High-throughput detection of microeukaryotic parasites in insects using Nanopore sequencing

Area of Science:

• * Entomology and Parasitology
• * Molecular Biology and Genomics
• * Food and Feed Production

Background:

• * The expanding insect farming industry faces challenges in identifying pathogens, particularly overlooked protists and microsporidia.
• * Current detection methods often focus on viruses, bacteria, and fungi, neglecting other significant entomopathogens.
• * Economic losses in insect mass rearing can be substantial due to undetected diseases.

Purpose of the Study:

• * To explore and phylogenetically analyze microsporidian and protist parasites in insects using Nanopore sequencing.
• * To develop improved molecular diagnostic tools for detecting overlooked entomopathogens.
• * To enhance the understanding of parasite evolution and biodiversity in commercially reared insects.

Main Methods:

• * Utilized Nanopore sequencing for pathogen detection in insect host DNA.
• * Employed group-specific primers and amplicon pooling for targeted amplification of protists (Gregarinasina), microsporidia, and fungi.
• * Generated small subunit and long subunit rRNA sequence data for phylogenetic analysis.

Main Results:

• * Successfully amplified and sequenced Gregarinasina, microsporidia, and fungal pathogens from insect DNA.
• * Generated novel molecular data for phylogenetic analysis, clarifying evolutionary relationships.
• * Identified limitations in existing primers and developed new Gregarine-specific primers.

Conclusions:

• * Nanopore sequencing is an effective tool for detecting overlooked protist and microsporidian entomopathogens.
• * The study provides valuable molecular data and improved primers for future research on insect parasites.
• * Advances the understanding of pathogen biodiversity and diagnostic capabilities in insect mass rearing.