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First published online January 31, 2006
Journal of Experimental Biology 209, 645-655 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02026
Effects of larval nutrition on the endocrinology of mosquito egg development
1 University of Arizona, Department of Biochemistry and Molecular
Biophysics, Tucson, AZ 85721, USA
2 Florida International University, Department of Biological Sciences,
Miami, FL 33199, USA
3 University of Georgia, Department of Entomology, Athens, GA 30602,
Greece
* Author for correspondence at present address: University of Georgia, Department of Entomology and Center for Tropical and Emerging Global Diseases, Biological Sciences Building, Athens, GA 30602, USA (e-mail: atelang{at}bugs.ent.uga.edu)
Accepted 30 November 2005
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Key words: autogeny, anautogeny, juvenile hormone, ecdysteroid, corpora allata, ovary, Aedes aegypti, Ochlerotatus atropalpus
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Raikhel et al., 1999). Soon
after female A. aegypti eclose, the corpora allata (CA) begin
secreting juvenile hormone (JH), which induces differentiation (competency) of
ovaries and fat body. Once this pre-vitellogenic phase is complete, oogenesis
is arrested until a blood meal is obtained. The act of feeding and presence of
blood in the midgut initiate an endocrine cascade that ultimately results in
egg maturation and oviposition. A steroidogenic gonadotropin released from the
brain stimulates A. aegypti ovaries to secrete ecdysteroids
(Klowden, 1997). Ecdysteroids
then act through nuclear hormone receptors to increase expression of the
vitellogenin genes in the fat body to support yolk synthesis
(Raikhel et al., 2002).
In addition to endocrine regulation, products of blood meal digestion play
a role in activating anautogenous oogenesis. The concentration of total free
amino acids in the haemolymph of the female house mosquito Culex pipiens
pallens reaches a maximum level 18 h after blood feeding
(Uchida et al., 1990), and
infusion of a balanced mixture of amino acids into the haemolymph activates
ovarian development in a number of mosquito species
(Uchida et al., 2001). In
A. aegypti, vitellogenic response by the fat body increases
substantially in the presence of both amino acids and 20-hydroxyecdysone, and
this response is transduced by the target of rapamycin (TOR) pathway
(Hansen et al., 2004). In
addition to a female's reliance on blood meal nutrients for anautogeny,
nutritional support is also present at eclosion (teneral) in the reserves she
carries from her larval stage. For A. aegypti and several
Anopheles species, the nutritional environment experienced by a
female larva dictates her adult body size and resulting teneral reserves
(Briegel, 1990a;
Briegel, 1990b). Teneral
reserves affect important female reproductive processes, such as utilization
of reserves, fecundity, longevity and blood meal consumption and utilization
(Briegel, 1990a;
Briegel, 1990b;
Briegel et al., 2002;
Naksathit et al., 1999a;
Naksathit et al., 1999b;
Takken et al., 1998;
Zhou et al., 2004). Thus, both
larval-derived teneral reserves and a blood meal in anautogenous females can
serve as sources of yolk precursors and as stimuli for hormonal regulation of
egg maturation.
Other mosquito species, such as the rock pool mosquito, Ochlerotatus
atropalpus, are autogenous, as they do not require a blood meal for at
least the first ovarian cycle and instead utilize teneral reserves as adults
to produce eggs. Autogeny has a strong genetic component, but its expression
is highly responsive to certain environmental factors, such as nutrition and
density in larval stages and host availability, mating and sugar-feeding in
the adult stage (Corbet, 1964;
Corbet, 1967;
Eberle and Reisen, 1986;
O'Meara, 1979;
Russell, 1979a;
Russell, 1979b;
Su and Mulla, 1997b). When
Oc. atropalpus females were given high amounts of food as larvae,
they emerged with a large body size and teneral reserves and produced a
greater number of eggs compared with females fed less food as larvae
(Telang and Wells, 2004).
Studies of Aedes albopictus and Culex tarsalis, species with
autogenous and anautogenous strains, have found that the key difference
between strains is the greater level of teneral metabolic reserves found in
autogenous females (Chambers and Klowden,
1994; Su and Mulla,
1997a).
While nutrition fuels oogenesis, hormones play a role in regulating this
process. How the nutritional condition of a female mosquito influences
hormones involved in oogenesis has not been adequately studied. The
nutritional condition of anautogenous females is determined by larval-derived
reserves, a sugar meal and a blood meal, whereas larval-derived teneral
reserves primarily determine that of autogenous Oc. atropalpus. To
understand how stage-specific nutrition affects endocrinology of oogenesis in
autogenous and anautogenous mosquitoes, we manipulated larval nutrition of
Oc. atropalpus and A. aegypti and measured their teneral
nutrient reserves and production of JH and ecdysteroids for the first few days
of adult emergence. During this time, sugar ingestion also affects female
reproduction (Foster, 1995)
and so its influence on hormonal activity was also examined. Past studies have
used ablation and implantation methods to determine the contribution of
endocrine tissues (Fuchs et al.,
1980; Lea, 1963;
Lea, 1964;
Lea, 1970) and ecdysteroids
(Kelly and Fuchs, 1980;
Masler et al., 1980) to
autogenous egg maturation. However, our study is the first to measure CA
biosynthesis of JH and ovarian ecdysteroidogenesis during autogenous egg
development in response to larval and adult nutrition. The first few days of
emergence are of interest given that oocytes are arrested in anautogenous
females but complete development in autogenous females over this time period.
Our study shows that differences in nutritional and hormonal profiles exist
between anautogenous and autogenous females during this critical time period
of emergence.
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Materials and methods |
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). Females of our colony strain are 100% autogenous for their
first ovarian cycle, as measured by primary follicles advancing beyond
Christopher's stage II, similar to Clement's stage 2b without the need for a
blood meal (Christophers,
1911; Clements,
1992). Routine colony maintenance of Aedes aegypti (L.)
(Rockefeller strain) was previously described
(Zhou et al., 2004). Adult
females feed ad libitum on 3% sucrose for 4-5 days prior to a blood
meal. Females are fed using an artificial blood feeder in which porcine blood,
supplemented with 100 mmol l-1 ATP per 1.0 ml blood for
phagostimulation, is added to a ParafilmTM-lined glass vessel. Feeding
stations are maintained at 37°C with the aid of a circulating water
bath.
Manipulation of nutrition for larval Oc. atropalpus and A. aegypti
Larval nutrition was manipulated in the same manner for both species using
rearing procedures previously described
(Telang and Wells, 2004).
Specifically, 24-h-old larvae of both species were placed in plastic trays
(27x16x6.5 cm) containing 1.0 litre of tapwater at a low larval
density of 50 per pan. For experiments, larvae were fed only 10% bovine liver
powder according to a schedule outlined in
Table 1. Thefeeding schedule
for A. aegypti and Oc. atropalpus was different to
accommodate their different larval development periods, but the species
received the same total number of feedings and amount of food for each
treatment over their respective larval stage. Earlier examination of larvae of
both species reared under the `low food quantity' treatment showed that this
amount of food was regularly depleted but still supported growth and
development to pupation whereas the `high food quantity' treatment provided
nourishment in excess of that required for maximal growth. The majority of
females began to pupate 1-2 days following the last day of feeding by larvae,
and only females eclosing from these pupae were examined. For both species,
the low food quantity treatment always produced some larvae that underwent an
extended developmental period, and these were not included in our
measurements. This experimental design uses a low larval density to remove any
effects of crowding while only varying food quantity and availability.
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Quantification of nutrient reserves
Newly emerged females (0-6 h old) reared as larvae on the two diet regimens
were immediately frozen for analysis of metabolic reserves. Whole-body
homogenates of 8-10 newly emerged females were made to extract glycogen,
storage lipids and proteins using a procedure first described by Van Handel
(1965) and modified for
Aedes aegypti (Zhou et al.,
2004), except that we omitted sections of the protocol leading to
the isolation of a sugar fraction. Fractions of glycogen, lipid and protein
were frozen until they could be quantified using colormetric-based assays. The
amount of storage lipid, triacylglycerol, was determined by a modified
vanillin reagent assay (Van Handel,
1985b). Total amount of glycogen was determined using a modified
anthrone-based assay (Van Handel,
1985a). Protein was quantified using the BCA protein assay reagent
kit (Pierce, Rockford, IL, USA). Complete details of our assay procedures were
previously described (Telang and Wells,
2004). All nutrients are reported on a microgram per mg dry mass
basis. Three replicates of each experiment were conducted for Oc.
atropalpus (N=12) and two replicates of each experiment were
conducted for A. aegypti (N=8).
Body size and fecundity
Another group of females from each replicate larval diet regimen was used
to measure body size and fecundity. Egg production in relation to larval food
amount was quantified for individual sugar-fed, mated Oc. atropalpus
females at 72 h post-emergence. Ovaries were dissected, and the number of
matured primary follicles was counted using a dissecting microscope
(N=36). Egg production in response to larval food amount was
quantified for individual A. aegypti females 72 h after blood feeding
(N=28). In both species, the number of mature primary follicles was
used because we were interested in a measure of potential fecundity. In
preliminary experiments with both species, we determined that there was no
significant difference between the number of mature follicles dissected and
the number of eggs oviposited (data not shown). The same females from which we
obtained fecundity data were also used to measure body size. Wing length was
used to assess body size (Nasci,
1990; O'Meara and Krasnick,
1970) and was measured from the point of attachment to the wing
tip, not including fringe, under a dissecting microscope using an ocular
micrometer.
In vitro radiochemical assay for CA activity
For both species, females emerging from larvae given high or low amounts of
food were maintained on either water or 3% sucrose, in association with males.
Corpora allata (CA) complexes consisting of CA + corpora cardiaca (CC) + aorta
+ brain + head capsule were isolated from five females of each larval and
adult diet treatment at specific times after emergence (0-6, 12, 24, 36 and 48
h). For female Oc. atropalpus, this time course covers an ovarian
cycle that ends at 72 h post eclosion with mature eggs ready to be oviposited.
For female A. aegypti, this time course reflects her previtellogenic
phase, during which her primary follicles develop to a point of arrested
growth awaiting a blood meal. Full details of assay methods and reagents were
previously described (Li et al.,
2003) and will only be summarized here; the same assay conditions
were applied to both Oc. atropalpus and A. aegypti
experiments. Briefly, CA complexes from individual females are transferred and
pre-incubated in tissue culture medium without methionine, so that
intraglandular methionine is consumed prior to assay. Complexes are then
transferred and incubated for 4 h in fresh medium containing
3H-labelled methionine. Under assay conditions, the incorporation
of 3H-labelled methionine into JH III was linear for at least 6 h
in both Oc. atropalpus and A. aegypti (data not shown).
After extraction and separation by thin-layer chromatography, the JH III band
is removed, placed into scintillation cocktail and assayed for3H.
The quantity of JH produced is calculated from the specific activity of the
3H-labelled methionine in the medium and averaged for one hour. One
replicate of this experiment was conducted.
Pre-vitellogenic follicle development in A. aegypti
Early follicular development in response to larval (high vs low
food amounts) and adult nourishment (water vs sugar) was measured in
A. aegypti 72 h post-emergence. Primary follicles were staged and
their lengths were measured under a dissecting microscope using an ocular
micrometer (N=60 for each larval and adult diet combination).
Ovarian ecdysteroid production in vitro, haemolymph ecdysteroid titer and the ecdysteroid radioimmunoassay
Oc. atropalpus females subjected to both nutritional regimens as
larvae were given access to males and given water or 3% sucrose prior to the
dissection of ovaries at different times after eclosion (0-6, 12, 24, 36 and
48 h). For the in vitro bioassay, four ovary pairs from identically
staged and treated females were dissected in saline solution (128 mmol
l-1 NaCl, 4.7 mmol l-1 KCl and 1.9 mmol l-1
CaCl2) (Riehle and Brown,
1999) and then transferred to and incubated in 60 µl of
buffered medium (139 mmol l-1 NaCl, 4.05 mmol l-1 KCl,
1.85 mmol l-1 CaCl2, 12.5 mmol l-1 Hepes, 2.5
mmol l-1 trehalose, 0.3 mmol l-1 MgCl2 and
0.9 mmol l-1 NaHCO3; pH 6.5, adjusted with NaOH)
(Riehle and Brown, 1999) in a
polypropylene tube lid for 6 h at 27°C. After incubation, 50 µl of
medium was collected and analyzed for ecdysteroid content using a
radioimmunoassay (RIA) with an ecdysteroid antiserum at a 1:45 000 final
dilution (Sieglaff et al.,
2005). Haemolymph was collected from the same set of females prior
to ovary removal for the in vitro bioassay. To collect haemolymph,
the last two abdominal segments of four females were excised while abdomens
were immersed in 75 µl of saline solution on ice, and then gentle pressure
was applied to each female to facilitate haemolymph diffusion into the
solution. After 5 min incubation, 50 µl of the saline solution was removed
and stored at -80°C for the ecdysteroid RIA.
For each experiment, triplicates of four ovary pairs and four body haemolymph collections were analyzed for all time points and for each nutritional regimen in the same RIA. Each experiment was replicated with females from three different cohorts. Values for each tissue sample types are reported as `ecdysteroid pg', because the secreted ecdysteroid species are unknown. Values reported are means of triplicate treatments from three experiments (N=9 per treatment).
Data analyses
Individual dry mass and percent dry mass protein, lipid and glycogen
(calculated as µg nutrient per mg insect mass) were analyzed using analysis
of variance (ANOVA), with larval diet, species and an interaction term
included in our statistical model. Both wing length and egg production were
analyzed using ANOVA, with larval diet as the explanatory variable. Follicle
length was analyzed using two-way ANOVA and we incorporated larval diet, adult
diet and an interaction term in our model. The biosynthesis of JH was analyzed
within each species using two-way ANOVA, with the inclusion of diet treatment,
time post-eclosion and an interaction term in our statistical model.
Ecdysteroid production by ovaries and ecdysteroid levels in haemolymph were
analyzed using two-way ANOVA with the inclusion of diet treatment, time
post-eclosion and an interaction term in our statistical model. When
necessary, differences between means were further analyzed using linear
contrasts of treatment effects, and only tests found to be non-significant,
based on P>0.01, are reported in this paper. All data were
statistically analyzed using JMP IN (version 4.0.3, SAS Institute Inc.).
Adjusted mean values (± standard errors of mean) were obtained from
statistical models and used in all graphical illustrations.
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Body dry masses attained by both autogenous Oc. atropalpus and anautogenous A. aegypti females strongly reflected the amount of larval food available (two-way ANOVA, P=0.005). While A. aegypti and Oc. atropalpus females of low-food larvae weighed the same at emergence (P=1.0, from a linear contrast; Fig. 1A), Oc. atropalpus of low-food larvae accumulated significantly less lipid, but significantly more glycogen and protein, than A. aegypti females from low-food larvae (Fig. 1B-D; P=0.001, from a linear contrast in all cases). Female Oc. atropalpus were significantly (37%) heavier relative to A. aegypti when both were derived from high-food larvae (Fig. 1A; P<0.001, from a linear contrast). On a per mg dry mass basis, Oc. atropalpus females of high-food larvae accumulated similar lipid levels (P=0.70, from a linear contrast), but significantly more glycogen and protein (P<0.0001, from a linear contrast in both cases), compared with A. aegypti of high-food larvae (Fig. 1B-D).
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Overall, both A. aegypti and Oc. atropalpus females derived from high-food larvae emerged with a larger size and body mass compared with females from low-food larvae. As a result, both A. aegypti and Oc. atropalpus females from high-food larvae contained greater total amounts (µg per individual) of lipid, glycogen and protein due to their heavier dry mass. For subsequent sections of our report, females from high-food larvae will be referred to as `high-reserve' and females from low-food larvae as `low-reserve' for both species.
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; A.T.,
personal observations).
Ovarian ecdysteroid production in relation to teneral reserves in female Oc. atropalpus
An in vitro bioassay was used to determine ecdysteroid production
by ovaries dissected from female Oc. atropalpus with high and low
reserves at different times during their first ovarian cycle. In addition,
haemolymph was collected from the same set of females before dissection of
ovaries used in the in vitro bioassay. Levels of ecdysteroid
production differed over the first 48 h of post-emergent ovarian development
(two-way ANOVA, P<0.0001) and were significantly influenced by
levels of teneral reserves in females (two-way ANOVA, P<0.0001)
(Fig. 5A). Ovaries of
high-reserve females produce a detectable level of ecdysteroids at emergence
(Fig. 5A). This capacity
increased at 12 h PE, peaked at 62-65 pg at 24 and 36 h PE and fell to a basal
level at 48 h PE (Tukey-Kramer HSD, P
0.05). After 12 h PE, yolk
uptake is evident in developing oocytes of high-reserve females. As observed
with ovarian ecdysteroidogenesis, haemolymph ecdysteroid titre was also
strongly influenced by the level of teneral reserves (two-way ANOVA,
P=0.008) and showed a similar rise and fall pattern in high-reserve
females, with the highest level at 36 h PE (Tukey-Kramer HSD,
P0.05) (Fig.
5B).
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Ovaries of low-reserve females, given only water upon eclosion, exhibited
basal levels of ecdysteroid production at eclosion
(Fig. 5A). This capacity
incrementally increased at 12 h and 24 h PE to peak at 36 h PE before
returning to a basal level at 48 h PE, though differences between time points
were not significant (Tukey-Kramer HSD, P0.05). By contrast, when
low-reserve females were given 3% sucrose throughout their first ovarian
cycle, ecdysteroid production by ovaries was significantly greater at 24 and
36 h PE compared with low-reserve females given only water
(P<0.0001, from a linear contrast;
Fig. 5A). By 36 h PE, ovaries
of sugar-fed, low-reserve females produced ecdysteroids at levels comparable
to ovaries of high-reserve females (Tukey-Kramer HSD, P0.05;
Fig. 5A).
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;
Briegel, 1990b;
Telang and Wells, 2004;
present study). Although A. aegypti females require a blood meal to
provision and mature eggs, fecundity for the first ovarian cycle is determined
by reserves derived from larval nourishment. High-reserve female A.
aegypti produced significantly more eggs, after a blood meal, than
blood-fed low-reserve females. Autogenous Oc. atropalpus do not need
blood and, instead, utilize larval-derived nutrient reserves to mature the
first egg batch. Oc. atropalpus females of high-food larvae attained
a larger body size and greater levels of metabolic reserves that allowed for
much higher fecundity compared with blood-fed A. aegypti females.
Although similar in dry mass, low-reserve A. aegypti females required
a blood meal to produce a similar number of eggs to that produced by
low-reserve Oc. atropalpus females without a blood meal
(Fig. 2). Overall, Oc.
atropalpus females accumulated significantly more metabolic reserves
compared with A. aegypti females. Therefore, one crucial element that
may favour anautogeny in A. aegypti is low larval-derived reserves,
so females must rely on a blood meal to complete oogenesis. In Oc.
atropalpus females, reserves are sufficient at adult emergence to enable
oogenesis within 12 h thereafter.
Because egg development is dependent on a female's reserve of nutrients, it
is important that she does not proceed with oogenesis until sufficient
nutrients are available. Presumably, the endocrine and nervous systems monitor
these reserves, and accordingly regulate physiological, developmental and
behavioural processes that rely on these reserves. Our study focused on the
effect of larval nutrition on hormonally regulated oocyte maturation in
mosquitoes. To start, we examined the effect of high and low levels of
larval-derived metabolic reserves on adult CA biosynthesis of JH in A.
aegypti and Oc. atropalpus. The highest level of JH biosynthesis
after eclosion was by CA from high-reserve A. aegypti females, and
high levels of JH biosynthesis are also reflected in whole-body extracts
during pre-vitellogenesis (Shapiro et al.,
1986). In addition, we cannot rule out the possibility of JH
biosynthesis by ovaries and other tissues in this species
(Borovsky et al., 1994). Using
the same in vitro assay for JH synthesis, but a different larval
rearing procedure, Caroci et al.
(2004) measured comparably
high levels of JH biosynthesis by CA isolated from large A. aegypti.
In the present study, JH biosynthesis by CA from low-reserve female A.
aegypti did not increase when given 3% sucrose, but in the Caroci et al.
study, it did so in small females given 15% sucrose
(Caroci et al., 2004). High JH
levels may serve a regulatory role of halting oocyte maturation in
anautogenous females and may be a second key element maintaining
anautogeny.
In comparison to A. aegypti, JH biosynthesis by CA was significantly lower in autogenous Oc. atropalpus regardless of her level of larval-derived reserves or adult sugar feeding. Results for Oc. atropalpus indicate that low levels of JH biosynthesis by CA after eclosion do not inhibit the activation of oogenesis, but we cannot exclude the possibility of higher JH biosynthesis by CA during the pharate adult stage.
In contrast to CA activity, in vitro ecdysteroid production by
ovaries is activated in Oc. atropalpus at eclosion and rises and
falls within 48 h in both high-reserve females and in sugar-fed, low-reserve
females. Ecdysteroid titres in haemolymph from high-reserve females showed a
similar rise and fall pattern and were present at a physiological range of
2-9x10-8 mol l-1 [assuming 1 µl total
haemolymph volume per female (Shapiro et
al., 1986) and calculated from results in
Fig. 5B]. Yolk uptake is
evident in oocytes of these females after 12 h. By comparison, ovarian
ecdysteroid secretion is low but detectable in non-blood-fed A.
aegypti females derived from well-nourished, standard colony-reared
larvae, but for ovaries taken from females at 18 h post-blood meal, in
vitro ecdysteroid production ranged from 100 to 140 pg per 6 h
(Sieglaff et al., 2005).
Ovaries of low-reserve Oc. atropalpus females exhibited only basal
levels of in vitro ecdysteroid production when these females were
denied a sugar meal. As observed in these females, yolk uptake in the
developing oocytes is delayed by 12-24 h in comparison with high-reserve
females, but low-reserve females still mature and deposit viable eggs
autogenously (Telang and Wells,
2004). Low levels of in vitro ecdysteroid production have
been found to occur by non-ovarian tissues in blood-fed A. aegypti
(Sieglaff et al., 2005); the
possibility that low-reserve Oc. atropalpus females rely on
ecdysteroid production by extra-ovarian tissues to produce viable eggs will be
investigated.
Ovaries of sugar-fed, low-reserve Oc. atropalpus females showed a
similar capacity for in vitro ecdysteroid production as that of
non-sugar-fed, high-reserve females (Fig.
5A). Activation of ovarian ecdysteroid production in low-reserve,
sugar-fed females may be explained by two hypotheses. First, sugar feeding
increased energy availability, which supplemented that possibly obtained from
low teneral glycogen and lipid reserves. Second, abdominal distention
occurring with sucrose ingestion may have accelerated the timing of ovarian
secretion of ecdysone similar to that of high-reserve females. Support for the
latter hypothesis comes from a study on A. aegypti that showed that a
blood meal triggers release of a head factor important for activating ovarian
ecdysteroidogenesis, and its release is accelerated by abdominal distention
(Klowden, 1987).
In our present report, in vitro CA biosynthesis of JH was directly
measured in autogenous Oc. atropalpus for the first time. JH
biosynthesis by CA from female Oc. atropalpus, regardless of larval
or adult nourishment, was found to be at lower levels throughout her
autogenous cycle. On the other hand, ecdysteroid production by ovaries from
Oc. atropalpus was triggered soon after eclosion in both high-reserve
and sugar-fed, low-reserve females. Additional support for an early role of
ecdysteroids in autogenous egg production comes from a study of A.
detritus and A. caspius females
(Guilvard et al., 1984). These
researchers observed a peak of ecdysteroids at 40 h PE, followed by a peak of
juvenile hormone 8 h later, in whole-body extracts of both autogenous species.
Vitellogenesis was initiated in these species when ecdysteroid levels began to
rise and yolk uptake continued during JH increase. Only one other study has
quantified ecdysteroid production by ovaries in Oc. atropalpus
(Birnbaum et al., 1984).
Ovarian ecdysteroid production was minimal in females decapitated soon after
emergence, but normal levels were attained in decapitated females given a
physiologically high dose of JH. Given that the CA is considered to be the
primary source of JH in insects
(Feyereisen, 1985) and its
role in the synthesis of JH in mosquitoes has been well established
(Li et al., 2003), it is
significant that in our study in vitro CA biosynthesis of JH remained
at low levels throughout egg production by autogenous Oc.
atropalpus.
In many insects, the CA synthesizes and secretes JH to regulate egg
production in response to nutrition levels
(Wheeler, 1996). In these
insects, JH activates and regulates oocyte maturation and vitellogenesis,
whereas ecdysteroids secreted by ovaries regulate final stages of egg
maturation such as chorionation and oviposition
(Davey, 1997;
Schal et al., 1997;
Strambi et al., 1997). In
dipterans studied to date, JH may prime fat body machinery for vitellogenin
synthesis, but ovarian ecdysteroids are the principal regulator that increases
the rate of vitellogenin synthesis and release into the haemolymph. In
dipteran females that take a protein meal, such as a blood meal for
anautogenous mosquitoes and stable flies, release of ecdysteroids from ovaries
and a corresponding rise in haemolymph ecdysteroid levels is triggered in
response to a protein meal (Adams et al.,
1985; Adams et al.,
1988; Chen and Kelly,
1993; Kelly and Chen,
1997; Kozlova and Thummel,
2000; Schwartz et al.,
1989; Yin et al.,
1990). These studies have shown both ovarian and haemolymph
ecdysteroid levels to correlate with vitellogenesis. Likewise, our data
suggest that the greater protein reserves present at eclosion in autogenous
Oc. atropalpus trigger early ecdysteroid production. This early
ecdysteroid stimulation seems to be sufficient to spur continued oocyte
development in Oc. atropalpus and other autogenous species in which
ecdysteroid levels have been examined
(Guilvard et al., 1984).
Based on the results reported in this paper, the following model is offered to explain how a female's nutritional condition and hormones interact to regulate oocyte development in autogenous and anautogenous mosquitoes (Fig. 6). The nervous and endocrine systems, ovaries and fat body are known to play key roles in mosquito egg development, and these tissues likely use diverse molecules to monitor and manipulate nutrient reserves. In this model, high levels of glycogen and protein (presumably stored or processed in the fat body) surpass a threshold set in the nervous system that activates ovarian ecdysteroid production and inhibits JH biosynthesis by the CA, which together allow vitellogenesis and egg maturation. When glycogen and protein levels are below this threshold, the CA secretes high JH levels, ovarian ecdysteroid production is low, and egg maturation is delayed or arrested.
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).
This threshold is particularly evident in anautogenous females - only a
blood meal initiates the low rate of JH biosynthesis and high ovarian
ecdysteroid production required to complete oogenesis in A. aegypti.
In high-reserve A. aegypti, teneral glycogen and protein reserves are
below the threshold, and other studies, including a recent one
(Sieglaff et al., 2005),
report that ovarian ecdysteroid production is generally low in non-blood-fed
females. As found in this study, JH synthesis by CA is high, and oocyte
development is arrested in high-reserve females. After females in this state
ingest a blood meal, its protein presumably exceeds the threshold and, as
reported in a related study, both JH biosynthesis and haemolymph titres drop
to undetectable levels (Li et al.,
2003; Shapiro et al.,
1986), while ovaries are stimulated to produce high levels of
ecdysteroids (Sieglaff et al.,
2005), all of which lead to the activation of vitellogenesis in
the fat body and the completion of egg maturation.
Once sufficient levels of nutrients are available for females to commit to
oogenesis, this information must be conveyed from diverse tissues (e.g. fat
body or blood-filled midgut) to the nervous system, which then secretes
neuropeptides to regulate JH biosynthesis and ovarian ecdysteroid production.
In A. aegypti, one such neuropeptide, ovary ecdysteroidogenic hormone
(OEH), is released from brain neurosecretory cells after a blood meal and
directly stimulates ovarian ecdysteroid production
(Brown and Cao, 2001;
Brown et al., 1998). JH
synthesis by the CA is regulated by allatotropins, which enhance synthesis,
and allatostatins, which are inhibitory
(Noriega, 2004), but evidence
for a response to a blood meal is lacking. Presumably, these same
neuropeptides with comparable functions are present in Oc.
atropalpus. Most importantly, studies are needed to discover how the fat
body and other tissues convey information about nutrient levels to the nervous
system, which then coordinates JH and ecdysteroid production for
vitellogenesis and egg maturation in mosquitoes.
This paper benefited from the comments of two anonymous reviewers. This collaborative work was funded by NIH training grant 1 K12 Gm00708 (Center for Insect Science, University of Arizona) to A.T., NIH/NIAID grant AI 45545 to F.G.N. and NIH grant AI33108 to M.R.B.
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