Functional Analysis of Insecticide Inhibition and Metabolism of Six Glutathione S-Transferases in the Rice Stem Borer, Chilo suppressalis.

The glutathione S-transferases (GSTs) are important detoxifying enzymes in insects. Our previous studies found that the susceptibility of Chilo suppressalis to abamectin was significantly increased when the CsGST activity was inhibited by glutathione (GSH) depletory. In this study, the potential detoxification mechanisms of CsGSTs to abamectin were explored. Six CsGSTs of C. suppressalis were expressed in vitro. Enzymatic kinetic parameters including Km and Vmax of recombinant CsGSTs were determined, and results showed that all of the six CsGSTs were catalytically active and displaying glutathione transferase activity. Insecticide inhibitions revealed that a low concentration of abamectin could effectively inhibit the activities of CsGSTs including CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1. However, the in vitro metabolism assay found that the six CsGSTs could not metabolize abamectin directly. Additionally, the glutathione transferase activity of CsGSTs in C. suppressalis was significantly increased post-treatment with abamectin. Comprehensive analysis of the results in present and our previous studies demonstrated that CsGSTs play an important role in detoxification of abamectin by catalyzing the conjugation of GSH to abamectin in C. suppressalis, and the high binding affinities of CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1 with abamectin might also suggest the involvement of CsGSTs in detoxification of abamectin via the noncatalytic passive binding and sequestration instead of direct metabolism. These studies are helpful to better understand the detoxification mechanisms of GSTs in insects.

Identification and Validation of ATP-Binding Cassette Transporters Involved in the Detoxification of Abamectin in Rice Stem Borer, Chilo suppressalis.

Chilo suppressalis has developed high levels of resistance to abamectin in many areas of China, while the underline resistance mechanisms are largely unclear. ATP-binding cassette (ABC) transporters function in transporting a large diversity of substrates including insecticides and play important roles in the detoxification metabolism of insects. In this study, synergism bioassay revealed that the ABC transporters were involved in the detoxification of C. suppressalis to abamectin. Six ABC transporter genes were upregulated in C. suppressalis after abamectin exposure, among which five genes CsABCC8, CsABCE1, CsABCF1, CsABCF2, and CsABCH1 were induced in the detoxification-related tissues. In addition, the five ABC transporters were recombinantly expressed in Sf9 cells, and the cytotoxicity assay showed that the viabilities of cells expressing CsABCC8 or CsABCH1 were significantly increased when compared with the viabilities of cells expressing EGFP after abamectin, chlorantraniliprole, cyantraniliprole, fipronil, and chlorpyrifos treatment, respectively. Overexpression of CsABCE1 significantly increased the viabilities of cells to abamectin, chlorantraniliprole, deltamethrin, and indoxacarb exposure, respectively. These results suggested that CsABCC8, CsABCE1, and CsABCH1 might participate in the detoxification and transport of abamectin and several other classes of insecticides in C. suppressalis. Our study provides valuable insights into the transport-related detoxification mechanisms in C. suppressalis and other insects.

Identification and characterization of glutathione S-transferases and their potential roles in detoxification of abamectin in the rice stem borer, Chilo suppressalis.

The glutathione S-transferases (GSTs) are a kind of metabolic enzymes and participate in the detoxification metabolism of xenobiotics in various organisms. In insects, GSTs play important roles in the development of insecticide resistance and antioxidant protection. The rice stem borer Chilo suppressalis is one of the most damaging pests in rice and has developed high levels of resistance to abamectin in many areas of China, whereas the potential resistance mechanisms of C suppressalis to abamectin are still unclear. In the present study, a total of 23 CsGSTs genes were identified from the C. suppressalis transcriptome and genome, including 21 cytosolic and two microsomal CsGSTs. The cytosolic CsGSTs were further classified into seven categories based on phylogenetic analysis, and their sequence characteristics and genome structures were also analyzed. Synergism study revealed that the susceptibility of C. suppressalis to abamectin was increased significantly when the CsGSTs were inhibited by diethyl maleate (DEM). Sixteen CsGSTs genes were up-regulated in C. suppressalis larvae after treatment with abamectin, among which four CsGSTs genes including CsGSTe2, CsGSTe4, CsGSTo4 and CsGSTu1 were significantly induced in the midgut and fat body tissues. These results indicated that CsGSTs were associated with the detoxification of C. suppressalis to abamectin, and CsGSTe2, CsGSTe4, CsGSTo4 and CsGSTu1 might play important roles in the insecticide detoxification or antioxidant protection in C. suppressalis. Our present study provides valuable information on C. suppressalis GSTs, and are helpful in understanding the contributions of GSTs in abamectin detoxification in C. suppressalis and other insects.

Flonicamid and knockdown of inward rectifier potassium channel gene CsKir2B adversely affect the feeding and development of Chilo suppressalis.

BACKGROUND: The selective insecticide flonicamid shows highly insecticidal activities against piercing-sucking insects and has been widely used for the control of Hemipteran insect pests, whereas its effects on Lepidopteran insect pests remain largely unknown. Recently, inward rectifier potassium (Kir) channel has been verified to be a target of flonicamid, however, functional characterization of Lepidopteran Kir genes is still lacking. RESULTS: Flonicamid shows no insecticidal toxicity against Chilo suppressalis larvae. However, the feeding and growth of larvae were reversibly inhibited by flonicamid (50-1200 mg L-1 ). Flonicamid treatment also remarkably reduced and delayed the pupation and eclosion of Chilo suppressalis. Additionally, five distinct Kir channel genes (CsKir1, CsKir2A, CsKir2B, CsKir3A and CsKir3B) were cloned from Chilo suppressalis. Expression profiles analysis revealed that CsKir2A was predominately expressed in the hindgut of larvae, whereas CsKir2B had high expressions in the Malpighian tubules and hindgut. RNA interference (RNAi)-mediated knockdown of CsKir2B significantly reduced the growth and increased the mortalities of larvae, whereas silencing of CsKir2A had no obvious effects on Chilo suppressalis. CONCLUSION: Flonicamid exhibits adverse effects on the growth and development of Chilo suppressalis. CsKir2B might be involved in the feeding behavior of Chilo suppressalis. These results provide valuable information on the effects of flonicamid on non-target insects as well as the function of insect Kir channels, and are helpful in developing new insecticide targeting insect Kir channels. © 2020 Society of Chemical Industry.

Identification and transcriptional response of ATP-binding cassette transporters to chlorantraniliprole in the rice striped stem borer, Chilo suppressalis.

BACKGROUND: As the largest transporter gene family in metazoans, ATP-binding cassette (ABC) transporters regulate the efflux of a broad spectrum of substrates from the cytoplasm to the outside of the cell. In arthropods, ABC transporters are involved in phase III of the detoxification process, and play important roles in the metabolism and transport of insecticides. RESULTS: We identified 54 ABC transporters from the genome and transcriptome of Chilo suppressalis, one of the most damaging pests of rice in China. The identified ABC transporters were classified into eight subfamilies (ABCA to ABCH) based on NCBI BLAST and phylogenetic analysis. Synergism studies showed that treatment with verapamil, a potent inhibitor of ABC transporters, resulted in significantly increased toxicity of chlorantraniliprole against C. suppressalis larvae. Among the 21 tested ABC genes, three ABC transporter genes including CsABCC8, CsABCG1C and CsABCH1 were significantly upregulated after chlorantraniliprole treatment. CONCLUSION: ABC transporters play important roles in the detoxification and transport of chlorantraniliprole in C. suppressalis. The results from our study provide valuable information on C. suppressalis ABC transporters, and are helpful in understanding the roles of ABC transporters in chlorantraniliprole resistance mechanisms in C. suppressalis and other insect pests. © 2020 Society of Chemical Industry.

Transcriptome analysis reveals global gene expression changes of Chilo suppressalis in response to sublethal dose of chlorantraniliprole.

The anthranilic diamide insecticide chlorantraniliprole was widely used for the controlling of Chilo suppressalis in China. Previous studies have revealed sublethal effects of chlorantraniliprole on the development and reproduction of C. suppressalis. In the present study, a comparative transcriptome analysis was performed to investigate the global gene expression changes in third-instar larvae of C. suppressalis after exposure to LC30 of chlorantraniliprole. A total of 908 differentially expressed genes (DEGs) were identified including 441 up-regulated and 467 down-regulated unigenes. Gene enrichment analysis revealed that the down-regulated DEGs were mainly linked to carbohydrate, energy, lipid and amino acid metabolisms as well as posttranslational modification, while most of the DEGs involved in signal transduction were up-regulated. Specifically, the DEGs encoding detoxification related genes were identified and validated by RT-qPCR. Our results provide a basis for understanding the molecular mechanisms of chlorantraniliprole action and detoxification in C. suppressalis and other insect pests.

Molecular characterization of glutamate-gated chloride channel and its possible roles in development and abamectin susceptibility in the rice stem borer, Chilo suppressalis.

Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems, and are of considerable interest in insecticide discovery. The full length cDNA encoding CsGluCl was cloned from the rice stem borer Chilo suppressalis (Walker). Multiple cDNA sequence alignment revealed three variants of CsGluCl generated by alternative splicing of exon 3 and exon 9. While all the transcripts were predominantly expressed in both nerve cord and brain, the expression patterns of these three variants differed among other tissues and developmental stages. Specifically, the expression level of CsGluCl C in cuticle was similar to that in nerve cord and brain, and was the predominant variant in late pupae and early adult stages. Both injection and oral delivery of dsGluCl significantly reduced the mRNA level of CsGluCl. Increased susceptibility to abamectin and reduced larvae growth and pupation rate were observed in dsGluCl-treated larvae. Thus, our results provide the evidence that in addition to act as the target of abamectin, GluCls also play important physiological roles in the development of insects.

Author Correction: Identifying long-term stable refugia for relict plant species in East Asia.

In the original version of this Article, an incorrect sample size was provided for the number of relict species (443 instead of 442) and the number of relict forests (423 instead of 422). These errors have been corrected in both the PDF and HTML versions of the Article.

Identifying long-term stable refugia for relict plant species in East Asia.

Today East Asia harbors many "relict" plant species whose ranges were much larger during the Paleogene-Neogene and earlier. The ecological and climatic conditions suitable for these relict species have not been identified. Here, we map the abundance and distribution patterns of relict species, showing high abundance in the humid subtropical/warm-temperate forest regions. We further use Ecological Niche Modeling to show that these patterns align with maps of climate refugia, and we predict species' chances of persistence given the future climatic changes expected for East Asia. By 2070, potentially suitable areas with high richness of relict species will decrease, although the areas as a whole will probably expand. We identify areas in southwestern China and northern Vietnam as long-term climatically stable refugia likely to preserve ancient lineages, highlighting areas that could be prioritized for conservation of such species.

Factors influencing phototaxis in nocturnal migrating birds.

Many migratory bird species fly during the night (nocturnal migrants) and have been shown to display some phototaxis to artificial light. During 2006 to 2009, we investigated phototaxis in nocturnal migrants at Jinshan Yakou in Xinping County (N23°56', E101°30'; 2400 m above sea-level), and at the Niaowang Mountain in Funing County (N23°30', E105°35'; 1400 m above sea-level), both in the Yunnan Province of Southwest China. A total of 5069 birds, representing 129 species, were captured by mist-netting and artificial light. The extent of phototaxis effect on bird migration was examined during all four seasons, three phases of the moon, and under two weather conditions (mist and wind). Data were statistically analyzed to determine the extent to which these factors may impact phototaxis of nocturnal migrants. The results point to phototaxis in birds migrating in the spring and autumn, especially in the autumn. Furthermore, migrating birds were more readily attracted to artificial lights during nights with little moonlight, mist, and a headwind. Regardless of the initial orientation in which birds flew, either following the wind or against the wind, birds would always fly against the wind when flying towards the light. This study broadens our understanding of the nocturnal bird migration, potentially resulting in improved bird ringing practices, increased awareness, and better policies regarding bird protection.

[Impacts of human disturbance on the species composition of higher plants in the wetlands around Dianchi Lake, Yunnan Province of Southwest China].

Introducing higher plants to build semi-natural wetland ecosystem is one of the key approaches to restore the wetlands and lakes that suffered from serious pollution and destruction. Based on the investigation data from 128 quadrats at 26 sampling sites in the wetlands around Dianchi Lake in December 2011-October 2012, and in combining with the references published in the 1960s, this paper discussed the impacts of human activities on the species composition of higher plants in the wetlands around the Lake. In 2012, there were 299 species of 88 families in the wetlands, of which, 181 species were native species, and 118 species were alien ones (including 32 invasive species). Of the 42 species of hydrophytes in the total species, 13 species were alien ones (including 2 invasive species). In comparing with the species data recorded in the 1960s, 232 plants were newly recorded and 43 species disappeared in 2012. Aquatic plants changed obviously. The decreased species were 2 submerged plants, 2 floating plants, and 5 floating leaved plants, and the increased species were 8 emergent plants. Fourteen community types were identified by cluster analysis, of which, the main communities were those dominated by alien species including Pistia stratiotes and Alternanthera philoxeroides. As compared with the data in the 1960s, the plant communities dominated by native species such as Ottelia acuminate and Vallisneria natans were not found presently. Therefore, in the practice of introducing higher plants to restore the degraded wetlands and lakes, it would be necessary to scientifically and appropriately select and blend plant species to avoid the wetland degradation by human activities.

Evidence for the persistence of wild Ginkgo biloba (Ginkgoaceae) populations in the Dalou Mountains, southwestern China.

PREMISE OF THE STUDY: The possible persistence of wild Ginkgo biloba populations in China has long been debated but never scientifically confirmed. We test our hypothesis that the extant Ginkgo populations in the Dalou Mountains (SW China) represent fragments of the original natural Ginkgo range and offer a range of pertinent perspectives on the living fossil Ginkgo's history, prehistory, ecology, and place in human culture-all important aspects of this highly valued species. METHODS: We analyzed the vegetation of the study area, determined the population age structure of Ginkgo, and compared it to existing fossil records. For supporting material, we also examined records of the lack of human presence before the mid-17th century in the area, the local people's beliefs regarding preservation of the forests and existing genetic studies. KEY RESULTS: Current species composition of Ginkgo forests in the Dalou Mountains agrees closely with floristic assemblages from fossil records bearing G. biloba. Current populations are found in habitats similar to those of fossil Ginkgo, which, as today, favored rock crevices. Female to male ratios are 3:2. Estimated ages for many of the trees show that Ginkgo was present in this area prior to human settlement and indigenous peoples of this area are unlikely to have planted Ginkgo because of traditional beliefs. Our results agree with existing genetic studies that show that these mountains were glacial refugia for G. biloba. CONCLUSIONS: The corroborative evidence confirms the finding that these populations represent fragments of the original natural Ginkgo in the valley and lower mountain slopes of the Dalou Mountains.

[Ecological issues and loss risk of cold-resistant rubber germplasm resource in Xishuangbanna].

Based on the field investigation data and related literatures, this paper analyzed the ecological issues in the breeding and utilization of highly cold-resistant and high-yielding rubber germplasm in Xishuangbanna, and the loss risk of this germplasm. The rubber farmers in Xishuangbanna had built a cycle pattern of establishing rubber nursery and managing rubber plantations to develop the local rubber industry, but the unlimited utilization of this germplasm made the rubber plantations expand to the highland with an elevation of 1300 m, resulting in the ecological issues such as the destruction of natural forests and species, decline of soil fertility, regional drought, rubber diseases and insect pests, and human-elephant conflicts. This germplsm and its intellectual property were under transnational loss without any control, making even more potential ecological and economic risks. Great attentions should be paid by the related organizations to take effective measures to conserve this germplsm resource to reduce its potential risks.

[Perception and attitude of rural community to the construction of Asian elephant conservation corridors in Xishuangbanna].

By using contingent valuation method (CVM), an investigation was made from November 2007 to March 2008 on the perception and attitude of 196 households in 5 villages within 2 planned Asian elephant conservation corridors in Xishuangbanna to the construction of the corridors. 80.61% of the interviewees conditionally supported the corridors construction. The main factors affecting the interviewees' support willingness included their education level, per capita income, and perceptions to Asian elephant protection, human-elephant relations, and corridor utilization patterns and its beneficiaries, among which, the interviewees' awareness of Asian elephant conservation, corridor utilization patterns, and corridor beneficiaries had strong influence on the support willingness, with the correlation coefficient being 0.231, 0.236, and -0.304, respectively. The rural community holding the land tenure played a key role in the corridors construction. To effectively design and planning the construction of biological conservation corridor, it is necessary to have a deep understanding on the perceptions and attitudes of rural community to the construction of the corridor and to obtain their support and participation for this construction.