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Laboratory Effects ofIngestion of Azadirachtin byTwo Pests ( Ceratitis capitataand Spodoptera exigua )and Three Natural Enemies( Chrysoperla carnea ,Opius concolor and Podisusmaculiventris )E. Vinuela , A. Adan , G. Smagghe , M.Gonzalez , Ma. P. Medina , F. Budia , H. Vogt & P.Del EstalPublished online: 28 Jun 2010.

To cite this article: E. Vinuela , A. Adan , G. Smagghe , M. Gonzalez , Ma. P.Medina , F. Budia , H. Vogt & P. Del Estal (2000) Laboratory Effects of Ingestionof Azadirachtin by Two Pests ( Ceratitis capitata and Spodoptera exigua ) andThree Natural Enemies ( Chrysoperla carnea , Opius concolor and Podisusmaculiventris ), Biocontrol Science and Technology, 10:2, 165-177, DOI:10.1080/09583150029305

To link to this article: http://dx.doi.org/10.1080/09583150029305

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Biocontrol Science and Technology (2000) 10, 165 ± 177

Laboratory EVects of Ingestion of Azadirachtin by TwoPests (Ceratitis capitata and Spodoptera exigua) and ThreeNatural Enemies (Chrysoperla carnea, Opius concolor and

Podisus maculiventris)

E. VINÄ UELA,É A. ADAÂ N,É G. SMAGGHE,Ê M. GONZAÂ LEZ,ÉMa

Å . P. MEDINA,É F. BUDIA,É H. VOGTË and P. DEL ESTAL1

1 Proteccio n de Cultivos, Escuela Te cnica Superior de Ingenieros Agro nomos,E-28040 Madrid, Spain; 2 Laboratory of Agrozoology, Department of Crop

Protection, Faculty of Agricultural and Applied Biological Sciences, Universityof Gent, Coupure Links 653, B-9000 Gent, Belgium; 3 Federal Biological

Research Centre for Agriculture and Forestry, Institute for Plant Protection inFruit Crops, Schwabenheimer Str. 101, D-69221 Dossenheim, Germany

(Received for publication 14 September 1998; revised manuscript accepted 5 December 1999)

The eVects of azadirachtin on two pests: neonate larvae and newly emerged adults of Ceratitiscapitata (Wiedemann) and last instar larvae of Spodoptera exigua (HuÈ bner); and threenatural enemies: newly emerged adults of Opius concolor SzeÁ pligeti, second instar larvae ofChrysoperla carnea (Stephens), and Wfth instar nymphs of Podisus maculiventris (Say) werestudied in laboratory. Adult insects were exposed to a non-oil formulation of azadirachtin(Align , emulsiWable concentrate, 3.2% azadirachtin , Sipcam Inagra, Spain) via their drinkingwater and immature instars were reared in the presence of the insecticide-treated diet. Thenatural enemies were exposed to at least the maximum Weld recommended concentration ofthe insecticide (0.15% v/v). Azadirachtin was highly toxic to neonate larvae of C. capitataand prevented adult emergence at a concentration of 1 mg a.i. l 2 1. When adults were fed theinsecticide at the maximum recommended concentration, their survival was not aVected butegg laying was totally inhibited. Last instar S. exigua larvae were also very susceptible(LC50 5 7.7 mg a.i. l 2 1) and at a concentration of 10 mg a.i. l 2 1 fecundity of surviving adults,and egg fertility, were reduced by 72 and 85%, respectively. EVects on O. concolor were large,and signiWcant reductions in longevity, percentage of attacked hosts, and progeny size perfemale, were recorded. The predator P. maculiventris was much less sensitive to azadirachtin,but slight reductions in survival of emerged adults and of reproductive parameters occurred.The insecticide had no signiWcant eVect on C. carnea larvae fed with treated Sitotrogacerealella (Oliver) eggs, probably because of its inability to penetrate inside the egg.

Keywords: azadirachtin , toxicity, side-eVects, Ceratitis capitata, Spodoptera exigua, Chryso-perla carnea, Opius concolor, Podisus maculiventris

Correspondence to: E.VinÄ uela. Tel: +34 91 33657 74;Fax: +34 91 54348 79; E-mail: evinuela@pvb.etsia.upm.es

ISSN 0958-3157 (print)/ISSN 1360-0478 (online)/00/020165-13 � 2000 Taylor & Francis Ltd

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INTRODUCTION

Biological control is an essential component of integrated pest management (IPM) andintegrated production of crops. However, because a sole method of pest control is seldominsuYcient to keep pests under economic threshold levels, requirements of this control tacticmust be integrated with the needs of diVerent techniques such as chemical pesticides(Hokkanen, 1997). An essential premise for the use of pesticides in IPM is to ascertain theircompatibility with bene® cial organisms, because it is known that pesticides can modify theinter-relationships of species in ecosystems, and that frequently parasitoids and predatorssuVer greater mortality than do their phytophagous hosts (Pimentel, 1992). But not allpesticides produce the same harmful eVects on bene® cials, and the simultaneous use ofenemies and pesticides can be achieved by applying selective control agents or by makingan appropriate timing of those products that only aVect certain life stages of the bene® cial(Jacas & VinÄ uela, 1994a).

Among the more environmentally friendly alternatives to the use of broad-spectrumchemicals for the control of pests of economic importance are naturally derived controlproducts. Azadirachtin, a highly oxidized limonoid, is a botanical pesticide formed by agroup of closely related isomers mainly obtained from the seed kernels of the neem treeAzadirachta indica A. Juss (Schmutterer, 1990). Its main mode of action seems to beinhibition of release of prothoracicotropi c hormones and allatotropin s (Banken & Stark,1997). The compound has gained more and more attention in recent years as a componentof integrated pest management programmes because it has been reported to be less toxic tobene® cials than to pests (Stark et al., 1992; Sipcam Inagra, 1996; Banken & Stark, 1997).

The Mediterranean fruit ¯ y Ceratitis capitata (Wiedemann) (Diptera, Tephritidae ) andthe beet armyworm Spodoptera exigua (HuÈ bner) (Lepidoptera, Noctuidae) are two pests ofworldwide importance. The former attacks more than 250 subtropical and deciduous fruitsin the Mediterranean region (Fimiani, 1989), and in Spain since 1955 the severity of damagecaused to citrus crops led to the establishment of mandatory control measures (Ada n et al.,1996). The beet armyworm S. exigua is a polyphagou s noctuid of worldwide importance inagriculture, horticulture and ornamentals. This pest is very much feared in western Europeand the Mediterranean region due to the severe losses it causes in greenhouses (Van de Vrie,1977; Marco & VinÄ uela, 1994). Moreover, during the last decade, the extensive use ofclassical insecticides has resulted in failure of control due to development of resistance(VinÄ uela, 1998; Smagghe et al., 1999).

Opius concolor SzeÁ pligeti (Hymenoptera, Braconidae) is an endoparasitoid of the olive ¯ yBactrocera oleae (Gmelin), one of the key pest of this crop in the Mediterranean region;O. concolor is easily mass-reared in laboratory in the host C. capitata (Jacas & VinÄ uela, 1994b).

Chrysoperla carnea (Stephens) (Neuroptera, Chrysopidae) is a general entomophagouspredator, which is commercially produced in many countries for use as a biocontrol agentagainst aphids. This species has been selected as one of the relevant bene® cials to be testedfor pesticide registration in the European Union (Barret et al., 1994).

Podisus maculiventris (Say) (Hemiptera, Pentatomidae) is a generalist predatory pentato-mid that feeds actively on larvae of many lepidopterans of economic importance (De Clercqet al., 1995). Its commercial use aiming at controlling S. exigua larvae has been recentlystarted in European greenhouses.

In this study we have examined the eVects of azadirachtin on two pests and three naturalenemies, representatives of diVerent orders, families and genera. The compound was appliedvia ingestion, and we studied toxicity and changes in development in the life stage treated,and eVects on reproduction by evaluating fecundity and egg viability.

MATERIALS AND METHODS

InsecticidesThe non-oil commercial formulation of azadirachtin, Align (3.2% azadirachtin, emulsi® able

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AZADIRACHTIN EFFECTS ON NATURAL ENEMIES AND PESTS 167

concentrate, Sipcam Inagra, Spain), was used in all experiments. In all the assays, freshsolutions were prepared in distilled water.

InsectsThe C. capitata and O. concolor specimens used in the tests were obtained from laboratorycultures maintained in Madrid following the standard procedures of Albajes and Santiago-AÂ lvarez (1980) and Jacas and VinÄ uela (1994b).

Colonies of C. carnea and P. maculiventris originated from eggs obtained from theInstitute for Plant Protection in Fruit Crops of Dossenheim (Germany) and University ofGent (Belgium), respectively, and were mass-reared in Madrid for at least three generationsprior to the assays. C. carnea larvae were reared on Sitotroga cerealella (Oliver) eggs, andadults were fed a nutritive mixture of 1 egg, 1 yolk, 30 g honey, 30 g brewer’s yeast ¯ akes,50 g wheat germ, 20 g fructose, 15 ml condensed milk and 50 ml distilled water as describedby Vogt et al. (1998a,b) . Nymphs and adults of P. maculiventris were fed Spodopteralittoralis (Boisduval) larvae according to De Clercq et al. (1988) and VinÄ uela et al.(1998). Rearing conditions were 25 6 2ë C, 75 6 5% relative humidity (RH) and 16 h lightphotoperiod.

All developmental stages of a continuous colony of S. exigua were maintained at 23 6 2ë C,70 6 5% RH and 16 h light photoperiod in Gent. Larvae were fed a Poitout based arti® cialdiet and adults a 15% solution of honey in water according to Smagghe and Degheele(1994).

Test MethodsEvery experiment consisted of at least three replicates of 10± 15 insects per concentrationlevel (expressed in active ingredient), and control specimens were treated with distilled water.For pests, concentrations and treatment methods were chosen in accord with our previousexperience and biological characteristics of the species. Following IOBC recommendations(Hassan, 1994), bene® cials were exposed to a concentration of at least 48 mg a.i. l - 1 whichis equivalent to the maximum ® eld recommended concentration of Align (150 ml hl - 1).Experiments were always done with the most exposed life stage of the enemies: adults ofthe parasitoid and larvae or nymphs of the predators.

In reproductive studies, fecundity was based on scores of four replicates of 3± 5 pairs/cagefor C. capitata, at least two replicates of 2± 3 pairs/cage for S. exigua and of eight replicatesof individua l pairs for the other species. Fertility was based on the hatching of about 100eggs per concentration level on two diVerent days. Adult survival was also monitoredthroughout the life span in O. concolor and during a 7 to 10-day period for the other species.

Ceratitis capitata. Our study focused on two of the vulnerable stages of this ¯ y: neonatelarvae (0± 12 h), that can be found in recently stored fruits, and adults, which are the currenttarget of ® eld treatments (Ada n et al., 1996). EVects on neonate larvae and newly emergedadults (0± 24 h) of this species were studied following the methods of VinÄ uela et al. (1993)for larvae, and of Budia and VinÄ uela (1996) for adults. Larvae were reared in the presenceof azadirachtin-treated diet at concentrations of 0.1 and 1 mg l - 1 . Azadirachtin was alsofed ad libitum to groups of ® ve newly emerged pairs of ¯ ies for a 7-day period, atconcentrations of 48 and 100 mg l - 1 . Insecticide solutions were oVered continuously fromadult emergence in glass troughs covered by Para® lm with a piece of Spontex wiper. Amixture of sucrose and autolysed brewer’s yeast was supplied as food in plastic containers.

Spodoptera exigua. Newly moulted (0± 12 h) last (L5 )-instar larvae were orally treated inaccordance with Smagghe and Degheele (1994). Ten diVerent concentrations of azadirachtinranging from 0.1 up to 100 mg l - 1 were prepared in water, and the surface of the arti® cialdiet was uniformly treated with 50 l l. Controls were treated with water alone. Mortalitywas scored 7 days after treatment. Control specimens had entered the pupal stage at this

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168 E. VINÄ UELA ET AL.

time. In addition, the fresh weight gain of larvae and pupae was measured using an analyticalSartorius balance, and means and SE were expressed as percentages of the control groups.Adult eclosion of surviving pupae was recorded, and expressed as a percentage of the totalnumber of larvae treated. Groups of 2± 3 pairs were kept in plastic containers with the insidewalls covered with paper providing oviposition places (Smagghe & Degheele, 1994) andadult longevity, fecundity (calculated as the number of eggs deposited during 10 days ofoviposition ) and fertility were evaluated.

Opius concolor. Groups of 15 newly emerged (0± 12 h) females per concentration wereexposed continuously to azadirachtin in their drinking water at a dose of 48 mg l - 1 . Insectswere also provided with a mixture of brewer’s yeast and sugar (1:4). Longevity and bene® cialcapacity of the wasps (measured as the percentage of attacked hosts and number of progenyper female), were studied following the method of Jacas and VinÄ uela (1994b).

Chrysoperla carnea Newly moulted (0± 12 h) L2 -larvae were individuall y placed in anuncovered 9 cm Petri dish with Fluon coated walls to prevent insect escape, with a ® lterpaper on the bottom. Insects were fed continuously with S. cerealella eggs treated atconcentrations ranging from 10 to 10 000 mg l - 1 azadirachtin following a modi® cation ofthe method described by Vogt et al. (1998a,b) . Eggs were treated under a Potter PrecisionSpray Tower, with a standard deposit of 1.89 6 0.05 mg cm - 2 (1 ml; 50 kPa) and wereallowed to dry at room temperature in a fume hood for 24 h before being oVered to larvae.Application rates were calculated using the PIEC formula (the predicted initial environmentalconcentration of a pesticide) developed by Barret et al. (1994), considering a 0.4 factor and1000 l ha - 1 .

Podisus maculiventris. Newly moulted (0± 12 h) N5 -nymphs were placed individuall y in a9 cm Petri dish lined with a ® lter paper and were fed the insecticide continuously in theirdrinking water at a concentration of 48 mg l - 1 . After the ® rst 24 h (to ensure that they hadtaken the insecticide initially) , the predatory nymphs were provided with S. littoralis L5

larvae ad libitum (De Clercq et al., 1995; VinÄ uela et al., 1998).

Statistical AnalysisDepending on the species, the following parameters were recorded: mortality during larval,nymphal, pupal and/or adult stage; duration of the instar; weight; malformations, adultlongevity, fecundity and fertility. In C. capitata, larval mobility was also scored by countingthe number of popped pupae (pupae which jump oV the diet to pupate in a drier substratum)as a percentage of the total number of pupae recorded. Data, presented in tables as meanswith standard errors, were analysed by one-way analysis of variance using Statgraphics(STSC, 1987). Where appropriate, percentages were transformed and means were separatedby the least signi® cant diVerence (LSD) option (P< 0.05). If the F from ANOVA was notsigni® cant, a Bonferroni test was applied.

Mortality data of S. exigua larvae was subjected to POLO-PC to estimate probitregressions (LeOra Software, 1994). Lethal concentrations in mg l - 1 (LC1 0 , LC5 0 andLC9 0 ) and 95% ® ducial limits were calculated for records taken after 7 days of feeding onthe insecticide, which coincided with control moulting into the pupal stage.

RESULTS AND DISCUSSION

Azadirachtin has been reported to have deterrent, anti-ovipositional , antifeeding, growth-regulating, fecundity and ® tness-reducing properties on insects (Schmutterer, 1990), andseveral of these detrimental eVects were observed in our assays.

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AZADIRACHTIN EFFECTS ON NATURAL ENEMIES AND PESTS 169

TABLE 1. In¯ uence of the developmental stage on the susceptibility of C. capitata toazadirachtin

C. capitataNeonate larvae fed on treated diet

Concentrations % Larval % Pupal % Popped % Adultmg a.i. l - 1 mortalitya mortalitya pupaea* emergencea*

Control 20.7 6 3.6a 1.3 6 1.4a 93.1 6 2.0a 78.0 6 4.7a0.1 24.7 6 6.8a 4.0 6 2.0ab 91.9 6 0.8a 71.3 6 4.5a1 86.7 6 4.1b 13.3 6 4.1b 11.4 6 6.0b 0 6 0b

Adults exposed via ingestion in their drinking water

Concentrations Eggs/female,mg a.i. /l % Mortality at 7 daysb 8 daysa** % Eclosion

Control 10.0 6 4.1a 235.4 6 28.8a 87.5 6 1.348 16.0 6 7.1a 0 6 0b Ð100 15.0 6 2.9a 1.7 6 1.7b 0 6 0

Within the same column, data followed by the same letter do not diVer signi® cantly.*Compared with total number of larvae.**Data represent the mean of four replicates of 3± 5 pairs of ¯ ies /cage.(P 5 0.05; aLSD; bBonferroni mean separation.)

C. capitataThe results of our studies indicated that Align is a good larvicide against C. capitata. Atthe concentrations tested, azadirachtin was highly toxic to neonate larvae (Table 1) andexhibited a potency similar to that of the organophosphat e fenthion (Ada n et al., 1996). Ata concentration much lower than the maximum recommended, 1 mg l - 1 , a signi® cantincrease in larval and pupal mortality, a decrease in mobility (measured as number ofpopped pupae) and a complete lack of adult emergence were observed.

The adults of C. capitata were less susceptible to azadirachtin. When they ingested theinsecticide continuously from emergence, the mortality scored at a concentration as high as100 mg l - 1 (twice the maximum ® eld recommended concentration), was not signi® cantlydiVerent from that of controls. By contrast, Ada n et al. (1998) had previously reported thatNeem-e , which is an oil-based formulation of azadirachtin (0.03%), was very eVective insuppressing adult survival at a concentration of 50 mg l - 1 .

However, Align was harmful to fruit ¯ y adults because egg laying was almost totallyinhibited at the two doses studied. The number of eggs obtained from treated individualswas so low that even though none of them hatched, it is diYcult to give a de® nitiveconclusion concerning fertility. The reduction in fecundity seems to be reversible becausepreliminary results have shown that there is recuperation in egg laying when adults areoVered the insecticide only during the preoviposition period (3 days in this insect) (Ada n,unpublished results).

In contrast with the above reported results, the only detrimental eVect reported by Starket al. (1990) in C. capitata, after treatment of the pupation medium with a neem seed extractcontaining 7.3 g of azadirachtin, was a higher mortality. Therefore, for a given species, thetoxicity and the life stages controlled by azadirachtin seem to be highly dependent on theformulation applied and on the application method. Two factors that could account forsuch a diVerence are the presence of more than one active ingredient in the formulatedproduct, and the type of formulation applied, aqueous or oil-based (Ermel & Kleeberg,1995; Ada n et al., 1998).

Spodoptera exiguaLarval toxicity assays demonstrated that Align was toxic at relatively low concentrationsto last-instars of the beet armyworm. Probit analysis of mortality percentages (Figure 1(a))

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170 E. VINÄ UELA ET AL.

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AZADIRACHTIN EFFECTS ON NATURAL ENEMIES AND PESTS 171

estimated an LC5 0 of 7.7 mg l - 1 (® ducial limitis 4.1± 13.7) and the slope was 2.2 6 0.4(v 2 5 17.1, df 5 7). The LC1 0 value was estimated to be 2.0 (0.4± 3.8), and the LC90

29.7 (16.1± 81.9).Surviving larvae showed inhibition of larval growth, partially due to the potent antifeedant

eVect of azadirachtin. Typically, larvae tried to escape from the treated arti® cial diet surfaceby biting a hole in the corner of the well and then eating underneath and avoiding thetreated surface. This eVect was obvious at concentrations of 10± 100 mg l - 1 . As shown inFigure 1(b), such larvae had signi® cantly reduced fresh weight, which in turn led to highpupal mortality.

It has also been documented that for many caterpillars, disruption of moulting at thetime of pupation is perhaps the most dramatic physiological eVect of azadirachtin. Larvaefail to initiate the larval± pupal moult, or moult into larval ± pupal intermediates (Schmuttereret al., 1983; Isman, 1995). In some noctuids such as Peridroma saucia (HuÈ bner), Heliothisvirescens (Fabricius) and Spodoptera exempta (Walker), pupation was disrupted by azadirach-tin at doses as low as 0.5± 1 l g per larva (2± 4 l g g - 1 ). The current observations of pupalabnormalities in the beet armyworm give rise to the suggestion that azadirachtin interfereswith the neuroendocrine control of the moulting process.

Pupal weight measurements of surviving pupae following treatment at the last-larval stagewith diVerent doses of Align are shown in Figure 1(c). It was clear that among the treatedlarvae which pupated successfully, those which showed a signi® cant lower fresh weightgenerally failed to develop into normal adults.

In the current assay, it was clear that adult emergence of S. exigua was aVected (Figure1(d)), because only 36% of larvae treated with 10 mg l - 1 , showed normal emergence. Higherconcentrations resulted in no emergence or abnormal lethal adult formation. So, in additionto eVects on larval± pupal moulting, signi® cant mortality was noted among pupae resultingfrom concentrations that permitted normal pupation. Furthermore, many of these pupaegave rise to deformed adults. The most common deformities that we observed were those ofthe wings, which failed to expand properly. This agrees with the observations reported byIsman (1995) in normal-looking pupae of the diamondblack moth, Plutella xylostella (L.),from which moths failed to emerge. In addition, dissection of these pupae by the latterauthor revealed fully-formed moths, several alive, but apparently unable to emerge. All theseeVects strengthen the suggestion that the toxic eVect of azadirachtin is via neuroendocrinedeteriorations related to moulting and metamorphosis. Apparently, recent observations intreated larvae and pupae of S. exigua with the nonsteroidal ecdysone agonist tebufenozideshowed a similar eVect of miniature and abnormally curled wings (Carton et al., 1998). Thelatter insecticide is known to interfere with the receptor of the natural insect moultinghormone, but treatment with tebufenozide in lepidoptera may also lead to inhibition of theinsect eclosion hormone (Truman, 1992). As reviewed by Riddiford (1985), this eclosionhormone is produced in the brain neurosecretory cells and is necessary to initiate normalecdysis.

The number of eggs produced by surviving adults (Figure 1(e)) and the percentage of egghatching (Figure 1(f)) were recorded. In the control groups, mean fecundity was 169 eggsper female (SE 5 29) and fertility was 88% (SE 5 5). For the insecticide treatments, data onegg-laying are expressed as a percentage of the average controls. Azadirachtin treatment inlarval stages had a pronounced eVect on egg laying and fertility. A concentration of10 mg l - 1 caused a reduction of about 72% of oviposition and of about 85% of egg hatching.With lower concentrations, the eVect on reproduction was not signi® cantly diVerent fromthe control groups. However, at 10 mg l - 1 only nine adults had developed normally. Atpresent, no clear details are known on the mechanism of action of azadirachtin in females.In addition, azadirachtin may have reduced the egg fertility via the male adult. Shimizu(1988) reported that this compound interferes with spermatogenesis resulting in sterility asdemonstrated in the cabbage armyworm, Mamestra brassicae (L.).

It is clear from these results that Align gave a strong activity against last instar larvae.

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172 E. VINÄ UELA ET AL.

TABLE 2. EVects of feeding azadirachtin in water to adult O. concolor on the longevityand the bene® cial capacity of the parasitoid

Opius concolorAdults exposed via ingestion in drinking water

Bene® cial capacitya

Concentrationsmg a.i. l - 1 Longevity (days) % Attacked hosts % Progeny

Control 31.4 6 20.7a 87.5 6 4.8a 65.0 6 7.9a48 4.9 6 0.1b 64.1 6 8.2b 43.9 6 4.8b

Within the same column, data followed by the same letter do not diVer signi® cantly(P 5 0.05; LSD mean separation).

aData represent the mean of eight replicates of 3-day pesticide-exposed and isolatedfemales. Twenty L3 C. capitata larvae were oVered daily to them for 2 h during a periodof 4 days.

This should open possibilitie s to use azadirachtin for controlling Spodoptera pests. Thisconcurs with Saxena (1987) who reported on the usefulness of azadirachtin to controlseveral pests in rice from laboratory and small-scale ® eld trials. In addition, we observed inthe study here that azadirachtin applied to the last-larval instar aVected pupal and adultdevelopment and caused a signi® cant reduction in egg laying and fertility in surviving adults.

Opius concolorOur study revealed that azadirachtin was toxic to this parasitoid when adults were fed theinsecticide continuously during their life span (Table 2). Align gave a large reduction(84.4%) in the longevity of treated wasps, which could be related to the reported antifeedingeVect of this product (Schmutterer, 1990). We did not observe any change in the behaviourof treated wasps but we know that the ingestion of liquids is a limiting factor for the survivalof this bene® cial hymenopteran The bene® cial capacity of O. concolor females was alsoimpaired, in that the number of attacked hosts and progeny size were decreased by 15.6 and32.5%, respectively. Based on these laboratory results, the product was classi® ed accordingto the IOBC categories (Hassan, 1994) as moderately harmful (class 3) based on eVects onlongevity, and slightly harmful (class 2) according to eVects on the bene® cial capacity of thewasps.

Align has been described as a safe product for many bene® cials (Sipcam Inagra, 1996)but hymenopterans seem to be among the most sensitive species. Negative eVects onlongevity and on reproduction of parasitoids after azadirachtin treatment, have beendescribed in the literature (Schmutterer, 1995; Ruiz et al., 1998). Moreover, O. concolor wasfound to be the most sensitive natural enemy out of a list of 22 studied species, representingdiVerent insect orders and families (Croft, 1990).

Chrysoperla carneaAlign was totally harmless for this predator when larvae were fed treated S. cerealella eggsand we did not detect any signi® cant alteration of the parameters measured even at thehighest doses (Table 3). The product was classi® ed as slightly harmful (class 2) due to thereduction in adult emergence at a concentration as high as 10 000 mg l - 1 (41.4%), and asharmless (class 1) according to reproductive eVects at all the doses studied.

Contrary to these results and under similar conditions, Neem-Azal-T/S, an oil formulationof azadirachtin (1% a.i.) (Trifolio, Germany), signi® cantly reduced both the number ofpupae and adults emerged from treated insects, and slightly aVected the reproduction ofadults (Vogt, unpublished data). Moreover Align applied via residual contact at themaximum ® eld recommended concentration to young larva of C. carnea, was also harmful

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AZADIRACHTIN EFFECTS ON NATURAL ENEMIES AND PESTS 173

TABLE 3. Toxicity of azadirachtin to the predator Chrysoperla carnea when L2 larvae were fed continuouslySitotroga cerealella treated eggs

Chrysoperla carneaL2 larvae fed on Sitotroga cerealella treated eggs

Concentrations % Larval % Pupal % Adult Eggs/female,mg a.i. l - 1 mortality mortality emergencea 10 daysb % Eclosion

Control 13.3 6 6.6a 10.0 6 6.8a 82.1 6 11.8a 501.4 6 56.7a 92.2 6 3.5a10 6.6 6 4.2a 30.0 6 4.4a 65.0 6 3.1a 523.0 6 56.2a 92.0 6 6.1a100 10.0 6 4.4a 23.3 6 12.0a 60.0 6 9.2a 555.5 6 108.5a 83.5 6 7.5a1000 13.3 6 4.2a 16.6 6 6.1a 65.8 6 8.7a 618.6 6 38.5a 87.2 6 7.8a10 000 30.0 6 8.4a 26.6 6 4.2a 48.1 6 12.0a

Within the same column, data followed by the same letter do not diVer signi® cantly (P 5 0.05; Bonferronimean separation).

aCompared with pupae.bData represent the mean of eight replicates of pairs kept individually.

and caused malformations of their cuticle and muscles visible under the electron microscope,and totally prevented adult emergence (Vogt et al., 1998b).

The reason for these contradictory results could be related to the inability of Align topenetrate the S. cerealella egg cuticular barriers, and to the feeding habits of the lacewinglarvae which pierce and suck out the prey with their sharp mandibles without contactingthe surface of the prey.

Podisus maculiventrisThe predatory bug was slightly sensitive to Align , and a small direct mortality in treatednymphs, and several delayed eVects on adults were observed (Tables 4 and 5). According to

TABLE 4. EVects of azadirachtin on N5 nymphs of the predatory bug Podisus maculi-ventris exposed via ingestion in the drinking water

Podisus maculiventrisN5 nymphs exposed via ingestion in the drinking water

Concentrations Nymphal weight at Duration of themg a.i. l - 1 % Nymphal mortalitya 5 days (mg)b instar (days)b

Control 0a 76.0 6 3.3a 7.0 6 0.2a48 12.5 6 6.3b 88.6 6 4.2a 7.4 6 0.1a

Within the same column, data followed by the same letter do not diVer signi® cantly.(P 5 0.05; aLSD; bBonferroni mean separation.)

TABLE 5. EVects of azadirachtin on Podisus maculiventris adults when N5 nymphs were fed the insecticidein the drinking water

Podisus maculiventrisN5 nymphs exposed via ingestion in the drinking water

% AdultConcentrations % Adult % Malformed mortality at 8 Eggs/female 8 % Fertilemg a.i. l - 1 emergence adultsa*

daysa daysb**females % Eclosionb

Control 100 6 0.0a 0a 10.0 6 4.1a 119.7 6 14.8a 100 60.4 6 10.4a48 87.5 6 6.3b 36.6 6 3.5b 46.7 6 8.7b 58.0 6 27.3a 44 33.8 6 11.3a

Within the same column, data followed by the same letter do not diVer signi® cantly.*Compared with total number of emerged adults.**Data represent the mean of 8 replicates of pairs kept individually.(P 5 0.05; aLSD mean separation; bBonferroni mean separation.)

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174 E. VINÄ UELA ET AL.

FIGURE 2. Daily cumulative mortality in Podisus maculiventris adults emerged from N5 nymphs fed Alignad libitum at 45 mg l - 1 via the drinking water.

the reduction in adult emergence (12.5%), the product was classi® ed as harmless (class 1),while due to its eVects on reproduction, it was classi® ed as slightly harmful (class 2)(reductions of 51.5, 44 and 56% in fecundity, number of fertile females and eclosion,respectively).

Inhibition of weight gain, and prolonged developmental periods in insects treated withazadirachtin, seem to be rather common, but in P. maculiventris no statistically signi® cantdiVerence in these two parameters was scored between control and treated insects (Table 4).

Predators aVected by azadirachtin did not die during the moulting process, but theemerged adults exhibited several malformations, had a limited mobility, lower ovipositionand egg hatching rates, and a reduced survival (Table 5). In accordance with observationsmade in other species (Stark et al., 1990), daily mortality in P. maculiventris adults wasconsiderably higher than in controls (Figure 2).

Malformations varied, and aVected adults had a sunken pronotum, a wrinkledmesonotum, incompletely formed or twisted wings, abnormal body melanization or hadfailed to shed the old cuticle. All these malformations are rather similar to those describedafter the application of the juvenoid pyriproxyfen to this insect (De Clerq et al., 1995) andare in agreement with the primary mode of action of azadirachtin: the blockage ofmorphogenetic peptid hormones (Banken & Stark, 1997).

Fecundity and fertility were reduced in treated insects as compared to controls (Table 5),but diVerences were not statistically signi® cant, probably because of the high intrinsicvariabilit y detected in these parameters, even in controls. Numbers of fertile females derivedfrom treated larvae were reduced to less than half, but those females that survived theinsecticide treatment exhibited egg laying rates similar to control females.

CONCLUSION

Our studies have shown that Align has a good potency against C. capitata neonate larvaeand S. exigua last instar larvae. According to these results, the insecticide may be a goodcandidate for the control of the fruit ¯ y in post-harvest disinfection because Ada n et al.(1998) have also reported its good ovicidal activity (CL5 0 5 1.4 mg l - 1), but studies shouldbe conducted to determine its penetrating characteristics inside the fruits. Azadirachtin mayalso be a promising insecticide to control Spodoptera pests but its performance under ® eldconditions should be investigated. Some advantages of using azadirachtin are that this

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AZADIRACHTIN EFFECTS ON NATURAL ENEMIES AND PESTS 175

insecticide represents little hazard to operatives (oral LD5 0 in female rats > 5000 mg kg - 1)and is environmentally friendly, being in EPA toxicity class IV (Copping, 1998).

The product was rather harmful in laboratory tests for adults of O. concolor, so its jointuse together with the susceptible life stage of the enemy only seems to be possible with anappropriate timing [Jacas & VinÄ uela (1994a) have reported that pesticides are totally harmlessfor the protected life stage of this enemy]. However, before reaching ® nal conclusions, it isnecessary to complete the whole IOBC sequential testing scheme for adults (Hassan, 1994).A ® rst series of preliminary semi-® eld tests recently indicated that Align was compatiblewith adults of the bene® cial (Gonza lez, unpublished results); however, further testing isrequired before publication .

The insecticide was harmless for fully grown nymphs of P. maculiventris, but severaldelayed eVects on emerged adults were observed. Hence, it is necessary to determine morecompletely its eVects on the reproduction of the bug before reaching a conclusion on itssuitability for use with this predator.

No conclusive results could be drawn from the results scored with C. carnea, but Aligncould be compatible with the predator, as NeemAzal-T/S, an oil formulation of azadirachtin,was found to be totally harmless in the ® eld for C. carnea L2 -larvae (VinÄ uela et al., 1996;Vogt et al., 1998a).

In summary it can be concluded that Align could be compatible with the natural enemiesstudied, but further studies should investigate the total risk of using this compound togetherwith them, because the insecticide can exhibit diVerential toxicity to diVerent life stages andages of the targeted species.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the research support provided by the Spanish Ministryof Education and Culture (Concerted action Spain ± Germany HA97-0005 and projectAGF98-0715) and the Autonomous Community of Madrid (project 06M/022/96) toE. VinÄ uela, and by the DAAD (Deutscher Akademischer Austauschdienst ) to H. Vogt.M. Gonza lez and Ma

Å . P. Medina are recipients of grants from the Autonomous Communityof Madrid. G. Smagghe acknowledges the Belgian National Fund of Scienti® c Research(Brussels) for a post-doctoral fellowship. We also thank Dr J. Jacas for suggesting improve-ments to the manuscript.

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