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- Widmer, Timothy
- Norrington, Bryan
- 2023
- 2022
- Crop Protection & Quarantine (304)

Research Project: Exploration for Natural Enemies in China for Citrus psyllid in the U.S. **Location:
                            Office of International Research Engagement and Cooperation** 2023 Annual Report **Objectives**
                                

Objective 1: Discover, identify and initiate the development of new biological control agents for citrus psyllid [NP304, C3, PS 3A]
Sub-objective 1.A: Setup malaise traps to collect insects in representative geographical locations in Yunnan and Guangdong provinces in China.
Sub-objective 1.B: Apply DNA metabarcoding technology for high throughput analysis of the collected samples, establish corresponding DNA barcode database.
Sub-objective 1.C: Use sentinel and wild egg masses/ nymphs of citrus psyllid to examine parasitism and the species composition of natural enemies.
Sub-objective 1.D: Field survey and collecting predators observed to be actively feeding on ACP, and parasitoids emerging from ACP.

Objective 2: Evaluate the biocontrol potential of biocontrol agents of citrus psyllid under laboratory and field conditions and develop the technology of mass rearing. [NP304, C3, PS 3B 3C]
Sub-objective 2.A: Evaluate the biocontrol potential of biocontrol agents of citrus psyllid under laboratory and field conditions.
Suitability and preference of predators.
Sub-objective 2.B: Develop the technology for mass rearing of some biocontrol agents. **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**
                                

3a. Under Objective 1, Sub-Objective 1A- Continuous insect sample collection. local collaborators and Sino-ABCL research scientists have set up in the main citrus growing areas of Yunan and Guangdong provinces in China.  Guangdong and Yunnan are the major citrus-producing provinces in China. There is rich insect biodiversity in Kunming city in Yunnan and Guangzhou city in Guangdong, where humid climatic conditions predominate.  Malaise traps have been set up in main citrus growing areas of Kunming and Guangzhou in China. Citrus orchards without a history of pesticide use will be selected as the experimental sites. Insects were sampled using Malaise traps deployed in citrus orchards producing a total of 300 pooled weekly samples.

3b. Under Objective 1, Sub-Objective 1B- Insect sample DNA metabarcoding data analysis. Sino-ABCL started sequencing and DNA metabarcoding the samples collected from citrus orchards. Widespread interest in metabarcoding has resulted in data proliferation and the development of computational tools to aid data analysis. One such tool, mBRAVE, the Multiplex Barcode Research and Visualization Environment, is a data storage and analytics platform with standardized pipelines and a sophisticated web interface designed to transform raw HTS data into biological insights. mBRAVE integrates common analytical methods and links to the BOLD System for access to reference datasets. The current study analyzed the composition of insect communities in citrus orchards using DNA metabarcoding followed by data analysis on BOLD and mBRAVE. The species revealed by BIN matches on BOLD were then searched in the literature to allow their classification into pest, parasitoid, predator, or pollinator.

3c. Under Objective 1, Sub-Objective 1C: Use sentinel and wild egg masses/ nymphs of citrus psyllid to examine parasitism and the species composition of natural enemies. We have established citrus psyllid populations in the laboratory by screening the host plants, that can produce enough sentinel egg masses of ACP for the field experiments. We used Murraya exotica and citrus as the host plants for rearing the citrus psyllid in our laboratory. The population of citrus psyllid has been maintained in the laboratory.

3d. Under Objective 1, Sub-objective 1.D: Field survey and collecting predators observed to be actively feeding on ACP, and parasitoids emerging from ACP and Sub-objective 2.A: Evaluate the biocontrol potential of biocontrol agents of citrus psyllid under laboratory and field conditions. Tamarixia radiata (Hymenoptera: Eulophidae) is an ectoparasitoid of the Asian citrus psyllid. We collected T. radiata in 5th instar ACP mummies and collected the specimens for morphological research and evaluation of biocontrol potential. In addition, we also collected predators including lady beetle, lacewing and Arma chinensis for the research of predation and evaluation of biocontrol potential as well as synergistic use with insecticides.

3e. Under Objective 2, Sub-objective 2.B: Develop the technology for mass rearing of some biocontrol agents, we continue to develop the technology of mass rearing of biological agents.  Large numbers of high-quality biological control agents are fundamental prerequisites to successful integration of augmentative biological control and thus, to the success of mass rearing programs. Several predator colonies are maintained in Sino-ABCL, including A. chinensis, lacewing, and lady beetle. We developed artificial diets for these predators and improved the rearing apparatus and environmental conditions accordingly. Studies on evaluation of the quality indicators for the mass rearing of biological agents were also conducted. **Accomplishments**
                                

1. Using DNA metabarcoding to assess insect diversity in citrus orchards. A large number of insect species, both pest and beneficial taxa occur on citrus but their identification is difficult. Cryptic morphology and lack of taxonomic experts are the major challenges in large-scale insect diversity assessments. Our study analyzed the composition of insect communities in citrus orchards using DNA metabarcoding followed by data analysis on two cloud databases and analytical platforms, the Barcode of Life Data System (BOLD) and Multiplex Barcode Research and Visualization Environment (mBRAVE). The species revealed on BOLD were then searched in citrus pest database and the literature to allow their classification into pest, parasitoid, predator, or pollinator. Our results provide a valuable resource for research on citrus pest management and beneficial insect exploration. 2. Detoxification and neurotransmitter clearance drive the recovery of Arma chinensis from ß-cypermethrin–triggered knockdown. Natural enemies of arthropods contribute considerably to agriculture by suppressing citrus pests, particularly when combined with chemical control. Studies show that insect recovery after insecticide application is rare. Here, we discovered the recovery of the predatory bug Arma chinensis from knockdown following the application of ß-cypermethrin. We verified metabolic detoxification by trans-regulated detoxifying genes in the A. chinensis recovery process and discovered a novel mechanism, the neurotransmitter clearance, in vivo during the recovery process. To reduce mortality caused by an influx of dopamine, A. chinensis rebalanced the excitatory neurotransmission by increasing ebony expression to remove the excessive dopamine during the recovery process. The data shows that screening for insecticide-resistant predators and parasitoids will be more conducive to promoting compatibility in pest management programs and ensuring the successful use of biological control agents in combination with pesticides. 3. Predaceous and Phytophagous Pentatomidae insects exhibit contrasting susceptibilities to imidacloprid. Stink bugs (Pentatomidae) are one of the few heteropteran families containing both plant feeders and predators in the suborder Heteroptera (Hemiptera) and comprise a range of invasive agricultural pests and natural enemies of great economic importance. We examined the acute toxicity of imidacloprid to the phytophagous Halyomorpha halys and predaceous Arma chinensis. Based on the results from bioassays, H. halys is significantly more sensitive to imidacloprid than A. chinensis, and female adults of both species showed higher insecticide resistance than male adults. Decreased reproduction performance indicated that the recovery of A. chinensis from imidacloprid-triggered knockdown carried fitness cost. Based on acute toxicity and molecular docking analysis results, imidacloprid is effective against the phytophagous H. halys, but potentially safe for the predatory A. chinensis when employed as a biological control agent. This is of particular interest for integrated pest control.


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