Single-cell whole-genome sequencing and 18S barcoding of gregarines (Apicomplexa) in Orthoptera: Paving the route toward the understanding of gregarine diversity in insects

Recent phylogeny-aware high-throughput metabarcoding 18S eDNA data have prompted the idea that gregarines are one of the most diverse eukaryotic lineages. Gregarines belong to the Apicomplexa, a phylum that is better known for including vertebrate parasites of medical importance. They, however, do not infect vertebrates but instead inhabit a broad array of invertebrates including insects, which is thought to explain its great diversity. However, this hypothesis has not been tested because we virtually ignore gregarine molecular biology, hindering the understanding of their phylogenetic diversity.

Here, we present a pipeline that facilitates the generation of whole-genome sequences coming from gregarine single-cell isolates. This approach involves the use of multiple displacement amplification and Illumina sequencing. This pipeline retrieves genomes with a variable degree of completeness, which can be improved by merging different single-cell libraries. Additionally, we develop a nested PCR approach that allows the generation of a nearly complete 18S fragment from gregarine isolates as small as 4 cells. This approach uses a pair of newly designed primers that are gregarine generalist in silico simulations, eliminates the need for molecular cloning, and can be readily used to molecularly delineate gregarine morphospecies. Using this nested PCR approach, we have been able to sequence the 18S loci from gregarines infecting different species in the two Orthoptera suborders. Combining both methods will benefit the understanding of the evolution and the great diversity of the gregarines in Insecta as revealed by eDNA data.