PHYSIOLOGY, BIOCHEMISTRY, AND TOXICOLOGY
Effects of Temperature and Controlled Atmospheres on Codling
Moth Metabolism
LISA G. NEVEN1
AND
LEE D. HANSEN2
Ann. Entomol. Soc. Am. 103(3): 418Ð423 (2010); DOI: 10.1603/AN09133
ABSTRACT Although controlled atmosphere temperature treatments are effective in controlling
codling moth, Cydia pomonella (L.), in fruit, the mechanism by which this combination treatment kills
the larvae is unknown. Differential scanning calorimetry was used to determine the effects of elevated
temperatures, low O2, and high CO2 on the metabolic heat rate of Þfth-instar codling moths. Total ATP
levels also were determined. Metabolic heat rates in air increased from 0 to 30⬚C and decreased above
30⬚C. Heat rates measured isothermally at 23⬚C under decreased O2 or increased CO2 were lower than
those in air with the lowest in 1 kPa O2 and 1 kPa O2 ⫹ 15 kPa CO2. Continuous temperature scans
from 23 to 44.5⬚C under low O2 and high CO2 atmospheres produced lower metabolic heat rates than
scans under air. Low O2 atmospheres produced the lowest ATP levels, and high concentrations of CO2
produced the highest ATP levels. These results indicate that heat treatments under controlled
atmospheres have a dramatic effect on codling moth metabolism, low O2 prevents ATP synthesis, and
high CO2 prevents use of ATP.
KEY WORDS differential scanning calorimetry, codling moth, ATP, metabolism, controlled atmospheres
The codling moth, Cydia pomonella (L.), is a cosmopolitan pest of apples (Malus spp.) throughout most of
the apple-growing regions of the world (Barnes 1991).
The presence or potential presence of this pest in
harvested apples has been the source of many quarantine restrictions to prevent accidental spread to
countries that do not yet have this pest. Although the
systems approach (Jang and MofÞtt 1994) and methyl
bromide fumigation (Hansen et al. 2000a,b) have been
proven to be effective to meet quarantine restrictions
for this pest, alternative quarantine treatments
(Neven 2005, Neven et al. 2006; Neven and RehÞeldRay 2006a,b) have been developed where those procedures are either not attainable, as is the case for the
systems approach, or not desirable, as is the case for
methyl bromide.
Alternative quarantine treatments, developed to
control codling moth in apples, peaches, nectarines,
and sweet cherries, use a combination of short term
high temperatures and controlled atmospheres
(Neven 2005; Neven et al. 2006; Neven and RehÞeldRay 2006a,b). This technology is called Controlled
Atmosphere Temperature Treatment System (CATTS)
(Neven and Mitcham 1996) and was developed to
optimize the difference between commodity tolerance and insect intolerance. Years of testing has
shown that these delicate deciduous fruits can tolerate
1 Corresponding author: USDAÐARS, Yakima Agricultural Research Laboratory, 5230 Konnowac Pass Rd., Wapato, WA 98951
(e-mail: [email protected]).
2 Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602.
CATTS treatments provided the heating rate is optimized (Mitcham et al. 1999, Neven and Drake 2000,
Obenland et al. 2005) and the total treatment time is
short (Neven et al. 2001, Shellie et al. 2001). Additional
testing demonstrated that codling moth could not
withstand heat treatments under a low O2, high CO2
environment (Neven 2005; Neven et al. 2006; Neven
and RehÞeld-Ray 2006a,b).
Although CATTS treatments are effective in controlling codling moth in fruit, the mechanism by which
this combination treatment kills the larvae is unknown. Work by Edwards (1968), Fleurat-Lessard
(1990), Friedlander (1983), and Zhou et al. (2000,
2001) indicates that the net effect of high CO2 environments on insect respiratory metabolism is similar to
that of reduced O2. Both low O2 and high CO2 reduce
oxidative phosphorylation even though the target sites
may be different. Reduced O2 limits a substrate (O2)
of respiratory metabolism, whereas elevated CO2 inhibits respiratory enzymes such as succinate dehydrogenase (Edwards 1968) and malic enzyme (FleuratLessard, 1990). Under anoxic conditions, insects can
generate ATP by converting glycogen to lactate and
alanine (Wegener 1993, Hoback and Stanley 2001). As
ATP levels fall, there is an accumulation of AMP and
IMP (Wegener 1993). However, it is not known how
elevated levels of CO2 effect this conversion of glycogen or ATP use. Because ATP use is directly related
to metabolism, measuring metabolic heat rates under
various atmospheric conditions would help in describing the effects of controlled atmospheres on insects.
May 2010
NEVEN AND HANSEN: CODLING MOTH METABOLISM
Isothermal and differential scanning calorimetry
(DSC) have been used to determine metabolic rates
of numerous organisms (Hansen and Criddle 1990,
Kemp 1999). Isothermal calorimetry measures the rate
of heat produced at a constant temperature and DSC
measures the heat rate as a function of temperature as
the temperature is continuously changed or scanned.
Some calorimeters, such as the calorimeter used in this
study, can be operated in either mode, isothermal or
temperature scanning. Metabolic heat rate is directly
related to the O2 use rate in aerobic organisms (Hansen et al. 2004), but heat rate is easier to measure,
particularly in small nonaquatic organisms, and it can
provide additional information on metabolism.
Changes in metabolic heat rates can indicate the
response of an organism to various environmental
stresses, such as temperature extremes and anoxia.
Many calorimetric studies have been done on insects
(Lamprecht and Schmolz 1999; Acar et al. 2001, 2004,
2005; Joyal et al. 2005). Of particular interest here,
Downes et al. (2003) studied the effects of temperature and atmosphere on green peach aphid, Myzus
persicae (Sulzer), and Zhou et al. (2000, 2001) used
DSC to determine the effects of various levels of O2
and CO2 on Platynota stultana Walsingham pupae.
Downes et al. (2003) found that heat rates declined
rapidly at 40⬚C and higher temperatures and that
aphids did not recover when returned to lower temperatures. Anoxia caused metabolic arrest and at 20⬚C,
anoxia for ⬎6 h led to death. Zhou et al. (2000) found
that omnivorous leafroller pupae used metabolic arrest as a major response to hypoxia. Pupal O2 consumption rate and metabolic heat rate decreased
slightly with decreasing O2 concentration until a critical concentration below which the decrease became
rapid. The critical concentration points were 10, 8, and
6 kPa at 30, 20, and 10⬚C, respectively. Although pupal
metabolism decreased quickly below the critical concentration points, the pupae did not initiate anaerobic
metabolism until the O2 concentration was ⬍2 kPa at
20⬚C. Concentrations of O2 below the anaerobic compensation point seem to be in the insecticidal range.
Here, we describe the effects of low O2 and high
CO2, separately and in combination, on metabolic heat
rate and ATP levels in Þfth-instar codling moth in an
effort to elucidate the pathway by which CATTS treatments kill this pest.
Materials and Methods
Test Insects. Codling moths used in these studies
were obtained from a laboratory colony originally
established in 1971 and reared on artiÞcial diet (Toba
and Howell 1991). Moths were reared at 25Ð27⬚C,
50 Ð 60% RH, and a photoperiod of 16:8 (L:D) h. Feeding Þfth instars were removed from the diet just before
use.
Water Bath Treatments. Treatments of Þfth-instar
codling moths for ATP analyses were conducted in the
CA water bath system (Neven 2008). Fifth instar codling moths were placed into 16- by 150-mm test tubes
in the treatment container (Neven 2008). Insects were
419
conÞned in the lower 1 cm of the chamber by a blunt
cut and mite cloth (Econet 0.15- by 0.35-mm mesh,
Hummert International, Earth City, MO)-sealed 5-ml
plastic pipette tip (Neven 2008). The water baths were
set to 23⬚C for equilibration of temperature and atmospheric gases. One treatment container was used
for atmospheric air treatments only, and the other
container was used for application of the controlled
atmosphere (low O2 and/or high CO2). Nonheated
treatments were performed at a water bath temperature of 23⬚C. The water bath was programmed to heat
at a linear rate of 24⬚C/h to a Þnal bath temperature
of 45.5⬚C, which took 57.5 min, and remained at that
temperature for another 2.5 min for a total treatment
time of 1 h. The Þnal bath temperature correlated to
a test tube Þnal temperature of 44.5⬚C. Thermocouples
in two randomly selected test tubes and one thermocouple in each water bath system recorded temperatures at 5-s intervals throughout the treatments
(Neven 2008).
A gas mixing board (Dwyer 0-2.0 SCFH, Dwyer
Instruments, Michigan City, IN) controlled the inlet of
house air, house nitrogen, and cylinder CO2, combined the gases, and regulated the output to the insect
treatment container. Gases from house air and nitrogen were regulated from the house source by a simple
house valve and Þne-tuned with a thumb screw valve.
Cylinder CO2 was regulated by a two stage regulator
in series with a thumb screw valve. A separate house
air line was used to supply the regular air container.
Flow rates in both containers were monitored by simple ßow meters (0 Ð3,000 ml/min; Gilmont Inc., St.
Louis, MO). The gas composition in the CA chamber
was monitored by an O2/CO2 analyzer (PaciÞc CA
Systems by Techni-Systems, Chelan, WA). Humidity
in the sealed containers was maintained by watersoaked sponges attached to the sides of the Plexiglas
container. House air was used with a ßow rate of 1
liter/min. For the CA container, gases were mixed in
the mixing board and ßowed into the chamber at a rate
of 1 liter/min. The gas analyzer and pump were turned
on to monitor O2/CO2 levels in the CA container.
Once the levels had stabilized to 1 kPa O2, 15 kPa CO2,
which normally took 5Ð10 min, the test was ready to
begin. Oxygen levels in the chamber varied by 0.2 kPa
and CO2 levels varied by 1.0 kPa during the treatments. Treatments are listed in Table 1. After treatments, larvae were carefully removed from the test
tubes with a Þne camel hair brush and immersed in
liquid nitrogen. A fresh, sterile razor blade was used to
sever the heads from the bodies. Bodies were stored
in separate 1.5-ml microfuge tubes and stored at
⫺80⬚C before use. In total, 50 larvae were used for
each treatment combination.
DSC. Fifth-instar codling moths were extracted
from artiÞcial media and placed into a 100-cm2 petri
dish. For treatments requiring altered atmospheric gas
levels, the larvae, soft forceps, and open 1-ml DSC
ampoules were placed into an air tight AtmosBag (Aldrich Chemical, Milwaukee, WI). The inlet and outlet
ports of the bag had airtight quick connect tubing
connectors. The bag was sealed and subjected to a
ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA
Table 1. Conditions of treatments used on fifth-instar codling
moth and resultant metabolic heat rates and ATP levelsa
Treatment Heat
A
B
C
D
E
F
G
H
No
Yes
Yes
Yes
Yes
No
No
No
CO2
O2
(kPa) (kPa)
21.5
21.5
1.0
18.0
1.0
1.0
18.0
1.0
0.0
0.0
0.0
15.0
15.0
0.0
15.0
15.0
Metabolic heat
ATP
rate at 23⬚C
(ng mg⫺1 FW)c
(␮W mg⫺1 FW)b
3.30 ⫾ 0.79A
9.51 ⫾ 1.02C
⫺0.86 ⫾ 0.25D
1.19 ⫾ 1.35D
0.35 ⫾ 0.51D
0.45 ⫾ 0.04B
3.2 ⫾ 0.47A
0.71 ⫾ 0.11B
50.3 ⫾ 9.4B
43.4 ⫾ 7.7B
16.4 ⫾ 2.5C
68.0 ⫾ 10.0AB
33.4 ⫾ 4.2C
34.0 ⫾ 4.7C
127.3 ⫾ 28.8A
66.9 ⫾ 9.9B
a
High CO2-only treatments used CO2 with the balance of the gas
being air; therefore, O2 levels in these treatments averaged 18 kPa.
Heat treatments consisted of a linear heating rate of 24⬚C/h from 23⬚C
to a Þnal temperature of 44.5⬚C.
b
From isothermal measurements. Values (mean ⫾ SEM) represent
the average of 12 measurements. Values followed by the same letter
are not signiÞcantly different from one another (DuncanÕs multiple
range test: F7 ⫽ 99.94, P ⬍ 0.0001).
c
Values (mean ⫾ SEM) represent the average of 15 individuals per
test. Values followed by the same letter are not signiÞcantly different
from one another (DuncanÕs multiple range test: F7 ⫽ 14.48, P ⬍
0.0001).
light vacuum. The gas mixing station was set to desired
gas levels and attached to the bag. Once the bag was
half Þlled, the output quick connect was attached to
the O2/CO2 analyzer, and the levels of gas monitored
to conÞrm the atmospheric gas levels in the bag. After
the desired gas levels were achieved, the bag was
disconnected and the larvae were individually placed
into the 1-ml ampoules and the lids were sealed. The
larvae in ampoules were removed from the bag and
placed into the DSC (model 4100, CSC Calorimetry
Sciences Corporation, Lindon, UT). For isothermal
measurements on larvae with treatments A and FÐH
(Table 1), the DSC was equilibrated to 23⬚C. Isothermal measurements on larvae in air were made at 2.5⬚C
intervals from 0 to 45⬚C. Continuous temperature
scanning measurements of metabolic heat rates were
made from 23 to 44.5⬚C at 0.4⬚C/min. Only one larva
was used in each ampoule for each measurement in all
the tests. After measurement of metabolic heat rates
of larvae, the ampoules were removed from the DSC,
the larvae removed from the ampoules, placed into a
60-ml plastic cup with a clear lid and assessed for
survivorship for up to 1 h after removal from the
ampoule. Survivorship was determined by movement
of the larva either independently or with gentle prodding with a Þne camelÕs hair paint brush.
ATP Analysis. Individual bodies without heads were
used for assessment of ATP levels using a luciferinbased assay system (ATP analysis kit, Roche Applied
Science, Indianapolis, IN). Each body was weighed
before the addition of homogenization buffer. In total,
6 ␮l of homogenization buffer was added for each
microgram of fresh weight. Samples were hand homogenized and centrifuged at 10,000 rpm for 10 min
to pellet insoluble material. After homogenization,
duplicate aliquots were used in the ATP assay. ATP
levels were determined by measuring luciferin luminescence on a Fluroskan Ascent FL microplate reader
Vol. 103, no. 3
Metabolic Heat Rates
Heat Rate uW/mg FW
420
8
7
6
5
4
3
2
1
0
0
5
10
15
20
25
30
35
40
45
50
Temperature °C
Fig. 1. Metabolic heat rates of Þfth-instar codling moth
measured at isothermal temperatures from 0 to 45⬚C. Values
(⫾SEM) represent the average of 12 individuals (six females
and six males).
(Thermo Fisher ScientiÞc, Sacramento, CA). Each
sample, consisting of a single insect, was tested twice
in a single assay. In total, three individuals from one
treatment group were tested in a single assay. Assays
were repeated Þve times.
Theory and Calculation. Metabolic heat rates during continuous temperature scanning were calculated
following the equation from Hansen and Criddle
(1990):
(dQ/dt)metabolism ⫽ (dQ/dt)measured
⫺ (dQ/dt)baseline ⫹ (Csample)(scan rate)
[1]
where Csample is the heat capacity of the sample.
Csample was calculated with the equation
SpeciÞc heat capacity
⫻ mass of sample or (2.10 J ⬚C⫺1 g⫺1)
(mass of sample)
[2]
where the speciÞc heat capacity was determined with
a scan from 23 to 44.5⬚C of three ampoules each containing an average 330 mg of dead nondiapausing Þfthinstar codling moths. SpeciÞc heat capacity was calculated as follows:
(dQ/dt)measured ⫺ (dQ/dt)baseline/(scan rate
⫻ g sample)
[3]
Statistics. The differences in ATP levels and heat
rates for the eight different treatments were analyzed
by analysis of variance (ANOVA) with the SAS version 9.0 (SAS Institute, Cary, NC). Means were separated using DuncanÕs multiple range test (SAS version 9.0). Basic scanning and isothermal data were
analyzed with Excel 2007 (Microsoft, Redmond, WA).
Results
Metabolic Heat Rate. Isothermal measurements of
metabolic heat rate beginning at 0⬚C and ending at
44.5⬚C under air are shown in Fig. 1. Heat rates increase with increasing temperature up to 30⬚C, and
above that, metabolic heat rate declines. At 44.5⬚C,
signiÞcant larval mortality occurred, indicating lack of
ability to tolerate the heat load.
May 2010
NEVEN AND HANSEN: CODLING MOTH METABOLISM
Fig. 2. Metabolic heat rates of Þfth-instar codling moth
measured with continuous temperature scans from 23 to
44.5⬚C under air (treatment B), 1 kPa O2 (treatment C), 15
kPa CO2 (treatment D), and 1 kPa O2 ⫹ 15 kPa CO2 (treatment E). Slightly negative rates are probably a consequence
of a small systematic error in Csample, but the response to
temperature is not affected by this error.
Metabolic heat rates measured isothermally at 23⬚C
under treatments F, G, and H were lower than metabolic heat rates in air (Table 1), with the lowest rates
in treatments F and H, 1 kPa O2 and 1 kPa O2 ⫹ 15 kPa
CO2, respectively. Isothermal heat rates for treatments A and G, air, and air ⫹ 15 kPa CO2 were similar.
Metabolic heat rates measured with continuous
scans from 23 to 44.5⬚C at a rate of 0.4⬚C/min under
low O2 and high CO2 showed a reduction of metabolic
heat rate (treatments CÐE, Fig. 2) compared with
scans done with larvae in air. Treatment B gave slightly
higher metabolic heat rates than isothermal measurements at comparable temperatures, possibly indicating metabolic compensation for the increase in heat
load. Treatments C and E, both under 1 kPa O2 atmospheres, gave low metabolic heat rates. Treatment
D, under air ⫹ 15 kPa CO2, gave lower metabolic heat
rates than treatment B but higher than treatments C
and E.
ATP Levels. ATP levels in the air only controls,
treatments A and B, and in the combination 1 kPa O2 ⫹
15 kPa CO2 atmosphere at room temperature, treatment H, were not signiÞcantly different from one
another (F7 ⫽ 14.48, P ⬍ 0.0001) (Table 1). The addition of 1 kPa O2 alone, treatments C and F, or with
a 15 kPa CO2 atmosphere in a heat treatment, treatment E, resulted in the lowest ATP levels that were
signiÞcantly different from all the other treatments
(F7 ⫽ 14.48, P ⬍ 0.0001). Treatments with high levels
of CO2, treatments D and G, had the highest ATP
levels, with only treatment G being signiÞcantly higher
than treatments A, B, and H (F7 ⫽ 14.48, P ⬍ 0.0001).
Discussion
Metabolic Heat Rate. The changes in metabolic heat
rate at isothermal temperatures (Fig. 1) closely resemble the changes in respiration under a simulated
heat treatment (Neven 1998). In previous studies,
CO2 output (respiration) increased as temperature
increased, but after larvae were at the soak temperature (⬎42.5⬚C) for ⬎15-min respiration began to de-
421
crease. It was presumed that the decrease in CO2
output was due to metabolic arrest once the insect had
used up most of its energy reserves. If the larvae were
returned to room temperature before 15 min into the
metabolic arrest, they recovered. After that point,
they die.
The differences in metabolic heat rates in heat treatments compared with isothermal measurements indicate there is a metabolic adjustment made when the
insects are experiencing rapid temperature changes.
The reduced metabolic heat rate in low O2 and high
CO2 indicates that metabolic arrest (Zhou et al. 2000,
2001) may be occurring. Lack of O2 most likely impairs
the ability of the insect to regulate metabolism in
relation to the increased heat load. High CO2 may
decrease internal pH through the formation of carbonic acid and thereby alter enzymatic and glycolytic
pathways (Fleurat-Lessard 1990). Reduced pH can
increase intercellular Ca2⫹ concentration, which
causes the cell and mitochondrial membranes to become more permeable, suggesting that high CO2 can
increase membrane permeability (Fleurat-Lessard
1990). High CO2 levels can alter the ratio of pyruvate
to lactate by 25% of normal, changing the redox potential and causing a lesion in the electron transport
chain, presumably by a modiÞcation in the permeability of mitochondrial membranes (Friedlander
1983). It would be interesting to measure the levels of
pyruvate and lactate in treatments D and E to see
whether the ratios are similar to previous work with
high CO2 performed under low O2 environments.
ATP Levels. The reduction of ATP levels after heat
treatment under low O2 environments was expected
because the presence of O2 is required for energy
production. What was unexpected was the higher levels of ATP under high CO2 atmospheres. High levels
of CO2 may inhibit the conversion of ATP to ADP by
blocking a critical pathway in glycolysis or the tricarboxylic acid (TCA) cycle. Hoback and Stanley (2001)
stated that reduced O2 consumption leads to a decreased rate of ATP production. Friedlander (1983)
and Fleurat-Lessard (1990) stated that as a result of
energy insufÞciency, the membrane ion pumps fail,
leading to K⫹ efßux, Na⫹ inßux, and membrane depolarization. The voltage-dependent Ca2⫹ gates then
open, causing Ca2⫹ inßux. The high Ca2⫹ concentrations in the cytosol activate phospholipases and lead to
increased membrane phospholipid hydrolysis (Friedlander 1983, Fleurat-Lessard 1990). The cell and mitochondrial membranes become further permeable,
causing cell damage or death (Zhou et al. 2000, 2001).
Because the high CO2 treatments (treatments D
and G) probably had enough O2 to support aerobic
metabolism, 18 kPa O2, it is likely that the TCA cycle
was inhibited at a point where ATP could be produced
through the metabolism of glucose to pyruvate but not
converted to ADP or AMP. Through the TCA cycle,
pyruvate is converted to CO2. If CO2 levels are elevated, then the cycle can be inhibited and ATP cannot
be used. The most likely enzymes to be inhibited by
elevated CO2 levels would be isocitrate dehydrogenase and ␣-ketoglutarate dehydrogenase. Friedlander
422
ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA
(1983) reported that high levels of CO2 can reduce
oxidative phosphorylation by inhibiting respiratory enzymes such as succinate dehydrogenase and malic enzyme. Friedlander (1983) also stated that reduced oxidative phosphorylation leads to reduced ATP
generation, which in turn leads to a failure of membrane
ion pumps, membrane depolarization and eventual cell
death, as described for hypoxia. However, these experiments were performed under low O2 environments.
Our research indicates that ATP can be produced in
CO2-enriched environments if there is sufÞcient O2
available for oxidative phosphorylation. It is unclear
whether the ATP produced under CO2-enriched environments can be used, thus the elevated levels of ATP
under these conditions. An examination of ADP, AMP,
NAD, and NADH is needed to fully elucidate the effects
of controlled atmospheres on codling moth metabolism.
Acknowledgments
We thank Benjamin Groves and Aram Langhans for technical assistance in this project. We also thank Drs. Judy
Johnson, George Yocum, and Peter Follett for peer reviews
of this paper.
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Received 8 September 2009; accepted 4 February 2010.