Reduced juvenile hormone synthesis in mosquitoes with low teneral reserves reduces ovarian previtellogenic development in Aedes aegypti
1 Departamento de Parasitologia, Instituto de Ciencias Biomedicas,
Universidade de Sao Paulo, Sao Paulo, SP, CEP 05,508-900, Brazil
2 Department of Biological Sciences, Florida International University,
Miami, FL 33199, USA
* Author for correspondence (e-mail: noriegaf{at}fiu.edu)
Accepted 18 May 2004
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Summary |
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Key words: Aedes aegypti, mosquito, juvenile hormone, corpora allata, nutrition, ovary
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Introduction |
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Juvenile hormone (JH) is the key hormone regulating previtellogenic ovarian
development in mosquitoes (Hagedorn,
1994; Klowden,
1997
). In newly emerged adult female mosquitoes (teneral females),
an increase in JH signals that ecdysis of the adult has finished and
reproductive processes should begin. JH levels in Ae. aegypti
increase during the first day after adult emergence
(Shapiro et al., 1986
). This
initial rise in JH is essential for reproductive maturation. JH acts on
several tissues, including ovaries, fat body and midgut, making them competent
to perform their adult-specific functions
(Klowden, 1997
).
JH is synthesized and released from the corpora allata (CA), a pair of
endocrine glands with nervous connections to the brain
(Stay, 2000). An increase in
rates of JH biosynthesis is responsible for the rise in JH levels observed
during the first day after eclosion (Li et
al., 2003a
).
Is there a relationship between the increase of JH synthesis in the CA and the presence of nutrient reserves? In this study, we report that low teneral nutrient reserves reduced JH biosynthesis and ovarian previtellogenic development in Ae. aegypti females.
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Materials and methods |
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Insects
Aedes aegypti L. of the Rockefeller strain were reared at 28°C
and 80% relative humidity under a photoperiod of 16 h:8 h light:dark. The
larval diet was a 10% solution of bovine liver powder (ICN, Aurora, OH, USA).
Five hundred larvae were raised in pans (23 cmx35 cmx13 cm)
containing 1 liter of distilled water. Large or small mosquitoes were obtained
by changing the amount of diet added to the pan. Large mosquitoes were
produced when larvae were reared in pans adding the following amount of diet:
1.5 ml on days 1, 3, 4, 5 and 6 and 0.75 ml on day 2. Small mosquitoes were
produced when larvae were reared in pans with the following diet: 0.75 ml on
days 1, 3, 5 and 7. Under these rearing conditions, most larvae pupate at day
8. Virgin adult females were offered a cotton wool pad soaked in water or a
15% sucrose solution. We refer to the cotton wool pad sucrose-fed females as
`sugar-fed'.
Wing length and ovarian development measurements
Ovaries were isolated by tearing the soft cuticle between the fifth and
sixth abdominal sternites, pulling off and placing the terminal segments in a
drop of saline. Ovary and wing lengths were measured under a dissecting
microscope using an ocular micrometer. Wing length, measured at adult
emergence, describes the distance between the point of articulation and the
wing tip, excluding the fringe scales
(Nasci, 1990).
Microseparation and analysis of nutrients
Microseparation of glycogen, lipids and proteins from the same mosquito
samples was accomplished as described by Van Handel
(1965) and modified by Zhou et
al. (2004
). Protein data were
obtained using the BCA protocol (Pierce, Rockford, IL, USA). Glycogen was
measured using the hot anthrone protocol
(Van Handel, 1985a
) and lipids
using vanillin as described by Van Handel
(1985b
). All biochemical
analyses were carried out using triplicate groups of five females.
In vitro radiochemical assay for CA activity
Preparation of CA complexes (CA attached to the corpora cardiaca and
connected to the brain) from adult Ae. aegypti females has been
previously described (Li et al.,
2003a). Rates of JH biosynthesis were estimated by the in
vitro radiochemical assay, as described by Feyereisen and Tobe
(1981
) and Feyereisen
(1985
) and modified by Li et
al. (2003a
). The glands were
incubated in 100 µl M-199 assay medium with labeled
L-[methyl-3H]methionine (specific activity
2.963.11 TBq mmol1;
8084 Ci
mmol1; Amersham Pharmacia, Piscataway, NJ, USA). The final
concentration of methionine in the medium was 50 µmol l1
and the specific activity was 0.56 TBq mmol l1 (15 Ci mmol
l1). Under these conditions, the incorporation of
L-[methyl-3H]-methionine into JH was linear for at least
6 h (Li et al., 2003a
). At the
end of the experimental period, incubations were terminated by the addition of
100 µl 1% EDTA, and 100 µl methanol containing 25 µg of unlabeled JH
as carrier and internal standard. The incubation medium and the gland were
extracted together with 1 ml of hexane and separated by thin-layer
chromatography (TLC). After TLC separation [developed in 2:1 (v/v)
hexane:ethyl acetate], the JH band was detected under UV light, cut, put into
10 ml scintillation cocktail overnight and assayed for 3H. The
quantities of JH produced in the experiment were calculated from the specific
activity of the L-[methyl-3H]methionine in the medium
with assumption of a specific incorporation ratio of 1 (non-isotopic
dilution). JH degradation by esterases was checked by incubating
[3H]JH in medium in the presence or absence of CA complexes and
analyzing the recovery of labeled JH. 9599% of the hormone was
recovered intact after 4 h of incubation (results not shown). In some
experiments, the JH esterase inhibitor OTFP
(Hammock et al., 1984
) was
added to the incubation medium. Results of incubations in the presence or
absence of OTFP were not significantly different.
Statistical analysis
Statistical analysis of the data was performed by t-test or
one-way analysis of variance (ANOVA) with Tukey's post-test using GraphPad
Prism version 3.00 for Windows (GraphPad Software, San Diego, CA, USA). The
results were expressed as means ±
S.E.M. and were considered significantly
different at P<0.05.
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Results |
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Groups of small and large females after eclosion were kept on water or sugar, and the total amount of lipids, glycogen and protein was measured 24 and 48 h after eclosion (Fig. 2). Lipids and glycogen showed significant increases in both small and large sugar-fed females.
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Effect of larval rearing conditions on adult ovarian development
Fig. 3 shows the length of
the terminal follicle of ovaries dissected from water-fed and sugar-fed small
and large females. Follicles from newly emerged small and large females were
40 µm long (Fig. 3A).
24 h after emergence, the terminal follicles of large females raised on water
or sugar had doubled their size. The follicles of small females raised in
water had grown very little, while those of sugar-fed small females increased
their size by
30% (Fig.
3B). Similar results were observed at 48 h after emergence; while
follicles of small females raised on water had grown to only 65 µm, those
of sugar-fed small females increased their size to
83 µm but were
still significantly smaller than the follicles from large females
(Fig. 3C).
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Effect of topically applied methoprene on adult ovarian development
To confirm a role for JH in previtellogenic ovary development, methoprene
(a JH analogue) was topically applied to newly emerged small mosquitoes (500
ng per 0.5 µl of acetone). Acetone alone was applied to controls. After 24
h, the length of the terminal follicles from methoprene-treated small females
was significantly larger than that from small females that were topically
applied with acetone (Fig. 4).
Topically applied methoprene induced the development of previtellogenic
ovaries in small mosquitoes to levels comparable with those observed in large
sugar-fed females.
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JH synthesis in small and large females
The basal rate of JH biosynthesis by CA dissected from small and large
mosquitoes was measured at different times after emergence using the in
vitro radiochemical assay. The biosynthetic activity of the Ae.
aegypti CA was significantly reduced in females emerged with low teneral
reserves (Fig. 5A). Raising the
small females on a high sugar diet (15%) resulted in a significant increase of
JH synthesis compared with small females raised on water
(Fig. 5B).
|
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Discussion |
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Growth of the primary follicle to the resting stage in Ae. aegypti
is linear and reaches maximum development about 60 h after adult eclosion
(Hagedorn et al., 1977).
Follicles of small mosquitoes did not develop much beyond the teneral stage
when they were kept on water for 48 h. By contrast, sugar feeding permitted
partial growth of ovarian follicles in small mosquitoes. Feinsod and Spielman
(1980
) also reported that a
sucrose meal stimulates partial development of follicles from
nutrient-deprived adult Ae. aegypti.
There are several reports describing correlations between teneral
nutritional reserves, JH activity and previtellogenic egg maturation in
anautogenous mosquitoes (Lea,
1963; Gwadz and Spielman,
1973
; Hagedorn et al.,
1977
; Feinsod and Spielman,
1980
). Decapitations, CA removal and abdominal ligations were used
to prove that the growth of the previtellogenic follicles is under the control
of factors from the brain and CA (Lea,
1963
; Gwadz and Spielman,
1973
; Hagedorn et al.,
1977
). Decapitation within 1 h of emergence or CA removal soon
after eclosion prevents ovarian previtellogenic growth. Topical application of
JH analogs stimulates normal growth of previtellogenic ovaries in decapitated
or CA-ablated teneral females (Gwadz and
Spielman, 1973
; Hagedorn et
al., 1977
). We confirmed the role of JH on ovary development. A
topically applied JH analogue stimulated previtellogenic ovarian development
in nutrient-deprived mosquitoes. Follicles of methoprene-treated small females
attained normal previtellogenic growth.
JH levels in Ae. aegypti are low at eclosion, increase during the
first day after adult emergence and remain high in sugar-fed females
(Shapiro et al., 1986). This
initial rise in JH is essential for female's reproductive maturation
(Klowden, 1997
). Rates of JH
biosynthesis by the CA in vitro closely reflect the levels of JH in
the mosquito; biosynthesis of JH is very low in newly emerged females and
increases dramatically during the first 24 h after adult eclosion
(Li et al., 2003a
). We have
investigated for the first time the effect of larval diet on synthesis of JH
by the CA of teneral females. We observed that biosynthesis of JH by the
Ae. aegypti CA was significantly reduced in females emerged with low
teneral reserves (small females). When small females were fed on a high sugar
diet, JH synthesis was significantly increased.
How do nutrients activate JH synthesis after adult eclosion? Allatotropins
(AT) are peptides that stimulate JH synthesis by the CA
(Kataoka et al., 1989;
Veenstra and Costes, 1999
). We
have previously described that the CA of a newly emerged mosquito needs to be
exposed to Ae. aegypti AT before it is capable of synthesizing JH
(Li et al., 2003b
).
Studies using antibodies against Ae. aegypti AT showed that the
peptide is present in cells of the brain of Aedes and
Anopheles mosquitoes (S. Hernández-Martínez and F.G.N.,
unpublished observations). Removal of the medial neurosecretory cells (mnc)
from Ae. aegypti immediately after adult emergence suppresses egg
maturation; in older females, mnc ablation has little effect
(Lea, 1967). It is reasonable
to hypothesize that AT is one of the factors from the head that is essential
for reproductive maturation. When the amount of nutrients is appropriate, the
brain would release AT, and the CA would become capable of synthesizing enough
JH to activate reproductive maturation. Therefore, the previtellogenic
maturation of ovaries seems to depend exclusively on the capacity of the CA to
produce high amounts of JH during the first day after the imaginal molt.
A similar mechanism controls diapause in mosquitoes. Readio et al.
(1999) reported that diapause
in the mosquito Culex pipiens is caused by inhibition of JH synthesis
by the CA. Nondiapausing adult females synthesize four times more JH than do
diapausing mosquitoes. Although small quantities of JH are produced during
diapause, diapausing females lack sufficient JH to stimulate growth of ovarian
follicles to the resting stage.
How are nutrients sensed? A recent paper by Colombani et al.
(2003) describes a nutrient
sensor mechanism that controls Drosophila growth. These authors
propose a model where the amino-acid-responsive serine/threonine protein
kinase target of rapamycin (TOR) signaling in the fat body modulates insulin
signaling and nutrient-dependent responses in peripheral tissues. The brain
mnc sense high circulating carbohydrate levels and secrete insulin-like
peptides (ILPs), which interact with factors released by the fat body in
response to amino acids and increase insulin signaling in the peripheral
tissues. According to this model, the sensing of amino acids in the fat body
and the sensing of carbohydrates in the brain are important for the regulation
of nutrient-dependent responses.
One of the most difficult challenges in the future will be to discover whether one type of nutrient is more important than another in triggering JH secretion, as well as the mechanisms by which mosquitoes sense and respond to changes in their nutritional reserves.
In summary, CA activity is promoted by the presence of enough nutrient stores; inadequate larval nutrition prevented the early peak of JH biosynthesis that we detected in large females. There seems to be a mechanism involving a minimum threshold of nutrients that elicits the release of AT by the brain.
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Acknowledgments |
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