Location: Beneficial Insects Introduction Research Unit
Project Number: 8010-22000-032-001-I
Project Type: Interagency Reimbursable Agreement
Start Date: Apr 15, 2017
End Date: Apr 14, 2021
Objective:
The introduction of parasitoids can provide effective control of invasive pests, but critics have argued that introduced parasitoids may evolve to attack non-target species. The likelihood of such evolution depends on the number of genes involved and how they interact. We will test alterative hypotheses about the genetics of host shifts: (1) many genes interact epistatically so that changes must happen simultaneously to reach a new adaptive peak; (2) few genes interact additively so a new adaptive peak could be reached more easily. Evolutionary shifts are much less likely under hypothesis 1 than under hypothesis 2. We will study the genetics of differences in host specificity among species of aphid parasitoids in the genus Aphelinus. We propose quantitative trait loci (QTL) and candidate gene mapping, as well as transcriptome analysis to identify genes that have diverged in sequence or expression. Our objectives are as follows: 1. For two species (Aphelinus atriplicis and Aphelinus certus) that hybridize in the laboratory, map quantitative trait loci (QTL) affecting parasitism of a novel host species to determine the number and interactions among QTL; 2. Test whether genes that diverge in sequence and/or expression between A. atriplicis and A. certus are associated with the QTL found under Objective 1 and whether those genes associated with QTL are expressed in the predicted tissues, which are sensilla on antennae or ovipositor; 3. Compare transcriptomes of 12 Aphelinus species that differ in host specificity, and analyze the relationship between divergence in gene sequences and/or expression levels and patterns of host acceptance and suitability.
Approach:
1. Map quantitative trait loci (QTL) affecting differences in parasitism of novel hosts to determine the number and interactions among these QTL.
We will cross four male A. atriplicis with four female A. certus and then cross the resulting recombinant F1 females from each of these crosses to "F1" males. We will intercross these lineages for four generations, and then mate brothers with sisters within each of 400 lines for ten generations to produce 400 recombinant inbred lines (RILs). We will then assay four females from each of the 400 RILs for parasitism of D. noxia. We will assay parasitism by exposing four females from each RIL to D. noxia nymphs on barley and scoring the number of aphids parasitized. After 24 hours, female parasitoids will be recovered, killed in ethanol, and kept at -20 C for later DNA extraction. Exposed aphids will be held on host plants until they mummify. We will then harvest the plants and store the mummies until adults emerge; upon emergence, we will kill them in ethanol and keep them at -20 °C for later DNA extraction.
2. Test whether genes that diverge in sequence and/or expression between A. atriplicis and A. certus are associated with the QTL found under Objective 1 and whether those genes associated with QTL are expressed in the predicted tissues.
To determine whether candidate genes map to QTL, we will use KASP technology (LGC Tissue-specific expression of candidate genes. We will use whole-mount in-situ hybridization and microscopy to determine whether candidate genes associated with QTL are expressed in sensilla on antennae or ovipositors. We will use probes for both proteins and RNA.
3. Compare transcriptomes of Aphelinus species that differ in host specificity, and analyze the relationship between divergence in gene sequence and/or expression levels and host acceptance and suitability.
We will extract RNA from ~200 individuals from each of two additional Aphelinus species (A. asychis and A. abdominalis). A total of four RNA extractions will be needed for analysis of male and female transcriptomes. One ug of RNA from each of the four samples will be used to generate indexed whole-transcriptome libraries using Illumina TruSeq RNA Sample Prep Kits. To test whether differences numbers of aphids parasitized arises from host acceptance versus host suitability, we will directly observe female parasitoids exposed to aphids, and for those aphids in which parasitoids insert their ovipositor, we will dissect some to find whether eggs were laid and rear others to find whether mummies are formed. We will extend previous measurements to seven additional species: A. abdominalis, A. asychis, A. atriplicis, A. daucicola, A. kurdjumovi, A. nigritus, and A. varipes. We will analyze the relationship between divergence in genes sequence or expression level and host acceptance and host suitability, using generalized linear models for each aphid species, as well as multivariate analysis of variance for patterns across aphid species, correcting for false-discovery rate. We will also determine whether divergent genes include those found to be associated with QTL in Objective 1.
