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 University of Khartoum 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 …………………………………………..
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Chapter Two Literature Review ………………………………………….…….
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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 ……………………………..
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2.6 Lymphatic Filariases transmission ………………………….
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2.7 Important species of mosquitoes in Sudan ………………….
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2.8 Life cycle of the mosquitoes ………………………………..
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2.9 Mosquito bionomics ………………………………………..
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2.10 The breeding sites of mosquitoes ………………………….
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2.10.1 Anopheles species ………………………………………
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2.10.2 Culex species ……………………………………………
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2.11 Mosquito larval control ………………………………. …… 18 2.11.1 Source reduction …………………………………… …… 18 2.11.2 Larviciding …………………………………………… …
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2.11.2.1 Chemical control …………………………………. …… 19 2.11.2.2 Biological control ………………………………... ……
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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 ……………………………… …..
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Chapter Three Materials and Methods …………………………………………..
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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 …………………………………..……
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3.2.5 Eclipta prostrata ……………………………………..……
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3.3 preparation of stock solution ………………………….……..
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3.4 Mosquito rearing …………………………………….………
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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 ……………….………
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3.6.2.1 Data analysis …………………………………..………… 46 3.6.3 Oviposition deterrent activity ……………………...……… 47 3.6.4 Ovicidal activity ……………………………….…………..
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4.7 Statistical analysis ……………………………….…..………
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Chapter Four Results ………………………………………………………
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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 …………………………………………
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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 ﻓﻲ ﺣﺎل وﺟﻮد ﺧﻴﺎر ﺁﺧﺮ ﻟﻠﺘﻜﺎﺛﺮ ﻏﻴﺮ ﻣﻌﺎﻟﺞ
) ﻣﻌﺎﻟﺞ -ﻏﻴﺮ ﻣﻌﺎﻟﺞ ( .ﺑﻴﻨﻤﺎ ﺗﺠﻨﺐ وﺿﻊ اﻹﻧﺎث
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 Table ( 4.2 )
Susceptibility of larvae of Anopheles arabiensis exposed as 3rd larval instar to different concentration of leaves extract of Calotropis procera after 24 hours ……………………………………………….. 54
Table ( 4.3 )
Susceptibility of larvae of Anopheles arabiensis 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
exposed
as
2nd
larval
instar
to
different
concentration of leaves extract of Calotropis procera after 24 hours ……………………………. Table ( 4.5 )
Susceptibility of larvae of Culex quinquefasciatus exposed
as
3rd
larval
instar
to
different
concentration of leaves extract of Calotropis procera after 24 hours ……………………………... 58 Table ( 4.6 )
Susceptibility of larvae of Culex quinquefasciatus exposed as 4th larval instar to different concentration 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
Table ( 4.8 )
Susceptibility of larvae of Anopheles arabiensis exposed
as
2nd
larval
instar
to
different
concentration of leaves extract of Ricinus communis after 24 hours ……...... …………………………… Table ( 4.9 )
66
Susceptibility of larvae of Anopheles arabiensis exposed as 3rd larval instar to different concentration of leaves extract of Ricinus communis after 24 hours ……………………………………………………… 67
Table ( 4.10 ) Susceptibility of larvae of Anopheles arabiensis exposed as 4th larval instar to different concentration of leaves extract of Ricinus communis after 24 hours ……………………………………………………… 68
Table ( 4.11 ) Susceptibility of larvae of Culex quinquefasciatus exposed
2nd
as
larval
instar
to
different
concentration of leaves extract of Ricinus communis after 24 hours ……………………………………..
70
Table ( 4.12 ) Susceptibility of larvae of Culex quinquefasciatus exposed
3rd
as
larval
instar
to
different
concentration of leaves extract of Ricinus communis after 24 hours ………………… ………..………...
71
Table ( 4.13 ) Susceptibility of larvae of Culex quinquefasciatus exposed
as
4th
larval
instar
to
different
concentration of leaves extract of Ricinus communis after 24 hours ……………………………………..
72
Table ( 4.14 ) Larvicidal activity of leaves extract of Ricinus communis against 2nd, 3rd and 4th instars larvae of Anopheles arabiensis and Culex quinquefasciatus 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 Table ( 4-16 ) The adult emergence inhibition activity of leaves extract of Calotropis procera against 3rd larval instar
of
Culex
quinquefsciatus
at
different
concentration …………………… ………………… 80 Table ( 4-17 ) The adult emergence inhibition activity of leaves extract of Ricinus communis against 3rd larval instar 84
of Anopheles arabiensis at different concentration ……………………………………………………… Table ( 4-18 ) The adult emergence inhibition activity of leaves extract of Ricinus communis against 3rd larval instar of Culex quinquefsciatus at different concentration ……………………………………………………… 86 Table ( 4-19 ) 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 .. 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 Table ( 4-23 ) Oviposition deterrent activity of leaves extract of Calotropis procera against gravid, female Culex quinquefasciatus …………………………………… 98 Table ( 4-24 ) Oviposition deterrent activity of leaves extract of Ricinus communis against gravid, female Anopheles arabiensis ………………………………………….. 101 Table ( 4-25 ) Oviposition deterrent activity of leaves extract of Ricinus communis against gravid, female Culex quinquefasciatus ….................................................... 102
List of Figures 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...
Figure 4.2
56
Larvicidal activity of leaves extract of calotropis procera against 2nd, 3rd and 4th instars larvae of Culex quinquefasciatus expressed as linear regression.......…
Figure 4.3
60
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 values .........................
Figure 4.4
62
Larvicidal activity of leaves extract of Ricinus communis against 2nd, 3rd and 4th instars larvae of Anopheles arabiensis expressed as linear regression...
69
Figure 4.5
Larvicidal activity of leaves extract of Ricinus communis against 2nd, 3rd and 4th instars larvae of Culex quinquefasciatus expressed as linear regression ........................................................................................ 73
Figure 4.6
Larvicidal activity of leaves extract of Ricinus communis against 2nd, 3rd and 4th instars larvae of Anopheles arabiensis and Culex quinquefasciatus 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
Figure 4.8
The adult emergence inhibition activity of leaves extract of Calotropis procera against 3rd larval instar of Culex quinquefsciatus at different concentration, expressed as linear regression ....................................... 81
Figure 4.9
The adult emergence inhibition activity of leaves extract of Ricinus communis against 3rd larval instar of Anopheles arabiensis at different concentration, expressed as linear regression ....................................... 85
Figure 4.10
The adult emergence inhibition activity of leaves extract of Ricinus communis against 3rd larval instar of Culex quinquefsciatus at different concentration, expressed as linear regression ....................................... 87
Figure 4.11
The adult emergence inhibition activity of leaves 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 . Plasmodium that cause malaria in humans are
Four species of 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 longlasting 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 dphenothrin (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 32 ), 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 :
Mortality (%) =
X-Y X
100
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 :
EI (%) = 100 -
T × 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 ( 2528 °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 : ER ( % ) =
NC - NT × 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 =
Total number of eggs in treated water
× 100
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) , (43) , (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 ( ppm )
Mortality
Log
Total
(%)
Probit 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
Table (4 – 2 ) : Susceptibility of larvae of Anopheles arabiensis exposed as third larval instar to different concentration of leaves extract of Calotropis procera after 24 hours .
Concentration ( ppm )
Mortality
Log
Total
(%)
Probit Tabulated Calculated
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
-
-
Number of larvae tested for each concentration = 100 Regression equation : Y = 2.883X – 2.393 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
Table (4 – 3 ) : Susceptibility of larvae of Anopheles arabiensis exposed as fourth larval instar to different concentration of leaves extract of Calotropis procera after 24 hours .
Concentration ( ppm )
Mortality
Log
Total
(%)
Probit Tabulated Calculated
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
Number of larvae tested for each concentration = 100 Regression equation : Y = 2.977X – 2.913 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
Probit of mortality
6.0 5.5 5.0 4.5 4.0 3.5 3.0 1.8
2.0
2.2
2.4
2.6
2.8
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
3.0
3.2
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 .
Concentration ( ppm )
Mortality
Log
Total
(%)
Probit Tabulated Calculated
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
Number of larvae tested for each concentration = 100 Regression equation : Y = 3.528X – 3.024 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
Table (4 – 5 ) : Susceptibility of larvae of Culex quinquefasciatus exposed as third larval instar to different concentration of leaves extract of Calotropis procera after 24 hours .
Concentration ( ppm )
Mortality
Log
Total
(%)
Probit Tabulated Calculated
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
Number of larvae tested for each concentration = 100 Regression equation : Y = 3.261X – 2.626 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
Table (4 – 6 ) : Susceptibility of larvae of Culex quinquefasciatus exposed as fourth larval instar to different concentration of leaves extract of Calotropis procera after 24 hours .
Concentration ( ppm )
Mortality
Log
Total
(%)
Probit Tabulated Calculated
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
-
-
Number of larvae tested for each concentration = 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
Figure 4.2 Larvicidal activity of leaves extract of calotropis procera against 2nd, 3rd and 4th instars larvae of Culex quinquefasciatus expressed as linear regression .
7.5 7.0
Probit of mortality
6.5 6.0 5.5 5.0 4.5 4.0 3.5 1.8
2.0
2.2
2.4
2.6
Log (dose)
▲ second larval instar : Y = 3.528X – 3.024 □ third larval instar : Y = 3.261X – 2.626 О fourth larval instar : Y = 2.77x – 1.713
2.8
3.0
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
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
218.27
538.27
Y= 3.261X - 2.626
769.13
Y= 2.77X - 1.713
Anopheles arabiensis
Culex quinquefas. III
264.85 IV
Figure 4.3 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 .
1400
1224.62 Concentration of plant extract (ppm)
1200
1018.59 1000
783.43
769.13 800
538.27
600
454.99
433.51 366.44
400
273.53
264.85 218.27
187.93
200
0
Culex
Anophoeles
4th instar
Culex
Anophoeles
3rd instar
Culex
Anophoeles
2nd instar
Species of mosquito and larval instars LC50
4-2-
LC90
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 ) . 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 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 .
Table (4 – 8 ) : Susceptibility of larvae of Anopheles arabiensis exposed as second larval instar to different concentration of leaves extract of Ricinus communis after 24 hours .
Concentration ( ppm )
Mortality
Log
Total
(%)
Probit Tabulated Calculated
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
Number of larvae tested for each concentration = 100 Regression equation : Y = 3.572x – 4.308 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
Table (4 – 9 ) : Susceptibility of larvae of Anopheles arabiensis exposed as third larval instar to different concentration of leaves extract of communis after 24 hours .
Concentration ( ppm )
Mortality
Log
Total
(%)
Probit Tabulated Calculated
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
Ricinus
1200
3.079
95
95
6.64
6.38
Number of larvae tested for each concentration = 100 Regression equation : Y = 3.217x – 3.521 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 communis after 24 hours .
Concentration ( ppm )
Mortality
Log
Total
(%)
Probit Tabulated Calculated
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
Number of larvae tested for each concentration = 100 Regression equation : Y = 2.935X – 2.92 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
Probit of mortality
6.5 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 О fourth larval instar : Y = 2.935X – 2.92
Table (4 – 11 ) : Susceptibility of larvae of Culex quinquefasciatus exposed as second larval instar to different concentration of leaves extract of Ricinus communis after 24 hours .
Concentration ( ppm )
Mortality
Log
Total
(%)
Probit Tabulated Calculated
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
-
-
Number of larvae tested for each concentration = 100 Regression equation : Y = 6.208X – 13.858 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
Table (4 – 12 ) : Susceptibility of larvae of Culex quinquefasciatus exposed as third larval instar to different concentration of leaves extract of Ricinus communis after 24 hours .
Concentration ( ppm )
Mortality
Log
Total
(%)
Probit Tabulated Calculated
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
Number of larvae tested for each concentration = 100 Regression equation : Y = 7.308X – 17.914 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
Table (4 – 13 ) : Susceptibility of larvae of Culex quinquefasciatus exposed as fourth larval instar to different concentration of leaves extract of Ricinus communis after 24 hours .
Concentration
Mortality
Probit
( ppm )
Log
Total
(%)
Tabulated Calculated
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
Number of larvae tested for each concentration = 100 Regression equation : Y = 7.101X – 17.436 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
Figure 4.5 Larvicidal activity of leaves extract of Ricinus communis against 2nd, 3rd and 4th instars larvae of Culex quinquefasciatus expressed as linear regression .
6.5
Probit of mortality
6.0
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)
▲ second larval instar : Y = 6.208X – 13.858 □ third larval instar : Y = 7.308X – 17.914 О fourth larval instar : Y = 7.101X – 17.436
Table (4 – 14 ) : Larvicidal activity of leaves extract of Ricinus communis against 2nd, 3rd and 4th instars larvae of Anopheles arabiensis and Culex quinquefasciatus expressed as LC50 and LC90 .
Species of Larvae
LC50
LC90
mosquitoes instar
ppm
ppm
Regression equation
II
403.65
920.45
Y= 3.572X – 4.308
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
quinquefas. III
1364.58 2046.44
Y= 7.308X – 17.914
IV
1445.44 2187.76
Y= 7.101X - 17.436
Anopheles arabiensis
Culex
Figure 4.6 Larvicidal activity of leaves extract of Ricinus communis against 2nd, 3rd and 4th instars larvae of Anopheles arabiensis and Culex quinquefasciatus expressed as LC50 and LC90 .
2500
2187.76 Concentration of plant extract (ppm)
2046.44 2000
1753.88
1500
1445.44 1364.58
1364.58 1114.29
1091.44 920.45
1000
489.88
445.66
403.65
500
0
Culex
Anophoeles
4th instar
Culex
Anophoeles
3rd instar
Culex
Anophoeles
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 (ppm)
Log
Control -
Mortality Total 0
(%)
Survival or emergence Total (%)
EI
Probit
(%)
Tabu. Calcu.
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
800 1000
2.903 86
86
5
5
95
6.64
6.52
3.000 100
100
0
0
100
-
-
EI = Emergence inhibition of adult Number of larvae tested for each concentration = 100 Regression equation : Y = 3.307X – 3.081 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
probit of mortality
6.5
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
Table (4 – 16 ) : The adult emergence inhibition activity of leaves extract of Calotropis procera against 3rd larval instar of Culex quinquefsciatus at different concentration . Concentration (ppm)
Log
Mortality Total (%)
Survival or emergence Total (%)
EI
Probit
(%)
Tabu. Calcu.
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
-
-
EI = Emergence inhibition of adult Number of larvae tested for each concentration = 100 Regression equation : Y = 3.258X – 2.376 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
Figure 4.8 : The adult emergence inhibition activity of leaves extract of Calotropis procera against 3rd larval instar of Culex quinquefsciatus at different concentration, expressed as linear regression .
6.5
Probit of mortality
6.0
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 .
Table (4 – 17 ) : The adult emergence inhibition activity of leaves extract of Ricinus communis against 3rd larval instar of Anopheles arabiensis at different concentration . Concentration (ppm)
Log
Control -
Mortality Total
(%)
Survival or emergence Total (%)
EI
Probit
(%)
Tabu. Calcu.
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
-
-
EI = Emergence inhibition of adult Number of larvae tested for each concentration = 100 Regression equation : Y = 3.071X – 2.904 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
Figure 4.9 : The adult emergence inhibition activity of leaves extract of Ricinus communis against 3rd larval instar of Anopheles arabiensis at different concentration, expressed as linear regression.
6.5
Probit of mortality
6.0
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
Table (4 – 18 ) : The adult emergence inhibition activity of leaves extract of Ricinus communis against 3rd larval instar of Culex quinquefsciatus at different concentration . Concentration (ppm)
Log
Mortality Total
(%)
Survival or emergence Total (%)
EI
Probit
(%)
Tabu. Calcu.
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
0
0
100
-
-
100
EI = Emergence inhibition of adult Number of larvae tested for each concentration = 100 Regression equation : Y = 6.578X – 15.209 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
Figure 4.10 : The adult emergence inhibition activity of leaves extract of Ricinus communis against 3rd larval instar of Culex quinquefsciatus at different concentrations, expressed as linear regression .
6.4 6.2
Probit of mortality
6.0 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
Table (4 – 19 ) : 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 .
Plant sp.
Mosqui.
EI50
EI90
Regression
sp.
ppm
ppm
equation
677.60
Y=3.307X-3.081
453.94
Y=3.258X-2.376
979.49
Y=3.071X-2.904
1849.27
Y=6.578X-15.209
An. Calotropis
arabiensis 277.90
procera
Culex quinquefa. 183.65 An.
Ricinus
arabiensis 374.97
communis
Culex quinquefa. 1180.32
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
Concentration of plant extract (ppm)
1800 1600 1400 1180.32 1200
979.49
1000 677.6
800 453.94
600
374.97 277.9
400
183.65
200 0 Culex
Anophoeles
Culex
Ricinus communis
Anophoeles
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 ppm
Egg mortality
Log
Total
(%)
Probit 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 Regression equation : Y = 3.014X – 1.882 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
Probit of mortality
6.5
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 Regression equation : Y = 4.758X – 9.193 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
Probit of mortality
6.0
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 Dose ppm Eggs laid within 48 hrs
A
B
C
C
1000
C
500
C
280
0
390
0
480
D 200 1000 500 0
0
115
E 200
C
C
250
290
260
100
ER %
ER ( % ) =
100
100
NC - NT × 100 NC
ER = effective repellency NC = number of eggs in control . NT = number of eggs in treated . C = Control
Table (4 – 23 ) : Oviposition deterrent activity of leaves extract of Calotropis procera against gravid, female Culex quinquefasciatus
Dose ppm Eggs laid within 48 hrs
C
B
A
Cage C
1000
C
500
C
405
0
490
0
520
E
D 100 1000 500
100
C
C
20
304
370
410
56
173
100
ER %
ER ( % ) =
100
90.6
NC - NT × 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 .
Table (4 – 24 ) : Oviposition deterrent activity of leaves extract of Ricinus communis against gravid , female Anopheles arabiensis
Cage Dose ppm Eggs laid within 48 hrs ER %
ER ( % ) =
A C 350
1200 0
B C
600
410
100
NC - NT × 100 NC
C
0
100
D
E
C
200
1200 600
200
C
C
505
25
60
290
310
340
90.5
140
ER = percent effective repellency NC = number of eggs in control . NT = number of eggs in treated . C = Control
Table (4 – 25 ) : Oviposition deterrent activity of leaves extract of Ricinus communis against gravid, female Culex quinquefasciatus
Dose ppm
C
1600
Eggs 345 0 laid within 48 hrs 100 ER %
ER ( % ) =
C
B
A
Cage
C
1200
425 0
NC - NT × 100 NC
100
C
E
D 600 1600 1200 600
490 40 90.1
45
165
C
C
385 350 330
ER = percent effective repellency NC = number of eggs in control . NT = number of eggs in treated . 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
quinquefasciatus .
the
mosquitoes
Anopheles
arabiensis
and
Culex
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 ) . 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 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 ) . From LC50 and LC90 values it was evident that 2nd instars were more 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 ( AlDoghairi 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
Callosobruchus chinensis L ( Coleoptera : Bruchidae )
activities
against
( 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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