Genome Wide Association Studies of Phagocytosis and the Cellular Immune Response in Drosophila melanogaster.

Phagocytosis of bacteria by specialized blood cells, known as hemocytes, is a vital component of Drosophila cellular immunity. To identify novel genes that mediate the cellular response to bacteria, we conducted three separate genetic screens using the Drosophila Genetic Reference Panel (DGRP). Adult DGRP lines were tested for the ability of their hemocytes to phagocytose the Gram-positive bacteria Staphylococcus aureus or the Gram-negative bacteria Escherichia coli. The DGRP lines were also screened for the ability of their hemocytes to clear S. aureus infection through the process of phagosome maturation. Genome-wide association analyses were performed to identify potentially relevant single nucleotide polymorphisms (SNPs) associated with the cellular immune phenotypes. The S. aureus phagosome maturation screen identified SNPs near or in 528 candidate genes, many of which have no known role in immunity. Three genes, dpr10, fred, and CG42673, were identified whose loss-of-function in blood cells significantly impaired the innate immune response to S. aureus. The DGRP S. aureus screens identified variants in the gene, Ataxin 2 Binding Protein-1 (A2bp1) as important for the cellular immune response to S. aureus. A2bp1 belongs to the highly conserved Fox-1 family of RNA-binding proteins. Genetic studies revealed that A2bp1 transcript levels must be tightly controlled for hemocytes to successfully phagocytose S. aureus. The transcriptome of infected and uninfected hemocytes from wild type and A2bp1 mutant flies was analyzed and it was found that A2bp1 negatively regulates the expression of the Immunoglobulin-superfamily member Down syndrome adhesion molecule 4 (Dscam4). Silencing of A2bp1 and Dscam4 in hemocytes rescues the fly’s immune response to S. aureus indicating that Dscam4 negatively regulates S. aureus phagocytosis. Overall, we present an examination of the cellular immune response to bacteria with the aim of identifying and characterizing roles for novel mediators of innate immunity in Drosophila. By screening panel of lines in which all genetic variants are known, we successfully identified a large set of candidate genes that could provide a basis for future studies of Drosophila cellular immunity. Finally, we describe a novel, immune-specific role for the highly conserved Fox-1 family member, A2bp1.

TAXONOMY, MORPHOLOGY, AND RNA-SEQ TRANSCRIPTOMICS OF THE CUBOZOAN ALATINA ALATA, AN EMERGING MODEL CNIDARIAN

Cnidarians are often considered simple animals, but the more than 13,000 estimated species (e.g., corals, hydroids and jellyfish) of the early diverging phylum exhibit a broad diversity of forms, functions and behaviors, some of which are demonstrably complex. In particular, cubozoans (box jellyfish) are cnidarians that have evolved a number of distinguishing features. Some cubozoan species possess complex mating behaviors or particularly potent stings, and all possess well-developed light sensation involving image-forming eyes. Like all cnidarians, cubozoans have specialized subcellular structures called nematocysts that are used in prey capture and defense. The objective of this study is to contribute to the development of the box jellyfish Alatina alata as a model cnidarian. This cubozoan species offers numerous advantages for investigating morphological and molecular traits underlying complex processes and coordinated behavior in free-living medusozoans (i.e., jellyfish), and more broadly throughout Metazoa. First, I provide an overview of Cnidaria with an emphasis on the current understanding of genes and proteins implicated in complex biological processes in a few select cnidarians. Second, to further develop resources for A. alata, I provide a formal redescription of this cubozoan and establish a neotype specimen voucher, which serve to stabilize the taxonomy of the species. Third, I generate the first functionally annotated transcriptome of adult and larval A. alata tissue and apply preliminary differential expression analyses to identify candidate genes implicated broadly in biological processes related to prey capture and defense, vision and the phototransduction pathway and sexual reproduction and gametogenesis. Fourth, to better understand venom diversity and mechanisms controlling venom synthesis in A. alata, I use bioinformatics to investigate gene candidates with dual roles in venom and digestion, and review the biology of prey capture and digestion in cubozoans. The morphological and molecular resources presented herein contribute to understanding the evolution of cubozoan characteristics and serve to facilitate further research on this emerging cubozoan model.