Natural infection of a holometabolous insect with gregarines and their detection in pupae

Gregarines are symbionts of the gut or other body cavities associated with numerous insect species. Knowledge about natural gregarine infection levels and across metamorphosis in holometabolous hosts is sparse. Individuals of the mustard leaf beetle (Phaedon cochleariae, Coleoptera: Chrysomelidae) were collected from eight populations and dissected to estimate the prevalence and load of infection with gregarines. In the laboratory, the effect of larval density (single individuals versus groups of five) on subsequent larval gametocyst load and adult infection with Gregarina cochlearium was tested using a stereomicroscope. The presence of gregarine DNA on the surface and in pupae was investigated by performing PCR on pupal surface swabs, swabbed pupae and washed pupae. Histological sections were used to visualize gregarine life stages in larvae and pupae. Gregarines were found in all of the examined populations, with high variation in prevalence and load among the populations and between the sexes, being higher in female beetles. The likelihood and intensity of gregarine infection in adults was influenced by their age and their experienced larval density. Gregarine DNA was present in pupal surface swabs, swabbed pupae and washed pupae. In the histological sections, gamontocysts (encysted stage of paired gamonts in syzygy) were visible only in the guts of the larvae, while they were not confined to the digestive system in pupae. High prevalence in the field and the presence of gregarines in pupae are indicators of a tight relationship between this gregarine species and its host.