Pest Management Science Pest Manag Sci 59:1250–1254 (online: 2003)DOI: 10.1002/ps.748

Acaricidal effects of cardiac glycosides,azadirachtin and neem oil against the cameltick, Hyalomma dromedarii (Acari: Ixodidae)DiefAlla H Al-Rajhy, Azzam M Alahmed, Hamdy I Hussein∗ and Salah M KheirDepartment of Plant Protection, College of Agriculture, King Saud University, PO Box 2460, Riyadh 11451, Saudi Arabia

Abstract: The cardiac glycoside, digitoxin, from Digitalis purpurea L (Scrophulariaceae), a cardiacglycosidal (cardenolide) extract from Calotropis procera (Ait) R Br (Asclepiadaceae), azadirachtin andneem oil from Azadirachta indica A Juss (Meliaceae) were tested for their effects against larvae and adultstages of the camel tick, Hyalomma dromedarii Koch (Acari: Ixodidae). The contact LC50 values of thefirst three materials against adults were 4.08, 9.63 and >40.7 µg cm−2, respectively, whereas the dippingLC50 values of the four materials were 409.9, 1096, >5000 and >5000 mg litre−1, respectively. Contact anddipping LC50 values of the extract and azadirachtin against larvae were 6.16, >20.3 µg cm−2 and 587.7 and>2500 mg litre−1, respectively. Azadirachtin had no effects on egg production or feeding of adults up to5000 mg litre−1; however at 2500 mg litre−1, it caused significant reduction in feeding activity of larve,prolonged the period for moulting to nymphal stage, and caused 60% reduction in moultability. Resultsof the two cardiac glycoside materials are comparable with those of several commercial acaricides. Therisks and benefits associated with the use of cardiac glycosides are considered. 2003 Society of Chemical Industry

Keywords: ticks; acaricides; digitoxin; cardiac glycosides; cardenolides; Calotropis procera; Digitalis purpurea;Hyalomma dromedarii; azadirachtin; neem oil

1 INTRODUCTIONThe use of pesticides has increased the level offood production. However, pests usually developresistance to pesticides, and it is therefore necessary tosearch continuously for alternatives to those currentlyavailable, for use when resistance appears or to beused in sequence with existing pesticides to delay theappearance of resistance. The camel tick, Hyalommadromedarii Koch, is an important economic pest inSaudi Arabia as well as in many other countries;this and many other tick species attack camels, cattleand sheep, which can cause great economic lossesto such livestock.1–7 Different groups of pesticidesare used to control ticks,8–11 but, like other pests,many populations of some tick species have developedresistance to all classes of acaricide.12–20 Azadirachtin,neem oil and other botanical materials have beentested for tick control, but their rates of application aretoo high for them to be used on large scale.21–25 Thereis a great deal in the literature on pesticidal effectsof Calotropis procera (Ait) R Br (latex, fresh plantand crude extracts) against different kinds of pests,for example, insects,26,27 nematodes,28 bacteria,29

fungi,30 however, few data on pesticidal properties

of digitoxin and other cardenolides derived fromC procera have been reported.31–36 In this study, wereport our results on the effects of digitoxin fromDigitalis purpurea L, a cardiac glycoside extract fromC procera, azadirachtin and neem oil from Azadirachtaindica A Juss on the camel tick, H dromedarii. Thiswork is a continuation of our efforts to find usefulpesticidal properties of cardiac glycosides derived fromplants.32,33,35,36

2 MATERIALS AND METHODS2.1 ChemicalsDigitoxin was purchased from Winlab (Leicestershire,UK), Neemix 4.5, a 45 g AI litre−1 azadirachtin ECformulation, and Trilogy, a 700 g litre−1 neem oilEC formulation, were purchased from ThermoTrilogy(Columbia, MD, USA). The cardiac glycosidal extractwas isolated from latex of C procera according to ourprevious method,36 using lead acetate precipitationfor clean-up of the ethanolic extract. The isolatedextract gave positive tests with reagents specific forcardenolides, Kedde and Raymond reagents; Keddereagent is also used for colorimetric estimation of

∗ Correspondence to: Hamdy I Hussein, Department of Plant Protection, College of Agriculture, King Saud University, PO Box 2460, Riyadh11451, Saudi ArabiaE-mail: hhussein@ksu.edu.saContract/grant sponsor: Agricultural Research Centre, College of Agriculture, King Saud University, Saudi Arabia(Received 15 January 2003; revised version received 10 March 2003; accepted 8 April 2003)Published online 18 June 2003

2003 Society of Chemical Industry. Pest Manag Sci 1526–498X/2003/$30.00 1250

Acaricidal effects of cardiac glycosides, azadirachtin, neem oil on camel tick

cardenolide content in crude extracts.37 Classificationof the plant was carried out by Professor Ahmed Al-Farhan, Botany Department, College of Science, KingSaud University (KSU), Riyadh.

2.2 Test animalsEngorged females of H dromedarii were collected ina farm near Riyadh, where pesticides had not beenused before, and were incubated at 28 ◦C and 85%RH to oviposit; this strain was reared on white NewZealand rabbits for several generations before usedin this study; classification of the ticks was carriedout by Professor Mohammed Al-Khalifa, ZoologyDepartment, KSU.

Adults of H dromedarii have a pointed and triangulargenital covering, with longer lateral margins. The bodyis depressed gradually, the scutum is as broad as/orbroader than long; capitulum length:breadth ratio is1.2:1. Larvae are much smaller than adults, with onlythree pairs of legs; the scutum is not prominent.

2.3 Contact trialsAzadirachtin EC and neem oil EC were dissolvedin acetone to obtain the highest test concentra-tions of azadirachtin and neem oil, respectively,ie 5000 mg litre−1; the highest test concentrationsof digitoxin and Calotropis extract were 1500 and2000 mg litre−1, respectively; lower concentrationswere made by further dilution with acetone. The testconcentration ranges were 250–1500, 250–2000 and250–5000 mg litre−1 for digitoxin, Calotropis extract,and both azadirachtin and neem oil, respectively. Onemillilitre of each concentration was distributed on thesurface of a Whatman No 1 filter paper (12.5 cmdiameter); the treated area was calculated as 123 cm2

and thus, for example, 1 ml of a 502 mg litre−1 gave adeposit of 4.08 µg cm−2. Filter papers were air-dried,folded through the edges, and fixed with clips in a waythat permitted ticks to move freely inside; ticks wereintroduced into the folded filter papers and securedwith clips. Dead ticks were detected 24 h after treat-ment by direct stimulation (either breath or gentleprodding with a camel-hair brush), using 10× mag-nification in cases of doubt; animals that were ableto move or crawl were counted as alive, otherwisethey were counted as dead. Controls were made usingsolvents alone.

2.4 Dipping trialsAzadirachtin 45 g litre−1 EC and neem oil 700 g litre−1

EC were diluted with distilled water, digitoxin andC procera extract were dissolved in ethanol + Tween80 (99 + 1 by volume) and then diluted with water(5 + 95 by volume). Ten adults or 100 larvae wereimmersed in the test concentrations for one minute,test solutions were decanted, treated ticks were driedover filter papers and kept at 28 ◦C and 85% RH in20 ml glass vials. Three replicates were used in eachtreatment, according to Gupta et al.23 Digitoxin wastested only against adults.

2.5 Delayed effects on feeding, moultability andegg productionSamples of larvae (three replicates, 30 each) and adults(three replicates, 10 each), that survived the Calotropisextract and azadirachtin treatments (see Table 3),along with those of controls, were transferred to feedon rabbits. In the case of larvae treatments, periodsrequired for moulting to nymphal stage, percentagemoultability and average weight of engorged nymphswere recorded. In the case of adult treatments, averageweights of engorged females were recorded, andfemales were allowed to oviposit in secured glasstubes, kept at 28 ◦C and 85% RH, and averages ofegg number and egg weight were estimated. Weightsof engorged nymphs or females were taken as afunction of feeding activity, and periods of moultingand percentage moulting as functions of moultability.

2.6 Statistical analysisProbit analysis of results of acute toxicity wascarried out according to Finney,38 after correctionfor mortality in controls according to Abbott’sformula,39 and effects on feeding, moultability andegg production were compared using t-tests.

3 RESULTS AND DISCUSSION3.1 Acute toxicityResults of acute toxicity testing by the contactor the dipping method (Table 1) indicated thatdigitoxin was the most potent material testedagainst adult H dromedarii, followed by the glycosidalextract. The LC50 values of digitoxin and theextract against adults were, by contact, 502 and1185 mg litre−1 (4.08 and 9.63 µg cm−2, respectively),and 409.9 and 1096 mg litre−1, respectively, bydipping. Both materials were slightly more effectiveby dipping than by contact. Digitoxin was abouttwo times more effective than the Calotropis extract.The LC50 for both azadirachtin and neem oilagainst adults, either by contact or by dipping,was >5000 mg litre−1. The cardenolide extract ofC procera was more toxic to larvae than azadirachtin,the contact and dipping LC50 values for the extractand azadirachtin being 6.16, >20.3 µg cm−2 and587.7 and >2500 mg litre−1, respectively. Table 2shows that digitoxin and Calotropis extract wereeffective against adult H dromedarii at 2363 and2539 mg litre−1 respectively, while the acaricidescoumaphos, carbaryl, malathion, chlorpyrifos andphosalone (used as commercial formulations) arereported to be effective against Hyalomma spp at1500, 5000, >7500, 2500 and 5000 mg AI litre−1,respectively. The botanical preparations pestobanand the multi-component AV/EPP/14 (Table 2),had reasonable effects against Hyalomma spp, butonly at unusually high concentrations (1 × 105 and2.5 × 105 mg litre−1).8,22,40,41 At 25 000 mg litre−1, anethanolic crude extract of C procera (leaves and stems)was not effective against the tropical cattle tick,

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Table 1. Acute contact and dipping toxicity of digitoxin, Calotropis extract, azadirachtin and neem oil to adults and larvae of Hyalomma dromedarii

Stage (method) MaterialLC50 (mg litre−1)

(95% FL)LC95 (mg litre−1)

(95% FL) Slope

Adult (dipping) Digitoxin 409.9 (355.3–472.7) 1827 (1402–2383) 2.53Calotropis extract 1096 (1024–1173) 2239 (1981–2531) 5.3Azadirachtin >5000Neem oil >5000

Adult (contact) Digitoxin 502 (439–574) 2363 (1759–3177) 2.45Calotropis extract 1185 (1106–1270) 2539 (2207–2922) 4.97Azadirachtin >5000

Larvae (dipping) Calotropis extract 587.7 (536.1–644.3) 1555 (1318–1835) 3.89Azadirachtin >2500

Larvae (contact) Calotropis extract 757.2 (699.6–819.5) 1654 (1439–1902) 4.85Azadirachtin >2500

Table 2. Effective concentrationsa of different materials against Hyalomma spp

Material Species Stage(s) (method)Concentrationa

(mg litre−1) Reference

Digitoxin H dromedarii Adults (contact) 2363 Table 1Calotropis extract H dromedarii Adults (contact) 2539 Table 1Chlorpyrifos H dromedarii Mixedb (spray) 2500 Khan and Srivastava41

Phosalone H dromedarii Mixedb (spray) 5000 Khan and Srivastava41

AV/EPP/14 H dromedarii Mixedb (spray) 2.5 × 105 Ravindra et al40

Pestoban H a anatolicum Larvae (spray) 1 × 105 Maske et al22

Malathion H a anatolicum Adult (dipping)c >7500 Bagherwal and Sisodia8

Coumaphos H a anatolicum Adult (dipping)c 1500 Bagherwal and Sisodia8

Flumethrin H a anatolicum Adult (dipping)c 30 and 45 Bagherwal and Sisodia8

Carbaryl H a anatolicum Mixed (spray) 5000 Basu and Halder17

a The concentration required to cause ≥95% mortality in test ticks.b Animals infested with mixed stages were sprayed with mentioned materials; the number of live ticks was counted on the animal’s body before andafter treatment.c In vitro dipping trials were carried out by the method used in the present work.

Boophilus microplus Canestrini, (46% mortality),21

although the cardenolide content of this particularextract was very low because the authors used thewhole ethanol extract, not a purified cardenolidefraction; the cardenolide content of C procera freshleaves was found to be only 0.0408% (0.204%, basedon dry weight).42

3.2 Delayed effects on feeding, moultability andegg productionLarvae that survived the 1000 mg litre−1 treatment ofCalotropis extract developed normally as control larvae,neither the moultability nor the weight of engorgedfemales being affected by this concentration (Table 3).

At this concentration, azadirachtin showed someeffects on larvae, and caused significant reduction inboth moultability and weight of engorged nymphs. At2500 mg litre−1, azadirachtin caused 60 and 29.4%reduction in moultability and weight of engorgednymphs, respectively; the period required for moultingto nymphal stage was prolonged in comparisonwith the control group, 24–31 and 15–21 days,respectively. However, azadirachtin had no effect onthe parameters tested in the case of adult females, upto 5000 mg litre−1.

Neem oil (technical grade) had previously beenshown to be only 60–75% effective on cattle infestedwith Hyalomma anatolicum anatolicum Koch, andfemales treated with it oviposited;24 however, it wasreported that control of Hyalomma anatolicum excava-tum Koch could be achieved at 3.2% concentrate ofthe neem seed oil, Neem Azal F.25 An ether extractof neem seeds affected the reproductive efficacy of thecattle tick, B microplus, at a 1:5 dilution,43 but neemoil, azadirachtin and related compounds did not causeany mortality in this species.23 Mixtures of neem oilwith other plant oils were used at 1 × 105 mg litre−1 tocontrol B microplus under field conditions;44 howeverthese concentrations are too high to be used in prac-tice. It seems that the effects of cardiac glycosides onticks are acute; we have noticed a similar phenomenonduring a previous study on land snails.35

4 CONCLUSIONThe effects of both digitoxin and the glycosidalextract are considered promising when comparedwith the effects of commercial pesticides and otherbotanical materials that have been tried againstHyalomma spp. Azadirachtin is a very potent insecti-cide which affects feeding, development and moulting

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Table 3. Delayed effects of azadirachtin and Calotropis procera extract on adults and larvae of Hyalomma dromedarii

Larvae Adults

MaterialaConcentration(mg litre−1) Method

Moultability(days)

Moultabilityb

(%)Engorged wtb,c

(mg) (±SD)Number of

eggsb (±SD)Egg weightb

(mg) (±SD)Engorged wtb,c

(mg) (±SD)

Aza 5000 Dipping ntd nt nt 6124 (±740) a 340 (±56) a 970 (±215) aContact nt nt nt 5980 (±640) a 390 (±48) a 1030 (±270) a

Aza 2500 Dipping 24–31 40 b 24 (±3.8) b nt nt ntContact 22–28 60 b 27 (±4.6) b nt nt nt

Aza 1000 Dipping 23–30 45 b 25 (±4.5) b nt nt ntContact 21–28 60 b 28 (±4.5) b nt nt nt

Extract 1000 Dipping 14–19 98 a 33 (±4) a 6120 (±712) a 360 (±40) a 920 (±136) aContact 14–19 100 a 36 (±5) a 5870 (±624) a 380 (±45) a 980 (±110) a

Aza 500 Dipping 20–26 60 b 28 (±4.6) b nt nt ntContact 18–25 75 a 30 (±5.1) a nt nt nt

Aza 250 Dipping 18–25 75 a 30 (±5.1) a nt nt ntContact 15–22 85 a 32 (±5.6) a nt nt nt

Control 0.0 Dipping 15–21 100 a 34 (±5.8) a nt nt ntContact 14–21 100 a 35.5 (±6.1) a 6850 (±720) a 420 (±65) a 1120 (±205) a

a Aza = azadirachtin.b Means followed by similar letters within the same column are not significantly different at P = 0.05.c Weight of engorged nymphs or females.d nt = not tested.

of insect larvae from different orders at very lowconcentrations;36,45–48 it has no or negligible effectson the ticks tested, however. Pestoban, AV/EPP/14and most of the botanicals that have been triedagainst Hyalomma spp are not effective against ticksat the usual concentrations used to control pests.Cardiac glycosides and plants containing them aretoxic to mammals.49–51 The latex of C procera causeddeath of goats treated at 1 ml kg−1 body weight viathe oral route or at 0.005 ml kg−1 body weight perday via the intravenous or intraperitoneal route atbetween 20 min and 4 days;51 however, our previ-ous findings of the promising pesticidal properties ofcardiac glycosides,32,33,35,36 together with the presenteffects of these materials against ticks attract atten-tion to these compounds; these compounds maybe leads for the synthesis of new effective pesti-cides (eg by modifying glycosidic group, analoguesof cardiac glycosides); the structure of cardiac gly-cosides in C procera is described elsewhere.42,52 Themode of action of these cardenolides, the inhibitionof Na+, K+-ATPase, may be exploited in detectingother, structurally simpler, compounds with the samemode of action.53 We expect such compounds tohave broad-spectrum pesticidal activity against ticks,snails and insects. To our knowledge, the presentwork is the first record for the acaricidal propertiesof digitoxin and cardenolide extracts derived fromC procera.

ACKNOWLEDGEMENTSThe Agricultural Research Centre, College of Agricul-ture, King Saud University, Saudi Arabia, supportedthis work.

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