TOXICITY AND BIOLOGICAL ACTIVITIES OF THREE PLANT
EXTRACTS ON CULCX PIPIENS LARVAE (DIPTERA: CULICIDAE).
Salam S. Teleb1 and Sameh A. Amin2
1- Zoology Department, Faculty of Science, Zagazig
University, Egypt.
2- Botany Department, Faculty of Science, El-Fayoum
University, Egypt.
Key Words: Larvicidal activity; Ovicidal; Pupicidal;
Oviposition; Hibiscus rosa-sinensis L., Anagallis arvensis L.;
Melilotus indica (L.); Culex pipiens larvae; Diptera;
Culicidae.
Abstract
Larvicidal activity of three plant extracts namely:
Hibiscus rosa-sinensis, Anagallis arvensis and Melilotus indica were
evaluated as a larvicidal potential against 2nd and 4th
instars larvae of Culex pipiens by using different
solvents; water, ethyl alcohol, acetone and petroleum
1
ether (40-60). The ethyl alcohol and acetone extracts
of Anagallis arvensis showed the highest larvicidal
activity (Lc50 was 6.4 and 14.5 ppm for the 2nd
13.4and 23.5ppm for the 4th, respectively) followed
by acetone and ethyl alcohol extracts of Melilotus indica
(Lc50 was 27.5 and 44.8 ppm for the 2nd and 46.3 and
66.5 for the 4th, respectively). The acetone extracts
of Hibiscus rosa-sinensis showed moderate larvicidal
activity (Lc50 was 235.0 and 825.0 ppm for the 2nd and
the 4th, respectively). The hot water extracts of
Hibiscus rosa-sinensis exhibited the lowest larvicidal
activity (Lc50 was 2225.0 and 3750.0 ppm for the 2nd
and the 4th, respectively). Followed by petroleum
ether extracts of Anagallis arvensis (Lc50 was 1362.5 and
2775.0 ppm for the 2nd and the 4th, respectively). and
water extracts for Melilotus indica (Lc50 was 622.5 and
1050.0 ppm for the 2nd and the 4th, respectively).
Ethanol extracts of Anagallis arvensis had completely
2
ovicidal action at 300ppm while at 400ppm for acetone
extracts of Hibiscus rosa-sinensis and 600ppm for Melilotus
indica. Acetone extracts of Hibiscus rosa-sinensis completely
inhibited adult emergence at 100ppm while at 1000ppm
for Melilotus indica and 3000ppm for ethanol extracts of
Anagallis arvensis. The ethanol extracts of Anagallis arvensis
completely inhibited oviposition by gravid females
till 300ppm, 1000ppm for Hibiscus rosa-sinensis and 7000ppm
for Melilotus indica.
Introduction
Culex pipiens is a mosquito species widely spread
in western districts of Saudi Arabia (Buttiker, 1979
and Abdullah & Merdan, 1995). Culex are implicated in
the transmission of pathogens that and arboviral
disease e.g., Wuchereria bancrofti, Rift valley fever,
Malaria, West Nile, Sindbis and Japanese encephalitis
viruses.
3
Extracts or essential oils from plants
are a rich source for bioactive compounds and non
toxic products which are suitable for use in control
of mosquito larvae. In fact, many researchers have
reported on the effectiveness of plant extracts or
essential oils against mosquito larvae (Teleb, 1995;
Teleb& Farag, 1999; Amer & Mehlhorn, 2006 and Rahuman
et al., 2008).
The herb Hibiscus rosa-sinensis L. is widely
distributed in tropical and sub tropical regions of
the world and reported to posses various medicinal
properties ;anti tumor, antihypertensive,
antioxidant, ant-implantation and antispermatogenic
activities (Kholkute & Udupa 1976; Sachdewa et al.,
2001; Herrera et al., 2004 and Hou et al., 2005). It was
also found to have a protective effect against the
tumor promotion stage of cancer development (Sharma &
Sultana, 2004). In addition to that extracts have
4
shown larvicidal effects to control the filarial
vector, Culex quinquefasciatus Say (Rahuman et al., 2009),
Aedes albopictus, C. quinquefasciatus, Anopheles culicifacies
and A. stephensi (Dua et al., 2006 and Nath et al., 2006).
The Anagallis arvensis L. is annual plant,
growing in fields, roadsides, etc., and reported to
posses various medicinal properties viz: antifungal
(Ali and Abu Ghdeib, 1999), antiviral activity against
several viruses including herpes simplex type 1,
adenovirus type 6, vaccinia, vesicular stomatitis and
poliovirus (Amoros et al., 1987). It also induced 100%
mortality on Oreochromis niloticus after 48 hrs exposure and
after 24 hrs for Gambusia affinis (Mostafa et al., 2005) and
Outbreaks and poisoning of cattle and sheep in Uruguay
(Rivero et al., 2001).
Melilotus indica (L.). or Sweet clover, long popular
as food for grazing animals, is used medicinally as
in the treatment of bowel complaints and infantile
5
diarrhea, used externally as a poultice or plaster on
swellings. The plant is discutient, emollient,
astringent, strongly laxative and narcotic. The plant
contains coumarin, which is an anticoagulant and also
contains dicumarol, which is a broad spectrum
bactericide (Knight et al., 1991 and Blumenthal, 1999)
The present investigation is undertaken to
find the Larvicidal, Ovicidal, and Oviposition
deterrents of Hibiscus rosa-sinensis L., Anagallis arvensis L,
and Melilotus indica (L.) extracts on Culex pipiens larvae.
Materials and methods
Collection and identification of plant species:
The leaves of Hibiscus rosa-sinensis L.
(Malvaceae), shoot system of Melilotus indica (L.) All.
(Fabaceae) and the whole plant of Anagallis arvensis L.
(Primulaceae) were collected from Dammam city and its
vicinities.
Insect rearing:
6
Culex pipiens (Diptera: Culicidae) larvae were
collected from a stagnant water and identified
according to Harbach (1985), larvae were reared on
dried fish meal and yeast 3:1, respectively and
adult males were fed on 10% sugar solution. Adult
females were given pigeon blood. All stages were
maintained in laboratory at 28± 2°C and 65± 5% R.H.
Preparation of plant extracts:
The dried leaves of Hibiscus rosa-sinensis shoot system
of Melilotus indica and whole plant of Anagallis arvensis were
grounded mechanically using electrical stainless
steel blender and extracted with Ethyl alcohol,
acetone , and petroleum ether (40–60°C). The aqueous
and hot water extracts were prepared following the
procedure of Chowdhury et al., (2008). The extracts
were concentrated and stored in refrigerator at 4°C.
7
Larvicidal test:
The larvicidal activity of Culex pipiens was
assessed by the procedure of WHO (1996) after some
modification with the method of Teleb (1994). From the
stock solution, different concentrations were
prepared. In each test 25 larvae were put in plastic
cups containing different concentrations of tested
plant extracts. The number of dead larvae were
counted after 24 h. of exposure, and the percentage
mortality was reported from the average of four
replicates.
Biological studies:
In the case of ovicidal action: Ten
replicates of egg rafts (0-6h.old )were counted and
transferred each in separate plastic cups (10cm
height) half filled with different concentrations of
ethanolic extracts of Anagallis arvensis and acetone
extracts of Hibiscus rosa-sinensis and Melilotus indica and
8
observed daily for hatching. Egg hatchability was
expressed as the percentage of hatched eggs from
total oviposited eggs.
For pupicidal action: Ten replicates of newly emerged
pupae were transferred into plastic cups 10cm height
(4/cup) half filled with different concentrations of
ethanolic extracts of Anagallis arvensis and acetone
extracts of Hibiscus rosa-sinensis and Melilotus indica and
observed daily for adult emergence. The number of
emerging adults was calculated and compared with the
control (untreated ).
For oviposition behavior: Four replicate cohorts of
20 gravid females of Culex pipiens were maintained each
in a cage (40x40x40 cm )equipped with plastic cups
filled with different contractions of ethanolic
extracts of Anagallis arvensis and acetone extracts of
Hibiscus rosa-sinensis L and Melilotus indica and the control
9
(water). The cages were observed daily, the egg rafts
were counted and compared with the control.
Statistical analysis:
The average larval mortality data were
subjected to probit analysis for calculating LC50 and
LC90 All results were expressed as mean ± standard
error, and the data were analyzed using student T-
test. Results with p<0.05 were considered to be
statistically significant.
Results and discussion
1- Larvicidal activity.
Results persistent in Table 1 indicated
that, the highest larval mortality were those from
ethanol and acetone extracts of Anagallis arvensis
(LC50=6.4 and 14.5 ppm; LC90=30.8 and, 68.3 ppm against
2nd instar larvae, and LC50=13.4 and 23.5 ppm;
LC90=32.8 and 85.5 ppm against 4th instar larvae,
respectively) followed by acetone and ethanol shoot
10
extracts of Melilotus indica (LC50=27.5, 44.8 ppm;
LC90=102.5, 165.0 ppm against 2nd instar larvae and
LC50=46.3, 66.5 ppm; LC90=205.0 and 325.0 ppm) against
4th instar larvae of Culex pipiens, respectively). The
acetone and ethanol leaf extracts of Hibiscus rosa-sinensis
exhibited moderate larvicidal effect (LC50=235.0,
395.0 ppm; LC90=655.0, 2675.0ppm against 2nd instar
larvae and LC50=825.0, 685.0 ppm; LC90= 3400.0,
4500.0ppm against 4th instar larvae of Culex pipiens,
respectively).
The petroleum ether (40–60°C) and the
aqueous or hot water extracts exhibited the lowest
larval mortality (LC50=775.0 and 2225.0ppm LC90 =
5325.0 and 9775.0ppm) against 2nd instar larvae ((LC50
= 880.0 and 3750.0ppm, LC90= 6025.0 and 10525.0ppm)
against 4th instar larvae for Hibiscus rosa-sinensis; (LC50 =
1362.5 and 1250.0 ppm, LC90 = 4450.0 and 7725.0 ppm
against 2nd instar larvae (LC50=2775.0 and 1727.0 ppm,
11
LC90=7850.0 and 8100.0 ppm against 4th instar larvae
for Anagallis arvensis and (LC50=425.0 ppm and 622.5ppm,
LC90=1425.0 ppm and 2600.0ppm against 2nd instar larvae
(LC50=927.5.0ppm and 1050.0ppm, LC90=3925.0 ppm and
2975.0 ppm against 4th instar larvae for Melilotus indica.
It has been observed earlier that the
crude leaf acetone, chloroform, hot water, methanol,
petroleum ether (60–80°C), and water extracts of
Calotropis procera (Ait.) R. Br., Canna indica L., Hibiscus rosa-
sinensis L., Ipomoea carnea Jacq. sp. fistulosa Choisy, and
Sarcostemma brevistigma showed moderate larvicidal effects
after 24 h of exposure at 1000ppm against second and
fourth instars larvae of, Culex quinquefasciatus (Rahuman et al.,
2009). Dua et al., (2006) have reported that mean median
lethal concentration of the aqueous extract from the
roots of Hibiscus abelmoschus against the larvae of Anopheles
culicifacies, An. stephensi, and Culex quinquefasciatus were 52.3,
52.6, and 43.8 ppm, respectively. Ali and Abu Ghdeib
12
(1999) reported that the most active extracts (90-100%
inhibition) were those of Anagallis arvensis, C. spinosa, J. regia,
Pistacia lentiscus and Ruta chalapensis against M. canis; Inula
viscosa, J. regia and P. lentiscus.
2- Biological activities:
2-1 Ovicidal toxicity
As shown in Table (2) the ethanolic
extracts of Anagallis arvensis and acetone extracts of
Hibiscus rosa-sinensis and Melilotus indica exhibited complete
ovicidal effect at 300 ppm for Anagallis arvensis, at 400
ppm for Hibiscus rosa-sinensis and 600 ppm for Melilotus indica
The hatchability increased by diluting with water.
Mullai and Jebanesa (2007) showed that 100% of
mortality at 450ppm for Citrullus colocynthis and 600ppm
for Cucurbita maxima on Culex quinquefasciatus.
2-2 – Pupicidal toxicity:
Table (3) shows that the acetone extracts of
Hibiscus rosa-sinensis L completely inhibited the emergence
13
of adults at 100ppm, at 1000 ppm for Melilotus indica and
at 3000ppm for Anagallis arvensis, respectively. The
emergence of adults are increasing by dilution. Elhag
(1999) shows that the methanolic extracts of
Azadirachta indica, Rhazya stricta and Syzygium aromaticum are
reducing pupation and inhibiting adult emergence of
Culex pipiens.
2-3- Oviposition behavior of gravid females:
Data in Table (4) indicate that gravid
females of Culex pipiens were completely inhibited for
oviposition at 300 ppm for Anagallis arvensis ethanolic
extracts, at 1000 ppm for Hibiscus rosa-sinensis L. and at
7000 ppm for Melilotus indica. At lower concentrations,
the repellency of gravid females decreased.
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23
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