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Lipid accumulation and utilization by oocytes and eggs of Rhodnius prolixus.

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A r t i c l e
LIPID ACCUMULATION AND
UTILIZATION BY OOCYTES AND
EGGS OF Rhodnius prolixus
Rachel Santos, Rafael Rosas-Oliveira, Felipe B. Saraiva,
and David Majerowicz
Instituto de Bioquı´mica Me´dica, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, RJ, Brazil
Katia C. Gondim
Instituto de Bioquı´mica Me´dica, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Cieˆncia e
Tecnologia em Entomologia Molecular, Brazil
Insect eggs must contain the necessary nutrients for embryonic growth. In
this article, we investigated the accumulation of triacylglycerol (TAG) in
growing oocytes and its utilization during embryonic development. TAG
makes up about 60% of the neutral lipids in oocytes and accumulates as
oocytes grow, from 2.270.1 mg in follicles containing 1.0 mm length
oocytes to 10.270.8 mg in 2.0 mm length oocytes. Lipophorin (Lp), the
hemolymphatic lipoprotein, radioactively labeled in free fatty acid (FFA)
or diacylglycerol (DAG), was used to follow the transport of these lipids to
the ovary. Radioactivity from both lipid classes accumulated in the
oocytes, which was abolished at 41C. The capacity of the ovary to receive
FFA or DAG from Lp varied according to time after a blood meal and
reached a maximum around the second day. 3H-DAG supplied by Lp to
the ovaries was used in the synthesis of TAG as, 48 hr after injection,
most of the radioactivity was found in TAG (85.7% of labeling in
neutral lipids). During embryogenesis, lipid stores were mobilized, and
the TAG content decreased from 16.472.1 mg/egg on the first day to
10.071.3 mg on day 15, just before hatching. Of these, 7.470.9 mg
were found in the newly emerged nymphs. In unfertilized eggs, the TAG
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); Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM).
Correspondence to: Katia C. Gondim, Instituto de Bioquı́mica Médica, Universidade Federal do Rio de Janeiro,
CCS, Bloco H, Ilha do Fundão, 21941-902, Rio de Janeiro, RJ, Brazil. E-mail: katia@bioqmed.ufrj.br
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 77, No. 1, 1–16 (2011)
Published online in Wiley Online Library (wileyonlinelibrary.com).
& 2011 Wiley Periodicals, Inc. DOI: 10.1002/arch.20414
2
Archives of Insect Biochemistry and Physiology, May 2011
content did not change. Although the TAG content decreased during
embryogenesis, the relative lipid composition of the egg did not change.
The amount of TAG in the nymph slowly decreased during the days after
C 2011 Wiley Periodicals, Inc.
hatching. Keywords: Rhodnius prolixus; lipid; oogenesis; lipophorin; triacylglycerol;
oocyte; egg
INTRODUCTION
Insect eggs must contain all the nutrients necessary for the developing embryo. Yolk
protein accumulation in insect oocytes has been extensively described (Oliveira et al.,
1986; Raikhel and Dhadialla, 1992). However, information on lipid accumulation is
scarce, even though they are a major constituent of oocytes (Ziegler and Van
Antwerpen, 2006). In Manduca sexta, for example, lipids represent around 40% of the
dry weight of a mature oocyte (Kawooya and Law, 1988), and in Culex quinquefasciatus,
90% of the energy required for embryogenesis is supplied by lipids (Van Handel,
1993). Lipids are mainly stored as triacylglycerol (TAG) in insect oocytes, but
phospholipids and cholesterol are also found (Troy et al., 1975; Kawooya and Law,
1988; Briegel, 1990). The ability of insect oocytes to obtain fatty acids by de novo
synthesis seems to be very small. In M. sexta and Aedes aegypti, about 1% of total lipids
were synthesized by oocytes in vitro (Kawooya et al., 1988; Ziegler, 1997). Therefore,
most of the lipids in oocytes must originate from the diet and/or storage tissues, such as
fat body, and lipoproteins are the most important vehicle of lipid delivery to the
ovaries (Kawooya and Law, 1988; Ziegler and Van Antwerpen, 2006).
Lipophorin (Lp), a major lipoprotein in the insect hemolymph, transports diverse
lipids, such as diacylglycerol (DAG), phospholipids, cholesterol, free fatty acids (FFA), and
hydrocarbons, between insect organs (Chino et al., 1981; Blacklock and Ryan, 1994;
Soulages and Wells, 1994a; Ryan and Van der Horst, 2000). This lipoprotein is classified as
low-density lipophorin (LDLp), high-density lipophorin (HDLp), or very high-density
lipophorin (VHDLp) according to its density (Beenakkers et al., 1988). Generally, Lp acts
as a reusable lipid shuttle; it delivers lipids to the organs, but the apoproteins are not
accumulated or degraded, and the lipoprotein can be reloaded (Downer and Chino, 1985;
Van Heusden et al., 1987). However, in some cases, the whole Lp particle can be taken up
by endocytosis (Kawooya and Law, 1988; Dantuma et al., 1997). In the ovary of M. sexta,
both mechanisms may occur. In this moth, two classes of Lp are found in the hemolymph,
HDLp, and LDLp. They both supply lipids to the ovaries, but LDLp particles are not
taken up, whereas HDLp is incorporated into the oocytes and converted to VHDLp after
the removal of the lipids (Kawooya and Law, 1988; Kawooya et al., 1988). In mosquitoes,
Lp apoproteins are incorporated by the ovaries, although not in sufficient quantity to
account for all the lipids accumulated there (Sun et al., 2000; Atella et al., 2006).
Lipids, similar to other substrates that are accumulated during oogenesis, are
necessary for embryogenesis; in oviparous organisms, no exogenous nutrients are
used by the egg. However, the mobilization of reserves by the embryos of insects and
other arthropods is only poorly understood, and even a simple description of nutrient
utilization is scarce.
In the hematophagous hemipteran Rhodnius prolixus, Lp delivers phospholipids to
vitellogenic oocytes by interacting with binding sites at the cell surface (Gondim et al.,
Archives of Insect Biochemistry and Physiology
Lipid Reserves in Rhodnius prolixus Oocytes
3
1989b; Machado et al., 1996), and the lipoprotein can be reloaded at the fat body and
midgut (Atella et al., 1992, 1995). These phospholipids are possibly used by the
oocytes for membrane synthesis. The formation of neutral lipid reserves by the
growing oocytes of R. prolixus has not been investigated. In a previous study,
radioactive fatty acids absorbed from a blood meal were transferred to hemolymphatic
Lp, mostly as DAGs, but also as other lipid classes such as phospholipids and FFAs.
Labeled lipids were then found in the fat body and oocytes (Grillo et al., 2007).
In this study, the lipid composition of the oocytes and eggs of R. prolixus was
determined, and the transfer of both DAG and FFA from Lp to the ovary was
investigated to follow the formation of lipid reserves during oogenesis in R. prolixus.
The utilization of these stores after egg laying was followed in fertilized and
nonfertilized eggs. This is the first description of lipid utilization by oocytes and eggs
of this insect.
MATERIALS AND METHODS
Insects
A colony of R. prolixus was maintained at 281C and 70–80% relative humidity and fed
on rabbit blood. The experimental animals were adult-mated females at their first
meal as adults; females and eggs were kept at 281C unless otherwise stated. To obtain
virgin females, female insects were separated from males during the fifth instar.
Determination of Oocyte TAG Content
On the third or seventh day after a blood meal, the ovaries were removed from mated
females. Ovarioles were dissected from the ovarian sheath, and the follicles were
isolated and their length determined in cold 0.15 M NaCl under a stereomicroscope.
Follicles were separated into three groups according to the oocyte development: 1.0,
1.5, and 2.0 mm length. Eight, six, and four follicles, respectively, were homogenized
in 200 ml of cold 0.15 M NaCl in a glass Potter–Elvehjem homogenizer and frozen
( 201C) until analysis. The samples were subjected to lipid extraction (Bligh and Dyer,
1959), and the equivalent of 0.8, 0.6, and 0.4 follicles, respectively, were used to
determine the TAG content by thin-layer chromatography (TLC) on silica gel plates
for neutral lipids using hexane–ethyl ether–acetic acid (60:40:1 by volume) as a solvent
(Kawooya and Law, 1988). For visualization, lipids were charred by dipping the plate
in a solution of 10% CuSO4 in 8% H3PO4 for 30 sec. After wiping the glass support dry,
the plate was thoroughly dried under a stream of hot air until the lipid spots became
visible, and the plate was immediately heated at 2001C for approximately 5 min
(Bitman and Wood, 1982). The spots corresponding to TAG in the samples and the
standard curve of TAG (glycerol trioleate; Sigma-Aldrich Co, St. Louis, MO) were
subjected to densitometric analysis (Ruiz and Ochoa, 1997) using ImageMaster
TotalLab v1.11 software (Amersham Pharmacia Biotech, England).
Determination of Neutral Lipid Composition of Oocytes
Follicles were homogenized as described above. The equivalent of four 1.0 mm
oocytes, three 1.5 mm oocytes, and two 2.0 mm oocytes were used to determine the
neutral lipid composition by TLC, developed in two sequential solvent systems:
benzene–ethyl ether–ethanol–acetic acid (50:40:2:0.2 by volume) and hexane–ethyl
Archives of Insect Biochemistry and Physiology
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Archives of Insect Biochemistry and Physiology, May 2011
ether–acetic acid (80:20:2 by volume), as described by Fan et al. (2004). For
visualization, the lipids were charred, and the relative lipid composition of each
follicle size group was determined by densitometry, as just described. For the
identification of lipid classes, the following commercial standards were used: 1-oleoylrac-glycerol, 1,3-diolein, glycerol trioleate, cholesterol, cholesteryl oleate, and oleic
acid (Sigma-Aldrich Co). A representative TLC is shown in Figure 1, in which the
separation of lipid classes can be observed.
Analysis of Lipids in Eggs, Embryos, and Nymphs
Seven days after a blood meal, laid eggs were collected (day zero) from mated and
virgin females (to obtain unfertilized eggs). Embryogenesis was allowed to proceed for
the desired time period (up to 15 days). At the desired time, groups of four eggs were
homogenized in 200 ml of cold 0.15 M NaCl and frozen ( 201C) until analysis. To
Figure 1. Separation of lipid classes by TLC. Eggs were collected on the day they were laid and
homogenized, and the lipids were extracted. Lipids were analyzed by TLC developed in two subsequent
solvent systems: benzene–ethyl ether–ethanol–acetic acid (50:40:2:0.2) and hexane–ethyl ether–acetic acid
(80:20:2). Lipid composition was determined by densitometry after staining the plate. 1, 2 and 3 indicate
samples containing lipids from two eggs each. Standards: CHOE, cholesteryl ester; TAG, triacylglycerol;
FFA, free fatty acid; DAG, diacylglycerol; CHOL, cholesterol; MAG, monoacylglycerol. Phospholipids (PL)
are at the origin (OR). Other experimental conditions are as described in M&Ms. TLC, thin-layer
chromatography.
Archives of Insect Biochemistry and Physiology
Lipid Reserves in Rhodnius prolixus Oocytes
5
obtain the embryos, eggs (from mated females) on the 7th, 10th, or 15th day after
oviposition were placed in a solution containing 1.5% Triton X-100 and 1% sodium
hypochlorite for 10 min and then washed with 0.15 M NaCl. Embryos were isolated in
cold 0.15 M NaCl under a stereomicroscope. Four embryos were homogenized in
200 ml of cold 0.15 M NaCl in a glass Potter–Elvehjem homogenizer and frozen
( 201C) until analysis. Additionally, four nymphs of each group (newly hatched and at
the indicated time point) were homogenized as described for the embryos. Samples
were subjected to lipid extraction, and the equivalent of 0.4 egg (fertilized and
unfertilized), embryo, and nymph, were used to determine the TAG content by TLC
followed by densitometry, as just described. To determine the relative lipid
composition, lipids extracted from two laid eggs (fertilized and unfertilized groups)
or two embryos were used, and TLC analysis was performed as described above.
In Vivo Transfer of Fatty Acids from Lp to the Ovary
3
H-Palmitic acid (Perkin-Elmer, Waltham, MA) in ethanol was injected into the
abdominal hemocoel of adult females (1 ml; 200,000 cpm/insect) 3 days after a blood
meal, unless otherwise stated. As previously described (Atella et al., 2000; Pontes et al.,
2008), injected FFAs immediately associate with hemolymphatic Lp. After injection,
females were maintained at 28 or 41C. For the 41C group, females were cooled to this
temperature before injection. At the desired time, the hemolymph was collected and
immediately transferred (1 ml) to scintillation liquid. The ovaries were dissected,
incubated for 30 min at 281C in 1 ml of R. prolixus saline (Maddrell, 1969) to wash out
contaminating hemolymph, and then homogenized in 100 ml of cold 0.15 M NaCl. The
radioactivity present in the hemolymph and ovaries was determined by scintillation
counting.
Preparation of Lp Labeled in DAG
Lp radioactively labeled in DAG (3H-DAG-Lp) was obtained as previously described
(Oliveira et al., 2006). 3H-Palmitic acid (1 ml, 5 mCi; 4,500 Ci/mmol; Perkin-Elmer) was
injected into the hemocoel of adult females on the third day after a blood meal with a
10-ml syringe (Hamilton Company, Reno, NV). Two hours later, the hemolymph was
collected, and Lp was purified by a KBr ultracentrifugation gradient as described by
Gondim et al. (1989a). Purified Lp was dialyzed against PBS (10 mM sodium
phosphate, 0.15 M NaCl, pH 7.4) and was subjected to lipid extraction and TLC as
described just above to determine the distribution of radioactivity in Lp lipids. The
TLC plate was stained with iodine vapor for lipid visualization. The spots were scraped
from the plates, and the lipids were eluted three times from the silica with 0.5 ml
chloroform–methanol–water (1:2:1 by volume). Radioactivity in each fraction was
determined by scintillation counting. Most of the radioactivity (90%) associated with Lp
was found in DAG (3H-DAG-Lp).
In Vivo Transfer of DAG From Lp to the Ovary
Purified 3H-DAG-Lp was injected (20,000 cpm) into the hemocoel of vitellogenic
females with a 10-ml syringe (Hamilton Company) 3 days after a blood meal, unless
otherwise stated. After injection, females were maintained at 28 or 41C. In the 41C
group, females were cooled before injection. At selected times, the hemolymph was
collected, and the ovaries were dissected. The presence of radioactivity was
determined as just described.
Archives of Insect Biochemistry and Physiology
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Archives of Insect Biochemistry and Physiology, May 2011
Fate of DAG Incorporated by the Ovary
Females were injected with 3H-DAG-Lp (30,000 cpm) on the second day after a blood
meal. Forty-eight hours later, a group of females was dissected, and mature oocytes
(2.0 mm length) were dissected and homogenized in cold 0.15 M NaCl (10 oocytes in
200 ml). From the other group, eggs were collected after oviposition and were either
immediately separated (day 0) or were allowed to develop until day 15. Oocytes and
eggs were homogenized in cold 0.15 M NaCl (10 oocytes or eggs in 200 ml). The
homogenates were subjected to lipid extraction, and neutral lipids were analyzed by
TLC as just described. The distribution of radioactivity in different lipids was
determined as described above.
Statistical Analysis
Data are expressed as the mean7SD. Comparisons among groups were performed by
the Mann–Whitney test or by one-way analysis of variance (ANOVA) followed by
Tukey’s multiple comparison test. When more than one factor was analyzed, the data
were compared by two-way ANOVA followed by the Bonferroni posttest. The tests
used in each case are stated in the figure legends. Significance level was 0.05.
RESULTS
TAG was the major lipid detected in developing oocytes, accounting for about 60% of
neutral lipids (Fig. 2A). DAG, FFAs, and cholesterol were also found. During oocyte
growth, the TAG content increased from 2.270.1 mg in 1.0-mm length follicles to
10.270.8 mg in 2.0-mm follicles (Fig. 2B). To investigate the role of Lp in the formation
of TAG store, the transport of both FFAs and DAG by this lipoprotein to the ovary was
analyzed.
It was previously demonstrated in R. prolixus that injected fatty acids immediately
associate with hemolymphatic Lp (Atella et al., 2000; Pontes et al., 2008). 3H-Palmitic
acid was injected into adult females to follow its transport by Lp to the ovary. The
radioactivity in the hemolymph was determined at different times after injection; it
rapidly decreased in the initial 10 min after injection (Fig. 3A). The incorporation
of 3H-palmitic acid by the ovary was very fast during the first 15 min after injection
(Fig. 3B). About 60 min after injection, the amount of radioactivity in the ovary leveled
off, in accordance with the already low level of radioactivity present in hemolymph. At
41C, the transfer of palmitic acid to the ovary was inhibited, which is similar to what has
been previously shown for the incorporation of phospholipids from Lp into the ovary
in this insect (Gondim et al., 1989a; Machado et al., 1996).
The capacity of the ovary to incorporate fatty acids from Lp varied as a function of
time (Fig. 4). It increased soon after the blood meal and reached a maximum around
day two.
To follow the transport of DAG to the ovary, Lp containing labeled DAG (3H-DAGLp) was injected into vitellogenic females. Radioactivity in the hemolymph decreased
much more slowly than for fatty acids (Fig. 5A), and was incorporated by the ovary,
when insects were kept at 281C (Fig. 5B). This incorporation increased up to 6 hr after
injection and was impaired at 41C. Because this experiment lasted 6 hr, it is possible
that a product of 3H-DAG metabolism was released back to the hemolymph. The
capacity of the ovary to take up DAG from Lp after a blood meal was determined. It
Archives of Insect Biochemistry and Physiology
Lipid Reserves in Rhodnius prolixus Oocytes
75
7
A
TAG
FFA
Lipids (%)
DAG
50
CHOL
25
0
1.0
1.5
2.0
Follicle (mm)
TAG ( µg / follicle)
12.5
B
10.0
7.5
5.0
2.5
0.0
1.0
1.5
Follicle (mm)
2.0
Figure 2. Accumulation of TAG in Rhodnius prolixus oocytes. Ovaries were dissected from adult females.
Follicles containing oocytes of different lengths (1.0, 1.5, and 2.0 mm) were separated in cold 0.15 M NaCl
under a stereomicroscope. Follicles were homogenized, and the lipids were extracted. Lipid composition
(A) and TAG content (B) were determined by TLC followed by densitometry, as described in Materials and
Methods. TAG, triacylglycerol; FFA, free fatty acid; DAG, diacylglycerol; CHOL, cholesterol. The results are
mean7SD for 4 (A) or 7–9 (B) determinations.
increased after feeding, was maximal at days 2 through 4, and then started to decrease
(Fig. 6), similar to what was observed for the transfer of fatty acids (Fig. 4).
The fate of lipids accumulated by oocytes was determined 48 hr after the injection
of 3H-DAG-Lp into females (Fig. 7). In mature oocytes (2.0 mm length), some
radioactivity (13.271.9%) was present in phospholipids, and the remaining 86.8 % was
in neutral lipids (data not shown). Of these, most of the incorporated radioactivity was
found in TAG (85.772.3%), while some was present in DAG (5.570.8%) and in FFAs
(5.371.2%). Some labeling, although at very low level, was present in monoacylglycerols and cholesteryl-esters (Fig. 7). These results show that the remodeling of lipids
and the synthesis of glycerolipids, such as TAG, occur inside growing oocytes. The fate
of lipids donated by Lp to the growing oocytes was also followed in eggs immediately
after oviposition and at 15 days, just before hatching. The relative distribution of
radioactivity in lipid classes was very similar to that observed in oocytes (Fig. 7). The
same result was also observed for phospholipids (data not shown).
To follow the utilization of lipid reserves during embryonic development, TAG
content was measured after oviposition (Fig. 8). TAG levels decreased from
16.472.1 mg when the eggs were laid to 10.071.3 mg at day 15, just before hatching.
Archives of Insect Biochemistry and Physiology
8
Archives of Insect Biochemistry and Physiology, May 2011
A
DPM/ µl Hemolymph
9000
*
6000
3000
0
0
1
2
3
4
Time (h)
B
DPM / Ovary
12000
9000
6000
3000
***
0
0
1
2
3
4
Time (h)
Figure 3. Transfer of FFA from lipophorin to the ovary. 3H-Palmitic acid was injected into adult females on
the third day after a blood meal and, after different times at 281C ( & ), the hemolymph was collected and
immediately transferred to scintillation liquid (A). Ovaries were dissected, washed, and homogenized, and
the incorporated radioactivity was determined by scintillation counting (B). Control females were kept at 41C
for 4 hr (&), and the amount of radioactivity in the hemolymph and ovaries was measured. Experimental
conditions were as described in the Materials and Methods section. The results are from a representative
experiment and are shown as the mean7SD for at least three determinations. The results for females at 41C
for 4 hr were different than those at 281C; Po0.05 (hemolymph) and Po0.001 (ovary), as determined by the
Mann–Whitney test. FFA, free fatty acid.
On the seventh day, about 50% of the total TAG was found in embryos (7.471.0 mg/
embryo), and the same amount (7.470.9 mg) was present in nymphs soon after
hatching, which corresponded to 74% of total TAG at this point. In nonfertilized eggs,
the TAG content remained constant after egg laying.
Although the amount of TAG in laid fertilized eggs decreased as the embryo
developed, the relative lipid composition remained the same, as TAG accounted for
about 70% of neutral lipids in eggs until the end of embryogenesis (Fig. 9A).
Nonfertilized eggs showed a similar and also constant lipid composition (Fig. 9B).
Embryos were removed from the eggs, and their lipid composition was analyzed
(Fig. 9C). The lipid composition of the embryos differed somewhat from that of the eggs
with TAG accounting for a smaller percentage of neutral lipids (59.376.3% in day 7 and
48.874.5% in day 15) compared with the whole egg (Fig. 9A). DAG was present in larger
amounts, accounting for 24.274.5% of lipids at day 15 compared with 11.970.9% in the
whole egg. This same tendency was also observed for FFAs and cholesterol (Fig. 9).
As a great part of the lipids from the egg is associated with the embryo (Fig. 8),
TAG was quantified in the nymph soon after hatching and in the days following
Archives of Insect Biochemistry and Physiology
Lipid Reserves in Rhodnius prolixus Oocytes
9
DPM / Ovary
20000
15000
10000
5000
0
0
3
6
9
12
15
Days after blood meal
Figure 4. 3H-Palmitic acid incorporation in the ovary after feeding. At different days after a blood meal,
3
H-palmitic acid was injected into females. One hour later, the ovaries were dissected, washed, and
homogenized, and the incorporated radioactivity was determined by scintillation counting. Experimental
conditions were as described in the Materials and Methods section. The results shown are from a representative
experiment and are shown as the mean7SD for at least five determinations.
A
DPM / µl Hemolymph
3000
*
2000
1000
0
0
1
2
3
4
5
6
Time (h)
B
DPM / Ovary
15000
10000
***
5000
0
0
1
2
3
4
5
6
Time (h)
Figure 5. Transfer of DAG from lipophorin to the ovary. Lipophorin with labeled DAG (3H-DAG-Lp) was
injected into adult females on the third day after a blood meal. The insects were kept at 281C ( & ), and the
hemolymph was collected at the times indicated and immediately transferred to scintillation liquid (A).
Ovaries were dissected, washed, and homogenized, and the incorporated radioactivity was determined by
scintillation counting (B). Control females were kept at 41C (&), and the level of radioactivity in the
hemolymph and ovaries was determined. Experimental conditions were as described in the Materials and
Methods section. The results shown are from a representative experiment and are shown as the mean7SD
for at least six determinations. The results for females at 41C for 6 hr were different than those at 281C.
Po0.05 (hemolymph) and Po0.001 (ovary), as determined by the Mann–Whitney test. DAG, diacylglycerol;
Lp, lipophorin.
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Archives of Insect Biochemistry and Physiology, May 2011
DPM / Ovary
14000
10500
7000
3500
0
0
3
6
9
12
15
Days after blood meal
Figure 6. DAG incorporation by the ovary at different days after feeding. On different days after a blood
meal, 3H-DAG-Lp was injected into females. Two hours later, the ovaries were dissected, washed, and
homogenized, and the incorporated radioactivity was determined by scintillation counting. Experimental
conditions were as described in the Materials and Methods section. The results shown are from a
representative experiment and are shown as the mean7SD for at least four determinations. DAG,
diacylglycerol; Lp, lipophorin.
100
TAG
FFA
Lipids (%)
80
DAG
CHOE
60
MAG
40
10
5
0
Oocyte
Egg 0
Egg 15
3
Figure 7. Fate of lipids incorporated in the ovary. H-DAG-Lp was injected into females on the second day
after a blood meal; 48 hr later, the ovaries were dissected from a group of females, and the mature oocytes
(2.0 mm length) were separated. From the other group of females, eggs were collected immediately after
oviposition (day 0) or collected and allowed to develop until day 15. Oocytes and eggs were homogenized
and subjected to lipid extraction and TLC for lipid identification. The radioactivity associated with various
lipid classes was determined by scintillation counting. TAG, triacylglycerol; FFA, free fatty acid; DAG,
diacylglycerol; MAG, monoacylglycerol; CHOE, cholesteryl ester. Experimental conditions were as described
in the Materials and Methods section, and the results are shown as the mean7SD for six to nine
determinations. DAG, diacylglycerol; Lp, lipophorin.
(Fig. 10). In accordance with the data obtained for embryos (Fig. 8), 9.171.2 mg of TAG
was present in newly hatched nymphs, and it decreased to 3.470.4 mg 15 days later.
DISCUSSION
In insects studied so far, lipids are found in oocytes mainly as TAG (Troy et al., 1975;
Wiemerslage, 1976; Kawooya and Law, 1988; Briegel, 1990), which was the case for
R. prolixus oocytes, in which the amount of stored TAG increased as the follicles grew.
Although Lp does not contain a high amount of FFA, FFA is efficiently transported
in the insect hemolymph by this lipoprotein (Soulages et al., 1988; Soulages and Wells,
Archives of Insect Biochemistry and Physiology
Lipid Reserves in Rhodnius prolixus Oocytes
11
25
TAG (µg)
20
15
10
5
0
0
3
6
9
12
15
Days after oviposition
Figure 8. TAG content in laid eggs, embryos, and nymphs of Rhodnius prolixus. TAG content was
determined in laid fertilized ( & ) and unfertilized (&) eggs after oviposition and in embryos. On the 7th day
after oviposition, fertilized eggs were dissected, and the TAG amount associated with the embryo () was
determined. The levels of TAG present in nymphs immediately after hatching (J) were also measured. Eggs,
embryos, and nymphs were homogenized and subjected to lipid extraction, and TAG content was
determined by TLC followed by densitometry. Experimental conditions were as described in the Materials
and Methods section, and the results are presented as the mean7SD for at least three determinations. In
fertilized eggs, the TAG content decreased (Po0.001), while in unfertilized eggs, the TAG content did not
change significantly (P40.05), according to the analysis by one-way ANOVA. When conditions (fertilized and
unfertilized eggs) were analyzed by two-way ANOVA plus the Bonferroni test, the TAG content in fertilized
eggs was different than that in unfertilized eggs at day 15 (Po0.01). TAG, triacylglycerol.
1994a; Atella et al., 2000). In M. sexta, the half life of FFA bound to Lp in the
hemolymph was about 2 min (Soulages and Wells, 1994b), and similar results were
obtained for adult females of R. prolixus on the 10th day after feeding (Atella et al.,
2000). In this article, females on the third day after a blood meal were analyzed, and a
rapid incorporation of fatty acids into the ovary after the injection of 3H-palmitic acid
was observed, corresponding to a decrease in fatty acids in the hemolymph. However,
30–60 min after injection, the radioactivity accumulated by the oocytes plateaued,
probably because fatty acids were very rapidly removed from the hemolymph and the
radioactivity had been depleted. This high rate of fatty acid incorporation was
expected due to the very intense oogenesis at this time (Oliveira et al., 1986).
DAG is the most abundant neutral lipid in Lp of most insects, including R. prolixus
(Blacklock and Ryan, 1994; Soulages and Wells, 1994a). As described for the ovary of
M. sexta, the decay of radioactivity in the hemolymph and its incorporation by the
ovary was observed, although the rate of incorporation was not as fast as that of FFA.
DAG is present in very high amounts in Lp particles, while FFAs are very scarce;
therefore, DAG is considered as a major source of fatty acids for insect organs (Ryan
and Van der Horst, 2000; Canavoso et al., 2001). However, the contribution of DAG
and FFA to the lipid content of the ovaries is not known (Ziegler and Van Antwerpen,
2006). In M. sexta, for example, Lp supplies DAG to growing oocytes, where they are
converted to TAG (Kawooya and Law, 1988). In this article, it was shown that both
FFAs and DAGs are supplied by Lp to developing ovaries in addition to phospholipids
(Gondim et al., 1989b). It is interesting to note that the previously described time
course for the incorporation of phospholipids in the ovary (Gondim et al., 1989b) was
very similar to that observed for DAG. The capacity of the ovary to take up both FFA
and DAG from Lp was maximal around the second day after feeding and was similar to
the previously shown rate for the incorporation of phospholipids by the ovary
Archives of Insect Biochemistry and Physiology
12
Archives of Insect Biochemistry and Physiology, May 2011
A
80
TAG
FFA
DAG
60
Lipids (%)
CHOL
40
20
0
0
80
1
2
7
Days after oviposition
15
B
Lipids (%)
60
40
20
0
0
15
Days after oviposition
80
C
Lipids (%)
60
40
20
0
7
10
Days after oviposition
15
Figure 9. Lipid composition of laid eggs and embryos of Rhodnius prolixus. Fertilized (A) and unfertilized
(B) laid eggs were collected at different days after oviposition, homogenized, and subjected to lipid
extraction. At days 7, 10, and 15 after oviposition, embryos (C) were also dissected from fertilized eggs, and
the lipids were extracted. The lipid composition was determined by TLC followed by densitometry, as
described in the Material and Methods section. TAG, triacylglycerol; FFA, free fatty acid; DAG,
diacylglycerol; CHOL, cholesterol. The results are shown as the mean7SD for three to six (A and B) or
four to five (C) determinations.
(Gondim et al., 1989b). These data are in accordance with the fact that all these lipid
groups are transported by Lp. Moreover, on the second and third day after a blood
meal, the ovary also shows a high rate of vitellogenin endocytosis, as the oocytes are
Archives of Insect Biochemistry and Physiology
Lipid Reserves in Rhodnius prolixus Oocytes
13
14
TAG ( µg / nymph)
12
10
8
6
4
2
0
0
1
5
Days after eclosion
15
Figure 10. TAG content in nymphs after hatching. Immediately after hatching (day 0) and on subsequent
days, nymphs were homogenized, and the lipids were extracted. TAG content was determined by TLC
followed by densitometry, as described in the Material and Methods section. The results are shown as the
mean7SD for at least three determinations. TAG, triacylglycerol; TLC, thin-layer chromatography.
growing very rapidly (Oliveira et al., 1986). Therefore, lipids and proteins are
simultaneously stored to be used by the embryo. In contrast to the ovary, the midgut of
R. prolixus has its greatest capacity to incorporate fatty acids from the hemolymph
around the 10th day after feeding, when digestion is almost finished (Atella et al.,
2000). Soon after the meal, during the first 3 days, when the ovary and fat body are
accumulating lipids (Gondim et al., 1989b; Pontes et al., 2008), the midgut is very
active in donating lipids from the diet to circulating Lp (Atella et al., 1995; Coelho
et al., 1997). These results show that insect organs acquire lipids at different times,
according to the physiological demands of each one.
Similar to the observations in oocytes of M. sexta (Kawooya and Law, 1988), lipids
that were incorporated into the ovaries of R. prolixus were primarily used to synthesize
TAG, which, in insect oocytes, is stored in lipid droplets (Ziegler and Van Antwerpen,
2006) and used to support embryogenesis, along with other nutrients. It is noteworthy
that just after oviposition in eggs of R. prolixus, the TAG content (70.4%) was slightly
higher than in mature oocytes (60%), while the DAG content (9.6%) was lower than in
2.0 mm oocytes (13.9%). Although these differences are small, they suggest that a
rearrangement of lipid composition occurs in the later stages of oocyte maturation,
after chorion deposition.
After oviposition, about 40% of TAG reserves were used during embryogenesis.
Previous reports have noted that glycogen is also consumed as an embryo develops,
with a similar time course to what we observed with TAG (Santos et al., 2008).
Therefore, it seems that lipids and carbohydrates are simultaneously used as fuel. The
fact that TAG was not consumed in the eggs of virgin females indicated that
fertilization is important for substrate degradation, as was also shown for glycogen
mobilization (Santos et al., 2008).
The observation that less than half of the TAG content in the egg was consumed
before hatching was similar to that of eggs of other arthropods such as
C. quinquefasciatus, the butterfly Bicyclus anynana, and the tick Boophilus microplus
(Van Handel, 1993; Campos et al., 2006; Geister et al., 2009). In these animals, a large
portion of the egg lipids are still present in the hatchlings. In the mosquito,
C. quinquefasciatus, for example, 46% of lipids are consumed during embryogenesis
Archives of Insect Biochemistry and Physiology
14
Archives of Insect Biochemistry and Physiology, May 2011
despite the fact that about 90% of the energy required for embryo growth is supported
by lipids (Van Handel, 1993).
On the seventh day after oviposition, about half of the TAG in the egg was
associated with the embryo, which is the same amount of lipids found in newly hatched
nymphs. It seems to be a common fact in R. prolixus that part of the yolk, including
proteins, glycogen and lipids, remains in the digestive tract of the nymphs and
provides nutrients during the initial days after hatching (Oliveira et al., 1989; Santos
et al., 2008). The amount of TAG was determined in unfed nymphs, and therefore,
any lipids in the nymph must have originated from the egg. This probably occurs
during dorsal closure when the yolk is internalized by the embryo (Kelly and Huebner,
1986). After hatching, lipids found in the nymph are slowly used by the insect in an
efficient and controlled mechanism. Fifteen days after emergence, about 30% of the
TAG is still present.
This study is the first description of TAG accumulation and mobilization by the
oocytes of R. prolixus. It is an initial step toward further investigations on the pathways
and regulatory mechanisms that are involved in these processes.
ACKNOWLEDGMENTS
The authors thank Heloisa S. L. Coelho and Lilian S. da C. Gomes for the excellent
technical assistance and José de S. Lima Junior and Litiane M. Rodrigues for insect
care.
LITERATURE CITED
Atella GC, Gondim KC, Masuda H. 1992. Transfer of phospholipids from fat body to lipophorin
in Rhodnius prolixus. Arch Insect Biochem Physiol 19:133–144.
Atella GC, Gondim KC, Masuda H. 1995. Loading of lipophorin particles with phospholipids at
the midgut of Rhodnius prolixus. Arch Insect Biochem Physiol 30:337–350.
Atella GC, Arruda MABCF, Masuda H, Gondim KC. 2000. Fatty acid incorporation by Rhodnius
prolixus midgut. Arch Insect Biochem Physiol 43:99–107.
Atella GC, Silva-Neto MA, Golodne DM, Arefin S, Shahabuddin M. 2006. Anopheles gambiae
lipophorin: characterization and role in lipid transport to developing oocyte. Insect
Biochem Mol Biol 36:375–386.
Beenakkers AMT, Chino H, Law JH. 1988. Lipophorin nomenclature. Insect Biochem 18:1–2.
Bitman J, Wood DL. 1982. An improved copper reagent for quantitative densitometric thinlayer chromatography of lipids. J Liquid Chromatogr 5:1155–1162.
Blacklock BJ, Ryan RO. 1994. Hemolymph lipid transport. Insect Biochem Mol Biol 24:
855–873.
Bligh EG, Dyer WJ. 1959. A rapid method of total lipid extraction and purification. Can J
Biochem Physiol 37:911–917.
Briegel H. 1990. Metabolic relationship between female body size, reserves, and fecundity of
Aedes aegypti. J Insect Physiol 36:165–172.
Campos E, Moraes J, Fac- anha AR, Moreira E, Valle D, Abreu L, Manso PP, Nascimento A, PelajoMachado M, Lenzi H, Masuda A, Vaz Jr IS, Logullo C. 2006. Kinetics of energy utilization
in Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) embryonic development. Vet
Parasitol 138:349–357.
Canavoso LE, Jouni ZE, Karnas KJ, Pennington JE, Wells MA. 2001. Fat metabolism in insects.
Annu Rev Nutr 21:23–46.
Archives of Insect Biochemistry and Physiology
Lipid Reserves in Rhodnius prolixus Oocytes
15
Chino H, Downer RGH, Wyatt GR, Gilbert LI. 1981. Lipophorins, a major class of lipoproteins
of insect hemolymph. Insect Biochem 11:491.
Coelho HSL, Atella GC, Moreira MF, Gondim KC, Masuda H. 1997. Lipophorin density
variation during oogenesis in Rhodnius prolixus. Arch Insect Biochem Physiol 35:301–313.
Dantuma NP, Pijnenburg MAP, Diederen JHB, Van der Horst DJ. 1997. Developmental downregulation of receptor-mediated endocytosis of an insect lipoprotein. J Lipid Res 38:
254–265.
Downer RGH, Chino H. 1985. Turnover of protein and diacylglycerol components of
lipophorin in insect hemolymph. Insect Biochem 15:627–630.
Fan Y, Schal C, Vargo EL, Bagnères AG. 2004. Characterization of termite lipophorin and its
involvement in hydrocarbon transport. J Insect Physiol 50:609–620.
Geister TL, Lorenz MW, Hoffmann KH, Fischer K. 2009. Energetics of embryonic development:
effects of temperature on egg amd hatchling composition in a butterfly. J Comp Physiol [B]
179:87–98.
Gondim KC, Oliveira PL, Coelho HSL, Masuda H. 1989a. Lipophorin from Rhodnius prolixus:
purification and partial characterization. Insect Biochem 19:153–161.
Gondim KC, Oliveira PL, Masuda H. 1989b. Lipophorin and oogenesis in Rhodnius prolixus:
transfer of phospholipids. J Insect Physiol 35:19–27.
Grillo LAM, Majerowicz D, Gondim KC. 2007. Lipid metabolism in Rhodnius prolixus
(Hemiptera: Reduviidae): role of a midgut triacylglycerol-lipase. Insect Biochem Mol Biol
37:579–588.
Kawooya JK, Law JH. 1988. Role of lipophorin in lipid transport to the insect egg. J Biol Chem
263:8748–8753.
Kawooya JK, Osir EO, Law JH. 1988. Uptake of the major hemolymph liporprotein and its
transformation in the insect egg. J Biol Chem 263:8740–8747.
Kelly GM, Huebner E. 1986. Experimental analysis of Rhodnius prolixus (Insecta, Hemiptera)
embryogenesis. Prog Clin Biol Res 217A:423–426.
Machado EA, Atella GC, Gondim KC, de Souza W, Masuda H. 1996. Characterization and
immunocytochemical localization of lipophorin binding sites in the oocytes of Rhodnius
prolixus. Arch Insect Biochem Physiol 31:185–196.
Maddrell SHP. 1969. Secretion by the Malpighian tubules of Rhodnius: the movements of ions
and water. J Exp Biol 51:71–97.
Oliveira PL, Gondim KC, Guedes DM, Masuda H. 1986. Uptake of yolk proteins in Rhodnius
prolixus. J Insect Physiol 32:859–866.
Oliveira PL, Petretski MDA, Masuda H. 1989. Vitellin processing and degradation during
embryogenesis in Rhodnius prolixus. Insect Biochem 19:489–498.
Oliveira GA, Baptista DL, Guimarães-Motta H, Almeida IC, Masuda H, Atella GC. 2006. Flightoogenesis syndrome in a blood-sucking bug: biochemical aspects of lipid metabolism. Arch
Insect Biochem Physiol 62:164–175.
Pontes EG, Leite P, Majerowicz D, Atella GC, Gondim KC. 2008. Dynamics of lipid accumulation
by the fat body of Rhodnius prolixus: the involvement of lipophorin binding sites. J Insect
Physiol 54:790–797.
Raikhel AS, Dhadialla TS. 1992. Accumulation of yolk proteins in insect oocytes. Annu Rev
Entomol 37:217–251.
Ruiz JI, Ochoa B. 1997. Quantification in the subnanomolar range of phospholipids and neutral
lipids by monodimensional thin-layer chromatography and image analysis. J Lipid Res 38:
1482–1489.
Ryan RO, Van der Horst DJ. 2000. Lipid transport biochemistry and its role in energy
production. Annu Rev Entomol 45:233–260.
Archives of Insect Biochemistry and Physiology
16
Archives of Insect Biochemistry and Physiology, May 2011
Santos R, Mariano AC, Rosas-Oliveira R, Pascarelli B, Machado EA, Meyer-Fernandes JR,
Gondim KC. 2008. Carbohydrate accumulation and utilization by oocytes of Rhodnius
prolixus. Arch Insect Biochem Physiol 67:55–62.
Soulages JL, Wells MA. 1994a. Lipophorin: the structure of an insect lipoprotein and its role in
lipid transport in insects. Adv Protein Chem 45:371–415.
Soulages JL, Wells MA. 1994b. Metabolic fate and turnover rate of hemolymph free fatty acids in
adult Manduca sexta. Insect Biochem Mol Biol 24:79–86.
Soulages JL, Rimoldi OJ, Peluffo OR, Brenner RR. 1988. Transport and utilization of free fatty
acids in Triatoma infestans. Biochem Biophys Res Commun 157:465–471.
Sun JX, Hiraoka T, Dittmer NT, Cho KH, Raikhel AS. 2000. Lipophorin as a yolk protein
precursor in the mosquito, Aedes aegypti. Insect Biochem Mol Biol 30:1161–1171.
Troy S, Anderson WA, Spielman A. 1975. Lipid content of maturing ovaries of Aedes aegypti
mosquitoes. Comp Biochem Physiol B 50:457–461.
Van Handel E. 1993. Fuel metabolism of the mosquito (Culex quinquefascitus) embryo. J Insect
Physiol 39:831–833.
Van Heusden MC, Van der Horst DJ, Voshol J, Beenakkers AMT. 1987. The recycling of protein
components of the flight-specific lipophorin in Locusta migratoria. Insect Biochem 17:
771–776.
Wiemerslage LJ. 1976. Lipid droplet formation during vitellogenesis in the cecropia moth.
J Insect Physiol 22:41–50.
Ziegler R. 1997. Lipid synthesis by ovaries and fat body of Aedes aegypti (Diptera: Culicidae). Eur
J Entomol 94:385–391.
Ziegler R, Van Antwerpen R. 2006. Lipid uptake by insect oocytes. Insect Biochem Mol Biol 36:
264–272.
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