Larvicidal, Ovicidal, Oviposition Deterrence and Emergence Inhibition Activity of Selected Sudanese Plants Against

Larvicidal, ovicidal, oviposition deterrence and emergence
inhibition activity of selected Sudanese plants against

Anopheles arabiensis and Culex quinquefasciatus .
By
Abd Alla Musa Ali Elimam
B. Sc. & Ed. (Hons. ) Biology, 1994
M. Sc. Biology ( Medical Entomology ) , 1998
Faculty of Education University of Khartoum
A thesis Submitted in Fulfillment of the Requirement for the Degree of

Doctor of Philosophy in Biology (Medical Entomology )
Supervisor
Dr. Khitma Hassan Elmalik
Department of Preventive Medicine , Faculty of Veterinary Medicine
University of Khartoum
Co-Supervisor
Dr. Faysal Sawi Ali
Department of Biology , Faculty of Education
December 2007
Contents
Dedication …………………………………….…………………. V
Abstract ………………………………………….………………. VI
Acknowledgement ……………………………….……………… XI
List of Tables …………………………………….……………… XII
List of Figures ………………………….……….……………….. XVI
List of Plates ……………………………….………………XVIII
Chapter One
Introduction …………………………………………………..… 1
Objectives of the study ………………………………………….. 4
Chapter Two
Literature Review ………………………………………….……. 5

2.1 Global burden of malaria …………………………….……… 5
2.2 Malaria in Sudan ……………………………………………. 6
2.3 Malaria transmission ……………………………………….. 6
2.4 Global burden of Lymphatic Filariases …………………….. 8
2.5 Lymphatic Filariases in Sudan …………………………….. 8
2.6 Lymphatic Filariases transmission …………………………. 9
2.7 Important species of mosquitoes in Sudan …………………. 10
2.8 Life cycle of the mosquitoes ……………………………….. 12
2.9 Mosquito bionomics ……………………………………….. 15
2.10 The breeding sites of mosquitoes …………………………. 16
2.10.1 Anopheles species ……………………………………… 16
2.10.2 Culex species …………………………………………… 17
2.11 Mosquito larval control ………………………………. …… 18
2.11.1 Source reduction …………………………………… …… 18
2.11.2 Larviciding …………………………………………… … 19
2.11.2.1 Chemical control …………………………………. …… 19
2.11.2.2 Biological control ………………………………... …… 22
2.11.2.2.1 Larvivorous fish …………………………………... … 23
2.12 Malaria vectors control in Sudan………………... ………… 26
2.13 Plants mosquitocides …………………………………. …… 27
2.13.1 Urge and advantage of using plants mosquitocides …... … 27
2.14 Some plants mosquitocides …………………………... …… 28
2.14.1 Larvicidal activity ……………………………….. ……... 29
2.14.2 Adults emergence inhibition activity ………………. ….... 33
2.14.3 Oviposition deterrent activity ……………………… …… 34
2.14.4 Eggs hatching activity ……………………………… ….. 34
Chapter Three
Materials and Methods ………………………………………….. 36

3.1 The study area ……………………………………….….…… 36
3.2 Plants used ……………………………………………...…… 36
3.2.1 Calotropis procera (Ait) ……………………………...…… 36
3.2.2 Ricinus communis L. ……………………………. …..….… 37
3.2.3 Euphorbia hirta L. …………………………………..….…. 37
3.2.4 Sonchus oleraceous …………………………………..…… 37
3.2.5 Eclipta prostrata ……………………………………..…… 38
3.3 preparation of stock solution ………………………….…….. 38
3.4 Mosquito rearing …………………………………….……… 39
3.5 Preparation of test concentration …………………………..... 40
3.6 Bioassay procedure …………………………………..……… 42
3.6.1 Larvicidal and pupicidal activity ……………………..…… 42
3.6.1.1 Data analysis …………………………………….….…… 43
3.6.2 Adult emergence inhibition activity ……………….……… 45
3.6.2.1 Data analysis …………………………………..………… 46
3.6.3 Oviposition deterrent activity ……………………...……… 47
3.6.4 Ovicidal activity ……………………………….………….. 48
4.7 Statistical analysis ……………………………….…..……… 49
Chapter Four
Results ……………………………………………………… 51
4.1 Larvicidal and pupicidal activity of leaves extract of
Calotropis procera against Anopheles arabiensis and Culex
quinquefasciatus …………………………………………….. …. 51
4.2 Larvicidal and pupicidal activity of leaves extract of
Ricinus communis L against Anopheles arabiensis and
Culex quinquefasciatus …………………………………….….… 63
4.3 Larvicidal and pupicidal activity of leaves extract of Eclipta
prostrata , Sonchus oleraceus ,and Euphorbia hirta against
Anopheles arabiensis and Culex quinquefasciatus ………..…….. 65
4-4- The adult emergence inhibition activity of leaves extract of 76
Calotropis procera against 3rd larval instar of Anopheles
arabiensis and Culex quinquefsciatus …………………………...
4-5- The adult emergence inhibition activity of leaves extract of
Ricinus communis against 3rd larval instar of Anopheles
arabiensis and Culex quinquefsciatus …………………………… 82
4.6 The ovicidal activity of leaves extract of Calotropis procera
against Culex quinquefasciatus ………………………………. … 90
4.7 The ovicidal activity of leaves extract of Ricinus communis
against Culex quinquefasciatus ………………………………….. 90
4.8 Oviposition deterrent activity of leaves extract of Calotropis
procera against gravid, female Anopheles arabiensis and Culex
quinquefasciatus ………………….……………………………... 95
4.9 Oviposition deterrent activity of leaves extract of Ricinus
communis against gravid, female Anopheles arabiensis and
Culex quinquefasciatus ………………………………………… 99
Chapter Five
Discussion ……………………………………………..………… 103

Conclusion ..................................................................................... 121
References …………………………………………..…………… 123
Dedication
To MY PARENTS
To
MY BROTHERS AND SISTERS

AND TO
MY WIFE WITH TRUE LOVE AND REGARD
MY KIDS
( MUSTAFA, HIND, LAMYA AND SAHAR )
WITH MY LOVE
Abstract
Malaria and Filariases are prevalent in Sudan , the control of these diseases
depends largely on preventive measures against mosquito vectors . Previous
control efforts targeting all stages of mosquitoes, but has focused almost on
adult (flying stage) control, by using conventional insecticides .
The present study aimed to investigate, the potential larvicidal
activity, and its subsequent effects upon egg hatching, adult emergence
inhibition and oviposition deterrent activity of aqueous leaves extract of five
selected indigenous plant against Anopheles arabiensis (the main vector of
malaria in Sudan) and Culex quinquefasciatus (the main vector of Filariasis)
as biological control .
Laboratory experiment were conducted as follows : The larvicidal
activity of each plant extract was studded against 2nd, 3rd and 4th instars
larvae of each mosquito . The larval mortality was observed after 24 hours .
The adult emergence inhibition activity was tested by exposing 3rd instar
larvae of each mosquito to different concentration of each plant extract . The
oviposition deterrent activity was tested by using three different
concentration of extract that cause high, moderate and low larvae mortality
in the larvicidal experiment . The results obtained were :
It was found that the three plants Euphorbia hirta Umlebien, Sonchus
oleraceous Molita and Eclipta prostrata Tamr Elganm have not shown any
larvicidal activity against both mosquito species studied, up to a
concentration as high as 10000ppm of the extract .
The plant Calotropis procera (Ushar) and Ricinus communis
(Khirwi) showed high level of toxicity against both mosquitoes larvae . The
pupal stage was not affected till a concentration of 10000 ppm . In all cases
2nd instar was more susceptible than 3rd instar and the later was more
susceptible than 4th instar . The extracts of C. procera was more potent than
that of R. communis against the two species of mosquito. Culex
quinquefasciatus was more susceptible than Anopheles arabiensis with
respect to C. procera, and Anopheles arabiensis was more susceptible than
Culex quinquefasciatus with respect to R. communis . Both plants showed
remarkable effect on egg hatching and larval development. Eggs were found
more susceptible than 3rd and 4th instar larvae . The concentration that cause
50% of adult emergence inhibition (EI50) was less than that cause 50%
larvae mortality ( LC50 ) .
The leaves extract of both plants showed 100% oviposition deterrent
and effective repellence against both mosquitoes at different larvicidal
concentration (high, moderate and low) when there is a choice of control
(treated – control ) . But the avoidance of eggs laying was not shown when
the control was not offered (treated water only), with the observation on that
maximum of eggs laying was preferred in the low larvicidal concentration .
In all cases at high larvicidal concentration the eggs laying was avoided or at
least reduced to very low number by female mosquito . The results suggest
that the leaves extract of C. procera and R. communis possess remarkable
larvicidal, adult emergence inhibitor, ovicidal and oviposition deterrent
properties against An. Arabiensis and Culex quinquefasciatus, and might be
used as natural biocides for mosquito control .
‫ﻣﻠﺨﺺ اﻟﺪراﺳﺔ ﺑﺎﻟﻠﻐﺔ اﻟﻌﺮﺑﻴﺔ‬
‫‪Abstract‬‬
‫اﻟﻤﻼرﻳﺎ واﻟﻔﻴﻼرﻳﻴﺴﻴﺲ ﻣﻦ اﻷﻣﺮاض اﻟﻤﻨﺘﺸﺮة ﻓﻲ اﻟﺴﻮدان ‪ .‬اﻟﺘﺤﻜﻢ ﻓﻲ هﺬﻩ اﻷﻣﺮاض‬
‫ﻳﻌﺘﻤﺪ ﺑﺼﻮرة آﺒﻴﺮة ﻋﻠﻰ درﺟﺔ اﻟﺤﻤﺎﻳﺔ ﻣﻦ اﻟﺒﻌﻮض اﻟﻨﺎﻗﻞ ‪ .‬اﻟﺠﻬﻮد اﻟﻤﺒﺬوﻟﺔ ﻟﻠﺘﺤﻜﻢ ﻓﻲ هﺬﻩ‬
‫اﻷﻣﺮاض ﺗﺴﺘﻬﺪف آﻞ أﻃﻮار اﻟﺒﻌﻮض اﻟﻨﺎﻗﻞ ﻟﻜﻨﻬﺎ ﻓﻲ اﻟﻐﺎﻟﺐ ﻣﺮآﺰة ﻋﻠﻰ ﻣﻜﺎﻓﺤﺔ اﻟﻄﻮر اﻟﺒﺎﻟﻎ‬
‫)اﻟﻄﺎﺋﺮ( ﺑﺎﺳﺘﻌﻤﺎل اﻟﻤﺒﻴﺪات اﻟﺘﻘﻠﻴﺪﻳﺔ ‪.‬‬
‫هﺪﻓﺖ اﻟﺪراﺳﺔ ﻟﺒﺤﺚ ﻓﺎﻋﻠﻴﺔ اﻟﻤﺴﺘﺨﻠﺺ اﻟﻤﺎﺋﻲ ﻷوراق ﺧﻤﺲ ﻧﺒﺎﺗﺎت ﻃﺒﻴﻌﻴﺔ ﻓﻲ ﻣﻜﺎﻓﺤﺔ‬
‫اﻟﻄﻮر اﻟﻴﺮﻗﻲ ‪ ،‬واﻵﺛﺎر اﻟﻼﺣﻘﺔ ﻟﺬﻟﻚ ﻣﻦ ﺧﻼل ﺗﺄﺛﻴﺮهﺎ ﻋﻠﻰ ﻣﻌﺪل ﻓﻘﺲ اﻟﺒﻴﺾ ‪ ،‬ﻣﻨﻊ اﻟﻴﺮﻗﺎت ﻣﻦ‬
‫اﻟﻮﺻﻮل إﻟﻰ اﻟﻄﻮر اﻟﺒﺎﻟﻎ وﻣﻨﻊ اﻹﻧﺎث ﻣﻦ وﺿﻊ اﻟﺒﻴﺾ ﻓﻲ اﻟﻤﻴﺎﻩ اﻟﻤﻌﺎﻟﺠﺔ آﻤﻜﺎﻓﺤﺔ ﺣﻴﻮﻳﺔ ﺿﺪ‬
‫ﺑﻌﻮض اﻻﻧﻮﻓﻠﺲ اراﺑﻴﻨﺴﻴﺲ ‪ ) An. arabiensis‬اﻟﻨﺎﻗﻞ اﻟﺮﺋﻴﺴﻲ ﻟﻠﻤﻼرﻳﺎ ﻓﻲ اﻟﺴﻮدان ( و اﻟﻜﻴﻮﻟﻜﺲ‬
‫آﻮﻳﻨﻜﻮﻳﻔﺎﺳﻴﺘﺲ ‪ ) Culex quinquefasciatus‬اﻟﻨﺎﻗﻞ اﻟﺮﺋﻴﺴﻲ ﻟﻠﻔﻴﻼرﻳﺴﻴﺲ ( ‪.‬‬
‫ﺗﻢ إﺟﺮاء اﻟﺘﺠﺎرب اﻟﻤﻌﻤﻠﻴﺔ آﺎﻵﺗﻲ ‪ :‬درﺳﺖ ﻓﺎﻋﻠﻴﺔ آﻞ ﻣﺴﺘﺨﻠﺺ ﻧﺒﺎﺗﻲ ﺿﺪ اﻟﻌﻤﺮ اﻟﻴﺮﻗﻲ‬
‫اﻟﺜﺎﻧﻲ )‪ (2nd instar‬و اﻟﺜﺎﻟﺚ )‪ (3rd instar‬واﻟﺮاﺑﻊ)‪ (4th instar‬ﻟﻜﻞ ﻧﻮع ﻣﻦ ﻧﻮﻋﻲ اﻟﺒﻌﻮض‬
‫ﻋﻠﻰ ﺣﺪﻩ وﻟﻮﺣﻆ ﻣﻌﺪل اﻟﻤﻮت ﺧﻼل ‪ 24‬ﺳﺎﻋﺔ ‪ .‬اﺧﺘﺒﺮ ﻣﻌﺪل ﻣﻨﻊ اﻟﻴﺮﻗﺎت ﻣﻦ اﻟﻮﺻﻮل إﻟﻰ اﻟﻄﻮر‬
‫اﻟﺒﺎﻟﻎ ﺑﺘﻌﺮﻳﺾ اﻟﻄﻮر اﻟﻴﺮﻗﻲ اﻟﺜﺎﻟﺚ ﻟﻜﻞ ﻧﻮع ﻣﻦ اﻟﺒﻌﻮض ﻟﻌﺪة ﺗﺮاآﻴﺰ ﻣﺨﺘﻠﻔﺔ ﻟﻜﻞ ﻣﺴﺘﺨﻠﺺ ﻧﺒﺎﺗﻲ‬
‫‪ .‬و اﺧﺘﺒﺮت ﻓﻌﺎﻟﻴﺔ اﻟﻤﺴﺘﺨﻠﺼﺎت ﻓﻲ ﻣﻨﻊ وﺿﻊ اﻟﺒﻴﺾ ﺑﻮاﺳﻄﺔ اﻹﻧﺎث‪ ،‬ﺑﺎﺳﺘﺨﺪام ﺛﻼث ﺗﺮاآﻴﺰ‬
‫ﻣﺨﺘﻠﻔﺔ وهﻲ اﻟﺘﻲ ﺗﺴﺒﺒﺖ ﻓﻲ ﻣﻮت ﻋﺪد آﺜﻴﺮ وﻋﺪد ﻣﺘﻮﺳﻂ وﻋﺪد ﻗﻠﻴﻞ ﻣﻦ اﻟﻴﺮﻗﺎت ﻓﻲ ﺗﺠﺮﺑﺔ ﻓﺎﻋﻠﻴﺔ‬
‫اﻟﻤﺴﺘﺨﻠﺺ ﺿﺪ اﻟﻄﻮر اﻟﻴﺮﻗﻲ ﺳﺎﺑﻘﺎ ‪.‬‬
‫وﻗﺪ ﺗﻢ اﻟﺘﻮﺻﻞ إﻟﻰ اﻟﻨﺘﺎﺋﺞ اﻟﺘﺎﻟﻴﺔ ‪:‬‬
‫وﺟﺪ أن اﻟﻨﺒﺎﺗﺎت اﻟﺜﻼث أم ﻟﺒﻴﻨﺔ )‪ (Euphorbia hirta‬و اﻟﻤﻮﻟﻴﺘﺔ )‪(Sonchus oleraceous‬‬
‫وﺗﻤﺮ اﻟﻐﻨﻢ ) ‪ (Eclipta prostrata‬ﻟﻴﺲ ﻟﻬﺎ أي ﺗﺄﺛﻴﺮ ﻋﻠﻰ ﻳﺮﻗﺎت ﻧﻮﻋﻲ اﻟﺒﻌﻮض ﺣﺘﻰ ﺗﺮآﻴﺰ‬
‫‪ 10000‬ﺟﺰء ﻣﻦ اﻟﻤﻠﻴﻮن ‪.‬‬
‫أﻇﻬﺮ ﻧﺒﺎﺗﻲ اﻟﻌﺸﺮ)‪ (Calotropis procera‬واﻟﺨﺮوع )‪ (Ricinus communis‬ﺳﻤﻴﺔ ﻋﺎﻟﻴﺔ ﺗﺠﺎﻩ‬
‫ﻳﺮﻗﺎت ﻧﻮﻋﻲ اﻟﺒﻌﻮض ‪ .‬ﺑﻴﻨﻤﺎ ﻟﻢ ﻳﺘﺄﺛﺮ ﻃﻮر اﻟﻌﺬراء )‪ (pupa‬ﺑﺎﻟﻤﺴﺘﺨﻠﺺ اﻟﻨﺒﺎﺗﻲ ﺣﺘﻰ ﺗﺮآﻴﺰ‬
‫‪ 10000‬ﺟﺰء ﻣﻦ اﻟﻤﻠﻴﻮن ‪ .‬ﻓﻲ آﻞ اﻟﺤﺎﻻت وﺟﺪ أن اﻟﻌﻤﺮ اﻟﻴﺮﻗﻲ اﻟﺜﺎﻧﻲ اآﺜﺮ اﺳﺘﺠﺎﺑﺔ ﻣﻦ اﻟﻌﻤﺮ‬
‫اﻟﻴﺮﻗﻲ اﻟﺜﺎﻟﺚ واﻷﺧﻴﺮ أآﺜﺮ اﺳﺘﺠﺎﺑﺔ ﻣﻦ اﻟﻌﻤﺮ اﻟﻴﺮﻗﻲ اﻟﺮاﺑﻊ ‪ .‬آﻤﺎ وﺟﺪ أن ﻧﺒﺎت اﻟﻌﺸﺮ أآﺜﺮ ﺳﻤﻴﺔ‬
‫ﻣﻦ ﻧﺒﺎت اﻟﺨﺮوع ﺿﺪ ﻧﻮﻋﻲ اﻟﺒﻌﻮض ‪ .‬آﻤﺎ وﺟﺪ أن ﺑﻌﻮض اﻟﻜﻴﻮﻟﻜﺲ آﻮﻳﻨﻜﻮﻳﻔﺎﺳﻴﺘﺲ اآﺜﺮ اﺳﺘﺠﺎﺑﺔ‬
‫ﻣﻦ ﺑﻌﻮض اﻻﻧﻮﻓﻠﺲ اراﺑﻴﻨﺴﻴﺲ ﺗﺠﺎﻩ ﻣﺴﺘﺨﻠﺺ ﻧﺒﺎت اﻟﻌﺸﺮ ‪ ،‬وﺑﻌﻮض اﻻﻧﻮﻓﻠﺲ اراﺑﻴﻨﺴﻴﺲ أآﺜﺮ‬
‫اﺳﺘﺠﺎﺑﺔ ﻣﻦ ﺑﻌﻮض اﻟﻜﻴﻮﻟﻜﺲ آﻮﻳﻨﻜﻮﻳﻔﺎﺳﻴﺘﺲ ﺗﺠﺎﻩ ﻣﺴﺘﺨﻠﺺ ﻧﺒﺎت اﻟﺨﺮوع ‪.‬‬
‫أﻇﻬﺮ آﻞ ﻣﻦ ﻣﺴﺘﺨﻠﺺ اﻟﻨﺒﺎﺗﻴﻦ ﺗﺄﺛﻴﺮا واﺿﺤﺎ ﻋﻠﻰ ﻣﻌﺪل ﻓﻘﺲ اﻟﺒﻴﺾ وﻣﻌﺪل ﻧﻤﺆ اﻟﻴﺮﻗﺎت ‪.‬‬
‫وﺟﺪ أن اﻟﺒﻴﺾ اآﺜﺮ اﺳﺘﺠﺎﺑﺔ ﻣﻦ اﻟﻌﻤﺮ اﻟﻴﺮﻗﻲ اﻟﺜﺎﻟﺚ و اﻟﺮاﺑﻊ ‪ .‬و أن اﻟﺘﺮآﻴﺰ اﻟﻼزم ﻟﻤﻨﻊ ‪ %50‬ﻣﻦ‬
‫اﻟﻴﺮﻗﺎت ﻣﻦ اﻟﻮﺻﻮل إﻟﻰ اﻟﻄﻮر اﻟﺒﺎﻟﻎ اﻗﻞ ﻣﻦ اﻟﺘﺮآﻴﺰ اﻟﻼزم ﻟﻤﻮت ‪ %50‬ﻣﻦ اﻟﻴﺮﻗﺎت ‪.‬‬
‫ﻓﻲ ﻣﺴﺘﺨﻠﺺ اﻷوراق ﻟﻠﻨﺒﺎﺗﻴﻦ ‪ ،‬وﺟﺪ أن ﺟﻤﻴﻊ اﻟﺘﺮاآﻴﺰ اﻟﻤﺴﺘﺨﺪﻣﺔ و اﻟﻤﺴﺒﺒﺔ ﻟﻤﻮت ﻋﺪد )‬
‫آﺜﻴﺮ و ﻣﺘﻮﺳﻂ و ﻗﻠﻴﻞ ( ﻣﻦ اﻟﻴﺮﻗﺎت ﻟﻬﺎ أﺛﺮا ﻃﺎردا و ﺗﻤﻨﻊ اﻹﻧﺎث ﻣﻦ وﺿﻊ اﻟﺒﻴﺾ ﺑﺪرﺟﺔ ‪، %100‬‬
‫) ﻣﻌﺎﻟﺞ ‪ -‬ﻏﻴﺮ ﻣﻌﺎﻟﺞ ( ‪ .‬ﺑﻴﻨﻤﺎ ﺗﺠﻨﺐ وﺿﻊ اﻹﻧﺎث‬ ‫ﻓﻲ ﺣﺎل وﺟﻮد ﺧﻴﺎر ﺁﺧﺮ ﻟﻠﺘﻜﺎﺛﺮ ﻏﻴﺮ ﻣﻌﺎﻟﺞ‬
‫ﻟﻠﺒﻴﺾ ﻟﻢ ﻳﺤﺪث ﻓﻲ ﺣﺎل ﻋﺪم وﺟﻮد ﻣﻜﺎن ﺁﺧﺮ ﻟﻠﺘﻜﺎﺛﺮ ﻏﻴﺮ ﻣﻌﺎﻟﺞ )وﺟﻮد اﻟﻤﺎء اﻟﻤﻌﺎﻟﺞ ﻓﻘﻂ( ﻣﻊ‬
‫ ﻓﻲ ﺣﺎﻟﺔ اﻟﺘﺮآﻴﺰ اﻟﻤﺴﺒﺐ‬. ‫ﻣﻼﺣﻈﺔ أن اﻹﻧﺎث ﺗﻔﻀﻞ اﻟﺘﺮآﻴﺰ اﻟﻤﻨﺨﻔﺾ ﻟﻮﺿﻊ اآﺒﺮ ﻋﺪد ﻣﻦ اﻟﺒﻴﺾ‬
‫ﻟﻤﻮت ﻋﺪد آﺜﻴﺮ ﻣﻦ اﻟﻴﺮﻗﺎت وﺟﺪ أن إﻧﺎث اﻟﺒﻌﻮض ﺗﺘﺠﻨﺐ وﺿﻊ اﻟﺒﻴﺾ ﺗﻤﺎﻣﺎ أو ﻋﻠﻰ اﻷﻗﻞ ﻳﺨﺘﺰل‬
. ‫وﺿﻊ اﻟﺒﻴﺾ إﻟﻰ اﻗﻞ ﻋﺪد‬
‫ﻧﺴﺘﻨﺘﺞ ﻣﻦ هﺬﻩ اﻟﺪراﺳﺔ أن ﻣﺴﺘﺨﻠﺺ أوراق ﻧﺒﺎﺗﻲ اﻟﻌﺸﺮ واﻟﺨﺮوع ﻳﻤﺘﻠﻜﺎن ﺧﺎﺻﻴﺔ ﻣﺒﻴﺪ‬
‫ﻟﻠﻴﺮﻗﺎت واﻟﺒﻴﺾ وﻣﻨﻊ اﻟﻴﺮﻗﺎت ﻣﻦ اﻟﻮﺻﻮل إﻟﻰ اﻟﺤﺸﺮة اﻟﺒﺎﻟﻐﺔ وﻣﻨﻊ اﻹﻧﺎث ﻣﻦ وﺿﻊ اﻟﺒﻴﺾ ﺿﺪ‬
‫ﺑﻌﻮض اﻻﻧﻮﻓﻠﺲ اراﺑﻴﻨﺴﻴﺲ و اﻟﻜﻴﻮﻟﻜﺲ آﻮﻳﻨﻜﻮﻳﻔﺎﺳﻴﺘﺲ وﻳﻤﻜﻦ اﺳﺘﺨﺪاﻣﻬﻤﺎ آﻤﺒﻴﺪ ﺣﻴﻮي ﻃﺒﻴﻌﻲ‬
. ‫ﻟﻤﻜﺎﻓﺤﺔ اﻟﺒﻌﻮض‬
Acknowledgement
Firstly , thanks and praise to Allah for giving me the health, patience
and for his limitless grace . Then I would like to express my sincere
gratitude and appreciation to Dr. Khitma Hassan Elmalik, Department of
Preventive Medicine, Faculty of Veterinary Medicine for her supervision,
encouragement, guidance and the interest she had in this work .
My deep gratitude and particular thanks to my co-supervisor Dr.
Faysal Sawi Ali, department of Biology, Faculty of Education , University
of Khartoum for his supervision, encouragement and advice .
Thanks are due to the members of Medical Entomology Department,
National Health Laboratory and Khartoum Without Malaria Centre for the
help they offered during samples collection.
I am also grateful to Dr. Abd Elgabar Nasir, Department of Biology,
Faculty of Education, University of Khartoum for Identifying and
classification of the selected plants used, help, encouragement and advices .
Thanks to the members of Biology Department, Faculty of Education and
Preventive medicine Department, Faculty of Veterinary Medicine,
University of Khartoum for their helpful aids and advice .With especiall
thanks to Prof. Mahmoud Musa, Mr. Ahmed Abd Elwahid, Dr. Aatif, Mr.
Husien, Mr. Babo and Mrs. Ilham from preventive Medicine . My gratitude
are extended to my friends and colleagues Mr. Alnour Abd Elmajeed for his
help in Statistical Analysis, Mr. Huzyfa Abd Elrahman and Mohamed Belo.
List of Tables
Table ( 4.1 ) Susceptibility of larvae of Anopheles arabiensis

exposed as 2nd larval instar to different
concentration of leaves extract of Calotropis
procera after 24 hours……………………………… 53
exposed as 3rd larval instar to different concentration
of leaves extract of Calotropis procera after 24
hours ……………………………………………….. 54
exposed as 4th larval instar to different concentration
of leaves extract of Calotropis procera after 24
hours………………………………………………... 55
Table ( 4.4 ) Susceptibility of larvae of Culex quinquefasciatus 57
procera after 24 hours …………………………….
Table ( 4.5 ) Susceptibility of larvae of Culex quinquefasciatus
exposed as 3rd larval instar to different
procera after 24 hours ……………………………... 58
of leaves extract of C. procera after24 hours……… 59
Table ( 4.7 ) Larvicidal activity of leaves extract of calotropis
procera against 2nd, 3rd and 4th instars larvae of
Anopheles arabiensis and Culex quinquefasciatus
expressed as LC50 and LC90 …………………… .. 61
concentration of leaves extract of Ricinus communis
after 24 hours ……...... …………………………… 66
exposed as 3rd larval instar to different concentration
of leaves extract of Ricinus communis after 24 hours
……………………………………………………… 67
of leaves extract of Ricinus communis after 24 hours
……………………………………………………… 68
after 24 hours …………………………………….. 70
exposed as 3rd larval instar to different
after 24 hours ………………… ………..………... 71
exposed as 4th larval instar to different
after 24 hours …………………………………….. 72
Table ( 4.14 ) Larvicidal activity of leaves extract of Ricinus
communis against 2nd, 3rd and 4th instars larvae of
expressed as LC50 and LC90 ……………………... 74
Table ( 4-15 ) The adult emergence inhibition activity of leaves
extract of Calotropis procera against 3rd larval
instar of Anopheles arabiensis at different
concentration ………………………………………. 78
extract of Calotropis procera against 3rd larval
instar of Culex quinquefsciatus at different
concentration …………………… ………………… 80
extract of Ricinus communis against 3rd larval instar 84
of Anopheles arabiensis at different concentration
………………………………………………………
extract of Ricinus communis against 3rd larval instar
of Culex quinquefsciatus at different concentration
……………………………………………………… 86
extract of Calotropis procera and Ricinus communis
against 3rd larval instar of Anopheles arabiensis and
Culex quinquefsciatus expressed as EI50 and EI90 .. 88
Table ( 4-20 ) The ovicidal activity of leaves extract of Calotropis
procera against Culex quinquefasciatus …………... 91
Table ( 4-21 ) The ovicidal activity of leaves extract of Ricinus
communis against Culex quinquefasciatus ………… 93
Table ( 4-22 ) Oviposition deterrent activity of leaves extract of
Calotropis procera against gravid, female
Anopheles arabiensis ………………………………. 97
Calotropis procera against gravid, female Culex
quinquefasciatus …………………………………… 98
Ricinus communis against gravid, female Anopheles
arabiensis ………………………………………….. 101
Ricinus communis against gravid, female Culex
quinquefasciatus ….................................................... 102
List of Figures
Figure 4.1 Larvicidal activity of leaves extract of calotropis

Anopheles arabiensis expressed as linear regression... 56
procera against 2nd, 3rd and 4th instars larvae of Culex
quinquefasciatus expressed as linear regression.......… 60
expressed as LC50 and LC90 values ......................... 62
Figure 4.4 Larvicidal activity of leaves extract of Ricinus
Anopheles arabiensis expressed as linear regression... 69
Culex quinquefasciatus expressed as linear regression
........................................................................................ 73
expressed as LC50 and LC90 values ...............………. 75
Figure 4.7 The adult emergence inhibition activity of leaves
extract of Calotropis procera against 3rd larval instar
of Anopheles arabiensis at different concentration,
expressed as linear regression ………………………... 79
extract of Calotropis procera against 3rd larval instar
of Culex quinquefsciatus at different concentration,
expressed as linear regression ....................................... 81
extract of Ricinus communis against 3rd larval instar of
Anopheles arabiensis at different concentration,
extract of Ricinus communis against 3rd larval instar of
Culex quinquefsciatus at different concentration,
extract of Calotropis procera and Ricinus communis 89
against 3rd larval instar of Anopheles arabiensis and
Culex quinquefsciatus expressed as EI50 and EI90 ......
Figure 4.12 Ovicidal activity of leaves extract of Calotropis
procera against Culex quinquefsciatus expressed as
linear regression ............................................................ 92
Figure 4.13 Ovicidal activity of leaves extract of Ricinus
communis against Culex quinquefsciatus expressed as
linear regression ............................................................ 94
List of Plates
Plate 3.1 The Bioassay cup ………………………………………… 41

Plate 3.2 Cage for Mosquito rearing ………………………………... 44
CHAPTER ONE
INTRODUCTION
Mosquitoes ( Diptera, Culicidae ) pose the greatest threat to public
health because of their ability to act as vectors of pathogens causing malaria,
filariasis, dengue, yellow fever, etc. which affect many millions of people all
over the world (WHO 1984 ; 1995 ) .
Malaria and Filariasis rank amongst the world most prevalent tropical
infectious diseases . An estimated 300-500 million people are infected with
malaria annually, resulting in 1.5-3 million deaths (WHO, 2000) . Malaria
remains a major health problem in Sudan, accordingly about 20-40% of out
patient clinic visits and approximately 30% of total hospital admissions are
due to malaria ( WHO and UNICEF, 2005 ) . Lymphatic filariases ( LF ) is
probably the fastest spreading insect-borne disease of human in the tropic,
about 30% ( 394 million ) of the global at risk population is estimated to be
in the LF-endemic countries of the African region ( WHO, 2006 ) .
Lymphatic filariasis is a significant public health and economic problem in
many tropical and subtropical regions of the world, including Sudan ( Satti
& Abdel Nur 1974 ; Elsetouhy & Ramzy 2003 ; Aiah et al, 2005 ) . One of
the methods to control these diseases is to control the vectors for the
interruption of disease transmission .
Beside early diagnosis and prompt treatment with effective drugs, it is

clear that any possible mean of reducing man-vector contact should be
employed , either through vertical, but preferably through horizontally
(participatory) staged programmes ( Rozendaal , 1997 ) .
Vector control in Africa can target all stages of the mosquito life
cycle, but has historically focused almost exclusively on adult control based
on indoor residual house spraying (Mnzava et al, 1993 ; Curtis, 1994 ) or
more recently , the use of insecticide treated bed nets or curtains (Lengler,
2001 ) . The use of residual Anopheline vector control , either through
indoor house spraying ( Mabaso et al, 2004 ) or for bed net impregnation
(Lengeler, 2001) has proven highly effective in various parts of Africa, but
is not without obstacles . Emergence and spread of insecticide resistance in
Anophelines (Chandre et al, 1999; Hargreaves et al, 2000 ) , limited
susceptibility and rapid build-up of resistance to synthetic pyrethroids by
Culicine filariases vectors (Chandre et al, 1998), environmental pollution
(Ariese et al, 2001) and unresolved issues pertaining to their toxicity to
humans and non-target organisms , prevent progressive use and broad
acceptance of these tools . Therefore, control of mosquito at the larval stage
is necessary and efficient in integrated pest management of mosquitoes .
During the immature stage, mosquitoes are relatively immobile , remaining
more concentrated than they are in the adult stage ( Rutledge et al, 2003 ) .
Larval control strategies against the vectors of malaria in sub-Saharan Africa
could be highly effective ,complementary to adult control interventions, and
should be prioritized for further development, evaluation and
implementation as an integral part of rolling back malaria ( Killeen et al,
2002 ) . Larval control of mosquitoes either by source reduction , use of
larvicides or a combination of these , is a preferred method for reducing
adult mosquitoes in many areas of the world ( Mulla et al, 2001 ) .
Larviciding and source reduction have a major advantage in that they

control mosquitoes before they disperse and transmit disease ( Killeen et al,
2002 ) . In general, it is believed that area-wide control programmes of
mosquito larvae also result in the control of adult mosquito populations that
are responsible for annoyance and the transmission of pathogens ( Mulla et
al, 2001 ) .
Since the discovery of DDT , mosquito control approach has been

almost completely based on synthetic organic insecticides . But the extensive
use of synthetic organic insecticides during the last five decades have
resulted in environmental pollution and also in the development of
physiological resistance in major vector species in addition to the increased
costs of insecticides . This has necessitated the need for search and
development of environmentally safer , low cost , indigenous methods for
vector control . During the last decade, various studies on natural plant
products against mosquito vectors indicate them as possible alternatives to
synthetic chemical insecticides ( Mittal & Subbarao 2003 ; Rajkumar and
Jebanesan 2005a ; 2005b ; Promsiri et al, 2006) . Plant product can be
obtained either from the whole plant or from specific part by extraction with
different types of solvents such as aqueous , methanol, choloroform ,
hexane, etc. , depending on the polarity of the phytochemicals (Mittal &
Subbarao, 2003 ) . The bioactive constituents of these plants could be either
a single substance or a mixture of substances . The separation of the mixture
is neither practical nor advantageous in the insect economic control
strategies .
Objective of the study :
The overall objective of this work is to investigate the activity of

aqueous leaves extract of five Sudanese indigenous plant species against the
aquatic stages of Culex quinquefasciatus ( the main vector of filariasis )
and Anopheles arabienses ( the main malaria vector in Sudan ), so
as to introduce indigenous method for vector control , which is
environmentally safe ( natural products ), low cost ( actually free ), efficient
, practically easy and can be used with minimum care by individual and
communities .
Specific objectives are :
1- To study the larvicidal activity of water extracts of some selected

plants against mosquitoes Culex quinquefasciatus and Anopheles
arabienses .
2- To study the subsequent effects of the plant extracts which had a

significant effect on adult emergence, egg hatching , and oviposition
deterrent .
3- To compare between the different species of mosquitoes on their

susceptibility to different plants extracts .
CHAPTER TWO
REVIEW OF LITERATURE
2-1 Global burden of malaria

At the end of 2004, 107 countries and regions had areas at risk of
malaria transmission . Some 3.2 billion people lived in areas at risk of
malaria transmission . An estimated 350- 500 million clinical malaria
episodes occur annually ; most of these are caused by infection with
Plasmodium falciparum and P. vivax . Falciparum malaria causes more than
one million deaths each year. It also contributes indirectly to many
additional deaths, mainly in young children, through synergy with other
infections and illness ( WHO and UNICEF 2005 ) .
Patterns of malaria transmission and disease vary markedly between
regions and even within individual countries . This diversity results from
variation between malaria parasites and mosquito vectors, ecological
conditions that affect malaria transmission and socioeconomic factors , such
as poverty and access to effective health care and prevention services .
About 60% of the cases of malaria worldwide , about 75% of global
falciparum malaria cases and more than 80% of malaria deaths occur in
Africa south of the Sahara . P falciparum causes the vast majority of
infectious in this region and about 18% of death in children under 5 years
of age . Malaria is also a major cause of anemia in children and pregnant
women, low birth weight , premature birth and infant mortality . In endemic
African countries, malaria accounts for 25 - 35% of all out patient visits, 20 -
45% of hospital admissions and 15 - 35% of hospital deaths, having
presence a great burden on already weak health care systems ( WHO and
UNICEF 2005 ) .
2-2 Malaria in Sudan

Malaria is one of the major causes of morbidity and mortality in Sudan . An
estimated 7.5 million patients suffer from malaria each year and 35,000 die
from this disease which account for up to 20% of hospital deaths ( Roll
Back Malaria (RBM), 2002 ) . Symptomatic malaria accounts for 20 - 40%
of out patient clinic visits and approximately 30% of hospital admission .
The entire population of Sudan is at risk of malaria, although to different
degrees . In the northern, eastern and western states malaria is mainly low to
moderate with predominantly seasonal transmission and epidemic outbreaks
. In southern Sudan, malaria is moderate to high or highly intense, generally
with perennial transmission . Plasmodium falciparum is by far the
predominant parasite species ( WHO and UNICEF 2005) .
2-3 Malaria transmission
Malaria is caused by the Plasmodium parasite which spends its life cycle in
both human and certain species of mosquitoes . Four species of
Plasmodium that cause malaria in humans are P. falciparum , P. vivax ,
P. malariae and P. ovale . Of these , P. falciparum is the most important in
most parts of the tropics , being responsible for most severe illnesses and
deaths . Malaria parasites are transmitted by female mosquitoes belonging to
the genus Anopheles . Only female anopheline mosquitoes bite and take up
blood . Male feed on plant juices and nectar, rather than blood , and there
fore cannot transmit malaria . If the right stages of the parasite (the
gametocytes) are ingested by the mosquito when it take up a blood meal,
these will form gametes within the mosquito’s stomach (midgut) . The male
and female gametes unite to form the zygote, which can move and is called
the ookinete .The ookinete penetrates the wall of the midgut and becomes a
round oocyst . In side the oocyst , the nucleus divides repeatedly, a large
number of sporozoites are formed and the oocyst enlarges . When the
sporozoites are fully formed, the oocyst bursts, releasing the sporozoites into
the mosquito’s body cavity . The sporozoites migrate to the salivary glands
of the mosquito . The time necessary for the development of the sporozoites
varies with temperature and to a smaller extent with the species of the
malaria parasite and with humidity, but generally it is about 8-15 days (
WHO 1994 ; 2002 ).
The sporozoites are the infective stages, it is injected with the saliva
when the mosquito next feeds . The sporozoites inter the persons blood and
reach the liver cells where they multiply. Over a period of 7-12 days, the
parasite multiplies until the infected liver cell bursts . Then the parasites (
merozoites ) are released into the blood stream and invade the red blood cell
where they multiply again . The infected red blood cells are destroyed , the
parasites invade fresh red blood cells and the cycle is repeated . A female
mosquito needs to take a blood meal so that her eggs can mature , and since
it lay several batches of eggs during its lifetime, it will have several
opportunities to take up and to transmit malaria parasites ( WHO 1994 ;
2002 ) .
2-4 Global burden of Lymphatic filariasis
The total population at risk is currently estimated to be 1307 million people
in 83 lymphatic filariasis-endemic countries and areas . 65% in the WHO
Southeast Asia region and 30% in the WHO African Region . The remaining
5% is distributed among the three other WHO regions except the WHO
European Region . Of the 83 lymphatic filariasis (LF) endemic countries, 59
have completed mapping, 11 are in progress and 13 have to start (
WHO, 2006 ) .
Some 30% of the global at-risk population is estimated to be in the
LF-endemic countries of the African region . Of these 39 endemic countries,
21 have completed the initial estimation and mapping of implementation
units for endemic status and another 6 are in progress . In the countries
where mapping has been completed , 72% out of a total population of 205
million have been identified as at risk of filariasis . Of the 3 LF-endemic
countries (Egypt, Sudan and Yemen ) in the WHO Eastern Mediterranean
Region, Egypt and Yemen continued mass drug administration in 2005
targeting the entire at-risk population . Mapping of implementation units in
Sudan is in progress ( WHO, 2006 ) .
2-5 Lymphatic filariasis in Sudan
Lymphatic filariasis (LF) is evidently endemic in Sudan based on
previous published data ( Satti & Abdel Nur 1974 ) and un published data of
scattered spot surveys and hospital records . Of the 25 Sudanese states, 12
states are considered LF-endemic areas . The disease is focally endemic in
all southern states ( Upper Nile, Unity, Jongli, Eastern Equatoria, Bahr Al
Jabal, Western Equatoria, El Buheirat, Warab, Western Bahr El Ghazal, and
Northern Bahr El Ghazal ) ; Darfur and Blue Nile states in central Sudan . In
addition, five more states ( Sinnar, Gedaref, Northern, El Gezira and
Khartoum ) which are suspected to be endemic of LF . However, since no
recent systematic epidemiological surveys have been done, other states
cannot be considered free of Lymphatic filariasis ( Elsetouhy & Ramzy 2003
) . Maping of Lymphatic filariasis-endemic in Sudan continued in 6 states in
2005 . A total of 141 administrative units in 33 localities were surveyed , of
which 86 were found to be endemic for the disease (WHO, 2006 ) .
2-6 Lymphatic filariasis transmission
Lymphatic filariasis ( LF ) , also known as elephantiasis , is a major
disease of tropical and subtropical regions worldwide . Lymphatic filariasis
is a mosquito-born parasitic disease caused by three nematode worms of the
family Filariidae : Wuchereria bancrofti , Brugia malayi and B. timori .
Wuchereria bancrofti is responsible for 90% of worldwide infections , with
9% caused by B. malayi in southeast and eastern Asia, whereas 1% result
from infection with B. timori in the Pacific region ( Michael and Bundy
1997 ) .
Bancroftian filariasis is caused primarily by adult worms ( known
as macrofilariae ) that live in the lymphatic vessels . Female worms release
embryonic microfilariae (MF) that in many endemic areas are characterized
by nocturnal periodicity and thus circulate in the peripheral blood at night
(21.00-02.00) . The disease is transmitted by Anopheles, Culex and to a
lesser extent by Aedes and Mansonia mosquito species. When mosquito
vectors feed on infected subjects, they ingest MF along with their blood
meal . In the vector, MF develop into infective larvae within 10-15 days .
Mosquitoes transmit the infection from person to another . Infective larvae
inter the human skin through the wound made by the biting mosquito, moult
and develop into adult worms in the affected lymphatic vessels, causing
severe distortion of the lymphatic system . Adult Wuchereria are often
lodged in the lymphatic of the spermatic cord, causing scrotal damage and
swelling . Elephantiasis (painful, disfiguring swelling of the limbs) is a
classic sign of late-stage diseas ( Elsetouhy and Ramzy 2003 )
2-7 Important species of mosquitoes in Sudan
Malaria is transmitted from one person to another by the female mosquito of
the genus Anopheles . There are many species of Anopheles in different
parts of the world but only a relatively small proportion of them are
important as vectors ( WHO 1992 , WHO 1994 ). Several species of
Anopheles are involved in malaria transmission in tropical and subtropical
Africa ( Macdonald 1957; Gilles and De Meillon 1968; Brayan1979 ) .
Anopheles gambiae ( Gilles ) and Anopheles arabiensis are
simultaneously distributed over about 70% of sub-Sahara Africa ( Brayan,
1979 ; Service, 1980 ) . Anopheles arabiensis is the only member of the An.
gambiae Comblex that has so far been identified in many parts of the
Ethiopian Zoogeographical region ( Zahar, 1974 ) . In the Sudan An.
arabiensis is the main malaria vector and is generally known to be
anthropophilic ( Haridi, 1972 ; Akood, 1980 ; Elsafi, 1992 ; Elimam, 1997 ) .
The mosquito, Culex quinquefaciatus Say , is one of the potential
vectors of Wuchereria bancrofti , the causative agent of human lymphatic
filariasis all over the world ( Ana et al, 1997 ; Palley et al, 1995 ; Pedersen
et al, 1999 ; Ramaiah et al, 2003) . Lymphatic filariasis also known as
elephantiasis, is transmitted in Africa mainly by female mosquitoes of the
Anopheles and Culex quinquefaciatus species ( WHO, 2004 ) .
Culex quinquefaciatus is widely distributed in tropical and
subtropical areas . It was more predominant in Lagos ( Nigeria ) area and in
Zanzibar ( Tanzania ) constituting 90% and 95% of the species present in
these area respectively (Pedersen et al, 1999; Oduola and Awe, 2006) .
Culex quinquefaciatus and Aedes aegypti are important species of the genera
Culex and Aedes mosquitoes in Sudan ( Rathor, 2000 ). Culex
quinquefaciatus is the most common species of mosquitoes in Khartoum (
Sudan ) area (Elamin Elrayah & Nawal, 1983 ) . Culex quinquefaciatus is a
domestic mosquito and over 50% of adults rest in the day-time on
nonsprayable surfaces in the house, such as mosquito nets, hanging clothes,
and furniture so that interior treatment with insecticides is of limited use,
particularly when the insecticide involved has low volatile or fumigant
properties . Furthermore the adults of Culex quinquefaciatus have developed
high level of resistance to organochlorine insecticides, and to some
organophosphrous ones, larviciding is now the principle method of control
for most species of Culex, especially in urban and semi urban areas ( WHO
1984 ) .
2- 8 Life cycle of the mosquitoes .
Mosquitoes have four different stages in their life cycle : the egg , larva ,
pupa and adult . The time taken for the various stages to develop depends on
the water temperature and other factors ; this time period is shorter at higher
temperatures ( WHO, 1992; 1994 ).
Eggs :
A female mosquito normally mates only once in its lifetime . Usually after
mating, it requires a blood meal before the eggs can develop . A blood meal
is generally taken every two to three days , before the next batch of eggs is
laid . Anophelines lay about 100 to 150 eggs separately on the surface of
water in each batch of eggs, and each egg has lateral air floats to keep it float
. Culicines of the genus Culex lay several eggs cemented together as an eggs
raft on the water, whereas those of the genus Aedes are laid separately often
on dry hollows or containers which become flooded after rain. The eggs of
many Aedes species are able to retain their viability without water for long
periods ( WHO, 1992; 1994; 2002 ) . The oviposition sites vary from small
amounts of water in hoof-prints and rain pools to larger bodies of water such
as streams, swamps , canals , rivers , ponds , lakes and rice fields . Each
species of mosquito prefers different kinds of water surfaces on which to lay
eggs . A female mosquito continues to lay eggs throughout its lifetime .
Most females will lay between one and three egg batches during their life,
though some may lay as many as 5 to 7 batches . Under the best conditions
in the tropics, the average lifespan of female anopheline mosquitoes is about
3 to 4 weeks( WHO, 1992; 1994; 2002 ) .
Larvae :
Eggs of mosquitoes generally hatch after two or three days in contact with
water . Some transient pool or flood water species e.g., Aedes may hatch
within one-half hour of submersion in water . The larva of most species is
about 1.5 mm long when newly hatched and about 10 mm when fully grown
. The larvae cast their skins four times, as they mature and finally become
pupae . The larva of mosquito has a head , thorax , and abdomen, the later
have eighth distinct segments . A mosquito larva breathes through a pair of
orifices at the terminal end of the body called spiracles ; those of the
anophiline larvae are situated on the eighth abdominal segment so that in
breathing it rests in horizontal position at the surface of the water (parallel
under the water surface just below it , as it needs to breath air )
In culicine larva , the spiracle are situated at the end of tabular organ ,
called the siphon , which extend from the eighth abdominal segment . The
culicine larva hangs down from the water surface by the tip of its siphon in
order to breath . The larvae feed by taking up food from the water . When
disturbed, the larva quickly swims towards the bottom but soon needs to
return to the surface to breath .
There are four larval stages or instars . The small larva emerging from
the egg is called the first instar . After one to two days it sheds its skin and
becomes the second instar, followed by the third and fourth instars at further
intervals of about two days each . The larva remains in the fourth instar stage
for three or four days before changing to a pupa . The total time spent in the
larval stage is generally eight to ten days at normal tropical water
temperatures . At lower temperatures, the aquatic stages take longer time to
develop ( WHO 1988 ; 1992 ; 1994 ; 2002 ) .
Pupae :
The pupa is a stage during which a major transformation takes place, from
living on water to become a flying adult mosquito . The pupa is comma
shaped . It stays under the surface and swims down when disturbed , it is a
none feeding stage . Breathing is carried out , at the surface of the water , by
a pair of respiratory trumpets extending from the thoracic area . The pupal
stage lasts for 2 to 3 days after which the skin of the pupa splits , the adult
mosquito emerge and rests temporarily on the water surface until it is able to
fly .
Adult :
Mating takes place soon after the adults emerge from the pupa . The female
usually mates only once because it receives sufficient sperms for all
subsequent egg batches from a single mating . Normally the female takes its
first blood meal only after mating , but sometimes the first blood meal can
be taken by young virgin females . The first batch of eggs develops often
after two blood meals, while successive batches usually require only one
blood meal ( WHO, 1988 ; WHO, 1992 ; WHO, 1994 ; WHO, 2002 ) .
The egg-adult life-cycle of the malaria mosquito takes from two to four
weeks depending on temperature, An. maculipennis develops in 30 days at
mean daily temperature of 16 to 19 °C ,18 days at 20 to 22 °C and 14 days at
24 to 27 °C ( Kassirsky and Poltnikov 1969 ) .
The thermal death point of An. gambiae larvae is about 42 °C and that
of An. funestus is 40 °C and the lower limit of larval activity is 16.5°C . An.
gambiae develops rapidly at the temperature of about 37 °C ( Jepson et al

1947 ) .
In the Sudan the duration of the egg-adult life-cycle of An.
arabiensis under field conditions , was observed to vary from 8 to 21 days
depending on seasonal temperature ( Omer 1968 ) . A maximum of 24 days
for the life cycle of An. arabiensis had been reported in winter time in
northern Sudan ( Dukeen 1981 ; Dukeen and Omer 1986 ) . In Culex
quinquefaciatus survival from egg-adult emergence was highest (85–90%)
in the range of temperature from 20- 30 °C and dropped drastically ( 38% )
at 15 °C . In Ades aegypti survival to adult stage was high ( 90-92%) at
temperature from 20 – 27 °C , and lowest ( 3% ) at 15 °C ( Ruedal et al 1990

).
2-9 Mosquito bionomics

Climatic factors play an important role in species distribution,
behavior, survival, and vectorial capacity . Water is an essential component
of the mosquito environment . The characteristics of the water habitat,
whether it is running or standing, clean or polluted, shaded or sunlit,
permanent or intermittent, is the predominant factor determining which
species of mosquito breed in it . The environment of the immature stages
and the adult mosquito are interdependent, since the adult mosquito must
have access to water for egg laying ( WHO, 1988 ).
Rate of growth of the immature stages of the mosquito depends in
part on the temperature of the water. This range is lower for species living in
temperate rather than in tropical zones and varies somewhat between
different species living in the same geographical zone, thus temperature is
one of the limiting factors for geographical distribution of a species ( WHO,
1988 ) .
2-10 The breeding sites of mosquitoes .
2-10-1 Anopheles Species :
Each species of mosquito prefers a particular kind of water surface to
lay its eggs ( WHO, 1992 ; WHO, 1994 ) . Many studies had shown that , the
larval habitats of An. arabiensis were varied . Dukeen ( 1981 ) reported that
the under ground source of water ( matara and wells) in the northern
Sudan was the most important breeding site during the period of the Nile
flood which washes away the old breeding sites . Shallow wells, hoof prints
and house hold storage tanks were reported as breeding sites for An.
arabiensis ( Omer 1968 ; Omer and Cloudsly-Thampson 1970 ; Elkhawam
1989 ) . The main breeding sites for An. arabiensis in the Gezira , central
Sudan , were abu-eshreens and the smaller ditches (abu-sittas) , temporary
pools created by rain water during the rainy season and seepage pools
forming along the side of canals and from overflow ( Elsafi, 1983 ) . In
Khartoum-north , Shambat area the main breeding sits for An. arabiensis
were man made pools for brick making, pools made by the receding of the
Nile water , rain pools, irrigation canals , seepage from water pipes and
cement , metal water storage containers ( Elimam, 1997 ) .
2-10-2 Culex Species :
Culex species breed in a large variety of stagnant waters, ranging from
artificial containers and catchments basins of drainage systems to large
bodies of permanent water . The most common species, Culex
quinquefaciatus, a major nuisance and vector of bancroftian filariasis breeds
in water specially polluted with organic material, such as refuse and excreta
or rotting plants . Examples of such breeding sites are soak away pits, septic
tanks, pit latrines, blocked drains, canals and abandoned wells . In many
developing countries Culex quinquefaciatus is common in rapidly expanding
urban areas where drainage and sanitation are inadequate ( Rozendaal, 1997)
. Culex mosquitoes can breed in almost any kind of water collection ( WHO,
1984 ) . The breeding of Culex quinquefaciatus was recorded in canals,
pools, paddy and marsh in rice cultivation area in Kenya ( Benjamin et al
2006 ) . The land use land cover change contributed to changes in
abundance, and distribution of Culex quinquefaciatus larval habitats . There
was higher preponderance of aquatic habitats positive for Culex
quinquefaciatus larvae in land use land cover change sites than non land use
land cover changes ( Benjamin et al 2006 ) . In Khartoum the water coolers
provide excellent breeding sites for both Culex quinquefaciatus and An.
arabiensis during the dry winter season ( Abdelaal et al 2006 ) .
2-11 Mosquito Larval control .
Controlling mosquitoes in the larval stage can be accomplished by two
methods , source reduction and larviciding . Source reduction is a means of
permanent control that involves physically modifying , improving the
quality , or removing a water source for larval mosquito development .
Larviciding involves the application of pesticides to the water where
mosquitoes are developing to kill mosquito larvae (stomach toxins, contact
pesticides) or prevent them from emerging as adults (surface control agents,
insect growth regulators, and natural agents), ( Rutledge, et al 2003 ) .
2-11-1 Source reduction .
The term source reduction refers to any measure that prevent the breeding of
mosquitoes or eliminates their breeding sites . If such measures are long-
lasting or cause permanent changes on land , water or vegetation ,they are
referred to as environmental modification (e.g. Filling, drainage, closing or
covering breeding sites). When such measures have temporary effect and
need to be repeated, they are known as environmental manipulation
(e.g. water level fluctuation, intermittent irrigation, flushing, changing water
salinity, clearing vegetation in streams, irrigation canals etc) ( WHO, 2002 )
.
2-11-2 Larviciding .
Larviciding includes the use of chemicals or biological agents toxins to kill
mosquito larvae and pupae or prevent them from emerging as adults .
Larvicides are used on breeding sites that can not be drained or filled and
where other source reduction would be too expensive or difficult ( WHO,
2002 ; Rozendaal, 1997 ) .
2-11-2-1 Chemical control .
Chemicals have been used successfully as mosquito larvicides . Petroleum
oil was widely used before the commercialization of DDT .The discovery in
the 1940 of the organochlorine insecticides led to abandonment in most
places of traditional mosquito control methods and the adoption of the
spraying of breeding sites with the new compounds . In the course of the
1950 the organochlorine insecticides lost most of their effectiveness in many
places as a result of the development of resistance by some mosquito species
. It also emerged that the organochlorines were very persistent in the soil and
in tissues of plants and animals ( Rozendaal, 1997 ) .
Due to uncontrolled use for several decades , DDT , probably the
best known and most useful insecticide in the world , has damaged wildlife
and might have negative effects on human health . Due to its stability and its
capacity to accumulate in adipose tissue , it is found in human tissues , and
there is no single living organism on the planet that does not contain DDT
now . The possible contribution of DDT to increasing the risks for cancers
at various sites and its possible role as an endocrine disruptor deserve
further investigation . The presence and persistence of DDT and its
metabolites worldwide are still problems of great relevance to public health (
Valdimir et al, 2002 ) . These insecticides are no longer recommended by
WHO for the control of mosquito larvae , although with the exception of
dieldrin they can still be used safely for spraying walls in houses . DDT
was followed by several organophosphorus compounds, the carbamates and
the pyrethroids are less persistent, breaking down quickly in the
environment, they are therefore recommended as larvicides . However, the
pyrethroides are very toxic to fish and should not be used where there are
fish or crustaceans . Water contamination with these larvicides is temporary
and most of the chemicals disappear from water within a day, although the
organophosphorus compounds may persist much longer . Among the most
commonly used larvicides are the organophosphorus compounds, such as
temephos , fenthion and chlorpyrifos ( Rozendaal, 1997 ) .
The Organophosphate insecticides are most widely used despite
increasing levels of resistance in some areas . Temephos which has a very
low mammalian toxicity has been the most widely used mosquito larvicide
world wide ( WHO, 2002 ) .
The change from one insecticide to another has been increasingly
guided by epidemiological impact following detection of resistance . In
situations where mosquitoes have developed resistance to all the
conventional larvicides, consideration may be given to using larvicidal oils,
the more expensive insect growth regulators, or bacterial larvicides as
alternatives ( Rozendaal 1997).
Insect growth regulator are compounds that are highly toxic to
mosquito larvae by preventing their development into adults . Their use has
generally been limited by their high cost . Insect growth regulator can be
divided into:(a) Juvenile hormone analogues, which prevent the
development of larvae into viable pupae or of pupae into adults (they do not
kill larvae) e.g. Methoprene ; and (b) chitin synthesis inhibitors, which
interferes with the moulting process, killing the larvae when they moult, e.g.
Diflubenzuron ( WHO, 2002 ) . They have very low toxicity to mammals,
birds, fish and adult insects, but are highly toxic to crustaceans and
immature stages of aquatic insects . They break down rapidly in the
environment but they may last between several weeks and several months
when applied as granules, microcapsules or briquettes . Their use is limited
by their high cost and restricted availability, but they may be of particular
interest where target insects have developed resistance to the
organophosphorus larvicides or where these compounds cannot be used
because of their effect on the environment .
The main disadvantage of chemical larvicides are toxicity to non
target organisms in the environment, including the natural enemies of
mosquito larvae, and to humans ; consequently training in technique and
safety precautions is necessary for those who apply them ( Rozendaal, 1997
) . Chemical methods of control, within the present major classes, have also
been extended by the finding that while resistance to one particular
organophosphorus insecticide may confer resistance to some others in the
same class it does not necessarily imply resistance to all
organophosphorus insecticides ( WHO, 1984 ) .
If chemical larvicides were used intensively, resistance to these
compounds might develop in mosquito vector . Chemical larvicides also
may create environmental problems if they are lethal to non-target species
( Shililu, 2001 ) .
2-11-2-2 Biological control
Biological control is the use of living organisms or their products to
control vector and pest insects . The organisms used include viruses,
bacteria, protozoa, fungi, plants, parasitic warms, predatory mosquitoes and
fish ( Rozendaal, 1997 ) .
Insect vectors of human diseases are subject to diseases of their
own caused by viruses, bacteria, fungi, protozoan, and nematodes . Over the
past 30 years, many members of these groups have been evaluated as vector
control agents, particularly for mosquito control . Of these, the only one
considered an operational success is the bacterium, Bacillus thuringiensis
israelensis ( Bti ), which has proven useful for control of mosquito and
black fly larvae in programs where larviciding has been traditionally
employed as vector control tactic . The reason for the success of Bti are its
cost effectiveness and relative ease of use, which are due, respectively, to
ability of Bti to be grown on artificial media and the development of
formulations that can be applied using conventional insecticide application
technology . Because few microbial insecticides are cost effective, and
those that are only effective against larvae, these agents will likely play only
a minor, but in some cases important, role in most future vector control
programs ( Federici, 1995 ) .
At present the principal biological control agents that have been
successfully employed against anopheles are predators, particularly fish, and
the bacterial pathogens Bacillus thuringiensis israelensis (Bti ) and Bacillus
sphaericus ( Bs ) that attack the larval stages of the mosquito ( Das and
Amalraj, 1997 ) .
2-11-2-2-1 Larvivorous fish :
Larvivorous fish feed on mosquito larvae . some of the most successful
species have been introduced in different countries are the top minnow or
mosquito fish (Gambusia affinis ) and the guppy ( Poecilia reticulate ) .
Gambusia is more efficient in clean water, while Poecilia can be used
successfully in organically polluted water (WHO, 2002 ) .
The two main factors determining the efficacy of the fish are the
suitability of the fish species to the water bodies where the vector species
breed and the ability of the fish to eat enough larvae to significantly reduce
the number of infective bites that people receive ( Kathleen, 2002 ) . Also
the use of pesticides and fertilizers can negatively impact fish stocked in
irrigated fields ( Lacey and Lacey, 1990 ) .
2-11-2-2-2 Bacterial larvicides :

To meet the challenges of vector resistance to chemical larvicides and
environmental safety, the National Malaria control program in Eritrea under
took an evaluation of two alternative bacterial larvicides ( Shililu, 2001 ) .
Two different species of bacteria of the genus Bacillus, Bacillus
thuringiensis israelensis (Bti) and B. sphaericus (Bs), have been widely
demonstrated to be effective larvicides against both anopheline and other
mosquito species . Both Bti and Bs function as stomach poisons in the
mosquito larva midgut . Since the discovery of the mosquito larvicidal
activity of Bti spores (serotype H-14) in 1977, different formulations of Bti
have been found effective against larvae of many mosquito species
( Das and Amalraj, 1997 ; Lacey and Lacey, 1990 ) .
The bacterium Bti (serotype H-14 ) produces toxins which are very
effective in killing mosquito and black fly larvae after ingestion . At normal
dosages it is harmless to other insects, fish, higher animals and human and is
suitable for use in water used for drinking or for the irrigation of food crops .
It is effective against insects that have developed resistance to chemical
larvicides ( Rozendaal, 1997 ; WHO, 2002 ) . It breaks down quickly
in the environment and must be reapplied periodically . The product contains
mainly dead bacteria, living spores and toxic crystals in the spores which do
not multiply, and it could therefore be considered as biologically produced
insecticide .
Another bacterium, Bacillus sphaericus ( Bs ) also produces toxin . It
has characteristics similar to those of Bti but is more effective in polluted
water while Bti is more effective in clean water ( Rozendaal 1997 ; WHO
2002 ) . It is not effective against black flies or Aedes aegypti . Unlike Bti it
is produced as formulation containing living bacteria that multiply even in
polluted water . Bs usually has longer action than Bti . It is considered very
suitable for the treatment of breeding sites of Culex mosquitoes in polluted
water . It has a higher residual effectiveness in such habitats than most other
larvicides and offer the added advantage of safety to non target organisms
and lack of resistance . This method is still being developed but some
products have already reached the market ( Rozendaal, 1997 ) .
There are many factors affecting the efficacy of Bti and Bs ;
Susceptibility to Bt of mosquito larvae is affected by feeding behavior and
nutritional value of the available food . Reduced mortality is attributed to
feeding inhibition and dilution of the pathogen in the presence of nutritional
and inert particles, which limit the amount of ingested toxin ( Ben-Dov et
al, 2003 ) . Many environmental factors can reduce the efficacy or effective
life span of Bti and Bs products . Natural breakdown or inactivation
processes are accelerated by heat, ultraviolet light, and water with high
organic matter (Consoli et al, 1995 ; Lacey and Lacey, 1990 ).
2-12 Malaria vectors control in Sudan :
DDT was introduced last century in the fifties but by the early
seventies, resistance to DDT was reported in irrigation areas. Nevertheless
DDT is still being applied in parts of the country whenever it is still effective
at a dose of 2gm/m² of internal house walls surfaces . Malathion replaced
DDT at a dose of 2gm/m² as a residual insecticide .The vector ( Anopheles
arabiensis ) developed resistance against Malathion in some irrigated areas
and by 1979 it was virtually confirmed in many parts of the Sudan . Another
organophosphorous compound, Fentrothion, came into use with a residual
effect of about 12 weeks . The main disadvantage of this insecticide is that it
is highly toxic and more expensive as compared to the others . The best anti
larval under use now is Abate (Temphos, an organophosphorous compound )
which is very effective and quite safe ( Abd Elnur and Dukeen, 1992 ) .
From 1975 to 1980 malaria was put under control through an annual
round of house spraying with malathion . Resistance to this compound by
adult Anopheles arabiensis was first detected in 1978 . Fenitrothion was
used since 1981 and is still a practical alternative ( Elgaddal et al, 1985 ;
Hemingway, 1983 ) .
In Khartoum State two rounds of house spraying were planned to cover

the months of high transmission during the rainy season and the rise and fall
of the Nile : Ultra low volume space spraying with vehicle mounted
applicators and knapsak sprayers was planned to be implemented daily
between 6 - 8 a.m for eight months with a mixture of d-allethrin and d-
phenothrin (Pesguard) . Larviciding was used to cover all breeding sites on a
weekly basis, by Abate larvicide ( Malaria control programme for
Khartoum State 1991 ) .
2-13 Plants Mosquitocides :

2-13-1 Urge and advantages of using plant mosquitocides
The problem of high cost and development of resistance in many mosquito
vector species to several of the synthetic insecticides have revived interest in
exploiting the pest control potential of plants ( Mittal and Subbarao 2003;
Singh et al 2006; Sivagnaname and Kalyanasundaram 2004 ) . In addition to
application as general toxicants against mosquito larvae, plant insecticides
also have potential uses as growth and reproduction inhibitors, repellents,
and ovipossition deterrents ( Prajapati et al 2005 ; Rajkumar and Jebanesan,
2005a ; 2005b ; Pushpanathan et al, 2006 ) . The botanical insecticide are
generally pest specific and are relatively harmless to non target organism
including man . They are also biodegradable and harmless to environment .
One plant species may possess substances with a wide range of activities, for
example extract from the neem tree Azadirachta indica showed antifeedant,
antioviposition, repellent and growth-regulating activities ( Schmutterrer,
1990 ) . Plant products can be used, either as insecticides for killing larvae or
adult mosquitoes or as repellent for protection against mosquito bites ,
depending on the type of activity they possess . A large number of plant
extracts have been reported to have mosquitocidal or repellent activity
against mosquito vectors ( Sukumar et al, 1991 ), but very few plant
products have shown practical utility for mosquito control . Plant product
can be obtained either from the whole plant or from specific part by
extraction with different types of solvents such as water, methanol,
choloroform, hexane, etc., depending on the polarity of the phytochemicals (
Mittal and Subbarao, 2003 ) .
2-14 Some plants mosquitocides
Some indigenous plant are very promising against mosquitoes and can be
used as insecticides and/or repellents . These plants are probable sources of
some biologically active agents for mosquito control in the future .
During the last decade, various studies on natural plant products
against mosquito vectors indicate them as possible alternatives to synthetic
chemical insecticides . However, more concerted efforts have to go into
these studies to make these environment friendly compounds viable for field
use and for large-scale vector control operations .
Zaridah et al, ( 2006 ) reported that extracts from about 30 species of
plant were tested for their ability to kill the larvae or to repel or knock down
the adult of Ades agypti the mosquito vector for dengue . Of these plants
three species showed high larvicidal activity, and two species are very
effective for repelling against adult mosquitoes, where knock down ability
was best with two other species .
A preliminary study revealed that extracts of 14 species (
collected from southern part of Thailand ) from 112 medicinal plant tested
showed evidence of larvicidal activity . Eight out of 14 plant species showed
100% mosquito larvae mortality ( Promsiri et al 2006 ) . Of 51
species of plant from State of Oaxaca, Mexico, evaluated for toxicity against
Culex quinquefaciatus larvae, three species of plant presented the greatest
larvicidal action as water and acetone extracts ( Rafael et al 2004 ).
Many studies indicated that, plant mosquitocides have potential uses
as larvicidal, growth regulator inhibitors, ovicidal , repellents, and
ovipossition deterrent .
2-14-1 larvicidal activity
The leaf extract of Centella asiatica plant is promising as larvicides and
adult emergence inhibitor against Culex quinquefaciatus and might be used
directly in small volume aquatic habitats or breeding sites of limited size
around human dwellings . The toxicity of this extract increased with
temperatures, the 50% medium lethal concentration ( LC50 ) ranged
between 6.84 ppm at 19 °C and 1.12 ppm at 31 °C . A similar trend was

observed for the LC90 which varied from 9.12 to 3.36 ppm at two
temperatures respectively ( Rajkumar and Jebanesan 2005 ) .
Methanolic extract of the leafs of Atlantia monophyla was found to be
effective against early fourth instars larvae and pupae of Culex
quinquefaciatus and Aedes aegypti and pupae of Anopheles stephensi (
Sivagnaname and Kalyanasundaram, 2004 ) .
Several species of plants have demonstrated toxic effects on
mosquitoes .
The essential oils of the plant Ipomoea cairica Linn. possess

remarkable larvicidal properties as it could induce 100% mortality in the
larvae of Culex tritaeniorhynchus ( 100 ppm ), Aedes aegypti (120ppm ),
Anopheles stephensi( 120 ppm ), and Culex quinquefaciatus ( 170 ppm )
mosquitoes . The LC50 and LC90 values estimated for these mosquitoes
were 14.8 and 78.3, 22.3 and 92.7, 14.9 and 109.9, and 58.9 and 161.6 ppm,
respectively ( Thekkevilayil et al, 2004 ) .
The leaf extract of Pavonia zeylanica and Acacia ferruginea showed
larval mortality at LC50 of 2214.7 and 5362.6 ppm respectively against the
third instars larvae of Culex quinquefaciatus after 24 hours treatment (
Vahitha et al, 2002 ) .
The leaf extracts of five species of Cucurbitacious plants,
Momordica charantia , Trichosanthes anguina , Luffa acutangula,
Benincasa cerifera and Citrullus vulgaris showed larval mortality after 24
hours at LC50 of 465.85, 567.81, 839.81, 1189.30 and 1636.04 ppm
repectively against the third instars larvae of Culex quinquefaciatus (
Prabakar and Jebanesan, 2004 ) . The hexane extract of Momordica
charantia Linn (Cucurbitaceae) fruits showed more potent larvicidal activity
than the crude extract . The LC50 values of hexane extract against fourth
instar larvae of Anopheles stephensi, Culex quinquefaciatus, and Aedes
aegypti were 66.05, 96.11, and 122.45 ppm, respectively ( Singh et al, 2006
) .
The potential larvicidal activity of three selected indigenous

medicinal plants indicate that Thevetia peruviana was the most potent,
followed by Pueraria mirifica, and Butea superba was the least effective . In
all cases, the late 3rd instar was more susceptible than the early 4th instar
larvae, and the 48 hours exposure yielded more potent larvicidal activity
than 24 hours exposure ( Lapcharoen et al, 2005 ) .
Water extracts of nine medicinal plants against larvae of Culex
quinquefaciatus Say and Aedes aegypti (L.) indicated that the plant Piper
retrofractum Vahi ( Piperaceae ) among these plant showed the highest level
of activity against mosquito larvae (Chansang et al, 2005 ) . Essential oils
extracted from dried leaves of three spontaneous plants naturally growing in
Burkina Faso, Cymbopogon proximus, Lippia multiflora and Ocimum
canum, exhibited larvicidal activity against 3rd and 4th instar larvae of Field
collected mosquitoes vectors of human disease, namely Aedes agypti and
members of the Anopheles gambiae complex, An. arabiensis and An.
gambiae . The median lethal concentration ( LC50 ) for Ae. aegypti and
An. gambiae S. L. larvae ranged between 53.5 – 258.5 ppm and 61.9 –
301.6 ppm , respectively . The LC90 estimates ranged between 74.8 – 334.8
ppm for Ae. aegypti and 121.6 – 582.9 ppm for An. gambiae S. L (
Bassole et al, 2003 ) .
Ethanol extracts of 83 plants species belonging to the Asteraceae (
Compositae ) family, collected in the State of Minas Gerais, Brazil, were
tested for larvicidal activity against the mosquito Aedes fluviatilis ( Diptera
: Culicidae ) . The result show that the extract from Tagetes minuta was the
most active with a LC90 of 1.5 mg/L and LC50 of 1.0 mg/L . This plant has
been the object of several studies by other groups and its active components
have already been identified as thiophene derivatives, a class of compounds
present in many Asteraceae species . The extract of Eclipta paniculata was
also significantly active with a LC90 of 17.2 mg/L and LC50 of 3.3 mg/L .
The extract of other plants were less active ( Macedo et al, 1997 ) .
Earlier studies with a common medicinal plant of Calotrpis
procera ( Asclepiadaceae ) deal with the fraction of the latex produced by
the green parts of the plant . The whole latex of Calotrpis procera was
shown to cause 100% mortality of 3rd instar larvae of Aedes aegypti within
five minutes ( Marcio et al, 2006 ) . The methanolic extract
from leaves of milkweed (Calotrpis procera ) showed larvicidal properties
against mosquito larvae of Anopheles stephensi, Aedes aegypti and Culex
quinquefaciatus ( Singh et al, 2005 ) .
Preliminary evaluation of larvicidal activity of aqueous extracts from
leaves of Ricinus communis L. and from wood of Tetraclinis articulata
(Vahl ) Mast. on larvae of four mosquito species , Culex pipiens (Linne),
Aedes caspius (Pallas), Culiseta longiareolata (Aitken) and Anopheles
maculipennis (Meigen), after 24 hours exposition showed strong toxic
activity against larvae of four mosquito species ( Brahim et al 2006 ) .
2-14-2 Adult emergence inhibition activity

The ethanolic extract of leaf of Centella asiatica ( Umbelliferae ) plant is
promising as adult emergence inhibitor against Culex quinquefaciatus . The
adult emergence inhibition (EI ) activity of this extract at LC50 of different
temperature was generally more pronounced in increased temperature (
Rajkumar and Jebanesan, 2005 ) .
Insect growth regulating activity of methanolic extract of leafs of
Atlantia monophylla ( Rutaceae ) was more pronounced against Aedes
aegypti with EI50 value of 0.002 mg/L . The extract was found safe to
aquatic mosquito predators Gambusia affinis with LC50 value of 23.4 mg/L
( Sivagnaname and Kalyanasundaram 2004 ) .
The potential insect growth regulator ( IGR ) properties of three
selected indigenous medicinal Thailand plants , indicated that Thevetia
peruviana did not show any IGR properties ; whereas Pueraria mirifica
and Butea superba seemed to exhibit the juvenile hormone type activity
which resulted in abnormal death at various stage of development . Butea
superba was more promising than Pueraria mirifica , and Aedes aegypti was
about twice more susceptible than Culex quinquefaciatus . In addition 3rd
instar was always more susceptible than 4th instar with both mosquito
species ( Lapcharoen et al, 2005 ) .
2-14-3 Oviposition deterrent activity
The acetone leaf extract of Solanum trilobatum ( Solanaceae ) was tested
under laboratory conditions for ovipostion deterrent and skin repellent
activities against the adult mosquito Anopheles stephensi. The result
suggested an effective ovipostion deterrent and skin repellent activities (
Rajkumar and Jebanesan, 2005 ) .
The latex of Calotrpis procera has shown a remarkable effect as a
larvicide, against Aedes aegypti, however at 0.7% concentration of latex , the
oviposition was avoided by the gravid female mosquitoes in the treated
plates and the same concentration was observed to be ovicidal (Manju et al,
2004; Marcio et al, 2006 ) .
2-14-4 Eggs hatching activity
Essential oils extracted from Cymbopogan citratus plant showed larvicidal
, ovicidal, and repellent activity against the filarial tranmitting mosquito,
Culex quinquefaciatus . Hundred percent ovicidal activity was observed at
300 ppm ( Pushpanathan et al 2006 ) .
Essential oils extracted from 10 medicinal plants were evaluated for
larvicidal , adulticidal , ovicidal , oviposition deterrent and repellent
activities against three mosquito species ; Anopheles stephensi , Ades agypti
and Culex quinquefaciatus . From these plants the essential oils of
Juniperus macropoda and Pimpinella anisum were highly effective as both
larvicidal and ovicidal . Essential oils of Zingiber officinale was found to be
ovicidal against the three mosquito species (Prajapati et al, 2005) .
Essential oils extracted from dried leaves of three spontaneous plants
naturally growing in Burkina Faso, Cymbopogon proximus , Lippia
multiflora and Ocimum canum, exhibited ovicidal activity against eggs of
Anopheles gambiae S. L. The LC50 values for Anopheles gambiae S. L.
eggs ranged between 17.1 - 188.7 ppm, while LC90 values ranged between
33.5 – 488 ppm . Lippia multiflora showed the highest activity against
Anopheles gambiae S. L eggs and Aedes aegypti larvae . Of the three plants,
essential oils from Ocimum canum had the lowest activity against both eggs
and larvae . Eggs were more susceptible than larvae ( Bassole et al, 2003 ) .
CHAPTER THREE
MATERIAL AND METHODS
3-1 The study area :

The selected area for this study ( Shambat Village ) lies in the western part
of Khartoum North town, on eastern bank of the River Nile between latitude
15.40N and longitude 32.32E . It is bound by the river Nile from the west,
Kober ( Omer El Mukhtar ) and industrial area from the east and Halfaya
from the north . The period of the study was between June 2005 to
September 2007 .
3-2 Plants used :
The following plants were collected from within the study area ( the
plants were abundant in the area round the Faculty of Agriculture,
University of Khartoum ) . They were identified at the university of
Khartoum, Faculty of Education, Department of Biology .
3-2-1 Calotropis procera (Ait) . Ushar
Family : Asclepiadaceae
Tomentose woody shrubs or small trees . Leaves opposite, pale green,
sessile, obovate-elliptical, tomentose . Inflorescences Sub-umbellate cymes ;
flowers purple ; calyx 5, imbricate, free ; corolla campanulate with 5 lobes ,
greenish white outside, tips purple in side : corona hairy ; ovary of 2 free
carples ; carple 1-locular ; ovules numerous with marginal placentation .
Fruit follicles, green, subglobose; seeds brown with long white sikly hairs .
Latex is used against scorpion bites ( Abusam 1998 ) .
3-2-2 Ricinus communis L. Khirwi
Family : Euphorbiaceae
Glabrous erect shrubs . Leaves alternate , orbicular-peltate , deeply
palmately lobed , lobes 7 – 9, dark green ; stipules large . Inflorescences
axillary , erect , large panicles ; flowers unisexual ; calyx 3-5 , united ;
corolla absent ; stamens much branched, united , numerous . Fruit capsules ,
spiny , oval , 3-valved ; seeds oblong , dark brown , smooth , mottled with
black , rich in oil .
Seeds oil used as laxative ( Abusam 1998 ) .
3-2-3 Euphorbia hirta L. Umlebien
Family : Euphorbiaceae
Annual hairy herbs . Leaves opposite , lanceolate or oblong, reddish beneath
, pubescent on both surfaces . Inflorescences axillary and terminal , dense
globose heads or umbellate or capitate; flowers purple ; ovary angled ; seeds
reddish , oblong minute ( Abusam 1998 ) .
3-2-4 Sonchus oleraceous . Molita
Family : Compositae
Erect herbs . Leaves crowded near the base of the stem, sessile, oblanceolate
or oblong . Inflorescences terminal heads, in lax racemes ; Flowers yellow ;
achenes transversely rugose ( Abusam, 1998 ) .
3-2-5 Eclipta prostrata . Tamr Elganam

Family : Compositae
Erect or decumbent annual or biennial herb ; leaves shortly petiolate,
narrowly elliptic, base tapering, margin serrate ; capitula hemispheric,
stalked, ray florets in conspicuous, white ; disc florets white ; occurring
naturally around pans and flood plains, preferring damp or swampy
situation, often a weed of irrigation schemes ( Burrows and Willis, 2005
).
3-3 Preparation of stock solutions .
Fully developed leaves of the plants , Ricinus communis, Khirwi and
Euphorbia hirita, Umlebien ( Euphorbiaceae ), Calotropis procera, Ushar,
(Ascelpiadaceae), Eclipta prostrata, Tamr Elganam and Sonchus oleraceus ,
Molita (Compositae ) were included . They were collected during the
flowering season of the plants , dried under shade and finely ground to
powder . Each plant powder was kept in plastic bag and labeled .
Five grams from the powder leaves of each plant was soaked in
separate plastic bottle ( 500ml ) containing 250ml distilled water . Each
solution was allowed to stand for 24 hours with vigorous occasional shaking
, the suspension of each was filtered with filter paper (Whatman2 ) . The
marc was washed several times with distilled water and filtered . The final
volume was adjusted to 500ml by adding distilled water to prepare stock
solution of 1% .
3-4 Mosquito rearing :

Larvae of the mosquito were collected from breeding sites within the study
area, and reared under laboratory condition in the laboratory of the
Department of Preventive Medicine, Faculty of Veterinary Medicine,
Khartoum university, at 25 – 28°C . The larvae were fed once daily by
adding finely ground powdered yeast on the surface of the water . Water
was changed every day to avoid scum formation; which might create toxicity
. Pupae were collected daily, and transferred to small bowls containing clean
water . The bowls were placed in net cage 30×30×30 cm for adult
emergence ( plate 3-1 ) .
Adult mosquito were kept in standard 30x30x30 cm net cages ( plate
3.1 ) . From the day of emergence, Adult mosquitoes were provided with
cotton soaked with a 10% sugar solution as a carbohydrate source . On the
third day post emergence from pupae ( this period was enough for mating )
the female mosquitoes were fed on pigeons for at least 10 hours during the
night . On the following day, Petri-dishes provided with moist cotton or
filter paper were fitted at the bottom of each cage for oviposition . The
females will lay eggs within 48 hours . To rear larvae for toxicity assays
single egg rafts were placed in a number of 2 liter plates ( 30 cm diameter
) containing 1liter of de chlorinated tap water . After the egg hatching
larvae were fed once daily by adding powdered yeast ( finely ground ) and
the water was changed every day . Pupae were collected daily, and
transferred to small bowls containing clean water . The bowls were placed in
net cages 30x30x30 cm for adult emergence ( plate 3-1 ) . The life cycle
continued as mentioned above .
3-5 Preparation of test concentration :
The volume of stock solution was 500ml of 1% , obtained by weighing
5gms from the powder leaves of each plant and adding 500ml of distilled
water . The stock solution was then serially diluted as follows :
To prepare a concentration of ( 5000 ppm ) 50 ml from stock
solution were Added to 50 ml of dechlorinated tap water . Thirty ml of
stock solution were added to 70 ml of dechlorinated tap water to prepare a
concentration of ( 3000 ppm ) . Then 12, 10, 8, 6, 4 and 2 ml of each stock
solution were separately completed to 100 ml ( in 250ml plastic cup ) by
adding de chlorinated tap water, the dose of test concentration obtained
were 1200, 1000, 800, 600, 400 and 200 ppm respectively.
Plate 3-1 : Cage for mosquito rearing
3-6 Bioassay procedure :
3-6-1 Larvicidal and pupicidal activity
Larvicidal and pupicidal activities of water extract of plants mentioned
above were determined by following the WHO standard procedure (
WHO, 2005 ) . Initially, the mosquito larvae were exposed to a wide range
of test concentrations and a control to find out the activity range of the water
extract of plants under test . After determining the mortality of larvae in this
wide range of concentrations, a narrower range of 4-6 concentrations,
yielding between 10% and 95% mortality in 24 hours was used, to determine
the lethal concentration of 50% ( LC50 ) and the lethal concentration of 90%
( LC90 ) values .
Twenty-five laboratory reared second , third and fourth instars
larvae , and twenty-five pupae of vector mosquito species were transferred
by means of droppers to the small test cups ( 250 ml plastic cups ) ( plate 3-
2 ), each containing 100ml of de chlorinated tap water to which the required
concentration were added . Small, unhealthy, or damaged larvae were
removed and replaced. Four replicates were setup for each test concentration
. In each replicate 25 larvae were used, with four replicate of control
The experiment was performed under laboratory conditions at 25-28
C° . To determine pupicidal activity , the mouth of each cup containing

pupae was covered with mosquito net to prevent the escape of any emerged
adult mosquitoes . Mortality in larvae and pupae was recorded 24 hours post
treatment . Moribund larvae were counted and added to dead larvae for
calculating percentage mortality . Dead larvae were those that cannot be
induced to move when they were probed with a needle in the siphon or the
cervical region . Moribund larvae were those incapable of rising to the
surface or not showing the characteristic diving reaction when the water is
disturbed .
If more than 10% of the control larvae pupate in the course of the
experiment, the test is discarded and repeated . If the control mortality is
between 5% and 20% , the mortalities of treated groups should be corrected
according to Abbott`s (1925) formula :
X-Y 100
Mortality (%) =
X
Where X = percentage survival in the untreated control .

and Y = percentage survival in the treated sample .
3-6-1-1 Data analysis
Data from all replicates were pooled for analysis . LC50 and LC90 values
were calculated from a log dosage-probit mortality regression line using
computer software programs , or estimated using log-probit paper .
Plate 3-2 : The Bioassay cup
3-6-2 Adult emergence inhibition activity
Water extracts of plant leaves were also tested for adult emergence
inhibition ( EI ) activity against the two mosquito species . Twenty-five third
instar larvae of each mosquito species were transferred by a dropper to the
test cups ( plate 3.2 ), each containing 100 ml of dechlorinated tap water to
which the required concentration were added . The plant extract was tested
at a range of five to six concentrations . Four replicates were setup for each
test concentration . Each replicate was used for 25 larvae, with four
replicates of control . Because of the long duration of the test the larvae
were provided with small amount of finely ground yeast extract as a nutrient
source, at concentration of 10 mg/l at two day intervals until mortality
counts were made . The yeast powder was prepared as stock suspension in
water from which one or two drops added per cup . The larvae in the control
were fed in the same manner as those in the treated batches . All the treated
and control cups containing pupae were kept separately in the net cage to
prevent successfully emerged adults from escaping into the environment .
Mortality of the larvae and pupae was recorded at 24 hours intervals .
Observation was continued in treated and control cups until the complete
emergence of adults . The test cups were held at 25-28 °C . At the end of
observation period, the impact is expressed as EI% based on the number of
larvae that do not develop successfully into viable adults . In recording EI%
for each concentration, moribund and dead larvae and pupae, as well as adult
mosquitoes not completely separated from the pupal case, were considered
as dead . The experiment stop when all the larvae or pupae in the controls
have died or emerged as adults .
3-6-2-1 Data analysis
The data from all replicates of each concentration were combined . Total or
mean emergence inhibition can be calculated on the bases of the number of
third stage larvae exposed . The overall emergence of adults reflects activity
. EI% is calculated using the following formula :
T × 100
EI (%) = 100 -
C
Where T = percentage survival or emergence in treated batches and C =

percentage survival or emergence in the control .
If adult emergence in the control is less than 80% , the test was discarded
and repeated . Where the percentage is between 80% and 95% , the data are
corrected using Abbott's formula ( see section 3-6-1) . EI values obtained at
each concentration should be subjected to probit regression analysis to
determine EI50 and EI90 values ( using computer software programs or
estimated from log-probit paper ) . The data analysis procedures stated in
section 3-6-1-1 were followed .
3-6-3 Oviposition deterrent activity

The oviposition deterrent test was performed using the method of Xue
et al, (2001) which has been used by Rajkumar & Jebanesan (2005) . Five
cages were designed and placed side by side A, B, C, D and E for each
bioassay . Fifteen gravid female of Anopheles arabiensis and Culex
quinquefaciatus were ( 2 days after blood feeding ) transferred to each
mosquito cage 30 × 30 × 30 cm ( plate 3.1 ) . A 10% sucrose solution was
available at all times . The concentration of leaf extract of of Ricinus
communis and Calotrpis procera which showed the highest, moderate and
lowest mortality in the larvicidal activity (against 3rd instar) were prepared ,
from each concentration 100 ml were taken and put in the test cup in cage A,
B and C . Three test cups ( plate 3-2 ) each containing 100 ml of de
chlorinated tap water were prepared and put in cage A, B and C on the
opposite place of the treated cup as control . The positions of the cups were
alternated between the different replicates so as to nullify any effect of
position on oviposition . In cage D all the experimental concentration ( high ,
moderate and low ) were placed without control . While in cage E two cups
of control were placed without any treated cup . Three replicates for each
concentration were run . All experiments were run at room temperature ( 25-
28 °C ) . After 48 hours, the number of eggs laid in treated and control cups
was recorded .
The percent effective repellency for each plant leaf extract

concentration was calculated using the following formula :
NC - NT
ER ( % ) = × 100
NC
Where ER = percent effective repellency ; NC = number of eggs in control ;

and NT = number of eggs in treatment .
In the case of Anopheles arabiensis the same experiment was conducted
except that the test cup was replaced by petridshes with filter paper in the
bottom .
3-6-4 Ovicidal activity
The method of Su & Mulla ( 1998 ) and Pushpanathan et al (2006 ) was
followed for the ovicidal activity . Hundred freshly laid eggs of Culex
quinquefaciatus were exposed to five concentrations of leaf extract of
Ricinus communis , and Calotrpis procera in de chlorinated tap water . Each
concentration was replicated three times . De chlorinated tap water served as
control . The hatching rate was assessed 5 days post treatment by the
following formula .
Number of hatched larvae

The hatch rate = × 100
Total number of eggs in treated water
The LC50 , LC90 values were calculated from a log dosage-probit mortality
regression line ( method 3-6-1-1) .
3-7 Statistical Analysis

Dosage-mortality curves are the subject matter of an entire field of
biometric analysis and bioassay is one of the technique used in this field .
The results of such analysis are plotted on probit paper . A regression line is
fitted to dosage-mortality data graphed on probit paper .
For the present study, probit data were computed using a programme
especially designed for this type of analysis .The regression analysis data
obtained the following :
Firstly the regression line equation , Y = a + bx where :
Y = the calculated ( empirical ) probit .
a = the intercept ( the intersection of the regression line with
the vertical axis when x equal zero ) .
b = the slope ( the tangent of the regression line made with the
horizontal axis ) .
x = log-concentration producing Y probit .
Secondly the standard error of X-coefficient ( SE – X ) .
Thirdly the standard error of Y-coefficient ( SE – Y ) .
Fourthly regression coefficient ( r² ) .
The data obtained were presented in tabular form in terms of
concentration ( ppm and log- concentration ) , mortality ( total and
percentage mortality ) , and probit ( tabulated and calculated ) . Each table
contained the following statistics : The regression line equation , ( SE – X ) ,
( SE – Y ), the lethal concentration that kills 50% of the population ( LC50 )
, the lethal concentration that kills 90% of the population ( LC90 ), the
fiducial limits with 95% confidence limits ( F. L. with 95% C.L. ) to
determine the upper and lower limit of action , and the regression
coefficient ( r² ) to show the homogeneity between concentration and
mortality was recorded in each table .
CHAPTER FOUR
RESULTS
4-1– Larvicidal and pupicidal activity of leaves extract of Calotropis

procera against Anopheles arabiensis and Culex quinquefasciatus .
The larvicidal activity of leaf extract of Calotropis procera against 2nd , 3rd
and 4th instar larvae of each of the selected mosquitoes species was studied
by exposing each instar of each mosquitoes species to at least five
concentration of the extract , the mortality was recorded after 24 hours .
The water leaf extract of Calotropis procera showed high level of
toxicity against the mosquitoes Anopheles arabiensis and Culex
quinquefasciatus larvae . The results are presented in tables (4-1) , (4-2) , (4-
3) , (4-4) , (4-5) , (4-6) , figure (4.1) and figure (4.2) as a toxicity against 2nd
, 3rd and 4th instar larvae of the selected mosquitoes species respectively,
and summarized in table ( 4 - 7 ) and figure ( 4.3 ) as LC50 and LC90
values .
The results showed that , the 50% mortality (LC50 values ) was
shown at 273.53 , 366.44 and 454.99 ppm for 2nd , 3rd and 4th instar larvae
respectively of Anopheles arabiensis and 187.93 , 218.27 and 264.85 ppm
for 2nd , 3rd and 4th instar larvae respectively of Culex quinquefasciatus as it
was shown in figure ( 4.3 ) .
The LC90 values ( 90% mortality ) were shown at 783.43 , 1018.59
and 1224.62 ppm for 2nd , 3rd and 4th instar larvae respectively of Anopheles
arabiensis and 433.51 , 538.27 and 769.13 ppm for 2nd , 3rd and 4th instar
larvae respectively of Culex quinquefasciatus . as it is shown in table ( 4- 7
) and figure ( 4.3 ).
From LC50 and LC90 values it was evident that 2nd instars were more
susceptible than 3rd and 4th instar , the higher concentration was required for
3rd and 4th instars of the two species of mosquitoes and 3rd instar was more
susceptible than 4th instar . The susceptibility of 2nd , 3rd and 4th istars of
Anopheles arabiensis and Culex quinquefasciatus to the extract of
Calotropis procera was shown in figure (4.1) and figure (4.2) . Also the two
species of selected mosquitoes larvae showed different susceptibility to the
leaf extract of Calotropis procera, higher concentration was required for
Anopheles arabiensis and lower concentration for Culex quinquefasciatus ,
with LC50 of 273.53 and 187.93 ppm respectively for the 2nd instars of the
two mosquitoes species and LC 90 of 783.43 and 433.51 ppm respectively,
this is clear in table (4-7) and figure (4.3) . Culex quinquefasciatus was more
susceptible than An. arabiensis to the leaf extract of Calotropis procera .
The leaf extract of Calotropis procera did not show any pupicidal
activity at higher concentration of ( 10000 ppm ) against the two species of
mosquitoes .
Table (4 – 1 ) : Susceptibility of larvae of Anopheles arabiensis exposed as

second larval instar to different concentration of leaves extract of Calotropis
procera after 24 hours .
Concentration Mortality Probit

( ppm ) Log Total (%) Tabulated Calculated
Control - 0 0 - -
100 2.000 10 10 3.72 3.78
200 2.301 40 40 4.75 4.62
400 2.602 65 65 5.39 5.46
600 2.778 82 82 5.92 5.96
800 2.903 91 91 6.34 6.31
1000 3.000 100 100 - -
Number of larvae tested for each concentration = 100

Regression equation : Y = 2.799X – 1.820
SE – X = 0.131
SE – Y = 0.334
LC 50 = 273.53 ppm
LC 90 = 783.43 ppm
F. L with 95% C. L. = +- 2.675
r² = 0.997

third larval instar to different concentration of leaves extract of Calotropis

Control - 0 0 - -
200 2.301 22 22 4.23 4.24
400 2.602 54 54 5.10 5.12
600 2.778 76 76 5.71 5.62
800 2.903 81 81 5.88 5.98
1000 3.000 90 90 6.28 6.26
1200 3.079 100 100 - -

SE – X = 0.144
SE – Y = 0.392
LC 50 = 366.44 ppm
LC 90 = 1018.59 ppm
F. L with 95% C. L. = +- 2.335
r² = 0.993

fourth larval instar to different concentration of leaves extract of Calotropis

Control - 0 0 - -
200 2.301 16 16 4.01 3.94
400 2.602 45 45 4.87 4.83
600 2.778 60 60 5.25 5.36
800 2.903 75 75 5.67 5.73
1000 3.000 80 80 5.84 6.02
1200 3.079 88 88 6.18 6.25
1400 3.146 96 96 6.75 6.45

SE – X = 0.235
SE – Y = 0.669
LC 50 = 454.99 ppm
LC 90 = 1224.62 ppm
F. L with 95% C. L. = +- 2.400
r² = 0.970
Figure 4.1 Larvicidal activity of leaves extract of calotropis procera against

2nd, 3rd and 4th instars larvae of Anopheles arabiensis expressed as linear
regression .
7.0
6.5
6.0
Probit of mortality
5.5
5.0
4.5
4.0
3.5
3.0
1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2
Log (dose)
▲ second larval instar : Y = 2.799X – 1.820
□ third larval instar : Y = 2.883X – 2.393
О fourth larval instar : Y = 2.977X – 2.913

Table (4 – 4 ) : Susceptibility of larvae of Culex quinquefasciatus exposed
as second larval instar to different concentration of leaves extract of
Calotropis procera after 24 hours .

Control 0 0 0 - -
100 2.000 20 20 4.16 4.03
200 2.301 52 52 5.05 5.09
300 2.477 70 70 5.52 5.71
400 2.602 86 86 6.08 6.16
500 2.699 92 92 6.41 6.50
600 2.778 98 98 7.05 6.78

SE – X = 0.295
SE – Y = 0.735
LC 50 = 187.93 ppm
LC 90 = 433.51 ppm
F. L with 95% C. L. = +- 2.372
r² = 0.973
as third larval instar to different concentration of leaves extract of Calotropis

Control 0 0 0 - -
100 2.000 14 14 3.92 3.90
200 2.301 48 48 4.95 4.88
400 2.602 76 76 5.71 5.86
600 2.778 90 90 6.28 6.43
800 2.903 98 98 7.05 6.84

SE – X = 0.243
SE – Y = 0.617
LC 50 = 218.27 ppm
LC 90 = 538.27 ppm
F. L with 95% C. L. = +- 2.675
r² = 0.984
as fourth larval instar to different concentration of leaves extract of
Calotropis procera after 24 hours .

Control 0 0 0 - -
100 2 11 11 3.77 3.83
200 2.301 40 40 4.75 4.66
400 2.602 69 69 5.51 5.49
600 2.778 82 82 5.92 5.98
800 2.903 91 91 6.34 6.33
1000 3 100 100 - -

Regression equation : Y = 2.77x – 1.713
SE – X = 0.098
SE – Y = 0.248
LC 50 = 264.85
LC 90 = 769.13
F. L with 95% C. L. = +- 2.675
r² = 0.996
2nd, 3rd and 4th instars larvae of Culex quinquefasciatus expressed as linear
regression .
7.5
7.0
6.5
Probit of mortality
6.0
5.5
5.0
4.5
4.0
3.5
1.8 2.0 2.2 2.4 2.6 2.8 3.0
Log (dose)
О fourth larval instar : Y = 2.77x – 1.713

Table (4 – 7 ) : Larvicidal activity of leaves extract of Calotropis procera
against 2nd, 3rd and 4th instars larvae of Anopheles arabiensis and Culex
quinquefasciatus expressed as LC50 and LC90 .
Species of Larva LC50 LC90

mosquitoes 1nstar ppm ppm Regresstion equation
II 273.53 783.43 Y= 2.799X - 1.820

Anopheles
arabiensis III 366.44 1018.59 Y= 2.883X - 2.393
IV 454.99 1224.62 Y= 2.977 - 2.913
II 187.93 433.51 Y= 3.528X - 3.024

Culex
quinquefas. III 218.27 538.27 Y= 3.261X - 2.626
264.85
IV 769.13 Y= 2.77X - 1.713
2nd, 3rd and 4th instars larvae of Anopheles arabiensis and Culex
1400
1224.62
1200
Concentration of plant extract (ppm)
1018.59
1000
769.13 783.43
800
600
538.27
454.99 433.51
366.44
400
264.85 273.53
218.27 187.93
200
Culex Anophoeles Culex Anophoeles Culex Anophoeles
4th instar 3rd instar 2nd instar

Species of mosquito and larval instars
LC50 LC90
4-2- Larvicidal and pupicidal activity of leaves extract of Ricinus

communis against Anopheles arabiensis and Culex quinquefasciatus .
The larvicidal activity of leaf extract of Ricinus communis against 2nd , 3rd
and 4th instar larvae of each of the selected mosquitoes species was studied
by exposing each instar of each mosquito species to at least five
concentrations of the extract , the mortality was recorded after 24 hours .
The water leaf extract of Ricinus communis showed high level of
toxicity against the mosquitoes Anopheles arabiensis and Culex
quinquefasciatus larvae . The results are presented in tables (4 – 8), (4 – 9 ),
(4 – 10 ), (4 – 11 ), (4 – 12 ), ( 4 – 13 ), figure ( 4.4 ) and figure ( 4.5 ) as
a toxicity against 2nd , 3rd and 4th instar larvae of the selected mosquitoes
species, and summarized in table (4 –14) and figure ( 4.6 ) as LC50 and
LC90 values .
The results showed that , the 50% mortality (LC50 values ) was
shown at 403.65 , 445.66 and 498.88 ppm for 2nd , 3rd and 4th instar larvae
respectively of Anopheles arabiensis and 1091.44 , 1364.58 and 1445.44
ppm for 2nd , 3rd and 4th instar respectively of Culex quinquefasciatus .
The LC90 values ( 90% mortality ) were shown at 920.45 , 1114.29
and 1364.58 ppm for 2nd , 3rd and 4th instar larvae respectively of Anopheles
arabiensis and 1753.88 , 2046.44 and 2187.76 ppm for 2nd , 3rd and 4th
instars larvae respectively of Culex quinquefasciatus , as it is shown in table
(4 – 14 ) and figure ( 4.6 ) .
susceptible than 3rd and 4th instar, the higher concentration was required for
3rd and 4th instars of the two species of mosquitoes, and 3rd instar was more
susceptible than 4th instar . The susceptibility of 2nd, 3rd and 4th larval instar
of Anopheles arabiensis and Culex quinquefasciatus to the leaves extract of
Ricinus communis was shown in figure ( 4.1 ) and figure ( 4.2 ) . Also the
two species of selected mosquitoes larvae showed different susceptibility to
the leaf extract of Ricinus communis , higher concentration was required for
Culex quinquefasciatus and lower concentration for Anopheles arabiensis
as it is shown in table (4 – 14) and figure ( 4.6 ) . It was observed that An.
arabiensis was more susceptible than Culex quinquefasciatus to the leaf
extract of Ricinus communis .
The leaf extract of Ricinus communis did not show any pupicidal
activity at higher concentration of ( 10000 ppm ) against the two species of
mosquitoes studded .
The overall results was that the leaf extract of Calotropis procera and
Ricinus communis showed larvicidal potentialities in controlling , An.
Arabiensis and Culex quinquefasciatus, with the observation on that
Calotropis procera was more potent than Ricinus communis against the two
species of mosquitoes tested, with different activity of the plant extract
against different mosquito species . Both plant extracts did not show any
pupicidal activity against the two species of mosquitoes .
4-3 – Larvicidal and pupicidal activity of leaves extract of Eclipta

prostrata , Sonchus oleraceus and Euphorbia hirita against Anopheles
arabiensis and Culex quinquefasciatus .
The larvicidal activity of the water leaves extracts of Eclipta prostrate

, Sonchus oleraceus and Euphorbia hirita were studied against the 3rd larval
instar of the two species of mosquitoes .
The three species of plant above did not show any larvicidal activity
against Anopheles arabiensis and Culex quinquefasciatus at higher
concentration of ( 10000 ppm ) after 24 hours .
second larval instar to different concentration of leaves extract of Ricinus
communis after 24 hours .

Control 0 0 0 - -
200 2.301 18 18 4.08 3.91
400 2.602 46 46 4.90 4.99
600 2.778 68 68 5.47 5.62
800 2.903 80 80 5.84 6.06
1000 3.000 91 91 6.34 6.41
1200 3.079 98 98 7.05 6.69

SE – X = 0.381
SE – Y = 1.062
LC 50 = 403.65
LC 90 = 920.45
F. L with 95% C. L. = +- 2.373
r² = 0.957

third larval instar to different concentration of leaves extract of Ricinus

Control 0 0 0 - -
200 2.301 16 16 4.01 3.88
400 2.602 41 41 4.77 4.85
600 2.778 62 62 5.31 5.42
800 2.903 75 75 5.67 5.82
1000 3.000 86 86 6.08 6.13
1200 3.079 95 95 6.64 6.38

SE – X = 0.274
SE – Y = 0.763
LC 50 = 445.66 ppm
LC 90 = 1114.29 ppm
F. L with 95% C. L. = +- 2.373
r² = 0.972
Table (4 – 10 ) : Susceptibility of larvae of Anopheles arabiensis exposed

as fourth larval instar to different concentration of leaves extract of Ricinus

Control 0 0 0 - -
200 2.301 15 15 3.96 3.83
400 2.602 34 34 4.59 4.72
600 2.778 56 56 5.15 5.23
800 2.903 70 70 5.52 5.60
1000 3.000 81 81 5.88 5.89
1200 3.079 90 90 6.28 6.12
SE – X = 0.209
SE – Y = 0.582
LC 50 = 498.88 ppm
LC 90 = 1364.58
F. L with 95% C. L. = +- 2.373
r² = 0.980
Figure 4.4 Larvicidal activity of leaves extract of Ricinus communis against

2nd, 3rd and 4th instars larvae of Anopheles arabiensis expressed as linear
regression .
7.5
7.0
6.5
Probit of mortality
6.0
5.5
5.0
4.5
4.0
3.5
2.2 2.4 2.6 2.8 3.0 3.2
Log (dose)
▲ second larval instar : Y = 3.572x – 4.308
□ third larval instar : Y = 3.217x – 3.521

as second larval instar to different concentration of leaves extract of Ricinus
Control 0 0 0 - -
800 2.903 24 24 4.29 4.16
1000 3.000 36 36 4.64 4.77
1200 3.079 58 58 5.20 5.26
1400 3.146 72 72 5.58 5.67
1600 3.204 88 88 6.18 6.03
1800 3.255 100 100 - -

SE – X = 0.620
SE – Y = 1.904
LC 50 = 1091.44
LC 90 = 1753.88 ppm
F. L with 95% C. L. = +- 2.024
r² = 0.971

as third larval instar to different concentration of leaves extract of Ricinus
Control - 0 0 - -
1000 3.000 20 20 4.16 4.01
1200 3.079 30 30 4.48 4.59
1400 3.146 49 49 4.97 5.08
1600 3.204 68 68 5.47 5.50
1800 3.255 80 80 5.84 5.87
2000 3.301 91 91 6.34 6.21

SE – X = 0.506
SE – Y = 1.601
LC 50 = 1364.58 ppm
LC 90 = 2046.44 ppm
F. L with 95% C. L. = +- 2.020
r² = 0.981

as fourth larval instar to different concentration of leaves extract of Ricinus

Control - 0 0 - -
1000 3.000 16 16 4.01 3.87
1200 3.079 24 24 4.29 4.43
1400 3.146 43 43 4.82 4.90
1600 3.204 63 63 5.33 5.32
1800 3.255 75 75 5.67 5.68
2000 3.301 86 86 6.08 6.00

SE – X = 0.456
SE – Y = 1.444
LC 50 = 1445.44 ppm
LC 90 = 2187.76 ppm
F. L with 95% C. L. = +- 2.020
r² = 0.984

2nd, 3rd and 4th instars larvae of Culex quinquefasciatus expressed as linear
regression .
6.5
6.0
Probit of mortality
5.5
5.0
4.5
4.0
3.5
2.90 2.95 3.00 3.05 3.10 3.15 3.20 3.25 3.30 3.35
Log (dose)
Table (4 – 14 ) : Larvicidal activity of leaves extract of Ricinus communis

against 2nd, 3rd and 4th instars larvae of Anopheles arabiensis and Culex
Species of Larvae LC50 LC90
mosquitoes instar ppm ppm Regression equation
II 403.65 920.45 Y= 3.572X – 4.308

Anopheles
arabiensis III 445.66 1114.29 Y= 3.217X – 3.521
IV 498.88 1364.58 Y= 2.935X – 2.920
II 1091.44 1753.88 Y= 6.208X – 13.858

Culex
quinquefas. III 1364.58 2046.44 Y= 7.308X – 17.914
IV 1445.44 2187.76 Y= 7.101X - 17.436

2nd, 3rd and 4th instars larvae of Anopheles arabiensis and Culex
2500
2187.76
2046.44
2000
1753.88
1445.44
1500 1364.58 1364.58
1114.29 1091.44
920.45
1000
489.88 445.66
403.65
500
0
Culex Anophoeles Culex Anophoeles Culex Anophoeles
4th instar 3rd instar 2nd instar

Species of m osquito and larval instars
LC50 LC90
4-4- The adult emergence inhibition activity of leaves extract of

Calotropis procera against 3rd larval instar of Anopheles arabiensis and
Culex quinquefsciatus .
The adult emergence inhibition of Anopheles arabiensis by
Calotropis procera leaves extract presented in table (4 – 15 ) and figure ( 4.7
) . Hundred percent emergence inhibition was shown at 1000 ppm , fifty
percent emergence inhibition ( EI 50% ) was shown at 277.90 ppm and ( EI
90% ) was shown at 677.64 ppm .
The adult emergence inhibition of Culex quinquefsciatus by
Calotropis procera leaves extract presented in table (4 – 16 ) and figure ( 4.8
) . Fifty percent emergence inhibition ( EI 50% ) was shown at 183.65 ppm .
The EI 90% was shown at 453.94 ppm. The EI50 and EI90 of Anopheles
arabiensis and Culex quinquefsciatus by Calotropis procera was shown in
table ( 4 – 19 ) and figure ( 4.11 ) . Therefore, it was shown that the
leaf extract of Calotropis procera also act as adult emergence inhibitions
against the two species of mosquito studied . Also higher concentration was
required for Anopheles arabiensis and lower concentration for Culex
quinquefsciatus as the same as it was shown in the larvicidal activity . Also
it was evident that EI50 and EI90 values for the two species of mosquitoes
studied was less than LC50 and LC90 for the 3rd larval instar of the same
mosquitoes species, with EI50 – EI90 of 277.90 – 677.60 ppm for
Anopheles arabiensis and 183.65 – 453.94 ppm for Culex quinquefsciatus as
it shown in table ( 4 – 19 ) and figure ( 4.11 ), and the LC50 – LC90 of
366.44 – 1018.59 ppm for Anopheles arabiensis and 218.27 – 538.27 ppm
for Culex quinquefsciatus . as it is shown in table (4 – 7) and figure ( 4.3 ) .
This indicated that lower concentration of the leaf extract was required for
the adult emergence inhibition than larvicidal . This also reflects the activity
of Calotropis procera leaves extract as possible insect growth regulator
against the two species of mosquito .
Table (4 – 15 ) : The adult emergence inhibition activity of leaves extract of
Calotropis procera against 3rd larval instar of Anopheles arabiensis at
different concentration .
Concentration Mortality Survival or EI Probit

emergence
(ppm) Log Total (%) Total (%) (%) Tabu. Calcu.
Control - 0 0 100 100 0 - -
200 2.301 30 30 65 65 35 4.61 4.53
400 2.602 55 55 35 35 65 5.39 5.52
600 2.778 80 80 15 15 85 6.04 6.11

2.903 86 86 5 5 95 6.64 6.52
800
1000 3.000 100 100 0 0 100 - -
EI = Emergence inhibition of adult

SE – X = 0.326
SE – Y = 0.866
EI 50 = 277.90 ppm
EI 90 = 677.641 ppm
F. L with 95% C. L. = +- 2.292
r² = 0.981
Figure 4.7 : The adult emergence inhibition activity of leaves extract of

Calotropis procera against 3rd larval instar of Anopheles arabiensis at
different concentration , expressed as linear regression .
7.0
6.5
probit of mortality
6.0
5.5
5.0
4.5
2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0
Log (dose)
Y = 3.307X – 3.081

Calotropis procera against 3rd larval instar of Culex quinquefsciatus at

emergence
Control - 0 0 100 100 0 - -
100 2.000 15 15 82 82 18 4.08 4.14
200 2.301 51 51 41 41 59 5.23 5.12
300 2.477 75 75 24 24 76 5.71 5.69
400 2.602 85 85 15 15 85 6.04 6.10
500 2.699 92 92 8 8 92 6.41 6.42
600 2.778 100 100 0 0 100 - -

SE – X = 0.147
SE – Y = 0.356
EI 50 = 183.65 ppm
EI 90 = 453.94
F. L with 95% C. L. = +- 2.335
r² = 0.994
Calotropis procera against 3rd larval instar of Culex quinquefsciatus at
different concentration, expressed as linear regression .
6.5
6.0
Probit of mortality
5.5
5.0
4.5
4.0
1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8
Log (dose)
Y = 3.258X – 2.376
4-5- The adult emergence inhibition activity of leaves extract of Ricinus

communis against 3rd larval instar of Anopheles arabiensis and Culex
quinquefsciatus .
The adult emergence inhibition of Anopheles arabiensis by Ricinus
communis leaves extract are presented in table (4 – 17 ) and figure ( 4.9 ) .
Hundred percent emergence inhibition was shown at 1200 ppm, fifty percent
emergence inhibition ( EI 50% ) was shown at 374.97 ppm and EI 90% was
shown at 979.49 ppm .
The adult emergence inhibition of Culex quinquefsciatus by Ricinus
communis leaves extract are presented in table (4 – 18 ) and figure ( 4.10 ) .
Fifty percent emergence inhibition ( EI 50% ) was shown at 1180.32 ppm .
The EI 90% was shown at 1849.27 ppm The EI50 and EI90 of Anopheles
arabiensis and Culex quinquefsciatus by Ricinus communis was shown in
table ( 4 – 19 ) and figure ( 4.11 ) . Therefore, it was shown that the leaf
extract of Ricinus communis also acts as adult emergence inhibition against
the two species of mosquito studied . Also higher concentration was required
for Culex quinquefsciatus and lower concentration for Anopheles arabiensis
similar to that shown in the larvicidal activity of the leaf extract . Also it
was evident that EI50 value for the two species of mosquitoes studied was
less than LC50 for the 3rd larval instar of the same mosquito species, with
EI50 of 374.97 for Anopheles arabiensis and 1180.32 ppm for Culex
quinquefsciatus as it shown in table (4 – 19 ) and Figure ( 4.11 ), and the
LC50 was 445.66 ppm for the 3rd larval instar of Anopheles arabiensis and
1364.58 ppm for 3rd larval instar of Culex quinquefsciatus as it was shown
in Table ( 4 – 14 ) and Figure ( 4.6 ) . This indicated that lower
concentration of the leaf extract was required for adult emergence inhibition
than larvicidal . This is also reflects the activity of Ricinus communis leaves
extract to act as a possible insect growth regulator against the two species of
mosquito .
Ricinus communis against 3rd larval instar of Anopheles arabiensis at

emergence
Control - 0 0 100 100 0 - -
200 2.301 25 25 75 75 25 4.33 4.16
400 2.602 40 40 55 55 45 4.87 5.09

600 2.778 62 62 30 30 70 5.52 5.63
800 2.903 76 76 18 18 82 5.92 6.01
1000 3.000 90 90 6 6 94 6.55 6.31
1200 3.079 100 100 0 0 100 - -

SE – X = 0.409
SE – Y = 1.116
EI 50 = 374.97 ppm
EI 90 = 979.49 ppm
F. L with 95% C. L. = +- 2.335
r² = 0.949
Ricinus communis against 3rd larval instar of Anopheles arabiensis at
different concentration, expressed as linear regression.
6.5
6.0
Probit of mortality
5.5
5.0
4.5
4.0
2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1
Log (dose)
Y = 3.071X – 2.904

Ricinus communis against 3rd larval instar of Culex quinquefsciatus at

emergence
Control - 0 0 100 100 0 - -
1000 3.000 32 32 65 65 35 4.61 4.53
1200 3.079 41 41 50 50 50 5.00 5.04
1400 3.146 58 48 34 34 66 5.41 5.49
1600 3.204 73 65 22 22 78 5.77 5.87
1800 3.255 90 90 9 9 91 6.34 6.20
2000 - 100 100 0 0 100 - -

SE – X = 0.596
SE – Y = 1.869
EI 50 = 1180.32 ppm
EI 90 = 1849.27 ppm
F. L with 95% C. L. = +- 2.006
r² = 0.976
Ricinus communis against 3rd larval instar of Culex quinquefsciatus at
different concentrations, expressed as linear regression .
6.4
6.2
6.0
Probit of mortality
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
3.00 3.05 3.10 3.15 3.20 3.25 3.30
Log (dose)
Y = 6.578X – 15.209

Calotropis procera and Ricinus communis against 3rd larval instar of
Anopheles arabiensis and Culex quinquefsciatus expressed as EI50 and EI90
.
Plant sp. Mosqui. EI50 EI90 Regression
sp. ppm ppm equation
An.
Calotropis arabiensis 277.90 677.60 Y=3.307X-3.081
procera Culex
quinquefa. 183.65 453.94 Y=3.258X-2.376
An.
Ricinus arabiensis 374.97 979.49 Y=3.071X-2.904
communis Culex
quinquefa. 1180.32 1849.27 Y=6.578X-15.209
EI = Emergence inhibition of adult .
Figure 4.11: The adult emergence inhibition activity of leaves extract of

Calotropis procera and Ricinus communis against 3rd larval instar of
Anopheles arabiensis and Culex quinquefsciatus expressed as EI50 and EI90
.
2000 1849.27
1800
1600
1400
1180.32
1200 979.49
1000
677.6
800
600 453.94
374.97
400 277.9
183.65
200
0
Culex Anophoeles Culex Anophoeles
Ricinus communis Calotropis procera

mosquito and plant species
EI50 EI90
4-6- The ovicidal activity of leaves extract of Calotropis procera against

Culex quinquefasciatus .
The ovicidal activity of leaves extract of Calotropis procera against
Culex quinquefasciatus was shown in table (4 – 20 ) and figure ( 4 .12 ) .
97% eggs mortality was shown at 600 ppm . The LC50 - LC90 values were
191.87 – 510.51 ppm respectively . The result indicated that Eggs were
more susceptible than 3rd and 4th istar larvae .
4-7- The ovicidal activity of leaves extract of Ricinus communis against
Culex quinquefasciatus .
The ovicidal activity of leaves extract of Ricinus communis against
Culex quinquefasciatus is shown in Table ( 4 – 21 ) and Figure ( 4.13 ) .
The LC50 was shown at 961.612 ppm and the LC90 was 1786.488 ppm .
The result indicated that Eggs were more susceptible than 3rd and 4th instar
larvae . The leaves extract of Calotropis procera was very effective in
preventing eggs hatching than the extract of Ricinus communis . Also the
water leaf extract of both plants were more potent against eggs than larvae .
Table (4 – 20 ) : Ovicidal activity of leaves extract of Calotropis procera

against Culex quinquefsciatus .
Concentration Egg mortality Probit

ppm Log Total (%) Tabulated Calculated
Control - 6 2 - -
100 2.000 73 24.3 4.29 4.15
200 2.301 153 51 5.03 5.05
300 2.477 202 67.3 5.44 5.58
400 2.602 225 75 5.67 5.96
500 2.699 263 87.7 6.18 6.25
600 2.778 292 97.3 6.88 6.49
Number of eggs tested for each concentration = 300

SE – X = 0.413
SE – Y = 1.029
LC 50 = 191.867 ppm
LC 90 = 510.505 ppm
F. L with 95% C. L. = +- 2.373
r² = 0.930
Figure 4.12 : Ovicidal activity of leaves extract of Calotropis procera

against Culex quinquefsciatus expressed as linear regression .
7.0
6.5
Probit of mortality
6.0
5.5
5.0
4.5
4.0
2.0 2.2 2.4 2.6 2.8 3.0
Log (dose)
Y = 3.014X – 1.882
Table (4 – 21 ) : Ovicidal activity of leaves extract of Ricinus communis

against Culex quinquefsciatus .
Concentration Egg mortality Probit

ppm Log Total (%) Tabulated Calculated
Control - 7 2.3 - -
600 2.778 61 20.3 4.16 4.02
800 2.903 97 32.3 4.53 4.62
1000 3.000 153 51 5.03 5.08
1200 3.079 188 62.6 5.33 5.46
1400 3.146 224 74.6 5.67 5.78
1600 3.204 270 90 6.28 6.05
Number of eggs tested for each concentration = 300

SE – X = 0.464
SE – Y = 1.402
LC 50 = 961.612 ppm
LC 90 = 1786.488 ppm
F. L with 95% C. L. = +- 2.076
r² = 0.975
Figure 4.13 : Ovicidal activity of leaves extract of Ricinus communis

against Culex quinquefsciatus expressed as linear regression .
6.5
6.0
Probit of mortality
5.5
5.0
4.5
4.0
2.7 2.8 2.9 3.0 3.1 3.2
Log (dose)
Y = 4.758X – 9.193
4-8- Oviposition deterrent activity of water leaves extract of Calotropis

procera against gravid, female Anopheles arabiensis and Culex
quinquefasciatus .
The oviposition deterrence was tested by using three different concentrations
of the extract that cause high , moderate and low larvae mortality in the
larvicidal experiment .
The water leaf extract of Calotropis procera showed important
observations on oviposition deterrent against the mosquitoes Anopheles
arabiensis and Culex quinquefasciatus at different concentrations of
larvicidal activity .
Table ( 4 – 22 ) show the effect of different concentrations ( high ,
moderate and low larvicidal activity ) of leaf extract of Calotropis procera
on the oviposition deterrence of female Anopheles arabiensis . In
experimental cage A , 280 eggs were laid in the control cup , while in the
corresponding treated cup ( 1000 ppm ) in the same cage no eggs were
laid . A similar observation was shown in cage B and C , 390 and 480 eggs
were laid in the control cup of the cage B and C respectively , and no eggs
were laid in the corresponding treated cup in the same cages B and C . In
cage D where choice of control was not offered , maximum of eggs laying (
250 eggs ) was shown in lowest concentration ( 200 ppm ) , and no eggs
were laid in the highest concentration ( 1000 ppm ) , while in the moderate
larvicidal concentration ( 500 ppm ) , 115 eggs were laid . In cage E where
only control was offered about 550 eggs were laid .
The water leaf extract of Calotropis procera at different
concentrations of larvicidal activity ( high, moderate ,low ) showed hundred
percent oviposition deterrence and hundred percent effective repellence
against Anopheles arabiensis when the extract is to be used as material of
choice (treated – control ) . However, when all the larvicidal concentrations
were offered without control ( choice ) the avoidance of eggs laying
was not shown except in the high larvicidal concentration (1000 ppm), and
the maximum eggs laying was preferred in the low larvicidal concentration
( 200 ppm ) . A similar observation was shown on Culex quinquefasciatus,
with relative difference in that Culex quinquefasciatus showed 90.6%
oviposition deterrent and effective repellency at low larvicidal concentration
( 100 ppm ) . The effect of leaf extract of Calotropis procera at different
concentrations of larvicidal activity (high, moderate, low) on the oviposition
deterrent and repellent activity against Culex quinquefasciatus was shown in
table ( 4 – 23 ) .
Table (4 – 22 ) : Oviposition deterrent activity of leaves extract of

Calotropis procera against gravid , female Anopheles arabiensis
Cage A B C D E
Dose
ppm C 1000 C 500 C 200 1000 500 200 C C
Eggs
laid 280 0 390 0 480 0 0 115 250 290 260
within
48 hrs
ER % 100 100 100
NC - NT
ER ( % ) = × 100
NC
ER = effective repellency
NC = number of eggs in control .
NT = number of eggs in treated .
C = Control

Calotropis procera against gravid, female Culex quinquefasciatus
Cage A B C D E
Dose
ppm C 1000 C 500 C 100 1000 500 100 C C
Eggs
laid 405 0 490 0 520 20 56 173 304 370 410
within
48 hrs
ER % 100 100 90.6
NC - NT
ER ( % ) = × 100
NC
ER = effective repellency
NC = number of eggs in control
NT = number of eggs in treated
C = Control
4-9- Oviposition deterrent activity of water leaves extract of Ricinus

communis against gravid, female Anopheles arabiensis and Culex
quinquefasciatus .
The water leaf extract of Ricinus communis also showed important
observations on the oviposition deterrence against the mosquitoes Anopheles
arabiensis and Culex quinquefasciatus at different concentrations of
larvicidal activity .
Table ( 4 – 24 ) shows the effect of different concentrations ( high,
moderate and low larvicidal activity ) of leaf extract of Ricinus communis on
the oviposition deterrent of female Anopheles arabiensis . In experimental
cage A, 350 eggs were laid in the control cup , while in the corresponding
treated cup ( 1200 ppm ) in the same cage no eggs were laid . Similar
observation was shown in cage B, 410 eggs were laid in the control cup ,
and no eggs were laid in the corresponding treated cup ( 600 ppm ) in the
same cages . In cage C 505 eggs were laid in the control cup, and 25 eggs
were laid in the corresponding treated cup ( 200 ppm ) in the same cage . In
cage D where choice of control was not offered, maximum of eggs laying (
290 eggs ) was shown in the lowest concentration ( 200 ppm ), and
minimum of eggs ( 60 eggs ) were laid in the highest concentration (1200
ppm), while in the moderate larvicidal concentration ( 600 ppm ), 140 eggs
were laid . In cage E where only control was offered about 650 eggs were
laid .
The water leaf extract of Ricinus communis at high (1200 ppm) and
moderate (600 ppm) concentrations of larvicidal activity showed hundred
percent oviposition deterrent and hundred percent effective repellency
against Anopheles arabiensis, while at low concentration of larvicidal
activity ( 200 ppm ) showed 90.5% effective repellency and oviposition
deterrent , when the extract is to be used as material of choice ( treated –
control ) . However when all the larvicidal concentrations were offered
without control ( choice ) the avoidance of eggs laying was not
shown, and the maximum eggs laying was preferred in the low larvicidal
concentration ( 200 ppm ) . Similar observation was shown on Culex
quinquefasciatus . The effect of leaf extract of Ricinus communis at
different concentrations of larvicidal activity ( high, moderate, low) on the
oviposition deterrent and repellent activity against Culex quinquefasciatus
was shown in table ( 4 – 25 ) . It was concluded that the leaf extract of both
plants showed hundred percent oviposition deterrent at low larvicidal
concentration , when there is a choice of control, and when the control was
not offered (the treated water only) the mosquitoes can lay eggs but at the
lowest number in comparison to the number of egg laid in control , when the
treated cup was not offered . Also the reduced number of eggs laid may lose
its viability to hatch, by the effect of the concentration used .

Ricinus communis against gravid , female Anopheles arabiensis
Cage A B C D E
Dose
ppm C 1200 C 600 C 200 1200 600 200 C C
Eggs
laid 350 0 410 0 505 25 60 140 290 310 340
within
48 hrs
ER % 100 100 90.5
NC - NT
ER ( % ) = × 100
NC
ER = percent effective repellency
C = Control

Ricinus communis against gravid, female Culex quinquefasciatus
Cage A B C D E
Dose
ppm C 1600 C 1200 C 600 1600 1200 600 C C
Eggs
laid 345 0 425 0 490 40 45 165 385 350 330
within
48 hrs
ER % 100 100 90.1
NC - NT
ER ( % ) = × 100
NC
ER = percent effective repellency
C = Control
CHAPTER FIVE
Discussion
To day , the environmental safety of an insecticides is considered to be of

paramount importance . An insecticide does not have to cause high mortality
on target organisms in order to be acceptable .
Indigenous plants, may serve as suitable alternatives to synthetic
insecticides in the future as they are relatively safe , inexpensive , and are
readily available in many areas of the world . The screening of locally
available medicinal plants for mosquito control would generate local
employment, reduce dependence on expensive important products and
stimulate local efforts to enhance puplic health .
In this study it was observed that, the crude extract of the leaves of
Calotropis procera and Ricinus communis has been found to possess
larvicidal , adult emergence inhibition , ovicidal and oviposition deterrent
activity against the mosquitoes Anopheles arabiensis and Culex
quinquefasciatus .
The biological activity of these plants extracts may be due to various
compounds . Including phenolics , terpenoides and alkaloids existing in
plants , these compounds may jointly or independently contribute to produce
larvicidal , adult emergence inhibition , ovicidal and oviposition deterrent
activity against the above species of mosquitoes .
The obtained results agree with some previous studies . In addition to
application as general toxicant against mosquito larvae, plants insecticides
also have potential uses as growth and reproduction inhibitors, repellents,
ovicidal and oviposition deterrents ( Prajapati et al, 2005 ; Rajkumar and
Jebanesan 2005a ; 2005b ; Pushpanathan et al, 2006 ) . One plant species
may possess substances with a wide range of activities, e.g. Neem
(Azadirachta indica) products showed anti feedant, oviposition deterrence ,
repellency, growth disruption, sterility and larvicidal action against insects (
Schmutterrer, 1990 ; Mulla and Su, 1999 ) .
The present results showed that the plants Eclipta prostrata, Sonchus
oleraceous and Euphorbia hirta did not show larvicidal activity against the
two species of mosquitoes after 24 hours exposure . This suggests that these
plants do not posses larvicidal properties or that, the active ingredient of
these plants can not be extracted by 24 hours soaking time or may be the
effects of the plants extract on the larvae may appear after more than 24
hours exposure . This is agree with ( Lapcharoen, 2005 ) who tested three
selected indigenous Thai plants for their larvicidal activity and insect growth
regulating properties against two species of mosquitoes . He found that the
48 hours exposure had yielded more potent larvicidal activity than 24 hours
exposure , and with ( Gopiesh and Kannabiran, 2007) they
reported that,The aqueous extract of three plant Hemidesmus indicus roots,
Gymnema sylvesre and Eclipta prostrata leaves were tested against Culex
quinquefasciatus larvae . In all cases the three days exposure yield more
potent larvicidal activity than one day exposure . The water leaves extract of
Eclipta prostrata showed 3.3% larvae mortality at concentration of 1% after
one day exposure and 28.3% mortality at the same concentration after two
days exposure and 36.6% mortality after three days exposure at the same
concentration ( Gopiesh and Kannabiran, 2007) .
In this study, the leaf extract of Calotropis procera and Ricinus
communis showed larvicidal potentialities in controlling , Anopheles
arabiensis and Culex quinquefasciatus . It was concluded that Calotropis
procera was more potent than Ricinus communis against the two species of
above mosquito , with different activity of plant extract against different
mosquito species .
In the case of Calotropis procera The results showed that , the 50%
mortality (LC50 values ) was at 273.53 , 366.44 and 454.99 ppm for 2nd , 3rd
and 4th instar larvae of Anopheles arabiensis respectively and 187.93 ,
218.27 and 264.85 ppm for 2nd , 3rd and 4th instar larvae of Culex
quinquefasciatus respectively .
The LC90 values ( 90% mortality ) were shown at 783.43 , 1018.59 and
1224.62 ppm for 2nd , 3rd and 4th instar larvae of Anopheles arabiensis
respectively and 433.51 , 538.27 and 769.13 ppm for 2nd , 3rd and 4th instar
larvae respectively of Culex quinquefasciatus , as it is shown in table ( 4 –
7 ) .
susceptible than 3rd and 4th instar, the higher concentration was required for
3rd and 4th instars of the two species of mosquitoes and 3rd instar was
susceptible than 4th instar . Also the two species of selected mosquitoes
larvae showed different susceptibilities to the leaf extract of Calotropis
procera . A higher concentration was required for Anopheles arabiensis and
lower concentration for Culex quinquefasciatus , with LC50 of 273.53 and
187.93 ppm respectively for the 2nd larval instars of the two mosquito
species and LC 90 of 783.43 and 433.51ppm respectively see table ( 4 – 7 )
. So it can be said that Culex quinquefasciatus was more susceptible than
An. arabiensis to the leaf extract of Calotropis procera . The leaf extract of
Calotropis procera did not show any pupicidal activity at the high
concentration of (10000 ppm ) against the two species of mosquitoes .
In the case of Ricinus communis The results showed that , the 50%
mortality (LC50 values ) was shown at 403.65 , 445.66 and 498.88 ppm for
2nd , 3rd and 4th instar larvae respectively of Anopheles arabiensis and
1091.44 , 1364.58 and 1445.44 ppm for 2nd, 3rd and 4th larval instar
respectively of Culex quinquefasciatus . The LC90
values ( 90% mortality ) were shown at 920.45, 1114.29 and 1364.58 ppm
for 2nd , 3rd and 4th instar larvae respectively of Anopheles arabiensis and
1753.88 , 2046.44 and 2187.76 ppm for 2nd, 3rd and 4th instars larvae
respectively of Culex quinquefasciatus as it is shown in table ( 4 – 14 ) .
susceptible than 3rd and 4th instar, higher concentrations were required for 3rd
and 4th instars of the two species of mosquitoes, and 3rd instar was more
susceptible than 4th instar . Also the two species of the selected mosquitoes
larvae showed different susceptibilities to the leaf extract of Ricinus
communis , higher concentration was required for Culex quinquefasciatus
and lower concentration for Anopheles arabiensis as it is shown in table (4
– 14 ) . It was concluded that An. arabiensis was more susceptible than
Culex quinquefasciatus to the leaf extract of Ricinus communis .
The leaf extract of Ricinus communis has not shown to exhibit any
pupicidal activity at the high concentration of ( 10000ppm ) against
the two species of mosquitoes .
It was concluded that the leaf extract of Calotropis procera and
Ricinus communis showed larvicidal potentialities in controlling , An.
Arabiensis and Culex quinquefasciatus , with the observation on that
Calotropis procera was more potent than Ricinus communis against the two
species of mosquitoes above , with different activity of the plant extract
against different mosquito species . Both plant extracts did not show any
pupal mortality against the two species of mosquitoes after 24 hours
exposure . Suggesting that the effects of the extract of both plants on the
pupal stage appear after more than 24 hours exposure , as growth regulator .
The results are comparable with the earlier studies of Prabakar
and Jebanesan, ( 2004 ) they had reported that the leaf extract of fives
species of Cucurbitacious plants , Momordica charntia , Trichosanthes
anguina , Luffa acutangula , Benincasa cerifera and Citrullus vulgaris
showed larvicidal activity at LC50 of 465.85 , 567.81, 839.81 , 1189.30 and
1636.04 ppm respectively ( after 24 hours treatment) against the 3rd instar
larvae of Culex quinquefasciatus . Culex quinquefasciatus showed different
susceptibility against different plant species .
The leaf extracts of Pavonia zeylanica and Acacia ferrugginea
showed larval mortality at LC50 of 2214.7 and 5362.6 ppm respectively
against the third instars larvae of Culex quinquefasciatus after 24 hours
treatment ( Vahitha et al, 2002 ) .
Water extracts of nine medicinal plants against larvae of Culex
quinquefasciatus and Aedes agypti indicate that the plant Piper retrofractum
( Piperaceae ) among these plant showed the highest level of larvicidal
activity ( Chansang et al, 2005 ) . Essential oils extracted
from dried leaves of three spontaneous plants naturally growing in Burkina
Faso, Cymbopogon proximus, Lippia multiflora and Ocimum canum ,
exhibited larvicidal activity against 3rd and 4th instars larvae of field
collected mosquitoes vectors of human disease , Aedes agypti and Anopheles
gambiae . The LC50 values ranged between 53.5 – 258.5 ppm for Aedes
agypti and 61.9 – 301.6 ppm for Anopheles gambiae . The LC90 estimated
ranged between 74.8 – 334.8 ppm for Aedes agypti and 121.6 – 582.9 ppm
for Anopheles gambiae ( Bassole et al, 2003 ) .
A preliminary study revealed that extract of 14 species from 112 medicinal
plant collected from southern part of Thailand, showed evidence of
larvicidal activity . Eight out of 14 plant species showed 100% mosquito
larvae mortality ( Promsiri et al, 2006 ) . Of 51 species of plants from state
of Oaxaca, Mexico, evaluated for toxicity against Culex quinquefasciatus
larvae, three species of plants presented the greatest larvicidal action as
water and acetone extracts ( Rafael et al, 2004 ) .
In this study it was found that Calotropis procera was more potent
than Ricinus communis and the 2nd instar was more susceptible than 3rd instar
and the later was more susceptible than 4th instar . This agree with (
Lapcharoen et al, 2005 ) who reported that the potential larvicidal activity of
three selected indigenous medicinal plants against Aedes agypti and Culex
quinquefasciatus indicated that Thevetia peruviana was the most potent,
followed by Pueraria mirifica, and Butea superba was the least effective . In
all cases, the late 3rd instar was more susceptiple than the early 4th instar .
Also the result agree with the finding of Pushpanathan et al, ( 2006 )
who had reported that 2nd instar larvae of Culex quinquefasciatus was more
susceptible than 3rd instar, and the later was more susceptible than 4th instar
larvae to the essential oils extracted from Cymbopogan citratus plant , with
LC50 – LC90 of 144.54 – 284.27 ppm , 165.70 – 318.48 ppm and 184.18 –
359.01 ppm for 2nd , 3rd and 4th instar respectively .
In this study the two species of selected mosquitoes larvae showed
different susceptibility to the leaf extract of Ricinus communis, higher
concentration was required for Culex quinquefasciatus and lower
concentration for Anopheles arabiensis as it is shown in table ( 4 – 14 ) .
Also we find that Culex quinquefasciatus was more susceptible than
An. arabiensis to the leaf extract of Calotropis procera . with the
observation on that Calotropis procera was more potent than Ricinus
communis against the two species of mosquitoes above, with different
activity of the plant extract against different mosquito species . The varying
larvicidal potency of the two plants, and the varying susceptibility of the two
species of mosquitoes are probably due to differences in levels of toxicity
among the active insecticidal ingredient of each plant and in the
physiological characteristics of the two species of mosquito . This agree with
( Thekkevilayil et al, 2004 ) who had reported that the four mosquitoes
Culex tritaeniorhynchus, Anopheles stphensi, Aedes aegypti and Culex
quinquefasciatus larvae showed different susceptibility to the oils extract of
Ipomoea cairica Linn. , higher concentration was required for Culex
quinquefasciatus followed by Aedes agypti , Anopheles stphensi and lower
concentration for Culex tritaeniorhynchus, with the LC50 – LC90 of 58.9 –
161.6 ppm for Culex quinquefasciatus, 22.3 – 92.7 ppm for Aedes aegypti,
14.9 – 109.9 ppm for Anopheles stphensi, and 14.8 – 78.3 ppm for Culex
tritaeniorhynchus .
Also the results agree with, ( Monzon et al, 1994 ) who reported that
five Philippine plants were screened and assayed for their larvicidal
potentialities against Aedes aegypti and Culex quinquefasciatus . By
exposing 3rd and 4th instars larvae to different concentration of the crude
aqueous extract derived from fresh leaves . Also to compare the larvicidal
potency of the five plants and to compare the susceptibility of the two
species of mosquito . He found that Lansium domisticum and Anona
squamosa was the most effective against larvae of Aedes aegypti and Culex
quinquefasciatus respectively . Aedes aegypti was more susceptible than
Culex quinquefasciatus with respect to Lansium domisticum and
Azadirachta indica but Culex quinquefasciatus more susceptible than Aedes
aegypti with respect to Eucalyptus globulus , Codiaeum variegatum and
Anona squamosa plant .
The whole latex of Calotropis procera was shown to cause 100%
mortality of 3rd instar larvae of Aedes agypti with in five minutes, and most
of individual growing under experimental conditions died before reaching
2nd instars or stayed in 1st instars . It may be possible that the highly toxic
effects of the whole latex from Calotropis procera upon egg hatching and
larvae development should be at least in part due to its protein content .
However the toxicity seems also to involve none protein molecules ( Marcio
et al, 2006 ) .
Fresh leaf extract of Calotropis procera showed larvicidal properties
against mosquito larvae of Anopheles stphensi, Culex quinquefasciatus and
Aedes aegypti . However methanolic extracts were more effective as
larvicide ( Singh et al, 2005 ) . The effect of alkaloid extracts of Calotropis
procera leaves at the vegetative stage on survival of fifth instar larvae and
on ovarian growth of Shistocerca gregaria have been studied . The results
reveal that a mortality rate of 100% was reached in the hoppers on the 15th
day after the beginning of the treatment . In the adult it had been observed
the arrest of ovarian growth in females and the absence of sexual maturity in
males . Furthermore it was observed in the individuals treated a hyper
excitability broken with moments of immobility, the trembling of the legs
and mouth appendages and abdominal segments, a reduction of weight, a
decrease in food intake, water loss and intense transpiration ( Abbassi et al,
2004 ) .
evaluation of larvicidal activity of aqueous extracts from leaves of
Ricinus communis L. and from wood of Tetraclinis articulata ( Vahl ) Mast.
against four mosquito species , after 24 hours exposition showed strong
toxic activity against larvae of four mosquito species ( Brahim et al, 2006 ) .
Aqueous leaf extract of Ricinus communis L (
Euphorbiaceae ) a cultivated plant in tropical countries showed excellent
insecticidal activity against Callosobruchus Chinensis L ( Coleoptera :
Bruchidae ) , ( Upasani et al, 2003 ) .
The results showed that the leaf extract of Calotropis procera also act
as adult emergence inhibitor against the two species of mosquito studied .
Also higher concentration was required for Anopheles arabiensis and lower
concentration for Culex quinquefsciatus the same as shown in the larvicidal
activity . Also it was evident that EI50 – EI90 values for the two species of
mosquitoes studied were less than LC50 – LC90 for the 3rd larval instar of
the same mosquitoes species, with EI50 – EI90 of 277.90 – 677.60 ppm for
Anopheles arabiensis and 183.65 – 453.94 ppm for Culex quinquefsciatus as
it shown in table (4 - 19), and the LC50 – LC90 of 366.44 – 1018.59 ppm
for Anopheles arabiensis and 218.27 – 538.27 ppm for Culex
quinquefsciatus as it shown in table (4 – 7) . This indicated that lower
concentration of the leaf extract was required for the adult emergence
inhibition than larvicidal, reflecting the activity of Calotropis procera to act
as insect growth regulator.
This study also showed that the leaf extract of Ricinus communis also
act as adult emergence inhibitions against the two species of mosquito
studied . The higher concentration was required for Culex quinquefsciatus
and lower concentration for Anopheles arabiensis similar to the larvicidal
activity of the leaf extract . Also it was evident that EI50 value for the
different species of mosquitoes studied was less than LC50 for the 3rd instar
larvae of the same mosquitoes species, with EI50 of 374.97 for Anopheles
arabiensis and 1180.32 ppm for Culex quinquefsciatus ( Table 4 – 19 ), and
the LC50 of 445.66 ppm for Anopheles arabiensis and 1364.58 ppm for
Culex quinquefsciatus as it shown in table( 4 – 14 ) . This indicated that
lower concentration of the leaf extract was needed for the adult emergence
inhibition than larvicidal . Reflecting the activity of Ricinus communis as
insect growth regulator .
The results agree with the previous studies that suggest the utility of
the plants extract as adult emergence inhibitions against mosquito larvae .
The Ethanol extract of the leaves of Centella asiatica (
Umbelliferae ) plant is promising as adult emergence inhibitor against Culex
quinquefaciatus . The adult emergence inhibition activity of this extract at
LC50 at different temperatures was generally more pronounced in increased
temperature ( Rajkumar and Jebanesan, 2005a ) .
Insect growth regulator activity of Methanol extract of leafs of
Atlantia monophylla ( Rutaceae ) was more pronounced against Aedes
aegypti with EI50 value of 0.002 mg/L . The extract was found safe to
aquatic mosquito predators Gambusia affinis with LC50 value of 23.4 mg/L
( Sivagnaname and Kalyanasundaram, 2004 ) .
The insect growth regulator ( IGR ) activities of three selected
Thailand plants , indicated that Thevetia peruviana did not show any IGR
properties ; whereas Pueraria mirifica and Butea superba exhibited high
properties as IGR . Butea superba was more promising than Pueraria
mirifica, and Aedes aegypti was about twice more susceptible than Culex
quinquefaciatus . In addition 3rd instar was always more susceptible than 4th
instar with both mosquito species ( Lapcharoen et al, 2005 ) .
The latex produced by the green part of Calotropis procera, was
found very effective as insect growth regulator against Aedes aegypti,
resulting in that most of individual growing under experimental condition
died before reaching 2nd instars or stayed in 1st instars ( Marcio et al, 2006 )
.
Only 33.3% of the larvae reared in media containing 0.025 percent of
the Calotropis procera leaves extract, completed development to the pupa
stage , and only 20% reached the adult stage of Culex pipiens mosquito ( Al-
Doghairi and Elhag, 2002 ) .
The ovicidal activity of leaves extract of Calotropis procera against
Culex quinquefasciatus was shown in table (4 – 20) .
The LC50 - LC90 values for Culex quinquefsciatus eggs were 191.87
– 510.51 ppm respectively . The result indicated that Eggs were more
susceptible than 3rd and 4th istar larvae of the mosquito Culex
quinquefasciatus .
The ovicidal activity of leaves extract of Ricinus communis against
Culex quinquefasciatus was shown in table ( 4 – 21 ) .The LC50 - LC90
values were 961.61 – 1786.49 ppm respectively . The result indicated that
eggs were more susceptible than 3rd and 4th instar larvae . The leaves extract
of Calotropis procera was more effective in preventing eggs hatching than
the extract of Ricinus communis . Also the water leaf extract of both plants
were more potent against eggs than larvae . The results agree with similar
previous studies .
Essential oils extracted from Cymbopogan citratus plant showed
larvicidal, ovicidal, and repellent activity against the filarial tranmitting
mosquito, Culex quinquefaciatus . Hundred percent ovicidal activity was
observed at 300 ppm, while LC90 of the 3rd istar larvae was 318.48 ppm,
indicating that eggs were more susceptible than larvae ( Pushpanathan et al,
2006 ) .
Essential oils extracted from 10 medicinal plants were evaluated for
larvicidal, adulticidal, ovicidal, oviposition deterrent and repellent activities
against three mosquito species ; Anopheles stephensi, Ades agypti and Culex
quinquefaciatus . From these plants the essential oils of Juniperus
macropoda and Pimpinella anisum were highly effective as both larvicidal
and ovicidal . Essential oils of Zingiber officinale was found to be ovicidal
against the three mosquito species (Prajapati et al, 2005) .
Essential oils extracted from dried leaves of three spontaneous plants
naturally growing in Burkina Faso, Cymbopogon proximus, Lippia
multiflora and Ocimum canum, exhibited ovicidal activity against eggs of
Anopheles gambiae S. L. The LC50 values for Anopheles gambiae S. L.
eggs ranged between 17.1 - 188.7 ppm , while LC90 values ranged between
33.5 – 488 ppm . Lippia multiflora showed the highest activity against
Anopheles gambiae S. L eggs and Aedes aegypti larvae . Of the three plants ,
essential oils from Ocimum canum had the lowest activity against both eggs
and larvae . Eggs were more susceptible than larvae ( Bassole et al, 2003 ) .
The latex of Calotropis procera, was studied to evaluate its toxic
effect upon eggs hatching against Aedes aegypti . It was found very
effective to prevent eggs from hatching and larval development ( Marcio et
al, 2006 ) .
The ( 0.1% ) concentration of Calotropis procera latex in water which
had showed no mortality effect in the larvicidal efficacy experiment , was
shown to cause about 94.1% mortality of Aedes aegypti eggs ( Manju et al,
2004 ) .
The isolated flavonoids from Ricinus communis L leaves showed
potential insecticidal , ovicidal and oviposition deterrent activities against
Callosobruchus chinensis L ( Coleoptera : Bruchidae ) . HPLC and HPTCL
chromatograms suggested quercetin to be the major flavonoid present in the
hydrolyzed aqueous leaf extract of Ricinus communis ( Upasani et al, 2003 )
.
The toxic activity of fractions of leaf extracts of Ricinus communis L (
Euphorbiaceae ) and isolated active compounds against the leaf-cutting ant
Atta , Sexdens rubropilosa (Forel) and its symbiotic fungus Leucoagaricus
gongylophorus (Singer) Moller have been studied . The main compounds
responsible for activity against the fungus and ant in leaf extract of Ricinus
communis were found to be fatty acids for the former and ricinine for the
ant ( Bigi et al, 2004 ) .
The water leaf extract of Calotropis procera at different
concentration of larvicidal activity ( high , moderate ,low ) showed hundred
percent oviposition deterrence and hundred percent effective repellency of
Anopheles arabiensis when the extract is to be used as material of choice
(treaed – control ) . However when all the larvicidal concentrations were
offered without control ( choice ) the avoidance of eggs laying was
not shown except in the high larvicidal concentration (1000 ppm) , and the
maximum eggs laying was preferred in the low larvicidal concentration (
200 ppm ) as it shown in table ( 4-22 ) . Similar observation was shown on
Culex quinquefasciatus, with relative difference in that Culex
quinquefasciatus showed 90.6% oviposition deterrence and effective
repellency at low larvicidal concentration ( 100 ppm ) . The effect of leaf
extract of Calotropis procera at different concentrations of larvicidal
activity ( high , moderate , low ) on the oviposition deterrent and repellent
activity against Culex quinquefasciatus was shown in table ( 4 – 23 ) .
The water leaf extract of Ricinus communis at high (1200 ppm) and
moderate (600 ppm ) concentration of larvicidal activity showed hundred
percent oviposition deterrence and hundred percent effective repellency
against Anopheles arabiensis , while at low concentration of larvicidal
activity ( 200 ppm ) showed 90.5% oviposition deterrence and effective
repellency, when the extract is to be used as material of choice ( treated –
control ), However when all the larvicidal concentrations were offered
without control ( choice ) the avoidance of eggs laying was not
shown , and the maximum eggs laying was preferred in the low larvicidal
concentration ( 200 ppm ) as it was shown in table ( 4 – 24 ) . Similar
observation was shown on Culex quinquefasciatus . The effect of leaf extract
of Ricinus communis at different concentrations of larvicidal activity ( high,
moderate, low) on the oviposition deterrent and repellent activity against
Culex quinquefasciatus was shown in table ( 4 – 25 ) .
it was concluded that the leaf extract of both plants showed hundred
percent oviposition deterrent at low larvicidal concentration , when there is a
choice of control and when the control was not offered ( the treated water
only ) the mosquitoes can lay eggs but at the lowest number in comparison
to the number of egg laid in control , when the treated cup was not offered .
Also the reduced number of eggs laid may lose it is viability to hatch, by the
effect of the concentration used . The results are comparable with related
studies .
The acetone leaf extract of Solanum trilobatum (Solanaceae) was
tested for ovipostion deterrent and skin repellent activities against the adult
mosquito Anopheles stephensi . Concentrations of 0.01, 0.025, 0.05 , 0.075 ,
and 0.1% showed oviposition deterrent and percent effective repellency (
ER% ) of 18.4 , 44 , 66.6 , 89.8 , and 99.4% respectively . Both oviposition
deterrent and skin repellent activity were dose dependent . The result
suggested that the leaf extract of S. trilobatum is an effective oviposition
deterrent and skin repellent against An. stephensi ( Rajkumar and Jebanesan,
2005 b ) .
The latex of Calotrpis procera has shown a remarkable effect as
larvicides, against Aedes aegypti, and at 0.7% concentration of latex, the
oviposition was avoided by the gravid female mosquitoes and this behavior
continued till three gonotrophic cycles . However at low concentrations (
0.2% and 0.1% ) of the larvicide latex, the refractory behavior of
ovipositioning could not be retained up to the third gonotrophic cycle .
When the three concentrations (0.1% , 0.2% , 0.7% ) of latex were made
available without control the maximum of eggs laying was preferred in 0.1%
of latex , and the concentrations of latex such as 0.7% and 0.2% were
observed as ovicidal also and this effect continued across all gonotrophic
cycles ( Manju et al, 2004 ) .
Calotropis species are perennial herb with a long history of use in
traditional medicinal especially in the tropical and subtropical regions . A
wide range of chemical compounds including cardiac glysocisdes ,
flavonoides , phenolic compounds, terpenoids have been isolated from this
species . Extract and metabolites from this plant have been found to possess
various pharmacological activities ( Mueen Ahmed et al, 2005 ) .
The isolated flavonoids from Ricinus communis L leaves showed
potential insecticidal, and oviposition deterrent activities against
Callosobruchus chinensis L ( Coleoptera : Bruchidae ) ( Upasani et al,
2003 ) .
Conclusion
The finding of the present investigation revealed that the aqueous
leaves extract of C. procera and R. communis possess remarkable larvicidal,
adult emergence inhibition, ovicidal and oviposition deterrent activity
against mosquitoes An. Arabiensis and Culex quinquefasciatus . These plants
might be used as natural biocides, for many reasons firstly : ecologically
acceptable (natural products), secondly: economically ( actually free) the
plants are well known and available in many areas of Sudan, the active
ingredient highly located in leaves and extractable with water. Further
studies are needed to investigate the toxicity of these plants extracts against
wide range of none target organisms, e.g. Gambusia affinis, Orechromis
niloticus, Tadpole and higher organisms such as mice , and to identify the
active compounds of the extracts responsible for larvicidal, adult emergence
inhibition, ovicidal and oviposition deterrent activity against An. Arabiensis
and Culex quinquefasciatus .
The results showed that the plants Eclipta prostrata, Sonchus
oleraceous and Euphorbia hirta had not shown larvicidal activity against the
two species of mosquitoes after 24 hours exposure . This suggests that these
plants do not posses larvicidal properties or the active ingredient of these
plants can not be extracted by 24 hours soaking time or that the effects of
the plants extract on the larvae may appear after more than 24 hours
exposure, or the active ingredient of these plants can not be extracted by
water . Further investigation are needed to investigate these Suggestion .
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Larvicidal, Ovicidal, Oviposition Deterrence and Emergence Inhibition Activity of Selected Sudanese Plants Against

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Larvicidal, ovicidal, oviposition deterrence and emergence

inhibition activity of selected Sudanese plants against

A thesis Submitted in Fulfillment of the Requirement for the Degree of

Literature Review ………………………………………….……. 5

Materials and Methods ………………………………………….. 36

Discussion ……………………………………………..………… 103

MY BROTHERS AND SISTERS

Table ( 4.1 ) Susceptibility of larvae of Anopheles arabiensis

Figure 4.1 Larvicidal activity of leaves extract of calotropis

Plate 3.1 The Bioassay cup ………………………………………… 41

Beside early diagnosis and prompt treatment with effective drugs, it is

Larviciding and source reduction have a major advantage in that they

Since the discovery of DDT , mosquito control approach has been

Objective of the study :

The overall objective of this work is to investigate the activity of

Specific objectives are :

1- To study the larvicidal activity of water extracts of some selected

2- To study the subsequent effects of the plant extracts which had a

3- To compare between the different species of mosquitoes on their

2-1 Global burden of malaria

2-2 Malaria in Sudan

mean daily temperature of 16 to 19 °C ,18 days at 20 to 22 °C and 14 days at

24 to 27 °C ( Kassirsky and Poltnikov 1969 ) .

gambiae develops rapidly at the temperature of about 37 °C ( Jepson et al

at 15 °C . In Ades aegypti survival to adult stage was high ( 90-92%) at

temperature from 20 – 27 °C , and lowest ( 3% ) at 15 °C ( Ruedal et al 1990

2-9 Mosquito bionomics

2-11-2-2-2 Bacterial larvicides :

In Khartoum State two rounds of house spraying were planned to cover

2-13 Plants Mosquitocides :

between 6.84 ppm at 19 °C and 1.12 ppm at 31 °C . A similar trend was

The essential oils of the plant Ipomoea cairica Linn. possess

The potential larvicidal activity of three selected indigenous

2-14-2 Adult emergence inhibition activity

MATERIAL AND METHODS

3-1 The study area :

3-2-5 Eclipta prostrata . Tamr Elganam

3-4 Mosquito rearing :

C° . To determine pupicidal activity , the mouth of each cup containing

Where X = percentage survival in the untreated control .

Where T = percentage survival or emergence in treated batches and C =

3-6-3 Oviposition deterrent activity

The percent effective repellency for each plant leaf extract

Where ER = percent effective repellency ; NC = number of eggs in control ;

Number of hatched larvae

3-7 Statistical Analysis

4-1– Larvicidal and pupicidal activity of leaves extract of Calotropis

Table (4 – 1 ) : Susceptibility of larvae of Anopheles arabiensis exposed as

Concentration Mortality Probit

Number of larvae tested for each concentration = 100

Table (4 – 2 ) : Susceptibility of larvae of Anopheles arabiensis exposed as

Concentration Mortality Probit

Number of larvae tested for each concentration = 100

Table (4 – 3 ) : Susceptibility of larvae of Anopheles arabiensis exposed as

Concentration Mortality Probit

Number of larvae tested for each concentration = 100

Figure 4.1 Larvicidal activity of leaves extract of calotropis procera against

▲ second larval instar : Y = 2.799X – 1.820

□ third larval instar : Y = 2.883X – 2.393

О fourth larval instar : Y = 2.977X – 2.913

Concentration Mortality Probit

Number of larvae tested for each concentration = 100

Concentration Mortality Probit