1. Animals rely on a balance of endogenous and exogenous sources of immunity to mitigate parasite attack. Understanding how environmental context affects that balance is increasingly urgent under rapid environmental change. In herbivores, immunity is determined, in part, by phytochemistry which is plastic in response to environmental conditions. Monarch butterflies, Danaus plexippus, consistently experience infection by a virulent parasite, Ophryocystis elektroscirrha, and some medicinal milkweed (Asclepias) species, with high concentrations of toxic steroids (cardenolides), provide a potent source of exogenous immunity. 2. We investigated plant-mediated influences of elevated CO₂ (eCO₂) on endogenous immune responses of monarch larvae to infection by O. elektroscirrha. Recently, transcriptomics have revealed that infection by O. elektroscirrha does not alter monarch immune gene regulation in larvae, corroborating that monarchs rely more on exogenous than endogenous immunity. However, monarchs feeding on medicinal milkweed grown under eCO₂ lose tolerance to the parasite, associated with changes in phytochemistry. Whether changes in milkweed phytochemistry induced by eCO₂ alter the balance between exogenous and endogenous sources of immunity remains unknown. 3. We fed monarchs two species of milkweed; A. curassavica (medicinal) and A. incarnata (non-medicinal) grown under ambient CO₂ (aCO2) or eCO₂. We then measured endogenous immune responses (phenoloxidase activity, hemocyte concentration, and melanization strength), along with foliar chemistry, to assess mechanisms of monarch immunity under future atmospheric conditions. 4. The melanization response of late-instar larvae was reduced on medicinal milkweed in comparison to non-medicinal milkweed. Moreover, the endogenous immune responses of early-instar larvae to infection by O. elektroscirrha were generally lower in larvae reared on foliage from aCO₂ plants and higher in larvae reared on foliage from eCO₂ plants. When grown under eCO₂, milkweed plants exhibited lower cardenolide concentrations, lower phytochemical diversity, and lower nutritional quality (higher C:N ratios). Together, these results suggest that the loss of exogenous immunity from foliage under eCO₂ results in increased endogenous immune function. 5. Animal populations face multiple threats induced by anthropogenic environmental change. Our results suggest that shifts in the balance between exogenous and endogenous sources of immunity to parasite attack may represent an underappreciated consequence of environmental change. 

We grew two species of milkweed, Asclepias curassavica (medicinal) and Asclepias incarnata (non-medicinal), under ambient CO₂ (aCO₂, 410 ppm) or elevated CO₂ (eCO₂, 810 ppm) using an open top chamber array at the University of Michigan Biological Station. After two months, we took leaf samples for chemical analysis and attached monarch eggs, Danaus plexippus, to excised leaves kept in individual plastic containers. Monarchs hatched and fed on their assigned plant, and at second instar were designated as either uninfected controls or inoculated with the protozoan parasite, Ophryocystis elektroscirrha. Monarchs were then divided into three treatments: assay control group, late-instar immune assay group, and early-instar immune assay group. Monarchs in the assay control group were fed cuttings from their assigned plants ad libitum until pupation and reared to the end of adulthood. Monarchs in the late-instar immune assay group were also fed ad libitum until the fourth instar, and then we inserted a sterile nylon filament above the final proleg to measure the strength of the encapsulation strength. Filaments were removed after 24 hours and monarchs were then reared to the end of adulthood. Finally, the early-instar immune assay group was sacrificed at the third instar to determine endogenous immune function (PO activity and hemocyte density) at this life stage.

Please review the "Metadata" tab included in the datafile. Because monarch larvae were divided into three treatment groups, there are many cells in the spreadsheet without values. This is because those variables are specific to one treatment group, thus, the remaining individuals not in that group do not have assay values.