DOI: 10.5061/dryad.dr7sqv9ww
title: Elevated atmospheric concentrations of CO2 increase endogenous immune function in a specialist herbivore

description: 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. 
description: We grew two species of milkweed, <i>Asclepias curassavica
 </i>(medicinal) and <i>Asclepias incarnata
 </i>(non-medicinal), under ambient CO<sub>2</sub>
 (aCO<sub>2</sub>, 410 ppm) or elevated
 CO<sub>2</sub> (eCO<sub>2</sub>, 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, <i>Danaus plexippus</i>, 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, <i>Ophryocystis
 elektroscirrha</i>. 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 <i>ad libitum</i>
 until pupation and reared to the end of adulthood. Monarchs in the
 late-instar immune assay group were also fed <i>ad libitum</i>
 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.
description: 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.