Biology subjects

Guney, G.

Publications and source records attributed to Guney, G..

7 recordsLinked to original sources

bioRxiv2026

Identification of insecticidal double-stranded RNAs targeting the proteasome pathway in the hop flea beetle Psylliodes attenuata using dsRIP

The hop flea beetle, Psylliodes attenuata, is a major pest of hop (Humulus lupulus), an important crop for the brewing industry. Restrictions on chemical insecticides and insecticide resistance create a need for alternative control strategies. RNA interference (RNAi) uses double-stranded RNA (dsRNA) to silence essential pest genes and cause insecticidal effects. In this study, we aimed to establish P. attenuata as an RNAi-based pest control target. We generated a transcriptome assembly using a public RNA sequencing dataset and used the dsRIP web platform to identify essential gene targets and design effective dsRNAs. Three dsRNAs targeting proteasome subunits were tested in feeding bioassays by applying them to hop leaf disks. The treatments caused significant mortality of up to 64% and feeding inhibition of up to 45%. Sequencing of small RNAs bound to the RNA-induced silencing complex identified predominantly 21-nucleotide small interfering RNAs produced from the orally delivered dsRNAs, supporting activation of the RNAi pathway. Additional RNA sequencing of adults enabled assembly of a higher-quality transcriptome with 89% BUSCO completeness for future RNAi studies. Overall, the feeding bioassays and molecular resources establish P. attenuata as a promising target for RNAi-based pest control and demonstrate the usefulness of dsRIP for RNAi research in insects previously unstudied at the molecular level.

zoology↗

bioRxiv2026

RISC-Bound Small RNA Sequencing Provides Insights into Guide Strand Selection and siRNA Trimming and Tailing Following Insecticidal dsRNA Delivery

RNA interference (RNAi) offers a sequence-specific approach to pest control. In insects, Dicer-2 processes double-stranded RNA (dsRNA) into small interfering RNA (siRNA) duplexes, from which the RNA-induced silencing complex (RISC) retains a guide strand. Only antisense-loaded RISC can mediate cleavage of the target transcript. However, how sequence features shape the RISC-bound siRNA pool in pests remains poorly understood, limiting opportunities for sequence optimization. Here, we profiled RISC-bound siRNAs following injection of 34 insecticidal dsRNAs targeting 11 essential genes in Tribolium castaneum larvae. We computationally reconstructed 7,879 siRNA pairs and examined associations between sequence features and strand bias. Differences in GC identity at terminal paired positions 1 to 5, used as a proxy for local thermodynamic asymmetry, correlated with strand bias, with the strongest correlations at the first two paired positions. ORF targeting and reduced predicted antisense self-folding were also associated with higher antisense fractions. Analysis of non-templated terminal additions revealed predominantly 3-prime uridylation, a known signature of small RNA turnover, along with putative 3-prime trimming. Among ORF-associated siRNA pairs, sense strands showed higher relative U-tailing abundance, based on 3-prime uridylated and putatively trimmed-and-3-prime-uridylated reads relative to perfect 21-nt reads, than antisense strands. Antisense strands with the least predicted self-folding also showed low relative U-tailing abundance. These observations are consistent with sequence-dependent contributions from both guide-strand selection and differential post-RISC-loading siRNA retention, although a causal link remains to be established. The identified associations provide a basis for testing whether dsRNA sequence optimization can improve pest control efficacy and reduce off-target activity.

zoology↗

bioRxiv2025

RNA degradomics and proteomics reveal the mechanism of dsProsβ1-mediated proteasome targeting in the cabbage stem flea beetle

BackgroundThe cabbage stem flea beetle (CSFB, Psylliodes chrysocephala) is a major threat to oilseed rape crops. Management of CSFB has become increasingly challenging due to the European Unions ban on neonicotinoids and the emergence of pyrethroid-resistant populations. Recently, RNA interference (RNAi) has shown potential as an environmentally friendly alternative for the management of CSFB, and proteasome subunits have been identified as very effective RNAi targets. However, the mechanism of action of proteasome-targeting RNAi strategies remains to be fully characterized at the molecular level in CSFB and other pests. Here, we used CSFB to investigate the mechanism of action of dsPros{beta}1, which is a double-stranded RNA targeting a proteasome subunit. ResultsRNA degradome sequencing identified siRNA-mediated cleavage events in the target transcript, with cleavage events occurring at higher rates between uracil-guanine and adenine-adenine pairs. RISC-bound small RNA sequencing confirmed the presence of mature siRNAs guiding these cleavage events while revealing discrepancies between siRNA abundance and cleavage patterns. Proteomics analysis identified changes in protein levels caused by proteasome inhibition, including an increase in mitochondria- and cytoskeleton-related proteins and a decrease in central dogma-associated proteins. ConclusionThis study demonstrates that combining RNA degradomics, RISC-bound sRNA-seq, and proteomics is an insightful approach to investigating the mechanism of RNAi-based pest control at the molecular level. The insights gained from these methods can be used to enhance proteasome-targeting RNAi strategies against insect pests. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/642439v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@79f29org.highwire.dtl.DTLVardef@62773borg.highwire.dtl.DTLVardef@81ecbeorg.highwire.dtl.DTLVardef@1ddce01_HPS_FORMAT_FIGEXP M_FIG C_FIG RNA degradomics revealed dsPros{beta}1-derived siRNA-mediated mRNA cleavage events, mainly at uracil-guanine and adenine-adenine pairs. Proteasome inhibition via dsPros{beta}1 increased mitochondrial and cytoskeletal proteins while reducing translation-related and mRNA-binding proteins.

zoology↗

bioRxiv2025

The MicroRNA pathway regulates obligatory aestivation in a flea beetle

Aestivation is a dormant state that allows animals to withstand hot and dry summer conditions and requires complex gene regulation. Nevertheless, the mechanisms involved in the regulation of genes necessary for aestivation remain unclear. MicroRNA (miRNA) are known to fine-tune gene expression at the post-transcriptional level and are important for various biological processes. In this study, we investigated the role of the miRNA pathway in the regulation of the obligatory aestivation stage in the cabbage stem flea beetle, a major pest of oilseed rape. Small RNA sequencing showed that [~]25% of miRNAs were differentially abundant during aestivation. The inhibition of the miRNA pathway deregulated 116 proteins in aestivation, which were mainly associated with metabolism and catabolism, including peroxisome activity. Most proteins regulated by miRNA exhibited lower transcript levels during aestivation. RNA degradome sequencing confirmed the miRNA-mediated exonucleolytic decay of several transcripts. Furthermore, inhibiting the miRNA pathway resulted in altered body composition, compromised metabolic suppression, and lower resilience to high temperature during aestivation. Also, beetles could not suppress their feeding activity during the transition into aestivation. Our findings highlight the critical role of miRNA in regulating aestivation in the cabbage stem flea beetle, with important implications for climate change.

zoology↗

bioRxiv2025

Optimizing dsRNA sequences for RNAi in pest control and research with the dsRIP Web-Platform

BackgroundRNA interference (RNAi) is a tool for studying gene function and has emerged as a promising eco-friendly alternative to chemical pesticides. RNAi relies on delivering double-stranded RNA (dsRNA), which is processed into small interfering RNA (siRNA) to silence genes. However, so far, knowledge and tools for optimizing the dsRNA sequences for maximum efficacy are based on human data, which might not be optimal for insects and pest control. ResultsHere, we systematically tested different siRNA sequences in the red flour beetle Tribolium castaneum to identify sequence features that correlated with high efficacy using pest control as a study case. Thermodynamic asymmetry, the absence of secondary structures, and Adenine at the 10th position in antisense siRNA were most predictive of insecticidal efficacy. Interestingly, we also found that in contrast to results from human data, high, rather than low GC content from the 9th to 14th nucleotides of antisense was associated with high efficacy. Consideration of these features for the design of insecticidal dsRNAs targeting essential genes in three insect species improved the efficacy of the treatment. The improvement was associated with a higher ratio of the antisense, rather than sense, siRNA strand bound to the RNA-induced silencing complex. Finally, we developed a web-platform named dsRIP (https://dsrip.uni-goettingen.de), which offers tools for optimizing dsRNA sequences, identifying effective RNAi target genes for pest control, and minimizing risk to non-target species. ConclusionsThe identified sequence features and the dsRIP web-platform allow optimizing dsRNA sequences for both application of RNAi for pest control and research.

zoology↗

bioRxiv2024

Effective target genes for RNA interference-based management of the cabbage stem flea beetle

The cabbage stem flea beetle (CSFB, Psylliodes chrysocephala) is a key pest of oilseed rape. The ban on neonicotinoids in the European Union due to environmental concerns and the emergence of pyrethroid-resistant populations have made the control of CSFB extremely challenging. In search of a solution, we have recently shown that RNA interference (RNAi) has potential in the management of CSFB. However, the previously tested target genes for RNAi-mediated pest control (subsequently called target genes) exhibited moderate and slow-acting lethal effects. In this study, 27 double-stranded RNAs (dsRNAs) were orally delivered to identify highly effective target genes in CSFB adults by leveraging the findings of a genome-wide RNAi screen in Tribolium castaneum. Our screen using 500 ng of dsRNA identified 10 moderately effective (> 50% mortality) and 4 highly effective target genes (100% mortality in 8-13 days). The latter mainly included proteasome subunits. RT-qPCR experiments confirmed target gene silencing and dose-response studies revealed LD50 values as low as [~]20 ng in 14 days following a single exposure to dsRNA. Four highly effective dsRNAs also inhibited leaf damage (up to [~]75%) and one affected locomotion. The sequences of promising target genes were subjected to in silico target prediction in non-target organisms, e.g., beneficials such as honeybees, to design environmentally friendly dsRNAs. Overall, the study provides valuable insights for the development of dsRNA-based insecticides against CSFB.

zoology↗

bioRxiv2024

Physiological and transcriptional changes associated with obligate aestivation in the cabbage stem flea beetle (Psylliodes chrysocephala)

1Aestivation is a form of seasonal dormancy observed in various insect species, usually coinciding with the summer season. Psylliodes chrysocephala (Coleoptera: Chrysomelidae), the cabbage stem flea beetle, is a key pest of oilseed rape and obligatorily aestivates as adult in late summer. At present, our understanding of the physiological and transcriptional changes linked to aestivation in P. chrysocephala is still limited. In this study, physiological parameters and RNA-seq analyses were performed with laboratory-reared beetles at pre-aestivation, aestivation, and post-aestivation stages. Measurements of CO2 production supported the notion that aestivating beetles dramatically reduce their metabolic rate and, together with assessments of reproductive maturation, allowed precise discrimination between the three adult stages. Aestivating beetles showed a reduction in carbohydrate reserves and an increase in lipid reserves compared to pre-aestivating beetles, indicating that aestivation is associated with drastic changes in energy metabolism. In agreement with these findings, we found that genes involved in carbohydrate and lipid metabolism, digestion, and mitochondrial activity are differentially expressed between the three stages. Furthermore, RNA-seq analysis suggested the regulation of transcription factors associated with aestivation maintenance and the involvement of cytochrome P450s in conferring a summer-resistant phenotype during the aestivation period. In conclusion, this study represents the first exploration of the transcriptomic and physiological aspects of the aestivation response in P. chrysocephala. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/588545v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@10d486forg.highwire.dtl.DTLVardef@10e9e1corg.highwire.dtl.DTLVardef@ae63aforg.highwire.dtl.DTLVardef@112985c_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIP. chrysocephala obligatorily aestivate as sexually immature adults in summer C_LIO_LIAestivation entails metabolic suppression, body composition changes and resistance C_LIO_LIAccordingly, metabolism and stress genes were differentially expressed C_LIO_LIThe findings can support the development of innovative pest management strategies C_LI

zoology↗