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First published online June 12, 2009
Journal of Experimental Biology 212, 2075-2084 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.027367
Embryonic diapause highlighted by differential expression of mRNAs for ecdysteroidogenesis, transcription and lipid sparing in the cricket Allonemobius socius
Division of Cellular, Developmental and Integrative Biology, Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA
Author for correspondence (e-mail:
reynolds.473{at}osu.edu)
Accepted 28 March 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: diapause, metabolic depression, ecdysone, cell cycle, lipid metabolism, qRT-PCR
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Tauber and Tauber, 1976;
Denlinger, 1986;
Hand, 1991;
Hand et al., 2001;
Denlinger, 2002). Diapause is
endogenously controlled, and this dormancy typically begins well before
conditions become too harsh to support normal growth and development. Entry
into diapause is widespread among insects and can occur at any stage of the
life cycle, although the specific stage at which diapause is initiated depends
on the species. Some physiological changes that occur during diapause have
been characterized for a variety of species (e.g.
Rakshpal, 1962a;
Rakshpal, 1962b;
Izumigama and Suzuki, 1986;
Loomis et al., 1996;
Podrabsky and Hand, 1999).
However, there have only been a few studies that address the molecular
mechanisms that regulate diapause
(Flannagan et al., 1998;
Blitvich et al., 2001;
Jones et al., 2001;
Denlinger, 2002;
Hong et al., 2006;
Kim et al., 2006;
Robich et al., 2007), and the
majority of these studies have looked at molecular regulation of diapause in
larvae, pupae and adults. The molecular regulation of embryonic diapause has
received less attention, probably because of the difficulty of extracting the
limited amounts of RNA from a developmental stage that has comparatively
little tissue and large amounts of protein and lipid. Because of the extreme
differences in complexity of tissues in adults, pupae, larvae and embryos, it
is unlikely that the molecular regulation of diapause is the same for all life
stages. The goal of this study is to reveal genes that may have a role in
diapause entry for embryos of the southern ground cricket, Allonemobius
socius (Scudder). We used PCR-based subtractive hybridization and
quantitative real-time PCR (qRT-PCR) to identify genes that are significantly
up- or downregulated in embryos that are preparing to enter diapause or that
have been in diapause for 10 days.
The only insect embryo for which gene expression during diapause has been
studied extensively is the silkworm, Bombyx mori (e.g.
Dorel and Coulon, 1988;
Suzuki et al., 1999;
Hong et al., 2006). In this
species, the initiation of diapause is accomplished through the action of a
`diapause hormone' which targets the ovaries of females that have received the
appropriate cues and initiates the diapause program by altering the
carbohydrate composition of the eggs produced
(Xu et al., 1995). However,
diapause hormone is not widespread among insects; in fact, this peptide has
only been found in a few Lepidoptera (Xu
et al., 1995). Therefore it is unlikely that the regulation of
diapause in this species is universal among insect embryos.
Allonemobius socius is an ideal animal for studying mechanisms
that regulate embryonic diapause because adult females can produce either
diapause or non-diapause embryos. Although this species is univoltine in the
northern part of its range (i.e. has only one generation per year and has an
obligate diapause), the species produces two or more generations per year in
the southern part of its range and has a facultative diapause
(Fulton, 1931;
Howard and Furth, 1986;
Mousseu and Roff, 1989). By manipulating the environmental conditions
experienced by lab colonies of A. socius, it is possible to alter the
proportion of diapause and non-diapause embryos produced by the population and
simultaneously obtain diapause and direct developing, non-diapause embryos.
This plasticity permits the direct comparison between diapause and
non-diapause embryos that are at a similar developmental stage and thus allows
us to separate ontogenetic changes from those that result from the initiation
of diapause.
Previous research on the physiological and biochemical characteristics of
embryonic diapause in various vertebrate and invertebrate species shows
dormancy at this stage is defined by arrest of cell proliferation/development
(Nakagaki et al., 1991;
Podrabsky and Hand, 2000),
depression of protein synthesis (Clegg et
al., 1996; Podrabsky and Hand,
2000), increased production of heat shock proteins and chaperones
(Clegg, 2005; Qui et al., 2007;
Qui and MacRae, 2007) and in many cases by depression of metabolism
(Clegg et al., 1996;
Podrabsky and Hand, 2000;
Loomis et al., 1996);
(Reynolds and Hand, 2004 and
references therein). Therefore, we hypothesized that there would be
significant upregulation of transcripts that encode for cell stress proteins
and cell cycle regulators in pre-diapause embryos compared with non-diapause
embryos. We also predicted there to be downregulation of genes encoding
products required for protein synthesis. A. socius appears to be
unusual in that there is not an acute metabolic depression during diapause
entry at the point when development ceases in the late gastrula stage
(4–5 days post-oviposition)
(Reynolds and Hand, 2009).
However, the ontogenetic increase in respiration observed for the non-diapause
embryos is fully blocked during diapause, such that metabolic rate is only 36%
of the rate measured for 15 days developing embryos. Thus, we also predicted
there to be differences in the mRNA abundance for genes encoding proteins that
regulate energy metabolism. This study looks at transcript abundance not only
in pre-diapause embryos but also in embryos that have been in diapause for
several days. Some transcripts present in pre-diapause embryos may be required
for initiating diapause, but not be necessary for maintaining the diapause
state. Thus, we expect there to be significant adjustments in transcript
abundance as diapause progresses.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Photoperiod was adjusted to encourage production
of either diapause or non-diapause embryos, which were collected as described
elsewhere (Reynolds and Hand,
2009).
RNA isolation and cDNA synthesis
Total RNA was isolated from pre-diapause and non-diapause A.
socius embryos that were reared at 29°C for 2–4 days
post-oviposition using RNAwiz reagent (Ambion, Austin, TX, USA). The
manufacturer's protocol was followed except for minor changes to help improve
recovery from limited starting tissue and to avoid interference from the large
amount of yolk. Briefly, approximately 100 eggs (i.e. approximately 30 mg
fresh mass) were homogenized in 500µl of RNAwiz with a Kontes Pellet Pestle
driven by a cordless Kontes Pellet Pestle motor (Kimble/Kontes, Vineland, NJ,
USA). The homogenate was drawn through an 18 gauge needle to further disrupt
the tissue and to improve mixing. After a 15 min incubation at room
temperature, a 0.2 volume of chloroform and a 0.1 volume of RNase-free water
were added to the homogenate. After an additional 15 min incubation at room
temperature the homogenate was centrifuged at 18,000 g for 30
min at 4°C to separate the RNA from DNA, protein and other cell debris.
Total RNA was precipitated from the supernatant with 0.5 volume of RNase-free
water and 1 volume of isopropanol and centrifugation at 18,000
g. The resulting RNA pellet was washed with cold 75% ethanol
and re-centrifuged. The final RNA pellet was resuspended in water, and the
purity and concentration of the RNA were assessed using a Nanodrop 1000
Spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Samples were
stored at –80°C until used for cDNA synthesis.
To obtain sufficient starting material for the cDNA subtraction, multiple
independent isolations (100 embryos each) were performed, and RNA samples were
later pooled. Since a single A. socius female may produce a mixture
of diapause-destined (pre-diapause) and non-diapause embryos, it was necessary
to verify the diapause status of the embryos before combining the RNA samples
into either pre-diapause or non-diapause batches. To do this, a subset of 15
embryos from each sample was returned to the 29°C incubator and allowed to
develop for an additional 10 days (the remaining embryos in each sample where
immediately homogenized as described above). After this extra incubation time,
embryos were chemically fixed and the chorion was cleared
(Hogan, 1959) as described by
Reynolds and Hand (Reynolds and Hand,
2009). Embryos within the egg were observed using a Leica MZ7
stereomicroscope, and the number of embryos exhibiting the diapause morphology
(see below) was counted. A. socius embryos that do not enter diapause
typically hatch after about 15 days; as such, after 10 days, non-diapause
embryos exhibited a distinctly different morphology than diapause embryos. An
embryo was considered to possess the diapause morphology if it was centered in
the middle of the yolk, was approximately 0.8 mm long, had a well developed
procephalon, and lacked obvious limb development. An RNA sample was
categorized as `pre-diapause' if at least 70% of the embryos from which it was
prepared showed the diapause morphology after 15 days. For 75% of all
pre-diapause samples, the composition was actually greater than 90%
pre-diapause embryos. Twenty-one confirmed pre-diapause samples were pooled to
yield 155 µg total RNA. A sample was classified as `non-diapause' if there
were at least 70% non-diapause embryos present; 57% of non-diapause samples
were composed of at least 80% non-diapause embryos. Fourteen confirmed
non-diapause samples were pooled to yield 100 µg total RNA.
Messenger RNA (mRNA) was isolated from the pooled, total RNA using OligotexTM beads (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. Equivalent quantities (0.5 µg) of mRNA from pre-diapause and non-diapause embryos were then used to synthesize cDNA using a SMART cDNA synthesis kit (Clontech, Mountain View, CA, USA) following the manufacturer's protocol.
Subtractive hybridization
Forward and reverse subtractions were performed using a PCR-based
subtractive hybridization kit (Clontech, Mountain View, CA, USA) according to
the manufacturer's protocol. The forward subtraction, intended to isolate
genes upregulated in pre-diapause embryos compared with non-diapause embryos,
was performed using driver cDNAs generated from pre-diapause mRNA and tester
cDNA from non-diapause embryos. The reverse subtraction, to identify genes
present only in non-diapause embryos (i.e. downregulated in pre-diapause
embryos), was performed using driver from cDNA from non-diapause RNA and
tester from cDNA from pre-diapause embryos. Briefly, double stranded (ds) cDNA
from tester and driver populations was digested with the restriction enzyme
RsaI to remove the oligonucleotides added during SMART cDNA synthesis
and to generate short cDNA molecules with blunt ends. The tester cDNA was
divided into two pools and each ligated to a different cDNA adaptor. As
recommended by the manufacturer's protocol, PCR and gel electrophoresis were
used to confirm at least 25% of ds-cDNA strands possessed adaptors (data not
shown). In these reactions, primers complementary to the adaptors were used in
combination with gene-specific primers for the COX I subunit. COX primers were
designed by aligning sequences from several Orthoptera species. The forward
primer sequence was 5'-AGCTCCTGATATAGCATTCCCACG-3' and the reverse
sequence was 5'-AGGGCTGTAATACCAACGGCTCAT-3'. Two hybridizations
were performed to (1) equalize high- and low-abundance molecules and (2)
enrich for cDNA molecules unique to the driver population. PCR using nested
primers was employed to further amplify only differentially expressed
sequences. These products were used to construct forward- and
reverse-subtracted libraries as described below. To evaluate the efficiency of
the subtractions, we compared the transcript abundance of actin in subtracted
and unsubtracted cDNA populations using degenerate primers (forward primer
5'-ACAATGGMTCYGGWATGTGCAARGCT-3'; reverse primer
5'-CCCAGTTKGTWACAATWCCRTGCT-3'). Gel electrophoresis of the PCR
products showed that the subtraction greatly reduced the amount of
actin, originally a highly abundant gene, relative to unsubtracted
controls.
Sequencing and bioinformatics analysis
The forward and reverse subtracted cDNAs, ligated into pGem vector, were
transfected into JM109 competent cells (Promega, Madison, WI, USA).
Transformed cells were grown overnight on LB plates containing ampicillin,
X-Gal, and IPTG for blue/white selection. For each library, 300–400
white colonies were isolated and grown overnight in LB-ampicillin broth at
37°C. Plasmid DNA was extracted and purified using a Qiaprep miniprep kit
(Qiagen, Valencia, CA, USA). Single pass sequencing was carried out using a
primer for the sp6 promoter and BigDye terminator chemistry on an ABI PRISM
3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences
were identified by homology to known genes using NCBI Blastx search of the
GenBank database. The functions of identified genes were evaluated with the
UniProt Knowledgebase (ExPasy proteomic server;
http://ca.expasy.org/).
Quantitative real-time PCR analysis
Quantitative real-time PCR (qRT-PCR) was used to assess the relative mRNA
abundance in pre-diapause and diapause embryos compared with non-diapause
embryos. Total RNA was isolated from pre-diapause and non-diapause embryos
that had developed for 2–4 days post-oviposition as described above, and
also from diapause embryos that were incubated for 15 days post-oviposition
(i.e. 10 days in diapause). As before, independently isolated RNA samples were
pooled to obtain sufficient total RNA. All `pre-diapause' samples for qRT-PCR
contained at least 83% confirmed pre-diapause embryos, whereas all
`non-diapause' samples for qRT-PCR contained 80% or more non-diapause embryos.
The `diapause' samples (15 days post-oviposition) contained 100% diapause
embryos, because non-diapause embryos could easily be distinguished and
removed at this late development point. For each of the three developmental
stages, 4 µg of total RNA, was reverse transcribed in an independent 20
µl reaction containing 500 ng of random primers (Integrated DNA Technology,
Coralville, IA, USA), 0.5 µmol l–1 dNTPs, 1x first
strand buffer, 5 mmol l–1 DTT, 1 µl RNaseOUT and 200 i.u.
of SuperScript IIITM Reverse Transcriptase (Invitrogen, Carlsbad, CA,
USA). Incubations were carried out according to the manufacturer's
instructions with an additional 5 min incubation at 25°C to promote
annealing of the random primers. After a 60 min incubation at 50°C,
samples were heated to 70°C for 15 min to inactivate the enzyme.
qRT-PCR was performed with a Bio-Rad iCycler (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Green mix (Roche, Indianapolis, IN, USA) to which 10 nmol l–1 fluorescein (Bio-Rad) was added to allow well-factor correction. The total reaction volume of 20 µl included 300 nmol l–1 of primers and 2 µl of template. PrimerQuest software (IDT DNA, Coralville, IA, USA) was used to design primer sequences (see supplementary material Table S1). Cycling parameters were 10 min at 95°C followed by 50 cycles of 95°C for 15 s, 58°C for 30 s and 72°C for 30 s. Analysis of melt curves verified that only one product was amplified in each reaction.
Calculations and statistical analysis
mRNA abundance for each developmental stage was evaluated in four to six
independent experiments, each with three technical replicates per primer pair.
Each experimental plate included three wells with primers for 18S rRNA, the
reference gene used for all analyses. After subtracting the baseline value
with software provided by Bio-Rad, the cycle threshold (Ct) values
were set to give a Ct value of approximately 9.5 for the wells
containing the reference gene, which allowed for easy comparison of results
across all experiments.
The 2–
Ct method was used to
calculate fold change relative to the non-diapause sample and to define
relative mRNA abundance for a particular gene of interest in relation to the
reference gene, 18S rRNA (Mimmack et al.,
2004). Values for fold change in mRNA abundance are given as means
± s.e.m. Under the 2–Ct
method, a fold change of 1 indicates there is no difference in the mRNA
abundance for a gene of interest in the experimental group compared with the
control group. Rather than arbitrarily select a fold-change cut off to
establish significance, we first used the one-way Student's t-test to
identify genes with a significant change in mRNA abundance (Minitab, State
College, PA, USA). Then, to reduce the probability of type I errors from
multiple comparisons, a q-value was calculated for each hypothesis
tested. The q-value is a measure based on the false discovery rate
(Benjamini and Hochberg, 1995)
and is an estimate of the number of false positives expected among the
significant tests. q-Values were calculated from P-values
using the QValue plug-in for R Statistical software
(Storey and Tibshirani, 2003);
a test was considered to be significant when q
0.015.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Eleven of the transcripts were from the upregulated library
(Table 1), and 22 were from the
downregulated library (Table
2). To facilitate the evaluation, these 33 transcripts were
separated into six functional groups.
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Stress proteins and chaperones
Eight transcripts were selected that encode proteins known to protect cells
from stressful conditions such as extreme temperature or oxidative damage.
Pyrroline 5-carboxylate reductase (P5cr) is involved in the biosynthesis of
proline, an amino acid associated with increased cold tolerance in
Drosophila (Misener et al.,
2001). This mRNA increased by 58±16% (q=0.006) in
pre-diapause embryos and was downregulated by 69±4.4% in diapause
(q=0.000) when compared with non-diapause (control) embryos
(Fig. 1). In addition, mRNA
encoding acyl-CoA delta 9-desaturase (desaturase), which converts saturated
fatty acids to monounsaturated fatty acids (MUFAs), was upregulated by 240%
(q=0.007) in pre-diapause embryos compared with non-diapause embryos
(Fig. 1). There was not a
significant change in the abundance of this transcript in diapause embryos
compared with non-diapause embryos (q=0.039).
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), and its
transcript was downregulated by 32±6.9% (q=0.007) in
pre-diapuse embryos and 80±4.0% (q=0.00) in diapause embryos.
Bax inhibitor-1 (BI-1) is also reported to protect against oxidative stress by
inhibiting Bax-induced apoptosis and protecting against cell death induced by
heat shock or oxidative stress
(Huckelhoven, 2004;
Chae et al., 2003). There was
no difference in the mRNA abundance of a BI-1 homolog in pre-diapause embryos
compared with non-diapause embryos (q=0.064), but an 86±3.0%
(q=0.002) reduction in the abundance of this transcript was observed
in diapause embryos (Fig.
1).
Fig. 1 also illustrates the relative transcript abundance of six heat shock proteins in pre-diapause and diapause embryos. Heat shock protein 20.7 (Hsp20.7), Hsp70, Hsp90, endoplasmin (an Hsp90 variant), and CHORD-containing protein (a homolog of the Hsp90 co-chaperone p23; CHORD) are included in this group. Compared with non-diapause embryos only CHORD showed a significant change in mRNA abundance in pre-diapause embryos, where it was upregulated by 44±8.9% (q=0.006). Comparable levels of Hsp20.7 (q=0.123), Hsp70 (q=0.145), Hsp90 (q=0.058) and endoplasmin (q=0.091) mRNAs were present in pre-diapause and non-diapause embryos. In diapause embryos, Hsp20 and Hsp90 mRNAs were reduced by approximately 60% (q=0.002 and q=0.003, respectively), whereas the abundance of endoplasmin mRNA was reduced by 86±3.0% (q=0.001) compared with non-diapause embryos. The abundance of Hsp70 mRNA was the same in diapause and non-diapause embryos (q=0.021).
Energy production and conversion proteins
Cytochrome c oxidase subunits II (COX II), IV (COX IV) and VII
(COX VII), and arginine kinase (AK) are involved in the production of ATP; and
cathpesin B-like protease, ATP-citrate lyase (ACLY), Niemann-Pick type C_2
like protein (NPC2), lipid metabolism protein (LMP) and male-sterility protein
(MSP) are all predicted to have roles in fatty acid and/or lipid metabolism
(see Discussion). Compared with non-diapause embryos, the transcript
abundances of COX II and AK were reduced in pre-diapause embryos by 31%
(q=0.001) and 36% (q=0.003), respectively
(Fig. 2). There was no change
in the amount of COX VII transcript (q=0.176) in pre-diapause
embryos, but the mRNA abundance of COX IV increased 40±8.4%
(q=0.006) in this early stage. When mRNA levels in diapause embryos
were compared with values for non-diapause controls, it was found that all
four of these genes are downregulated by values ranging from about 36% for COX
II to 86% for AK (COX II q=0.001, COX IV q=0.001, COX VII
q=0.004 and AK q=0.000).
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DNA replication, cell cycle regulation and transcription
Developmental arrest is a hallmark feature of embryonic diapause in this
species as noted in our companion paper
(Reynolds and Hand, 2009), and
thus four genes with some role in cell division were evaluated. Histone 2A is
part of the nucleosome and is important for chromosome packaging. As seen in
Fig. 3 there was a modest but
significant 20±2.0% decrease in the amount of histone 2A mRNA
(q=0.006) in pre-diapause embryos, and there was an 86±1.0%
decrease (q=0.01) in this mRNA in embryos that had been in diapause
for 10 days. In Drosophila spaghetti squash encodes a cytoplasmic
myosin that is required for cytokinesis in dividing cells of
(Young et al., 1993;
Wheatley et al., 1995).
However, the abundance of this transcript was not significantly different in
pre-diapause (q=0.09) or diapause embryos (q=0.014) compared
with non-diapause embryos.
|
). There was a 99±1.5% increase in TFDp2
(q=0.002) in pre-diapause embryos
(Fig. 3), but the abundance of
this transcript in diapause embryos was not significantly different from that
of non-diapause embryos (q=0.07). Reptin is a protein that can
repress transcription through its association with c-Myc
(Etard et al., 2005) or
histone acyltransferases (Qi et al.,
2006). In pre-diapause embryos there was a 76±1.8% increase
in Reptin mRNA (q=0.007), and a 71±3.0% drop
(q=0.000) was observed in diapause embryos compared with non-diapause
embryos.
Transcription, translation and post-translational modification
Protein synthesis is an energetically expensive process that has been
previously shown to be depressed under conditions of cell stasis and metabolic
depression (Hochachka and Guppy,
1987; Hand and Hardewig,
1996; Guppy and Withers,
1999; Storey and Storey,
2007). RNA polymerase III (Pol III), which transcribes small
ribosomal RNAs and transfer RNAs that carry amino acids to ribosomes during
translation increased almost 70% (q=0.006) in pre-diapause embryos
compared with non-diapause embryos (Fig.
4) and decreased 47±2.0% (q=0.000) in diapause
embryos. There was a significant decrease in mRNA abundance for only one of
the six proteins involved in translation and post-translational modification
in pre-diapause embryos (Fig.
4). Ribosomal protein L35a (RpL35a) was reduced approximately 40%
(q=0.008). There were no significant changes in the mRNA levels of
translation elongation factor 1B-
(eEF1B-; q=0.17),
translation initiation factor 4- (eIF4-; q=0.06),
ribosomal protein L45 (RpL45; q=0.18), Nedd8 (q=0.09) or MPP
(q=0.013). However, all of the transcripts related to protein
synthesis were significantly depressed by 47–80% in diapause embryos
compared with non-diapause embryos. eEF1B- was reduced by 61±3%
(q=0.001), eIF4- by 57±6.0% (q=0.004), RpL35a
by 80±1.2% (q=0.000), RpL45 by 56±1.2%
(q=0.003), Nedd8 by 75±3.0% (q=0.000) and MPP by
60±3.0% (q=0.002).
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), was 40±13% higher (q=0.009) in pre-diapause
embryos. Activated protein kinase c receptor (RACK1), which is reported to
have a role in ecdysone signaling (Quan et
al., 2006), was moderately, but significantly, upregulated
23±2.1% (q=0.003). In diapause embryos, the abundance of
CYP450 was reduced by 50% (q=0.009), and AKR was reduced by 98%
(q=0.000) compared with non-diapause embryos. IFRD was reduced by
45±2.2% (q=0.000), and RACK1 was downregulated by
67±1.0% (q=0.000) in diapause embryos compared with
non-diapause embryos.
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|
DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Robich et al., 2007). One
interpretation of our finding for A. socius is that genes upregulated
in pre-diapause embryos (i.e. those with the potential to regulate diapause
entry) are transient and are not required to maintain diapause. It could be
argued that an overall depression in transcription (and presumably
translation) as diapause proceeds is consistent with a physiological state
where energy conservation and developmental arrest are advantageous
(Hand and Hardewig, 1996;
Hand et al., 2001;
Hahn and Denlinger, 2007).
Stress proteins and chaperones
Numerous studies have shown that animals in diapause are more resistant to
environmental insults such as cold stress, heat shock and oxidative damage
than con-specific individuals that are not in diapause (e.g.
Podrabsky et al., 2001;
Rinehart et al., 2006;
Hand et al., 2007;
Podrabsky et al., 2007).
However, upregulation of stress proteins is not a general feature of diapause
in A. socius because only three of nine transcripts analyzed
increased in pre-diapause embryos. Two of the genes that are upregulated in
pre-diapause embryos, encoding P5cr and 9 desaturase, are also
upregulated in some overwintering insects. P5cr is involved in the final step
in proline biosynthesis, and this amino acid has been correlated with
increased cold acclimation and tolerance in species ranging from crickets to
flies (Shimada and Riihima,
1990; Ramlov,
1999; Miesner et al., 2001). P5cr expression and the proline pool
are known to be upregulated in cold-adapted Drosophila melanogaster
(Miesner et al., 2001). 9 desaturases are also associated with cold
tolerance in a number of species (Tiku et
al., 1996; Cossins et al.,
2006). The 9 desaturases are lipogenic enzymes that produce
monounsaturated fatty acids (MUFAs), which are components of membranes and
energy storage molecules (Brock et al.,
2006). Increasing the proportion of unsaturated fatty acids is
correlated with the intrinsic fluidity of the membranes and helps maintain the
function of these structures during exposure to low temperatures
(Hazel, 1995;
Hochachka and Somero, 2002;
Michaud and Denlinger, 2006).
Since diapause is an overwintering strategy in A. socius, we predict
that upregulating P5cr and 9 desaturase expression is required to guard
against damage from low temperatures.
Glyoxylase is reported to protect cells from oxidative damage by
enzymatically detoxifying 
-oxoaldehydes, which are produced through the
degradation of glycolytic intermediates and are known to cause mutations and
induce apoptosis (Sommer et al.,
2001). Given its protective function, it is surprising that the
mRNA for this enzyme is downregulated in pre-diapause and diapause embryos.
Perhaps a reduction in glycolytic activity decreases the need for this
protection in dormant embryos.
Expression profiles for heat shock proteins such as Hsp70, Hsp90 and Hsp23
have been studied in numerous animals that enter diapause. In general, the
expression patterns of these genes are species specific. For example, Hsp70
and Hsp23 are upregulated in brain tissue of Sarcophaga crassipalpis
whereas Hsp90 is downregulated (Denlinger,
2002). By contrast, in larvae of Chilo suppressalis,
Hsp90 is upregulated during diapause
(Sonoda et al., 2006); and
none of the heat shock proteins analyzed show differential expression in
diapause larvae of Lucilia sericata
(Tachibana et al., 2005). In
A. socius, only CHORD, a homolog of the Hsp90 co-chaperone p23, is
upregulated in pre-diapause embryos. There is no change in mRNA abundances of
Hsp20.7, Hsp70, Hsp90 or endoplasmin in pre-diapause or diapause embryos
compared with non-diapause embryos; thus upregulation of these genes is not a
requirement for diapause in this species.
Energy conversion and metabolism
Normally metabolic depression is one defining characteristic of diapause,
but for A. socius an acute metabolic downregulation does not occur
during diapause entry at the point when developmental ceases
(Reynolds and Hand, 2009).
Instead, the ontogenetic increase in respiration that is observed during
development of non-diapause embryos is completely prevented by diapause.
Presumably this latter phenomenon would still require a substantial
restriction of transcription and translation of selected metabolic genes at
the onset of diapause, and thus it was of interest to evaluate differential
expression during this transition from growth to maintenance. Certainly other
mechanisms including allosteric inhibition and covalent modification could be
at work, but for long-term adjustments, we predicted that alterations in mRNA
abundance profiles would also occur.
Functionally, the metabolic genes analyzed fall into two groups –
genes involved in oxidative phosphorylation and ATP metabolism and genes
involved in the interconversion of cellular energy reserves. AK is a
phosphagen kinase and is part of the system that buffers ATP levels in insects
(Tanaka et al., 2007).
Downregulation of transcripts for this enzyme
(Fig. 2) is consistent with
data that show low levels of ATP in pre-diapause and diapause A.
socius (Reynolds and Hand,
2009). COX II is one of two subunits forming the catalytic core
cytochrome c oxidase, the terminal complex in the mitochondrial
electron transport chain (Nicholls and
Ferguson, 2002), and COX IV and COX VII are required for proper
assembly and function of the COX complex in mammals and yeast
(Aggler and Capaldi, 1990;
Li et al., 2006). With the
exception of COX IV, the general trend is a decrease in the amount of mRNA
encoding COX subunits in pre-diapause and diapause embryos, a pattern that is
in keeping with the respiration profiles described above. The majority of
genes involved in inter-conversion of cellular energy reserves are
downregulated in pre-diapause embryos. Cathpesin B participates in the
digestion of yolk proteins in a number of insects
(Medina et al., 1988;
Ribolla and De Mianchi, 1995;
Cho et al., 1999;
Zhao et al., 2005).
Downregulation of this gene in A. socius (25% in pre-diapause) could
slow digestion of the yolk proteins during a time when it is critical to
preserve the fuel stores that will be required to complete post-diapause
development. Consistent with lipid sparing, ACLY, LMP and MSP (i.e. acyl-CoA
reductase) are enzymes that promote fatty acid/lipid usage, and all are
downregulated 20–40% in pre-diapause. The trend continues and is more
pronounced with Cathepsin B, ACLY and MSP being more strongly downregulated
(about 80%) in diapause (Fig.
2B). The NPC2 protein, which is involved in sterol metabolism and
homeostasis (Ory, 2004), does
not show differential expression in pre-diapause embryos. With this one
exception, the data are consistent with conservation of lipids during
diapause, a hypothesis supported by previous studies on two Orthoptera
species, Melanoplus differentialis
(Kaocharern, 1958) and
Aulocara elliotti (Visscher,
1976), which show accumulation of lipids in diapause embryos
(Kaocharern, 1958). Finally,
it is notable that ACLY and NPC2 also have been implicated in the synthesis of
juvenile hormone and ecdysone, respectively
(Noriega et al., 2006;
Ioannou, 2007). Both of these
hormones are known regulators of diapause in a number of insect species (see
Endocrine and signal transduction, below).
DNA replication, cell cycle regulators and transcription factors
Development is arrested 4–5 days post-oviposition when A.
socius embryos enter diapause
(Reynolds and Hand, 2009).
Histone 2A, which is downregulated in pre-diapause embryos, is a core histone
protein and is essential to the structure of condensed chromatin. Abundance of
histone mRNA typically increases only during the DNA synthesis phase of the
cell cycle, and mRNA is degraded if DNA synthesis is inhibited
(Anderson and Lengyel, 1980;
Kaygun and Marzulff, 2005).
Downregulation of histone 2A mRNA is consistent with developmental arrest in
general and cell cycle arrest in particular.
TFDp2 and Reptin are transcription factors that play a role in the
regulation of cell proliferation. TFDp2 (DP) is a dimerization partner with
E2F, a transcription factor that can activate or repress the G1 to
S cell-cycle transition (Duronio et al.,
1998; Zheng et al.,
1999). Targets of this heterodimer include cyclinE, cyclinA,
cdc2, c-myc, and growth-regulatory proteins. Activity of E2F-DP depends
on the presence or absence of a retinoblastoma (Rb) family protein. Rb-E2F-DP
complexes arrest the cell cycle at G1 and thus are characteristic
of quiescent cells (Duronio et al.,
1998; Zheng et al.,
1999). Reptin is an evolutionarily conserved protein that
represses transcription through its interactions with c-Myc
(Etard et al., 2005) and
histone acyltransferases (HATs) (Qi et
al., 2006). Because TFDp2 and Reptin are both upregulated almost
twofold in pre-diapause embryos, and Reptin is later downregulated in
diapause, analysis of the transcript abundance of genes encoding E2F, Rb,
c-Myc, HATs and other proteins in these pathways could prove useful for
highlighting mechanisms underlying developmental arrest.
Transcription, translation and protein processing
Although protein synthesis is energetically expensive
(Hand and Hardewig, 1996) and
is depressed during diapause in a number of animals including flies
(Joplin and Denlinger, 1989)
and fish (Podrabsky and Hand,
2000), genes that encode proteins involved in protein synthesis
and post-translational modification are not downregulated in pre-diapause
embryos, which is consistent with the lack of acute metabolic depression at
the onset of diapause in A. socius. Specifically, the mRNA abundance
of four genes, eIF4-, eEF1B-, RpL45
and Nedd8, is the same in pre-diapause and non-diapause embryos. The
exception to this general trend is RpL35A which is downregulated by
almost 50% in pre-diapause embryos. Overexpression of this gene in Jurkat
cells confers resistance to apoptosis-stimulating chemicals
(Lopez et al., 2002), but it
is unlikely that this protein has a protective role in diapause embryos.
Significant upregulation was observed for two genes, RNA Pol III and MPP. Pol III encodes a polymerase that transcribes 5S ribosomal RNA, tRNAs and other small RNAs. MPP is a protein located in the mitochondrial matrix that removes the leader sequence from proteins targeted to these organelles (Muhopadhyay et al., 2002). Upregulation of these genes suggests an increase in translation and protein processing in pre-diapause embryos rather than the expected downregulation. However, all genes in this category are significantly downregulated during late diapause (Fig. 4).
Endocrine and signal transduction
Diapause in insects is, at least in part, regulated by the endocrine system
(Nijhout, 1984;
Yamashita and Hasagawa, 1985;
Denlinger, 2002). In many
species, embryonic diapause results from changes in the titre of ecdysone
(e.g. Gharib et al., 1981)
and/or juvenile hormone, a sesquiterpenoid (e.g.
Visscher, 1976). Three genes
thought to be part of the ecdysone signaling and/or biosynthesis pathways,
RACK1, AKR and CYP450, show an upregulation of mRNA in
pre-diapause embryos and a substantial decrease in mRNA abundance in diapause
embryos compared with non-diapause embryos. RACK1, also known as activated
protein kinase c receptor, is predicted to be activated by 20-hydroxyecdysone,
which results in the phosphorylation and activation of additional components
of the ecdysone receptor and, ultimately, stimulation of genes containing an
ecdysone response element (Quan et al.,
2006). AKR is a member of a protein family that includes more than
120 members with a wide range of physiological functions including polyol
synthesis and steroid metabolism (Luccio
et al., 2006). AKRs are monomeric proteins with NADPH-dependent
catalytic activity. One member of this family is known to be upregulated
during diapause initiation in the heteropteran bug, Pyrrhocoris
apterus (Ko
tál et al.,
2008). Biochemical studies on ecdysteroid metabolism in insects
and crustaceans suggest that aldo-ketoreductase enzymes may be involved in
this pathway (Maibeche-Coisne et al.,
2001; Sieglaff et al.,
2005). AKR transcript is upregulated in Aedes aegypti
ovary at the time ecdysone synthesis peaks, and the authors conclude that
early expression of the AKR gene may be required for activation of
ecdysteroidogenesis (Sieglaff et al.,
2005). CYP450 is part of a large family of enzymes with
mono-oxygenase activity. Numerous members of this family are required for
ecdysone biosynthesis (Gilbert,
2004; Feyereisen,
2005; Huang et al.,
2008) and are known to be expressed in Drosophila
(Chávez et al., 2000)
and Bombyx (Horike et al.,
2000) embryos. Although CYP450s can have a variety of functions,
including the detoxification of xenobiotics, it seems unlikely that a
pre-diapause embryo, which is isolated from the environment by a network of
extra-embryonic membranes as well as a chorion, would have a need for a
detoxification enzyme. Thus we predict that the CYP450 identified in this
study has a function in ecdysteroidogenesis. However, further analysis,
including 3' and 5' RACE to determine the full-length sequence of
the gene as well as enzymatic assays to determine the substrate of this
enzyme, are required to verify the role of this gene in embryonic development
and diapause entry.
It is surprising that the expression of RACK1 (upregulated 1.2-fold),
CYP450 (up 2.4-fold) and AKR (up 1.7-fold) is increased in pre-diapause
embryos of A. socius, because embryonic diapause in insects is
typically correlated with a decrease in, or lack, of ecdysone [e.g. Bombyx
mori (Horike and Sonobe,
1999), Locusta migratoria
(Tawfik et al., 2002),
Chortoicetes terminfera (Gregg et
al., 1987) and Oxya yezoensis
(Kidokoro et al., 2006)].
However, the endocrine events associated with embryogenesis are species
specific and there is considerable variation in ecdysteroid titres even
between species from the same order
(Whiting and Dinan, 1988). In
addition, previous studies on the endocrine events associated with diapause
have looked at species that arrest development at a later point in
embryogenesis (e.g. Wardhaugh,
2006). Thus it is possible that diapause is characterized by an
increase in ecdysone in A. socius rather than a decrease as reported
for the species above. For example in Lymantria dispar, which enters
diapause as a pharate first instar larva, an increase in ecdysone titre is
required for diapause induction and maintenance, and a decrease in ecdysone
occurs upon diapause termination (Lee and
Denlinger, 1996; Lee et al.,
1997). Furthermore, ecdysone has been shown to cause cell cycle
arrest in cultured cells from Aedes albopictus
(Gerenday and Fallon, 2004).
The abundances of RACK1, CYP450 and AKR transcripts are depressed at least 50%
during diapause in A. socius, with AKR being the most dramatically
decreased (98% relative to non-diapause embryos). Coupled with its significant
upregulation during pre-diapause, AKR has the largest differential expression
of any gene in the study – an 85-fold swing between pre-diapause and
late diapause. Consequently, it appears that the functions of these gene
products are necessary for diapause entry but not for the maintenance of the
dormant state. Future studies that quantify the amount of ecdysone,
20-hydroxyecdysone and their conjugates are clearly required to solidify the
roles of RACK1, CYP450 and AKR in diapause entry for A. socius
embryos.
In summary, eight candidate genes have been identified that show promise as
regulators of diapause entry in A. socius embryos and warrant
additional study. Our designations are based both on the magnitude/consistency
of differential mRNA expression and our ability to place the projected
functions of these genes into rational context by considering the
physiological and biochemical events of diapause that we have experimentally
characterized for A. socius
(Reynolds and Hand, 2009). The
functional categories into which the products of these genes fall are
ecdysteroid synthesis and signaling (CYP450, AKR and RACK1), transcription and
cell cycle control (Reptin, TFDp2) and lipid sparing (Cathpesin B, ACYL, MSP).
mRNAs for CYP450, AKR and RACK1 are consistently upregulated in pre-diapause,
followed by major downregulation later in diapause. This initial upregulation
would suggest that elevated ecdysone may facilitate onset of diapause in
A. socius. Our observed upregulation of Reptin and TFDp2 mRNAs may
serve to depress transcription and cell cycle progression. Finally,
transcripts for Cathpesin B-like protease, ACLY and MSP are downregulated,
which may serve to promote lipid sparing during diapause.
|
Footnotes |
|---|
This study was supported by DARPA grant N00173-01-1-G011 and NIH grant 1-R01-GM071345-01. J.A.R. received GIARs from Sigma Xi and the Orthoptera Society. We thank Dana Jones, Joy Norris, and Drs. Michael Menze (Assistant Professor, Research, Louisiana State University) and Scott Herke (Genomics Facility, Louisiana State University) for assistance with EST sequencing. Dr M. Rob Michaud (Postdoctoral Researcher, Ohio State University) assisted with statistical analysis. Deposited in PMC for release after 12 months.
* Present address: Department of Entomology, Ohio State University, Columbus,
OH 43210, USA 
|
References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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