Action of neem oil (Azadirachta indica A. Juss) on cocoon spinningin Ceraeochrysa claveri (Neuroptera: Chrysopidae)

Elton Luiz Scudeler a,n, Ana Silvia Gimenes Garcia a, Carlos Roberto Padovani b,Daniela Carvalho Santos a,c

a Laboratory of Insects, Department of Morphology, Bioscience Institute, UNESP—São Paulo State University, Botucatu, SP 18618-970, Brazilb Department of Biostatistics, Biosciences Institute, UNESP—São Paulo State University, Botucatu, SP 18618-970, Brazilc Electron Microscopy Center, Bioscience Institute, UNESP—São Paulo State University, Botucatu, SP 18618-970, Brazil

a r t i c l e i n f o

Article history:Received 16 April 2013Received in revised form29 July 2013Accepted 9 August 2013Available online 29 August 2013

Keywords:BiopesticideCocoonLacewingNonwovenUltrastructure

a b s t r a c t

Neem oil is a biopesticide that disturbs the endocrine and neuroendocrine systems of pests and mayinterfere with molting, metamorphosis and cocoon spinning. The cocoon serves protective functions forthe pupa during metamorphosis, and these functions are dependent on cocoon structure. To assess thechanges in cocoon spinning caused by neem oil ingestion, Ceraeochrysa claveri larvae, a commonpolyphagous predator, were fed with neem oil throughout the larval period. When treated with neem oil,changes were observed on the outer and inner surfaces of the C. claveri cocoon, such as decreased wallthickness and impaired ability to attach to a substrate. These negative effects may reduce theeffectiveness of the mechanical and protective functions of cocoons during pupation, which makes thespecimen more vulnerable to natural enemies and environmental factors.

& 2013 Elsevier Inc. All rights reserved.

1. Introduction

Biopesticide selectivity and impact on non-target species, such aspredatory insects, are both concerns that have received worldwideinterest due to the potentially desirable properties of effectiveness,safety and ecological acceptability of biopesticides (Schmutterer, 1997;Aggarwal and Brar, 2006; Cordeiro et al., 2010). Natural pesticidesbased on products of the Indian neem tree (Azadirachta indica A. Juss)(Meliaceae) have been used successfully in agroecosystems againstinsect pests; in particular, neem oil, which is obtained by cold pressingneem seeds, is an ingredient in such pesticides (Schmutterer, 1990;Mordue (Luntz) and Blackwell, 1993; Isman, 2006).

The limonoids present in neem oil are responsible for its anti-feedant and toxic effects, which cause interference with growth andmolting, as well as repellency, sterility and mortality. These effectsresult from cytotoxicity in different tissues and organs and disruptionof the endocrine and neuroendocrine systems. Azadirachtin, a tetra-nortriterpenoid, is the predominant limonoid component of neemseeds and blocks the release of the neurosecretory peptides thatregulate the synthesis and release of molting hormones (ecdysteroids)and juvenile hormones, leading to incomplete ecdysis in immature

insects and all of the effects listed previously (Rembold, 1989;Schmutterer, 1990; Mordue (Luntz) and Blackwell, 1993; Meurantet al., 1994; Mitchell et al., 1997; Mordue (Luntz) et al., 1998; Reed andMajumdar, 1998; Mordue (Luntz) and Nisbet, 2000; Sayah, 2002;Morgan, 2009).

Recent studies have questioned the safety of neem products fornon-target insects such as lacewings (Qi et al., 2001; Medina et al.,2003; Aggarwal and Brar, 2006; Cordeiro et al., 2010; Scudeler andSantos, 2013). Lacewing species are one of the most commongroups of polyphagous predators that occur worldwide, feeding onseveral pests of economic importance. Among the species oflacewing, Ceraeochrysa claveri is commonly present in neotropicalagroecosystems, where they are recognized as biological controlagents of several arthropod pests such as aphids, leafhoppers,whiteflies, thrips, mites, and the eggs and small larvae of severallepidopterans (Principi and Canard, 1984; Albuquerque et al.,2001; De Freitas and Penny, 2001; Pappas et al., 2011).

C. claveri has three larval instars, at the end of which the larvaspins a silk cocoon that is produced by silk precursors that flowfrom its Malpighian tubules into the hindgut and are extrudedfrom the anus during cocoon spinning. When the third instar larvafinishes its cocoon (prepupa), it remains inside the cocoon andtransforms into a decticous pupa. A pharate adult emerges fromthe cocoon, fixes on a substrate and goes through imaginal ecdysis,emerging as an adult (Gepp, 1984; Canard and Principi, 1984;LaMunyon, 1988; Bortolotti et al., 2005; Sutherland et al., 2010).

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Ecotoxicology and Environmental Safety

0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ecoenv.2013.08.008

n Corresponding author. Fax: þ55 14 38153744.E-mail addresses: escudeler@ibb.unesp.br,

eltonscudeler@hotmail.com (E.L. Scudeler), daniela@ibb.unesp.br (D.C. Santos).

Ecotoxicology and Environmental Safety 97 (2013) 176–182

The silk cocoon is a natural polymer composite that hasdeveloped important biological functions through evolutionarypressures, providing mechanical properties and protective func-tions for the pupa against potential predators and parasitoids; forexample, the cocoon helps prevent abrasion of the pupal water-proof external cuticle, microbial degradation and other harshenvironmental conditions during metamorphosis (Tagawa, 1996;Danks, 2002, 2004; Zhao et al., 2007; Weisman et al., 2008; Chenet al., 2012; Horrocks et al., 2013).

The maintenance of these properties during pupal metamor-phosis depends on the cocoon structure as it was spun by thelarva. It is important to assess the adverse effects of biopesticideson natural enemies, as lacewings, as they have been assessed for

chemical insecticides, and it was observed that larvae were unableto metamorphose and spin a cocoon (Bortolotti et al., 2005). Thus,the aim of this work was to analyze the morphology and ultra-structure of cocoons spun by C. claveri that were fed with neem oil.

2. Material and methods

2.1. Insects

Newly hatched larvae of C. claveri were obtained from the stock of theLaboratory of Insects in the Department of Morphology at the Bioscience Instituteat UNESP, Botucatu, Brazil. C. claveri were fed with Diatraea saccharalis eggs(Lepidoptera: Crambidae) and maintained in an environmental chamber with

Fig. 1. Morphology of the C. claveri cocoon. (A) The cocoon of untreated larvae was regular with a rounded shape. (B) SEM micrograph of the entire cocoon wall (W) in cross-section, where the outer layer (O) is composed of a nonwoven structure of fibers and the inner layer (I) is composed of fibers embedded within a solid matrix.

Fig. 2. Outer surfaces of C. claveri cocoons. (A and D) Control group. The cocoons exhibited a nonwoven structure, with a few loose fibers and most fibers bonded and embeddedwithin a solid matrix wall, producing greater uniformity in the surface of the cocoon. (B, C and E) Neem oil concentrations of 0.5 percent, one percent and two precent,respectively. Fibers of the cocoons were densely loose and chaotically arranged, with a high degree of nonwoven structure, leaving the cocoon surface with a rough appearance.

E.L. Scudeler et al. / Ecotoxicology and Environmental Safety 97 (2013) 176–182 177

a controlled temperature of 2571 1C, a relative humidity of 70710 percent and aconstant photoperiod of 12 L:12D.

2.2. Neem oil bioassays

The neem oil was a commercial formulation called Natuneems (Natural RuralInd. e Com. de Produtos Orgânicos e Biológicos Ltda, Araraquara-SP, Brazil), a purecold-pressed neem oil extracted from neem seeds that was diluted in distilledwater to three concentrations of 0.5 percent (5 ml/l), one percent (10 ml/l) and twopercent (20 ml/l) and prepared fresh daily for use. We used the recommended at itslabel for use in Brazilian agricultural fields and concentrations previously tested byScudeler and Santos (2013). All dilutions were prepared starting from the stockcommercial formulation Natuneems. Fresh egg clusters that were recently depos-ited by females of D. saccharalis were collected and dipped once in neem oilsolution for 5 s and air-dried at room temperature for 1 h. Egg clusters dipped onlyin distilled water were used as controls (Scudeler and Santos, 2013).

Newly hatched lacewings were kept individually in polyethylene boxes (2 cmheight�6 cm diameter). The insects were selected randomly and divided into fourexperimental groups (n¼30/group) that were classified according to neem oilconcentration being assayed. In the control group, larvae fed on D. saccharalis eggstreated with water provided ad libitum. In the treated groups (0.5 percent, onepercent and two precent), larvae were fed on eggs treated with neem oil providedad libitum. For all groups, larvae were fed in these conditions throughout the larvalperiod (twelve consecutive days). For both treatments, insects were kept in thesame climate chamber at 2571 1C, with a relative humidity of approximately 70percent and a 12 h photoperiod during pupation, and after cocoon spinning,specimens remained in the same boxes with the same climate conditions untilpharate adult emergence (fourteen days). Bioassays had a total duration of 26 days.Two replicates were performed for each experimental group, resulting in 30cocoons per concentration.

The morphology and ultrastructure of the cocoons spun by untreated and treatedlarvae were studied and documented using an Olympus SZX16 stereo microscopewith cellD imaging software (Olympus Soft Imaging Solutions GmBH, Münster,Germany) and an FEI Quanta 200 scanning electron microscope (SEM) (FEI Company,

Eindhoven, Netherlands). Ten samples of silk cocoons from each treatment groupwere collected and processed for ultrastructural study. The samples were desiccatedovernight, mounted on stubs, and then sputter coated with gold (Bal-Tec SCD 050).They were examined in an FEI Quanta 200 SEM with an accelerating voltage of12.5 kV at the Electron Microscopy Center of the Bioscience Institute, UNESP,Botucatu-SP.

Both the inner and outer sides of the cocoons were analyzed. Cocoons were cutinto cross-sections using a sharp blade for assessing thickness. Ten cocoons weresampled six times at random locations, and the cocoon thickness was measuredusing Scandium, the SEM imaging platform (Olympus Soft Imaging SolutionsGmBH, Münster, Germany). To assess changes in the thickness of the cocoon, weused analysis of variance for the model with one incompletely randomized factor,which was complemented by Tukey's multiple comparison test (Zar, 2009).

3. Results

The cocoons had a round shape (Fig. 1A) with a heterogeneousstructure that consisted of two distinct layers, outer and inner,where the outer layer was made of a nonwoven structure fromfibers and the inner layer was composed of fibers embeddedwithin a solid matrix (Fig. 1B). Scanning electron microscopy(SEM) analysis of the outer surfaces of cocoons revealed ultra-structural differences between the control and neem oil treatmentgroups. SEM clearly showed that the outer layer surface in thecontrol group exhibited a nonwoven structure, with cross-bindingfibers and fewer loose fibers, mostly bonded and embedded withina solid matrix (Fig. 2A). This characteristic produced a greateruniformity on the surface of the cocoon (Fig. 2D).

However, in all neem oil treatments, many of the fibers wereloose and chaotically arranged on the surface, with a high degree

Fig. 3. Lateral surface of the cocoon attached to the substrate. (A) Control group. A small number of spun silk fibers can attach the cocoon to the surface. (B, C and D) Neem oilconcentrations of 0.5 percent, 1 percent and two precent, respectively. Larvae had difficulty in attaching the cocoon and spun more quantities of silk fibers that werechaotically arranged to attach the cocoon on the substrate.

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of nonwoven structure, causing a lack of uniformity and leaving thecocoon surfaces with a rough texture (Fig. 2B, C and E). This samechange was also found in the silk fibers that attached the cocoon tothe box surface, impairing the ability of the larvae to attach thecocoon (Fig. 3A–D). As a result, larvae that received neem oil had tosecrete more silk fibers than those in the control group.

On the inner surface of the control group samples, we foundwoven holes with the fibers bonded and embedded within a solidmatrix, forming uniform and smooth holes. The hole structureis slightly triangular in shape, with a central vent (Fig. 4A and B).For the treated groups, holes had an irregular shape and the

central vent was often obstructed by fibers or there were multiplevents (Fig. 4C–E). We noted in the treatments that there was animpaired ability to make these holes, as the fibers were randomlyarranged, generating irregular and rough structures (Fig. 4F). Theseeffects were shown to be dose-dependent.

The inner surface structure is composed of fibers that blend with amatrix, making them appear as a smooth continuous layer with lowporosity (Fig. 5A). In contrast, treated groups presented fibers that didnot blend in the matrix, giving a rough and fibrous appearance to theinner surface of the cocoon (Fig. 5B–D). Similar to the holes, neem oilaffected the inner layer spin in a dose-dependent manner.

Fig. 4. SEM pictures of woven holes in the inner surface in lacewing cocoons. (A and B) Control group. Holes were woven with bonded fibers and embedded within a solidmatrix, forming uniform and smooth holes with a central vent (arrowhead). (C and D) Neem oil concentrations of 0.5 percent and one percent, respectively. Holes had anirregular shape, and the central vent was often obstructed by fibers (arrow). (E and F) Neem oil, two percent. Holes with multiple vents (*) formed by fibers arranged atrandom, generating irregular and rough structures.

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The effect of neem oil on the thickness of the cocoons ispresented in Table 1. All of the treatment doses caused a significantreduction of the thickness (po0.001) compared to the control group,and this effect was more pronounced in the groups that received0.5 percent and two percent doses of neem oil. This indicates that theobserved change in thickness was not dose-dependent.

4. Discussion

Neem oil ingestion during the larval stage had an effect ongrowth regulation, which affected larval and pupal developmentduring the spinning process. These effects were caused primarilyby changes in the concentrations of ecdysteroid and juvenilehormones (Rembold, 1989; Schmutterer, 1990; Mordue (Luntz)and Blackwell, 1993; Mordue (Luntz) et al., 1998; Mordue (Luntz)and Nisbet, 2000; Morgan, 2009).

An insecticide with hormonal action (fenoxycarb, a juvenilehormone-mimicking compound with insecticidal effects) in Chry-soperla carnea (Neuroptera: Chrysopidae) also caused the inhibitionof cocoon spinning and irregular or incomplete, or even the absenceof metamorphosis. However, unlike C. claveri, Bortolotti et al. (2005)observed that at low doses of fenoxycarb, the inner wall ofirregularly spun cocoons was absent, and this was not observedfor any evaluated dose of neem oil. Only in cases where there wasan inhibition of the synthesis of the cocoon there was presence ofsilk fibers, confirming the report by Weisman et al. (2008) that the

fibrous components of the cocoon are spun before the internalarray, which acts as a scaffold for the inner wall (LaMunyon, 1988).

The effects caused in the outer layer, as well as the difficulty ofattaching the cocoon to a surface, may reflect deleterious effectson the synthesis of camouflage or fixing the cocoon to a substrate,generating less protection in a natural environment (Danks, 2002).The largest number of silk fibers secreted by larvae treated withneem oil can occur due to an attempted defense of the organism inorder to ensure the attachment of the cocoon to surface eventhough the intake of this oil affects the arrangement of thesefibers. Just as azadirachtin affects intestinal motility (Mordue(Luntz) et al., 1985; Trumm and Dorn, 2000), it may also haveaffected the secretion process of silk fibers by the Malpighian

Fig. 5. C. claveri inner cocoon surface. (A) Control group. The cocoon inner layer is a continuous smooth and solid layer composed of fibers that blend with a matrix. (B, C andD) Neem oil concentrations of 0.5 percent, one percent and two percent, respectively. Silk fibers not blended in the matrix, giving a rough and fibrous appearance to the innersurface. Irregular holes with central vents obstructed by fibers (arrows). (O) outer surface.

Table 1Wall thickness of the cocoons (mean and standard deviation) ofdifferent groups of C. claveri fed with neem oil throughout thelarval period. (n¼ten cocoons per concentration).

Treatment (percent) Thickness (mm)

Control 12.10672.621c

0.5 6.39072.783a

1 10.30573.480bc

2 7.96672.650ab

Two means followed by the same letter are not significantlydifferent based on the Tukey test (p40.05).

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tubules and their subsequent release by part of the hindgut,resulting in loose fibers that were densely and chaotically arrangedon the cocoon surface and generating a less compact and rigidcocoon. Such as azadirachtin or neem oil affects different organs ina dose-dependent form, it may have occurred in the Malpighiantubules, resulting in ultrastructural changes in a dose-dependentmanner on cocoon spinning (Nasiruddin and Mordue (Luntz),1993; Nisbet et al., 1996; Scudeler and Santos, 2013). Malpighiantubules are one of the main sites of retention of dihydroazadir-achtin, which has the same biological activity as azadirachtin(Rembold et al., 1988; Nisbet et al., 1995) and may affect thesecretion of silk fibers by C. claveri larvae.

The inner-most layer of the cocoon, also called the pelade, wasone of the cocoon structures most affected by the consumption ofneem oil. We know that the inner layer has a significant andmultifunctional role in the process of metamorphosis (LaMunyon,1988, Zhao et al., 2007; Weisman et al., 2008). The changesobserved here, such as rough irregular holes and multiple open-ings, affect the function of this layer. Damage to the structure, suchas the aforementioned roughness, could affect protection againstmechanical damage or abrasion to the waterproof layer cuticle(Goto et al., 1997; Danks, 2004). Weisman et al. (2008) report alipidic nature for the composition of the inner wall of the greenlacewing cocoon, which would help prevent water loss duringpupation.

The increase in porosity and decreased wall thickness of thecocoon may affect the rate of the diffusion of gases, includingwater vapor, which plays an important role in pupation (Tagawa,1996; Chen et al., 2012; Horrocks et al., 2013) and protects againstthe ingress of small organisms (Zhao et al. 2007). Changes found inthe wall thickness of the cocoon also affect protection againstnatural enemies, resulting in less resistance to penetration bypredators and parasitoids (Danks, 2004).

Our results indicate that ingestion of neem oil by C. clavericauses morphological and ultrastructural changes in cocoons. Thechanges observed externally and internally, such as the decrease inthe wall thickness of the cocoons and impairment of the attach-ment of the cocoon to a substrate, may reduce the effectiveness ofcocoon functions during pupation, which makes the specimenmore vulnerable to natural enemies and environmental factors.

Acknowledgments

We are grateful to the Centro de Microscopia Eletrônica (CME),Instituto de Biociências, UNESP. This work was supported by theFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP),Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq) and Coordenação de Aperfeiçoamento de Pessoal de NívelSuperior (CAPES).

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