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12 pages, 1515 KiB

Open AccessArticle

Expression of Heat Shock Protein 90 Genes Induced by High Temperature Mediated Sensitivity of Aphis glycines Matsumura (Hemiptera: Aphididae) to Insecticides

by Xue Han, Yulong Jia, Changchun Dai, Xiaoyun Wang, Jian Liu and Zhenqi Tian

Insects 2025, 16(8), 772; https://doi.org/10.3390/insects16080772 - 28 Jul 2025

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Abstract

Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a major pest of soybean fields. While high-temperature stress induced by global warming can initially suppress aphid populations, these pests may eventually adapt, leading to more severe infestations and crop damage. Heat shock proteins (HSPs), [...] Read more.

Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a major pest of soybean fields. While high-temperature stress induced by global warming can initially suppress aphid populations, these pests may eventually adapt, leading to more severe infestations and crop damage. Heat shock proteins (HSPs), which are upregulated in response to heat stress to protect aphid development, also confer tolerance to other abiotic stressors, including insecticides. To investigate the role of HSPs in insecticide resistance in A. glycines, we analyzed the expression profiles of three AgHsp90 genes (AgHsp75, AgHsp83, and AgGrp94) following exposure to high temperatures and insecticides. Functional validation was performed using RNA interference (RNAi) to silence AgHsp90 genes. Our results demonstrated that AgHsp90 genes were significantly upregulated under both heat and insecticide stress conditions. Furthermore, after feeding on dsRNA of AgHsp90 genes, mortality rates of A. glycines significantly increased when exposed to imidacloprid and lambda-cyhalothrin. This study provides evidence that AgHsp90 genes play a crucial role in mediating thermal tolerance and insecticide resistance in A. glycines. Full article

(This article belongs to the Special Issue RNAi in Insect Physiology)

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14 pages, 1508 KiB

Open AccessArticle

Effects of Trehalase on the Gene Expression of the Reproductive Regulation Pathway Network and Triglyceride Metabolism in Nilaparvata lugens (Stål)

by Bin Tang, Yuxin Ge, Yongkang Liu, Liwen Guan, Ye Han, Yang Zhu, Gao Hu and Yan Wu

Insects 2025, 16(7), 725; https://doi.org/10.3390/insects16070725 - 16 Jul 2025

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Abstract

The high reproductive performance of Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) is one of the key factors contributing to its serious damage. Studies have demonstrated that trehalose hydrolysis, catalyzed by trehalase (TRE), plays an important role in the reproductive regulation of N. lugens. [...] Read more.

The high reproductive performance of Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) is one of the key factors contributing to its serious damage. Studies have demonstrated that trehalose hydrolysis, catalyzed by trehalase (TRE), plays an important role in the reproductive regulation of N. lugens. However, it remains unclear how TRE regulates the reproduction of N. lugens. To address this question, the current study was designed to investigate the effects of TRE on the reproductive regulatory network of N. lugens. Specifically, RNA interference (RNAi) was conducted by injecting double-stranded RNA (dsRNA) targeting the TRE genes, and a trehalase inhibitor, validamycin, was used to suppress TRE activity. Subsequently, the relative expression levels of key genes involved in the reproductive regulatory pathway, as well as the triglyceride content in the fat body and ovary, were determined. The results indicated that both dsTREs and validamycin treatment decreased the relative expression of Vitellogenin (Vg) without affecting the expression of its receptor, VgR. However, their impact on the reproductive regulatory network differed: dsTREs injection did not affect the relative expression of JHAMT and Met in the juvenile hormone signaling pathway, but resulted in the upregulation of USP and TOR, and inhibition of InR2 and S6K. In contrast, validamycin injection inhibited the expression of Met, USP, EcR, InR1, TOR, and S6K. Both dsTREs and validamycin inhibited the expression of Fas, but only validamycin decreased the ovarian triglyceride content. These findings suggest that the inhibition of TRE leads to alterations in reproductive regulatory signals, thereby reducing the reproductive capacity of N. lugens. Full article

(This article belongs to the Special Issue RNAi in Insect Physiology)

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14 pages, 5582 KiB

Open AccessArticle

Silencing Miniature Gene Disrupts Elytral and Hindwing Structures in Leptinotarsa decemlineata

by Man-Hong Cheng, Kai-Yun Fu, Wei Zhou, Ji-Feng Shi and Wen-Chao Guo

Insects 2025, 16(7), 700; https://doi.org/10.3390/insects16070700 - 8 Jul 2025

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Abstract

The Colorado potato beetle (Leptinotarsa decemlineata, CPB) is a major pest in potato crops, notorious for its rapid dispersal and insecticide resistance, which are enabled by its robust elytra and flight-capable hindwings. The Miniature (Mi) gene, encoding a protein [...] Read more.

The Colorado potato beetle (Leptinotarsa decemlineata, CPB) is a major pest in potato crops, notorious for its rapid dispersal and insecticide resistance, which are enabled by its robust elytra and flight-capable hindwings. The Miniature (Mi) gene, encoding a protein with a zona pellucida (ZP) domain, is involved in wing development and cuticle integrity, yet its functional role in beetles remains underexplored. In this study, we cloned and characterized the LdMi gene in the CPB and investigated its function using RNA interference (RNAi), morphological analyses, and spectroscopy. LdMi encodes a 146.35 kDa transmembrane protein with a conserved ZP domain, clusters with coleopteran homologs, and exhibits relative conservation across insect species. Expression profiling showed high LdMi transcript levels in the hindwings, the elytra, and the pupal stages. RNAi knockdown in fourth-instar larvae resulted in severe eclosion defects, including malformed wings and reduced adult weight. Scanning electron microscopy (SEM) revealed disrupted elytral patterns and deformed hindwing veins in knockdown individuals. Spectroscopic analyses using Fourier-transform infrared (FTIR) and Raman spectroscopy indicated a reduction in protein–chitin crosslinking and diminished hydrogen bonding, suggesting compromised cuticular integrity. These results highlight the essential role of LdMi in cuticle formation and the surface morphology of the elytra and hindwings, offering new insights into ZP domain proteins in insects. Full article

(This article belongs to the Special Issue RNAi in Insect Physiology)

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16 pages, 2174 KiB

Open AccessArticle

Development of a Stage- and Species-Specific RNAi System for Molecular Insights in Trichogramma Wasps

by Zelong Yang, Yan Lu, Zhuo Jiang, Xilin Jiao, Han Lin, Wanning Jiang, Wenmei Du, Xue Zhang, Zhao Peng, Junjie Zhang, Xiao Wang and Ying Hu

Insects 2025, 16(7), 673; https://doi.org/10.3390/insects16070673 - 27 Jun 2025

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Abstract

Trichogramma wasps, egg parasitoids widely used to control lepidopteran pests, have long eluded in-depth molecular mechanistic studies due to their minute size and genetic tool scarcity. While previous RNAi efforts were restricted to T. dendrolimi, we developed the first cross-species RNAi system [...] Read more.

Trichogramma wasps, egg parasitoids widely used to control lepidopteran pests, have long eluded in-depth molecular mechanistic studies due to their minute size and genetic tool scarcity. While previous RNAi efforts were restricted to T. dendrolimi, we developed the first cross-species RNAi system for both T. dendrolimi and the previously intractable T. ostriniae. Temporal expression profiling identified white and laccase 2 as stage-specific RNAi targets, peaking during prepupal/pupal stages, which were tested across species and developmental stages using microinjection and soaking dsRNA delivery methods. Survival analysis prioritized soaking for T. dendrolimi prepupae/pupae, while microinjection was essential for T. ostriniae to bypass prepupal mortality during soaking. Concentration-dependent RNAi targeting the white gene achieved 85.61% transcript reduction in T. dendrolimi via soaking and 89.36% in T. ostriniae via microinjection at 2000 ng/μL, correlating with 64.06% and 32.09% white-eyed pupae, causing a significant reduction in eye pigments. For the laccase 2 gene, soaking at 2000 ng/μL induced 88.35% transcript reduction in T. dendrolimi and 73.31% in T. ostriniae, leading to incomplete cuticle tanning and sclerotization. This study resolves the long-standing challenge of genetic manipulation in Trichogramma wasps, providing a universally applicable framework to decipher parasitoid–host interactions at the molecular scale, which is useful for sustainable pest management strategies. Full article

(This article belongs to the Special Issue RNAi in Insect Physiology)

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16 pages, 1963 KiB

Open AccessArticle

Characterization and Functional Analysis of Small Heat Shock Protein Genes (Hsp22.2 and Hsp26.7) in Sitodiplosis mosellana Diapause

by Qitong Huang, Qian Ma, Xiaobin Liu, Keyan Zhu-Salzman and Weining Cheng

Insects 2025, 16(7), 649; https://doi.org/10.3390/insects16070649 - 20 Jun 2025

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Abstract

Small heat shock proteins (sHsps) play crucial roles in organismal adaptation to stress tolerance. Sitodiplosis mosellana, a devastating insect wheat pest, undergoes long obligatory larval diapause to survive temperature extremes during summer and winter. To elucidate the function of sHsps in this [...] Read more.

Small heat shock proteins (sHsps) play crucial roles in organismal adaptation to stress tolerance. Sitodiplosis mosellana, a devastating insect wheat pest, undergoes long obligatory larval diapause to survive temperature extremes during summer and winter. To elucidate the function of sHsps in this process, two sHsp-encoding genes (SmHsp22.2 and SmHsp26.7) were characterized from S. mosellana, and their responsiveness to diapause and thermal stress, as well as their roles in cold stress, was analyzed. Both SmHsp22.2 and SmHsp26.7 possessed the canonical α-crystallin domain and lacked introns. Quantitative PCR indicated significant upregulation of SmHsp22.2 and SmHsp26.7 during diapause, especially in summer and winter. Notably, SmHsp22.2 exhibited higher expression in summer relative to winter, whereas SmHsp26.7 showed the opposite profile. Moreover, short-term heat shock (≥35 °C) in over-summering larvae or cold shock (≤−10 °C) in over-wintering larvae was found to trigger transcriptional upregulation of both genes, while prolonged temperature extremes (i.e., 45–50 °C or −15 °C) did not elicit a comparable response. RNA interference-mediated knockdown of both genes significantly increased the mortality of S. mosellana larvae under cold stress. These findings indicate the importance of both SmHsps in diapause and environmental adaptation in S. mosellana. Full article

(This article belongs to the Special Issue RNAi in Insect Physiology)

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12 pages, 1087 KiB

Open AccessArticle

Activation of BmToll9-1 in Silkworm (Bombyx mori) Larval Midgut by Escherichia coli and Regulation of Growth

by Jisheng Liu, Weijian Chen, Minchun Lai, Jiahua Chen and Luc Swevers

Insects 2025, 16(6), 621; https://doi.org/10.3390/insects16060621 - 11 Jun 2025

Cited by 1 | Viewed by 1419

Abstract

Insects rely on their innate immune system to defend against pathogens, and the Toll signaling pathway plays an important role in immune regulation. Our previous studies have shown that BmToll9-1 functions as a positive regulator in the Toll pathway. This study seeks to [...] Read more.

Insects rely on their innate immune system to defend against pathogens, and the Toll signaling pathway plays an important role in immune regulation. Our previous studies have shown that BmToll9-1 functions as a positive regulator in the Toll pathway. This study seeks to elucidate the role of BmToll9-1, as a sensor to bacterial challenge, in modulating larval development and downstream Toll signaling pathways. Silkworm larvae were subjected to infection with either Gram-negative Escherichia coli or Gram-positive Staphylococcus aureus bacteria following silencing of BmToll9-1 by RNA interference (RNAi). This bacterial challenge triggered a compensatory re-induction of BmToll9-1 expression, which resulted in the recovery of larval weight and size to levels observed in untreated controls. Furthermore, upon bacterial infection of BmToll9-1-silenced larvae, there was an up-regulation in the expression of both signaling genes in the Toll pathway and downstream effector genes, with a marked preference for Gram-negative bacteria. These results highlight the involvement of BmToll9-1 in the Toll signaling pathway as a positive regulator, influencing silkworm development. Additionally, BmToll9-1 and BmToll9-2 were cross-validated to be genetically distinct genes, even though they were confirmed to be functionally analogous in the silkworm. Full article

(This article belongs to the Special Issue RNAi in Insect Physiology)

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14 pages, 2253 KiB

Open AccessArticle

Risk Assessment of RNAi-Based Potential Pesticide dsNlAtg3 and Its Homologues for Nilaparvata lugens and Non-Target Organisms

by Kai Li, Tongtong Chen, Yuliang Li, Kai Sun, Kun Pang, Xiaoping Yu and Peiying Hao

Insects 2025, 16(2), 225; https://doi.org/10.3390/insects16020225 - 19 Feb 2025

Cited by 1 | Viewed by 878

Abstract

The brown planthopper (Nilaparvata lugens) is an insect pest of rice, which mainly feeds on the phloem sap of the leaf sheath. RNA interference (RNAi) has application prospects in pest control, but it is necessary to select target genes and design [...] Read more.

The brown planthopper (Nilaparvata lugens) is an insect pest of rice, which mainly feeds on the phloem sap of the leaf sheath. RNA interference (RNAi) has application prospects in pest control, but it is necessary to select target genes and design suitable dsRNA fragments for RNAi so that it can achieve effective pest control and avoid risks to non-target organisms. NlAtg3 is a key protein in the autophagy pathway of N. lugens. Three kinds of dsRNA fragments of the NlAtg3 gene (dsNlAtg3-474×1, dsNlAtg3-138×3 and dsNlAtg3-47×10) were designed to compare the RNAi efficiency and specificity against the target insect N. lugens and non-target organisms through microinjection. The results showed that the fragment dsNlAtg3-474×1 showed strong inhibitory effects on the survival of N. lugens, which resulted in the survival rate decreasing to zero on the fifth day, while the survival rate of a closely related species, Sogatella furcifera, dropped to 2.22%. In contrast, dsNlAtg3-47×10 specifically designed against N. lugens only showed slight or no inhibitory effects on S. furcifera and other non-target organisms such as Drosophila melanogaster, but still showed good lethal effects against N. lugens, with the survival rate dropping to 18.89% on the ninth day. In addition, after being fed N. lugens injected with dsNlAtg3-47×10 fragments, the survival rate of the natural enemies Dolomedes sulfureus and Tytthus chinensis did not show significant change, compared with those treated with the dsGFP control. Our results suggest that the NlAtg3 gene can serve as a potential target for controlling N. lugens. Moreover, by designing suitable RNAi fragments, it is possible to avoid harm to non-target organisms while effectively inhibiting the target insect N. lugens. Full article

(This article belongs to the Special Issue RNAi in Insect Physiology)

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Review

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25 pages, 2052 KiB

Open AccessReview

Perspectives of RNAi, CUADb and CRISPR/Cas as Innovative Antisense Technologies for Insect Pest Control: From Discovery to Practice

by Hemant Kumar, Nikita Gal’chinsky, Verma Sweta, Nikita Negi, Roman Filatov, Anamika Chandel, Jamin Ali, Vol Oberemok and Kate Laikova

Insects 2025, 16(7), 746; https://doi.org/10.3390/insects16070746 - 21 Jul 2025

Viewed by 453

Abstract

Pest management is undergoing a transformative shift with the development of the cutting-edge antisense technologies: RNA interference (RNAi), contact unmodified antisense DNA biotechnology (CUADb), and the CRISPR-associated proteins (CRISPR/Cas). These approaches function by facilitating sequence-specific pairing of nucleic acids followed by nuclease-mediated cleavage, [...] Read more.

Pest management is undergoing a transformative shift with the development of the cutting-edge antisense technologies: RNA interference (RNAi), contact unmodified antisense DNA biotechnology (CUADb), and the CRISPR-associated proteins (CRISPR/Cas). These approaches function by facilitating sequence-specific pairing of nucleic acids followed by nuclease-mediated cleavage, offering exceptional precision for targeted pest control. While RNA-guided mechanisms such as RNAi and CRISPR/Cas were initially characterized in non-insect systems, primarily as innate defenses against viral infections, the DNA-guided CUADb pathway was first identified in insect pests as a functional pest control strategy. Its broader role in ribosomal RNA (rRNA) biogenesis was recognized later. Together, these discoveries have revealed an entirely new dimension of gene regulation, with profound implications for sustainable pest management. Despite sharing a common principle of sequence-specific targeting RNAi, CUADb, and CRISPR/Cas differ in several key aspects, including their mechanisms of action, target specificity, and applicability. Rather than serving as universal solutions, each technology is likely to be optimally effective against specific pest groups. Moreover, these technologies allow for rapid adaptation of control strategies to overcome target-site resistance, ensuring long-term efficacy. This review summarizes the core functional characteristics, potential applications, and current limitations of each antisense technology, emphasizing their complementary roles in advancing environmentally sustainable pest control. By integrating foundational biological discoveries with applied innovations, this work provides a new perspectives on incorporating antisense-based strategies into next-generation integrated pest management systems. Full article

(This article belongs to the Special Issue RNAi in Insect Physiology)

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