Archivesol lnsectBiochemistryand Physiology48:199-205(2001)
IntracellularFreezing,Viability,and Composition
Larvae
of Fat Body Cells From Freeze-lntolerant
of Sarcophaga crassiPalPis
Diana J. Davis and Richard E. Lee, Jr..
Department of Zoology, Miami University, Oxford, Ohio
Although it is often assumed that survival of freezing requires
compart'
must be restricted to extracellular
that ice formation
larvae of the gall fly'
ments, fat body cells from freeze-tolerant
Eurosta solidaginis (Diptera, Tephritidae) survive intracellu'
lar freezing, Furthermore, these cells are highly susceptible to
inoculative freezing by external ice, undergo extensive lipid
coalescence upon thawing, and survive freezing better when
glycerol is added to the suspension medium' To determine
freeze tolwhether these traits are required for intracellular
erance or whether they are incidental and possessed by fat
body cells in general, we investigated the capacity of fat body
(i.e',
and diapause-destined
cells from nondiapause-destined
flesh fly Sorcophago
cold-hardy) larvae ofthe freeze-intolerant
cra.ssipalpis (Diptera, Sarcophagidae) to survive intracellular
freezing. Fat body cells from both types of larvae were highly
susceptible to inoculative freezing; all cells froze between -3.7
freez.
to -6.2"C. The highest rates for survival of intracellular
of glycerol to the media
at -5"C. The addition
ing occurred
markedly increased survival rates. Upon thawing, the fat body
cells showed little or no lipid coalescence. Fat body cells from
had a water content of only 357o compared to
E, solidaginis
larvae that had 52-55Vo; cells with
cells from S. crassipalpie
less water may be less likely to be damaged by mechanical
freezing. Arch, Insect Biochem.
forces during intracellular
Physiol. 481199-205, 2001. @2o0l wiley-Liss,Inc.
freeze tolerance; cold-hardiness;
Key words: intracellular
protection; fat body
INTRODUCTION
A variety of insects and other invertebrates
well
as a few lower vertebrates naturaliy suras
vive freezing (see reviews Lee, 1991; Storey and
Storey, 1988; Costanzo et al., 1995; Lee and
Costanzo, 1998).However, it is generally assumed
that survival is possible only if ice formation is
restricted to the extracellular fluid compartments.
Nonetheless,in 1,959and 1962 R.W. Salt demonO 20Ol Wiley-Liss, Inc.
diapause; cryo-
Contract grant sponsor: USDA CSRS; Contract grant number: 96-35302-3419; Contract grant sponsor: NSF; Contract
grant number: IBN-0090204.
Diana J. Davis's present address is Department of Chemistry and Physical Science, College of Mount St. Joseph, Cincinnati, OH 42533.
* C o r r e s p o n d e n c et o : R i c h a r d E . L e e , J r . , D e p a r t m e n t o f Z o ology, Miami University, Oxford, OH 45056.
E-mail: [email protected]
Received 25 February 2001; Accepted 4 July 2001
200
Davis and Lee
strated that fat body cells of the freeze-tolerant
the water and lipid content of fat body cells from
larvae of Eurosta solid,aginis(Diptera, Tephritidae)
diapause-destined and nondiapause-destined S.
survive intracellular freezing (salt, 1959, 1962).
uaisipotpiu larvae, and from freeze-tolerantlarvae
These reports are especially noteworthy since the
of E. ioridaginis.
insect fat body is a critical tissue that plays a primary role in carbohydrare, protein,
i11t1*"MATERIALS AND METHODS
""ia l i
t a b o l i s m s i m i l a r t!u
o tu'41
h a t ur
o f urarrlrrlallan
mamm
ilver
Insect Rearing
(Keeley,1985).
our research gror'rp confirmed and extended
Sarcophaga crassipalpis were reared accordSalt's investigations of intracellul ar freezetolerance ing to the
methods described by Lee and Denlinger
n E. solidaginis (Lee et al., 1993). Using fluores- (1g8b).During
the first 6 days iort_u-".g"rrce, flies
cent vital dyes, we determined that many fat
body were provid.ea*itt beef liver as a protein sourceto
cells survive freezing to -80'C and thai survival promote normal
oogenesisand embryonic developincreased when glyceroi was added to the cryo- ment. Eleven
days after the adult emergence,a 50preservation medium' However, we were
surprised g packet of livei was providea
u substrate for
to frnd that internal freezing was readii-v iniiiated
".
laiviposition. on the following day,
approximately
when fat body cells contacted external ice, even
at b0 larvae were transferred to iresh packets ofliver.
high subzero temperatures (_4.6"C).This suscepti_ Thesepackets
were piacedin plastic tubs lined with
bility to inoculative freezing differs consideraily
a 2-cm layer of sawdust into which the larvae were
from the case with mammalian cells that gunerally allowed.to *andur
and pupariate. roilo*lrrg pupariresist inoculation by ice to -15'C or below (Mazua ation, puparia
were sifted from the sawdust and
1984)' we also observedextensive coalescenceof
lipl- stored in Petri dishes. To avoid diapause, animals
d droplets into larger droplets or even a single
large were maintained at 25oC under a long day photodroplet occupying the center of the cell (LJe
et aI., period (LD, 1b:9_h),while diafaur" *u, induced by
1993; Morason et al., 1994). Consequently,consid_ rearing
flies at LD l2:I2h and 20.C. Freeze_toler_
ering the novelty of this phenomenon, we wondered ant larvae
of E. sotid.aginls were field-collected near
whether these distinctive characteristics are
re_ Oxford, ohio, rn mid_January 1gg6 and stored fro_
quired for intracellular freeze tolerance.
or are they zen at -22c until used.
incidental traits also possessedby other fat
bodv
cells including ones from freeze-intolerantspeciesi Intracellular Freezing
Toaddressthese,questions,weinvestrgatedthe
Fat body cells from s. crassipalplis nondiacapacityof fat body cells from nondiapause--destined pause-destined
and diapause-destinedlarvae were
and diapause-destined(i.e., the typelhat gives
rise placed in 860 or 1,000 mosmol schneider,s
media,
to the cold-hardy overwintering pupae) laivae
from iespectively. A small amount or.-t ll"a preparasarcophaga crassipalpis. (Diptera,-sarcophagrdae)
tion of ice nucleating active pseud.on,onassyringae
to survive chilling and intracellular freezing.
Dia- (provided by Genencor International, Rochester,
N$
pause-destinedlarvae refer to ones that
will become was added to the media t" pr;;;;;;percooling
and
diapause pupae' the overwintering stage for
this insure that ice formed at temperatures slightly
bespecies'Diapause-destinedlarvae are substantially
low 0'c, ceils were cooled at a rate of
2oc/min.
more cold tolerant than nondiapause-destined
ones, Freezing of the cells, identified as an abrupt
darkalthough neither larval type survives freezing ening
of the cytoplasm, was visualized b.y cryo(Adedokun and Denlingea 1984;
Lee and Denlingei
micrlscopy using a Linkham computer-controlled
1985)' consequently, investigation of the
fat body conduction cryostage after Lee et al. (19g3).
cells from these two types of larvae should prove
useful-in determining whether the traits
observed Viability of Frozen Fat Body Cels
in fat body cells from E' solidaginls are specific
to
whole nondiapause-destined and diapausefreeze tolerant cells' we also determined whether
destined larvae were placed in plastic
1.5-ml
these cells were susceptible to inoculative
freezing microcentrifuge tubes and heid tar
is nat -5, -10,
and whether the addition of glvcerol to the
medium -22, or -sg"c: Larvae *"r" t;;;;?;ed
direcgy to
enhanced freezing survival' Finally, we
measured room temperature and the fat body
cells were dis-
lntracellularFreezingof Fat Body Cells
201
sectedfrom these larvae and placed into either 360 (Sigma Chemical Co.) until dried to a consr,anr
mosmol Schneider'smedia (nondiapause-destined) weight. To determine lipid content, the lipids were
or 1,000 mosmol Schneider'smedia (diapause-des- extracted from dried fat body cells according to
tined) and stained with a 1:50 mixture of 0.02Vo the method of Folch et al. (19b2).
acridine orange and 0.0lvo ethidium bromide afber
Lee et al. (1993). Cells were examined with fluoRESULTS
rescencemicroscopy(Olyrnpus BH-2), and live and
Intracellular Freezing
dead cellswere counted,Cells with bright green nuclei were scored as live, and those with bright orAs groups of fat body cells were cooled on
ange nuclei were scored as dead. In a second the cryostage, ice began to form in the surroundexperiment, fat body cells irom 5 individuals were ing media at approximately -3.3.C (Fig. 1). Howremoved, pooled, and placed in 1 ml of media in ever, fat body cells only began to freeze after the
microcentrifuge tubes befbre placing them at the gtowing ice iattice in the surrounding medium
treatrnent temperatures.
contacted the cells. Intracellular freezing was
Fat body ceils from five nondiapause-des- clearly indicated by an abrupt darkening of the
tined lalvae were pooled and half were placed in ey'toplasmdue to the rapid growth of ice. Within
1 ml 360 mosmol Schneider's insect media and the larvae, the fat body cells are arrayed in chains
half into 1 ml 360 mosmol Schneider'sinsect me- with close contact between adjacent cells. Nonedia with 1 M glycerol. Aliquots of 200 pl were theless, cells froze independently of each other
incubated for 24}r at -5, -10, -22, or -88"C. Fat (e.g., the time at which a particular cell froze did
body cells from diapause-destined larvae were not influence when nearby or touching cells froze),
treated in the same way but were placed in 1,000 suggestingthat ice within one cell couid not propamosmol Schneider's insect media. After incuba- gate to the next ceil in the chain. The temperature
tion, the survival status of these cells was as- of crystallization was determined by observing
s e s s e d b y a c r i d i n e o r a n g e / e t h i d i u m b r o m i d e when the cells froze and recording the temperastaining as described above. We conrpared sur- ture indicated by a thermocouple that monitored
vivai among the various treatments using mul- the sample. The temperature of crystallization
(-4.61 t 0.06'C, mean t SEM) for the nondiatiple linear regressionusing SAS version 6.12.
pause-destined larvae was significantly different
Composition of Fat Body Cells
from the diapause-destinedlarvae, -4.11 t 0.04.C
Groups of fat body cells were harvested from (P < 0.001)(Fig, 1). However,the overall range of
larvae, lightly blotted, weighed immediately to values for individual cells, -8.7 to -6,2oC, was
0.01 mg, and placed in a desiccator over P2O5 nearly identical for the two types oflarvae.
60
N S O
60
Non-diapause
50
X T S E M = - 4 . 6 1t 0 . 0 6
n=105
40
q ? n
30
a J v
0
5O rrt
q
H
o
20
. ^
r0
IU
n
-J.5
-3.5 -4.O
-4.tJ -4.5 -5.0 -5.5 _6.0
0
-3.5 -4.0 -4.5 -5.0 -5.5 _6.0
Temperarure
of crystallization
("C)
Fig l
Temperature at which intracellular freezing occurred
in isolated fat body cells from nondiapause-destined larvae
(n = 105) and diapause-destined larvae (n = 131) of Sorco-
phaga crassipolpis in Schneider's insect media. Cooling rate
*ur 2"c/roln. Airo*. indicate the approximate temperature
(-3.3.C) at which the media froze.
2O2
Davis and Lee
Viability of Frozen Fat Body Cells
The survival curves were similar for fat body
cells dissectedfrom intact non-diapauseand diapause-destinedwhole larvae of S. crassipalpis
that were exposed to various subzero temperatures (Fig. 2). The highest rates of fat body cell
survival occurred at -5'C for both larval types.
Survival rates decreased abruptly and few cells
survived when they were exposed to -10.C or
= 19.94,P = 0.0001).
lower (F23,rs3
A very similar pattern of survival was evident for the treatment in which the fat body cells
first were dissected from the two types of larvae,
and then held in the Schneider'sinsect media during subzero exposures. Once again the highest
rates of'survival occurred at -5.C with few cells
surviving exposure to temperatures between -10
and -83oC. No difference was detected between
100
Non-diapause
destinedlarvae
80
Composition of Fat Body Cells
--+-
40
F)
Whole larvae
_.FBC
--r-- FBC+lMglycerol
100
Diapausedestinedlarvae
a
80
60
40
20
0
0
the overall survival rates of cells from diapausedestined iarvae and nondiapause-destinedlarvae.
Augmenting the Schneider's insect media
with the cryoprotectant glycerol signifrcantly increased the survival rates for isolated fat body
cells from both larval types compared to the other
two treatments (Fig. 2). At -b"C, cells from diapause-destinedlarvae had a survival rate of g7.B
t 0.8 Vo(mean t SEM), while 85 t 5.8Vosurvived
24 h of exposure to -83oC. Fat body cells from
nondiapause-destined larvae also exhibited high
rates of survival when the media was supplemented with glycerol.
For all treatments in which cells were judged
to be dead based on the vital dye criteria, the cell
membranes appeared to be intact and unbroken.
Also it appeared that few, if any, lipid droplets
within the cytoplasm had coalesced into larger
droplets following intracellular freezing as was
reported previously in fat body cells from larvae
of E. solidaginis (Lee et al., 1993).
-20
-40
-60
-80
-100
Temperature ("C)
Fig. 2. Effect of 24 h of exposur€ to various subzero temperatures on the survival offat body cells from nondiapausedestrned (top) and diapause-destined (bottom) larvae of
Sarcophaga crassipalpis. The whole larvae treatment group
refers to intact Iarvae that were first exposed to subzero
temperature before removing their fat body cells and testing them for survival. The two additional tieatment groups
used isolated fat body cells held in Schneider,s insect medla
or Schneider's insect media supplemented with 1 M glycerol priol to subzero exposure.
Fat body cells of nondiapause-destined larvae of S, crassipalpis contained slightly, but significantly (P < 0.001), more lipids and less water
than diapause-destinedlarvae (Table 1). For comparative purposes, we also determined the lipid
and water contents for fat body cells from freezetolerant E. solidaginls larvae. Fat body cells of ,8.
solidaginis larvae had only 34.6Vowater, significantly less (P < 0.001) than the bZ-bSVo water
content of either nondiapause- or diapause-destined larvae. The lipid content (47.SVo)for these
freeze-tolerant larvae was approximately twice
that of cells from either type of S. crassipalpis
larvae.
DISCUSSION
lntracellular Freezing
Our previous study of intracellular freeze
tolerance in E. solidaginis larvae demonstrated.
that their fat body cells were susceptible to inoculative freezing at high subzero temperatures,
-4,6'C (Lee et al., 1998).At that time, we speculated that susceptibility to inoculative freezing
might be an adaptation for survival of intracellular freezing. Here, fat body cells from both types
of S. crasslpalpis larvae were also highly suscep-
IntracellularFreezingof Fat Body Cells
TABLE l. Relative Water and Lipid Composition
crassip alpis and Eurosta s olid.aginie
203
(Mean t SEM) of Fat Body Cells From Larvae of Sarcophogo
Species and diapause status
S. cras sipalp is (nondiapause-destlned larvae)
S. crassipal p is (diapause-destined larvae)
E. solidtginis (freeze-tolerant, diapausrng larvae)
tible to inoculative freezing at temperatures as
high as -3.5"C (Fie'. 1). Celis from both nondiapause- and diapause-destinedlarvae froze on average between -4.1 and -4.6"C suggesting that
their membranes did not differ markedly in their
susceptibility to inoculative freezing, and that ice
nucleating agents with activity above -.4oC were
nol present in their ceils.
The susceptibility of fat body cell membranes
to ice penetration contrasts markedly with the
behavior of mammalian cell membranes, which
prevent inoculative freezing at these high subzero temperatures (Mazur, 1984).When mammalian cells are cooied slowly in the presence of ice
in the surrounding medium, inoculative freezing
of their cytoplasmic water typically does not occur until temperatures decreaseto at least -10'C
and often to considerably lower temperatures.
This response is particulariy trre for cells that
have not been produced in tissue culture or isolated by enzymatic digestion of their extracellular matrix (Karlsson et al., 1993). The fact that
the fat body cells E. solidaginis and S. crosslpalpis did not begin to freeze until external ice
had contacted the cells indicates that ice nucleation was initiated by an inoculative process.The
oniy other animal known to survive intracellular
freezing is the Antarctic nematode Panagrolaimus
dauidi (Wharton and Ferns, 1995). The cells of
this speciesare also highly susceptibleto inoculative freezing.
Cryopreservationprotocolsgenerally seek to
avoid intracellular ice formation, while cryosur,
gical procedures are designed to promote its occurrence in, for example, tumors (Karlsson et al.,
1993). Despite its fundamental importance in
cryobiology, the mechanism(s) by which extracellular ice initiates intracellular freezing remains
Iargely unknown, but may involve growth of the
ice lattice through membrane pores, membrane
failure, or surface-catalyzed nucleation in which
external ice changes the nature of the plasma
Relative composition offat body cells (7o)
Water content
Lipid content
n
52.4 r 0 . 3
ID
3 4 , 6t 0 . 8
24
30
23.9 1 0.8
2 0 . 3t 0 . 7
4 7 . 3r 1 . 8
8
8
9
membrane such that it induces ice nucleation
within the cytoplasm (Karlsson et al,, 1993).The
excep*"ionallyhigh susceptibility of these insects
to inoculative freezing suggests that they may be
valuable models for future study of this important cryobiological phenomenon.
These results for fat body cells from S.
crassipalpis and E. solidaginis differ notably from
those for the weta, Hemideina muori (Sinclair et
a1., 1999). Cells from the fat body and the Malpighian tubule ofthis speciesresisted inoculative
freezing during cooling to -15oC, while assuming
a shrunken and dehydrated appearance as ice
formed extracellularly. Although this species tolerates the freezing of a remarkably large amount
of its body water (827a),this responseimplies that
its survival offreezing depends on its capacity to
restrict ice formation to extracellular compartments (Sinclair and Wharton, 1997).
Viability ol Frozen Fat Body Cells
Aithough tolerance of intracellular freezing
may be promoted by susceptibility to inoculative
freezing at high subzero temperatures, this trait
was not sufficient to confer high levels of freeze
tolerance on the fat body cells of S. crassipalpis
larvae. In the absenceofglycerol, only at the highest test temperature of -5oC were significant
numbers ofcells from nondiapause- and diapausedestined larvae able to survive freezing (Fig. 2).
When the treatment temperature was decreased
to -10"C few, if any, cells survived. This response
is consistent with the fact that fewer than L}Vo
of nondiapause- and diapause-destinedlarvae can
survive 2 h at -10'C (Lee et aI., 1987a).In contrast, fat body cells from E. solidaginis exhibit
rates of survival greater than 807a at this temperature (Lee et al., 1993; Bennett and Lee, 1997).
The addition of 1 M glycerol, a common cryoprotectlve compound in cold-hardy insects, to the
medium greatly enhanced fat body cell survival
for both types of S. crassipalpis larvae with many
204
Davis and Lee
cells surviving exposureto *83.C (Fig. 2). Changes LITERATURE
CITED
in glycerol levels are associatedwith a rapid cold.A d e d o k u n T A , D e n l i n g e r D L , 1 9 8 4 . C o l d - h a r d i n e s s :a c o m hardening response,low temperature acclimation,
ponent ofthe diapause syndrome in pupae ofthe flesh
and developmentalpatterns in this species(Chen
flies, Sarcophaga crassipalpis and,S. bullata. phvsiol
Entomol 9:361-364.
e t a l . , 1 9 8 7 ;L e e e t a l . , 1 9 g 7 a , b ) T
. he addirionof
glycerol also enhances freezing tolerance in a Bennett VA, Lee
RE. 1997. Modeling seasonal changes rn
simiiar fashion in fat body ceils of E. solidaginis
intracellular freeze-tolerance of fat br:dv cells of the
gall fly, Eurosta solidaginis (Diptera: Tephritidael. J
(Leeet al.. 1993).
Exp Biol 200:18b-192.
Other freeze tolerant plants and animals
avoid the damaging effects of excessive ice for_ Chen C-P, Denlinger DL, Lee RE. 19g7. Responses of nondiapausing flesh flies (Diptera: Sarcophagidae) to low
mation by dehydrating sensitive tissues (Lee and
rearing temperatures: developmental rate, cold toler_
Costanzo, 1998). For example, in the wood frog
ance, and glycerol concentrations. Ann Entomol Soc
(Rana syluatica),water is translocatedfrom body
Am 80:790-796.
or6l'ansto subdermal lymph sacs and the coeio_
mic cavity where it freezes;some organs lose more Costanzo JP, Lee RE, Wright Mn 1992. Cooling rate influ_
ences cryoprotectant distribution and organ dehydra_
than 60Voof their initial water mass (Lee et al.,
tion in freezing wood frogs. J Exp Zool 261:373_87g.
1992). Another advantage of dehydration is that
removal of water effectively increases the concen_ Costanzo JP, Lee RE, DeVries AL, Wang ! Layne JR. 1995.
Survival mechanisms of vertebrate ectotirerms at sub_
tration, and therefore the potential benefit of
temperatures: applications in cryomedicine.
t::r:fg
cryoprotectants (Costanzo et al., 19g2). A numFASEB J 9:Bb1-358
ber of freeze tolerant insects markedly decrease
their total body water content (Ring, 19g2).pre- Folch J, Lees M, Sloane Stanley GH. 1952. A simple method
for the isolation and purification of total iioid" f.osumably, both internal translocation of water and
a n i m a l t i s s u e s .J B i o l C h e m 2 2 6 : 4 9 7 _ 5 0 9 .
overall reduction in total body water would promote freeze tolerance by reducing cellular water Karisson JOM, Cravalho EG, Toner M. 1993. Intracellular
ice formation: causes and consequences. Cryoletters
content.
14:323-334.
Consequently,another possible reason why
fat body cells from E. solid.aginls larvae survlve Keelcy LI,. 19,85.Physiology and biochemistry ofthe fat body.
In: Kerkut GA, Giibert LI, editors. Comprehensive in_
intracellular freezing is their intrinsicaliy low
sect physiology biochemistry and pharmacology: rnwater content (Table 1). Although it is well docutegument, respiration and circulation. New york:
Pergamon Press. p ZtI-249.
mented in many organisms that intracellular
freezing is damaging, as with the mechanism of
Lee RE. 1991. Principles of insect lolv temperature tolerance.
inoculative freezing of cells, the specific nature
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of injury caused by freezing remains largeiy un_
perature. New York: Chapman and Hall. p 1746.
known (seereview by Karlsson et al., 1g93).None_
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theless, several lines of evidence suggest that
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mechanical damage caused by growing ice crys_
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and non-_diapausingstages ofthe flesh l|y, Sariophagi
tent of only 357owhile cells from S. crassipalpis
crassipalpis. Physiol Entomol 10:809_815.
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ter may be less likely to be damaged by mechani_ Lee RE, Chen C-P, Meacham MH, Denlinger DL. 1982a. On_
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in Sarcophaga crassipalpis. J Insect physiol
!gcjr^1n
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ACKNOWLEDGMENTS
We thank Jason Irwin, John Mugnano, Jon
Kelty, and Shala Hankison for technical and statistical assistance,and Jon Costanzofor revlewing an earlier version of the manuscript.
LeeRE, Chen C-P,DenlingerDL. 1987b.A rapid cold_hardeningprocessin insects.Science2Bg:l4lE_1417.
Lee RE, CostanzoJP, Davidson EC, Layne JR. 1992. Dynamicsof body water during freezingand thawing in
a freeze-tolerantfrog (Rana sytuatii). J Therm Eiol
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IntracellularFreezingol Fat Body Cells
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fieeze-tolerant Eurosta solidaginis larvae afier cheurical fixation and high pressure freezing. J Insect
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2Os
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