Dissertations, Theses, and Capstone Projects

Date of Degree

2-2021

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Shubha Govind

Committee Members

Sanna Goyert

Karen Hubbard

Shannen Singh

Keith Hopper

Subject Categories

Bioinformatics | Cell Biology | Computational Biology | Genetics | Genetics and Genomics | Genomics | Immunology and Infectious Disease | Molecular Genetics | Parasitology

Keywords

MSEV, VLP, parasitoid, Leptopilina heterotoma, Leptopilina boulardi

Abstract

Host-parasite interactions influence the biology of each over the course of evolution. Parasite success allows for the passage of potent virulence strategies from generation to generation. Host success passes stronger immunity and resistance strategies to the following generations as well. Only by studying both partners within their natural contexts can we begin to understand the relationship between the two and how immune mechanisms and virulence strategies interact as a molecular arms race.

In this work, we focus on a natural host-parasite pair, the Drosophila-Leptopilina model. Leptopilina species are parasites of several fruit fly species, including Drosophila melanogaster. This model offers many advantages, including the well-annotated Drosophila genome, the genetic and transgenic tools available for Drosophila, and the ease of culturing these insects in the lab.

The wasps Leptopilina heterotoma, Leptopilina boulardi, and Leptopilina victoriae utilize somewhat distinct immune-suppressive strategies to suppress the host defenses. These are mediated in part through virus-like particles (VLPs) that are injected into the host larva along with the wasp egg. VLPs target hosts’ blood cells. The primary Leptopilina species studied within this work is L. heterotoma, a wasp that is successful against a wide range of Drosophila species. Its infection leads to the destruction of almost all larval blood cells, blocking encapsulation and antimicrobial peptide production in D. melanogaster. L. heterotoma’s VLPs contain ~400 proteins; some are predicted to carry out conserved cellular processes or may modulate the immune response. To understand the nature of VLPs and L. heterotoma’s unique immune suppression strategy, this work has utilized mixed-omics, transgenic, and RNA interference (RNAi) approaches.

In Chapter 1, we characterized the expanded particle proteome, the whole-body transcriptome, and sequenced the L. heterotoma genome to search for evidence of virus-related proteins within the particle’s expanded proteome. We found no viral coat proteins within the expanded particle proteome. In addition, more than 90% of VLP proteins had coding regions within the wasp genome.

In an Addendum to Chapter 1, we compared the recently published L. boulardi VLP proteome to that of the L. heterotoma VLP/MSEV proteome to better understand the similarities of both particles and their extracellular vesicle character. While both wasps only share approximately 30% of their particle proteins, the overall extracellular vesicle profile and distribution of proteins across classes is similar. A search of highly assembled wasp genomes improved our published assessment of the proportion of L. heterotoma MSEV proteins that are encoded by the wasp genome. This observation also held for the L. boulardi VLP proteins, reinforcing the extracellular nature of these particles.

In Chapter 2, we focus on the virulence function of a key L. heterotoma spike protein, p40. In transgenic expression, a full-length construct of p40 localized to cell membranes while a truncated construct (without the putative transmembrane domain) was found to be secreted, supporting previous structural predictions of p40. The secreted protein prevented encapsulation of eggs of a closely related wasp L. victoriae, which normally elicits strong encapsulation in D. melanogaster. RNA interference-mediated knockdown of p40 significantly reduced L. heterotoma’s ability to suppress encapsulation as a larger proportion of p40RNAi-infected wild type hosts showed strong encapsulation. Together, these results underscore the importance of p40 in L. heterotoma’s ability to prevent encapsulation and ensure offspring survival.

Parasitic wasps are keystone species and some species are utilized to control agricultural pests. By providing genomics, transcriptomics and proteomics analyses, this work expands the utility of the Drosophila-Leptopilina model; the released data will facilitate studies in novel areas of host-parasite biology. It also illuminates virulence strategies and the evolution of virulence factors of these wasps.

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