Cold Hardiness of the Multicolored Asian Lady Beetle (Coleoptera: Coccinellidae)

Abstract

A classical biological control agent, Harmonia axyridis (Pallas), is having both beneficial and detrimental impacts in North America. The objective of this study was to evaluate the cold hardiness of H. axyridis in North America. Supercooling points and survival at subzero temperatures of field-collected and insectary-reared H. axyridis were examined. The mean (±SE) supercooling points for eggs and pupae (i.e., nonfeeding stages) were −27.0 ± 0.18°C and −21.3 ± 0.52°C, respectively. The mean supercooling points for larvae and adults (i.e., feeding stages) were −14.17 ± 0.33 and −11.9 ± 0.53°C, respectively. Sex and color morph (i.e., red: f. succinea versus black: f. spectabilis) had no effect on the supercooling point of H. axyridis adults. Mean supercooling points of H. axyridis adults from Minnesota and Georgia were significantly lower during winter months than summer months. The mortality of H. axyridis increased significantly after individuals were exposed to temperatures below the mean supercooling point of the population. Supercooling point was a good predictor of cold hardiness. However, the cold hardiness of H. axyridis appears to be a poor predictor of its northern distribution.

Full text

PHYSIOLOGICAL ECOLOGY
Cold Hardiness of the Multicolored Asian Lady Beetle
(Coleoptera: Coccinellidae)
R. L. KOCH,
1
M. A. CARRILLO, R. C. VENETTE, C. A. CANNON, and W. D. HUTCHISON
Department of Entomology, 219 Hodson Hall, 1980 Folwell Avenue, University of Minnesota, St. Paul, MN 55108
Environ. Entomol. 33(4): 815Ð822 (2004)
ABSTRACT A classical biological control agent, Harmonia axyridis (Pallas), is having both beneÞcial
and detrimental impacts in North America. The objective of this study was to evaluate the cold
hardiness of H. axyridis in North America. Supercooling points and survival at subzero temperatures
of Þeld-collected and insectary-reared H. axyridis were examined. The mean (⫾SE) supercooling
points for eggs and pupae (i.e., nonfeeding stages) were ⫺27.0 ⫾ 0.18⬚C and ⫺21.3 ⫾ 0.52⬚C,
respectively. The mean supercooling points for larvae and adults (i.e., feeding stages) were ⫺14.17 ⫾
0.33 and ⫺11.9 ⫾ 0.53⬚C, respectively. Sex and color morph (i.e., red: f. succinea versus black:
f. spectabilis) had no effect on the supercooling point of H. axyridis adults. Mean supercooling points
of H. axyridis adults from Minnesota and Georgia were signiÞcantly lower during winter months than
summer months. The mortality of H. axyridis increased signiÞcantly after individuals were exposed
to temperatures below the mean supercooling point of the population. Supercooling point was a good
predictor of cold hardiness. However, the cold hardiness of H. axyridis appears to be a poor predictor
of its northern distribution.
KEY WORDS Harmonia axyridis, cold hardiness, supercooling point, overwintering, low temper-
ature
THE MULTICOLORED ASIAN LADY beetle, Harmonia axyri-
dis (Pallas), most likely entered North America
through intentional releases for biological control
(Gordon 1985). However, Day et al. (1994) argue that
H. axyridis may have entered through accidental sea-
port introductions. The Þrst established population of
H. axyridis in North America was recorded in 1988
(Chapin and Brou 1991). The establishment of this
exotic coccinellid has had both beneÞcial and detri-
mental consequences. As a beneÞt, H. axyridis feeds
on pest insects of numerous crops, such as pecan
(Tedders and Schaefer 1994), apple (Brown and
Miller 1998), citrus (Michaud 1999, Michaud 2000,
Stuart et al. 2002), and corn (Musser and Shelton
2003). Conversely, evidence suggests that H. axyridis
may be adversely affecting native natural enemies
(Koch 2003) and other nonpest insects, such as the
monarch butterßy, Danaus plexippus (L.) (Koch et al.
2003). In addition, two unexpected adverse effects of
H. axyridis are its status as a household nuisance pest
(Huelsman et al. 2002), and as an emerging pest in fruit
production (Ejbich 2003, Koch et al. 2004).
The potential geographic extent of impacts by an
exotic organism, such as H. axyridis, depends on the
capacity of the organism to withstand unfavorable
environmental conditions, such as temperature ex-
tremes (Tauber et al. 1986). Insects in cold regions
require some degree of cold hardiness to protect them
from low temperatures (Bale 1987, Salt 1961). In gen-
eral, insects that survive freezing temperatures can be
classiÞed as freeze tolerant or freeze intolerant (Salt
1961). Freeze-tolerant insects withstand extracellular
ice formation, while freeze-intolerant insects avoid ice
formation by supercooling (Baust and Rojas 1985).
The supercooling point is deÞned as the temperature
at which body ßuids spontaneously freeze when
cooled below the melting point (Zachariassen 1985).
The supercooling point generally represents the ab-
solute lower lethal temperature for freeze-intolerant
individuals, although death may also occur at temper-
atures above this point as a result of chill injury (Lee
1991, Lee and Denlinger 1985). In addition to the
supercooling point, lower lethal temperature and le-
thal time have been used as indices of cold hardiness
(Watanabe 2002).
Adult H. axyridis overwinter in mass aggregations in
or near prominent objects on the horizon (reviewed
by Koch 2003). Watanabe (2002) suggested that
H. axyridis in Japan might be freeze intolerant with a
degree of chill tolerance. Concentrations of a known
cryoprotectant, myo-inositol, increased concurrently
with a seasonal decrease in the supercooling point of
adult H. axyridis (Watanabe 2002). However, cold
hardiness is affected by several other factors, includ-
ing geographic location, environmental conditions,
1
Corresponding author: R. L. Koch (e-mail address:
koch0125@umn.edu).
0046-225X/04/0815Ð0822$04.00/0 䉷 2004 Entomological Society of America

developmental stage, sex, and age (Sømme 1982, Turn-
ock et al. 1990, Renault et al. 2002). The objective of
the current study was to measure the effect of devel-
opmental stage, season, color morph (i.e., red: f. suc-
cinea versus black: f. spectabilis), and sex on the su-
percooling point. Survival at subzero temperatures
also was analyzed as an index of cold hardiness of
H. axyridis in the United States.
Materials and Methods
Supercooling Point Determinations
Supercooling points were measured using surface-
contact thermometry, as described by Carrillo et al.
(2004). Individuals were attached to a 24-gauge cop-
per-constantan thermocouple using high vacuum
grease (Dow Corning, Midland, MI). Insect-thermo-
couple arrangements were placed inside a solid 19 ⫻
19 ⫻ 19-cm polystyrene cube, and these into a
⫺80⬚C freezer to cool insects at ⬇1⬚C min
⫺1
(Car
-
rillo et al. 2004). Supercooling points were deter-
mined as the lowest temperature reached before the
release of latent heat of fusion. The release of latent
heat is observed as an abrupt increase of the body
temperature.
Effect of Developmental Stage on Supercooling Point
The relative cold hardiness of H. axyridis was ex-
amined by determining the supercooling point of each
developmental stage. Individuals used in this study
were laboratory-reared F
1
progeny of Þeld-collected
overwintering adults from St. Paul, Minnesota. Over-
wintering adults were placed into 60 ⫻ 15-mm plastic
petri dishes and held at 22⬚C with a photoperiod of 16:8
(L:D) h. Insects were provisioned with an ad libitum
supply of drone honey bee diet (Okada and Matsuka
1973) and water. Mating was observed within5dof
warming. Individual mated females were placed into
60 ⫻ 15-mm plastic petri dishes and were provisioned
with an ad libitum supply of pea aphids, Acyrthosiphon
pisum (Harris), and water. Offspring of the mated
females were individually reared to the desired stage
of development in 60 ⫻ 15-mm plastic petri dishes
provisioned with an ad libitum supply of drone honey
bee diet and water. Eggs used for supercooling-point
measurements were removed from the petri dish
using a small camelhair brush. Eggs, Þrst through
fourth instars, and pupae were used in the study
24Ð36 h after molting to the desired stage. Adults
were used in the study 72 h after eclosion. Super-
cooling-point measurements were recorded from 18
eggs, 20 Þrst instars, 17 second instars, 18 third
instars, 17 fourth instars, 20 pupae, and 20 adults.
Data were not analyzed statistically because each
stage of development was measured at separate
times because of difÞculty in synchronizing the de-
velopment of all stages tested.
Effect of Sex and Color Morph on Supercooling Point
The effect of sex on the supercooling point of adult
H. axyridis was examined for Þeld-collected individ-
uals. On 16 April, 15 August, and 9 and 26 September
2002, adult H. axyridis were collected near Rose-
mount, Minnesota. One day after collection, individ-
uals were sexed and their supercooling points were
determined. Supercooling points were determined for
10Ð21 individuals of each sex for each collection date.
To determine the effect of color morph on supercool-
ing point, red adults with black spots (f. succinea) and
black adults with four red spots (f. spectabilis) were
obtained from The Green Spot (Nottingham, NH) on
29 May 2003. On the following day, the supercooling
point was determined for 17 and 16 individuals of the
red and black color morphs, respectively. Individuals
from The Green Spot were from an insectary-reared
colony that was ⬃5 yr old and frequently supple-
mented with Þeld-collected individuals. Because the
black color morphs of H. axyridis are rare in North
America (LaMana and Miller 1996), particularly in
Minnesota (R. L. Koch, unpublished data), we had to
rely on the insectary-reared individuals. Data for sex
or color morph comparisons were analyzed with
analysis of variance (ANOVA) (Proc GLM, SAS
Institute 1995). For the sex comparison, sex, collec-
tion date, and the interaction between sex and col-
lection date were included in the model. For the
color morph comparison, color was the sole predic-
tor in the model.
Effect of Season on Supercooling Point
The effect of season on the supercooling point of
adult H. axyridis was examined for populations from
Minnesota and Georgia. Adult H. axyridis used in this
study were Þeld collected 24Ð72 h before measuring
the supercooling point. Adults were collected from
various locations near St. Paul and Rosemount, Minne-
sota, and from the United States Department of Agri-
culture-Agricultural Research Service Fruit and Tree
Nut Research Laboratory in Byron, Georgia. Super-
cooling points of adults from Minnesota were measured
on 16 April, 15 August, 26 September, 8 November,
26 November 2002, 7 February, 26 February, 9 April,
26 August, and 15 December 2003. Supercooling points
of adults from Georgia were measured on 19 September,
23 November 2002, and 7 February 2003. For each
date, supercooling-point measurements were deter-
mined from 18 to 42 individuals. The seasonal change of
mean supercooling points of individuals from each lo-
cation was analyzed using ANOVA (Proc GLM, SAS
Institute 1995) and the TukeyÕs Studentized Range test
(honestly signiÞcant difference [HSD]). Month was the
sole predictor used in the ANOVA models. When mul-
tiple supercooling-point measurements were taken
within a month, the measurements for that month were
pooled.
816 E
NVIRONMENTAL ENTOMOLOGY Vol. 33, no. 4

Effect of Subzero Temperatures on Adult Survival
The effect of subzero temperatures on the survival
of adult H. axyridis was examined for Þeld-collected
and insectary-reared individuals. Adults used in this
study were either collected near Rosemount, Minne-
sota, on 26 August 2003 or obtained from The Green
Spot on 31 May 2003. Groups of 10 adults were placed
into 16 ⫻ 150-mm glass test tubes that were closed with
a small piece of foam. Test tubes containing adults
were placed into the core of a 35 ⫻ 35 ⫻ 35-cm
polystyrene cube with a starting temperature of 27⬚C.
A 24-gauge copper-constantan thermocouple also was
placed into each test tube to monitor temperature.
The polystyrene cube was then placed into a ⫺80⬚C
freezer to cool the insects at a rate of ⬇0.3⬚C min.
⫺1
Insectary-reared individuals were cooled to 0, ⫺5,
⫺10, ⫺15, ⫺20, ⫺25, or ⫺30⬚C; and Þeld-collected
individuals were cooled to 0, ⫺5, ⫺10, ⫺15, or ⫺20⬚C.
Test tubes were removed from the cube 1 min after
reaching the desired temperature and immediately
placed into a programmable growth chamber set at
0⬚C. Ten minutes after the Þnal tube was placed into
the growth chamber, insects were warmed to 22⬚Cat
a rate of 0.3⬚C min.
⫺1
Preliminary data indicated that
a 10-min exposure to 0⬚C was sufÞcient to equilibrate
adults from all temperature treatments to 0⬚C (data
not shown). One test tube was withheld from the
cooling treatment and was placed directly into the
growth chamber at 22⬚C as a control. Adults from each
treatment and the control were transferred to separate
150 ⫻ 15-mm plastic petri dishes provisioned with an
ad libitum supply of drone honey bee diet and water.
The dishes were held at 22⬚C and 60 Ð70% RH under
a photoperiod of 16:8 (L:D) h. Adult survival was
assessed at 24 h after treatment by ßipping individuals
onto their backs. Individuals were considered func-
tionally dead if they were unable to right themselves
within an additional 24 h (i.e., 48 h after treatment).
Both trials of this experiment were replicated three
times, except for the trial with H. axyridis from Min-
nesota, in which ⫺20⬚C exposure treatment was rep-
licated twice. The control showed no mortality, so a
correction (Abbott 1925) was not necessary. An
ANOVA (Proc GLM, SAS Institute 1995) and the
TukeyÕs Studentized Range test (HSD) were used to test
for differences in the arcsine square root transformed
proportionate mortality for each treatment. The super-
cooling points of 28 Þeld-collected and 73 insectary-
reared individuals were determined for comparison with
the results of the survival study. For individuals from
each source, the cumulative percentage of individuals
supercooling was calculated by summing the number of
individuals that supercooled at and above each 1-degree
temperature step, and dividing each resulting sum by the
total number of individuals measured.
Survival at Various Points on the Supercooling Curve
To examine survival of adult H. axyridis at various
points along their supercooling curves, we modiÞed
the method from Carrillo et al. (2004). Instead of using
high vacuum grease to attach individuals to the ther-
mocouples, a modiÞed 6-ml plastic syringe was used to
hold individuals in contact with the thermocouples
(Brunnhofer et al. 1991). The cooling rate was
achieved, as described above, for the supercooling
point studies. Individual body temperatures were
monitored using an Omega 2809 digital thermometer
(Omega Engineering, Stanford, CT) and graphed us-
ing a Fischer Graph Recordall (Fischer ScientiÞc,
SpringÞeld, NJ) (Schmid 1988). H. axyridis used in this
study were laboratory reared under the conditions
described above in the study examining the effect of
developmental stage on supercooling point. Individ-
uals were cooled to either their supercooling point
(i.e., the lowest temperature attained before the re-
lease of latent heat of fusion), the peak of the exo-
therm (i.e., the maximum temperature attained after
the release of latent heat of fusion), or the end of the
exotherm (i.e., the temperature equal to the super-
cooling point) (Block et al. 1988). The removal point
was randomly determined before each run. Seven
individuals were cooled to each of these three points
on the supercooling curve. When an individual
reached the desired point along the supercooling
curve, it was immediately removed from the freezer
and placed into 150 ⫻ 15-mm plastic petri dishes pro-
visioned with drone honey bee diet and water. The
dishes were then placed into a programmable growth
chamber at 0⬚C for 10 min and warmed to 22⬚C at a rate
of 0.3⬚C min.
⫺1
After 24 h, survival was assessed as in
the study examining the effect of subzero tempera-
tures on adult survival. The percentage of mortality at
each removal point was compared with zero based on
the overlap of 95% conÞdence intervals for population
proportions with small sample sizes (Kvanli 1988).
Results
Effect of Developmental Stage on Supercooling Point
In rank order, the mean (⫾SE) supercooling point
for eggs was ⫺27.0 ⫾ 0.18⬚C; pupae, ⫺21.3 ⫾ 0.52⬚C;
Þrst instars, ⫺15.4 ⫾ 0.82⬚C; third instars, ⫺13.9 ⫾
0.53⬚C; fourth instars, ⫺13.8 ⫾ 0.60⬚C; second instars,
⫺13.3 ⫾ 0.45⬚C; and adults, ⫺11.9 ⫾ 0.53⬚C (Fig. 1).
Effect of Sex and Color Morph on Supercooling Point
The mean supercooling point of adult H. axyridis
was not signiÞcantly affected by sex (F ⫽ 0.51; df ⫽ 1,
116; P ⫽ 0.4783) (Table 1). In addition, the interaction
of sex and date was not signiÞcant (F ⫽ 0.90; df ⫽ 3,
116; P ⫽ 0.4455). However, the effect of date was
signiÞcant (F ⫽ 13.18; df ⫽ 3, 116; P ⬍ 0.0001). Color
morph did not signiÞcantly affect the mean super-
cooling point of adult H. axyridis (F ⫽ 1.13; df ⫽ 1, 31;
P ⫽ 0.2967) (Table 1).
Effect of Season on Supercooling Point
The mean supercooling points of H. axyridis signif-
icantly changed through time for adults from Minne-
August 2004 KOCH ET AL.: COLD HARDINESS OF H. axyridis 817

sota (F ⫽ 73.64; df ⫽ 7, 257; P ⬍ 0.0001) and Georgia
(F ⫽ 6.50; df ⫽ 2, 71; P ⫽ 0.0026) (Fig. 2, A and B). For
individuals from Minnesota, the mean supercooling
point decreased ⬇13⬚C from August 2002 to February
2003, and ⬇11⬚C from August 2003 to December 2003
(Fig. 2A). For individuals from Georgia, the mean
supercooling point decreased ⬇4⬚C from November
2002 to February 2003 (Fig. 2B).
Effect of Subzero Temperatures on Adult Mortality
SigniÞcant differences in the percentage of mortal-
ity were observed among treatments for Þeld-col-
lected individuals from Minnesota (F ⫽ 24.64; df ⫽ 4,
8; P ⫽ 0.0001) and insectary-reared individuals from
The Green Spot (F ⫽ 36.93; df ⫽ 6, 14; P ⬍ 0.0001). The
insectary-reared H. axyridis had a mean supercooling
point of ⫺15.9 ⫾ 0.40⬚C, with values ranging from
⫺23.5⬚Cto⫺8.0⬚C. The Þeld-collected H. axyridis had
a mean supercooling point of ⫺8.0 ⫾ 0.54⬚C, with
values ranging from ⫺18.5⬚Cto⫺6.7⬚C. From either
source, the percentage of H. axyridis that died was not
signiÞcantly ⬎0% at temperatures above or near the
mean supercooling point (Fig. 3, a and b). After the
mean supercooling point was surpassed, a signiÞcant
increase in mortality (⬎50%) was observed for indi-
viduals from both locations (Fig. 3, a and b). Mortality
reached 100% when the minimum value of the super-
cooling point range was surpassed (Fig. 3, a and b).
The curves for the cumulative percentage of individ-
uals supercooling were consistently shifted to the right
of the mortality curves (Fig. 3, a and b).
Mortality at Various Points on the Supercooling Curve
The mean supercooling point for individuals tested
in this portion of the study was ⫺11.7 ⫾ 0.79⬚C. In-
dividuals cooled to their supercooling point showed
no mortality (Fig. 4). However, the percentage of
mortality was signiÞcantly greater than zero (P ⬍
Fig. 1. Effect of developmental stage on the supercooling point of laboratory-reared H. axyridis. The center bars of the
box plots represent the median; the upper and lower ends of the boxes represent the 25th and 75th percentiles; the whiskers
represent the 10th and 90th percentiles; circles represent outliers; and the squares represent the mean.
Table 1. Effect of sex and color morph on the supercooling point (SCP) of adult Harmonia axyridis
Date Comparison n Mean SCP (⬚C) ⫾ SE
c
Range (⬚C)
Sex
a
16 April 2002 Female 13 ⫺14.2 ⫾ 1.02 ⫺18.8, ⫺8.2
Male 14 ⫺12.4 ⫾ 0.95 ⫺18.0, ⫺7.6
15 Aug. 2002 Female 21 ⫺9.8 ⫾ 0.56 ⫺17.2, ⫺5.2
Male 21 ⫺10.1 ⫾ 0.38 ⫺15.3, ⫺8.2
9 Sept. 2002 Female 15 ⫺13.6 ⫾ 0.77 ⫺16.9, ⫺8.2
Male 15 ⫺13.8 ⫾ 0.68 ⫺17.1, ⫺8.3
26 Sept. 2002 Female 15 ⫺11.3 ⫾ 0.78 ⫺16.4, ⫺6.6
Male 10 ⫺11.1 ⫾ 0.83 ⫺14.4, ⫺6.2
Color
b
29 May 2003 Red 17 ⫺16.8 ⫾ 0.53 ⫺23.5, ⫺10.8
Black 16 ⫺17.8 ⫾ 0.73 ⫺20.1, ⫺10.0
a
Individuals were Þeld-collected near Rosemount, MN.
b
Individuals were insectary-reared and obtained from The Green Spot, Ltd., Nottingham, NH. The red color morph was f. succinea, and the
black color morph was f. spectabilis.
c
Mean SCPÕs for sex within dates and color morph did not differ signiÞcantly (P ⬎ 0.05); analysis of variance (Proc GLM, SAS 1995).
818 ENVIRONMENTAL ENTOMOLOGY Vol. 33, no. 4

0.05) at the peak (43%) and at the end (57%) of the
exotherm (Fig. 4).
Discussion
The ability of H. axyridis to overwinter has re-
ceived attention in the northeastern United States
(McClure 1987) and in Japan (Watanabe 2002). Re-
ports of ⬎90% overwintering mortality (McClure
1987) suggest that cold winter temperatures may be an
important factor regulating populations of H. axyridis
from one year to the next. Watanabe (2002) suggested
that the ability of H. axyridis to survive in more north-
ern latitudes (i.e., colder locations) than Tsukuba,
Japan, would be dependent upon the ability of this
insect to increase its cold hardiness (e.g., depress the
supercooling point) during winter months.
Our results indicate that the supercooling point of
H. axyridis, as with other freeze-intolerant insects,
signiÞcantly changes with season (e.g., Fig. 2, a and b).
In addition, it appears that developmental stage also
may have an effect on the supercooling point of this
coccinellid (e.g., Fig. 1). The mean supercooling
points for nonfeeding stages (i.e., eggs and pupae) of
H. axyridis remained below ⫺20⬚C, while the mean
supercooling points of the feeding stages (i.e., larvae
and adults) were warmer than ⫺16⬚C (Fig. 1). These
results suggest that food in the digestive tract may
induce ice nucleation, and increase the supercooling
point (Salt 1953). Therefore, the lower supercooling
points of Þeld-collected adult H. axyridis during win-
ter (Fig. 2) (Watanabe 2002) may be partially because
of an absence of food in the digestive tract (Iperti and
Be´rtand 2001).
Freeze-intolerant insects die when exposed to tem-
peratures at or below the supercooling point, but some
may die at temperatures above the supercooling point,
because of chill injury (Lee 1991). For H. axyridis, the
supercooling point appears to be a good indicator of
cold hardiness when mortality is assessed after an
exposure period of 1 min. However, Watanabe (2002)
found that some prefreeze mortality occurred when
adult H. axyridis where exposed for a longer period of
time (i.e., 24 h). The results of our studies indicate that
Fig. 2. Effect of season on the supercooling point of Þeld-collected adult H. axyridis from (A) Minnesota and (B) Georgia.
The center bars of the box plots represent the median; the upper and lower ends of the boxes represent the 25th and 75th
percentiles; the whiskers represent the 10th and 90th percentiles; circles represent outliers; and the squares represent the
mean. Different lower case letters above box plots indicate signiÞcant differences among mean supercooling points
(Minnesota, P ⬍ 0.0001; Georgia, P ⫽ 0.0026) based on ANOVA and TukeyÕs Studentized Range test (HSD).
August 2004 KOCH ET AL.: COLD HARDINESS OF H. axyridis 819

mortality of H. axyridis did not occur immediately at
the supercooling point, but increased with time after
the supercooling point was reached. Similar results
have been observed for other freeze-intolerant insects
in which mortality was proportional to the amount of
ice formed inside the body (Salt 1953, Block et al.
1988). In our study examining the effect of subzero
temperatures on mortality of H. axyridis adults, the
difference in the shapes of the curves for cumulative
percentage of individuals supercooling from Minne-
sota compared with Georgia (Fig. 3) may be because
of the difference in the number of individuals used to
characterize the distribution (73 individuals from
Minnesota versus 28 individuals from Georgia). The
ability of H. axyridis to survive after exposure to their
supercooling point may be an artifact of minimal ex-
posure times used in laboratory studies, as recognized
by Salt (1953) while working with other freeze-intol-
erant insects. In other words, survival of individuals
after a short-duration exposure to the supercooling
point in laboratory studies does not necessarily indi-
cate freeze tolerance, and further investigation (e.g.,
increased exposure time) may be required to catego-
rize cold hardiness.
The presence of H. axyridis has been conÞrmed in
parts of the northern United States (Koch and Hutchi-
son 2003) and southern Canada (Coderre et al. 1995,
McCorquodale 1998). In these locations, minimum air
temperatures (Kaliyan et al. 2003) can exceed the
minimum supercooling points observed in this study
and should be lethal to H. axyridis. Thus, local air
temperature alone appears to be a poor predictor of
the distribution of H. axyridis. H. axyridis adults
most likely Þnd microclimates for overwintering that
provide protection from extreme low temperatures.
H. axyridis generally shows hypsotactic orientation
(i.e., movement toward prominent objects) during
its fall movement to overwintering sites (Obata 1986).
At the overwintering sites, mass aggregations of
H. axyridis adults are formed (Tanigishi 1976) in dark,
Fig. 3. Mean (⫾SE) percentage of mortality and cumulative percentage of adult H. axyridis supercooling at different
subzero temperatures for: (A) insectary-reared individuals from The Green Spot; (B) Þeld-collected individuals from
Rosemount, Minnesota. For each location, different lower case letters indicate signiÞcant differences among means within
each mortality curve (Þeld collected, P ⫽ 0.0001; insectary reared, P ⬍ 0.0001) based on ANOVA and TukeyÕs Studentized
Range test (HSD).
820 ENVIRONMENTAL ENTOMOLOGY Vol. 33, no. 4

concealed locations (Sakurai et al. 1993). H. axyridis
adults also overwinter in leaf litter (Obata 1986; R. L.
Koch, unpublished data), where temperatures may be
less extreme (Leather et al. 1993). Therefore, the
capacity of H. axyridis to survive winter conditions in
northern locations may be more related to the avail-
ability of quality overwintering sites than to its capac-
ity to increase cold hardiness.
Acknowledgments
We thank T. Cottrell (United States Department of
Agriculture-Agricultural Research Service, Byron, GA),
R. Moon (University of Minnesota), and P. OÕRourke (Uni-
versity of Minnesota) for supplying H. axyridis. This work
was funded by a University of Minnesota Doctoral Disser-
tation Fellowship and the University of Minnesota Experi-
ment Station.
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Received 7 January 2004; accepted 10 April 2004.
822 ENVIRONMENTAL ENTOMOLOGY Vol. 33, no. 4