Pseudolycoriella hygida: enzymes and a microbiome for living in the soil

• Grant number: 24/23632-7
• Support Opportunities: Regular Research Grants
• Start date: July 01, 2025
• End date: June 30, 2028
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Nadia Monesi
• Grantee: Nadia Monesi
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Our laboratory employs Pseudolycoriella hygida (Diptera: Sciaridae) as a model. This sciarid was isolated on the Ribeirão Preto campus in 1965 and has been kept in the laboratory ever since. Sciarids live in the soil, where they act as decomposers of partially decomposed plant material. To study Psl. hygida, we have several tools at our disposal, including a high-quality cytogenetic map and nucleic acid extraction methods, among others. Our laboratory recently deposited an annotated version of the genome of this sciarid in GenBank. The genomic data allowed us to expand our studies on this unconventional model organism, which provided new information about embryonic development, gene expression patterns in the salivary gland at the time of DNA puff formation, mitochondrial function, and the biochemical basis associated with the decomposer role of sciarids in soil food chains. The general objective of this proposal is to expand the knowledge about the molecular basis associated with the life of sciarids in soil, with emphasis on the characterization of peptidases, the microbiome and the mitochondrial enzyme alternative oxidase (AOX) of Psl. hygida. The project has three main objectives. The saliva of Psl. hygida larvae is rich in peptidases and their relevance to the life of sciarids in soil has not been determined. Thus, the first objective is the identification and biochemical characterization of the peptidases present in the saliva of 12-day-old larvae (subproject 1). Specifically, we propose: a) manual curation and identification of the peptidases involved in larval digestion; b) identification of the activities of peptidases present in the intestine, saliva and diet consumed by 12-day-old larvae; and c) cloning, expression, purification and characterization of three peptidases from Psl. hygida. The roles played by the gut microbiome in the life of sciarid larvae in the soil constitutes another important gap in sciarid biology. Thus, the second objective of this project is the characterization of the gut microbiome of 12-day-old larvae of Psl. hygida (subproject 2), which will be carried out in two stages. In the exploratory stage, we propose to sequence and analyze sequencing data from amplicon libraries of the 16S ribosomal RNA gene and ITS (Internal transcribed sequence). For the second stage, which constitutes the characterization of the microbiome itself, we propose: a) sequencing and analyzing metagenome data (DNA shotgun library) and b) sequencing and analyzing metatranscriptome data (RNAseq). The third objective is to expand the molecular and functional characterization of the alternative oxidase (AOX) of Psl. hygida (subproject 3). In the sciarid, AOX is activated in situations of inhibition of complexes III or IV and it is possible that AOX confers protection against respiratory chain inhibitors found in soil, such as cyanide and antimycin A. To expand the molecular and functional characterization of AOX of Psl. hygida, we propose: a) to expand the characterization of mitochondrial function and the expression pattern of AOX in development; b) to investigate the effects of AOX knockdown on mitochondrial function and development; c) to investigate the formation of supercomplexes in the mitochondria of Psl. hygida. The development of this project will result in the characterization of the activity and functions performed by the peptidases found in the saliva of larvae, will provide information on the functions of the intestinal microbiome and will expand the functional characterization of AOX of Psl. hygida. Together, the development of these subprojects, whose objectives are apparently distant, will result in intersecting data that will expand the knowledge about the molecular bases associated with the life of sciarids in soil. (AU)