Understanding the host range, patterns, and consequences of parasite infection is essential for detecting and responding to parasite-mediated insect declines. The debilitating protozoan Ophryocystis elektroscirrha (OE; Neogregarinorida: Ophryocystidae) was identified as a parasite of monarch (Danaus plexippus) and queen (D. gilippus) butterflies in the late 1960s in Florida, USA. Recently, similar parasites were identified in other Danaus species, but their occurrence among the diverse clade of milkweed butterflies (Nymphalidae: Danainae) remains unexplored. This study used museum collections to systematically investigate the host range and global occurrence of OE and similar Ophryocystis parasites in milkweed butterflies. We examined 2,727 museum specimens from 86 countries/areas, spanning 61 milkweed butterfly species across 10 genera, to document parasitism. Our findings revealed that, among milkweed butterflies, Ophryocystis parasites were confined to hosts in the genus Danaus and were widespread across five continents. Positive specimens dated back to 1909. Lesser wanderers (D. petilia) exhibited the highest percentage of positive specimens (16%), followed by monarchs (12%), plain tigers (D. chrysippus, 11%), and queens (4%). We also present the first documented infections in Jamaican monarchs (D. cleophile, 4%). The patterns of parasite variation in size reflected the relatedness of their host species but not their size. Overall, this research underscores the value of museum collections for investigating geographic and temporal host-parasite associations and provides foundational ecological data on the relationships between Ophryocystis parasites and their milkweed butterfly hosts.

We used archived specimens to determine infection status and the temporal and geographic infection patterns among milkweed butterflies. Butterfly species targeted were milkweed butterflies in the subfamily Danainae. We recorded the specimen information from its label. Pictures of the dorsal side of each pinned specimen were taken. When no species information was included on the specimen’s label, pictures were used for identification. A subset of pictures alongside label information was shared with an expert on milkweed butterfly taxonomy for identification or confirmation. We followed Ackery and Vane-Wright (1984) to standardize the taxonomic nomenclature and resolve synonyms at the species level except where more recent taxonomic work was available. Specimens were swabed and screened for the presence of spores of OE and morphologically similar parasites. We used digital photographs to estimate the forewing length (mm) of a subset of parasite-positive and negative specimens. We also estimated the median spore size (two-dimensional area, µm2) for each positive butterfly measured.