Synergistic effects of Fe-based nanomaterial catalyst on humic substances formation and microplastics mitigation during sewage sludge composting.

In this study, a novel Fe-based nanomaterial catalyst (Fe0/FeS) was synthesized via a self-heating process and employed to explore its impact on the formation of humic substances and the mitigation of microplastics. The results reveal that Fe0/FeS exhibited a significant increase in humic acid content (71.01 mg kg-1). Similarly, the formation of humic substances resulted in a higher humification index (4.91). Moreover, the addition of Fe0/FeS accelerated the degradation of microplastics (MPs), resulting in a lower concentration of MPs (9487 particles/kg) compared to the control experiments (22792 particles/kg). Fe0/FeS significantly increased the abundance of medium-sized MPs (50-200 µm) and reduced the abundance of small-sized (10-50 µm) and large-sized MPs (>1000 µm). These results can be attributed to the Fe0/FeS regulating the ▪OH production and specific microorganisms to promote humic substance formation and the degradation of MPs. This study proposes a feasible strategy to improve composting characteristics and reduce contaminants.

Natural Coumarin Shows Toxicity to Spodoptera litura by Inhibiting Detoxification Enzymes and Glycometabolism.

Coumarin and its derivatives are plant-derived compounds that exhibit potent insecticidal properties. In this study, we found that natural coumarin significantly inhibited the growth and development of Spodoptera litura larvae through toxicological assay. By transcriptomic sequencing, 80 and 45 differentially expressed genes (DEGs) related to detoxification were identified from 0 to 24 h and 24 to 48 h in S. litura after coumarin treatment, respectively. Enzyme activity analysis showed that CYP450 and acetylcholinesterase (AChE) activities significantly decreased at 48 h after coumarin treatment, while glutathione S-transferases (GST) activity increased at 24 h. Silencing of SlCYP324A16 gene by RNA interference significantly increased S. litura larval mortality and decreased individual weight after treatment with coumarin. Additionally, the expression levels of DEGs involved in glycolysis and tricarboxylic acid (TCA) cycle were inhibited at 24 h after coumarin treatment, while their expression levels were upregulated at 48 h. Furthermore, metabonomics analysis identified 391 differential metabolites involved in purine metabolism, amino acid metabolism, and TCA cycle from 0 to 24 h after treated with coumarin and 352 differential metabolites associated with ATP-binding cassette (ABC) transporters and amino acid metabolism. These results provide an in-depth understanding of the toxicological mechanism of coumarin on S. litura.

Alternative splicing of chitin deacetylase 2 regulates chitin and fatty acid metabolism in Asian citrus psyllid, Diaphorina citri.

Chitin plays an important role in the development and molting of insects. The key genes involved in chitin metabolism were considered promising targets for pest control. In this study, two splice variants of chitin deacetylase 2 (CDA2) from Diaphorina citri were identified, including DcCDA2a and DcCDA2b. Bioinformatics analysis revealed that DcCDA2a and DcCDA2b encoded 550 and 544 amino acid residues with a signal peptide, respectively. Spatio-temporal expression patterns analysis showed that DcCDA2a and DcCDA2b were highly expressed in D. citri wing and nymph stages. Moreover, DcCDA2a and DcCDA2b expression levels were induced by 20-hydroxyecdysone (20E). Silencing DcCDA2a by RNA interference (RNAi) significantly disrupted the D. citri molting and increased D. citri mortality and malformation rate, whereas inhibition of DcCDA2b resulted in a semimolting phenotype. Furthermore, silencing DcCDA2a and DcCDA2b significantly suppressed D. citri chitin and fatty acid metabolism. Our results indicated that DcCDA2 might play crucial roles in regulating D. citri chitin and fatty acid metabolism, and it could be used as a potential target for controlling D. citri.

New Insights into the Plutella xylostella Detoxifying Enzymes: Sequence Evolution, Structural Similarity, Functional Diversity, and Application Prospects of Glucosinolate Sulfatases.

Brassica plants have glucosinolate (GLs)-myrosinase defense mechanisms to deter herbivores. However, Plutella xylostella specifically feeds on Brassica vegetables. The larvae possess three glucosinolate sulfatases (PxGSS1-3) that compete with plant myrosinase for shared GLs substrates and produce nontoxic desulfo-GLs (deGLs). Although PxGSSs are considered potential targets for pest control, the lack of a comprehensive review has hindered the development of PxGSSs-targeted pest control methods. Recent advances in integrative multi-omics analysis, substrate-enzyme kinetics, and molecular biological techniques have elucidated the evolutionary origin and functional diversity of these three PxGSSs. This review summarizes research progress on PxGSSs over the past 20 years, covering sequence properties, evolution, protein modification, enzyme activity, structural variation, substrate specificity, and interaction scenarios based on functional diversity. Finally, we discussed the potential applications of PxGSSs-targeted pest control technologies driven by artificial intelligence, including CRISPR/Cas9-mediated gene drive, transgenic plant-mediated RNAi, small-molecule inhibitors, and peptide inhibitors. These technologies have the potential to overcome current management challenges and promote the development and field application of PxGSSs-targeted pest control.

Exploring the mechanisms of humic acid mediated degradation of polystyrene microplastics under ultraviolet light conditions.

Microplastics (MPs) are emerging pollutants that interact extensively with dissolved organic matter (DOM) and this influences the environmental behavior of MPs in aqueous ecosystems. However, the effect of DOM on the photodegradation of MPs in aqueous systems is still unclear. The photodegradation characteristics of polystyrene microplastics (PS-MPs) in an aqueous system in the presence of humic acid (HA, a signature compound of DOM) under ultraviolet light conditions were investigated in this study through Fourier transform infrared spectroscopy coupled with two-dimensional correlation analysis, electron paramagnetic resonance, and gas chromatography-mass spectrometry (GC/MS). HA was found to promote higher levels of reactive oxygen species (0.631 mM of ▪OH), which accelerated the photodegradation of PS-MPs, with a higher degree of weight loss (4.3%), higher level of oxygen-containing functional groups, and lower average particle size (89.5 µm). Likewise, GC/MS analysis showed that HA contributed to a higher content of oxygen-containing compounds (42.62%) in the photodegradation of PS-MPs. Moreover, the intermediates and final degradation products of PS-MPs with HA were significantly different in the absence of HA during 40 days of irradiation. These results provide an insight into the co-existing compounds on the degradation and migration processes of MP and also support further research toward the remediation of MPs pollution in aqueous ecosystems.

Sex-specific transcription and DNA methylation landscapes of the Asian citrus psyllid, a vector of huanglongbing pathogens.

The relationship of DNA methylation and sex-biased gene expression is of high interest, it allows research into mechanisms of sexual dimorphism and the development of potential novel strategies for insect pest control. The Asian citrus psyllid, Diaphorina citri Kuwayama, is a major vector for the causative agents of Huanglongbing (HLB), which presents an unparalleled challenge to citrus production worldwide. Here, we identify the X chromosome of D. citri and investigate differences in the transcription and DNA methylation landscapes between adult virgin males and females. We find a large number of male-biased genes on the autosomes and a depletion of such on the X chromosome. We have also characterized the methylome of D. citri, finding low genome-wide levels, which is unusual for an hemipteran species, as well as evidence for both promoter and TE methylation. Overall, DNA methylation profiles are similar between the sexes but with a small number of differentially methylated genes found to be involved in sex differentiation. There also appears to be no direct relationship between differential DNA methylation and differential gene expression. Our findings lay the groundwork for the development of novel epigenetic-based pest control methods, and given the similarity of the D. citri methylome to some other insect species, these methods could be applicable across agricultural insect pests.

Single-molecular insights into the breakpoint of cellulose nanofibers assembly during saccharification.

Plant cellulose microfibrils are increasingly employed to produce functional nanofibers and nanocrystals for biomaterials, but their catalytic formation and conversion mechanisms remain elusive. Here, we characterize length-reduced cellulose nanofibers assembly in situ accounting for the high density of amorphous cellulose regions in the natural rice fragile culm 16 (Osfc16) mutant defective in cellulose biosynthesis using both classic and advanced atomic force microscopy (AFM) techniques equipped with a single-molecular recognition system. By employing individual types of cellulases, we observe efficient enzymatic catalysis modes in the mutant, due to amorphous and inner-broken cellulose chains elevated as breakpoints for initiating and completing cellulose hydrolyses into higher-yield fermentable sugars. Furthermore, effective chemical catalysis mode is examined in vitro for cellulose nanofibers conversion into nanocrystals with reduced dimensions. Our study addresses how plant cellulose substrates are digestible and convertible, revealing a strategy for precise engineering of cellulose substrates toward cost-effective biofuels and high-quality bioproducts.

Quantitative ubiquitylome crosstalk with proteome analysis revealed cytoskeleton proteins influence CLas pathogen infection in Diaphorina citri.

Huanglongbing (HLB), also known as citrus greening disease, is caused by Candidatus Liberbacter asiaticus (CLas) and transmitted by Diaphorina citri. Previous studies reported that CLas infection significantly influences the structure of the D. citri cytoskeleton. However, the mechanisms through which CLas manipulates cytoskeleton-related proteins remain unclear. In this study, we performed quantitative ubiquitylome crosstalk with the proteome to reveal the roles of cytoskeleton-related proteins during the infection of D. citri by CLas. Western blotting revealed a significant difference in ubiquitination levels between the CLas-free and CLas-infected groups. According to ubiquitylome and 4D label-free proteome analysis, 343 quantified lysine ubiquitination (Kub) sites and 666 differentially expressed proteins (DEPs) were identified in CLas-infected groups compared with CLas-free groups. A total of 53 sites in 51 DEPs were upregulated, while 290 sites in 192 DEPs were downregulated. Furthermore, functional enrichment analysis indicated that 18 DEPs and 21 lysine ubiquitinated proteins were associated with the cytoskeleton, showing an obvious interaction. Ubiquitination of D. citri tropomyosin was confirmed by immunoprecipitation, Western blotting, and LC-MS/MS. RNAi-mediated knockdown of tropomyosin significantly increased CLas bacterial content in D. citri. In summary, we provided the most comprehensive lysine ubiquitinome analysis of the D. citri response to CLas infection, thus furthering our understanding of the role of the ubiquitination of cytoskeleton proteins in CLas infection.

Silencing of Glycogen Synthase Kinase 3 Significantly Inhibits Chitin and Fatty Acid Metabolism in Asian Citrus Psyllid, Diaphorina citri.

Glycogen is a predominant carbohydrate reserve in various organisms, which provides energy for different life activities. Glycogen synthase kinase 3 (GSK3) is a central player that catalyzes glucose and converts it into glycogen. In this study, a GSK3 gene was identified from the D. citri genome database and named DcGSK3. A reverse transcription quantitative PCR (RT-qPCR) analysis showed that DcGSK3 was expressed at a high level in the head and egg. The silencing of DcGSK3 by RNA interference (RNAi) led to a loss-of-function phenotype. In addition, DcGSK3 knockdown decreased trehalase activity, glycogen, trehalose, glucose and free fatty acid content. Moreover, the expression levels of the genes associated with chitin and fatty acid synthesis were significantly downregulated after the silencing of DcGSK3. According to a comparative transcriptomics analysis, 991 differentially expressed genes (DEGs) were identified in dsDcGSK3 groups compared with dsGFP groups. A KEGG enrichment analysis suggested that these DEGs were primarily involved in carbon and fatty acid metabolism. The clustering analysis of DEGs further confirmed that chitin and fatty acid metabolism-related DEGs were upregulated at 24 h and were downregulated at 48 h. Our results suggest that DcGSK3 plays an important role in regulating the chitin and fatty acid metabolism of D. citri.

New insights into the distribution and interaction mechanism of microplastics with humic acid in river sediments.

Information on the distribution and interaction of microplastics (MPs) and humic acids (HAs) in river sediment has not been fully explored. This study assessed the distribution and interaction of MPs with HAs at different depths in river sediments. The results delineated that the average abundance of MPs in the 0-10 cm layer (190 ± 20 items/kg) was significantly lower than that in the 11-20 cm and 21-30 cm layers (211 ± 10 items/kg and 238 ± 18 items/kg, respectively). Likewise, the large MP particles mainly existed in the 0-10 cm layer (31.53%-37.87%), while small MP particles were found in the 21-30 cm layers (73.23%-100%). Moreover, HAs in MPs showed a transformation from low molecular weight to high molecular weight with an increase in depth from 0-10 cm to 21-30 cm, which may contribute to the distribution of MPs in the river sediments. These results provide new insight into the migration of MP pollution in river sediments, but further research needs to assess the interaction of MP with HA for mitigating MP pollution in river sediment.

Bombyx mori ferritin heavy-chain homolog facilitates BmNPV proliferation by inhibiting reactive oxygen species-mediated apoptosis.

Ferritin heavy-chain homolog (FerHCH), an iron-binding protein, plays an important role in the host defense against oxidative stress and pathogen infections. In our previous research, Bombyx mori native ferritin had an interaction with B. mori nucleopolyhedrovirus (BmNPV). However, the underlying molecular mechanism of single ferritin homolog responses to BmNPV infection remains unclear. In this study, we found that BmNPV titer and B. mori FerHCH (BmFerHCH) expression were positively correlated with the ferric iron concentration. We performed RNA interference (RNAi) and overexpression experiments to investigate the effects of BmFerHCH on BmNPV proliferation. BmFerHCH knockdown suppressed BmNPV proliferation in vivo and in vitro, whereas BmFerHCH overexpression facilitated BmNPV proliferation. In addition, the oxidative stress level was increased significantly in BmN cells after budded virus infection, while BmFerHCH could neutralize the increased ROS production induced by BmNPV. Of note, we found that ROS was involved in BmNPV-induced apoptosis. Through inhibiting ROS, apoptosis was suppressed by BmFerHCH, whereas BmFerHCH knockdown facilitated apoptosis. Therefore, we hypothesize that BmFerHCH-mediated inhibition of virus-induced apoptosis depends on suppressing ROS accumulation and, thereby, facilitates virus replication. These results suggest that BmFerHCH plays an important role in facilitating BmNPV proliferation and modulating BmFerHCH is potential strategy for studying host-pathogen interactions.

Silencing of Chitin-Binding Protein with PYPV-Rich Domain Impairs Cuticle and Wing Development in the Asian Citrus Psyllid, Diaphorina citri.

Chitin is a major component of the arthropod exoskeleton, always working together with chitin-binding proteins to maintain the functions of extracellular structures. In the present study, we identified a cuticle protein 64 from Diaphorina citri using a chitin-binding assay. Bioinformatics analysis revealed that DcCP64 contained eight conserved PYPV motifs but lacked a Rebers-Riddiford (R-R) consensus and other chitin-binding domains. RT-qPCR analysis suggested that DcCP64 had the highest expression level in the wing and fifth-instar nymph stage. Knockdown of DcCP64 by RNA interference (RNAi) resulted in a malformed-wing phenotype, higher mortality and decreased molting rate. Furthermore, transcriptomics analysis revealed that 1244 differentially expressed genes (DEGs) were up-regulated and 580 DEGs were down-regulated, compared with dsDcCP64 groups and dsGFP groups. KEGG enrichment analysis revealed that up-regulated DEGs were mainly related to oxidative phosphorylation, whereas down-regulated DEGs were mainly involved in the MAPK and FoxO signaling pathways. Moreover, inhibition of DcCP64 significantly affected the cuticle surface, and increased the permeability of the abdomen and wings. Further chitin- and cellulose-binding assay confirmed the chitin-binding properties of recombinant DcCP64 in vitro. These results indicate that DcCP64 might play an important role in the cuticle and wing development of D. citri.

Functional analysis of a peptidoglycan recognition protein involved in the immune response in the common cutworm, Spodoptera litura.

Peptidoglycan recognition proteins (GRPs) are family of pattern recognition receptors (PRRs), which can recognize the peptidoglycan and trigger the innate immune system against the microorganisms in insects. In this study, we identified a GRP-LB from Spodoptera litura genome database and named SlGRP-LB, which contained a complete open reading frame (ORF) of 639 bp, encoding a protein of 212 amino acids with a signal peptide and GRP domain. Phylogenetic tree analysis suggested that the SlGRP-LB has a close relationship with Helicoverpa armigera GRP-LB (HaGRP-LB). Tissue expression analysis revealed that SlGRP-LB had a high expression level in the fat body. The expression levels of SlGRP-LB were significantly upregulated in the hemolymph, fat body, and midgut from 3 to 12 h after injection of Escherichia coli and Staphylococcus aureus, while the expression levels were not downregulated at 24 h postinfection. Knockdown of SlGRP-LB expression by RNA interference reduced the expression of antibacterial peptide-related genes in the fat body and midgut, while their expression levels were upregulated in the hemolymph. In addition, the recombinant SlGRP-LB was expressed by using E. coli expression system, and it exhibited binding activity to E. coli. Taken together, the data suggest that S. litura GRP-LB might play a crucial role in regulating immune response in S. litura.

Insights into pectin dominated enhancements for elimination of toxic Cd and dye coupled with ethanol production in desirable lignocelluloses.

Pectin is a minor wall polysaccharide with potential applications for bioproducts. Despite the application of specific plants and biomass-based sorbents for environmental remediation, little has been reported about characteristic roles of pectin. Using the natural rice mutant (Osfc16) treated with Cd, this study explored that pectin could predominately enhance Cd accumulation with lignocellulose, mainly due to remarkably raised uronic acids deposition. The Cd-treatment further reduced lignocellulose recalcitrance for significantly enhanced biomass saccharification and bioethanol production along with almost complete Cd release. Using all remaining fermentation rice residues that are of typical ribbon-structure and large surface, this study generated novel biosorbents by optimal chemical oxidation with the pectin extraction from citrus peels, and examined consistently raised Cd and methylene blue (MB) adsorption capacities. Therefore, this work has proposed a mechanism model about multiple pectin enrichment roles for Cd and MB removals in agricultural and industry locations with full lignocellulose utilization towards bioethanol production.

Validamycin treatment significantly inhibits the glycometabolism and chitin synthesis in the common cutworm, Spodoptera litura.

Validamycin, as a broadly applied antibiotic, has been used to control rice sheath blight disease. Furthermore, validamycin was considered as an insecticide to control agricultural pests. Insight into the mechanism of validamycin's action on insects can provide molecular targets for the control of agricultural pests. In this study, a toxicological test analysis revealed that Spodoptera litura larval growth and development was significantly inhibited and the pupation rate was significantly reduced with the increase of the concentration of validamycin. According to the NMR-based metabolomic analysis, a total of 15 metabolites involved in glycolysis and tricarboxylic acid cycle (TCA) pathways were identified. Additionally, trehalase activities, glucose and chitin contents were significantly downregulated, but the trehalose content was upregulated after exposure to validamycin. Reverse transcription quantitative PCR analysis revealed that the expression level of genes involved in glycolysis, TCA and chitin synthesis were upregulated after treating with validamycin. Further chitin staining also confirmed that chitin content was downregulated at 12 h after validamycin treatment. Our results indicated that validamycin worked via two different molecular mechanisms, one through inhibiting glycometabolism and the other by inhibiting chitin synthesis in S. litura. The information lays a theoretical foundation for further control of S. litura.

Functional Characterization of a Trehalose-6-Phosphate Synthase in Diaphorina citri Revealed by RNA Interference and Transcriptome Sequencing.

Trehalose-6-phosphate synthase (TPS) plays an important role in the synthesis of trehalose. In the current study, a TPS gene was obtained from Diaphorina citri, and named as DcTPS1 which encoded a protein of 833 amino acid residues. Real-time quantitative PCR (qPCR) analysis revealed that DcTPS1 had the highest expression level in the midgut and fifth-instar nymph stage. Knockdown of DcTPS1 by RNA interference (RNAi) induced an abnormal phenotype and increased mortality and malformation rate with a decreased molting rate. In addition, silencing of DcTPS1 significantly inhibited D. citri chitin metabolism and fatty acid metabolism, while the expression levels of fatty acid decomposition-related genes were downregulated. Furthermore, comparative transcriptomics analysis revealed that 791 differentially expressed genes (DEGs) were upregulated and 678 DEGs were downregulated when comparing dsDcTPS1 groups with dsGFP groups. Bioinformatics analysis showed that upregulated DEGs were mainly involved in oxidative phosphorylation, whereas downregulated DEGs were mainly attributed to the lysosome and ribosome. These results indicated that DcTPS1 played an important role in the growth and development of D. citri.

Down-regulation of OsMYB103L distinctively alters beta-1,4-glucan polymerization and cellulose microfibers assembly for enhanced biomass enzymatic saccharification in rice.

BACKGROUND: As a major component of plant cell walls, cellulose provides the most abundant biomass resource convertible for biofuels. Since cellulose crystallinity and polymerization have been characterized as two major features accounting for lignocellulose recalcitrance against biomass enzymatic saccharification, genetic engineering of cellulose biosynthesis is increasingly considered as a promising solution in bioenergy crops. Although several transcription factors have been identified to regulate cellulose biosynthesis and plant cell wall formation, much remains unknown about its potential roles for genetic improvement of lignocellulose recalcitrance. RESULTS: In this study, we identified a novel rice mutant (Osfc9/myb103) encoded a R2R3-MYB transcription factor, and meanwhile generated OsMYB103L-RNAi-silenced transgenic lines. We determined significantly reduced cellulose levels with other major wall polymers (hemicellulose, lignin) slightly altered in mature rice straws of the myb103 mutant and RNAi line, compared to their wild type (NPB). Notably, the rice mutant and RNAi line were of significantly reduced cellulose features (crystalline index/CrI, degree of polymerization/DP) and distinct cellulose nanofibers assembly. These alterations consequently improved lignocellulose recalcitrance for significantly enhanced biomass enzymatic saccharification by 10-28% at p < 0.01 levels (n = 3) after liquid hot water and chemical (1% H2SO4, 1% NaOH) pretreatments with mature rice straws. In addition, integrated RNA sequencing with DNA affinity purification sequencing (DAP-seq) analyses revealed that the OsMYB103L might specifically mediate cellulose biosynthesis and deposition by regulating OsCesAs and other genes associated with microfibril assembly. CONCLUSIONS: This study has demonstrated that down-regulation of OsMYB103L could specifically improve cellulose features and cellulose nanofibers assembly to significantly enhance biomass enzymatic saccharification under green-like and mild chemical pretreatments in rice. It has not only indicated a powerful strategy for genetic modification of plant cell walls in bioenergy crops, but also provided insights into transcriptional regulation of cellulose biosynthesis in plants.

An odorant-binding protein of Asian citrus psyllid, Diaphorina citri, participates in the response of host plant volatiles.

BACKGROUND: Odorant-binding proteins (OBPs) in insects contribute to the sensitivity of the olfactory system and connect external odorants to olfactory receptor neurons. Determination of the chemosensory functions in Diaphorina citri, a vector of the citrus Huanglongbing pathogen, may help in developing a potential target for pest management. RESULTS: Diaphorina citri showed dose-dependent electroantennogram recording (EAG) responses to 12 host plant volatiles. A two-choice behavioral trap experiment showed that four compounds (methyl salicylate, linalool, citral and R-(+)-limonene) that elicited high EAG responses also had significant attraction to adults. The expression profiles induced by these compounds were detected in nine OBP genes, DcitOBP1-9. DcitOBP3, DcitOBP6 and DcitOBP7 commonly showed significant upregulation or downregulation compared with the control. Microscale thermophoresis (MST) showed that the recombinant protein DcitOBP7 had high in vitro binding affinities (Kd < 10 µm) to methyl salicylate, linalool and R-(+)-limonene, and moderate binding affinity to citral with a Kd value of 15.95 µm. Furthermore, RNA interference (RNAi)-suppressed messenger RNA (mRNA) expression of DcitOBP7 resulted in a significant reduction in EAG activity and in adult D. citri behavioral responses to tested volatiles and the preferred host, Murraya paniculata. The hydrophilic residue Arg107 of DcitOBP7 may have a key role in binding odorants via formation of hydrogen bonds. CONCLUSION: These results show that DcitOBP7 plays an important role in the olfactory response. This finding may provide new insight into the functions of OBP families in D. citri and aid in the development of safe strategies for managing D. citri populations. © 2021 Society of Chemical Industry.

Integrated transcriptome sequencing and RNA interference reveals molecular changes in Diaphorina citri after exposure to validamycin.

Validamycin has been widely used as a specific competitive inhibitor of trehalase. In our previous research, validamycin significantly inhibited trehalase activity and chitin synthesis in Diaphorina citri, resulting in abnormal phenotypes. However, the mechanism of validamycin's action on D. citri remains unclear. Here, using a comparative transcriptome analysis, 464 differentially expressed genes (DEGs) in D. citri were identified after validamycin treatment. A Gene Ontology enrichment analysis revealed that these DEGs were mainly involved in "small molecule process", "structural molecule activity" and "transition metal ion binding". DEGs involved in chitin metabolism, cuticle synthesis and insecticide detoxification were validated by reverse transcription quantitative polymerase chain reaction. The RNA interference of D. citri chitinase-like protein ENO3 and D. citri cuticle protein 7 genes significantly affected D. citri molting. Moreover, the recombinant chitinase-like protein ENO3 exhibited a chitin-binding property, and an antimicrobial activity against Bacillus subtilis. This study provides a first insight into the molecular changes in D. citri after exposure to validamycin and identifies two effective RNA interference targets for D. citri control.