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Seedling Extracts Upon Ct]Lex Pipiens Larvae

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Seedling Extracts Upon Ct]Lex Pipiens Larvae Jounr.iel. oF THE Arr,rnnIclNMosqurro CoNrnor, AssocrlrroN Vol. 6. No. S LARVICIDAL EFFECTS OF GRAIN SORGHUM (SORGHUMBICOLOR) SEEDLING EXTRACTS UPON CT]LEX PIPIENS LARVAE F. L. C. JACKSONI, S. S. BEHKEIT,, S. M. EL ETR, auo N. K. QUACHI . ABSTRACT. A study .of.laboratory and field reared 2nd and 3rd instar Cul.expipiens larvae suggests that extracts from 2 varieties of Sorghum bicolor seedlingsare significant (P <0.05) larvicides ir"nder labo_ratoryconditions. These plant extracts contain the organic cyinogen dhurrin and were calibrated to produce 9OZo-mortalityin 2nd instar Culexpipiens larvae-at 0.8i ppm and,90%mortality in 3rd instar larvae at 1.12 ppm. A,preliminary behavioral assessmentof late 3rd instar larvae expo.ed to 1.42 ppm suggeststlrat these plant extracts produce 80% mortality after only 4-5 h of contact. Plant extr-acts appear stable when stored at up to 32'C in a closed container. Once the extracts are infused in water and_exposedto air, however,they biodegradeaf\er 24 h. These laboratory results emphasizethe needfor field tests against natural populations of Culexpipiens and nontarget organisms. INTRODUCTION mosquitoes and thus be an effective mosquito control agent, In Eg5pt, Culex pipiens (Linn.) mosquitoes remain major diseasevectors of bancroftian fi- lariasis (Gad et al. 1988),Rift Valley fever virus MATERIALS AND METHODS (Bres 1978), West Nile and Sindbis viruses Plant extracts were prepared from 4- to 7- (Saminaet al. 1986),as well as seriousnusiance day-old seedlings grown from pregerminated pests (World Health Organization 1983).The sorghum which was cultivated at 25'C and at control of this mosquito vector through the use 55% RH in 3 cm of sterile humus soil, given 20- of effective,biodegradable, economically feasible ml deionized water and exposedto 24-h cycles botanical extracts is an important alternative of white fluorescent light. Seedling leaveswere control strategy. We report on laboratory tox- harvested and the leaves pressed in a hand icity tests of the application of grain sorghum grinder to extract the liquid. A 60% yield by (Sorghum bicolor Moench) seedling ex- [Linn.] weight of leaves to liquid was obtained using tract to 2nd and 3rd instar larvae of Culcx pi- piens. this method.The extract was storedat23-32"C and later centrifuged, filtered and assayedfor The grain sorghum extracts evaluated as lar- its organic cyanogencontent using a cyanogenic vicides were derived from seedling varieties glycosidase specific enzymatic assay (Cooke widely cultivated in the United States and in 1979).All assayswere done in triplicate. Egypt for local human food and animal fodder Two sourcesof grain sorghum provided the use. Our rationale for testing these extracts was basisof our extract stocks.Extract I was derived the presenceof the organic cyanogen dhurrin, from Beefbuilder T3 variety and contained 357.5 [(S)-p-hydroxymandelonitrile B-D-glucopyran- ppm organic cyanogen; extract II was derived osidel in grain sorghum. This cyanogenic gly- from Giza 15 and Local 129'varietiesand con- coside(depicted along with its hydrolysis in Fig. tained 195 ppm organic cyanogen. Serial dilu- 1) is an endogenous precursor biological of cya- tions of these extract stocks (ranging in lUVo nide, is present in high concentrations in the intervals from 0 to t00Vo\were used for mortal- plant (Nartey immature 1981),and is the most ity testing against the mosquitoes.The organic grain likely toxic constituent in sorghumcapable cyanogencontent ofthese dilutions was assayed (Branson of larvicidal effects et al. 1969).Most and rangedfrom 0 to 1.42ppm organic cyanogen. of the plant discussions effects of organic cyan- Secondand 3rd instar larvae of Culexpipiens on humans and ogens animals have focusedon were used and represented both laboratory potential of these the toxins to initiate signifi- rearedand field collectedspecimens. Laboratory physiological cant modifications within the hu- rearedlarvae were maintained following a stand- plant man or animal consuming a cyanogenic ardized protocol at the Research Institute for (Osuntokun 1981, Jackson et al. 1988). Our re- Medical Entomology in Dokki, Cairo, Egypt. search objective in this case, however, was to Field reared laryae were collected from nearby test the hypothesis that extracts of this cyano- untreated sites in and around Cairo. These field genic plant can impair the viability of immature larvae were then transported to the laboratory, maintained in water from their original breeding I Laboratory in Biological Anthropology, 1350Tur- lington Hall, University of Florida, Gainesville, FL 3Beefbuilder T is distributed by Asgrow SeedCom- 32611. pany,USA. 2 Research Institute of Medical Entomology, 1 Ma- a Giza 15 and Local 129 were provided by the Agri- thaf El Zarai Street, Dokki, Cairo, Egypt (A.R.E.). cultural ResearchCenter, Giza Egypt. SEPTEMBER1990 Soacnuu Extnects AS CULEr LARvrcrDE HoCH2 p-flucoenrscs + p-D4LITOOPYnANOSE DtIUXruN + o -.{gl:^fln H pttYDROXYBENALDEIIYDG +I HC=N TiTffiH Fig. 1. Hydrolysis of dhurrin, (S)-p-hydroxymandelonitrileB-D-glucopyranoside. sites and subsequentlyused for toxicity testing RESULTS AND DISCUSSION within 24 h of initial collection. Following the World Health Organization Both extracts killed 100%ofboth 2nd and 3rd guidelines for insecticide assessment (World instat Culexpipiens law ae under laboratory con- Health Organization1981), 1 ml of each serial ditions of assessment.The calculated r and F dilution of Sorghum bicolor extracts was added values for 2nd instar larvae were significant (r to 225 ml of water in a 500-ml glassbeaker and : 0.79;Fo.zot:33.30). The calculatedr and F stirred vigorously with a glassrod. To this were values for 3rd instar larvae were also significant added25 Culexpipiens larvae (either 2nd instar (r : 0.93;Fo.,zo) : 126.18). The dilutions of plant or late 3rd instar) in 25 ml of water, bringing derived organic cyanogenscapable of killing 2nd the total volume of water to 250 ml. All experi- instar larvae were less than those required to mental and control waters were maintained at produce a similar level of mortality among 3rd 23-32"C and 24-h mortality recorded.The con- instar larvae. Combining data from Extract I centrations of organic cyanogen in the treated and II, the linear regressionmodel predicted that waters was evaluatedinitially and again after 24 the LCso and LCso for 2nd instar larvae were h. Since behavior is an establishedcriterion of 0.30 ppm and 0.82 ppm, respectively.The cali- toxicity (Weiss1978), to complementour studies brated LCsoand LCsofor 3rd instar larvae (com- of absolute mortality in the mosquitoes,hourly bining data from extract I and II) were 0.67 ppm behavioral observationswere made of a subsam- and 1.12 ppm, respectively. Figures 2 and 3 ple of 50 late 3rd instar larvae exposedto 1.42 display the linear regressionof mortality for 2nd ppm organic cyanogen. and 3rd instar Culexpipiens lawae, respectively, Data were initially plotted logarithmically as exposedto sequential dilutions of extract I and recommendedby the World Health Organiza- II plant organic cyanogens.In both extract I and tion (World Health Organization1981). Follow- extract II, the assayedlevels of organic cyano- ing this, data were subjectedto linear regression gens were well below the amounts identified as and r values calculated for the correlation of toxic for humans and other mammals (Mont- organic cyanogen concentration (independent gomery1965). variable) and vector mortality (dependentvari- After 24 h of exposureto air, the water infused able). An ANOVA was also performed and the with the Sorghum bicolor seedling extract no F statistic evaluated relative to an alpha value longer contained detectable organic cyanogen of 0.05. Probability analysis was used to cali- and also lost its larvicidal properties. This ap- brate LCsosand LCgosfor 2nd and 3rd instar parent lossoftoxicity was due to the degradation Iarvae exposed to extract I and extract II of of the organic cyanogensand proceededinde- Sorghum bicolnr. pendent of the initial concentration of the ex- JouRNer,oF THEArrannrcnN Moseurro Cournor,Assocrltror.r VoL.6,No.3 E J * u o -t6tt' J 4 J s t)-tt' '{ 6 hb PPM ORGAMC CYANOCEN Fig. 2. Linear regression of various dilutions of plant organic cyanogens (X) on 2nd instar Culex pipiens larval mortality (Y) (after 24 h) with 95% confidence bands. -t' -tt' F -2? J 11s -':-::-:'. 'l . 'r:- ,-tt' J s -"" PPM ORCANIC CYANOGEN Fig. 3. Linear regression of various dilutions of plant organic cyanogens (X) on 3rd instar Culex pipiens larval mortality (Y) (after 24 h) with 95% confidence bands. tract or the density of the mosquito larvae pres- beaker and again on the bottom. After 2 h more ent. Dhurrin is the most likely, although perhaps ofexposureto the plant extracts, larger numbers not the only, insecticidalcomponent of our plant of larvae began to lie very still on the surface extracts. and then drift downward with erect siphons. The results of our preliminary behavior as- These larvae remained dormant on the bottom sessrnentssuggested that the exposure of late and appearedtolack the energyto remain afloat. 3rd instar larvaeto 1.42ppm organiccyanogen Four and one-half hours post-initial contact initiated a rapid sequenceof morbidity associ- with the plant toxin, 80% of the remaining lar- ated behavioral changes.Initially, all 50 larvae vae appearedmoribund and resisted movement moved vigorously and appearedto be quite vi- in responseto being gently poked in the thorax. bration sensitive.Within 17 min following
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