Frankliniella occidentalis, the western flower thrips, is among the most destructive agricultural pests worldwide and a major vector of orthotospoviruses. Escalating insecticide resistance has intensified efforts to develop molecularly informed control strategies. RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing have emerged as principal tools for functional genomics in this species, enabling targeted analysis of genes involved in development, metabolism, and insecticide resistance. This review synthesizes recent advances in RNAi and CRISPR applications in F. occidentalis, with particular emphasis on delivery strategies, editing efficiencies, and resulting phenotypes. RNAi studies demonstrate growing technical feasibility through injection, feeding-based approaches, and plant-mediated systems, and emerging CRISPR/Cas investigations confirm the possibility of targeted gene knockouts affecting resistance traits and visible genetic markers. Despite these advances, genetic manipulation of F. occidentalis remains technically challenging, primarily due to biological and technical constraints, including inefficient delivery of ribonucleoprotein to the germline, the extremely small and fragile nature of eggs, and narrow developmental windows for manipulation. We discuss these barriers and highlight methodological innovations required to improve RNAi stability, embryo manipulation, and genome editing efficiency. Finally, we propose how these molecular and genetic tools may be incorporated into existing integrated pest management, emphasizing their potential as complementary, species-specific strategies rather than standalone control solutions toward sustainable F. occidentalis management. © 2026 Society of Chemical Industry.

Thrips are cosmopolitan agricultural pests and important vectors of plant viruses, and the increasing coexistence of multiple morphologically similar species has intensified the demand for species-specific molecular identification. However, traditional morphological identification and PCR assays using universal primers are often inadequate for mixed-species samples and field-adaptable application. In this study, we developed a species-specific molecular identification framework targeting a polymorphism-rich region of the mitochondrial cytochrome c oxidase subunit I (COI) gene, which is more time-efficient than sequencing-based COI DNA barcoding, for four economically important thrips species in southern China, including the globally invasive Frankliniella occidentalis. By aligning COI sequences, polymorphism-rich regions were identified and used to design four species-specific primer pairs, each containing a diagnostic 3'-terminal nucleotide. These primers were combined with a PBS-based DNA extraction workflow optimized for single-insect samples that minimizes dependence on column-based purification. The assay achieved a practical detection limit of 1 ng per reaction, demonstrated species-specific amplification, and maintained reproducible amplification at DNA inputs of ≥1 ng per reaction. Notably, PCR inhibition caused by crude extracts was effectively alleviated by fivefold dilution. Although the chemical identities of the inhibitors remain unknown, interspecific variation in inhibition strength was observed, with T. hawaiiensis exhibiting the strongest suppression, possibly due to differences in lysate composition. This integrated framework balances target specificity, operational simplicity, and dilution-mitigated inhibition, providing a field-adaptable tool for thrips species identification and invasive species monitoring. Moreover, it provides a species-specific molecular foundation for downstream integration with visual nucleic acid detection platforms, such as the CRISPR/Cas12a system, thereby facilitating the future development of portable molecular identification workflows for small agricultural pests.

Yolk protein genes (YPs) are central to insect reproduction, yet the extent of functional divergence among members of the same gene family remains poorly understood in many pest species. Here, we identified five YP genes in Spodoptera frugiperda. Gene structure analysis showed that four YP genes are intronless, whereas YP3 possesses a two-exon structure. Conserved domain analysis revealed that all five YP proteins contain a PFM_spherulin-2a-like domain. We then examined their developmental expression profiles to assess potential stage-specific specialization. YP1 and YP2 were highly expressed in eggs, whereas YP4 and YP5 were more strongly expressed during the pupal and adult stages, with YP3 showing no marked enrichment at either targeted stage. Based on these expression patterns, four genes were selected for RNA interference assays at two developmental stages to evaluate their functions during embryogenesis and reproductive development. Knockdown of YP1 and YP2 in eggs significantly reduced hatchability, whereas pupal-stage knockdown of these genes did not affect fecundity or offspring hatchability. In contrast, knockdown of YP4 and YP5 in eggs caused little or no effect on hatchability, whereas pupal-stage knockdown reduced fecundity and offspring hatchability and impaired ovarian development. In addition, YP4 knockdown disrupted adult emergence, leading to incomplete eclosion and malformed adults. These results show that YP genes in S. frugiperda are not functionally redundant, but instead exhibit clear stage-specific specialization. YP1 and YP2 are primarily associated with embryonic development and egg viability, whereas YP4 and YP5 contribute more to later reproductive performance, with YP4 additionally affecting successful adult emergence. This study provides new evidence for stage-specific functional divergence within the YP gene family in a major agricultural pest, which may pave the ways for developing RNAi-based strategies for green pest control in S. frugiperda.

Dongshan Bay is a typical subtropical semi-enclosed bay characterized by abundant fish resources. We aimed to assess fish diversity and its seasonal variation in Dongshan Bay and to provide a scientific basis for the sustainable management and conservation of the fishery’s resources. In this study, we employed environmental DNA (eDNA) metabarcoding technology to analyze fish diversity in the bay during winter 2023 and summer 2024. A total of 76 fish species were detected across 12 sampling sites, with 43 species identified in summer and 45 species seen in winter. Overall, 13 species were detected in both the winter and summer. Non-significant differences were observed in Alpha diversity among the sampling sites. Fish species richness at the HXH2 site was the lowest among all the sampling sites for the reason that this sampling site was near to the effluent outlet of the Zhangzhou nuclear power plant and notably influenced by the thermal discharge. In general, fish diversity and abundance were higher in winter than in summer. RDA test analysis revealed that water temperature and dissolved oxygen were the primary environmental factors influencing fish distribution in summer. In winter, the influence of various factors is relatively balanced, with chlorophyll and Blue Green Algae Phycoerythrin (BGA PE) having a relatively greater impact than other factors. Our results offer valuable insights into enhancing fish diversity management in Dongshan Bay.

The diamondback moth (Plutella xylostella), a major lepidopteran pest with a wide host range, presents persistent challenges to sustainable agriculture due to its high adaptability to cruciferous host plants. Although glyoxylate/hydroxypyruvate reductases (GRHPRs) have been well-characterized in plants and humans, their functional role in insects, particularly in host plant adaptation, remains largely unexplored. In this study, we characterized PxGRHPR2, a member of the GRHPR gene family, using a bioinformatics analysis, expression profiling, and CRISPR/Cas9-mediated gene knockout. RT-qPCR analysis showed that PxGRHPR2 was predominantly expressed in larval stage, with the highest transcript levels observed in the second instar and larval midgut tissues. Three homozygous PxGRHPR2 knockout strains were successfully generated using CRISPR/Cas9 system. Mutation of PxGRHPR2 led to significant reductions in larval weight, survival, and eclosion rates when larvae were fed on radish seedlings, whereas no such effects were observed under artificial diet conditions. These findings suggest that PxGRHPR2 plays a critical role in detoxification and metabolic regulation, thereby facilitating host plant adaptability in P. xylostella. Overall, this study provides new insights into insect-plant interactions and identifies PxGRHPR2 as a potential molecular target for developing sustainable pest management strategies.

The diamondback moth (Plutella xylostella), a globally destructive pest, has Brassicaceae as its long-term co-evolved host and can also utilize Fabaceae as an alternative field host. The primary differential factor between these plant families is glucosinolates (GLs). Conventional transcriptome data revealed high midgut expression of glucosinolate sulfatases (GSSs) in response to glucosinolates. However, due to the inability of conventional transcriptomics to resolve cellular heterogeneity, it remained unclear which specific cell types express GSSs and whether exposure to different field hosts triggers distinct differentiation fates in these cells. To address these questions, we constructed a high-resolution atlas of 28,451 midgut cells from larvae reared on radish and pea leaves. Marker gene-based clustering identified 16 distinct cell types, including enterocytes (ECs), enteroendocrine cells (EEs), and intestinal stem cells (ISCs). ScRNA-seq and qRT-PCR analyses revealed host-dependent differential expression patterns of three GSS genes (GSS1-GSS3) among ECs, EEs, and ISCs. Notably, elevated GSS3 protein levels in radish-fed larvae were confirmed by Western blot (WB) and Immunohistochemistry (IHC), while GSS1 and GSS2 exhibited more variable expression patterns. Pseudo-time trajectory analysis further demonstrated that all three GSSs followed differentiation pathways from ISCs into EEs or ECs, but dietary conditions led to distinct differentiation trajectories. This study elucidates the diet-dependent regulatory landscape of GSSs in P. xylostella and delineates the differentiation trajectory of GSS-expressing cells.

The co-evolution between plants and herbivorous insects has led to a continuous arms race on defense and anti-defense mechanisms. In this process, insect-derived effectors are crucial for suppressing plant defense. Despite considerable progress in plant–insect interaction studies, the functional role of heat shock cognate protein 70 (HSC70) as an effector in herbivorous insects remains poorly characterized. This study provides evidence that HSC70-3 functions as an effector in interactions between the cruciferous specialist diamondback moth (Plutella xylostella) and its host plant radish (Raphanus sativus ‘Nanpan Prefecture’). Using immunofluorescence labeling and in situ Western blot (WB), we demonstrated that HSC70-3 is secreted into plant wound sites through larval gut regurgitant during feeding. Short-term host transfer experiments revealed tissue-specific hsc70-3 expression changes, indicating a dynamic response to plant-derived challenges. These findings suggest hsc70-3 is differentially regulated at transcriptional and translational levels to facilitate insect adaptation to host plant shifts. Knockout of hsc70-3 using CRISPR/Cas9 technology significantly impaired larval growth, prolonged development duration, and reduced pupal weight on host plants, indicating its involvement in host adaptation. However, knockout mutants exhibited no significant developmental defects when reared on an artificial diet, suggesting that hsc70-3 primarily functions in modulating plant-induced defense responses rather than directly affecting insect physiology. Collectively, these findings provide evidence for the functional roles of HSC70-3 in P. xylostella and plant interactions, laying a foundation for further investigations into insect effectors and their mechanisms in modulating plant defense responses.

Plants perceive proteins from insect-derived oral secretion (OS) and regulate the classical endogenous hormone jasmonic acid to resist insects, but the role of abscisic acid (ABA) in this process is poorly understood. In this study, we used the specialist herbivorous caterpillar Plutella xylostella and cruciferous plants as a model to investigate how the ABA hormone responds to the OS and its core peptide from the insect. Through proteomics and Western blotting analysis, glucosinolate sulfatase 1 (GSS1) was identified in OS. Yeast library screening revealed that GSS1 and its 28-amino-acid core peptide (GSS1-P1) interact with ABA biosynthetic enzyme ABA1. Arabidopsis overexpressing GSS1 and plants treated with synthetic GSS1-P1 showed elevated ABA levels. Transcriptome analysis and RT-qPCR confirmed that GSS1-P1 upregulates WRKY18 and ABA1 expression, modulating ABA production. Both GSS1-P1 application and optimal ABA concentrations enhanced plant resistance to herbivory. Our study shows that GSS1 and its peptide stimulate ABA production, boosting plant-insect resistance and highlighting ABA's potential role in pest-stress response.

Environmental stresses diversely affect multiple processes related to the growth, development, and yield of many crops worldwide. In response, plants have developed numerous sophisticated defense mechanisms at the cellular and subcellular levels to react and adapt to biotic and abiotic stressors. RNA silencing, which is an innate immune mechanism, mediates sequence-specific gene expression regulation in higher eukaryotes. ARGONAUTE (AGO) proteins are essential components of the RNA-induced silencing complex (RISC). They bind to small noncoding RNAs (sRNAs) and target complementary RNAs, causing translational repression or triggering endonucleolytic cleavage pathways. In this review, we aim to illustrate the recently published molecular functions, regulatory mechanisms, and biological roles of AGO family proteins in model plants and cash crops, especially in the defense against diverse biotic and abiotic stresses, which could be helpful in crop improvement and stress tolerance in various plants.

The fall armyworm (Spodoptera frugiperda) poses a substantial threat to many important crops worldwide, emphasizing the need to develop and implement advanced technologies for effective pest control. CRISPR/Cas9, derived from the bacterial adaptive immune system, is a prominent tool used for genome editing in living organisms. Due to its high specificity and adaptability, the CRISPR/Cas9 system has been used in various functional gene studies through gene knockout and applied in research to engineer phenotypes that may cause economical losses. The practical application of CRISPR/Cas9 in diverse insect orders has also provided opportunities for developing strategies for genetic pest control, such as gene drive and the precision-guided sterile insect technique (pgSIT). In this review, a comprehensive overview of the recent progress in the application of the CRISPR/Cas9 system for functional gene studies in S. frugiperda is presented. We outline the fundamental principles of applying CRISPR/Cas9 in S. frugiperda through embryonic microinjection and highlight the application of CRISPR/Cas9 in the study of genes associated with diverse biological aspects, including body color, insecticide resistance, olfactory behavior, sex determination, development, and RNAi. The ability of CRISPR/Cas9 technology to induce sterility, disrupt developmental stages, and influence mating behaviors illustrates its comprehensive roles in pest management strategies. Furthermore, this review addresses the limitations of the CRISPR/Cas9 system in studying gene function in S. frugiperda and explores its future potential as a promising tool for controlling this insect pest.

Genetic pest control strategies based on precise sex separation and only releasing sterile males can be accomplished by site-specific genome editing. In the current study, we showed that the mutation of single-allele Pxfl(2)d can significantly impair the normal mating behavior and testis development in male adults of the notorious cruciferous insect pest Plutella xylostella, in addition to its known functions in the ovarian development in female adults and egg hatching. Subsequent CRISPR/Cas9-based knock-in experiments revealed that site-specific integration of an exogenous green fluorescent protein (GFP) gene into autosomal Pxfl(2)d for labelling mutants could be achieved. However, this gene is not a suitable target for GFP insertion to establish a genetically stable knock-in strain because of the severe decline in reproductive capacity. We further screened for the W-chromosome-linked and Z-chromosome-linked regions to test the knock-in efficiency mediated by CRISPR/Cas9. The results verified that both types of chromosomes can be targeted for the site-specific insertion of exogenous sequences. We ultimately obtained a homozygous knock-in strain with the integration of both Cas9 and cyan fluorescent protein (CFP) expression cassettes on a Z-linked region in P. xylostella, which can also be used for early sex detection. By injecting the sgRNA targeting Pxfl(2)d alone into the eggs laid by female adults of the Z-Cas9-CFP strain, the gene editing efficiency reached 29.73%, confirming the success of expressing a functional Cas9 gene. Taken together, we demonstrated the feasibility of the knock-in of an exogenous gene to different genomic regions in P. xylostella, while the establishment of a heritable strain required the positioning of appropriate sites. This study provides an important working basis and technical support for further developing genetic strategies for insect pest control.

The ongoing interplay among plants, insects, and bacteria underscores the intricate balance of defense mechanisms in ecosystems. Regurgitant bacteria directly/indirectly impact plant immune responses, but the underlying mechanism is unclear. Here, we focus on the interaction between regurgitant bacteria, diamondback moth (DBM), and plant. Six culturable bacteria were isolated from DBM gut regurgitant, including three Enterobacter strains (RB1-3), Micrococcus sp. (RB4), Staphylococcus haemolyticus (RB5), and Bacillus cereus (RB6). These RB strains suppressed genes related to jasmonic acid and glucosinolate signaling pathways but had little effect on salicylic acid signaling pathway genes in Arabidopsis thaliana wounds. RB1 and RB5 inhibited DBM development on A. thaliana but not on an artificial diet. RB1 and RB5 significantly suppressed GOX genes and proteins in DBMs. However, the Pxgox2 insect mutant strain inoculated with RB1 or RB5 did not significantly affect DBM feeding on A. thaliana compared to the wild type. Six RB have been functionally identified, with RB1 and RB5 negatively regulating GOX-mediated host adaptability. The deliberate addition of RB1 and RB5 can negatively affect DBM herbivory and fitness. Our study provides a molecular basis for the further application of RB for insect pest management by modulating insect–plant interactions.

Brassica plants have glucosinolate (GLs)-myrosinase defense mechanisms to deter herbivores. However, Plutella xylostella specifically feeds on Brassica vegetables. The larvae possess three glucosinolate sulfatases (PxGSS1-3) that compete with plant myrosinase for shared GLs substrates and produce nontoxic desulfo-GLs (deGLs). Although PxGSSs are considered potential targets for pest control, the lack of a comprehensive review has hindered the development of PxGSSs-targeted pest control methods. Recent advances in integrative multi-omics analysis, substrate-enzyme kinetics, and molecular biological techniques have elucidated the evolutionary origin and functional diversity of these three PxGSSs. This review summarizes research progress on PxGSSs over the past 20 years, covering sequence properties, evolution, protein modification, enzyme activity, structural variation, substrate specificity, and interaction scenarios based on functional diversity. Finally, we discussed the potential applications of PxGSSs-targeted pest control technologies driven by artificial intelligence, including CRISPR/Cas9-mediated gene drive, transgenic plant-mediated RNAi, small-molecule inhibitors, and peptide inhibitors. These technologies have the potential to overcome current management challenges and promote the development and field application of PxGSSs-targeted pest control.

Glucose oxidase (GOX) is a representative compound found in most insect saliva that can suppress plant-defensive responses. However, little is known about the origin and role of GOX in the crucifer-specialized pest Plutella xylostella. In this study, we showed obvious regurgitation from the larval gut of P. xylostella and identified abundant peptides highly similar to known GOX. Three PxGOX genes were verified with PxGOX2 preferentially expressed in the gut. The heterologously expressed PxGOX2 confirmed its function to be a GOX, and it was detected in plant wounds together with the gut regurgitant. Further experiments revealed that PxGOX2 functioned as an effector and may suppress defensive responses in plant through the production of H2O2, which modulates levels of antagonistic salicylic acid and jasmonic acid. However, excessive H2O2 in the host plant may be neutralized by peroxidase, thus forming defensive feedback. Our findings provided new insights into understanding the GOX-mediated insect-plant interactions.

The glucosinolates (GLs) and myrosinase defensive systems in cruciferous plants were circumvented by Plutella xylostella using glucosinolate sulfatases (PxGSSs) during pest-plant interaction. Despite identifying three duplicated GSS-encoding genes in P. xylostella, limited information regarding their spatiotemporal and induced expression is available. Here, we investigated the tissue- and stage-specific expression and induction in response to GLs of PxGSS1 and PxGSS2 (PxGSS1/2) at the protein level, which shares a high degree of similarity in protein sequences. Western blotting (WB) analysis showed that PxGSS1/2 exhibited a higher protein level in mature larvae, their guts, and gut content. A significantly high protein and transcript levels of PxGSS1/2 were also detected in the salivary glands using WB and qRT-PCR. The immunofluorescence (IF) and immunohistochemistry (IHC) results confirmed that PxGSS1/2 is widely expressed in the larval body. The IHC was more appropriate than IF when autofluorescence interference was present in collected samples. Furthermore, the content of PxGSS1/2 did not change significantly under treatments of GL mixture from Arabidopsis thaliana ecotype Col-0, or commercial ally (sinigrin), 4-(methylsulfinyl)butyl, 3-(methylsulfinyl)propyl, and indol-3-ylmethyl GLs indicating that the major GLs from leaves of A. thaliana Col-0 failed to induce the expression of proteins for both PxGSS1 and PxGSS2. Our study systemically characterized the expression properties of PxGSS1/2 at the protein level, which improves our understanding of PxGSS1/2-center adaptation in P. xylostella during long-term insect-plant interaction.

Tea green leafhopper (TGL), Empoasca onukii, is of biological and economic interest. Despite numerous studies, the mechanisms underlying its adaptation and evolution remain enigmatic. Here, we use previously untapped genome and population genetics approaches to examine how the pest adapted to different environmental variables and thus has expanded geographically. We complete a chromosome-level assembly and annotation of the E. onukii genome, showing notable expansions of gene families associated with adaptation to chemoreception and detoxification. Genomic signals indicating balancing selection highlight metabolic pathways involved in adaptation to a wide range of tea varieties grown across ecologically diverse regions. Patterns of genetic variations among 54 E. onukii samples unveil the population structure and evolutionary history across different tea-growing regions in China. Our results demonstrate that the genomic changes in key pathways, including those linked to metabolism, circadian rhythms, and immune system functions, may underlie the successful spread and adaptation of E. onukii. This work highlights the genetic and molecular basis underlying the evolutionary success of a species with broad economic impacts, and provides insights into insect adaptation to host plants, which will ultimately facilitate more sustainable pest management.

Numerous herbivores orally secrete defense compounds to detoxify plant toxins. However, little is known about the role of orally secreted enzymes by a specialized pest, Plutella xylostella, in the detoxification of plant defense compounds. Three glucosinolate sulfatases (GSSs) or two sulfatase-modifying factors (SUMF1s) mutant strains were established on the basis of CRISPR/Cas9 technology to validate the existence of a species-specific GSSs-SUMF1s system. In comparison to the bioassay data from mutant strains of GSS1/GSS2 or SUMF1a/SUMF1b, GSS3 had a minimal role because no significant change was found in GSS3-/- under different feeding contexts. Antibody-based technologies were used to examine GSSs-related deficient strains, and the results showed that the GSS1 protein was primarily released through larval oral secretion. On the basis of high-performance liquid chromatography, we found that GSS1 was secreted to pre-desulfate the typical plant defensive glucosinolates known as 4-(methylsulfinyl)butyl glucosinolate (4MSOB-GL) to suppress the production of the toxic substance, which is referred to as pre-detoxification strategy. These findings highlighted that the GSSs-SUMF1s system is the key factor for counteradaptation of P. xylostella to cruciferous plants, which strengthens the concept that herbivores deploy pre-detoxification strategies to disrupt the plant chemical defenses to facilitate the colonization process.

N6-methyladenosine (m6A) is one of the major epigenetic modifications in eukaryotes. Although increasing functions of m6A have been identified in insects, its role in Plutella xylostella L. for host plant adaptation remains unclear. In the current study, we show that the m6A content of P. xylostella was relatively low in different developmental stages and tissues, with no significant differences. Two RNA methyltransferase genes, PxMETTL3 (methyltransferase-like 3) and PxMETTL14 (methyltransferase-like 14), were identified and characterized. PxMETTL3 could be transcribed into two transcripts, and PxMETTL14 had only one transcript; both of these genes were highly expressed in egg and adult stages and reproductive tissues. The CRISPR/Cas9-mediated knockout of PxMETTL3 (ΔPxMETTL3-2) or PxMETTL14 (ΔPxMETTL14-14) confirmed their function in m6A installation into RNA. Furthermore, upon transfer from an artificial diet to the host plant, the mutant strains were affected in terms of larval and pupal weight or adult emergence rate, while the wildtype (WT) strain did not exhibit any difference. In addition, the fecundity and egg hatching rate of the WT strain decreased significantly, whereas only the ΔPxMETTL14-14 mutant strain displayed significantly decreased fecundity. There seemed to be a tradeoff between the stress adaptation and reproduction in P. xylostella mediated by m6A modification. During host transfer, the expression of PxMETTL14 was consistent with the change in m6A content, which implied that PxMETTL14 could respond to host plant defense effectively, and may regulate m6A content. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed transcripts with changes in m6A levels revealed that the potential functions of m6A-related genes may be involved in steroid biosynthesis for larval performance and metabolic pathways for adult reproduction. Overall, our work reveals an epigenetic regulation mechanism for the rapid adaptation of P. xylostella to variations in the host environment.

Liriomyza sativae is a globally distributed quarantine pest. We isolated polymorphic microsatellite loci from transcriptomic data for this species and tested their potential application in population genetics and cross-species transferability in other Agromyzidae species. Of the 130 microsatellite loci selected for validation, 40 were successfully amplified, and 18 were verified as polymorphic in L. sativae . The number of alleles (Na) ranged from two to ten, with polymorphism information content (PIC) values between 0.289 and 0.818. Observed ( H O ) and expected ( H E ) heterozygosity varied from 0.250 to 0.900 and 0.314 to 0.859, respectively. Low among-population differentiation was also indicated by pairwise F ST (0.032–0.045). Structure analysis and principal coordinate analysis (PCoA) suggested three clusters for the 94 samples from three different locations (FZNT, SMSX, and QZYC) in Fujian Province, China. The low genetic differentiation and clear genetic structure among populations of this invasive species may result from extensive human activity. In additional, cross-species tests validated the amplification of 16, 19, 11 and 9 of 40 loci in Liriomyza huidobrensis , Liriomyza trifolii , Chromatomyia horticola and Ophiomyia phaseoli , respectively, showed the potential application of these markers for population genetic analysis in these species.

MicroRNAs (miRNAs) are important regulatory noncoding RNAs (ncRNAs) at the posttranscriptional level of gene expression. Linear long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) can function as competing endogenous RNAs (ceRNAs) of miRNAs and regulate the expression of protein-coding genes. This chapter presents a procedure for the bioinformatic analysis of these three ncRNAs that are differentially expressed during insect development. In the first step, lncRNAs and circRNAs are identified based on RNA-sequencing data. In the second step, miRNAs are identified based on small RNA-sequencing data and combined with the two ncRNAs from the previous step for functional characterization.