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