motility, decreased sperm counts, and abnormal development of fertilized eggs. Furthermore, analysis of the lncRNA transcriptome identified as lnc17962 significantly expressed in testis. Notably, lnc17962 was found to competitively bind with miR-3960, regulating the expression of TSSKs and ultimately causing sterility in offspring. These findings emphasize the crucial role of TSSKs and lnc17962 in sperm function and suggest their potential as targets for the applications of gSIT technology in C. pomonella control. Materials and Methods Insect The experimental insects were reared in a controlled environment with a temperature of 26 ± 1°C, relative humidity of 60 ± 5% RH, and a photoperiod of 16 h light and 8 h dark. The adult was fed with 10% honey water. A detailed description of the feeding method has been previously described by Wang et al 41 . RNA sample preparation and sequencing Freshly emerged adult males were placed in an 85 mL plastic cup (40 mm in diameter × 60 mm in height), and their testes were collected on the second day (CpA2T) using phosphate-buffered saline (PBS) at pH 7.5. Controls were established using the entire male moths, excluding the spermatozoa (CpA2U). On the fifth day after mating, the testes were dissected (CpA5T). This allowed for the use of the whole male moths, from which the testicular tissue had been removed (CpA5U), as a control. Each sample comprised of tissue from 50 individuals and there were three biological replicates per sample. Total RNA was extracted from the samples using Trizol (TaKaRa, Tokyo, Japan) following the provided instructions. The quality of the RNA samples was assessed using NanoDrop (Thermo Scientific, Wilmington, USA), and the integrity of the RNA samples was evaluated through 1% agar gel electrophoresis. The RNA-seq datasets were obtained and analyzed using the aforementioned four samples. RNA sequencing An Illumina TruseqTM RNA sample preparation kit (Illumina, San Diego, CA, USA) was used for library preparation. Before library construction, the quality of the RNA samples was assessed using an Agilent 2100 instrument (Agilent Technologies, Santa Clara, CA, USA). Paired-end sequencing was performed on an Illumina Novaseq 6000 sequencing platform. The library was then subjected to the removal of N-containing error bases and reads of low quality. Annotation of testis-specific expression genes and quantitative analyses The clean read was obtained by analyzing the library with the default parameter of the HISAT2 analysis using the reference genome ( http://v2.insect-genome.com/Pcg ). Clean reads were defined as those that had a unique match with a maximum of two mismatched bases and no deletions or insertions. The quality of each RNA read was evaluated by comparing its localization ratio to the reference genome. Subsequently, RNA sequencing (RNA-seq) was performed on the clean reads and aligned to the genomes using the expectation-maximization method (RSEM, version 1.3.3). These aligned reads were then used for gene expression analysis. The expression levels of all genes were quantified as transcripts per million (TPM) in repeats. Standardized average TPM values were employed to compare the relative expression of each gene in the tissues. Differentially expressed genes (DEGs) were identified using DESeq (version 1.30.0) based on the criteria of corrected P 2. By comparing gene expression levels across all tissues, genes with high expression in the testes were identified, suggesting their potential role in fertility. Full-length confirmation and sequence analysis of TSSKs The complete open reading frame (ORF) of the TSSKs gene was verified using reverse transcriptase PCR (RT-PCR) with primers designed by Oligo7. Each PCR reaction was conducted in a 20 µL volume, consisting of 10 µL 2x Primer STAR Max Premix (TaKaRa), 7.4 µL RNase-free water, 1.0 µL testicular cDNA, and 0.8 µL (10 µmol L − 1 ) forward and reverse primers (Table S1). The thermal cycler was programmed as followings: initial denaturation at 98°C for 2 min, followed by 39 cycles of denaturation at 98°C for 5 s, annealing at 55°C for 15 s, and extension at 72°C for 1 min. Subsequently, electrophoresis analysis on a 1% agarose gel was performed on the PCR-amplified products. The resulting target products were then ligated to the pESI-Blunt simple vector (YEASEN, shanghai, China) and transformed into Escherichia coli DH5α. After transformation, the bacteria were cultured in an LB medium, and positive colonies were confirmed through PCR and Sanger sequencing executed by GENEWIZ (Tianjin, China). Homologous protein amino acid sequences from TSSKs and other insect species were obtained using NCBI BlasP, and sequence comparison analysis was conducted using DNAMAN. The results were visualized graphically using espript3 ( https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi ). Additionally, a phylogenetic tree was generated using MEGA11.0 software with the neighbor-joining (NJ) method and 1000 bootstrap replicates for increased reliability of the results. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of TSSKs in C. pomonella The expression of the TSSKs genes was validated through real-time quantitative polymerase chain reaction (RT-qPCR). The primer used for this purpose can be found in Table S2. Briefly, in accordance with the procedures outlined in a previous study 42 , tissues and stages were collected. Following the extraction of total RNA, the first strand of complementary DNA (cDNA) was synthesized using approximately 1 µg of RNA per sample following the instructions provided by the manufacturer of the PrimeScriptTM RT reagent kit witn gDNA eraser (TaKaRa, Dalian, China).As previously described 42 , the qPCR reaction was conducted with a total volume of 20 µL, comprising 10 µL GoTaq qPCR Master Mix (TaKaRa, Dalian, China), 1 µL template cDNA, 0.8 µL of each primer (10 µM), and 7.4 µL nuclease-free water. The CFX 384 Real-Time System (Bio-Rad, Singapore) was employed for all qPCR analyses, with the following settings: initial denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 30 s. To ensure the specificity of the amplification reaction within the temperature range of 60 ~ 95 ° C, a melting curve analysis was performed on all samples. Each qPCR included three biological replicates, with three technical replicates for each of the following. The expression level of target gene was normalized with the geometric mean of EF-1α and RP12 using the 2 −△△ct method 43 . Functional analysis of the role of TSSKs in male fertility of C. pomonella The dsRNA was synthesized and purified following the protocol described in the T7 RiboMAXTM Express RNAi system (Promega, USA). The concentration of dsRNA was determined using a Nanodrop (Thermo Scientific, Wilmington, USA), while the integrity of the dsRNA was evaluated through 1% agarose gel electrophoresis. Male pupae in the advanced stage of development, distinguished by their dark pigmentation, were selected for the experimental procedure. These pupae were injected with 1 µL of dsRNA at a concentration of 3000 ng/µL. To enhance the durability of RNAi following injection, the same dosage of dsRNA was administered once more after the emergence of the adults (Double injections should be administered within a 24 h period). Insects injected with double-stranded green fluorescent protein (dsGFP) were used as negative control. To assess the efficacy of RNAi, nine moth were sampled at 24, 48, and 72 h post-injection. Furthermore, the sperm count was examined in nine male moths. Specifically, the testicles of each male were collected in PBS, punctured with tweezers in 100 µL of 1× PBS solution, gently shaken, and then stained with 10 µL of 4',6-diamidino-2-phenylindole (DAPI) for 15 min to stain the nuclei of the resulting sperm solution. The 10 µL sperm suspension was placed on a slide and the sperm were counted using an Olympus FV1000S fluorescence microscope (Olympus, Tokyo, Japan). The total sperm count was obtained by averaging the counts from five microscope fields. The testicles from 2-day-old male moths were dissected in petri dishes containing Hayes solution (composed of 0.2 g CaCl 2 , 9.0 g NaCl, 0.1 g NaHCO 3 , and 0.2 g KCl in 1000 mL H 2 O). The motility of sperm was assessed using a sperm motility kit (Thermofisher, Vilnius, Lithuania), which utilizes two fluorescent dyes to differentiate between dead (red propyl iodide) and live (SYBR-14, green glow) sperm cells. Spermatozoa were diluted 5 µL and incubated with 5 µL SYBR-14 working solution (2 µL SYBR-14 stock solution plus 98 µL Hayes solution) on a glass slide at 25℃ for 10 min, followed by 7 min of incubation with propyl iodide. All experiments were performed in triplicate. The 45 male moths injected with dsRNA were placed in an 85 mL plastic cup (40 mm in diameter × 60 mm in height) and allowed to mate with female moths of the same age to lay eggs. The hatching rate of the eggs was subsequently determined. sgRNA synthesis and embryo microinjection In accordance with the screening criteria "5-GG-N18-NGG-3" (where N represents one of the four foundations), target locations were selected by the Crispor website ( http://crispor.tefor.net/ ) based on a comprehensive score calculation. The templates for sgRNA synthesis were amplified through PCR using a set of primers (Table S2) and the MEGA shortscript™ T7 Transcription Kit (Invitrogen, Carlsbad, CA). The PCR was carried out using PrimeSTAR® Max DNA Polymerase (TaKaRa, Dalian, China). Eggs at the pre-embryonic stage were promptly collected within one hour of being laid and securely placed on a microscope slide with double-sided adhesive tape. The embryos were then injected using a Leica DM2700 M stereomicroscope (Leica, Berlin, Germany) equipped with a FemtoJet4i microinjector (Eppendorf, Berlin, Germany). The injection involved 300 ng/µL sgRNA and 150 ng/µL TrueCut™Cas9 protein v2 (Thermofisher, Vilnius, Lithuania). All procedures, including embryo collection and microinjection, needed to be completed within a two-hour timeframe. Following microinjection, the eggs remained attached to the slide and were incubated at 26°C until hatching, after which they were transferred to an artificial diet. lncRNA sequencing and analyses of lncRNA expression Transcriptome sequencing was employed to generate four RNA-seq libraries, which consisted of testes samples from 1-day-old (CpT1D) and 3-day-old (CpT3D) individuals. The expression levels of lncRNAs were quantified using the Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) value and Cuffdiff software (version 2.1.1). To identify specific lncRNA expression, a screening process was conducted on the four testis samples, utilizing the criteria of log2FoldChange > 5 and Q-value < 0.05. To validate the expression of 12 randomly selected testicular-specific lncRNAs, their expression profiles in various tissues were analyzed through RT-qPCR. from the tissues examined included accessory glands, vas deferens, seminal vesicles, and testes from both 1-day and 3-day male adults, along with the previously transcriptomically sequenced samples. All samples were replicated three times for biological accuracy. Total RNA was isolated using Trizol (TaKaRa, Tokyo, Japan) following the manufacturer's instructions. The cDNA for the first lncRNA was synthesized using RR037A (TaKaRa, Japan). RT-qPCR primers (Table S2) were designed using Primer 5 software. EF-1α and RPL12 were used as internal reference genes, and data analysis was conducted using the 2 −△△ct method 43 . Fluorescence in situ hybridization The FAM-labeled probe was synthesized through the in vitro synthesis method and obtained from GefanBio (Shanghai, China). To conduct fluorescence in situ hybridization (FISH) analysis, the testes of 5-day-old male moths were dissected in PBS and fixed overnight at 4°C using 4% paraformaldehydefan. The WISH in situ hybridization kit (GefanBio, Shanghai, China) was used for this analysis, following the procedure described below: the samples were dehydrated using an alcohol gradient (50%, 60%, 70%, 80%, 90%, 100% alcohol for 30 min), dried with DEPC-PBST (0.1% Tween-20) for 1 h, bleached with 6% H 2 O 2 for 1 h, washed three times with PBS, and then washed three times with PBST (5 min/wash). Subsequently, the samples were permeated with protease K at 37°C for 20 min. Probes (diluted 1,000 times) were added and incubated for 48 h. The slides were rinsed three times with the wash solution provided in the kit and stained with DAPI (Sigma, St. Louis, Missouri, USA) for 10 min. Finally, the samples were examined using a Zeiss LSM780 confocal microscope (Zeiss, Jena, Germany). Functional analysis of the role of lnc117962 in male fertility The primer design, synthesis, quality detection, and RNAi of lincRNA were executed following the procedures described in the preceding section. The lncRNA overexpression vector was procured from GenePharma (Shanghai, China). To begin, male moths were subjected to continuous injection of dsRNA, following which their testes were dissected in petri dishes containing PBS after a 48 h interval. The testes were then punctured using forceps, and 2 µL of motile spermatozoa were expeditiously collected. Subsequently, the motility and hatchability of the spermatozoa were assessed using the aforementioned methodologies. Mating competitiveness analysis of sterile males To evaluate the mating competitiveness of lnc117962 knockdown males in the presence of wt males, the latter were chosen as an appropriate control group due to their shared genetic background with the lnc117962 knockdown males. For this purpose, an 85 mL plastic cup (40 mm in diameter × 60 mm in height) was employed to accommodate one wt male, one lnc117962 knockdown male, and one wt virgin female. This configuration, covered with plastic wrap, facilitated the process of egg laying. Likewise, a plastic box (10.4 cm × 17.3 cm × 6.6 cm) containing 10 wt males, 10 lnc117962 knockdown males, and 20 wt virgin male moths, was employed in an apple orchard (41.83'N, 123.57'E) to ensure mating and egg laying. The competition mating index was calculated by analyzing statistical data about fecundity per female and hatching rates (Zhang et al., 2023). Detection of the interaction of miRNAs with lnc117962 and TSSKs To identify potential miRNA that may target lnc117962 using pre-existing miRNA, we employed three established miRNA target gene prediction software programs: miRanda, PITA, and RNAhybrid. The final prediction result was obtained by comparing the outcomes from these three software programs, taking into account their individual preferences during the prediction process. The candidate miRNAs were screened to ensure that no more than 2 GUs were matched between 2 and 8 binding positions, and that the absolute critical strength exceeded 25. The mature miRNA sequences were then sent to GenePharma (Shanghai, China) for the design and synthesis of agomir/antagomir, as well as the synthesis of the corresponding stable negative control and miRNA inhibitor negative control. After synthesizing the agomir and antagomir, RNase-free H 2 O was added to achieve a final concentration of 20 µM. Male adults of newly emerged were collected and injected with agomir and antagomir in their abdomens, using an injection volume of 1 µL/moth. Stable negative control and microRNA inhibitor were injected as a negative control. Total RNA was extracted from randomly selected samples 48 h after the injection. Three biological replicates were obtained for each treatment, and 10 samples were obtained for each replicate. The Mir-X miRNA first-strand Synthesis Kit (Promega, Madison, USA) was used to synthesize the first cDNA of the miRNA, following the provided instructions. To examine the interaction between miRNA and lncRNA, a dual luciferase reporter system was employed. This experimental procedure adhered to the protocol provided by GenePharma (Shanghai, China). Specifically, a luciferase reporter vector containing the target gene was constructed. Subsequently, lnc117962 and miR3960 were linked to the pmirGLO dual luciferase reporter vector (Promega, Madison, USA). The resulting vector was designated as pmirGLO-Lnc117962-3960-mutant. Well-conditioned 293T cells were digested, resuspended, and plated in 12-well plates at appropriate cell densities. The cells were then incubated overnight at 37℃. The 293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) (1.5 mg/mL glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin). The cells were maintained in a 5% CO2-saturated humidity incubator at 37ºC. Subsequently, the interaction between miR-3960 and lnc17962 was examined. Four groups of cells were transfected, including ( 1 ) pmirGlO-Lnc117962-3960-wt and mimic negative control, ( 2 ) pmirGlO-Lnc117962-3960-wt and miR-3960 mimic, ( 3 ) pmirGlO-Lnc117962-3960-mut and mimic NC, ( 4 ) pmirGlO-Lnc117962-3960-mut and miR-3960 mimic. After 24 h of transfection, the cells were lysed and the fluorescence values of the samples were measured using the Dual-Glo® Luciferase Assay System (Promega, Wisconsin, USA). The relative activity was determined by normalizing to the Renilla luciferase. Three independent experiments were performed, with each sample being replicated three times. Statistical analysis The statistical analysis was conducted using JMP 8.0.2 by IBM SPSS Statistics 26. Biological replicates were used to generate statistical means for the purpose of comparisons. P values were calculated through a two-sample Student’s t-test with unequal variance, where the significance level was set at of P < 0.05. The results are presented as the mean value of the triplicates ± standard deviation (SD). Results TSSKs are specifically expressed in male testis of C. pomonella During the larval stage, the C. pomonella , like other Lepidopteran insects, possesses two separate testes. However, as the insect enters the prepupal stage, these testes gradually move closer to each other. By the time the insect reaches the pupal stage, the two testes fuse together to form a single mature testis in the adult stage (Fig. 1 A). To investigate the genes that are specifically expressed in the testes of adult males and their role in mating, RNA-seq was conducted on the testes of 2-day-old (presexual maturity, CpA2T) and 5-day-old (postsexual maturity, CpA5T) male of C. pomonella . Other parts of the testes were used as controls (CpA2U and CpA5U, respectively). This analysis identified a total of 7736 DEGs in the comparison of presexual maturity and control samples, with 4240 upregulated and 3496 downregulated genes. Similarly, 7677 DEGs, with 4378 upregulated and 3299 downregulated genes, were identified in the comparison of postsexual maturity and control samples (Fig. 1 B; Figure S1A and S1B). Notably, the testes exhibited high expression of serine protease genes (Figure S2), tektin (Figure S3A), cyclin (Figure S3B), ubiquitin (Figure S3C), and tubulin (Figure S3D). These DEGs may serve as potential candidates for modulating the fecundity of male C. pomonella . Furthermore, GO enrichment analysis highlighted the crucial role played by the differential genes (Figure S1C and S1D), while KEGG enrichment analysis showed their close relationship to the metabolic pathway (Figure S1E and S1F), particularly the testicle-specific serine/threonine protein kinase ( TSSK ), which displayed high expression (Figure S2). Members of the TSSK gene family, including TSSK1 , TSSK1a , TSSK2 , TSSK2a , and TSSK4 , were selected for further investigation to elucidate their role in spermatogenesis. Phylogenetic analysis revealed the clustering of these genes with TSSK from other Lepidoptera species (Fig. 1 C; Table S1), and collinearity analysis demonstrated their presence among Lepidoptera insects (Figure S4). Multiple sequence comparisons confirmed the conservation of TSSK residues in the S-TKc region, as well as the adenosine triphosphate (ATP) and substrate binding domains (Fig. 1 D). Additionally, RT-qPCR analysis determined the expression levels of TSSKs in the developmental stages and tissues of C. pomonella , showing moderate expression during the pupal stages and increased expression as the insects reached sexual maturity. TSSKs were found to be expressed only in the male testis, with TSSK4 exhibiting the highest expression level (Fig. 1 E). These findings suggest that these testis-specifically expressed TSSKs play an important role in the m ale fertility of C. pomonella . Loss function of TSSKs influence male fertility of C. pomonella Following a 48-h period of injection, the efficiency of RNAi was observed to increase from 37.32% (Figure S5) to 86.09% (Fig. 2 A), suggesting that double injections method enhances the interference efficiency over time. Upon injecting adults subjects with dsTSSKs and conducting subsequent observations after 24 h, it was found that the expression levels of TSSK1 , TSSK1a , TSSK2 , TSSK2a , and TSSK4 were significantly reduced by 56.13% ( p = 0.001)、86.09% ( p < 0.001)、42.67% ( p = 0.038)、72.08% ( p < 0.001), and 75.62% ( p < 0.001), respectively, compared to control treatment with dsGFP (Fig. 2 A). Although the number of eggs produced by the females mated with dsTSSKs treated males remained unchanged (Fig. 2 B), the hatching rate of these eggs was significantly lower compared to dsGFP treatment (Fig. 2 C). The sterility rates of TSSK1 , TSSK1a , TSSK2 , TSSK2a , and TSSK4 knockdown lines were found to be 90.61%, 83.93%, 100%, 100%, and 100%, respectively (Fig. 2 C). The unhatched eggs exhibited progressive desiccation during development, with no evidence of reaching the blackhead stage (Fig. 2 D). To ascertain whether the underdeveloped eggs were a result of lack of fertilization in the female, a spermatophore examination was conducted, ruling out this possibility (Figure S6). Sperm viability was then assessed in both the dsGFP and dsTSSKs treatment groups, revealing a higher number of dead eupyrene and apyrene sperm bundles in the dsTSSKs treatment group compared to the dsGFP group (Fig. 2 E). In males, the number of spermatozoa was reduced by 28.61% (dsTSSK1, p = 0.0014), 63.03% (dsTSSK1a, p < 0.001), 39.00% (dsTSSK2, p = 0.0 030), 46.77% (dsTSSK2a, p < 0.001), and 30.38% (dsTSSK4, p = 0.0024) in the dsTSSKs treatment groups compared to the control (Fig. 2 F). In addition, we employed the CRISPR/Cas9 gene editing system to induce mutation in the TSSK genes (Fig. 2 G), which led to deletions at the desired location (Fig. 2 H and I). The mutation rate of the TSSK genes varied from 9.38–12.46% (Table S3), and these genetic modifications did not have any impact on the insect′s developmental period (Table S4). When TSSKs −/− males were mated with wt females, the egg-laying levels were similar to the results of RNAi (Fig. 2 J). However, a significant decrease in hatchability, consistent with the results of RNAi approach, was observed (Fig. 2 K). Most embryos derived from TSSKs −/− males failed to develop properly, even after seven days following ovulation. Consequently, TSSKs −/− males exhibit normal sexual behavior but are ultimately sterile. Lnc117962 is specifically expressed in male testis of C. pomonella Distinctive expression patterns of lncRNAs have been observed in testis, accessory gland, seminal vesicles and vas deferens., indicating the presence of tissue-specific lncRNAs (Fig. 3 A). By analyzing the Venn diagram, it was found that 6361 lncRNAs were specifically expressed in the testis (CpT1D vs CpT1DC). Furthermore, the testis exhibited specific expression of 7390 lncRNAs in CpT3D compared to CpT3DC, with 4430 lncRNAs being expressed in both CpT1D vs CpT1DC and CpT3D vs CpT3DC (Fig. 3 B). Twelve lncRNAs with differential expression (log2fold change ≥ 4 threshold) were identified, and their tissue-specific expression patterns in different developmental stages were confirmed using RT-qPCR. The RT-qPCR results revealed that all 12 lncRNAs were highly expressed in the testis, while exhibiting negligible expression in the vas deferens, accessory glands, and seminal vesicles (Fig. 3 C). Notably, lnc117962showed the highest expression level in the testes, with subsequent decreased expression as sperm matured and shifted. Furthermore, fluorescent in FISH revealed a localization signal in the testes of C. pomonella (Fig. 3 D). Results from Gene Ontology (GO) pathway analysis suggest that lnc117962 has the potential to modulate various gene enrichments associated with ATP activity and serine/threonine protein kinase pathways. This indicates that lnc117962 might play a crucial role in regulating the expression of TSSKs and could be significant in the post-transcriptional control of male fertility (Figure S7). Loss function of lnc117962 influences male fertility of C. pomonella The role of lnc117962 in the regulation of male fertility was investigated using RNAi technology. Silencing lnc117962 with ds117962 resulted in a 74.83% efficacy after 48 h (Fig. 4 A). Assessment of TSSK family gene expression levels following ds117962 treatment showed reductions of 71.94%, 42.87%, 47.96%, 91.58%, and 63.36% compared to dsGFP control (Fig. 4 B). Overexpression of lnc117962 with pcDNA3.1-lnc117962 resulted in a 2.47-fold increase in lnc117962 expression compared to pcDNA3.1-GFP (Fig. 4 C). Additionally, TSSK1 , TSSK1a , and TSSK4 expression decreased by 39.28%, 26.61%, and 19.93%, respectively, while TSSK2a expression increased by 1.26-fold with no significant change in TSSK2 expression (Fig. 4 D). Fertility response to dslnc117962 treatment was assessed based on fertilization and hatching rates, showing no significant difference in egg laying but a 28.5% reduction in hatching rate (Fig. 4 E, 4 F). Male longevity was unaffected (Figure S8). The dslnc117962 treatment group exhibited more dead sperm in eupyrene and apyrene sperm bundles compared to the control group (Fig. 4 G), suggesting lnc117962 involvement in C. pomonella male spermatogenesis and its role in male fertility. The insignificance of the overall fitness of dslnc117962 knockdown males was anticipated and evaluated using a mating competitiveness assay (Fig. 4 H). The results indicated that dslnc117962 knockdown males displayed the capacity for courtship, mating, and successful competition with wt females. Specifically, the egg hatch rate was found to be 52.47% ± 15.96% for a pair of one wt male, one dslnc117962 knockdown male and one wt female, compared to 25.52% ± 9.55% for a pair of one wt male and one wt female, and 75.26% ± 4.28% for a pair of one dslnc117962 treated male and one wt female, confirming the mating competitiveness (0.84) of dslnc117962 knockdown males (Table 1 ). Furthermore, the mating performance of lnc117962 knockdown males was assessed through field cage experiments conducted in the first two weeks of June 2023, under controlled humidity and temperature conditions (Figure S9). Results obtained from the apple orchard (Fig. 4 J) indicated no significant disparity in egg production between 10 wt females mated with 10 dslnc117962 knockdown males and those mated with 20 wt males (Fig. 4 K). However, the hatch rates were 39.91% ± 7.46% and 83.13% ± 2.7%, respectively (Fig. 4 L). Notably, the hatching rate of the F1 generation was determined to be 67.56 ± 5.41% ( p = 0.0029), suggesting that the RNAi treatment predominantly affected the parents and had a lasting impact on the offspring (Figure S10). These results indicate that lnc117962 regulates the activity of TSSKs through post-transcriptional mechanisms, exerting a suppressive effect on the population development of C. pomonella . Table 1 Effect of dslnc117962 knockdown males on the mating competitiveness of C. pomonella. Matching ratio (wtM♂: lnc117962KDM♂: wtF♀) Egg laid per female Hatching rate (%) competition mating index (C) 1:0:1 103.33 ± 18.67 a 75.26 ± 4.28 a 0.84 1:1:1 101.00 ± 17.59 a 54.47 ± 15.96 b 0:1:1 105.40 ± 22.64 a 25.52 ± 9.55 c Lnc117962 knockdown males (lnc117962KDM), wild-type males (wtM) and wild-type females (wtF) were introduced for mating in the ratios 0:1:1, 1:0:1 and 1:1:1, respectively. A total of 15 replicate experiments were evaluated. The table shows mean ± standard deviation (SD) data. Letters following data indicate significant differences analyzed by one-way analysis of variance (ANOVA) using Duncan's test ( p < 0.05). Lnc117962 regulates TSSKs through competitive bindings to miR-3960 In order to further elucidate the regulatory mechanism of lnc117962 on TSSKs gene expression, we predicted its potential miRNA partner. The miR-3960 emerged as the top candidate among the predicted targets (Table S5 and Figure S11), suggesting its possible interaction with lnc17962. The resemblance in their seed sequences indicates that miR-3960 could plausibly act as a target of lnc117962 (Fig. 5 A). To confirm this interaction, a dual luciferase assay demonstrated a 44.33% reduction in luciferase activity in cells transfected with miR-3960 and pmirGLO-lnc117962 mimics compared to mimic negative control (Fig. 5 B). Additionally, computational tools including miRanda, PITA, and RNAhybrid were utilized to predict the binding potential of miR-3960 to the 3’UTR of TSSKs , revealing it capability to interact with the 3'UTR of TSSKs (Table S6). Further analysis was conducted to ascertain if lnc117962 functions as a "sponge" or decoy ceRNA of miR-3960. The synthesis of agomir/antagomir-miR-3960 and subsequent RT-qPCR assessment of miR-3960, lnc117962, and TSSKs revealed noteworthy. Following the injection of agomir-miR-3960, the expression level of miR-3960 increased by 3.4 times, along with a significant increase in the expression level of lnc117962, TSSK1a , and TSSK2a , while TSSK1 , TSSK2 , and TSSK4 levels decreased ( P < 0.05, Fig. 5 C). Conversely, antagomir-miR-3960 injection led to reduced expression levels of miR-3960, lnc117962, TSSK1 , TSSK1a , TSSK2a , and TSSK4 ( P < 0.05, Figure. 5D). These findings indicate that lnc117962 acts as a ceRNA by suppressing miR-3960, thereby positively regulating the expression of the TSSK2a (Figure. 5E). However, further research is required to explore the regulatory mechanisms of TSSK1 , TSSK1a , TSSK2 , and TSSK4 . Discussion The utilization of SIT has been proven to be effective and eco-friendly for managing the invasive pest, C. pomonella 13,15 . In this study, we discovered TSSKs genes which are exclusively expressed in the testis, serving as molecular targets for the development of the gSIT strategy in managing this invasive pest. Spermatogenesis is a meticulously orchestrated process involving a series of maturation stages that induce notable structural and biochemical changes in spermatogonia within the mature testis 44,45 . This intricate process encompasses both mitotic and meiotic divisions, alongside cellular remodeling throughout the cell cycle 26 . This remodeling necessitates the synchronized activation and deactivation of specific serine/threonine protein kinase (s) that regulate the recombination of sperm chromatin 45,46 . Previous studies consistently affirms the pivotal role of serine/threonine protein kinases in spermatogenesis 44 . The TSSKs belongs to the 5'-adenosine monophosphate-activated protein kinase (AMPK) family, characterized by the serine/threonine protein kinase catalytic (S-TKc) domain. Although typically comprising six members, TSSK1 , TSSK2 , TSSK3 , TSSK4 , and TSSK6 , the exact number of TSSK family members in insects remains undisclosed. In this study, 5 TSSK genes were identified in the genome of C. pomonella , namely TSSK1 , TSSK1a , TSSK2 , TSSK2a , and TSSK4 . The emergence of TSSK1a and TSSK2a as novel TSSK copies in distinct regulatory contexts may elucidate the substitution of TSSK3, TSSK5 , and TSSK6 within this group 47 . Shang et al 48 posit that the greater frequency of sequence mutations in the C-terminal domain of TSSK1/TSSK1B compared to TSSK2 could be attributed to positive evolutionary selection, potentially influenced by variations in protein partner and substrate preferences. The C-terminal domain plays a crucial role in determining the accurate spatial configuration of the serine/threonine protein kinase activity 49 , facilitating the transfer of γ-phosphate residues from ATP to the hydroxyl group of serine, threonine, or tyrosine residues on the target protein 50 . Through bioinformatics analysis, it was revealed that TSSK1 , TSSK1a , TSSK2 , TSSK2a , and TSSK4 genes exhibit homology, with a highly conserved S-TKc domain. All TSSK members habor essential lysine residues crucial for ATP binding, as well as conserved aspartate residues vital for catalytic function. Phylogenetic analysis supports the close relationship between C. pomonella TSSKs and other Lepidoptera TSSK proteins, underscoring their significant involvement in spermatogenesis. The progress in genomics, gSIT technology, and the implementation of a large-scale sterile system have become profoundly influential in current research endeavors 16 . Nevertheless, challenges such as off-target effects and the insufficient knockdown efficacy of RNAi have acted as constraining factors, despite the productive application of RNAi in numerous studies on Lepidoptera 51,52 . Enhancing RNAi efficiency in Lepidoptera is essential, the RNAi efficiency of TSSKs was successfully improved through double injections during the pupal and adult stages, making the first attempt in C. pomonella . In Rhodnius prolixus and Nilaparvata lugens , double injections of dsRNA increase RNAi efficiency from 38–75% and 25–50%, respectively, indicating that double injection is a suitable method to improve RNAi efficiency 53,54 . In recent years, the utilization of CRISPR/Cas9 technology has emerged as a prominent method for investigating gene functionality model insects have made significant advancements, particularly through the implementation of the precision-guided sterile insect technique (pgSIT), as evidenced in studies involving D.melanogaster 16 and Anopheles gambiae 55 . Nevertheless, it is important to recognize that investigations pertaining to gene editing in C. pomonella are still in the nascent stages. A study conducted by Garczynski et al 56 . In 2017, demonstrated that the introduction of a combination of sgRNA and Cas9, targeting the CpomOR1 gene in C. pomonella resulted in notable impacts on fertility, with female moths producing inviable eggs 56 . In this study, we have successfully established a functional TSSKs genes system using CRISPR/Cas9. TSSKs genes were knocked out through the introduction of a combination of sgRNA and Cas9 protein, resulting in an efficiency rate ranging from 9.38–12.46%. Prior studies by Chen et al. 57 and Ye et al. 58 demonstrated knockout efficiencies of 6.5–13.9% for opsin genes in Plutella xylostella and a 36% efficiency for the pheromone binding protein 1 ( PBP1 ) gene in Helicoverpa armigera , respectively. However, the effictiveness of this system in facilitating targeted gene disruption varies significantly among species due to species-specific distinctions 52 . Research on mice has revealed that mutations in the TSSK1 and TSSK2 genes lead to male sterility, attributed to issues in spermatogenesis, absence of elongated spermatids, elevated apoptosis rate, and increased round spermatogenic cells in the epididymis 59 . The loss of TSSK expression in mice results in defective sperm cell development, abnormal sperm cell structure, and an inability of these cells to fertilize eggs 60 . In this study, we observed a marked rise in deceased spermatozoa and a decline in both spermatozoa count and offspring fertility following TSSK gene fysfunction. These findings align with prior investigations on B. tryoni and B. dorsalis 29 . Phenotypic abnormalities during spermatogenesis have been also observed, including spermatogonial membrane rupture due to the TSSK4 mutation and sperm chromosome disarray caused by TSSK6 mutation in mice 61 . Interestingly, studies on TSSK mutants in D.melanogaster indicated unsuccessful formation of the individualization complex (IC) during spermatogenesis 10 . Furthermore, the absence of the N-terminal or C-terminal domain of TSSK hindered the typical IC structure formation, resulting in male infertility 16 . Nevertheless, gaps remain in understanding the post-transcriptional regulation of TSSKs in spermatids during spermiogenesis. The application of advanced deep transcript sequencing methodologies has spurred an escalation in the exploration of post-transcriptional regulation in insects 32 . At the post-transcriptional level, lncRNAs and miRNAs play crucial roles as regulators of gene expression. Notably, lncRNAs demonstrate tissues-specific expression, particularly exhibiting higher abundance in the testicular tissues 38 . Studies conducted on Z. cucurbitae have identified a considerable number of highly expressed lncRNAs in the testis 55 . Similarly, examination of RNA-seq data in B. mori has unveiled a predominant expression of lncRNAs in the testicular tissues 62 . In this study, a comprehensive analysis of lncRNAs expression profiles in the testes of C. pomonella was carried out, leading to the identification of an upregulation of a specific lncRNA, named lnc17962. Through FISH analysis, the precise localization of lnc117962 in the apical region of the testis, characterized by the presence of mature spermatozoa, was successfully achieved. This localization pattern mirrors the findings from a prior study on Bactrocera cucurbitae , where mature sperm was observed 52 . The involvement of lncRNAs in the development of male germ cells in diverse species has been postulated, with some displaying distinctive expression patterns in fertile and infertile spermatozoa 63 . Notably, the knockout of murine testis-specific lncRNA Tslrn1 using CRISPR/Cas9 technology resulted in a significant decrease in spermatozoa count 64 . In this study, RNAi was utilized to silence lnc117962, resulting in a marked reduction in sperm count and diminished embryo hatching rate compared to the control group. The findings presented in this study provide additional evidence supporting the involvement of lnc117962 in the spermatogenesis process in C. pomonella . Furthermore, the depletion of CR42858, a testis-specific lncRNA in Drosophila, led to a decline in sperm count and male fertility 38 . The findings reported in B. dorsalis also indicate impaired male fertility, possibly due to defective spermatogenesis and reduced sperm count 29 . It is widely recognized that many lncRNAs habor binding sites for miRNA 65 . This observation suggests that numerous lncRNA may regulate gene expression by sequestering miRNAs, thereby reducing their cellular concentration 66 . Known as the "competing endogenous RNA (ceRNA)" hypothesis, this mechanism proposes that lncRNAs act as negative regulators of miRNA activity, ultimately promoting gene expression 67 . Additionally, our research indicates that lnc117962 can act as a ceRNA for miR-3960, controlling the expression of TSSK2a and impacting spermatogenesis in C. pomonella . Interestingly, this regulatory pattern resembles the reproductive mechanism involving lncR26319/miR-2834/EndophilinA in B. mori 68 , suggesting a potential universality of this regulatory mode. It is noteworthy that miR-3960 has been found to directly regulate the expression of TSSK1a , although other factors may be involved in the regulatory process of lnc117962 on TSSK1a. While our study identifies lnc117962 as a ceRNA of miR-3960, the precise mechanism by which it regulates TSSK1 , TSSK2 , and TSSK4 remains unclear, and there may be multiple pathways through which their expression is controlled. The conventional irradiated-based SIT is known to generate sterile males with reduced mating competitiveness as a result of mutations across multiple genes 69 . Consequently, an alternative approach focusing on the production of highly competitive sterile males has emerged as an appealing strategy for managing C. pomonella populations 27 . In this study, we evaluated low-fertility males exhibiting comparable traits to wt males under both laboratory and apple orchard settings, following the suppression or elimination of TSSK genes. The findings indicate that these males cold potentially possess the same level of safety and resilience as wt counterparts. Furthermore, unlike the previous SIT method where maintaining sterile males as homozygous lines was challenging, mutants with TSSK s could generate offspring with decreased hatchability rates. This parallels the disruption of leucine aminopeptidase 1 in Aedes aegypti , resulting in reduced hatching rates among descendants while preserving their competitiveness edge relative to wt males 70 . Ideally, genetic modifications induced by sterility-inducing genes should naturally disseminate through pest populations without necessitating continuous mass releases of sterile insects 71 . Consequently, mating with TSSKs −/− males offers an effective means to propagate genetic alterations within C. pomonella populations through a reduced number of viable offspring. As a result, successful population management was achieved through the utilization of TSSKs −/− males in mating scenarios. Overall, TSSKs were identified as promising candidates for gSIT owing to their notable level of conservation, rendering them applicable across various insect species. The utilization of gSIT stands to offer a secure, eco-friendly, and effictive approach for managing field pest populations, thereby facilitating the adoption of sustainable agricultural practices. Declarations Acknowledgements This work was supported by the National Key R&D Program of China (2021YFD1400200). Author contributions statement X.Q.Y. conceived and designed experiments, and writing ; Z.H.W. performed molecular analysis of TSSKs and writing; Y.Q.W. performed molecular analysis of lncRNA; K.W.Z. p.c.w and R.H.L. performed feeder population; Z.P.W. performed gene editing experiments; Y.Li. and P.G. performed experimental guidance. All authors discussed the results and declared that they had no conflicts of interest. interest statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. References Stork, N.E. How many species of insects and other terrestrial arthropods are there on earth? Annu. Rev. Entomol. 63 , 31-45 (2018). Niassy, S., & Ekesi, S. 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Complexities of post-transcriptional regulation and the modeling of ceRNA crosstalk. Crit. Rev. Biochem. Mol. Biol. 53 , 231-245 (2018). Dykes, I.M., & Emanueli, C. Transcriptional and post-transcriptional gene regulation by long non-coding RNA. Genomics. Proteomics. Bioinformatics. 15 , 177-186 (2017). Sahu, A., Singhal, U., & Chinnaiyan, A.M. Long noncoding RNAs in cancer: from function to translation. Trends. Cancer. 1 , 93-109 (2015). Wang, Y. et al. lncR26319/miR‐2834/ EndophilinA axis regulates oogenesis of the silkworm, Bombyx mori . Insect. Sci. 30 , 65-80 (2023). Dame, D. A., Curtis, C.F., Benedict, M.Q., Robinson, A.S., & Knols, B.G. Historical applications of induced sterilisation in field populations of mosquitoes. Malar. J. 8(2) , 1-10, (2009). Sun, X. et al. Leucine aminopeptidase1 controls egg deposition and hatchability in male Aedes aegypti mosquitoes. Nat. Commun. 15(1) , 106 (2024). Harris, A.F. et al. Field performance of engineered male mosquitoes. Nat. Biotechnol. 29(11) , 1034-1037 (2011). Additional Declarations There is NO Competing Interest. Supplementary Files supportinginformation.pdf supporting information Cite Share Download PDF Status: Published Journal Publication published 04 Oct, 2024 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4156281","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":286994275,"identity":"485b5a48-5154-40fc-aa3a-6c249816d485","order_by":0,"name":"Xueqing Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYDACdsYGBoaKGjt+CTBXQoawFmaQljPHkiVnMIBYEjxEaAFixjZmxg03wFoYCGuRb2Zue/iFjY3Z+Hbz8Uc3aix4GNgPH92AT4vBYcZ2YxkeGT6zO8cSm3OOAR3Gk5Z2A68WZsY2aQkJNmazGzmGzTlsQC0SPGZ4tcg3g7QANW6eAdLyjwgtDIcZ2yQ/JAC9LwHUkttGhBagX9qkGQ4cS5a4kZY4O7dPgoeNkF/k29ufSf78B4zKGckHPud8q5PjZz98DL/DgIAZJS7YCCkHAcYfxKgaBaNgFIyCkQsA54NC0zdySLQAAAAASUVORK5CYII=","orcid":"","institution":"College of Plant Protection, Shenyang Agricultural University, Shenyang","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xueqing","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-03-24 02:50:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4156281/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4156281/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-024-06961-5","type":"published","date":"2024-10-04T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55100419,"identity":"3b396a68-bcba-45d6-992c-71cd970c9347","added_by":"auto","created_at":"2024-04-22 15:19:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2339267,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of testis specific expression genes. (A) Testis development dynamics; 2T, 5T: second and fifth day adult female testis. (B) The number of differentially expressed genes (DEGs) in the testis as compared to other tissues in adult male of \u003cem\u003eC.pomonella\u003c/em\u003e. (C) Phylogenetic analysis of TSSK gene family of \u003cem\u003eC.pomonella\u003c/em\u003e and other insects. (D) Multiple amino acid sequence alignment of TSSK family proteins. ATP binding sites are respectively represented with black triangles. (E). RT-qPCR verification of TSSKS expression in different development stages and tissues of \u003cem\u003eC.pomonella\u003c/em\u003e. All data were normalized using the log2 ratio.\u003c/p\u003e","description":"","filename":"Figure.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4156281/v1/58db32ce5137f1fc385ec048.png"},{"id":55101450,"identity":"287a4366-c6e5-4ded-8d58-83fe1e55659f","added_by":"auto","created_at":"2024-04-22 15:27:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3018930,"visible":true,"origin":"","legend":"\u003cp\u003eThe role of TSSKs as a regulator of male fertility in \u003cem\u003eC.pomonella\u003c/em\u003e were analyzed using gene silencing. (A) The efficiency of TSSKs knockdown was analyzed after 24 h, 48 h, and 72 h of continuous dsRNA inject. The effect of TSSK knockdown on oviposition (B), egg hatching rates (C), developmental dynamics of eggs (D)in \u003cem\u003eC.pomonella\u003c/em\u003e is being investigated. (E) Represents the number of eupyrene sperm bundle and apyrene sperm bundle per male were observed after injection dsRNA. Dead and live sperm bundle are indicated by red and green respectively. (F) Changes in the number of spermatozoa were observed after the RNAi treatment. (G) Schematic of TSSKs gene structure and sgRNA target sites. The gray box indicates the exon. Red arrows indicate the target sites of sgRNA. The target sequence and PAM sequence are highlighted in black and red, respectively. (H) Representative chromatograms of direct sequencing of the PCR products for genotyping the TSSKs mutation. (I) Verification of TSSKs\u003csup\u003e− /− \u003c/sup\u003emutations by Sanger sequencing. PCR products spanning the target site were amplified from gDNA of TSSK\u003csup\u003e− /−\u003c/sup\u003e mutations and subjected to Sanger sequencing. The target sequence was in red font, while the PAM sequence was shown in purple, deleted bases are underlined in red. The effect of TSSK-KO on oviposition (J), egg hatching rates (K). The asterisks on the bars indicate the significant differences analyzed by the independent samples t-test. (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4156281/v1/35b83305bd6294128788648b.png"},{"id":55100414,"identity":"7b7e21a1-e59e-460a-abe5-2872f6af9b2a","added_by":"auto","created_at":"2024-04-22 15:19:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":802895,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of testis-specific expression of lncRNA. (A) Reproductive system of \u003cem\u003eC.pomonella\u003c/em\u003e. 1: testis; 2: accessory gland; 3: seminal vesicles and vas deferens. (B) The number of differentially expressed genes (DEGs) in the testis compared to other tissues in adult males of \u003cem\u003eC.pomonella\u003c/em\u003e. Localization of lnc117962 in the testis of \u003cem\u003eC. pomonella\u003c/em\u003e by fluorescence in situ hybridization; D1, D3: first and third-day adult female. (C) Result of the negative control in the whole testis; Whole testis showing the lnc117962 fluorescent signal (lnc117962); (D) RT-qPCR verification of 12 testis-specific lncRNAs expression in different development stages and tissues of \u003cem\u003eC.pomonella\u003c/em\u003e. All data were normalized using the log2 ratio.\u003c/p\u003e","description":"","filename":"Figure.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4156281/v1/86b3a7aed82627a2b45a624b.png"},{"id":55100416,"identity":"1f5a4725-1e91-4b66-ac95-622c0be36705","added_by":"auto","created_at":"2024-04-22 15:19:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1119136,"visible":true,"origin":"","legend":"\u003cp\u003eThe role of lnc117962 as a regulator of male fertility in \u003cem\u003eC.pomonella\u003c/em\u003e. (A) The lnc117962 knockdown efficiency was analyzed after 48 h, 72 h, and 96 h continuous dsRNA injection. (B) Effect of pcDNA3.1-lnc117962 injection on the relative expression of lnc117962 (C) and \u003cem\u003eTSSKs\u003c/em\u003e (D) in \u003cem\u003eC.pomonella\u003c/em\u003e. Data is mean ± SD; asterisks indicate the significance. The effect of lnc117962 knockdown on oviposition (E) and egg hatching rates (F) in \u003cem\u003eC.pomonella\u003c/em\u003e is being investigated. Data are means ± standard error. The asterisks on the bars indicate the significant differences analyzed by the independent samples \u003cem\u003et\u003c/em\u003e-test. (*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). Represents the number of eupyrene sperm bundle (G) and apyrene sperm bundle (H) per male were observed after injection ds117962. Dead and live sperm bundle are indicated by red and green respectively. (I) An experimental design to assess the competitiveness of ds117962 males competing with wt females. The mating success of sterile males was assessed by fertility reduction. (J) An experimental design to Assess the competitiveness of male ds117962 males in the cage. (K) The graph shows the proportions of oviposition. (L) The bar graph shows the proportions of hatched eggs. The presence of dslnc117962 males resulted in a significant decrease in female fertility. Bars represent means ± SD for three replicates.\u003c/p\u003e","description":"","filename":"Figure.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4156281/v1/4324fb4d77e945d4ac7f0878.png"},{"id":55101449,"identity":"fd0b0b5d-f0c4-4dc8-833f-875aa6641354","added_by":"auto","created_at":"2024-04-22 15:27:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":594746,"visible":true,"origin":"","legend":"\u003cp\u003eLnc117962 regulates TSSKs through competitive bindings to miR-3960. (A) Target prediction of lnc117962. (B) Validation of the interaction of miR-3960 and lnc117962 by luciferase assay. NC: negative control of miR-3960 mimics; miR-3960 mimics: 293Tcells that were co-transfected with miR-3960 and pmiGlo-lnc117962; miR-3960 mutant: 293Tcells that were co-transfected with mutant mimics of miR-3960 and pmiGlo-lnc117962. Data were presented as the relative ratio of firefly to Renilla luciferase activity. Significance analysis was conducted with independent samples \u003cem\u003et-\u003c/em\u003etest. SD: standard error of the mean. Effect of miRNA agomir (C) /antagomir (D) injection on the relative expression of lnc117962 and \u003cem\u003eTSSKs\u003c/em\u003e in \u003cem\u003eC.pomonella\u003c/em\u003e. (E) Schematic diagram of \u003cem\u003eTSSKs\u003c/em\u003epost-transcriptional regulation. Data is mean ± SD. Three independent experiments were performed, and each sample was repeated 3 times. The significances were indicated by asterisks (* \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4156281/v1/c4877ac26fafd130ea1be1bf.png"},{"id":65898514,"identity":"b12334a7-63ff-4d9a-a150-3d410a25144e","added_by":"auto","created_at":"2024-10-04 07:05:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8585864,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4156281/v1/73ad748b-bc3f-4b87-ae5a-f192cf9b2e93.pdf"},{"id":55101892,"identity":"e72edeef-3aca-45d5-b5b1-6d579e4840b1","added_by":"auto","created_at":"2024-04-22 15:35:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1998852,"visible":true,"origin":"","legend":"supporting information","description":"","filename":"supportinginformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4156281/v1/059e5d9dbaa5e87d5353687c.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Loss-of-function in testis-specific serine/threonine protein kinase (TSSKs) triggers male infertility in an invasive moth","fulltext":[{"header":"Introduction","content":"\u003cp\u003eArthropods, constituting approximately 1.1 to 1.2\u0026nbsp;million species, are widely distributed in various habitats, including soil and plant surfaces, and are known to parasitize both humans and animals\u003csup\u003e1\u003c/sup\u003e. Insects, the largest class among arthropods, have been extensively studied with a recorded count exceeding one million species \u003csup\u003e2\u003c/sup\u003e. According to the Food and Agriculture Organization of the United Nations (FAO), insect pests contribute to nearly 40% of annual global crop yield losses, resulting in losses of over \u003cspan\u003e$\u003c/span\u003e70\u0026nbsp;billion primarily due to invasive species. The challenges posed by insects have heightened in recent years due to factors such as climate change, rapid globalization and urbanization, and the proliferation of invasive species \u003csup\u003e3,4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIntegrated Pest Management (IPM) is a methodology for pest control that emphasizes technical integration of various strategies to maintain pest populations below economically acceptable thresholds\u003csup\u003e5\u003c/sup\u003e. In light of the imperative for increased food production driven by population growth, the utilization of pesticides remains pivotal within the framework of IPM\u003csup\u003e6,7,8\u003c/sup\u003e. Nevertheless, challenges arose following the initiation of insecticide-based control measures, leading to detrimental consequences such as insect outbreaks, ecological disruption, and the persistence of pesticide residues\u003csup\u003e9\u003c/sup\u003e. The adverse effects were linked to the eradication of natural enemies and the development of insecticide resistance\u003csup\u003e10\u003c/sup\u003e. Hence, there is a critical need to explore sustainable and eco-friendly approaches to pest management in agricultural and forestry practices.\u003c/p\u003e \u003cp\u003eThe sterile insect technique (SIT) has become a crucial method for producing and releasing sterile males targeting specific species\u003csup\u003e11,12,13\u003c/sup\u003e. Initially developed in the mid-1930s, SIT utilizes radiation to induce dominant lethal mutations, as seen in the successful eradication of \u003cem\u003eCochliomyia hominivorax\u003c/em\u003e in United States\u003csup\u003e11\u003c/sup\u003e. The Okanagan-Kootenay Insect Sterile Release (OK SIR) project is a prominent example of regional management for \u003cem\u003eC. pomonella\u003c/em\u003e, covering extensive orchards and neighboring areas urban in British Columbia, Canada\u003csup\u003e14,15\u003c/sup\u003e. Traditional sterilization methods using DNA-damaging agents can reduce the fitness and mating competitiveness of released males\u003csup\u003e16\u003c/sup\u003e. Newer approaches include microbial-mediated of SIT variations like the Wolbachia-based incompatible insect technique (IIT)\u003csup\u003e17\u003c/sup\u003e, genetically similar SIT-like systems such as release of insects carrying dominant lethal genes (RIDL)\u003csup\u003e18\u003c/sup\u003e, and methods targeting female elimination like female-specific RIDL (fsRIDL)\u003csup\u003e19\u003c/sup\u003e and autosomal linked x-chromosome powder machine\u003csup\u003e17\u003c/sup\u003e. While these first-generation gene SIT technologies present a notable advancement, but each approach has its limitations. For instance, IIT requires that infected females cannot be released, posing practical challenges in field settings\u003csup\u003e20\u003c/sup\u003e. The use of tetracycline, an antibiotic in the microbiome, could impact the fitness of RIDL/fsRIDL males\u003csup\u003e16\u003c/sup\u003e. X-chromosome shredders are confined to species with sex chromosome heterogamy, restricting their application to other species\u003csup\u003e21\u003c/sup\u003e. Recently, genetic modification sterile technology strategies (gSIT) have emerged to suppress insect populations\u003csup\u003e22\u003c/sup\u003e. gSIT offers advantages like enhanced individual competitiveness and survival, successfully applied in managing agricultural pests such as fruit flies, silkworms, and diamondback moths\u003csup\u003e23\u003c/sup\u003e. Therefore, the deployment of more efficient gSIT technologies that sterilize males without compromising their fitness significantly is beneficial\u003csup\u003e24\u003c/sup\u003e. To achieve this, identifying molecular targets linked to male sterility is essential for the optimal development of genetic technologies.\u003c/p\u003e \u003cp\u003eNumerous studies have been conducted to explore the genetic and molecular processes that govern spermatogenesis to identify potential targets for gene-specific treatments for infertility\u003csup\u003e25,26\u003c/sup\u003e. The testicular-specific serine/threonine protein kinases (\u003cem\u003eTSSKs\u003c/em\u003e) are exclusively expressed after sperm cell meiosis, and their distinct patterns of expression across developmental stages and in various tissues suggest their involvement in the regulation of spermatogenesis\u003csup\u003e27,28\u003c/sup\u003e. For example, experiments involving the knockdown of \u003cem\u003eTSSK1\u003c/em\u003e in \u003cem\u003eD rosophila melanogaster\u003c/em\u003e and \u003cem\u003eZeugodacus cucurbitae\u003c/em\u003e have demonstrated a significant decrease in sperm viability and a detrimental effect on male fertility, resulting in a decrease in the hatching of eggs\u003csup\u003e29,30\u003c/sup\u003e. Similarly, the use of CRISPR/Cas9 technology to knockout the serine protease 2 (\u003cem\u003eser2\u003c/em\u003e) gene in \u003cem\u003eBombyx mori\u003c/em\u003e has resulted in the inability of sperm and eggs to fertilize, ultimately leading to infertility in subsequent generations\u003csup\u003e23\u003c/sup\u003e. Furthermore, the reduction of the \u003cem\u003etektin\u003c/em\u003e gene, which is associated with sperm flagella, has been found to cause a significant decrease in the hatching rate of offspring\u003csup\u003e29\u003c/sup\u003e. These results suggest that genes specifically expressed in testis play a crucial role in male reproduction, although the exact regulatory mechanism remains uncertain.\u003c/p\u003e \u003cp\u003eEukaryotic gene expression has traditionally been assessed through steady-state mRNA levels. However, this fails to consider translational activation variability or the stability of specific mRNA types. Consequently, post-transcriptional regulation plays a pivotal role in the overall coordination of gene expression\u003csup\u003e31\u003c/sup\u003e. It is important to highlight that the maturation of insect sperm involves extensive post-transcriptional regulation, whereby a significant number of transcripts are preserved for translation at a later stage\u003csup\u003e32\u003c/sup\u003e. In recent years, long non-coding RNAs (lncRNAs) have emerged as crucial factors in post-transcriptional regulatio\u003csup\u003e33,34\u003c/sup\u003e. These lncRNAs play a significant role in regulating gene expression through various mechanisms, including chromatin modification, RNA decoying, transcriptional co-activation, ribonucleoprotein complex formation, and microRNA sequestration\u003csup\u003e35,36,37\u003c/sup\u003e. LncRNAs exhibit a multifaceted modus operandi involving various regulatory modalities, encompassing direct inhibition of gene expression by lncRNAs, as well as their role as competitive endogenous RNAs (ceRNAs), sequestering microRNAs (miRNAs) to modulate the expression of messenger RNAs (mRNAs)\u003csup\u003e34\u003c/sup\u003e. Notably, the knockdown of 128 testicle-specific lncRNAs in fruit flies demonstrated that silencing nine of these lncRNAs led to sterility or reduced fertility\u003csup\u003e38\u003c/sup\u003e. The \u003cem\u003elnc94638\u003c/em\u003e is crucial for spermatogenesis in \u003cem\u003eZ. cucurbitae\u003c/em\u003e and significantly affects male fertility\u003csup\u003e39\u003c/sup\u003e. Therefore, further investigation of the post-transcriptional regulatory mechanisms involved in spermatogenesis is necessary to advance the development of male sterile technology.\u003c/p\u003e \u003cp\u003eThe invasive agricultural pest, \u003cem\u003eC. pomonella\u003c/em\u003e, has been listed among the world's 20 most resistant pests to insecticides by the Insecticide resistance action committee (IRAC)\u003csup\u003e40\u003c/sup\u003e. To address resistance problem, the use of SIT as part of area-wide integrated pest management (AW-IPM) has been proposed as an environmentally friendly control technology\u003csup\u003e12\u003c/sup\u003e. Previous studies have shown the effectiveness of SIT in controlling \u003cem\u003eC. pomonella\u003c/em\u003e\u003csup\u003e15\u003c/sup\u003e. However, the most commonly used radiation-based sterile technology (rSIT) technology is not aligned with the current trend of green agriculture development due to issues such as cobalt source waste, waste source treatment and radioactive safety hazards\u003csup\u003e23\u003c/sup\u003e. In order to explore the concept of gSIT for pest control, this study focused on investigating genes related to male reproduction. Through genome and testis transcriptome analysis, five \u003cem\u003eTSSKs\u003c/em\u003e genes were identified. To understand the functional role of \u003cem\u003eTSSKs\u003c/em\u003e, an RNA interference (RNAi) and CRISPR/Cas9-based loss-of-function approach was employed, comparing \u003cem\u003eC. pomonella\u003c/em\u003e mutant and wild-type (wt) individuals. The study revealed that \u003cem\u003eTSSKs\u003c/em\u003e are highly expressed in the testes of adult males. Surprisingly, disruption of the \u003cem\u003eTSSKs\u003c/em\u003e gene in males resulted in sterility, reduced sperm motility, decreased sperm counts, and abnormal development of fertilized eggs. Furthermore, analysis of the lncRNA transcriptome identified as lnc17962 significantly expressed in testis. Notably, lnc17962 was found to competitively bind with miR-3960, regulating the expression of \u003cem\u003eTSSKs\u003c/em\u003e and ultimately causing sterility in offspring. These findings emphasize the crucial role of \u003cem\u003eTSSKs\u003c/em\u003e and lnc17962 in sperm function and suggest their potential as targets for the applications of gSIT technology in \u003cem\u003eC. pomonella\u003c/em\u003e control.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInsect\u003c/h2\u003e \u003cp\u003eThe experimental insects were reared in a controlled environment with a temperature of 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, relative humidity of 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH, and a photoperiod of 16 h light and 8 h dark. The adult was fed with 10% honey water. A detailed description of the feeding method has been previously described by Wang et al\u003csup\u003e41\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRNA sample preparation and sequencing\u003c/h2\u003e \u003cp\u003eFreshly emerged adult males were placed in an 85 mL plastic cup (40 mm in diameter \u0026times; 60 mm in height), and their testes were collected on the second day (CpA2T) using phosphate-buffered saline (PBS) at pH 7.5. Controls were established using the entire male moths, excluding the spermatozoa (CpA2U). On the fifth day after mating, the testes were dissected (CpA5T). This allowed for the use of the whole male moths, from which the testicular tissue had been removed (CpA5U), as a control. Each sample comprised of tissue from 50 individuals and there were three biological replicates per sample. Total RNA was extracted from the samples using Trizol (TaKaRa, Tokyo, Japan) following the provided instructions. The quality of the RNA samples was assessed using NanoDrop (Thermo Scientific, Wilmington, USA), and the integrity of the RNA samples was evaluated through 1% agar gel electrophoresis. The RNA-seq datasets were obtained and analyzed using the aforementioned four samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing\u003c/h2\u003e \u003cp\u003eAn Illumina TruseqTM RNA sample preparation kit (Illumina, San Diego, CA, USA) was used for library preparation. Before library construction, the quality of the RNA samples was assessed using an Agilent 2100 instrument (Agilent Technologies, Santa Clara, CA, USA). Paired-end sequencing was performed on an Illumina Novaseq 6000 sequencing platform. The library was then subjected to the removal of N-containing error bases and reads of low quality.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eAnnotation of testis-specific expression genes and quantitative analyses\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe clean read was obtained by analyzing the library with the default parameter of the HISAT2 analysis using the reference genome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://v2.insect-genome.com/Pcg\u003c/span\u003e\u003cspan address=\"http://v2.insect-genome.com/Pcg\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Clean reads were defined as those that had a unique match with a maximum of two mismatched bases and no deletions or insertions. The quality of each RNA read was evaluated by comparing its localization ratio to the reference genome. Subsequently, RNA sequencing (RNA-seq) was performed on the clean reads and aligned to the genomes using the expectation-maximization method (RSEM, version 1.3.3). These aligned reads were then used for gene expression analysis. The expression levels of all genes were quantified as transcripts per million (TPM) in repeats. Standardized average TPM values were employed to compare the relative expression of each gene in the tissues. Differentially expressed genes (DEGs) were identified using DESeq (version 1.30.0) based on the criteria of corrected \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log2 expression ratio\u0026thinsp;\u0026gt;\u0026thinsp;2. By comparing gene expression levels across all tissues, genes with high expression in the testes were identified, suggesting their potential role in fertility.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFull-length confirmation and sequence analysis of TSSKs\u003c/h2\u003e \u003cp\u003eThe complete open reading frame (ORF) of the \u003cem\u003eTSSKs\u003c/em\u003e gene was verified using reverse transcriptase PCR (RT-PCR) with primers designed by Oligo7. Each PCR reaction was conducted in a 20 \u0026micro;L volume, consisting of 10 \u0026micro;L 2x Primer STAR Max Premix (TaKaRa), 7.4 \u0026micro;L RNase-free water, 1.0 \u0026micro;L testicular cDNA, and 0.8 \u0026micro;L (10 \u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) forward and reverse primers (Table S1). The thermal cycler was programmed as followings: initial denaturation at 98\u0026deg;C for 2 min, followed by 39 cycles of denaturation at 98\u0026deg;C for 5 s, annealing at 55\u0026deg;C for 15 s, and extension at 72\u0026deg;C for 1 min. Subsequently, electrophoresis analysis on a 1% agarose gel was performed on the PCR-amplified products. The resulting target products were then ligated to the pESI-Blunt simple vector (YEASEN, shanghai, China) and transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α. After transformation, the bacteria were cultured in an LB medium, and positive colonies were confirmed through PCR and Sanger sequencing executed by GENEWIZ (Tianjin, China). Homologous protein amino acid sequences from \u003cem\u003eTSSKs\u003c/em\u003e and other insect species were obtained using NCBI BlasP, and sequence comparison analysis was conducted using DNAMAN. The results were visualized graphically using espript3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi\u003c/span\u003e\u003cspan address=\"https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Additionally, a phylogenetic tree was generated using MEGA11.0 software with the neighbor-joining (NJ) method and 1000 bootstrap replicates for increased reliability of the results.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReal-time quantitative polymerase chain reaction (RT-qPCR) analysis of\u003c/b\u003e \u003cb\u003eTSSKs\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eC. pomonella\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe expression of the \u003cem\u003eTSSKs\u003c/em\u003e genes was validated through real-time quantitative polymerase chain reaction (RT-qPCR). The primer used for this purpose can be found in Table S2. Briefly, in accordance with the procedures outlined in a previous study\u003csup\u003e42\u003c/sup\u003e, tissues and stages were collected. Following the extraction of total RNA, the first strand of complementary DNA (cDNA) was synthesized using approximately 1 \u0026micro;g of RNA per sample following the instructions provided by the manufacturer of the PrimeScriptTM RT reagent kit witn gDNA eraser (TaKaRa, Dalian, China).As previously described\u003csup\u003e42\u003c/sup\u003e, the qPCR reaction was conducted with a total volume of 20 \u0026micro;L, comprising 10 \u0026micro;L GoTaq qPCR Master Mix (TaKaRa, Dalian, China), 1 \u0026micro;L template cDNA, 0.8 \u0026micro;L of each primer (10 \u0026micro;M), and 7.4 \u0026micro;L nuclease-free water. The CFX 384 Real-Time System (Bio-Rad, Singapore) was employed for all qPCR analyses, with the following settings: initial denaturation at 95\u0026deg;C for 2 min, followed by 40 cycles of denaturation at 95\u0026deg;C for 15 s and annealing at 60\u0026deg;C for 30 s. To ensure the specificity of the amplification reaction within the temperature range of 60\u0026thinsp;~\u0026thinsp;95 \u0026deg; C, a melting curve analysis was performed on all samples. Each qPCR included three biological replicates, with three technical replicates for each of the following. The expression level of target gene was normalized with the geometric mean of \u003cem\u003eEF-1α\u003c/em\u003e and \u003cem\u003eRP12\u003c/em\u003e using the 2\u003csup\u003e\u0026minus;△△ct\u003c/sup\u003e method\u003csup\u003e43\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFunctional analysis of the role of\u003c/b\u003e \u003cb\u003eTSSKs\u003c/b\u003e \u003cb\u003ein male fertility of\u003c/b\u003e \u003cb\u003eC. pomonella\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe dsRNA was synthesized and purified following the protocol described in the T7 RiboMAXTM Express RNAi system (Promega, USA). The concentration of dsRNA was determined using a Nanodrop (Thermo Scientific, Wilmington, USA), while the integrity of the dsRNA was evaluated through 1% agarose gel electrophoresis. Male pupae in the advanced stage of development, distinguished by their dark pigmentation, were selected for the experimental procedure. These pupae were injected with 1 \u0026micro;L of dsRNA at a concentration of 3000 ng/\u0026micro;L. To enhance the durability of RNAi following injection, the same dosage of dsRNA was administered once more after the emergence of the adults (Double injections should be administered within a 24 h period). Insects injected with double-stranded green fluorescent protein (dsGFP) were used as negative control. To assess the efficacy of RNAi, nine moth were sampled at 24, 48, and 72 h post-injection. Furthermore, the sperm count was examined in nine male moths. Specifically, the testicles of each male were collected in PBS, punctured with tweezers in 100 \u0026micro;L of 1\u0026times; PBS solution, gently shaken, and then stained with 10 \u0026micro;L of 4',6-diamidino-2-phenylindole (DAPI) for 15 min to stain the nuclei of the resulting sperm solution. The 10 \u0026micro;L sperm suspension was placed on a slide and the sperm were counted using an Olympus FV1000S fluorescence microscope (Olympus, Tokyo, Japan). The total sperm count was obtained by averaging the counts from five microscope fields. The testicles from 2-day-old male moths were dissected in petri dishes containing Hayes solution (composed of 0.2 g CaCl\u003csub\u003e2\u003c/sub\u003e, 9.0 g NaCl, 0.1 g NaHCO\u003csub\u003e3\u003c/sub\u003e, and 0.2 g KCl in 1000 mL H\u003csub\u003e2\u003c/sub\u003eO). The motility of sperm was assessed using a sperm motility kit (Thermofisher, Vilnius, Lithuania), which utilizes two fluorescent dyes to differentiate between dead (red propyl iodide) and live (SYBR-14, green glow) sperm cells. Spermatozoa were diluted 5 \u0026micro;L and incubated with 5 \u0026micro;L SYBR-14 working solution (2 \u0026micro;L SYBR-14 stock solution plus 98 \u0026micro;L Hayes solution) on a glass slide at 25℃ for 10 min, followed by 7 min of incubation with propyl iodide. All experiments were performed in triplicate. The 45 male moths injected with dsRNA were placed in an 85 mL plastic cup (40 mm in diameter \u0026times; 60 mm in height) and allowed to mate with female moths of the same age to lay eggs. The hatching rate of the eggs was subsequently determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003esgRNA synthesis and embryo microinjection\u003c/h2\u003e \u003cp\u003eIn accordance with the screening criteria \"5-GG-N18-NGG-3\" (where N represents one of the four foundations), target locations were selected by the Crispor website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispor.tefor.net/\u003c/span\u003e\u003cspan address=\"http://crispor.tefor.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) based on a comprehensive score calculation. The templates for sgRNA synthesis were amplified through PCR using a set of primers (Table S2) and the MEGA shortscript\u0026trade; T7 Transcription Kit (Invitrogen, Carlsbad, CA). The PCR was carried out using PrimeSTAR\u0026reg; Max DNA Polymerase (TaKaRa, Dalian, China). Eggs at the pre-embryonic stage were promptly collected within one hour of being laid and securely placed on a microscope slide with double-sided adhesive tape. The embryos were then injected using a Leica DM2700 M stereomicroscope (Leica, Berlin, Germany) equipped with a FemtoJet4i microinjector (Eppendorf, Berlin, Germany). The injection involved 300 ng/\u0026micro;L sgRNA and 150 ng/\u0026micro;L TrueCut\u0026trade;Cas9 protein v2 (Thermofisher, Vilnius, Lithuania). All procedures, including embryo collection and microinjection, needed to be completed within a two-hour timeframe. Following microinjection, the eggs remained attached to the slide and were incubated at 26\u0026deg;C until hatching, after which they were transferred to an artificial diet.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003elncRNA sequencing and analyses of lncRNA expression\u003c/h2\u003e \u003cp\u003eTranscriptome sequencing was employed to generate four RNA-seq libraries, which consisted of testes samples from 1-day-old (CpT1D) and 3-day-old (CpT3D) individuals. The expression levels of lncRNAs were quantified using the Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) value and Cuffdiff software (version 2.1.1). To identify specific lncRNA expression, a screening process was conducted on the four testis samples, utilizing the criteria of log2FoldChange\u0026thinsp;\u0026gt;\u0026thinsp;5 and Q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. To validate the expression of 12 randomly selected testicular-specific lncRNAs, their expression profiles in various tissues were analyzed through RT-qPCR. from the tissues examined included accessory glands, vas deferens, seminal vesicles, and testes from both 1-day and 3-day male adults, along with the previously transcriptomically sequenced samples. All samples were replicated three times for biological accuracy. Total RNA was isolated using Trizol (TaKaRa, Tokyo, Japan) following the manufacturer's instructions. The cDNA for the first lncRNA was synthesized using RR037A (TaKaRa, Japan). RT-qPCR primers (Table S2) were designed using Primer 5 software. \u003cem\u003eEF-1α\u003c/em\u003e and \u003cem\u003eRPL12\u003c/em\u003e were used as internal reference genes, and data analysis was conducted using the 2\u003csup\u003e\u0026minus;△△ct\u003c/sup\u003e method\u003csup\u003e43\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence in situ hybridization\u003c/h2\u003e \u003cp\u003eThe FAM-labeled probe was synthesized through the \u003cem\u003ein vitro\u003c/em\u003e synthesis method and obtained from GefanBio (Shanghai, China). To conduct fluorescence in situ hybridization (FISH) analysis, the testes of 5-day-old male moths were dissected in PBS and fixed overnight at 4\u0026deg;C using 4% paraformaldehydefan. The WISH in situ hybridization kit (GefanBio, Shanghai, China) was used for this analysis, following the procedure described below: the samples were dehydrated using an alcohol gradient (50%, 60%, 70%, 80%, 90%, 100% alcohol for 30 min), dried with DEPC-PBST (0.1% Tween-20) for 1 h, bleached with 6% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 1 h, washed three times with PBS, and then washed three times with PBST (5 min/wash). Subsequently, the samples were permeated with protease K at 37\u0026deg;C for 20 min. Probes (diluted 1,000 times) were added and incubated for 48 h. The slides were rinsed three times with the wash solution provided in the kit and stained with DAPI (Sigma, St. Louis, Missouri, USA) for 10 min. Finally, the samples were examined using a Zeiss LSM780 confocal microscope (Zeiss, Jena, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFunctional analysis of the role of lnc117962 in male fertility\u003c/h2\u003e \u003cp\u003eThe primer design, synthesis, quality detection, and RNAi of lincRNA were executed following the procedures described in the preceding section. The lncRNA overexpression vector was procured from GenePharma (Shanghai, China). To begin, male moths were subjected to continuous injection of dsRNA, following which their testes were dissected in petri dishes containing PBS after a 48 h interval. The testes were then punctured using forceps, and 2 \u0026micro;L of motile spermatozoa were expeditiously collected. Subsequently, the motility and hatchability of the spermatozoa were assessed using the aforementioned methodologies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMating competitiveness analysis of sterile males\u003c/h2\u003e \u003cp\u003eTo evaluate the mating competitiveness of lnc117962 knockdown males in the presence of wt males, the latter were chosen as an appropriate control group due to their shared genetic background with the lnc117962 knockdown males. For this purpose, an 85 mL plastic cup (40 mm in diameter \u0026times; 60 mm in height) was employed to accommodate one wt male, one lnc117962 knockdown male, and one wt virgin female. This configuration, covered with plastic wrap, facilitated the process of egg laying. Likewise, a plastic box (10.4 cm \u0026times; 17.3 cm \u0026times; 6.6 cm) containing 10 wt males, 10 lnc117962 knockdown males, and 20 wt virgin male moths, was employed in an apple orchard (41.83'N, 123.57'E) to ensure mating and egg laying. The competition mating index was calculated by analyzing statistical data about fecundity per female and hatching rates (Zhang et al., 2023).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetection of the interaction of miRNAs with lnc117962 and TSSKs\u003c/h2\u003e \u003cp\u003eTo identify potential miRNA that may target lnc117962 using pre-existing miRNA, we employed three established miRNA target gene prediction software programs: miRanda, PITA, and RNAhybrid. The final prediction result was obtained by comparing the outcomes from these three software programs, taking into account their individual preferences during the prediction process. The candidate miRNAs were screened to ensure that no more than 2 GUs were matched between 2 and 8 binding positions, and that the absolute critical strength exceeded 25. The mature miRNA sequences were then sent to GenePharma (Shanghai, China) for the design and synthesis of agomir/antagomir, as well as the synthesis of the corresponding stable negative control and miRNA inhibitor negative control. After synthesizing the agomir and antagomir, RNase-free H\u003csub\u003e2\u003c/sub\u003eO was added to achieve a final concentration of 20 \u0026micro;M. Male adults of newly emerged were collected and injected with agomir and antagomir in their abdomens, using an injection volume of 1 \u0026micro;L/moth. Stable negative control and microRNA inhibitor were injected as a negative control. Total RNA was extracted from randomly selected samples 48 h after the injection. Three biological replicates were obtained for each treatment, and 10 samples were obtained for each replicate. The Mir-X miRNA first-strand Synthesis Kit (Promega, Madison, USA) was used to synthesize the first cDNA of the miRNA, following the provided instructions.\u003c/p\u003e \u003cp\u003eTo examine the interaction between miRNA and lncRNA, a dual luciferase reporter system was employed. This experimental procedure adhered to the protocol provided by GenePharma (Shanghai, China). Specifically, a luciferase reporter vector containing the target gene was constructed. Subsequently, lnc117962 and miR3960 were linked to the pmirGLO dual luciferase reporter vector (Promega, Madison, USA). The resulting vector was designated as pmirGLO-Lnc117962-3960-mutant. Well-conditioned 293T cells were digested, resuspended, and plated in 12-well plates at appropriate cell densities. The cells were then incubated overnight at 37℃. The 293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) (1.5 mg/mL glutamine, 100 U/ml penicillin, 100 \u0026micro;g/ml streptomycin). The cells were maintained in a 5% CO2-saturated humidity incubator at 37\u0026ordm;C. Subsequently, the interaction between miR-3960 and lnc17962 was examined. Four groups of cells were transfected, including (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) pmirGlO-Lnc117962-3960-wt and mimic negative control, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) pmirGlO-Lnc117962-3960-wt and miR-3960 mimic, (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) pmirGlO-Lnc117962-3960-mut and mimic NC, (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) pmirGlO-Lnc117962-3960-mut and miR-3960 mimic. After 24 h of transfection, the cells were lysed and the fluorescence values of the samples were measured using the Dual-Glo\u0026reg; Luciferase Assay System (Promega, Wisconsin, USA). The relative activity was determined by normalizing to the Renilla luciferase. Three independent experiments were performed, with each sample being replicated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was conducted using JMP 8.0.2 by IBM SPSS Statistics 26. Biological replicates were used to generate statistical means for the purpose of comparisons. \u003cem\u003eP\u003c/em\u003e values were calculated through a two-sample Student\u0026rsquo;s t-test with unequal variance, where the significance level was set at of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The results are presented as the mean value of the triplicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTSSKs\u003c/strong\u003e \u003cstrong\u003eare specifically expressed in male testis of\u003c/strong\u003e \u003cstrong\u003eC. pomonella\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the larval stage, the \u003cem\u003eC. pomonella\u003c/em\u003e, like other Lepidopteran insects, possesses two separate testes. However, as the insect enters the prepupal stage, these testes gradually move closer to each other. By the time the insect reaches the pupal stage, the two testes fuse together to form a single mature testis in the adult stage (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eA). To investigate the genes that are specifically expressed in the testes of adult males and their role in mating, RNA-seq was conducted on the testes of 2-day-old (presexual maturity, CpA2T) and 5-day-old (postsexual maturity, CpA5T) male of \u003cem\u003eC. pomonella\u003c/em\u003e. Other parts of the testes were used as controls (CpA2U and CpA5U, respectively). This analysis identified a total of 7736 DEGs in the comparison of presexual maturity and control samples, with 4240 upregulated and 3496 downregulated genes. Similarly, 7677 DEGs, with 4378 upregulated and 3299 downregulated genes, were identified in the comparison of postsexual maturity and control samples (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eB; Figure S1A and S1B). Notably, the testes exhibited high expression of serine protease genes (Figure S2), tektin (Figure S3A), cyclin (Figure S3B), ubiquitin (Figure S3C), and tubulin (Figure S3D). These DEGs may serve as potential candidates for modulating the fecundity of male \u003cem\u003eC. pomonella\u003c/em\u003e. Furthermore, GO enrichment analysis highlighted the crucial role played by the differential genes (Figure S1C and S1D), while KEGG enrichment analysis showed their close relationship to the metabolic pathway (Figure S1E and S1F), particularly the testicle-specific serine/threonine protein kinase (\u003cem\u003eTSSK\u003c/em\u003e), which displayed high expression (Figure S2). Members of the \u003cem\u003eTSSK\u003c/em\u003e gene family, including \u003cem\u003eTSSK1\u003c/em\u003e, \u003cem\u003eTSSK1a\u003c/em\u003e, \u003cem\u003eTSSK2\u003c/em\u003e, \u003cem\u003eTSSK2a\u003c/em\u003e, and \u003cem\u003eTSSK4\u003c/em\u003e, were selected for further investigation to elucidate their role in spermatogenesis. Phylogenetic analysis revealed the clustering of these genes with \u003cem\u003eTSSK\u003c/em\u003e from other Lepidoptera species (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eC; Table S1), and collinearity analysis demonstrated their presence among Lepidoptera insects (Figure S4). Multiple sequence comparisons confirmed the conservation of TSSK residues in the S-TKc region, as well as the adenosine triphosphate (ATP) and substrate binding domains (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eD). Additionally, RT-qPCR analysis determined the expression levels of \u003cem\u003eTSSKs\u003c/em\u003e in the developmental stages and tissues of \u003cem\u003eC. pomonella\u003c/em\u003e, showing moderate expression during the pupal stages and increased expression as the insects reached sexual maturity. \u003cem\u003eTSSKs\u003c/em\u003e were found to be expressed only in the male testis, with \u003cem\u003eTSSK4\u003c/em\u003e exhibiting the highest expression level (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eE). These findings suggest that these testis-specifically expressed \u003cem\u003eTSSKs\u003c/em\u003e play an important role in the m ale fertility of \u003cem\u003eC. pomonella\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLoss function of\u003c/strong\u003e \u003cstrong\u003eTSSKs\u003c/strong\u003e \u003cstrong\u003einfluence male fertility of\u003c/strong\u003e \u003cstrong\u003eC. pomonella\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing a 48-h period of injection, the efficiency of RNAi was observed to increase from 37.32% (Figure S5) to 86.09% (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eA), suggesting that double injections method enhances the interference efficiency over time. Upon injecting adults subjects with dsTSSKs and conducting subsequent observations after 24 h, it was found that the expression levels of \u003cem\u003eTSSK1\u003c/em\u003e, \u003cem\u003eTSSK1a\u003c/em\u003e, \u003cem\u003eTSSK2\u003c/em\u003e, \u003cem\u003eTSSK2a\u003c/em\u003e, and \u003cem\u003eTSSK4\u003c/em\u003e were significantly reduced by 56.13% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001)、86.09% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001)、42.67% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.038)、72.08% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and 75.62% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), respectively, compared to control treatment with dsGFP (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eA). Although the number of eggs produced by the females mated with dsTSSKs treated males remained unchanged (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eB), the hatching rate of these eggs was significantly lower compared to dsGFP treatment (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eC). The sterility rates of \u003cem\u003eTSSK1\u003c/em\u003e, \u003cem\u003eTSSK1a\u003c/em\u003e, \u003cem\u003eTSSK2\u003c/em\u003e, \u003cem\u003eTSSK2a\u003c/em\u003e, and \u003cem\u003eTSSK4\u003c/em\u003e knockdown lines were found to be 90.61%, 83.93%, 100%, 100%, and 100%, respectively (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eC). The unhatched eggs exhibited progressive desiccation during development, with no evidence of reaching the blackhead stage (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eD). To ascertain whether the underdeveloped eggs were a result of lack of fertilization in the female, a spermatophore examination was conducted, ruling out this possibility (Figure S6). Sperm viability was then assessed in both the dsGFP and dsTSSKs treatment groups, revealing a higher number of dead eupyrene and apyrene sperm bundles in the dsTSSKs treatment group compared to the dsGFP group (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eE). In males, the number of spermatozoa was reduced by 28.61% (dsTSSK1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0014), 63.03% (dsTSSK1a, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 39.00% (dsTSSK2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0 030), 46.77% (dsTSSK2a, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and 30.38% (dsTSSK4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0024) in the dsTSSKs treatment groups compared to the control (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eF).\u003c/p\u003e\n\u003cp\u003eIn addition, we employed the CRISPR/Cas9 gene editing system to induce mutation in the \u003cem\u003eTSSK\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eG), which led to deletions at the desired location (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eH and I). The mutation rate of the \u003cem\u003eTSSK\u003c/em\u003e genes varied from 9.38\u0026ndash;12.46% (Table S3), and these genetic modifications did not have any impact on the insect\u0026prime;s developmental period (Table S4). When TSSKs\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e males were mated with wt females, the egg-laying levels were similar to the results of RNAi (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eJ). However, a significant decrease in hatchability, consistent with the results of RNAi approach, was observed (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eK). Most embryos derived from TSSKs\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e males failed to develop properly, even after seven days following ovulation. Consequently, TSSKs\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e males exhibit normal sexual behavior but are ultimately sterile.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLnc117962 is specifically expressed in male testis of\u003c/strong\u003e \u003cstrong\u003eC. pomonella\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDistinctive expression patterns of lncRNAs have been observed in testis, accessory gland, seminal vesicles and vas deferens., indicating the presence of tissue-specific lncRNAs (Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eA). By analyzing the Venn diagram, it was found that 6361 lncRNAs were specifically expressed in the testis (CpT1D vs CpT1DC). Furthermore, the testis exhibited specific expression of 7390 lncRNAs in CpT3D compared to CpT3DC, with 4430 lncRNAs being expressed in both CpT1D vs CpT1DC and CpT3D vs CpT3DC (Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eB). Twelve lncRNAs with differential expression (log2fold change\u0026thinsp;\u0026ge;\u0026thinsp;4 threshold) were identified, and their tissue-specific expression patterns in different developmental stages were confirmed using RT-qPCR. The RT-qPCR results revealed that all 12 lncRNAs were highly expressed in the testis, while exhibiting negligible expression in the vas deferens, accessory glands, and seminal vesicles (Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eC). Notably, lnc117962showed the highest expression level in the testes, with subsequent decreased expression as sperm matured and shifted. Furthermore, fluorescent in FISH revealed a localization signal in the testes of \u003cem\u003eC. pomonella\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eD). Results from Gene Ontology (GO) pathway analysis suggest that lnc117962 has the potential to modulate various gene enrichments associated with ATP activity and serine/threonine protein kinase pathways. This indicates that lnc117962 might play a crucial role in regulating the expression of \u003cem\u003eTSSKs\u003c/em\u003e and could be significant in the post-transcriptional control of male fertility (Figure S7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLoss function of lnc117962 influences male fertility of\u003c/strong\u003e \u003cstrong\u003eC. pomonella\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe role of lnc117962 in the regulation of male fertility was investigated using RNAi technology. Silencing lnc117962 with ds117962 resulted in a 74.83% efficacy after 48 h (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eA). Assessment of \u003cem\u003eTSSK\u003c/em\u003e family gene expression levels following ds117962 treatment showed reductions of 71.94%, 42.87%, 47.96%, 91.58%, and 63.36% compared to dsGFP control (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eB). Overexpression of lnc117962 with pcDNA3.1-lnc117962 resulted in a 2.47-fold increase in lnc117962 expression compared to pcDNA3.1-GFP (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eC). Additionally, \u003cem\u003eTSSK1\u003c/em\u003e, \u003cem\u003eTSSK1a\u003c/em\u003e, and \u003cem\u003eTSSK4\u003c/em\u003e expression decreased by 39.28%, 26.61%, and 19.93%, respectively, while \u003cem\u003eTSSK2a\u003c/em\u003e expression increased by 1.26-fold with no significant change in \u003cem\u003eTSSK2\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eD). Fertility response to dslnc117962 treatment was assessed based on fertilization and hatching rates, showing no significant difference in egg laying but a 28.5% reduction in hatching rate (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eE, \u003cspan\u003e4\u003c/span\u003eF). Male longevity was unaffected (Figure S8). The dslnc117962 treatment group exhibited more dead sperm in eupyrene and apyrene sperm bundles compared to the control group (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eG), suggesting lnc117962 involvement in \u003cem\u003eC. pomonella\u003c/em\u003e male spermatogenesis and its role in male fertility.\u003c/p\u003e\n\u003cp\u003eThe insignificance of the overall fitness of dslnc117962 knockdown males was anticipated and evaluated using a mating competitiveness assay (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eH). The results indicated that dslnc117962 knockdown males displayed the capacity for courtship, mating, and successful competition with wt females. Specifically, the egg hatch rate was found to be 52.47% \u0026plusmn; 15.96% for a pair of one wt male, one dslnc117962 knockdown male and one wt female, compared to 25.52% \u0026plusmn; 9.55% for a pair of one wt male and one wt female, and 75.26% \u0026plusmn; 4.28% for a pair of one dslnc117962 treated male and one wt female, confirming the mating competitiveness (0.84) of dslnc117962 knockdown males (Table\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e). Furthermore, the mating performance of lnc117962 knockdown males was assessed through field cage experiments conducted in the first two weeks of June 2023, under controlled humidity and temperature conditions (Figure S9). Results obtained from the apple orchard (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eJ) indicated no significant disparity in egg production between 10 wt females mated with 10 dslnc117962 knockdown males and those mated with 20 wt males (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eK). However, the hatch rates were 39.91% \u0026plusmn; 7.46% and 83.13% \u0026plusmn; 2.7%, respectively (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eL). Notably, the hatching rate of the F1 generation was determined to be 67.56\u0026thinsp;\u0026plusmn;\u0026thinsp;5.41% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0029), suggesting that the RNAi treatment predominantly affected the parents and had a lasting impact on the offspring (Figure S10). These results indicate that lnc117962 regulates the activity of \u003cem\u003eTSSKs\u003c/em\u003e through post-transcriptional mechanisms, exerting a suppressive effect on the population development of \u003cem\u003eC. pomonella\u003c/em\u003e.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEffect of dslnc117962 knockdown males on the mating competitiveness of \u003cem\u003eC. pomonella.\u003c/em\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMatching ratio\u003c/p\u003e\n \u003cp\u003e(wtM♂: lnc117962KDM♂: wtF♀)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEgg laid per female\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHatching rate\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ecompetition mating\u003c/p\u003e\n \u003cp\u003eindex (C)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:0:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e103.33\u0026thinsp;\u0026plusmn;\u0026thinsp;18.67 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75.26\u0026thinsp;\u0026plusmn;\u0026thinsp;4.28 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"3\"\u003e\n \u003cp\u003e0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:1:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e101.00\u0026thinsp;\u0026plusmn;\u0026thinsp;17.59 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.47\u0026thinsp;\u0026plusmn;\u0026thinsp;15.96 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0:1:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e105.40\u0026thinsp;\u0026plusmn;\u0026thinsp;22.64 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.52\u0026thinsp;\u0026plusmn;\u0026thinsp;9.55 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eLnc117962 knockdown males (lnc117962KDM), wild-type males (wtM) and wild-type females (wtF) were introduced for mating in the ratios 0:1:1, 1:0:1 and 1:1:1, respectively. A total of 15 replicate experiments were evaluated. The table shows mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) data. Letters following data indicate significant differences analyzed by one-way analysis of variance (ANOVA) using Duncan\u0026apos;s test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eLnc117962 regulates\u003c/strong\u003e \u003cstrong\u003eTSSKs\u003c/strong\u003e \u003cstrong\u003ethrough competitive bindings to miR-3960\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to further elucidate the regulatory mechanism of lnc117962 on \u003cem\u003eTSSKs\u003c/em\u003e gene expression, we predicted its potential miRNA partner. The miR-3960 emerged as the top candidate among the predicted targets (Table S5 and Figure S11), suggesting its possible interaction with lnc17962. The resemblance in their seed sequences indicates that miR-3960 could plausibly act as a target of lnc117962 (Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003eA). To confirm this interaction, a dual luciferase assay demonstrated a 44.33% reduction in luciferase activity in cells transfected with miR-3960 and pmirGLO-lnc117962 mimics compared to mimic negative control (Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003eB). Additionally, computational tools including miRanda, PITA, and RNAhybrid were utilized to predict the binding potential of miR-3960 to the 3\u0026rsquo;UTR of \u003cem\u003eTSSKs\u003c/em\u003e, revealing it capability to interact with the 3\u0026apos;UTR of \u003cem\u003eTSSKs\u003c/em\u003e (Table S6). Further analysis was conducted to ascertain if lnc117962 functions as a \u0026quot;sponge\u0026quot; or decoy ceRNA of miR-3960. The synthesis of agomir/antagomir-miR-3960 and subsequent RT-qPCR assessment of miR-3960, lnc117962, and TSSKs revealed noteworthy. Following the injection of agomir-miR-3960, the expression level of miR-3960 increased by 3.4 times, along with a significant increase in the expression level of lnc117962, \u003cem\u003eTSSK1a\u003c/em\u003e, and \u003cem\u003eTSSK2a\u003c/em\u003e, while \u003cem\u003eTSSK1\u003c/em\u003e, \u003cem\u003eTSSK2\u003c/em\u003e, and \u003cem\u003eTSSK4\u003c/em\u003e levels decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003eC). Conversely, antagomir-miR-3960 injection led to reduced expression levels of miR-3960, lnc117962, \u003cem\u003eTSSK1\u003c/em\u003e, \u003cem\u003eTSSK1a\u003c/em\u003e, \u003cem\u003eTSSK2a\u003c/em\u003e, and \u003cem\u003eTSSK4\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Figure. 5D). These findings indicate that lnc117962 acts as a ceRNA by suppressing miR-3960, thereby positively regulating the expression of the \u003cem\u003eTSSK2a\u003c/em\u003e (Figure. 5E). However, further research is required to explore the regulatory mechanisms of \u003cem\u003eTSSK1\u003c/em\u003e, \u003cem\u003eTSSK1a\u003c/em\u003e, \u003cem\u003eTSSK2\u003c/em\u003e, and \u003cem\u003eTSSK4\u003c/em\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe utilization of SIT has been proven to be effective and eco-friendly for managing the invasive pest, \u003cem\u003eC. pomonella\u003c/em\u003e\u003csup\u003e13,15\u003c/sup\u003e. In this study, we discovered \u003cem\u003eTSSKs\u003c/em\u003e genes which are exclusively expressed in the testis, serving as molecular targets for the development of the gSIT strategy in managing this invasive pest.\u003c/p\u003e \u003cp\u003eSpermatogenesis is a meticulously orchestrated process involving a series of maturation stages that induce notable structural and biochemical changes in spermatogonia within the mature testis\u003csup\u003e44,45\u003c/sup\u003e. This intricate process encompasses both mitotic and meiotic divisions, alongside cellular remodeling throughout the cell cycle\u003csup\u003e26\u003c/sup\u003e. This remodeling necessitates the synchronized activation and deactivation of specific serine/threonine protein kinase (s) that regulate the recombination of sperm chromatin \u003csup\u003e45,46\u003c/sup\u003e. Previous studies consistently affirms the pivotal role of serine/threonine protein kinases in spermatogenesis\u003csup\u003e44\u003c/sup\u003e. The \u003cem\u003eTSSKs\u003c/em\u003e belongs to the 5'-adenosine monophosphate-activated protein kinase (AMPK) family, characterized by the serine/threonine protein kinase catalytic (S-TKc) domain. Although typically comprising six members, \u003cem\u003eTSSK1\u003c/em\u003e, \u003cem\u003eTSSK2\u003c/em\u003e, \u003cem\u003eTSSK3\u003c/em\u003e, \u003cem\u003eTSSK4\u003c/em\u003e, and \u003cem\u003eTSSK6\u003c/em\u003e, the exact number of \u003cem\u003eTSSK\u003c/em\u003e family members in insects remains undisclosed. In this study, 5 \u003cem\u003eTSSK\u003c/em\u003e genes were identified in the genome of \u003cem\u003eC. pomonella\u003c/em\u003e, namely \u003cem\u003eTSSK1\u003c/em\u003e, \u003cem\u003eTSSK1a\u003c/em\u003e, \u003cem\u003eTSSK2\u003c/em\u003e, \u003cem\u003eTSSK2a\u003c/em\u003e, and \u003cem\u003eTSSK4\u003c/em\u003e. The emergence of \u003cem\u003eTSSK1a\u003c/em\u003e and \u003cem\u003eTSSK2a\u003c/em\u003e as novel \u003cem\u003eTSSK\u003c/em\u003e copies in distinct regulatory contexts may elucidate the substitution of \u003cem\u003eTSSK3, TSSK5\u003c/em\u003e, and \u003cem\u003eTSSK6\u003c/em\u003e within this group\u003csup\u003e47\u003c/sup\u003e. Shang et al\u003csup\u003e48\u003c/sup\u003e posit that the greater frequency of sequence mutations in the C-terminal domain of \u003cem\u003eTSSK1/TSSK1B\u003c/em\u003e compared to \u003cem\u003eTSSK2\u003c/em\u003e could be attributed to positive evolutionary selection, potentially influenced by variations in protein partner and substrate preferences. The C-terminal domain plays a crucial role in determining the accurate spatial configuration of the serine/threonine protein kinase activity\u003csup\u003e49\u003c/sup\u003e, facilitating the transfer of γ-phosphate residues from ATP to the hydroxyl group of serine, threonine, or tyrosine residues on the target protein\u003csup\u003e50\u003c/sup\u003e. Through bioinformatics analysis, it was revealed that \u003cem\u003eTSSK1\u003c/em\u003e, \u003cem\u003eTSSK1a\u003c/em\u003e, \u003cem\u003eTSSK2\u003c/em\u003e, \u003cem\u003eTSSK2a\u003c/em\u003e, and \u003cem\u003eTSSK4\u003c/em\u003e genes exhibit homology, with a highly conserved S-TKc domain. All TSSK members habor essential lysine residues crucial for ATP binding, as well as conserved aspartate residues vital for catalytic function. Phylogenetic analysis supports the close relationship between \u003cem\u003eC. pomonella\u003c/em\u003e TSSKs and other Lepidoptera TSSK proteins, underscoring their significant involvement in spermatogenesis.\u003c/p\u003e \u003cp\u003eThe progress in genomics, gSIT technology, and the implementation of a large-scale sterile system have become profoundly influential in current research endeavors \u003csup\u003e16\u003c/sup\u003e. Nevertheless, challenges such as off-target effects and the insufficient knockdown efficacy of RNAi have acted as constraining factors, despite the productive application of RNAi in numerous studies on Lepidoptera\u003csup\u003e51,52\u003c/sup\u003e. Enhancing RNAi efficiency in Lepidoptera is essential, the RNAi efficiency of \u003cem\u003eTSSKs\u003c/em\u003e was successfully improved through double injections during the pupal and adult stages, making the first attempt in \u003cem\u003eC. pomonella\u003c/em\u003e. In \u003cem\u003eRhodnius prolixus\u003c/em\u003e and \u003cem\u003eNilaparvata lugens\u003c/em\u003e, double injections of dsRNA increase RNAi efficiency from 38\u0026ndash;75% and 25\u0026ndash;50%, respectively, indicating that double injection is a suitable method to improve RNAi efficiency\u003csup\u003e53,54\u003c/sup\u003e. In recent years, the utilization of CRISPR/Cas9 technology has emerged as a prominent method for investigating gene functionality model insects have made significant advancements, particularly through the implementation of the precision-guided sterile insect technique (pgSIT), as evidenced in studies involving \u003cem\u003eD.melanogaster\u003c/em\u003e\u003csup\u003e16\u003c/sup\u003e and \u003cem\u003eAnopheles gambiae\u003c/em\u003e\u003csup\u003e55\u003c/sup\u003e. Nevertheless, it is important to recognize that investigations pertaining to gene editing in \u003cem\u003eC. pomonella\u003c/em\u003e are still in the nascent stages. A study conducted by Garczynski et al\u003csup\u003e56\u003c/sup\u003e. In 2017, demonstrated that the introduction of a combination of sgRNA and Cas9, targeting the \u003cem\u003eCpomOR1\u003c/em\u003e gene in \u003cem\u003eC. pomonella\u003c/em\u003e resulted in notable impacts on fertility, with female moths producing inviable eggs\u003csup\u003e56\u003c/sup\u003e. In this study, we have successfully established a functional \u003cem\u003eTSSKs\u003c/em\u003e genes system using CRISPR/Cas9. \u003cem\u003eTSSKs\u003c/em\u003e genes were knocked out through the introduction of a combination of sgRNA and Cas9 protein, resulting in an efficiency rate ranging from 9.38\u0026ndash;12.46%. Prior studies by Chen et al.\u003csup\u003e57\u003c/sup\u003e and Ye et al.\u003csup\u003e58\u003c/sup\u003e demonstrated knockout efficiencies of 6.5\u0026ndash;13.9% for opsin genes in \u003cem\u003ePlutella xylostella\u003c/em\u003e and a 36% efficiency for the pheromone binding protein 1 (\u003cem\u003ePBP1\u003c/em\u003e) gene in \u003cem\u003eHelicoverpa armigera\u003c/em\u003e, respectively. However, the effictiveness of this system in facilitating targeted gene disruption varies significantly among species due to species-specific distinctions\u003csup\u003e52\u003c/sup\u003e. Research on mice has revealed that mutations in the \u003cem\u003eTSSK1\u003c/em\u003e and \u003cem\u003eTSSK2\u003c/em\u003e genes lead to male sterility, attributed to issues in spermatogenesis, absence of elongated spermatids, elevated apoptosis rate, and increased round spermatogenic cells in the epididymis\u003csup\u003e59\u003c/sup\u003e. The loss of \u003cem\u003eTSSK\u003c/em\u003e expression in mice results in defective sperm cell development, abnormal sperm cell structure, and an inability of these cells to fertilize eggs\u003csup\u003e60\u003c/sup\u003e. In this study, we observed a marked rise in deceased spermatozoa and a decline in both spermatozoa count and offspring fertility following \u003cem\u003eTSSK\u003c/em\u003e gene fysfunction. These findings align with prior investigations on \u003cem\u003eB. tryoni\u003c/em\u003e and \u003cem\u003eB. dorsalis\u003c/em\u003e\u003csup\u003e29\u003c/sup\u003e. Phenotypic abnormalities during spermatogenesis have been also observed, including spermatogonial membrane rupture due to the \u003cem\u003eTSSK4\u003c/em\u003e mutation and sperm chromosome disarray caused by \u003cem\u003eTSSK6\u003c/em\u003e mutation in mice\u003csup\u003e61\u003c/sup\u003e. Interestingly, studies on \u003cem\u003eTSSK\u003c/em\u003e mutants in \u003cem\u003eD.melanogaster\u003c/em\u003e indicated unsuccessful formation of the individualization complex (IC) during spermatogenesis\u003csup\u003e10\u003c/sup\u003e. Furthermore, the absence of the N-terminal or C-terminal domain of \u003cem\u003eTSSK\u003c/em\u003e hindered the typical IC structure formation, resulting in male infertility\u003csup\u003e16\u003c/sup\u003e. Nevertheless, gaps remain in understanding the post-transcriptional regulation of \u003cem\u003eTSSKs\u003c/em\u003e in spermatids during spermiogenesis.\u003c/p\u003e \u003cp\u003eThe application of advanced deep transcript sequencing methodologies has spurred an escalation in the exploration of post-transcriptional regulation in insects\u003csup\u003e32\u003c/sup\u003e. At the post-transcriptional level, lncRNAs and miRNAs play crucial roles as regulators of gene expression. Notably, lncRNAs demonstrate tissues-specific expression, particularly exhibiting higher abundance in the testicular tissues\u003csup\u003e38\u003c/sup\u003e. Studies conducted on \u003cem\u003eZ. cucurbitae\u003c/em\u003e have identified a considerable number of highly expressed lncRNAs in the testis\u003csup\u003e55\u003c/sup\u003e. Similarly, examination of RNA-seq data in \u003cem\u003eB. mori\u003c/em\u003e has unveiled a predominant expression of lncRNAs in the testicular tissues\u003csup\u003e62\u003c/sup\u003e. In this study, a comprehensive analysis of lncRNAs expression profiles in the testes of \u003cem\u003eC. pomonella\u003c/em\u003e was carried out, leading to the identification of an upregulation of a specific lncRNA, named lnc17962. Through FISH analysis, the precise localization of lnc117962 in the apical region of the testis, characterized by the presence of mature spermatozoa, was successfully achieved. This localization pattern mirrors the findings from a prior study on \u003cem\u003eBactrocera cucurbitae\u003c/em\u003e, where mature sperm was observed\u003csup\u003e52\u003c/sup\u003e. The involvement of lncRNAs in the development of male germ cells in diverse species has been postulated, with some displaying distinctive expression patterns in fertile and infertile spermatozoa\u003csup\u003e63\u003c/sup\u003e. Notably, the knockout of murine testis-specific lncRNA Tslrn1 using CRISPR/Cas9 technology resulted in a significant decrease in spermatozoa count\u003csup\u003e64\u003c/sup\u003e. In this study, RNAi was utilized to silence lnc117962, resulting in a marked reduction in sperm count and diminished embryo hatching rate compared to the control group. The findings presented in this study provide additional evidence supporting the involvement of lnc117962 in the spermatogenesis process in \u003cem\u003eC. pomonella\u003c/em\u003e. Furthermore, the depletion of CR42858, a testis-specific lncRNA in Drosophila, led to a decline in sperm count and male fertility\u003csup\u003e38\u003c/sup\u003e. The findings reported in \u003cem\u003eB. dorsalis\u003c/em\u003e also indicate impaired male fertility, possibly due to defective spermatogenesis and reduced sperm count\u003csup\u003e29\u003c/sup\u003e. It is widely recognized that many lncRNAs habor binding sites for miRNA\u003csup\u003e65\u003c/sup\u003e. This observation suggests that numerous lncRNA may regulate gene expression by sequestering miRNAs, thereby reducing their cellular concentration\u003csup\u003e66\u003c/sup\u003e. Known as the \"competing endogenous RNA (ceRNA)\" hypothesis, this mechanism proposes that lncRNAs act as negative regulators of miRNA activity, ultimately promoting gene expression\u003csup\u003e67\u003c/sup\u003e. Additionally, our research indicates that lnc117962 can act as a ceRNA for miR-3960, controlling the expression of \u003cem\u003eTSSK2a\u003c/em\u003e and impacting spermatogenesis in \u003cem\u003eC. pomonella\u003c/em\u003e. Interestingly, this regulatory pattern resembles the reproductive mechanism involving lncR26319/miR-2834/EndophilinA in \u003cem\u003eB. mori\u003c/em\u003e\u003csup\u003e68\u003c/sup\u003e, suggesting a potential universality of this regulatory mode. It is noteworthy that miR-3960 has been found to directly regulate the expression of \u003cem\u003eTSSK1a\u003c/em\u003e, although other factors may be involved in the regulatory process of lnc117962 on \u003cem\u003eTSSK1a.\u003c/em\u003e While our study identifies lnc117962 as a ceRNA of miR-3960, the precise mechanism by which it regulates \u003cem\u003eTSSK1\u003c/em\u003e, \u003cem\u003eTSSK2\u003c/em\u003e, and \u003cem\u003eTSSK4\u003c/em\u003e remains unclear, and there may be multiple pathways through which their expression is controlled.\u003c/p\u003e \u003cp\u003eThe conventional irradiated-based SIT is known to generate sterile males with reduced mating competitiveness as a result of mutations across multiple genes\u003csup\u003e69\u003c/sup\u003e. Consequently, an alternative approach focusing on the production of highly competitive sterile males has emerged as an appealing strategy for managing \u003cem\u003eC. pomonella\u003c/em\u003e populations\u003csup\u003e27\u003c/sup\u003e. In this study, we evaluated low-fertility males exhibiting comparable traits to wt males under both laboratory and apple orchard settings, following the suppression or elimination of \u003cem\u003eTSSK\u003c/em\u003e genes. The findings indicate that these males cold potentially possess the same level of safety and resilience as wt counterparts. Furthermore, unlike the previous SIT method where maintaining sterile males as homozygous lines was challenging, mutants with \u003cem\u003eTSSK\u003c/em\u003es could generate offspring with decreased hatchability rates. This parallels the disruption of leucine aminopeptidase 1 in \u003cem\u003eAedes aegypti\u003c/em\u003e, resulting in reduced hatching rates among descendants while preserving their competitiveness edge relative to wt males\u003csup\u003e70\u003c/sup\u003e. Ideally, genetic modifications induced by sterility-inducing genes should naturally disseminate through pest populations without necessitating continuous mass releases of sterile insects\u003csup\u003e71\u003c/sup\u003e. Consequently, mating with \u003cem\u003eTSSKs\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e males offers an effective means to propagate genetic alterations within \u003cem\u003eC. pomonella\u003c/em\u003e populations through a reduced number of viable offspring. As a result, successful population management was achieved through the utilization of \u003cem\u003eTSSKs\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e males in mating scenarios.\u003c/p\u003e \u003cp\u003eOverall, \u003cem\u003eTSSKs\u003c/em\u003e were identified as promising candidates for gSIT owing to their notable level of conservation, rendering them applicable across various insect species. The utilization of gSIT stands to offer a secure, eco-friendly, and effictive approach for managing field pest populations, thereby facilitating the adoption of sustainable agricultural practices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key R\u0026amp;D Program of China (2021YFD1400200).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eX.Q.Y.\u0026nbsp;\u003c/strong\u003econceived and designed experiments, and writing\u003cstrong\u003e; Z.H.W.\u0026nbsp;\u003c/strong\u003eperformed molecular analysis of \u003cem\u003eTSSKs\u003c/em\u003e and writing;\u003cstrong\u003e\u0026nbsp;Y.Q.W.\u0026nbsp;\u003c/strong\u003eperformed molecular analysis of lncRNA;\u003cstrong\u003e\u0026nbsp;K.W.Z. p.c.w\u0026nbsp;and R.H.L.\u0026nbsp;\u003c/strong\u003eperformed feeder population; \u003cstrong\u003eZ.P.W.\u0026nbsp;\u003c/strong\u003eperformed gene editing experiments;\u003cstrong\u003e\u0026nbsp;Y.Li. and P.G.\u003c/strong\u003e performed experimental guidance.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll authors discussed the results and declared that they had no conflicts of interest.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003einterest statement\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eWe declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eStork, N.E. 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Biotechnol. \u003c/em\u003e\u003cstrong\u003e29(11)\u003c/strong\u003e, 1034-1037 (2011).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"TSSK, Cydia pomonella, CRISPR/Cas9, SIT, Post-transcriptional regulation","lastPublishedDoi":"10.21203/rs.3.rs-4156281/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4156281/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGenetic control system at molecular level presents a promising and eco-friendly strategy for the management of pest and insect-transmitted diseases. Although considerable advancements have been achieved in gene drive applications targeting mosquitoes, endeavors to combat agricultural pests have been somewhat restricted. Here, we identified that the testis-specific serine/threonine kinases (\u003cem\u003eTSSKs\u003c/em\u003e) family is uniquely expressed in the testes of \u003cem\u003eCydia pomonella\u003c/em\u003e, a prominent global invasive species. We further generated male moths with disrupted the expression of \u003cem\u003eTSSKs\u003c/em\u003e and those with \u003cem\u003eTSSKs\u003c/em\u003e completely knocked out using RNA interference and CRISPR/Cas 9 genetic editing techniques, resulting in significant disruptions in spermiogenesis, decreased sperm motility, and hindered development of fertilized oocytes. Further explorations into the underlying post-transcriptional regulatory mechanisms have revealed the involvement of lnc117962 as a competing endogenous RNA (ceRNA) for miR-3960, thereby regulating \u003cem\u003eTSSKs\u003c/em\u003e. Notably, orchard trials have demonstrated that the release of male strains can effectively suppress population growth. Our findings indicate that targeting \u003cem\u003eTSSKs\u003c/em\u003e could serve as a feasible avenue for managing \u003cem\u003eC. pomonella\u003c/em\u003e populations, offering significant insights and potential strategies for controlling invasive pests through genetic sterile insect technique (gSIT) technology.\u003c/p\u003e","manuscriptTitle":"Loss-of-function in testis-specific serine/threonine protein kinase (TSSKs) triggers male infertility in an invasive moth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-22 15:19:29","doi":"10.21203/rs.3.rs-4156281/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"10d72bc5-6d01-46be-a3fa-cd08292c54eb","owner":[],"postedDate":"April 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-04T07:05:26+00:00","versionOfRecord":{"articleIdentity":"rs-4156281","link":"https://doi.org/10.1038/s42003-024-06961-5","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2024-10-04 04:00:00","publishedOnDateReadable":"October 4th, 2024"},"versionCreatedAt":"2024-04-22 15:19:29","video":"","vorDoi":"10.1038/s42003-024-06961-5","vorDoiUrl":"https://doi.org/10.1038/s42003-024-06961-5","workflowStages":[]},"version":"v1","identity":"rs-4156281","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4156281","identity":"rs-4156281","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}