Differential partitioning of maternal fatty acid and phospholipid in neonate mosquito larvae
Laboratory of Malaria and Vector Research, National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD
20892, USA
* Present address: Universidade Federal do Rio de Janeiro, UFRJ, Centro de
Ciências da Saúde, Instituto de Ciências Biomédicas,
Departamento de Bioquímica Médica, Ilha da Ciudade
Universitária, Rio de Janeiro, CEP 21941-590, Brazil
Author for correspondence (e-mail:
mshahabudd{at}niaid.nih.gov)
Accepted 13 August 2002
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Summary |
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Key words: Malaria vector, mosquito, lipid metabolism, lipophorin, fatty acid, phospholipid, neonate
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Introduction |
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Studies with chicken eggs described lipid metabolism in the developing
embryo (Speake et al., 1998;
Noble and Cocchi, 1990
).
During development, especially in the second half, there is an increased lipid
transfer from the yolk to the embryo
(Noble and Cocchi, 1990
).
Lizards are also an interesting model for studying lipid transport to
offspring, because in this family there are both live-bearing and egg-laying
species (Speake and Thompson,
2000
). Lipid composition in eggs from alligators
(Speake and Thompson, 1999
)
and from some turtles (Rowe et al.,
1995
) have also been studied.
Studies with such insects as Manduca sexta and Locusta
migratoria have described insect lipid transport processes
(Ryan et al., 1986;
Arrese et al., 2001
). Lipid is
carried by a lipoprotein complex, called lipophorin, which picks up lipid from
the gut and delivers it to tissues such as developing eggs
(Shapiro et al., 1984
;
Kawooya et al., 1988
;
Gondim et al., 1989
; Atella et
al., 1992
,
1995
). In M. sexta,
approximately 40% of the dry mass of the mature egg is lipid. Most of these
lipids are imported from the maternal lipid pool, biosynthesis accounting for
only about 1% of the lipid in the egg
(Canavoso et al., 2001
).
Among the lipids, triacylglycerols consist of three molecules of fatty acids and are the most concentrated form of energy present in the yolk. Phospholipids and free cholesterols are poor sources of energy. Phospholipids are major components of cell plasma membranes. Because lipids serve functionally different purposes, segregation of lipids in appropriate tissues in embryos of egg-laying animals is essential for development of healthy neonates. However, little is known about how imported lipids are distributed during embryogenesis and in neonates.
Unlike imported proteins and nucleic acids that usually degrade to monomers
before utilization, most imported lipids remain intact and can be easily
traced in live animals. Farber et al.
(2001) used fluorescently
labeled phosphatidylcholine substrate to trace phospholipase enzyme in live
zebrafish, where labeled lipids accumulated in the digestive organs and the
gall bladders that fluoresced intensely
(Hendrickson et al., 1999
;
Farber et al., 2001
). In the
present work, we used fluorescently labeled fatty acids and phospholipids to
investigate maternal lipid uptake and distribution, both in the developing egg
and in the neonate mosquitoe larvae. Because mosquitoes need blood feeding to
trigger egg development (Clements,
1992
), they provide an opportunity to monitor of a batch of
synchronously developing oocytes with precision. Our results show a novel
differential partitioning of fatty acid and phospholipid in the newly hatched
larvae.
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Materials and methods |
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Purification of lipophorin
Mosquito lipophorins were purified using potassium bromide gradient
ultracentrifugation as described earlier
(Gondim et al., 1989).
Mosquitoes (4 g) were homogenized using a glass hand grinder (Thomas
Scientific, Swedesboro, NJ, USA) on an ice bath in the presence of 20
mmoll-1 glutathione, 5 mmoll-1 EDTA, 2
mmoll-1 PMSF, 0.5 µg µl-1 antipain, 5
µmoll-1 pepstatin and 0.5 µg µl-1 leupeptin in
PBS (10 mmoll-1 sodium phosphate, 0.15 moll-1 NaCl, pH
7.4). The homogenate was then centrifuged (100 000 g, 30 min,
4°C) to remove insoluble material. Solid potassium bromide was added to
the supernatant to a final concentration of 0.4 g ml-1, and the
mixture was again centrifuged at 125 000 g in a Beckman 50.2 Ti rotor
at 4°C for 20 h. The gradient was then fractionated from top to bottom,
and lipophorin fractions were pooled, extensively dialyzed against PBS, and
stored under liquid nitrogen until use. The degree of purification was
monitored by SDS-PAGE, and protein concentration was estimated using a
microBCA kit (Pierce, Rockford, IL, USA) in the presence of 0.5% SDS, using
bovine serum albumin as standard.
Preparation and measurement of 32P-phospholipid-labeled
lipophorin
Into the hemolymph of adult female A. gambiae, 0.1 µl of
32Pi (1 µCi, 3700 Bq, Amersham, Piscataway, NJ, USA) was
injected using a PLI-100 microinjector (Harvard Apparatus, Holliston, MA,
USA). 1 day later, 32P-phospholipid-labeled lipophorin
(32P-lipophorin) was purified from the total homogenate of the
injected mosquitoes using potassium bromide gradient ultracentrifugation as
described above (Gondim et al.,
1989). 32P-labeled lipophorin was injected into
mosquito hemolymphs at different days after a blood meal using a PLI-100
microinjector. Ovaries were dissected, separated according to the stages
described by Christophers
(1911
), homogenized, and the
radioactivity measured by scintillation counting.
In vitro labeling of lipophorin with fluorescent lipid
analogs
Purified lipophorin was labeled with Texas Red-conjugated
phosphatidylethanolamine, N-Texas Red®
sulfonyl-1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium
salt (Texas Red-PE), BODIPY-labeled palmitic acid,
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid
(BODIPY-FA), or BODIPY-labeled phosphatidyl choline,
2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine
(BODIPY-PC) (Molecular Probes) as described by Martin-Nizard et al.
(1987) with slight
modifications. Briefly, fluorescent lipid-coated glass beads (0.2 mg) were
incubated with 2 mg of pure lipophorin. The mixture was gently stirred at room
temperature for 1 h, and the glass beads removed by centrifugation (5 min at
9500 g). Fluorescently labeled lipophorin was re-isolated by gel
chromatography on a column of Sephacryl S-200 concentrated by Microcon filters
(Amicon, Bedford, MA, USA). Lipophorin was also labeled with fluorescein
5'-isothiocyanate using a Fluorescein-EX Protein labeling kit
(FluoReporter®; Molecular Probes).
Observation of fluorescent lipids in live oocytes and larvae
2 days after a blood meal, fluorescently-labeled lipophorin was injected
into the hemolymph of vitellogenic females. 3 days later, egg laying was
induced by transferring the mosquitoes to a Petri dish with a 80 mm filter
paper disk soaked with distilled deionized water. The Petri dish was protected
from the light until the larval hatch. Approximately 36 h later, the larvae
emerged from the eggs and were collected immediately and maintained in ice
protected against the light. To visualize the lipids in live oocytes and
larvae, a 0.5-mm thick plastic -ring sticker was placed on a glass
microscope slide, into the middle of which dissected oocytes or newly hatched
larvae were placed and covered with a coverslip. The O-ring protects the
oocytes and larvae from being damaged by the coverslip. The slide was
immediately transferred to the microscope stage for observation. Fluorescence
was analyzed by a confocal microscopy (Leica DMIRBE microscope, Leica
TCS-NT/SP confocal; Leica Microsystems GmbH, Heidelberg, Germany) equipped
with argon laser. Additional observations were made by epifluorescence
microscopy (Carl Ziess, Inc., Thornwood, NJ, USA) with monochromatic filters
for BODIPY (excitation 505 nm/emission 515 nm) or for Texas Red (excitation
582 nm/emission 601 nm). High-resolution digital images were captured and
processed using Adobe Photoshop (Adobe Systems, Inc., San José, CA,
USA).
Thin-layer chromatography of lipids
Lipid extraction was performed according to Blight and Dyer
(1959) for 2 h in a stoppered
tube in 5 ml of chloroformmethanolwater solution (2:1:0.8, v/v)
with intermittent agitation. After centrifugation, the supernatant was
collected and the precipitate subjected to a second lipid extraction (1 h). To
the pooled extract, 5 ml of water and 5 ml of chloroform were added; the
mixture was shaken and, after centrifugation, the organic phase was removed
and dried under nitrogen. The amount of total lipid was determined
gravimetrically. Extracted lipids were analyzed by thin-layer chromatography
as described for phospholipids (Horwitz
and Perlman, 1987
) on silica gel plates (Merck KGaA, Darmstadt,
Germany). Lipids were visualized by illuminating the plates with a UV
lamp.
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Results |
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To examine whether the entire lipophorin complex is taken up or only lipids
are delivered as the lipophorin binds to the oocyte, we labeled the
apolipoprotein moiety of purified lipophorin with fluorescein isothiocyanate.
The fluorescent lipophorin was injected into mosquito hemolymph. 2 days after
injection, fluorescence was observed surrounding the follicular cell lining
and in the micovillar region, similar to the sites where vitellogenin binding
had been observed earlier (Sappington and
Raikhel, 1998). Lipophorins were also found in spherical yolk
bodies in the cortex, except in the nurse cell region
(Fig. 1B). Similar distribution
of lipophorin was observed earlier with A. aegypti oocytes
(Sun et al., 2000
).
Distribution of imported lipids in oocytes was also examined by labeling lipophorins with fluorescently tagged fatty acid and phospholipids. Purified lipophorin was loaded with a mixture of Texas Red-tagged phosphatidyl-ethanolamine (Texas Red-PE) and BODIPY-labeled palmitic acid (BODIPY-FA) (Fig. 1C,D) and injected into the hemolymph of blood-fed mosquitoes. When developing eggs from these mosquitoes were examined, both lipids were found in oocytes (Fig. 1E,F) colocalized within the same yolk vesicles with lipophorin (Fig. 1G), demonstrating that imported lipids are stored in the same compartment in developing oocytes.
Maternal phospholipid and fatty acid segregate differently in neonate
larvae
To examine the imported maternal lipids in newly hatched mosquito larvae,
eggs from the mosquitoes previously injected with lipophorin fluorescently
labeled with phospholipid (Texas Red-PE;
Fig. 1C) and fatty acid analog
(BODIPY-FA; Fig. 1D) were
collected. 2 days later when the eggs hatched, live larvae were examined for
maternal lipid within 30 min after hatching. Both red and green fluorescence
were found in the newly hatched larvae
(Fig. 2), indicating that at
least some of the imported lipids survived embryogenesis.
|
Distribution of the lipid types, however, was remarkably different in the
larvae. With a setup of higher gain of fluorescence, fatty acids were seen in
most parts of the body, but when gain was adjusted to a lower setting to
examine the site of maximum accumulation, the majority of the fatty acid
(BODIPY-FA) was found segregated in spherical vesicles in the thorax and along
the side of the body cavity (Fig.
2A). Accumulation of fatty acid or phospholipids in head was
remarkably low. Along the body, fatty acid-containing vesicles also
concentrated at the base of the body segment hair. In contrast to the fatty
acid, however, imported phosphatidylethanolamine was segregated differently
and mostly found associated with the upper intestine and the gastric caeca. In
mosquito larvae, gastric caeca are eight pouch-like structures located
immediately behind the cardia and opening into the anterior end of the midgut
(Volkmann and Peters, 1989).
They are located within the thorax of the neonate larvae
(Fig. 2B). In every larva, the
gastric caeca was found to be filled with the phospholipid (Texas Red-PE). In
the intestine, although the phospholipids were always seen near the upper gut
region close to the gastric caeca, the length along the gut where fluorescence
was found was variable. In most cases, this was limited to one third of the
upper gut only, but phospholipids sometimes filled most of intestine from the
gastric caeca to the lower midgut. Movement of the phospholipid from the caeca
to intestine was also frequently observed. There was little overlap in the
localization of stored fatty acids and phospholipids seen in the neonate
larvae (Fig. 2C,D). The unique
segregation of the fatty acid and the phospholipid was reproducible in all
experiments (N=32) and in all larvae examined from each batch of eggs
(Fig. 2E,F) obtained from
labeled lipophorin-injected mosquitoes (15 mosquitoes in each
experiments).
Similar partitioning of lipid also occurs in A. aegypti larvae
To investigate whether differential partitioning of lipids occurs in
mosquitoes other than Anopheles, we injected Texas Red-PE- and
BODIPY-FA-labeled lipophorin into the hemolymph of blood-fed A.
aegypti mosquitoes (order Culicidae). Aedes was evolutionarily
separated from Anopheles at least 150 million years ago
(Kwiatowski et al., 1995). The
geographic distribution of these mosquitoes is also different, and they are
vectors of different blood-borne human pathogens
(Lehane, 1991
). Maternal
lipids were seen in neonate larvae of both mosquitoes, suggesting that,
similar to Anopheles, some maternal lipids also remain intact during
Aedes embryogenesis. Furthermore, the fatty acid and phospholipid
were distributed in A. aegypti larvae with a pattern similar to that
seen in A. gambiae (Fig.
2G,H), showing that differential partitioning of phospholipid and
fatty acid is a common phenomenon in neonates of both of these mosquitoes.
We then examined whether the fluorescence in the larvae is due to the accumulation of a metabolized form of Texas Red-PE instead of the intact molecule. For this, we extracted total lipids from the labeled neonate larvae and fractionated them on thin-layer chromatography. Fig. 3 shows that the phosphatidylethanolamine analogs from the larvae had identical relative mobility to that of the Texas Red-PE originally injected into the mosquito, which indicates that the fluorescence in the gastric caeca and intestine of neonate larvae was due to the intact phosphatidylethanolamine analog and probably not due to any metabolized fluorescent product.
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Both phosphatidylcholine and phosphatidylethanolamine segregate in
gastric caeca
To verify whether accumulation in the gastric caeca is unique to
phosphatidylethanolamine or common to other phospholipids as well, mosquitoes
were injected with lipophorin loaded with a mixture of two different
phospholipids fluorescently tagged at different positions. The
phosphatidylcholine analog (BODIPY-PC) was labeled with BODIPY at the gamma
position of one of the fatty acid chains
(Fig. 4A), and Texas Red-PE
(Fig. 1C) was labeled with
Texas Red moiety at the ethanolamine domain. When the newly hatched larvae
from these mosquitoes were examined (Fig.
4D), phosphatidylcholine was found in the gastric caeca
(Fig. 4B) along with the
phosphatidylethanolamine (Fig.
4C). This demonstrated that both phospholipids accumulate at the
same structure in the mosquito, which is different from the structure where
fatty acid accumulates. Notably, some green fluorescence derived from
BODIPY-PC, where BODIPY was attached to a fatty acid, was also found along the
body with a pattern similar to the fatty acid distribution
(Fig. 4B). Such fluorescence
was not seen when the ethanolamine domain of PE was tagged with Texas Red.
This suggested that the fatty acid moiety of some phospholipids is exchanged
with other lipid forms during embryogenesis.
|
Partitioning of the maternal lipids occurs during embryogenesis
To investigate the process of lipid segregation and to determine the stage
at which the maternal lipid partitioning occurs in the larvae, we examined
live developing embryos dissected from the eggs laid by mosquitoes previously
injected with Texas Red-PE and BODIPY-FA. At 26°C and 80% relative
humidity, it takes approximately 36h for A. gambiae eggs to hatch. We
dissected eggs approximately 30h after laying to obtain intact embryonic
larvae (Fig. 4E). At this
stage, most larval structures were formed and easily identifiable, although
these premature larvae were not motile. When examined for lipid distribution,
fatty acids were found distributed in the entire body of the embryonic
mosquito larvae including the developing head
(Fig. 4F). The phospholipids,
however, were confined within the intestine
(Fig. 4G). Some fatty acids
were also present in the developing intestine of the embryo with the
phospholipids. This suggests that, although incomplete, the separation of the
lipids occurs during embryogenesis prior to hatching of the mature larvae.
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Discussion |
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Biologically active fluorescent lipids are commonly used to elucidate
complex biological phenomena in live animals. To track cells in vivo,
surface-labeling fluorescent fatty acid analogues, such as PKH26, have been
used (Horan et al., 1990;
Teare et al., 1991
). PKH26 was
also used to track malaria parasite P. gallinaceum ookinetes in
mosquitoes, to study parasitevector interactions
(Shahabuddin et al., 1998
;
Zieler et al., 1999
).
Fluorescent lipids also have been used to label intracellular organelles
(Cole et al., 2000
;
Moreno et al., 2000
;
Buckman et al., 2001
) and to
locate lipids in live animals (Hendrickson
et al., 1999
; Farber et al.,
2001
). In this study, we have used fluorescently labeled lipids to
examine maternal fatty acid and phospholipid transport and distribution in
mosquito eggs and larvae.
Fatty acids and glucose are the primary sources of energy in animals. In
exclusively egg-laying animals such as chickens and mosquitoes, the entire
embryogenesis process takes place outside the mother's body; for these
animals, fatty acids are the major energy source
(Speake and Thompson, 2000).
Phospholipids, however, play more complex roles: they are major components of
the cell wall, precursors of cell signaling molecules
(Payrastre et al., 2001
), and
biological lubricants (Hills,
2000
) and food emulsifiers
(Hasenhuettl and Hartel,
1997
). Phospholipids also act as surfactants in normal mammalian
pulmonary function (Veldhuizen et al.,
2000
). Phospholipids such as lecithin, when added to food, act as
emulsifiers and enhance assimilation
(Hasenhuettl and Hartel,
1997
). Lecithins are a mixture of phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol.
What roles might the uniquely segregated fatty acids and phospholipids play in neonatal larvae? They may be related to finding and ingesting the food that the newly born larvae need to survive immediately after hatching. Stored maternal fatty acids may provide the energy for the initial rapid movements needed for finding food. Fatty acid accumulation along the body and thorax, where muscles for these movements are located, makes it a convenient, ready-made energy source. Localization of the fatty acid-containing vesicles at the thorax and at the base of the body segment hairs also supports the possibility that energy required for rapid muscle contractions and hair movement in neonate mosquito larvae is supplied by the stored maternal fatty acids (Fig. 4H). This is corroborated by the observation that neonate larvae of both A. aegypti and A. gambiae, when left in distilled water, move about vigorously, slowing as the fluorescence of fatty acids is depleted (data not shown). The larvae eventually die if food is not provided at this stage. The first ingested food of neonate larvae also needs to be digested and absorbed quickly, with rapid dissolution of food particles. As mentioned above, phospholipids are excellent emulsifiers and lubricants that, when released into the intestine, may facilitate digestion of the first-ingested food particles.
We have therefore described a novel process for differential partitioning and storage of maternal fatty acids and phospholipids that may allow neonate mosquito larvae to deal with the first few hours of life. Better understanding of this mechanism may lead to development of more efficient insecticide delivery tools, where larvicidal toxins can be more effectively released and absorbed by larvae. This may contribute to improved strategies for controlling mosquito-borne diseases such as malaria.
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Acknowledgments |
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