Biology subjects

Scholten, S.

Publications and source records attributed to Scholten, S..

9 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

Proteomic changes associated with the initiation and termination of aestivation in the cabbage stem flea beetle

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/655265v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@18535d2org.highwire.dtl.DTLVardef@908d8borg.highwire.dtl.DTLVardef@3cdc76org.highwire.dtl.DTLVardef@1ade6e6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAestivation initiation is associated with a reduction in translation proteins C_LIO_LIAestivation termination involves changes in proteolysis-related proteins C_LIO_LI23-30% of the changes at the protein level were coherent with the RNA-seq study C_LIO_LITAG and chitin peak, while ATP and glucose get depleted during aestivation C_LI The cabbage stem flea beetle (CSFB, Psylliodes chrysocephala) is a major pest of oilseed rape crops and exhibits obligatory adult aestivation, summer diapause, that coincides with the summer season. The aestivation in CSFB is characterized by metabolic suppression, cessation of feeding, and reproductive activities. Previous investigations have employed RNA-seq to explore gene expression changes associated with aestivation, providing initial insights into the molecular pathways. However, studies assessing proteomic changes during aestivation in this species and insects more broadly have been lacking. In this study, we conducted a comprehensive quantitative proteomic analysis of adult CSFB at four time points: pre-aestivation (day 5), aestivation initiation (day 15), aestivation maintenance (day 30), and post-aestivation (day 55), to investigate proteomic changes associated with aestivation. We found that proteins related to the central dogma decreased in abundance, and metabolism-related proteins were altered during the initiation of aestivation. The proteomic changes during aestivation were minor and included reduction in mitochondrial proteins. Notably, the proteolysis-related proteins were enriched at the termination of aestivation. Interestingly, we observed discrepancies between our previous RNA-seq results and the proteomic data, particularly in the genes that increased in abundance during aestivation compared to post-aestivation. This highlights the importance of proteomic analysis for a more complete understanding of molecular mechanisms underlying aestivation. Body composition measurements showed that triglyceride and chitin levels peaked, while ATP and glucose were depleted during aestivation, following the changes in proteins belonging to different biological pathways.

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

Reciprocal roles of two trehalose transporters in aestivating cabbage stem flea beetles (Psylliodes chrysocephala)

The cabbage stem flea beetle (Psylliodes chrysocephala, CSFB) is a significant pest of winter oilseed rape crops in northern Europe. CSFB adults aestivate during the summer to protect themselves from heat and desiccation stress. Trehalose, the primary hemolymph sugar, has been linked to energy homeostasis and stress resilience, but its regulation and function during aestivation remain poorly understood. Here, we investigated the roles of two trehalose transporters, Tret-1 and Tret-2, in modulating trehalose dynamics across different adult stages in CSFB. Through spatiotemporal transcript profiling, we found that Tret-1 was predominantly expressed in the fat body, where it facilitates trehalose export to the hemolymph, whereas Tret-2 expression was higher in the Malpighian tubules, mediating trehalose uptake from the hemolymph. RNA interference experiments revealed that Tret-1 is involved in transporting trehalose from the fat body into the hemolymph, while Tret-2 works reciprocally to transport trehalose from the hemolymph into the Malpighian tubules. The disruption of trehalose transportation resulted in excess glucose, glycogen, and triglyceride levels, mainly in pre-aestivation beetles. Furthermore, the knockdown of either trehalose transporter caused a compensatory increase in feeding activity in pre-aestivation beetles, while the knockdown of Tret-2 compromised resilience to heat stress. Our findings uncover the reciprocal functions of Tret-1 and Tret-2 in regulating trehalose distribution and maintaining metabolic stability during aestivation, offering insights into the physiological strategies underpinning insect survival during aestivation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/639621v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1ec0817org.highwire.dtl.DTLVardef@107534eorg.highwire.dtl.DTLVardef@1ee4322org.highwire.dtl.DTLVardef@1797559_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LITwo Trehalose transporters were investigated in aestivating P. chrysocephala C_LIO_LITret-1 mainly transports trehalose out of fat body C_LIO_LITret-2 mainly transports trehalose into Malpighian tubules C_LIO_LIDynamic trehalose transportation regulates other metabolites, including Glucose C_LIO_LITret-2, but not Tret-1, might be necessary for heat stress resilience C_LI

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↗

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↗

bioRxiv2017

Omics-Based Hybrid Prediction In Maize

Accurate prediction of traits with complex genetic architecture is crucial for selecting superior candidates in animal and plant breeding and for guiding decisions in personalized medicine. Whole-genome prediction (WGP) has revolutionized these areas but has inherent limitations in incorporating intricate epistatic interactions. Downstream \"omics\" data are expected to integrate interactions within and between different biological strata and provide the opportunity to improve trait prediction. Yet, predicting traits from parents to progeny has not been addressed by a combination of \"omics\" data. Here, we evaluate several \"omics\" predictors -- genomic, transcriptomic and metabolic data -- measured on parent lines at early developmental stages, and demonstrate that the integration of transcriptomic with genomic data leads to higher success rates in the correct prediction of untested hybrid combinations in maize. Despite the high predictive ability of genomic data, transcriptomic data alone outperformed them and other predictors for the most complex heterotic trait, dry matter yield. An eQTL analysis revealed that transcriptomic data integrate genomic information from both, adjacent and distant sites relative to the expressed genes. Together, these findings suggest that downstream predictors capture physiological epistasis that is transmitted from parents to their hybrid offspring. We conclude that the use of downstream \"omics\" data in prediction can exploit important information beyond structural genomics for leveraging the efficiency of hybrid breeding.\n\nKey messageComplementing genomic data with other \"omics\" predictors can increase the probability of success for predicting the best hybrid combinations using complex agronomic traits.\n\nConflict of InterestThe authors declare that they have no conflict of interest.

genetics↗