Time-resolved transcriptomics of haemocyte discrimination between challenges by nematodes and inert material in the...

Insects defend against large metazoan parasites such as nematodes through haemocyte-mediated encapsulation, but how haemocytes respond to living parasites remains poorly understood. This study examined the cellular and transcriptional responses of haemocytes to living nematodes in the lepidopteran insect Mythimna separata.

Encapsulation assays were combined with time-course transcriptomic analyses to compare haemocyte responses to inert polystyrene beads and the free-living nematode Caenorhabditis elegans. Haemocytes were collected following injection treatments, and RNA sequencing was used to characterise temporal changes in gene expression.

Inert polystyrene beads were rapidly encapsulated, whereas C. elegans induced a slower, progressive encapsulation response. Time-resolved RNA sequencing showed that injection alone established a structured physiological baseline, while nematode exposure consistently drove haemocytes into distinct transcriptional states not observed with inert beads. Nematode-associated responses were enriched for regulatory, immune, and membrane-related processes, with biased and temporally stabilised engagement of the Toll signalling pathway. Importantly, Toll pathway activation also occurred in response to axenically prepared nematodes, demonstrating that living nematodes alone are sufficient to influence canonical innate immune signalling.

These findings show that haemocytes distinguish living nematodes from inert particles not merely through their physical presence, but by mounting sustained and coordinated transcriptional responses. This study provides a temporal and mechanistic framework for understanding how insect immune systems recognise and respond to large metazoan parasites.