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Synthesis and mobilization of glycogen and trehalose in adult male Rhodnius prolixus.

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A r t i c l e
SYNTHESIS AND MOBILIZATION
OF GLYCOGEN AND TREHALOSE
IN ADULT MALE Rhodnius prolixus
Ana C. Mariano
Instituto de Bioquı´mica Me´dica, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, Brazil
Rachel Santos
Instituto de Bioquı´mica Me´dica, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, Brazil; Instituto Nacional de Cieˆncia e
Tecnologia de Entomologia Molecular, CCS, Bloco H, Cidade
Universitária, Ilha do Fundão, Rio de Janeiro, RJ,
Brazil
Marcelo S. Gonzalez and Denise Feder
Departamento de Biologia Geral, Instituto de Biologia, Universidade
Federal Fluminense, Nitero´i, Rio de Janeiro, Brazil
Ednildo A. Machado
Laborato´rio de Entomologia Me´dica, Instituto de Biofı´sica Carlos
Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro,
Brazil; Instituto Nacional de Cieˆncia e Tecnologia de Entomologia
Molecular, CCS, Bloco H, Cidade Universitária, Ilha do Fundão, Rio de
Janeiro, RJ, Brazil
Bernardo Pascarelli
Departamento de Histologia, Instituto de Cieˆncias Biome´dicas,
Universidade Federal do Rio de Janeiro, Brazil
Katia C. Gondim
Instituto de Bioquı´mica Me´dica, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, Brazil; Instituto Nacional de Cieˆncia e
Tecnologia de Entomologia Molecular, CCS, Bloco H, Cidade
Universitária, Ilha do Fundão, Rio de Janeiro, RJ, Brazil
Grant sponsors: Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq); Coordenac- ão de
Aperfeic- oamento de Pessoal de Nı́vel Superior (CAPES); Fundac- ão de Amparo à Pesquisa do Estado do Rio de
Janeiro (FAPERJ).
Correspondence to: José R. Meyer-Fernandes, Instituto de Bioquı́mica Médica, Universidade Federal do Rio de
Janeiro, CCS, Bloco H, Ilha do Fundão, 21541–590, Rio de Janeiro, RJ, Brazil.
E-mail: meyer@bioqmed.ufrj.br
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 72, No. 1, 1–15 (2009)
Published online in Wiley InterScience (www.interscience.wiley.com).
& 2009 Wiley Periodicals, Inc. DOI: 10.1002/arch.20319
2
Archives of Insect Biochemistry and Physiology, September 2009
José R. Meyer-Fernandes
Instituto de Bioquı´mica Me´dica, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, Brazil; Instituto Nacional de Cieˆncia e
Tecnologia de Biologia Estrutural e Bioimagem (INCTBEB), CCS,
Bloco H, Cidade Universita´ria, Ilha do Fundão, Rio de Janeiro, RJ,
Brazil
The vector of Chagas’ disease, Rhodnius prolixus, feeds exclusively on
blood. The blood meals are slowly digested, and these insects wait some
weeks before the next meal. During the life of an insect, energy-requiring
processes such as moulting, adult gonadal and reproductive growth,
vitellogenesis, muscular activity, and fasting, lead to increased
metabolism. Carbohydrates are a major source of energy and their
mobilization is important. We determined the amounts of glycogen,
trehalose, and glucose present in the fat body and/or hemolymph of adult
males of R. prolixus and recorded the processes of accumulation and
mobilization of these carbohydrates. We also tested our hypothesis that
these processes are under endocrine control. The amount of glycogen in
the fat body progressively increased until the fourth day after feeding
(from 9.372.2 to 77.377.5 mg/fat body), then declined to values
around 36.374.9 mg/fat body on the fifteenth day after the blood meal.
Glycogen synthesis was eliminated in decapitated insects and headtransplanted insects synthesized glycogen. The amount of trehalose in the
fat body increased until the sixth day after feeding (from 16. 671.7 to
40. 675.3 nmol/fat body), decreased abruptly, and stabilized between
days 7 and 15 at values ranging around 15–19 nmol/fat body.
Decapitated insects did not synthesize trehalose after feeding, and this
effect was reversed in head-transplanted insects. The concentration of
trehalose in the hemolymph increased after the blood meal until the
third day (from 0.0770.01 to 0.7570.05 mM) and at the fourth
day it decreased until the ninth day (0.2170.01 mM), when it
increased again until the fourteenth day (0.7970.06 mM) after the
blood meal, and then declined again. In decapitated insects, trehalose
concentrations did not increase soon after the blood meal and at the
third day it was very low, but on the fourteenth day it was close to the
control values. The concentration of glucose in the hemolymph of
untreated insects remained low and constant (0.1870.01 mM)
during the 15 days after feeding, but in decapitated insects it
progressively increased until the fifteenth day (2.0070.10 mM). We
recorded the highest trehalase activity in midgut, which was
maximal at the eighth day after feeding (2,8307320 nmol of
glucose/organ/h). We infer that in Rhodnius prolixus, the
metabolism of glycogen, glucose, and trehalose are controlled by
factors from the brain, according to physiological demands at
C 2009 Wiley Periodicals, Inc.
different days after the blood meal. Archives of Insect Biochemistry and Physiology
Glycogen and Trehalose in R. prolixus
3
Keywords: Rhodnius prolixus; brain; fat body; glycogen; glucose;
trehalose
INTRODUCTION
Rhodnius prolixus is an important vector of Trypanosoma cruzi, the etiologic agent of
Chagas disease. In insects, as in other organisms, glycogen serves as a glucose
reserve for utilization at different points of the life cycle. In insects, glycogen is most
abundant in fat body and flight muscles (Lohr and Gäde, 1983; Ziegler, 1991;
Siegert, 1995). Compared to vertebrates, only scarce information is available
concerning glycogen synthesis and its control in insects. In the medfly Ceratitis capitata,
the synthesis and utilization of glycogen was determined during the larva-toadult transition, and the presence of a glycogenin-like protein was determined
for the first time in an arthropod (Tolmasky et al., 2001). The biosynthesis of
glycogen by the fat body of Musca domestica is under neuroendocrine control, as it
was lower when the corpus allatum-cardiacum complex was removed (Liu, 1974).
Regulation of carbohydrate reserves has been studied in several insect species
and there is considerable variation in regulatory mechanisms (Gäde and Auerswald,
2003).
Glycogen contents are maintained during periods of hyperglycemia and
these stores are mobilized during situations such as flight and starvation (Van der
Horst et al., 2001). The fat body generates, for most insects, trehalose, the
predominant hemolymph sugar (Meyer-Fernandes et al., 2000, 2001). The first
evidence that carbohydrate metabolism is under hormonal control was provided by
Steele (1961) in cockroaches. Later, it was demonstrated in a number of insect species
that fluctuations in hemolymph trehalose concentration and/or in fat body glycogen
are controlled by peptide hormones of the adipokinetic hormone/red pigmentconcentrating hormone (AKH/RPCH) family (Siegert and Ziegler, 1983; Gäde et al.,
1997). Mechanisms involved in the mobilization of stored reserves have been
extensively studied during flight and starvation. When AKH is released, the fat body
glycogen phosphorylase is activated (Van der Horst et al., 2001) and there is an
increase in trehalose biosynthesis and release (Becker et al., 1996). During glycogen
breakdown, glucose 1-phosphate and glucose 6-phosphate are produced and used for
the synthesis of trehalose (Becker et al., 1996). Although glycolysis and trehalose
synthesis compete for the same substrate, glycolytic flux is reduced by hypertrehalosemic hormones, so that trehalose can be supplied to the hemolymph (Sevala and
Steele, 1991).
In insects, the adipokinetic/hypertrehalosemic hormones, released from the
corpora cardiaca, stimulate the degradation of glycogen and the synthesis of trehalose
in the fat body (Van der Horst et al., 2001). Although trehalose is the
main carbohydrate in insect hemolymph, there is also a substantial concentration of
glucose (Friedman, 1985). It was proposed for larval Manduca sexta that hemolymph
glucose concentration exerts a direct influence on the content of fructose-2,6bisphosphate in the fat body and also the glycogen phosphorylase activity
(Meyer-Fernandes et al., 2001). In R. prolixus, as oogenesis occurs, oocytes store
glycogen to be used during embryonic development (Santos et al., 2008). However, the
role of carbohydrates in energetic balance in nymphs and adults has not been studied.
Here, we report the accumulation and mobilization of glycogen and trehalose during
Archives of Insect Biochemistry and Physiology
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Archives of Insect Biochemistry and Physiology, September 2009
feeding and fasting in adult males. We also report on the outcomes of experiments
designed to test the hypothesis that these processes are regulated by factors released by
the brain.
MATERIALS AND METHODS
Insects
Insects were taken from a colony of R. prolixus maintained at 281C and 70–80% relative
humidity. Adults males fed on rabbit blood at 3-week intervals were used in all
experiments.
Chemicals
Enzymes, coenzymes, and substrates for enzymatic assays were obtained from SigmaAldrich Co (St. Louis, MO). For glucose determination, the specific enzymatic kit
Glucox 500, based on glucose oxidase activity, was used following the manufacturer’s
protocol (Doles Reagentes e Equipamentos para Laboratórios Ltda., Brazil).
Determination of Glucose and Trehalose Concentrations in the Hemolymph
Hemolymph (25 ml) from three insects for each point was collected in the presence of
N-phenylthiourea (3–13 mg/ml) and kept on ice. The samples were centrifuged
(5 min, 13,000g) and the supernatants (15 ml) were used for determination of glucose
and trehalose concentrations, as described (Santos et al., 2008). For trehalose
determinations, samples were incubated with trehalase (0.1 U; EC 3.2.1.28) in
40 mM phosphate buffer, pH 5.5 (200 ml final volume) for 4 h at 401C (Parrou and
Franc- ois, 1997). After incubation, 100 ml was taken to determine the amount of glucose
generated from trehalose hydrolysis. To control for glucose in hemolymph, blanks
were run in the absence of trehalase in the incubation. To verify the accuracy of the kit,
glucose was also quantified by coupling the production of glucose-6-phosphate to
reduction of NADP using hexokinase and glucose-6-phosphate dehydrogenase
(Siegert, 1987). The values obtained for glucose contents measured using both
methods were comparable.
Preparation of Fat Body Samples
Fat bodies were dissected, washed in 0.15 M NaCl and homogenized in buffer
containing 50 mM Tris-HCl pH 7.2, 5 mM NaF, 5 mM EDTA, 5 mM EGTA, and 1 mM
DTT (2 fat bodies in 200 ml). The homogenates were stored at 201C until use.
Control experiments showed that storage did not affect the amounts of carbohydrates
or proteins even after 15 days (data not shown). For determination of trehalase activity,
tissue homogenates were immediately used.
Determination of Trehalose Content in the Fat Body
An aliquot (50 ml) of the fat body homogenate was processed as just described. Because
this value included the amount of intrinsic glucose, the trehalose content was corrected
for the glucose present in an aliquot from the same sample that was not incubated with
trehalase.
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Glycogen and Trehalose in R. prolixus
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Determination of Glycogen Content in the Fat Body
For glycogen quantification, a method based on the enzymatic hydrolysis of glycogen
by amyloglucosidase (EC 3.2.1.3), was used (Parrou and Franc- ois, 1997). An aliquot
(50 ml) of the fat body homogenate was mixed with 20 ml (1 U) of amyloglucosidase
diluted in 0.1 M sodium acetate, pH 5.5. The reaction components were incubated for
4 h at 401C. After incubation, 100 ml was separated and the amount of glucose
generated from glycogen was determined as described. Because the value included the
amount of intrinsic glucose, the glycogen amount was corrected for the glucose
content in samples that were not incubated with amyloglucosidase.
Determination of Protein Content in the Midgut
At selected days post blood meal (PBM), whole midguts were dissected, washed in
0.15 M NaCl, and homogenized in buffer containing 50 mM Tris-HCl, pH 7.2, 5 mM
NaF, 5 mM EDTA, 5 mM EGTA, and 1 mM DTT. Protein contents of midgut
homogenates were determined (Lowry et al., 1951) using bovine serum albumin as
standard.
Assays of Trehalase Activity
Midguts, fat bodies, thoracic muscles, and Malphigian tubules were dissected, washed
in 0.15 M NaCl, and homogenized in cold PBS (phosphate-buffered saline–10 mM
sodium phosphate, 0.15 M NaCl, pH 7.4). Luminal contents from midguts were
removed, as described (Grillo et al., 2007). An aliquot (20 ml) of fresh tissue
homogenate was mixed with 20 ml of 0.1 M trehalose and incubated at 371C, in
40 mM phosphate buffer, pH 5.5 (200 ml final volume) for 1 h. The reactions were
stopped by heating samples at 1001C for 5 min. Samples were centrifuged at 13,000g
for 10 min and glucose liberated by trehalase activity was determined in 100 ml of
supernatants. Because this value included the amount of intrinsic glucose present in
the different tissues, the trehalase activity was calculated by subtracting the glucose
present in the same tissue samples that were heating at 1001C for 5 min before the
addition of substrate trehalose.
Assays of Endocrine Manipulation (Decapitation and Head Transplantation)
Operations were performed according to Knobloch and Steel (1989). Immediately
after feeding, insects were decapitated with a small razor blade and the wound was
sealed with liquid wax melted at 451C. The success of decapitation was tested by
checking the withdrawal reflex after application of a delicate pressure to the legs with
forceps (Gonzalez et al., 1998). For transplantation, the head from a donor insect was
removed and immediately placed in the hole left in the host body after decapitation,
and a wax collar was melted around the base of the head with a small warm dropper.
The success of the operation was evaluated by checking that the wound was sealed and
by ensuring that there was a flow of hemolymph from the body to the head by
applying a gentle pressure on the abdomen and observing that the antennae were
extended (Garcia et al., 1990).
Histological Preparations
Fat bodies before and at different days PBM were dissected, washed in 0.15 M NaCl,
and fixed in 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M sodium phosphate
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Glycogen and Trehalose in R. prolixus
7
buffer, pH 7.2, at 41C overnight. The organs were oriented under a dissecting
microscope, in an aluminum chamber filled with Optimal Cutting Temperature
(Sakura Finetec) embedding medium, and transverse sections were cut in a cryostat
(Rehen et al., 1996). The sections were counterstained with the periodic acid Schiff ’s
(PAS) reaction (Tolmasky et al., 2001).
Statistical Analysis
All experiments were performed in triplicate with similar results obtained from at least
three independent assays. Data are expressed as mean7S.E.M. Data were analyzed by
means of ANOVA One-way followed by the Turkey test using the Sigma. Stat
Computer software (SYSTAT Software Inc., San Jose, CA), and significance was set at
Po0.05.
RESULTS
Before feeding, the fat body exhibited no PAS-staining, demonstrating that glycogen
reserve was exhausted (Fig. 1A and B). At day 4 PBM, when substrates are abundant, a
strong PAS-positive staining was observed (Fig. 1C and D). At 7 days PBM, staining,
weaker than at day 4, was present (Fig. 1E and F). At 14 days, we registered little
staining (Fig. 1G and H).
The amount of fat body glycogen was low before feeding and progressively
increased until day 4 PBM (from 9.072.2 to 77.077.5 mg/fat body). Glycogen content
declined to approximately 36.0 mg/fat body on day 14 PBM. Figure 2 shows that in
decapitated insects the synthesis of glycogen was significantly lower than in intact
males.
Figure 3 shows that decapitation did not interfere with the digestion of blood and
decapitated insects did not significantly synthesize glycogen. Head transplantation
(Fig. 4) reversed this situation at day 4 PBM.
The amount of trehalose in the fat body progressively increased PBM until day 6
after feeding (from 16.071.7 to 40.075.3 nmol/fat body). After day 6, the content of
trehalose began to decline and reached 19.173.2 nmol/fat body on day 15 PBM
(Fig. 5). In order to know whether the metabolism of trehalose is under control of
brain factors, the amount of trehalose in the fat body of decapitated and headtransplanted insects was determined before feeding and 3 days PBM. Figure 6 shows
that the increase in fat body trehalose content observed in untreated males on the third
day after feeding did not occur in decapitated insects, and this effect was suppressed in
transplanted insects.
After blood meal, trehalose content increased until day 3 PBM (from 0.0770.01
to 0.7570.05 mM) and at day 4, it decreased until day 9 (0.2170.01 mM),
when it increased again reaching maximal values again on day 14 PBM
(0.6970.06 mM) (Fig. 7). In decapitated insects, the hemolymph trehalose
Figure 1. Periodic acid Schiff ’s (PAS) stain of fat bodies of adult males before and at different days after
blood meal. After dissection, fat bodies were washed and prepared for analysis by optical microscopy. Tissue
sections were stained with the PAS reaction. A, B: Before feeding. C, D: Four days after blood meal. E, F:
Seven days after blood meal. G, H: Fourteen days after blood meal. Amplification: 50 (A, C, E, G); 630 (B, D, F, H). LD, lipid droplet; arrowheads indicate nuclei.
Archives of Insect Biochemistry and Physiology
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Archives of Insect Biochemistry and Physiology, September 2009
Figure 2. Glycogen content in the fat body of control and decapitated adult males at different days after
blood meal. Immediately after feeding, insects were decapitated and, at different days after blood meal, fat
bodies were dissected from control () and decapitated (3) males and homogenized in buffer (50 mM
Tris-HCl, pH 7.2, 5 mM NaF, 5 mM EDTA, and 1 mM DTT). Samples were incubated at 401C for 4 h in the
presence of amyloglucosidase. After incubation, glucose content was determined as described in Materials
and Methods. Vertical bars represent the S.E.M. for 3 independent determinations. Significant difference
from controls.
Figure 3. Protein content in the midgut of control and decapitated adult males at different days after blood
meal. Immediately after feeding, insects were decapitated, and at different days after blood meal, midguts of
control () and decapitated (3) insects were dissected and homogenized in buffer (50 mM Tris-HCl, pH 7.2,
5 mM NaF, 5 mM EDTA, and 1 mM DTT). Protein content was determined by the method of Lowry et al.
(1951). Values are means7S.E.M. of 3 independent determinations. No significant differences were
recorded.
concentration did not increase after feeding and at day 3 it was very low (Fig. 8).
However, at day 14 PBM, the trehalose level was similar to the value found in control
insects (Fig. 8).
In untreated insects, the hemolymph glucose concentration remained constant
during 15 days PBM. In decapitated insects, this concentration increased and reached
1.9570.12 mM at day 15 PBM (Fig. 9).
Archives of Insect Biochemistry and Physiology
Glycogen and Trehalose in R. prolixus
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Figure 4. Glycogen content in the fat body of decapitated and head-transplanted adult males at day 4 after
blood meal. Insects were decapitated and head-transplanted immediately after feeding. At day 4, fat bodies
were dissected from control (C), decapitated (D), and head transplanted (HT) insects, and were
homogenized in buffer (50 mM Tris-HCl, pH 7.2, 5 mM NaF, 5 mM EDTA, and 1 mM DTT). Samples
were incubated at 401C for 4 h in the presence of amyloglucosidase. After incubation, the glucose contents
were determined as described in Materials and Methods. Values are means7S.E.M. of 3 independent
determinations. Significant differences from controls.
Figure 5. Trehalose content in the fat body of adult males at indicated days after blood meal. Fat bodies
were dissected and homogenized in buffer (50 mM Tris-HCl, pH 7.2, 5 mM NaF, 5 mM EDTA, and 1 mM
DTT). Samples were incubated with trehalase for 4 h at 401C and glucose content was determined as
described in Materials and Methods. Values are means7S.E.M. of 3 independent determinations.
Significant differences from controls (unfed insects).
Trehalase activity in the midgut was 1,4007100 nmol glucose/organ/h at day 1
PBM, increased to 2,8307320 nmol glucose/organ/h by day 8, and decreased to
1,120729 nmol glucose/organ/h by day 15 PBM (Fig. 10A). In the fat body, this activity
remained constant and much lower when compared to the midgut. At day 8, the
trehalase activity in the intestinal tract was much higher than that found in fat body,
Malpighian tubules, and muscle (Fig. 10B).
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Archives of Insect Biochemistry and Physiology, September 2009
Figure 6. Trehalose content in the fat body of decapitated and head-transplanted adult males. Insects were
decapitated and head-transplanted immediately after feeding. Fat bodies were dissected before feeding (BF)
and 3 days after (D3) blood meal, from control (open bars), decapitated (hatched bars), and headtransplanted (black bars) insects, and were homogenized in buffer (50 mM Tris-HCl, pH 7.2, 5 mM NaF,
5 mM EDTA, and 1 mM DTT). Samples were incubated with trehalase for 4 h at 401C and glucose content
was determined as described in Materials and Methods. Values are means7S.E.M. of 3 independent
determinations. Significant differences from controls (unfed insects).
Figure 7. Trehalose concentration in the hemolymph of adult males at the indicated days after blood meal.
Hemolymph was collected and centrifuged at 13,000 g for 5 min. Supernatants were incubated with trehalase
for 4 h at 401C and glucose content was determined as described in Materials and Methods. Values are
means7S.E.M. of 3 independent determinations. Significant differences from controls.
DISCUSSION
In this study, we analyzed some aspects of carbohydrate metabolism in adult male
R. prolixus, and the effect of factors released by the head on glycogen, trehalose, and
glucose levels.
Trehalose is present in all insects (at least in adults) studied in this respect, and in
many insects it is present in high concentrations and constitutes the major hemolymph
sugar (Becker et al., 1996). In the last larval instar of Bombyx mori, the trehalose
concentration is around 10 mM (Oda et al., 1997), and in Omphisa fuscidentalis it may
Archives of Insect Biochemistry and Physiology
Glycogen and Trehalose in R. prolixus
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Figure 8. Trehalose concentration in the hemolymph of decapitated adult males. Immediately after
feeding, insects were decapitated and, at day 1 (D1), day 3 (D3), and day 14 (D14) days PBM, hemolymph
was collected from control (open bars) and decapitated (hatched bars) males. After centrifugation at 13,000 g
for 5 min, supernatants were incubated with trehalase for 4 h at 401C and glucose content was determined as
described in Materials and Methods. Values are means7S.E.M. of 3 independent determinations.
Significant differences from whole insects after 1 day PBM. No significant differences were observed
between control and decapitated insects at 14 days.
Figure 9. Glucose concentration in the hemolymph of control and decapitated adult males at the indicated
days after blood meal. Immediately after feeding, insects were decapitated and hemolymph was collected
from control () and decapitated (3) males. After centrifugation, glucose content was determined as described
in Materials and Methods. Values are means7S.E.M. of 3 independent determinations. Glucose increased
significantly in decapitated insects () but not in control insects ().
reach values between 40 and 50 mM (Singtripop et al., 2002). Although in R. prolixus
the concentration of trehalose in the hemolymph was much lower than in
non-hematophagous species, it is the predominant hemolymph carbohydrate (Figs. 7
and 9).
The amounts of glycogen stored in the fat body of R. prolixus (80 mg/fat body) were
small compared to other insects, Locusta migratoria, at the end of the last larval instar,
and adults of Periplaneta americana and Manduca sexta may contain between 4 and 22 mg
of glycogen in the fat body (Hill and Goldsworthy, 1968; Hanaoka and Takahashi,
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Archives of Insect Biochemistry and Physiology, September 2009
Figure 10. A: Trehalase activity in the midgut and fat body of adult males at the indicated days after blood
meal. Insects were dissected and midgut () and fat bodies (3) were washed and homogenized. B: Trehalase
activity at day 8 in the midgut (MG), fat body (FB), thoracic muscle (M), and Malpighi tubules (MT) were
dissected, washed, and homogenized. Samples were incubated in the presence of trehalose at 371C for 1 h
and the concentration of produced glucose was determined as described in Materials and Methods. Values
are means7S.E.M. of 3 independent determinations. Significant increases in trehalase activity from midgut
(). Significant decreases in trehalase activity from midgut ().
1976; Ziegler, 1991). This small glycogen content in the fat body of R. prolixus may be
because it does not ingest large amounts of carbohydrates. In females of Aedes aegypti
and Aedes sollicitans, energy reserves accumulate according to the type of meal they
imbibe, their age when they feed on blood, and the time they are examined after the
blood meal (Briegel, 1990; Ziegler and Ibrahim, 2001). Naksathit et al. (1999) showed
that mosquitoes fed on sugar contained more glycogen than those fed only on blood,
and Zhou et al. (2004) demonstrated in Aedes aegypti females that gluconeogenesis from
amino acids was rapidly activated after a blood meal. After feeding, R. prolixus
synthesized glycogen and trehalose, probably from carbohydrates obtained after the
digestion of blood and also from non-carbohydrate precursors, as happens in other
insects (Thompson, 1995; Becker et al., 2001; Zhou et al., 2004).
The first evidence that energy reserves in insects are under hormonal control was
provided by Steele (1961) for carbohydrate mobilization in cockroaches and by Mayer
and Candy (1969) and Beenakkers and Van den Broek (1974) for lipid breakdown in
locusts. In R. prolixus, the synthesis of trehalose and glycogen by the fat body were
affected by decapitation. We infer from this finding that energy storage in R. prolixus
also is regulated by endocrine moieties.
Influence of starvation upon carbohydrate metabolism has been widely reported.
In the phytophagous hemimetabolous Carausius morosus, glycogen content decreased
after 15 h of starvation and virtually disappeared after 24 h (Lohr and Gäde, 1983). In
Manduca sexta after 5 days of starvation, glycogen reserves were exhausted (Ziegler,
1991). Fasting also influenced carbohydrate metabolism in R. prolixus. About four days
after feeding, the content of glycogen in the fat body began to decline. This decrease in
glycogen content was concomitant with a decreased hemolymph trehalose concentration. Possibly, the breakdown of glycogen supplied substrates for trehalose synthesis,
and resulted in the second increase in hemolymph trehalose concentration in
R. prolixus (Fig. 7). In other insects, a major source of hemolymph trehalose is glycogen
from the fat body. Hemolymph trehalose is homeostatically regulated at the expense of
tissue glycogen during starvation or exercise (Siegert and Ziegler, 1983; Becker et al.,
1996) and is under the control of hypertrehalosemic hormones (Sun et al., 2002;
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Glycogen and Trehalose in R. prolixus
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Michitsch and Steele, 2008). In the last larval instar of Manduca sexta, a ‘‘glycogen
phosphorylase activating hormone’’ is responsible for glycogen mobilization during
starvation (Siegert and Ziegler, 1983). Hypertrehalosaemic neuropeptides activate
glycogen phosphorylase in the fat body to break down glycogen (Gies et al., 1988;
Ziegler et al., 1990).
The concentration of glucose in the hemolymph of R. prolixus was much lower
than that found in other insect species (Siegert, 1995; Meyer-Fernandes et al., 2001)
and remained constant during 15 days PBM. This profile was altered and glucose
accumulated in decapitated insects from which we infer glucose concentration is under
the control of head factors.
The influence of decapitation on hemolymph trehalose concentration, like the
concentration of glucose, depended on the day PBM. We interpret this result to
suggest that although trehalose concentration in the hemolymph may be regulated by
head factors, it seemed to be regulated also by mechanism(s) unrelated to head factors.
The intestinal tract of insects, especially the midgut, contains high trehalase activity
(Becker et al., 1996). In the larval Omphisa fuscidentalis, for instance, at the end of
diapause, trehalase activity is stimulated by ecdysone, probably by induction of its
expression (Tatun et al., 2008). Our data indicate that the R. prolixus midgut is the
main organ responsible for the hydrolysis of hemolymph trehalose. However, the
mechanisms involved in the regulation of its activity in the midgut at days after feeding
are not known. Other organs, fat body inclusive, showed very low trehalase activity.
Trehalase activity is present in vitellogenic oocytes, where it is probably involved with
the synthesis of glycogen (Santos et al., 2008).
We conclude that adults of R. prolixus are able to synthesize glycogen and trehalose
soon after feeding and mobilize these stores during fasting between meals. In these
insects, the metabolism of carbohydrates is under the control of head factors.
ACKNOWLEDGMENTS
The authors thank Fabiano F. Esteves, Heloisa S.L. Coelho, Lilian S. da C. Gomes, and
Rosângela R. de Araújo for excellent technical assistance; José de S. Lima Junior and
Litiane M. Rodrigues for insect care; and Dr. Wanderley de Souza for the use of the
optical microscope.
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Archives of Insect Biochemistry and Physiology
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