Comparative Biochemistry and Physiology, Part B 227 (2019) 56–63

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Comparative Biochemistry and Physiology, Part B

journal homepage: www.elsevier.com/locate/cbpb

Hypometabolic strategy and glucose metabolism maintenance of Aedes T

aegypti egg desiccation
Renato Martins da Silvaa, Wagner Oliveira Vitala, Rodrigo Nunes da Fonsecaa,
Yolanda Porto Muniz Martinsb, Francisco José Alves Lemosb, Itabajara da Silva Vaz Jrc,
⁎
Carlos Logulloa,b,
a
Laboratório Integrado de Bioquímica Hatisaburo Masuda (NUPEM), Universidade Federal do Rio de Janeiro (UFRJ), Campus Macaé, Macaé, RJ, Brazil
b
Laboratório de Sanidade Animal/CCTA, Unidade de Experimentação Animal, Laboratório de Biotecnologia/CBB, Universidade Estadual do Norte Fluminense Darcy
Ribeiro (UENF), Campos dos Goytacazes, RJ, Brazil
c
Centro de Biotecnologia and Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: The mosquito Aedes aegypti is vector of several viruses including yellow fever virus, dengue virus chikungunya
Desiccation virus and Zika virus. One of the major problems involving these diseases transmission is that A. aegypti embryos
Glucose metabolism are resistant to desiccation at the end of embryogenesis, surviving and remaining viable for several months
Gluconeogenesis inside the egg. Therefore, a fine metabolism control is essential to support these organisms throughout this
Glycolysis
period of resistance. The carbohydrate metabolism has been shown to be of great importance during arthropod
Mosquito
embryogenesis, changing dramatically in order to promote growth and differentiation and in periods of re-
sistance. This study investigated fundamental aspects of glucose metabolism in three stages of A. aegypti egg
development: pre-desiccated, desiccated, and rehydrated. The activities of regulatory enzymes in carbohydrate
metabolism such as pyruvate kinase, hexokinase and glucose 6-phosphate dehydrogenase were evaluated. We
show that these activities were reduced in A. aegypti desiccated eggs, suggesting a decreased activity of glycolytic
and pentose phosphate pathway. In contrast, gluconeogenesis increased in desiccated eggs, which uses protein as
substrate to synthesize glucose. Accordingly, protein amount decreased during this stage, while glucose levels
increased. Glycogen content, a major carbohydrate reserve in mosquitoes, was evaluated and shown to be lower
in desiccated and rehydrated eggs, indicating it was used to supply energy metabolism. We observed a re-
activation of carbohydrate catabolism and an increased gluconeogenesis after rehydration, suggesting that
controlling glucose metabolism was essential not only to survive the period of desiccation, but also for sub-
sequent larvae hatch. Taken together, these results contribute to a better understanding of metabolism regula-
tion in A. aegypti eggs during desiccation periods. Such regulatory mechanisms enable higher survival rate and
consequently promote virus transmission by these important disease vectors, making them interesting subjects in
the search for novel control methods.

1. Introduction control methods, which can be aided by a better understanding of the

Aedes aegypti mosquito is a medically important vector of viruses
that cause yellow fever, zika, chikungunya and dengue, with strong
impact especially in Africa and Latin America (Gould and Solomon,
2008). Aedini mosquitoes, including A. aegypti, produce eggs that are
resistant to desiccation, increasing mosquito survival in seasonal per-
iods of drought (Christophers, 1960; Kliewer, 1961; Clements, 1992). A
great impact on human health has prompted the search for novel
mosquitoes physiology. In this context, the biochemical processes be-
hind A. aegypti development, and particularly the mechanisms of re-
sistance enabling survival and completion of the life-cycle during per-
iods of unfavorable environmental conditions, are an interesting subject
demanding investigation.
Animals need proper nutrition for growth and survival, as a source
of energy for cellular processes. However, the availability of nutrients
in the environment changes with seasonal climatic transitions. To

Abbreviations: HK, hexokinase; PK, pyruvate kinase; PEPCK, phosphoenolpyruvate carboxykinase; G6PDH, glucose-6-phosphate dehydrogenase; G6P, glucose-6
phosphate; PEP, phosphoenolpyruvate
⁎
Corresponding author at: Universidade Federal do Rio de Janeiro – UFRJ/NUPEM, Av. São José Barreto, 764 - São José do Barreto, Macaé, RJ 27965-045, Brazil.
E-mail address: logullo@uenf.br (C. Logullo).

https://doi.org/10.1016/j.cbpb.2018.09.005
Received 13 September 2018; Accepted 20 September 2018
Available online 25 September 2018
1096-4959/ © 2018 Elsevier Inc. All rights reserved.
R.M. da Silva et al.
overcome periods of unfavorable conditions, different species adopt
biological strategies which are classically named diapause, quiescence,
torpor or hibernation, depending on the species and the physiological/
metabolic aspects associated to each one (Denlinger and Armbruster,
2014). Arthropods can adopt diapause, quiescence, or simply resistance
to abiotic stress as survival strategies (Denlinger, 2002; Perez and
Noriega, 2013; Rezende et al., 2008). The hypometabolic stages of re-
sistance are normally characterized by a developmental arrest, which
allows survival under unfavorable conditions, synchronizing periods of
growth and reproduction only when conditions are optimal (Lees, 1955;
Tauber and Tauber, 1976; Denlinger, 1986; Denlinger, 2002). Species-
specific physiological changes occur during different forms of devel-
opmental arrest, including decreased metabolism, enhanced stress tol-
erance, and altered protein production. These changes ensure protec-
tion against numerous environmental stressors, such as desiccation,
extreme temperatures, and oxidative stress (Denlinger, 2002; Lee et al.,
2002; Clegg, 1965).
Metabolism depression, accompanied by a decrease in biosynthetic
activities (e.g. protein synthesis), is one of the hallmarks of a dormancy
period (Wigglesworth, 1957; Kunkel and Williams Jr., 1951). Larvae
and adult insects perform diapause or quiescence utilizing energy re-
serves obtained before the dormancy period (Denlinger, 2002; Hahn
and Denlinger, 2007). Likewise, all nutrients necessary for embryo
development are previously deposited in the eggs during oogenesis, and
embryogenesis occurs without exogenous supplies (Stewart and
Thompson, 1993), except for oxygen required for aerobic metabolism,
and water, in some species (Rahn et al., 1974; Vleck, 1991).
A. aegypti females lay their eggs in humid substrates near the water
surface (Funasa - Fundação Nacional de Saúde, 2001). During ovipo-
sition, these eggs are permeable to water; however, within around 20 h,
eggs develop the serosal cuticle, an impermeable layer that lends re-
sistance to desiccation (Rezende et al., 2008). Some studies have sug-
gested that the A. aegypti eggs may be quiescent after oviposition (Silva
and Silva, 1999; Perez and Noriega, 2013); however the purpose of the
present study is the dehydration tolerance of these eggs. Although they
can tolerate the absence of water, the eggs need appropriate energy
metabolism to survive for long periods, since the embryogenesis re-
quires high energy levels in order to sustain cell proliferation and de-
velopment and, subsequently, promote egg hatching (Thompson and
Stewart, 1997). Metabolic modulation during periods of stress varies
from species to species, and different organisms mobilize diverse classes
of nutrients in order to obtain the energy necessary during latency
(Adedokun and Denlinger, 1985; Yaginuma et al., 1990; Godlewski
et al., 2001). The beetle Leptinotarsa decemlineata uses carbohydrate
reserves primarily in the early diapause stage; however, if the latency
period extends for too long, the beetle starts to mobilize lipid reserves
(Lefevere, 1988). In contrast, the pupa of the Sarcophaga crassipalpis fly
preserves the carbohydrate reserves for the late diapause stage, for
arousing (Adedokun and Denlinger, 1985).
Thus, a fine metabolism control is essential to support these or-
ganisms throughout these processes. Carbohydrate metabolism has
been shown to be of great importance during arthropod embryogenesis,
changing dramatically in order to promote growth and differentiation
(Moraes et al., 2007). Our group previously demonstrated that glucose
metabolism undergoes a dramatic alteration during A. aegypti egg de-
velopment, where germ band retraction is a morphological landmark,
suggesting that carbohydrate metabolism is fundamental to regulate the
mosquito embryogenesis (Vital et al., 2010). Since carbohydrate re-
serves are mobilized by various organisms as a way to readily obtain
energy, it is reasonable to hypothesize that they are important during
dessication in A. aegypti eggs. In this work, key enzymes involved in the
gluconeogenic and glycolytic pathways were investigated in three
phases of A. aegypti egg development (pre-desiccated, desiccated, and
rehydrated). The results will be useful to understand the modulation of
energy metabolism during latent stages of the mosquito life-cycle.
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Comparative Biochemistry and Physiology, Part B 227 (2019) 56–63
2. Methods
2.1. Mosquito maintenance and the ethics committee
Aedes aegypti mosquitoes (Rockefeller strain) were reared and
maintained in our laboratory. Larvae were fed rat food, and adults were
fed sucrose solution 10% (w/v). For egg production, female mosquitoes
were blood-fed on mice. During oviposition assays, females were kept at
28 °C in a 80% humidity chamber, with a 12 h:12 h (light:dark) cycle,
for periods of 24 or 48 h after a blood meal.
The procedures were performed in the Animal Experimentation Unit
(UEA) Universidade Estadual do Norte Fluminense Darcy Ribeiro
(UENF) in Campos dos Goytacazes, RJ, Brazil. All experiments were
performed in accordance with the standards and ethics of animal ex-
perimentation, established by Brazilian federal law no. 11794/08 a er
approval of the Ethics Committee of Animal Use (CEUA) of the
Universidade Estadual do Norte Fluminense Darcy Ribeiro, under pro-
tocol number: 171/2012.
2.2. Synchronous egg-laying and preparation of egg homogenates
Adult A. aegypti females were handled for oviposition as described
previously (Rezende et al., 2008), with few modifications. The start of
egg development was defined as 30 min after egg laying. Eggs were kept
in an 80% humidity chamber at 28 °C until the end of embryogenesis
(62 h). A. aegypti eggs were assessed in three different conditions: pre-
desiccated (PrD), desiccated (D), and rehydrated (R), simulating dif-
ferent stages of the natural cycle. The PrD group included synchronized
62-h eggs that did not undergo desiccation. In turn, D eggs were syn-
chronized 62-h eggs that were dried and remained dehydrated for
2 weeks. Finally, R eggs included desiccated eggs that were rehydrated
by soaking in water for 10 min (maximum time before they start
hatching) and immediately used in the experiments.
Eggs homogenates (containing twenty milligrams of eggs) were
prepared by mechanical homogenization in appropriate buffer ac-
cording to the downstream assay (described below), followed by cen-
trifugation (200 ×g for 10 min). The supernatant of the eggs homo-
genates was used in biochemical tests. For enzyme assays, a protease
inhibitor cocktail was added to egg homogenates containing 1 μM
phenylmethylsulfonyl fluoride (PMSF), 1 mM ethylenediaminete-
traacetic acid (EDTA), 1 μM pepstatin A, and 10 μM leupeptin. The
protein content of samples was determined according to the Lowry
method (Lowry et al., 1951).
2.3. Mosquito eggshell clarification and morphological assessment
Eggs obtained from synchronous eggs laying (PrD, D, or R) were
fixed and clarified according to Trpisˇ (1973). This technique fixes the
embryo and turns the eggshell transparent. Eggs were viewed under a
magnifying glass with 10× magnification for the evaluation of embryo
morphology.
2.4. Determination of glucose, glycogen, and glucose-6-phosphate content
Eggs (20 mg, PrD, D, or R) were homogenized in 200 mM phosphate
buffered saline (PBS) pH 7.4 and glucose content was enzymatically
quantified by glucose oxidase reaction (Glucox® enzymatic Kit for
glucose dosage; Doles, Inc.). After 30 min of incubation at 37 °C, the
assays were read at 510 nm in a Shimadzu U1240 spectrophotometer,
according to the manufacturer's instructions.
For glycogen quantification, eggs (20 mg, PrD, D, or R) were
homogenized in 200 mM sodium acetate, pH 4.8, and supernatant ali-
quots were incubated with 1 unit of alpha-amyloglucosidase (Sigma
Chemicals) for 4 h at 40 °C. The newly generated glucose was en-
zymatically determined by glucose oxidase as described above. Free
glucose quantified in samples without alpha-amyloglucosidase was
R.M. da Silva et al.
subtracted from the results. Glycogen content was determined using a
standard curve submitted to the same conditions.
For determination of glucose-6-phosphate content, eggs (20 mg,
PrD, D or R) were homogenized in 55 mM Tris-HCl pH 7.8. Supernatant
aliquots (in triplicate) were assayed in 1 mL of 55 mM Tris-HCl, pH 7.8
containing 6 mM MgCl2, 100 mM, 0.5 mM beta-NADP+ and G6PDH
(300 U/mL). The reaction was started with sample addition. The for-
mation of beta-NADPH was monitored at 340 nm in a Shimadzu U1240
spectrophotometer during 5 min, using a molar extinction coefficient of
6.22 M−1 as described by Worthington (1998).
2.5. Phosphoenolpyruvate carboxykinase (PEPCK) activity assay
Eggs (20 mg, PrD, D, or R) were homogenized in extraction buffer
containing 100 mM HEPES buffer, pH 7.0. Supernatant aliquots (in
triplicates) were assayed in 400 μL of 100 mM HEPES buffer pH 7.0
containing 10 mM MnSO4, 100 mM KHCO3, 2 mM reduced glutathione,
10 mM PEP, 0.2 mM NADH, and 24 units of malate dehydrogenase
(Sigma Chemicals). The reaction was started by the addition of 10 μL
2.5 mM inosine diphosphate (IDP). The consumption of beta-NADH was
monitored at 340 nm, and PEPCK activity was determined as described
by Petersen et al. (2001).
2.6. Hexokinase (HK) activity assay
Eggs (20 mg, PrD, D, or R) were homogenized in extraction buffer
containing 20 mM Tris-HCl, pH 7.5 MgCl2. Supernatant aliquots (in
triplicates) were assayed in 20 mM Tris-HCl pH 7.5 containing 6 mM
MgCl2, 1 mM ATP, 0.5 mM NAD+ and 2 mM glucose. HK catalytic ac-
tivity was measured by adding Leuconostoc mesenteroides glucose 6-
phosphate dehydrogenase (Sigma-Aldrich Chemicals) (Worthington
Code: ZF or ZFL) dissolved to 1 UI/mL in Tris-MgCl2 buffer described
above (Tøien et al., 2011). The production of beta-NADH was mon-
itored at 340 nm in a Shimadzu U1240 spectrophotometer using a
molar extinction coefficient of 6.22 M−1, as described by Worthington
(1998).
2.7. Pyruvate kinase (PK) activity assay
Eggs (20 mg, PrD, D, or R) were homogenized in extraction buffer
containing 20 mM Tris-HCl pH 7.5 with 5.5 mM MgCl2. Supernatant
aliquots (in triplicate) were assayed in 1 mL of 20 mM Tris-HCl pH 7.5
containing 5.5 mM MgCl2, 1 mM ADP, 0.4 mM NADH, 1 unit/mL lactate
dehydrogenase and 1 mM phosphoenolpyruvate. Beta-NADH con-
sumption was monitored at 340 nm in a Shimadzu U1240 spectro-
photometer using a molar extinction coefficient of 6.22 M−1, as de-
scribed by Worthington (1998).
2.8. Glucose-6-phosphate dehydrogenase (G6PDH) activity assay
Eggs (20 mg, PrD, D, or R) were homogenized in extraction buffer
containing 55 mM Tris-HCl pH 7.8. Supernatant aliquots (in triplicate)
were assayed in 1 mL of 55 mM Tris-HCl, pH 7.8 containing 6 mM
MgCl2, 100 mM glucose 6-phosphate, and 0.5 mM beta-NADP+. The
reaction was started with sample addition. The formation of beta-
NADPH was monitored for 5 min at 340 nm in a Shimadzu U1240
spectrophotometer, using a molar extinction coefficient of 6.22 M−1 as
described by Worthington (1998).
2.9. Phylogenetic analysis of PEPCK in insects
Aedes aegypti PEPCK protein sequences were used as queries for
BLAST searches in other insect genomes. The best protein sequence
model was calculated with MEGA6 and maximum likelihood trees were
calculated. One hundred bootstraps were used for tree resampling. The
source of the PEPCK protein sequences is found in the Section 2.10.
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Comparative Biochemistry and Physiology, Part B 227 (2019) 56–63
2.10. Relative quantification of PEPCK transcripts by RT-qPCR
Total RNA was extracted from A. aegypti eggs using the Trizol re-
agent (Life Technologies) according to the manufacturer's instructions.
The amount of total RNA was estimated by spectrophotometry at 260/
280 nm in a Shimadzu spectrophotometer U1240.
Two micrograms of total RNA were reverse-transcribed using the
High-capacity cDNA Reverse Transcription synthesis kit according to
the manufacturer's instructions (Life Technologies). Amplification was
performed in a real-time PCR LightCycler 1.5 (Roche) that uses a ca-
pillary platform. Serial dilutions of cDNA were used to construct the
calibration curve for each pair of primers. Primer efficiencies between
85 and 100% were determined from calibration curves for each set of
primers in 10-μL reactions. There are three distinct sequences of A.
aegypti PEPCK genes deposited in GenBank with the accession numbers
XP_001647936.1, XP_001647935.1, XP_001647937.1, referred to as
PEPCK-25, PEPCK-6, and PEPCK-80, respectively. Specific primers were
designed for each of the three A. aegypti PEPCK genes as follows:
5′-TTCCGTAGCGGACAACCAAATCC-3′ (forward) and 5′-AGCGTGAAG
AGTATTAATCAGGGTAGCA-3′ (reverse) for PEPCK-25; 5′-GCCAAAGG
CCATCTACAACCACC-3′ (forward) and 5′-TCGGTGTAGCATCTTCAGT
AGTGTGTTG-3′ (reverse) for PEPCK-6; and 5′-CCCAAGTCGCAAAACG
TGATCC-3′ (forward) and 5′-GATGGTGCCTTGGGCCTGTA
GAG-3′(reverse) for PEPCK-80. Relative expression was determined
relative to pre-desiccated eggs (group PrD) using the crossing-point
(Cp) values obtained for each reaction. Analyses were done using the
Relative Expression Software Tool-REST (Pfaffl, 2001), and primers for
the constitutive rp49 gene as an internal reference gene (Gentile et al.,
2005). For this test, we used the Master SYBR Green I kit (Roche), in 10-
μL reactions according to the manufacturer's instructions.
2.11. Statistical analyses
The experiments were carried out in triplicate. All data values are
expressed as mean ± S.D. ANOVA was used to determine significant
differences between groups for normally distributed data. The Tukey's
test was used to compare data between three groups. Significance was
set at *p < .05.
3. Results
3.1. Glycolytic pathway decreases in A. aegypti desiccated eggs
The glycolytic pathway was evaluated in A. aegypti pre-desiccated,
desiccated, and rehydrated embryos by determining the enzymatic ac-
tivity of pyruvate kinase (PK) (Fig. 1A), hexokinase (HK) (Fig. 1B).
Comparing with the end of embryogenesis in pre-desiccated eggs, all
two enzymes activities significantly decrease after desiccation period.
In rehydrated embryos, PK and G6PDH activity returned to a level si-
milar to that observed in the pre-desiccated group (p < .05); in con-
trast, HK activity was lower than in the previous stages. Glucose 6-
phosphate levels were low in pre-desiccated eggs, increased in desic-
cated eggs, and decreased again after rehydration (Fig. 2).
3.2. Embryo external morphology upon desiccation
Embryo morphology was not affected by desiccation and rehydra-
tion processes (Fig. 3). All three groups analyzed showed typical size
and segmentation pattern previously described for A. aegypti embry-
ogenesis (Vital et al., 2010). The 2-week period of desiccation also did
not cause visual differences on eggshells.
Pentose phosphate pathway decreases in A. aegypti desiccated eggs
The Pentose phosphate pathway was evaluated in A. aegypti pre-
desiccated, desiccated, and rehydrated embryos by determining the
enzymatic activity of glucose-6-phosphate dehydrogenase (G6PDH)
(Fig. 4). Comparing with the end of embryogenesis in pre-desiccated
R.M. da Silva et al. Comparative Biochemistry and Physiology, Part B 227 (2019) 56–63

Fig. 1. Glycolysis decreases in A. aegypti desiccated

eggs. The canonical glycolytic pathway decreases in
the desiccated stage, as suggested by glycolytic key
enzymes, pyruvate kinase activity (A), and hex-
okinase activity (B), pointing to a hypometabolic
program activation, consuming less G6P. Enzyme
activities were determined spectrophotometrically
by the presence of beta-NADH (340 nm). Each ex-
periment was replicated three times and error bars
represent standard deviation of sample means
(*p < .05; ***p < .001 ANOVA).

Fig. 2. Glucose 6-phosphate content accumulates in A. aegypti desiccated eggs

Fig. 4. Pentose-phosphate pathway undergoes reduction when A. aegypti eggs
as a consequence of a hypometabolic program. G6P levels increase nearly 4
enter a period of desiccation. Glucose 6-phosphate dehydrogenase activity, a
times during desiccation due to glycolysis and pentose-phosphate pathway re-
key enzyme in the pentose-phosphate pathway, decreases by nearly 50% after
duction, as well as during glycogen degradation. Glucose concentration was
desiccation induction, as a consequence of a hypometabolic program activation.
enzymatically measured in A. aegypti pre-desiccated, desiccated, and rehy-
Enzyme activity was determined spectrophotometrically by the presence of
drated eggs. Each experiment was replicated three times and error bars re-
beta-NADH (340 nm). Each experiment was replicated three times and error
present standard deviation of sample means (*p < .05; ***p < .001 ANOVA).
bars represent standard deviation of sample means (*p < .05; ***p < .001
ANOVA).
eggs, both enzymes activities significantly decrease after desiccation
period. In rehydrated embryos, G6PDH activity returned to a level si-
milar to that observed in the pre-desiccated group (p < .05).

3.3. Gluconeogenesis increases in desiccated and rehydrated eggs

The glycogen levels was evaluated in A. aegypti in pre-desiccated,

desiccated, and rehydrated groups as well as the gluconeogenic
pathway, by measuring phosphoenolpyruvate carboxykinase (PEPCK)
enzymatic activity, total protein and glucose levels. The Glycogen re-
serves decreased continually in desiccated and rehydrated eggs (Fig. 5).
The protein level was high in pre-desiccated eggs, and decreased in
desiccated and rehydrated eggs (Fig. 6). Conversely, PEPCK activity
was low in pre-desiccated group, increasing in desiccated and rehy- Fig. 5. Glycogen degradation is necessary to arouse the A. aegypti desiccated
drated eggs (Fig. 7A). Glucose level also was low in pre-desiccated eggs, eggs. Egg homogenates of different egg stages (PrD, D, and R) were homo-
and increased in desiccated and rehydrated eggs (Fig. 7B). genized and glycogen content was assayed. Glycogen reserves help maintain
high-energy demand during rehydration, decreasing by > 50% after water
stimulus in these eggs, thus ensuring hatching. Glycogen was measured en-
3.4. Representation of glucose metabolism regulation in A. aegypti eggs
zymatically; each experiment was replicated three times and error bars re-
present standard deviation of sample means (*p < .05; ***p < .001 ANOVA).
The Fig. 8 summarizes the metabolic mechanisms presented in this
study. Here, the glycolysis is represented by the activities of HK and PK.
The gluconeogenesis is represented by the activity of PEPCK, and the

Fig. 3. No significant differences in external morphology

were observed in A. aegypti egg stages. Eggs of different stages
(PrD, D, and R) were clarified and viewed under a magnifying
glass (10×) in order to observe embryo morphology. Egg
morphology does not vary consistently between pre-desic-
cated, desiccated, and rehydrated eggs, showing that two
weeks of desiccation are not sufficient to alter eggs structure.
Scale bar 100 μm.

59
R.M. da Silva et al.
Fig. 6. Protein reserves support gluconeogenesis in A. aegypti desiccated and
rehydrated eggs. Protein degradation increases in desiccated (D) and rehy-
drated (R) eggs, supporting gluconeogenesis in these stages. Total protein
concentration was measured in A. aegypti pre-desiccated (PrD), desiccated (D),
and rehydrated (R) eggs, using the method of Lowry. Each experiment was
replicated three times and error bars represent standard deviation of sample
means (*p < .05; ***p < .001 ANOVA).
pentose phosphate pathway is represented by the activity of G6PDH.
The metabolites studied in this work are also highlighted to help un-
derstand their routes in pre-desiccated, desiccated, and rehydrated
groups.
3.5. Phylogenetic and transcriptional analysis of PEPCK in A. aegypti eggs
The three PEPCK genic sequences present in the A. aegypti genome
(PEPCK-6, PEPCK-25, and PEPCK-80) were phylogenetically similar to
other mosquito PEPCKs (Fig. 9A). The amounts of transcripts (mRNA)
of the three A. aegypti PEPCK was different among the studied condi-
tions. PEPCK-80 and PEPCK-25 was increased in pre-desiccated and
desiccated eggs, respectively (Fig. 9B and C). The PEPCK-6 showed no
transcription in any of the stages investigated in the present work.
4. Discussion
Carbohydrates are the most abundant biomolecules in living or-
ganisms, performing a wide range of structural and signaling functions.
They are important energy reserves, and play a central role in meta-
bolism in most organisms. Our group previously demonstrated that
glucose metabolism undergoes a dramatic alteration during A. aegypti
egg development, where germ band retraction is a morphological
landmark, suggesting that carbohydrate metabolism is fundamental to
regulate the mosquito embryogenesis (Vital et al., 2010). Therefore, the
study of the nutrient utilization dynamics in A. aegypti eggs during
desiccation stages complements the prior data on mosquito embry-
ogenesis, providing important insights on the mechanisms of resistance
to unfavorable conditions.
It is well established that quiescence and diapause prolong lifespan
of an insect, which therefore is better prepared to wait until the ap-
propriate stimulus is provided, when it is then able to conclude its
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Comparative Biochemistry and Physiology, Part B 227 (2019) 56–63
dormancy period (Denlinger, 2002; Danks, 1987; Zaslavski, 1988). This
strategy ensures the organism's reproductive success and the comple-
tion of its life cycle. Thus, modulation of energy metabolism is essential
for insect survival and for reinfestation. Although has not been de-
scribed that the A. aegypti enters in diapause stage, the resistance to
drought observed in Aedes aegypti also ensures a prolongued lifespan
(Silva and Silva, 1999), therefore, a refined metabolism modulation is
likely required for mosquito survival in the environment.
4.1. Glycolysis and pentose phosphate pathway during desiccation and
rehydration
Here we observed that, even though the external morphology of the
embryo remained unaltered, profound metabolic changes were in place
during desiccation and rehydration periods. The enzymatic activities of
PK and HK, key enzymes in glycolysis, decreased in desiccated eggs,
suggesting a reduction in glycolysis at this dormancy stage. This may be
an evolutionary strategy to reduce overall energy metabolism, and
ensure survival. Similar strategies have been reported for other species
in nature (Lyman and Chatfield, 1955). Dormancy periods have broad
metabolic implications, since ATP demand is reduced during such
times, mitigating the need for production. Additional evidence that
supports the hypothesis of metabolic rate reduction in A. aegypti mos-
quito during desiccation is decreased oxygen consumption, when
compared with pre-desiccated eggs (data not shown). To ensure egg
survival, energy conservation becomes indispensable, and it is neces-
sary to control the metabolism. The reduction of the main energy-
producing pathways is observed in A. aegypti desiccated eggs, strongly
suggesting the activation of a particular metabolic program that leads
to a drought-induced hypometabolic state. A schematic representation
of these pathways during egg development is shown in Fig. 8, and helps
to understand the metabolic alterations observed.
After egg rehydration, PK enzymatic activity increases, returning to
similar levels to those observed in pre-desiccated eggs. This change in
PK activity profile suggests a higher consumption of glucose-6-phos-
phate by glycolysis, sustaining the reactivation of larval metabolism
after exposure to water. In contrast, HK enzymatic activity remains low
in rehydrated eggs suggesting that glucose-6 phosphate is instead pro-
vided by glycogen degradation. The amount of glucose 6-phosphate is
lower in pre-desiccated and rehydrated eggs, whereas G6PDH activity,
like PK, increases (Fig. 4). The glycolytic and pentose phosphate
pathway utilize glucose 6-phosphate to produced intermediates and
ATP. In desiccated eggs we observed an increase in the amount of G6P,
possibly due to reduced carbohydrate catabolism at this stage, which
seems to be reversed by rehydration. Together, these results suggest an
important role for the pentose phosphate and glycolytic pathways after
metabolism reactivation in rehydrated eggs.
Environmental signals and energy metabolism regulation that pro-
mote arousal in dormancy periods vary between species (Tauber et al.,
1986; Danks, 1987; Harvey, 1962). Many studies monitored oxygen
consumption and reported an increased metabolic rate in insects at the
final moments of diapause (Wipking et al., 1995; Kostal et al., 1998;
Fig. 7. Gluconeogenesis is essential to sustain de-
siccation satage in A. aegypti mosquito.
Phosphoenolpyruvate carboxykinase activity (A), a
key gluconeogenic enzyme, increases in desiccated
and rehydrated eggs, when also the levels of this
pathway's end product, glucose, are the highest (B).
Each experiment was replicated three times and
error bars represent standard deviation of sample
means (*p < .05; ***p < .001 ANOVA).
R.M. da Silva et al.
Fig. 8. Metabolism regulation in A. aegypti pre-desiccated (PrD), desiccated (D), and rehydrated (R) eggs. The hypothesis of the glucose metabolism regulation and
routes of substrates is represented for each stage of A. aegypti egg development: pre-desiccated (PrD), desiccated (D), and rehydrated (R) eggs. The name of enzymes is
in bold.
Singtripop et al., 2007). However, few studies describe the changes in
the central pathways of glucose metabolism in the transition from a
dormancy pediod to awakening. Studies conducted in the 1980's
showed that mobilization of carbohydrates is essential for the moth
Bombyx mori and the fly Sarcophaga crassipalpis to wake from diapause
(Adedokun and Denlinger, 1985; Su et al., 1994). Nevertheless, these
studies lacked a more detailed description of glucose metabolism reg-
ulation during arousal.
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Comparative Biochemistry and Physiology, Part B 227 (2019) 56–63
4.2. Utilization of glycogen reserves
Glycogen is the main form of glucose stored in animal cells and is
considered as a primary form of energy in these organisms. Under low
caloric energy intake, the moths Manduca sexta and B. mori carry out
glycolysis by glycogen degradation (Steele, 1985; Danks, 1987). In
oviparous animals, carbohydrate reserves may change systematically
during the embryonic process, like observed in the tick Rhipicephalus
microplus (Moraes et al., 2007). During the first 9 days of embryogenesis
of this tick, glycogen levels dramatically decrease. After this period,
glycogen re-synthesis occurs, supported by intense gluconeogenesis
Fig. 9. Phylogenetic analysis and transcriptional
regulation of PEPCK in A. aegypti egg stages.
Maximum likelihood phylogenetic tree (A) and the
relative quantification (RQ) of PEPCK-25 (B) and
PEPCK-80 (C) in A. aegypti pre-desiccated (PrD),
desiccated (D), and rehydrated (R) eggs. Sequences,
species and the respective accession numbers are
represented in the figure.
R.M. da Silva et al.
(Moraes et al., 2007). Hence, carbohydrate mobilization from glycogen
reserves is a dynamic process, essential for normal embryonic devel-
opment. According to our present data, it is also important for meta-
bolic changes observed during desiccation-induced dormancy in A.
aegypti eggs. The amount of glycogen showed to be higher in pre-de-
siccated eggs and decreased in desiccated eggs. After rehydration,
glycogen levels in the mosquito eggs continued to decrease, reaching
the lowest values. Some insects that perform diapause, like the potato
beetle Leptinotarsa decemlineata and the fly Eurosta solidaginis, normally
use carbohydrate reserves primarily in early diapause (Lefevere, 1988;
Storey and Storey, 1986). In a similar way, carbohydrate reserves in A.
aegypti eggs are mobilized during desiccation and arousal (Fig. 8).
4.3. Protein reserves mobilization and gluconeogenesis
To maintain a stage of dormancy, various insects accumulate es-
sential energy reserves and protein (Danks et al., 1987). Like carbo-
hydrate reserves, protein content helps maintain energy homeostasis
during the dormancy period, as it proceeds. In the present study, we
observed that the total protein levels in A. aegypti eggs varied among
the different investigated stages. The amount of protein increased in
pre-desiccated eggs when compared with desiccated eggs. During de-
siccation, protein levels undergo a significant decrease, suggesting that
protein degradation, in addition to carbohydrate reserves, contributes
to latency period maintenance. Some insects performing diapause
usually store proteins in larval stages before the latency period for this
purpose (Telfer and Kunkel, 1991). The corn borer larvae, Diatraea
grandiosella, and the potato beetle, L. decemlineata, store amino acids in
specialized proteins before the unfavorable season. When these insects
enter diapause during winter, the levels of stored proteins decrease
(Chippendale, 1973; Lefevere, 1988). Proteins can be degraded during
diapause, and their amino acids may oxidize in order to generate energy
or produce other compounds, such as glucose through gluconeogenesis.
We believe that a similar process may occur in A. aegypti eggs during
desiccation.
Gluconeogenesis is an antagonistic pathway of glycolysis, and is the
central pathway that leads to glucose formation from non-carbohydrate
precursors such as lactate, glycerol, and amino acids. Interestingly, the
enzymatic activity of PEPCK, a key enzyme in gluconeogenesis, was at
its lowest in pre-desiccated eggs, when compared with desiccated or
rehydrated stages, suggesting an activation of the gluconeogenic
pathway in the latency period. The levels of free glucose increased
concurrently with PEPCK activity. Total protein content showed a
contrasting profile, compared to PEPCK activity. It is possible that
protein degradation during desiccation provides amino acids as gluco-
neogenic substrate for glucose synthesis (Fig. 8). As stored glycogen is
utilized and depleted during desiccation, glucose resynthesis from other
precursors is necessary for maintenance of the organisms' physiological
processes.
4.4. Phylogeny and expression of PEPCK isoforms
Changes in the transcription rate of the PEPCK gene are critical to
determine the total activity of the enzyme (Chakravarty et al., 2005).
After years of research on PEPCK, the consensus is that changes in
PEPCK transcription regulate the total activity of the enzyme (Hanson
and Reshef, 2003). It is known that eukaryotes have a cytoplasmic and a
mitochondrial isoform of this enzyme (Yang et al., 2009; Hanson and
Reshef, 2003). In this study we analyzed three isoforms of the PEPCK
gene in the mosquito A. aegypti (PEPCK-25 PEPCK-6 and PEPCK-80)
and compared them with other mosquito PEPCKs. Interestingly, PEPCK
duplications appear to be specific to mosquitoes, since Culex quinque-
fasciatus and Aedes aegypti PEPCKs were identified as a single group
separated from the duplicated PEPCKs in higher diptera, like Drosophila.
The transcriptional analysis of the three isoforms of the PEPCK gene in
the A. aegypti eggs was performed in order to observe if PEPCK
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Comparative Biochemistry and Physiology, Part B 227 (2019) 56–63
transcription was correlated to the enzymatic activity (gluconeogenesis
hypothesis). Transcription profile showed a higher transcript level of
isoform PEPCK-80 in pre-desiccated eggs, compared with desiccated
and rehydrated groups. This profile was in disagreement to that ob-
served for PEPCK activity, which has the lowest value in pre-desiccated
eggs, while the highest activity was observed in rehydrated eggs. On the
other hand, the isoform PEPCK-25 showed a high number of transcripts
in desiccated eggs, suggesting that PEPCK activity in this period may be
linked to the expression of this particular isoform. Abrupt changes
observed in the PEPCKs transcriptional levels might reflect the different
energetic needs of the embryos experienced in the three conditions,
since the transcription and synthesis of this enzyme only occurs if the
energetic needs of the cell requires (Hanson and Reshef, 2003). Thus,
PEPCK transcripts would be present at late embryogenesis (pre-la-
tency), but would only be reflected later on, in the desiccated and re-
hydrated stages as observed for the enzymatic activity (Fig. A), when a
greater demand for this enzyme is observed. Studies based on enzy-
matic activity and transcriptional profiles show that PEPCK-25 and
PEPCK-80 genes correspond to the mitochondrial and the cytosolic
isoforms, respectively (unpublished data). The third gene (PEPCK-6)
showed no transcription in any of the stages investigated in the present
work.
5. Concluding remarks
Taken together, the results described here provide an overview of
energy metabolism in A. aegypti eggs. Apart from the similarities ob-
served to quiescence or diapause in other organisms, this study showed
that A. aegypti eggs modulates its metabolism, as a strategy to survive
long periods of dryness, possibly ensuring reinfestation. Although this
stage is largely neglected, other studies on the embryogenesis of ar-
thropods, particularly focused on carbohydrate metabolism, have shed
new light on nutrient utilization dynamics, from the formation of the
oocyte to the mobilization of reserves and embryo formation (Vital
et al., 2010; Moraes et al., 2007; Campos et al., 2006). However, to our
knowledge, no study has been published before describing egg energy
metabolism during desiccation in the mosquito A. aegypti. The present
study contributes new knowledge on this topic, and indicates that
glucose metabolism regulation during A. aegypti eggs desiccation can be
a more dynamic process than previously thought. It is important to
mention that the study of lipid metabolism is also essential to elucidate
various cellular processes, including maintenance of the dormancy
periods (Adedokun and Denlinger, 1985; Hahn and Denlinger, 2007;
Mitchell and Briegel, 1989). Lipids are an important source of energy,
which is stored by animals as intracellular neutral lipid drops in spe-
cialized tissues such as adipose tissue in mammals and the fat body in
insects (Teixeira et al., 2003). The maintenance of a dormant period
requires a species-specific metabolic modulation, in which lipids, as
well as carbohydrates, may have an important role. Further investiga-
tion of energy metabolism will continue to improve our understanding
of the mechanisms that sustain A. aegypti eggs development during the
initial phase of desiccation.
Competing interests
The authors declare that they have no competing interests.
Conflict of interest
The authors declare that there is no conflict of interest.
Authors' contributions
CL directed the project and participated in the coordination and
management of the study. RMS performed the laboratory tests and the
data analysis and wrote the manuscript. WOV, RNF, MRF, YPM, and ISV
R.M. da Silva et al.
helped with various aspects of the experiments and manuscript re-
vising. FJAL helped in the colony mosquito maintenance. CL provided
new analytical reagents and tools. All authors read and approved the
final version of manuscript.
Acknowledgements
The authors are grateful to Tae Kwon Kim for aiding in the manu-
script revision and the Brazilian agencies FAPERJ, INCT-Entomologia
Molecular, CNPq and CAPES for supporting this project.
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