Alluvial fans are typically fragile and unstable landforms in arid regions, and their bioenvironmental coupling mechanisms remain unclear. The aims of this study were to test the hypothesis that elevation gradients drive beetle functional group differentiation through environmental filtering, reveal the core strategies of functional group adaptation to fragile habitats, and provide a theoretical basis for arid ecological restoration. Five transects were established along the alluvial fan elevation gradient, and ground-dwelling beetles were collected using pitfall trapping. Species and functional diversity analyses were conducted, and functional groups were classified using hierarchical clustering. The associations between functional traits and environmental factors were analyzed using RLQ and fourth-corner analysis. A total of 8251 ground-dwelling beetles were collected, and community diversity varied significantly with elevation. The first two axes of the RLQ explained 91.28% of total co-inertia. Feeding habits, activity rhythms, hind legs, antennae, and head width were significantly correlated with environmental variables. The five functional groups showed a gradient distribution from the fan apex to the fan margin: "broad-headed nocturnal saprophagous group → nocturnal carnivorous group → nocturnal phytophagous group → diurnal phytophagous group → long-legged and long-antennae diurnal phytophagous group." Combined with partial least squares structural equation modeling (PLS-SEM), this study confirmed the rule that alluvial fan instability regulates habitat heterogeneity through elevation gradients, which in turn drives beetle functional group differentiation. Functional groups can serve as indicators of habitat stability. This study deepens understanding of insect community assembly mechanisms in fragile landforms and provides scientific support for arid ecosystem protection and restoration.
Heritable endosymbionts will shape how many insect pests respond to climate warming. Aphids are globally important pests that almost universally depend on the obligate symbiont Buchnera aphidicola, which can constrain host thermal tolerance, while additional secondary symbionts may further modify heat responses. Importantly, aphid-symbiont interactions are developmentally dynamic, with physiological functions and phenotypic effects changing as aphids develop. However, it remains unclear whether aphids and their endosymbionts are most vulnerable to heat stress at the same developmental stages, or whether mismatches in thermal sensitivity shape fitness outcomes. We addressed this gap by testing how heatwaves typical of southern Australia affect the invasive cereal pest, the Russian wheat aphid (Diuraphis noxia), and two endosymbionts (Buchnera aphidicola and Rickettsiella viridis) across developmental stages. Using a factorial design (endosymbiont status × life stage × temperature), we exposed early nymphs or adults to either moderate or extreme heatwave conditions-representative of their invaded range-and quantified survival, post-exposure longevity, fecundity, and symbiont density in treated individuals and their offspring. Heatwaves had a disproportionately strong effect on the nymph stage compared with adults, primarily through reduced fecundity. In contrast, endosymbiont responses were strongest following adult exposure, including Buchnera aphidicola suppression, in adults and/or their offspring. These results reveal a life-stage mismatch in thermal vulnerability between hosts and symbionts, with implications for physiological responses to climate extremes.
Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a double-stranded RNA reovirus, causes significant losses in sericulture; however, the host factors that support its replication remain poorly understood. Here, we identify Tudor staphylococcal nuclease (TSN) as a critical pro-viral host factor in silkworms. TSN is a multifunctional, evolutionarily conserved protein involved in transcription and RNA processing. BmTSN expression was increased markedly in BmCPV-infected midgut tissues. RNAi-mediated knockdown in BmN cells suppresses viral replication, whereas overexpression enhances it. Mechanistically, BmTSN drives a metabolic shift by enhancing lipid accumulation; knockdown of BmTSN reduces the expression of BmSREBP1 and other lipid-regulatory genes. Disruption of either BmTSN or BmSREBP1 reduces BmCPV replication. In a reciprocal effect, RNAi-mediated knockdown of BmSREBP1 also reduces BmTSN expression. Furthermore, BmSREBP1 transcriptionally activates BmTSN expression by binding to the BmTSN promoter, suggesting a regulatory loop between these factors. Together, these findings reveal a virus-induced BmTSN-BmSREBP1 axis that links host lipid metabolism to BmCPV replication. This work identifies BmTSN as a key host-dependency factor and provides new insights into virus-host interactions in insects.
While G protein-coupled receptors (GPCRs) are recognized as key upstream regulators of insecticide resistance in various agricultural pests, effective strategies to exploit these receptors for practical pest control remain largely undeveloped. In this study, we screened and identified TMEM145 as a novel GPCR significantly upregulated in clothianidin resistant Nilaparvata lugens. RNAi-mediated knockdown of TMEM145 suppressed the expression of the detoxification gene CYP6ER1 and the transcription factor AP-1, successfully restoring clothianidin susceptibility with a lethal dose ratio (LCR50) of 1.78. Through structure-based virtual screening of 4000 bioactive molecules targeting TMEM145, we mined pectolinarin as a potential inhibitor exhibiting a high binding affinity of -10.945 kcal/mol. Its structural scaffold, apigenin, was identified as an alternative with a binding affinity of -8.223 kcal/mol. Experimental validation confirmed that chemical inhibition by these flavonoids significantly downregulated detoxification gene expression. In vivo bioassays demonstrated that pectolinarin markedly increased clothianidin susceptibility, achieving a synergistic effect with an LCR50 of 2.87. These results demonstrate that targeting upstream GPCR-mediated regulatory nodes with natural synergists offers a promising and sustainable approach to managing metabolic resistance in agricultural pests.
The Australian giant wood moth, Endoxyla cinereus (Cossidae), is a major forestry pest whose later instars bore into the trunks and branches of Eucalyptus, but the early larval stages have remained unknown for over a century. Using DNA analysis, we identified larvae collected from galls formed by scale insects (Apiomorpha spp.), as En. cinereus. This relationship was tested by rearing over 100 000 neonate larvae on alternative potential food resources including active galls of scale insects (Apiomorpha munita and A. crispa). Only larvae assigned to galls fed and developed into later instars. The complete life cycle of this moth was also confirmed by rearing a gall-fed larvae to adulthood over 2 years after establishment in a tree (Eu. tereticornis). This two-phase developmental strategy explains how larvae bridge the large size gap between hatching and tree establishment and provides a mechanism for the high reproductive output and dispersal bottlenecks of this species.
CRISPR/Cas9 has become a key tool for functional genomics and genetic control strategies in mosquitoes, with its efficiency highly dependent on precise spatiotemporal regulation of Cas9 expression. While germline-specific promoters help reduce resistance and improve drive efficiency, constitutive promoters confer higher editing efficiency due to their ubiquitous and sustained expression, making them more suitable for gene functional analysis and population-specific suppression. However, the scarcity of strong constitutive promoters in Aedes aegypti (Ae. aegypti) has limited the development of highly efficient editing systems. In this study, we cloned the promoter of the housekeeping gene eukaryotic translation elongation factor 1α (EF1Α, AAEL017096) and confirmed its transcriptional activity. RT-qPCR analysis revealed that the EF1Α promoter (EF1Αp) directed significantly higher Cas9 expression in mosquito ovaries and testes than did the germline-specific Exu promoter (Exup). Crossing EF1Ap-Cas9 transgenic lines with various gRNA-expressing lines achieved efficient editing of the marker gene white, the functional gene PNP, and the microRNA precursor miR-1174, with all double-positive progeny displaying expected loss-of-function phenotypes. To overcome vector capacity limitations, we truncated the U6 promoter. A truncated U6 promoter of only 235 bp (U6p235) supported editing efficiency comparable to the full-length 964 bp version. When U6p235-white gRNA lines were crossed with EF1Ap-Cas9 lines, all double-positive offspring exhibited a white-eye phenotype, confirming successful white gene disruption. Together, this work provides an efficient constitutive promoter and a compact U6p, expanding the genetic toolkit for Ae. aegypti and supporting advanced functional studies and multi-target control strategies.
Ticks are ectoparasites that can transmit zoonotic pathogens, causing significant economic losses worldwide. RNA interference (RNAi) offers a gene-specific alternative to chemical acaricides; however, its application is limited by double-stranded RNA (dsRNA) instability and poor cuticle penetration. Using Haemaphysalis longicornis (H. longicornis) as a model, we identified the ecdysone receptor gene (ECR) as an effective RNAi target. Microinjection of dsECR completely abolished engorgement rate (0%) and reduced body weight by ∼90%. To enable practical delivery, three nanocarrier systems-chitosan (CS), star polycation (SPc), and disulfide-crosslinked chitosan (CS-ss) were developed to improve the stability and uptake of dsRNA. All three nanocarrier systems enhanced dsRNA resistance to ribonuclease (RNase) degradation while exhibiting distinct physicochemical properties: CS-ss formed the smallest nanoparticles (∼175 nm), whereas SPc showed superior surface wettability. All nanocarrier-dsRNA complexes achieved comparable RNAi efficiency under microinjection. In contrast, immersion delivery of naked dsECR was ineffective, whereas SPc-dsECR and CS-ss-dsECR enabled significant ECR silencing, reduced engorgement rate, and decreased body weight. Similar RNAi efficiency levels were validated in Hyalomma asiaticum, supporting the cross-species applicability of this strategy. Notably, SPc-dsRNA exhibited lower contact angles on the tick cuticle, indicating enhanced spreading and suggesting improved penetration efficiency. Collectively, the results of this study demonstrate that nanocarrier encapsulation is effective for immersion-based RNAi control of ticks. SPc provides superior dispersion and surface interaction for consistent delivery, while CS-ss offers the potential for redox-responsive release. The findings establish a scalable and non-invasive RNAi strategy for sustainable tick control.
Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) is a widely distributed polyphagous pest, associated with soybean stay-green syndrome, causing substantial yield losses across Asia. Continued reliance on chemical control remains problematic, highlighting the need for sustainable biological alternatives. In this study, we comparatively evaluated the parasitism performance, host-feeding behavior, developmental traits, and host-age preference of five egg parasitoids of the genus Anastatus (A. dexingensis, A. fulloi, A. gansuensis, A. japonicus, and A. shichengensis) on R. pedestris eggs of different ages (0-, 2-, 4-, and 6-d-old). All five parasitoid species successfully completed development on R. pedestris eggs, with offspring emergence rates ranging from 82% to 100%. Parasitism and host-feeding were strongly influenced by host egg age, with all species showing a clear preference for 0- to 2-d-old. Anastatus dexingensis and A. japonicus parasitized the highest number of host eggs (13.5 and 12.3 eggs/24 h, respectively), whereas the thelytokous A. gansuensis exhibited the strongest host-feeding activity (3.8 eggs/24 h). In contrast, A. fulloi and A. shichengensis produced no female progeny, whereas A. dexingensis and A. japonicus produced less than 20% female offspring. Developmental duration generally increased with host age, indicating that older host eggs may be less suitable for parasitoid development. Overall, A. dexingensis and A. japonicus appear well suited for short-term suppression of R. pedestris through inundative releases, whereas the thelytokous A. gansuensis shows strong potential for long-term, sustainable population regulation, making it promising for augmentative biocontrol. These findings provide a scientific basis for selecting effective Anastatus spp. and optimizing egg-stage biocontrol strategies against R. pedestris.
Microsporidia are unicellular eukaryotic obligate intracellular parasites that primarily infect vertebrates, invertebrates, and some protists. However, our understanding of microsporidian-host interaction mechanisms remains insufficient, especially the mechanisms underlying the noncoding RNA response to microsporidian infection. To systematically explore the characteristics of the host noncoding RNA response to microsporidian infection, we used whole-transcriptome sequencing technology to identify noncoding RNAs in the Bombyx mori midgut at different time points after Nosema bombycis (N. bombycis) infection. We identified 51 novel microRNAs (miRNAs) and 269 novel circular RNAs (circRNAs) in the Bombyx mori midgut and constructed a differentially expressed (DE)-circRNA-miRNA regulatory interaction network. Furthermore, our qRT-PCR screening revealed that bmo_circ_0000157 (circ_0000157) facilitates N. bombycis proliferation. Ultimately, dual-luciferase reporter and cell-level rescue assays confirmed that circ_0000157 functions as a molecular sponge for bmo-miR-281-5p (miR-281-5p), thereby regulating N. bombycis proliferation. Overall, our results indicate that the novel regulatory target circ_0000157 facilitates N. bombycis proliferation by modulating miR-281-5p expression, thereby altering its effects on downstream target genes. This study expands our understanding of circRNA biological functions and provides an important foundation for elucidating N. bombycis infection mechanisms.
Zona pellucida (ZP)-domain proteins are critical for development and reproduction in insects; however, the underlying mechanisms remain largely unclear. Here we demonstrate that knockdown of papillote (pot), which encodes a ZP-domain protein, suppresses larval growth, induces premature metamorphosis, and impairs reproduction in Tribolium castaneum (T. castaneum). Pot silencing stimulated juvenile hormone (JH) degradation while reducing phosphorylated levels of insulin receptor β (InRβ) and FOXO, leading to nuclear location of FOXO. Methoprene treatment rescued larval growth and pupation defects as well as phosphorylated InRβ (p-InRβ) and phosphorylated FOXO (p-FOXO) levels inhibited by pot knockdown. Similarly, insulin injection reversed pot silencing-mediated reductions in larval growth, pupation, and p-InRβ and p-FOXO levels. Silencing pot induced ecdysone biosynthesis, whereas FOXO knockdown suppressed this effect. Interestingly, reducing ecdysone biosynthesis via phm silencing increased p-FOXO levels, repressed FOXO nuclear translocation, and reversed the decline in larval growth and pupation caused by pot RNAi. FOXO or phm knockdown rescued ovarian maturation and egg production decreased by pot silence, which inhibited vitellogenin1 (Vg1) and Vg2 expression via FOXO. These results indicate that Pot orchestrates development and reproduction via a positive hormone feedback axis. This study comprehensively elucidates the mechanism of ZP-domain protein regulating development and reproduction in insects.
Mimicry, a complex visual defensive strategy, shapes ecological dynamics and evolution by influencing predator-prey interactions. Insect mimicry of plant structures drives their co-evolution with plants. However, fossil evidence of insect-angiosperm mimicry is exceedingly scarce and has previously been confined to the Cenozoic, although the occurrence of angiosperms extends far earlier. Here, the earliest known insect mimicking angiosperm leaves, Dryadithone cretacea gen. et sp. nov. (Neuroptera: Ithonidae), is reported from the mid-Cretaceous Kachin amber. The sophisticated camouflage of this new species, achieved by mimicking the midvein, pinnate framework, and reticulate venation of angiosperm leaves through its forewing shape and venation, was further validated by the Siamese Neural Network. This mimicry deceived a wider range of visual predators and compensated for the disadvantages from large body size. Dryadithone cretacea extends the earliest record of insects mimicking angiosperms to the mid-Cretaceous, shedding light on complex insect behaviors and the intricate ecological interactions among plants, insects, and higher trophic-level predators during this period of flowering-plant proliferation.
The olfactory system is pivotal for insects to detect external chemical signals and regulate essential life processes. Dendroctonus valens, an invasive forest pest, displays strong chemotaxis toward the plant kairomone (+)-3-carene. To unravel the molecular mechanism underlying olfactory recognition, we identified 28 odorant-binding proteins (OBPs) from D. valens via transcriptome sequencing and identified DvalOBP6 and DvalOBP18 as potential key candidates for this host kairomone component using qRT-PCR. Tissue expression analysis revealed that these two Minus-C OBPs are predominantly expressed in olfactory-related tissues (antennae, legs, and wings) with distinct sexual dimorphism. Homology modeling and molecular docking showed that both proteins adopt a typical six-α-helix fold, and bind (+)-3-carene primarily via hydrophobic interactions with binding energies of -5.53 kcal/mol and -4.96 kcal/mol, respectively. RNA interference of DvalOBP6 or DvalOBP18 significantly abolished the olfactory preference of D. valens adults for (+)-3-carene. Collectively, our findings demonstrate that DvalOBP6 and DvalOBP18 play critical roles in (+)-3-carene perception, providing a theoretical basis for the development of green pest control technologies targeting insect olfactory communication.
This study presents a comprehensive three-dimensional anatomical atlas of the adult Nilaparvata lugens using micro-CT and FIB-SEM. The reconstructions reveal the spatial organization of the flight muscle system, digestive tract, reproductive organs, and nervous system. The indirect flight muscles, including dorsal longitudinal and dorsoventral muscles, are structurally similar between sexes but show size differences in certain components. The female reproductive system occupies most of the abdominal cavity, reflecting high fecundity, while the male reproductive system features a specialized ejaculatory duct associated with muscular control. Notably, the genital coupling during copulation involves a sophisticated interlocking structure, ensuring stable alignment and preventing separation. These structural insights offer a holistic framework for understanding dispersal and reproduction in N. lugens, with implications for developing novel pest management strategies.
The tomato leafminer, Tuta absoluta, poses a severe threat to tomato production in China, yet regional resistance patterns remain poorly characterized. This study investigates the insecticide resistance status and underlying molecular mechanisms in field populations of T. absoluta across major tomato-producing regions in China. Ten populations were evaluated for susceptibility to seven commonly used insecticides using standardized bioassays, alongside screening for resistance-associated target-site mutations in key genes. The results indicate that most populations remain susceptible to spinetoram, abamectin, and indoxacarb, supporting their continued effectiveness in field control. However, moderate resistance to emamectin benzoate was detected in several populations, with resistance ratios reaching up to 75.09 fold. Additionally, low to moderate resistance to Bacillus thuringiensis (Bt) and broflanilide was observed in certain regions. Although resistance ratios to bifenthrin were relatively low, consistently high LC50 values suggest a widespread reduction in baseline susceptibility. Molecular analyses revealed that the L1014F mutation in the voltage-gated sodium channel (VGSC) gene was fixed across all populations, while M918T and T929I mutations occurred at high frequencies. Notably, no significant correlation was found between these mutations and phenotypic resistance levels, and no resistance-associated mutations were detected in the nAChR gene. These findings highlight the complex and potentially multifactorial nature of resistance in Chinese T. absoluta populations and provide important guidance for optimizing insecticide rotation and resistance management strategies.
Transient dopaminergic signaling activation in the honeybee brain regulates food wanting and drives foraging behavior. However, the roles of endogenous microRNAs (miRNAs) in the regulation of food wanting by dopamine pathways remain poorly understood. Here, we report that miR-375-3p plays a critical role in the food-wanting system in honeybees. MicroRNA transcriptomic analysis revealed the marked downregulation of miR-375-3p expression in the brains of starved foragers, which exhibited increased appetitive responsiveness. Furthermore, miR-375-3p directly targeted DOPA decarboxylase (Ddc), a crucial enzyme in dopamine synthesis, thereby decreasing dopamine levels. Overexpression of miR-375-3p or Ddc RNA interference reduced dopamine production and suppressed food wanting in honeybees. Collectively, the results indicate that miR-375-3p acts as a negative regulator of food wanting by inhibiting Ddc expression and reducing dopamine-mediated appetitive behavior. This study reveals a molecular mechanism underlying miRNA-regulated food wanting and suggests potential strategies for miRNA-based intervention in managing honeybee health.
The remarkable host adaptability of Hyphantria cunea contributes to its invasiveness and destructive impact. This study investigates the molecular mechanisms underlying the multi-host adaptability in H. cunea larvae, focusing on UDP-glucuronosyltransferases (UGT). The results revealed a significant increase in UGT enzyme content in H. cunea larvae feeding on low-preference host plants compared to those on high-preference hosts. UGT inhibitor treatment markedly reduced larval body weight and food intake across all host plant groups. Additionally, qPCR analysis indicated distinct expression patterns of UGT family genes in larvae feeding on different host plants. Notably, HcUGT40R20 was highly expressed specifically in larvae consuming intermediate- and low-preference hosts. Functional analysis showed that silencing HcUGT40R20 led to a significant decrease in larval body weight and disrupted the expression of genes involved in energy metabolism and growth regulation across all host plant groups, along with a marked increase in larval mortality in intermediate- and low-preference hosts. Moreover, the bio-toxicity of the host plant defensive compounds, cytisine and coumarin, was significantly reduced in transgenic Drosophila and Sf9 cells overexpressing HcUGT40R20. Furthermore, an RNAi-based formulation, CS-dsHcUGT40R20, was successfully developed using chitosan (CS) nanomaterials and dsRNA, demonstrating stability in the gut fluid of H. cunea. CS-dsHcUGT40R20 treatment significantly reduced the body weight of larvae fed on different host plants and markedly increased the mortality rate of larvae under coumarin stress. In conclusion, HcUGT40R20 plays a critical role in the multi-host adaptability of H. cunea, and RNAi-based formulation targeting HcUGT40R20 provide an effective strategy for controlling H. cunea.
During biparental pre- and post-hatching care, parents take on energy-consuming tasks for the offspring's benefit and further reduce their own individual costs by specializing in different care aspects. But how is biparental care evolutionarily stable when biparental care is facultative, that is, when offspring survival does not obligately rely on post-hatching care? We examine this phenomenon in the carrion-breeding beetle Nicrophorus vespilloides, whose facultative biparental care involves microbiome control of the carcass nursery through continued application of antimicrobial exudates, shielding offspring from adverse environmental conditions. While evidence suggests synergistic effects of biparental care in Nicrophorus, any adaptive benefits in terms of social immunity are unknown in this genus. We presented Nicrophorus adults with a microbial challenge while manipulating parental care patterns during the period of post-hatching care, investigating consequences in parent and offspring performance. We found that microbial environment and parental care pattern influence larval development and survival. Additionally, we show for the first time that both factors affect personal immunity response in Nicrophorus offspring, responding to challenging conditions. Simultaneously, we show that biparentally caring beetles lose more weight during post-hatching care than uniparentally caring beetles, indicating higher investment and/or higher competition with mates or offspring. We present new evidence that burying beetle offspring adjust their personal immunity based on their microbial and social environment, and that biparental care may allow parents to sustain parental care under challenging conditions, raising further questions about the interplay of care patterns and the microbial environment on immune-regulatory and developmental processes in offspring.
The beet leafhopper (BLH), Neoaliturus tenellus, is a major agricultural pest and vector of beet curly top virus (BCTV), a pathogen responsible for severe economic losses in vegetable and field crops in the Western U.S. Despite its economic importance, the genetic basis of BLH survival, reproduction, and virus-vector interactions remains unexplored mainly due to the lack of functional genomics tools in this species. In this study, we assessed the feasibility of RNA interference (RNAi) in BLHs by targeting two genes, protein gustavus isoform X2 (Gus) and pumilio homolog 3 (Pum3), which were previously identified as differentially expressed following BCTV infection. Double-stranded RNA (dsRNA) targeting conserved domains of each gene was delivered via microinjection or star polycation nanoparticle-mediated topical spray. Our results show that microinjection of dsRNA resulted in >90% knockdown of both Gus and Pum3 by 5 days post-treatment. Silencing Pum3 significantly reduced BLH survival and fecundity, whereas silencing Gus reduced fecundity without affecting adult survival, suggesting gene-specific fitness consequences. Nanoparticle-mediated dsRNA spray resulted in 88% reduction in Gus and 62% reduction in Pum3 expression, yet both produced biological effects on survival and/or reproduction, comparable to those observed with microinjection. To our knowledge, this study provides the first demonstration of the effectiveness of microinjection and nanoparticle-mediated RNAi in the BLH. Together, these findings demonstrate the critical roles of Gus and Pum3 in vector fitness and establish microinjection and nanoparticle-based dsRNA spray as robust platforms for functional genomics and developing potential scalable, non-invasive RNAi strategies for sustainable vector and disease management.
Megalurothrips usitatus (bean flower thrips) has transitioned from a tropical Asian pest into a global threat to legumes, particularly cowpea (Vigna unguiculata). Driven by climate warming and trade, its recent expansion across the Americas causes 20%-30% yield losses and sometimes leads to crop failure. Heavy reliance on chemical control has rapidly selected for multi-class insecticide resistance. This review synthesizes current research on the pest's biological traits, invasion ecology, cryptic diversity, and the molecular mechanisms driving resistance to spinosyns, neonicotinoids, and pyrethroids. Our review also evaluates integrated pest management strategies, ranging from cultural controls to RNAi biotechnologies, while addressing the critical challenges of laboratory-to-field translation. Finally, we propose key research priorities, including intelligent monitoring, localized resistance management, and synergistic control packages, to provide a sustainable framework for global legume protection and biosecurity policy.
