Lethal and Sublethal Effects of Methoxyfenozide on the Development, Survival and Reproduction of the Fall Armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)

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Lethal and Sublethal Effects of Methoxyfenozide on the Development, Survival and Reproduction of the Fall Armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)

N Zarate^{I, II}; O Díaz^II; AM Martínez^I; JI Figueroa^I; MI Schneider^III; G Smagghe^IV; E Viñuela^V; F Budia^V; S Pineda^I

^IInstituto de Investigaciones Agropecuarias y Forestales, Univ Michoacana de San Nicolás de Hidalgo, Tarímbaro, Michoacán, Mexico

^IIFacultad de Agronomía, Univ Autónoma de San Luis Potosí, San Luis Potosí, Mexico

^IIICentro de Estudios Parasitológicos y de Vectores CEPAVE (CCT LaPlata CONICET), La Plata, Buenos Aires, Argentina

^IVLab of Agrozoology, Faculty of Bioscience Engineering, Ghent Univ, Ghent, Belgium

^VProtección de Cultivos, Escuela Técnica Superior de Ingenieros Agrónomos, Madrid, Spain

^{Correspondence}

ABSTRACT

The lethal and sublethal effects of the ecdysone agonist methoxyfenozide on the fall armyworm, Spodoptera frugiperda (J. E. Smith), were investigated by feeding a methoxyfenozide-treated diet to fifth instars until pupation in doses corresponding to the LC₁₀ and LC₂₅ for the compound. Larval mortality reached 8% and 26% in the low and high concentration groups, respectively, on the seventh day of the experiment. A progressive larval mortality of 12% for the LC₁₀ and 60% for the LC₂₅ was observed before pupation. Treated larvae exhibited lower pupal weights, higher pupal mortality, presence of deformed pupae, and more deformed adults than untreated larvae. The incorporation of methoxyfenozide into the diet had a significant effect on the timing of larval development. The development period for males and females was about seven days longer than the controls for both concentrations tested. In contrast, the compound affected neither pupae nor adult longevity. Finally, S. frugiperda adults that resulted from fifth instars treated with methoxyfenozide were not affected in their mean cumulative number of eggs laid per female (fecundity), nor percentages of eggs hatched (fertility), or the sex ratio. Our results suggest that the combination of lethal and sublethal effects of methoxyfenozide may have important implications for the population dynamics of the fall armyworm.

Keywords: Ecdysone agonist, insect growth regulator, reproductive parameter

Introduction

The compound 20-Hydroxyedysone (20HE) is one of the most active insect ecdysteroid hormones, acting at every stage of the insect's growth to regulate molting and metamorphosis. This hormone plays a crucial role in insect development, and it is theorised that its agonists or antagonists may disrupt the physiological processes of the target pest (Oberlander & Smagghe 2001, Yanagi et al 2006). Over the past four decades, efforts have been made to develop insecticides with selective properties that act specifically on biochemical sites that are present in particular insect groups but with properties that differ from other insecticides (Ishaaya et al 2005). This approach has led to the discovery of today's modern insect growth regulator (IGR) insecticides (Dhadialla et al 1998).

RH-5849 (the prototype compound) (1,2-dibenzoyl-1-tert-butylhydrazine) tebufenozide, halofenozide, methoxyfenozide, and chromafenozide are all ecdysone agonists (Dhadialla et al 1998, Palli & Retnakaran 2001, Yanagi et al 2006), a novel class of IGRs (Wing 1988). These compounds mimic the biological function of the natural insect molting hormone 20HE, inducing a premature and lethal larval molt by binding directly to the ecdysteroid receptors (Dhadialla et al 1998, Smagghe et al 2004). Ecdysone agonists are highly selective against lepidopteran larvae, with the result that other insects are often less affected by them (Silhacek et al 1990, Schneider et al 2003, 2008). In addition, their narrow spectrum of activity, positive ecotoxicological profile, and short persistence in the environment makes these compounds promising against many economically important agriculture and forest pests (Sundaram et al 2002, Smagghe et al 2003, Biddinger et al 2006, Osorio et al 2008).

Like other IGRs, ecdysone agonists act more slowly than neurotoxic insecticides because they disrupt the hormonal system or the physiological development of insects rather than directly killing them (Biddinger et al 2006). Although these effects may be important in the field, they have been poorly investigated compared to immediate lethality (Seth et al 2004). Since ecdysone agonists do not kill insects immediately, using the criterion of direct mortality caused by ecdysone agonists to assess control efficacy may underestimate their toxicity (Biddinger et al 2006).

Studies of the sublethal effects of ecdysone agonists in several important pests have been widely documented, both in larvae and adults. These effects include delayed or accelerated developmental time (Adel & Sehnal 2000, Biddinger et al 2006), weight loss of both larvae and pupae (Pineda et al 2007, Zamora et al 2008), mortality and pupae deformations (Gobbi et al 2000, Pineda et al 2004), wing deformities in adults (Trisyono & Chippendale 1998, Sundaram et al 2002), disturbed diapause (Eizaguirre et al 2007), impaired reproductive parameters (Seth et al 2004, Smagghe et al 2004), and changes in the adult sex ratio (Biddinger et al 2006). In addition, Hoelscher & Barret (2003) reported that methoxyfenozide interfered with the ability of Argyrotaenia velutinana (Walker) and Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae) adult males to respond to the pheromones of sexually mature females.

In this study, we investigated the lethal and sublethal effects of methoxyfenozide on S. frugiperda, the most serious maize pest throughout Latin America, when the larvae were continuously exposed to a methoxyfenozide-treated diet from the fifth instar until pupation.

Material and Methods

Insect rearing

A laboratory colony was started using S. frugiperda larvae collected in the summer of 2007 from a maize field at El Trebol, within a 9.5 km radius of the town of Morelia, Michoacan, Mexico. Following collection, the larvae were transported to the Instituto de Investigaciones Agropecuarias y Forestales, Universidad Michoacana de San Nicolas de Hidalgo, Tarimbaro, Michoacan, where all the experiments were performed. The larvae were reared on an artificial diet (Poitout & Bues 1974) in a controlled environmental chamber at 25 ± 2 ºC with 75 ± 5% RH and a photoperiod of 16:8 L:D h. Adults were fed a 15% honey solution. Brown paper was provided as a substrate for oviposition. The paper was replaced periodically.

Bioassays

Newly molted (0-8h) fifth instars of S. frugiperda were continuously fed a semi-synthetic diet containing 0.24 mg and 0.35 mg of active ingredient (AI)/kg diet of methoxyfenozide (Intrepid 2 F suspension concentrate, Dow Agrosciences, Zamora, Michoacan, Mexico). We used fifth instars because in other studies we have reported lethal and subethal effects on this developmental stage when ecdysone agonists were applied to different lepidopteran pests (Gobbi et al 2000, Sundaram et al 2002, Biddinger et al 2006). The quantities of methoxyfenozide used correspond to the LC₁₀ and LC₂₅ values for this insecticide, which were estimated from a preliminary study (Zarate 2008). Both concentrations were prepared in 20 ml water and were mixed into the artificial diet after it had cooled to about 45 ºC but shortly before it solidified.

Groups of 12 larvae were randomly selected. The larvae were individually placed into 2.5-cm² cylindrical wells of 12-well Castor tissue culture plates containing approximately 12 g of insecticide-treated diet. Untreated artificial diet was provided for certain specimens as a control. The diet was replaced periodically as necessary until all the larvae had completed the pupal molt. Prior to the bioassay, the larvae were starved for 6h in order to induce a high feeding rate. For the control, CL₁₀, and CL_25, we used 168, 384, and 600 larvae, respectively, and 14, 32, and 50 replicates were performed for the respective treatments. Untreated artificial diet was provided to controls.

Bioassays were carried out under the same environmental conditions, as detailed in the section entitled Insect Rearing. The larvae were checked at 24-h intervals for pupation or until mortality occurred. If no movement was observed, larvae were recorded as dead. The larval duration was also determined. The development of surviving individuals was tracked, and the sublethal effects on pupae and adult emergence were recorded.

To determine pupal weight, between 44-122 male and 38-125 female pupae for each treatment were individually weighed three days after pupation. Afterward, the pupae were individually placed into wells of tissue culture plates as detailed above. After seven days of pupation, the pupae were examined daily for adult emergence, and the adults were classified as normal or deformed. An adult was considered deformed if it was unable to shed from the pupal exuvium or if it did not have normal wings. Pupae were considered dead if an adult did not emerge after 12 d. In order to determine the duration of pupae and adult stages, both were checked daily for mortality. Sex ratio was also assessed.

Effects on reproduction

The sublethal effects of methoxyfenozide were evaluated in specimens derived from larvae that had been exposed to LC₁₀ and LC₂₅ of insecticide. Pupae were sexed based on examination of the seventh, eighth, and ninth sterno-abdominal segments (Sannino et al 1897) by using a stereoscopic microscope (40x) (Zeiss Stemi DV4). After emergence, adults (< 48h old) were placed, using a test tube (2 cm in diameter by 10 cm in height) in a separate oviposition container (7.5 cm in diameter, 5 cm in height) lined with tissue paper. To determine fecundity and fertility, a minimum of 16 and a maximum of 23 pairs of adults were used. They were only provided with a 15% honey solution that was administered using moist cotton and replaced every 2 d to prevent fungal growth. The tissue paper was replaced every 2 d, and fecundity was determined by counting the total number of eggs laid by each female during the first 12 d after the onset of oviposition. Pairs that failed to reproduce were discarded. The percentage of eggs that hatched from those collected 6 d after the first oviposition was used to evaluate fertility. The number of eggs that hatched was assessed 4 d after collection when egg hatching was complete in the control group.

Data analysis

Mortality data for larvae and pupae, pupal formation time, pupal weight, adult emergence time, duration of the larval, pupal, and adult stages, and adult fecundity and fertility were analyzed using one-way ANOVAs. The means were compared by the LSD test (P < 0.05) using Statgraphics (Graphic software system; STSC Inc., Rockville) (STSC 1987). In cases where assumptions of the analysis were violated even after performing an arcsine√x or log (x + 1) transformation, a non-parametric Kruskal-Wallis test was applied. The sex ratio in adults was analyzed using the General Linear Model (GLM) procedure with a binomial distribution by sex using SAS Version 8.1 (SAS Institute 2000).

Results

Larval mortality

Fifth instars of S. frugiperda that were fed a methoxyfenozide-treated diet exhibited the expected mortality rates for both concentrations when examined seven days after the beginning of the study [(8% for LC₁₀ (0.24 mg AI/kg diet) and 26% for LC₂₅ (0.35 mg AI/kg diet)] (Table 1). After seven days, we observed a progressive larval mortality of 4% to 34% until just before pupation. Because of the mortality rate after the initial seven days, cumulative larval mortality was 12% for the LC₁₀ and 60% for the LC₂₅. Mortality in the control groups did not exceed 5%.

Effects on pupae and adults

Over 70% of larvae that survived the methoxyfenozide treatment exhibited normal pupae (Table 2), which was significantly less than the 100% normal pupae found in the control. The compound also caused a significantly higher percentage of deformed pupae compared with the control (Table 2).

An increase in deformed and normal pupal mortality was observed as the insecticide concentration increased (Table 2). However, significant differences were only observed at the highest concentration (0.35 mg AI/kg diet), where pupal mortality was 2.6 times higher than the control.

It is important to point out that the combined mortality during larval and pupal stages was higher for both concentrations tested than for untreated larvae (Table 2). At the highest concentration (0.35 mg AI/kg diet), total mortality during these stages was 79%, whereas at the lower concentration (0.24 mg AI/kg diet) mortality was slightly less than half of that figure (35%). Both treatments were significantly different from control group (21%).

Pupae from larvae treated with methoxyfenozide weighed less that untreated larvae, with more pronounced reductions in females than in males (Table 3). At the lower concentration, male pupae underwent a 39% weight reduction while females experienced a 43% reduction. At the higher concentration, males underwent a 47% reduction while females experienced a 50% reduction.

The percentage of normal adults that emerged was reduced across both treatments. To our surprise, the 0.24 mg AI/kg concentration had a lower percentage (88%) of normal adults emerging than the 0.35 mg AI/kg concentration (91%); although small, this difference was significant (Table 3). Significant differences were observed in the rates of emergence of the two concentrations, but both were significantly lower than the control mean of 100%. For the percentage of deformed adults emerging, the opposite trend was observed.

Effects on development

The incorporation of methoxyfenozide into the diet had a significant effect on the duration of the larval stage (Table 4). The larval stage was 7-8 d longer for males and 5-7 d longer for female larvae in both concentrations than in the controls. For both males and females, the pupal stage lasted between 10 and 11 d and was not significantly different from that of the controls (10-11 d).

Neither concentration of methoxyfenozide impacted adult longevity (Table 5). Both male and female adults exhibited identical lifespan (11-12 d) that were not significantly different from those of the controls (11 d). In contrast, the lifespan from the fifth instar to adulthood was significantly affected in both sexes (Table 5). Males and females in both treatment groups lived almost twice as long as untreated larvae.

Effects on fecundity and fertility

Adults of S. frugiperda derived from fifth instars treated with methoxyfenozide did not exhibit alterations in reproductive parameters. The mean cumulative number of eggs laid per female was 264 for 0.24 mg of AI/kg diet and 356 for 0.35 mg of AI/kg diet. Neither was significantly different from the 393 eggs observed in the control (F = 1.68; d.f = 2, 34; P = 0.20).

Fertility was not affected by either concentration of methoxyfenozide. The percentage of eggs hatched was 95% for the low concentration and 86% for the high concentration. Neither was significantly different from the 85% found in the control (F = 0.80; d.f. = 2, 10; P = 0.48).

Sex ratio

Methoxyfenozide did not appear to impact the adult sex ratio. The male to female ratio for the low concentration treatment was 54:46, while the high concentration treatment group exhibited a ratio of 48:52. Neither was significantly different from the ratio of 54:46 in the control group (F = 1.08; d.f. = 2, 66; P = 0.34)

Discussion

Previous studies demonstrated that ecdysone agonists can cause progressive larval mortality in subsequent instars of the treated insects. These studies also suggested that the chemicals may cause several sublethal effects, such as weight loss in larvae and deformation, in both the pupal and adult stages of surviving individuals. In this study, S. frugiperda larvae treated with methoxyfenozide from the fifth instar until pupation showed also a feeding cessation and morphological changes such as double head capsule formation, extrusion of the hindgut, and incapability for shedding the old cuticle. A progressive larval mortality was observed in subsequents instars when early instars of Spodoptera littoralis (Boisduval) were treated with methoxyfenozide (Pineda et al 2007). Similar results were seen when Platinota idaeusalis (Walker) was treated with tebufenozide (Biddinger et al 2006). This effect may be due to some combination of a high metabolic stability of the compounds within the larval body tissue and the larval food, and the compounds' high affinities for the target sites. In addition, the long time period over which the compounds were continuously administered may have led to sufficient accumulation of the compound in the larval tissue to induce a lethal molting cycle (Trisyono & Chippendale 1997, 1998).

Spodoptera frugiperda larvae that did not appear to be immediately affected by methoxyfenozide exhibited abnormal or lethal pupation and frequently resulted in larva-pupa intermediates. These effects were also observed in the last instars of Spodoptera exempta (Walker), Spodoptera exigua (Hübner), S. littoralis, Mamestra brassicae (L.), Galleria mellonella (L.), and Mythimna unipuncta (Haworth) treated topically with RH-5849 or tebufenozide (Budia et al 1994, Smagghe & Degheele 1994, Gobbi et al 2000). In all these cases molt induction had lethal consequences. The morphological abnormalities occurred because the induction of a rapid molt did not provide enough time for the completion of larval-pupal transformation. Thus, the insects molted to nonviable forms between the life stages (Tateishi et al 1993). Molts induced during the early phase of the last instar produce larval-like individuals, while those formed in the late phase generate pupal-like individuals (Eizaguirre et al 2007), as was observed in our study.

Larvae that survived the methoxyfenozide treatment experienced high pupal mortality (27% and 46% for LC₁₀ and LC₂₅, respectively). Mortality of pupae derived from fourth instars of S. exigua and M. unipuncta reached 20% and 100%, respectively, when the larvae ingested a low concentration of tebufenozide (0.1 mg AI/kg diet) (Gobbi et al 2000). Similarly, when larvae were exposed to the CL₂₅ and CL₅₀ of this compound as neonates or third instars, the mortality of P. idaeusalis pupae was 10-23% for CL₂₅ and 22-37% for CL₅₀ (Biddinger et al 2006). These results, similar to our own, can be explained by the accumulation and persistence of ecdysone agonists in the larval tissue until the pupal molt, at which point the agonist kills the insect. In addition, Sundaram et al (2002) reported the presence of an ecdysone receptor complex in the lepidopteran pupae, so it is clear that this life stage is just as susceptible to ecdysone agonists as larval stage.

It is documented that ecdysone agonists cause larvae to stop feeding, what leads to a reduced larval body weight (Pineda et al 2006, Eizaguirre et al 2007). In our study, the effect of methoxyfenozide on pupal weight was dose-dependent and more pronounced in females than in males. Previously, Biddinger & Hull (1999) reported that pupal weight in both males and females of a susceptible strain of P. idaeusalis remained unaffected when neonates were fed tebufenozide at concentrations of CL₁₀ and CL₂₅. However, when concentrations of this compound were increased (up to CL₅₀), pupae from treated larvae weighed less than untreated larvae, with more pronounced weight reductions at higher concentrations for females (Biddinger et al 2006). As reported by these authors, the major weight reduction in female pupae suggests that females are more susceptible to ecdysone agonists than males. In addition, although a significant difference of deformed adults (2.3%) was observed between high (0.35 mg AI/kg diet) and low (0.24 mg AI/kg diet) concentration, the biological significance of this finding remains unclear.

We observed obvious malformations of the wings in adults that were treated with methoxyfenozide as larvae. Similarly, curled and poorly formed wings were seen in the Lepidoptera Heliothis zea (Boddie) (Noctuidae), Diatraea grandiosella (Dyar) (Pyralidae), S. exigua, S. littoralis, and Choristoneura fumiferana (Clemens) (Tortricidae) (Chandler et al 1992, Trisyono & Chippendale 1998, Carton et al 1998, Sundaram et al 2002, Pineda et al 2004) adults when exposed to tebufenozide, methoxyfenozide or a combination of both compounds as larvae or pupae. It is important to point out that in our study the hind wings were severely affected, and in some cases, they were even vestigial. No abnormalities were noted in the legs, antennae or any other adult body parts. Therefore, it appears that wing development is particularly sensitive to these ecdysone agonists. The affected individuals are functionally dead because the resulting adults are sterile (Sundaram et al 2002) or develop in an asynchronous manner to their host plant or normal breeding population (Biddinger et al 2006).

Methoxyfenozide caused a delay in the development of S. frugiperda larvae when they were exposed as fifth instars. The larval stage lasted 1.6 times longer for treated females and 1.7 times longer for treated males than for untreated larvae. This is very similar to the results seen when larvae of the same insect were exposed as neonates to tebufenozide (Chandler et al 1992). This delay increased the total developmental time from larva to adult emergence because treated individuals remained larvae for more than twice as long as the controls (Table 5).

We cannot exclude the possibility that the increase in the length of the larval stage for individuals treated with methoxyfenozide may be due to the fact that larvae in the study had one or two extra molts. For instance, when treated with ecdysone agonists, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae) (Trisyono & Chippendale 1997) and S. littoralis (Adel & Sehnal 2000) larvae underwent an additional ecdysis into a sixth or seventh instar. A similar study with S. exigua resulted in two additional larval molts (Smagghe & Degheele 1994). Unfortunately, this effect was not considered in our work, but additional studies are underway to examine this possibility. The increase in the length of the larval stage for specimens treated with ecdysone agonists is undesirable, because larvae can cause severe damage to crops. However, larvae receiving sublethal doses may suffer sublethal chronic effects, such as progressive larval mortality, a decrease in pupal weight, pupal mortality, and deformation in both pupae and adults. These effects may be important from a practical standpoint because they might negatively impact pest population dynamics in subsequent generations.

Here we have shown that methoxyfenozide did not have any effect on the longevity of S. frugiperda adults as seen in a study of S. exigua adults derived from third instars treated with the same compound (Martínez A M, unpublished data). The noctuid moth Spodoptera litura (F.) is to our knowledge the only other lepidopteran in which a 50% reduction in adult longevity was observed when exposed as fifth and sixth instars to RH-5849 (Seth et al 2004), but the reason for this effect is unclear. Therefore, further research is needed to elucidate the effects of ecdysone agonists on the longevity of adults derived from larvae treated at immature stages.

Most studies of the toxicity of ecdysone agonists on lepidopteran pests have been conducted during the larval stages, and little has been published regarding their effects on reproductive parameters in the surviving individuals. Our study found that methoxyfenozide did not affect the fecundity and fertility of S. frugiperda adults resulting from treated larvae, as occurred in P. idaeusalis and D. saccharalis (Rodríguez et al 2001, Biddinger et al 2006) adults when exposed to tebufenozide as third instars. In contrast, both reproductive parameters were negatively affected in S. littoralis when neonates were exposed to methoxyfenozide (Adel & Shenal 2000). In other studies, a decrease in either the percentage of the eggs hatching (in H. zea, Carpenter & Chandler 1994) or the number of eggs laid by the females (in P. idaeusalis, Biddinger et al 2006) was observed when individuals were treated with tebufenozide as neonates or third instars, respectively. Differences between the reproductive parameters in our study and those reported by other authors may be attributed to the fact that we only exposed the larvae during their fifth and sixth instars, whereas larvae used by Carpenter & Chandler (1994), Adel & Shenal (2000), and Biddinger et al (2006) were exposed during their entire larval stage.

Little information is available about the effects of ecdysone agonists on sex ratio. This parameter was not affected in S. frugiperda adults. In contrast, Biddinger et al (2006) reported a significant change in sex ratio (2:1 male:female) in P. idaeusalis adults derived from neonates and third instars exposed to tebufenozide. The mechanisms by which these effects occur are poorly understood. It is reasonable to assume that the differences in sex ratio detected in our study and those reported by Biddinger et al (2006) are probably due to a difference in concentrations used (LC₁₀ and LC₂₅ for S. frugiperda; LC₂₅ and LC₅₀ for P. idaeusalis) and the exposure period for the compounds. As reported for P. idaeusalis, because of the high mortality during the larval and pupal stages, it was impossible to determine the sex of many of the specimens. This may have rendered any sex ratio change caused by methoxyfenozide unobservable, such as the one seen in P. idaeusalis (Biddinger et al 2006).

Our studies clearly indicate that LC₁₀ and LC₂₅ of methoxyfenozide were harmful to S. frugiperda larvae. Moreover, survivors showed a great range of delayed sublethal effects such as an increase in larval longevity, a lower pupal weight, a higher pupal mortality and deformed pupae and adults. This is indicative that the combination of lethal and sublethal effects of methoxyfenozide has important implications in the population dynamics of the fall armyworm, contributing to enhance its control.

Acknowledgements

Authors are grateful to American Journal Experts for reviewing the English style, which has contributed to clarify the manuscript. This research was financially supported by the International Foundation for Science, Stockholm, Sweden, the Programa de Mejoramiento del Profesorado (PROMEP-PTC-101), and the Coordinacion de la Investigacion Cientifica, Universidad Michoacana de San Nicolas de Hidalgo through a grant to Samuel Pineda.

Received 01 April 2009 and accepted 10 August 2010

Edited by Raul N Guedes - UFV

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Publication Dates

• Publication in this collection
  14 Mar 2011
• Date of issue
  Feb 2011

History

• Received
  20 Jan 2008
• Accepted
  10 Aug 2010