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Copyrightri.)1993PergamonPressLtd
J. Insett Phl,siol. Vol. 39, No. 5, pp. 445-450, 1993
Printed in Great Britain. All rights reserved
Survival of Intracellular Freezitg,
and Osmotic Fragility in
Lipid Coalescence
Fat Body Cells of the Frceze-tolerant
Gall Fly Eurosta solidaginis
RICHARD E. LEE JR,* JOHN J. MCGRATH,i R. TODD MORASON,* RONALD M. TADDEO*
Receired 8 September 1992
Atthough it is generallybelievedthat under natural conditionsofreeze-tolerantorganismscan survive
only if ice formation is restrictedto the extracellularspace,in 1959R. W. Salt reportedthat fat body
cefls of the freeze-tolerant gall fly, Eurosta solidaginis (Diptera: Tephritidae)' survive intracellular
for cells in
freezing. Using cryomicroscopy,intratellular freezing was observedat -4.6*0.1oC
Grace'i media. Freezing was apparently causedby inoculative freezing from outside the cell, since fat
body cells in oil cooled to below - l5oc.without internal ice formation. Viability of cells was assessed
-5 and
using fluorescent vital dyes immediately following freezing for 24h at temperatures between
- 80oC.At - 25oC'or lower few cells survivedfreezingin Graceosmediumalone.At - 25 and - 80oC,
cells frozen in Grace's media supplementedwith I M glycerol exhibited rates of survival similar to
those in whole larvae. No larvae (n :2Olsurvived freezingto -80oC, but more-than 60oh of the fat
body cells survived this treatment. Most faf body cells survived osmotic concentrations from 0-25 to
2 times that of the normal concentration of Grace's media (340 mOsm) for ?Ah at 4"C. More than
ffio/o of the cells survived 5 M glycerol in Grace's media under these conditions. An unusual response
ofthese cells to freezing is the coalescenceofintracellular lipid droplets upon thawing. The magnitude
of coalescence increases with decreasing temperature and increasing duration of exposure, but
decreaseswith the addition of glycerol to the media. Coalescencein itself is not indicative of injury'
since larvae frozen under conditions that causeextensivecoalescencereadily survived to complete their
development and emerge as adults.
Cold-hardinessFreezetolerance Cryoprotection Glycerol
(Storey and Storey, 1988;Baust and Nishino, 1991)'In
the spring, adults emerge from ball galls of goldenrod
Although it is generallybelievedthat survival of freezing (Solidago spp), mate and females oviposit into the
under natural conditions occurs only if ice formation stem of young plants. Larvae pass through two instars
is restricted to the extracellular space (Mazur, 1984), during the summer and overwinter within the gall as
this assumption is based on an extrapolation from the a third instar. During the summer and early autumn,
cryopreservation of mammalian cells that would never E. solidagims is intolerant of freezing; however, in
naturally experiencesubzero temperatures.This premise response to environmental cues, larvae acquire freeze
has rarely been directly investigated in cells from tolerance in mid to late autumn (Morrissey and Baust,
naturally freeze-tolerant organisms. However, on two 1976).The supercooling point, sometimesreferred to as
occasionsSalt (1959, 1962)reported'intracellularfreeze the temperature of crystallization, of the freeze-tolerant
tolerance in the fat body cells of the goldenrod gall fly, larvae is approximately -8 to - l0"C (Baust and Lee,
Eurosta solidaginis. For more than 30 yr Salt's obser- l98l; Bale et al., 1989).Larval exposureto low temperavations on this novel phenomenon have been largely tures between 0 and 5'C trigger an accumulation of
sorbitol (Baust and Lee, 1981, 1982;Rojas et al.,19841'
ignored.
received
Storey and Storey, 1988). Desiccation may also play a
The goldenrod gall fly, E. solidaginis, has
role
in the accumulation of cryoprotectants; glycerol
model
insect
extensive study as a freeze-tolerant
accumulation in third instars is closely correlated with
the drying of the surrounding gall tissues as the plant
+Department of Zoology. Miami University, Oxford, OH 45056,
senesces(Rojas et al., 1986).
U.S.A.
In this report we have confirmed and extended the
tDepartment of Mechanical Engineering. Michigan State University,
initial observationsof Salt (1959, 1962)on survival of
E a s t L a n s i n g , M I 4 8 8 2 3 ,U . S . A .
INTRODUCTION
445
RICHARD
F.. LEE et al
intracellular {reezing in E. solidaglrlr.r.Specifically we
used a combination of in uir:oand,in t:itro approachesto
determinethe following: the tcmperatureat which intracellular freezing occurs, survival of the fat body cells
fiozcn with and without cryoprotectant,f-actorsinfluencing the coalescence
of cytoplasmiclipids <Juringfreezing
and osmotic fragility of fat body cells.
Cryomicroscopywas preformcd using a conduction
type cryomicroscopesystem as describedby McGrath
(1987). Fat body cells were frozen in Grace's insect
media (340mOsm) unlessotherwiseindicated. To prevent inoculation of cclls by extracellularice cells were
frozen in a light paraffinic oil called American White Oil
(Standard Oil Company).
Following various treatmentscell survival was assessed
using
fluoresccntvital dyesas follows (Haugland, 1992).
MATI]RIAI,S AND METHODS
A mixture clf acridine orange and ethidium bromide
Goldenrod galls were collectedin St Paul, Minnesota stainedlive celis and nuclei green, while dead cells
and
o n F e b r u a r y 1 6 , 1 9 9 1 . F o r t h e f i r s t 2 0 d a y s t h e y nuclei appearedorange. Propidium iodide stained the
were held at 4''C before transfer to - l5'C where they nuclei of dead cells orange. Stained cells were
viewed
were kept until they were used for experimentation. using an Olympus BH-2 microscopeequipped with
a
Supercoolingpoints ancl glycerol determinatlonswere rcflectcdlight ffuorescenceattachment.
determined as described previously (Baust and Lce.
Coalescence
was scoredon a relativescalefrom 0 to 4.
r 9 8r) .
A score of 0 was assigncd to unfrozcn cells that
FICURE I Representativecrvomicroscopic photographs of freezing and thawing in lat body cells of the freeze-tolerant
gall
fly, Eurosta xtlidaginis, in Grace's medium. (A) Unfrozen fat body cells at 5'C. (B) Inrracellular lieezing
ol'fat botly cells.
(C) Beginning ol' lipid coalescenceimmediately after tliawing. (D) Extensive lipid coalescencewithin
I min after. thawrnq.
E a c h m i n o r s c a l cd i v i s i o n - I 0 t r m .
FAT BODY INTRACELLULAR FREEZING
TABLE
l. Survival of third-instar Eurosta ,soliduglniifollowing 24 h
oI Ireezlng
Temperature
Adults fully formed
- 25'C
--80c
Adult cmergence
7 s %( t 5 i 2 0 )
0% (0120)
85o/"(17120)
0% (0120)
100
a
a
6 8 0
E
(n 6 0
6
tr
4a
= 2 0
contained a large number of small lipid droplets dis.E
persedevenly throughout the cytoplasm[seeFig. l(A)]'
n-80
-25
-10
-5
Cells scoredas I containedin excessof 15 medium-sized
Temperature(" C)
progressedthe number of dropdroplets.As coalescence
of f-at
lets decreased,but droplets becamelarger. Cells with FIGURE 3. Effect of subzero temperature on the viability
were either frozen in Grace's insect
Cells
h
freeze,
24
alter
a
body
cells
2-l5large dropletswere scoreda 2. Cells scoredas 3 or
m e d i a w i t h o r w i t h o u t t h e a d d i t i o n o f t M g l y c e r o l ,o r f o l l o w i n g l o w
4 contained a single targe lipid droplet which occupied lemperature exposure they were dissectedfiom larvae and tested for
the majority of the cell volume. In cellsscoredas 3, the viability. Bars representthe averagecell survival as determined using
singledroplet only partially occupiedthe cell, and those acridinc orange/ethidium bromide and propidium iodide fluorescence
(50 cells wcre used for cach assay).
scored as 4 contained the single lipid droplet occupied
cell
the
approached
most of the center of the cell and
causedby inoculativefreezingfrom outsidethe cell,since
membrane [seeFig. l(D)].
fat body cells in oil could be cooled to below * 15'C
rvithout internal ice formation.
RESULTS
Viability oJ'fat body cells t's lart'ae
Coltl-hardiness of gall flY lurt'ae
The viability of fat body cells dissectedfrom whole
The mean supercoolingpoint of larvae was - l0.l
larvae or isolatedcells frozen in Grace's media with or
+ 0.8'C (n : l4). Glycerol titers were 32.3pgimg live
without the addition of 1 M glycerol was determined
weight. The limit of freezing tolerance was assessedby
a 24-h freeze at various temperatures (Fig. 3).
freezinglarvaefor 24h at either - 25 or - 80.C and then after
all treatmentscells frozen to -5 or - l0'C had
holding then-rat 22-24'C until they emerged from the Under
rates basedupon our criteria for survival.
high
survival
galls (Table t). Of the larvae frozen at -25"C, 85o
survival of the fat body cells frozen in uitro
Furthermore,
successfullymetamorphosed from larvae to adults, and
subzero temperatures correlated closely
high
at
these
75%osuccessfullyemerged from the gall. In contrast,
of intact larvae frozen under these
survival
with
the
-80"C.
no adults formed from larvae frozen to
conditions. At -25"C or lower, few cells survived
freezingin Grace'smedium alone. At -25 and -80"C'
In t r uceII ular .freezing
frozen in Grace's plus glycerol exhibited rates of
Cryomicroscopy was used to directly observe the cells
similar to those from whole larvae. No larvae
effects of freezing and thawing on fat body cells in survival
(Table
1) survived freezing at -80"C, but more than
Grace's insectmedia. Intracellular freezing,visualizedas
the fat body cells dissectedfrom larvae that had
flashing, was identified as an abrupt darkening of the 60oh of
survived this temperature (Fig. 3).
frozen
been
cytoplasm [cf. Fig. l(A) vs (B)] (McGrath, 1987)'Cells
were cooled at 2"C/min. The mean onset temperatureof Lipid coalescencev,ithin
.fat bod1,-cells
intracellularfreezingwas -4.6 + 0.1'C (mean* SEM)'
Unfrozen cellswere filled with a large number of small
n :68 (Fig. 2). Somecells froze at temperaturesas high
lipid
droplets dispersedevenly throughout the cytoplasm
-8"C.
-3'C. while a few cells did not freezeuntil
Ice
ahvays formed in the surrounding media before intracellular flashingwas observed.Freezingwas apparently
f
€
*
g
N
c
o
o
xY 3 0
d)
o
c
u
I
6
o 4 v
l)
E
a
o
rv
n
z
0
l
-3
-4
-s
'6
-7
-8
Temperature of Crystallization(oC)
FIGURE 2. Temperature at which intracellular freezing occurred
in fat body cells (n = 68) frozen in Grace's insect media. Cooling rate
was 2'Clmin.
-5
-25
-10
( oC)
T€mperature
-80
of intracellularlipids alter freezing
FIGURE 4. Relativecoalescence
Cellswereeitherfrozenin Grace's
for 24h at varioustemperatures.
insectmediawith or withoutthe additionof I M glycerolor theywere
from larvaeand testedfor viability lollowinglow temperadissected
ture exposure.
RICHARD E. LEE er at
x
I
AcddlneO€ngo/€thiOumBrcnjdJ
A
Popldlum
lodtdo
AN
5
S 4 0
o
-ru."
Relative coalescenceof "t,
intracellular
,our.
24
FIGURE 5.
lipids durine 24 h
o f f r e e z i n ga t - 2 5 C . C e l l s w e r e e i l h e r f r o z e n i n C r a . , e . ,i n s e c t L c d i a
with or without the addition of I M glycerol or they were dissected
from larvae and tested for viability following low temperature
exposure.
0
.t
s
1o
GlycerolConcentration(M)
20
FIGURE 7. Osmotic fragility of fat body celts (r : 50) of third
rnstar larvae of Euro.stasolidaginisexposed to various concentratlons
of glycerol in Grace's insect media, pH 7.0 for 24h at 4.,C.
Osmotic .fragility of fat body cetts
[Fig. l(A)] Galls containing larvae were held at
Sinceunder natural conditionsfreezingis inextricably
- l5'C until they were used in this
study; thesestorage tied to soluteconcentration
in the unfrozenbody fluids,
conditions causedpartial coalescence
(index scoreof l) we also examined
the toleranceof isolatedfat body cells
of fat body cells.When cells from thesepreviously frozen
to osmotic stressat 4'C for 2a h (Fig. 6). As described
larvae were froz,enin Grace's media with or without
earlier cell survival was based the responseof cells to
glycerol at -5 or - 10"C, index scoresfor coalescence
treatment with fluorescentvital dyes. Even in the abremainednear I (Fig. 4). After a 24h freezeto _25,C
sence of cryoprotectant these cells survived osmotic
fat body cells frozen in Grace's media showed a
concentrationsthat ranged from 0.25 to 2 times that of
marked increasein coalescencecompared to ones frozen
Grace'smedia (340mOsm). The independentestimates
at warmer temperatures. In contrast, cells frozen in
of cell viability using the two sets of vital dyes gave
Grace's media plus glycerol or cells from larvae frozen
similar results.
to -25'C exhibited only slightly greater coalescence.
Fat body cell viability was also testeclin Grace'smedia
After the lowest temperature freeze to - g0.,C. nearlv
supplemented with several concentrations of glycerol
all cytoplasmic lipids were contained in a single
for 24 h at 4'C (Fig. 7). Slightly less rhan 70%oof the
droplet that occupied the majority of the cell volume
cells survived 5 M glycerol, while few cells survived
[Fig. l(D)]. At this temperature,cells dissecredfrom higher concentrations.
whole larvae had a greater degree of coalescencethan
cells frozen in Grace's media containing I M glycerol
DISCUSSION
(Fig. a).
We examined the time course of coalescence<iuring a
Few investigatorshave examinedcellular mechanisms
24h freezeat -25"C (Fig. 5). During the first hour of of chilling and freezing
injury in insects.Two forms of
freezingthe index of coalescence
increasedapproximately Iow temperatureinjury are evident: (l) injury resulting
2-fold in fat body cells frozen in Crace,s media alone from freezingand (2)
cold shock associatedwith chilling,
with a lesserincrease during the next 24h of freezing. but without ice
formation (Lee, l99l). We selectedfat
Slight increases in coalescenceoccurred for cells in body cells because
of their central role in intermediary
Grace's media with glycerol or in cells from intact larvae metabolism, parricularly
with respectto the synthesis
during the 24 h exposure.
of cryoprotectants. Fat body functions are comparable
to thoseof mammalianliver, servingas a primary site of
protein synthesisand secretion,and the synthesisand
100
ffihq€/AtrbmBEM
|
storageof lipids and carbohydrates(Locke, l9g4).
E Pqldum 6@
5a
s80
Suruiuol of intr acellular .freezing
It is generally believed that freeze tolerance at high
650
subzerotemperatureswith slow cooling rates is possible
only if ice formation is restrictedto the extracellular
E+o
space (Mazur, 1984). In contrast, our present study
confirmed the previousreports of Salt (1959, 1962)that
Ero
fat body cells ofthe freeze-tolerantgall fly larvae survlve
n
intracellular freezing.
0 0.1 0.250.5 1 2 5 10 20 40
A cryomicroscope was used to directly observe the
Concsntration
of Grac€'sInsectM€dia
FIGURE 6. Osmoricfragility of fat body cells (z = 50) of third effectsof freezing and thawing on fat body cells. Using
instarlarvaeof EurostasolidagiTtis
exposedto variousconcentrations this instrument we observed intraceilular freezing oi
of Grace'sinsectmedia,pH 7.0 for 24h at 4.C.
i n d i v i d u a lc e l l s a t - 4 . 6 + 0 . 1 " C . I n e v e r y i n s t a n c er c e
0)
FAT BODY INTRACELLULAR FREEZING
had already formed in the surrounding media before
intracellular freezingoccurred. Furthermore, when cells
were cooled to - l5"C in oil, to prevent inoculative
freezing,no flashing was observed.Theseresultsindicate
that efficient heterogeneousice nucleators are absent
in fat body cells and that intracellular freezing resulted
from inoculative freezing from ice in the surrounding
media.
In mammalian cells that are rapidly cooled, inoculative freezing of the intracellular fluid occurs between * 5
and -30'C depending on the cell type, however nucleation is typically blocked by the cell membrane at
temperaturesabove - l5'C (Mazur, 1984).In comparison the fat body cells were frozen at the high end of this
temperature range. The relative susceptibility of the fat
body cell to inoculative freezing may representan adaptation promoting intracellular freezetolerance' Since so
few cells have been examined for intracellular freeze-tolerance it is possible that this trait will be found in other
freeze tolerant organisms.
Another objective of our study was to compare the
low temperature limit of freeze tolerance for whole
larvae vs their fat body cells.Although greater than 60%
of the fat body cells survived freezing to - 80'C, no larvae survived freezing to this temperature. These results
suggestthat fat body cells are not the cell type within the
larvae that is most susceptibleto freezing injury.
Coalescenceqf lipid droplets within fat body cells
Asahina (1969) reported lipid coalescencein fat body
cells of insects that did not survive freezing. However,
Salt (1959) described coalescenceof intracellular lipid
droplets in fat body cells that survived intracellular
freezing.Prior to freezing,each cell contained many tens
of lipid droplets; however, intracellular freezing resulted
in the coalescenceof these into fewer, larger droplets.
At relatively low freezing temperatures the cytoplasm
appearedto be filled with a single large lipid unit following thawing. The magnitude of coalescenceis related to
the duration of exposureto subzerotemperatures:longer
exposuresproduced increasedlevels of coalescence.We
also observedthat the addition of glycerol to the Grace's
media greatly decreasedthe amount of coalescencein
cells frozen to - 80'C. Coalescencein itself was not
indicative of injury, sincelart,ae frozen under conditions
that causeextensivecoalescenceof lipid droplets within
their fat body cells readily survived to complete their
development and emerge as adults. It appears that the
presenceof coalescencemay be useful as a marker indicating that fat body cells have experiencedintracellular
freezing.
During freezingonly water moleculesjoin the growing
crystal, rejected solute becomes concentrated in the
remaining unfrozen body fluids. The resulting osmotic
gradient removeswater from the cells.This processmay
play a role in prornoting lipid coalescence.If water is
removed from the cell during freezingit should bring the
lipid droplets closer together, and thereby facilitate their
fusion with each other. It is also possiblethat the lipid
droplets may fuse due to their compression between
growing arms of the ice lattice within the fat body cell.
Mazur (1984) suggesteda similar process to explain
mechanical injury to cells within narrow channels of
unfrozen fluid during frcezing. Lipid coalescencemay
also be causedby a combination of freezing-inducedcell
dehydration and compressionof lipid droplets between
arms of the ice lattice.
Osmotic fragility of fat body cells
Freezing-inducedcellular dehydration is an important
mechanism of freezing injury (Mazur, 1984). The cell
membrane is commonly identified as the primary site
of freezing injury due to the action and interaction of
hypertonicity and/or the direct effectof low temperature.
Hypertonic stress may result in the actual loss of
membrane material predisposingthe cell to lethal injury
during thawing (Steponkus, 1988). Our preliminary
cryomicroscopoic observationsdid not suggestthat this
happened in the fat body cells that we examined' Our
study demonstrated that fat body cells are resistant to
a range of osmotic stresses(Figs 6 and 7), a trait that is
not surprising in a cell that survives both intra- and
extracellularice formation.
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Asahina E. (1969) Frost resistancein insects Adt:. Insect Physiol. 6,
t- 49.
Bale J. S.. HansenT. N., Nishino M. and Baust J. G. (1989)Etrect
of cooling rate on the survival of larvae, pupariation, and adult
emergenceofthe gallfly Eurosta solidaginis.Cryobiology 26,285 289.
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J. Insecr Physia!.27,485 490.
Baust J. G. and Lee R. E. (1982) Environmental triggers to cryoprotectanl modulation in separate populations of the gall fly'
Eurosta solidagrnis(Fitch). J. Insect Physiol.2S' 431'436.
Baust J. G. and Nishino M. (1991) Freezingtolerance in the goldenrod
gall fly (Eurosta solidaginis). In Insects al Low Temperature (Eds
Lre R. E. and Denlinger D. L.), pp. 260-275. Chapman and Hall,
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Chemicals,42l pp. Molecular Probes, Eugene, Oregon.
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lnsectsat Lov' Temperature(Eds Lee R. E. and Denlinger D. L.)'
pp. t7-46. Chapman and Hall, New York.
Locke M. (1984) The structure and the development of the vacuolar
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R . C . a n d A k a i H . ) , p p . l 5 l - 1 9 7 . P l e n u m P r e s s ,N e w Y o r k .
Mazur P. (1984) Freezingof living cells: mechanismsand implications.
Am. J. Physiol. 247, Cl25'{142.
McGrath J. J. (1987) Temperature-controlled cryogenic light
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o/ Low Temperature ott Biological Systems (Eds Grout B. W' W.
and Morris G. J.), pp. 234-26'1. Edward Arnold Press, London.
Morrissey R. and Baust J. G. (1976) The ontogeny of cold tolerance
in the gall fly, Eurosta solidaginis.J' Insect Physiol.22,43l-438.
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ture dependence-independence
solidaginis (Fitch). "/. Insect Physiol. 29, 865-869
Rojas R. R., Lee R. E. and Baust J. G. (1986) Relationshipof
environmental water content to glycerol accumulation in the freezing tolerant larvae of Eurosta solidagfuis (Fitch). Cryo-Letters 1'
234-245.
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RICI{ARD
S a l t R . W , ( 1 9 5 9 ) S u r v i v a l o f f r o z e n f a r b o d y c e l l s In an insect
Nature 184, 1426.
Salt R. W. ( 1962) Intracellular freezing in insects. Narure 193.
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E. LEE et at
Acknot'ledgement.r-Wc thank Bill Schmid for assistingin the
collection
of the galls and Peler Lortz fcrr help with the HpLC analysis.
Research
support was provided by the National ScienceFoundation, grant
DCB
N o . 8 8 l l 3 l 7 a n d b y a U n d e r g r a d u a t eS u m m e r R e s e a r c hI n t e r n s h i p
f r o n , M i a m i U n i v e r s i t y / H o w a r dH u g h e s M e d i c a l I n s t i t u t e
Grarrt to
RTM.