Location: European Biological Control Laboratory
2020 Annual Report
Objectives
To search for, identify, and evaluate the potential of natural enemies of pest insects, weeds, and plant diseases that affect Chinese and U.S. agriculture.
Objective 1: Explore for arthropod natural enemies of invasive arthropod pests and weedy plants identified as high priority targets by the ARS Office of National Programs, performing collections, importations and exportations in compliance with local and international regulations. High priority targets include, but are not limited to, giant cane, cogon grass, cattle fever tick, Asian longhorned beetle, emerald ash borer, brown marmorated stink bug, bean plataspid, spotted wing drosophila, spotted lantern fly, and wheat stem sawfly. (non-hypothesis driven) [NP304, Component 2, Problem Statements 2A3 and 2B3]
Objective 2: Perform taxonomic and phylogenetic studies of targets and their natural enemies, as needed to support development of biological control agents. (non-hypothesis driven) [NP304, Component 1, Problem Statement 1B]
Objective 3: Conduct studies to identify the biological and physical parameters that affect the efficacy and safety of potential agents, including climatic conditions, host specificity, effective rearing conditions, and biogeography. [NP304, Component 2, Problem Statements 2A3 and 2B3]
Approach
Survey for, collect and identify natural enemies of agricultural and landscape pests. Make field collections, establish laboratory colonies when needed, and ship candidate agents to the U.S. for further evaluation. Conduct preliminary host
specificity tests on promising parasites, predators, and beneficial microorganism. Collect, colonize, and ship to the U.S., candidates found to be safe. Cooperate with USDA and State laboratories in future collections for shipment and evaluation for release. Collect natural enemies attacking pests at differing host densities and climatic zones. All natural enemies selected for shipment will be sent directly to State or Federal quarantine facilities in the U.S. for further evaluation. Both Chinese and American scientists share the same right of obtaining biological control agents collected from the two countries. Assist in obtaining the necessary regulatory agency permissions for exporting live material from China.
Progress Report
Using DNA metabarcoding for assessing insect diversity in citrus orchards in China. The current study analyzed the composition of the insect communities in citrus orchards in Jiangxi province in China using DNA metabarcoding combined with two cloud-based data storage and analysis platforms (BOLD and mBRAVE). In addition, we compared the identified species with the insect barcodes reported from China. The results provided a valuable resource for research in a broad range of areas such as citrus pest management and beneficial insect exploration.
Determining the distribution and natural enemy complex of the Roseau cane scale in China. In 2020, Sino-ABCL continued to determine the distribution and natural enemy complex of the Roseau cane scale in China, including Beijing, Hebei, Guangdong and Yunnan. According to the survey plan, we will get 3 rounds of field survey. Up to now, Sino-ABCL together with local collaborators have completed the first round of field survey.
To identify potential toxin genes, toxin-like and venom auxiliary ploypeptides from Arma chinensis, all of protein sequences were compared using BLASTp to an exclusive database for toxins and venom proteins in the UniProt Animal Toxin Annotation Program (http://www.uniprot.org/program/Toxins). Based on the A. chinensis transcriptome data, we identified toxin gene transcripts in different developmental stages and proceeded with k-meas clustering analysis to ascertain toxin gene expression across the A. chinensis life cycle.
Application of A. chinesis for controlling fall armyworm. FAW invaded the western part of Yunnan province in January 2019. This species seemingly displays a very wide host range, with over 80 plants recorded. The most frequently consumed plants are field corn and sweet corn. In Yunnan, it caused great damage to fresh-eating maize. Sino-ABCL continued to release A. chinensis in maize field in Yunnan to control the FAW damage under local collaborators’ help.
Salivary gland secretome analysis reveals a novel mechanism of horizontal gene transfer in the brown marmorated stink bug. We aimed to investigate the composition and evolution of the secretory salivary gland proteins in the brown marmorated stink bug, Halyomorpha halys, and whether these proteins empower the insect to become a successful invader worldwide feeding across a wide host range. We re-sequenced its salivary gland transcriptome and predicted hundreds of secretory salivary gland proteins with the largest categories involved in metabolic and cellular processes, linking them to diet digestion and host manipulation. Interestingly, a group of secretory salivary gland exhibited homology to bacteria rather than to insects, and we subsequently demonstrated that H. halys acquired the genes through the mechanism of horizontal gene transfer (HGT) from bacteria.
Identification and characterization of microRNAs in immature stage of the beneficial predatory bug A. chinensis. Deep sequencing technology was used to examine the miRNAs associated with 5 distinct ages of stinkbug nymphs based on the genomic information of A. chinensis. Three genetic target prediction methodologies were used to predict the target genes of differentially expressed miRNAs, and these putative target genes were analyzed by GO annotation and KEGG pathway analysis to start to understand the miRNA regulation. In addition, some miRNAs were screened using qRT-PCR analysis to verify the consistency of deep sequencing and quantitative results.
Accomplishments
1. DNA metabarcoding. Using DNA metabarcoding for assessing insect diversity in citrus orchards in China. We identified citrus insect biodiversity by DNA metabarcoding combined with two cloud-based data storage and analysis platforms (BOLD and mBRAVE). We found more than 1300 bins that are corresponding to insects at species level. In addition, some insect species, which were not reported before in citrus planting areas in China, have been identified by DNA metabarcoding in current study.
2. Parasitoid species. Determining the distribution and natural enemy complex of the Roseau cane scale in China. Based on the collection in the summer in 2019, we found four species parasitoids emergence in Beijing, Guangzhou and Yunnan. One of the parasitoid species that has been recovered from the collections in China has tentatively been identified as Platencyrtus parkeri (another encyrtid species). To date, China is the only country from which we have recovered this species. In Beijing, they make up a relatively high proportion of what has emerged.
3. Genome Analysis. Genome wide analysis of A. chinensis to identify toxin gene family. Using the UniProt Animal Toxin Annotation Program, we finally identified 507 genes, which were assigned to 42 Pfam ID. One hundred and eighty-eight latrotoxin genes, including a-latrotoxin, a-latrocrustatoxin and d-latroinsectotoxin, accounted for the highest proportion of 37.08% of total identified A. chinensis toxin genes. By clustering analysis, we found that 67 latrotoxin genes were tandem-arrayed on six chromosomes, forming 26 gene clusters, suggesting that most A. chinensis latrotoxin genes evolved by gene duplication. The largest number of clusters is in Chromosome 5, including 34 latrotoxin genes. Based on the A. chinensis developmental transcriptome, we identified 505 toxin transcripts expressed across nymphal and adult stages and conducted k-meas clustering analysis. We found 82 up-regulated toxin genes from the 2nd instar nymph to adult stages, comparing to the 1st instar nymph stage. It provided a molecular evidence for A. chinensis predatory behavior that the 1st instar did not prey until it developed into the 2nd instar.
4. Salivary gland secretome analysis. Salivary gland secretome analysis reveals a novel mechanism of horizontal gene transfer in the brown marmorated stink bug. From the salivary gland secretome of H. halys, we identified several genes with bacterial homology, possibly gained by the insect through HGT. Despite that previous transcriptome and genome annotations allowed the detection of a number of HGT events in H. halys, likely with H. halys endosymbionts Wolbachia as gene donors, none was reported to be related with host-manipulating activities. Here, we discovered a new gene transfer event with a different bacterial lineage as the donor.
5. MicroRNAs. Identification and characterization of microRNAs in immature stage of the beneficial predatory bug A. chinensis. High throughput analyses identified a total of 1076 miRNAs from 107 raw reads, including 812 conserved miRNAs and 264 novel miRNAs with a small RNA length distribution peak of 22nt. Expression profiles showed a total of 225 miRNAs were differentially expressed among five A. chinensis nymphal instar libraries. 4303 potential gene targets for these 225 miRNAs were predicted by comprehensive analyses using miRanda, PITA and RNAhybrid. GO annotation of predicted A. chinensis target genes suggested key processes regulated by miRNAs involved biological regulation, cellular regulation, protein modification, RNA biosynthesis, and signal transduction. KEGG pathway analyses suggested A. chinensis genetic pathways regulated by miRNAs included longevity regulating, tryptophan metabolism, extracellular matrix (ECM)-receptor interaction, pentose phosphate, and sugar metabolism.
