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versão On-line ISSN 2521-9766versão impressa ISSN 1405-3195

Agrociencia vol.44 no.3 México Abr./Mai. 2010


Protección vegetal


Survivorship and development of immature Harmonia axyridis Pallas (Coleoptera: coccinellidae) exposed to diflubenzuron


Supervivencia y desarrollo de Harmonia axyridis Pallas (Coleoptera: coccinellidae) inmaduros expuestos a diflubenzuron


Esaú Ruiz–Sánchez1* , Lorenza Caamal–Eb1 , Jairo Cristóbal–Alejo1 , Ricardo Munguía–Rosales2 , Alfonzo Pérez–Gutiérrez1


1 Instituto Tecnológico de Conkal. Km. 16.3 antigua carretera Mérida–Motul. 97345, Conkal, Yucatán, México, *Autor responsable: (

2 Comité Estatal de Sanidad Vegetal del Estado de Yucatán. Calle 19 # 443, Colonia Ciudad Industrial. 97288, Mérida, Yucatán, México.


Received: August, 2009.
Approved: January, 2010.



The multicolored Asian lady beetle, Harmonia axyridis Pallas (Coleoptera: Coccinelliadae), is a voracious predator of soft–bodied insects that has been widely used for biological control. In this work, we examined the effects of dilflubenzuron, an insect growth regulator (IGR), on a young population of H. axyridis under laboratory conditions in order to evaluate the risk of using this IGR in integrated pest management programs. Larval and pupal stages of H. axyridis were dipped 3 s in diflubenzuron dilutions (50, 100 or 200 mg L–1) or pure distilled water (control). The percentage of survival and the number of days from immature stage treated to adult emergence were evaluated. The negative effects were related to the developmental stages of H. axyridis and the concentration of diflubenzuron used. All concentrations caused significant effects (p<0.05) on survival when first and second–instar larvae were treated. When third–instar larvae were treated, only the highest concentration caused a significant decrease on H. axyridis survival. No significant effects on survival were found when forth–instar larvae and pupae were treated. Developmental time (days) of H. axyridis from immature stage treated to adult emergence increased significantly when first and second–instar larvae were treated with 200 mg L–1 of diflubenzuron. Based on these results, the use of diflubenzuron might represent a risk for the population of H. axyridis in integrated pest management programs that combine the use of this IGR and predator in pest management strategies.

Key words: H. axyridis, diflubenzuron, insect growth regulator, predator.



La catarinita asiática, Harmonia axyridis Pallas (Coleoptera: Coccinelliadae), es un depredador voraz de insectos de cuerpo blando que ha sido ampliamente usado como agente de control biológico. En este estudio, se examinó los efectos del dilflubenzuron, como un regulador de crecimiento de insectos (RCI), en una población joven de H. axyridis en condiciones de laboratorio para evaluar el riesgo de usar este RCI en programas de manejo integrado de plagas. Larvas de diferentes estadios y pupas de H. axyridis se sumergieron 3 s en diluciones de diflubenzuron (50, 100 o 200 mg L–1) o agua pura destilada (testigo). Se evaluó el porcentaje de supervivencia y el número de días desde el estado inmaduro tratado hasta la emergencia de adultos. Los efectos negativos se relacionaron con los estados de desarrollo de H. axyridis y la concentración usada de diflubenzuron. Todas las concentraciones tuvieron efectos significativos (p<0.05) en la supervivencia cuando se trataron larvas de primer y segundo estadio. En larvas de tercer estadio tratadas, sólo la concentración más alta causó una disminución significativa en la supervivencia de H. axiridis. No hubo efectos significativos en la supervivencia cuando se trataron larvas y pupas de cuarto estadio. El tiempo de desarrollo (días) de H. axyridis desde la etapa inmadura tratada hasta la emergencia de adultos aumentó significativamente en larvas de primer y segundo estadio tratadas con 200 mg L–1 de diflubenzuron. Con base en estos resultados, el uso de diflubenzuron podría representar un riesgo para la población de H. axyridis en los programas de manejo integrado de plagas que combinan el uso de este RCI y el depredador en las estrategias del manejo de plagas.

Palabras clave: H. axyridis, diflubenzuron, regulador de crecimiento de insectos, depredador.



Diflubenzuron is a benzoylphenylurea chitin–synthesis–inhibitor classified as insect growth regulator (IGR) that disrupts molting (Grafton–Cardwell et al., 2005). This compound effectively controls immature individuals of many species of insect pests, especially Coleoptera, Diptera and Lepidoptera (Eisler, 1992). Like other IGRs, diflubenzuron plays a critical role in integrated pest management (IPM) programs, as this IGR is an attractive replacement for organophosphorates and carbamates due mostly to its low mammalian toxicity and high selectivity (Grafton–Cardwell et al., 2005). The latter characteristic, however, is questionable as it has been shown that many arthropods are susceptible to diflubenzuron, including some beneficial insects. In studies concerning the effects of this IGR on hymenopteran parasitoids exposed directly it has been shown that this compound can cause significant immature mortality in Allorhogas pyralophagus Marsh (Hymenoptera: Brachonidae) and Eulophus pennicornis Nees (Hymenoptera: Eulophidae) (Butaye and Degheele, 1995; Legaspi et al., 2000). It has been also shown that diflubenzuron causes deleterious effects on immature stages of some arthropod predators, such as Aleochara bilineata Gyll (Coleoptera: Staphylinidae) (Gordon and Cornect, 1986), Forfícula auricularia Linnaeus (Dermaptera: Forficulidae) (Sauphanor etal., 1993), Orius laevigatus Fieber (Heteroptera: Anthocoridae) (Delbeke et al., 1997) and Podisus maculiventris Say (Heteroptera: Pentatomiade) (De Clercq et al., 1994).

The multicolored Asian lady beetle, Harmonia axyridis Pallas (Coleoptera: Coccinellidae), is a highly effective predator of aphids and other soft–bodied insects and has been extensively utilized as biological control agent (Seo and Youn, 2000). The impact of various IGR on H. axyridis has been scarcely studied. Under laboratory conditions, it has been reported that diflubenzuron applied directly to larvae of H. axyridis or to its food has low impact on survival (Michaud, 2002a; 2002b). However, these works have been evaluated based on acute toxicity and restricted to a single developmental stage, but little attention has been paid to the susceptibility and developmental effects of this IGR on immature stages. The objective of this study was to evaluated the effects of dilfiubenzuron on survivorship and development of immature H. axyridis were examined to provide insights into the risk of using this IGR in combination with H. axyridis in integrated pest management programs.




Adult H. axyridis were obtained from the Centro de Reproducción de Organismos Benéficos (CROB; Centre for Reproduction of Beneficial Organisms) located in Mérida, Yucatán, México. Adults were kept individually in 60X15 mm petri dishes at 25 °C and a photoperiod of 14:10 h (light:darkness). To obtain immature individuals for the bioassays, eggs laid by the adults were allowed to hatch and larvae were reared in petri dishes and fed with the following diet: pollen 400 g, royal jelly 3 g, powdered milk 200 g, active dry yeast 50 g, spirulin 5 g, honey 200 mL, distilled water 300 mL.


Commercially formulated diflubenzuron (Dimilin® 25 P.H, Uniroyal Chemistry) was evaluated at three concentrations: 50, 100 and 200 mg L–1 . For the experiments H. axyridis were treated by dipping bioassay as indicated by Tong–Xiang and Tian–Ye (2001). Briefly, diflubenzuron was dissolved in distilled water and larvae or pupae were dipped for 3 s in the dilutions (treatments) or pure distilled water (control) and placed on paper tissue for 2 h to air–dry. Insects were then placed individually in 60 X 15 mm plastic petri dishes and kept as indicated above. Each treatment had four replications with 20 insects in each replicate. Survival rate was recorded as the percentage of adults that emerged relative to the number of immature individuals treated. Immature developmental time was recorded as the average days for each developmental stage treated until adult emergence.

Data analysis

The experimental design was completely randomized. An analysis of variance and mean comparison (Tukey; p<0.05) of survivorship rate and developmental time (d) for each immature stage treated were performed using GraphPad InStat (GraphPad Software Inc., La Jolla, CA, USA).



Survivorship of H. axyridis

The effects of diflubenzuron on survival of immature H. axyridis depended on the developmental stage treated and the concentration used (Table 1). A significant decrease (p<0.05; n = 4) in survival was observed when first–instar larvae were treated with all concentrations of this IGR. Survival rate decreased (p = 0.05; n=4) 30, 40 and 46.3 % in the larval groups treated with 50, 100 and 200 mg L–1 of diflubenzuron, relative to the control group (Table 1). When second–instar larvae were treated, survival rate decreased (p<0.05; n=4) 31.2 and 32.5 % in the larval groups treated with 100 and 200 mg L–1 of diflubenzuron, compared to the control group. Third–instar larvae were less susceptible than the early developmental stages, as survival rate decreased only 16.2 % (p<0.05; n=4) of 16.2 % on larvae treated with 200 mg L–1 of this IGR, as compared to the control group. No significant effects (p>0.05; n = 4) on survival rate were observed when fourth–instar larvae or when pupae were treated with any of the concentrations.

Developmental time of immature H. axyridis

Significant increase (p<0.05, n=4) in developmental time of immature H. axyridis was observed when first and second–instar larvae were treated with diflubenzuron 200 mg L–1 (Table 2). No significant effects relative to the control were observed when third and fouth–instar larvae or pupae were treated (Table 2).

Studies on the effects of diflubenzuron on nontarget arthropods have shown different outcomes. Butaye and Degheele (1995) mention that with few exceptions, this IGR has shown no appreciable effects on hymenopteran or dipteran parasitoids. De Clercq et al. (1994), however, conclude that the effects depend on the type of exposure, as this IGR caused no deleterious effects through direct or residual contact in some predatory insects, but was highly toxic when ingested.

In our work, it was found that the degree of the effects of diflubenzuron on immature H. axyridis treated by immersion depended on the developmental stage treated and the concentration used. In general, first and second–instar larvae were more susceptible than the late immature stages (Table 1). Other authors also point out that diflubenzuron is most toxic to early stages of insects (El–Gazzar et al., 1988; Eisler, 1992). Particularly on H. axyridis, the susceptibility to insecticides varies depending on the developmental stage. Adults are often less susceptible than immature stages (Michaud, 2002a, 2002b; Michaud and Grant, 2003). The differential susceptibility might be attributed to higher activity of detoxifying enzymes and less sensitivity on target–sites of the late developmental stages (Cho et al., 2002).

It was observed also that the highest concentration of diflubenzuron tested caused a higher rate of mortality (Table 1) and significantly increased developmental time (Table 2) of immature H. axyridis, particularly when first and second–instar larvae were treated. Label rates of this IGR for field, vegetable and fruit crops usually range from 50 to 100 mg L–1 (Michaud 2002a; Anonymous, 2007). The impact of these rates on survival, but not in developmental time of immature H. axyridis, might be critical in the field for the natural population with mixed developmental stage individuals if repeated applications of this IGR are carried out. In cases where H. axyridis is used in inundative biological control strategies, the use of this IGR might represent an important risk for the biological control program success.

Based on this study, diflubenzuron might be used in combination with H. axyridis in IPM programs, but the following considerations should be taken into account: 1) applications of this IGR should be carried out only when H. axyridis are massively liberated on late developmental stages; 2) rates of diflubenzuron higher than 100 mg L–1 should be avoided to preserve natural occurring or massively liberated H. axyridis in agroecosystems. Field evaluations of the effects of diflubenzuron on H. axyridis are needed to optimize the use of this specific IGR with this biological control agent.



The effects of diflubenzuron on immature H. axyridis depended on the developmental stage treated and the concentration used. Young larvae were more susceptible to this IGR than the late immature stages. Diflubenzuron at concentration of 200 mg L–1 caused significant mortality and increased the developmental time of immature H. axyridis.



The authors thank the technicians of the Centre for Reproduction of Beneficial Organisms from Mérida, Yucatán, for their assistance with H. axyridis rearing and colony keeping.



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