This article appeared in a journal published by Elsevier. The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/authorsrights
Author's personal copy
Journal of Asia-Pacific Entomology 17 (2014) 531–535
Contents lists available at ScienceDirect
Journal of Asia-Pacific Entomology
journal homepage: www.elsevier.com/locate/jape
Larvicidal activities of the stem bark extract and rotenoids of Millettia
usaramensis subspecies usaramensis on Aedes aegypti L.
(Diptera: Culicidae)
Carren M. Bosire a, Tsegaye Deyou b, Jacques M. Kabaru a, Dennis M. Kimata a, Abiy Yenesew b,⁎
a
b
School of Biological Sciences, University of Nairobi, P.O. Box 30197-00100, Nairobi, Kenya
Department of Chemistry, University of Nairobi, P.O. Box 30197-00100, Nairobi, Kenya
a r t i c l e
i n f o
Article history:
Received 22 October 2013
Revised 11 May 2014
Accepted 12 May 2014
Available online 22 May 2014
Keywords:
Millettia usaramensis subspecies usaramensis
Aedes aegypti
Larvae
Rotenoid
Usararotenoid-A
a b s t r a c t
The dichloromethane/methanol (1:1) extract of the stem bark of Millettia usaramensis subspecies usaramensis
was tested for its larvicidal activity against the 4th instar Aedes aegypti larvae and demonstrated activity with
LC50 value of 50.8 ± 0.06 μg/mL at 48 h. Compounds isolated from the extract were also tested for their larvicidal
activities, and the rotenoid usararotenoid-A (LC50 4.3 ± 0.8 μg/mL at 48 h) was identified as the most active
principle. This compound appears to be the first rotenoid having a trans-B/C ring junction and methylenedioxy
group at C-2/C-3 with high larvicidal activity. Related rotenoids with the same configuration at the B/C-ring
junction did not show significant activity at 100 μg/mL.
© 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection
Society. Published by Elsevier B.V. All rights reserved.
Introduction
Some of the many insects that proliferate in tropical environments
due to conduciveness of its weather conditions transmit diseases and
affect the health of both man and livestock. Mosquitoes are associated
with several public health problems. This includes malaria, yellow
fever, filariasis, dengue fever and Japanese encephalitis, which cause
millions of deaths every year (Vatandoost and Vaziri, 2001). Aedes
aegypti (Linnaeus 1762) (Diptera: Culicidae) is a vector for an arbovirus
responsible for yellow fever and dengue fever. The latter disease
sometimes leads to a complex and life-threatening stage called dengue
haemorrhagic fever and dengue shock syndrome which is fatal if not
treated. It has unusual manifestations such as central nervous system
involvement (Hendarto and Hadinegoro, 1992; Pancharoen et al.,
2002). About two-fifths of the world's population are at risk of catching
dengue (Kautner et al., 1997; Rigau, 1998).
A. aegypti originated from Africa but now is found in the tropics
worldwide (Womack, 1993; Mousson, 2005). It prefers breeding in
areas of stagnant water such as flower vases, uncovered barrels,
buckets, and discarded tyres, as well as wet shower floors and toilet
tanks in houses. Treating these breeding areas with larvicidal agents
remains an attractive strategy for the control of mosquitoes.
⁎ Corresponding author. Tel.: +254 733 832576; fax: +254 20 4446138.
E-mail address: ayenesew@uonbi.ac.ke (A. Yenesew).
The use of conventional chemical pesticides such as organochlorides
and organophosphates has resulted in the development of resistance
(Rawlins and Ragoonansingh, 1990; Severini et al., 1993; Wirth and
Georghiou, 1999; Macoris et al., 2007), undesirable effects on nontarget organisms and fostered environmental and human health
concerns (Forget, 1989). Therefore, the development of plant-derived
products that do not produce adverse effects on the non-target organisms and are easily biodegradable remains one of the top objectives
of scientists in search of alternative vector control agents (Redwane
et al., 2002; Ribeiro et al., 2009; Kannathasan et al., 2011; Sagnou
et al., 2012).
Insecticidal plants comprise of an array of secondary metabolites
that act in concert on both behavioural and physiological processes,
and hence the chances of pests to develop resistance to such insecticides
are less probable (Saxena, 1987). Furthermore, botanical insecticides
are less likely to bio-accumulate, as they are biodegradable. Amongst
the botanical insecticides, rotenone and other rotenoids are found in
some taxa of the family Leguminosae (Fabaceae), including the genus
Millettia Wight et Arn. (Ollis et al., 1967; Dagne et al., 1991). In our
earlier work we have reported the larvicidal activities of the crude
extract and the rotenoids from the seeds of Millettia dura (Yenesew
et al., 2003a). The rotenoids of Millettia dura having a cis-B/C
ring junction satisfy the structural requirement for insecticidal
activity (Fukami and Nakajima, 1971). The presence of unique 12ahydroxyrotenoids with a trans-B/C ring junction (Yenesew et al.,
1998; Yenesew et al., 2003b) from the stem bark of Millettia usaramensis
http://dx.doi.org/10.1016/j.aspen.2014.05.003
1226-8615/© 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
Author's personal copy
532
C.M. Bosire et al. / Journal of Asia-Pacific Entomology 17 (2014) 531–535
subspecies usaramensis Taub (Gillet et al., 1971) has also been reported.
The larvicidal activities of the extract and rotenoids obtained from the
stem bark of this plant against 4th instar larvae of Aedes aegypti are
presented here.
8.4 Hz, H-10), 7.80 (1H, d, J = 8.4 Hz, H-11), 5.08 (2H, s, H-6), 6.01
(2H, s, 2-OCH2O-3), 6.32 (2Hs, 8-OCH2O-9).
Materials and methods
The larvae of Aedes aegypti (L) were obtained from the School
of Biological Sciences, University of Nairobi. They were maintained
at 25 ± 2 °C, 70–80% relative humidity (RH) and 12:12 light and dark
photo period cycle. The larvae were fed on ground dog biscuit and
yeast powder in the ratio of 3:1. The adults were fed on 10% sucrose
solution and allowed to take blood meals from the blood vessels of the
ears of immobilized rabbits.
General
Column chromatography was performed on silica gel (70–230 mesh).
Analytical TLC was performed on Merck pre-coated silica gel 60F254
plates. 1H NMR (200 MHz) and 13C NMR (50 MHz) were recorded on
a Varian 200 MHz spectrometer using the residual solvent peak as a
reference.
Plant material
The stem bark of Millettia usaramensis subspecies usaramensis
Taub. was collected in February 2008 from Diani along the Kenyan
coast, geographical coordinates 4° 19′ 20″ S, 39° 34′ 30″ E. The plant
was identified by Mr Simon Mathenge of the Herbarium, School of Biological Sciences of the University of Nairobi, where a voucher specimen
(AYT-038-2008) was deposited. The sample was dried under shade and
ground to fine powder using a mill.
Extraction of plant material
The stem bark of M. usaramensis subspecies usaramensis (0.6 kg)
was extracted with dichloromethane/methanol (1:1) by cold percolation (3 × 24 h). The extract was filtered and concentrated in a rotary
evaporator at 35 °C to afford crude extract (38 g).
Isolation of compounds
The constituents of the stem bark of M. usaramensis subspecies
usaramensis including rotenoids (12a-epimillettosin, usararotenoid-A,
12-dihydrousararotenoid-A and usararotenoid-B), chalconoids (α,4,2′trihydroxy-4′-O-geranyldihydrochalcone, 4′-O-geranylisoliquirtigenin
and isoliquirtigenin) and isoflavones (jamaicin, norisojamaicin,
barbigerone and maximaisoflavone G) were isolated and identified as
described in Yenesew et al. (1998, 2003b). Deguelin was isolated from
the seeds of Millettia dura according to (Yenesew et al., 2003a).
Preparation of derivatives
To a solution of 12-dihydrousararotenoid-A (10 mg, in 50 mL of
acetone) four drops of conc. HCl were added and the solution was
kept overnight. Purification of the product on PTLC (eluent: dichloromethane/n-hexane; 4:1) yielded 6 mg of colourless solid of 12adeoxyusararotenoid-A. 1H NMR (CDCl3, 200 MHz): δ 6.69 (1H, s, H1), 6.44 (1H, s, H-4), 4.18 (1H, dd, J = 3.6, 12.0 Hz, H-6α), 4.65
(1H, dd, J = 3.0, 12.0 Hz, H-6β), 4.98 (1H, t, J = 3.8 Hz, H-6a), 6.58
(1H, d, J = 8.4 Hz, H-10), 7.60 (1H, d, J = 8.4 Hz, H-11), 3.86 (1H,
d, J = 3.8 Hz, H-12a), 5.83 (1H, d, J = 1.0 Hz, 2-OCH 2 O-3), 5.87
(1H, d, J = 1.0 Hz, 2-OCH2O-3), 6.01 (1H, d, J = 1.2 Hz, 8-OCH 2O9), 6.08 (1H, d, J = 1.2 Hz, 8-OCH2O-9). 13C NMR (CDCl3, 50 MHz):
δ 107.0 (C-1), 144.8 (C-2), 148.2 (C-3), 99.2 (C-4), 148.8 (C-4a),
66.3 (C-6), 72.8 (C-6a), 142.5 (C-7a), 134.6 (C-8), 154.9 (C-9),
104.0 (C-10), 123.7 (C-11), 115.7 (C-11a), 188.9 (C-12), 45.6 (C12a), 105.4 (C-12b), 101.4 (2-OCH2O-3), 103.0 (8-OCH2O-9).
A solution of 12-dihydrousararotenoid-A (15 mg) was refluxed
in methanol containing 10 drops of concentrated HCl over water bath
for 1 h. The product was purified by column chromatography
(silica gel, using dichloromethane/hexane as eluent) to give colourless
solid of 6a,12a-dehydrousararotenoid-A (9 mg). 1H NMR (CDCl3,
200 MHz): δ 8.27 (1H, s, H-1), 6.57 (1H, s, H-4), 7.09 (1H, d, J =
Mosquito cultures and larval rearing conditions
Larvicidal assays
The larvicidal bioassays followed the guidelines for laboratory and
field-testing of mosquito larvicides (WHO, 2005) with slight
modifications. Initially, twenty 4th instar mosquito larvae were exposed
to 5–1000 μg/mL of test solutions of M. usaramensis subspecies
usaramensis crude stem bark extract and the control. After determining
the mortality of larvae in this wide range of concentrations, a narrower
range of 8 concentrations, yielding between 10% and 95% mortality in
24 h or 48 h was used to determine LC50 values. Batches of twenty 4th
instar larvae were transferred by means of a dropper to glass jars each
containing 100 mL of tap water. The appropriate volume of stock solution, where each of the crude extract and pure compound was dissolved
in DMSO, was added to 100 mL water in the glass jars to obtain 0, 5, 10,
25, 50, 100, 200, 400, and 800 μg/mL (for crude extract), and 0, 6.25,
12.5, 25, 50, and 100 μg/mL (for pure compounds) dose levels. Six replicates were set up for each crude extract concentration (triplicates for
pure compounds) and an equal number of controls were set up simultaneously with tap water, to which 1 mL DMSO was added. Each test
was run three times on different days. Same quantity (20 mg) of larval
food was added to each glass jar. The photoperiod was 12 h light followed by 12 h dark (12 L:12D). Larval mortality was recorded at 24 h and
48 h after exposure.
Statistical analysis
The average larval mortality data were subjected to probit analysis
for calculating lethal concentrations (LC50 and LC90) and ANOVA at
95% confidence limits using SPSS 16.0 software. Results with p b0.05
were considered to be statistically significant. When the mortality in
control was between 5 and 20%, Abbot's formula was used to correct
the mortality to remove error due to other factors other than the toxicity
of the test samples extracts (Abbot, 1925).
Corrected %mortality = [(T − C) / (100 − C)] × 100 where, T = %
mortality in test concentration.
C = % mortality in control.
The values were expressed as mean ± standard deviation of
replicates. Log dosage-probit mortality was plotted with Minitab®
statistical software.
Results and discussion
The dichloromethane/methanol (1:1) extract of the stem bark
of Millettia usaramensis subspecies usaramensis was tested for
lethality against the 4th instar larvae of Aedes aegypti. This crude
extract showed marginal activity with LC 50 value of 167.0 μg/mL
at 24 h, which improved at 48 h to LC 50 value of 50.8 ±
0.06 μg/mL. The constituents of the stem bark (Yenesew et al.,
1998, 2003b), including rotenoids (12a-epimillettosin, usararotenoidA, 12-dihydrousararotenoid-A and usararotenoid-B, Fig. 1), chalconoids (α,4,2′-trihydroxy-4′-O-geranyldihydrochalcone, 4′-Ogeranylisoliquirtigenin and isoliquirtigenin) and isoflavones
(jamaicin, norisojamaicin, barbigerone and maximaisoflavone G)
Author's personal copy
C.M. Bosire et al. / Journal of Asia-Pacific Entomology 17 (2014) 531–535
533
Fig. 1. Structures of rotenoids.
were also tested against the 4th instar larvae of A. aegypti. Amongst
these, the rotenoid usararotenoid-A caused 100% mortality at
100 μg/mL at 48 h. The rest of the compounds did not show significant activities at this concentration at 48 h.
Usararotenoid-A along with 12a-epimillettosin (the major compound of the stem bark of this plant) and deguelin (insecticidal rotenoid
with cis-B/C ring junction) was tested at different concentrations; the
LC50 values with corresponding 95% confidence limits were calculated
for each bioassay and are shown in Table 1. The slopes of the concentration–mortality curves of the results varied considerably between 24 h
and 48 h post exposure (Fig. 2). The slopes were greater for the
48 hour than the 24 hour post exposure period indicating homogeneity
of response to the tested larvicides.
It has been reported that the activity of rotenoids such as rotenone
(Fig. 1), against insects is associated with the fused four-ring system
(rings A, B, C and D) – a chromanochromanone known as 6a,12adihydrorotoxen-12(6H)-one – where the B/C ring junction is cis.
Furthermore rotenoids with modified rings and a trans-B/C ring
junction were shown to be less insecticidal (Fukami and Nakajima,
1971; Joseph and Casida, 1992). We have earlier demonstrated the
importance of the methoxyl groups at C-2 and C-3 of ring A for larvicidal
activity amongst rotenoids with cis-B/C ring junction (Yenesew et al.,
2003a). In this study, it was of no surprise then that 12a-
epimillettosin (and other related rotenoids of the stem bark), having
trans-B/C ring junction and methylenedioxy at C-2/C-3, did not show
significant larvicidal activity at 100 μg/mL. The major rotenoid of this
plant, 12a-epimillettosin, was further tested at higher concentrations
to determine the LC50 value and was found to be 2037 ± 8.3 μg/mL at
48 h, against the 4th instar larvae (Table 1), which is over 800 times
less active than deguelin (LC50 2.6 ± 0.9 μg/mL at 48 h), a rotenoid
with known larvicidal activity (Yenesew et al., 2003a).
Unexpectedly, usararotenoid-A, despite having a trans-B/C ring
junction and methylenedioxy group at C-2/C-3 as 12a-epimillettosin,
showed high larvicidal activity (LC50 4.3 ± 0.8 μg/mL, at 48 h) against
the 4th instar A. aegypti larvae which is comparable with that of
deguelin (Table 1). On the other hand the structurally related compounds, 12-dihydrousararotenoid-A and usararotenoid-B (Fig. 1) were
inactive even at 100 μg/mL showing the importance of the 12-keto
group and the methylenedioxy group at C-8/C-9 for the activity of
usararotenoid-A.
In order to explore further the structural requirement for larvicidal
activity, 12a-deoxyusarotenoid-A and 6a,12a-dehydrousararotenoid-A
(Fig. 1) were prepared and tested for larvicidal activity. Both derivatives
were completely inactive at 100 μg/mL against the 4th instar A. aegypti
larvae at 48 h. The fact that usararotenoid-A having a trans-B/C ring
junction is active and the 12a-deoxyusararotenoid-A with cis-B/C ring
Author's personal copy
534
C.M. Bosire et al. / Journal of Asia-Pacific Entomology 17 (2014) 531–535
Table 1
Summary of Log probit analysis of the larvicidal activity of crude CH2Cl2/MeOH (1:1) stem bark extract and pure compounds from M. usaramensis subspecies usaramensis on the 4th instar
A. aegypti larvae.
Sample
Exposure period (h)
Crude stem bark extract
24
48
24
48
24
48
24
48
12a-Epimillettosin
Usararotenoid-A
Deguelin
R2 (%)
Regression equations
Y
Y
Y
Y
Y
Y
Y
Y
=
=
=
=
=
=
=
=
2.41
2.76
2.83
2.97
3.63
3.48
2.91
3.82
+
+
+
+
+
+
+
+
1.17X
1.31X
0.571X
0.612X
0.914X
2.42X
2.75X
2.82X
LC50 (μg/mL)
95% CI (μg/mL)
a
96.3
91.2
75.0
89.7
80.9
68.0
78.6
89.0
167.00
50.82a
6350.31b
2037.00b
31.77b
4.27b
5.75b
2.63b
LCL
UCL
142.82
49.91
6348.76
2035.68
18.79
0.799
2.15
0.25
196.28
67.72
6351.86
2038.32
70.16
8.05
9.21
5.02
Y, probit; X, log conc; R, coefficient of regression equation; LC, lethal concentration; arepresents mean of six replicates; brepresents mean of triplicates; calculated log LC50 transformed to
LC50; CI, confidence interval; LCL, lower confidence limit; UCL, upper confidence limit.
junction is inactive is the exact opposite to what has been reported for
rotenone and related rotenoids having methoxyl groups at C-1 and C2 (Fukami and Nakajima, 1971; Joseph and Casida, 1992). In light of
this finding, it is worth to investigate the insecticidal activity of
usararotenoid-A against a variety of insect species. In relation to
usararotenoid-A, the present finding supports the previous assertion
that the structure–activity relationship amongst rotenoids and
isoflavonoids is not entirely clear as structurally different isoflavonoids
have shown some activity against insects (Sreelatha et al., 2010; Morel
et al., 2013; Pluempanupat et al., 2013).
a
According to literature, the organophosphate synthetic insecticide
temephos, has shown an LC50 value of 2.3 μg/mL in larvicidal activity
assays performed against third-instar larvae of susceptible strains of
A. aegypti (Macoris et al., 2007). In this study, usararotenoid-A (LC50 of
4.3 ± 0.8 μg/mL) showed high activity (in a 48 hour period), comparable to that of deguelin (LC50 2.6 ± 0.9 μg/mL) and temephos. Therefore,
usararotenoid-A may be considered as a promising natural mosquito
larvicidal agent, in the era of resistance of A. aegypti populations to
organophosphate and other synthetic organic pesticides, as well as the
environmental safety and human health concerns associated to their use.
b
4.2
MORTALITY (PROBIT)
MORTALITY (PROBIT)
7
6
5
4
4.0
3.8
3.6
3.4
3.2
3.0
3
1.0
1.5
2.0
2.5
3.0
0.8
1.0
LOG CONC.
1.2
1.4
1.6
1.8
2.0
LOG CONC.
c
d
9
8
MORTALITY (PROBIT)
MORTALITY (PROBIT)
9
7
6
5
8
7
6
5
4
0.8
1.0
1.2
1.4
LOG CONC.
1.6
1.8
2.0
0.8
1.0
1.2
1.4
1.6
1.8
2.0
LOG CONC.
Fig. 2. Probit regression lines (LC-p lines) resulting from A. aegypti 4th instar larvae exposed to: M. usaramensis subspecies usaramensis crude stem bark extract (a), 12a-epimillettosin
(b), usararotenoid-A (c), deguelin (d); 24 h and; 48 h.
Author's personal copy
C.M. Bosire et al. / Journal of Asia-Pacific Entomology 17 (2014) 531–535
Conclusion
The crude extract of the stem bark of Millettia usaramensis is moderately larvicidal against the 4th instar larvae of Aedes aegypti, which
could be attributed mainly to usararotenoid-A. This compound appears
to be the first rotenoid with trans-B/C ring junction demonstrating
remarkable insecticidal activity, and offers promise as a potential biocontrol agent against A. aegypti. However, further studies on the
larvicidal mode of action, its effect on non-target organisms and
environment, and formulations for improving the larvicidal potency
and stability are needed for its practical use in mosquito control.
Acknowledgments
We thank the authorities of the Department of Zoology and the
Department of Chemistry, University of Nairobi, for providing facilities
for the undertaken research work. AY is grateful to the International Science Program (ISP, Sweden, KEN-02) and African Institute for Capacity
Development (AICAD, Japan, 2nd call-Phase III-Abiy Yenesew ) for financial support.
References
Abbot, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ.
Entomol. 18, 265–267.
Dagne, E., Mammo, W., Bekele, A., Odyek, O., Byaruhanga, M.A., 1991. Flavonoids of
Millettia dura. Bull. Chem. Soc. Ethiop. 5, 81–86.
Forget, O., 1989. Pesticides, necessary but dangerous poisons. IDRC Rep. 18, 7–13.
Fukami, H., Nakajima, M., 1971. Rotenone and rotenoids. In: Jacobson, M., Crosby, D.G.
(Eds.), Naturally Occurring Insecticides. Marcel Dekker, New York, pp. 71–79.
Gillet, J.B., Polhil, R.M., Verdcourt, B., 1971. Flora of Tropical East Africa-Leguminosae.
Whitefriars Press, London, pp. 123–144.
Hendarto, S.K., Hadinegoro, S.R., 1992. Dengue encephalopathy. Acta Paediatr. Jpn. 34,
350–357.
Joseph, J.L., Casida, J.E., 1992. The rotenoid core structure: modifications to define the
requirements of the toxophore. Biol. Org. Med. Chem. Lett. 2, 593–596.
Kannathasan, K., Senthilkumar, A., Venkatesalu, V., 2011. Mosquito larvicidal activity of
methyl-p-hydroxybenzoate isolated from the leaves of Vitex trifolia Linn. Acta Trop.
120, 115–118.
Kautner, I., Robinson, M., Kuhnle, U., 1997. Dengue virus infection: epidemiology,
pathogenesis, clinical presentation, diagnosis, and prevention. J. Pediatr. 131,
516–524.
Macoris, M.L.G., Andrighetti, M.T.M., Otrera, V.C.G., Carvalho, L.R., Caldas Jr., A.L., Brogdon,
W.G., 2007. Association of insecticide use and alteration on Aedes aegypti susceptibility
status. Mem. Inst. Oswaldo Cruz 102, 895–900.
Morel, S., Helesbeux, J.-J., Séraphin, D., Derbré, S., Gatto, J., Aumond, M.-C., Abatuci, Y.,
Grellier, P., Beniddir, M.A., Le, P.P., Pagniez, F., Litaudon, M., Landreau, A.,
Richomme, P., 2013. Anti-AGEs and antiparasitic activity of an original prenylated
isoflavonoid and flavanones isolated from Derris ferruginea. Phytochem. Lett. 6,
498–503.
535
Mousson, L., 2005. Phylogeography of Aedes (Stegomyia) aegypti (L.) and Aedes
(Stegomyia) albopictus (Skuse) (Diptera: Culicidae) based on mitochondrial DNA
variations. Genet. Res. 86, 1–11.
Ollis, W.D., Rhodes, C.A., Sutherland, I.O., 1967. The extractives of Millettia dura. Tetrahedron 23, 4741–4760.
Pancharoen, C., Kulwichit, W., Tantawichien, T., Thisyakorn, U., Thisyakorn, C., 2002.
Dengue infection: a global concern. J. Med. Assoc. Thail. 85, 25–33.
Pluempanupat, S., Kumrungsee, N., Pluempanupat, W., Ngamkitpinyo, K., Chavasiri, W.,
Bullangpoti, V., Koul, O., 2013. Laboratory evaluation of Dalbergia oliveri (Fabaceae:
Fabales) extracts and isolated isoflavonoids on Aedes aegypti (Diptera: Culicidae)
mosquitoes. Ind. Crops Prod. 44, 653–658.
Rawlins, S.C., Ragoonansingh, R., 1990. Comparative organophosphorus insecticide
susceptibility in Caribbean populations of Aedes aegypti and Toxorhynchites
moctezuma. J. Am. Mosq. Control 6, 315–317.
Redwane, A., Lazrek, H.B., Bouallam, S., Markouk, M., Amarouch, H., Jana, M., 2002.
Larvicidal activity of extracts from Quercus lusitanica var infectoria galls (Oliv). J.
Ethnopharmacol. 79, 261–263.
Ribeiro, K.A.L., de Carvalho, C.M., Molina, M.T., Lima, E.P., López-Monter, E., Reys, J.R.M., de
Oliveira, M.B.F., Pinto, A.V., Santana, A.E.G., Goulart, M.O.F., 2009. Activities of
naphthoquinones against Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae), vector
of dengue and Biomphalaria glabrata (Say, 1818), intermediate host of Schistosoma
mansoni. Acta Trop. 111, 44–50.
Rigau, P., 1998. Dengue and dengue haemorrhagic fever. Lancet 352, 971–977.
Sagnou, M., Mitsopoulou, K.P., Koliopoulos, G., Pelecanou, M., Couladouros, E.A.,
Michaelakis, A., 2012. Evaluation of naturally occurring curcuminoids and related
compounds against mosquito larvae. Acta Trop. 123, 190–195.
Saxena, R.C., 1987. Antifeedants in tropical pest management. Insect Sci. Applic. 8,
731–736.
Severini, C., Rom, R., Marinucci, M., Rajmond, M., 1993. Mechanisms of insecticide
resistance in field populations of Culex pipiens from Italy. J. Am. Mosq. Control
Assoc. 9, 164–168.
Sreelatha, T., Hymavathi, A., Rao, V.R.S., Devanand, P., Rani, P.U., Rao, J.M., Babu, K.S., 2010.
A new benzyl derivative from Derris scandens: structure-insecticidal activity. Bioorg.
Med. Chem. Lett. 20, 549–553.
Vatandoost, H., Vaziri, M., 2001. Larvicidal activity of neem extract (Azadirachta indica)
against mosquito larvae in Iran. Pestology 25, 69–72.
WHO/CDS/WHOPES, 2005. Guidelines for Laboratory and Field Testing of Mosquito
Larvicides, p. 13 (GCDPP/2005).
Wirth, M.C., Georghiou, G.P., 1999. Selection and characterization of temephos resistance
in population of Aedes aegypti from Tortola, British Virgin Islands. J. Am. Mosq.
Control 15, 315–320.
Womack, M., 1993. The yellow fever mosquito, Aedes aegypti. Wing Beats 5, 4.
http://www.rci.rutgers.edu/~insects/sp5.htm (Accessed on 16th October 2013).
Yenesew, A., Midiwo, J.O., Waterman, P.G., 1998. Rotenoids, isoflavones and chalcones
from the stem bark of Millettia usaramensis subspecies usaramensis. Phytochemistry
47, 295–300.
Yenesew, A., Derese, S., Midiwo, J.O., Hedenreich, M., Peter, M.G., 2003a. Effect of
rotenoids from the seeds of Millettia dura on larvae of Aedes aegypti. Pest Manag.
Sci. 59, 1157–1161.
Yenesew, A., Derese, S., Midiwo, J.O., Oketch-Rabah, H.A., Lisgarten, J., Palmer, R.,
Heydenreich, M., Peter, M.G., Akala, H., Liyala, P., Waters, N.C., 2003b. Antiplasmodial
activities and X-ray crystal structures of rotenoids from the stem bark of Millettia
usaramensis subspecies usaramensis. Phytochemistry 64, 773–779.