Spotted-wing drosophila, Drosophila suzukii (Diptera: Drosophilidae), is a destructive pest of ripening berries and stone fruits. D. suzukii is attracted to ripening fruit volatiles, allowing it to occupy a distinct ecological niche from other Drosophila species. Ripening fruit volatiles act as relatively specific cues, which can be leveraged for pest management. Here, we tested whether volatiles from damaged host fruit serve as attractive adult host-finding cues. Using multivariate analysis, we characterized the headspace of mechanically damaged or undamaged blueberries and strawberries, finding distinct volatile organic chemical profiles. In no-choice assays, D. suzukii adults responded similarly between damaged and undamaged treatments at 24 and 48 h; however, when presented with a choice, adults were more strongly attracted to damaged than undamaged fruit volatiles. Adults exhibited a preference for volatiles from an individual damaged fruit over those from many undamaged fruit, despite higher total volatile release by the undamaged treatment as quantified by chromatography. In oviposition assays, females held in constant darkness laid significantly more eggs on damaged than undamaged fruit, whereas those exposed to a light cycle showed no preference. Infestation by early-instar D. suzukii induced a volatile profile distinct from mechanical damage, which attracted females but not males. Together, these results suggest that adult D. suzukii exhibit a preference for the specific ratio of volatiles from mechanically damaged fruit, while additional sensory inputs likely modulate subsequent behavioral decisions. Practically, damaged fruit volatile profiles identified here provide a chemical basis for developing more attractive lures to improve D. suzukii control and monitoring.

Phloem-feeding insects, including the Asian citrus psyllid ( Diaphorina citri), navigate one of the most osmotically challenging diets in nature, the sugar-rich phloem sap. Maintaining osmotic homeostasis is critical for survival, development, and vector competence, particularly in transmitting ‘ Candidatus Liberibacter asiaticus’, the causal agent of citrus greening disease or huanglongbing (HLB). This review synthesizes current knowledge on the physiological, biochemical, and behavioral adaptations that enable D. citri to cope with extreme osmotic pressures. Key strategies include sucrose hydrolysis and conversion into glucose, fructose, and trehalose, as well as redistribution via the hemolymph, and efficient excretion of honeydew, which varies structurally and chemically across different developmental stages and sexes. These adaptations support prolonged phloem feeding, facilitate dual-lifestyle pathogen survival in both phloem and hemolymph, and directly enhance ‘ Ca. L. asiaticus’ acquisition and transmission efficiency. Honeydew as an indicator for feeding behavior, host suitability, and susceptibility to insecticides is also highlighted. Finally, emerging approaches to disrupt osmoregulation, including RNAi-mediated interference with sugar metabolism and water transport, which may provide innovative avenues for integrated management of D. citri and mitigation of HLB spread, are discussed.

Integrating oxytetracycline (OTC) in management programs of bacterial plant diseases, including citrus greening, remains controversial due to concerns related to antimicrobial resistance, environmental impact, and residue safety, highlighting the need for sensitive and reliable residue monitoring methods. This study reports the development and validation of an HPLC-PDA-based method for the quantitative determination of OTC and six structurally related analogues in citrus tissue. Chromatographic separation was achieved using a gradient mobile phase of 0.01 M oxalic acid (pH 2.5) and acetonitrile, providing baseline resolution, symmetrical peak shapes, and low tailing factors (<1.2) for all target analytes. Despite structural similarity among OTC analogues, UV spectral profiling enabled reliable compound discrimination, even in cases of minor co-elution. The method demonstrated strong linearity over a concentration range of 0.195-100 μg mL-1 (R2 > 0.99 for most analytes), with acceptable intra- and inter-day precision (RSD <5 %). Spiking studies exhibited reliable inter-matrix performance with extraction recoveries ranging from 84 to 108 % across multiple citrus matrices, including healthy and infected leaves, peel, pulp, and juice, with minimal matrix interference. The method showed high sensitivity across citrus tissues, with limits of detection (LOD) as low as 1.48 ng mL-1 and limits of quantification (LOQ) below the U.S. Environmental Protection Agency (EPA) default regulatory threshold for citrus. Application to field samples from OTC-injected commercial groves detected parent OTC residues in citrus leaves, but not other structural analogues, and no detectable OTC translocation into juice. Overall, this method provides a robust analytical platform that enables simultaneous, high-sensitivity quantification of OTC and multiple structural analogues across complex citrus matrices to support environmental exposure assessment, regulatory compliance, and food safety evaluation in disease-managed citrus production systems.

The Asian citrus psyllid, Diaphorina citri, transmits the putative causal agent of citrus greening disease, 'Candidatus Liberibacter asiaticus'. The transmission occurs in a propagative, circulative manner. Specific protein-protein interactions are required for the accomplishment of the transmission process. We employed the far-Western technique, also known as protein overlay assay, and mass spectrometry to identify D. citri proteins that specifically recognize membrane proteins of 'Ca. L. asiaticus'. Whole-body protein extracts of D. citri were separated by electrophoresis and blotted onto polyvinylidene difluoride membranes. The membranes were overlayed with sonicated leaf midrib extracts from 'Ca. L. asiaticus'-infected citrus plants. The protein complexes were detected using different antibodies against 'Ca. L. asiaticus' membrane proteins and peptidoglycans associated with lipoproteins. By comparison, spots were extracted from a polyacrylamide gel for liquid chromatography-tandem mass spectrometry analysis. Identified proteins included ATP synthase alpha and beta subunits, actin, alpha- and beta-tubulins, transitional endoplasmic reticulum ATPase TER94, 78 kDa glucose-regulated protein, and arginine kinase. These findings emphasize the involvement of energy machinery and muscle proteins in the 'Ca. L. asiaticus'-D. citri interactions. Understanding the pathogen-vector interactions will lead to better-designed control strategies based on the interference with specific interactions and/or blocking transmission.

Silicon dioxide nanoparticles (SiO2-NPs) have shown promise as plant biostimulants; however, their physiological, metabolic, and transcriptional effects in perennial fruit crops remain poorly understood. In this study, we evaluated the physiological, metabolic, and transcriptomic responses of Citrus sinensis leaves to repeated foliar spray applications of SiO2-NPs under greenhouse conditions. Plants were foliar-sprayed every 15 days with SiO2NPs at 0, 200, 400, and 600 mg L-1. SiO2-NPs treatment significantly enhanced chlorophyll and phenolics accumulation, recording total chlorophyll of 10.08 mgL- 1 and increased total phenolics to 171.0 mg g-1 FW. Volatile organic compound profiling revealed concentration-dependent modulation of terpene emissions, including significant induction of sesquiterpenes. Transcriptome analysis identified 2236 differentially expressed genes (1619 upregulated and 617 downregulated), with significant enrichment in pathways associated with cell wall biosynthesis, cytoskeleton organization, hormone signaling, and photosynthetic function. SiO2-NPs treatment significantly induces cell wall genes, including pectin methylesterases, expansins, arabinogalactan proteins, and kinesin motor proteins. Additionally, we recorded upregulation of cell cycle regulators, microtubuleassociated proteins, and auxin-responsive transcription factors. SiO2-NPs significantly enhanced primary metabolism, including a nearly ten-fold increase in glucose and accumulation of polyols and inositols. SiO2-NPs induced significant shifts in phytohormone profiles, elevating IAA from 19.8 to 56.4 & micro;g/g FW, IBA from 255.3 to 664.7 & micro;g/g FW, IPA from 176.4 to 292.4 & micro;g/g FW, tJA from 35.0 to 62.8 & micro;g/g FW, and ABA from 102.7 to 159.8 & micro;g/g FW, while cinnamic and salicylic acid levels remained unchanged. Our results suggest that the application of SiO2-NPs maintains plant integrity by coordinating cell wall remodeling, cytoskeletal organization, and hormone-mediated regulatory networks.

Citrus greening, also known as huanglongbing (HLB), is one of the most destructive citrus diseases worldwide. The current study investigated the metabolic and transcriptional mechanisms underlying citrus tolerance to ‘Candidatus Liberibacter asiaticus’, with a focus on melatonin-phytohormone crosstalk across eight citrus genotypes exhibiting varying degrees of HLB tolerance. Tolerant genotypes showed higher basal melatonin contents and upregulated melatonin biosynthetic genes, suggesting transcriptional activation of melatonin metabolism as a central adaptive feature. Likewise, auxin and its biosynthetic genes exhibited expression patterns similar to those of melatonin, revealing an integrated tryptophan-based metabolic flux that supports melatonin-auxin crosstalk. Tolerant citrus genotypes also accumulated higher basal levels of phenylalanine and its downstream salicylates, including trans-cinnamic acid (tCA), benzoic acid (BA), and salicylic acid (SA), along with upregulation of phenylalanine ammonia-lyase (PAL)-related genes, indicating the activation of the PAL-dependent SA biosynthetic pathway as a core mechanism of basal immune priming in tolerant citrus. The 3D response surfaces showed distinct tolerance-dependent crosstalk among phenylalanine, SA, and melatonin, suggesting a genotype-specific crosstalk between the SA biosynthesis pathway and melatonin accumulation under HLB-associated stress conditions. On the other hand, jasmonic acid (JA) biosynthesis exhibited a precursor-primed but flux-limited profile, with tolerant genotypes accumulating α-linolenic acid and upregulating early JA biosynthetic genes without excessive JA accumulation. Abscisic acid (ABA) analysis revealed clear genotype-dependent variation; however, this variation was not associated with HLB tolerance level, implying a general stress response. Collectively, these findings suggest that melatonin integrates auxin- and SA-mediated pathways to sustain growth-defense balance, providing a mechanistic basis for metabolic resilience and HLB tolerance in citrus.

While the role of succinic semialdehyde (SSA) dehydrogenase (SSADH; also known as gabD) is well-reported from model plants, the lack of functionality and structure of SSADH from citrus represents a significant knowledge gap. Herein, genome-wide analyses identified 17 high-confidence SSADH-like proteins from Citrus sinensis, among which three putative SSADHs have potential GABA dehydrogenase function. Sequence alignment, phylogenetic analyses, and domain architecture demonstrated high conservation among CsSSADHs (aka CsgabD) and their homologs across diverse plant taxa. Notably, CsSSADH-2 lacked a conserved QGIVC motif found in CsSSADH-1/-3. Secondary structure analyses indicated conserved aldehyde dehydrogenase domains. Homology-based 3D modeling predicted CsSSADH-1 and 2 as homo-tetramers; however, AlphaFold2-based modeling suggested their full-length monomer structures. PPI networks revealed CsSSADH-1 interacts with 10 proteins, primarily involved in GABA/succinate metabolism and the TCA cycle. Docking studies indicated that CsSSADH-1 displayed acceptable affinity and binding modes with GABA, SSA, and succinate. GABA supplementation enhances CsSSADH expression, GABA, and succinate content in a dose-dependent manner in both healthy and infected citrus plants under greenhouse conditions. CsSSADH was involved in citrus responses to 'Candidatus Liberibacter asiaticus' and/or its vector, Diaphorina citri. Nevertheless, GABA accumulation under biotic stress leads to condition-specific rerouting of GABA metabolism. Chemical inhibition of CsSSADH resulted in increased GABA accumulation but reduced succinate levels in both healthy and infected plants. This study offers the first comprehensive characterization of C. sinensis SSADH isoforms, providing insights into their evolutionary divergence, structural features, and potential functions, and enhancing our understanding of their possible roles in GABA metabolism and citrus defense responses.

Spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), is a destructive pest of ripening berries and stone fruits, where larval feeding rapidly renders fruit unmarketable. Larvae are widely assumed to remain inside their natal fruit until pupation, limiting exposure to external management tactics. Here, we challenge this paradigm by demonstrating that D. suzukii larvae exhibit complex chemotactic capabilities and dynamic interactions with host fruit throughout development. Third-instar larvae responded in a dose-dependent manner to fermentation-associated volatiles and discriminated between conspecific and heterospecific larval developmental cues, indicating the capacity for substrate assessment. Complementary to these chemotaxis assays, direct observations across multiple hosts revealed frequent larval emergence from fruit, including posterior spiracle, head, and whole-body exposure, as well as a previously undocumented behavior: secondary infestation of neighboring, uninfested fruit. Secondary infestation occurred across all hosts tested and throughout larval development, with cumulative movement into fresh berries exceeding 60% in some fruit types. Host species and larval density modulated the frequency and timing of emergence, and inter-fruit dispersal, highlighting the influence of natal environment on larval behavior. Together, these findings suggest that D. suzukii larvae are not confined to a single fruit but can interact repeatedly with fruit surfaces and move into neighboring hosts. This revised understanding of D. suzukii larval ecology exposes new vulnerabilities in the life cycle and has possible implications for the development of management strategies targeting immature stages.

Citrus Tristeza Virus (CTV) is the most economically significant viral pathogen of citrus worldwide, transmitted in a semipersistent manner by several aphid species, most efficiently by Aphis [Toxoptera] citricidus. Transmission efficiency varies substantially among CTV genotypes, but the viral determinants underlying these differences remain poorly understood. In this study, we investigated the roles of three viral genes, p33, p18, and p13, in aphid transmission using a genetically engineered CTV-T36 mutant (CTV-T36Δp33Δp18Δp13) as a backbone. This mutant is derived from the poorly transmissible T36 isolate and lacks three accessory genes implicated in host specificity and possibly vector interactions. To assess their contribution to transmissibility, we substituted the deleted genes with their counterparts from CTV-FS577, a highly transmissible isolate. The resulting chimeric virus exhibited a significant increase in transmission efficiency up to 50% compared to the original T36 background (∼0.6%). Sequence analysis revealed that among the three substituted genes, only p33 differs between T36 and FS577, with a single amino acid change (K174R). This residue is conserved in other highly transmissible isolates such as T68-1, supporting its potential role as a key determinant of transmission. Our findings highlight P33 as a critical viral protein in aphid-mediated transmission and suggest that efficient transmissibility requires coordinated function of multiple viral proteins, including P33, P61, and P65. This work provides new insights into the molecular basis of vector transmission and sets the stage for further studies on virus-vector interactions in CTV.

BACKGROUND:Citrus root weevil, Diaprepes abbreviatus is one of the most destructive pests of citrus, causing extensive root damage that predisposes trees to secondary infection with phytophthora and substantial yield losses. In the present study, we identified and functionally characterized the coatomer subunit beta gene (DaCOPB), a core component of the COPI vesicle trafficking machinery responsible for two-way transport between the endoplasmic reticulum and Golgi apparatus. RESULTS:The full-length DaCOPB transcript was obtained from the de novo transcriptome of D. abbreviatus and exhibited strong evolutionary conservation with insect homologs, including characteristic Armadillo repeat domains associated with protein-protein interactions. Developmental expression analysis revealed that DaCOPB is constitutively expressed across all life stages, suggesting an essential role in insect growth and development. To evaluate its functional importance, dsRNA targeting DaCOPB was orally delivered to larvae at multiple concentrations. RT-qPCR analysis performed 48 h post feeding confirmed significant suppression of DaCOPB transcripts relative to control treatments, demonstrating efficient RNAi-mediated gene silencing. Knockdown of DaCOPB resulted in severe developmental defects, including larval mortality, disrupted pupation, and malformed adults. Regression analysis further revealed a significant dose-dependent correlation between dsRNA exposure and the magnitude of gene suppression and phenotypic effects. CONCLUSION:These results demonstrate that DaCOPB is necessary for normal development and survival for D. abbreviatus, highlighting its potential as a promising molecular target for RNAi-based pest management strategies aimed at controlling citrus root weevil populations. © 2026 Society of Chemical Industry.

Micronutrient deficiencies, affecting over two billion people worldwide, pose significant health challenges. Recent advancements in biofortification and genetic engineering have shown considerable promise in enhancing the nutritional content of staple crops, thereby improving global nutrition and health outcomes. Biofortification, the process of increasing the nutrient content of crops through conventional breeding techniques and genetic engineering, has expanded the possibilities by enabling precise modifications to enhance nutrient profiles. These technologies have led to crops with increased levels of pigments, iron, zinc, vitamin A, and other critical micronutrients. Despite the potential benefits, several challenges hinder the widespread adoption of biofortified and genetically engineered crops. Regulatory hurdles, consumer acceptance, and socioeconomic factors continue to be significant barriers. Additionally, the long-term environmental impacts and ethical considerations of genetic modifications continue to spark debate. Continued research and collaboration among scientists, policymakers, and stakeholders are essential to overcome these challenges. This review reports the current state of biofortification through genetic engineering, while also addressing the barriers to its adoption. These plant-based solutions can play an essential role in advancing global food security and improving public health. This review highlights biotechnological strategies for enhancing crop nutritional value, including genetic engineering and genome editing. We focus on precise manipulation of metabolic and molecular pathways to increase micronutrient content and bioavailability, accelerating the development of nutrient-enriched crops for global food security.

BACKGROUND:Citrus production worldwide is severely impacted by a devastating disease known as citrus greening, or Huanglongbing (HLB). The phytopathogen causing HLB is transmitted between trees by the Asian citrus psyllid, Diaphorina citri, which acts as the primary vector. Currently, there is no cure for HLB, and management efforts primarily rely on the use of insecticides. However, there is a growing need for alternative, environmentally sustainable control methods, such as RNA interference (RNAi). RESULTS:We investigated the impact of gene silencing on the mortality of D. citri by targeting two genes, vacuolar-sorting protein/sucrose non-fermenting protein 7 (DcSnf7) and inhibitor of apoptosis 5 (DcIap5). Gene silencing was initially assessed by delivering synthesized double-stranded RNA (dsRNA) to D. citri through topical feeding. To further assess gene suppression in vivo, we used a virus-induced gene silencing (VIGS) approach to deliver RNAi to psyllids via citrus plants. Suppressing DcSnf7 and DcIap5 individually resulted in elevated nymph mortality; however, the combined suppression of both genes using dual dsRNA treatment did not yield an additive effect. We modified the infectious Citrus tristeza virus (CTV-T36) clone to individually and jointly carry the truncated genes, DcSnf7 and DcIap5. Over two successive generations, D. citri reared on plants inoculated with CTV-tSnf7, CTV-tIap5, or the combined construct CTV-tSnf7-tIap5 exhibited increased mortality at all life stages, as well as significantly reduced fecundity and fertility, compared to insects reared on non-infected or CTV-wt-inoculated control plants. In addition, these VIGS plants shortened the lifespan of D. citri. Notably, the dual construct CTV-tSnf7-tIap5 consistently produced the most pronounced reductions in survival, fecundity, fertility, and longevity of D. citri across all experiments, exceeding the effects observed with either single-gene construct. CONCLUSIONS:Our results suggest that silencing key genes of D. citri using RNAi mediated by VIGS represents a promising control strategy that could play a role in HLB management. © 2026 Society of Chemical Industry.

BACKGROUND:The citrus root weevil, Diaprepes abbreviatus, is a destructive agricultural pest for which molecular control options remain limited due to historically sparse genomic resources. Leveraging a comprehensive de novo transcriptome, we investigated developmental gene regulation across larval, pupal, and adult stages and identified essential targets for RNA interference (RNAi)-based intervention. RESULTS:Stage-resolved transcriptomic analyses revealed extensive transcriptional reprogramming associated with metabolism, detoxification, cuticle biosynthesis, endocrine signaling, and sensory perception. Among these, chitin synthase (DaCHS) emerged as a critical developmental gene, exhibiting pronounced up-regulation during late larval and pupal stages corresponding to intensive cuticle synthesis. Phylogenetic and structural analyses demonstrated that DaCHS is highly conserved among insects and retains canonical catalytic domains and transmembrane topology. Alpha Fold-based structural modeling and molecular docking confirmed stable interaction of DaCHS with its substrate, N-acetylglucosamine, supporting functional conservation of enzymatic activity. Oral delivery of DaCHS double-stranded RNA induced robust transcript suppression, leading to significant mortality and severe developmental defects, including larval and pupal abnormalities, and adults with disrupted wing and abdominal morphogenesis. CONCLUSION:These findings establish DaCHS as an indispensable gene for D. abbreviates development and validate transcriptome-guided RNAi as a powerful framework for target discovery. This work provides a strong molecular foundation for developing RNAi-based strategies that can be integrated into sustainable management programs for citrus root weevil control. © 2026 Society of Chemical Industry.

Salicylic acid (SA), the active component of aspirin, is a crucial defense phytohormone that coordinates both local and systemic acquired resistance. For decades, SA biosynthesis was attributed to two pathways: (i) the isochorismate synthase pathway, which is dominant in arabidopsis (Arabidopsis thaliana) and other Brassicaceae, and (ii) the phenylalanine ammonia-lyase (PAL) pathway, common in most other plants. However, the PAL pathway remained biochemically incomplete due to the lack of a molecularly identified benzoic acid 2-hydroxylase. Recent findings fill this gap by identifying a conserved post-PAL route that converts trans-cinnamic acid to SA through benzoyl-coenzyme A (CoA) intermediates, spanning multiple subcellular compartments. This pathway explains pathogen-induced SA accumulation in non-Brassicaceae and establishes post-PAL metabolism as an ancestral, conserved mechanism underlying plant immunity.

Although citrus tristeza virus (CTV; Closterovirus tristezae, family Closteroviridae) has historically been responsible for severe losses in the citrus industry, mild strains such as CTV-T36 have been successfully repurposed as powerful biological tools. The development of infectious cDNA clones from these mild strains has opened new avenues in citrus biotechnology. This review highlights the diverse applications of the CTV-T36 infectious clone, including its use as a platform for citrus functional genomics, the study of citrus physiology, RNA interference (RNAi) targeting both endogenous plant genes and pest genes, and the expression of antimicrobial peptides and bioinsecticidal proteins. These innovative applications position CTV-T36-based vectors as valuable tools in integrated strategies to combat major citrus threats such as Huanglongbing (HLB). Recent advancements, technical challenges, and prospects of this technology in citrus research and pest management are also discussed.

Suppression of δ-aminolevulinic acid dehydratase in citrus disrupts chloroplast development and photosynthetic stability, triggering broad transcriptional reprogramming across metabolic, stress, and hormone signaling pathways. Chlorosis is often the earliest visible indicator of plant stress, whether caused by abiotic or biotic factors. Complex stress networks can ultimately lead to the loss of photosynthetic organelles and pigments, resulting in reduced cell viability and eventually, cell death. However, plants possess coordinated mechanisms to maintain homeostasis under adverse conditions. δ-Aminolevulinic acid dehydratase (ALAD), a central enzyme in tetrapyrrole biosynthesis, is essential for chlorophyll production and photosynthetic function. Here, we examined the physiological and transcriptomic consequences of ALAD suppression in Citrus macrophylla using virus-induced gene silencing. At the cellular level, ALAD-deficient plants exhibited a reduced number of plastids and altered plastid morphology. Transcriptomic profiling revealed major alterations in genes associated with photosynthesis, Fe and Zn nutrient uptake, and stress- and defense-related pathways, including reactive oxygen species (ROS) detoxification, hormone signaling, and secondary metabolism. Integrating these transcriptional and physiological changes into predictive models for photosynthesis- and redox-related genes allowed the identification of key regulatory nodes. To achieve this, we implemented a machine-learning-ready pipeline using variance-stabilized expression of the most variable genes and Random Forest modeling to classify photosynthesis and redox states. ALAD suppression triggered upregulation of cytochrome P450s, lipoxygenases, annexins, and ferritin, ROS detoxification, and iron homeostasis. ALAD suppression induced a pronounced lipid peroxidation signature, as evidenced by elevated malondialdehyde accumulation and altered levels of linoleic and α-linolenic acids, accompanied by increased jasmonic acid (JA) accumulation. Together, the suppression of ALAD reveals a coordinated stress response integrating chloroplast architecture, photosynthetic efficiency, redox regulation, nutrient homeostasis, and defense pathways.

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.

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is the main vector for the bacterium ‘Candidatus Liberibacter asiaticus’, which is associated with citrus greening, also known as Huanglongbing. D. citri transmits ‘Ca. L. asiaticus’ during its feeding on citrus phloem sap. Transmission occurs in a circulative, propagative, and persistent manner. ‘Ca. L. asiaticus’ has a small genome (1.2 Mb). Therefore, it acquires most of its nutrients and energetic nucleotides from its hosts. The objective of this study was to assess the effect of ‘Ca. L. asiaticus’ infection on the level of the free fatty acids in its vector. The fatty acids were extracted from adult D. citri using ethyl acetate, derivatized with boron trifluoride-methanol, and analyzed using gas chromatography-mass spectrometry (GC-MS). Nine fatty acids were identified in the D. citri extracts. Oleic acid was the most predominant fatty acid followed by stearic and palmitic acid, whereas the rest of the fatty acids were present in low amounts. In general, the levels of the detected fatty acids in ‘Ca. L. asiaticus’-infected D. citri were lower than those found in healthy psyllids. Our findings showed that, the reduction of fatty acids in ‘Ca. L. asiaticus’-infected psyllids resulted from the higher activity of β-oxidation to generate acetyl-CoA, which causes more production of ATP. Our results indicated that ‘Ca. L. asiaticus’ may enhance the β-oxidation of fatty acids in its vector insect to fulfill its nutrient and energetic nucleotide requirements.

Diaprepes abbreviatus is a major pest causing severe root damage and yield loss in over 150 species, with a complex life cycle that hinders effective control. Limited genomic and transcriptomic resources have restricted molecular insights into its biology. De novo transcriptome assembly provides a practical approach for reconstructing the full set of transcripts in organisms without a reference genome, enabling the identification of novel genes and isoforms as well as the study of gene expression patterns. We performed RNA-seq across seven developmental stages of D. abbreviatus, generating a high-quality de novo transcriptome assembly comprising 991,860 unique transcripts and 505,007 unigenes with 99.2

BackgroundHuanglongbing (HLB) is a devastating bacterial disease caused by the bacterium Candidatus Liberibacter asiaticus (CaLas) that affects the citrus industry worldwide. This study investigated the response of two pummelo x finger lime hybrid siblings to natural infection with CaLas. The hybrids were identified primarily using leaf morphology and molecular marker assessments and were selected for further studies on the basis of the CaLas titers in leaf petioles.ResultsHLB-infected budwood from the selected hybrids (PFL 2-61 and PFL 1-11), as well as the two parental plants, were propagated by grafting onto Swingle citrumelo rootstocks for further evaluation. Plant samples were collected two years after grafting for analysis. Leaves of PFL2-61 exhibited decreased CaLas titers compared with those of PFL 1-11. Additionally, we recorded increased chlorophyll content, total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity in PFL 2-61 compared to PFL 1-11 and the parents. We subsequently conducted a detailed investigation of these two hybrid siblings using transcriptome analysis. Among the 20,675 differentially expressed genes (DEGs) identified, 1,416 were downregulated in PFL 2-61 compared with PFL 1-11, whereas 326 were upregulated. Transcriptome analysis revealed that many of the DEGs were associated with the cell wall structure, redox homeostasis, and biotic stress responses. Moreover, key genes related to the biosynthesis of secondary metabolites and phytohormones, including PAL1, jasmonate-related genes, and WRKY transcription factors, were upregulated in the tolerant hybrid (PFL 2-61). In contrast, three transcripts associated with the Sieve Element Occlusion N-Terminus (SEO_N) domain were downregulated in the tolerant hybrid (PFL 2-61).ConclusionsOur findings provide valuable insights into the molecular mechanisms of tolerance and susceptibility to HLB in finger lime derived hybrids, highlighting the potential of this citrus species towards developing disease-tolerant varieties.