Identification of constitutively-expressed immune effectors in the house fly (Musca domestica L.) and the transcription factors that regulate them

Authors: ASGARI, DANIAL - University Of Houston; SASKI, CHRISTOPHER - Clemson University; MEISEL, RICHARD - University Of Houston; Nayduch, Dana

Submitted to: Insect Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/21/2022
Publication Date: 7/25/2022
Citation: Asgari, D., Saski, C.A., Meisel, R.P., Nayduch, D. 2022. Identification of constitutively-expressed immune effectors in the house fly (Musca domestica L.) and the transcription factors that regulate them. Insect Molecular Biology. 31(6):782-797. https://doi.org/10.1111/imb.12804.
DOI: https://doi.org/10.1111/imb.12804

Interpretive Summary: Insects defend themselves against pathogens by using an innate immune response much like the primitive responses that exist in other animals. In most insects, these defense systems are activated only upon infection, and the end products of these immune pathways result in production effectors called antimicrobial peptides (AMPs) or lysozymes which bind and kill bacteria. In this study, we fed two groups of female house flies a bacterial species, either Pseudomonas aeruginosa or Escherichia coli, and collected flies after gene expression was activated (4 hours post-ingestion). Flies were dissected to separate their digestive tract ("gut") from the remainder of the body ("carcass"), RNA was extracted and sequenced to identify which of the house fly genes were upregulated or downregulated based on exposure to these bacteria. Our results showed that genes encoding digestive proteins were upregulated in the gut upon ingestion of P. aeruginosa, but surprisingly there was no induction of immune genes after bacterial ingestion. However, we identified constitutive induction of the immune genes coding for the bacteria-destroying enzyme lysozyme as well as a few AMP genes (i.e. they were not induced, but they were "turned on" at a high level naturally) in both treatment groups and control flies. Next, we used in silico approaches to identify factors involved inducing the constitutive expression of the AMPs and lysozyme genes. We identified the transcription factor Myc as the most likely candidate for controlling the constitutive expression of AMPs, and Klumpfuss for lysozymes. We speculate that this persistent or constitutive expression of AMPs benefits the flies by offering immediate protection without a delay in response. House flies also have a larger number of immune effectors like AMPs compared to other insects such as fruit flies, with their genome revealing massive expansions and duplications of genes coding for many classes of defense molecules. The ability to mount a rapid response along with an expanded immune repertoire are much-needed for house flies who live and thrive in septic environments like manure, garbage dumpsters and other microbe-rich resources. These insights into the house fly's defense system can be used to develop novel targets for controlling them. Alternatively, these immune defense molecules can be explored for their potential use as antimicrobial drug alternatives.

Technical Abstract: Insects possess innate immune defenses that are either induced upon infection or are constitutively active regardless of infection status. Constitutive defenses offer protection without a delay in response yet incur the cost of continuous activity. In flies, constitutive expression of immune molecules has been reported for some lysozymes and antimicrobial peptides (AMPs), but the extent of constitutive and induced immune responses vary across tissues and species. The size of the immune gene repertoire within the genome also varies across species. For example, the house fly (Musca domestica) which inhabits septic environments has an expanded suite of immune-related genes relative to Drosophila, including many duplicated and expanded families of AMPs and other effectors. In this study we fed groups of female house flies one of two Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) or control broth and used RNA-seq to determine differential expression of immune-related genes at 4 h post-feeding. Flies that ingested P. aeruginosa induced expression of genes encoding digestive proteins in the gut but did not induce AMPs. Flies in all treatment groups constitutively expressed some paralogs of lysozyme and AMP genes at the transcript level. in silico approaches were used to find potential transcription factors responsible for constitutive transcription of AMPs and lysozymes. Myc was identified as the most likely candidate for controlling the constitutive expression of AMPs, and Klumpfuss for lysozymes. Constitutive transcription of lysozymes and AMPs likely contributes to house fly survival by allowing for a rapid response to pathogens encountered in their microbe-rich niche.