Insecticidal Gene Silencing by RNAi in the Neotropical Region.

Insecticidal gene silencing by RNA interference (RNAi) involves a post-transcriptional mechanism with great potential for insect control. Here, we aim to summarize the progress on RNAi research toward control of insect pests in the Neotropical region and discuss factors determining its efficacy and prospects for pest management. We include an overview of the available RNAi information for Neotropical pests in the Lepidoptera, Coleoptera, Diptera, and Hemiptera orders. Emphasis is put on significant findings in the use of RNAi against relevant Neotropical pests, including diamondback moth (Plutella xylostella L.), Asian citrus psyllid (Diaphorina citri Kuwayama), and the cotton boll weevil (Anthonomus grandis Boheman). We also examine the main factors involved in insecticidal RNAi efficiency and major advances to improve screening of lethal genes, formulation, and delivery. Few studies detail resistance mechanisms to RNAi, demonstrating a need for more research. Advances in formulation, delivery, and resistance management tools for insecticidal RNAi in the Neotropics can provide a basis for efficient field application.

The Fitness and Economic Benefits of Rearing the Parasitoid Telenomus podisi Under Fluctuating Temperature Regime.

Successful biological control requires detailed knowledge about the mass rearing conditions of the control agents in order to ensure higher quality of field-released insects. Thus, we investigated whether rearing fluctuating thermal condition would affect the fitness and costs of the parasitoid wasp Telenomus podisi Ashmead (a biocontrol agent used for controlling the Neotropical brown stink bug Euschistus heros (Fabricius)) when compared with parasitoid reared at constant temperature condition, which is commonly used in insect facilities. Parasitoids were reared under either constant (continuous exposure at 25 ± 2°C) or fluctuating temperature conditions (i.e., 30 ± 2°C during day and 20 ± 2°C at night) during four consecutive generations. Our results indicated that tested fluctuating temperature is more suitable for rearing of T. podisi as such temperature condition not only resulted in fitness benefits (e.g., shorter developmental time, longer female longevity, higher fecundity/fertility) but also reduced (approximately 23.5%) the estimated costs for producing the parasitoids. Furthermore, rearing T. podisi under fluctuating temperatures improved tolerance to low constant temperatures (i.e., 20°C) without changing the tolerance to constant high temperatures (30°C) in the fourth generation. Surprisingly, even parasitoids that developed under fluctuating thermal conditions performed better than those reared at constant temperature of 25°C. Collectively, our findings suggest that T. podisi reared under fluctuating thermal condition can tolerate better fluctuating temperatures that normally occur both during long periods of transport and in agricultural ecosystems, which will increase the quality and productivity of mass-reared T. podisi for inundative releases.

Toxicity and Metabolism of Zeta-Cypermethrin in Field-Collected and Laboratory Strains of the Neotropical Predator Chrysoperla externa Hagen (Neuroptera: Chrysopidae).

Resistance to pesticides has been studied in several insect pests, but information on the natural enemies of pests-including the Neotropical predator Chrysoperla externa Hagen (Neuroptera: Chrysopidae), a major biological control agent in South America-is lacking. We report here a comparative study between a field-collected strain of C. externa subjected to monthly sprayings of pyrethroids and neonicotinoids and a laboratory strain without exposure to pesticides. The tolerance of both strains against zeta-cypermethrin was similar, and addition of the synergist piperonyl butoxide increased the toxicity by 30% in both strains. Gas-chromatography analyses and mixed-function-oxidase measurements indicated similar values in both strains and also confirmed the key role of oxidative metabolism in this species. Because C. externa has maintained a tolerance to zeta-cypermethrin without previous pesticide exposure, this species could potentially be mass-reared and released in fields in the presence of pesticide pressure.

Mitochondrial DNA COI characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) from Paraguay and Uruguay.

Since its detection in Brazil in 2013, the Old World cotton bollworm Helicoverpa armigera has been reported in Argentina, Paraguay, and Bolivia. Here we present evidence extending the South American range of H. armigera to Uruguay, using polymerase chain reaction and sequencing of the partial mitochondrial DNA (mtDNA) cytochrome oxidase I region. Molecular characterization of this gene region from individuals from Paraguay also supports previous morphological identification of H. armigera in Paraguay. Shared mtDNA haplotypes in H. armigera from Brazil, Uruguay, and Paraguay were identified. Additional surveying of populations in this region will be imperative to better monitor and understand factors that are underpinning its presence and successful adaptation in these South American regions. We discuss our findings with respect to the development of resistance pest management strategies of this invasive insect pest in a predominantly monoculture soybean crop landscape in the Southern Cone region.

RNAi Technology for Insect Management and Protection of Beneficial Insects from Diseases: Lessons, Challenges and Risk Assessments.

The time has passed for us to wonder whether RNA interference (RNAi) effectively controls pest insects or protects beneficial insects from diseases. The RNAi era in insect science began with studies of gene function and genetics that paved the way for the development of novel and highly specific approaches for the management of pest insects and, more recently, for the treatment and prevention of diseases in beneficial insects. The slight differences in components of RNAi pathways are sufficient to provide a high degree of variation in responsiveness among insects. The current framework to assess the negative effects of genetically modified (GM) plants on human health is adequate for RNAi-based GM plants. Because of the mode of action of RNAi and the lack of genomic data for most exposed non-target organisms, it becomes difficult to determine the environmental risks posed by RNAi-based technologies and the benefits provided for the protection of crops. A better understanding of the mechanisms that determine the variability in the sensitivity of insects would accelerate the worldwide release of commercial RNAi-based approaches.

INSECTICIDAL ACTIVITY OF PROTEIN EXTRACTS OBTAINED FROM BULBS OF CHILEAN AMARYLLIDACEAE AGAINST TRIALEURODES VAPORARIORUM WESTWOOD AND PSEUDOCOCCUS VIBURNI SIGNORET.

Entomotoxic proteins are produced by plants in defence against insect herbivory. Some carbohydrate-binding proteins exhibit strong insecticidal activity affecting the survival, growth, development and feeding behavior of phytophagous insects. The occurrence of entomotoxic lectins is well documented in the Amaryllidaceae, a plant family spread world-wide. In Chile, this family is represented by numerous species, many of which are also of high ornamental value. Protein extracts were obtained from bulbs of five different species of Chilean Amaryllidaceae. A dose-response assay was carried out with two important pests: the greenhouse whitefly Trialeurodes vaporariorum Westwood and the mealybug Pseudococcus viburni Signoret. The extracts were offered to insects in a liquid artificial diet for three days and the mortality was scored. The Phycella australis Ravenna extract caused the highest insecticidal activity (T. vaporariorum LC50: 7200 µg/mL; P. viburni LC50: 9500 µg/mL). Applied at 1000 µg/mL in the diet the P. australis extract did not repel feeding of these pests. A mannose-binding lectin isolated from the bulbs of P. australis proved to be moderately toxic for these pests (T. vaporariorum LC50: 1127 µg/mL; P. viburni LC50: 2320 µg/mL).

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.

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 percent and 26 percent in the low and high concentration groups, respectively, on the seventh day of the experiment. A progressive larval mortality of 12 percent for the LC10 and 60 percent 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 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.

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.