Insect

Molecular
Biology

Insect Molecular Biology (2012) 21(1), 3139 doi: 10.1111/j.1365-2583.2011.01108.x

A comparison of Frost expression among species and

life stages of Drosophila

X. Bing1, J. Zhang2 and B. J. Sinclair Introduction

Department of Biology, The University of Western At subzero temperatures, some insects can survive ice
Ontario, London, ON, Canada crystallization in their body tissues (freeze tolerant), or
keep their body fluids in a super cooled liquid state (freeze
Abstract imb_1108 31..39
avoiding) (Lee, 2010). However, the majority of insects are
chill susceptible, and die at low temperatures because of
Frost (Fst) is a gene associated with cold exposure in
accumulated damage unrelated to freezing (chilling injury)
Drosophila melanogaster. We used real-time PCR to
(Lee, 2010). Drosophila melanogaster (Diptera: Drosophil-
assess whether cold exposure induces expression
idae) is chill susceptible: adults and larvae suffer exten-
of Fst in 10 different life stages of D. melanogaster,
sive mortality after a 2 h exposure at -5 C, although
and adults of seven other Drosophila species. We
freezing does not occur until -20 C. Exposure to a prior
exposed groups of individuals to 0 C (2 h), followed
mild (sublethal) cold stress can increase the cold toler-
by 1 h recovery (22 C). Frost was significantly
ance of chill susceptible insects such as D. melanogaster
upregulated in response to cold in eggs, third instar
through rapid cold hardening (RCH) (Czajka & Lee, 1990).
larvae, and 2- and 5-day-old male and female adults in
Proposed mechanisms of RCH include elevated levels
D. melanogaster. Life stages in which cold did not
of glucose and trehalose, restructuring of plasma mem-
upregulate Fst had high constitutive expression. Frost
branes through changes in saturation composition
is located on the opposite strand of an intron of
(Overgaard et al., 2005, 2007; although MacMillan et al.,
Diuretic hormone (DH), but cold exposure did not
2009, found decreased glucose and no consistent
upregulate DH. Frost orthologues were identified in
changes in membrane composition after RCH pretreat-
six other species within the Melanogaster group
ments in D. melanogaster) and protection against cold-
(Drosophila sechellia, Drosophila simulans, Droso-
shock induced apoptosis caused by chilling injury (Yi
phila yakuba, Drosophila erecta, Drosophila ananas-
et al., 2007). Microarrays suggest significant differences
sae and Drosophila mauritiana). Frost orthologues
in constitutive global gene expression following selection
were upregulated in response to cold exposure in both
for cold tolerance in D. melanogaster (Telonis-Scott et al.,
sexes in adults of all of these species. The predicted
2009) or between populations with different cold exposure
structure of a putative Frost consensus protein shows
history in Drosophila subobscura (Laayouni et al., 2007).
highly conserved tandem repeats of motifs involved in
Microarrays have also identified a small number of genes
cell signalling (PEST and TRAF2), suggesting that Fst
upregulated by D. melanogaster during recovery from
might encode an adaptor protein involved in acute
cold exposure (Qin et al., 2005; Zhang, 2010), but there
stress or apoptosis signalling in vivo.
are few robust candidate genes associated with cold in
Keywords: Cold tolerance, gene transcription, Frost, Drosophila.
ontogeny. The most consistently reported cold-related candidate
gene is Frost (Fst), which is notably upregulated by adult
First published online 28 September 2011. D. melanogaster during recovery from cold exposure
Correspondence: Brent J. Sinclair, Department of Biology, The University (Goto, 2001; Qin et al., 2005; Sinclair et al., 2007). Goto
of Western Ontario, London, ON, Canada N6A 5B7. Tel.: + 1 519 661 2111 (2001) concluded from putative protein sequence that Fst
extension 83138; fax: + 1 519 661 3935; e-mail: bsincla7@uwo.ca is homologous to mucins, and that the presence of a
Present addresses: 1Department of Chemistry & Biochemistry, Laurentian putative signal peptide at the N-terminus suggested that
University, Sudbury, ON, Canada; 2Department of Human Genetics, McGill
University and Genome Quebec Innovation Centre, Montreal, QC, the Frost protein would be secreted extracellularly. Frost
Canada. has high constitutive expression in D. melanogaster

2011 The Authors

Insect Molecular Biology 2011 The Royal Entomological Society 31
32 X. Bing, J. Zhang and B. J. Sinclair
Figure 1. Location of Frost (Fst) on the opposite strand of an intron of
Diuretic hormone (DH) of the right arm of chromosome 3 in Drosophila
melanogaster. Based on the Flybase Genome Map (http://flybase.org).
Malpighian tubules and midgut (Wang et al., 2004).
There was no association between amongst-population
sequence variation at the Frost locus and variation in chill
coma recovery time in D. melanogaster from different
populations in Australia (Rako et al., 2007), but actin-
driven RNA interference (RNAi) knockdown of Fst expres-
sion did increase chill coma recovery time and mortality
and decrease climbing ability after 12 h exposure to 0 C
(Colinet et al., 2010). Thus, although its role is unknown, it
is clear that expression of Fst is associated with cold
recovery in adult D. melanogaster. Frost is located on the
opposite strand of an intron in the diuretic hormone (DH)
gene on the right arm of chromosome 3 (Fig. 1). Diuretic
hormone plays a significant role in water balance of
insects (Chown & Nicolson, 2004), so it is interesting to
note that the loss of ion homeostasis leading to water
imbalance across the gut epithelium appears to cause
chilling injury (MacMillan & Sinclair, 2011). Diuretic
hormone has not been identified as a candidate in any
previous studies of gene expression in response to cold,
and the expression of DH during and after cold exposure
has not been investigated.
To date, all measurements of gene expression in rela-
tion to cold exposure in Drosophila have been conducted
on adult flies. However, there are significant changes in
both cold tolerance and its plasticity during development
of D. melanogaster (eg Jensen et al., 2007; Rajamohan
& Sinclair, 2008). Understanding patterns of candidate
gene expression in other developmental stages is one
approach to determining whether responses to cold are
part of a general stress response. Thus, the first objective
of this work is to measure constitutive and cold-induced
expression of Frost in all life stages of D. melanogaster,
including a comparison of male and female adult flies of
differing ages. The second objective was to determine
whether expression of Fst and DH are correlated in adult
D. melanogaster, given their physical colocalization in the
D. melanogaster genome.
The availability of complete genome sequences for a
dozen species of Drosophila (Clark et al., 2007) makes
it possible to readily identify orthologues to Frost and
other candidate genes. Low temperature tolerance varies
amongst Drosophila species (eg Kimura et al., 1994;
Gibert & Huey, 2001; Gibert et al., 2001; Strachan et al.,
2011). Our third objective was to identify orthologues to
Insect Molecular Biology 2011 The Royal Entomological Society, 21, 3139
Frost in the Drosophila genomes, and to determine
whether expression of Fst orthologues is also upregulated
in response to cold exposure. Finally, the availability of Fst
orthologue sequences allows the conserved functional
elements of the Frost protein to be identified. Our fourth
objective was therefore to determine whether these con-
served elements provide clues to the function of the
protein and the possible role of Frost in Drosophila cold
tolerance.
Results
Survival of a 2 h exposure to 0 C was highest in D. mela-
nogaster adults and eggs, and lowest in third instar larvae
and prepupae (Fig. 2). Close to 100% of D. melanogaster
adults survived a 2 h exposure to 0 C, which was signifi-
cantly higher than other species of Drosophila, whereas
Drosophila willistoni had the lowest survival of the cold
exposure, with approximately 50% mortality (Fig. 2).
Frost was significantly upregulated in response to cold
exposure in eggs, third larval instar (L3), prepupae, pupae
and younger adults, but not in 1st or 2nd larval instars (L1
and L2), wandering third instar larvae, and 20-day-old
adults. There was some variance in the extent of upregu-
lation, with 5-day-old adults showing the greatest induc-
tion (Fig. 3A). Basal expression of Fst was relatively
similar to the egg amongst most life stages, although L1,
L2 and female 20-day-old adults had significantly higher
levels of constitutive expression than the egg, whereas
pupae and 5-day-old males had low levels of constitutive
expression relative to the egg (Fig. 3B).
Frost is located on the opposite strand of an intron of
DH in D. melanogaster, but DH was not upregulated in
either male or female 5-day-old adult flies that upregulated
Fst transcript following cold exposure (Fig. 4).
Potential Fst orthologues were identified in D. simulans,
D. mauritiana, D. erecta, D. sechellia, D. yakuba and D.
ananassae, all of which are in the Melanogaster group,
within the Sophophora subgenus of Drosophila (Fig. 5).
Putative Fst orthologues were significantly upregulated
following cold exposure in both males and females of all
species with the exception of D. erecta females (Fig. 6).
The outgroup gene found in D. willistoni was not signifi-
cantly upregulated in response to cold exposure (Fig. 6).
An amino acid profile was calculated, and predicted
structural features are summarized in Table 2. The maxi-
mum identity of the predicted amino acid alignment was
61.8%. Genes in other genome-sequenced Drosophila
species identified using BLAST failed to meet orthology
criteria (Hirsh & Fraser, 2001), and did not display synteny.
Furthermore, only a partial mRNA sequence for D. mau-
ritiana was available, which allowed analysis of expres-
sion, but did not allow comparison of predicted protein
structure. PREDICTPROTEIN and PSIPRED predicted a
2011 The Authors
 Frost expression in Drosophila 33

Figure 3. (A) Mean SE abundance of Frost mRNA in different life

stages of Drosophila melanogaster after a 2 h exposure to 0 C relative
to abundance in control individuals, measured with real-time PCR. Note
the logarithmic y-axis, where values > 1 indicate upregulation. Frost
expression was normalized to Actin79B. Asterisks indicate significant
upregulation compared to control groups of the same life stage,
calculated in REST software (* P < 0.05; ** P < 0.01; *** P < 0.001). (B)
Mean SE basal abundance of Frost mRNA, normalized to Actin79B,
relative to basal mRNA abundance in the egg. There is no significant
difference in mRNA abundance between columns with the same letter
(F11,35 = 92.658, P < 0.001; Tukeys post hoc P < 0.05). Asterisks
indicate significant up- or downregulation compared to egg (REST
software, P < 0.001 in all cases). Black bars: immature; white bars:
Figure 2. Mean SE survival of Drosophila after 2 h exposure to 0 C, adult males; grey bars: adult females.
measured 72 h post-exposure. (A) Survival of different life stages of
Drosophila melanogaster. L1, L2, L3, larval instars 13; W, wandering
third instar larvae; PP, prepupae; P, pupae; A2, A5, A20, adults 2, 5 and
20 days post-eclosion. Black bars: immature; white bars: adult males;
grey bars: adult females. The same letters above bars indicate that
survival is not significantly different amongst life stages (ANOVA on
arcsine-square root-transformed survival F12,40 = 11.37, P < 0.001,
Tukeys P < 0.05). (B) Survival by five-day post-eclosion adults of eight
different Drosophila species. White bars: adult males; grey bars: adult
females; D.mel, D. melanogaster; D.sim, D. simulans; D.mau,
D. mauritiana; D.yak, D. yakuba; D.sec, D. sechellia; D.ere, D. erecta;
D.ana, D. ananassae; D.wil, D. willistoni. ANOVA on arcsine-square
root-transformed survival indicated that survival was higher in females
than males (F1,40 = 4.3, P = 0.045), and differed significantly amongst
species (F7,40 = 13.5 P < 0.001; the same letters above bars indicate
that survival is not significantly different amongst species, Tukeys post
hoc P < 0.05), but there was no significant species sex interaction
(F7,40 = 0.58, P = 0.77).

conserved signal peptide, and a trans-membrane helix.
The Eukaryotic linear motifs (ELM) server revealed large
conserved sections of low-complexity regions within the
protein across species, with most of the protein being
disordered, and a high potential to be phosphorylated or
Figure 4. Mean SE mRNA abundance of Frost and Diuretic hormone
(DH) in 5-day-old adult Drosophila melanogaster during recovery from
2 h exposure at 0 C, normalized to Actin79B and relative to control flies
that were not exposed to cold. Note the logarithmic y-axis, where values
> 1 indicate upregulation relative to control. White bars: males; grey
bars: females.

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Insect Molecular Biology 2011 The Royal Entomological Society, 21, 3139
34 X. Bing, J. Zhang and B. J. Sinclair
glycosylated (Fig. 7). However, ELM did not predict a
trans-membrane helix in any of the Fst orthologues. Fst
orthologue predicted proteins have an average serine and
threonine content of more than 25%, and a P, E, S, T
content of 50% (Table 2). In addition, ELM predicted
well-conserved multiple tandem repeats of various
amino acid sequence motifs in the PEST region (Table 2,
Fig. 7), along with long segments of well-conserved low-
complexity (or disordered) regions.
Discussion
We have shown that Frost is upregulated in response to
cold exposure in most life stages of D. melanogaster, and
that Fst orthologues within the Melanogaster group of the
Sophophora subgenus are upregulated in response to
cold exposure in adults. Together, these imply that Fst has
Insect Molecular Biology 2011 The Royal Entomological Society, 21, 3139
Figure 5. Alignment of predicted amino acid
sequences, and the consensus amino acid
sequence, of Frost and its putative orthologues in
Drosophila. Dark shading indicates residues that are
100% conserved, grey shading indicates residues
that are 75% conserved amongst species. Alignment
was carried out using MEGA 5 (Tamura et al., 2007)
and the output was generated by DNAman (Lynnon,
Pointe-Claire, QC, Canada).
a conserved and possibly a consistent role in the
molecular processes associated with recovery from cold in
D. melanogaster and its relatives. We also demonstrate
that Fst expression is not coregulated with DH, and that
the latter is not upregulated in response to cold exposure,
even though there is a close link between cold injury and
water balance (MacMillan & Sinclair, 2011). We could not
identify strong orthologues outside the Melanogaster
group, and an apparently related gene in D. willistoni was
not upregulated in response to cold exposure we also
note that D. willistoni had the lowest cold tolerance of the
species examined.
There was variation in the extent of cold induction of Fst
transcription across the life stages of D. melanogaster.
Some stages, such as Wandering third instar and L1 and
L2 larvae and older adults did not upregulate Fst in
response to cold, although it is interesting to note that
2011 The Authors
 Frost expression in Drosophila 35

Figure 6. Relationships amongst Frost orthologues in Drosophila, and the upregulation of their mRNA abundance after cold exposure. A
neighbour-joining tree of Drosophila Frost orthologues was generated from a genomic DNA alignment. The scale indicates the distance as calculated
from multiple alignments, expressed as substitutions per site. Bootstrap values (1000 replicates) are shown at nodes. BLAST-identified Frost orthologues,
including all Drosophila species, were aligned using ClustalW, and the phylogeny was subsequently created using Mega5 (Tamura et al., 2007). Mean
SE abundance of Frost and Frost orthologue mRNA in 5-day-old adult Drosophila, normalized to Actin79B after 2 h exposure to 0 C, and expressed
relative to control flies, which were not exposed to cold. White bars: adult males; grey bars: adult females; D.mel, D. melanogaster; D.sim, D. simulans;
D.mau, D. mauritiana; D.yak, D. yakuba; D.sec, D. sechellia; D.ere, D. erecta; D.ana, D. ananassae; D.wil, D. willistoni. ns indicates that no significant
upregulation of the gene was detected after cold exposure, all other values are significantly different from 1 (P < 0.01 in all cases, calculated by REST
software), indicating upregulation relative to the control.

20-day-old female adults and L2 larvae had the highest
basal expression. If Fst is integral to recovery from a 0 C
exposure, then this increased basal expression could
explain the high survival in these stages (although we note
that male 20-day-old adults did not upregulate Fst in
response to cold or have elevated basal expression, yet
their cold tolerance did not differ from females at the same
stage). Global expression profiling also suggests con-
stitutive upregulation of Fst during some larval instars
(Chintapalli et al., 2007). Nevertheless, the extent of
upregulation or constitutive expression of Frost did not
appear to be closely related to cold tolerance across life
stages or species, which is consistent with the observation
that clinal variation in cold tolerance is not associated with
Fst genotypic variation in adult D. melanogaster (Rako
et al., 2007). As all life stages had some transcription of
Fst, this observation is not inconsistent with the necessity
Figure 7. Motifs of the consensus predicted Frost protein based on
sequence information from six species of Drosophila. Predictions were
made using PREDICTPROTEIN (Rost et al., 2004), PSIPRED (McGuffin
et al., 2000), SignalP 3.0 server (Emanuelsson et al., 2007), and the
Eukaryotic linear motifs server (Gould et al., 2010). Mucin-like regions
previously identified in BLAST searches (eg Goto, 2001) all reside in the
tandem repeat region of TRAF2 and PEST motifs. aa, amino acids.
of Fst for survival of, and recovery from, cold tolerance
(Colinet et al., 2010). An alternative explanation for the
lack of upregulation may be that some life stages have
different kinetics of transcriptional response to cold.
Frost and its orthologues were upregulated in response
to cold exposure in all of the Melanogaster-subgroup
species that we investigated, and the predicted amino
acid sequence and structure were also fairly highly con-
served amongst these species. However, it is unclear how
and why Fst became an integral part of the cold recovery
process in this subgroup, which comprises largely chill-
susceptible species with relatively limited cold tolerance
(Nyamukondiwa et al., 2011; Strachan et al., 2011). There
were no orthologues found in the other genome-
sequenced Drosophila species, which include more cold-
hardy species (Nyamukondiwa et al., 2011; Strachan
et al., 2011). Thus, although Fst is clearly associated with
the response to cold in the Melanogaster group, it is not
associated with any substantial difference in cold toler-
ance between species in the Melanogaster group and
other species of Drosophila.
Frost is thus established as a transcript clearly associ-
ated with recovery from cold exposure in these species,
but transcription data shed little light on the actual function
of the protein. Previous studies (eg Qin et al., 2005;
Colinet et al., 2010) have suggested that the D. melano-
gaster Frost protein might be a mucin-like protein, based
on the presence of PTS motifs (high in proline, serine and
threonine), trans-membrane helices and the presence of
an N-terminal signal peptide. As Fst is highly transcribed
in adult and larval Malpighian tubules and the midgut
(Wang et al., 2004; Chintapalli et al., 2007), Frost might be
a mucin-like protein that plays a role in protecting the gut

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Insect Molecular Biology 2011 The Royal Entomological Society, 21, 3139
36 X. Bing, J. Zhang and B. J. Sinclair

following cold exposure. However, Syed et al. (2008) did
not identify Frost as a potential mucin in their genome-
wide analysis, and our analysis of consensus sequences
suggest that the PTS motifs that had been previously
reported are more likely to be PEST motifs (incorporating
a glutamine as well, Rogers et al., 1986), and the addition
of this glutamine is likely to change the dynamics of the
repeats such that they probably cannot be considered
PTS analogues. Furthermore, a transmembrane helix was
predicted by only one piece of protein prediction soft-
ware (PSIPRED), and only in D. melanogaster. Thus, with
neither the characteristic PTS region nor trans-membrane
helices of mucins, the consensus Frost protein does not
appear to be a mucin-like protein.
The predicted protein sequences of Frost orthologues
(and the consensus sequence) suggest high net charge
and low hydrophobic amino acid content typical of disor-
dered regions (flexible regions of proteins that lack
secondary structure under physiological conditions and
which typically contain PEST motifs; Tompa, 2002; Dyson
& Wright, 2005). The structural plasticity of disordered
regions allows them to be highly reactive, for example by
glycosylation or phosphorylation or by proteolysis through
calpain cleavage and ubiquitin-proteasome degradation
(Dyson & Wright, 2005). Tumour necrosis factor receptor
consensus motifs are in tandem with the PEST regions in
Fst and its orthologues, and these motifs are generally
associated with apoptosis and cell signalling during the
stress response (Bradley & Pober, 2001; Tompa, 2002),
which is consistent with a potential role of apoptosis in
cold shock injury in D. melanogaster (Yi et al., 2007). Goto
(2001) noted that PEST motifs often indicate rapid degra-
dation, suggesting that the responses mediated by Frost
may be rapid and transient. Together, these suggest that
Frost might play a role as a signalling protein during acute
stress response and apoptosis. Further elucidation of
the role of Frost and its orthologues could include RNAi
knockdown experiments (eg Colinet et al., 2010) or
tissue-specific expression studies, but to allow significant
improvements in understanding, the development of an
antibody or green fluorescent protein fusion protein would
allow the Frost protein to be investigated.
Experimental procedures
Fly rearing and sample collection
Flies were mass-reared on banana-yeast-proprionic acid medium
at 22 C, 50%RH, 13:11 light : dark as described previously (Nya-
mukondiwa et al., 2011). We used eight species of Drosophila,
primarily from long-standing laboratory colonies (Table 1). Droso-
phila melanogaster were used at 10 stages of development:
egg (8 h after oviposition), larval instars I (L1), II (L2), feeding (L3)
and wandering (W) instar III, prepupae (PP), pupae (P), adult
male and female two (A2M or A2F for males and females,
respectively), five (A5M or A5F) and 20 (A20M or A20F) days

Table 1. Primer pairs for quantitative real-time PCR in this paper

Primer sequence Source (collection location) Product length (bp)

Frost
Drosophila melanogaster (Left) 5-CGATTCTTCAGCGGTCTAGG-3 Sinclair et al., 2007 92
(Right) 5-CTCGGAAACGCCAAATTTTA-3 (London, ON, Canada)
Drosophila sechellia (Left) 5-GAATCCGTGGAATCCAAATG-3 GM26233 237
(Right) 5-ACAACATCATCCTCGGTGGT-3 (Cousin Island)
Drosophila simulans (Left) 5-CACCACCGACTCTGAGGACGGT-3 CG9493 Line KY237 134
(Right) 5-TGGATTCTTCGGGAGCCTCGGT-3 (Madagascar)
Drosophila yakuba (Left) 5-TGGTAATGGTCATGGTCACG-3 GE24753 269
(Right) 5-GTTCCTGAGTGGTGGATTGC-3 (Liberia)
Drosophila mauritiana (Left) 5-CATCATGGCAACAATCATGG-3 G105 CG9434 219
(Right) 5-ACCGCTACCTTCTTCAGTGG-3 (Mauritius)
Drosophila erecta (Left) 5-CACGGAAACAACATTCATGG-3 GG17348 262
(Right) 5-CCAAGGTGGTGCTATCTTCC-3 (Tucson)
Drosophila ananassae (Left) 5-TTGGAGGCAATTGGATTAGG-3 GF18404 195
(Right) 5-CGTGATTGTGACCGTTTCC-3 (Kagoshima)
Drosophila willistoni (Left) 5-GGTGCCCGCTGGAAGGCAAT-3 GK11989 195
(Right) 5-TTCAGCTCGCCGATTGCGCA-3 (Guadeloupe)
Actin 79B
D. melanogaster (Left) 5-CCAGGTATCGCTGACCGTAT-3 CG7478 158
(Right) 5-TTGGAGATCCACATCTGCTG-3 (London, ON)
All other species (Left) 5-CACACCTTCTACAATGAGCTGCG-3 CG7478 120
(Actin 79B orthologues) (Right) 5-CATGATCTGGGTCATCTTCTCGCG-3 (London, ON)
Diuretic hormone
D. melanogaster (Left) 5-GCTCTGAATGATGAAAGCCACAGCG-3 CG8348/ 176
(Right) 5-CATGATCTGGGTCATCTTCTCGCG-3 (London, ON)

Primers were obtained from the literature or designed from database sequences using PRIMER-3.
Product length was calculated by PRIMER-3.
Source numbers refer to the transcript identities in Flybase (http://www.flybase.org) or a published primer.

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Insect Molecular Biology 2011 The Royal Entomological Society, 21, 3139
 Frost expression in Drosophila 37

Table 2. Amino acid composition of putative

Frost orthologues in Drosophila Conserved motifs Signal
Species S-T-P-E % (number of repeats) peptide

Drosophila simulans 52 PEST (15) Yes

TRAF (12)
SH2 (5)
Drosophila sechellia 50 PEST (16) Yes
TRAF (16)
SH2 (5)
Drosophila melanogaster 50 PEST (11) Yes
TRAF (11)
SH2 (5)
Drosophila erecta 48 PEST (11) Yes
TRAF (7)
SH2 (5)
Drosophila yakuba 49 PEST (12) Yes
TRAF (12)
SH2 (5)
Drosophila ananassae 46 PEST (13) Yes
TRAF (13)
SH2 (6)
Drosophila willistoni 46 PEST (18) No
TRAF (16)
SH2 (18)

Compositions were calculated from predicted amino acid sequences of Frost orthologues using
MEGA v. 5 (Tamura et al., 2007).
PEST motifs [serine (S) + threonine (T) + proline (P) + glutamic acid (E)] comprise approximately
half of the total amino acid sequence in Frost and its orthologues.
The presence of signal peptides was determined with the SignalP 3.0 server (Emanuelsson et al.,
2007).

post-eclosion. Five-day-old males and females of the other

Drosophila species were used.
Eggs, larvae and pupae were collected under the microscope,
rinsed of food with de-ionized water and blotted dry with a
tissue. Larval stages were confirmed for a subsample by the
morphology of anterior spiracles and size of the mouth hook
(Ashburner, 1989). Prior to cold exposure, eggs and larvae
were placed back into fresh 35 ml vials with 4 ml media to
reflect rearing conditions and for practical reasons (Jensen
et al., 2007), and sealed in plastic bags. Pupae were treated
similarly, but without food in the vial. Virgin male and female
flies were collected within 8 h of eclosion under CO2 anaesthe-
sia, transferred in groups of 30 to 35 ml vials with food and held
at 22 C for at least 48 h before treatment (Nilson et al., 2006),
and were not subsequently anaesthetized. Adult flies were cold-
exposed in 35 ml vials, with food. Vials containing Drosophila
were placed in sealed plastic bags, which were submerged in
ice-water slurry (0 C) for 2 h cold exposure, and then allowed
1 h to recover at 22 C (Sinclair et al., 2007). Control groups of
animals were kept in food vials at 22 C for the full period of
exposure/recovery, and otherwise treated exactly as the treat-
ment groups of animals. For larvae and adults, only individuals
that were moving at the end of the recovery period were col-
lected for RNA extraction. Samples for RNA extraction were
snap-frozen in liquid nitrogen and stored at -80 C. To obtain
biological replicates, each experiment was repeated three times
on separate generations.
Survival of a 2 h exposure to 0 C was measured by removing
three to eight vials of individuals to 22 C. Survival was measured
as the ability for adults to move in a coordinated fashion 72 h
post-exposure. For juvenile stages, survival was assessed as
successful eclosion of adults.
RNA extraction and quality control
Total RNA was isolated using the RNeasy Kit (Qiagen, Valencia,
CA, USA) according to the manufacturers instructions. Extracted
RNA was re-suspended in DEPC-treated water. The purity and
quantity of RNA was assessed using Nanodrop ND-1000 (A260/
A280 and A260/A230 > 1.8; Nanodrop Technologies, Wilmington,
DE, USA). RNA samples were treated with DNase I (Invitrogen,
Carlsbad, CA, USA) to remove genomic DNA and isolated RNA
was stored at -80 C until reverse transcription. A total of 5 mg
of RNA was used for single-stranded cDNA synthesis from
polyadenylated mRNA, using Oligo-dT primers and SuperScript
II Reverse Transcriptase (Invitrogen). Three to five treatment
groups each of 100 eggs through second larval instar, 30 third
larval instar and adults were used for real-time PCR (RT-PCR),
giving cDNA extracts from at least three different generations per
treatment group. Expression of DH following cold exposure rela-
tive to control was determined in 5-day-old adult male and female
flies only.
Real-time PCR
Primer sequences for RT-PCR were either sourced from the
literature or designed from transcript sequences from Flybase
(http://flybase.org/), using Primer3 (http://frodo.wi.mit.edu/
primer3/), with DNA primer length of 2025 bases and G/C
content around 60% (Table 1). Primers were synthesized by Invit-
rogen, and were tested on cDNA using conventional PCR ampli-
fied by Taq polymerase (Invitrogen) to ensure that there was a
single product of the desired length. All cDNA samples from the
same life stage or species were pooled and separated into sub-
samples to generate a standard concentration curve from 1.6 to

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Insect Molecular Biology 2011 The Royal Entomological Society, 21, 3139
38 X. Bing, J. Zhang and B. J. Sinclair
1000 mM for each primer pair. The reaction efficiencies of the
standard curves were close to 1, and r2 > 0.99, with unique peaks
in melting curves indicating unique products. Expression of each
target gene was normalized to Actin-79B throughout, using a
published primer for D. melanogaster, and a single general
primer designed to work for all of the other species (Table 1).
RT-PCR was performed using the SYBR Green PCR Master
Mix (Applied Biosystems, Warrington, UK) on a Rotor-Gene 6000
Cycler (Corbett, San Francisco, CA, USA) with the following
programme: 10 min at 95 C; up to 45 repeats of 15 s at 95 C,
15 s at 55 C and 30 s at 72 C followed by a melting curve.
Analysis was performed for each biological replicate in technical
triplicate, alongside a negative control and a triplicate of pooled
sample used to generate a standard curve for calibration. Both the
target gene and reference gene were amplified in the same run for
each sample. Variation in threshold cycle (Ct) values of technical
replicates did not exceed 0.9 cycles in any sample, and the
average difference in Ct value was 0.2 0.3 cycles per sample.
Threshold cycles were normalized to Actin-79B expression
using the efficiency correction method in REST 2009 software
(Qiagen). Relative expression was calculated in two ways accord-
ing to the general approaches of Pfaffl (2001). To detect upregu-
lation of target genes in response to cold exposure, normalized
expression of cold-exposed groups was compared to control
groups of the same life stage and species. To compare constitu-
tive expression of Fst between life stages of D. melanogaster,
normalized expression of controls of each life stage was normal-
ized to that of control eggs.
Data analysis
Relative gene expression is reported as mean SE throughout.
Upregulation of transcript expression was compared between
control and cold-exposed treatments by a pairwise fixed-
reallocation randomization test in the REST software. Relative
expression was compared among developmental stages using
ANOVA and Tukeys post hoc test (SPSS, ver. 16.0 for Windows,
SPSS, Chicago, IL, USA). Arcsine-square root-transformed
survival was compared amongst developmental stages, sexes
and species using ANOVA followed by Tukeys post-hoc tests in R
2.11 (R Development Core Team, 2010).
A tBLASTx search for Fst orthologues was conducted on the
NCBI website amongst all other organisms (http://blast.
ncbi.nlm.nih.gov/). Matches were identified as potential ortho-
logues based on the reciprocal best hit method (Hirsh & Fraser,
2001), and confirmed by synteny of putative orthologues. Droso-
phila willistoni was selected as an outgroup because although it
had a matching region, the gene did not fulfil the criteria to be a
functional orthologue of Fst. Multiple sequence alignment of pre-
dicted amino acid sequences was conducted using CLUSTALW
in MEGA v. 5.0 (Tamura et al., 2007), and output was generated
using DNAman for Windows (v. 5.2.2, Lynnon Biosoft, Canada).
The alignments were compared with orthologues identified in
the genome sequences in FLYBASE (http://flybase.org/). Protein
structures were predicted using PREDICTPROTEIN (Rost et al.,
2004) and PSIPRED (McGuffin et al., 2000). Signal peptides
were predicted with the SignalP 3.0 Server (Emanuelsson
et al., 2007). Linear motifs, regions of protein that are indepen-
dently responsible for mediating some sort of function (ie inter-
action with another protein), were predicted with ELM (Gould
et al., 2010).
Insect Molecular Biology 2011 The Royal Entomological Society, 21, 3139
Acknowledgements
Thanks to Drs Chris Guglielmo, Amanda Moehring, Greg
Thorn and Nusha Keyghobadi for access to equipment,
Drs Andr Lachance, Anthony Percival-Smith and Alex-
ander Timoshenko for advice and Dr Hiroko Udaka and
Heath MacMillan for assistance and advice in the labora-
tory. The manuscript was improved thanks to critical and
constructive comments from several referees. This work
was supported by funding to B. J. S. from the Canadian
Foundation for Innovation, an Early Researcher award
from the Ontario Ministry for Research and Innovation and
an NSERC Discovery grant.
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