Extreme resistance to desiccation and microclimate-related differences in cold-hardiness of gall wasps (Hymenoptera: Cynipidae) overwintering on roses in southern Canada
1 Department of Zoology, Miami University, Oxford, OH 45056, USA
2 Department of Biology, Laurentian University, Sudbury, Ontario, Canada P3E
2C6
* e-mail: willia37{at}muohio.edu
Accepted 2 May 2002
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Summary |
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Key words: desiccation, cold-hardiness, water loss, permeability, overwintering, gall wasp, Diplolepis sp., Periclistus sp., hibernaculum
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Introduction |
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Most insects cannot survive internal ice formation and are termed
freeze-intolerant. The seasonal accumulation of glycerol and other
low-molecular-mass polyols and sugars, termed cryoprotectants, promotes
increased cold-hardiness and resistance to water loss
(Lee, 1991). These substances
colligatively increase the insect's hemolymph osmolality, which enhances the
ability of its body fluids to supercool (i.e. to remain in the liquid state
below the melting point). These substances also act in a similar colligative
manner to reduce the vapor pressure deficit between the insect's hemolymph and
the environment and, thus, decrease its rate of water loss. Other insects
achieve the same result by decreasing their body water content, which
concentrates their hemolymph (Ring,
1981
; Rickards et al.,
1987
).
Insects used in studies of adaptation to cold have been collected in a
variety of habitats and associated environmental conditions such as in plant
stems, under bark and rocks, in soil and within plant galls
(Leather et al., 1995).
Several species of gall-inducing insect, including the tephritid Eurosta
solidaginis (Fitch) (Lee et al.,
1995
), the olethreutid moth Epiblema scudderiana
(Clemens) (Rickards et al.,
1987
) and cynipids of the genus Diplolepis
(Rickards and Shorthouse,
1989
; Shorthouse et al.,
1980
; Sømme,
1964
), have been used in cold-hardiness studies, partly because it
is easy to collect large numbers in mid-winter. Wasps of the genus
Diplolepis, all of which induce galls on wild roses
(Shorthouse, 1993
), are
particularly useful for comparative studies of cold-hardiness because some
species overwinter above the snow (supranivean) while others overwinter below
it (subnivean).
Approximately 30 species of nearctic Diplolepis are known, and
each induces galls of a distinctive size and shape on the leaves, stems, buds
or tips of stems arising from rhizomes
(Shorthouse, 1993). All
species are univoltine, and galls are initiated in the spring; larvae feed on
specialized nutritive cells lining larval chambers, and they overwinter within
their galls as freeze-intolerant prepupae
(Sømme, 1964
;
Rickards and Shorthouse,
1989
). Diapause ends and development resumes in early spring. The
pupal stage lasts for approximately 15 days before the adult exits its gall
and searches for oviposition sites (Brooks
and Shorthouse, 1997
).
Galls of some species of Diplolepis are inhabited and structurally
modified by inquiline cynipids of the genus Periclistus (Brooks and
Shorthouse, 1997,
1998
;
Shorthouse, 1998
). The life
cycles of Periclistus are similar to those of Diplolepis
spp. except that Periclistus spp. kill the inducer with their
ovipositors as they lay their eggs into developing Diplolepis galls.
Periclistus spp. larvae then feed on nutritive cells they induce from
tissues of the host gall (Shorthouse,
1998
).
Depending upon the location of the gall on the host rose, overwintering Diplolepis and Periclistus prepupae may experience very different microclimatic conditions. Prepupae in ground-level galls on shoots or rhizomes are insulated from harsh conditions by overlying snow. Prepupae in leaf galls may occupy similar subnivean hibernacula as rose leaves abscise and fall to the ground before winter. In contrast, prepupae in stem galls may be supranivean and exposed to extreme cold and desiccation above the snowpack.
The Diplolepis complex and its Periclistus inquilines are
unique among gall-inducers in that groups of species overwinter in distinctly
different sites. Here, we report on the cold-hardiness and resistance to
desiccation of the supranivean galler D. spinosa and the inquiline
P. pirata, found in the gall of D. nodulosa (Brooks and
Shorthouse, 1997,
1998
), the subnivean leaf
gallers D. polita, D. gracilis and an unnamed Periclistus
inquiline, found in the gall of D. polita
(Shorthouse, 1998
), and the
shoot tip galler D. radicum found at ground level. We examined
cold-hardiness and desiccation-resistance in prepupae of these species exposed
to a simulated autumn-to-winter transition and in winter-acclimated
individuals by measuring supercooling points, glycerol concentrations,
hemolymph osmolalities, resistance to water loss, transition temperatures, the
effects of treatment with various solvents on rates of water loss and the
ability to absorb atmospheric water vapor.
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Materials and methods |
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Gall collections
Maturing galls containing prepupae of Diplolepis spp. and
Periclistus spp. were collected from mid-August to the beginning of
October 1999 at four sites in southern Canada. Galls of D. radicum
and D. polita and those containing P. pirata and
Periclistus sp. were collected near Sudbury, Ontario, Canada. Galls
containing D. spinosa were found at two sites; those collected near
Medicine Hat, Alberta, Canada, were termed D. spinosa AB and those
collected within the Cypress Hills Provincial Park south of Maple Creek,
Saskatchewan, Canada, were termed D. spinosa SK. Galls of D.
gracilis were collected within the Douglas Provincial Park northwest of
Moose Jaw, Saskatchewan, Canada.
Galls were held at 15°C until all samples had been collected. In late October, prepupae were removed from their galls, placed individually in culture plates and held in an incubator at 15°C and 65% RH in the dark. To simulate winter conditions, all species were transferred on 11 November 1999 to desiccators containing saturated solutions of NaCl (75% RH) and held at 5°C.
Desiccation measurements after cold acclimation
After 2 months at 5°C, the specimens were weighed to ±0.1 µg
before and after desiccation using a Mettler Toledo UMT2 balance. Animals were
held in ELISA plastic well plates while desiccated over Drierite (W. A.
Hammond Drierite Co., Ohio, USA) with an RH of 0% (1.5x10-2%
RH; Toolson, 1980) until a
measurable water loss of 2-5% of fresh mass was detected
(Hadley, 1994
).
The surface area of the prepupae was estimated by using the equation of the
best-fitting line derived from individuals of known mass and surface area. The
surface area was calculated for five individuals of each species by making a
small incision on the insect's cuticle, expressing the internal contents and
then flattening them on millimeter-squared paper. Equations for the six
species were, y=5.65x-1.19 for D. gracilis, where
y is predicted surface area in mm2 and x is the
mass of the organism in mg, y=2.59x+2.99 for D. polita,
y=1.41x+8.51 for D. radicum, y=1.54x+11.52 for
D. spinosa, y=3.41x-2.07 for P. pirata and
y=2.98x+1.10 for Periclistus sp. The strength of
linear association, as measured by r2, for these
regression equations ranged from 0.78 to 0.91, averaging 0.86. Meeh's formula
(S=kW0.667, where S is surface area, k
is a constant and W is mass;
Wigglesworth, 1945;
Hadley, 1994
) for estimating
surface area was also used. In all species, Meeh's formula estimated a 4-10%
larger surface area compared with the best-fitting line method; it was not
used for any subsequent calculations.
The difference in vapor pressure, P, between the hemolymph
of the animal and the surrounding air was calculated using the formula:
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Body fluid osmolality was determined using the psychrometric vapor pressure
depression technique described by Hølmstrup and Westh
(1994). For each species, five
groups of 2-5 individual prepupae, with a combined mass of approximately 10
mg, were placed in the sample holder, lanced open with fine probes to expose
their hemolymph and quickly inserted into a Wescor C-52 sample chamber (Logan,
UT, USA). Samples were allowed to equilibrate for 1 h before osmolality was
determined using a Wescor HR33T dew-point microvoltmeter operated in the
dew-point mode.
The temperature at which there is an abrupt and dramatic increase in the
rate of cuticular transpiration is termed the critical transition temperature.
This was determined by exposing live prepupae to temperatures of 5, 20, 27,
33, 40 and 45 °C over Drierite until a measurable water loss of 2-5% of
fresh mass was detected (Hadley,
1994). Eight individuals of each species were used for the 5
°C trial, while five specimens were used in the remaining temperature
treatments.
To determine whether water loss was under physiological control, five prepupae of each species were killed by exposure to cyanide for 24 h at 22 °C and desiccated over Drierite at 5 °C. Total body water was measured by placing eight individuals of each species in an oven at 65 °C for 24 h followed by a final weighing to determine dry mass.
To investigate the effects of solvents on the rate of water loss, live prepupae (five per species) were gently washed with hexane, methanol/chloroform (1:2), acetone or water for 1 min. Specimens were then carefully blotted with filter paper and air-dried for 5 min before being weighed and exposed to 0% RH at 20 °C for 12-24 h to obtain a water loss rate.
To ascertain whether these species were capable of absorbing atmospheric water vapor, individuals (five per species) were desiccated at 0% RH and 20 °C until they lost 5-10% of their original body mass. The specimens were then placed over a saturated sodium phosphate solution (95% RH) at 20 °C for 72 h and reweighed.
Measurements of cold tolerance after cold acclimation
The cold-hardiness of the six species was assessed by measuring their
supercooling points and glycerol concentrations. Prepupae (eight per species)
were placed in submerged vials in an alcohol bath and cooled at 1 °C
min-1 until an exotherm, indicating the supercooling point, was
detected by a copperconstantan thermocouple placed near the animal.
During supercooling point determinations, prepupae were cooled to as much as 5
°C below their supercooling point. Prepupae were then warmed at 1 °C
min-1 to 5 °C, where they were held for 24 h before being
judged to be alive if they responded to tactile stimulation. Glycerol
concentration was measured on the same individuals by enzymatic assay (Sigma
no. 337), as described by Hølmstrup et al.
(1999).
We believe that the supercooling point is a good measure of cold tolerance because preliminary data collected for five of the six cynipid species, D. gracilis being the absent group, had survival rates of above 50% for 10 prepupae per species held within 5 °C above their mean supercooling point for 24 h. Furthermore, no individual survived freezing during supercooling point determination.
Accumulation of resistance to cold and desiccating events
The rhizome shoot galler D. radicum, the stem galler D.
spinosa SK and the leaf galler D. gracilis were used to examine
changes in rates of water loss and parameters related to cold hardening of the
prepupa from late fall to mid-winter conditions through exposure to a constant
low temperature. These species were selected because they belong to the same
genus and they include representatives that overwinter in both supranivean and
subnivean hibernacula. Acclimating procedures were followed as described
above, with prepupae extracted from the gall and directly transferred from 15
to 5 °C on 11 November 1999. Using the techniques described above,
measurements of rates of water loss, percentages of total body water, glycerol
concentrations and supercooling points were taken after 0, 20, 40 and 62 days
of exposure to 5 °C. Day zero corresponds to the day when the holding
temperature was lowered on 11 November.
Statistical analyses
Analyses of the data included one-way analysis of variance (ANOVA) followed
by a Fisher's protected least significant difference (PLSD) test
(Sokal and Rohlf, 1995). These
analyses were used to identify differences within a species over time when
examining the accumulation of resistance to cold and desiccation as well as
between species when examining measures of cold-hardiness and
desiccation-resistance in mid-winter-acclimated individuals. A significance
level of
=0.05 was used for all tests, and values are presented as
means ± S.E.M. Linear regression analysis was used to estimate the
surface area of the cynipid prepupae, as stated above, and also in estimating
the critical transition temperature of mid-winter-acclimated individuals.
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Results |
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Even with the milder conditions, differences between supranivean and subnivean microclimates were readily apparent from a vertical temperature transect located near the study site, which was 25 km south of Sudbury airport. From September to late December and from late February to the end of May, weekly mean temperatures at ground level were only 1.1 °C higher than temperatures recorded 25 cm above the ground (Fig. 1A). However, during the coldest period of the winter, from late December to late February, there was a continuous snowpack of at least 10 cm. This snow cover resulted in weekly mean temperatures for the subnivean microclimate that were 9.1 °C higher than for the supranivean microclimate. During this period, subnivean temperatures never fell below -2.8 °C, with a daily temperature range averaging only 0.4 °C (Fig. 1B). In contrast, supranivean temperatures were below -20 °C on 14 different days and had an average daily temperature range of 9.9 °C.
|
Time course of cold-tolerance and desiccation-resistance during cold
acclimation
Throughout the 62 days of cold acclimation at 5 °C, D. spinosa, D.
gracilis and D. radicum supercooled extensively within the range
-26 to -40 °C (Fig. 2A).
These species were judged to be freeze-intolerant because no individuals
survived supercooling point determinations. During cold-acclimation,
supercooling points remained unchanged for D. gracilis and D.
radicum, species that overwinter in subnivean hibernacula. In contrast,
D. spinosa, which overwinters in supranivean hibernacula,
significantly reduced its supercooling point by more than 13 °C from day 0
(-27.4±3.0 °C; N=8) to day 20 (P<0.05).
Because of this large decrease, D. spinosa had significantly lower
supercooling points than D. gracilis and D. radicum from day
20 until the end of the study (P<0.05).
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Decreases in supercooling points were paralleled by increases in glycerol concentrations for D. spinosa (Fig. 2B). Levels of the cryoprotectant glycerol significantly increased more than threefold during cold-acclimation, reaching a final value of 0.98±0.11 mol l-1 (N=8, P<0.05). At the beginning of the acclimation period, D. radicum already contained considerable amounts of glycerol (0.17±0.04 mol l-1; N=8) which was significantly higher than was found in D. gracilis (P<0.05). However, neither of these species increased their glycerol level during cold-acclimation.
Body water content remained relatively constant for all species during cold-acclimation (Fig. 2D). The supranivean D. spinosa had a significantly higher body water content (P<0.05) than the subnivean D. gracilis and D. radicum throughout the acclimation period. Similarly, there were no significant changes in the rates of water loss (P>0.05) between or among the three species tested (Fig. 2C). D. spinosa averaged 0.034 mg h-1 mm-2 of water loss compared with 0.030 mg h-1 mm-2 for both D. gracilis and D. radicum.
Comparison of cold-tolerance and desiccation-resistance for
cold-acclimated cynipid prepupae
Aspects of cold-tolerance and desiccation-resistance were examined for six
species of cynipid prepupae after 2 months of exposure to 5 °C. The mean
mass of the prepupae ranged widely from 1.5 mg for Periclistus sp. to
9.1 mg for D. spinosa AB, while body water contents were between 53.3
and 62.3 % for all species (Table
1). Statistically, body water content separated the prepupae into
three groups. The two populations of D. spinosa had significantly
higher body water contents than did D. polita, D. gracilis and D.
radicum. In turn, these three species had significantly higher water
contents than the two Periclistus species (P<0.05).
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In addition to prepupae tested during cold acclimation Diplolepis spinosa AB, P. pirata, Periclistus sp. and D. polita prepupae also supercooled extensively and were unable to survive freezing (Fig. 3). There was no significant difference in the mean supercooling points among the supranivean species, with values ranging between -38 and -40°C. However, their supercooling points were significantly lower (P<0.05) by 6-8 °C than those of all four subnivean species.
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Within each genus, hemolymph osmolality was significantly higher (P<0.05) for species overwintering in supranivean hibernacula compared with those overwintering beneath the snow (Fig. 4). The supranivean inquiline P. pirata had the highest overall mean osmolality at 1849±65 mosmol kg-1 (N=5), and the leaf gall inquiline Periclistus sp. had the lowest overall osmolality at 977±84 mosmol kg-1 (N=5). The D. spinosa populations averaged 1796 mosmol kg-1 compared with the subnivean Diplolepis species, which had hemolymph osmolalities that did not exceed 1464 mosmol kg-1.
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As with hemolymph osmolality, concentrations of glycerol showed similar trends between supra- and subnivean species within each genus (Fig. 4). The supranivean D. spinosa had substantial levels of glycerol that were 5-7 times higher than that of any subnivean species (P<0.05). Likewise, glycerol levels for the supranivean P. pirata (300 mosmol kg-1) were significantly higher than those of its subnivean Periclistus congener (P<0.05).
Cuticular permeabilities for the Diplolepis species were extremely low and did not differ markedly among themselves, ranging between 0.33 and 0.54 µg h-1 cm-2 mmHg-1 (Table 2). However, the two Periclistus species had significantly higher cuticular permeabilities at 5 °C and 0 % RH (0.71 µg h-1 cm-2 mmHg-1 for P. pirata and 1.00 µg h-1 cm-2 mmHg-1 for Periclistus sp.) than the Diplolepis species (P<0.05). To distinguish cuticular versus respiratory contributions to water loss, we also determined the cuticular permeability of dead insects. Rates of water loss increased dramatically, by 1.5- to twofold, for dead individuals, suggesting that a substantial component of the resistance to water loss is under physiological/ventilatory control.
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To assess further the role of the cuticle in desiccation-resistance, the integuments of the prepupae were treated with water and three organic solvents (Table 3). Washing with acetone did not increase water loss rates significantly compared with control values (washed with water) for any species (P>0.05). However, washing with hexane increased water loss rates significantly for all but two test species, D. spinosa SK and Periclistus sp., while washing with the methanol/chloroform (1:2) mixture increased water loss rates significantly compared with control values for all species in the study (P<0.05).
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Cuticular permeability increased gradually as temperature increased (Table 4). Between 5 and 27°C, the Q10 values ranged from 1.87 for D. spinosa SK to 1.13 for D. gracilis. The critical transition temperature, marked by a dramatic increase in cuticular permeability, was estimated graphically to be 33.5°C for D. spinosa AB (Fig. 5). The same procedure was used to estimate transition temperatures for the remaining test groups, whose values ranged between 32.3 and 34.6°C (Table 4).
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To determine whether these species can absorb atmospheric water vapor, individuals from each species were desiccated until they had lost 5-10% of their original wet mass; they were then exposed to 95% RH at 15°C for 72h. Over this interval, no species showed a net gain in mass. In fact, there was a mean loss of 0.5-2% of their body mass, suggesting that these organisms did not absorb water vapor under these conditions.
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Discussion |
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The results obtained in the present study support the above scenario. All
species of Diplolepis and Periclistus examined were highly
cold-tolerant, with those species overwintering in supranivean galls having
supercooling points 6-9°C lower than those overwintering beneath the snow.
Since the supercooling point approximates the lower lethal temperature for
these species, the results suggest that supranivean species are more
cold-tolerant than subnivean ones. Furthermore, the values we measured closely
matched those of Rickards and Shorthouse
(1989) for D. spinosa
(-38°C) also collected near Sudbury, Ontario, and Sømme
(1964
) for D. radicum
(-33°C) collected near Lethbridge, Alberta. Although our acclimation
protocol exposed prepupae to somewhat milder conditions than they may
experience in the field, it was sufficient to induce high levels of
cold-tolerance.
The mean supercooling points for the cynipid species collected near Sudbury (P. pirata, Periclistus sp., D. radicum and D. polita) corresponded to the environmental temperatures recorded during the winter of 1999-2000. The daily minimum supranivean temperatures were below -30°C on four occasions, with the lowest recorded winter temperature being -36.4°C (Fig. 1B); however, even the lowest temperature was not below the mean supercooling point of the supranivean P. pirata (-39°C). In contrast, subnivean species supercooled to approximately -32°C, well below the minimum temperature recorded (-6.4°C) at ground level, which occurred prior to the formation of the snowpack (Fig. 1A).
Increases in the supercooling capacity of the supranivean species were
associated with increases in hemolymph osmolality. Theoretically, a 1000
mosmol kg-1 increase in solute concentration depresses the melting
point by 1.86°C and also decreases the supercooling point by 2-3 times
that of the melting point (Duman et al.,
1991). By this measure, the mean difference in hemolymph
osmolality between the supranivean and subnivean species accounted for
approximately 3°C of the 7°C difference in their mean supercooling
points (Figs 3,
4).
Glycerol was the major solute in the hemolymph of the two populations of
the supranivean D. spinosa, constituting 56 and 74% of their overall
osmolality (Fig. 4). However,
the remaining five species all had low levels of glycerol ranging from 4 to
23% of the overall osmolality. With such a large proportion of the hemolymph
solutes unaccounted for by glycerol, other cryoprotectants such as sorbitol,
mannitol or trehalose may constitute the remaining and major solutes for these
species (Lee, 1991).
Compared with literature reports for other insects, these cynipid wasps had
extremely low rates of water loss (Table
2). These rates are as low as those of the most xeric-adapted
species, including the heavily sclerotized adult desert beetle Onymacris
laeviceps, which Hadley
(1994) reported as having the
lowest known cuticular permeability. Such high resistance to desiccation for
these cynipid species suggests that they experience extreme desiccation stress
in winter. Consistent with this pattern is information for the stem-galling
tephritid Eurosta solidaginis, whose northern range overlaps with
that of the cynipid populations we studied
(Lee et al., 1995
) and which
is also highly resistant to water loss
(Ramløv and Lee, 2000
),
suggesting that insects in exposed supranivean hibernacula and in particular
those within gall chambers experience extreme water stress.
Even though there were differences in cold-tolerance between the
supranivean and subnivean species of cynipids, there were no differences in
rates of water loss among them (Table
2). The similarity of their well-developed resistance to water
loss implies that they experience similar desiccating conditions. Anatomical
studies have shown that, during the late summer and early autumn, tissues of
rose galls senesce and dry (Shorthouse,
1993; Brooks and Shorthouse,
1998
) and that, once the larvae cease feeding, thick-walled
sclerenchyma cells line the interior surface of all gall chambers. The
formation of this layer of sclerenchyma undoubtedly provides galls with
structural support and perhaps gives protection from parasitoids to the gall
former. More importantly, the sclerenchyma layer might also prevent the
absorption of water (Rickards and
Shorthouse, 1989
), which could benefit the prepupae by decreasing
the chances of fungal penetration and/or inoculative freezing. We suspect that
rose gall cynipids cannot obtain or do not come into contact with free water
within their galls until the adults exit 8-9 months later. Consequently, rose
gall cynipids may experience desiccation stress for much longer than the
winter months. The extremely low rates of water loss, which were similar to
mid-winter levels, for D. spinosa, D. gracilis and D.
radicum prior to cold-hardening (Fig.
2) support this conclusion.
Several parameters related to water conservation reflect the xeric
microhabitat and the extended period of dormancy of these wasps. At
temperatures below the transition temperature, Q10 values for
cuticular permeability were relatively low
(Table 4), ranging from 1.13
and 1.87, compared with an average for insects of approximately 2
(Hadley, 1994). These
Q10 values indicate that large diurnal and seasonal changes in
temperature would have little effect on rates of water loss. Similarly,
critical transition temperatures for all prepupae (32.3-34.6°C) were at
the low end of the range for terrestrial arthropods (40-60°C;
Hadley, 1994
), possibly
reflecting the relatively cooler conditions found in their northern climate.
These rose gall wasps also appear to conserve water by regulating ventilatory
losses. As reported for other insects
(Hadley, 1994
), rates of water
loss increased dramatically for dead individuals, indicating that active
physiological control of their spiracles was important for reducing
ventilatory losses (Hadley,
1994
). The removal of epicuticular lipids by organic solvents
illustrated the importance of the cuticle in water conservation for these
species. The average 11-fold increase in water permeability due to hexane
washing and 33-fold increase in permeability due to methanol:chloroform (1:2)
washing were similar to values reported for the larvae of E.
solidaginis (Ramløv and Lee,
2000
).
Lundheim and Zachariassen
(1993) concluded that the
lower vapor pressure deficit of the frozen larvae of Pytho depressus
and adults of the beetles Upis ceramboides beetles contributed to
their lower rates of water loss compared with that of supercooled individuals.
A reduction in the vapor pressure deficit between the insect's hemolymph and
the surrounding air elicited by an increase in hemolymph osmolality was one
factor that led Ring and Danks
(1994
) to suggest that
adaptations primarily associated with cold-hardiness, such as increased
hemolymph solute levels, might be even more important for water
conservation.
As discussed above, the species with the highest hemolymph osmolalities had
the greatest capacity to supercool and were therefore the most cold-tolerant.
However, the effect of elevated hemolymph osmolalities on water conservation
was less clear. Supranivean cynipids acclimated to mid-winter conditions had
the highest hemolymph osmolalities (Fig.
4), but their rates of water loss were not significantly lower
than those of subnivean species (Table
2). Edney (1977)
contends that even a large increase in osmolality would have a minimal effect
on reducing a large vapor pressure deficit and, thus, would have little effect
on water loss. The approximate range of hemolymph osmolalities for the wasps
in this study (977-1849 mosmol kg-1) would create a corresponding
range in vapor pressure deficits of only 6.42-6.33 mmHg (at 5°C and 0%
RH), a difference of 1.4%. Experimentally, differences in rates of water loss
due to such small decreases in vapor pressure deficit would be difficult to
demonstrate because of individual variability and because cuticular
waterproofing may differ among the species. However, compared with typical
hemolymph osmolalities for summer insects (300 mosmol kg-1;
Edney, 1977
), it is possible
that a three- to sixfold increase in hemolymph osmolality may have a
significant effect on water conservation over the varied conditions and
extended periods in which rose cynipids inhabit their galls. Furthermore, the
fact that supranivean species within a genus had higher levels of glycerol in
their hemolymph compared with subnivean species gives issue to the possibility
that the well-known hydroscopic properties of this polyhydric alcohol
(Crowe and Clegg, 1973
) are
important for water conservation.
We suggest that traits associated with water conservation may represent
pre-adaptations that facilitated the evolution of an increased capacity for
cold-tolerance. This, in turn, could have allowed for dispersal of these
species from southern to northern climates. The prepupal stages of D.
spinosa and D. radicum had relatively high glycerol
concentrations (Lee, 1991)
even prior to cold exposure (Fig.
2), suggesting that these solutes may be associated with water
retention. The presence of biochemical pathways for synthesizing and
accumulating glycerol and other polyhydric alcohols associated with water
retention in warmer climates may have been selected for because quantitative
increases in the production of these solutes would promote supercooling and
cold-hardiness. Increased hemolymph solute levels together with other
physiological and morphological factors that also promote supercooling and
coldhardiness, such as a relatively small body size compared with other
insects (Lee and Costanzo,
1998
), may have facilitated the radiation of these species
northwards.
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Acknowledgments |
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References |
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