Desiccation and rehydration elicit distinct heat shock protein transcript responses in flesh fly pupae
Ohio State University, Department of Entomology, 318 W. 12th Ave, Columbus, OH 43210, USA
* Author for correspondence (e-mail: hayward.23{at}osu.edu)
Accepted 15 December 2003
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Summary |
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Key words: diapause, insect, Sarcophaga crassipalpis, stress, water loss, heat shock protein, flesh fly, desiccation, rehydration
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Introduction |
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Among insects, dehydration is known to induce the expression of a novel
desiccation protein (dsp28) in the beetle Tenebrio molitor
(Graham et al., 1996) and to
increase the abundance of heat shock protein (Hsp) transcripts in several
species (Tammariello et al.,
1999
; Bayley et al.,
2001
). Hsps are well known for their role as molecular chaperones
and they function in the cellular stress response in organisms as diverse as
bacteria, yeast, plants and humans (Feder
and Hofmann, 1999
; Fink,
1999
). Based on their molecular mass, three size categories of
Hsps are well documented in insects: small Hsps (
2030 kDa), the
Hsp70 group (
70 kDa) and the 90 kDa group. Representatives of each group
have been cloned and sequenced from the flesh fly Sarcophaga
crassipalpis, and patterns of expression have been monitored in response
to a variety of environmental insults
(Yocum et al., 1998
;
Rinehart and Denlinger, 2000
;
Rinehart et al., 2000
).
Desiccation stress increases the abundance of hsp23 and
hsp70 transcripts in nondiapausing pupae of this species
(Tammariello et al., 1999
),
but little more is known about this response. What other Hsps are responsive
to dehydration in S. crassipalpis? At what level of water loss is Hsp
transcription initiated? Do transcription thresholds differ between Hsps or
between different rates of water loss?
The desiccation response during diapause is also of considerable interest
because pupae in diapause have no access to free water for long periods of
time (Yoder and Denlinger,
1991) and certain Hsps are developmentally upregulated throughout
this period (Yocum et al.,
1998
; Rinehart et al.,
2000
). Increased Hsp expression during diapause has also been
reported in several other invertebrates
(Liang and MacRae, 1999
;
Yocum, 2001
;
Denlinger, 2002
). In S.
crassipalpis, hsp23 and hsp70 transcripts are maximally
expressed during diapause, even in the absence of stress
(Yocum et al., 1998
;
Rinehart et al., 2000
).
Neither heat (45°C) nor cold (10°C) treatment further enhances
this response. Constitutively expressed Hsp90 is downregulated during diapause
yet remains responsive to both heat and cold stress
(Rinehart and Denlinger,
2000
). Another constitutive Hsp, heat shock cognate 70 (Hsc70), is
unchanged by diapause and seems more responsive to cold than heat stress
(Rinehart et al., 2000
). The
Hsp response to desiccation during diapause in S. crassipalpis has
not yet been examined. Hsc70 may be particularly interesting in this respect
as adaptations to cold and desiccation often have overlapping characteristics
(Ring and Danks, 1994
;
Block, 1996
).
In addition to the stress caused by desiccation, the rapid uptake of water
during rehydration has the potential to elicit cell damage. Consequently,
physiological and molecular responses to rehydration are arguably as important
as those to desiccation. In the moss Tortula ruralis, most of the
molecular repair mechanisms are thought to be initiated during rehydration
rather than desiccation (Oliver,
1991; Bewley and Oliver,
1992
). Rehydration-specific gene expression has also been reported
in higher plants, in conjunction with the decline of dehydration-specific gene
products (Bernacchia et al.,
1996
). Although Hsps have not been identified as key components of
the rehydration response in plants, their known chaperone function suggests a
potentially important role during the re-initiation of `normal' protein
synthesis upon removal of stress. Certain Hsps do indeed appear to function
during stress recovery (Van Nieuwenhoven
et al., 2001
), including recovery from cold shock in S.
crassipalpis (Rinehart et al.,
2000
). Thus far, molecular responses to rehydration in insects
have not been examined.
To determine how Hsps influence stress tolerance at the organismal level, it is useful to characterize the responses of different Hsps across stress gradients. Desiccation studies are particularly instructive in this respect, as they provide the opportunity to quantify both the level of stress imposed (e.g. relative humidity) and the level of stress experienced (e.g. the amount and/or rate of water loss), which is not possible with thermal stress. Desiccation stress was utilized in this study to identify differences in the Hsp response of nondiapausing and diapausing pupae of the flesh fly S. crassipalpis, as well as changes in Hsp transcription thresholds in relation to different rates of water loss. We report distinct differences in the Hsp response to desiccation and rehydration in this species that suggest different functions for the constitutive and inducible groups of Hsps.
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Materials and methods |
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Desiccation treatments
Three days after pupariation, nondiapausing pupae were transferred to
constant 75% relative humidity (RH) conditions, maintained using saturated
NaCl solutions, at 25°C for 24 h to synchronise their hydrated state prior
to desiccation. The desiccation assessment was conducted with both intact day
4 nondiapausing pupae and samples from which the operculum of the puparium
(anterior cap) had been removed. Samples were placed inside 2 ml centrifuge
tubes (N=5 per tube) perforated with 20x1 mm holes, which in
turn were placed in 15 ml containers filled with anhydrous calcium sulphate
(Drierite®) and maintained at <5% RH/25°C. Samples were removed at
24 h intervals over a 7 day period. The following characteristics were
assessed:
Survival and emergence time
Only individuals completely extricated from the puparium were considered
survivors. Emergence time was recorded as the number of days between
pupariation and emergence. Eclosion patterns within the long-day L:D cycle
were also noted. Emergence was monitored at 25°C by placing pupae within
an eclosion counting device (Yocum et al.,
1994) set to record eclosion events every 2 h. RH conditions
during this period were 59.5±1.4% RH (mean ±
S.E.M.; N=20).
Water loss
Five pupae were selected for monitoring daily water loss. Individuals were
weighed prior to desiccation (fresh mass) and upon removal from the
Drierite® (desiccated mass) at each 24 h interval. Samples were then dried
to constant mass at 50°C and their dry mass was noted. From these values,
mean initial water content and percentage water loss were calculated. Four
replicates were conducted; thus, for each value, N=20. Linear
regression analysis determined the relationship between percentage water loss
(arcsine transformed) and time at <5% RH/25°C. Differences between
regression coefficients were calculated following Fowler and Cohen
(1990).
hsp expression
Total RNA was extracted by homogenizing samples in TRIzol® reagent
using standard protocol. Only live animals, i.e. those showing no tissue
discoloration, were used. RNA from three animals was pooled for each sample,
and hsp expression was assessed using northern blot hybridization.
Northern blot hybridization
Total RNA from each sample (20 µg) was loaded on a 1.5% agarose, 0.41
mol l1 formaldehyde gel for electrophoresis. Samples were
transferred to a positively charged nylon membrane (Roche Diagnostics GmbH,
Mannheim, Germany) by downward capillary action using alkaline transfer buffer
(Schleicher and Schuell, Inc., Keene, NH, USA). Northern blot hybridization
was performed following Sambrook et al.
(1989), using the following
S. crassipalpis clones as templates to make DNA probes: Hsp23
(GenBank accession no. U96099; Yocum et
al., 1998
), Hsp70 and Hsc70 (GenBank accession no. AF107338 and
AF107339, respectively; Rinehart et al.,
2000
) and Hsp90 (GenBank accession no. AF261773;
Rinehart and Denlinger, 2000
).
28s ribosomal RNA was used as a control gene. Each probe was labeled with
digoxigenin-11dUTP using DIG High Prime (Roche Molecular Biochemicals,
Mannheim, Germany). Hybridization, washing and detection were undertaken
following the DIG High Prime labeling and detection standard protocol.
Membranes were exposed to X-ray film (Fuji) for 2530 min at room
temperature.
Diapause samples
Day 10 diapausing pupae were transferred to 25°C (75% RH) and allowed
to acclimatize for 5 days (day 15 of diapause). The operculum of the puparium
was removed from all day 15 diapause samples, which were then desiccated at
<5% RH/25°C as described for nondiapausing pupae. Samples were removed
at 3 day intervals over a 15 day period to assess survival, water loss and hsp
expression as previously described. This procedure was repeated with day 30
diapausing pupae. Thus, day 15 diapause samples were desiccated from day 15 to
day 30 of diapause, and day 30 diapause samples were desiccated from day 30 to
day 45 of diapause.
Diapause termination under desiccation stress
hsp transcript expression was also monitored following diapause
termination, elicited by hexane application
(Denlinger et al., 1980),
while individuals were under desiccation stress. Day 15 diapausing pupae (with
operculum removed) were placed under <5% RH/25°C conditions, and after
9 days of desiccation the first RNA sample was collected. The remaining pupae
were treated with hexane to terminate diapause under desiccating conditions by
applying 5 µl of hexane to the head of each pupa. RNA samples were
collected 3, 6, 9, 12 and 24 h post diapause termination, and hsp expression
was noted as previously described.
Rehydration experiments
Day 4 nondiapausing pupae (operculum removed) were desiccated for 4 days at
<5% RH/25°C and then transferred to either 75% or 100% RH conditions.
After 24 h and 48 h of rehydration, total RNA was extracted and hsp expression
assessed. Ten pupae were randomly selected to represent each condition and
their mass monitored as previously described to determine initial water
content, water loss and water gain through rehydration. This procedure was
repeated with day 15 diapausing pupae (operculum removed).
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Results |
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The mean mass of diapausing pupae at the outset of the experiment was 110.8±1.7 mg (mean ± S.E.M.; N=120), with a mean water content of 67.0±0.2% (mean ± S.E.M.; N=120). Water loss increased throughout the 15 day desiccation period for diapausing pupae of both ages (Fig. 1B). The relationship between water loss and duration of desiccation was significant for both diapause groups (Table 1) but did not differ significantly between day 15 and day 30 samples (t=0.18, d.f.=10). Survival of both day 15 and day 30 diapausing pupae declined at a similar rate as a result of desiccation (Fig. 1B).
hsp transcript expression
In nondiapausing flies, transcripts of hsp23 and hsp70
became abundant in response to desiccation after 2 days at <5% RH
(Fig. 2A) in pupae encased in
an opened puparium. Transcript abundance remained elevated throughout the
desiccation treatment at a level equivalent to that resulting from heat shock.
The upregulation of hsp23 and hsp70 was not noted in pupae encased in an
intact puparium (Fig.
2B).Expression of hsc70 and hsp90 transcripts
was unaltered by this level of desiccation stress in pupae encased in either
open or intact puparia (Fig.
2).
|
Transcript levels of all Hsps investigated remained unresponsive to
desiccation stress in day 15 diapausing pupae
(Fig. 3A). In day 30 diapause
samples, the expression of hsp70 and hsp23 transcripts
remained high until day 6 of desiccation, disappeared on day 9 and day 12 but
reappeared on day 15 at <5% RH (Fig.
3B). hsp90 transcripts were upregulated on day 12 and day
15 of desiccation in these day 30 pupae, while levels of hsc70
transcripts remained constant. The conspicuous drop in expression of hsp23 and
hsp70 after day 6 of desiccation in day 30 diapause samples coincided with
diapause termination in these pupae. The upregulation of hsp90 on day 12 and
day 15, i.e. day 42 and day 45 pupae, is also consistent with diapause
termination (Rinehart and Denlinger,
2000). The renewed expression of hsp23 and hsp70 on day 15 of
desiccation suggests that, once development has been reinitiated, the fly
again responds to desiccation stress by expressing these Hsps.
|
Diapause termination under desiccation stress
The association noted above between the onset of development and the
expression of Hsps under desiccation stress prompted a second approach to
testing our hypothesis that Hsp23 and Hsp70 must be turned off to initiate
development, even under stressful conditions. This was achieved by using
hexane, as described by Denlinger et al.
(1980), which promptly
terminates diapause without causing mortality. In response to hexane,
diapausing pupae break diapause immediately; within 12 h (20°C), the
transcript abundance of Hsps that have been upregulated during diapause (Hsp23
and Hsp70) is undetectable (Yocum et al.,
1998
; Rinehart et al.,
2000
), and Hsp90, an Hsp that is downregulated during diapause, is
upregulated. When this hexane tool was applied to terminate diapause under
desiccation stress, the abundance of both hsp23 and hsp70
transcripts declined within 3 h of hexane application
(Fig. 4). After 6 h,
hsp23 and hsp70 transcripts were highly expressed again, but
after 9 h were greatly diminished (hsp23) or were undetectable
(hsp70). Consistent with earlier observations, hsp90
transcript abundance increased 6 h after hexane application. These results
thus imply that in order to initiate adult development both Hsp23 and Hsp70
must be downregulated, even under severe desiccation stress.
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Water content and survival following rehydration
As anticipated from an earlier study on S. crassipalpis
(Yoder and Denlinger, 1991),
pupae were capable of absorbing atmospheric water vapor. One day of
rehydration at either constant 75% or 100% RH returned the water content of
nondiapausing pupae to values recorded prior to desiccation and, in the case
of diapausing pupae, the rehydrated water content actually exceeded the
initial predesiccated level (Fig.
5). Survival of both nondiapausing and diapausing pupae increased
as a result of rehydration and reached levels similar to the survival noted in
nondesiccated controls (Fig.
5).
|
hsp transcript expression in response to rehydration
hsp23 and hsp70 transcripts were undetectable during
rehydration in nondiapausing pupae until 2 days at 100% RH
(Fig. 6A). In diapausing pupae,
both hsp23 and hsp70 continued to be expressed during rehydration
(Fig. 6B). In other words, the
expression patterns were the same as in the nondesiccated controls reared
under these two different developmental programs. By contrast, hsp90 and hsc70
were both upregulated by rehydration in both nondiapausing
(Fig. 6A) and diapausing
(Fig. 6B) pupae.
|
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Discussion |
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While many of the Hsps are simultaneously upregulated in response to heat
(Lindquist and Craig, 1988),
other forms of stress may selectively induce different Hsps. For example, in
S. crassipalpis, constitutively expressed Hsp90 is further
upregulated in response to heat shock (40°C) but does not respond directly
to cold treatment (10°C;
Rinehart and Denlinger, 2000
).
By comparison, Hsc70, which is also constitutively expressed, is not
immediately upregulated by either cold or heat treatment in S.
crassipalpis (Rinehart et al.,
2000
). However, both of these constitutive Hsps are upregulated
during the recovery from cold shock, demonstrating considerable similarity to
their response to rehydration/desiccation recovery identified in the present
study. This result indicates another common link between adaptations to cold
and desiccation stress.
Desiccation and rehydration create contrasting cellular environments, which
could help explain their different Hsp responses. To compensate for the
deficit in hydrogen bonding with water caused by cellular dehydration,
hydrogen bonding with other molecules can occur, which may lead to protein
aggregation and/or denaturation
(Pestrelski et al., 1993).
Water loss from membrane phospholipids also leads to phase transitions from
the biologically active fluid phase to the gel phase
(Crowe et al., 1992
), which in
turn influences transmembrane ion and protein activity. A principal role for
both small Hsps and Hsp70 is to bind to denatured proteins and inhibit
aggregation (Fink, 1999
).
Small Hsps are also thought to function in stabilizing the liquid crystalline
state of membranes (Tsvetkova et al.,
2002
), and membrane lipid interactions with Hsp70 have been
identified (Arispe et al.,
2002
). By contrast, rehydration will increase cellular turgor
pressure and re-establish membrane fluidity, as well as reinitiate productive
protein folding pathways, in which both Hsc70
(Leung and Hightower, 1997
)
and Hsp90 (Young et al., 2001
)
play a fundamental role. Despite rehydrated samples returning to their
pre-desiccated water content within a 24 h period
(Fig. 5), the reversal of
membrane disruption and the reestablishment of protein folding often takes
considerably longer (Bernacchia et al.,
1996
). This could explain the prolonged expression of Hsp90 and
Hsc70 during the rehydration phase.
Clearly, Hsps are not the only players likely to be involved in
desiccation/rehydration responses of insects. A desiccation-specific protein
has been reported from the mealworm Tenebrio molitor (Kroeker and
Walker,
1991a,b
).
Although its identity remains unknown, it does not appear to be an Hsp. Plants
also possess different classes of desiccation and rehydration responsive
proteins, termed dehydrins and rehydrins, respectively, along with their seed
counterparts, the late-embryogenesis-abundant (LEA) proteins
(Bohnert, 2000
). While these
proteins have functional similarity to certain Hsps
(Mtwisha et al., 1998
), they
are distinct and give an indication of diverse molecular responses to
desiccation/rehydration stress in terrestrial organisms.
Polyols, sugars and other such cryoprotectants are also likely to
contribute to the insect's response to desiccation
(Bayley and Holmstrup 1999).
This appears to be true for S. crassipalpis: glycerol synthesis
increases in response to desiccation in nondiapausing pupae (S.A.L.H.,
unpublished observations). Interactions between glycerol/trehalose and Hsps
have been well documented in systems as diverse as human cell cultures
(Brown et al., 1996
) and the
brine shrimp Artemia franciscana
(Viner and Clegg, 2001
).
Interactions between membrane lipids and Hsps are also well documented
(Tsvetkova et al., 2002
;
Arispe et al., 2002
), thus
underscoring the likelihood that Hsps are unlikely to be responding to
desiccation/rehydration stress in isolation from other cell processes.
An interaction between Hsps and other physiological stress mechanisms could
help explain the difference in Hsp transcription thresholds between
desiccated, intact, nondiapausing pupae and nondiapausing samples desiccated
with the operculum of their puparium removed. After 6 days at <5%
RH/25°C, intact, nondiapausing pupae lose more than 10% of their initial
water content (Fig. 1A). This
is beyond the water loss threshold that initiates hsp transcription
in nondiapause samples with an open puparium, yet neither Hsp23 nor Hsp70 is
upregulated in the intact group (Fig.
2B). Thus, the threshold of Hsp expression increases in response
to a reduced rate of desiccation. The rate of water loss is known to influence
cryoprotectant synthesis (Bayley and
Holmstrup, 1999) and cell membrane adaptation
(Holmstrup et al., 2002
) in
some terrestrial arthropods, but how this may influence Hsp expression has not
been investigated. Biochemical analysis of cryoprotectant synthesis and
membrane lipid composition during dehydration in insects, and its influence on
Hsp expression, therefore seems an appropriate avenue for future research.
Our experiments with S. crassipalpis also revealed major
differences in responses to desiccation stress between diapausing and
nondiapausing pupae and underscore the role of the puparium in offering
protection against dehydration. Under natural conditions, these flies enter
diapause in the early autumn and the adult flies do not emerge from the
puparium until the following spring
(Denlinger, 1972). During
these many months, the fly does not have access to free water, and the
maintenance of water balance emerges as a critical issue
(Yoder and Denlinger, 1991
).
The addition of an extra layer of hydrocarbons on the surface of the puparium
provides an important barrier to water loss
(Yoder et al., 1992
), and from
the present study we can see the impact of breaching that barrier by removing
the operculum and thus exposing the pupal body to the atmosphere. The rate of
water loss was dramatically increased in these opened puparia, yet the impact
of opening the puparium differs between diapausing and nondiapausing pupae.
Water loss rates remained far lower in diapausing pupae with an opened
puparium than in their nondiapausing counterparts, presumably due to the
suppressed metabolism (Denlinger et al.,
1972
) and elevated glycerol content
(Lee et al., 1987
) inherent in
the diapause program.
Differences in the Hsp response to desiccation and rehydration are also
evident between these two types of pupae. While the abundance of
hsp23 and hsp70 transcripts increased in response to the
desiccation of nondiapausing pupae (operculum removed), both transcripts were
unresponsive to desiccation in diapausing pupae. This is probably because both
hsp23 and hsp70 are already highly expressed in diapausing pupae by virtue of
their being in diapause (Denlinger et al.,
2001). The developmental upregulation of these Hsps during
diapause possibly represents a prophylactic response for diapausing pupae
against desiccation, temperature extremes and other stresses to which the
overwintering pupa may be exposed
(Denlinger et al., 2001
). The
unresponsiveness of hsp23 and hsp70 during diapause is further highlighted by
their sustained expression during the rehydration of desiccated diapausing
pupae (Fig. 6B). This contrasts
with rehydrated nondiapausing pupae, in which both hsp23 and
hsp70 transcripts decline (Fig.
6A).
Desiccation also elicited another, somewhat unexpected, response on
development time. Three or more days at <5% RH extended the interval
between pupariation and adult eclosion by several days in nondiapausing flies
(Fig. 1A). This duration of
stress was sufficient to elicit expression of hsp23 and hsp70, and we thus
anticipate that this upregulation of Hsps could contribute to the delay in
adult eclosion, based on previous work indicating that the expression of Hsps
is not compatible with the progression of development
(Feder and Hofmann, 1999).
Indeed, one of the first events associated with pupal diapause termination and
the initiation of adult development in flesh flies is downregulation of the
Hsps (Yocum et al., 1998
;
Rinehart et al., 2000
). A
similar response was noted in the present study when older diapausing pupae
started to break diapause during desiccation. In such pupae, Hsp70 and Hsp23
synthesis was interrupted during diapause termination despite samples being
under desiccation stress. A couple of days later, after development had been
initiated, the flies again expressed the hsp transcripts if they
remained in a desiccating environment (Fig.
3). This association was tested further using hexane as a tool to
terminate diapause under desiccating conditions. The fact that desiccated
diapausing pupae that were stimulated to break diapause with hexane ceased to
express Hsps (Fig. 4), even
though they continued to be exposed to desiccation stress, is consistent with
the idea that synthesis of stress-induced Hsps must stop before development
can ensue.
In summary, this paper demonstrates that the expression of some, but not all, Hsps is elicited by desiccation, and a different set of Hsps responds to rehydration. Pupae in diapause already express the Hsps elicited by desiccation and no further upregulation is noted. Several lines of evidence also suggest a causal relationship between the desiccation-stimulated upregulation of Hsps and the delay in development observed in such pupae.
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Acknowledgments |
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