UNIVERSIDADE ESTADUAL PAULISTA 

“Júlio de Mesquita Filho” 

INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU  

 

 

TOXICIDADE E GENOTOXICIDADE DO BIOPESTICIDA 

AZAMAX™ EM OVÁRIO E INTESTINO DE Ceraeochrysa claveri 

(NEUROPTERA:CHRYSOPIDAE) 

 

 

BERTHA IRINA GASTELBONDO PASTRANA 

 

PROFA. DRA. DANIELA CARVALHO DOS SANTOS 

 

PROF. DR. FÁBIO HENRIQUE FERNANDES 

 

 

Tese apresentada ao Instituto de Biociências, 

Campus de Botucatu, UNESP, para obtenção do 

título de Doutora no Programa de Pós-

Graduação em Biotecnologia, Área de 

concentração Biotecnologia aplicada à saúde 

humana e animal. 

 

  

       Profa. Dra. Daniela Carvalho dos Santos 

 
 

 

 

 

 

 

 

BOTUCATU – SP 

2019 
  



 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

 

 

 

 

 

 

 

 

 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

...Muito obrigada Deus, por não ter me dado tudo o que eu quis, 
mas, por ter me dado o necessário para eu chegasse até aqui...  

 
“E sabemos que todas as coisas contribuem juntamente para o bem 

daqueles que amam a Deus, daqueles que são chamados segundo o seu 
propósito.” 

 
Romanos 8:28  



 

 

 

 

Dedico este trabalho... 

 

 

Aos meus pais Concepción e Alcibiades, 

e aos meus irmãos Alcibiades, Camilo Ernesto e Jorge Arturo. 

 

 
  



 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

AGRADECIMENTOS 

 

 



 
 

 

❖ A Deus, rei da minha vida...por ser o meu suporte sempre que eu tinha vontade de 

cair.  

 

❖ À Professora Daniela, por acreditar em mim, por apoiar o meu propósito de 

estudar danos no DNA no seu modelo biológico e por se arriscar em me orientar 

ainda sem me conhecer. Pelo exemplo, a compreensão e o conhecimento 

compartilhado, muito obrigada. Você além de uma orientadora sempre foi para 

mim a mãe que me acompanhou de pertinho em todos esses anos de doutorado 

longe da minha família. Ah e obrigada também pela paciência com meu 

portunhol!! 

 

❖ Ao meu co-orientador Fábio, quem sempre esteve disposto a me ajudar em tudo, 

por ter sempre palavras de encorajamento e otimismo, pela valiosa amizade e pela 

valiosa ajuda no desenvolvimento deste trabalho. Agradeço de coração por tudo, 

ficarei grata a vida toda! 

 

❖ À Alcibiades e Concepción, meus pais, meus eternos cúmplices e o motor da minha 

vida, pelos sacrifícios e pelas palavras de encorajamento. Obrigada por aguentar 

todo este tempo longe de mim, pela paciência, e por ir junto comigo neste sonho. 

Essa conquista é de vocês!!! 

 

❖ Aos meus irmãos, Alcy, Cami e Jorge Arturo. Cada um com seu jeito, foi peça 

chave para eu não desistir deste sonho. Agradeço especialmente ao meu irmão 

Camilo e sua esposa Lorena, os que foram uma grande ajuda nos momentos difíceis 

que eu passei quando estava tentando concorrer para a bolsa deste doutorado. 

 

❖ À Karen Franco, minha amiga de tantas lutas e risadas, a amiga que a pesquisa 

me deu...obrigada pela ajuda acadêmica e emocional, você tem sido uma grande 

ajuda nesses quatro anos de doutorado no Brasil, foram muitas ligações que 

fizeram a diferença para eu continuar focada no meu sonho...agradeço a Deus tua 

presença na minha vida cada dia. Te quiero mucho Franco! 

 

❖ À Marilúcia Santorum, a grande amiga que o Brasil me deu, minha parceira de 

tantos momentos de lutas acadêmicas e pessoais...obrigada querida Mari, pelas 

viagens, pela amizade, pelas lagrimas compartilhadas, os finais de semana na sua 

casa estudando, comendo, e assistindo filmes. Você é uma pessoa maravilhosa, 

uma excelente amiga, e uma “inteligentona” que sempre me deu excelentes 



 
 

 

contribuições para os resultados deste trabalho. Na verdade, eu não tenho 

palavras para lhe agradecer tudo o que você tem feito por mim durante este tempo, 

eu ficarei eternamente grata com você, com o Júnior e com sua família toda.  

 

❖ À Professora Daisy Fávero Salvadori, pelo apoio, a ajuda constante e as 

excelentes contribuições ao trabalho. Você é um grande exemplo para mim. 

Obrigada pela confiança para deixar trabalhar a esta estrangeira no seu 

laboratório ainda sem conhecê-la, fico grata pela sua disposição para participar 

nesse estudo. 

 

❖ Aos outros pais que a vida me deu, Carlos e Denis, pela amizade sincera e o apoio 

em cada passo.  

 

❖ À Silvia Galván, minha colega do mestrado, minha amiga de tantos momentos, 

minha companhia colombiana que esteve comigo sempre que eu precisei, obrigada 

por me brindar sua casa em Rio Claro e por me fazer sentir sempre como se 

estivesse na minha. Sempre foi bom compartilhar tempo juntas, fazer comida 

colombiana, dar risadas, assistir filmes e sentir de pertinho um pouco do nosso 

Sampués.  

 

❖ Aos meus colegas do Laboratório de Insetos: Elton, Ana e Marilúcia, obrigada 

pelo conhecimento compartilhado, pelas aulas de português, pelo companheirismo e 

pela amizade. Agradeço muito as suas importantes sugestões para a realização 

deste trabalho.  

 

❖ Ao pessoal do Laboratório OMICS, pela amizade, ajuda e apoio constante.  

 

❖ À professora Angélica Guerrero, por incentivar em mim os desejos de pesquisar e 

estudar sem importar as barreiras, pelas palavras de alento, pela ajuda acadêmica 

para entender a Bioquímica, pelas assessorias virtuais, e pela amizade...Gracias mi 

Doc!!  

 

❖ Ao pessoal do Laboratório de Mutagênese ambiental da UNESP-Rio Claro/SP, 

especialmente Yadi e Jorge, pela colaboração, pela boa disposição e as valiosas 

contribuições no processo de padronização da técnica do ensaio cometa.  



 
 

 

❖ À todo o corpo de funcionários do Centro de Microscopia Eletrônica: Shelly, 

Luciana, Claudete, Carol, Maria Helena e Tiago, que sempre foram de muita 

ajuda. Obrigada pelo carinho e pela amizade. 

 

❖ Aos amigos que o Brasil me deu: Marilúcia, Adrielli, Junior, Shelly, Márcio, Ana, 

Bruno, Elton, Sarah e Katielle, muito obrigada pelo carinho, pelos momentos de 

descontração, por tentar sempre entender meu portunhol e pela amizade sincera. E 

ao Gabriel e o Pedrinho, os amiguinhos mais fofos que o Brasil meu deu...sempre 

vou sentir saudades!!!  

 

❖ À Clary, minha única amiga colombiana em Botucatu, obrigada pelo apoio, pelos 

bons momentos, pelos jogos da Colômbia compartilhados e pela ajuda nos 

momentos mais difíceis que eu já passei aqui.  

 

❖ À minha amiga, Lesvi...a amiga Cubana que o Brasil me deu. Obrigada amiga por 

tantos momentos bons que a gente compartilhou e que me ajudaram para não 

desistir, você mora no meu coração.  

 

❖ Aos meus tios e tias, pelo apoio, carinho e as palavras sempre certas, especialmente 

ao Lazaro e ao Jorge, meus segundos pais. Ao meu tio Ricardo in memoriam, 

aonde você estiver eu sei que você está feliz por mim...Obrigada por ser parte da 

minha vida!!!  

 

❖ Ao governo do estado de Sucre na Colômbia, à Fundação de Amparo à Pesquisa do 

Estado de São Paulo – FAPESP (Processo 2014/15016-2, 2017/15120-2), ao 

CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) pelo 

apoio financeiro para o desenvolvimento da referida tese. Ao pessoal da 

universidade CECAR (Colômbia) e à fundação de pesquisa colombiana 

“COLCIENCIAS”, pelo apoio técnico para a realização do meu doutorado.  

 

❖ Aos meus sobrinhos Camilo José, Lorena Lucía, Ana Sofía, Simón Arturo, Isaac 

Arturo, Samuel de Jesús, Dulce María e Marina que são as coisas que mais amo 

na vida.  

 

❖ Aos meus primos, especialmente a Yenis e Henry, Noris, Guillermina, Lácides e 

Lazaro “El Kalvo”, que estão me esperando com os braços abertos!! 

 



 
 

 

❖ Aos meus queridos compadres, Manuel “el negrito” e Vane. Muito obrigada porque 

ainda na distância sempre estiveram torcendo por mim, me apoiando e guardando 

todo seu carinho e amor por mim até o fim desse processo de doutorado. Seus filhos 

Santi e meu Thiaguinho são fonte de felicidade na minha vida, amo vocês!!  

 

❖ Aos meus amigos Natalia Vergara, Daniel Mestra e Sandy Hoyos, muito obrigada 

pelo carinho, pelos bons momentos ainda de longe, e por não desistir da nossa 

amizade durante estes 4 anos que eu esteve longe!!! 

 

❖ Aos outros grandes amigos que a vida me tem dado, os que ficaram na Colômbia 

ansiosos com minha volta: Anderson Ortega, Margarita Verbel, Diana Villalobos, 

Lina Berthel, Liliana Carranza, Yuliana Vargas, Rúben Guzmán, Rafael Bertel, 

Martha Luz Fernandez, Carmen Isabel Ojeda, Alfaro Torres, Joselyn Orozco, 

Avid Torres, Álvaro José Castillo Buelvas, Ercília Osorio, Irina Tirado, Daniel 

Romero, Alexandra Núñez, Luis Hernán Sandoval, Gabriel  Jaime Atehortúa, 

Carito Muñoz e Cesar David Atehortúa. Muito obrigada pelo carinho, amizade 

verdadeira e por estar sempre ali para mim. 

 

❖ Agradeço muito à minha amiga Yina, quem parece que conheço de faz muito tempo 

e quem ainda de longe soube me alegrar em muitos momentos tristes, com quem 

compartilhei risadas em Rio Claro, e quem muitas vezes me deu ânimos para não 

desistir deste meu sonho.  

 

❖ Aos amigos que Botucatu me deu, meus queridos Eliane e Don Samuel, Dona 

Maria, Senhor Luiz, Cris, Giovana, Angélica e Roberta. Obrigada por cada um me 

acolher como parte da sua família.  

 

❖ A todos os funcionários da Seção de Pós-Graduação do Instituto de Biociências de 

     Botucatu, pelo suporte, em especial Davi Müller e Fábio Sugano. 

 

❖ As crianças da catequese e a Dona Antonina, por alegrar as minhas quartas 

falando da vida de Jesus e por me fazer sentir muito amada!! 

 

❖ Aos membros titulares e suplentes da banca, pelo aceite e disponibilidade. E a 

todos aqueles que são parte da minha vida, e que foram parte deste processo e que 

sempre estão ali para mim... obrigada!!!! 

 



 
 

 

SUMÁRIO 

 

RESUMO............................................................................................................................10 

ABSTRACT........................................................................................................................11 

1. INTRODUÇÃO ....................................................................................................... 12 

2. OBJETIVOS ............................................................................................................ 21 

3. REFERÊNCIAS ...................................................................................................... 23 

4. RESULTADOS ........................................................................................................ 31 

4.1. CAPÍTULO 1........................................................................................................ 32 

4.2. CAPÍTULO 2........................................................................................................ 35 

5. CONCLUSÕES ....................................................................................................... 71 

6. ANEXO .................................................................................................................... 73 

 

  
 



10 
 

RESUMO 

O biopesticida Azamax™ tem sido utilizado como uma alternativa aos inseticidas sintéticos 

para o controle de pragas em plantações. Além disso, este produto tem obtido destaque por 

poder se associar ao uso de predadores naturais no controle biológico previsto pelo Manejo 

Integrado de Pragas (MIP). Dentre os modelos biológicos, destaca-se a espécie 

Ceraeochrysa claveri, sobretudo devido sua característica predadora na fase de larva com 

ampla variedade de presas e alto potencial reprodutivo na fase adulta. Embora a azadiractina, 

ingrediente ativo do Azamax™, já tenha sido relacionada com a indução de efeitos 

citotóxicos em insetos, ainda não há dados na literatura sobre os possíveis efeitos da sua 

ingestão indireta em células germinativas e intestinais de C. claveri. Desta forma, o objetivo 

deste estudo foi avaliar o efeito tóxico e genotóxico do Azamax™ em células do ovário e do 

intestino de fêmeas de C. claveri. As larvas foram alimentadas ad libitum, com ovos de 

Diatraea saccharalis tratados com duas diferentes concentrações do Azamax™ (0,3% e 

0,5%) durante todo o seu período larval (n=150 larvas/grupo). As análises morfológicas, 

ultraestruturais e de genotoxicidade foram realizadas após os indivíduos se desenvolverem 

até a fase adulta. Os resultados indicaram que a exposição ao Azamax™ somente na fase 

larval, induziu sinais de toxicidade em todas as fases do desenvolvimento do inseto, como: 

(a) significante taxa de mortalidade a partir do 3º instar larval;  (b) prepupa e pupas inviáveis; 

(c) alterações na duração das fases (larva, prepupa e pupa); (d) marcantes malformações nos 

adultos emergidos; (e) danos primários no DNA de células intestinais e células dos ovários; 

(f) alterações ultraestruturais significantes nas células ovarianas (desorganização dos 

cistos/folículos ovarianos, dilatação do envelope nuclear e do retículo endoplasmático das 

células ovarianas e grande número de células com morfologia sugestiva de morte celular 

apoptótica. Nas condições experimentais utilizadas, os presentes dados indicam que o 

Azamax™ afeta o desenvolvimento e o potencial reprodutivo de Ceraeochrysa claveri, o 

que pode afetar a manutenção da espécie no ecossistema e, consequentemente, a 

continuidade do controle biológico natural exercido por esta espécie predadora de pragas. 

  

 

Palavras-chave: Pesticida natural; reprodução; insetos não alvo; crisopídeo; danos no DNA.



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ABSTRACT 

Azamax ™ biopesticide has been used as an alternative to synthetic insecticides for pest 

control in several crops. Besides, this product has been highlighted because this association 

use with natural predators for biological control in the integrated pest management programs 

(IPM). Among the biological models, is commonly use the specie Ceraeochrysa claveri, 

above all its predatory characteristic at the larval stage with a wide variety of prey and high 

reproductive potential in the adult stage. Azadirachtin, active ingredient of Azamax ™, has 

been related to the induction of cytotoxic effects in insects, although it has not been published 

in literature about the possible effects of Azamax and its indirect ingestion in germ and 

intestinal cells of C. claveri. From this, the aim of this study, is to evaluate the toxic and 

genotoxic effect of Azamax ™ in cells of the ovary and intestine of females of C. claveri. 

Larvae were fed ad libitum with treated eggs of Diatraea saccharalis at different 

concentrations of Azamax ™ (0.3% and 0.5%) during all or their larval period (n = 150 

larvae/group). Morphological, ultrastructural and genotoxic analyzes carried out when 

individuals reached adult stage. The results indicated that exposure to Azamax ™ only in 

the larval phase induced signs of toxicity at all stages of insect development, such as: (a) 

significant mortality rate from 3rd instar larval; (b) prepupa and pupae inviable; (c) changes 

in the duration of the phases (larva, prepupa and pupa); (d) marked malformations in the 

emergent adults; (e) primary damage to the DNA of intestinal cells and ovarian cells; (f) 

significant ultrastructural alterations in ovarian cells (disorganization of ovarian 

cysts/follicles, dilatation of nuclear envelope and endoplasmic reticulum of ovarian cells, 

and large numbers of cells with morphology suggestive of apoptotic cell death. Under 

experimental conditions used in this study, Azamax ™ affects development and the 

reproductive potential of Ceraeochrysa claveri, and it can affect the maintenance of species 

in the ecosystem, and consequently, the natural biological control exercised by this predator 

species of pests. 

 

Keywords: Natural pesticide, reproduction, non-target insects, green lacewings, DNA 

damage.     



 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

1. INTRODUÇÃO 

 



13 
 

 

1.1. Considerações iniciais 

 

 A poluição ambiental, o aumento do uso de inseticidas sintéticos e os seus riscos à 

saúde humana são temas atuais que norteiam a discussão sobre o desenvolvimento de uma 

agricultura sustentável (GARZÓN et al. 2015; SANTOS et al. 2015). A agricultura é hoje 

um dos setores mais importantes da economia brasileira. No entanto, a incidência de 

artrópodes pragas nos sistemas de produção agrícola tem sido o principal desafio para a 

manutenção da produtividade das diferentes culturas (ANSANTE et al., 2015). Neste 

sentido, tem sido necessário o controle químico das pragas por meio de inseticidas sintéticos, 

mas o aumento da resistência a estes compostos convencionais, a persistência de resíduos 

químicos nos ecossistemas, e a crescente demanda para o consumo de alimentos livres de 

agrotóxicos têm mobilizado a criação de novas metodologias que garantam a 

sustentabilidade (TIWARI et al., 2011; SANTOS et al., 2015; CARVALHO et al., 2013). 

Isto tem motivado a implementação de novas e diferentes estratégias para o controle de 

insetos-pragas, como o uso do sistema de Manejo Integrado de Pragas (MIP), no qual é 

utilizado o controle biológico por meio de predadores naturais com ou sem associação ao 

controle químico (ROGERS et al., 2007). No entanto, o MIP recomenda o uso de produtos 

que preservem, ou ao menos sejam compatíveis com os inimigos naturais das pragas, 

pertencentes ou inseridos aos agroecossistemas (WIKTELIUS et al., 1999: ROGERS et al., 

2007).  

 

1.2. Predadores naturais e Ceraeochrysa claveri 

 

Dentre os diversos grupos de insetos considerados predadores naturais, destaca-se a 

família Chrysopidae (Ordem: Neuroptera), a qual inclui 1.200 espécies de insetos, 

comumente conhecidos no Brasil como crisopídeos ou bichos-lixeiros, pelo fato de algumas 

espécies terem o comportamento de carregar detritos no seu dorso como método de 

camuflagem ou barreira física de proteção (FREITAS; PENNY, 2001; ALBUQUERQUE, 

2009). Os crisopídeos, por serem predadores na fase larval com alta voracidade e ampla 

variedade de presas, além da sua plasticidade ecológica e alto potencial reprodutivo na fase 

adulta, são atualmente considerados agentes de controle biológico por excelência (PAPPAS 

et al., 2011). Estes insetos, na sua fase de larva, tem preferência na predação de diversos 



14 
 

 

tipos de insetos pragas de várias ordens e famílias, como pulgões, cochonilha, mosca-branca, 

cigarrinhas, tripés, psilideos, ovos e larvas de Lepidoptera, Coleoptera e Diptera; 

determinando assim, um importante papel no controle biológico natural dos 

agroecossistemas que englobam as diferentes culturas de grande importância econômica no 

Brasil e no mundo (CANARD; PRINCIPI, 1984; FREITAS;PENNY, 2001; 

ALBUQUERQUE, 2009; PAPPAS et al., 2011). No Brasil, de acordo com Freitas e Penny 

(2001), ocorrem importantes gêneros da família Chrysopidae, com grande potencial para 

serem usados nos programas de controle biológico de pragas, como são os gêneros 

Crysoperla e Ceraeochrysa (ALBUQUERQUE et al., 2001). 

O gênero Ceraeochrysa possui 56 espécies descritas no mundo inteiro, distribuindo-

se no continente americano desde o Canadá até a Argentina e tem principal ocorrência nas 

regiões neotropicais (FREITAS; PENNY, 2001), sendo 15 espécies identificadas nos 

ecossistemas agrícolas brasileiros, entre as quais a espécie Ceraeochrysa claveri (Navás, 

1911). Há na literatura poucas informações sobre C. claveri, embora esta espécie já esteja 

estabelecida como agente de controle biológico de várias pragas agrícolas (FREITAS; 

PENNY, 2001). C. claveri é um inseto considerado holometábolo por apresentar aparência 

e hábitos alimentares diferentes durante as fases do seu ciclo de vida: ovo, larva, pupa e 

adulto (Fig. 1) (CANARD et al., 1984; FREITAS; PENNY, 2001).  

 

Figura 1. Ciclo de vida de Ceraeochrysa claveri. Fonte: Própria. 

 

Larva: 3 instares

Pupa: dentro de 

casulo

Adulto

Ovos



15 
 

 

 Os ovos têm forma esférica e são dispostos na extremidade de um pedicelo fino e 

longo, que tem tamanho variando entre 2 mm e 20 mm, e coloração amarelada ou verde-

azulada no período de oviposição, embora tendo a escurecer ao se aproximar o momento de 

eclosão da larva (GEPP, 1984; ALBUQUERQUE, 2009). Na fase de larva, o inseto passa 

por três instares, nos quais as larvas apresentam atividade predadora, tendo mandíbulas 

longas e afiadas. Nessa fase, as larvas também podem predar ovos e larvas recém-eclodidas 

da mesma espécie, fato que as torna canibais. No entanto, essa característica é comum apenas 

quando há escassez de alimento (FREITAS; PENNY, 2001; ALBUQUERQUE, 2009).  

As larvas de C. claveri são terrestres campodeiformes (com pernas torácicas longas 

e ambulatórias), apresentam corpo oval e um pouco achatado dorso-ventralmente, recoberto 

por cerdas longas (ALBUQUERQUE, 2009). Após completar o desenvolvimento larval, os 

indivíduos do 3º instar confeccionam um casulo esférico que contém numerosas camadas de 

fios finos de seda firmemente aderidos em função de proteger no seu interior a fase de pupa 

do inseto (GEPP, 1984; CANARD et al., 1984). A pupa é exarada (com apêndices visíveis) 

e de cor verde, mas tem anteriormente a etapa de prepupa, a qual compreende o período 

desde a confecção do casulo até a última ecdise larval ocorrida ainda no interior deste, e é 

caracterizada pela presença da exúvia, vista como um enegrecimento em forma de disco em 

uma das extremidades do casulo; que evidencian o acúmulo de resíduos metabólicos 

produzidos durante o desenvolvimento larval-pupal (ALBUQUERQUE, 2009).  

Ao completar a fase de pupa, o adulto farato ou pupa móvel, emerge do casulo por 

meio de uma abertura circular, e passa pela última ecdise ou estágio imaginal, na qual o 

adulto atinge sua maturidade, expulsa a exúvia larval em forma de mecônio dentro do casulo 

pupal e finalmente, adquire asas expandidas e funcionais (ALBUQUERQUE, 2009).  Os 

crisopídeos adultos, medem de 10 a 15 mm de comprimento, são predominantemente verdes, 

com dois pares de asas membranosas apresentando nervosidades, antenas finas e longas 

comumente maiores que as asas, três pares de pernas ambulatórias e olhos grandes 

iridescentes (ALBUQUERQUE, 2009).  

A transição dos estágios imaturos até a fase adulta é controlada por dois hormônios 

morfogenéticos, o hormônio juvenil (HJ) e a 20-hidroxiecdisona (20E), os quais regulam 

todos os processos de desenvolvimento pós-embrionário, incluindo ecdises, metamorfose e 

transformações pupa-adulto. Especificamente, o hormônio 20E, do tipo ecdisteróide, induz 

a ecdise enquanto o HJ determina o tipo de ecdise (larval-larval ou larval-pupal) (TRUMAN; 

RIDDIFORD, 2002; YAMANAKA et al., 2013). 



16 
 

 

É importante destacar que o inseto apresenta diferentes hábitos alimentares durante 

o seu ciclo de vida. No estágio adulto, os Crisopídeos são glicopolinívoros, ou seja, 

alimentam-se de pólen, néctar e/ou “honeydew” (uma excreção dos membros da subordem 

Sternorrhyncha: Hemiptera) (SOUZA et al., 1996; ALBUQUERQUE, 2009). No estágio de 

larva, o seu hábito alimentar é unicamente polífago, tendo que consumir insetos pragas para 

completar o desenvolvimento dos seus estágios imaturos, sendo então considerados 

eficientes predadores (ALBUQUERQUE, 2009).  

Pelo fato de C. claveri ser um importante predador de pragas fitófagas pertencentes 

aos agroecossistemas neotropicais, esses insetos contribuem para o controle da densidade 

populacional dessas pragas no campo e a sua preservação para manter o equilíbrio ecológico 

se faz muito necessária (ONO et al., 2017). Assim, ao ser incluído dentro dos programas de 

MIP, juntamente com o controle químico, busca-se o uso de produtos mais seguros para o 

ambiente e que não causem a eliminação das espécies predadoras (SCHMUTTERER, 1990; 

LIU; CHEN, 2000; ONO et al., 2017).  Neste sentido, inseticidas de origem natural, também 

chamados de biopesticidas, têm sido muito estudados e têm alcançado boa aceitação devido 

à contribuição para a redução da dependência aos pesticidas sintéticos convencionais, 

sobretudo pela rápida degradação no ambiente (REGNAULT-ROGER et al., 2012).   

 

1.3. Bioinseticidas e azadiractina 
 

Entre os inseticidas de origem vegetal atualmente utilizados na agricultura, 

destacam-se aqueles que contêm a azadiractina (Fig. 2) como princípio ativo, substância 

obtida das folhas e sementes de Azadirachta indica, popularmente conhecida por “Nim”. 

Desde a sua descoberta em 1966, a azadiractina tem sido estudada por apresentar elevado 

potencial de inseticida e acaricida, com diferentes mecanismos de ação sobre insetos pragas 

(MORGAN; THORTON, 1973; MORDUE (LUNTZ); NISBET, 2000; SIDDIQUI et al., 

2004; MORGAN, 2009). Devido à mínima ou nula toxicidade induzida em vertebrados de 

modo geral, e baixa persistência no ambiente, esta substância é considerada “eco-saudável”. 

Os principais efeitos inseticidas são: atividade anti-alimentar e a ação direta de regulador do 

crescimento (MITCHELL et al., 1997; MORDUE (LUNTZ); NISBET, 2000; 

AGGARWAL; BRAR, 2006; MORGAN, 2009).   

 



17 
 

 

 

Figura 2. Estrutura química 2D da Azadiractina. Fonte: PUBCHEM/NCBI. 

 

O inseto que ingere azadiractina não morre imediatamente, mas devido ao efeito anti-

alimentar, o inseto cessa sua alimentação, o que o leva a morte por inanição (MORDUE 

(LUNTZ et al., 1996). Desta forma, o composto pode causar diversos efeitos sub-letais, tais 

quais: atraso no desenvolvimento larval e/ou pupal, ecdise incompleta, presença de larvas 

permanentes, de pupas e adultos malformados, e diferentes efeitos sobre o ciclo reprodutivo 

dos insetos (SCHMUTTERER, 1990; NASIRUDDIN; MORDUE (LUNTZ), 1993; 

MORDUE (LUNTZ); NISBET, 2000; AGGARWAL; BRAR, 2006; MORGAN, 2009; 

ALMEHMADI, 2011).  

De modo geral, a azadiractina pode causar desregulação generalizada dos processos 

fisiológicos do inseto (MORGAN, 2009). O efeito patológico ocorre principalmente, pela 

indução de interferência no funcionamento normal dos sistemas endócrino e neuroendócrino 

(MEURANT et al., 1994; SAYAH, 2002). Tem sido demonstrado, que o mecanismo de ação 

pode ser considerado multifatorial, por envolver vários outros mecanismos subsequentes 

(Fig. 3). Um dos efeitos baseia-se no bloqueio da liberação de neuropeptídios precursores de 

hormônios, que tem a função do controle da síntese e liberação dos hormônios por parte das 

glândulas endócrinas, especificamente do 20E, que é regulado pelas glândulas pro-torácicas 

do inseto. Portanto, a azadiractina, bloqueia paralelamente a síntese e a liberação do HJ na 

hemolinfa, a qual também é dependente da liberação desses neuropeptídios (MORDUE et 

al., 2005). A azadiractina também interfere na biosíntese de ecdisteroides, pois afeta a 

conversão do hormônio ecdisônio (20E) em 20-hidroxiecdisona, sua forma mais ativa 



18 
 

 

fisiologicamente.  Além disso, os diferentes compostos do óleo de nim, inibem a atividade 

da ecdisona 20-monooxigenase, enzima responsável pela conversão da 20E (MITCHELL et 

al., 1997; MORDUE et al., 2005).   

A alteração no sistema hormonal do inseto tem sido associada à ação da azadiractina, 

a qual atua como antagonista do hormônio ecdisônio, além de interagir com o receptor 

celular da ecdisona. Portanto, essa interação produz alteração em todos os eventos 

moleculares subsequentes, os quais ainda não foram totalmente esclarecidos (SOIN et al., 

2010; LI et al., 2004). Desta forma, as alterações no sistema endócrino e neuroendócrino do 

inseto leva a muitas alterações nos órgãos vitais dependentes deste controle hormonal, dentre 

eles, os órgãos reprodutores (MORGAN, 2009; LAI et al., 2014). Alguns autores classificam 

o conjunto dessas manifestações/alterações morfológicas à síndrome de toxicidade 

generalizada, causada pela azadiractina na ecdise do inseto (LAI et al., 2014; BEZZAR-

BENDJAZIA et al., 2016).  

 

Figura 3. Esquematização do modo de ação da azadiractina após ingestão segundo 

Mordue et al. (2005). Fonte: Própria. 

 

Os outros efeitos associados à azadiractina, acontecem em dois tipos de células que 

são principalmente consideradas as células alvo da molécula. A azadiractina inibe a atividade 

celular em regiões dos órgãos responsáveis pela síntese de enzimas, no caso do intestino atua 

inibindo a produção de enzimas digestivas e de detoxificação por parte das células colunares 

(MORDUE et al., 2005). Este composto também atua sobre as células com alta taxa de 



19 
 

 

divisão celular como é o caso de células ovarianas, inibindo também a atividade desse tipo 

de células (MORDUE et al., 2005).  

A toxicidade da azadiractina tem direcionado diversas pesquisas em insetos não-

alvos como os crisopídeos, tendo em vista os diferentes efeitos subletais demonstrados 

nessas espécies de insetos (MEDINA et al, 2004; GHAZAWI et al., 2007). Existe a 

recomendação de que o uso desses compostos naturais seja altamente controlado em culturas 

nas quais se utiliza o biopesticida associado ao controle biológico de pragas no MIP 

(SANTOS et al., 2015). Alterações morfofisiológicas em órgãos reprodutores e no intestino 

de várias espécies de insetos têm permitido demonstrar que o efeito generalizado desse 

biopesticida pode também se apresentar nos inimigos naturais (MEDINA et al., 2003; 

MEDINA et al., 2004; GAZHAWI et al., 2007; SCUDELER; SANTOS, 2013; SCUDELER; 

SANTOS, 2014). O óleo de nim mostrou-se tóxico à espécie Ceraeochrysa claveri em 

diferentes abordagens (SCUDELER; SANTOS, 2013; SCUDELER; SANTOS, 2014; 

SCUDELER et al., 2014; SCUDELER et al., 2016; GARCIA et al., 2019).  

Embora estudos demonstrem a eficácia de biopesticidas com o princípio ativo 

azadiractina (SCHMUTTERER, 1990; NASIRUDDIN; MORDUE (LUNTZ), 1993; 

MORDUE (LUNTZ); NISBET, 2000; AGGARWAL & BRAR, 2006; MORGAN, 2009; 

ALMEHMADI, 2011; JASMINE et al., 2012, RIBEIRO et al., 2014; CÉSPEDES et al., 

2014), poucos são aqueles voltados para os efeitos deste composto ativo no desenvolvimento 

de insetos não-alvos. Desta forma, a investigação e o conhecimento dos efeitos de 

biopesticidas em espécies não-alvos sob diferentes condições (laboratoriais, semi-campo e 

no campo) são de grande importância para o conhecimento do mecanismo de ação desses 

compostos nas culturas e nos agroecossistema em que se faz uso do controle biológico de 

pragas. 

A molécula da azadiractina tem sido estudada também pelo seu potencial genotóxico; 

sugerindo danos no DNA e aberrações cromossômicas após exposição à produtos associados 

ao neem (AWASTHY et al., 1995; KHAN; AWASTHY, 2003; CHANDRA; KHUDA-

BUKHSH, 2004; PACKIAM et al., 2015; DUMAN; ALTUNTAŞ, 2018). Um dos 

mecanismos moleculares de ação da azadiractina é a inibição ou alteração da transcrição ou 

tradução de proteínas expressas em estágios específicos do ciclo celular (MORDUE et al., 

2005). Ao inibir especificamente a transcrição de proteínas que participam da síntese e 

montagem do sistema de microtúbulos celular altera-se consequentemente diferentes 

eventos mitóticos (MORDUE et al., 2005). Neste sentido, considera-se que azadiractina 



20 
 

 

inibe a proliferação celular; pois ao não se dar a montagem correta dos microtúbulos as 

células são induzidas à apoptose (MORDUE (LUNTZ) et al., 2005).  Essas alterações no 

ciclo celular induzem a quebras na fita cromossômica ou podem também produzir distúrbios 

do fuso mitótico levando consequentemente ao desenvolvimento de efeitos genotóxicos 

tardios (AWASTHY et al., 1995). 

A maioria dos métodos utilizados para identificar uma resposta celular induzida pela 

azadiractina é de avaliação toxicológica clássica utilizando-se biomarcadores histológicos, 

morfológicos e fisiológicos (LAI et al., 2014), Contudo, a técnica de avaliação de danos no 

DNA (teste do cometa) vem sendo uma opção para elucidar o mecanismo de ação de 

compostos ricos em azadiractina, tais como o Azamax™ (JHA, 2008; AUGUSTYNIAK et 

al., 2016). 

Atualmente, várias formulações comerciais com base na azadiractina estão 

disponíveis para utilização na agricultura, entre as quais Azadirex®, Neemix®, Neemix® nos 

Estados Unidos e NeemAzal T/S e o NeemAzal F disponíveis na Alemanha, Austria, Italia, 

Espanha, Holanda e India, onde existem mais de 20 formulações comerciais derivadas do 

óleo de nim (MORGAN, 2009). No Brasil, utiliza-se uma formulação de concentrado 

emulsionável denominada Azamax™, (UPL e United Phosphorus do Brasil Ltda., 

Indianapólis, SP, Brasil) que é autorizada para o controle de pragas artrópodes em diferentes 

culturas e sistemas de produção (Agrofit, 2016). A formulação do Azamax™ tem tido 

crescente aceitação no mercado pelo fato de reduzir as desvantagens do extrato puro da 

azadiractina, como o curto período residual e a possível resistência para certas insetos pragas. 

O Azamax™ é um concentrado emulsificável composto por limonóides, tendo a azadiractina 

como o principal, e o 3-tigloylazadirachtol em menor quantidade (SANTOS et al., 2015).   

O direcionamento de novas pesquisas que permitam detalhar as características 

toxicológicas do Azamax™, hoje considerado “moderadamente tóxico e com mecanismo de 

ação desconhecido” (MAPA, 2018), é fundamental, segundo o Guidelines for Reproductive 

Toxicity Risk Assessment da EPA (US Environmental Protection Agency) (1996), para 

avaliar o risco toxicológico e estabelecer o potencial reprotóxico do composto químico, 

considerando, quando for possível, outras manifestações de toxicidade como a 

genotoxicidade ou mutagênese e outras formas de toxicidade sistêmica geral (EPA, 1996). 



 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

2. OBJETIVOS 

 



22 
 

 

Diante do exposto, o atual estudo tem como objetivo geral investigar os efeitos do 

Azamax™ na biologia reprodutiva e entérica de C. claveri.  

 

Objetivos específicos: 

-   Padronizar a técnica do ensaio cometa nos ovários e intestino de C. claveri.   

- Avaliar os efeitos do biopesticida Azamax™ quanto aos níveis de danos primários 

(quebras de cadeia e sítios álcali-lábeis) no DNA de células intestinais e ovarianas de fêmeas 

adultas de C. claveri. 

- Avaliar os efeitos do biopesticida Azamax™ no desenvolvimento dos insetos (duração 

das fases do ciclo de vida), assim como na morfologia e ultraestrutura dos ovários de fêmeas 

adultas de C. claveri. 

 

 



 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

3. REFERÊNCIAS 

 



24 
 

 

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29 
 

 

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30 
 

 

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African agroecosystems: a case for establishing regional testing programmes. Agriculture, 

ecosystems & environment, v.75(1-2), p. 121-131, 1999. 

 

YAMANAKA, N.; REWITZ, K. F.; O'CONNOR, M. B. Ecdysone control of developmental 

transitions: lessons from Drosophila research. Annual review of entomology, v. 58, p. 497-

516, 2013. 



 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

4. RESULTADOS 

 



 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

4.1. CAPÍTULO 1 

 



33 
 

 

Artigo publicado 

 Revista Chemosphere (Impact factor:5.108) 

The comet assay in Ceraeochrysa claveri (Neuroptera: Chrysopidae): a suitable approach 

for detecting somatic and germ cell genotoxicity induced by agrochemicals 

DOI: 10.1016/j.chemosphere.2019.06.142 

 

 

Bertha Irina Gastelbondo-Pastranaa*, Fábio Henrique Fernandesb, Daisy Maria Fávero 

Salvadorib, Daniela Carvalho dos Santosa,c 

 

 

aLaboratory of Insects, Department of Morphology, Institute of Biosciences of Botucatu, 

UNESP - São Paulo State University, Botucatu, SP, Brazil. 

bLaboratory of Toxicogenomic and Nutrigenomic, Department of Pathology, Medical 

School, UNESP - São Paulo State University, Botucatu, SP, Brazil. 

cElectron Microscopy Center, Institute of Biosciences of Botucatu, UNESP - São Paulo State 

University, Botucatu, SP, Brazil. 

 

 

*Corresponding Author: Bertha Irina Gastelbondo Pastrana.  

E-mail: berthairinagastelbondo@gmail.com 

Laboratory of Insects, Department of Morphology, Institute of Biosciences of Botucatu, 

UNESP - São Paulo State University, Botucatu, SP 18618-689, Brazil.  

Phone: +55 (14) 3880 0479 

 

 

 

 

 

 

 

 

 

 



34 
 

 

 

Abstract 

 

Some agrochemicals are genotoxic to several organisms. Nevertheless, few protocols are 

currently available for measuring the toxicogenetic effects of these compounds in target 

and non-target field-collected species of insects important to agriculture. Herein, we used 

the species Ceraeochrysa claveri (Neuroptera: Chrysopidae), a non-target predator insect, 

to investigate the ability of an azadirachtin-based biopesticide (Azamax™) to induce DNA 

damage. The alkaline version of the comet assay was standardized to evaluate genetic 

instability caused by the toxicant in somatic (gut) and germ (nurse cells and oocytes) cells 

of C. claveri. For this, C. claveri larvae were distributed into three groups (10/each) and 

treated with Azamax™ at 0, 0.3% or 0.5% throughout the larval stage. DNA damage (tail 

intensity) was measured in adult insects, four days after emerged. The data showed that 

both doses of Azamax™ (0.3% and 0.5%) were able to significantly (p<0.05) increase 

DNA damage in somatic and germ cells of C. claveri. In conclusion, C. claveri (intestinal 

and ovarian cells) was a sensitive bioindicator for identifying Azamax™ genotoxic 

potential, whereas the comet assay was a useful tool for detecting the genotoxic hazard of 

the pesticide in the field-collected insect species. Given that estimation of adverse effects 

of pollutants on ecosystems is an essential component of environmental risk assessment, 

the approach used can be recommended to estimate the ecotoxicity of agricultural 

chemicals. 

 

Keywords: azadirachtin, biopesticide, DNA damage, ecotoxicity, ovary, gut. 

 

 

 

 



35 
 

 

 

 
 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

4.2. CAPÍTULO 2 

 



36 
 

 

Artigo sometido  

Revista Environmental pollution. Impact Factor: 5.71 

Azadirachtin-based biopesticide acts as a hazardous product by affecting fitness and 

ovarian development in the natural enemy Ceraeochrysa claveri (Neuroptera: 

Chrysopidae)  

Authors 

Bertha Irina Gastelbondo-Pastranaa*, Marilucia Santorum a, Ana Silvia Gimenes Garcia a, 

Elton Luiz Scudelera, Fábio Henrique Fernandesc, Daniela Carvalho dos Santosa,b 

 

Authors Affiliations 

a Laboratory of Insects, Department of Morphology, Institute of Biosciences of Botucatu, 

UNESP—São Paulo State University, Botucatu, SP, Brazil. 

bElectron Microscopy Center, Institute of Biosciences of Botucatu, UNESP - São Paulo 

State University, Botucatu, SP, Brazil. 

cLaboratory of Toxicogenomic and Nutrigenomic, Department of Pathology, Medical 

School, UNESP - São Paulo State University, Botucatu, SP, Brazil. 

 

 

 

 

* Corresponding Author: Bertha Irina Gastelbondo Pastrana. Ph.D.-Student. Laboratory 

of Insects. Department of Morphology. Institute of Biosciences of Botucatu. UNESP-São 

Paulo State University. Botucatu, SP, Brazil. Fax: +55 14 38153744. E-mail: 

gastelbondo.pastrana@unesp.br  

 

 

 

 

 

 

 

 

 

 

 

 

mailto:gastelbondo.pastrana@unesp.br


37 
 

 

Abstract 

Ceraeochrysa claveri is an important non-target predator insect. Searches for new control 

methods that minimize the adverse effects of synthetic insecticides have initiated a 

resurgence of the use of botanical insecticides. Azadirachtin-based biopesticides are the 

more prominent botanical products commercialized today and represent an alternative to 

these compounds. Despite extensive studies of the physiological effects on pest insects, little 

attention has been given to multiple toxic effects of azadirachtin under authorized 

concentrations in natural enemies of insects important to agriculture. In the present study, 

we used the C. claveri, to assessment the toxic effect of an azadirachtin-based biopesticide 

(Azamax™) on fitness and ovarian development. C. claveri larvae were distributed in 3 

groups (150 larvae per group) and treated with Azamax™ at 0 (control), 0.3 and 0.5 % during 

all the larval stage (15 days) to evaluate the effects of the biopesticide on the survival, 

behavior, external morphology, development, and the impairment of ovarian follicles of C. 

claveri. The two doses tested corresponding at the minimum and maximum concentrations 

that is used in the field. Results showed that Azamax™ (i) reduced survival (ii) change larval 

and pupal developmental time, (iii) induced impairment in the animal body and (iv) in the 

ultrastructure of adult ovaries – thinner ovariolar sheath, complete shrinkage, alterations in 

cellular organization and cell death –. Together, these effects could potentially compromise 

the health of C. claveri. In conclusion, Azamax™, known as a reduced risk insecticide, 

induces hazard effects in lifespan and ovarian development of C. claveri, an economically 

important non-target insect. 

 

Keywords: ovary, non-target insects, growth regulation, natural pesticides. 

 

 

 

 

 



38 
 

 

1. Introduction 

Ceraeochrysa species are being studied due to their current use in commercial agriculture 

(Pappas et al., 2011). Many of these are distributed over variety of ecosystems and a 

restricted number have been reported from agroecosystems (Albuquerque et al., 2001). 

Among these species, Ceraeochrysa claveri is considered a predator insect with great 

potential for the biological control in neotropical agroecosystems; it has been recognized due 

to the key effect of natural enemy on a certain pest species (Albuquerque et al., 2001; Freitas 

and Penny, 2001). At larval stage, C. claveri has a broad range of prey, such as aphids, thrips, 

mites, witheflies, eggs and larvae of economically important insect pests. Due to larval 

feeding habits of green lacewings such as C. claveri, they are recommended to be included 

in integrated pest management (IPM) programs (Albuquerque et al., 2001; Freitas and 

Penny, 2001; Pappas et al., 2011). 

Considering the use of these natural enemies in IPM schemes, associated with conventional 

synthetic insecticides for rotation or botanical products as a substitute, is an important 

alternative for pest control (Garzón et al., 2015; Santos et al., 2015). This strategy is 

necessary to establish and maintaining a sustainable agriculture, decreasing pesticide 

dependency, reducing environmental pollution and human contamination. Therefore, 

preserving these non-target beneficial insects in the agroecosystems could contribute to an 

effective and natural pest management (Wiktelius et al., 1999; Rogers et al., 2007).  

However, to thoroughly verify the compatibility of the conventional pesticide and 

biopesticides with beneficial insects is necessary testing the side effects of these preparations 

for guaranteeing sustainability, safety and credibility of this technology (Santos, 2015). 

Reduced-risk pesticides are generally compatible with natural enemies (Rimoldi et al., 2012; 

Guedes et al., 2016), but still previous to their joint use, it is necessary to assessment the side 

effects in non-target insects. The results of different research could be used by agronomist 



39 
 

 

and environmental agencies to choose safe products for use in IPM programs and to facilitate 

the registration of new enable products for use in agriculture (Garzón et al., 2015). 

Among the reduced-risk products, the interest is high for natural compounds, primarily 

derived from plants. The natural pesticides obtained from the Indian neem tree (Azadirachta 

indica A. Juss) (Meliaceae) are used worldwide in agricultural crops against arthropod pests 

(Charbonneau et al., 2007; Grimalt et al., 2011; Biondi et al., 2012). The most promisoring 

and effective insecticide extracted of the neem tree is Azadirachtin, a limonoid with different 

modes of action, acts mainly as a repellent, antifeedant and insect growth inhibitor, and it 

interferes with the mating behaviour, fecundity and fertility of female arthropod pests with 

different eating habits (Weathersbee and McKenzie, 2005; Abedi et al., 2014; Sanchez-

Ramos et al., 2014). It combines antifeedant action (Blaney et al., 1990) with growth 

regulatory and sterilant effects, caused mainly by alterations of ecdysteroid and juvenile 

hormone titers (Mordue (Luntz) et al., 1998). Negative effects of extracts from the neem tree 

on reproduction have also been reported in a number of insects (Schmutterer and Rembold, 

1995). Thus, taken together, it is assumed that azadirachtin has direct effects on a variety of 

tissues and organs and, as a consequence, several modes and sites of action. These multiple 

activities of azadirachtin involve several pathways (Lai et al., 2014); however, previous 

studies have tended to focus on single physiological effects of azadirachtin, with little 

attention on multiple and combined toxicity effects or possible interactions.  

The increasing demand of these biopesticides has led to a strong interest in studying their 

potential to cause deleterious effects on non-target species. Besides activity against 

arthropod pests, azadirachtin-based products may also cause deleterious effects on beneficial 

insects, such as C. claveri (Scudeler and Santos, 2013; Scudeler and Santos, 2014; Scudeler 

et al., 2014; Scudeler et al., 2016; Garcia et al., 2018; Scudeler et al., 2019; Gastelbondo-

Pastrana et al., 2019). Several azadirachtin-based commercial formulations are 



40 
 

 

commercially available for use in agriculture. In Brazil, the biopesticide is available in an 

emulsificable concentrate formulation termed Azamax™, and it is authorized for use in the 

control of arthropod pests in different crops and production systems (Agrofit, 2018).  

Prior to large-scale application of this novel biopesticide for the control of insect pests, it is 

important to confirm its multiple sublethal effects in non-target insects, as part of an effective 

pesticide risk assessment (Bernardes et al., 2018; Zhang et al., 2018). In this sense, we 

evaluate the effects of this azadirachtin-based product in the biological parameters and on 

ovarian development in C. claveri. It is important to assess the adverse effects of 

biopesticides on natural enemies, and besides is particularly necessary evaluate defects can 

be passed onto the next generations.  From this, in this study was assessment effects not only 

the animal directly affected with the biopesticide.  

 

2. Materials and methods 

 

2.1. Chemicals 

The biopesticide analyzed was available as an emulsifiable concentrate formulation, 

Azamax™ (active ingredient 12 g/L; 1.2% m/m) obtained from UPL e United Phosphorus 

do Brazil Ltd. (Sao Paulo, Brazil) containing azadirachtin (main component) (Santos et al., 

2015).  

 

2.2. Insects maintenance  

The newly hatched Ceraeochrysa claveri larvae (0–12 h old) used in the bioassays were 

obtained from the continuous laboratory colony of the Laboratory of Insects in the 

Department of Morphology at the Institute of Biosciences of Botucatu at UNESP, Brazil. 

The C. claveri colony was reared with Diatraea saccharalis (Lepidoptera: Crambidae) eggs 

during the larval stage and with an artificial diet (1:1 honey/yeast solution) for adults. The 



41 
 

 

insects were maintained in an environmental chamber under controlled conditions (25±1 °C; 

70±10% RH; 12L:12D photoperiod) (Scudeler et al., 2016). 

 

2.4. Bioassays 

Fresh egg clusters recently deposited by females of D. saccharalis (Lepidoptera: Crambidae) 

were collected and dipped once in each dose tested of Azamax™, establishing a minimal 

dose of 36 mg/L (0,3%) and a high dose of 60 mg/L (0,5%) according to the official 

regulation for the phytosanitary pesticide system of the Ministry of Agriculture, Livestock 

and Food Supply of Brazil for use in the field in agricultural crops in which the lacewings 

occur (MAPA, 2018). Eggs of each dose of treatment (diluted in distilled water to obtain the 

desired concentrations) were immersed for 5 s and air-dried at room temperature for 1 h. For 

control group, egg clusters were dipped in distilled water (Scudeler and Santos, 2013). 

Newly hatched larvae were selected randomly and placed in individual polyethylene cups (2 

cm height x 6 cm diameter), these were divided into three experimental groups). The groups 

were tested under the same environmental conditions as described for rearing. In the control 

group (n = 150), larvae were fed ad libitum on D. saccharalis eggs treated with distilled 

water. In the experimental groups of Azamax™ at 0,3 % (n = 150) and 0,5% (n = 150) doses, 

larvae were fed ad libitum on eggs treated with the biopesticide throughout the larval period 

(15 days) until pupation. After cocoon spinning, specimens remained in the same 

polyethylene cups under the same controlled conditions for the entire experiment and until 

the adult emerged. Adults were kept in a polyethylene box (9 cm height x 18 cm diameter) 

and fed with an artificial diet (1:1 honey/yeast solution). One day after they have emerged, 

female insects were used to obtain the ovaries to perform the morphological analysis. 

 

2.5. Toxicity analysis 



42 
 

 

For larval instars, the specimens were checked daily by monitoring larval mortality for 

fifteen days. Individuals without movement move after being touched with a fine brush, were 

considered dead as reported by Scudeler et al. (2016). The number of individuals used per 

treatment (i.e., biopesticide dose, including the control) for this analysis was 150, totaling 

450 larvae sampled. At the prepupal period, when the larva finished a cocoon, the eventual 

inability of a larva to spin the cocoon to start the prepupal period were considered dead 

prepupa (Fig. 1b). And at the pupal stage, in which larva remained inside the cocoon and 

transformed into an exarate pupa (Fig. 1c), the pupal mortality was considered when it dead 

inside the cocoon (nonviable pupa). Larval mortality, percentage of pupae formed and 

successful adult emergence from those pupae were recorded. Cumulative mortality was the 

percentage of total individuals who failed to reach the complete adult form.  

 

To assist in the description of the change in the prepupae, pupae and adults, the morphology 

of the normal and the malformed cocoons spun by larvae in the prepupal period, the viable 

and the nonviable pupae, the malformations of adults emerged from the larvae untreated and 

treated with Azamax were studied and documented with an Olympus SZX16 stereo 

microscope with cellD imaging software (Olympus Soft Imaging Solutions GmBH, 

Münster, Germany) at the Electron Microscopy Center of the Institute of Biosciences, 

UNESP, Botucatu-SP according to reported by Scudeler et al. (2016). 

 

 

2.6. Development time  

The developmental time of the larval, prepupa and the pupal stages were monitored daily. 

The total duration of lifecycle was also recorded. The transition of larval instars was 



43 
 

 

confirmed by the exuviae left after each ecdysis and by the color of the larvae as reported by 

Scudeler et al. (2016).  

 

2.7. Morphology of the female reproductive system 

2.7.1 Transmission Electron Microscopy (TEM) 

For TEM, Adult ovarioles were collected 24 h after emergence and were fixed in 2.5% 

glutaraldehyde and 4% paraformaldehyde solution in 0.1 M phosphate buffer (pH 7.3) 

(Karnovsky, 1965) for 24 h at room temperature and then postfixed in 1% osmium tetroxide 

in the same buffer for 2 h. After being washed with distilled water, the samples were 

contrasted with an aqueous solution of 0.5% uranyl acetate for 2 h at room temperature, 

dehydrated in a graded acetone series (50%, 70%, 90% and 100%), and embedded in 

Araldite resin. Ultrathin sections were contrasted with uranyl acetate and lead citrate and 

were then observed and photographed with a Tecnai Spirit transmission electron microscope 

(FEI Company, Eindhoven, Netherlands) at the Electron Microscopy Center of the Institute 

of Biosciences of Botucatu, SP, Brazil. 

2.7.2 Scanning Electron Microscopy (SEM) 

The ovaries samples were fixed for 48 h at room temperature in 2.5% glutaraldehyde in 0.1 

M phosphate buffer (pH 7.3). Thereafter, the samples were washed in distilled water, 

postfixed in 1% osmium tetroxide diluted in distilled water for 30 min at room temperature, 

dehydrated through a graded series of ethanol, critical point-dried with CO2 and coated with 

gold. The samples were examined and photographed using an FEI Quanta 200 scanning 

electron microscope (FEI Company, Eindhoven, Netherlands) at the Electron Microscopy 

Center of the Institute of Biosciences of Botucatu, SP, Brazil. 



44 
 

 

The wings of C. claveri adults were also analyzed and documented by SEM. Ten samples of 

wings of three experimental groups, were collected and processed for SEM. The samples 

were desiccated overnight, mounted on stubs, and critical point-dried with CO2 and then 

sputter coated with gold (Bal-Tec SCD 050). They were examined in an FEI Quanta 200 

SEM with an accelerating voltage of 12.5 kV at the Electron Microscopy Center of the 

Bioscience Institute, UNESP, Botucatu-SP. Both wings of insects were analyzed.  

 

2.8. Statistics analysis 

The study of association relative to the mortality of larvae, percentage of pupation and 

emergence and cumulative mortality were conducted using the Chi-square test that involved 

contrasts among and within multinomial populations. All tests were performed at a 

significance level of 5%. 

To assess the developmental time of the larvae, prepupae, pupae and total duration of life 

cycle data are presented as mean ± standard error. ANOVA was used to evaluate mean 

differences groups, previously checking for normality and variance homogeneity, using 

Kolmogorov–Smirnov and Bartlett tests, respectively. When normality was not achieved, 

the non-parametric test Kruskal-Wallis, complemented with the Dunn test was used instead. 

The criterion of significance was set at P<0.05. 

3. Results and discussion 

In the present study, we examined the potential effects of Azamax™ on insect fitness and its 

developmental consequences on whole insect and on ovarian follicles development, after 

indirect ingestion (prey poisoning) during larval period of C. claveri. Results show that the 

mortality of immature stage increases when tested both doses of this biopesticide and the 

prepupal and pupal stages represents the most sensitive parts of the life cycle of C. claveri 

leading to an inhibition of adult emergence. 



45 
 

 

 

Evaluation of azadirachtin toxicity in immature stages 

 

In larval period, accumulated mortality was significantly more increased at third instar, as 

demonstrated in Table 1. These larvae died at about the time of the molting to pre-pupa. At 

this stage, the percentage mortality recorded in controls was only 6.8%. When treated third 

instar remaining larvae trying to molt to the pre-pupa few individuals were able to ecdyse, 

at both doses of treatment (0.3% and 0.5) a high percentage of the larvae of last instar were 

unable to undergo or terminate ecdysis.  

These treated larvae remained in this condition. The inability of treated larvae to spin a 

cocoon was the main effect of Azamax in this period; it was documented by the presence of 

nonviable prepupae like a resultant deformed pre-pupa without cocoon that finally died or 

with incomplete cocoons (Fig.1a). 

The quantitative effect of Azamax on the prepupal survival is shown in table 2 and it was 

represented by percentage of pupation (percentage of pupae formed). Significant differences 

were found among treatments and control in pupation. Both doses of Azamax (0.3% and 

0.5%) reduced significantly the percentage of pupation when compared by the control 

(92%); 54.7% and 40.0% of treated larvae were able to moult into the pupa, respectively. As 

occurred at the prepupal stage, at undergo on the pupal development treated insects also have 

shown several malformations and finally dying at this stage (Fig. 1b). At the pupal stage 

were demonstrated other differences in external morphology between the Azamax 

treatments and the control, the treated pupae were not able to complete ecdyse and 

metamorphosis inside de cocoon (malformed pupa) and others died inside the cocoon. 

Malformed pupa and died pupa are represented in the Figure 1b. The malformed specimens 



46 
 

 

at the end of the pupal development presented characteristics of both pupa and adult 

indicating incomplete metamorphosis. 

To explain the innocuous effects of this biopesticide at freshly eclodding larvae (first and 

second instar), it is likely that at this period the larvae had been exposed by lower time than 

at became third instar larval. According to Garzón et al. (2015) the higher accumulative 

larval mortality at third instar larvae was probably, higher because these larvae can be more 

easily damaged due to they walk on treated surfaces and cannot fly, and this overexposure 

can be by ingestion and inhalation at the same time. Scudeler et al. (2019) also observed the 

toxic effect of azadirachtin compounds by ingestion exposure in the third larvae of C. claveri. 

These compounds induce antifeedant effect, a feeding deterrent or antifeedant has been 

defined as a chemical which inhibits feeding but does not kill the insect directly, the insect 

often remaining near the treated plant and possibly dying through starvation (Morgan, 2009). 

The antifeedant effect could also explain the mortality at the last larval stage; 0.3% and 0.5% 

of Azamax seems to be no lethal for C. claveri larvae. Certain doses are lower than required 

for feeding deterrence; some insects ingesting azadirachtin not die immediately but soon 

stop feeding and will show developmental toxicity (Morgan, 2009). Moreover, azadirachtin 

deterrent feeding activity has also been reported in other insect species (Mordue (Luntz) and 

Nisbet, 2000; Morgan, 2009). 

Despite its slightly toxic effect on larvae of third instar, this developmental stage showed 

less susceptibility than the prepupa and pupa. The significant alterations showed during 

larval-pupal development of C. claveri treated insects, demonstrate that Azamax™ mainly 

affect prepupal and pupal stage. Deleterious metamorphic effects and prevent to pupation 

seems to be associated with the interference of azadirachtin on the endocrine system 

(Mordue et al., 2005). These actions of azadirachtin present in Azamax™ probably resulting 

in growth inhibition and molting defects in the insect (Min-li et al., 1987; Feder et al., 1988). 



47 
 

 

This compound is known to cause degenerative structural changes of the nuclei in all 

endocrine glands (prothoracic gland, corpus allatum and corpus cardiacum) responsible for 

controlling moulting and ecdysis in insect which would contribute to a generalized 

disruption of neuroendocrine function (Mordue (Luntz) & Nisbet, 2000). Bezzar-Bendjazia 

et al. (2016) examined the lethal and sublethal effects of azadirachtin on Drosophila 

melanogaster and reported that the mortality of immature stage increases when the doses of 

azadirachtin are increased and the pupae represent the most affected stage as observed in our 

results. In addition, Scudeler et al. (2013) and (2016) showed similar results about the no 

formation of pupae with increase percentage of non-viable prepupa due to no cocoon 

formation when C. claveri larvae were exposed to different treatments of neem oil that 

contained 1500 ppm of azadirachtin A as the active ingredient. These authors confirmed that 

azadirachtin interfere with the molting and cocoon spinning, decreasing wall thickness and 

impairing ability to attach to a substrate. These negative effects may reduce the effectiveness 

of the mechanical and protective functions of cocoons during pupation, which makes the 

specimen more vulnerable to environmental factors (Scudeler et al., 2013).  

Medina et al. (2003) also reported toxicological effects in the pupal stage in a green 

lacewing. These authors noted that Azadirachtin was harmful at higher doses during the 

formation the pupa when the predator Chysoperla carnea was exposed by topical 

application.  Pupae of queens of Partamona helleri that ingested azadirachtin-contaminated 

diet during the post-embryonic development also presented malformations and most of them 

were unable to walk or feed (Bernardes et al., 2018). In contrast, Medina et al. (2001) 

demonstrated not deleterious activities in pupae of C. carnea after topical azadirachtin 

treatment of young and old pupae. They explain these results due to the silken ovoid cocoon 

that covers the decticous pupae might inhibit uptake in the insect body after topical 

application. In the same study, Azadirachtin, however, significantly reduced the percentage 



48 
 

 

of pupae when last-instar C carnea larvae were exposed by residual contact (Medina et al., 

2001). Indicating that susceptibility to azadirachtin varies with application method and 

duration of exposure as reported by Zhang et al. (2018). 

Regarding cumulative mortality represented by the percentage of individuals on inmature 

stages who failed to reach the complete adult form, the results show significant differences 

compared with the control as showed in the Table 2. At the dose of 0.5% of Azamax more 

than half (64.7%) of treated insects no reached to adult stage. In a quantitative sense, because 

the cumulative prepupal and pupal mortality in the treated groups were increase compared 

with the control, cumulative mortality total were also increased; from this, at both doses 

tested of the Azamax, this compound is considered substantially toxic to C. claveri on the 

immature stages. Conversely, at increasing Azamax™ doses, it was decreased emergence 

rate at adult stage as observed in Table 2. 

Indeed, azadirachtin affects various biological processes including larval and pupal 

development by regulating the genes involved in insect growth (Lai et al., 2014). In 

holometabolous insects, such as C. claveri, pupal stage encountering a drastic remodeling of 

most tissues and organs and represent a critical phase for adult formation (Ureña et al., 2014). 

Because, this stage is a vulnerable part of the lifecycle of C. claveri which azadirachtin can 

induce various morphogenetic alterations on the puparium preventing the complete 

emerging of the adult (Bezzar-Bendjazia et al., 2016). 

The molecular mechanisms of sublethal effects of ingested azadirachtin is unclear, but 

studies in D. melanogaster larvae have demonstrated that azadirachtin appears to mainly 

affect post-transcriptional enzyme regulation, proteins involved in cytoskeleton 

development, and transcription, translation, and regulation of hormones, and energy 

metabolism (Lai et al., 2014; Asaduzzaman et al., 2016). In addition, a recent study 



49 
 

 

demonstrated genotoxicity of Azamax treatment in female adults of C. claveri after larval 

exposure to this product (Gastelbondo-Pastrana et al., 2019). 

On the other hand, a great percent of adult emerged of treated groups presented 

malformations as detailed by Gastelbondo-Pastrana et al. (2019) in other part of this study. 

Adults emerged with malformations were considered non-viable adults and consequently 

died. The malformations including insects with incomplete metamorphosis, insects with 

parts of the old edcyse remained adhering to the adult body, lower size, contorted antennae, 

abnormal legs and wings as showed in Figure 1c. Morphology of wings are detailed in Figure 

2. Other authors support these findings: Ghazawi et al. (2007), Barbosa et al. (2015), 

Bernardes et al. (2018). In accordance with Zhang et al. (2018), Azadirachtin reduce the 

lifespan.  

 

Effects on developmental time  

 

The duration of larval development is given in Figure 3. Treatment of freshly larvae altered 

the duration of larval development at the two tested doses. At the first instar, the results did 

not show significant differences; in contrast, at second instar Azamax™ at the minimal dose 

induce delay of development and a high dose it reduces developmental time. Conversely, at 

the last instar Azamax™ reduces the duration of the period at 0.3% (minimal dose) and 

increased this time at 0.5% (maximum dose). In the prepupal stage was observed decreased 

of developmental time at 0.3% of Azamax but the same group of treated insects at pupal 

stage showed delay in the duration of the stage as observed inf the Figure 4. And the pupal 

period of the treated insect at 0.5% presented a reduction on the duration when it was 

compared with the control insects. No statistically significant differences were found for 

total time of life cycle among treatments.  



50 
 

 

In the current experiments, azadirachtin treatment not altered the total of life cycle but it 

affects the development of each stage of the insect . Previously, azadirachtin is reported to 

impair development and survival of different insect's species (Kraiss and Cullen, 2008; Lai 

et al., 2014; Asaduzzaman et al., 2016; Scudeler et al., 2016). Furthermore, azadirachtin was 

found to induce robust developmental delays in the larva-to-pupa and the pupae-to-adult 

transition. Lai et al. (2014) previously reported a delay in pupariation time in azadirachtin 

treated third-instar of D.melanogaster and showed that azadirachtin down regulated 

expression of genes related to hormonal regulation which may be related to the development 

and the deleterious metamorphic effect observed in our results. Similar with our results, 

Bernardes et al. (2018) reported that azadirachtin treatment resulted in reduced survival, 

altered development time and caused deformations. Moreover, Qiao et al. (2013) reported 

that the neurotoxic effect of azadirachtin might interfere with different endocrinological and 

physiological actions in insects. 

 

Effects on ovarian development 

The reproductive system of female adults of C. claveri showed morphological differences 

when compared treated insects with the control insects. The azadirachtin treatment impaired 

development of the reproductive system as recently reported by Bernardes et al. (2018). 

Several effects of azadirachtin on reproduction have been reported in several insects (Medina 

et al., 2004; Ghazawi et al., 2007; Bezzar- Bendjazia et al., 2016; Scudeler et al., 2016; 

Bernardes et al., 2018; Garcia et al., 2018; Gastelbondo-Pastrana et al., 2019). In the presente 

study was observed external abnormalities on the reproductive system and significant 

alterations on the normal ovarian follicle.  

Effects of Azamax™ on external superficie of ovarioles 



51 
 

 

In control group, normal ovarioles are elongated and externally covered by a connective 

tissue sheath which contains a reticulum of muscle, the ovariole sheath (Garbiec and 

Kubrakiewicz, 2012). According to Ruickshank (1972) the ovariole sheath in insects, 

building by a cellular network with small and large meshes seems to be continuous over the 

ovarian follicles cells but discontinuous over the areas between these structures. This author 

was observed that large pores were present in the sheath only between the oocyte chambers 

and externally large pores were observed between strands of sheath. In the current study, we 

observed a type of structure like a large pores in the external superficie on the ovariole sheat 

of the control ovarioles (Fig. 5A,C). In C. claveri ovaries, each pair of ovarioles are 

connected by one unique terminal filament (Fig. 5A). The terminal filament is shorter than 

the proximal region to the oviduct have a larger size as observed in the Figue 5A.   

At both doses of treatment, ovarioles of females treated with Azamax™ presented amount 

of protuberances like as large pores on the ovariolar sheat and subsequently the sheath seems 

to be thinner than the observed and control females ovarioles as detailed in the Figure 5D. 

The external morphology of treated ovarioles are detailed in the Figure 5D. In accordence to 

reported by Ruickshank (1972), this increase of ovariolar large pores on the sheath likely to 

be associated with larger intercellular spaces between the cells that compose ovarian 

follicles. These changes can be comparables to citronela oil induced damages on ovarioles 

of Spodoptera frugiperda reported by Dos santos et al. (2015). Oil-treatment ovarioles 

showed follicular cell stratification and removal, and a thinner conjunctiva sheath. Giorgi et 

al. (1990) proposed that the enclosure and closeness exhibited by developing ovarioles 

through sheaths is designed for guaranteeing protection to the oocyte and to allow a 

directional flow of hemolymph through the insect ovary. From this, the external 

disorganisation observed in ovarioles of Azamax™ treated insects can result in diminished 

reproduction.  



52 
 

 

Moreover, as observed in the figure 5B, ovarioles of treated insect at both doses presented 

shrinkage of ovarioles represented by larger terminal filament and the vitellarium and 

germarium region seems to be smaller when compared to the control ovarioles. The 

Grasshopper Heteracris littoralis treated with azadirachtin suffered shrinkage of the ovary 

as showed by Ghazawi et al. (2007). These authors associated this fact to azadirachtin 

interference with the vitellogenesis process at the ovary. Vitellogenesis is a rather 

complicated process, involving the deposition of yolk in the oocyte, resulting in a very rapid 

increase in size (Ghazawi et al., 2007). Bernardes et al. (2018), also described this effect of 

azadirachtin demonstrating reduced size of the queens' reproductive organs after treatment 

with a azadirachtin-based biopesticide on stingless bee, Partamona helleri.  

 

Effects of Azamax on ovarian follicle organization 

The pair of ovaries of this insect are formed by polytrophic meroistic ovarioles, which 

containing ovarian follicles in different stages of development (Garbiec and Kubrakiewicz 

et al., 2012). Ultrastructure analyzes showed ovarian follicles composed by the oocyte, nurse 

cells and follicular cells in different stages of development. In control group we observed 

germ cells (oocyte and nurse cells) surrounded by follicle cells. The nuclei of the germ cells 

are round with regular contour and decondensed chromatin and the follicle cells exhibit small 

nuclei with irregular contour as observed in the Figure 6A-B.  

At both doses, Azamax™ treatment seems to be toxic to the adult ovaries, as the 

ultrastructure results revealed significant alterations in cellular organization into the ovaries. 

In group treated with 0.3% it is possible to see a large dilatation of the endoplasmic reticulum 

cisterns and also an important nuclear envelope dilation. Moreover, we also detected 

condensed nucleus and many autophagosomes with heterogeneous material and part of 

organelles. The alterations of this group are detailed in the Figure 7A-D. 



53 
 

 

At 0.5% of Azamax™ the intercellular spaces detected in control group between somatic 

and germ cells and also between adjacent germ cells, seems to present a discrete 

enlargement. Finally, we also observed many apoptotic bodies indicating apoptotic cell death 

process as observed at Figure 7E-F. Azamax™ seems to be more aggressive to the ovaries 

at maximum dose, since the cellular alterations were more prominent in 0,5% treated group. 

However, these alterations suggested the absent of oocytes reaching to the egg stage at both 

doses of treatment. The ovarian follicle failed to complete its development as compared with 

the normal cells in different stages of development observed in the control ovaries.  

The inhibition of ovarian follicle development was also reported by Ghazawi et al. (2007). 

They reported that when azadirachtin is administered at an appropriate time, it can cause 

severe damage to the oocytes. Medina et al. (2004) pointed out that a commercial product 

based on azadirachtin (Align) interfered with vitellogenin synthesis and/or its uptake by 

developing oocytes; thus, in this study growing follicles in treated females were significantly 

smaller than those of the controls. Azadirachtin may inhibit vitellogenin synthesis or 

absorption which eventually leads to: inhibition of both oogenesis and ovarian ecdysteroid 

synthesis, inhibition of ovarian development, delaying of the vitellogenin synthesis process 

and interference with vitellogenin synthesis and its absorption by the follicles (Schmutterer, 

1990; Mordue et al., 2005). Lower ovarian proteins levels like vitellogenin suggesting a 

reduced level of vitellogenesis as reported in Spodoptera exempta (Tanzubil and McCaffery, 

1990) and D. melanogaster (Boulahbel et al., 2015) treated with azadirachtin. This 

compound also alters or prevents the formation of new actin cytoskeleton resulting in 

disruption of cell division and blocked transport which may affect the process of dumping 

of the cytoplasmic contents of nurse cells to the oocyte (Mordue et al., 2005). Indeed, 

Anuradha et al. (2007) reported that azadirachtin induces in D. melanogaster, a 

depolymerisation of actin leading to arrest of cells and subsequently apoptosis.  



54 
 

 

Bernardes et al. (2018) in accordance with their results suggest that azadirachtin treatment 

impaired development of the reproductive system, which might be critical for insect 

maintenance and reproduction. The impact of azadirachtin on female reproduction could be 

explained by this interference of Juvenile Hormone and ecdysteroids (Mordue et al., 2005), 

and eventually lead to abnormal ovaries and an associated decrease in fecundity (Zhang et 

al., 2018).  

 

4. Conclusions 

 

In conclusion, our results suggest that the natural enemy C. claveri may be extremely harmed 

by indirect ingestion of an azadirachtin-based biopesticide, eliciting a range of sub-lethal 

effects in this beneficial insect, including mortality, disturbance on fitness, development and 

reproduction. So, the application of these botanical products must be considered carefully 

for use in IPM programs involving populations of C. claveri. Assessment of the side-effects 

of natural products on different developmental stages of non-target species is particularly 

important for guaranteeing safety for use together with predator insects such as C. claveri. 

 

Acknowledgements 

The authors thank to the Scholarship Program for Doctoral Formation of the government of 

Department of Sucre, Colombia and The Administrative National Department of Science, 

Technology and Innovation of Colombia, Bogota, Colombia (COLCIENCIAS, 678-2015). 

We are also grateful to the Electron microscopy Center (CME) of Biosciences Institute of 

Botucatu at UNESP-Botucatu/SP.  

 

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