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

The mass rearing of insects for food and feed is increasing significantly, but it presents challenges in detecting and characterizing pathogens which may cause severe economic losses. Much attention is placed on detecting viruses, bacteria and fungi; however, other significant entomopathogens remain overlooked, such as protist and microsporidian parasites. This study employed Nanopore sequencing to explore and phylogenetically analyse microsporidian and protist parasites in reared insects. Using group-specific primers and amplicon pooling strategies, we successfully amplified and sequenced a key protist group (Gregarinasina), microsporidia and fungal pathogens from insect host DNA, providing data for their future detection. Our approach generated novel molecular data, enabling the inference of small subunit and long subunit rRNA phylogenies which enhance the understanding of the evolutionary origin of these parasites. Furthermore, we highlight potential shortcomings of currently available primers and provide new Gregarine-specific primers to aid in further research of these overlooked entomopathogens. This research demonstrates the effectiveness of third-generation sequencing technologies in improving diagnostic tools and advances the study of pathogen biodiversity within economically important insect hosts.