Effects of insecticides used in strawberries on stingless bees Melipona quadrifasciata and Tetragonisca fiebrigi (Hymenoptera: Apidae).

The use of pesticides is considered one of the most important threats to pollinators, especially since they are widely used in agriculture for pest control. In the last years, several studies have reported severe secondary effects on various bee species, including exotic and native bees. In this study, lethal (mortality) and sublethal (locomotor activity) effects of insecticides and acaricides used in strawberries in Brazil (abamectin, novaluron, spinetoram, and thiamethoxam) were evaluated on the native stingless bees Melipona quadrifasciata and Tetragonisca fiebrigi. The results showed that the effects varied significantly according to the pesticide, type of exposure (oral or topical), and bee species. Through oral exposure, M. quadrifasciata was more susceptible to all insecticides except for abamectin, while in topical exposure, T. fiebrigi was more sensitive. Thiamethoxam followed by spinetoram and abamectin were the most lethal, regardless of species or exposure route; novaluron was not harmful at the highest tested dose. The locomotor activity of bees was altered in the presence of sublethal doses (LC10 and LC50) of all insecticides. Spinetoram and abamectin can be as much as toxic as thiamethoxam against M. quadrifasciata and T. fiebrigi in laboratory experiments. These findings should be confirmed in field experiments to define possibilities to combine pest control and pollinator management. In crops like strawberries, the selectivity of native pollinators should be considered.

First transcriptome of the Neotropical pest Euschistus heros (Hemiptera: Pentatomidae) with dissection of its siRNA machinery.

Over the past few years, the use of RNA interference (RNAi) for insect pest management has attracted considerable interest in academia and industry as a pest-specific and environment-friendly strategy for pest control. For the success of this technique, the presence of core RNAi genes and a functional silencing machinery is essential. Therefore, the aim of this study was to test whether the Neotropical brown stinkbug Euschistus heros has the main RNAi core genes and whether the supply of dsRNA could generate an efficient gene silencing response. To do this, total mRNA of all developmental stages was sequenced on an Illumina platform, followed by a de novo assembly, gene annotation and RNAi-related gene identification. Once RNAi-related genes were identified, nuclease activities in hemolymph were investigated through an ex vivo assay. To test the functionality of the siRNA machinery, E. heros adults were microinjected with ~28 ng per mg of insect of a dsRNA targeting the V-ATPase-A gene. Mortality, relative transcript levels of V-ATPase-A, and the expression of the genes involved in the siRNA machinery, Dicer-2 (DCR-2) and Argonaute 2 (AGO-2), were analyzed. Transcriptome sequencing generated more than 126 million sequenced reads, and these were annotated in approximately 80,000 contigs. The search of RNAi-related genes resulted in 47 genes involved in the three major RNAi pathways, with the absence of sid-like homologous. Although ex vivo incubation of dsRNA in E. heros hemolymph showed rapid degradation, there was 35% mortality at 4 days after treatment and a significant reduction in V-ATPase-A gene expression. These results indicated that although sid-like genes are lacking, the dsRNA uptake mechanism was very efficient. Also, 2-fold and 4-fold overexpression of DCR-2 and AGO-2, respectively, after dsRNA supply indicated the activation of the siRNA machinery. Consequently, E. heros has proven to be sensitive to RNAi upon injection of dsRNA into its hemocoel. We believe that this finding together with a publically available transcriptome and the validation of a responsive RNAi machinery provide a starting point for future field applications against one of the most important soybean pests in South America.

Management of Pest Insects and Plant Diseases by Non-Transformative RNAi.

Since the discovery of RNA interference (RNAi), scientists have made significant progress towards the development of this unique technology for crop protection. The RNAi mechanism works at the mRNA level by exploiting a sequence-dependent mode of action with high target specificity due to the design of complementary dsRNA molecules, allowing growers to target pests more precisely compared to conventional agrochemicals. The delivery of RNAi through transgenic plants is now a reality with some products currently in the market. Conversely, it is also expected that more RNA-based products reach the market as non-transformative alternatives. For instance, topically applied dsRNA/siRNA (SIGS - Spray Induced Gene Silencing) has attracted attention due to its feasibility and low cost compared to transgenic plants. Once on the leaf surface, dsRNAs can move directly to target pest cells (e.g., insects or pathogens) or can be taken up indirectly by plant cells to then be transferred into the pest cells. Water-soluble formulations containing pesticidal dsRNA provide alternatives, especially in some cases where plant transformation is not possible or takes years and cost millions to be developed (e.g., perennial crops). The ever-growing understanding of the RNAi mechanism and its limitations has allowed scientists to develop non-transgenic approaches such as trunk injection, soaking, and irrigation. While the technology has been considered promising for pest management, some issues such as RNAi efficiency, dsRNA degradation, environmental risk assessments, and resistance evolution still need to be addressed. Here, our main goal is to review some possible strategies for non-transgenic delivery systems, addressing important issues related to the use of this technology.

Toxicity, attraction, and repellency of toxic baits to stingless bees Plebeia emerina (Friese) and Tetragonisca fiebrigi (Schwarz) (Hymenoptera: Apidae: Meliponini).

Toxic bait formulations have been one of the main strategies used in apple orchards in southern Brazil for the control of South American fruit fly. However, its effects on the stingless bees Plebeia emerina (Friese) and Tetragonisca fiebrigi (Schwarz) are unknown. This study aimed to assess the toxicity, attraction and repellency of food lures and toxic baits on P. emerina and T. fiebrigi. We evaluated the food lures Anamed® (pure), Biofruit® (3%), Flyral® (1.25%), Sugarcane molasses (7%) and Samaritá Tradicional® (3%), in toxic baits formulations associated with spinosad (Tracer® 480SC) and malathion (Malathion® 1000EC), and the ready-to-use toxic baits Success® 0.02CB and Gelsura®. We obtained the mean lethal concentration (LC50) and the mean survival of workers after exposure to toxic bait formulations. In the field, we carried out attraction and repellency tests of toxic baits to foraging. The food lures associated with malathion and spinosad showed different levels of toxicity to P. emerina and T. fiebrigi. Sugarcane molasses and Samaritá Tradicional® associated with spinosad showed high toxicity, with LC50 values of 6.92 and 10.61 ng/µL diet to P. emerina, and of 4.37 and 15.48 ng/µL diet to T. fiebrigi, respectively. Gelsura® and food lures with malathion caused rapid workers mortality, with mean survival less than 3 h after exposure. No toxic bait formulation was attractive to P. emerina foragers in the field. Anamed®, Gelsura®, and Success® were repellent to P. emerina foragers.

Differential impacts of pesticides on Euschistus heros (Hem.: Pentatomidae) and its parasitoid Telenomus podisi (Hym.: Platygastridae).

Euschistus heros (Fabricius) (Hemiptera: Pentatomidae) primarily attack the pods and seeds of soybean plants, causing severe economic losses in Neotropical Region, and chemical control is essential to avoid these losses. Thus, insecticides more effective against this pest and less toxic to Telenomus podisi Ashmead (Hymenoptera: Platygastridae) - the main biological control agent of E. heros - should be used. In this report, we studied the differential acute impacts of pesticides used in Brazilian soybean against E. heros and T. podisi and evaluated their sublethal effects on the parasitoid to identify effective pesticides towards the pest with less harmful effect to the natural enemy. The LC50 of the insecticides to E. heros ranged from 1.20 to 533.74 ng a.i./cm2; the order of toxicity was thiamethoxam + lambda-cyhalothrin > acetamiprid + fenpropathrin > zeta-cypermethrin > acephate > imidacloprid. All pesticides were classified as slightly to moderately toxic to T. podisi based on the risk quotient. The exposure of T. podisi females to imidacloprid and the insecticide pre-formulated mixtures reduced the emergence of the offspring parasitoids by up to 40% whereas zeta-cypermethrin and the insecticides pre-formulated mixtures reduced offspring survival. The preferred order of choice of insecticides for the management of E. heros according to agronomic, toxicological, and environmental feasibility was the following: thiamethoxam + lambda-cyhalothrin > zeta-cypermethrin > acetamiprid + fenpropathrin > acephate > imidacloprid. Our study provides important and pioneer information to select insecticides for effective control of E. heros with lower impacts on T. podisi.

Compared activity of agonist molecules towards ecdysone receptor in insect cell-based screening system / Comparação da atividade de moléculas agonistas em relação ao receptor de ecdisona em um sistema de triagem baseado em linhagens celulares de insetos

The ecdysone receptor, naturally activated by steroidal hormones, is a key protein for molting and reproduction processes of insects. Artificial activation of such receptor by specific pesticides induces an anomalous process of ecdysis, causing death of insects by desiccation and starvation. In this paper, we established a protocol for screening agonistic molecules towards ecdysone receptor of insect cells line S2 (Diptera) and Sf9 (Lepidoptera), transfected with the reporter plasmid ere.b.act.luc. Therefore, we set dose-response curves with the ecdysteroid 20-hydroxyecdysone, the phytoecdysteroid ponasterone-A, and tebufenozide, a pesticide belonging to the class of diacylhydrazines. In both cell lines, the median effective concentration values on reporter gene induction (EC50) of ponasterone-A was the smallest, meaning the most active agonist molecule. In Sf9 cells, tebufenozide had as smaller EC50 than 20-hydroxyecdysone, indicating the high agonistic capability and lepidopteran specificity. The protocol established in this study can be useful for a quick screening and rational research of site-specific pesticides.(AU)

O receptor de ecdisona, naturalmente ativado por hormônios esteroidais, é uma proteína-chave nos processos de muda e reprodução de insetos. A ativação artificial desse receptor por meio de pesticidas específicos induz um processo de ecdise anômala, levando o inseto à morte por dessecação e inanição. Neste trabalho, foi estabelecido um protocolo para a triagem de moléculas agonistas em relação ao receptor de ecdisona nas linhagens celulares responsivas S2 (Diptera) e Sf9 (Lepidoptera), transfectadas com o plasmídeo repórter ere.b.act.luc. Para tanto, curvas de dose-resposta foram estabelecidas com o ecdisteroide 20-hidroxiecdisona, o fitoecdisteroide ponasterona-A e tebufenozida, um pesticida pertencente à classe das diacilhidrazinas. Em ambas linhagens celulares, os valores médios de concentração efetiva para indução gênica (EC50) ponasterona-A foram menores, significando que este é o agonista mais potente. Em células Sf9, a tebufenozida apresentou EC50 menor que a 20-hidroxiecdisona, indicando uma alta atividade agonista e especificidade deste inseticida a lepidópteros. O protocolo estabelecido neste trabalho pode ser utilizado para uma rápida triagem e busca racional de pesticidas de alvo bioquímico específico.(AU)

Flexibility and extracellular opening determine the interaction between ligands and insect sulfakinin receptors.

Despite their fundamental importance for growth, the mechanisms that regulate food intake are poorly understood. Our previous work demonstrated that insect sulfakinin (SK) signaling is involved in inhibiting feeding in an important model and pest insect, the red flour beetle Tribolium castaneum. Because the interaction of SK peptide and SK receptors (SKR) initiates the SK signaling, we have special interest on the structural factors that influence the SK-SKR interaction. First, the three-dimensional structures of the two T. castaneum SKRs (TcSKR1 and TcSKR2) were generated from molecular modeling and they displayed significance in terms of the outer opening of the cavity and protein flexibility. TcSKR1 contained a larger outer opening of the cavity than that in TcSKR2, which allows ligands a deep access into the cavity through cell membrane. Second, normal mode analysis revealed that TcSKR1 was more flexible than TcSKR2 during receptor-ligand interaction. Third, the sulfated SK (sSK) and sSK-related peptides were more potent than the nonsulfated SK, suggesting the importance of the sulfate moiety.

Analogs of sulfakinin-related peptides demonstrate reduction in food intake in the red flour beetle, Tribolium castaneum, while putative antagonists increase consumption.

The insect sulfakinins (SKs) constitute a family of neuropeptides that display both structural and functional similarities to the mammalian hormones gastrin and cholecystokinin (CCK). As a multifunctional neuropeptide, SKs are involved in muscle contractions as well as food intake regulation in many insects. In the red flour beetle Tribolium castaneum, the action on food intake by a series of synthetic SK analogs and one putative antagonist was investigated by injection in beetle adults. The most remarkable result was that both sulfated and non-sulfated SKs [FDDY(SO3H)GHMRFamide] inhibited food intake by about 70%. Strong activity observed for SK analogs featuring a residue that mimics the acidic nature of Tyr(SO3H) but lack the phenyl ring of Tyr, indicate that aromaticity is not a critical characteristic for this position of the peptide. SK demonstrated considerable tolerance to Ser and Ala substitution in position 8 (basic Arg), as analogs featuring these uncharged substitutions retained almost all of the food intake inhibitory activity. Also, the Phe in position 1 could be replaced by Ser without complete loss of activity. Conversely, substitution of Met by Nle in position 7 led to inactive compounds. Finally, the Caenorhabditis elegans sulfated neuropeptide-like protein-12 (NLP-12), that shares some sequence similarities with the SKs but features a Gln-Phe-amide rather than an Arg-Phe-amide at the C-terminus, elicited increased food intake in T. castaneum, which may indicate an antagonist activity. Co-injection of NLP-12 with nsSK blocked the food intake inhibitory effects of nsSK.

Sequencing and structural homology modeling of the ecdysone receptor in two chrysopids used in biological control of pest insects.

In insects, the process of molting and metamorphosis are mainly regulated by a steroidal hormone 20-hydroxyecdysone (20E) and its analogs (ecdysteroids) that specifically bind to the ecdysone receptor ligand-binding domain (EcR-LBD). Currently, several synthetic non-steroidal ecdysone agonists, including tebufenozide, are commercially available as insecticides. Tebufenozide exerts its activity by binding to the 20E-binding site and thus activating EcR permanently. It appears that subtle differences in the architecture among LBDs may underpin the differential binding affinity of tebufenozide across taxonomic orders. In brief, first we demonstrated the harmlessness of tebufenozide towards Chrysoperla externa (Ce). Then, a molecular analysis of EcR-LBD of two neuropteran insects Chrysoperla carnea and Ce was presented. Finally, we constructed a chrysopid in silico homology model docked ponasterone A (PonA) and tebufenozide into the binding pocket and analyzed the amino acids indentified as critical for binding to PonA and tebufenozide. Due to a restrict extent in the cavity at the bottom of the ecdysone-binding pocket a steric clash occurred upon docking of tebufenozide. The absence of harm biological effect and the docking results suggest that tebufenozide is prevented of any deleterious effects on chrysopids.

Susceptibilidade de lagartas dos biótipos milho e arroz de Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) a inseticidas com diferentes modos de acão / Susceptibility of caterpillars of the biotypes corn and rice of Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) to insecticides with different action manners

O objetivo do trabalho foi avaliar a susceptibilidade de lagartas dos biótipos milho e arroz de Spodoptera frugiperda, a inseticidas com diferentes modos de acão. Os insetos foram coletados em milho e em arroz irrigado no agroecossistema de várzea, município de Pelotas, região que produz milho e arroz irrigado (lado a lado). Os experimentos foram realizados, em condicões controladas de temperatura (25 n 1ºC), umidade relativa (70 n 10 por cento) e fotofase (14 horas), utilizando-se folhas do híbrido de milho Pionner 30F33 (40 dias após a emergência). As folhas pulverizadas em torre de Potter calibrada para aplicacão de um volume de calda de 1,7 n 0,305mg cm-2, foram colocadas em recipientes de plásticos com tampa, sendo individualizadas 25 lagartas de 3º ínstar de cada biótipo de S. frugiperda. Os inseticidas e concentracões avaliados foram: clorpirifós [Lorsban 480 BR, 0,960g i.a. L-1 (Organofosforado)], lambda-cialotrina [Karate Zeon 50 CE, 0,003g i.a. L-1 (Piretróide sintético)], lufenuron [Match CE, 0,006g i.a. L-1 (Aciluréia)], methoxifenozide [Intrepid 240 SC, 0,158g i.a. L-1 (Diacilhidrazina)] e spinosad [Tracer, 0,960g i.a. L-1 (Naturalyte)]. A avaliacão da mortalidade foi realizada 24, 48, 72, 96 e 120 horas após o tratamento. O biótipo milho de S. frugiperda foi menos suscetível aos inseticidas lambda-cialotrina, lufenuron e methoxifenozide. Os inseticidas clorpirifós e spinosad foram eficientes no controle das lagartas dos biótipos milho e arroz de S. frugiperda.