Location: Systematic Entomology Laboratory
Project Number: 8042-30400-004-014-I
Project Type: Interagency Reimbursable Agreement
Start Date: Aug 25, 2026
End Date: Aug 31, 2027
Objective:
1. Collect, prepare, and sequence Brassica-feeding aphid and slide mount specimens and add them to the National Aphidoidea Collection.
2. Develop and test repeatable and reproducible bioinformatics pipelines to process raw genomic data, generate the reference alignments, species tree, and ultimately produce the diagnostic species identification tool.
Approach:
The primary goal for the first year of this agreement is to develop a molecular diagnostic tool for Brassica-feeding aphids that uses genome data and open-source software. This work will be completed with the use of tissues and specimens from the National Collection of Aphidoidea housed at the Smithsonian National Museum of Natural History.
Currently, there are 22 species of aphids known from North America that feed on Brassica species. These taxa belong to two subfamilies (i.e., Aphidinae & Eriosomatinae) and 14 genera. Taxon sampling will include many specimens for each species throughout its range to incorporate the genetic diversity of each. DNA extraction process will produce high-quality DNA, enough to sequence using NGS platforms and standard PCR with Sanger Sequencing. Furthermore, a non-destructive DNA extraction will be used to preserve each specimen for post-sequencing examination and ultimately add each to the National Aphidoidea Collection. This is imperative for aphids because it has been shown that interspecific hybridization and introgression are common in agriculturally important aphids. Moreover, a single locus barcode diagnosis may lead to false positives due to evolutionary processes such as hybridization and introgression and maternally sharing mitochondrial DNA from different species.
The genomic data for all species will be used as the framework for the molecular diagnostic tool. Traditional bioinformatic pipelines will be used to identify thousands of single-copy orthologs among the species. From these data, a species tree will be estimated. Moreover, the phylogeny will provide a means to diagnose unknown species feeding on Brassica. Open-source software will be repurposed with novel bioninformatics scripts to perform the test. Once the unknown Brassica-feeding aphids are placed on the phylogeny, three pieces of data will be reported to the scientist. First, the phylogeny with the new specimens depicted in a different color. Next, whether the unknown specimen falls in a monophyletic clade with Brassica feeding aphid species. This information is valuable because monophyly may suggest to users the confirmation of a species according to the Phylogenetic Species Concept. The last pieces of data that will be reported are the nearest neighbor on the phylogeny and the patristic distance from the nearest neighbor. Pairwise sequence divergence has long been used to identify species and using patristic distance (i.e., tip-to-tip length on a phylogeny) is no different; therefore, the identity of the closest taxon and the patristic distance. Given that mitochondrial barcode region is the most used locus for aphid species delimitation, it will be incorporated into the results from the nuclear genome.
Each specimen used for the genomic tool will be slide mounted and added to the National Aphidoidea Collection. This will be performed using standard approaches that include using permanent Canada balsam mounts with label data that include DNA repository accession numbers, taxon name, host, date, and locality data.