Mitochondrial function is fundamental to organismal performance, health, and fitness – especially during energetically challenging events, such as migration. With this investigation, we evaluated mitochondrial sensitivity to ecologically relevant stressors. We focused on an iconic migrant, the North American monarch butterfly (Danaus plexippus), and examined the effects of two stressors: seven days of food deprivation, and infection by the protozoan parasite Ophryocystis elektroscirrha (known to reduce survival and flight performance). We measured whole-animal resting metabolic rate (RMR) and peak flight metabolic rate, and mitochondrial respiration of isolated mitochondria from the flight muscles. Food deprivation reduced mass-independent RMR and peak flight metabolic rate, whereas infection did not. Fed monarchs used mainly lipids in flight (respiratory quotient 0.73), but the respiratory quotient dropped in food-deprived individuals, possibly indicating switching to alternative energy sources, such as ketone bodies. Food deprivation decreased mitochondrial maximum oxygen consumption but not basal respiration, resulting in lower respiratory control ratio (RCR). Furthermore, food deprivation decreased mitochondrial complex III activity, but increased complex IV activity. Infection did not result in any changes in these mitochondrial variables. Mitochondrial maximum respiration rate correlated positively with mass-independent RMR and flight metabolic rate, suggesting a link between mitochondria and whole-animal performance. In conclusion, low food availability negatively affects mitochondrial function and flight performance, with potential implications for migration, fitness, and population dynamics. Although previous studies have reported poor flight performance in infected monarchs, we found no differences in physiological performance, suggesting that reduced flight capacity may be due to structural differences or low energy stores.

Rearing of butterflies and inoculation with parasite spores

The monarchs used in this investigation were offspring of outbred crosses of butterflies collected in Georgia, USA. Eggs were collected on milkweed (Asclepias incarnata). The rearing took place at Emory University. Two days after hatching, caterpillars were either infected with the parasite Ophryocystis elektroscirrha or left uninfected. They were placed in a 10 cm Petri dish with a wet filter paper and a small milkweed leaf disk (8 mm diameter), to which 10 parasite spores were manually added (uninfected caterpillars received a leaf disk without parasites). The parasite used was a revived isolate of the parasite clone E10, originally obtained from the wild in eastern USA (De Roode et al., 2008). After finishing their leaf disk in the next 48 hours, caterpillars were transferred to a live potted milkweed plant, which was covered with a mesh bag (13 x 57 cm). The larvae were maintained in a greenhouse with natural light at an average temperature of 26 °C. Following pupation, the pupae were transported to Auburn University where they were glued onto the lid of individual 500 ml solo cups and maintained at a daylight cycle of 12L:12D and a 28 °C:24 °C temperature cycle. Upon eclosion, individuals were weighed, sexed and marked. The individuals were allocated into four treatment groups: uninfected, fed (n=9), infected, fed (n=6), uninfected, food-deprived (n=9), and infected, food-deprived (n=7). 

Food deprivation treatment

We focused on females only in this study. In both feeding treatments, females were kept in large indoor cages (90 cm x 90 cm x 210 cm) under a 12L:12D light cycle and 28 °C:24 °C temperature cycle. Infected and uninfected females were kept in the same cages, as parasite transmission occurs from adult to larval offspring, and adults cannot become infected. Control females had constant access to 10% honey water provided from saturated sponges placed in small plastic cups. Females in the food deprivation treatment were provided with water only, served from similar sponges in cups.

Whole-animal metabolic rates

We measured resting metabolic rate (RMR) and flight metabolic rates using flow-through respirometry following similar procedures as described for other Lepidoptera (Niitepõld and Boggs, 2015) and the monarch (Zhan et al., 2014). In short, on the evening before the measurements, monarchs were individually placed in covered plastic cups and kept at room temperature overnight. The next day, postabsorptive monarchs were provided with water before being placed in a cylindrical 1-litre respirometry chamber that was covered with a black cloth ca. 20 min before the measurement of RMR. The chamber was continuously flushed with dry, CO2-free air, obtained from a Whatman purge-gas generator (Whatman, Haverhill, MA, USA) at a flow rate of 1.5 l min-1 and subsampled at a flow rate of 0.75 l min-1 using two Sable Systems Mass Flow Systems (Sable Systems, Las Vegas, NV, USA). We pulled the excurrent air through a column filled with magnesium perchlorate to remove traces of humidity and then through a Li-Cor 6262 CO2 analyser (Li-Cor, Lincoln, NE, USA) and finally, an Oxzilla II O2 analyser (Sable Systems). Individuals remained calm and motionless when kept in the dark, but if an individual became restless, this could immediately be seen in the CO2 production curve. In these rare cases, we allowed the individual to settle before continuing with the experiment. We took the average of 1.5 min of stable CO2 production to represent RMR. Flight metabolic rate was measured when the cover was removed, and the butterfly was stimulated to fly by gently shaking the chamber each time the butterfly attempted to land. The stimulation continued for 7 min, after which the chamber was covered again. We used standard respirometry equations (Lighton, 2008), and converted CO2 and O2 concentrations  to metabolic rates expressed in millilitres per hour. We used peak metabolic rate, the highest rate of CO2 production and O2 consumption, to represent flight capacity. Peak metabolic rate was typically recorded during the first minutes of the flight trial. We calculated RQ by dividing peak CO2 production rate with O2 consumption rate from the same time point. As the respirometry setup was optimised for flight measurements, we do not report O2 consumption rate and RQ for RMR.

We aimed at a balanced age distribution among the treatment groups, although this was challenging due to the limited material and the number of individuals that could be processed each day. The mean ages and standard deviations for the groups were: uninfected, fed 5.8±1.1 days; uninfected, starved 6.6±1.1 days; infected, fed 6.0±2 days; and infected, starved 6.7±2.9 days. After the measurement, the butterflies were weighed, and individuals in the feeding treatment were given honey-water solution, starved individuals were given water, and all butterflies were placed in individual glassine envelopes and stored at room temperature overnight. On the next day, butterflies were sacrificed and used for mitochondrial assays.

Isolation of mitochondria

We first decapitated the individual with scissors, then quickly separated the body parts, and weighed the thorax without the legs and wings. Half of the thorax was placed in 10 ml of mitochondrial isolation solution (100 mM KCl, 40 mM Tris HCl, 10 mM Tris base, 1 mM MgSO4, 0.1 mM EDTA, 0.2 mM ATP, and 0.15% (wt/vol) free fatty acid bovine serum albumin (BSA)) and was minced with scissors, after which we removed remnants of the exoskeleton. We homogenized the muscle tissue with a VirTis homogeniser for 5 sec. We added Trypsin (5 mg/g of muscle) and repeatedly mixed the solution for 7 min and finally, added isolation solution (10 ml) to terminate the reaction. The solution was then centrifuged at 500g for 10 min. The supernatant was transferred through a cheesecloth filter into a new centrifuge tube and centrifuged at 3500g for 10 min. The supernatant was discarded, and we resuspended the mitochondrial pellet in isolation solution (10 ml) and centrifuged the sample for the second time at 3500g for 10 min. The supernatant was again discarded, and the mitochondrial pellet was resuspended in isolation solution, as above, but with no BSA (10 ml), and the sample was centrifuged for the third time at 3500g for 10 min. The final mitochondrial pellet was resuspended in mannitol/sucrose solution (220 mM mannitol, 70 mM sucrose, 10 mM Tris HCl, and 1 mM EGTA) with a Dounce homogeniser

Mitochondrial respiration

Mitochondrial respiration was measured polarographically (Oxytherm, Hansatech Instruments, Norfolk, UK) following procedures (with minor modifications) described by Hyatt et al. (2017). Isolated mitochondria (20 µL) were incubated in a final volume of 1 ml of respiration buffer (100 mM KCL, 50 mM MOPS, 10 mM KH2PO4, 20 mM glucose, 10 mM MgCl2, 1 mM EGTA, and 0.2% fatty acid free BSA; pH = 7.0) adapted from Wanders et al. (1984) at 37°C with continuous stirring. The measurement temperature corresponds to the upper end of the range of thoracic temperatures measured in monarchs under field conditions (Masters et al., 1988). We used the complex I substrates of 2 mM malate, 10 mM glutamate, and 2 mM pyruvate (final concentrations). We measured state 3 respiration (maximal respiration) after adding 5.0 μL of 50 mM solution of ADP to the chamber, and state 4 (basal) respiration was recorded following the phosphorylation of the added ADP as described by Estabrook (1967). State 3 and state 4 were normalised to total protein content that was measured by the Bradford assay (Bradford, 1976). We calculated the respiratory control ratio (RCR) by dividing state 3 by state 4.

Complex activity

Microplate spectrophotometric enzymatic assays using isolated mitochondria were performed using the protocols (with minor modifications) described by Trounce et al. (1996). Due to limited tissue material and working with a novel study system, only the activities of complexes III and IV were successfully quantified for all individuals.

Briefly, complex III activity (EC 1.10.2.2) was obtained (30°C) by recording the reduction of cytochrome c at 550 nm catalysed by the presence of reduced decylubiquinone. The reaction mixture (250 µl) contained 250 mM sucrose, 1 mM EDTA, 50 mM Tris-HCl (pH 7.4), 50 µM cytochrome c, 2 mM KCN and the reaction was initiated by adding 1.4 µl of reduced decylubiquinone (10 mM) to each well.

Briefly, complex IV activity (EC 1.9.3.1) was obtained (~22°C) by recording the oxidation of reduced cytochrome c at 550 nm. The reaction mixture (200 µl) contained 10 mM potassium phosphate buffer (pH 7.4), 20 µM reduced cytochrome c, and 0.025% lauryl maltoside and the reaction was initiated by adding mitochondria to each well.

Complex III and IV activities were normalised to citrate synthase activity (EC 4.1.3.7) that was obtained (30°C) by monitoring the reduction of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) at 412 nm. The reaction mixture (250 µl) contained 125 mM Tris-HCl (pH 8.0), 0.3 mM Acetyl-CoA, and 1 mM DTNB. The reaction was initiated by adding 5 µl of oxaloacetate (50 mM) to each well.

Confirmation of infection success

We quantified the spore load of infected females based on the abdomen. The abdomen was placed in a 5 ml scintillation vial, and vortexed at max speed with a Vortex Genie. We counted parasite spores on a haemocytometer slide and calculated the original number of spores in the 5 ml vial.