Identification and Functional Characterization of an Omega-Class Glutathione S-Transferase Gene PcGSTO1 Associated with Cyetpyrafen Resistance in Panonychus citri (McGregor).

Cyetpyrafen is a recently developed acaricide. The citrus red mite, Panonychus citri (McGregor), has developed significant resistance to cyetpyrafen. However, the molecular mechanism underlying the cyetpyrafen resistance in P. citri remains unclear. Glutathione S-transferases (GSTs) play a critical role in arthropod pesticide resistance. This study showed that GSTs were potentially related to the resistance of P. citri to cyetpyrafen through synergistic experiments and enzyme activity analysis. An omega-family GST gene, PcGSTO1, was significantly up-regulated in the egg, nymph, and adult stages of the cyetpyrafen-resistant strain. Additionally, silencing of PcGSTO1 significantly increased the mortality of P. citri to cyetpyrafen and recombinant PcGSTO1 demonstrated the ability to metabolize cyetpyrafen. Our results indicated that the overexpression of PcGSTO1 is associated with cyetpyrafen resistance in P. citri, and they also provided valuable information for managing resistance in P. citri.

Chromosome-level genome assembly of the Asian citrus psyllid, Diaphorina citri.

Diaphorina citri is a global citrus pest. As a vector insect, it can transmit the causative agents of citrus huanglongbing, causing irreversible losses to the citrus industry. The acquisition of genomic information can provide a molecular genetic basis for effective control of D. citri. Here, the DNBSEQ™ , Oxford Nanopore Technologies, and Hi-C technologies are applied to generate a high-quality chromosome-level genome of D. citri. The genome size of D. citri was 523.78 Mb with a scaffold N50 of 47.05 Mb distributed on 13 chromosomes. A total of 250.64 Mb (47.85%) repeat sequences and 24 048 protein-coding genes were predicted. Genome resequencing of female and male individuals indicated that the sex chromosome system of D. citri is XO. Phylogenetic analysis demonstrated that D. citri and Pachypsylla venusta, which separated from their most recent common ancestor about 336.62 million years ago, were the most closely related. Additionally, we identified genes potentially involved in detoxification metabolism, pathogen transmission, and honeydew secretion for further investigation. The high-quality genome provides an important reference for developing effective management strategies of D. citri.

Effects of Candidatus Liberibacter asiaticus infection on metagenome of Diaphorina citri gut endosymbiont.

Asian citrus psyllid (Diaphorina citri, D. citri) is the important vector of "Candidatus Liberibacter asiaticus" (CLas), associated with Huanglongbing, the most devastating citrus disease worldwide. CLas can affect endosymbiont abundance of D. citri. Here, we generated the high-quality gut endosymbiont metagenomes of Diaphorina citri on the condition of CLas infected and uninfected. The dataset comprised 6616.74 M and 6586.04 M raw reads, on overage, from CLas uninfected and infected psyllid strains, respectively. Taxonomic analysis revealed that a total of 1046 species were annotated with 10 Archaea, 733 Bacteria, 234 Eukaryota, and 69 Viruses. 80 unique genera in CLas infected D. citri were identified. DIAMOND software was used for complement function research against various functional databases, including Nr, KEGG, eggNOG, and CAZy, which annotated 84543 protein-coding genes. These datasets provided an avenue for further study of the interaction mechanism between CLas and D. citri.

Accurate prediction of huanglongbing occurrence in citrus plants by machine learning-based analysis of symbiotic bacteria.

Huanglongbing (HLB), the most prevalent citrus disease worldwide, is responsible for substantial yield and economic losses. Phytobiomes, which have critical effects on plant health, are associated with HLB outcomes. The development of a refined model for predicting HLB outbreaks based on phytobiome markers may facilitate early disease detection, thus enabling growers to minimize damages. Although some investigations have focused on differences in the phytobiomes of HLB-infected citrus plants and healthy ones, individual studies are inappropriate for generating common biomarkers useful for detecting HLB on a global scale. In this study, we therefore obtained bacterial information from several independent datasets representing hundreds of citrus samples from six continents and used these data to construct HLB prediction models based on 10 machine learning algorithms. We detected clear differences in the phyllosphere and rhizosphere microbiomes of HLB-infected and healthy citrus samples. Moreover, phytobiome alpha diversity indices were consistently higher for healthy samples. Furthermore, the contribution of stochastic processes to citrus rhizosphere and phyllosphere microbiome assemblies decreased in response to HLB. Comparison of all constructed models indicated that a random forest model based on 28 bacterial genera in the rhizosphere and a bagging model based on 17 bacterial species in the phyllosphere predicted the health status of citrus plants with almost 100% accuracy. Our results thus demonstrate that machine learning models and phytobiome biomarkers may be applied to evaluate the health status of citrus plants.

Biochemical and Molecular Analysis of Field Resistance to Spirodiclofen in Panonychus citri (McGregor).

Spirodiclofen is one of the most widely used acaricides in China. The citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), is one of the most destructive citrus pests worldwide and has developed a high resistance to spirodiclofen. However, the molecular mechanism of spirodiclofen resistance in P. citri is still unknown. In this study, we identified a field spirodiclofen-resistant strain (DL-SC) that showed 712-fold resistance to spirodiclofen by egg bioassay compared to the susceptible strain. Target-site resistance was not detected as non-synonymous mutations were not found by amplification and sequencing of the ACCase gene of resistant and susceptible strains; in addition, the mRNA expression levels of ACCase were similar in both resistant and susceptible strains. The activity of detoxifying enzymes P450s and CCEs in the resistant strain was significantly higher than in the susceptible strain. The transcriptome expression data showed 19 xenobiotic metabolisms genes that were upregulated. Stage-specific expression profiling revealed that the most prominent upregulated gene, CYP385C10, in transcriptome data was significantly higher in resistant strains in all stages. Furthermore, functional analysis by RNAi indicated that the mortality caused by spirodiclofen was significantly increased by silencing the P450 gene CYP385C10. The current results suggest that overexpression of the P450 gene, CYP385C10, may be involved in spirodiclofen resistance in P. citri.

Whole genome sequencing and bulked segregant analysis suggest a new mechanism of amitraz resistance in the citrus red mite, Panonychus citri (Acari: Tetranychidae).

BACKGROUND: Amitraz is a broad-spectrum insecticide/acaricide for the control of aphids, psyllids, ticks and mites. Current evidence suggests that ticks and phytophagous mites have developed strong resistance to amitraz. Previous studies have shown that multiple mechanisms are associated with amitraz resistance in ticks, but very few reports have involved Panonychus citri. We therefore used whole genome sequencing and bulked segregant analysis (BSA) to identify the mechanism underlying P. citri's resistance to amitraz. RESULTS: High-quality assembly of the whole P. citri genome was completed, resulting in a genome of approximately 83.97 Mb and a contig N50 of approximately 1.81 Mb. Gene structure predictions revealed 11 577 genes, of which 10 940 genes were annotated. Trait-associated regions in the genome were mapped with bulked segregant analysis and 38 candidate SNPs were obtained, of which T752C had the strongest correlation with the resistant trait, located at the 5' untranslated region (UTR) of the ß-2R adrenergic-like octopamine receptor gene. The mutation resulted in the formation of a short hairpin loop structure in mRNA and gene expression was down-regulated by more than 50% in the amitraz-resistant strain. Validation of the T752C mutation in field populations of P. citri found that the correlation between the resistance ratio and the base mutation was 94.40%. CONCLUSION: Our results suggest that this 5' UTR mutation of the ß-2R octopamine receptor gene, confers amitraz resistance in P. citri. This discovery provides a new explanation for the mechanism of pest resistance: base mutations in the 5' untranslated region of target gene may regulate the susceptibility of pests to pesticides.

A Y(III)-based Metal-Organic Framework as a Carrier in Chemodynamic Therapy.

A biocompatible Y(III)-based metal-organic framework [Y4(TATB)2]·(DMF)3.5·(H2O) (ZJU-16, H3TATB= 4,4',4''-(1,3,5-triazine-2,4,6-triyl) tribenzoic acid) was synthesized, and it was adopted to load Mn2+ for chemodynamic therapy. Meanwhile, ibuprofen sodium (IBUNa), an anti-inflammatory drug, was introduced to increase the amount of Mn2+ (about 5.66 wt %) due to the low loading capacity of Mn2+. Mn&IBUNa@ZJU-16 which was loaded by Mn2+ and IBUNa exhibited significant effects of chemodynamic therapy and excellent inhibition of the 4T1 tumor cell growth, implying its long-term prospects in chemodynamic therapy and its possibility in bimodal cancer therapy.

Genetic analysis and screening of detoxification-related genes in an amitraz-resistant strain of Panonychus citri.

Panonychus citri (McGregor) is the most common pest in citrus-producing regions. Special low-toxicity acaricides, such as spirocyclic tetronic acids and mite growth inhibitors, have been used for a long time in China. However, pesticide resistance in mites is a growing problem due to the lack of new acaricide development. Wide-spectrum insecticides, such as amitraz have gained acceptance among fruit growers. An amitraz-resistant strain of P. citri was obtained by indoor screening to examine field resistance monitoring of mites to acaricides and to explore the resistant mechanism of mites against amitraz. The amitraz-resistant strain of P. citri had an LC50 value of 2361.45 mg l-1. The resistance ratio was 81.35 times higher in the resistant strain of P. citri compared with the sensitive strain. Crossing experiments between the sensitive and resistant strains of P. citri were conducted, resulting in a D value of 0.11 for F1 SS♀×RS♂ and 0.06 for F1 RS♀×SS♂. Reciprocal cross experiments showed that the dose-mortality curves for the F1 generations coincided, indicating that the resistance trait was not affected by cytoplasmic inheritance. The dose-expected response relationship was evaluated in the backcross generation and a significant difference was observed compared with the actual value. The above results indicate that the inheritance of resistance trait was incompletely dominant, governed by polygenes on the chromosome. Synergism studies demonstrated that cytochrome P450s and esterase may play important roles in the detoxification of amitraz. Based on differential gene analysis, 23 metabolism-related genes of P. citri were identified, consistent with the results of synergism studies. Real-time PCR verification implied that P450s, ABC transporters, and acetylcholinesterase might influence the detoxification of amitraz by P. citri. These results provide the genetic and molecular foundation for the management of pest mite resistance.

Expression of exogenous dsRNA by Lecanicillium attenuatum enhances its virulence to Dialeurodes citri.

BACKGROUND: Dialeurodes citri is an important pest in citrus-producing areas of the world. Lecanicillium attenuatum parasitizes D. citri and kills it, suggesting a potential approach for the biological control of pests. However, the low virulence of the fungus and its slow rate of killing have limited its commercial competitiveness. The objective reason for these disadvantages is immunological rejection by the host. Our strategy was to use fungi to express the double-stranded RNA (dsRNA) of the host immune genes. The fungal hyphae release siRNA at the time of infection, thus interfering with the expression of immune genes in the host and facilitating fungal invasion. RESULTS: We selected prophenoloxidase (DcPPO), prophenoloxidase-activating factor (DcPPO-AF), and lysozyme (DcLZM) as target genes to construct intron-splicing hairpin RNA expression vectors and to successfully obtain transgenic fungi. Two days after infection, the immune genes of D. citri showed varying degrees of silencing compared with those in the positive control group. The median lethal concentration (LC50 ; spores mL-1 ) values of La::GFP, La::DcPPO, La::DcPPO-AF, and La::DcLZM were 9.63 × 104 , 2.66 × 104 , 1.21 × 105 , and 3.31 × 104 , respectively. The 50% lethal time (LT50 ) values of these fungi were 5.15, 3.60, 5.34, and 4.04 days, respectively. The virulence of La::DcPPO and La::DcLZM increased 3.62- and 2.91-fold, respectively, and their LT50 decreased by 30.10% and 21.55%, respectively. CONCLUSIONS: The results indicate that this method, which uses tens of thousands of hyphae to inject dsRNA to improve the virulence of transgenic fungi, can play a greater role in the prevention and control of pests in the future. © 2018 Society of Chemical Industry.

Expression of fascin in thyroid neoplasms: a novel diagnostic marker.

PURPOSE: Fascin, an actin-bundling protein, is markedly upregulated in several epithelial tumors and its expression often correlates with high-grade, extensive invasion, and distant metastasis. However, reports about fascin expression in endocrine tumors remain rare. The aim of the present study was to assess the diagnostic significance of fascin in thyroid neoplasms. METHODS: Thyroid samples from 177 cases were examined for fascin and Ki-67 expression by immunohistochemistry. RESULTS: Fascin immunoreactivity was negative in normal follicles and nodular goiter. Fascin immunostaining was positive in 62.1% (41/66) of thyroid carcinomas and 26.4% (19/72) of thyroid adenomas; the difference being significant (P < 0.0001). In thyroid papillary carcinoma, upregulation of fascin was associated with both the Ki-67 labeling index and the occurrence of lymph node metastasis. CONCLUSION: Fascin may be a novel marker to distinguish thyroid carcinoma from benign lesions and may be involved in the proliferation and metastasis of papillary carcinoma.

[Study on N-nitroso compound in food and its relevant risk factors for esophageal cancer].

OBJECTIVE: To study multiple risk factors of N-nitroso compounds (NOC) in high- and low-risk areas for esophageal cancer in southern China. METHOD: The samples of 24-hr diets and 12-hr overnight urine were collected from 120 male healthy subjects (35-64 years old) selected by a 3-stage random cluster sample procedure in each of the high-risk area (Nanao County) and low-risk area (Lufeng County) for esophageal cancer. The urinary samples were respectively collected from undosed subjects, subjects ingested 500 mg L-proline (together with 200 mg ascorbic acid ) and subjects ingested 500 mg proline. The levels of total NOC (TNOC), N-nitrosamino acids (NAAs), volatile N-nitroso compounds and reductive ascorbic acid (VC) in the samples were measured. By unconditional logistic stepwise regression model, we analyzed the association between the multiple factors of NOC and esophageal cancer mortality. The factors included the intake and excretion levels of various kinds of NOC, the ability of NAAs endogenous formation and its inhibition by VC, and nutrition status of VC in the body. RESULTS: The results of unconditional logistic stepwise regression showed that risk factors entered the model were diet TNOC content (OR 9.613, 95% CI 1.921-48.115) and urinary NAAs level after ingested VC (OR 1.137, 95 % CI 1.001-1.298). CONCLUSION: The higher level of diet TNOC and the lower inhibition ability of NOC endogenous formation by VC were important risk factors on NOC etiology of esophageal cancer in southern China.