1a. Objectives (from AD-416):
Objective 1: Determine the physiological, behavioral, ecological, and genetic basis of host ranges of noctuid moths and parasitoids of pest insects, such as soybean aphid, Russian wheat aphid, sugar cane aphid, and spotted-wing Drosophila, with a focus on using molecular genetic methods to elucidate factors responsible for the evolution of host specificity. Subobjective 1.1 – Determine the genetic basis of host ranges of noctuid moths and of parasitoids of pest insects. Subobjective 1.2 – Test whether bacterial endosymbionts affect acceptance and suitability of hosts and determine mechanisms of these effects. Subobjective 1.3 – Test whether the host specificity of Aphelinus species changes with stress or experience. Objective 2: Determine interactions between biological control and host plant resistance in their effects on survival, reproduction, and population dynamics of pest insects, such as soybean aphid, Russian wheat aphid, sugar cane aphid, and spotted-wing Drosophila, in laboratory and field experiments. Objective 3: Determine molecular phylogenetic relationships, test host specificity, and introduce parasitoids for biological control of pest insects, such as soybean aphid, Russian wheat aphid, sugar cane aphid, and spotted-wing Drosophila, and determine the impact of the introduced parasitoids on the abundance and distribution of target and non-target species. Subobjective 3.1 – Determine phylogenetic relationships among parasitoids whose members are candidates for biological control introductions. Subobjective 3.2 – Measure host specificity of parasitoids that are candidates for biological control introductions. Subobjective 3.3 – Introduce parasitoids to control pest insects, such as soybean aphid, Russian wheat aphid, sugar cane aphid, and spotted-wing Drosophila, and measure the impact of the introduced parasitoids on the abundance and distribution of target and non-target species.
1b. Approach (from AD-416):
We will use analysis of genomes for genes that are divergent in sequence or expression, QTL mapping, co-localization of probes for QTL markers and divergent genes with chromosomal fluorescence in-situ hybridization and allele genotyping, analysis of tissue-specific expression (antenna, ovipositor), and gene knock-out with CRISPR/Cas9 and RNAi technology to identify genes involved in host recognition and acceptance. To test whether defensive bacterial endosymbionts affect acceptance and suitability of hosts of parasitoids and to determine mechanisms underlying these effects, we will assay more species of parasitoids on more species of aphids with and without their defensive endosymbionts. To test whether host ranges of Aphelinus species are ever dynamic, we will test the effects of starvation, age, and experience on parasitism of sub-optimal hosts by parasitoid species with broad host ranges. We will do additional experiments on the interactions between host plant resistance and parasitism by Aphelinus species. Continued development of the molecular phylogeny of Aphelinus species will provide a framework for other results. We will conduct host specificity testing of parasitoids for release against D. noxia, M. sacchari and D. suzukii. We will introduce parasitoid species with narrow host ranges and monitor their impact on target and non-target species.
3. Progress Report:
To find candidate genes underlying differences in host specificity, we sequenced, assembled, and completed preliminary annotation for five more genomes of Aphelinus species, which include aphid parasitoids being used in biological control. We also estimated their genome sizes using flow cytometry. We have now sequenced the genomes of 15 Aphelinus species that vary in host specificity. These include Aphelinus glycinis and Aphelinus rhamni from China that are being introduced to against soybean aphid, Aphelinus coreae from Korea that is a candidate for introduction against soybean aphid, Aphelinus hordei that is a candidate for introduction against Russian wheat aphid, and Aphelinus near Nigritus that is a candidate for use against sugarcane aphid on sorghum. We are now analyzing genes in these genomes for homology, sequence/expression divergence, and the association of divergences with differences in host specificity. We extracted large amounts of high molecular weight DNA from Aphelinus atriplicis and Heliothis virescens. However, a preliminary assembly of the A. atriplicis sequences was very fragmented and was only 67% of the genome size estimated from flow cytometry. We will re-assemble these genomes using a different assembler, which is designed to handle genetic variation well. To map genes involved in parasitism on a new host species by Aphelinus ramni, we made and sequenced reduced representation libraries for 382 F1 and backcross progeny for crosses between control lines and lines selected to parasitize Rhopalosiphum padi, an aphid rarely parasitized by A. rhamni. This research relates to objective 1.1 of the project. With University of Illinois, experiments on the effects on parasitism of three soybean genes (RAG1, 2 and 3) providing resistance to soybean aphid showed that most of the eight combinations of these genes reduced the parasitism of soybean aphid by A. certus, but only two reduced parasitism of soybean aphid by A. glycinis, and indeed two combinations increased parasitism of soybean aphid. This research relates to objective 2 of the project. With Texas A&M University, we continued revision of the systematics of Aphelinus, for which we received funding from a National Science Foundation grant. With amino acid differences in a set of 100 genes, we made evolutionary trees for 14 species of Aphelinus using Aphytis melinus and Nasonia vitripennis as outgroups. Trees based on different methods of analysis were almost identical. These trees provide the framework for analyzing the evolution of differences in host specificity. This research relates to objective 3.1 of the project. We finished host specificity testing of A. hordei and are now preparing a NAPPO (North American Plant Protection Organization) petition for its introduction against the Russian wheat aphid, Diuraphis noxia. In a laboratory experiment on parasitoid behavior with various aphid species, A. hordei rarely laid eggs in aphid species outside the genus Diuraphis and within Diuraphis, produced the greatest number of parasitized aphids on D. noxia. This research relates to objective 3.2 of the project. As part of an ARS Areawide Project in collaboration with Iowa State University, University of Minnesota, Ohio State University, and University of Illinois, ~300,000 Aphelinus glycinis were reared and released in 20 sites in Iowa and 20 sites in Minnesota. We are monitoring parasitism of soybean aphid on resistant and susceptible soybean and the effects of parasitism on the frequencies of avirulent versus virulent soybean aphid. The results will show whether these introduced parasitoids can slow the spread of soybean aphid that can overcome host plant resistance and will provide the basis of management of soybean aphid without the use of insecticides. In the summer of 2015, releases of Aphelinus glycinis were made at the University of Minnesota Agricultural Experiment Station in Rosemount, Minnesota. A total of 104,000 parasitoids were release in 16 sites, and sampling during the season showed a high abundance of aphids parasitized by Aphelinus, peaking at over 50 parasitized aphids per plant. This research relates to objective 3.3 of the project.
5. Significant Activities that Support Special Target Populations:
Yao, Y., Duan, J.J., Hopper, K.R., Moltern, J.L., Gates, M.W. 2016. A new species of oobius trjapitzin (hymenoptera:encyrtidae) from the russian far east that parasitizes eggs of emerald ash borer (coleoptera:buprestidae). Annals of the Entomological Society of America. 109(4):629-638. doi: 10.1093/aesa/saw022.