Partial link between the seasonal acquisition of cold-tolerance and desiccation resistance in the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae)
Department of Zoology, Miami University, Oxford, Ohio 45056, USA
* Author for correspondence (e-mail: willia37{at}muohio.edu)
Accepted 30 September 2004
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Summary |
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Key words: cold-tolerance, desiccation resistance, goldenrod gall fly, cryoprotectants
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Introduction |
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Larvae of the goldenrod gall fly, Eurosta solidaginis Fitch
(Diptera, Tephritidae), have been used extensively as an insect model for
studying freeze tolerance. The gall fly ranges throughout much of the United
States and southern Canada where they induce stem galls on goldenrod plants
(Solidago spp.; Uhler,
1951). Larvae feed on the mature, moist gall tissue throughout the
summer. In early autumn, larvae cease feeding (as the goldenrod plant
senesces) and overwinter as freeze-tolerant third instar larvae within their
dried galls. Gall tissue offers little protection against winter extremes
(Layne, 1993
). Overwintering
larvae experience ambient air temperatures and extremely desiccating
conditions above the snow pack, although hydric parameters of the gall may
change depending on precipitation (Layne,
1993
).
To survive the low temperatures and desiccating conditions of winter,
larvae of E. solidaginis increase their cold-tolerance during the
autumn and have extremely low rates of water loss. In early autumn, few larvae
can survive 6°C for 24 h, but as the season progresses larvae
readily survive freezing at 20°C
(Lee and Hankinson, 2003). The
seasonal increase in cold-tolerance is correlated with the accumulation of the
cryoprotectants glycerol and sorbitol, whose synthesis is triggered,
respectively, by drying of the gall tissue and low temperature
(Baust and Lee, 1982
;
Rojas et al., 1986
;
Storey and Storey, 1992
).
Recently, mid-winter E. solidaginis larvae were shown to have
extremely low rates of water loss, rates comparable to heavily sclerotized
desert beetles (Ramløv and Lee,
2000
). However, it is unknown if resistance to water loss changes
in this species from early autumn, when gall tissue is fully hydrated, to
mid-winter, when their galls can be extremely dry. Also, if seasonal changes
in the rate of water loss exist, are these reductions in rate of water loss
linked to physiological process of increasing cold-tolerance?
Previous studies examined several parameters of cold-tolerance and their
possible link to desiccation resistance in cold-hardy insects collected in
mid-winter (c.f. Williams et al.,
2002). By contrast, the purpose of the present study was to
characterize seasonal changes in cold-tolerance and resistance to water loss
in E. solidaginis larvae to determine if the acquisition of
desiccation resistance is linked to increases in cold-tolerance. To
investigate this question we measured survival after exposure to subzero
temperatures, hemolymph osmolality as a measure of cryoprotectant production,
resistance to water loss, and body water content of field collected larvae
from early autumn to mid-winter. To identify possible environmental cues for
seasonal increases in cold-tolerance and enhanced desiccation resistance we
monitored ambient temperature, gall water content and gall water activity. In
conjunction with the field study, we also examined the effect of mild
desiccation stress on rates of water loss and cold-tolerance, prior to and
after plant senescence and gall drying in the autumn.
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Materials and methods |
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Environmental and gall measurements
Beginning 1 September 2001, air temperature was monitored by the Miami
University weather station located at the Miami University Ecology Research
Center approximately 0.20.4 km from the collection sites. Because the
dried gall tissue offers little insulative value, larval body temperature
should closely track ambient air temperatures, particularly on cloudy days or
at night (Layne, 1993).
Water activity of the galls was assessed by measuring the total water
content of each gall and the water vapor potential of the gall tissue
immediately surrounding the larvae. Gall water content was determined by
weighing 10 galls that had contained larvae to ±0.1 mg using a Mettler
Toledo AG245 balance (Mettler-Toledo Inc., Hightstown, NJ, USA), before and
after drying in an oven at 65°C until they reached a constant mass. Water
vapor potential of the gall tissue was determined by the psychrometric vapor
pressure depression technique described by Hølmstrup and Westh
(1994). Immediately after
opening an occupied gall, 1020 mg of gall tissue directly surrounding
the larval chamber was transferred to a Wescor C-52 sample chamber (Wescor,
Logan, UT, USA) and allowed to equilibrate for 30 min. Water potential was
then determined with a Wescor HR 33T Dewpoint Microvoltmeter operated in the
dewpoint mode. Measurements were taken on 10 randomly selected galls for the
first three testing dates. However, only five of the 10 randomly selected
galls on 30 October were moist enough to obtain a reading. No vapor pressures
were measured after 30 October because gall tissues were too dry to
measure.
Measurement of cold-tolerance
Larval cold-tolerance was assessed by measuring survival rates after
exposure to various subzero temperatures. Ten larvae were placed in
temperature-controlled baths and cooled at 1°C min1 to
either 2, 4, 8, or 12°C. A fifth group was
placed in an insulated container that provided a cooling rate of approximately
1°C min1 until it reached equilibrium in a
20°C freezer. After 24 h exposure to a treatment temperature,
larvae were warmed to room temperature (23°C) at 1°C
min1. Larvae were then held for 24 h at room temperature and
considered alive if they moved after being gently touched with a blunt probe.
The 12°C experimental group was added on 3 October 2001 to increase
sensitivity for detecting changes in cold-hardiness. Cold-tolerance tests were
not done after 30 October when all larvae survived 20°C for 24 h
and were considered to be highly cold-tolerant.
Hemolymph osmolality provided a measure of the seasonal accumulation of cryoprotectants. Hemolymph osmolality (N=10) was determined by drawing 710 µl of hemolymph into a capillary tube through a small incision in the larva's cuticle. The hemolymph was then analyzed in a Wescor Vapro 550 Hemolymph Osmometer.
Measurement of desiccation resistance
Resistance to desiccation was examined using measures of water loss rate in
units of µg mm2 h1, and body water
content as a ratio of wet mass to dry mass. To determine water loss rates, 10
individuals per test date were weighed to ±0.01 mg to obtain a fresh
mass. Larvae were then re-weighed after being desiccated at 5°C over
Drierite (W. A. Hammond Drierite Co., Ohio, USA), providing a 4% RH, until
they lost 510% of their fresh mass. Body water content was determined
by placing the desiccated larvae in an oven at 65°C until a constant dry
mass was obtained.
Cuticular surface area was estimated from initial wet mass using an
equation derived from the best fit line for larvae of known mass and surface
area. Surface area was calculated for 10 individuals of varying mass by
puncturing the cuticle, expelling the internal contents by gently flattening
the cuticle on millimeter-squared paper, and estimating the surface area
(Williams et al., 2002;
Ramløv and Lee, 2000
).
The derived equation was y=0.912x+4.204,
r2=0.804, where y=surface area in mm2
and x=mass in mg.
Effect of moderate desiccation stress on cold-tolerance and desiccation resistance
Larvae collected on 5 October and 2 November were used to determine the
effects of mild water stress on cold-tolerance and desiccation resistance.
Larvae were either held over a saturated solution of sodium sulfate producing
a RH of 95% or over a saturated solution of sodium chloride producing a RH of
76% at 15°C. After 10 days of exposure to these conditions, larval
cold-tolerance, water loss rate and body water content were measured using the
techniques described previously. In contrast to the previous techniques, 15
larvae per treatment were used in these experiments as opposed to 10, and
cold-tolerance was determined using only the 8 and 12°C
treatment conditions. Larvae collected on 3 October and 30 October as
described in the previous sections, were compared with the 95% and 76% RH
experimental groups and referred to as field groups.
Statistical analyses
Seasonal data were analyzed using a one-way analysis of variance (ANOVA)
followed by StudentNewmanKeuls test. To identify differences
between gall and larval water activity for a given date, unpaired
t-tests were used. When determining the effect of moderate
desiccation on cold-tolerance and desiccation resistance, a one-way ANOVA
followed by StudentNewmanKeuls test were used to indicate
significant differences between treatment groups for a given date. To identify
differences in survival between larvae subjected to moderate desiccation
stress a chi-squared analysis was used. A significance level of
P=0.05 was used for all tests. Linear regression analyses were used
to estimate surface area of the larvae, as well as the relationships between
events of cold hardening and acquisition of desiccation resistance.
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Results |
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In contrast to the gradual decrease in air temperature, gall water content decreased dramatically during two weeks in October as the goldenrod plants senesced. Galls were well hydrated from 20 September to 16 October, ranging from 1.9 to 2.1 mg water per mg dry mass (Fig. 2A). However, between 16 October and 30 October, gall water content decreased significantly (P<0.05) to 0.4 mg water per mg dry mass. Gall water content reached a minimum value of 0.2 mg water per mg dry mass on 15 January.
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Even though gall water content decreased markedly during the study period, larval body water content remained statistically unchanged (Fig. 2B). No trends were evident in values for body water content, which ranged between 1.44 and 1.71 mg water per mg dry mass.
As air temperatures decreased through the autumn and winter, larval cold-tolerance gradually increased (Fig. 3).Larvae collected in September already had a modest level of cold-tolerance, as all larvae survived a 24 h exposure to 2°C and 90% survived 4°C; however, no larvae survived 20°C. Larvae were judged to be extremely cold-tolerant on 30 October, as all individuals survived 20°C for 24 h. Notably, throughout the study larvae tolerated temperatures that were 1020°C lower than were measured in the field.
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The gradual increase in cold-tolerance of E. solidaginis larvae was mirrored by steady increases in hemolymph osmolality (Fig. 2C). Values for hemolymph osmolality increased significantly (P<0.05) at each successive testing date after 16 October and ranged from an initial value of 488 mOsmol kg1 to the final measure of 967 mOsmol kg1.
In contrast to the gradual increase in larval cold-tolerance, there were
two distinct periods in which water loss rates decreased during the autumn.
The first phase of reduced rates of water loss was a substantial sixfold
decrease that occurred between 3 October (3.5 µg mm2
h1) and 16 October (0.6 µg mm2
h1; Fig. 2D).
This initial reduction in the rate of water loss was followed by a second
phase in which rates of water loss decreased more slowly over an 8 week period
(Fig. 2D). Even though the
second phase of reduced rates of water loss was not as dramatic as the one
that occurred in early October, the 3.9-fold decrease was significantly
different when the 16 October, 30 October, 14 November and 11 December data
were analyzed using an ANOVA followed by StudentNewmanKeuls test
(Fig. 2D). Interestingly, the
decrease in rates of water loss during this period correlated strongly with
increases in hemolymph osmolality levels, r2= 0.94
(Fig. 4). It is important to
note that the data for the larvae collected on 15 January were excluded from
this analysis because these individuals were probably no longer in the
refractory phase of diapause (Irwin et
al., 2001).
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The water potential of the gall tissue decreased from 20 September to 16 October, ranging between 9.1 and 12.7 bars (Fig. 5). By 30 October, gall tissue was considerably drier, only 5 of the 10 randomly selected galls were moist enough to obtain a measure of water potential. Water potential of the gall tissue was significantly higher (P<0.05) than the water potential of larval hemolymph on 20 September and 3 October, suggesting larvae were not subjected to desiccation stress at this time. However, the water potential of the gall tissue and the insect's hemolymph did not differ on 16 October, indicating the gall was transitioning between a non-desiccating and desiccating environment for the larvae.
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Cold-tolerance and desiccation resistance after moderate desiccation stress
To determine whether desiccation stress could induce changes in rates of
water loss and cold-tolerance, larvae were collected on two different dates
and were subjected to desiccating conditions in the laboratory. One group of
larvae were collected on 5 October, when the goldenrod plant tissue was green
and moist, and a second group on 2 November, after the plant had senesced and
dried.
Larvae collected on 5 October were subjected to either 95% RH or 76% RH at 15°C for 10 days prior to assessing their cold-tolerance and desiccation resistance. Even though there was an apparent trend toward increased survival at 8°C and 12°C for larvae in both the 76% and 95% RH treatment groups, these differences were not significant when compared with field samples taken on 3 October (Fig. 6). Body water content for all larval groups for the 5 October treatments were the same, averaging 1.49 mg water mass mg1 dry mass (Fig. 7A). In contrast, moderate desiccation stress enhanced desiccation resistance as rates of water loss were significantly lower (P<0.05) for larvae in the 95% RH and the 76% RH groups, 2.24 and 0.83 µg mm2 h1 respectively, than the field group (Fig. 7B). These data suggests that mild desiccation stress induced an enhanced desiccation resistance in the larvae.
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A second group of larvae collected on 2 November were tested for
desiccation resistance after 10 days exposure to 95 or 76% RH at 15°C. As
with the 5 October collection, the November-collected control group had the
same body water content as the 95% and 76% RH experimental groups, 1.68
mg water mass mg dry mass1
(Fig. 7A). In contrast to the 5
October collection, water loss rates were very low and there were no
differences in rates of water loss between larvae in the control and
experimental groups (Fig. 7B). These results suggest that larvae were highly resistant to desiccation prior
to being collected and subjected to these conditions on 2 November.
Cold-tolerance was not examined for this collection date as the larvae were
previously deemed to be extremely cold-tolerant on 30 October.
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Discussion |
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The first phase of increased resistance to water loss is probably due to
decreased respiratory transpiration as larvae entered diapause. Water loss
through transpiration is positively linked to the activity level of a given
insect. High levels of metabolic activity due to flight
(Nicolson and Louw, 1982) or
elevated temperatures (Ahearn,
1970
), increase respiratory water loss. Irwin et al.
(2001
) showed that E.
solidaginis larvae from southwest Ohio reduce their metabolic rate by
more than 75% between 1 and 15 October, when they enter diapause. Diapause is
defined as a genetically determined state of low metabolic activity,
suppressed development and heightened resistance to environmental extremes
that lasts longer than the adverse conditions
(Danks, 1987
;
Tauber et al., 1986
). A
reduction of metabolic rate and consequent decrease in respiratory
transpiration, as larvae entered diapause, most likely contributed importantly
to the rapid decrease in rates of water loss between 3 and 16 October.
In addition to reduced transpiration, increased levels of cuticular lipids
may have contributed to the first phase of reduced rates of water loss. Water
loss for dormant insects primarily occurs as water diffuses across their
cuticle and during respiratory transpiration
(Edney, 1977;
Hadley, 1994
). Cuticular water
loss is primarily regulated by the amount and type of epicuticular lipids on
the integumental surface (see references in
Hadley, 1994
;
Gibbs, 1998
). Dormant stages
of insects, which are at risk of dehydration, such as larvae of the flesh fly
Sarcophaga crassipalpis (Yoder et
al., 1992
), the tobacco hornworm, Manduca sexta
(Bell et al., 1975
;
Coudron and Nelson, 1981
) and
the moth Mamestra configurata
(Hegdekar, 1979
), increase the
amount of their epicuticular lipids to diminish water loss. Epicuticular
hydrocarbons increase 40-fold in E. solidaginis from late summer to
mid-winter (D. R. Nelson and R. E. Lee,
2004
) and may have contributed to the rapid phase one decrease
rates of water loss (Fig. 2D).
However, it is unknown if epicuticular lipids increased over the 13-day period
that constituted phase one in the present study.
Gall water content has been used as the primary indicator of desiccation
stress in galling insects (Irwin et al.,
2001; Layne and Medwith,
1997
; Lee and Hankinson,
2003
). However, this technique is unable to detect slight changes
in water potential of the gall tissue immediately surrounding the insect that
could profoundly impact its physiology and water balance. The springtail
Folsomia candida increases its drought tolerance after being exposed
to a water potential deficit between its environment and hemolymph of only 17
bars (Sjursen et al., 2001
).
Small changes in water potential between the body fluids of E.
solidaginis larvae and its gall tissue may also influence its resistance
to water loss. For instance, on 20 September and 3 October the water potential
of larval hemolymph was significantly lower than the water potential of the
surrounding gall tissue (Fig.
5), indicating the larvae were in a potentially hydrating
environment. By contrast, between 16 and 30 October the water potential of the
gall tissue decreased markedly, indicating a shift to a dehydrating
environment. This small change in water potential deficit between the gall
tissue and larval hemolymph correlates closely with the phase one reduction in
rates of water loss (Fig. 2D)
and may be a cue that triggers larvae to increase their resistance to
desiccation and to enter into diapause.
We found no correlation between increased desiccation resistance and
increased cold-tolerance early in the study. Between late September and 30
October, larvae exhibited a gradual increase in cold-tolerance
(Fig. 3) that correlates well
with other studies performed on this species in southwest Ohio
(Lee and Hankinson, 2003) as
well as in western Pennsylvania (Layne,
1991
). This seasonal increase in cold-tolerance is due to the
concomitant increase in cryoprotectants levels
(Baust and Lee, 1981
;
Storey and Storey, 1992
), as
evidenced by hemolymph osmolality, which increased by 30% from 20 September to
30 October (Fig. 2C). However,
cold-tolerance only gradually increased and hemolymph osmolality remained
unchanged between 3 and 16 October when larval water loss rates decreased
rapidly (Fig. 2D). In addition,
larval rates of water loss were significantly lowered after being subjected to
mild desiccation stress in early October
(Fig. 7B), although larval
cold-tolerance did not change (Fig.
6). Taken together, these data suggest that different mechanisms
regulate desiccation resistance and cold-tolerance during this period.
In contrast to phase one, the second phase of increased desiccation
resistance correlated closely with increases in hemolymph osmolality and
suggests a link between desiccation resistance and cold-tolerance in E.
solidaginis (Fig. 4). It
is unlikely that the decrease in the rate of water loss during the second
phase was caused by changes in respiratory water loss because E.
solidaginis larvae remain in diapause, with a depressed metabolic rate
until mid-January (Irwin et al.,
2001). As mentioned previously, epicuticular hydrocarbons increase
40-fold in E. solidaginis from late summer to mid-winter
(D. R. Nelson and R. E. Lee,
2004
). Therefore, increased levels of epicuticular hydrocarbons
may be partly responsible for the increased desiccation resistance between 16
October and 11 December. However, it is likely that most cuticular
hydrocarbons were added prior to experiencing desiccating conditions as the
gall tissue senesced and dried in early October.
The manner in which the elevated cryoprotectant concentrations could have
affected water loss rates is unknown, however it is unlikely that it is was
due to a colligative reduction in the water potential deficit between the
insect's hemolymph and environmental water vapor
(Edney, 1977;
Williams et al., 2002
). In
response to desiccating conditions, the springtail F. candida rapidly
synthesizes osmolytes, predominantly myoinositol and glucose, which
colligatively lowers its hemolymph water activity and consequently reduces or
even eliminates organismal water loss
(Bayley and Holmstrup, 1999
;
Sjursen et al., 2001
). The
production of these solutes can reduce water loss colligatively only because
the desiccating conditions the springtails experience are quite mild
(Av
0.984) with a water potential deficit between the insect's
hemolymph and environmental water vapor of only
17 bars
(Bayley and Homlstrup, 1999
).
By contrast, gall fly larvae experience much drier conditions during winter.
For example, the water potential deficit between larval hemolymph and their
environment of 14,400 bars(simulated in the water loss trials of
Fig. 2D) is commonly
experienced by these insects in mid-winter. Between 16 October and 11
December, larvae increased their hemolymph osmolality by 302 mOsmol
kg1 (Fig.
2C); this increase in solutes would reduce the water potential
deficit between the hemolymph and the environment by only
7 bars. Such a
small reduction of the water potential deficit through colligative actions of
increased solutes would have a negligible effect on rates of water loss over
that period.
Multiple lines of evidence indicate that carbohydrates influence arthropod
water relations in a non-colligative manner. Trehalose, glycerol and sorbitol
can protect cell membranes against severe desiccation stress and increase
organismal tolerance to desiccation (Crowe
et al., 1984; Bryszewska and
Epand, 1988
; Crowe,
2002
; Oliver et al.,
2002
). Certain cryoprotectants, like glycerol and sorbitol, are
effective at binding water (Storey et al.,
1981
; Storey,
1983
). Bound water differs markedly from bulk water as it is less
likely to freeze than bulk water and is also highly resistant to removal when
dried at biologically relevant temperatures (see references in
Danks, 2000
; Block,
1996
,
2003
). Intracellular bound
water may increase post-freeze survival for freeze-tolerant organisms
(Storey et al., 1981
;
Storey, 1983
), however, little
is known about the effect of extracellular bound water.
The insect cuticle, a multi-layered structure with a single basal layer of
epidermal cells, is the primary barrier to organismal water loss
(Hadley, 1994). The cuticle
also functions as the main barrier by which freeze-susceptible insects resist
inoculative freezing (Somme,
1982
). Several insects seasonally increase their resistance to
inoculative freezing (Duman,
2001
). Winter-acclimated larvae of the beetle, Dendroides
canadensis, resist inoculative freezing better than summer larvae, in
part because they produce antifreeze proteins that adhere to the epidermis
(Olsen et al., 1998
).
Antifreeze proteins lower the non-equilibrium freezing point of a solution
without affecting the melting point (Duman
et al., 1991
; Duman,
2001
). Recently, Duman
(2002
) found that the
cryoprotectant glycerol interacts synergistically with antifreeze proteins to
increase their activity, apparently by stabilizing the protein.
We speculate that cryoprotectants associate with proteins on the surface of
the epidermal layer and, thereby, enhance resistance to desiccation. Although
no antifreeze proteins are known to be produced by E. solidaginis,
they do produce a novel, dehydrin-like protein during natural cold hardening
(Pruitt and Shapiro, 2001).
Dehydrins are a family of proteins produced in response to desiccation.
Certain dehydrins are localized to leaf epidermal tissue in cold acclimated
barley (Bravo et al., 2003
) and
may interact with low molecular mass cellular components (see references in
Allagulova et al., 2003
). Thus,
it is possible that an epidermal proteins associates with cryoprotectants in
E. solidaginis. Glycerol and sorbitol substantially increase the
amount of bound water in E. solidaginis during natural
cold-hardening; 10 to 20% of total body water is bound due to sorbitol and
glycerol in mid-winter larvae (Storey et
al., 1981
; Storey,
1983
). Taken together, bound water associated with epidermal
cryoprotectants may collectively thicken the cuticular barrier, resulting in
decreased rates of water loss (Fig.
4).
In summary, hemolymph osmolality and cold-tolerance of E. solidaginis larvae steadily increased during the autumn. An initial rapid decrease in seasonal rates of water loss was correlated with drying of the gall tissue surrounding the larvae that was probably caused by decreased respiratory water loss as larval metabolism fell upon entering diapause. Later in the autumn, cryoprotectant accumulation may have affected water conservation through non-colligative actions.
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Acknowledgments |
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahearn, G. A. (1970). The control of water loss in desert tenebrionid beetles. J. Exp. Zool. 53,573 -595.
Allagulova, C. R., Gimalov, F. R., Shakirova, F. M. and Vakhitov, V. A. (2003). The plant dehydrins: structure and putative functions. Biochem. Moscow 68,945 -951.
Baust, J. G. and Lee, R. E. (1981). Divergent mechanisms of frost-hardiness in two populations of the gall fly, Eurosta solidaginsis. J. Insect Physiol. 27,485 -490.[CrossRef]
Baust, J. G. and Lee, R. E. (1982). Environmental triggers to cryoprotectant modulation in separate populations of the gall fly, Eurosta solidaginis (Fitch). J. Insect Physiol. 28,431 -436.[CrossRef]
Bayley, M. and Holmstrup, M. (1999). Water
vapor absorption in arthropods by accumulation of myoinositol and glucose.
Science 285,1909
-1911.
Bell, R. A., Nelson, D. R. and Borg, T. K. (1975). Wax secretion in non-diapausing and diapausing pupae of the tobacco hornworm, Manduca sexta. J. Insect Physiol. 21,1725 -1729.[CrossRef]
Block, W. (1996). Cold or drought the lesser of two evils for terrestrial arthropods? Eur. J. Entomol. 93,325 -339.
Block, W. (2003). Water or ice? The challenge for invertebrate cold survival. Sci. Prog. 86, 77-101.[Medline]
Bravo, L. A., Gallardo, J., Navarrete, A., Olave, N., Martinez, J.,Alberdi, M., Close, T. J. and Corcuera, L. J. (2003). Cryoprotective activity of a cold-induced dehydrin purified from barley. Physiol. Plant. 118,262 -269.[CrossRef]
Bryszewska, M. and Epand, R. M. (1988). Effects of sugar alcohols and disaccharides in inducing the hexagonal phase and altering membrane-properties implications for diabetes-mellitus. Biochim. Biophys. Acta 943,485 -492.[Medline]
Coudron, T. A. and Nelson, D. R. (1981). Characterization and distribution of the hydrocarbons found in diapausing pupae tissues of the tobacco hornworm, Manduca sexta (L). J. Lipid Res. 22,103 -112.[Abstract]
Crowe, L. M. (2002). Lessons from nature: the role of sugars in anhydrobiosis. Comp. Biochem. Physiol. A 131,505 -513.
Crowe, L. M., Mouradian, R., Crowe, J. H., Jackson, S. A. and Womersley, C. (1984). Effects of carbohydrates on membrane stability at low water activities. Biochim. Biophys. Acta 769,141 -150.[Medline]
Danks, H. V. (1987). Insect Dormancy: An Ecological Perspective. Ottawa: Biological Survey of Canada (Terrestrial Artropods).
Danks, H. V. (2000). Dehydration in dormant insects. J. Insect Physiol. 46,837 -852.[CrossRef][Medline]
Duman, J. G. (2001). Antifreeze and ice nucleator proteins in terrestrial arthropods. Annu. Rev. Physiol. 63,327 -357.[CrossRef][Medline]
Duman, J. G. (2002). The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate. J. Comp. Physiol. B 172,163 -168.[Medline]
Duman, J. G., Xu, L., Neven, L. G., Tursman, D. and Wu, D. W. (1991). Hemolymph proteins involved in insects subzero-temperature tolerance: ice nucleators and anti-freeze protiens. In Insects at Low Temperature, (ed. R. E. Lee and D. L. Denlinger), pp. 94-127. New York: Chapman and Hall.
Edney, E. B. (1977). Water Balance In Land Arthropods. New York: Springer-Verlag.
Gibbs, A. G. (1998). Water-proofing properties of cuticular lipids. Am. Zool. 38,471 -482.
Hadley, N. F. (1994). Water relations of terrestrial arthropods. San Diego: Academic Press.
Hegdekar, B. M. (1979). Epicuticular wax secretion in diapause and non-diapause pupae of the Bertha armyworm Mamestra configurata. Annu. Entomol. Soc. Am. 72, 13-15.
Hølmstrup, M. and Westh, P. (1994). Dehydration of earthworm cocoons exposed to cold a novel cold-hardiness mechanism. J. Comp. Physiol. B 164,312 -315.
Irwin, J. T., Bennett, V. A. and Lee, R. E., Jr (2001). Diapause development in frozen larvae of the goldenrod gall fly, Eurosta solidaginis Fitch (Diptera: Tephritidae). J. Comp. Physiol. B 171,181 -188.[Medline]
Layne, J. R. (1991). Microclimate variability and the eurythermic nature of goldenrod gall fly (Eurosta solidaginis) larvae (Diptera, Tephritidae). Can. J. Zool. 69,614 -617.
Layne, J. R. (1993). Winter microclimate of goldenrod spherical galls and its effects on the gall inhabitant Eurosta solidaginis (Diptera, Tephritidae). J. Therm. Biol. 18,125 -130.[CrossRef]
Layne, J. R. and Medwith, R. E. (1997). Winter conditioning of third instars of the gall fly Eurosta solidaginis (Diptera:Tephritidae) from western Pennsylvania. Physiol. Chem. Ecol. 26,1378 -1384.
Lee, R. E. and Hankison, S. J. (2003). Acquisition of freezing tolerance in early autumn and seasonal changes in gall water content influence inoculative freezing of gall fly larvae, Eurosta solidaginis (Diptera, Tephritidae). J. Insect Physiol. 49,385 -393.[CrossRef][Medline]
Nelson, D. R. and Lee, R. E. (2004) Accumulation of cuticular lipids and increased desiccation resistance in overwintering larvae of the gall fly Eurosta solidaginis (Diptera: Tephritidae). Comp. Biochem. Physiol. B. 138,313 -320.[CrossRef][Medline]
Nicolson, S. W. and Louw, G. N. (1982). Simultaneous measurement of evaporative water-loss, oxygen-consumption, and thoracic temperature during flight in a carpenter bee. J. Exp. Zool. 222,287 -296.
Oliver, A. E., Hincha, D. K. and Crowe, J. H. (2002). Looking beyond sugars: the role of amphiphilic solutes in preventing adventitious reactions in anhydrobiotes at low water contents. Comp. Biochem. Physiol. A 131,515 -525.
Olsen, T. M., Sass, S. J., Li, N. and Duman, J. G.
(1998). Factors contributing to seasonal increases in inoculative
freezing resistance in overwintering fire-colored beetle larvae Dendroides
canadensis (Pyrochroidae). J. Exp. Biol.
201,1585
-1594.
Pruitt, N. L. and Shapiro, C. (2001). Evidence for a cryoprotective protein in freeze tolerant larvae of the goldenrod gall fly, Eurosta solidaginis. Am. Zool. 41,1561 -1561.
Ramløv, H. and Lee, R. E. (2000).
Extreme resistance to desiccation in overwintering larvae of the gall fly
Eurosta solidaginis (Diptera, Tephritidae). J. Exp.
Biol. 203,783
-789.
Ring, R. A. and Danks, H. V. (1994). Desiccation and cryoprotection overlapping adaptations. Cryo-Lett. 15,181 -190.
Rojas, R. R., Lee, R. E. and Baust, J. G. (1986). Relationship of environmental water-content to glycerol accumulation in the freezing tolerant larvae of Eurosta solidaginis (Fitch). Cryobiol. 23,564 -564.
Sjursen, H., Bayley, M. and Holmstrup, M. (2001). Enhanced drought tolerance of a soil-dwelling springtail by pre-acclimation to a mild drought stress. J. Insect Physiol. 47,1021 -1027.[CrossRef][Medline]
Somme, L. (1982). Supercooling and winter survival in terrestrial arthropods. Comp. Biochem. Physiol. A 73,519 -543.[CrossRef]
Storey, K. B. (1983). Metabolism and bound water in overwintering insects. Cryobiol. 20,365 -379.[Medline]
Storey, K. B., Baust, J. G. and Buescher, P. (1981). Determination of water "bound" by soluble subcellular components during low-temperature acclimation in the gall fly larva, Eurosta solidagensis. Cryobiol. 18,315 -321.[Medline]
Storey, K. B. and Storey, J. M. (1992). Biochemical adaptations for winter survival in insects. In Advances in Low-Temperature Biology, vol. 1 (ed. P. L. Steponkus), pp. 101-140. London: JAI Press.
Tauber, M. J., Tauber, C. A. and Masaki, S. (1986). Seasonal Adaptations of Insects. New York: Oxford University Press.
Uhler, L. D. (1951). Biology and ecology of the goldenrod gall fly, Eurosta solidaginis (Fitch). New York: New York State College of Agriculture.
Williams, J. B., Shorthouse, J. D. and Lee, R. E. (2002). Extreme resistance to desiccation and microclimate-related differences in cold-hardiness of gall wasps (Hymenoptera: Cynipidae) overwintering on roses in southern Canada. J. Exp. Biol. 205,2115 -2124.[Medline]
Yoder, J. A., Denlinger, D. L., Dennis, M. W. and Kolattukudy, P. E. (1992). Enhancement of diapausing flesh fly puparia with additional hydrocarbons and evidence for alkane biosynthesis by a decarbonylation mechanism. Insect Biochem. Mol. Biol. 22,237 -243.[CrossRef]
Zachariassen, K. E. (1991). The water relations of overwintering insects. In Insects at Low Temperature (ed. R. E. Lee and D. L. Denlinger), pp.47 -63. New York: Chapman and Hall.
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