Recent assessment and characterization of Tuta absoluta resistance to cartap hydrochloride.

Tuta absoluta can cause 100% loss in tomato yield in Brazil and chemical control, which uses cartap hydrochloride (nereistoxin derivative), is still the most used tactic against T. absoluta populations. Despite the long use of cartap hydrochloride, the genetic and physiological bases underlying the resistance are not known. Resistance to cartap hydrochloride among field populations varied from very low (RR = 2.3 fold) to very high (RR = 537 fold). The Gameleira 2 (GML 2-Res) population was exposed to cartap hydrochloride (up to 500 mg L-1) for few rounds of selection to clean extrinsic factors before used in downstream experiments after 2.5 years without selection in laboratory. Resistance to cartap hydrochloride was autosomal, incompletely recessive, and polyfactorial. The effective dominance (dominance level of survival at a given insecticide dose) at 60 mg of cartap hydrochloride L-1 (which killed 100% of heterozygous individuals) discriminated resistant from susceptible phenotypes. Hydrolases and glutathione S-transferase appear to detoxify cartap hydrochloride as TPP and DEM synergized its toxicity, but CYP450-dependent monooxygenases are as well implicated. Cross-resistance was significant between cartap hydrochloride and methoxyfenozide (RR = 6.99 fold), deltamethrin (RR = 3.57 fold), chlorfenapyr (RR = 3.21 fold), or chlorantraniliprole (RR = 2.83 fold). The characterization of T. absoluta resistance to cartap hydrochloride provides valuable information to refine the management of resistance to insecticides (MRI) program in Brazil with cross resistance pattern very favorable to the rotation of active ingredients that will impair survival of this pest to that insecticide in the field.

Field-Evolved Resistance and Cross-Resistance of Brazilian Tuta absoluta (Lepidoptera: Gelechiidae) Populations to Diamide Insecticides.

Tuta absoluta (Meyrick), one of the most important tomato pests worldwide, is heavily controlled by the application of insecticides. Diamide insecticides represent a new class of products recently registered to control T. absoluta After 6 yr of use, control failures have been reported in populations of this pest, suggesting a hypothetical resistance development. Therefore, a resistance survey was performed using nine populations of T. absoluta that were collected in open fields, including from areas with reports of a reduced efficacy of diamides in the Northeast and Central regions of Brazil. Initial surveys with diagnostic and label doses proved the reduced efficacy of diamides against most populations. The LC50 values of chlorantraniliprole varied from 0.0044 (Brasília) to 1,263 (América Dourada) mg AI liter-1 (the resistance ratios [RR50] ranged from 1.0- to 288,995-fold), whereas the LC50 values for cyantraniliprole and flubendiamide, respectively, varied from 0.015 (Brasília) to 281 (América Dourada) mg AI liter-1 and from 0.038 (Guaraciaba do Norte) to 3,018 (Gameleira 1) mg AI liter-1 The resistance ratios (RR50) ranged from 1.0- to 18,423-fold for cyantraniliprole and from 1.0- to 80,413-fold for flubendiamide. The log LC50 values of pairwise diamides were strongly and significantly correlated, which denoted cross-resistance among them. Very high resistance to diamides in T. absoluta was observed in this study, suggesting that strategies to mitigate resistance and thereby control the pest must not include only insecticides. Other control tactics must be carefully implemented over time to increase the life span of diamides, including rotational practices with other molecules.

Mutation (G275E) of the nicotinic acetylcholine receptor α6 subunit is associated with high levels of resistance to spinosyns in Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae).

The tomato leafminer, Tuta absoluta, now a major pest of tomato crops worldwide, is primarily controlled using chemical insecticides. Recently, high levels of resistance to the insecticide spinosad have been described in T. absoluta populations in Brazil. Selection of a resistant field-collected strain led to very high levels of resistance to spinosad and cross-resistance to spinetoram, but not to other insecticides that target the nicotinic acetylcholine receptor (nAChR). In this study the mechanisms underlying resistance to spinosad were investigated using toxicological, biochemical and molecular approaches. Inhibition of metabolic enzymes using synergists and biochemical assessment of detoxification enzyme activity provided little evidence of metabolic resistance in the selected strain. Cloning and sequencing of the nAChR α6 subunit from T. absoluta, the spinosad target-site, from susceptible and spinosad-resistant strains were done to investigate the role of a target-site mechanism in resistance. A single nucleotide change was identified in exon 9 of the α6 subunit of the resistant strain, resulting in the replacement of the glycine (G) residue at position 275 observed in susceptible T. absoluta strains with a glutamic acid (E). A high-throughput DNA-based diagnostic assay was developed and used to assess the prevalence of the G275E mutation in 17 field populations collected from different geographical regions of Brazil. The resistant allele was found at low frequency, and in the heterozygous form, in seven of these populations but at much higher frequency and in the homozygous form in a population collected in the Iraquara municipality. The frequency of the mutation was significantly correlated with the mortality of these populations in discriminating dose bioassays. In summary our results provide evidence that the G275E mutation is an important mechanism of resistance to spinosyns in T. absoluta, and may be used as a marker for resistance monitoring in field populations.

Status of pyrethroid resistance and mechanisms in Brazilian populations of Tuta absoluta.

The tomato leafminer, Tuta absoluta, is a major pest of tomato crops worldwide. This study surveyed the resistance of T. absoluta populations from four regions in Brazil to pyrethroid insecticides, the frequencies of L1014F, T929I and M918T Na channel mutations, and the role of detoxification metabolism in the resistance. Resistance ratios varied from 1- to 11-times among populations and insecticides, but control failure likelihood assays showed that all pyrethroids assessed exhibited no efficacy at all (and thus, 98-100% control failure likelihood) against all T. absoluta populations. The activity of glutathione S-transferase and cytochrome P450-mediated N-demethylation in biochemical assays was significantly correlated with the level of resistance to deltamethrin and permethrin suggesting that these enzymes may play a role in resistance. TaqMan assays were used to screen for the presence of knockdown resistance (kdr) mutations and revealed that the L1014F kdr mutation was fixed in all populations and associated with two super-kdr mutations, M918T and particularly T929I, at high frequency. Altogether, results suggest that control failures are because of mutations in the domain II of the sodium channel, as a prevailing mechanism of resistance to pyrethroids in populations of T. absoluta in Brazil. But, enhanced cytochrome P450-dependent monooxygenases and GST activities also play an important role in the resistance of some populations, which reinforce that pyrethroids must not be used overall to control T. absoluta.

Susceptibility of Tuta absoluta (Lepidoptera: Gelechiidae) Brazilian populations to ryanodine receptor modulators.

BACKGROUND: Phthalic and anthranilic diamides comprise a new insecticide class recently registered in Brazil to control Lepidoptera such as Tuta absoluta (Meyrick). Therefore, the baseline of susceptibility was determined for eight representative field populations of this species to establish a resistance monitoring programme. The potential for cross-resistance as well as detoxification metabolism was assessed in order to fine-tune the resistance management programme. RESULTS: Brazilian populations were very susceptible to chlorantraniliprole (LC50 values varied from 3.17 to 29.64 µg AI L(-1) ), cyantraniliprole (LC50 values varied from 8.61 to 28.95 µg AI L(-1) ) and flubendiamide (LC50 values varied from 94 to 230 µg AI L(-1) ), with respective resistance ratios of 9.33-, 3.36- and 2.45-fold between most susceptible and tolerant populations. Anthranilic diamides showed significant correlations between log LC50 values among themselves, suggesting a high risk of cross-resistance. However, the log LC50 values of T. absoluta to phthalic diamide did not show any correlation with anthranilic diamides. Cytochrome- P450-dependent monooxygenase activity showed a weak correlation with log LC50 values of T. absoluta populations to anthranilic diamides, which suggests a potential route for evolving resistance to anthranilic diamides. CONCLUSION: The diamides were highly effective against T. absoluta, with populations showing a homogeneous response to them. Cross-resistance is very likely between anthranilic diamides in T. absoluta. Populations of this pest may evolve resistance by increasing cytochrome- P450-dependent monooxygenases.

Resistance and behavioural response of Plutella xylostella (Lepidoptera: Plutellidae) populations to Bacillus thuringiensis formulations.

BACKGROUND: Insecticide resistance is probably the major cause of control failure of Plutella xylostella (L.) in Brazil. In most production regions, the use of chemicals has been the prevalent method of control, with reduced efficacy through cropping seasons, even for the most recent use of products based on Bacillus thuringiensis (Bt). The current status of the resistance to these products was assessed, as well as the behavioural response of P. xylostella populations to Bt sprays. RESULTS: Most populations of P. xylostella were resistant to Bt products, particularly to Xentari®WDG (2-54-fold). Differences in walking characteristics of larvae were variable for most populations, for both Dipel®WP and Xentari®WDG, but not associated with resistance. Most females preferred to lay eggs on untreated surfaces and showed a reduced proportion of oviposition on treated surfaces that only correlated with resistance to Dipel®WP (r = -0.74, P = 0.02). CONCLUSION: Broad and indiscriminate use of Bt-based products has selected Brazilian P. xylostella populations to resistance. Larval movement appears to be a resistance-independent mechanism. Most populations of P. xylostella preferred to lay eggs on Bt-free surfaces, which might be a result of growers' practice of spraying the cabbage head. Reduced oviposition on treated surfaces correlated with physiological resistance, suggesting a behavioural response among the Bt-resistant colonies to Dipel®WP.

Response of different populations of seven lady beetle species to lambda-cyhalothrin with record of resistance.

Simultaneous use of biological and chemical controls is a valued and historic goal of integrated pest management, but has rarely been achieved. One explanation for this failure may be the inadequate documentation of field populations of natural enemies for insecticide tolerance or resistance because natural enemies surviving insecticide application do not create problems like resistant pest species. Therefore, this study investigated 31 populations of lady beetles (Coleoptera: Coccinellidae) regarding their susceptibility to lambda-cyhalothrin, a pyrethroid insecticide that is widely used in cotton and other crops to control lepidopteran and coleopteran pests that are not targeted as prey by lady beetles. The study focused on seven coccinellid species common in cotton fields Coleomegilla maculata De Geer, Cycloneda sanguinea (L.), Eriopis connexa Germar, Harmonia axyridis (Pallas), Hippodamia convergens Guérin-Méneville, Olla v-nigrum (Mulsant), and Brumoides foudrasi (Mulsant) and one lady beetle species [Curinus coeruleus Mulsant] from a non-cotton ecosystem for comparisons. Dose-mortality curves were estimated after topical treatment of adult lady beetles with lambda-cyhalothrin. Statistically significant variations in lady beetle susceptibility were observed between species and between populations of a given species. Seven and eighteen populations of lady beetles exhibited greater values of LD50 and LD90, respectively, than the highest recommended field rate of lambda-cyhalothrin (20g a.i./hectare≈0.2g a.i./L) for cotton fields in Brazil. Furthermore, based on LD50 values, 29 out of 30 tested populations of lady beetles exhibited ratios of relative tolerance varying from 2- to 215-fold compared to the toxicity of lambda-cyhalothrin to the boll weevil, Anthonomus grandis Boh. (Coleoptera: Curculionidae). Four populations of E. connexa were 10.5-37.7 times more tolerant than the most susceptible population and thus were considered to be resistant to lambda-cyhalothrin, the first record of resistance for this species. These findings demonstrate that field selection for resistance to lambda-cyhalothrin in common lady beetles is occurring, opening up possibilities to effectively integrate biological control where the popular insecticide lambda-cyhalothrin is used.

Effect of sub-lethal doses of Bacillus thuringiensis subsp. Aizawai and deltamethrin with regard to fertility and organ toxicity in pregnant albino rats.

Products with Bacillus thuringiensis (Bt) and synthetic insecticides have been widely used against important vectors of human diseases. However, few studies have addressed the application of these substances on the female reproduction apparatus during pregnancy at doses that do not cause clinical symptoms of intoxication. Seventy pregnant albino rats were analyzed with regard to fertility and histopathology of the kidneys, liver and lungs as well as the morphology of the neonates. The rats were submitted to three sub-lethal doses of the biological insecticide XenTari(®) WG (B. thuringiensis subsp. Aizawai) and the synthetic insecticide deltamethrin (Decis(®) 25CE). After the confirmation of copulation, the insecticides were administered orally for either seven days or during the entire pregnancy. The analysis revealed histopathological alterations in all organs analyzed in both treatments. No miscarriages occurred and the neonates did not exhibit signs of malformation of the head, limbs, thorax or abdomen. However, there were a smaller number of pups in the groups that received higher doses of the insecticides in comparison to the control group. Both insecticides produced similar lesions in the kidneys, liver and lungs and reduced the fertility of rats when administered at sub-lethal doses with no clinical signs of intoxication. Thus, this study suggests that sublethal doses of both insecticides can provide chronic toxicity in humans.

Survival and behavioural response to acaricides of the coconut mite predator Neoseiulus baraki.

The coconut mite, Aceria guerreronis Keifer, is a major pest of coconut palm in the world. The control of this pest species is done through acaricide applications at short time intervals. However, the predators of this pest may also be affected by acaricides. Among the predators of A. guerreronis, Neoseiulus baraki (Athias-Henriot) has potential for biological control. The objective of this study was to assess the effect of acaricides on the survival and behavior of N. baraki. The survivorship of N. baraki was recorded in surface-impregnated arenas. Choice and no-choice behavioral bioassays were carried out using a video tracking system to assess the walking behavior of the predator under acaricide exposure. Although all acaricides negatively affected the survival of N. baraki, chlorfenapyr and azadirachtin caused lower effect than the other acaricides. No significant differences in walking behavior were observed under exposure to fenpyroximate, chlorfenapyr and chlorpyrifos on fully-contaminated arenas. Azadirachtin and chlorpyrifos caused repellence. Irritability was observed for all acaricides, except for abamectin. Chlorfenapyr was the most suitable product for managing the coconut mite because of its low effect on survival and behavior of N. baraki.

Residual bioassay to assess the toxicity of Acaricides against Aceria guerreronis (Acari: Eriophyidae) under laboratory conditions.

Aceria guerreronis Keifer (Acari: Eriophyidae) is considered a major pest of the coconut (Cocos nucifera L.), and the use of pesticides is the current method to control it. However, no standard toxicological tests exist to select and assess the efficiency of molecules against the coconut mite. The aim of this study was to develop a methodology that allows for the evaluation of the relative toxicity of acaricides to A. guerreronis through rapid laboratory procedures. We confined A. guerreronis on arenas made out of coconut leaflets and tested two application methods: immersing the leaf fragments in acaricides and spraying acaricides on the leaf fragments under a Potter spray tower. In the latter application method, we sprayed leaf fragments both populated with and devoid of mites. We evaluated the comparative toxicity of two populations (Itamaracá and Petrolina, Pernambuco, Brazil) by spraying on leaflets without mites and submitted the mortality data to probit analysis after 24 h of exposure. No difference was observed in the LC50, regardless of whether the leaflets were immersed or sprayed with acaricide (abamectin, chlorfenapyr or fenpyroximate). The toxicity of chlorfenapyr and fenpyroximate did not differ, irrespective of whether it was applied directly to the leaflet or to the mite; however, the toxicity of abamectin was higher when applied directly to the mite. Chlorpyrifos and abamectin toxicities were lower for the Petrolina population than for the Itamaracá population. Immersing and spraying coconut leaflets can be used to assess the mortality of A. guerreronis under laboratory conditions.

Measurements of Cry1F binding and activity of luminal gut proteases in susceptible and Cry1F resistant Ostrinia nubilalis larvae (Lepidoptera: Crambidae).

The biochemical mechanism of resistance to the Bacillus thuringiensis Cry1F toxin was studied in a laboratory-selected strain of Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) showing more than 3000-fold resistance to Cry1F and limited cross resistance to other Cry toxins. Analyses of Cry1F binding to brush border membrane vesicles of midgut epithelia from susceptible and resistant larvae using ligand immunoblotting and Surface Plasmon Resonance (SPR) suggested that reduced binding of Cry1F to insect receptors was not associated with resistance. Additionally, no differences in activity of luminal gut proteases or altered proteolytic processing of the toxin were observed in the resistant strain. Considering these results along with previous evidence of relatively narrow spectrum of cross resistance and monogenic inheritance, the resistance mechanism in this Cry1F selected strain of O. nubilalis appears to be specific and may be distinct from previously identified resistance mechanisms reported in other Lepidoptera.

Podisus nigrispinus requer cópulas longas para o sucesso reprodutivo / Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae) requires long matings for successful reproduction

The occurrence of multiple and long matings seem to play an important role in the reproduction of the predatory stinkbugs such as Podisus nigrispinus (Dallas). However, the mechanisms underlying these behaviors remain unclear. In this study, the reproduction of P. nigrispinus was investigated as function of females' choice for their mating pair based on male body size and previous mating status, risk of predation and duration of mating. The female mating behavior was studied with female having multiple, partial or no partner choice based on male body size. Time to initiate a mating and its duration was observed after pairing males and females under risk of predation imposed by the presence of the predatory wasp Polistes versicolor Oliver. In addition, aiming to determine the reasons for long lasting matings, female had their mating interrupted at different intervals. Our data indicated that P. nigrispinus females do not select male partners as function of their body size and mating status. Duration of mating, mating partners' choice, and female fecundity and fertility were not influenced by the male size and risk of predation imposed by the predatory wasps. Mating interrupted after 30, 60, 120 and 240 min resulted in only 0, 3.1, 7.7 and 34 percent of egg hatching compared to 74.2 percent under uninterrupted mating (338 to 671 min long). Therefore, the reproductive success of P. nigrispinus females does not depend on male size and male mating status, but requires long-lasting mating as a condition for adequate spermatozoa transference to females instead.

A realização de múltiplas e longas cópulas parece ser importante para a reprodução de percevejos predadores como Podisus nigrispinus (Dallas), mas o comportamento governando essas características permanece desconhecido. Neste estudo foram investigados o comportamento de cópula e a reprodução de P. nigrispinus em função do tamanho do macho, seu status prévio de acasalamento, risco de predação e interrupção de cópulas. A escolha da fêmea para o acasalamento foi estudada sob múltipla escolha, parcial ou sem chance de escolha por machos grandes ou pequenos. O comportamento para o início do acasalamento e tempo de cópula sob risco de predação quando na presença de Polistes versicolor Oliver foi comparado àquele de casais sem risco de predação. Machos e fêmeas de P. nigrispinus acasalaram independentemente do tamanho e status prévio de acasalamento dos machos, além de não apresentarem preferência por parceiros. A duração da cópula, escolha do parceiro e fertilidade das fêmeas não foram influenciadas pelo tamanho do macho, nem a presença do predador alterou o comportamento de acasalamento e duração de cópula. Fêmeas com cópulas interrompidas após 30, 60, 120 e 240 min apresentaram baixa viabilidade de ovos (0; 3,1; 7,7 e 34 por cento, respectivamente), enquanto aquelas com tempo natural de cópula (338 a 671 min) obtiveram 74,2 por cento de viabilidade. Portanto, o sucesso reprodutivo de P. nigrispinus não depende do tamanho do macho ou de seu status de acasalamento, mas requer longas cópulas para a transferência de espermatozóides.

Ultrastructure of the Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) midgut.

The insect midgut has ultimately been the focus of researches tempting to control insect pests because alterations in the insect gut may affect not only its development, but also physiological events such as nutrient absorption and transformation. The objective of the present work was to describe morphologically, histochemically, and ultrastructurally the larva midgut of Alabama argillacea (Hübner) (Lepidoptera: Noctuidae), a cotton key pest in Brazil. Light and electronic transmission microscopy was used to obtain images from midgut sections of late fourth-instar larvae of A. argillacea. In general, the morphology, histochemistry, and ultrastructure characteristics of A. argillacea midgut follow that described in the literature for other lepidopteran species. However, the results showed a mitochondrial polymorphism and branched microvilli, which suggest an ultrastrucutural and physiological modification possibly associated with a high absorption and secretion activity by the columnar cells of this species. This intense activity may favor a faster response related to the action of ingested microbial agents and/or toxins, and can explain the high susceptibility of A. argillacea to the agents of control such as the toxin of Bacillus thuringiensis.

[Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae) requires long matings for successful reproduction]. / Podisus nigrispinus requer cópulas longas para o sucesso reprodutivo.

The occurrence of multiple and long matings seem to play an important role in the reproduction of the predatory stinkbugs such as Podisus nigrispinus (Dallas). However, the mechanisms underlying these behaviors remain unclear. In this study, the reproduction of P. nigrispinus was investigated as function of females' choice for their mating pair based on male body size and previous mating status, risk of predation and duration of mating. The female mating behavior was studied with female having multiple, partial or no partner choice based on male body size. Time to initiate a mating and its duration was observed after pairing males and females under risk of predation imposed by the presence of the predatory wasp Polistes versicolor Oliver. In addition, aiming to determine the reasons for long lasting matings, female had their mating interrupted at different intervals. Our data indicated that P. nigrispinus females do not select male partners as function of their body size and mating status. Duration of mating, mating partners' choice, and female fecundity and fertility were not influenced by the male size and risk of predation imposed by the predatory wasps. Mating interrupted after 30, 60, 120 and 240 min resulted in only 0, 3.1, 7.7 and 34% of egg hatching compared to 74.2% under uninterrupted mating (338 to 671 min long). Therefore, the reproductive success of P. nigrispinus females does not depend on male size and male mating status, but requires long-lasting mating as a condition for adequate spermatozoa transference to females instead.

Determining the genotoxicity of an aqueous infusion of Bauhinia monandra leaves / Determinação da genotoxicidade do infuso aquoso das folhas da Bauhinia monandra

Bauhinia monandra, commonly known as "cow's-foot", is native from Asia and widely used all over the world to treat a variety of illnesses, in particular diabetes. The high usage of the plant in Brazil and to fulfill the need of medicinal plant regulation by the Ministry of Public Health, we aimed at determining the genotoxicity, cytotoxicity and mutagenicity of an aqueous infusion from B. monandra leaves. The results were correlated to the chemical compounds found after phytochemical selection. Tests were performed in an in vitro system with plasmid DNA, in the presence and absence of exonuclease III, and in vivo system employing prokaryotic (transformation into competent DH10B bacteria) and eukaryotic (Allium cepa) assays. The infusion concentrations were 0.8 µg/µL, 4 µg/µL, 20 µg/µL and 100 µg/µL. The infusion concentrations did not cause mutagenicity or cytotoxicity, however the highest concentrations were able to induce breaks in DNA phosphodiester bonds and form abasic sites, an effect suggested by the presence of phenolic hydroxyls. The results revealed risks and benefits of plant extracts for therapeutic use and their effect on genetic integrity, especially when commonly employed as a hypoglycemic.

Bauhinia monandra, popularmente conhecida como "pata-de-vaca", é nativa da Ásia e amplamente utilizada em todo o mundo para o tratamento de várias doenças, em especial diabetes. Diante da grande utilização dessa planta no Brasil, e de forma a satisfazer a necessidade de regulamentação das plantas medicinais pelo Ministério Público de Saúde, objetivamos determinar a genotoxicidade, citotoxicidade e mutagenicidade do infuso aquoso das folhas da B. monandra. Os resultados foram correlacionados com os compostos químicos encontrados após realização de uma triagem fitoquímica. Os testes foram realizados em sistema in vitro como o DNA plasmidial, na presença e ausência de exonuclease III, e in vivo empregando sistema procarioto (transformação com bactérias competente DH10B) e eucarioto (teste Allium cepa). As concentrações do infuso foram 0,8 µg/µL, 4 µg/µL, 20 µg/µL e 100 µg/µL. Nessas concentrações o infuso não causou mutagenicidade ou citotoxicidade, porém as concentrações mais elevadas foram capazes de induzir quebra nas ligações fosfodiéster do DNA e formar sítios abásicos, efeito sugerido pela presença de hidroxilas fenólicas. Os resultados revelaram riscos e benefícios desse extrato vegetal para uso terapêutico e seus efeitos sobre integridade do material genético, especialmente quando empregados como hipoglicêmico.