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The effects of azadirachtin on two pests: neonate larvae and newly emerged adults of Ceratitis capitata(Wiedemann) and last instar larvae of Spodoptera exigua(Hubner); and three natural enemies: newly emerged adults of Opius concolorSzepligeti, second instar larvae of Chrysoperla carnea(Stephens), and fifth instar nymphs of Podisus maculiventris(Say) were studied in laboratory. Adult insects were exposed to a non-oil formulation of azadirachtin (Align, emulsifiable concentrate, 3.2% azadirachtin, Sipcam Inagra, Spain) via their drinking water and immature instars were reared in the presence of the insecticide-treated diet. The natural enemies were exposed to at least the maximum field recommended concentration of the insecticide (0.15% v/v). Azadirachtin was highly toxic to neonate larvae of C. capitataand prevented adult emergence at a concentration of 1 mg a.i. l[sup -1] . When adults were fed the insecticide at the maximum recommended concentration, their survival was not affected but egg laying was totally inhibited. Last instar S. exigualarvae were also very susceptible (LC[sub 50] = 7.7 mg a.i. l[sup -1] ) and at a concentration of 10 mg a.i. l[sup -1] fecundity of surviving adults, and egg fertility, were reduced by 72 and 85%, respectively. Effects on O. concolorwere large, and significant reductions in longevity, percentage of attacked hosts, and progeny size per female, were recorded. The predator P. maculiventriswas much less sensitive to azadirachtin, but slight reductions in survival of emerged adults and of reproductive parameters occurred. The insecticide had no significant effect on C. carnealarvae fed with treated Sitotroga cerealella(Oliver) eggs, probably because of its inability to penetrate inside the egg. [ABSTRACT FROM AUTHOR]

Studies of the effects of a novel insect growth regulator (tebufenozide, RH 5992) in pest lepidopterans are extended to its influence on pre- and post-diapause larvae of the pine-tree lappet, Dendrolimus pini (L.) (Lep., Lasiocampidae). Larval diapause was induced under experimental conditions. The relation between the width of head capsule and each larval instar of the experimental populations is presented. The toxicity of tebufenozide applied topically and per os (orally), and its effects on feeding activity were examined. High doses and concentrations (≥100 ng/larva and ≥0.001%) of tebufenozide were toxic and reduced the feeding intensity in larvae of D. pini, however, post-diapause larvae were less sensitive to oral treatment with tebufenozide then pre-diapause insects. In pre-diapausing insects of the fourth instar a greater than four-fold fall in mortality was observed among the insects treated with low doses/ concentrations (0.1–10 ng/larva and 0.00001%–0.001%, respectively). A remarkable stimulatory effect on feeding behaviour was observed in pre-diapausing IV instar larvae when treated with tebufenozide topically, in a dose of 10 ng/larva. A possible ecdysteroid mimicking effect of tebufenozide onto feeding and mortality among diapause-destined D. pini is discussed. [ABSTRACT FROM AUTHOR]

Insecticidal activity against the Colorado potato beetle, Leptinotarsa decemlineata, was measured for a series of substituted N-tert-butyl-dibenzoylhydrazines, in which one of the benzoyl moieties closer tothe tert-butyl group was fixed as being 2-chloro-substitued and the other variously substituted singly or doubly. The effects of substituents on the activity were quantitatively analysed using the classicalquantitative structure-activity relationship (QSAR) procedure. The activity against the Colorado potato beetle increases with the molecular hydrophobicity. In addition, various types of steric effect are atwork, depending upon the positions. Hydrogen-bonding acceptor substituents at the para position enhance the activity. There seem to be threshold (or optimum) values, albeit position-dependent, in the molecular hydrophobicity, above which the activity starts to decrease. Thisbiphasic contribution of the molecular hydrophobicity to activity against coleopterous larvae is the most conspicuous difference in substituent effects from those found for similar compounds against lepidopterous pest insects, and may be the basis of the variations in the activity spectrum for certain compounds in this series. The introduction of bulkier substituents into the meta- and para-positions of the benzene ring, apart from the tert-butyl group, is unfavorable to activity. LD50 values against Colorado potato beetle larvae of methoxyfenozide (RH-2485) and tebufenozide (RH-5992) were in the orderof 10-7 mol per insect, whereas those of RH-5849, and halofenozide (RH-0345) were very low, 10-9-10-10 mol per insect being selective to the coleopterous larvae. [ABSTRACT FROM AUTHOR]

Aims: The aims of this study were to design universal markers for different protozoan parasites of Bombus spp. based on the phylogenetic position of two important bumblebee parasites Crithidia bombi and Apicystis bombi. Methods and Results: Standard PCR and extraction techniques were used to amplify and sequence 18S rDNA. Phylogenetic analysis of the rDNA was performed in order to predict the parasite range of the primers. Conclusions: Crithidia bombi phylogenetically clusters with the trypanosomatids with slowly-evolving SSU-rRNA sequences (SE), while A. bombi is the closest sister group of Mattesia. A multiplex was designed containing an internal control and two broad-range primer pairs, detecting C. bombi and other SE trypanosomatids and also A. bombi and other neogregarines. Significance and Impact of the Study: Sequence data generated will further improve the current systematics of insect trypanosomatids and gregarines that remain troublesome. Broad-range markers for bumblebee parasites are necessary tools enabling the screening of commercially imported colonies and thus controlling their worldwide distribution and to discover related emerging parasites. [ABSTRACT FROM AUTHOR]

Abstract: KK-42, an imidazole derivative considered as potent inhibitor of ecdysteroid biosynthesis, was tested on adult females of Tenebrio molitor. The compound was applied topically (0.1, 1, 5, and 10μg/insect) on 0- or 2-day-old adult females or added to the culture medium (1 and 10μM) of ovaries. To test the effect of KK-42 on the reproduction capacity, ovaries were explanted from 2- and 4-day-old females corresponding, respectively, to follicles at the beginning and the end of vitellogenesis. KK-42 treatment reduced the fecundity and the longevity of females, and in addition, it delayed oviposition and affected growth and development of oocytes as evidenced by measurements of the ovarian weight, the number of oocytes per ovaries, and the size of the basal follicle. Hence, the effects were stronger in newly emerged females compared to 2-day-old females. Interestingly, RH-0345, an ecdysteroid agonist, could partly restore the effects on reproductive events induced by KK-42. In a second series of experiments using an enzyme immunoassay to measure ecdysteroid titers, KK-42 reduced the amounts of ecdysteroids in ovaries after application on newly emerged adult females. In 2-day-old females, it caused a significant effect with the highest doses. In addition, the compound significantly reduced the amounts of ecdysteroid released into the culture medium by ovaries explanted from 2- or 4-day-old females. The results are discussed in relation to the mode of action of KK-42 on reproduction. [Copyright &y& Elsevier]

The activity of RH-0345 (halofenozide), a novel benzoylhydrazine analogue, was investigated on ecdysteroid production, cuticle secretion, and hemolymph protein concentrations in Tenebrio molitor pupae. The compound was applied topically (5 and 10 μg/pupa) on newly ecdysed pupae or added to the culture medium (1 and 10 μM) of sternal integuments explanted from newly ecdysed pupae. Enzyme immunoassay measurements of ecdysteroid titers in the hemolymph revealed that RH-0345 applied topically on newly ecdysed pupae had no effect on the peak position of ecdysteroids in the hemolymph, but it caused a significant increase of the peak values. With in vitro cultured integument explants, RH-0345 caused a significant increase of the amounts of ecdysteroids released into the culture medium. Further, integuments were cultured in medium containing RH-0345 and/or 20-hydroxyecdysone (20E) and the effects on apolysis and cuticle growth were tested. In the presence of 20E alone, the epidermis was able to secrete a new cuticle under the apolyzed pupal cuticle. Similarly, integument explants from RH-0345-treated series cultured without 20E underwent apolysis and then secreted a new cuticle. The thickness of such a newly induced cuticle did not change as a function of the concentration of RH-0345 nor of the incubation period. In contrast, integument explants from RH-0345-treated series cultured in the presence of 20E secreted a new significantly thicker cuticle. The observed differences in cuticle thickness could be explained by differences in the protein content. [Copyright &y& Elsevier]

Biotransformation of mycotoxins in animals comprises phase I and phase II metabolisation reactions. For the trichothecene deoxynivalenol (DON), several phase II biotransformation reactions have been described resulting in DON-glutathiones, DON-glucuronides and DON-sulfates made by glutathione-S-transferases, uridine-diphosphoglucuronyl transferases and sulfotransferases, respectively. These metabolites can be easily excreted and are less toxic than their free compounds. Here, we demonstrate for the first time in the animal kingdom the conversion of DON to DON-3-glucoside (DON-3G) via a model system with plant pathogenic aphids. This phase II biotransformation mechanism has only been reported in plants. As the DON-3G metabolite was less toxic for aphids than DON, this conversion is considered a detoxification reaction. Remarkably, English grain aphids (Sitobion avenae) which co-occur with the DON producer Fusarium graminearum on wheat during the development of fusarium symptoms, tolerate DON much better and convert DON to DON-3G more efficiently than pea aphids (Acyrthosiphon pisum), the latter being known to feed on legumes which are no host for F. graminearum. Using a non-targeted high resolution mass spectrometric approach, we detected DON-diglucosides in aphids probably as a result of sequential glucosylation reactions. Data are discussed in the light of an eventual co-evolutionary adaptation of S. avenae to DON. [ABSTRACT FROM AUTHOR]

Feeding and molting are particularly important physiological processes for insects, and it has been reported that neuropeptides are involved in the nervous regulation of these 2 processes. Sulfakinin (SK) is an important neuropeptide that is widely distributed among insects and plays a pivotal role in regulating feeding, courtship, aggression, and locomotion. In this study, we investigated the involvement of SK in feeding and molting on a highly notorious pest insect, the fall armyworm, Spodoptera frugiperda. SK transcript levels were found in all larval stages and there was a predominant expression of SK in the brain of 5th instar larvae. By immunostaining, SK was detected in 2 pairs of cells in the median protocerebrum. But during prolonged periods of starvation, there was a significant reduction in SK messenger RNA levels; however, subsequent refeeding led to a notable increase. To investigate the role of SK in feeding and molting, SK was silenced in S. frugiperda larvae through RNA interference. This resulted in a significant increase in food intake, weight gain, and the molting process happened more rapidly in the double-stranded SK-treated larvae compared to the controls. Conversely, injection of sulfated SK peptide (sSK) caused opposite effects. Interestingly, SK-knockdown in larvae resulted in increased levels of 20-hydroxyecdysone and also of the expression of some of it signaling pathway genes. Altogether, this study highlights the important role played by SK in regulating feeding and molting in S. frugiperda., (© 2024 Institute of Zoology, Chinese Academy of Sciences.)

Sophora davidii is a cross-pollinated plant with important ecological protection and medicinal value in China, but its seed yield is low due to backward and nonstandard production technology. Therefore, we divide the flowering period of Sophora davidii into initial, full and final flowering period, measuring the floral morphology, pollen viability, stigma receptivity, nectar volume and nectar concentration, foraging behavior of pollinators, fertilization physiology, seed yield and quality through field observation and indoor testing to explore whether the flowering period affects the floral traits, pollinator behavior and seed production of plants. Our results revealed that the nectar volume, nectar concentration, pollen viability and stigma receptivity at full flowering period were the highest. The single visit time and visit time per flower of Chinese honey bees were the longest in the full flowering period, while the number of transfer, visit frequency and number of touching stigma were the least. The visiting number of the bees was the most and the most active in the full flowering period. The bees pollination not only improved the pollen amount, germination rate, pollen tube length and the ovule number of S. davidii , but also their effect was the most obvious in full flowering period. The principal component analysis showed that pollination by Chinese honey bees during the whole flowering period of S. davidii was the best way to produce seeds. We can conclude that flowering period affects flower traits, foraging behavior of pollinators, seed yield and quality of S. davidii .

Mosquito control still relies heavily on synthetic molecules, which can lead to the selection of resistant populations and undesirable environmental problems. This study described the preparation of a nanoparticle of the plant-derived molecule, β -myrcene, with chitosan, and the assessment of its toxicity against larvae of the yellow fever mosquito, Aedes aegypti . By producing fluorescent chitosan nanoparticles, we were able to observe their distribution in the digestive tract of larvae of Ae. aegypti . Chitosan-based nanoparticles containing β -myrcene (238 mg/L) could kill 100% of the larvae tested, whereas the blank control (i.e., the nanoparticle without β -myrcene) showed no larvicidal activity. The chitosan nanoparticles with β -myrcene had a zeta potential of +15 mV and a hydrodynamic diameter ranging from 30 to 2800 nm. The blank control, without β -myrcene, had a zeta potential of +26 mV and a diameter of 30 to 830 nm. Fluorescence analysis showed that the nanoparticles were efficiently absorbed and distributed in the digestive tract organs of the Ae. aegypti larvae. In short, our results reinforce the benefits of using chitosan to carry molecules of plant-derived-molecules, such as β -myrcene, in mosquito control, suggesting a broad internal distribution that contributes to their toxicity.

Agricultural pesticides have historically been a critical tool in controlling pests and diseases, preventing widespread suffering and crop losses that led to catastrophes such as the Great Irish Famine (1845-1852) and the Cotton Boll Weevil Infestation (1915-1916). However, their usage has brought challenges, including resistance development, secondary pest outbreaks, harm to non-target organisms like pollinators, and environmental contamination. In response to these concerns, integrated pest management (IPM) has emerged as a comprehensive approach, emphasizing non-chemical pest control methods such as cultural practices, biological control, and crop rotation, with pesticides as the last resort. IPM has evolved, influenced by regulations like the Food Quality Protection Act (FQPA), which prioritizes human health protection, especially for children. The development of systemic pesticides, particularly neonicotinoids, introduced a more efficient and targeted pest control method within the IPM framework. However, they have also raised concerns due to their potential adverse effects on pollinators. In recent years, integrated pest and pollinator management (IPPM) has emerged as an enhanced approach, integrating pollinator health considerations into pest management strategies. In this article, we discuss this new approach, and briefly present an example of a modifying pesticide program in Pennsylvania apple orchards to illustrate the application of IPPM, in order to highlight the importance of IPPM in sustaining agriculture, protecting vital pollinators, and maintaining effective pest control practices. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Liposcelis tricolor (Psocoptera: Liposcelididae) is a significant pest affecting stored products globally. However, due to the lack of a detailed genomic reference, the mechanisms of sex determination, stress resistance, and potential control methods for this booklouse remain poorly understood. In this study, the chromosome-level genome of L. tricolor was assembled by employing Illumina, Nanopore, and Hi-C sequencing technologies. The final genome size was determined to be 229.33 Mb, anchored to 9 pseudo-chromosomes. BUSCO analysis showed that 99.2% of complete BUSCOs were identified, suggesting the high completeness of the genome. A total of 91.49 Mb of repetitive sequences, accounting for 38.84% of the total genome, were annotated, and 15,647 protein-coding genes were predicted, with 88.17% functionally annotated. Additionally, we identified 25 typical sex-determining genes based on the genomic data. This high-quality genome assembly provides a crucial foundation for advancing our comprehension of the molecular biology, genetics, and potential control strategies for psocid L. tricolor., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The application of exogenous RNA for gene-silencing strategies has gained significant traction in agriculture, offering a highly efficient and eco-friendly alternative to conventional plant protection methods. This success has been driven by advances in biotechnology, from the design of long double-stranded RNA (dsRNA) and small interfering RNA (siRNA) molecules to the development of nanocarrier systems that address the challenge of RNA delivery into plant cells. In particular, polymer-based nanocarriers have emerged as a promising solution for enhancing the stability and delivery efficiency of RNA molecules. This review provides a comprehensive overview of the current state of research on the use of polymeric nanocarriers in RNA interference (RNAi) systems for crop protection. It examines key technological developments that have enabled the effective delivery of dsRNA/siRNA to target organisms, with a focus on the unique advantages polymers offer as carriers. Recent studies highlight significant progress in the preparation, characterization, and application of polymeric nanocarriers for RNA encapsulation and delivery. The review also explores the environmental and health challenges posed by these technologies, emphasizing the need for sustainable approaches in their development. Specifically, the production of nanocarriers must adhere to the principles of green chemistry, prioritizing chemical modification routes that reduce harmful residues, such as toxic solvents. Finally, this paper discusses both the current challenges and future prospects of using polymer-based nanocarriers in sustainable agriculture, offering critical insights into their potential to transform crop protection through RNAi technologies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bumblebees play an important ecological economic role as pollinators in nature and agriculture. For reasons of biosecurity, many countries promote the cultivation of native bumblebee species for crop pollination instead of importing "alien" species. In South China, a few bumblebee species are considered useful in this way, particularly, Bombus atripes, Bombus bicoloratus and Bombus breviceps. However, whether they are suitable for artificial rearing and forming healthy colonies for pollination, remains unknown. In this project, queens from the 3 native species of Guizhou Province were collected and colonies were started under standardized conditions. The colonies were scored based on 19 parameters, including the stage of colony development, number and weight of offspring, and diet consumed. The data revealed that B. breviceps had the best performance, produced more workers and consumed the smallest diet. Next, we performed 16S rDNA sequencing of the bacterial communities found in the guts of offspring workers, and then a correlation analysis between colony performance and gut bacteria was conducted. Here, B. breviceps showed the highest diversity in gut bacterial composition, dominated by the bacteria Gilliamella, Snodgrassella, Enterobacter, and Lactobacillus Firm5. The higher the abundance of Snodgrassella, the better the performance of the colony in the foundation stage, and later Lactobacillus Firm5, Apibacter and Bifidobacterium were beneficial during the stages of rapid growth and colony decline. Although we do not understand all of the interactions yet, these correlations explain why B. breviceps demonstrated better colony performance. Our data provide valuable information for breeding local Bombus species and will contribute to developing strong colonies for crop pollination., (© 2024 The Authors. Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.)

RNA interference (RNAi)-based products have the potential to significantly contribute to insect pest control. However, RNAi efficiency varies widely among different insect orders, particularly in Lepidoptera, where it is often low. One key factor affecting RNAi efficiency is the presence of double-stranded ribonuclease (dsRNase) in the digestive tract, which can degrade dsRNA prior to uptake by gut cells. In this study, four dsRNases were identified in the beet armyworm, Spdoptera exigua, of which two were highly expressed gut dsRNases, SedsRNase1 and SedsRNase2. To assess their effect on dsRNA degradation activity via the oral route, CRISPR/Cas9-based gene editing was employed to knock out these gut dsRNases. The results indicate that all mutant strains, including SeKO1 (knockout SedsRNase1), SeKO2 (knockout SedsRNase2), and SeKO1KO2 (knockout SedsRNase1 and SedsRNase2), showed significantly decreased dsRNA degradation activity, particularly in the SeKO1KO2 mutant strain, where the weakest degradation occurred in both the gut and whole body. Additionally, we noticed that the lack of gut SedsRNases led to a slight extended developmental period and reduced reproductive capacity in S. exigua. Collectively, these findings deepen our understanding of gut SedsRNases and how they can impact the biology of the beet armyworm and can support the exploration dsRNA-based approaches for pest control., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Neuropeptide CCHamide-1 (abbreviated as CCHa1 ) is a recently discovered peptide that is present in many arthropods and is the ligand of the CCHa1R , a member of the G protein-coupled receptors (GPCRs) superfamily, which plays a regulatory role in diverse physiological processes such as feeding, circadian rhythm, insulin production, lipid metabolism, growth, and reproduction. However, the function of this gene in aphids is still unknown. Here, we characterized and determined the potential role of CCHa1 / CCHa1R signaling in the pea aphid, Acyrthosiphon pisum , which is a notorious pest in agriculture. The docking analysis revealed that the CCHa1 peptide binds to its receptor CCHa1R through specific amino acid residues, which are critical for maintaining the structural and functional integrity of the peptide-receptor complex. Quantitative real-time reverse transcription-PCR (qRT-PCR) revealed the expression levels of CCHa1 / CCHa1R transcripts in different development stages and different tissues, indicating that the CCHa1 expression was high in the first nymphal instar compared to the upcoming nymphal instars and adults, and was predominantly high in the brain. The CCHa1 / CCHa1R transcript levels were significantly upregulated in starved aphids compared to fed aphids. Moreover, RNAi knockdown by the injection of dsRNA- CCHa1 and dsRNA- CCHa1R significantly reduced the corresponding expression of the target gene and reduced their food intake in adult aphids, as revealed by the electrical penetration graph results. CCHa1 / CCHa1R -silencing also reduced the reproduction, but not the survival, in A. pisum . Our data demonstrated that CCHa1/CCHa1R play a role in the regulation of feeding in A. pisum , suggesting a role of the CCHa1 signaling pathway in the aphids relating to their nutritional status.

Background: The German cockroach Blattella germanica is a notorious urban health pest that has developed resistance to multiple pesticides. Thus, novel non-lethal pest control agents are urgently needed. Olfaction interference via disruption of sex pheromone recognition-related genes offers a promising approach. The German cockroach has a unique courtship behavior in which female adults emit contact sex pheromones (CSPs) in response to antennal touching, which subsequently triggers distinctive male sex behavioral responses. Due to the limited volatility of CSPs, the molecular mechanisms underlying their recognition and the specific olfactory pathways activated remain poorly defined. Although the odorant receptor coreceptor (Orco) is critical for most insect olfaction, sensory neuron membrane proteins (SNMPs), in particular SNMP1, also play crucial roles in sex pheromone recognition in moths and flies. While multiple SNMP1 homologs have been identified in multiple insect species, they have yet to be fully functionally characterized in cockroaches., Results: In this study, RNA-interference (RNAi)-mediated knockdown of BgerOrco reduced both the electrophysiology responses and courtship behaviors of males, indicating CSP perception proceeds via an olfaction pathway. Similar RNAi knockdown of BgerSNMP1e and BgerSNMP1d, which are predominantly expressed in male antennae, revealed critical roles in perceiving the major component of the Blattella germanica CSP blend. Unlike BgerSNMP1e, BgerSNMP1d was also found to function in the perception of the minor CSP component. Molecular docking analyses revealed no differences in the binding affinities of BgerSNMP1d for the major and minor CSP components, whereas the binding affinities of BgerSNMP1e displayed clear selectivity for the major component., Conclusion: Our results show that the olfactory pathway is critical for CSP recognition and that two male-enriched SNMP genes, BgerSNMP1e and BgerSNMP1d, are crucial factors mediating the male response to CSP stimulation in German cockroaches. This study lays a foundation for studying the mechanisms of CSP recognition and provides novel molecular targets with potential to be exploited as disruptors of courtship behavior. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Background: Sensing dietary components in the gut is important to ensure an appropriate hormonal response and metabolic regulation after food intake. The fall webworm, Hyphantria cunea, is a major invasive pest in China and has led to significant economic losses and ecosystem disruption. The larvae's broad host range and voracious appetite for leaves make H. cunea a primary cause of serious damage to both forests and crops. To date, however, the gustatory receptors (Grs) of H. cunea and their regulatory function remain largely unknown., Results: We identified the fall webworm gustatory receptor HcGr76 as a fructose and chlorogenic acid receptor using Ca 2+ imaging and determination of intracellular Ca 2+ concentration by a microplate reader. Moreover, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis revealed that HcGr76 is highly expressed in the anterior and middle midgut. Knockdown of HcGr76 caused a significant reduction in the expression of neuropeptide F 1 (NPF1) and CCHamide-2, and led to a decrease in carbohydrate and lipid levels in the hemolymph., Conclusion: Our studies provide circumstantial evidence that HcGr76 expressed in the midgut is involved in sensing dietary components, and regulates the expression of relevant peptide hormones to alter metabolism in H. cunea larvae, thus providing a promising molecular target for the development of new insect-specific control products. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

To elucidate phylogenetic relationships within the leafhopper's subfamily Megophthalminae (Hemiptera: Cicadellidae), mitogenomes of 12 species of the subfamily were sequenced and assembled. These were added to the mitogenomes of the eight other species that are currently available. Mitogenome size ranged from 15,193 bp in Onukigallia onukii (Matsumura, 1912) to 15,986 bp in Multinervis guangxiensis (Li and Li, 2013), they all contained 37 genes, and gene order was similar to that in other leafhoppers. Nucleotide composition analysis showed that the AT content was higher than that of GC, and the protein-coding genes usually ended with A/T at the 3rd codon position. The Ka/Ks ratio showed that the CYTB gene has the slowest evolutionary rate, while ND4 is the gene with the fastest evolutionary rate. Relative synonymous codon usage analysis revealed the most frequently used codon was UUA (L), followed by CGA (R), and the least frequently used codon was CCG (P). Parity plot and neutrality plot analyses showed that the codon usage bias of mitochondrial genes was influenced by natural selection and mutation pressure. However, natural selection plays a major role, while the effect of mutation pressure was small. Effective number of codons values were 40.15-49.17, which represented relatively low codon bias. Phylogenetic analyses based on three datasets (AA, 13PCG, 13PCG_2rRNA) using two methods (maximum likelihood and Bayesian inference). In the obtained topology, the Megophthalminae species were clustered into a monophyletic group. In conclusion, our results clarify structural modules of the mitochondrial genes and confirm the monophyly of Megophthalminae within Cicadellidae., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)

The rice leaf folder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), is a major migratory pest in rice agriculture. This pest is characterised by its larvae's ability to fold rice leaves using silk, a behaviour that culminates in the formation of a silken cocoon during the pupal stage. The fibroin light chain (CmFib-L) gene is crucial for silk production, yet its specific function in C. medinalis has reminded elusive. This study presents a comprehensive analysis of the CmFib-L gene, revealing its complete open reading frame (ORF) and expression patterns. Notably, the gene is highly expressed in the fifth-instar larvae and the silk gland, which are critical stages for silk production. Our experiments demonstrate that silencing the CmFib-L gene leads to a reduction in pupal weight, an extension of the pupal stage and a disorganised silk cocoon. Furthermore, the larval behaviour of leaf folding and spinning is significantly impaired when the expression of CmFib-L is downregulated. These findings not only show the importance of fibroin light chain in silk production but also reveal a new target gene to regulate and control the behaviour and development of C. medinalis., (© 2024 The Royal Entomological Society.)

Chikungunya and Mayaro fevers are viral infectious diseases characterized by fever and arthralgia, for which there are currently no effective vaccines or treatments. The urgent need for novel antiviral agents against Chikungunya virus (CHIKV) and Mayaro virus (MAYV) has led to interest in plant-based compounds that can disrupt the viral replication cycle. Chiococca alba (L.) Hitchc., a Neotropical plant traditionally used by Yucatec Maya healers as an antipyretic and antirheumatic, may hold potential as a source of antiviral agents. This study aimed to evaluate the antiviral potential of C. alba methanolic extracts (CAH21 and CAH24) against CHIKV and MAYV through preliminary in vitro and in silico analyses. The cytotoxicity of two methanolic extracts from C. alba roots was assessed in Vero cells using the neutral red assay, and their viral activity was determined via plaque assay post-treatment. Given the observed antiviral effects, we used computational predictions to explore interactions between the multifunctional nsP2 proteases and secondary metabolites identified in C. alba extracts. The metabolites were identified using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). Phytochemical analysis revealed the presence of flavonoids, coumarins, and phenolic acids in the C. alba extracts. In vitro assays demonstrated that both extracts inhibited over 70% of activity against CHIKV and MAYV at a concentration of 60 µg/mL. In silico predictions suggested that the flavonoids naringin and vitexin had the highest affinity for the nsP2 proteases of CHIKV and MAYV, indicating their potential as viral inhibitors. Our findings revealed that C. alba extract represents a promising source of novel antiviral compounds.

The takeout (TO) gene family impacts diverse physiological and behavioral functions in insects, yet specific olfactory-associated roles for the family have yet to be fully elucidated. To provide insights into TO function in rice planthoppers, the genomes of three rice planthoppers (white-backed planthopper, brown planthopper and small brown planthopper) were searched for TO homologs and their degree of conservation assessed via chromosomal localization, exon-intron boundaries, phylogenetic relationships and protein domains/motifs. We performed a tissue-specific expression analysis of the 20 TO genes in the white-backed planthopper (WBPH, Sogatella furcifera) and found that SfTO17 is enriched in adult antennae. RNAi-mediated knockdown of SfTO17 impaired WBPH olfaction and reduced host-seeking responses following exposure to rice plants. The binding profile of β-ionone, hexyl benzoate and benzyl benzoate with recombinant SfTO17 was evaluated via competitive fluorescence binding assays. Conformational prediction of SfTO17 coupled with molecular docking analyses revealed several amino acid residues potentially critical for odorant binding. This study demonstrates the olfactory function of SfTO17 in WBPH and highlights its potential as a target for controlling this rice pest. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Background: The two-spotted spider mite, Tetranychus urticae, is an important pest mite in agriculture worldwide. E78, as a member of the nuclear receptor superfamily and a downstream responsive gene of ecdysteroids, plays a crucial role in regulating physiological behaviors such as development and reproduction in insects. However, its function in mites remains unclear. The aim of this study was to explore how E78 functions in the molting process of spider mites., Results: In this study, reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments to analyze the expression pattern of TuE78 during the development of Tetranychus urticae, demonstrated that the expression level of TuE78 was higher during the molting state than that after the completion of molting, and it reached a peak expression level when the deutonymph mites entered the molting stage. RNA interference (RNAi)-mediated gene-silencing of TuE78 resulted in 95% deutonymph mite molt failure. A series of analysis under a light microscope, and scanning and transmission electron microscopy revealed that RNAi mites died within the exuvium without ecdysis, and that apolysis had started but the new cuticle was thin and the typical cuticular lamellae were absent, indicating blockage of the post-apolysial processes and explaining molt failure. Hence, transcriptome sequencing confirmed that the expression of cuticle protein and lipid metabolism-related genes was significantly affected after TuE78 silencing., Conclusion: This study demonstrated that TuE78 participates in the molting process of Tetranychus urticae by regulating the post-apolysial processes with the formation of new cuticle and successful ecdysis. This in turn suggests the potential of TuE78 as a target for pest mite control and provides a theoretical basis for further exploration of the molecular regulatory mechanism of spider mite molting. © 2024 Society of Chemical Industry. Published by John Wiley & Sons Ltd., (© 2024 Society of Chemical Industry. Published by John Wiley & Sons Ltd.)

Macropsini is a tribe of Eurymelinae in the family Cicadellidae that is widely distributed worldwide. Still, its taxonomic status has been unstable, and the classification of certain clades at the genus level has been controversial. The aim of this study is to address the patterns and processes that explain the structure and the evolution of the mitogenomes of Macropsini, while contributing to the resolution of systematic issues involving five of their genera. To this task, the mitogenomes of 26 species of the tribe were sequenced and characterized, and their phylogenetic relationships were reconstructed. The results revealed that the nucleotide composition of mitochondrial genes in these 26 species was significantly skewed toward A and T. Codons ending with T or A in relative synonymous codon usage were significantly more prevalent than those ending with C or G. The parity plot, neutrality plot, and correspondence analysis revealed that mutation and selective pressure affect codon usage patterns. In the phylogenetic relationships of the Macropsini, the monophyly of Pedionis and Macropsis was well-supported. Meanwhile, Oncopsis revealed paraphyletic regarding Pediopsoides . In conclusion, this research not only contributes the valuable data to the understanding of the mitogenome of the Macropsini but also provides a reference for future investigations on codon usage patterns, potential adaptive evolution, and the phylogeny of the mitogenome within the subfamily Eurymelinae., Competing Interests: All authors declare no conflict of interest., (© 2024 The Author(s). Ecology and Evolution published by John Wiley & Sons Ltd.)

Background: The melon fly, Zeugodacus cucurbitae (Coquillett), is an invasive Tephritidae pest with robust fertility. The male accessory glands (MAGs) form a vital organ that ensures insect reproductive efficiency. Most of the secreted proteins by MAGs exhibit a male bias expression. Takeout, one of these proteins, is abundantly present in the MAGs of many insects., Results: In this study, we identified 32 takeout genes in Z. cucurbitae. The phylogenetic analysis and multiple sequence alignment results showed that Zctakeout1 is the most related homolog to the MAGs-specific takeout in Tephritidae. The real-time quantitative PCR results showed that Zctakeout1 was exclusively expressed in the male adult stage, and its expression level gradually increased with the increase in age and then remained stable at the sexually matured stage. The distribution among tissues demonstrated the specific expression of Zctakeout1 in the MAGs, and fluorescence immunohistochemical results confirmed the presence of Zctakeout1 in close proximity to binuclear cells of the mesoderm epidermal MAGs. In continuation, CRISPR/Cas9-mediated genome editing was employed, resulting in successfully generating a homozygous strain with an +8 bp insertion. The mating experiments with the Zctakeout1 -/- males resulted in significant reductions in both the mating rate and egg production of females., Conclusion: These findings prove that the MAGs-specific Zctakeout1 is essential in regulating fecundity in female Z. cucurbitae fruit flies. Our data suggests its utilization in future essential insect-specific gene-directed sterility insect technique (SIT) by the genetic manipulation to keep these important Tephritidae populations under control. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Anisopteromalus calandrae (Howard) shows great promise as an ectoparasitoid for controlling various coleopteran pests in warehouses. However, for a large-scale release, it is crucial to establish an ample supply of A. calandrae while carefully maintaining their quality and effectiveness. Appropriate cold storage techniques are the key to achieving these goals. Previous studies on cold storage have focused on specific developmental stages and explored cold storage conditions that can be applied only to those stages. Herein, we examined the development, survival and reproductive capacity of A. calandrae at different temperatures (13, 16, and 19 °C) and storage durations (30, 60, and 90 d) and evaluated the fitness of the offspring. A. calandrae completed its egg-to-larva development and pupated at 16 °C, but its development was arrested at an early pupal stage. Even after 90 d of cold storage at 16 °C, the survival rate of A. calandrae remained high at 77%, with no significant impact on reproductive capacity. Furthermore, cold storage showed no negative effect on the F 1 generation. In contrast, eggs stored at 13 °C failed to hatch, whereas those stored at 19 °C developed. Adults emerged after > 60 d. This indicates that storage at 19 °C is only suitable for short durations. Our findings highlight the developmental pattern of A. calandrae at 16 °C, indicating that the parasitic wasp can be stored for a long time at this temperature across all stages of development before pupation, substantially facilitating its mass reproduction and industrial production., (© 2024 Institute of Zoology, Chinese Academy of Sciences.)

Biopesticides based on RNA interference (RNAi) took a major step forward with the first registration of a sprayable RNAi product, which targets the world's most damaging potato pest. Proactive resistance management is needed to delay the evolution of resistance by pests and sustain the efficacy of RNAi biopesticides., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The greater wax moth, Galleria mellonella (Lepidoptera, Pyralidae), is a major bee pest that inflicts considerable harm on beehives, leading to economic losses. It also serves as a valuable resource insect and a model organism. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system plays a crucial role in improving economic insect breeding and developing efficient agricultural pest management systems in Lepidoptera. However, the CRISPR/Cas9 protocols have not been developed for G. mellonella. Here, the Gmebony knockout (KO) strain was established using the CRISPR/Cas9 genome editing system. We obtained Gmebony KO strain in the G4 generation, which took approximately 10 months. When compared with wild-type, the head, notum, and the terminal abdominal surface of 1st to 4th instar larvae in the KO strain changed from yellow to brown, and these regions of the KO strain gradually transformed into a black color from the 5th instar larvae, and the body color of the adult moth in the KO strain changed to black. The developmental period of the early larval and the following larval instars extended. The embryonic hatchability of the Gmebony KO strain was significantly decreased. The pupal body weight of the Gmebony KO strain was not affected. The feasibility of the CRISPR/Cas9 methodology was validated by single-target editing of Gmebony. Our findings provide the first evidence that the ebony gene can serve as a pigmentation reference gene for genetic modifications of G. mellonella. Meanwhile, it can be utilized in the development of genome editing control strategies and for gene function analyses in G. mellonella., (© 2024 Institute of Zoology, Chinese Academy of Sciences.)

Antimicrobial peptides (AMPs) in insects are endogenous peptides that are effector components of the innate defense system of the insect. AMPs may serve as antimicrobial agents because of their small molecular weight and broad-spectrum antimicrobial activity. In this study, we performed transcriptome analysis of cigarette beetle (Lasioderma serricorne) larvae, parasitized by the ectoparasitic wasp, Anisopteromalus calandrae. Several AMP genes were significantly upregulated following A. calandrae parasitism, postulating the hypothesis that the parasitization enhanced the host's resistance against pathogenic microorganisms through the regulation of host AMP genes. Specifically, 3 AMP genes (LsDef1, LsDef2, and LsCole) were significantly upregulated and we studied their immune function in L. serricorne. Immune challenge and functional analysis showed that LsCole was responsible for the immune response against Gram-negative and Gram-positive bacteria, while LsDef1 and LsDef2 were involved in insect defense against Gram-positive bacteria. Purified recombinant LsCole exhibited antimicrobial activities against the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. LsDef2 showed an antibacterial effect against S. aureus. LsCole and LsDef2 exhibited antibiofilm activity against S. aureus. The 2 AMPs disrupted cell membranes and caused leakage of S. aureus cell contents. The results indicated that the 3 AMPs in L. serricorne are involved in the innate immunity of this pest insect. These AMPs may have potential as antimicrobial agents for bacterial infection chemotherapy. Hence, data are discussed in relation to new control strategies with greater biosafety against pest insects with use of microbial biocontrol agents in combination with RNA interference against the insect's defensive AMP genes., (© 2023 Institute of Zoology, Chinese Academy of Sciences.)

The pink stem borer, Sesamia inferens Walker (Lepidoptera: Noctuidae), is one of the most notorious pest insects of rice and maize crops in the world. Here, we generated a high-quality chromosome-level genome assembly of S. inferens, using a combination of Illumina, PacBio HiFi and Hi-C technologies. The total assembly size was 973.18 Mb with a contig N50 of 33.39 Mb, anchored to 31 chromosomes, revealing a karyotype of 30 + Z. The BUSCO analysis indicated a high completeness of 98.90% (n = 5286), including 5172 (97.8%) single-copy BUSCOs and 58 (1.1%) duplicated BUSCOs. The genome contains 58.59% (564.58 Mb) repeat elements and 26628 predicted protein-coding genes. The chromosome-level genome assembly of S. inferens provides in-depth knowledge and will be a helpful resource for the Lepidoptera and pest control research communities., (© 2024. The Author(s).)

Natalisin (NTL) is a conserved neuropeptide, only present in insects, that has been reported to regulate their sexual activity. In this study, we investigated the involvement of NTL in the reproductive behaviors of a major invasive pest, Spodoptera frugiperda. We identified NTL precursor-encoded transcripts, and evaluated their transcript levels in different stages and tissues of S. frugiperda. The results showed that the NTL transcript level was expressed in both male and female pupae and both male and female adults in the later stage. It was highly expressed in male pupae, 3-day-old male and female adults, and 5-day-old male adults. In different tissues, the expression level is higher in the male and female adult brain and male testis. Immunohistochemical staining of the brain of S. frugiperda female and male adults revealed that three pairs of brain neurons of S. frugiperda adults of both sexes secreted and expressed NTL. To study the role of NTL in reproductive behaviors, NTL was silenced in S. frugiperda male and female adults by RNA interference (RNAi) technology, the results showed that silencing NTL could significantly affect the sexual activity behavior of the adults, reducing the calling rate of females, the courtship rate of males, and the mating rate. In summary, this study emphasizes the important role of NTL in regulating the mating behavior and sexual activity of S. frugiperda in both male and female adults, potentially laying a foundation to employ NTL as a new insect-specific target to control populations of pest insects., (© 2024. The Author(s).)

Background: The melon fly, Zeugodacus cucurbitae Coquillett, is one of the major pests attacking Cucurbitaceae crops. Identifying critical genes or proteins regulating fertility is essential for sustainable pest control and a research hotspot in insect physiology. MicroRNAs (miRNAs) are short RNAs that do not directly participate in protein translation, but instead function in post-transcriptional regulation of gene expression involved in male fertility., Results: We found that miR-927-5p is highly expressed in the testes and investigated its function in spermatogenesis in Z. cucurbitae. Fluorescence in situ hybridization (FISH) showed miR-927-5p in the transformation and maturation region of the testis, and overexpression of miR-927-5p reduced the number of sperms by 53%. In continuation, we predicted 12 target genes of miR-927-5p using bioinformatics combined with transcriptome sequencing data, and found that miR-927-5p targets the new gene Stalky in insects, which was validated by quantitative real-time PCR, RNA pull-down and dual luciferase reporter assays. FISH also confirmed the co-localization of miR-927-5p and the transcript Stalky_1 in the testis. Moreover, silencing of Stalky_1 by RNA interference reduced the number of sperms by 32% and reduced sperm viability by 39% in physiologically mature male adults. Meanwhile, the silencing of Stalky_1 also resulted in low hatchability., Conclusion: Our work not only presents a new, so far unreported mechanism regulating spermatogenesis by miR-927-5p targeting a new unknown target, Stalky, which is providing new knowledge on the regulatory network of insect spermatogenesis, but also lays a foundation for the development of SIT against important tephritid fly pests. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Blister beetles of Epicauta impressicornis have attracted attention because they contain a large amount of cantharidin (CTD). To date, however, the synthesis and transfer of CTD in adults of E. impressicornis are largely unknown. Here, we showed that the larvae E. impressicornis are capable of synthesizing CTD and they consume CTD during pupation. Before sexual maturity, both male and female adults synthesized a small amount of CTD, while after sexual maturity, males produced larger amounts of CTD, but females did not. The newly synthesized CTD in males first appeared in the hemolymph and then accumulated in the reproductive system. During the mating, the males transferred CTD to the reproductive system of females. In addition, a farnesyl pyrophosphate synthase (FPPS) gene was identified in male E. impressicornis . RNA-seq analysis, quantitative RT-PCR, and RNA interference analyses were conducted to investigate expression patterns and the functional roles of E. impressicornis FPPS ( EiFPPS ). Our results indicate that EiFPPS is highly expressed in the fat body of males. Moreover, the knock-down of EiFPPS led to a significant decrease in CTD synthesis. The current study indicates that EiFPPS is expressed in the fat body to regulate CTD synthesis in male E. impressicornis blister beetles.

Neuropeptides are crucial in regulation of a rich variety of developmental, physiological, and behavioral functions throughout the life cycle of insects. Using an integrated approach of multiomics, we identified neuropeptide precursors in the greater wax moth Galleria mellonella, which is a harmful pest of honeybee hives with a worldwide distribution. Here, a total of 63 and 67 neuropeptide precursors were predicted and annotated in the G. mellonella genome and transcriptome, in which 40 neuropeptide precursors were confirmed in the G. mellonella peptidome. Interestingly, we identified 12 neuropeptide precursor genes present in G. mellonella but absent in honeybees, which may be potential novel pesticide target sites. Honeybee hives were contaminated with heavy metals such as lead, enabling its bioaccumulation in G. mellonella bodies through the food chain, we performed transcriptome sequencing to analyze the effects of Pb stress on the mRNA expression level of G. mellonella neuropeptide precursors. After treatment by Pb, the expression of neuropeptide F1 was found to be significantly downregulated, implying that this neuropeptide might be associated with responding to the heavy metal stress in G. mellonella. This study comprehensively identified neuropeptide precursors in G. mellonella, and discussed the effects of heavy metals on insect neuropeptides, with the example of G. mellonella. The results are valuable for future elucidation of how neuropeptides regulate physiological functions in G. mellonella and contribute to our understanding of the insect's environmental plasticity and identify potential new biomarkers to assess heavy metal toxicity in insects., (© 2023 Institute of Zoology, Chinese Academy of Sciences.)

Vacuolar-type (H + )-ATPase (vATPase) is a conserved multi-subunit eukaryotic enzyme composed of 14 subunits that form a functional complex consisting of an ATP-hydrolytic domain (V1) and a proton-translocation domain (V0). ATP hydrolysis and subsequent H + translocation rely heavily on a fully assembled V1/V0 complex. Since vATPase is crucial for insect survival, it is a viable molecular target for pest control. However, detailed functional analyses of the 14 subunits and their suitability for pest control have not been fully explored in a single insect species. In this study, we identified 22 vATPase subunit transcripts that correspond to 13 subunits (A1, A2, B, C, D, E, F, G, H, a1, a2, c and d) in the white-backed planthopper (WBPH), Sogatella furcifera, a major hemipteran pest of rice. RNAi screens using microinjection and spray-based methods revealed that the SfVHA-F, SfVHA-a2 and SfVHA-c2 subunits are critical. Furthermore, star polymer (SPc) nanoparticles were utilized to conduct spray-induced and nanoparticle-delivered gene silencing (SI-NDGS) to evaluate the pest control efficacy of RNAi targeting the SfVHA-F, SfVHA-a2 and SfVHA-c2 transcripts. Target mRNA levels and vATPase enzymatic activity were both reduced. Honeydew excreta was likewise reduced in WBPH treated with dsRNAs targeting SfVHA-F, SfVHA-a2 and SfVHA-c2. To assess the environmental safety of the nanoparticle-wrapped dsRNAs, Cyrtorhinus lividipennis Reuter, a major natural enemy of planthoppers, was also sprayed with dsRNAs targeting SfVHA-F, SfVHA-a2 and SfVHA-c2. Post-spray effects of dsSfVHA-a2 and dsSfVHA-c2 on C. lividipennis were innocuous. This study identifies SfVHA-a2 and SfVHA-c2 as promising targets for biorational control of WBPH and lays the foundation for developing environment-friendly RNAi biopesticides., Competing Interests: Declaration of competing interest Peng He reports financial support was provided by Guizhou University. Peng He reports a relationship with Guizhou University that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The increase in studies on bee microbiomes is prompted by concerns over global pollinator declines. Bumble bees host core and non-core microbiota which may contribute to increased lifetime fitness. The presence of Fructobacillus in the gut microbiomes of bumble bee workers, or the replacement of core symbionts with Fructobacillus bacteria, has been considered a marker of dysbiosis. A phylogenomic analysis and functional genomic characterization of the genomes of 21 Fructobacillus isolates from bumble bees demonstrated that they represented four species, i.e. Fructobacillus cardui, Fructobacillus fructosus, Fructobacillus tropaeoli, and the novel species Fructobacillus evanidus sp. nov. Our results confirmed and substantiated the presence of two phylogenetically and functionally distinct Fructobacillus species clades that differ in genome size, percentage G + C content, the number of coding DNA sequences and metabolic characteristics. Clade 1 and clade 2 species differed in amino acid and, to a lesser extent, in carbohydrate metabolism, with F. evanidus and F. tropaeoli genomes featuring a higher number of complete metabolic pathways. While Fructobacillus genomes encoded genes that allow adhesion, biofilm formation, antibacterial activity and detoxification, other bacteria isolated from the bumble bee gut appeared better equipped to co-exist with the bumble bee host. The isolation and identification of multiple Fructobacillus species from several bumble bee gut samples in the present study also argued against a specific partnership between Fructobacillus species and their bumble bee hosts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Background: Agricultural landscapes provide resources for arthropod pests as well as their natural enemies. To develop integrated pest management (IPM) practices, it is important to understand how spatiotemporal location influences crop colonization and damage severity. We performed a 3-year (2016-2018) field experiment in winter oilseed rape (OSR, Brassica napus) fields in Estonia, where half of the fields were within 500 m of the location of the previous year's winter OSR field and half were outside this zone. We investigated how distance from the previous year's OSR crop influences the infestation and parasitism rates of two of its most important pests: the pollen beetle (Brassicogethes aeneus) and the cabbage seed weevil (Ceutorhynchus obstrictus)., Results: When the distance from the previous year's OSR crop was >500 m, we recorded significantly reduced pest pressure by both B. aeneus and C. obstrictus in the study fields. Biocontrol of both pests, provided by parasitic wasps, was high in each study year and commonly not affected by distance. Mean parasitism rates of B. aeneus were >31%, occasionally reaching >70%; for C. obstrictus, mean parasitism was >46%, reaching up to 79%, thereby providing effective biocontrol for both pest species., Conclusion: Spatiotemporal separation of OSR fields can reduce pest pressure without resulting in reduced parasitism of OSR pests. This supports a spatiotemporal field separation concept as an effective and sustainable technique for IPM in OSR. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)