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:
Using genetic mapping, we have identified 50 genes that genes are associated with differences between Aphelinus atriplicis and Aphelinus certus in parasitism of Diuraphis noxia. Many of these genes do not have homologs in the Refseq database at the National Center for Biotechnology Information (NCBI), but our hypothesis is that they code for chemosensory proteins involved in host recognition. To test this hypothesis, we have used fluorescent in-situ hybridization with antibody and mRNA probes to test whether several candidate genes are expressed in sensilla on antennae or ovipositors. The first few of these genes we examined are indeed expressed on sensilla of antennae and/or ovipositors of A. atriplicis. This research relates to objective 1.1 of the project. (Agreement number 8010-22000-029-16R) Rapid evolution of species introduced for biological control of pests can have important consequences for biological control safety (non-target effects) and effectiveness (establishment and target effects). To better understand, manage and predict rapid adaptation of biological control agents to novel host plants and climates, we need to know more about how genotypes are linked with the adaptive phenotypic traits, and how those traits impact performance and fitness. With colleagues at Oregon State University, we are investigating adaptation of Tyria jacobaeae, cinnabar moth, which was introduced to control Senecio jacobaea, tansy ragwort, a toxic rangeland weed. To assemble genomes and compare sequence differences between mountain and valley habitats, we extracted DNA from 30-225 individuals in 5 mountain populations and 55-165 individuals in 4 valley populations. For each population, separate libraries were prepared and sequenced on the Illumina Hiseq 2500 platform (paired-end reads of 150 nucleotides). With the de novo assembly program in the CLC Genomics Workbench, we made separate assemblies of the genomes of the insects from mountain and valley habitats, using 50 gigabases (370 million reads) and 36 gigabases of sequence data (265 million reads), respectively. We searched these assemblies for genes using Augustus, and compared the amino acid sequences the proteins that these genes produced with those the Refseq database at NCBI using blastp. For the mountain and valley habitats, 66 and 62 percent of the proteins had homologs in the Refseq database, respectively. We mapped reads from one habitat to the assemblies and to the coding sequences for genes from the other habitat, and analyzed for sequence divergence in assemblies and genes. There were 36 variants per kilobase between mountain and valley habitats when reads were mapped to assemblies, and 72-79 percent of genes diverged in coding sequence between mountain and valley habitats. Highly divergent genes, i.e. diverged by greater than 30 percent, were 0.1 percent of all genes. Compared to all genes, these highly divergent genes had far fewer homologs in the Refseq database than all genes (13 versus 66 percent for the mountain habitat and 25 versus 66 percent for the valley habitat). The few genes with functional annotations were either transcript factors for genes of unknown function or transposon-associated sequences. These results resemble those being found for other rapidly evolving genes: homologs are difficult to find, and those that are found are either transcription factors or transposon-associated sequences, the latter being a major source of genetic variation upon which selection can act. This research relates to objective 1.1 of the project. (Agreement number 8010-22000-029-12R) With colleagues at University of Illinois, Iowa State University, and Ohio State University, we continued research on the interaction between host plant resistance in soybean, virulence in Aphis glycines, the soybean aphid, and parasitism by Aphelinus species. We did a laboratory experiment with resistant (LD14-8003, Rag1,2,3) and susceptible (LD14-8007) soybean accessions, avirulent (biotype 1) and virulent (biotype 2) soybean aphid, and two parasitoid species (Aphelinus glycinis and Aphelinus certus), Aphelinus glycinis is being introduced to control soybean aphid and Aphelinus certus is an adventive species that heavily parasitizes soybean aphid so understanding their interaction with host plant resistance and aphid virulence is important for integrated control of this pest. We found that both parasitoid species were able to parasitize both aphid genotypes on both resistant and susceptible soybean. Although parasitism by Aphelinus glycinis did not vary with aphid genotype or soybean resistance, Aphelinus certus tended to parasitize the virulent genotype more than the avirulent genotype, perhaps because the virulent genotype was more abundant. Such density or frequency dependent mortality may slow the spread and reduce the prevalence of virulent genotypes. This research relates to Objective 2 of the project. With colleagues at Texas A&M University, Kansas State University, and Pennsylvania State University, we continued research on the interaction between host plant resistance in sorghum and parasitism of Melanaphis sacchari, the sugarcane aphid. In laboratory experiments on the effect of host plant resistance on parasitism, using cultures of the sugarcane aphid and a species of Aphelinus from sorghum in Texas, on sorghum accessions (PI 550610, DKS 37-07, PAR-3, PAW-1) with different levels of resistance, we found that this species of Aphelinus is able to parasitize sugarcane aphid on both resistant and susceptible sorghum accessions. This research relates to Objective 2 of the project. With colleagues at Colorado State University, we have begun research on the interaction between host plant resistance in wheat, virulence in Diuraphis noxia, the Russian wheat aphid, and parasitism by Aphelinus species. We are now counting the samples from a laboratory experiment on the effects of resistant (YumaR-Dn4) versus susceptible (Yuma) wheat accessions and avirulent (RWA1) versus virulent (RWA2) Russian wheat aphid biotypes on parasitism by Aphelinus hordei, which is a candidate for introduction against this pest. This research relates to objective 2 of the project. We prepared and submitted a petition to the North American Plant Protection Organization to introduce Aphelinus hordei for biological control of Diuraphis noxia, the Russian wheat aphid. The petition was approved, an environmental assessment has been prepared, and we expect to receive a field-release permit soon. Diuraphis noxia has become a major pest of wheat and barley since it was detected in the western USA in 1986. Wheat varieties resistant to D. noxia began to be used in 1996, and research has revealed multiple genes in wheat and barley that can provide resistance to D. noxia. However, aphid genotypes able to overcome one or more of these resistance genes began appearing by 2003, and four virulent genotypes of D. noxia have been discovered, some of which have become widespread. Diuraphis noxia is rarely a pest in Eurasia, and field experiments have shown that natural enemies have a large impact there. Parasitoids in the genus Aphelinus are among the most important natural enemies of D. noxia in Eurasia, and A. hordei should have a significant impact on this pest in the U.S. This research relates to Objective 3.2 of the project.
Desneux, N., Monticelli, L., Luo, C., Asplen, M.K., Brady, C.M., Heimpel, G.E., Hopper, K.R., Oliver, K.M., White, J.A. 2017. Intraspecific variation in facultative symbiont infection among native and exotic pest populations: potential implications for biological control. Biological Control. https://doi.org/10.1016/j.biocontrol.2017.1006.1007.