Apis mellifera filamentous virus (AmFV) is an emerging DNA virus significantly affecting honey bee health. AmFV infections weaken bee resistance to other pathogens, and can cause tissue lysis and death. Early, accurate detection of AmFV is crucial for timely intervention and preventing large-scale outbreaks. Current AmFV detection relies largely on polymerase chain reaction (PCR)-based methods. To enable rapid field detection of AmFV, we developed a rapid and ultrasensitive detection platform using recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 12a (Cas12a) technology. A CRISPR RNA (crRNA1) specifically targeting the AmFV Bro gene was designed, ensuring no cross-reactivity with other insect DNA viruses or uninfected honey bees. After optimization of the reaction time, the platform generated results within 35 min: 20 min for the RPA reaction and 15 min for CRISPR-mediated cleavage. Two visualization approaches, fluorescence-based and lateral flow dipstick, were used to display the detection results. The detection sensitivity of both approaches was as few as 10 copies of the AmFV genome. Validation with field-collected honey bee samples demonstrated consistency with conventional PCR, revealing widespread latent AmFV infections in the field. Taken together, we successfully developed an RPA-CRISPR/Cas12 platform for rapid, specific, and sensitive detection of AmFV in Apis mellifera and Apis cerana. This platform holds promise as a simple, accurate, and cost-effective tool for point-of-care AmFV diagnosis in the field.

Tissue growth in Drosophila is regulated by various factors, with microRNAs (miRNAs) emerging as key players over the past decade. However, the precise roles of miRNAs in growth regulation remain incompletely understood. In this study, we explored the biological role of miR-315 in wing growth regulation. Inhibition of miR-315-5p activity using a miR-315 sponge led to an increase in wing size, whereas its overexpression resulted in reduced wing size, primarily through a decrease in wing cell size. We identified ribosomal protein kinase p-70-S6k (S6k) as a target of miR-315-5p in relation to wing growth control. Overexpression of miR-315 reduced both total S6k and phosphorylated S6k protein levels in Drosophila S2 cells and wing discs. Additionally, a luciferase reporter assay confirmed that miR-315-5p directly binds to the 3′-untranslated region of S6k. Consistently, RNAi-mediated depletion of S6k led to smaller wings, primarily due to a reduction in cell size. Notably, co-overexpression of active S6k rescued the wing defects caused by miR-315 overexpression. Overall, these findings demonstrate that miR-315 regulates wing growth by suppressing S6k expression.

The basic helix–loop–helix (bHLH) domain transcription factors precisely regulate various developmental processes in insects. Dimm, a specific bHLH transcription factor, integrates the insulin/insulin-like growth factor signaling (IIS) and juvenile hormone signaling (JHS) pathways to modulate larval development in silkworms. However, the molecular mechanisms underlying this regulation are not yet fully understood. This study aimed to determine the targets of Dimm through which it regulates larval development. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed ganglioside-induced differentiation-associated protein 2 (GDAP2) as a direct downstream target gene of Dimm. Further study showed that Dimm directly binds to an enhancer element located in the second intron of the GDAP2 gene to promote its transcription. GDAP2 exhibited widespread expression across different stages and tissues of silkworms, regulated by both the IIS and the JHS pathways. The systemic knockout of GDAP2 leads to delayed larval development with a significant reduction in body weight; moreover, larval development was arrested at the 4th-instar stage. Further investigation unveiled that the inhibition of the ecdysone and innate immune signaling pathways in the mutant line led to abnormal larval development. A systematic investigation of the biological functions of GDAP2 offers valuable insights into the mechanism by which Dimm integrates IIS and JHS pathways to regulate the larval development of silkworms.

Endoreplication plays a crucial role in the morphogenesis of various organs. The silk gland of Bombyx mori, which produces silk after maturation, engages in a unique cell cycle transition, mitosis-to-endoreplication, in the early stages of formation. This plays an essential role in the secretion of silk protein from silk gland cells. In this study, we identified and analyzed the expression characteristics of BmCdc25. The functional domain of BmCdc25 is conserved across species. BmCdc25 is mainly expressed in the G2/M phase of the cell cycle, and is expressed at low levels in the silk gland during active endoreplication. Overexpression of BmCdc25 enhanced cell proliferation, while inhibition of BmCdc25 caused cells to accumulate in the G2/M phase, thereby inhibiting cell proliferation. Additionally, BmCdc25 overexpression prevented DNA replication during silk gland cell endoreplication, while BmCdc25 inhibition had the opposite effect. BmZFP67 was confirmed to regulate BmCdc25 transcription by chromatin immunoprecipitation and electrophoretic mobility shift assays, while aberrant cytokinesis caused by knockout of BmZFP67 was shown to involve BmCdc25. Our findings expand the regulatory network of BmZFP67 from the perspective of transcriptional regulation and protein interaction and provide insight into the roles of BmCdc25 in the silkworm cell cycle.

Silkworm silk gland cells undergo multiple rounds of endoreplication, a process in which the genome is duplicated without cell division, leading to cellular polyploidization. This results in the accumulation of genomic DNA, serving as the foundation for rapid silk proteins synthesis. For the first time, we report a previously uncharacterized gene, SGDAcn, in the silkworm silk gland that clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9-mediated SGDAcn knockout in the posterior silk gland increased cell size and enhanced silk production. SGDAcn knockout facilitated the progression of endoreplication by upregulating the expression of various cyclin genes and promoting energy metabolism, leading to a substantial increase in fibroin gene expression and its transcription factor Dimm, as well as the stimulation of ribosome biogenesis for messenger RNA translation and enhancement of eukaryotic translation initiation factors for protein synthesis. Our findings demonstrate that SGDAcn influences endoreplication, cell growth, and nucleolus size through SGDAcn-EGFR/PI3K/AKT and SGDAcn-NF-κB signaling pathways. Overall, SGDAcn acts as a negative regulator of silk gland development, affecting cell size and protein synthesis, thus modulating silk production. These mechanisms might be conserved in cell growth and progression, making an attractive target for genetic editing to improve silk yield in silkworms and potentially in mammalian cell growth regulation.

LIM-HD (homeodomain) protein is essential for the specific differentiation of various cells and tissues due to its conserved HD. Within the LIM subclass, the LIM-HD transcription factor 1 alpha (Lmx1a) remains poorly characterized in insects. This study investigates the expression patterns of HvLmx1a in the 28-spotted lady beetle, Henosepilachna vigintioctopunctata, at both developmental stage and tissue levels. Furthermore, RNA interference (RNAi)-mediated knockdown of HvLmx1a resulted in increased mortality during the early larval stage. Injection of dsHvLmx1a at the beginning of the 4th instar caused a reduction in 20E titer, disrupted normal molting and pupation processes, and led to the emergence of larval-pupal abnormalities. Notably, HvLmx1a expression was significantly down-regulated on d 2 and 4 post-injection, which coincided with a marked inhibition of HvCHS1 and genes associated with 20E and bursicon signaling pathways. Additionally, silencing HvLmx1a induced qualitative changes in male testes and female ovaries, resulting in infertility and increased mortality. Ultimately, these findings suggest that HvLmx1a influences ovarian morphology and development through lipid metabolism. In conclusion, this study provides the first evidence of the diverse physiological roles of Lmx1a in H. vigintioctopunctata and highlight its potential as a target for RNAi-based biological control strategies.

Insect pigmentation patterns are critical for ecological adaptation and serve as an excellent model for studying the genetic basis of phenotypic diversity. The Wnt family is a conserved group of genes that play crucial roles in various biological processes across species. While Wnt1 and WntA have been extensively characterized for their roles in insect coloration, particularly in Drosophila and butterflies, the functions of other Wnt family members, such as Wnt6 and Wnt10, remain poorly understood. These genes originated from a common ancestor with Wnt1 and are arranged in tandem with it in the genome. In this study, we investigated the roles of Wnt6 and Wnt10 in spot formation in silkworm larvae using 3 spot-marking mutants with similar phenotypes. Through gene expression analysis, RNA interference, and overexpression studies, we discovered that Wnt1, Wnt6, and Wnt10 each regulate spot formation via the Armadillo-dependent canonical Wnt pathway. This highlights their distinct and nonredundant functional attributes despite their common origin and genomic arrangement. Additionally, we identified a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) gene contributes to caterpillar pigmentation related to the canonical Wnt pathway. Our findings underscore the complexity and conservation of the Wnt signaling pathway in insect coloration.

Leptinotarsa decemlineata (Say) (Colorado potato beetle, CPB) is a notorious pest of potatoes. Its hardened forewings, known as elytra, protect the hindwings and the abdomen against enemies and chemical pesticides. Ubx plays a crucial role in differentiating insect wings, but the molecular mechanism remains unclear. Our research showed that Ubx is highly expressed in the T3 thoracic segment (T3) and appendages, while Ubx is absent in the forewings. RNA interference for Ubx resulted in the dorsal plate of the T3 being shifted toward the T2, hindwings were sheathed from the membranous in adults, and forewings were curling. The hindwing development-related genes downstream of Ubx, including LdDpp, LdIro, LdVg, LdWg, and LdASH, were significantly up-regulated in Ubx-inhibited insects compared with the control, by 2.40, 1.88, 1.81, 1.89, and 2.81 times, respectively. After feeding 20-hydrosyecdysone (20E), the forewing was flatter and more stretched than the control, leading to increased wing area, which indicates Ubx regulation is associated with the feedback of ecdysteroids. The expression levels of 3 ecdysteroid receptor genes EcR, EcRA, and EcRB were significantly lower in knocking down LdUbx insects than the control and was able to be recovered by 20E feeding, suggesting that ecdysteroid is associated with LdUbx-regulated wing development. Cosilencing LdUbx and LdAkt genes significantly inhibited the growth of forewings and hindwings, reducing the area of the wings. Together, our findings suggested that LdUbx regulates wing development along with ecdysteroid and insulin-like peptide signaling pathways, which provides a novel molecular mechanism of wing development and differentiation in insects.

Insects can effortlessly walk on various substrate surfaces using attachment pads on their feet, even upside down on smooth surfaces. These pads undergo frequent deformation throughout the lifecycle of insects to achieve stable adhesion. Interest in insect adhesion spans 100s of years; studies have indicated the special mechanical properties and intricate structural design of insect attachment pads. However, the genetic basis of their function remains unclear. Resilin, an elastic protein widely distributed in insect exoskeletons, plays a crucial role in many physiological activities. Here, we identified a resilin-like protein in the Lepidopteran insect Bombyx mori and generated mutants for this gene. The resulting adults exhibited “slipping” phenotype and reduced attachment ability. Further behavioral investigations, microscopic observations and mechanical performance tests, confirmed that the mutation stiffened the attachment pads, reducing flexibility and effective contact area, which ultimately led to decreased adhesive ability in adults. Molecular-level analyses revealed that the resilin-like mutation led to differential expression of melanin metabolism-related genes, potentially explaining the hardening and abnormal pigmentation of the attachment pads. Our results provide genetic and phenotypic evidence demonstrating the significant role of resilin in insect attachment.

Silkworm is a typical monophagous insect that can only feed on fresh mulberry leaves. The mechanism for this monophagous nature is not fully understood. One bitter gustatory receptor (GR) GR66 located on the maxilla of the mouthpart has been reported to be an important factor influencing the feeding preference of silkworm. However, the preference of GR66 mutants for a nonhost plant was very low, suggesting that other factors related to silkworm monophagy need to be further explored. In this study, 10 bitter GRs were screened out based on their specific high expression in the maxilla of silkworm, and the 4 most expressed GRs (GR15, GR43, GR69, and GR66) were knocked out by clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9. Feeding experiments showed that except for GR69^−/−, the host range of GR43^−/−, GR15^−/−, and GR66^−/− expanded remarkably, and both GR43^−/− and GR66^−/− revealed the highest preference for the nonhost plants. Moreover, the strict preference for mulberry leaves was almost eliminated in the double mutant silkworms of GR43^−/− and GR66^−/−. These results imply that it is likely that a variety of different bitter GRs are involved in determining the monophagy of silkworm larvae. In addition, GR15 mutation significantly inhibited ovarian development, resulting in a significant decrease in oviposition. These findings enhance our understanding of the monophagous nature of silkworms and provide a possibility for the molecular breeding of polyphagous silkworms in the future.

Insects possess an advanced olfactory system capable of detecting a wide range of odors through seven-transmembrane olfactory receptors (ORs). These ORs form heteromeric complexes with olfactory receptor co-receptor, Orco, and upon binding to specific ligands, they trigger the intracellular influx of ions such as sodium and calcium. Identifying ORs that respond to chemical molecules released from explosives holds significant importance for the development of biosensors for security and humanitarian purposes. In this study, screening of 196 mosquito ORs in HEK293FT cells for intracellular calcium flux on nitro compound administrations identified ORs as sensors for 2,4-dinitrotoluene, 2-nitroaniline, 2,3-dinitrotoluene, 2,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene. The different odor response profiles exhibited by naturally occurring polymorphisms or indels in the single OR gene that we had cloned were also explored. Sequence comparisons on these natural genetic variations and heterologous expression of each variant resulted in the identification of the amino acid positions involved critically in the gain and loss of odor sensitivity. Furthermore, we found that various combinations of the identified positions and different amino acid residues artificially evolve the OR with a higher sensitivity to nitro compounds. Our findings pave the way for the development of high-performance explosive detection biosensors, significantly contributing to technological advancements in landmine clearance and other areas. Additionally, our established screening system suggests the potential for identifying insect ORs that could serve as elements for various biosensors beyond explosive detection.

As a Toll receptor ligand, Spätzle (Spz) plays a crucial role in activating the Toll pathway and participating in the innate immune response of insects. However, the immune function of Spz in Myzus persicae remains poorly understood. In this study, we identified and cloned 7 Spz genes from M. persicae, all containing a Spz domain (cystine-knot domain). Phylogenetic analysis revealed that the 7 different MpSpzs were divided into 6 groups within a single cluster with each Spz of Drosophila melanogaster and Acyrthosiphon pisum. These genes were mainly expressed in 1st-instar nymphs, hemolymph, and embryos and showed varying levels of positive response to infection with Escherichia coli, Staphylococcus aureus, and Beauveria bassiana. After gene-silencing of MpSpzs by RNA interference with injection of target gene-specific double-stranded RNA, microbial infection significantly increased the mortality of M. persicae compared to control groups. Further studies revealed that the suppression of MpSpz resulted in a significant reduction in lysozyme expression. The present study offers novel insights into the role of Spätzle in the innate immune response against microbial infection in M. persicae.

The presence of microsporidian infections in the animal industry could result in substantial economic losses. Nosema bombycis, as the first identified species of microsporidia, poses a significant threat to the silkworm industry. Currently, there is no strain of silkworm with obvious resistance that can inhibit the proliferation of N. bombycis in silkworm rearing. In this study, we developed a microsporidia-inducible GAL4/UAS-RTA (Ricin toxin A chain) system in silkworms that confers resistance against N. bombycis. This system utilizes the microsporidia-inducible promoters of BmUGT2 and BmUGT3 genes (PUGT2 and PUGT3) to drive the expression of GAL4 gene, while RTA is driven by a UAS cis-acting element. We generated hybrid silkworms through crosses between GAL4 transgenic silkworms (PUGT2-GAL4 or PUGT3-GAL4) and UAS-RTA transgenic silkworms. Under normal conditions, these hybrid lines exhibited unaltered fundamental economic characteristics compared to wild-type silkworms. However, when exposed to N. bombycis infection, they displayed significantly enhanced resistance against microsporidia. Our research successfully demonstrated mitigation of microsporidia proliferation in transgenic individuals using the microsporidia-inducible GAL4/UAS-RTA system in silkworms. This approach not only provides a novel strategy for developing resistant strains against microsporidia but also serves as an important reference for genetically enhancing resistance against intracellular pathogens in other economically significant insects.

Insects have evolved a diversity of regulatory mechanisms to determine their sex. Understanding the molecular regulation mechanisms of insect sex determination is of great significance in revealing the general law of insect sex determination and providing potential routes for the genetic manipulation of pest species. Although the sex determination cascade and doublesex (dsx) gene functions have been well described in some holometabolous insects, little is known about this cascade in hemimetabolous insects. In this study, we identified the dsx homolog in Gryllus bimaculatus, which belongs to the Orthoptera order and is an important model for developmental and evolutionary biology. We found that Gbdsx has two alternative splicing isoforms (male-specific Gbdsx^M and non-sex-specific Gbdsx^C). Using RNAi-mediated knock-down of Gbdsx^M in 6th-instar nymphs resulted in adult male forewings showing feminized vein development and abnormal external genitalia. CRISPR/Cas9 knockout of Gbdsx in embryos resulted in adult males becoming pseudofemales, with feminized forewings and abnormal external and internal genitalia. Additionally, the pseudofemales created by Gbdsx knockout demonstrated normal courtship trends and aggressive behavior but no actual mating behavior. However, the knockout and knock-down of Gbdsx in female crickets does not affect their sexual traits or fertility. Our results suggest that Gbdsx^M plays a critical role in the development of male cricket sexual traits, courtship and mating behavior, which furthers our understanding of sex determination in hemimetabolous insects.

The ovary plays a crucial role in the female reproductive system of insects and is vital for the perpetuation of insect populations. The insulin/insulin-like growth factor signaling (IIS) and target of rapamycin (TOR) signaling network play a key role in controlling ovarian development and maturation during the adult stage. However, the pupal stage is a critical developmental phase in the reproductive system of holometabolous insects, and the regulatory role of the IIS/TOR network during this stage remains to be elucidated. In this study, we observed that during the pupal stage, the enlargement of ovarian development was accompanied by a corresponding increase in messenger RNA expression levels of genes within the IIS/TOR signaling pathway and insulin levels in the hemolymph. Decapitation experiments, serving as a pivotal approach to assess the regulatory role of the IIS signaling pathway in ovarian development, demonstrated that decapitation markedly inhibited ovarian development at this critical stage. Exogenous insulin administration significantly increased the phosphorylation levels of FOXO, S6K, and 4E-BP, thereby enhancing ovarian development and resulting in significant elongation of the ovarioles. In contrast, the injection of IIS/TOR pathway inhibitors, LY294002 and Rapamycin, reduced the phosphorylation of these proteins, inhibiting ovarian development during the pupal stage and leading to a significant shortening of the ovarioles, negatively impacting the reproductive performance of adult females. The current findings indicate that the IIS/TOR signaling network significantly regulates ovarian development during the pupal stage of Spodoptera frugiperda, providing new molecular insights for the development of pest management strategies.

Fatty acids are important intermediates in lipid metabolic pathways, and fatty acid metabolism is a complex process. As one of the hormones regulating insect lipid metabolism, the precise regulatory mechanisms of 20-hydroxyecdysone (20E), particularly its relationship with fatty acid metabolism, remain unclear. Using the rice pest Chilo suppressalis as a model, we investigated the effects of 20E on fatty acid metabolism in the fat body during the pupal stage. Our study found that high levels of 20E disrupts normal fat body development during the pupal stage, resulting in reduced expression of lipid metabolism-related genes. Lipidomic analysis revealed that 20E induces fatty acid accumulation in the fat body. Transcriptomic analysis indicates that differentially expressed genes are enriched in the fatty acid synthesis pathway. These genes are highly expressed during the late pupal stage, which correlates positively with the development of C. suppressalis female pupae. After knocking down C. suppressalis fatty acid synthase (CsFAS) and C. suppressalis fatty acyl-CoA reductase (CsFAR), we observed increased levels of triglyceride and free fatty acids in the fat body, along with evidence that 20E inhibits the expression levels of CsFAS and CsFAR. Our study indicates that 20E regulates lipid metabolism during the pupal stage by suppressing the transcription levels of key metabolic genes, CsFAS and CsFAR, in the fatty acid metabolism pathway. This helps to better understand how hormones regulate lipid metabolism in the insect fat body and suggests that the discovery of FAS and FAR may provide new molecular targets for pest control.

The eukaryotic translation initiation factor 4E binding protein (4EBP) represses protein translation as a nonphosphorylated form by interacting with the eukaryotic translation initiation factor 4E (eIF4E). However, the upstream regulator of 4EBP is unclear. Using the major agricultural pest, Helicoverpa armigera, cotton bollworm, as a research model, we observed higher expression levels of 4Ebp at the 5th instar molting stage and metamorphosis. 20-hydroxyecdysone (20E) significantly upregulated the expression of 4Ebp and inhibited its phosphorylation. 20E promoted the binding of ecdysone receptor B (EcRB) to the ecdysone response element (EcRE) in the promoter sequence of 4Ebp, thus facilitating 4Ebp transcription. Knocked down 4Ebp by RNA interference caused abnormal molting of the 5th instar larvae, and overexpression of 4EBP in the H. armigera epidermal cell line inhibited cell proliferation. These data suggested that 20E repressed cell proliferation temporarily by upregulating 4Ebp expression level and inhibiting its phosphorylation, which is necessary for insect larval molting.

Apolipophorin-III (ApoLp-III), a multifunctional protein with lipid transport and immune defense functions, widely exists in insects. Although the function of ApoLp-III as a pattern recognition receptor (PRR) in immunity has been relatively studied, the immune response mediated by ApoLp-III is still vague. To understand whether ApoLp-III is involved in the activation of the prophenoloxidase-activating system (PPO-AS), we examined the production of nitric oxide (NO), and the synthesis of antimicrobial peptides after immune recognition. The larvae of lepidopteran pest Ostrinia furnacalis were used as a model to address these questions by detecting the changes of phenoloxidase (PO) activity and NO concentration after the knockdown of OfApoLp-III and bacterial infections. In the present study, we reported the cloning and characterization of the OfApoLp-III complementary DNA, and found that OfApoLp-III is mainly expressed in the larval fat body. These investigations revealed that OfApoLp-III was an immune-related gene, its knockdown reduced the PO activity by 41.9%, and NO concentration reached 2.7-fold higher level than that after double-stranded GFP treatment. Our data indicated that OfApoLp-III was involved in increased expression of Moricin, activation of PPO, and reduction of NO production in O. furnacalis larvae after different bacterial infections, which were required for innate immunity. ApoLp-III is a candidate target for an integrated pest control strategy using the combined application of double-stranded RNA and biocontrol bacteria.

Transglutaminase (TGase) is a Ca²⁺-dependent acyltransferase that catalyzes protein cross-linking. We previously reported that the TGases of Mythimna separata, that is, MysTGase1 and MysTGase2, contribute to insect immune responses against microbial invaders. In this study, we aimed to elucidate the involvement of MysTGases in the encapsulation immune responses that target large invaders such as parasitoid eggs and nematodes. The expression levels of mystgases in hemocytes forming capsules around beads and nematodes were significantly higher than those in circulating hemocytes and increased in a size-dependent manner with increasing bead size. The expression of both mystgases was detected in plasmatocytes and granulocytes, which are the capsule components. RNA interference targeting mystgase1 or mystgase2 led to a significant decrease in the encapsulation rate of the beads, whereas treatment with recombinant MysTGases had the opposite effect. Furthermore, the knockdown of mystgase2 inhibited plasmatocyte spreading, whereas mystgase1 did not. This study highlights the potential significance of TGase in the encapsulation process and presents a notable molecular discovery of the encapsulation mechanism. This study also provides a scientific foundation for future research on insect immunity and may contribute to the development of innovative pest control strategies.

The Asian citrus psyllid (ACP), Diaphorina citri, serves as the primary vector for Candidatus Liberibacter asiaticus (CLas), the pathogen responsible for citrus Huanglongbing (HLB). D. citri modulates the expression of its key proteins in response to CLas infection. Previous research has revealed that CLas infection significantly alters the expression levels of E3 ubiquitin ligases in D. citri; however, the specific functions of these E3 ligases remain largely uncharacterized. In this study, a total of 11 E3 ubiquitin ligases were identified from the proteomics database of D. citri, among which E3 ubiquitin ligase RNF115 was significantly upregulated following CLas infection. RING finger protein 115 (RNF115) consists of 156 amino acids and contains a RING finger domain at its N-terminus. Silencing RNF115 via RNA interference (RNAi) and injecting the inhibitor disulfiram, which targets RNF115, significantly increased CLas bacterial content in D. citri. In contrast, injection of recombinant RNF115 protein markedly inhibited CLas bacterial proliferation. Furthermore, interaction between RNF115 and D. citri histone H1 was confirmed using yeast 2-hybrid assay, pull-down experiments and molecular docking analysis. Knockdown of histone H1 via RNAi significantly reduced CLas bacterial content, whereas injection of recombinant histone H1 protein led to an increase in CLas content within D. citri. These findings suggest that CLas infection may induce an upregulation of RNF115 expression in D. citri, leading to subsequent interactions with histone H1 that facilitate the ubiquitination of histone H1, ultimately resulting in reduced expression levels and inhibiting CLas proliferation within D. citri.

Non-immune defenses against pathogens and parasites are of particular importance for animals that have incomplete immune systems, such as aphids. Production of winged offspring that are able to fly away from danger in response to infection is one of the non-immune defensing strategies. However, the signaling mediating winged offspring production induced by infection is largely unknown so far. In this study, we found that the pea aphids Acyrthosiphon pisum produced more winged offspring when they were challenged by Micrococcus luteus and Beauveria bassiana. The target of rapamycin (TOR) pathway was up-regulated after infections, and inhibition of TOR activity or knockdown of TOR expression resulted in fewer winged offspring produced by the infected aphids. Similarly, inhibition of Jun N-terminal kinase (JNK) or knockdown of JNK expression had same effect. We further provide evidence showing that JNK regulates the TOR pathway. Taken together, our study suggests the JNK-TOR signaling pathway mediates induction of winged offspring as a non-immune response to infection in aphids.

Migratory insects are capable of long-distance flight and strong fecundity, but often have finite amounts of resources available for these energy-demanding traits. Although the trade-off between flight and reproduction has been reported in migratory insects, the optimal timing of flight to reproduction transition remains largely unknown. Here, using the gregarious phase of migratory locust Locusta migratoria, we report that 4-d-old adult females possessed the strongest flight capacity in the first gonadotrophic cycle. Tethered flight assays demonstrated that the timing point between ending of previtellogenesis and beginning of vitellogenesis, when vitellogenin (Vg) was not yet massively synthesized, was optimal for locust flight. Transcriptome and metabolome analyses showed that glycogen and triglyceride were primarily synthesized in the fat body of adult females during previtellogenic stage. Sustained flight of adult females significantly reduced Vg expression levels accompanied by blocked oocyte growth, prolonged preoviposition period and declined egg number. In addition, long-term flight led to significantly reduced expression of juvenile hormone (JH) synthesis genes JHAMT, HMGR, and allatotropin, but not JH metabolism genes JHE and JHEH. Application of JH mimic to JH-deprived 4-d-old adult females at a lower dose was conducive to flight. In contrast, JH administration at higher doses stimulated vitellogenesis and egg production but suppressed flight capacity. Our results suggest that JH along with energy metabolism regulate the optimal timing of flight to reproduction switch in adult females of migratory locust. The findings shed new light on the regulation of trade-off between flight and reproduction, as well as the sustainable control of migratory locusts.

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a serious invasive pest, which has attracted concern regarding the effectiveness of environmental bioinsecticide as a substitution for synthetic insecticide in controlling its damage to numerous agricultural crops in recent years. Hence, laboratory and field experiments have investigated insecticidal toxicity in S. frugiperda by 5 insecticides and have determined the activity of acetylcholinesterase (AChE) and detoxifying enzymes, namely mixed function oxidase (MFO), carboxylesterase (CarE), and glutathione S-transferase (GST) on 3rd instar larvae at 25% lethal concentration (LC₂₅) to explore detoxification mechanisms. The results showed that the most effective insecticides were 3.0% emamectin benzoate (0.024 mg/L), 60 g/L spinetoram (0.282 mg/L), and 1.3% matrine (0.380 mg/L) at 48 h depending on LC₅₀ values. The field efficiencies of emamectin benzoate and spinetoram were over 80% at 24 h and 90% at 72 h, indicating that both insecticides had acute and long-lasting toxicity on S. frugiperda; matrine had extended-release toxicity with 90% field efficiency at 72 h. Emamectin benzoate and spinetoram activated the activities of AChE, MFO and GST, and spinetoram decreased in CarE activity; cyantraniliprole induced an increase of 4 detoxifying enzymes; spinosad increased the activities of AChE, MFO, and CarE but not GST. Furthermore, matrine had an inhibiting effect on AChE and acceleration on CarE and GST. Overall results obviously depicted that semi-synthetic insecticide spinetoram and bioinsecticide matrine were recommended to control S. frugiperda with effective and long-lasting toxicity. Moreover, this study will provide basic information for sustainable control of S. frugiperda under field conditions in China.

Fungus-farming termites efficiently degrade recalcitrant lignocellulose through a symbiotic relationship with Termitomyces and the gut microbiome, making them successful key decomposers in (sub)tropical ecosystems. Despite extensive research on plant biomass decomposition, the mechanisms of lignin degradation in fungus-farming termites remain elusive. In view of this information gap, the present study employed several analytical approaches and ligninolytic enzyme assays to investigate lignin modification in the symbiotic system of a fungus-farming termite, Macrotermes barneyi. The results revealed the structural modification of lignin across different points of the degradation process. Enzyme assays of termite guts and fungus combs showed the obvious differences in ligninolytic enzyme activity at different sites of decomposition, likely initiating the modification of lignin. The findings of the current study support the hypothesis that although young workers start the modification of lignin to some extent, they largely leave the lignin monomers p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) intact. Most of the lignin-derived compounds are transferred to the fresh comb, where the majority of lignin modification begins and continues in mature and older parts of the comb. This study provides new insights into biomass degradation within the microsymbiotic system of an insect. A better understanding of these mechanisms has the promising potential for unlocking new lignin-degrading agents for the production of renewable energy.

Obligate endosymbionts of sap-sucking auchenorrhynchan insects of Hemiptera colonize the bacteriomes and are transmitted vertically through the ovaries to the offspring of host insects, but the critical time of symbiont transmission and molecular mechanisms underlying the process remain unknown. We used histological and transmission electron microscopy, 16S rDNA amplification sequencing and transcriptome analyses to explore the vertical transmission of bacteriome-associated symbionts in the cicada Hyalessa maculaticollis. We find that the symbiont Candidatus Karelsulcia muelleri (hereafter Karelsulcia) proliferates and changes shape after the adult cicadas emerged for 3 h, which is then extruded to the hemolymph from the basal membrane of bacteriome units. The yeast-like fungal symbiont (YLS) harbored in bacteriome sheath cells is released freely along with Karelsulcia. As ovaries mature, Karelsulcia and YLS infect oocytes of cicadas that had emerged for 60 h, and begin to gather at the posterior pole of oocytes, where they form a symbiont ball in each oocyte. Expressions of genes associated with cytoskeletal organization, endocytosis, amino acid transporter and lipid synthesis increase in the newly emerged adults, mediating the transport of substances during the transmission of symbionts. The amino acid-sensitive mechanistic target of the rapamycin pathway is one of the crucial pathways coordinating the vesicle-mediated symbiotic transmission. The insulin signaling pathway potentially together with insect hormones synergically regulate insect fertility and affect yolk deposition, which is closely related to the symbiont infection of ovaries. This study highlights the importance of signaling pathways in regulating the vertical transmission of symbionts in sap-feeding auchenorrhynchan insects.

Aphids are important herbivores in natural and managed environments. We studied the response of aphids and their associated microbiota to the presence of the fungal endophyte Epichloë sp. LpTG-3 strain AR37, and the AR37-derived alkaloids in plants. We hypothesized that AR37 and/or AR37-derived alkaloids would reduce the aphid performance, and that this reduction would be associated with endophyte-mediated changes in the abundance, composition, and diversity of beneficial bacterial endosymbionts of aphids (e.g., Buchnera). Plants of Lolium perenne associated with AR37 variants able (wild type and ∆idtA) and unable (∆idtM) to produce indole diterpene alkaloids were challenged with Rhopalosiphum padi aphids. We measured aphid population size, plant biomass, and the abundance, composition and diversity of the aphid's bacterial microbiota. The presence of AR37 increased the resistance of plants against R. padi aphids via the production of indole diterpene alkaloids, and this effect was independent of the plant biomass. The endophyte-mediated reduction in aphid performance was not associated with changes in the abundance, composition and diversity of the insect's bacterial microbiota. However, we cannot rule out that the reduction in aphid performance could be associated with a putative endophyte effect on the bacterial provision of benefits to aphids. Our study highlighted the protective role of endophyte-derived indole diterpene alkaloids against aphids. Further investigations will be needed to determine if there is a link between the endophyte-mediated aphid resistance and the integrity of the insect's bacterial microbiota.

Hoverflies are essential to ecosystems, with adults serving as important pollinators and larvae preying on plant-feeding insects or recycling nutrients. Species like Sphaerophoria rueppellii are used in biocontrol programs to target aphid pests. To enhance these programs, markers can be used in a mark-release-recapture (MRR) method to track hoverfly feeding and oviposition sites. Effective markers must be persistent and not harm the hoverflies’ vital functions. This study evaluated three marking methods for S. rueppellii: rubidium (RbCl), fluorescein, and fluorescent dust. Laboratory experiments assessed the effects of these markers on female hoverfly fecundity, mating behavior and marking persistence. Results showed no significant differences in egg-laying or survival time between marked and unmarked females. Rubidium and fluorescein did not affect mating behavior, but dust-treated females mated significantly less than untreated females. In terms of marking persistence, rubidium and fluorescent dusts remained detectable throughout the hoverflies’ adult lifespan, while fluorescein markings faded within 24 h. Fluorescent dusts were easy-to-use, durable, and cost-effective, but careful application and further study are needed to avoid potential effects on insect activity and mating ability. Fluorescein showed no adverse effects on insect biology, was economical and quick to apply, but had short persistence, making it unsuitable for long-term field studies. Rubidium was harmless to insects and detectable for long periods, but its detection required financial investment, time, and specialized equipment. This research provides valuable insights into the potential of hoverflies as biocontrol agents and offers new tools for their effective management in agricultural settings.

Nutritional ecology examines the environmental effects on nutritional needs, food intake and foraging behaviors, and the use of nutrients ingested by animals. Adults of many insects’ species feed on nectars rich in sugars allowing them to match the nutritional needs necessary for reproduction. Among insects, Lepidoptera are often considered opportunistic foragers that visit a wide variety of available flowers, although with some preferences. While nutritional ecology of diurnal Lepidoptera is beginning to be explored, very little work focuses on nocturnal species because they are complicated to study in the wild. To address this, we used new laboratory approaches to study feeding behaviors (number and duration of visits to artificial flowers, food preferences associated with the texture and odors of the flowers) as well as gustatory detection by antennae (proboscis extension reflex) in the male crop pest moth Agrotis ipsilon. We showed that (i) food responsiveness is age-dependent and increases mainly with sugar quantity and marginally with sugar quality, (ii) diet quality impacts feeding behaviors in the first days of adulthood, and (iii) male moths choose their food through floral cues. Taken together, these data allow to define this species as a generalist forager with a preference for flowers with sugary nectars rich in sucrose, fructose, and glucose. Our results thus provide considerable information on the close links between food sources and nutritional ecology in this species, which is important for guiding future studies on their behavioral ecology, population dynamics, as well as for population monitoring and for regional pest management.

Spiders are important predatory enemies that control a range of insect pests in the rice ecosystem. m-Aminophenylacetylene (m-A) has been demonstrated to induce maternal care behaviors in Pardosa pseudoannulata, protecting young spiders and promoting population growth. Here the roles of m-A on other reproduction stages were evaluated by injecting m-A into virgin and postreproductive female spiders. When injected into virgin females, m-A can prolong the first pulli-carrying stage of the females after mating and breeding offsprings. In postreproductive females, m-A injection shortened the eggsac-carrying stages, especially in the second and third stages. Transcriptomic analysis showed that differentially expressed genes between the control female and m-A-injected female were enriched in the arachidonic acid (AA) metabolism pathway. Real-time quantitative PCR (qPCR) confirmed the upregulation of cyclooxygenase-II (COX-II), prostaglandin E synthase 2 (PGES2), and prostaglandin E synthase 3 (PGES3) genes involved in PGE₂ (prostaglandin E₂) synthesis. In a reverse validation experiment, the sequential injection of m-A followed by a COX inhibitor aspirin restored the shortened eggsac-carrying stage to normal level. The findings indicated that m-A enhanced PGs synthesis, leading to a reduced eggsac-carrying stage. The eggsac-carrying stage can be fatal when female spiders encounter their enemies, so the shortening of the stage by m-A would reduce the potential threat. These findings are significant for developing environmentally friendly control strategies.

How phenotype varies across geographic space constitutes a key question in current evolutionary biology. Species with widespread geographic ranges are helpful models for examining phenotype variation patterns. Here we examined body size and tegmen shape variation in 12 populations of widespread agricultural pest Dichroplus elongatus collected in Central-West Argentina. D. elongatus showed significant phenotypic variation. Combining linear and geometric morphometric techniques reveals differences in body size and tegmen shape between sexes. General linear mixed models (GLMMs) analyses showed that body size is mainly associated with variables related to temperature of the warmest trimester and temperature annual range along a latitudinal gradient. Individuals achieve larger body sizes at colder areas with lower thermal amplitude. The GLMMs procedures to test allometries demonstrated that most of the analyzed traits scaled isometrically in both sexes indicating that the shape of the individual will not modify with changes in body size. Thorax length is the only trait that scaled hypoallometrically although there were sex-specific differences. Climatic conditions, mainly temperature and precipitations, molded isometric and allometric trait patterns. The detected clinal patterns of phenotypic variation of D. elongatus from Central-West Argentina is likely to be the result of local adaptation to temperature and phenotype plasticity along climatic gradients, and identify geographic areas in where this species showed bigger body size and would undergo greater dispersal capacity. Finally, our results offer insights into how environmental changes may impact the species dispersal and adaptability, advising targeted control and management strategies for this agricultural pest.

Life tables allow the exploration of insects’ and terrestrial arthropods’ biology, and how they respond to external factors. Data collection process has been partially standardized, but the presentation of results mainly depends on the purpose of the study. Two different data representations can be obtained from the raw dataset: the differential representation provides the distribution of the stage-development times, while the integral representation provides the number of individuals into the different life stages, over time. The representations provide relevant biological information, but they lead to a loss of information with respect to the raw dataset. To date, a conceptual explanation of how the two representations can be obtained from the raw data, and of their respective properties, is still missing; moreover, providing the raw dataset as supporting information of the published papers is still not customary. This paper highlights three main points: (i) how the two representations are obtained from life tables raw dataset; (ii) without raw data, it is not possible to switch between the two representations, with a subsequent loss of information; and (iii) why there is the need for a data collection standard. The conceptual explanation is further completed by an electronic file that could support data collection and sharing, and that automatically transform the data in the two representations. We believe that this study is a first step toward a more efficient diffusion of the information among the scientific community, maximizing the efforts made by scholars during the experimental and data analysis process.

Halyomorpha halys is an invasive pest affecting a wide range of crops in many regions of the world. Rapid and cost-effective methods to reconstruct its invasion routes are crucial for implementing strategies to prevent further spread. The mitochondrial markers COI and COII and the pseudogene ΔybgF of the primary symbiont “Candidatus Pantoea carbekii” have been analyzed to track the spread of H. halys. However, these markers do not provide sufficient resolution to fully elucidate invasion routes. Here, H. halys individuals from native and invasive populations were analyzed to identify new DNA markers and evaluate their effectiveness in a multilocus sequence typing (MLST) framework. Three new nuclear markers for H. halys (Hh_KsPi, Hh_UP1, Hh_D3PDh) and three new markers for P. carbekii (Pc_TamA, Pc_SucA, Pc_SurA) were identified. Hh_D3PDh was the most informative marker for H. halys, describing two more haplotypes than COI. By integrating Hh_D3PDh with mitochondrial markers, 30 distinct haplotypes were identified, with each of the populations studied exhibiting multiple haplotypes. Pc_SucA was the most informative symbiont marker, and when all P. carbekii markers were combined, symbiont diversity was greatly increased. The low network specialization between the novel nuclear markers and both mitochondrial and symbiont markers underlined the higher power of nuclear markers. Interestingly, perfect network specialization between H. halys COI and symbiont markers was found in populations from invaded areas, suggesting that some holobiont variants may contribute to enhanced invasive ability. A MLST workflow is proposed as a new tool for population genetics analysis and reconstruction of H. halys invasion.