Evaluation of imidacloprid (Confidor OD®) genotoxicity in Chrysoperla externa eggs (Neuroptera: Chrysopidae) through comet assay.

The comet assay allows the analysis of DNA damage caused by different genotoxins. This assay has recently gained interest because of its ease of studying the interactions of xenobiotics with different organisms. Chrysoperla externa (Hagen, 1861) is a species of great economic relevance because it is a predator of major agricultural pests during its larval stage. Neonicotinoids are the most important chemical class of insecticides introduced into markets. A previous imidacloprid toxicity assessment on C. externa showed that this neonicotinoid insecticide reduced the egg viability. The objective of this study was to analyze the genotoxicity of Confidor OD® (imidacloprid 20% a.i., LS, Bayer CropScience) on the biological control agent C. externa at DNA level using the comet assay as an ecotoxicological biomarker. A comet assay protocol has been developed for this species at first time. For the bioassays, the commercial product formulated Confidor OD® was used at two concentrations: 100 and 180 mg/l of the active ingredient. Selected eggs were dipped in a Confidor OD® solution for 15 s. Descriptors evaluated in the comet assay were damage index, % DNA damage, and tail length. The damage index did not show any significant differences between the different concentrations evaluated, but differences were observed for tail length, because at higher concentrations of Confidor OD®, there were greater DNA breaks. The DNA of the cells from treated eggs analyzed at 48 h and 96 h of development showed the same % DNA damage; that is, they had no recovery capacity. Application of Confidor OD® to C. externa eggs produced irreparable breaks at the DNA level. The technique adjusted for C. externa can be used in other beneficial insects to study pesticide genotoxicity using a comet assay.

Biological activity of three alkyl cinnamates on young larvae of Tuta absoluta.

The "tomato moth," Tuto absoluta (Meyrick) (Lepidoptera: Gelechiidae), is a neotropical oligophagous insect considered a key pest of tomato crops. It was detected in Europe seven years ago and quickly spread to several regions of Asia and North Africa. In Argentina, its control is done by chemical pesticides mainly. The indiscriminate use of chemical broad spectrum pesticides have caused several problems in the control of this pest due to resistance mechanism involved to several insecticides including pyrethroids, organophosphates and biopesticides. In this context, the searching and evaluation of new compounds- compatible with integrated pest management programs- has become relevant. Within the phenylpropanoids, alkyl cinnamates, whether natural or synthetic, have been reported with biological activity: repellence, antifeedant and insecticide. Therefore, the cinnamates could be a valuable alternative to replace the conventional insecticides. The aim of this work was to study the antifeedant effect of three alkyl cinnamates (methyl, ethyl and propyl cinnamate) on the consumption rate patterns of second instar larvae of T. absoluta. A series of concentrations of each compound (100, 250 and 500 micrograms/milliliter) were prepared using acetone (analytical grade) - distilled water as solvent and a surfactant (Tween 80) was added to each solution to improve the wet of leaves. Tomato leaf disks of 2 cm diameter were treated by immersion in each solution during 15 seconds. Afterward, the treated discs were dried under fume hood and each disk was placed in a plastic capsule. A larva with 6-8 hours of starvation was added to each experimental unit. Each treatment was replicated between 24-30 times. The area consumed by each larva was measured after 24, 48 and 72 hours post-treatment. We also evaluated development time, weight of the pupa, adult emergence, fecundity and fertility as sublethal effects. The results were analyzed using ANOVA test. None of the compounds tested at the aforementioned concentrations exhibited insecticidal effect. However, ethyl cinnamate showed a strong antifeedant effect. The results observed on the leaf consumption and other sublethal effects assessed will be discussed. According to these preliminary results, further studies are needed to complete its toxicological profile by other exposure methods.

Toxicity of cypermethrin on the neotropical lacewing Chrysoperla externa (Neuroptera: Chrysopidae).

The generalist predator Chrysoperla externa (Neuroptera: Chrysopidae) is distributed in the Neotropical Region and presents a strong potential as a biological control agent due to its easily rearing, high voracity and tolerance to several pesticides. This species feeds on relevant pests such as aphids, whiteflies, trips and various lepidopterans. In Argentina, an indiscriminate chemical control with broad spectrum pesticides is still the first option for controlling pests causing resistance problems and reduction of beneficial organisms. Therefore, it would be desirable that predators or parasitoids develop tolerance or resistance as phytophagous pests for their maintenance in the agricultural ecosystems. The objective of this work was to evaluate the tolerance of C. externo to the pyrethroid cypermethrin by topical exposure in laboratory conditions. The symptoms evaluated were tremors, movement, knockdown, inability to walk due to being glued. Recovery from poisoning symptoms was also evaluated. Six different concentrations of the pesticide, including the full field one, were tested. The exposure method was by topical applications on third larval instar. A low toxicity on larvae at 24 h post-treatment was observed, even at the highest concentration evaluated. Most of treated individuals looked normal at 24 h post-treatment; those who were knocked down showed a complete recovery between 48 and 72 h post-treatment. C externa demonstrated a high tolerance to pyrethroids. This could be due to the activity involved in pyrethroids biotransformation (for example, esterases and mixed-function oxidases), or to a low sensitivity of the molecular target. Future studies should be addressed at biochemical and molecular levels to complete our knowledge about insecticide effects on this predator.

Preliminary studies of effectiveness and selectivity of Movento on Bemisia tabaci and its parasitoid Eretmocerus mundus.

The Bemisia tabaci Gennadius (Homoptera:Aleyrodidae) biotype complex is a key pest of several worldwide crops. The management and control of this pest has become difficult mainly due to its high reproductive rate and capacity to develop resistance to broad spectrum insecticides. In Argentina B. tabaci whitefly, causes economic losses in most areas of agricultural production. Eretmocerus mundus Mercet (Hymenoptera:Aphelinidae) is the most important parasitoid of B. tabaci and is commercialized as a biocontrol agent, mainly in Europe. Conservation of this biological control agent in Argentinean orchards requires the adoption of sustainable pest management practices due the negative impact of traditional pesticides on non-target organisms. Spirotetramat (Movento) belongs to a new class of pesticides that acts as a lipid biosynthesis inhibitor and claims to be selective towards natural enemies. The objectives of this work were 1) to evaluate the effectiveness of spirotetramat on eggs and nymphs of B. tabaci and 2) to determine the selectivity of spirotetramat towards E. mundus. Whitefly's eggs and nymphs (first nymphal settled instar) were exposed to the insecticide by foliar immersion whereas parasitoid adults (6 days old) were exposed to the insecticide by residual method for one hour, to simulate exposure of the parasitoid to the insecticide in the field. Lethal and sublethal effects of the insecticide were recorded daily. These preliminary studies have shown a high effectiveness of spirotetramat on the first nymphal instar of B. tabaci as well as a high selectivity for the pest in comparison to the parasitoid adults showing a low acute toxicity to them. These results suggest Movento could be included in Integrated Pest Management programs although more studies are required to complete its ecotoxicological profile.

Lethal and sublethal effects of methoxyfenozide on the development, survival and reproduction of the fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).

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.

Side-effects of glyphosate on the life parameters of Eriopis connexa (Coleoptera: Coccinelidae) in Argentina.

In Argentina, transgenic soybean crop (Roundup Ready, RR) has undergone a major expansion over the last 15 years, with the consequent increase of glyphosate applications, a broad-spectrum and post emergence herbicide. Soybean crops are inhabited by several arthropods. Eriopis connexa Germar (Coleoptera: Coccinelidae) is a predator associated to soybean soft-bodies pest and have a Neotropical distribution. Nowadays, it is being considered a potentially biological control agent in South America. The objectives of this work were to evaluate the side-effects of glyphosate on larvae (third instar) and adults of this predator. Commercial compound and the maximum registered concentrations for field use were employed: GlifoGlex 48 (48% glyphosate, 192 mg a.i./litre, Gleba Argentina S.A.). The exposure was by ingestion through the treated prey (Rophalosiphum padi) or by drinking treated water during 48 h for treatment of the adult. The herbicide solutions were prepared using distilled water as solvent. The bioassays were carried out in the laboratory under controlled conditions: 23 +/- 0.5 degrees C, 75 +/- 5% RH and 16:8 (L:D) of photoperiod. Development time, weight of pupae, adult emergence, pre-oviposition period, fecundity and fertility were evaluated as endpoints. Larvae from glyphosate treatment molted earlier than controls. In addition, the weight of pupae, longevity, fecundity and fertility were drastically reduced in treated organisms. The reductions were more drastic when the treatments were performed at the third larval stage than as adult. The reproduction capacity of the predator was the most affected parameter and could be related to a hormonal disruption by glyphosate in the treated organisms. This work can confirm the deleterious effects of this herbicide on beneficial organisms. Also, it agrees with prior studies carried out on other predators associated to soybean pest, such as Chrysoperla externa (Neuroptera: Chrysopidae) and Alpaida veniliae (Araneae: Araneidae).

Compatibility of a Melia azedarach extract with Eriopis connexa (Coleoptera: Coccinellidae).

The integrated pest management (IPM) premises claim for a joint use of beneficial organisms (parasitoids and predators) and selective pesticides. Botanical pesticides are being considered as selective pesticides. During the last 20 years of the 20th century, the plant family Meliaceae was identified as a promising source of compounds with insect-control properties. The Chinaberry tree, Melia azedarach L., is a deciduous tree native to Persia, India, and China, but has been cultivated in many countries including Africa, Australia, and the Americas. It is a source of tetranortriterpenoid compounds called meliatoxins, which have similar structures and insecticidal activities compared to azadirachtin. Eriopis connexa Germar (Coleoptera: Coccinelidae) is a native predator from the Neotropical Region. It is a beneficial organism associated to several agro-ecosystems, feeding on soft-bodies pest such as aphids, whiteflies and thrips. In horticultural crops, it has been observed to feed on worldwide important pest such as Bemisia tabaci Gennadius (Heteroptera: Aleyrodeidae) and Myzus persicae L. (Heteroptera: Aphididae), both of them considered as virus vectors. The objectives of this work were to evaluate the side-effects of M. azaderach (Meliaceas) extract on eggs, larvae and adults of Eriopis connexa. Extracts from unripe fruits (1%, w/w, obtained by the CEPROCOR, Cordoba-Argentina) were employed for ecotoxicological bioassays. The exposure method was by immersion for eggs and by ingestion of drinking water for adults. The botanical extract solutions were prepared using distilled water as solvent and a tensioactive (Tween 20) for topical application. The bioassays were carried out in the laboratory under controlled conditions: 23 +/- 0.5 degrees C temperature, 75 +/- 5% RH and 16:8 (L:D) photoperiod. Hatching, development time, adult emergence, pre-oviposition period, fecundity and fertility were evaluated as endpoints. No detrimental effects were observed in treated organisms irrespective of the exposure way and development stage. According to these preliminary results, M. azaderach fruit extracts can be compatible with E. connexa for pest control. However, future studies should be addressed to conclude about its toxicity on beneficial organisms.

Effects of the herbicide glyphosate on biological attributes of Alpaida veniliae (Araneae, Araneidae), in laboratory.

In the past decades there has been increasing interest in the study of arthropod predators as effective potential natural enemies to be used in the biological control of agricultural pests. In Argentina, transgenic soybean crops (Round-up Ready, RR) are inhabit by many spider species, some of them in high abundance, being indicative of an import potential for pest predation. This crop is associated with the use of glyphosate, a broad-spectrum herbicide, with low environmental impact, even though since the 80's, several negative effects have been deeply documented on mammals, fishes, amphibians, snails, earthworms, insects, etc. Nowadays, the effects on arthropod physiology, behavior and life history traits as end-points in ecotoxicological evaluations are being recognized. In transgenic soybean crops of Buenos Aires province (Argentina), Alpaida veniliae (Araneae, Araneidae) is one of the most abundant orb web weaver spiders. The purpose of this study was to address the effects of glyphosate on some biological attributes of A. veniliae, in laboratory. Results of this study showed no lethal direct effects of Glifoglex on this spider, but it is the first report in literature about sublethal effects of this herbicide on a spider's biological attributes. Negative effects on prey consumption, web building, fecundity, fertility and developmental time of progeny were observed. Although sublethal effects have received less attention than direct lethal effects, they are relevant from an ecological point of view, since the reduction of the arthropod performance may create risks to arthropod biodiversity conservation in agroecosystems.

Impact of glyphosate on the development, fertility and demography of Chrysoperla externa (Neuroptera: Chrysopidae): ecological approach.

Few ecotoxicological studies have used life table analysis to evaluate the toxicity of pesticides on beneficial organisms. This study is the first report of the effect of the herbicide glyphosate on a predator insect, Chrysoperla externa, using a demographic approach. This predator is associated to soybean pests and has a potential role as a biological control agent in the Neotropical Region. The objective of this work was to evaluate the side-effects of glyphosate on the development, fertility and demography of C. externa, treated orally by ingestion of glyphosate-dipped eggs of Sitotroga cerealella in laboratory conditions. The data were analyzed using the age-stage, two-sex life table. Development from third larval instar to pupae and adult longevity were shorter in glyphosate-treatment than in the control. Adult pre-reproductive period was longer in glyphosate-treatment than in the control. Fecundity and fertility were deeply reduced, as well, being fertility greater affected. A high important reduction was registered in all population parameters. Most eggs from glyphosate-treated cohort looked abnormal, smaller than control, dehydrated and became black 2d after oviposition. In addition, adults developed tumours in the abdomen region at 20d after emergence, being the effect more drastic in females than males. It is beyond the scope of our study to speculate on the effects of this herbicide on C. externa field populations. However, it seems likely that populations under continuous use of glyphosate would be exposed at greater detrimental effects in the long term.

Effects of larval exposure to sublethal concentrations of methoxyfenozide in Spodoptera frugiperda (J.E. Smith).

Methoxyfenozide is an ecdysone agonist, the most new class of insect growth regulators. Most studies of the toxicity of ecdysone agonists on lepidopteran pests have been conducted during the larval stages, and little has been published regarding its sublethal effects in the surviving individuals. In this study, the lethal and sublethal effects of methoxyfenozide on the fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) were assessed. For this, fifth instars of this pest were continuously fed, until pupation, with artificial diet containing 0.24 and 0.35 mg of active ingredient/kg diet, which correspond to the LC10 and LC25 values, respectively, for this insecticide. 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. Both male and female treated larvae lived about seven days longer than the controls for both concentrations tested. Finally, S. frugiperda adults that resulted from fifth instars treated with methoxyfenozide were not affected in their 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.

Sublethal effects of two neurotoxican insecticides on Araneus pratensis (Araneae: Araneidae).

Spiders are important predators of several agricultural pests and they play an important role as indicators of ecosystem disturb. In Argentina, soybean crop has increased from the introduction of transgenic soybean resistant to glyphosate. This expansion produced an increase in the use of conventional and non-selective pesticides to control soybean pests. The objective of this work was to evaluate the side effects of subletal concentrations of two neurotoxican insecticides with a different mode of action: endosulfan (Glex, 35%, 25 mg/l a.i.) and spinosad (Tracer, 48%, 30 and 3 mg/l a.i) on Araneus pratensis. The insecticides were applied by ingestion of the treated prey (Musca domestica), and the effects on mortality, prey consumption, web building, mating, ootheca construction and fecundity were determined. Spinosad (30 mg/l a.i.) produced higher mortality than endosulfan (25 mg/l a.i.). Tremors and non-coordinated movements were observed in this treatment. The prey consumption was significantly reduced by the two insecticides (approximately 40% lower than control). The spider web building was significantly affected by the two insecticides, but spinosad had a greater effect. Though mating was not affected by both pesticides, abnormal oothecas and dehydrated eggs were observed. This work reports that sublethal concentrations representing approximately from 25 to 2.5% of the maximum field recommended concentrations (105 and 120 mg/l a.i., respectively) showed negative effects on A. pratensis. The consequences of these effects on role of A. pratensis as a natural mortality factor of soybean pests are discussed.

Side effects of conventional and non-conventional insecticides on eggs and larvae of Chrysoperla externa (Hagen) (Neuroptera: Chrysopidae) in Argentine.

The objective of this work was to evaluate the effect of two conventional insecticides commonly used in soybean crops of Argentina, endosulfan (Endosulfan 35 Glex, 105 ppm) and cypermethrin (Glextrin 25, 75 ppm) and two insecticides belonging to the new insecticides' generation, methoxyfenozide (Inteprid SC 24%, 144 ppm) and spinosad (Tracer 48%, 120 ppm), on eggs and larvae of Chrysoperla externa (Hagen) by dipping and topical application, respectively, under laboratory conditions. After egg treatment, the percentage of egg hatching, duration of the embryonic period and survival of first-instar larvae were evaluated. In the third-instar larvae assay, the survivals of the larvae and pupae, as well as eggs fertility were recorded. Endosulfan and cypermethrin were highly toxic towards eggs and larvae with mortality rates ranging from 80% to 100%. Toxicity of spinosad was intermediate causing short-term effects, but it did not cause any long-term effects. Methoxyfenozide was harmless to eggs and larvae of C. externa, showing good potential for use in integrated pest management in soybean crops. The selectivity of these insecticides and their inclusion in IPM programs is discussed.