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Biocontrol Sci Tech 10 (2) 175-187.

Laboratory Effects of Ingestion of Azadirachtin by Two pests (Ceratitis capitata and

Spodoptera exigua) and Three Natural Enemies (Chrysoperla carnea, Opius concolor and Podisus maculiventris)

E. VIÑUELA1, A. ADÁN1, G. SMAGGHE2, M. GONZÁLEZ1, Mª.P. MEDINA1, F. BUDIA1, H. VOGT3 AND P. DEL ESTAL1

1 Protección de Cultivos, Escuela Técnica Superior de Ingenieros Agrónomos, E-28040 Madrid, Spain. 2 Laboratory of Agrozoology, Department of Crop Protection, Faculty of Agricultural and Applied Biological Sciences, University of Gent, Coupure Links 653,B-9000 Gent, Belgium 3 Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection in Fruit Crops, Schwabenheimer Str. 101, D-69221 Dossenheim, Germany

Running title: Azadirachtin effects on natural enemies and pests

Key words: azadirachtin, toxicity, side-effects, Ceratitis capitata, Spodoptera exigua, Chrysoperla carnea, Opius concolor, Podisus maculiventris. Corresponding author: Professor Elisa Viñuela, Protección de Cultivos, E.T.S.I. Agrónomos, E-28040-Madrid, Spain. phone: 34 91 336 57 74 fax: 34 91 336 58 66 E-mail: evinuela@pvb.etsia.upm.es

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SUMMARY

The effects of azadirachtin on two pests: neonate larvae and newly emerged adults of

Ceratitis capitata (Wiedemann) and last-instar larvae of Spodoptera exigua (Hübner); and

three natural enemies: newly emerged adults of Opius concolor Szèpligeti, second-instar

larvae of Chrysoperla carnea (Stephens), and fifth-instar nymphs of Podisus maculiventris

(Say) were studied in laboratory. Adult insects were exposed to a non-oil formulation of

azadirachtin (Align®, emulsifiable concentrate, 3.2% azadirachtin, Sipcam Inagra, Spain) via

their drinking water and immature instars were reared in the presence of the insecticide-

treated diet. The natural enemies were exposed to at least, the maximum field recommended

concentration of the insecticide (0.15% v/v). Azadirachtin was highly toxic to neonate larvae

of C. capitata and prevented adult emergence at a concentration of 1 mg a.i./l totally. When

adults were fed the insecticide at the maximum recommended concentration, their survival

was not affected but egg laying was totally inhibited. Last-instar S. exigua larvae were also

very susceptible (LC50 = 7.7 mg a.i./l) and at a concentration of 10 mg a.i./l fecundity of

surviving adults and egg fertility were reduced by 72% and 85% respectively. Effects on O.

concolor were large and significant reductions in longevity, percentage of attacked hosts and

progeny size per female 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 significant effect on C. carnea

larvae fed with treated Sitotroga cerealella (Oliver) eggs, probably because of its inability to

penetrate inside the egg.

INTRODUCTION

Biological control is nowadays an essential component of integrated pest management

(IPM) and integrated production of crops. However because a sole method of pest control is

seldom insufficient to keep pests under economic threshold levels, requirements of this control

tactic must be integrated with the needs of different techniques such as chemical pesticides

(Hokkanen, 1997). An essential premise for the use of pesticides in IPM is to ascertain their

compatibility with beneficial organisms, because it is known that pesticides can modify the

interrelationships of species in ecosystems and that frequently parasitoids and predators suffer

greater mortality than do their phytophagous hosts (Pimentel, 1992). But not all pesticides

produce the same harmful effects on beneficials and the simultaneous use of enemies and

pesticides can be achieved by applying selective control agents or by making an appropriate

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timing of those products that only affect certain life stages of the beneficial (Jacas & Viñuela,

1994a).

Among the more environmentally friendly candidates to the use of broad-spectrum

chemicals for the control of pests of economic importance are the naturally derived control

products. Azadirachtin, a highly oxidized limonoid, is a botanical pesticide formed by a group of

closely related isomers mainly obtained from the seed kernels of the neem tree Azadirachta

indica A. Juss (Schmutterer, 1990). Its main mode of action seems to be inhibition of release of

prothoracicotropic hormones and allatotropins (Banken & Stark, 1997). The compound has

gained more and more attention in recent years as a component of integrated pest management

programmes because it has been reported to be less toxic to beneficials than to pests (Stark et al.,

1992; Sipcam Inagra, 1996; Banken & Stark, 1997).

The Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Diptera, Tephritidae) and

the beet armyworm Spodoptera exigua (Hübner) (Lepidoptera, Noctuidae) are two worldwide

important pests. The former attacks more than 250 subtropical and deciduous fruits in the

Mediterranean region (Fimiani, 1989) and in Spain since 1955, the severity of damage caused to

citrus crops led to the establishment of mandatory control measures (Adán et al.,1996). The beet

armyworm S. exigua is a polyphagous noctuid of worldwide importance in agriculture,

horticulture and ornamentals. This pest is very much feared in western Europe and the

Mediterranean region due to the severe losses it causes in greenhouses (Van de Vrie, 1977;

Marco & Viñuela, 1994). Moreover, during the last decades, the extensive use of classical

insecticides has resulted in failure of control due to development of resistance (Viñuela, 1998;

Smagghe et al., 1999).

Opius concolor Szèpligeti (Hymenoptera, Braconidae) is an endoparasitoid of the olive

fly Bactrocera oleae (Gmelin), one of the key pest of this crop in the Mediterranean region,

which is easily mass-reared in laboratory in the substitution host C. capitata (Jacas & Viñuela,

1994b).

Chrysoperla carnea (Stephens) (Neuroptera, Chrysopidae) is a general entomophagous

predator, which is commercially produced in many countries for use as a biocontrol agent against

aphids. This species has been selected as one of the relevant beneficials to be tested within

pesticide registration in the European Union (Barret et al.,1994).

Podisus maculiventris (Say) (Hemiptera, Pentatomidae) is a generalist predatory

pentatomid that feeds actively on larvae of many lepidopterans of economic importance (De

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Clercq et al.,1995). Its commercial use has been recently started in European greenhouses aiming

at controlling S. exigua larvae.

In this study we have examined the effects of azadirachtin on two pests and three natural

enemies, representatives of different orders, families and genera. The compound was applied via

ingestion and we studied toxicity and changes in development in the life stage treated and in

some of the insects, on reproduction by evaluating fecundity and egg viability.

MATERIALS AND METHODS

Insecticides

The non-oil commercial formulation of azadirachtin, Align® (3.2% azadirachtin,

emulsifiable concentrate, Sipcam Inagra, Spain), was used in all experiments. In all the assays,

fresh solutions were prepared in distilled water.

Insects

The C. capitata and O. concolor specimens used in the tests were obtained from

laboratory cultures maintained in Madrid following the standard procedures of Albajes and

Santiago-Álvarez (1980) and Jacas and Viñuela (1994b).

Colonies of C. carnea and P. maculiventris originated from eggs obtained from the

Institute for Plant Protection in Fruit Crops of Dossenheim (Germany) and University of Gent

(Belgium) respectively, and were mass-reared in Madrid for at least three generations prior to the

assays. C. carnea larvae were reared on Sitotroga cerealella (Oliver) eggs, and adults were fed a

nutritive mixture of 1egg, 1yolk, 30g honey, 30g brewer’s yeast flakes, 50g wheat germ, 20g

fructose, 15 ml condensed milk and 50ml distilled water as described by Vogt et al. (1998a,b).

Nymphs and adults of P. maculiventris were fed Spodoptera littoralis (Boisduval) larvae

according to De Clercq et al. (1988) and Viñuela et al. (1998). Rearing conditions were 25±2ºC,

75±5% R.H. and 16 h light photoperiod.

All developmental stages of a continuous colony of S.exigua were maintained at 23±2ºC,

70±5% R.H. and 16 h light photoperiod in Gent. Larvae were fed a Poitout based artificial diet

and adults a 15% solution of honey in water according to Smagghe and Degheele (1994).

Test methods

Every experiment consisted of at least three replicates of 10 to 15 insects per

concentration level (expressed in active ingredient), and control specimens were treated with

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distilled water. For pests, concentrations and treatment methods were chosen in accord with our

previous experience and biological characteristics of the species. Following IOBC

recommendations (Hassan, 1994), beneficials were exposed to a concentration of at least 48 mg

a.i./l which is equivalent to the maximum field recommended concentration of Align® (150

cc/hl). Experiments were always done with the most exposed life stage of the enemies: adults of

the parasitoid and larvae or nymphs of the predators.

In reproductive studies, fecundity was based on scores of 4 replicates of 3-5 pairs/cage

for C. capitata, at least two replicates of 2-3 pairs/cage for S. exigua and of 8 replicates of

individual pairs for the other species. Fertility was based on the hatching of about 100 eggs per

concentration level on two different days. Adult survival was also monitored during 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 fly: neonate larvae

(0-12 h), that can be found in recently stored fruits, and adults, which are the current target of

field treatments (Adán et al., 1996). Effects on neonate larvae and newly emerged adults (0-24 h)

of this species were studied following the methods of Viñuela et al. (1993) for larvae and of

Budia and Viñuela (1996) for adults. Larvae were reared in the presence of azadirachtin-treated

diet at concentrations of 0.1 and 1 mg/l. Azadirachtin was also fed ad libitum to groups of 5

newly emerged pairs of flies for a 7-day period, at concentrations of 48 and 100 mg/l. Insecticide

solutions were offered continuously from adult emergence in glass troughs covered by Parafilm®

with a piece of Spontex® wiper. A mixture 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 in

accordance with Smagghe and Degheele (1994). Ten different concentrations of azadirachtin

ranging from 0.1 up to 100 mg/l were prepared in water, and the surface of the artificial diet was

uniformly treated with 50 µl. Controls were treated with water alone. Mortality was scored 7

days after treatment. Control specimens had entered the pupal stage at this time. In addition, the

fresh weight gain of larvae and pupae was measured using an analytical Sartorius 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 total number of larvae treated. Groups

of 2-3 pairs were kept in plastic containers with the inside walls covered with paper providing

oviposition places (Smagghe & Degheele, 1994) and adult longevity, fecundity (calculated as the

number of eggs deposited during 10 days of oviposition) and fertility were evaluated.

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Opius concolor: Groups of 15 newly emerged (0-12 h) females per concentration were exposed

continuously to azadirachtin in their drinking water at a dose of 48 mg/l. Insects were also

provided with a mixture of brewer’s yeast and sugar (1:4). Longevity and beneficial capacity of

the wasps (measured as the percentage of attacked hosts and progeny size per female), were

studied following the method of Jacas and Viñuela (1994b).

Chrysoperla carnea: Newly moulted (0-12 h) L2-larvae were individually placed in an

uncovered 9 cm Petri dish with Fluon coated walls to avoid insect escape with a filter paper on

the bottom. Insects were fed continuously with S. cerealella eggs treated at concentrations

ranging from 10 to 10,000 mg/l azadirachtin following a modification of the method described

by Vogt et al. (1998a,b). Eggs were treated under the Potter Precision Spray Tower, with a

standard deposit of 1.89±0.05 mg/cm2 (1 ml; 50 kPa) and were allowed to dry at room

temperature in a fume hood for 24h before being offered to larvae. Applications rates were

calculated using the PIEC formula (the predicted initial environmental concentration of a

pesticide) developed by Barret et al. (1994), considering a 0.4 factor and 1,000 l/ha.

Podisus maculiventris: Newly moulted (0-12 h) N5-nymphs were placed individually in a 9 cm

Petri dish lined with a filter paper and were fed the insecticide continuously in their drinking

water at a concentration of 48 mg/l. After the first 24 h (to ensure that they had taken the

insecticide initially), the predatory nymphs were provided with S. littoralis L5 larvae ad libitum

(De Clercq et al., 1995; Viñuela et al., 1998).

Statistical analysis

Depending on the species, the following parameters were recorded: mortality during

larval, nymphal, pupal and/or adult stage; duration of the instar; weight; malformations, adult

longevity, fecundity and fertility. In C. capitata, larval mobility was also scored by counting the

number of popped pupae (pupae which jump off the diet to pupate in a drier substratum) as a

percentage of the total number of pupae recorded. Data, presented in tables as means with

standard errors, were analysed by 1-way analysis of variance using Statgraphics (STSC, 1994).

Where appropriate, percentages were transformed and means were separated by the least

significant difference (LSD) option (P<0.05). If the F from ANOVA was not significant, a

Bonferroni test was applied.

Mortality data of S. exigua larvae was subjected to POLO-PC to estimate probit

regressions (LeOra Software, 1994). Lethal concentrations in mg/l (LC10, LC50 and LC90) and

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95% fiducial limits were calculated for records taken after 7 days of feeding the 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 fitness-reducing properties on insects (Schmutterer, 1990), and several

of these detrimental effects were observed in our assays.

Ceratitis capitata

The results of our studies indicated that Align® is a good larvicide against C. capitata. At

the concentrations tested, azadirachtin was highly toxic to neonate larvae (Table 1) and exhibited

a potency similar to that of the organophosphate fenthion (Adán et al., 1996). At a concentration

much lower than the maximum recommended, 1 mg/l, both a significant increase in larval and

pupal mortality, a decrease in mobility (measured as number of popped pupae) and a complete

lack of adult emergence were observed.

The adults of C. capitata were less susceptible to azadirachtin. When they ingested the

insecticide continuously from emergence, the mortality scored at a concentration as high as 100

mg/l (twice the maximum field recommended concentration), was not significantly different

from that of controls. By contrast, Adán et al. (1998) had previously reported that Neem-e®,

which is an oil-based formulation of azadirachtin (0.03%), was very effective in suppressing

adult survival at a concentration of 50 mg/l.

However, Align® was harmful to fruit fly adults because egg laying was almost totally

inhibited at the two doses studied. The number of eggs obtained from treated individuals was so

low that even though none of them hatched, it is difficult to give a definitive conclusion

concerning fertility.The reduction in fecundity seems to be reversible because preliminary results

have shown that there is recuperation in egg laying when adults are offered the insecticide only

during the preoviposition period (3 days in this insect) (Adán, unpublished results).

In contrast with the aboved reported results, the only detrimental effect reported by Stark

et al. (1990) in C. capitata, after treatment of the pupation medium with a neem seed extract

containing 7.3 g of azadirachtin, was a higher mortality. Therefore, for a given species, the

toxicity and the life stages controlled by azadirachtin seem to be highly dependent on the

formulation applied and on the application method. Two factors that could account for such a

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difference are the presence of more than one active ingredient in the formulated product and the

type of formulation applied, aqueous or oil-based (Ermel & Kleeberg, 1995; Adán et al., 1998).

Spodoptera exigua

Larval toxicity assays demonstrated that Align® was toxic at relatively low

concentrations to last-instars of the beet armyworm. Probit analysis of mortality percentages

(Figure 1A) estimated an LC50 of 7.7 mg/l (fiducial limitis 4.1-13.7) and the slope was 2.2±0.4

(χ² = 17.1, d.f.= 7). The LC10 value was estimated to be 2.0 (0.4-3.8), and the LC90 29.7 (16.1-

81.9).

Intoxicated larvae showed inhibition of larval growth, partially due to the potent

antifeedant effect of azadirachtin. Typically, larvae tried to escape from the treated artificial diet

surface by biting a whole in the corner of the well and then eating underneath and avoiding the

treated surface. This effect was obvious at concentrations from 10 up to 100 mg/l. As shown in

Figure 1B, such larvae had significantly reduced fresh weight, which in turn led to high pupal

mortality.

It has also been documented that for many caterpillars, disruption of moulting at the time

of pupation is perhaps the most dramatic physiological effect of azadirachtin. Larvae fail to

initiate the larval-pupal moult or moult into larval-pupal intermediates (Schmutterer et al.,1983;

Isman, 1995). In some noctuids such as Peridroma saucia (Hübner), Heliothis virescens

(Fabricius) and Spodoptera exempta (Walker), pupation was disrupted by azadirachtin at doses

as low as 0.5 to 1 µg per larva (2-4 µg/g). The current observations of pupal abnormalities in the

beet armyworm give rise to the notion that azadirachtin interferes with the neuroendocrine

control of the moulting process.

Pupal weight measurements of surviving pupae following treatment at the last-larval

stage with different doses of Align® are shown in Figure 1C. It was clear that among the treated

larvae which pupated successfully, those which showed a significant lower fresh weight

generally failed to develop into normal adults.

In the current assay, it was clear that adult emergence of S. exigua was affected (Fig. 1D),

because only 36 percent of larvae treated with 10 mg/l, showed normal emergence. Higher

concentrations resulted in no emergence or abnormal lethal adult formation. So, in addition to

effects on larval-pupal moulting, significant mortality was noted among pupae resulting from

concentrations that permitted normal pupation. Furthermore, many of these pupae gave rise to

deformed adults. The most common deformities that we observed were those of the wings, which

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fail to expand properly. This agrees with the observations reported by Isman (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 latter author revealed fully-formed moths,

several alive, but apparently unable to emerge. All these effects may strengthen the notion that

the toxic effect of azadirachtin is via neuroendocrine deteriorations related to moulting and

metamorphosis. Apparently, recent observations in treated larvae and pupae of S. exigua with the

nonsteroidal ecdysone agonist tebufenozide showed a similar effect of miniature and abnormally

curled wings (Carton et al., 1998). The latter insecticide is known to interfere with the receptor

of the natural insect moulting hormone, but treatment with tebufenozide in Lepidoptera may also

lead to inhibition of the insect eclosion hormone (Truman, 1992). As reviewed by Riddiford

(1985), this eclosion hormone is produced in the brain neurosecretory cells and is necessary to

initiate normal ecdysis.

The number of eggs produced by surviving adults (Figure 1E) and the percentage of egg

hatching (Figure 1F) were recorded. In the control groups, mean fecundity was 169 eggs per

female (SE=29) and fertility was 88 % (SE=5). For the insecticide treatments, data on egg-laying

are expressed as a percentage of the average amount of controls. Azadirachtin treatment in larval

stages had a pronounced effect on egg laying and fertility. A concentration of 10 mg/l caused a

reduction of about 72% of oviposition and of about 85% of egg hatching. With lower

concentrations, the effect on reproduction was not significantly different from the control groups.

However, few normal appearing adults were obtained in the current assay because at 10 mg/l

only nine adults had developed normally. At present, no clear details are yet 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 as demonstrated in the cabbage armyworm, Mamestra

brassicae (L.).

Taken together, Align® gave a strong activity against last-instar larvae. This should open

possibilities to use azadirachtin for controlling Spodoptera pests. This concurs with Saxena

(1987) who reported on the usefulness of azadirachtin to control several pests in rice from

laboratory and small-scale field trials. In addition, we observed in the study here that azadirachtin

applied to the last-larval instar affected pupal and adult development and caused a significant

reduction in egg laying and fertility in surviving adults.

Opius concolor

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Our study revealed that azadirachtin was toxic to this parasitoid when adults were fed the

insecticide continuously during their life span (Table 2). Align® gave a large reduction (84.4%)

in the longevity of treated wasps that could be related to the reported antifeeding effect of this

product (Schmutterer, 1990). We did not observe any change in the behaviour of treated wasps

but we know that the ingestion of liquids is a limiting factor for the survival of this beneficial

hymenopteran. The beneficial capacity of O. concolor females was also impaired and the number

of attacked hosts and progeny size were decreased by 15.6% and 32.5%, respectively. Based on

these laboratory results, the product was classified according to the IOBC categories (Hassan,

1994), as moderately harmful (class 3) based on effects in longevity, and slightly harmful (class

2) according to those in the beneficial capacity of the wasps.

Align® has been described as a safe product for many beneficials (Sipcam Inagra, 1996)

but hymenopterans seem to be among the most sensitive species. As such, negative effects in

longevity and on reproduction of parasitoids after azadirachtin treatment, have been described in

literature (Schmutterer, 1995; Ruiz et al., 1998). Moreover, O. concolor was found to be the

most sensitive natural enemy out of a list of 22 studied species, representing different insect

orders and families (Croft, 1990).

Chrysoperla carnea

Align® was totally harmless for this predator when larvae were fed treated S. cerealella

eggs and we did not detect any significant alteration of the parameters measured even at the

highest doses (Table 3 ). The product was classified as slightly harmful (class 2) due to the

reduction in adult emergence scored at a concentration as high as 10,000 mg/l (41.4%), and as

harmless (class 1) due to reproductive effects at all the doses studied.

Contrary to these results and under similar conditions, NeemAzal-T/S, an oil formulation

of azadirachtin (1% a.i.) (Trifolio, Germany), significantly reduced both the number of pupae

and adults emerged from treated insects, and slightly affected the reproduction of adults (Vogt,

unpublished data). Moreover Align® applied via residual contact at the maximum field

recommended concentration to young larva of C. carnea, was also harmful and caused

malformations on their cuticle and muscles visible under the electron microscope, and totally

prevented adult emergence (Vogt et al., 1998b).

The reason of these contradictory results could be related to the inability of Align® to

penetrate the S. cerealella egg cuticular barriers, and to the feeding habits of the lacewing larvae

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who pierce and suck out the prey with their sharp mandibles without entering in contact with the

surface of the prey.

Podisus maculiventris

The predatory bug was slightly sensitive to Align® and both a small direct mortality in

treated nymphs, and several delayed effects on adults were observed (Tables 4 and 5). According

to the reduction in adult emergence (12.5%), the product was classified as harmless (class 1),

while due to its effects on reproduction, it was classified 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 with

azadirachtin, seem to be rather common, but in P. maculiventris no statistically significant

difference in these two parameters was scored between control and treated insects (Table 4).

Predators affected by azadirachtin did not die during the moulting process, but the

emerged adults exhibited several malformations, had a limited mobility, lower oviposition and

egg hatching rates and a reduced survival (Table 5). In accordance with observations made in

other species (Stark et al., 1990), daily mortality in P. maculiventris adults was considerably

higher than in controls (Figure 2).

Malformations varied and affected adults had a sunken pronotum, a wrinkled

mesonotum, incompletely formed or twisted wings, abnormal body melanization or had failed to

shed the old cuticle. All these malformations are rather similar to those described after the

application of the juvenoid pyriproxyfen to this insect (De Clercq et al., 1995) and are in

agreement with the primary mode of action of azadirachtin: the blockage of morphogenetic

peptid hormones (Banken & Stark, 1997).

Fecundity and fertility were reduced in treated insects as compared to controls (Table 5),

but differences were not statistically significant, probably because of the high intrinsic variability

detected in these parameters even in controls. Number of fertile females derived from treated

larvae was reduced to less than half but those females that survived the insecticide treatment,

exhibited egg laying rates similar to control females.

CONCLUSION

Our studies have shown that Align® has a good potency against C. capitata neonate

larvae and S.exigua last-instar larvae. According to these results, the insecticide may be a good

candidate for the control of the fruit fly in post-harvest disinfection because Adán et al. (1998)

has also reported its good ovicidal activity (CL50 = 1.4 mg/l), but studies should be conducted to

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determine its penetrating characteristics inside the fruits. Azadirachtin may also be a promising

insecticide to control Spodoptera pests but its performance under field conditions should be

investigated. Some advantages of using azadirachtin are that this insecticide is little hazardous to

applicators (oral LD50 in females rats > 5,000 mg/kg) and environmentally friendly, being its

EPA toxicity class IV (Copping, 1998).

The product was rather harmful in laboratory tests for adults of O. concolor, so its joint

use together with the susceptible life stage of the enemy only seems to be possible with an

appropriate timing (Jacas & Viñuela, 1994a have reported that pesticides are totally harmless for

the protected life stage of this enemy). However before reaching final conclusions, it seems to be

necessary to complete the whole IOBC sequential testing scheme for adults (Hassan, 1994). A

first series of preliminary semi-field tests recently indicated that Align® was compatible with

adults of the beneficial (González, unpublished results); however, further testing is required

before publication.

The insecticide was harmless for fully grown nymphs of P. maculiventris, but several

delayed effects on emerged adults were observed. Hence, it is necessary more completely

determine its effects on the reproduction of the bug before reaching a conclusion on its suitability

for use with this predator.

No conclusive results could be drawn from the results scored in C. carnea, but Align®

could be compatible with the predator as NeemAzal-T/S, an oil formulation of azadirachtin, was

found to be totally harmless in the field for C. carnea L2-larvae (Viñuela et al., 1996; Vogt et

al., 1998a).

In summary it can be concluded that Align® could be compatible with the natural

enemies studied but further studies should investigate the total risk of using this compound

together with them, because the insecticide can exhibit differential toxicity to different life stages

and ages of the targeted species.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the research support provided by the Spanish

Ministry of Education and Culture (Concerted action Spain-Germany HA97-0005 and project

AGF98-0715) and the Autonomous Community of Madrid (project 06M/022/96) to E.

Viñuela, and by the DAAD (Deutscher Akademischer Austauschdienst) to H. Vogt. M.

González and Mª.P. Medina are recipient of grants from the Autonomous Community of

Madrid. G. Smagghe acknowledges the Belgian National Fund of Scientific Research

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(Brussels) for a post-doctoral fellowship. We also thank Dr. J. Jacas for suggesting

improvements to the manuscript.

REFERENCES

ADÁN, A., DEL ESTAL, P., BUDIA, F., GONZÁLEZ, M. & VIÑUELA, E. (1996) Laboratory evaluation of the novel naturally derived compound spinosad against Ceratitis capitata. Pesticide Science. 48, 261-168.

ADÁN, A., SORIA, J., DEL ESTAL, P., SÁNCHEZ-BRUNETE, C. & VIÑUELA, E. (1998) Differential action of two azadirachtin formulations on the developmental stages of Ceratitis capitata. Boletín Sanidad Vegetal. Plagas 24, 1009-1018. (In Spanish).

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C e r a t i t i s c a p i t a t a Neonate larvae fed on treated diet

Concentrations mg a.i./l

% Larval mortality1

% Pupal mortality1

% Popped pupae1*

% Adult emergence1*

Control 20.7± 3.6a 1.3± 1.4a 93.1± 2.0a 78.0± 4.7a 0.1 24.7± 6.8a 4.0± 2.0ab 91.9± 0.8a 71.3± 4.5a 1 86.7± 4.1b 13.3± 4.1b 11.4± 6.0b 0±0b

Adults exposed via ingestion in their drinking water Concentrations

mg a.i./l % Mortality at 7 days2 Eggs/female,

8 days1** % Eclosion

Control 10.0± 4.1a 235.4± 28.8a 87.5± 1.3 48 16.0± 7.1a 0±0b - 100 15.0± 2.9a 1.7± 1.7b 0±0

Within the same column, data followed by the same letter do not differ significantly. (P=0.05; 1 LSD 2 Bonferroni mean separation). * Compared with total number of larvae. ** Data represent the mean of 4 replicates of 3-5 pairs of flies/ cage.

Table 1: Influence of the developmental stage on the susceptibility of Ceratitis capitata to azadirachtin.

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O p i u s c o n c o l o r Adults exposed via ingestion in drinking water

Concentrations Longevity Beneficial capacity* mg a.i./l (days) % Attacked hosts % Progeny Control 31.4± 20.7a 87.5± 4.8a 65.0± 7.9a

48 4.9± 0.1b 64.1± 8.2b 43.9± 4.8b

Within the same column, data followed by the same letter do not differ significantly (P=0.05; LSD mean separation). * Data represent the mean of 8 replicates of 3-day pesticide-exposed and isolated females. Twenty L3 C. capitata larvae were daily offered to them for 2 h during a period of 4 days.

Table 2: Effects of feeding azadirachtin in water to adult Opius concolor during life span on the longevity and the beneficial capacity of the parasitoid.

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C h r y s o p e r l a c a r n e a L2 larvae fed on Sitotroga cerealella treated eggs

Concentrations mg a.i./l

% Larval mortality

% Pupal mortality

% Adult emergence*

Eggs/female, 10 days**

% Eclosion

Control 13.3± 6.6a 10.0± 6.8 a 82.1± 11.8a 501.4± 56.7a 92.2± 3.5a 10 6.6± 4.2a 30.0± 4.4a 65.0± 3.1a 523.0± 56.2a 92.0± 6.1a 100 10.0± 4.4a 23.3± 12.0a 60.0± 9.2a 555.5± 108.5a 83.5± 7.5a

1,000 13.3± 4.2a 16.6± 6.1a 65.8± 8.7a 618.6± 38.5a 87.2± 7.8a 10,000 30.0± 8.4a 26.6± 4.2a 48.1± 12.0a

Within the same column, data followed by the same letter do not differ significantly (P=0.05; Bonferroni mean separation). * Compared with pupae. ** Data represent the mean of 8 replicates of pairs kept individually.

Table 3: Toxicity of azadirachtin to the predator Chrysoperla carnea when L2 larvae were fed continuously Sitotroga cerealella treated eggs.

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P o d i s u s m a c u l i v e n t r i s N5 nymphs exposed via ingestion in the drinking water

Concentrations mg a.i./l

% Nymphal mortality1

Nymphal weight at 5 days (mg)2

Duration of the instar (days)2

Control 0a 76.0± 3.3a 7.0± 0.2a 48 12.5± 6.3b 88.6± 4.2a 7.4± 0.1a

Within the same column, data followed by the same letter do not differ significantly (P=0.05; 1 LSD 2 Bonferroni mean separation)

Table 4: Effects of azadirachtin on N5 nymphs of the predatory bug Podisus maculiventris exposed via ingestion in the drinking water

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P o d i s u s m a c u l i v e n t r i s

N5 nymphs exposed via ingestion in the drinking water Concentrations

mg a.i./l % Adult

emergence % Malformed

adults1* % Adult mortality

at 8 days1 Eggs/female,

8 days2** % Fertile females

% Eclosion2

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

Within the same column, data followed by the same letter do not differ significantly. * Compared with total number of emerged adults. ** Data represent the mean of 8 replicates of pairs kept individually. (P=0.05; 1 LSD mean separation; 2 Bonferroni mean separation).

Table 5: Effects of azadirachtin on Podisus maculiventris adults when N5 nymphs were fed the insecticide in the drinking water.

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Figure 2: Daily cumulative mortality in Podisus maculiventris adults emerged from N5 nymphs fed Align® ad libium at 45 mg/l via the drinking water. Figure 1: Effects of Align® on larval mortality (A), larval weight gain (B), pupal weight (C), adult survival (D), fecundity of surviving adults (E), and fertility of eggs deposited (F) of Spodoptera exigua when treated in the last-larval instar. Data are expressed as means±SE.