Location: Beneficial Insects Introduction Research Unit
2019 Annual Report
Objectives
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.
Approach
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.
Progress Report
We continued research on the genetics of differences in host specificity among species of aphid parasitoids in the genus Aphelinus. A postdoc and technician were hired last fall under a NIFA-funded grant. We are mapping quantitative trait loci (QTL) and candidate genes, as well doing transcriptome analysis, to identify genes that have diverged in sequence or expression between specialist and generalist parasitoids. For two species (Aphelinus atriplicis and Aphelinus certus) that hybridize in the laboratory, we are developing recombinant inbred lines (RILs) from multi-parent advanced generation intercrosses (MAGIC) that we will use in mapping QTL and candidate genes. We have completed four generations of intercrosses and have started on four generations of random mating to be followed by ten generations of inbreeding. We made a combined assembly of the genome of A. atriplicis using Illumina paired-end and PacBio long-read data. The assembly is 461 Mb with 3,042 contigs and an N50 of 240 kb. The contigs are now long enough that we should be able to generate much longer scaffolds by using markers from the genetic map made with MAGIC-RILs. We have analyzed backcross progeny from selection and control lines of Aphelinus rhamni and found 35 markers out of 501 markers where associated with the response to selection of parasitism of a new host, Rhopalosiphum padi. We studied expression of 14 candidate genes associated with QTL affecting parasitism of Diuraphis noxia by backcross progeny of the cross between A. atriplicis and A. certus and found that 13 where expressed in sensilla on the ovipositor and 4 where expressed in sensilla on the mandibles. We have completed the annotation of the genomes and transcriptomes of 20 populations of Aphelinus in 16 species, and we are now analyzing the differences among them and how these differences relate to differences in host specificity among them. This research relates to Objective 1.1 of the project. (Agreement number 8010-22000-029-16R)
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. A major difference we have found between valley and mountain populations of T. jacobaeae is in the development time of larvae. Within valley populations larvae also differ in development time, and to determine whether these differences were associated with differences in gene expression, and if so, to find which genes differed in expression, we extracted mRNA from larvae that differed by 4 to 10 days in the time from egg to fourth instar. We prepared libraries from this RNA using Kappa RNAseq kits and had the libraries sequenced on an Illumina HiSeq 2500 at the core sequencing facility at the Delaware Biotechnology Institute. Sequencing yielded 362 million reads that assembled into contigs that coded for 64k putative protein sequences. With an overall Busco score of 95% (percent of core insect genes found in the assembly), this gene set is quite complete. Analysis of differences in gene expression revealed 20 genes that differed in expression between fast versus slow larvae. Gene functions included cuticle development, flight muscles, and regulation of development, which make sense, but also included molecular chaperones, immune response, retrotransposons, and regulation of transcription and translation, which make less sense. As in the Oregon, T. jacobaeae was also introduced into New Zealand to control S. jacobae, and as in the Oregon, the moth has spread to high altitudes. To determine whether the genetic differentiation between valley and mountain populations T. jacobaeae in Oregon has also occurred in New Zealand, we obtained 50 larvae each from three low altitude populations and two higher altitude populations. These insects were collected and shipped to us by Landcare Research, Palmerston North, New Zealand. We have extracted DNA from pools of larvae from each population using Qiagen DNeasy blood and tissue kits. To do this we dissected and pooled equal weights of tissue the from the anterior of each larva. DNA was submitted to core sequencing facility at the Delaware Biotechnology Institute that prepared and sequenced a library on an Illumina HiSeq 2500. This yielded 49 Gb of sequence in 165 million paired reads. We are using these data for genome assembly and analysis of genetic differences between low versus high altitude populations. This research relates to Objective 1.1 of the project. (Agreement number 8010-22000-029-12R)
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 hordei, which is a candidate for introduction against this pest. Parasitism by A. hordei did not vary with aphid genotype or wheat resistance, and parasitism was high on all treatments. Thus, host plant resistance in wheat and biological control with A. hordei will be compatible strategies for managing the Russian wheat aphid. This research relates to Objective 2 of the project.
With colleagues at University of California Riverside, Hebei University (Baoding, China), Indiana University, Zoologisches Forschungsmuseum Alexander Koenig (Bonn, Germany), University of Rochester, University of Georgia, Texas A&M University, Illinois Natural History Survey, State Museum of Natural History (Stuttgart, Germany), and University of Hohenheim (Stuttgart, Germany), we explored the transcriptome-based phylogeny of Chalcidoidea, and found that the poorly resolved relationships could only be marginally improved by adding more genes and taxa, proof-checking for errors of homology and contamination, and decreasing missing data. Results of concatenation analyses were consistent in supporting a hypothesis of egg parasitism as ancestral within Chalcidoidea (Trichogrammatidae sister to remaining Chalcidoidea after Mymaridae), whereas coalescent approaches provided a different hypothesis, unless filtered for the most highly supported loci. The results uncover a wide spectrum of gene discordance in the transcriptomic markers and identified a strong signal of functional bias in genes supporting alternative phylogenies that might be indicative of ancient adaptive introgression. An ancestral mode of egg parasitism remains the most highly supported hypothesis. However, the basal nodes of the phylogeny may be incorrectly biased by cascades of differential support from functional gene complexes. This study proposes that understanding and identifying mechanisms that result in gene tree discordance (incomplete lineage sorting or adaptive introgression) may be both beneficial and essential to sorting out the backbone relationships, especially for a group that has gone through a rapid post-Cretaceous radiation. This research relates to Objective 3 of the project.
The secretory structures on male antennomeres of parasitic Hymenoptera are diverse and widespread and function during antennation in courtship. With colleagues at Texas A&M University, we surveyed these secretory structures in ten species in six complexes of Aphelinus, as well as in two species, Aphytis melinus and Centrodora sp., that served as outgroups. The external structures differed in the number and position of the pores, the conformation of cuticle surrounding pores, and the shape of the carina delimiting the area around the pores. The morphology of these pores is diagnostic for the species groups of Aphelinus and maps well onto a molecular phylogeny of these species Aphelinus. This research relates to Objective 3 of the project. (Agreement number 8010-22000-029-03R)
With colleagues at the City University of New York, we studied vesicles in the venom of Leptopilina heterotoma, which is a parasitoid of Drosophila species. During oviposition, female wasps introduce venom containing discrete 300 nm-wide mixed-strategy extracellular vesicles (MSEVs) into the larval host’s body cavity that play a critical role in wasp success by destroying host blood cells. Here we report 246 MSEV proteins from the L. heterotoma proteome. An enrichment analysis of the entire proteome supports vesicular nature of these structures. Transcripts of more than 90% of these proteins were present in whole body Lh14 transcriptomes. Sequencing and assembly of the 460 Mb-sized Lh genome revealed at least wasp 30,000 genes, including 80% of the BUSCOs in the Insecta set. We identified genes for a majority of MSEV proteins within the genomic scaffolds. Altogether, these results explain the stable association of MSEVs with their wasps, and like other wasp structures, their vertical genetic pattern of inheritance. While our results do not rule out a viral origin of MSEVs, they support a vesicular nature and suggest that the strategy for co-opting cellular machinery for immune suppression may be shared by other wasps to gain advantage over their hosts. These results are relevant to our understanding of the evolution of figitid wasps. This research relates to Objective 3 of the project.
Accomplishments
1. Population genetics of host specificity documented. Levels of parasitism by populations of Aphelinus certus, a parasitoid of the soybean aphid, varied among aphid species, suggesting adaptation to locally abundant aphids. Differences in host specificity among parasitoid populations correlated with genetic differences among them. Whether different populations of the same natural enemy species vary in host specificity and the genetic basis of these differences are important because a major part of the search for natural enemies for introduction against invasive pests involves evaluating host specificity in laboratory experiments.
