DOI: 10.5061/dryad.d2547d84x
title: Flying on empty: Reduced mitochondrial function and flight capacity in food-deprived monarch butterflies

description: 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.
description: <strong>Rearing of
 butterflies and inoculation with parasite
 spores</strong> 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). 
 <strong>Food deprivation
 treatment</strong> 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. 
 <strong>Whole-animal
 metabolic rates</strong> 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.
 <strong>Isolation of
 mitochondria</strong> 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 <strong>Mitochondrial
 respiration</strong> 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. <strong>Complex
 activity</strong> 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. <strong>Confirmation of infection
 success</strong> 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.