Content uploaded by Christopher Newell
Author content
All content in this area was uploaded by Christopher Newell on Nov 29, 2017
Content may be subject to copyright.
Physiological Entomology (2016), DOI: 10.1111/phen.12127
Mitochondrial substrate specificity in beetle flight
muscle: assessing respiratory oxygen flux in small
samples from Dermestes maculatus and Tenebrio
molitor
CHRISTOPHER NEWELL1,2, CONSTANCE L. KANE2and DANIEL
A. KANE2
1Department of Medical Science, Cumming School of Medicine, University of Calgary, Calgary, Canada and 2Department of Human
Kinetics, St Francis Xavier University, Antigonish, Canada
Abstract. In the present study, the permeabilized bre approach is adapted to
investigate substrate utilization patterns in the ight muscle mitochondria of Der-
mestes maculatus (Coleoptera: Dermestidae; a carrion scavenger beetle) and Tene-
brio molitor (Coleoptera: Tenebrionidae; a phytophagous scavenger beetle). Respira-
tion in saponin-permeabilized bres is measured during titration of palmitoyl-carnitine
(Palm-C), pyruvate (Pyr) or -glycerol 3-phosphate (G3-P). Michaelis– Menten-type
enzyme kinetics for oxygen consumption are observed as a function of substrate con-
centration in Pyr and G3-P, from which substrate-specic apparent Km(sensitivity) and
Vmax (capacity) are determined. Compared with D. maculatus, the apparent Kmin T.
molitor is lower (P<0.001) for Pyr, and Vmax is greater for G3-P (P<0.001). In D.
maculatus, the apparent Kmfor G3-P is greater (P<0.001), and respiratory Vmax is
lower (P<0.001), than kinetics for Pyr. Robust respiration with -proline (Pro) is also
observed in both beetle species tested; however, it is over 2.5-fold greater in D. macula-
tus than T. molitor (P<0.05). These results demonstrate that respiration in beetle ight
muscle mitochondria can be assessed in small samples (i.e. at the individual beetle level)
using the approach adapted for the present study. The results of the present study also
highlight the substrate oxidative capacity patterns in both D. maculatus and T. molitor,
which rank Palm-C <G3-P <Pyr <Pro.
Key words. Insect, metabolism, permeabilized bres, skeletal muscle.
Introduction
Insect ight is one of the most energetically demanding
activities known in biological systems. Effective coupling of
energy supply and consumption within insect ight muscula-
ture enables high mass-specic rates of aerobic metabolism.
A primary role of mitochondria is to utilize the products of
fat, protein and carbohydrate metabolism to generate ATP
through the electron transport system, which necessarily con-
sumes oxygen. Generous oxygen provision, coupled with high
Correspondence: Christopher Newell, Department of Medical Sci-
ence, Cumming School of Medicine, University of Calgary, 2500
University Drive NW, Calgary, Alberta T2N 1 N4, Canada. Tel.:
+1 403 210 8932; e-mail: cnewell@ucalgary.ca
concentrations of metabolic enzymes and mitochondrial densi-
ties, allows such unique respiratory rates in insect ight muscle
(Rockstein, 1974).
The mealworm beetle Tenebrio molitor (Coleoptera: Tene-
brionidae) is a common pest found living in, and feeding on
stored grains. The utilization of T. molitor is popular in biologi-
cal research as a result of their ease of colonization, acquisition
and relatively large size. Previous immunological and evolu-
tionary biological research has examined T. molitor as a model
organism (Dobson et al., 2012). The ight musculature of the
adult T. molitor is noted for its high concentration of mitochon-
dria (Watanabe & Williams, 1951), and the species thus exhibits
potential as an experimental model organism for mitochondrial
research (Wolf & Joshi, 1995). Another common beetle main-
tained in research institutions is the carrion beetle Dermestes
© 2016 The Royal Entomological Society 1
2C. Newell et al.
maculatus (Coleoptera: Dermestidae), which is used predomi-
nantly in the preparation of skeletons for university and museum
displays (McManus, 1974).
Mitochondria have been studied for over a century (Ern-
ster & Schatz, 1981), including considerable research exam-
ining aerobic energy metabolism (Moyes et al., 1990). Two
commonly used means of studying muscle mitochondrial func-
tion include mitochondrial isolation (Chance & Williams,
1955) and permeabilized bre techniques (Kuznetsov et al.,
2008; Pesta & Gnaiger, 2012; Perry et al., 2013). Isolat-
ing mitochondria involves destroying the tissue to release
the mitochondria, and separation via centrifugation. Study-
ing the mitochondria intact and in situ through permeabi-
lization of the plasma membrane is a preferred method of
mitochondrial functional analysis, partly because it requires
a smaller amount of tissue for analysis than the isolated
technique.
Previous studies investigating beetle ight muscle mitochon-
dria report the use of isolated mitochondria from the pooled
muscle of multiple individuals (Hansford & Johnson, 1975;
Auerswald & Gäde, 1999b). However, assessing ight muscle
mitochondria from an individual beetle may be desirable for
some studies. Moreover, in mammalian mitochondria, the isola-
tion procedure itself confers potentially undesirable changes to
mitochondrial function. These deleterious artefacts of the isola-
tion procedure may be mitigated by use of a permeabilized bre
approach (Picard et al., 2011), which involves selectively per-
meabilizing the sarcolemma with a detergent, often a saponin,
and leaving the mitochondrial reticulum intact. Because far
less tissue is required for the analysis of permeabilized bres,
the assessment of small muscle samples from insects is ideal.
Indeed, a recent study reports the use of the permeabilized bre
technique for the investigation of mitochondrial respiration in
ight muscle from the fruit y Drosophila melanogaster (Cor-
rea et al., 2012; Pichaud et al., 2013), although this still requires
pooled samples of multiple ies for certain experimental
conditions.
The present study aims to adapt the permeablized bre
approach for comparing the respiratory substrate prole of
ight muscle mitochondria between T. molitor and D. macu-
latus beetles. Selected substrates are: the β-oxidation substrate
palmitoyl-carnitine (Palm-C), the metabolic intermediate pyru-
vate (Pyr), the glycolytic intermediate -glycerol 3-phosphate
(G3-P), and the amino acid -proline. We demonstrate that the
permeabilized bre technique can be adapted for the analysis of
mitochondrial respiration in small samples of insect ight mus-
cle from two beetle species.
Materials and methods
Insects
Hide beetles, D. maculatus, were obtained from the Biology
Department at St Francis Xavier University, where they are
commonly used for skeleton preparation. Beetles were reared in
darkness at 22 ∘C, with free access to food (carrion). Common
mealworm beetles, T. molitor, were purchased locally, and
housed in a cage (30 ×15 ×15 cm) at 22 ∘C. Beetles were
reared in darkness, except during the experimental proce-
dures, and had free access to food (standard, commercially
available oats). Ambient moisture was provided by misting a
paper-lined cage with puried water daily. These studies were
approved by St Francis Xavier University Animal Care prior to
experimentation.
Sample dissection
To maintain adequate mitochondrial function, adult beetles
were sacriced at 5–20 days; both T. molitor and D. maculatus
exhibit a decline in mean survival rate at approximately 100 days
(Bowler, 1967; Archer & Elgar, 1998). Immediately prior to
sacrice, beetles were captured in an enclosed Petri dish, and
placed in a freezer at −20 ∘C for 3– 5 min to induce anaesthesia.
Next, the beetles were decapitated and dissected superior to their
most anterior pair of legs and inferior to their most posterior pair
of legs using a scalpel. The nal pair of legs was dissected off
the remaining thoracic section, and the gut was removed using
needle-tipped forceps. The supercial exoskeletal coverings
and deep wing pairings on the dorsal portion of the thoracic
section were also removed. A scalpel incision was made by
gently inserting the blade into the gut tract, dorsal portion
oriented downwards, providing sufcient pressure to cut cleanly
through the specimen. An identical incision was then made
along the ventral thorax to halve the section. All incisions were
performed under magnication (×1.6; SteREO Discovery.V8
stereoscope; Carl Zeiss systems, Göttingen, Germany) with aid
of needle-tipped forceps for stabilization.
Preparation of permeabilized muscle bres
This technique was adapted from previous methods for
mammalian tissues (Kuznetsov et al., 2008; Pesta & Gnaiger,
2012; Perry et al., 2013). For T. molitor beetles, the remaining
exoskeletal shell fragments were removed from each thorax
half with needle-tipped forceps and scalpel in approximately
3–5 mL of ice-cold buffer X, containing (in m) 50 potassium
ethanesulfonic acid (K-MES), 7.23 potassium ethylene glycol
tetraacetic acid (EGTA), 2.77 calcium-potassium EGTA, 20
imidazole, 20 taurine, 5.7 ATP, 14.3 phosphocreatine and 6.56
MgCl2-6 H2O) (pH 7.1; 290 mOsm). Both portions of muscle
were then trimmed of remaining non-muscle tissue. Two small
muscle bundles were prepared from each beetle. As a result
of the smaller size of D. maculatus beetles, the remaining
exoskeleton was left attached to the muscle tissue. Tissue
bundles were then treated with 50 μgmL
−1saponin in cold
(4 ∘C) buffer X for 30 min to permeabilize the myobres. The
permeabilized bre bundles were then placed in buffer Z con-
taining (in m) 105 K-MES, 30 KCl, 1 EGTA, 10 KH2PO4and
5MgCl
2-6 H2O, with 5.0 mg mL−1bovine serum albumin (pH
7.1; 290 mOsm). Twenty millimolar of creatine was included
in the assay buffer Z. Permeabilized bundles remained within
the buffer Z solution at 4 ∘C until analysis (approximately
15 min).
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12127
Beetle ight muscle respiration 3
Mitochondrial respiration in permeabilized bres
High-resolution respirometric measurements were performed
in2mLofassaybufferZat25
∘C using the Oxygraph-2k
(Oroboros Instruments, Innsbruck, Austria). The permeabilized
bre preparation results in muscle tissue being removed from
its respective connective tissue, yielding a fragile product. To
account for potential tissue separation, bre bundles were nor-
malized to tissue wet weight. Immediately prior to respira-
tory assessment, muscle samples were gently patted dry with
tissue paper, and weighed, with weights ranging from 0.93
to 5.35 mg (mean ±SD, 2.73 ±1.18 mg). Respiration rate was
obtained from 20 data points in -proline-supported respira-
tion in D. maculatus; the rest of the conditions were com-
prised of 40 data points, and all were expressed as pmol
O2s−1mg−1wet weight. The same experimental protocol was
performed in both oxygraph chambers simultaneously on the
two half-sections of beetle brillar ight muscles. Immedi-
ately prior to the addition of each bre bundle to the oxy-
graph chamber, 20 μ blebbistatin (Bleb; Sigma-Aldrich, St
Louis, Missouri) was added to inhibit contraction (Perry et al.,
2011, 2013). All experiments were completed using oxygen con-
centrations in the range of (70 –300 nmol mL−1), with instru-
ment chambers being re-oxygenated when necessary. A rep-
resentative trace of oxygen consumption by permeabilized D.
maculatus bres is reported in the Supporting information,
Figure S1.
Because D. maculatus is a much smaller beetle than T.
molitor (0.6–1.0 cm versus 1.2–2.5 cm), the methods were
modied to assess D. maculatus mitochondrial respiration.
Dermestes maculatus beetle segments were blotted and weighed
as described previously. The muscle sample with attached
exoskeleton was then placed into the chamber and allowed to
spin for 20 min, at which point the majority of the muscle
tissue had detached from the exoskeleton. The stir bars were
then stopped and the exoskeleton pieces were retrieved from
the oxygraph chambers after visual inspection of the static
chambers. These were then blotted and weighed, with weights
ranging from 1.39 to 5.26 mg (mean ±SD, 3.21 ±1.14 mg). The
weight of the retrieved exoskeleton was subtracted from the
initial weight to determine the mass of muscle tissue in each
chamber. The stir bars were then restarted, the chambers closed
and the experiments were continued. Because of the technical
skill required to perform the T. molitor dissection, bre bundles
with accompanying exoskeletal fragments may also be removed
and subsequently weighed using the adapted method for D.
maculatus tissues.
After each bre bundle was sealed in its oxygraph chamber,
2 m ADP was added to each chamber to initiate oxidative
phosphorylation, and 2 m malate was added to promote oxida-
tion of any endogenous respiratory substrates present. Substrate
additions were selected to isolate the various metabolic inputs
to the electron transport system. Either 2.5 –100 μPalm-C,a
substrate for β-oxidation; 0.025–5 m Pyr, a substrate for pyru-
vate dehydrogenase complex and the tri-carboxylic acid cycle,
or 0.5– 48 m -G3-P, a glycolytic intermediate involved in the
shuttling of electrons to the mitochondria (i.e. the G3-P shuttle)
was titrated to each bre bundle. Each substrate was titrated in
stepwise increments to determine the enzyme kinetics of respi-
ration. Lack of a cytochrome c(10 μ) response at the end of the
oxygraph protocol was indicative of intact outer mitochondrial
membrane after tissue permeabilization (Gnaiger & Kuznetsov,
2002). For the kinetic analyses, the respiratory rates with
ADP +malate alone were subtracted from the respective respi-
ratory values for each substrate tested. Absolute respiratory rates
for both D. maculatus and T. molitor, expressed as a combination
of malate +ADP respiration plus that with each respec-
tive substrate, are presented in the Supporting information,
Figure S2.
Beetle ight muscle, as with tsetse ies and bees (Bursell &
Slack, 1976; Suarez et al., 2005b), favours -proline oxidation
(de Kort et al., 1973; Weeda et al., 1980; Auerswald et al.,
1998; Gäde & Auerswald, 2002). -proline was added in a
stepwise manner to establish a saturating concentration for
each species (2–48 m). Furthermore, a protocol was generated
to examine the impact of the contraction inhibitor, Bleb, and
phosphate acceptors, creatine and -arginine, on the oxygen
consumption rates of T. molitor. To compare the present results
with those available in the literature, respiration with 25 m
-proline was examined (Soares et al., 2015). Accordingly,
25 m -proline +2 m malate was included with 0.5 m of
subsaturating ADP before the addition of 20 m creatine and
20 m -arginine, respectively. Saturating ADP (2 m) was
then added before a nal 10 μ addition of cytochrome c.A
representative trace of oxygen consumption using the protocol
described above is presented in the Supporting information,
Figure S3.
Statistical analysis
Statistical analysis was performed with , version 5.02
(GraphPad Software, San Diego, California). Maximum res-
piratory velocity (Vmax) and substrate concentration at Vmax /2
(Km) were determined with curve-tting software (, ver-
sion 5.02). The apparent Kmfor each substrate was determined
by applying the Michaelis–Menten enzyme kinetics tting
model to Palm-C-, G3-P- and Pyr-supported respiration. Dif-
ferences between respiratory kinetics collected for T. molitor
and D. maculatus, and among the respiratory kinetics for
substrates, were determined by two-way analysis of variance
(), followed by a Bonferroni post-hoc test. The rate
of -proline-supported respiration between T. molitor and D.
maculatus were compared using Student’s independent t-test
with Welch’s correction for unequal sample SDs. To investigate
the effects of Bleb on respiration in beetle bres, a two-way
with repeated measures was used to compare ±Bleb,
with a Bonferroni post-hoc test to examine differences among
substrate additions. All data are reported as the mean ±SEM.
P<0.05 was considered statistically signicant.
Results
A representative Michaelis– Menten curve illustrates the
pyruvate-fuelled respiration of T. molitor and D. macula-
tus beetle ight muscle (Fig. 1). Additionally, mitochondria
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12127
4C. Newell et al.
0 500 1000 1500 2000 2500
0
20
40
60
80
100
D. maculatus
T. molitor
Rate of O2 consumption
(pmol · s–1 · mg–1 wet wt)
Pyruvate conc. (µM)
Fig. 1. Michaelis –Menten type enzyme kinetic curves for pyruvate
(Pyr) substrate additions to brillar ight muscle bres from Dermestes
maculatus and Tenebrio molitor. Data are the mean±SEM after sub-
traction of initial ADP +malate respiratory rates.
exhibited robust respiratory rates with -proline as a substrate;
-proline-supported respiration in D. maculatus was more than
2.7-fold greater than that of T. molitor (independent Student’s
t-test; P<0.05) (Fig. 2). Palm-C-fuelled respiration for both
species of beetle was not consistent with Michaelis– Menten
enzyme kinetics, and the substrate-dependent alterations in
respiratory O2ux were negligible. Among the values com-
puted for the substrate-specic apparent Km, signicant main
effects for beetle species (F1,19 =50.99, P<0.0001) and sub-
strate (F1,19 =86.74, P<0.0001) were detected, as well as
a signicant species-by-substrate interaction (F1,19 =46.41,
P<0.0001) (Table 1). The apparent Kmfor G3-P was signif-
icantly greater than Pyr in both T. molitor and D. maculatus
(P<0.001), suggesting a relatively low sensitivity to G3-P in
both species. Signicant main effects for the substrate-specic
Vmax were also observed for beetle species (F1,19 =34.60,
P<0.0001) and substrate (F1,19 =35.96, P<0.0001). The Vmax
for Pyr was signicantly greater than G3-P in D. maculatus
(P<0.0001) (Table 1), whereas a trend was observed for T.
molitor (P=0.09). The apparent Kmfor Pyr was lower in T.
molitor compared with D. maculatus (P<0.001) (Table 1),
suggesting a greater sensitivity to this substrate in T. molitor
ight muscle mitochondria. The Vmax for G3-P was greater in T.
molitor compared with D. maculatus (P<0.001), suggesting a
greater capacity for oxidation in the ight muscle mitochondria
of T. molitor for this substrate.
Tenebrio molitor mitochondria exhibited greater
-proline-supported respiration when treated with Bleb
(two-way with repeated measures; main effect for Bleb;
F1,3 =13.07, P=0.036) (Fig. 3). Post-hoc analysis revealed
signicant differences in respiration ±Bleb after the addition
of -arginine, 2 m ADP and cytochrome c(P<0.001). Titra-
tion of the phosphate acceptor -arginine increased respiration
from basal respiration (P<0.05). The addition of cytochrome
cyielded no increase in respiration, indicating an intact outer
mitochondrial membrane after tissue permeabilization. Because
of the apparent inuence of Bleb on respiration, it should
be noted that all of the results obtained in the present study
D. maculatus T. molitor
0
500
1000
1500
L-Proline-supported respiration
(pmol O2 · s–1 mg–1 wet wt)
Fig. 2. -Proline-supported respiration in Dermestes maculatus and
Tenebrio molitor.InT. molitor bres, 16, 32 and 48 m -proline
was titrated to elicit maximal respiration. Compared with D. maculatus
(n=4), respiratory oxygen ux was signicantly lower in T. molitor
(n=6; unpaired Student’s t-test with Welch’s correction for assumed
differences in SD; *P<0.05).
involving ADP-stimulated respiration may be inuenced by the
inclusion of Bleb (Fig. 3).
Discussion
The present study aims to adapt the permeabilized bre approach
to beetle ight muscle and enable the study of mitochon-
dria in a comparative physiology context. To this end, the
substrate-specic mitochondrial respiratory kinetics of T. moli-
tor versus D. maculatus are assessed.
Fatty acids such as palmitate constitute a primary energetic
fuel for the ight muscles of many species of insects, with their
oxidation being carnitine-dependent (Wegener, 1996; Hauner-
land, 1997). Initially, Palm-C is tested, an activated fatty acid
known to be particularly important in prolonged ight in locusts
(Wegener, 1996). Notably, the Palm-C data are inconsistent with
Michaelis–Menten enzyme kinetics and substrate-dependent
changes in O2ux are negligible (Table 1). Although lipid oxi-
dation is conrmed in several species of beetle (Thompson &
Bennett, 1971), T. molitor and D. maculatus are not known to
engage in prolonged or long-distance ight. The results of the
present study (Table 1) agree with previous research demonstrat-
ing that, relative to other substrates (e.g. -proline, pyruvate),
lipid oxidation in certain beetle ight muscle is of relatively lit-
tle importance (Hansford & Johnson, 1975; Khan & de Kort,
1978; Auerswald & Gäde, 1999a).
Carbohydrate metabolism is a focal point of some insect
research; for example, the high enzyme capacities for carbo-
hydrate oxidation observed in bees (Suarez et al., 2005a). In
the present study, the capacity for Pyr-supported respiration is
greater than either for Palm-C or G3-P in both beetle species
tested, highlighting the importance of carbohydrate metabolism
in beetle ight muscle. The results of the present study are in
agreement with previous studies demonstrating appreciable Pyr
oxidation in beetle ight muscle (Hansford & Johnson, 1975;
Weeda et al., 1980; Auerswald & Gäde, 1999b). Compared with
D. maculatus, the ight muscle mitochondria from T. molitor in
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12127
Beetle ight muscle respiration 5
Tabl e 1. Comparison of the apparent Km(μM) and Vmax (pmol O2s−1mg−1wet weight) of Dermestes maculatus versus Tenebrio molitor each for
palmitoyl-carnitine (Palm-C), pyruvate (Pyr) and -glycerol 3-phosphate (G3-P).
Palm-C Pyr G3-P
KmVmax KmVmax KmVmax
Dermestes maculatus NA NA (13) 298.7 ±55.3 105.7 ±5.0 (8) 9134.4 ±1505.3a27.9 ±1.0 (5)a
Tenebrio molitor NA NA (15) 42.2 ±11.6b139.7 ±8.2 (5) 4137.4 ±777.4b66.6 ±7.7 (5)b
aP<0.001 versus Pyr.
bP<0.001 versus D. maculatus.
Data are the mean ±SEM (n).
NA, not applicable. Data were not consistent with Michaelis– Menten enzyme kinetics and substrate-dependent changes in respiratory O2ux were negligible.
G3-P respiratory O2ux rates are those after the subtraction of malate +ADP rates (for details, see text).
Malate
Proline
0.5 mM ADP
Creatine
Arginine
2 mM ADP
Cytc
0
50
100
150
200
+ Bleb
- Bleb
*
**
JO
2
(pmol · s
–1
· mg
–1
wet wt)
Fig. 3. Effect of blebbistatin (Bleb) on -proline-supported respiration
in Tenebrio molitor. Mitochondrial respiration supported by 25 m
-proline, was signicantly greater in T. molitor permeabilized brillar
ight muscle in the presence of Bleb (+Bleb) versus without (−Bleb)
(two-way repeated measures analysis of variance with Bonferroni
post-hoc test; *P<0.001; n=four paired samples run in duplicate).
the present study exhibit greater sensitivity to Pyr, suggesting a
greater reliance on carbohydrate metabolism in T. molitor.
The ability of beetle ight muscle mitochondria to readily
oxidize G3-P is reported previously (Hansford & Johnson,
1975). This is conrmed in the two species tested in the present
study, and further supports the importance of the G3-P shuttle
and carbohydrate metabolism in beetle ight muscle. Although
G3-P oxidation remains an important energetic substrate for
ight muscle mitochondria, it is surpassed by the rate of pyruvate
oxidation in several studies, in addition to the data reported
here (Hansford & Johnson, 1975; Soares et al., 2015). The
results of the present study demonstrate that, compared with
D. maculatus, the ight muscle mitochondria of T. molitor
exhibit greater capacity for G3-P-supported respiration, as well
as greater sensitivity to the substrate (Table 1).
Various insects, including several species of beetle, are
known to readily oxidize -proline (Hansford & Johnson, 1975;
Auerswald & Gäde, 1999a). In the present study, both beetle
species exhibit far greater -proline-stimulated respiration
than with any other substrate (Fig. 2). These results support
the evidence-based contention by Gäde & Auerswald (2002),
proposing that beetles use -proline predominately, if not
exclusively, to fuel ight muscle contraction, with D. maculatus
tting this model better than T. molitor in the present study. It
is also notable that individuals of the T. molitor colony used in
the present study are never observed to y, whereas individuals
from the D. maculatus colony were observed ying on occasion.
As proposed by Gäde & Auerswald (2002), these observations
suggest that the action of ight may contribute to increased
-proline oxidation in D. maculatus ight muscle mitochondria.
Bleb is a myosin II ATPase inhibitor originally synthesized as
a small molecule for cellular and muscle physiology research
(Kovacs et al., 2004). In previous studies, permeabilized muscle
bres incubated with 25 μM Bleb are reported to show increased
mitochondrial respiratory rates compared with untreated con-
trols (Perry et al., 2011, 2013). Similarly, the present study
demonstrates a positive response to permeabilized muscle bres
treated with Bleb (Fig. 3). One mechanism explaining this phe-
nomenon is postulated by Perry et al. (2011, 2012), in which
contractile inhibition increases mitochondrial ADP sensitivity
through permeability of voltage-dependent anion channels on
the outer mitochondrial membrane. This is achieved through
restructuring of the mitochondrial-cytoskeletal architecture in
a tubulin-dependent manner (Tepp et al., 2014). Although only
T. molitor tissues are examined with respect to ±Bleb in the
present study, the data suggest that Bleb may increase mitochon-
drial respiration in other species.
Energy buffer systems are used by both vertebrates
(creatine/phosphocreatine) and invertebrates (argi-
nine/phosphoarginine) to maintain homeostasis and attenuate
declines in ΔGof ATP (Voncken et al., 2013). The media
prepared for mitochondrial respirometry measurements are
commonly supplemented with creatine or phosphocreatine to
saturate the phosphate buffering system across the mitochon-
drial membranes (Perry et al., 2013). Although invertebrates
utilize unique phosphate shuttling strategies, there is no known
recommendation for the use of arginine or phosphoarginine
when examining invertebrate mitochondrial respiration via
the isolated mitochondria or permeabilized bre approach.
Although the observation in the present study indicating that
-arginine may serve as an appropriate additive to media for the
analysis of some invertebrate mitochondria remains a notewor-
thy nding, the use of saturating levels of ADP may obviate the
inclusion of arginine or creatine (Fig. 3).
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12127
6C. Newell et al.
Historically, investigations of substrate-specic mitochondrial
respiration in beetle ight muscle use isolated mitochondria
from the pooled muscle from multiple individual beetles, rang-
ing from two individuals in the larger African fruit beetle Pachn-
oda sinuata (Auerswald & Gäde, 1999a) to 50 individuals for
the Japanese beetle Popillia japonica (Hansford & Johnson,
1975). By contrast, in the present study, respiratory measure-
ments can be readily achieved in individual beetles, or indeed
more than one experiment per individual (technical replicates
averaged per number of beetles). Interestingly, Soares et al.
(2015) and Pichaud et al. (2013) also report adapting the per-
meabilized bre technique to study insect ight muscle. The
study by Pichaud et al. (2013) utilizes pseudoreplicates, whereas
Soares et al. (2015) successfully assess mitochondrial physi-
ology using individually dissected thoraces from the yellow
fever mosquito Aedes aegypti. Although similar to the currently
adapted method, the study by Soares et al. (2015) focuses on
assessing the energetics of individual respiratory complexes as
opposed to enzyme kinetics and substrate specicity. However,
examination of individual sex differences, as is demonstrated by
Soares et al. (2015), is not practical in the present study, and thus
represents a potential limitation.
Although the kinetics results in the present study differ from
previous studies (Pichaud et al., 2013; Soares et al., 2015),
comparable rates of substrate utilization remain, regardless of
insect species and individual tissue sizes: Palm-C <G3-P <Pyr
(Soares et al., 2015). It appears that the permeabilized bre
approach may be useful for almost any substrate-specic assess-
ment of respiration in beetle ight muscle mitochondria. This
may be a favourable approach in many circumstances, particu-
larly because the permeabilized bre technique confers advan-
tages in the assessment of mitochondrial function (Kuznetsov
et al., 2008; Picard et al., 2011).
Supporting Information
Additional Supporting Information may be found in the
online version of this article under the DOI reference:
DOI: 10.1111/phen.12127
Figure S1. Typical trace of oxygen consumption after
permeabilized bre preparation using pyruvate (Pyr) as substrate
in Dermestes maculatus. The blue line represents the oxygen
concentration within the chamber. The red line is the negative
slope of the blue line (i.e. respiratory rate), normalized to tissue
mass.
Figure S2. Absolute respiratory rates for palmitoyl-
carnitine, pyruvate and glycerol 3-phosphate substrate additions
to brillar ight muscle bres from Dermestes maculatus and
Tenebrio molitor. Comparisons between each species are shown
as a combination of malate +ADP plus all other respective
substrates.
Figure S3. Typical trace of oxygen consumption after
permeabilized bre preparation comparing the impact of the
contractile inhibitor (blebbistatin) and the phosphate acceptors
(creatine and -arginine) on Tenebrio molitor brillar ight
muscle. Experiment including blebbistatin: red line. Experiment
lacking blebbistatin: green line.
Acknowledgements
The present study was supported by StFX University Council
for Research, the Natural Sciences and Engineering Research
Council of Canada, the Canada Foundation for Innovation and
the Nova Scotia Research and Innovation Trust. We especially
thank Mr. Randy Lauff of StFX Biology for kindly providing the
Dermestes maculatus samples. We also thank Mitchell George
for reading an earlier draft of this manuscript.
References
Archer, M.S. & Elgar, M.A. (1998) Cannibalism and delayed pupation
in hide beetles, Dermestes maculatus DeGeer (Coleoptera: Dermesti-
dae). Australian Journal of Entomology,37, 158–161.
Auerswald, L. & Gäde, G. (1999a) The fate of proline in the African fruit
beetle Pachnoda sinuata.Insect Biochemistry and Molecular Biology,
29, 687– 700.
Auerswald, L. & Gäde, G. (1999b) Effects of metabolic neuropeptides
from insect corpora cardiaca on proline metabolism of the African
fruit beetle, Pachnoda sinuata.Journal of Insect Physiology,45,
535– 543.
Auerswald, L., Schneider, P. & Gäde, G. (1998) Proline powers pre-ight
warm-up in the African fruit beetle Pachnoda sinuata.Journal of
Experimental Biology,201, 1651– 1657.
Bowler, K. (1967) Changes in temperature tolerance with adult age
in Tenebrio molitor.Entomologia Experimentalis et Applicata,10,
16– 22.
Bursell, E. & Slack, E. (1976) Oxidation of proline by sarcosomes of the
tsetse y, Glossina morsitans.Insect Biochemistry,6, 159–167.
Chance, B. & Williams, G.R. (1955) Respiratory enzymes in oxidative
phosphorylation I. kinetics of oxygen utilization. Journal of Biologi-
cal Chemistry,217, 383– 393.
Correa, C.C., Aw, W.C., Melvin, R.G. et al. (2012) Mitochondrial
DNA variants inuence mitochondrial bioenergetics in Drosophila
melanogaster.Mitochondrion,12, 459– 464.
Dobson, A.J., Johnston, P.R., Vilcinskas, A. & Rolff,J. (2012) Identica-
tion of immunological expressed sequence tags in the mealworm bee-
tle Tenebrio molitor.Journal of Insect Physiology,58, 1556–1561.
Ernster, L. & Schatz, G. (1981) Mitochondria: a historical review.
Journal of Cell Biology,91, 227– 255.
Gäde, G. & Auerswald, L. (2002) Beetles’ choice-proline for energy
output: control by AKHs. Comparative Biochemistry and Physiology
Part B: Biochemistry and Molecular Biology,132, 117–129.
Gnaiger, E. & Kuznetsov, A.V. (2002) Mitochondrial respiration at low
levels of oxygen and cytochrome c.Biochemical Society Transac-
tions,30, 252– 258.
Hansford, R.G. & Johnson, R.N. (1975) The nature and control of the
tricarboxylate cycle in beetle ight muscle. Biochemical Journal,148,
389– 401.
Haunerland, N.H. (1997) Transport and utilization of lipids in insect
ight muscles. Comparative Biochemistry and Physiology Part B:
Biochemistry and Molecular Biology,117, 475– 482.
Khan, M.A. & de Kort, C.A.D. (1978) Further evidence for the
signicance of proline as a substrate for ight in the Colorado potato
beetle, Leptinotarsa decemlineata.Comparative Biochemistry and
Physiology Part B: Comparative Biochemistry,60, 407– 411.
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12127
Beetle ight muscle respiration 7
de Kort, C.A.D., Bartelink, A.K.M. & Schuurmans, R.R. (1973) The
signicance of -proline for oxidative metabolism in the ight
muscles of the Colorado beetle, Leptinotarsa decemlineata.Insect
Biochemistry,3, 11– 17.
Kovacs, M., Toth, J., Hetenyi, C. et al. (2004) Mechanism of blebbis-
tatin inhibition of myosin II. Journal of Biological Chemistry,279,
557– 563.
Kuznetsov, A.V., Veksler, V., Gellerich, F.N. et al. (2008) Analysis of
mitochondrial function in situ in permeabilized muscle bers, tissues
and cells. Nature Protocols,3, 965–976.
McManus, J.J. (1974) Oxygen consumption of the beetle, Dermestes
maculatus.Comparative Biochemistry and Physiology Part A: Com-
parative Physiology,49, 169– 173.
Moyes, C.D., Suarez, R.K., Hochachka, P.W. & Ballantyne, J.S.
(1990) A comparison of fuel preferences of mitochondria from
vertebrates and invertebrates. Canadian Journal of Zoology,68,
1337– 1349.
Perry, C.G., Kane, D.A., Lin, C.T. et al. (2011) Inhibiting
myosin-ATPase reveals a dynamic range of mitochondrial res-
piratory control in skeletal muscle. Biochemical Journal,437,
215– 222.
Perry, C.G., Kane, D.A., Herbst, E.A. et al. (2012) Mitochondrial
creatine kinase activity and phosphate shuttling are acutely regulated
by exercise in human skeletal muscle. The Journal of Physiology,21,
5475– 5486.
Perry, C.G., Kane, D.A., Lanza, I.R. & Neufer, P.D. (2013) Meth-
ods for assessing mitochondrial function in diabetes. Diabetes,62,
1041– 1053.
Pesta, D. & Gnaiger, E. (2012) High-resolution respirometry: OXPHOS
protocols for human cells and permeabilized bers from small
biopsies of human muscle. Methods in Molecular Biology,810,
25– 58.
Picard, M., Taivassalo, T., Ritchie, D. etal. (2011) Mitochondrial
structure and function are disrupted by standard isolation methods.
PLoS ONE,6, e18317.
Pichaud, N., Messmer, M., Correa, C.C. & Ballard, J.W. (2013) Diet
inuences the intake target and mitochondrial functions of Drosophila
melanogaster males. Mitochondrion,13, 817– 822.
Rockstein, M. (1974) The Physiology of Insecta. Academic Press, New
York, New York and U.K.
Soares, J.B., Gaviraghi, A. & Oliveira, M.F. (2015) Mitochondrial
physiology in the major arbovirus vector Aedes aegypti: substrate
preferences and sexual differences dene respiratory capacity and
superoxide production. PLoS ONE,10, e0120600.
Suarez, R.K., Darveau, C.A. & Hochachka, P.W. (2005a) Roles of
hierarchical and metabolic regulation in the allometric scaling of
metabolism in Panamanian Orchid bees. Journal of Experimental
Biology,208, 3603– 3607.
Suarez, R.K., Darveau, C.A., Welch, K.C. Jr. et al. (2005b) Energy
metabolism in orchid bee ight muscles: carbohydrate fuels all.
Journal of Experimental Biology,208, 3573– 3579.
Tepp, K., Mado, K., Varikmaa, M. et al. (2014) The role of tubulin in the
mitochondrial metabolism and arrangement in muscle cells. Journal
of Bioenergetics and Biomembranes,46, 421– 434.
Thompson, S.N. & Bennett, R.B. (1971) Oxidation of fat during ight
of male Douglas-r beetles, Dendroctonus pseudotsugae.Journal of
Insect Physiology,17, 1555– 1563.
Voncken, F., Gao, F., Wadforth, C. et al. (2013) The phosphoarginine
energy-buffering system of Trypanosoma brucei involves multiple
arginine kinase isoforms with different subcellular locations. PLoS
ONE,8, e65908.
Watanabe, M.I. & Williams, C.M. (1951) Mitochondria in the ight
muscles of insects I. Chemical composition and enzymatic content.
Journal of General Physiology,34, 675– 689.
Weeda, E., de Kort, C.A.D. & Beenakkers, A.M. (1980) Oxidation of
proline and pyruvate by ight muscle mitochondria of the Colorado
beetle, Leptinotarsa decemlineata Say. Insect Biochemistry,10,
305– 311.
Wegener, G. (1996) Flying insects: model systems in exercise physiol-
ogy. Experientia,52, 404–412.
Wolf, K.W. & Joshi, H.C. (1995) Microtubule organization and the dis-
tribution of gamma-tubulin in spermatogenesis of a beetle, Ten eb ri o
molitor (Tenebrionidae, Coleoptera, Insecta). Journal of Cell Science,
108, 3855– 3865.
Accepted 3 December 2015
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12127
