A 2-Cys Peroxiredoxin in Response to Oxidative Stress in the Pine Wood Nematode, \u3cem\u3eBursaphelenchus xylophilus\u3c/em\u3e

The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease that has devastated pine forests in Asia. Parasitic nematodes are known to have evolved antioxidant stress responses that defend against host plant defenses. In this study, the infestation of whitebark pine, Pinus bungean, with B. xylophilus led to a significant increase in plant hydrogen peroxide (H2O2) and salicylic acid levels. Correspondingly, the expression of an antioxidative enzyme, 2-Cysteine peroxiredoxin (BxPrx), was elevated in B. xylophilus following the H2O2 treatments. Recombinant BxPrx, a thermal stabile and pH tolerant enzyme, exhibited high level of antioxidant activity against H2O2, suggesting that it is capable of protecting cells from free radical attacks. Immunohistochemical localization study showed that BxPrx was broadly expressed across different tissues and could be secreted outside the nematode. Finally, the number of BxPrx homologs in both dauer-like and fungi-feeding B. xylophilus were comparable based on bioinformatics analysis of existing EST libraries, indicating a potential role of BxPrx in both propagative and dispersal nematodes. These combined results suggest that BxPrx is a key genetic factor facilitating the infestation and distribution of B. xylophilus within pine hosts, and consequently the spread of pine wilt disease

Comparative Profiling of MicroRNAs in the Winged and Wingless English Grain Aphid, \u3cem\u3eSitobion avenae\u3c/em\u3e (F.) (Homoptera: Aphididae)

MicroRNAs (miRNAs) are short single-stranded non-coding RNAs that regulate gene expression, particularly during development. In this study, 345 miRNAs were identified from the English green aphid, Sitobion avenae (F.), of which 168 were conserved and 177 were S. avenae-specific. Quantitative comparison of miRNA expression levels indicated that 16 and 12 miRNAs were significantly up-regulated in winged and wingless S. avenae small RNA libraries, respectively. Differential expression of these miRNAs was confirmed by real-time quantitative RT-PCR validation. The putative transcript targets for these candidate miRNAs were predicted based on sequences from a model species Drosophila melanogaster and four aphid species Acyrthosiphon pisum, Myzus persicae, Toxoptera citricida, and Aphis gosspii. Gene Ontology and KEGG pathway analyses shed light on the potential functions of these miRNAs in the regulation of genes involved in the metabolism, development and wing polyphenism of S. avenae

Genome Editing of \u3cem\u3eWnt-1\u3c/em\u3e, a Gene Associated with Segmentation, via CRISPR/Cas9 in the Pine Caterpillar Moth, \u3cem\u3eDendrolimus punctatus\u3c/em\u3e

The pine caterpillar moth, Dendrolimus punctatus, is a devastating forest pest. Genetic manipulation of this insect pest is limited due to the lack of genomic and functional genomic toolsets. Recently, CRISPR/Cas9 technology has been demonstrated to be a promising approach to modify the genome. To investigate gene functions during the embryogenesis, we introduced CRISPR/Cas9 system in D. punctatus to precisely and effectively manipulate gene expressions inmutant embryos. Compared to controls, knocking out of DpWnt-1, a gene well known for its role in the early body planning, led to high embryonic mortality. Among these mutants, 32.9% of the embryos and larvae showed an abnormal development. DpWnt-1 mutants predominantly exhibited abnormal posterior segments. In addition, multiple phenotypes were observed, including the loss of limbs and the head deformation, suggesting that DpWnt-1 signaling pathway is necessary for anterior segmentation and appendage development. Overall, our results demonstrate that CRISPR/Cas9 system is feasible and efficient in inducing mutations at a specific locus in D. punctatus. This study not only lays the foundation for characterizing gene functions in a non-model species, but also facilitates the future development of pest control alternatives for a major defoliator

Cloning and Characterization of a Pyrethroid Pesticide Decomposing Esterase Gene, \u3cem\u3eEst3385\u3c/em\u3e, from \u3cem\u3eRhodopseudomonas palustris\u3c/em\u3e PSB-S

Full length open reading frame of pyrethroid detoxification gene, Est3385, contains 963 nucleotides. This gene was identified and cloned based on the genome sequence of Rhodopseudomonas palustris PSB-S available at the GneBank. The predicted amino acid sequence of Est3385 shared moderate identities (30–46%) with the known homologous esterases. Phylogenetic analysis revealed that Est3385 was a member in the esterase family I. Recombinant Est3385 was heterologous expressed in E. coli, purified and characterized for its substrate specificity, kinetics and stability under various conditions. The optimal temperature and pH for Est3385 were 35 °C and 6.0, respectively. This enzyme could detoxify various pyrethroid pesticides and degrade the optimal substrate fenpropathrin with a Km and Vmax value of 0.734 ± 0.013 mmol·l−1 and 0.918 ± 0.025 U·µg−1, respectively. No cofactor was found to affect Est3385 activity but substantial reduction of enzymatic activity was observed when metal ions were applied. Taken together, a new pyrethroid degradation esterase was identified and characterized. Modification of Est3385 with protein engineering toolsets should enhance its potential for field application to reduce the pesticide residue from agroecosystems

Bacterial Symbionts, \u3cem\u3eBuchnera\u3c/em\u3e, and Starvation on Wing Dimorphism in English Grain Aphid, \u3cem\u3eSitobion avenae\u3c/em\u3e (F.) (Homoptera: Aphididae)

Wing dimorphism in aphids can be affected by multiple cues, including both biotic (nutrition, crowding, interspecific interactions, the presence of natural enemies, maternal and transgenerational effects, and alarm pheromone) and abiotic factors (temperature, humidity, and photoperiod). The majority of the phloem-feeding aphids carry Buchnera, an obligate symbiotic proteobacteria. Buchnera has a highly reduced genome size, but encode key enzymes in the tryptophan biosynthetic pathway and is crucial for nutritional balance, development and reproduction in aphids. In this study, we investigated the impact of two nutritional-based biotic factors, symbionts and starvation, on the wing dimorphism in the English grain aphid, Sitobion avenae, a devastating insect pest of cereal crops (e.g., wheat) worldwide. Elimination of Buchnera using the antibiotic rifampicin significantly reduced the formation of winged morphs, body mass, and fecundity in S. avenae. Furthermore, the absence of this primary endosymbiont may disrupt the nutrient acquisition in aphids and alter transgenerational phenotypic expression. Similarly, both survival rate and the formation of winged morphs were substantially reduced after neonatal (\u3c24h \u3eold) offspring were starved for a period of time. The combined results shed light on the impact of two nutritional-based biotic factors on the phenotypic plasticity in aphids. A better understanding of the wing dimorphism in aphids will provide the theoretical basis for the prediction and integrated management of these phloem-feeding insect pests

Genome Editing of \u3cem\u3eWnt-1\u3c/em\u3e, a Gene Associated with Segmentation, via CRISPR/Cas9 in the Pine Caterpillar Moth, \u3cem\u3eDendrolimus punctatus\u3c/em\u3e

The pine caterpillar moth, Dendrolimus punctatus, is a devastating forest pest. Genetic manipulation of this insect pest is limited due to the lack of genomic and functional genomic toolsets. Recently, CRISPR/Cas9 technology has been demonstrated to be a promising approach to modify the genome. To investigate gene functions during the embryogenesis, we introduced CRISPR/Cas9 system in D. punctatus to precisely and effectively manipulate gene expressions inmutant embryos. Compared to controls, knocking out of DpWnt-1, a gene well known for its role in the early body planning, led to high embryonic mortality. Among these mutants, 32.9% of the embryos and larvae showed an abnormal development. DpWnt-1 mutants predominantly exhibited abnormal posterior segments. In addition, multiple phenotypes were observed, including the loss of limbs and the head deformation, suggesting that DpWnt-1 signaling pathway is necessary for anterior segmentation and appendage development. Overall, our results demonstrate that CRISPR/Cas9 system is feasible and efficient in inducing mutations at a specific locus in D. punctatus. This study not only lays the foundation for characterizing gene functions in a non-model species, but also facilitates the future development of pest control alternatives for a major defoliator

Reference Gene Selection for Transcriptional Profiling in \u3cem\u3eCryptocercus punctulatus\u3c/em\u3e, an Evolutionary Link between Isoptera and Blattodea

The subsocial life style and wood-feeding capability of Cryptocercus gives us an evolutionary key to unlock some outstanding questions in biology. With the advent of the Genomics Era, there is an unprecedented opportunity to address the evolution of eusociality and the acquisition of lignocellulases at the genetic level. However, to quantify gene expression, an appropriate normalization strategy is warranted to control for the non-specific variations among samples across different experimental conditions. To search for the internal references, 10 housekeeping genes from a gut transcriptome of a wood-feeding cockroach, Cryptocercus punctulatus, were selected as the candidates for the RT-qPCR analysis. The expression profiles of these candidates, including ACT, EF1α, GAPDH, HSP60, HSP70, αTUB, UBC, RPS18, ATPase and GST, were analyzed using a panel of analytical tools, including geNorm, NormFinder, BestKeeper, and comparative ΔCT method. RefFinder, a comprehensive ranking system integrating all four above-mentioned algorithms, rated ACT as the most stable reference gene for different developmental stages and tissues. Expression analysis of the target genes, Hex-1 and Cell-1, using the most or the least appropriate reference genes and a single or multiple normalizers signified this research. Our finding is the first step toward establishing a standardized RT-qPCR analysis in Cryptocercus

Selection of Reference Genes for RT-qPCR Analysis in a Predatory Biological Control Agent, \u3cem\u3eColeomegilla maculata\u3c/em\u3e (Coleoptera: Coccinellidae)

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying gene expression across various biological processes, of which requires a set of suited reference genes to normalize the expression data. Coleomegilla maculata (Coleoptera: Coccinellidae), is one of the most extensively used biological control agents in the field to manage arthropod pest species. In this study, expression profiles of 16 housekeeping genes selected from C. maculata were cloned and investigated. The performance of these candidates as endogenous controls under specific experimental conditions was evaluated by dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ΔCt method. In addition, RefFinder, a comprehensive platform integrating all the above-mentioned algorithms, ranked the overall stability of these candidate genes. As a result, various sets of suitable reference genes were recommended specifically for experiments involving different tissues, developmental stages, sex, and C. maculate larvae treated with dietary double stranded RNA. This study represents the critical first step to establish a standardized RT-qPCR protocol for the functional genomics research in a ladybeetle C. maculate. Furthermore, it lays the foundation for conducting ecological risk assessment of RNAi-based gene silencing biotechnologies on non-target organisms; in this case, a key predatory biological control agent

Detection and Epidemic Dynamic of ToCV and CCYV with \u3cem\u3eBemisia tabaci\u3c/em\u3e and Weed in Hainan of China

Background: In recent years, two of the crinivirus, Tomato chlorosis virus (ToCV) and Cucurbit chlorotic yellows virus (CCYV) have gained increasing attention due to their rapid spread and devastating impacts on vegetable production worldwide. Both of these viruses are transmitted by the sweet potato whitefly, Bemisia tabaci (Gennadius), in a semi-persistent manner. Up to now, there is still lack of report in Hainan, the south of China. Methods: We used observational and experimental methods to explore the prevalence and incidence dynamic of CCYV and ToCV transmitted by whiteflies in Hainan of China. Results: In 2016, the chlorosis symptom was observed in the tomato and cucumber plants with a large number of B. tabaci on the infected leaves in Hainan, China, with the incidence rate of 69.8% and 62.6% on tomato and cucumber, respectively. Based on molecular identification, Q biotype was determined with a viruliferous rate of 65.0% and 55.0% on the tomato and cucumber plants, respectively. The weed, Alternanthera philoxeroides near the tomato and cucumber was co-infected by the two viruses. Furthermore, incidence dynamic of ToCV and CCYV showed a close relationship with the weed, Alternanthera philoxeroides, which is widely distributed in Hainan. Conclusion: Our results firstly reveal that the weed, A. philoxeroides is infected by both ToCV and CCYV. Besides, whiteflies showed a high viruliferous rate of ToCV and CCYV. Hainan is an extremely important vegetable production and seed breeding center in China. If the whitefly can carry these two viruses concurrently, co-infection in their mutual host plants can lead to devastating losses in the near future