Changes in gut and Malpighian tubule transport during seasonal acclimatization and freezing in the gall fly Eurosta solidaginis
Department of Zoology, Miami University, Oxford, Ohio 45056, USA
Author for correspondence (e-mail: leere{at}muohio.edu)
Accepted 14 March 2005
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Summary |
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Key words: transport, cholesterol, epithelial membranes, freezing tolerance, cold tolerance, Malpighian tubule, gall fly, Eurosta solidaginis
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Introduction |
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The goldenrod gall fly Eurosta solidaginis is a naturally
freeze-tolerant insect that survives both intra- (as in the case of fat body
cells) and extra-cellular (Salt,
1959; Lee et al.,
1993
; Bennett and Lee,
1997
) freezing. During autumn, third instar larvae increase their
cold-hardiness and become tolerant of extensive internal ice formation, partly
owing to the accumulation of the cryoprotectants glycerol, sorbitol and
trehalose (Baust and Lee, 1981
;
Storey et al., 1981
;
Lee and Hankison, 2003
).
Concurrently, the larvae acquire extreme resistant to desiccation through the
deposition of large amounts of cuticular hydrocarbons and by metabolic
depression associated with entry into diapause
(Ramløv and Lee, 2000
;
Nelson and Lee, 2004
).
Since Malpighian tubules (MT) and the gut constitute the primary system for
ionoregulation, osmoregulation and excretion in insects
(O'Donnell and Spring, 2000),
these organs may be especially valuable models for studying the effects of
freezing injury and cold-hardening. When stimulated, some MT can transport
water and ions at rates higher than those of any other known tissue, resulting
in them being called `insect kidney tubules'
(Meulemans and De Loof, 1992
).
The high rate of fluid secretion depends crucially on the activity of a
V-ATPase located on the apical cell membrane
(Maddrell and O'Donnell,
1992
). Along with a large amount of fluid passing through the
cells, many small molecules, such as amino acids, sugars and ions also enter
the lumen, but they may be reabsorbed during their passage through the tubules
and the rectum (Maddrell and Gardiner,
1974
; Bradley,
1985
).
Most dipteran MT contain four tubules, two oriented anteriorly (MTA) and
two oriented posteriorly (MTP) in the abdomen
(Meulemans and De Loof, 1992;
Mugnano et al., 1996
). Each
pair of tubules forms a common ureter that opens at the junction between the
midgut and hindgut (Waterhouse,
1950
). In E. solidaginis larvae, the anterior pair of
tubules has two morphologically distinct parts, proximal and distal. The
distal region contains numerous clear spherules of
while the proximal region lacks crystals, and is yellow-green in color
(Mugnano et al., 1996
;
Yi and Lee, 2003
). The calcium
phosphate spherules have an ice-nucleating function that promotes freeze
tolerance by limiting the capacity of larvae to supercool
(Mugnano et al., 1996
).
However, little is known about the epithelial function of the MT in relation
to the seasonal acquisition of freeze tolerance and entry into diapause.
Cholesterol is an important component of biological membranes and the
precursor for the biosynthesis of steroid hormones in insects and other
animals (Waterman, 1995). This
molecule is believed to have dual roles in preserving membrane fluidity, which
is essential for cell survival and function, in response to changes in
environmental temperature: at high temperature cholesterol functions to make
membranes less fluid, while at low temperature it serves to maintain membrane
fluidity by preventing the acyl chains of lipids from binding to each other
and rigidifying the membrane (Crockett,
1998
). Although mechanisms involving cholesterol in the modulation
of membrane structure and function have been proposed
(Yeagle, 1991
;
Crockett, 1998
), little is
known about their role in insects.
Our primary objective was to characterize functional responses of the gut and MT to seasonal acclimatization and freezing tolerance in overwintering larvae of the goldenrod gall fly. Since little research has been done with these organs in E. solidaginis larvae, we began this project by characterizing the basic structure and transport functions of these organs.
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Materials and methods |
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Supercooling point
The supercooling point (SCP) was identified as the lowest body temperature
recorded immediately prior to the spontaneous release of the latent heat of
fusion as water froze within the insect
(Lee, 1991). The SCP was
measured by positioning a thermocouple on the larval surface within a 1.5 ml
plastic tube placed in a glass test tube (1.6 cmx15.0 cm) suspended in a
refrigerated bath (Neslab, model RTE-140, Portsmouth, NH, USA). Temperatures
were recorded on a chart recorder connected to a multichannel thermocouple
recorder (Omega, model RD3752, Stamford, CT, USA). Larvae (N=10) were
cooled from
22° to -20°C at a rate of 1°C
min-1.
Assessing freeze tolerance
Three groups of 30 larvae from each collection were placed individually in
a 0.5 ml plastic tube and all tubes were kept in a large plastic container.
Because larvae greatly increased their tolerance during the course of the
study it was necessary to increase the severity of the freezing treatment to
induce freezing injury and assess cold tolerance. Consequently, after field
collection larvae were frozen at either -20°C or -80°C for various
periods as follows: 48 h for larvae collected in September, 10 days for
October, 20 and 40 days for November and 2 months for December. Larvae were
checked for survival every 30 min for 5 h under a dissecting microscope after
they were removed from the freezers and thawed at 22°C. Movement in
response to tactile stimuli was used to identify viable larvae.
Tissue dissection and measurement
The larval gut and MT were dissected in Coast's solution
(Coast, 1988). Larvae were
pinned dorsal-side uppermost in a silicone elastomer-filled (Sylgard 184, Dow
Corning, Midland, MI, USA) Petri dish. A midline incision allowed removal of
the fat body to expose the gut and MT. The entire gut (foregut, midgut and
hindgut) and the attached MT were removed from the body. The length of each
tissue was measured, and the cells and crystals in the MT were counted under a
dissecting microscope. For fluid secretion assays, the MT was dissected free
from the gut and quickly transferred to a fluid secretion assay system
(Xu and Marshall, 2000
). For
experiments concerning transmembrane transport of ions, the gut-MT complex was
transferred into 1.0 ml of 0.5 mmol l-1 Chlorophenol Red-Coast's
solution in a tissue culture dish (35 mmx10 mm, Corning, NY, USA).
Fluid secretion by MT
In vitro fluid secretion by the MT was assayed by a procedure
modified from those of Ramsay
(1952), Spring and Hazelton
(1987
) and Xu and Marshall
(2000
) in a Coast's solution
saturated with oxygen by bubbling air through it at room temperature. An
isolated tubule was placed in 0.5 ml Coast's solution that was covered with
mineral oil in a Petri dish. The open (ureter) end of the MT was pulled out
into the oil and wrapped around a pin. Secreted fluid formed a droplet in the
oil. The droplet was then removed from the tubule every 30 min with a fine
hair and its diameter was measured with an ocular graticule
(Spring and Hazelton, 1987
).
The volume was calculated based on the assumption that it was spherical
(Xu and Marshall, 2000
). For
each tubule, the rate of secretion, expressed as nl min-1, was
determined by the cumulative volume secreted during a period of 30 min to 2
h.
Transepithelial transport
Chlorophenol Red is a pH sensitive dye that can be seen accumulating in the
MT lumen. The dye is carried in the water that follows the active transport of
potassium (Yurkiewicz, 1983;
Pritchard and Miller, 1993
).
To test the epithelial function of both the gut and MT, the gut-MT complex
from November-collected larvae was isolated and incubated in 1.0 ml of 0.5
mmol l-1 Chlorophenol Red-Coast's solution in a tissue culture
dish. To examine the effect of temperature on active transport, tissues were
dissected immediately from newly collected larvae and incubated under various
temperatures or from larvae held frozen at -20°C for 40 days and then
thawed at 22°C. The effects of metabolic (0.01 mol l-1 KCN) and
membrane (0.01 mol l-1 ouabain) inhibitors on transport were also
tested. Preliminary measurements indicated that pH varied by 0.2 units or less
among the different tissues, which did not cause color changes of the
Chlorophenol Red, and thus did not interfere with the dye transport assay.
Membrane preparation
MT dissected from control and freeze-treated groups were either immediately
homogenized in an ice-cold homogenization buffer containing 250 mmol
l-1 sucrose and 5 mmol l-1 imidazole, pH 7.4
(Al-Fifi et al., 1998) or
stored in 100 µl of the same buffer containing 10% DMSO (dimethyl
sulfoxide) at -80°C overnight. Membrane preparations were conducted with a
modified procedure based on the methods described by Al-Fifi et al.
(1998
), Crockett and Hazel
(1995), Fogg et al. (1991
) and
Sørensen (1981
,
1993
). Briefly, tissues were
first homogenized in a glass homogenizer with the homogenization buffer (1 mg
tissue in 10 µl buffer), and then processed with an Ultrasonic Processor
(Cole-Parmer Instrument Co., Vernon Hill, IL, USA) for 10 s, three times, with
an Amplitude setting at 40. All processes were carried out on ice. The
homogenate was centrifuged at 600 g, 4°C in an Eppendorf
centrifuge for 10 min to remove nuclei. The supernatant was then centrifuged
at 135,000 g in a Beckman L5-50 B Ultracentrifuge for 20 min
at 4°C to yield a supernatant and a membrane fraction. The membrane
fraction was suspended by homogenization in an appropriate volume of
homogenization buffer containing 10 mmol l-1 MgCl2 and
0.1 mol l-1 choline chloride for cholesterol and protein
assays.
Biochemical assays for cholesterol and proteins
A specific enzymatic cholesterol assay system provided by DCL (Diagnostic
Chemicals Limited, Oxford, CT, USA) was used in hemolymph and the membrane
preparations of MT, using a single reagent and a standard cholesterol
calibrator (Sigma, St Louis, MO, USA). The assay includes coupled enzymatic
reactions in which hydrogen peroxide (H2O2) is produced
from the sequential action of cholesterol esterase and cholesterol oxidase on
cholesterol and its esters. The H2O2 oxidatively couples
with 4-aminoantipyrine and p-hydroxybenzoate in the presence of
peroxidase to produce the chromogen, quinoneimine, measured at 505 nm. We
reduced the volumes of both sample and reagent to suit our purpose, i.e. in
separate test tubes, 10 µl buffer (as blank), calibrator (as standard) or
unknown samples were mixed respectively with 1.0 ml of reagent in a 1.5 ml
semi-micro cuvette. After incubation at room temperature for 20 min,
absorbance of the mixture (OD value) was measured at 505 nm using the reagent
blank as a background. Cholesterol content was calculated by division of the
OD value of an unknown sample by the OD value of the calibrator, and then
multiplying by a factor of 4.6 (concentration of the calibrator in mg/100
ml).
Protein contents in the MT membrane preparations were determined by a
Bio-Rad standard procedure (Bradford,
1976) with a reduced volume (1.02 ml), using BSA as the
standard.
Hemolymph osmolality
Hemolymph was collected individually from 10 larvae with a 10 µl glass
capillary micropipette (Drummond Scientific Co., Broomall, PA, USA). Osmotic
concentration of the hemolymph was determined with a Wescor 5500 vapor
pressure osmometer (Wescor, Logan, UT, USA).
Statistical analysis
SigmaPlot was used to conduct t-tests and ANOVA post hoc tests
used Statview from SAS Institution. A value of P<0.05 between
groups was considered as a significant difference. Values are reported as mean
± S.E.M., N=10.
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Results |
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Through the autumn, hemolymph osmolality and cold-hardiness increased
steadily (Fig. 2). The
hemolymph osmolality, which was used as a measure of cryoprotectant
accumulation (Baust and Lee,
1981), increased twofold from 455±16 mOsmol l-1
in September to 926±27 mOsmol l-1 in December
(Fig. 2A). During this period
the larvae also acquired a high level of freezing tolerance. In September, no
larvae survived freezing for as little as 48 h at -20°C
(Fig. 2B). In contrast, by
October 80% and 33% survived following 10 days of freezing at -20 and
-80°C, respectively, and by Nov. 13 all larvae survived freezing for 20
days at -20°C and 83% survived at -80°C.
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Freezing for extended periods reduced survival rates and increased the recovery time upon thawing. For example, the survival rate for November-collected larvae frozen for 40 days was lower by 43% at -20°C and 56% at -80°C compared to that in the 20-day-frozen group (Fig. 2B). Depending on the given conditions, larvae that were frozen for longer periods required more time to recover at 22°C (Fig. 2C); November-collected larvae needed 2.5 h to reach a 100% rate of survival after freezing at -80°C for 20 days, while the December-collected larvae required 5 h before reaching a 90% rate of survival after being frozen for 2 months.
Transport in the gut and MT
Since transport processes in the gut and MT of E. solidaginis
larvae have not previously been investigated, we began by characterizing their
basic anatomical and physiological characteristics (summarized in
Fig. 3) as a foundation for the
studies reported later in this section. Within the gut, the midgut was the
longest region (15.4±0.5 mm) compared to the foregut (6.0±0.5
mm) and hindgut (5.4±0.3 mm). In the MT, the length of the ureter was
1.2±0.1 mm and contained 49.7±2.0 epithelial cells. The distal
crystal-containing region of the anterior pair of MT was 8.2±0.9 mm in
length and contained 19.5±1.2 calcium phosphate spherules (see
Mugnano et al., 1996). The
proximal region of the anterior pair of tubules was 7.8±0.3 mm in
length and contained 185±11 cells. The posterior pair of tubules was
7.6±0.4 mm long and contained 188±12 cells.
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To investigate basic transport properties of the gut and MT we used
Chlorophenol Red, a pH-dependent colored substrate commonly used to study
organic anion transport systems (Pritchard
and Miller, 1993). The amount of dye being transported from the
bathing solution into the lumen can be estimated visually. This dye is a rosy
red at a pH (6.4) and turns yellow at pH 4.8. We first examined in
vitro transmembrane transport of Chlorophenol Red by the isolated gut and
MT in November-collected larvae (Table
1). Chlorophenol Red was readily transported into the lumen of
foregut, the posterior portion of the midgut, ureter, the proximal region of
the anterior MT, and the posterior MT, but not the anterior portion of the
midgut, the entire hindgut and the crystal-containing distal region of the
anterior MT (Fig. 3;
Table 1). When either 0.01 mol
l-1 of KCN or ouabain was present in the solution, the expected
inhibitory effect on transport was observed; this effect was especially
distinct in the MT compared to the control values
(Table 1). Although 0.01 mol
l-1 solutions of both inhibitors were used, KCN showed a greater
inhibitory effect on the ion transport by MT than did ouabain.
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Effect of temperature on transport in the gut and MT
Incubation temperature had a significant effect on the rate of dye
transport for in vitro preparations of the gut and MT
(Table 2). At 22°C, the dye
first appeared in the lumen of transporting regions of the gut and MT within 5
min and reached the highest level of coloration after 10 min of incubation.
However, at a lower temperature of 10°C, the dye was not secreted into the
lumen until 10 min after incubation began, and 30 min were required to reach
transport levels as high as that of the 10 min-incubation at 22°C, except
in the foregut, which did not reach the highest level even after 30 min. At
the lowest incubation temperature of 0°C, the rate of ion transport by
each tissue slowed still more, however, maximal transport levels were still
reached within 60 min in most tissues.
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Seasonal decreases in transport function in MT
The rate of fluid secretion by the MT decreased markedly from September to
December (Fig. 4). In
September-collected larvae, isolated MT from both the anterior (MTA) and
posterior pairs (MTP) secreted fluid at the highest rates (10.7±0.8 nl
min-1 for the MTA; 12.7±1.0 nl min-1 for the MTP)
when compared to the other collection times. During October and November, the
rates declined sharply, and continued to decrease until secretion could no
longer be detected in December-collected larvae
(Fig. 4A). The rate of
secretion from posterior tubules was greater than from the anterior pair in
both October (P<0.01) and November (P<0.05), although
no significant difference (P>0.05) was observed in September. For
each monthly collection, the amount of fluid secreted by the MTA and MTP was
linear for incubation periods between 30 and 120 min
(Fig. 4B). Over a period of 120
min, each MTA tubule secreted 1.26±0.07 µl and each MTP tubule
secreted 1.52±0.04 µl of fluid in September. In contrast, only
0.14±0.03 µl and 0.20±0.03 µl were secreted by the MTA and
MTP, respectively in November over the same period.
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Effect of freezing on transport in the gut and MT
The influence of freezing and larval survival on transport by the gut and
MT was examined using larvae collected on Nov. 13 and held frozen for 40 days
at -20°C (Table 3). Larvae
were judged to have survived if they responded to touch after 5 h at 22°C.
The gut and MT were then excised and dye transport measured as described
previously. Overall, tissues from surviving larvae retained better ion
transport capacity than from ones scored as dead
(Table 3). However, significant
levels of transport were evident in tissues removed from dead larvae. Compared
to rates of dye transport from unfrozen larvae (see
Table 2), freezing of the
larvae decreased the rate of ion transport by the gut and MT epithelia. For
example, in the posterior midgut of unfrozen larvae maximal transport rates
were achieved within 10 min (Table
2), while 30 min were required to reach this level in previously
frozen larvae (Table 3).
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Cholesterol and cold tolerance
Since cholesterol has been implicated in processes related to seasonal
cold-hardening, we monitored larvae for seasonal changes in cholesterol levels
in the hemolymph and the MT membranes. Through the autumn, cholesterol
concentrations in the hemolymph increased nearly fourfold
(Fig. 5A). From September to
October, hemolymph cholesterol level doubled, and doubled again from October
to November. In contrast to the hemolymph, the ratio of cholesterol to protein
content (nmol mg-1) in the MT membrane of untreated (control)
larvae remained relatively constant (2224 nmol mg-1 protein)
during the entire period (Fig.
5B).
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Larval freezing caused a significant decrease in cholesterol levels in the hemolymph and the MT membranes. November-collected larvae that had been frozen for 20 days at either -20 or -80°C had significantly lower levels of cholesterol in their hemolymph compared to unfrozen control larvae (Fig. 5A, insert). Freezing of larvae at either subzero temperature decreased cholesterol concentrations to a level equivalent to that found in October-collected larvae (Fig. 5A). Similarly, freezing at -20 or -80°C generally lowered membrane cholesterol content in MT compared to that of their respective unfrozen controls (Fig. 5B). In all cases, -20°C decreased membrane cholesterol levels significantly more than did treatment at -80°C.
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Discussion |
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The seasonal acquisition of increased cold tolerance was evidenced by
increases in hemolymph osmolality, elevated SCP values and survival rates
after freezing (Lee and Hankison,
2003). From September through late December
(Fig. 2A) hemolymph osmolality
more than doubled, reaching 926 mOsmol kg l-1. Since previous
studies (c.f. Baust and Lee,
1981
; Storey and Storey, 1986) have demonstrated that this
increase is due to the accumulation of low molecular mass cryoprotective
compounds, primarily glycerol, sorbitol and trehalose, monitoring hemolymph
osmolality provides a convenient way to monitor cryoprotectant accumulation.
Furthermore, since a primary mode of action for these compounds is
colligatively based, it also provides a direct measure of this property
(Lee, 1991
). From the time of
our first sampling date in September until mid December, SCP values were
already elevated and they remained constant at temperatures near -10°C
(Bennett and Lee, 1997
).
Elevation of the SCP promotes freezing tolerance and winter survival by
slowing the formation of extracellular ice, reducing the metabolic rate and
energy utilization, and decreasing the rate at which water is lost to
surrounding frozen microhabitats
(Zachariassen and Hammel,
1976
; Storey et al.,
1981
; Lundheim and
Zachariassen, 1993
). Large crystalloid spheres of calcium
phosphate within the Malpighian tubules serve as endogenous ice nucleators
that elevate the SCP (Mugnano et al.,
1996
).
Between September and December, larvae progressively increased their
tolerance of freezing, consistent with previous reports
(Bennett and Lee, 1997;
Lee and Hankison, 2003
). Fully
cold-hardened larvae are freeze-tolerant and survive freezing at -25°C
with 75% reaching adulthood (Lee et al.,
1993
). In the present study, we used larval responsiveness to
tactile stimuli as the criterion of survival. We found that all
November-collected larvae survived freezing for 20 days at -20°C but only
83% survived -80°C, although we did not follow their late development to
either pupariation or emergence (Fig.
2). Lee and Hankison
(2003
) reported that larvae
collected in early autumn can survive freezing treatments at -4°C.
Consequently, if September-collected larvae had been subjected to a milder
freezing treatment, it is probable they would have survived.
Our observations of the gut and MT transport in E. solidaginis
larvae are consistent with the general understanding of their function in
other insects (Maddrell and O'Donnell,
1992). Active transport of a wide range of organic metabolites,
including certain synthetic dyes is known
(Maddrell and Gardiner, 1974
).
In this study, we used a pH-sensitive dye, Chlorophenol Red as an indicator to
assess the transport function of the epithelial membrane from different
regions of the gut and the MT. The dye is carried in the water that follows
the active transport of potassium into the tubule lumen against a
concentration gradient (Pritchard and
Miller, 1993
). As summarized in
Fig. 3, the dye was readily
transported into the lumen of the foregut, posterior region of the midgut,
ureter, proximal region of anterior MT, and posterior MT, but not of the
anterior region of the midgut, the entire hindgut and the spherule-containing
distal region of anterior MT. This regional specificity in ion and water
transport reflects different physiological functions of the gut and MT
epithelial membranes in E. solidaginis
(Table 1). After feeding,
blood-sucking insects can secrete fluid across cells of the MT at phenomenally
high rates (Maddrell, 1991
),
however the fluid secretion in the MT of E. solidaginis occurred more
slowly (10.7-12.7 nl min-1; Fig.
4), which is typical for herbivorous insects
(Phillips, 1981
;
Neufeld and Leader,
1998c
).
Effects of the metabolic poison (KCN) and the specific
Na+/K+-ATPase inhibitor (ouabain) on dye uptake by gut
and MT are consistent with species-specific differences reported by other
investigators. In E. solidaginis, both inhibitors slowed the uptake
of dye, indicating reduced rates of active transport of potassium
(Table 1). However, KCN
inhibited transport to a greater extent than ouabain. Previous studies on
Locusta tubule cells showed that the trans-cellular gradients for
Na+, K+ and Cl- were altered by ouabain and
n-ethyl maletimide (Pivovarova et
al., 1994). In the New Zealand alpine weta Hemideina
maori, however, fluid secretion was unaffected by ouabain or bumetanide,
but the transport inhibitors Ba2+ and amiloride reduced secretion
by 79 and 57%, respectively (Neufeld and
Leader, 1998c
). The high sensitivity of the apical cation pump,
maintaining the cell interior as a K+-rich, Na+-poor
environment (see Maddrell and O'Donnell,
1992
), may explain why the function of most MT are not affected by
treatment with ouabain.
Temperature had a significant effect on gut and MT transport. Predictably,
transport occurred more rapidly at higher temperatures
(Table 2). Nicolson and
Isaacson (1996) reported that
warming the MT from 20 to 30°C increased the MT secretion rate in the
tsetse fly. Of special interest in our study was the fact that transport
occurred at significant rates even in larvae held at 0°C, although 60 min
were required before transport reached the same level as occurred after 30 min
at 10°C (Table 2).
Seasonal acclimatization of larvae to overwinter also had a major effect on
MT secretion. During the course of this study, larvae underwent a major
transition from actively feeding larvae with negligible cold tolerance to
diapausing larvae with well-developed freezing tolerance
(Lee and Hankison, 2003;
Bennett and Lee, 1997
). In
field-collected larvae, the rate of secretion by both the anterior and
posterior MT pairs decreased markedly from Septemeber 20 until December 11
when secretion was no longer detectable
(Fig. 4). This decrease matches
larval entry into diapause, when metabolic rates decrease two to threefold
(Irwin et al., 2001
). To our
knowledge these data are the first to show a seasonal decrease in MT transport
rates associated with diapause.
Freezing and thawing subjects cells and organs to myriad chemical,
mechanical and physiological stresses including cellular dehydration, anoxia,
extracellular solute concentration and extreme osmotic flux across organelles
and membranes (Pegg, 1987;
Storey and Storey, 1996
).
Injury is frequently manifest at the level of the cell membrane. As pointed
out by Neufeld and Leader
(1998a
) the single-cell-thick
tubules constituting the MT are an especially good model for studying the
effects of freezing in a relatively simple organ. They found that the MT of
the freeze-tolerant weta (H. maori) readily tolerated in
vitro freezing if high concentrations of trehalose or glucose were
present in the bathing saline. However, membrane potential and secretion rate
decreased markedly if MT were frozen in a saline solution lacking sugar
(Neufeld and Leader, 1998a
).
The MT of this species is also highly tolerant of hyperosmotic exposure
comparable to the osmotic shock experienced during freezing to -4°C in
nature (Neufeld and Leader,
1998b
).
In the present study, we examined gut and MT that were removed from E.
solidaginis larvae after 40 days of freezing at -20°C
(Table 3). For surviving
larvae, so judged for their capacity to move in response to touch, gut and MT
function returned within 30 min, although more slowly than for unfrozen larvae
(Table 2). Even gut and MT from
larvae that were scored as dead exhibited transport function, albeit at lower
rates than surviving larvae (Table
3). Death at cellular levels does not always correlate with
organismal mortality (Yi and Lee,
2003). Previously, we reported that fat body survived freezing at
significantly lower temperatures than did the intact E. solidaginis
larvae (Lee et al., 1993
).
Using fluorescent vital dyes to assess tissue viability in these larvae, Yi
and Lee (2003
) found that
integumentary muscle, hemocytes, trachea, MT, fat body and gut were more
tolerant of freezing than the whole animal. Ultrastructural manifestations of
lethal freezing in these larvae suggest that the brain exhibits greater
structural perturbations than muscle or MT (Collins et al.,
1996
,
1997
).
Membranes are highly complex structures, characterized by domains of
non-randomly distributed protein and lipid components; only recently has the
important functional role of these domains begun to be appreciated
(Brown and London, 1998;
Hazel, 1995
;
Williams, 1998
). Mounting
evidence makes it clear that cholesterol plays a multiplicity of roles in
membrane function including regulation of membrane fluidity and maintenance of
sphingolipid rafts (Hochachka and Somero,
2002
; van Meer,
2002
; Simons and Ikonen,
2000
). Evidence from diverse sources suggests that cholesterol
plays special roles in membrane function at low temperature. Crockett
(1998
) reviewed the diversity
of roles that membrane cholesterol plays in temperature adaptation. It is
commonly reported that membrane cholesterol increases with acclimation to
higher temperatures, consistent with the homeoviscous adaptation model, in
which cholesterol functions as a membrane stabilizer
(Hazel, 1995
). However, in
some tissues cold acclimation increases membrane cholesterol, as found in
brush border membranes from intestinal epithelia of trout (Crockett and Hazel,
1995). Furthermore, mammalian sperm membranes with naturally higher or
artificially elevated levels of cholesterol are more resistant to cold shock
(Drobnis et al., 1993
;
Zahn et al., 2002
).
Concomitantly with increases in hemolymph osmolality and cold tolerance, cholesterol levels in the hemolymph increased fourfold in the autumn (Fig. 5A). This result suggests the possibility that cholesterol plays a role in the seasonal acquisition of freeze tolerance in insects. We have evidence that increased membrane cholesterol also enhances chilling tolerance and rapid cold-hardening in Drosophila melanogaster (S.-X.Y. and R.E.L., unpublished data). Although the membrane ratio of cholesterol to protein in the MT epithelium remained relatively constant during the course of this study, we did not measure membrane levels in other types of cells. Insects cannot synthesize cholesterol and must obtain it in their diet. Since larvae of E. solidaginis cease feeding in September, the increase in hemolymph cholesterol, observed late in the autumn, must have come from other stores.
Freezing is known to cause changes in the composition and thermotropic
properties of cell membranes and the composition of membrane components
(McKersie et al., 1989;
Crowe et al., 1989
). In
Dunning prostate tumor cells, freezing increased the phase transition
temperature, elevated membrane fatty acids and caused membrane protein
denaturation (Bischof et al.,
2002
). In this study freezing of intact larvae decreased
cholesterol levels in the hemolymph and in MT membranes
(Fig. 5A,B). Freezing at
-20°C had the greatest effect on MT membrane levels, however both freezing
treatments caused a similar reduction in cholesterol concentrations in
hemolymph. These results suggest that future investigations of the role of
cholesterol in insect cold-hardiness will prove fruitful.
In summary, this study demonstrated changes in the functional responses of gut and Malpighian tubules to seasonal acclimatization, chilling and freezing in a freeze-tolerant insect. These results indicate that cold acclimatization occurs, not only at the cellular level, but also at the organ level in insects. Future studies of organ-level function should improve our understanding of fundamental mechanisms underlying freezing injury and cryoprotection.
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