Trypsin-modulating oostatic factor: a potential new larvicide for mosquito control
University of Florida-IFAS, Florida Medical Entomology Laboratory, 200 9th Street, SE Vero Beach, FL 332962,
(e-mail: dobo{at}mail.ifas.ufl.edu)
Accepted 9 July 2003
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Summary |
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Key words: TMOF, trypsin-modulating oostatic factor, mosquito, Aedes aegypti, larvicide, trypsin biosynthesis, TMOF receptor, 3-dimensional modeling
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Introduction |
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Chemical insecticides are the most important components of integrated
vector control. However, safe and cost-effective insecticides are rapidly
disappearing because of the development of resistance, abandonment of many
compounds for reasons of environmental safety, and new registration
requirements that are more stringent. A global economy that has suffered
setbacks in the past 10 years worldwide and limited investments into research
and development of new compounds that control vectors of public health
importance have slowed the development of new insecticides against mosquitoes.
Because it takes 7-10 years and more than $50 million to develop and register
a new insecticide (Rose,
2001), industry is very reluctant to take on new ventures that are
mainly aimed at third world countries and, thus, deemed nonprofitable.
In Africa, pyrethroids are used as the main insecticides for treating
mosquito nets. However, the use of pyrethroids, DDT, organophosphates and
carbamate has led to resistance in major malaria vectors worldwide
(Zaim and Guillet, 2002).
Since 1970, there has been a steady decrease in the development of alternative
insecticides for use in public health
(Tomlin, 2000
). The
pyrethroids, introduced in 1980 for indoor residual spraying and for treatment
of mosquito nets, induced resistance to this entire class of compounds, as
well as cross resistance to other compounds, limiting the number of effective
alternatives suitable for vector control. Industrial thrust of developing
more-selective compounds for agricultural use, either acting by ingestion or
cloned and expressed in transgenic crops, could be used as models to develop
new public health insecticides that are environmentally friendly and can be
used as effective larvicides.
Currently, there are two effective mosquito larvicides on the market;
methoprene, a juvenile hormone analogue that interferes with pupal-to-adult
development, and Bacillus thuringiensis subsp. israelensis
(Bti), a bacterium toxin that binds to the gut epithelial cells, forming
non-specific pores that lead to gut swelling and larval death
(Henrick et al., 1973;
Schnepf et al., 1998
). The
present review describes the physiological and biochemical roles of Aedes
aegypti trypsin-modulating oostatic factor (TMOF) in adults and larvae
and its potential as a future biorational larvicide.
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The discovery of TMOF |
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Borovsky (1985) reported
that the mosquito ovary is a rich source of `oostatic hormone'. Injections of
the hormone into female mosquitoes inhibited yolk deposition and vitellogenin
biosynthesis (Borovsky, 1985
).
The hormone did not block the release of EDNH from mosquito brain and, thus,
it was assumed that the hormone acted directly on the ovary, either by
preventing pinocytosis or by inhibiting ecdysteroid biosynthesis. When
partially purified oostatic hormone was injected into female Aedes
aegypti, both egg development and blood digestion were inhibited
(Borovsky, 1988
). Injections of
the hormone into decapitated and ovariectomized females (these females do not
synthesize ecdysteroids and do not develop eggs but synthesize protease in
their gut) inhibited trypsin-like enzyme biosynthesis and blood digestion in
their midgut. These results suggested that oostatic hormone inhibits trypsin
biosynthesis in cells of the midgut and not in the ovary or in the endocrine
system as was earlier suggested (Borovsky,
1988
). The hormone is not species specific, as injection of the
hormone caused inhibition of egg development and trypsin biosynthesis in the
mosquitoes Culex quinquefasciatus, Culex nigripalpus and
Anopheles albimanus (Borovsky,
1988
). Even though the target tissue of the hormone is the
mosquito midgut and not the ovary or the brain, the hormone was named
trypsin-modulating oostatic factor (TMOF). Borovsky and co-workers purified,
sequenced and, using mass spectrometry, characterized the hormone as an
unblocked decapeptide (NH2-YDPAPPPPPP-COOH;
Borovsky et al., 1990
). Several
peptide analogues were synthesized and shown to possess TMOF activity
(Borovsky et al., 1990
,
1991
,
1993
). The solution structure
of the hormone was determined by NMR studies
(Curto et al., 1993
), which
confirmed earlier suggestions (based upon computer modeling) that the
polyproline portion of TMOF is a left-handed
helix in solution
(Borovsky et al., 1990
,
1993
;
Fig. 1).
|
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Biological activity and mode of action of TMOF |
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|
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Inhibition of trypsin biosynthesis by TMOF in other insects |
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Synthesis and secretion of TMOF |
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Characterization and localization of TMOF receptors |
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Genetic characterization and expression of TMOF |
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The effect of TMOF and its analogues on mosquito larvae |
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|
|
To determine the shortest sequence of TMOF that binds its receptor, 25
analogues were synthesized and evaluated
(Table 2). First, the maximum
size of a TMOF analogue that will not traverse the gut into the hemolymph, and
thus would not bind to the TMOF receptor, was established. TMOF to which an
arginine was added at the C-terminus (Arg11; YDPAPPPPPPR) was not active
(Table 2). When the same
peptide was injected into female A. aegypti that were fed a blood
meal and immediately ligated (Borovsky et
al., 1993), trypsin biosynthesis in the midgut was 70% and 80%
inhibited with 100 ng and 250 ng of TMOF, respectively (D. Borovsky,
unpublished observations). These results indicate that the addition of Arg to
TMOF inhibited the transport of the hormone through the gut into the hemolymph
rather than its ability to bind to a TMOF gut receptor
(Borovsky et al., 1994a
).
Several longer analogues of 20 mer [(H)6IEGRYDPAPPPPPP and
(YDPAR)4] and 16 mer [(DPAR)4] to which trypsin cleavage
sites were added (IEGR, R and R, respectively) were cleaved in the midgut to
smaller peptides of 10, five and four amino acids each. These short peptides
traversed the gut into the hemolymph and inhibited trypsin biosynthesis. The
multiple peptides showed enhanced activities of 2-fold and 4-fold for
(YDPAR)4 and (DPAR)4, respectively
(Table 2). Although the six
prolines at the N-terminus have a very low biological activity (18%;
Table 2), removal of three
prolines from the C-terminus of TMOF lowered the activity to 45%, and removal
of five prolines lowered the activity to 31%. Alternatively, removal of the
six prolines at the C-terminus increased the activity to 95%, and the
biological activity was not significantly different from Aea-TMOF
activity (Table 2). Thus, it
seems that the smallest size of TMOF that binds a TMOF gut receptor and
maintains biological activity of the original decapeptide is the tetrapeptide
YDPA (Fig. 1). The six prolines
at the C-terminus form a left-handed helix (Borovsky et al.,
1990
,
1993
;
Curto et al., 1993
;
Fig. 1). When three prolines or
more are removed, the truncated molecule cannot form a stable left helix at
the C-terminus, and thus the binding of TMOF to its receptor and the
biological activity of the truncated molecule are reduced
(Table 2). When all the
prolines are removed, the tetrapeptide (YDPA) assumes a more stable
conformation, and the binding to the receptor is enhanced, bringing it to the
same level exhibited by the decapeptide
(Table 2). Similar results were
obtained when eightmer and fivemer TMOF analogues (YDPAPPPP and YDPAP,
respectively) were injected into female A. aegypti
(Borovsky et al., 1993
).
|
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Cloning and expression of Aea-TMOF in Saccharomyces cerevisiae |
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Acknowledgments |
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