Interactions between biomolecules and their target sites are fundamental to pest control, and such relationships can either intensify or inhibit their effects. A broader understanding of these complex interactions aids in interpreting toxicological responses, while physiological changes can elucidate insect metabolic pathways. Research involving terpinolene and 1-butyl-3,4-methylenedioxybenzene (BMDB) has demonstrated their insecticidal activity against Aedes aegypti. However, the effects of these substances on physiological and immunological parameters require further clarification; furthermore, the binary combination of these compounds may result in more significant and permanent damage. Thus, this study aimed to test the hypothesis that combining different modes of action would intensify histological and immunological alterations critical to insect survival, thereby enabling more effective control. To this end, LC50 values for the individual compounds were obtained from the literature (Silva et al. 2016), and a dose-response curve was established for the 1:1 combination, revealing an LC50 of 75.69 ± 2.37 ppm. The cytotoxic parameters evaluated included midgut histology, apoptosis, and cell proliferation in L4-stage larvae, while immunological parameters evaluated included phenoloxidase activity, nitric oxide levels, and oxidative stress markers (TBARS and GST). Histological changes in the midgut were observed in all treatment groups compared to the control; however, more severe histopathological alterations-such as vacuolization, cellular swelling, and loss of the peritrophic matrix-were observed in the combined treatment. Regarding apoptosis, a significant increase was observed only in treatments containing BMDB, whether alone (2.66 ± 0.66) or in combination (3.33 ± 0.33), compared to the control (0.5 ± 0.54). As for cell proliferation (PCNA), larvae in the combined group exhibited a lower rate (0.50 ± 0.11) compared to terpinolene (1.33 ± 0.12) and BMDB (1.40 ± 0.15). Reductions in phenoloxidase activity (1.026 ± 0.061 OD/min/mg) and nitric oxide levels (0.878 ± 0.034 µM/mL) were observed in the combined group. Regarding oxidative stress, there was no statistical difference in GST measurements (p > 0.05), whereas TBARS levels decreased in the combined group (2.497 ± 0.184 nmol MDA/mg/protein) compared to the control (3.401 ± 0.327 nmol MDA/mg/protein). Thus, despite exhibiting lower larvicidal activity compared to the isolated compounds, the binary combination caused more severe histopathological and immunological damage that compromises larval survival and recovery capacity, representing a promising strategy for integrated vector management.
For many insects, the ecdysteroid biosynthetic pathway that converts cholesterol to ecdysone includes multiple cytochrome P450 monooxygenases, a Rieske oxygenase, and a short-chain dehydrogenase. Collectively, the genes encoding these enzymes are referred to as Halloween genes. While the importance of these enzymes in oogenesis is well-documented in holometabolous insects, less is known about their oogenic role in hemipterans. To address this, we used RNA interference-mediated knockdown to determine the role that the Halloween transcripts neverland, spookiest, shroud, phantom, disembodied, shadow, and shade have on oogenesis and fertility in the western tarnished plant bug, Lygus hesperus Knight. Knockdown of neverland and spookiest severely curtailed oogenesis and resulted in little to no oviposition, phantom knockdown had no impact on oogenesis but reduced oviposition and egg hatch, and shadow knockdown resulted in reduced egg hatch. In contrast, knockdown of shroud, disembodied, and shade had no observable effects on oogenesis, oviposition, or hatch rate. While this study demonstrates the critical reproductive role of some Halloween genes in L. hesperus, it also raises questions regarding how broadly conserved the current ecdysteroid biosynthetic pathway is across insects.
The cardiac extracellular matrix provides structural support to the primary circulatory organ of an insect: a dorsal vessel that is divided into an aorta in the thorax and a heart in the abdomen. In the fruit fly, Drosophila melanogaster, the extracellular matrix components, Pericardin and Lonely heart, are needed for proper heart contractility, as they mediate adhesion between cardiomyocytes, pericardial cells, hemocytes, and alary muscles. In the mosquito, Anopheles gambiae, Pericardin and Lonely heart are necessary for the infection-induced migration of hemocytes to the heart. In these infected mosquitoes, hemocytes decrease the heart rate by releasing nitric oxide. Here, we demonstrate in mosquitoes that Pericardin is transcribed throughout the abdomen, but that Lonely heart is transcribed mainly in the heart. Moreover, we demonstrate that Pericardin and Lonely heart influence the infection-induced disproportional decrease in the anterograde and retrograde heart rates. We also demonstrate that, in infected mosquitoes, Pericardin and Lonely heart alter the directionality of heart contractions, shifting to more contractions propagating anterograde. These findings further explain how the immune and circulatory systems of insects are functionally integrated by demonstrating that the cardiac extracellular matrix, in addition to driving hemocyte organization on the heart, modulates heart rhythmicity in infected mosquitoes.
The citrus root weevil, Diaprepes abbreviatus, is an economically important pest of citrus and ornamental crops whose subterranean larval feeding damages roots and predisposes plants to secondary pathogen infection. Development of efficient RNA interference (RNAi) delivery methods for early larval stages is essential for functional genomics studies and the evaluation of RNAi-based pest management strategies. In this study, we developed a droplet-based feeding assay for oral delivery of double-stranded RNA (dsRNA) to neonates of D. abbreviatus using chitin synthase 2 (DaCHS2) as a model RNAi target to validate the assay. Feeding solutions containing dsRNA were supplemented with sucrose and bromophenol blue dye, with bromophenol blue used to visually confirm ingestion. Across three independent biological replicates, all neonates exposed to DaCHS2-dsRNA, GFP-dsRNA, and water control droplets were confirmed to have ingested the feeding solution (45/45 neonates per treatment; 100% feeding success). Oral delivery of dsRNA targeting DaCHS2 reduced transcript abundance and was associated with developmental abnormalities and mortality, including incomplete molting, abnormal pigmentation, cuticular deformities, defective pupation, and malformed adults. Regression analysis demonstrated moderate and significant relationship between dsRNA concentration and neonate mortality and developmental abnormalities. RT-qPCR further confirmed reduced DaCHS2 transcript abundance following oral dsRNA exposure. The developed assay provides a simple, reproducible, and minimally invasive proof-of-concept platform for oral dsRNA delivery to D. abbreviatus neonates. The assay requires only small dsRNA volumes, provides visual confirmation of ingestion, and may facilitate laboratory-based screening of additional RNAi target genes in D. abbreviatus and other coleopteran pests.
Wohlfahrtia magnifica, a worldwide pest, causes myiasis in many domestic animals. Its pheromones and volatiles from Bactrian camels were reported to attract or repel W. magnifica, but the molecular mechanisms are still unclear. A small protein in the insect olfactory system, called odorant binding protein (OBP), plays a crucial role in odor reception. In this study, two W. magnifica odorant-binding proteins (WmagOBPs) genes, WmagOBP42531 and WmagOBP39280, were cloned from the antennae of W. magnifica, and then expressed and purified by prokaryotic expression. Fluorescence binding assays demonstrated that WmagOBP39280 exhibits high binding affinity to methylheptenone, butylbutyrate, p-ethylacetophenone, acetophenone (Ki: 4.69, 4.15, 3.48, 4.06), while WmagOBP42531 exhibits high binding affinity to p-ethylacetophenone and dipentene (Ki: 4.85, 4.98) and moderate binding to methylheptenone, 1-octene-3-ol, acetophenone, ethylbenzene. o-xylene (Ki: 7.00, 5.96, 5.72, 7.86, 6.15). Molecular docking and molecular dynamics simulation results suggest that a stable complex could be formed between WmagOBP42531 and 1-octene-3-ol, acetophenone, p-ethylacetophenone, or o-xylene; between WmagOBP39280 and acetophenone, butylbutyrate, p-ethylacetophenone, or methylheptenone, with Gibbs free energy (ΔG) ranging from -26.84 to -14.7 kcal/mol. RMSD, RMSF, and radius of gyration suggested the spontaneous binding of WmagOBP42531 and WmagOBP39280 to odor ligands.
Bemisia tabaci damages crop plants by sucking plant sap and transmitting economically important viruses. For both processes, whitefly saliva plays an important role in establishing successful feeding contact. This study evaluated the effect of the Bta78 gene on whitefly survival, fecundity, and feeding activity by transiently expressing it in Nicotiana benthamiana plants using a Turnip mosaic virus (TuMV) expression vector. The Bta78 gene encodes phosphatidylethanolamine-binding protein (PEBP), previously reported as a B. tabaci salivary protein. A two-fold increase in whitefly feeding activity, measured by honeydew secretion, was observed on Bta78-expressing plants compared with green fluorescent protein (GFP)-expressing TuMV-GFP control plants. By contrast, no significant effects on whitefly survival or fecundity were detected. In this study, vitellogenin, a well-conserved protein in the insect phylum associated with insect fecundity, was used as a positive control. Whiteflies exposed to vitellogenin-expressing plants exhibited a 2-2.5-fold increase in egg production relative to those infesting TuMV-GFP control plants. To the best of our knowledge, these results provide the first evidence that the PEBP-domain-carrying salivary protein Bta78 promotes whitefly feeding.
The Neotropical stink bugs Euschistus heros and Diceraeus melacanthus are major pests of soybean and maize in South America, yet current chemical control strategies face widespread resistance, highlighting the urgent need for sustainable alternatives. This study investigates Ficus carica pruning residues as a source of selective botanical insecticides, integrating optimized extraction, phytochemical profiling, bioassays, and molecular modeling. Eight extracts were prepared under varying solvent, temperature, and acidity conditions, and analyzed for total phenolic content (TPC) and the key furanocoumarins psoralen and bergapten. Acidification enhanced overall mass yields, while mild ethanol extraction at room temperature selectively maximized furanocoumarin recovery, yielding psoralen concentrations up to 14.83 mg g-1, which is substantially higher than previously reported in leaves or fruit. Biological evaluation of the optimized ethanolic extract (ERA) revealed strong insecticidal activity, with 86% mortality of E. heros and 40% of D. melacanthus nymphs at 48 h, and a calculated LC50 of 1232 mg L-1 for E. heros. The differential susceptibility between species suggests both metabolic and cuticular factors influence efficacy. Computational docking and phylogenetic analyses suggested a potential mechanistic basis for the observed selectivity: furanocoumarins are predicted to bind hemipteran AChE via a compensatory polar scaffold, whereas binding to Apis mellifera AChE is predicted to be weaker due to lineage-specific differences in aromatic density within the catalytic gorge, potentially explaining the minimal off-target susceptibility. The molecular modeling results characterize these compounds as low-affinity, reversible inhibitors, combining effective pest control with a favorable safety profile for pollinators. The present work demonstrates that valorizing agro-industrial waste from F. carica can yield potent, selective, and environmentally safer insecticidal agents. The integration of extraction optimization, biological evaluation, and molecular modeling provides a robust framework for developing sustainable botanical insecticides, advancing circular economy principles in pest management and offering promising alternatives to synthetic neurotoxins.
Neurodegenerative disorders are characterized by progressive neuronal loss and functional decline, yet effective interventions remain limited. The polyamine spermidine was suggested to exert neuroprotective effects, but its concentration-dependent impact on longevity, neuronal integrity, and behavior remains still not well studied. Here, we investigated the effects of spermidine on lifespan, behavioral responses, brain tissue, target gene expression, and antioxidant status in Drosophila melanogaster model of age-dependent neurodegeneration. Wild-type flies and swiss cheese (sws1) mutants were exposed to 0.5, 1, and 5 mM spermidine from early adulthood. Lifespan analysis revealed that high-dose spermidine (5 mM) reduced survival in both wild-type and sws1 mutants, whereas lower doses (0.5 and 1 mM) significantly improved survival in mutants without affecting wild-type flies. Behavioral assays revealed that sws1 flies exhibited reduced climbing ability compared to controls, which was further decreased at 5 mM. Lower concentrations did not significantly affect locomotor performance. Taste preference for trehalose, impaired in untreated sws1 mutants, was partially restored by spermidine at all tested concentrations. Histological analysis of 10-13-day-old mutants showed a concentration-dependent reduction in degeneration zones within the lamina and medulla at 0.5 and 1 mM, whereas 5 mM had no effect. Biochemical assays indicated mild pro-oxidant effects at 5 mM, reflected by increased malondialdehyde (MDA) levels, while 0.5 mM enhanced antioxidant defenses, including catalase activity and Trolox equivalent antioxidant capacity (TEAC). Our results suggest that spermidine at low doses has the potential to be a general-purpose neuroprotector.
The whitefly Bemisia tabaci is an infamous agricultural pest that inflicts significant harm on crops globally by directly feeding on them and indirectly transmitting viruses. Although it is believed that whiteflies inject saliva into their host plants to modulate plant defenses, however, due to their small size, study on the salivary proteome of whiteflies is still sparse and the roles of many salivary proteins remain elusive. This research employed four-dimensional data-independent acquisition (4D DIA) based proteomic techniques to analyze whitefly secreted saliva. In total, 2625 salivary proteins were identified. Bioinformatic analysis results showed that the secreted saliva proteins may play roles in hydrolysis, transport, protein binding and metabolism, and significantly increased the levels of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) activities as well as malondialdehyde (MDA), soluble sugars (SS), soluble protein (SP), and proline (PRO) in cucumbers. The expression of the salicylic acid (SA) response genes (PR1a, PR3, and PR5) were significantly induced. Additionally, exogenous methyl-salicylate (MeSA) treatment of cucumbers can significantly reduce the reproductive capacity and survival rate of whiteflies, and decrease the preference of whiteflies for host plants. This study's outcomes provide valuable insights into the interaction between plants and insects. It has been proved that the salivary proteins of whitefly participate in host plant defense responses by activating the SA signaling pathway, and provides basic data on the functional study of whitefly saliva elicitors and effectors, which can be used for development of novel strategies for pest management.
Diapause in the Sunn pest, Eurygaster integriceps, is a key seasonal adaptation associated with migration, overwintering survival, and reproductive timing. However, the molecular components associated with this physiological state remain poorly characterized in this hemipteran pest. In this study, we identified and characterized a takeout-family transcript from E. integriceps (EiTO1; GenBank OP913374.1) and combined evolutionary, structural, and expression analyses to examine its association with seasonal physiological states. EiTO1 contained a 738-bp open reading frame encoding a 245-amino acid protein and was assigned to the juvenile hormone-binding protein (JHBP) superfamily based on conserved-domain analysis. Phylogenetic reconstruction recovered EiTO1 as sister to the Nilaparvata lugens-Bemisia tabaci clade; however, the low bootstrap support for this placement prevented confident inference of its closest evolutionary relationship. Structural comparison of the predicted EiTO1 model with experimentally resolved takeout/JHBP-family proteins indicated a conserved barrel-like fold and a corresponding internal cavity, consistent with structural features of this protein family. Quantitative PCR profiling revealed marked seasonal and tissue-specific variation in EiTO1 expression. In female whole-body samples, EiTO1 transcript abundance was low during spring field activity and in newly emerged adults sampled in June, reached its maximum during the refuge-associated phase in August (160-fold), and showed a secondary increase during the late-overwintering/premigration transition in March (21.5-fold). Tissue-resolved assays in females showed the highest expression in the head and intermediate expression in the fat body. Ovarian expression remained at or below the April baseline at most sampling points but showed a transient increase in August. Together, these findings identify EiTO1 as a takeout/JHBP-family gene whose expression is associated with seasonal physiological transitions in E. integriceps.
The larva-to-pupa transition in honey bees (Apis mellifera) involves extensive neural remodeling, yet the molecular dynamics of brain development and their relationship with the surrounding microbial environment remain poorly characterized. This study integrated brain transcriptomic profiling with comb-associated metagenomic analysis to characterize stage-specific molecular signatures during metamorphosis. RNA sequencing of larval and pupal brains was combined with honeycomb shotgun metagenomics from the same sample. Brain transcriptomes exhibited marked stage-specific divergence. Pupae displayed downregulation of transcriptional regulators, ecdysone and insulin signaling, and growth-related pathways, alongside upregulation of cuticular proteins, glutathione metabolism, and odorant-binding proteins. Notably, numerous poorly annotated, lineage-specific loci showed extreme stage-specific regulation. In contrast, comb-associated microbial communities remained globally stable across developmental stages, though supervised ordination identified stage-discriminatory taxa, including core symbionts and opportunistic pathogens. Integrative network analysis revealed significant correlations between comb potential bee pathogens' abundances and brain transcripts involved in translation, stress response, and metabolic regulation. Our data suggest that honey bee neural maturation is primarily driven by intrinsic transcriptional reprogramming, while structured variation in the external microbial milieu correlates with host neural gene expression. Honeycomb microbiome shift should be the consequence of the environmental conditions changes and host developmental shifts. Their roles in that process, as well as the brood immune system-comb microbiome interactions, may be part of future research.
The ecdysone receptor (EcR) is a key subunit of the heterodimeric EcR-USP (ultraspiracle) receptor, whose role in insect growth and development has been well established. In contrast, its role in regulating chitin biosynthesis, particularly at transcriptional level received less attention. Here, we identified the EcR from Hyphantria cunea and investigated its roles in larval growth and development, particularly in chitin biosynthesis, via dsRNA-mediated HcEcR knockdown. Bioassays showed that silencing of HcEcR significantly increased larval mortality and caused developmental defects in H. cunea. Meanwhile, HcEcR knockdown significantly downregulated the expression of genes in the chitin biosynthetic pathway. The yeast one-hybrid (Y1H) assay indicated that HcEcR alone did not interact with the HcCHSA or HcHK2 promoters, suggesting that, independent of HcUSP, HcEcR might not transcriptionally regulate chitin biosynthesis pathway genes. Additionally, HcEcR knockdown significantly suppressed the expression of 20E-induced response genes, but dramatically increased glucose, trehalose and glycogen levels in H. cunea larvae. Overall, HcEcR was pivotal for the growth, molting and chitin biosynthesis in H. cunea larvae. Meanwhile, HcEcR knockdown suppressed epidermal chitin synthesis probably by downregulating 20E signaling in H. cunea larvae. Furthermore, inhibiting chitin synthesis might reduce substrate consumption for chitin synthesis, thereby resulting in an increase of carbohydrate levels in the larvae. Moreover, HcEcR alone might not transcriptionally regulate chitin biosynthetic pathway genes. The findings provide novel insights into the physiological role of EcR in chitin biosynthesis and identify potential molecular targets for pest control.
This study investigated the temporal dynamics of extracellular signal-regulated kinase (ERK) phosphorylation, together with the expression profiles of its putative upstream ligands and downstream target genes, during embryonic development in Bombyx mori. The results revealed distinct ERK phosphorylation dynamics: significantly higher levels of phosphorylated ERK were observed between Days 1 and 6 after oviposition in developing eggs (non-diapause and HCl-treated eggs), whereas diapause eggs exhibited low levels. The temporal expression patterns of genes in the prothoracicotropic hormone (PTTH)/torso and epidermal growth factor (EGF) signaling pathways-spitz (spi), rhomboid (rho), and Egf receptor (Egfr)-did not differ between diapause and HCl-treated eggs. In contrast, the EGF pathway gene vein (vn) and genes in the transforming growth factor (TGF)-β signaling pathway-including the ligand myoglianin (myo) and its receptor babo-displayed distinct temporal expression patterns. Similarly, Jelly belly (Jeb)/anaplastic lymphoma kinase (Alk), fibroblast growth factor (Fgf)/Fgf receptor (Fgfr), neurotrophin (NT), and Toll-receptor ligands (spätzle family members (spz2, spz4, and spz5)) showed differential expression, with levels significantly elevated during the middle and late stages of embryonic development in HCl-treated eggs. Non-diapause and chilled eggs also exhibited increasing expression patterns of these genes, similar to those observed in HCl-treated eggs. Additionally, ERK target genes pointed and huckebein (hkb) showed higher and broader expression peaks in developing eggs compared to diapause eggs. This study provides a comprehensive analysis of the transcriptional regulation of upstream and downstream ERK signaling components during embryonic development and reveals new insights into the mechanisms underlying embryonic diapause.
Western flower thrips (WFT) Frankliniella occidentalis cause extensive damage to crops either by direct feeding injury or by transmitting diseases. Current management strategies against this pest rely heavily on synthetic insecticides, leading to issues such as resistance development and environmental concerns. Several safer alternatives including RNA interference (RNAi), also referred to as post-transcriptional gene silencing (PTGS), are being extensively researched to develop species-specific molecular pesticides for the management of this pest. However, the success of RNAi depends on the selection of lethal targets and the delivery of double-stranded RNA (dsRNA) to the target pest. In this study, we demonstrate the uptake of dsRNA by WFT through artificial diet feeding and its distribution within the insect. Several candidate gene targets were evaluated, leading to the identification of promising RNAi targets for thrips management. In addition, dsRNA production was successfully standardized using a recombinant bacterial expression system for large-scale applications. We also observed degradation of naked dsRNA following feeding, likely due to nuclease activity associated with thrips feeding processes, highlighting a key limitation for RNAi efficacy. Overall, our findings support the potential of RNAi as a targeted pest management strategy for WFT. The results emphasize the importance of strategic gene selection, improved delivery systems, and scalable dsRNA production for the development of effective RNAi-based molecular biopesticides.
In holometabolous insects, the pupal stage enables the transition from larva to adult and provides a useful system for studying the organization and evolution of ecdysis behavior. However, the motor programs underlying the formation of exarate pupae remain less well characterized than those of obtect and coarctate pupae. Here, we describe the pupal ecdysis sequence in Henosepilachna vigintioctomaculata at phase-resolved behavioral resolution. Pupal ecdysis was subdivided into pre-ecdysis, ecdysis, and post-ecdysis. During pre-ecdysis, prepupae exhibited slight dorso-ventral (D-V) contractions followed by progressively strengthened twitch contractions that loosened the old exoskeleton. During ecdysis, successive anteriorly directed peristaltic posterior-anterior (P-A) contractions, together with D-V contractions and lateral swinging, drove exuvial shedding and the freeing and stretching of pupal wings and cephalothoracic appendages. During post-ecdysis, posteriorly directed anterior-posterior (A-P) and D-V movements restored stretched appendages to their normal sizes and positions. RNA interference against the ecdysis-triggering hormone gene Hveth, delivered by microinjection of in vitro synthesized dsRNA or by oral administration of bacterially expressed dsRNA, impaired pupation. Knockdown of Hveth prolonged pre-ecdysis, reduced twitch frequency, and partially or completely disrupted ecdysis and post-ecdysis movements. Together, these results define the pupal ecdysis sequence of an exarate coleopteran pupa and support a requirement for ETH signaling in the proper execution and coordination of this motor program.
Glutathione S-transferases (GSTs) constitute a critical enzyme superfamily responsible for xenobiotic detoxification in insects. The green peach aphid, Myzus persicae, is a globally significant agricultural pest that has exhibited declining field susceptibility to λ-cyhalothrin (LCT), a widely applied pyrethroid insecticide. While elevated GST activity has been correlated with LCT tolerance in various aphid populations, the precise molecular mechanisms driving this process have not been fully elucidated. In this study, we functionally characterized a delta-class GST gene (designated MpGSTd1) from M. persicae. The encoded protein possesses hallmark structural motifs of the GST family, including distinct glutathione-binding (G-site) and hydrophobic substrate-binding (H-site) pockets. Transcriptional profiling revealed maximal MpGSTd1 expression during the late nymphal to adult stages, and its transcript levels were markedly induced following LCT challenge. Biochemical analysis of the recombinant MpGSTd1 protein confirmed its catalytic competency toward the model substrate CDNB, exhibiting a Vmax of 0.91 µmol/min/mg and a Km of 2.73 mM. Notably, LCT exposure inhibited the enzyme's activity in vitro with an IC50 of 0.02 mM, and high-performance liquid chromatography (HPLC) assays demonstrated that MpGSTd1 can directly deplete LCT by 11.4%. Furthermore, RNA interference (RNAi)-mediated suppression of MpGSTd1 in vivo significantly elevated aphid mortality upon subsequent LCT exposure. Collectively, these findings delineate an essential role for MpGSTd1 in modulating LCT susceptibility in M. persicae and underscore its potential utility as a molecular target for future aphid management strategies.
Spermatogenesis is a conserved process across animals, involving the maintenance and differentiation of germ stem cells, haploid spermatid production via meiosis, and morphologically distinct sperm formation during spermiogenesis. We previously demonstrated that Slmap (Sarcolemma-associated protein) knockdown causes male sterility with no sperm in the seminal vesicle. Here, we show that Slmap knockdown in the early Drosophila melanogaster germline led to severe spermiogenesis defects, including aberrant paracrystalline material deposition in both mitochondrial derivatives, fusion of the two derivatives and of spermatids, loss of the central pair of axonemal microtubules, and axoneme clustering. These phenotypes were supported by transcriptomic data showing that the most prominently affected genes were those involved in cuticle development and plasma-membrane adhesion, including 20 cuticular protein genes. In parallel, we detected upregulation of individualization complex genes (chic, ctp, WASp, Lasp) and significant association with the oxidative phosphorylation pathway. Consistently, KEGG analysis and functional assays showed that the sustained damage activated germ cell apoptosis, as indicated by increased TUNEL signals, elevated Hid levels, and reduced Diap1 expression. Together, these findings place Slmap at the intersection of structural integrity and cell survival, linking its loss to both morphogenetic failure and activation of the Hid-Diap1 apoptotic pathway.
Phloem-feeding insects such as aphids, planthoppers, and whiteflies use salivary proteins to aid feeding, adapt to hosts, and manipulate plant defenses, shaping a dynamic plant-herbivore conflict. Among them, the sweet potato whitefly, Bemisia tabaci, is a major global pest that threatens food security by causing feeding damage and transmitting plant viruses. The salivary secretory proteins of B. tabaci remain poorly characterized. We conducted a proteome-wide hypothesis-based screening of the B. tabaci MEAM1/B-biotype reference proteome using secretion, membrane-topology, localization, and effector-likelihood filters to prioritize candidate secreted proteins. Among 1404 proteins screened in EffectorP 3.0, 884 were identified as putative effector candidates and reported as computationally predicted rather than a validated effectorome. Most of these candidates were cytoplasmic (741), with fewer following the classical extracellular secretory pathway (143). Functional annotation showed strong enrichment of proteases (cathepsin B, cathepsin L, and serine proteases) and thioredoxin-related proteins, some of which resemble proteins implicated in feeding systems among hemipteran insects, supporting their prioritization for validation. Protein-association analyses highlighted protease-related candidates as central predicted network hubs. Transient validation of one prioritized candidate, BtApe, showed attenuation of chitin-associated oxidative staining and normalized defense-marker responses in tomato, supporting BtApe as an immune-modulatory candidate and defining a focused framework to validate salivary gland expression and secretion, as well as loss of function effects.
Lectins, the glycan-binding proteins that are known to function as essential pattern-recognition receptors (PRRs) in the innate immune system of insects, also act as strategic modulators of physiological stress responses. An attempt is made here to demonstrate how chronic stress affects the lectin from the larvae of Zophobas morio, an adapter molecule in physiological response. The haemagglutination (HA) titer used to measure the activity of lectin revealed discrepancies in the larval serum of Z. morio after exposure to various stress conditions. Among the stress-induced treatment groups, except the control, the treatments exhibited a marked reduction in the HA activity from the larval serum of Z. morio. Examination of the haemagglutination inhibition (HAI) activity, which is used to assess the ability of specific sugars that inhibit this process, clearly showed that lectin from the larval serum of stress-induced groups had deteriorated in its binding ability to d-fucose and lactose. Pyriproxyfen (a juvenile hormone analog) mimics hormones exclusive to insects and is extremely target-specific. It induces physiological and cellular alterations in insects. Although the effects of pyriproxyfen on insect lectins are indirect, it acts through hormonal signaling to affect lectins. The relative quantitation of d-fucose-binding lectin (FBL) mRNA under various stress conditions in the larval fat body of Z. morio, analyzed through RT-qPCR, clearly indicated significantly lower levels of its expression, except for pyriproxyfen treatment. Molecular docking and simulation were performed to recognize the stability of the interaction between FBL and pyriproxyfen. These results revealed a significant binding characterized by high affinity through a strong amide bridge, and other non-covalent interactions, showing evidence for increased expression of lectin at doses of pyriproxyfen treatments as a measure of neutralization. On the whole, stressful environments bring swift and dynamic changes in the activity of lectin, including its expression in Z. morio larvae, revealing lectin as a vital constituent in response to physiological challenges.
