Herbert A.A. Siqueira

Cross-Resistance of Cry1Ab-Selected Ostrinia nubilalis (Lepidoptera: Crambidae) to Bacillus thuringiensis δ-Endotoxins

Abstract Corn plants expressing the toxin from Bacillus thuringiensis (Berliner) have proven to be effective in controlling lepidopteran pests such as the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae). Several Bt toxins are being tested and incorporated into crop genomes, although tests for cross-resistance among different toxins have been limited by a lack of resistant colonies. Four different colonies of O. nubilalis selected with full-length Cry1Ab incorporated into artificial diet developed significant levels of resistance (2.0- to 10-fold) within 10 generations. Additionally, selection with Cry1Ab resulted in decreased susceptibility to a number of other toxins to which the selected colonies were not previously exposed. Significantly, levels of resistance were highest to Cry1Ac with resistance ratios up to 51.0-fold. Low levels (less than five-fold) of cross-resistance were detected with Cry1F. In contrast, Cry9C susceptibility was unaffected by selection with Cry1Ab. These results indicate that the availability of multiple toxins could improve resistance management strategies, provided cross-resistance among toxins is not a factor.

Spinosad and the tomato borer Tuta absoluta: a bioinsecticide, an invasive pest threat, and high insecticide resistance.

The introduction of an agricultural pest species into a new environment is a potential threat to agroecosystems of the invaded area. The phytosanitary concern is even greater if the introduced pest’s phenotype expresses traits that will impair the management of that species. The invasive tomato borer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is one such species and the characterization of the insecticide resistance prevailing in the area of origin is important to guide management efforts in new areas of introduction. The spinosad is one the main insecticides currently used in Brazil for control of the tomato borer; Brazil is the likely source of the introduction of the tomato borer into Europe. For this reason, spinosad resistance in Brazilian populations of this species was characterized. Spinosad resistance has been reported in Brazilian field populations of this pest species, and one resistant population that was used in this study was subjected to an additional seven generations of selection for spinosad resistance reaching levels over 180,000-fold. Inheritance studies indicated that spinosad resistance is monogenic, incompletely recessive and autosomal with high heritability (h 2 = 0.71). Spinosad resistance was unstable without selection pressure with a negative rate of change in the resistance level ( = −0.51) indicating an associated adaptive cost. Esterases and cytochrome P450-dependent monooxygenases titration decreased with spinosad selection, indicating that these detoxification enzymes are not the underlying resistance mechanism. Furthermore, the cross-resistance spectrum was restricted to the insecticide spinetoram, another spinosyn, suggesting that altered target site may be the mechanism involved. Therefore, the suspension of spinosyn use against the tomato borer would be a useful component in spinosad resistance management for this species. Spinosad use against this species in introduced areas should be carefully monitored to prevent rapid selection of high levels of resistance and the potential for its spread to new areas.

Cross-resistance and mechanism of resistance to Cry1Ab toxin from Bacillus thuringiensis in a field-derived strain of European corn borer, Ostrinia nubilalis.

The cross-resistance spectrum and biochemical mechanism of resistance to the Bacillus thuringiensis Cry1Ab toxin was studied in a field-derived strain of Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) that was further selected in the laboratory for high levels (>1000-fold) of resistance to Cry1Ab. The resistant strain exhibited high levels of cross-resistance to Cry1Ac and Cry1Aa but only low levels of cross-resistance (<4-fold) to Cry1F. In addition, there was no significant difference between the levels of resistance to full-length and trypsin-activated Cry1Ab protein. No differences in activity of luminal gut proteases or altered proteolytic processing of the toxin were observed in the resistant strain. Significantly reduced binding of radiolabeled Cry1Aa was observed in the resistant strain whereas binding of Cry1Ab and Cry1Ac was practically the same in both resistant and susceptible strains. The interpretation of the overall data seems to suggest the involvement of an alteration in the binding of Cry1A toxins to a common receptor, which is more clearly revealed by the binding assays using radiolabeled Cry1Aa.

Analyses of Cry1Ab Binding in Resistant and Susceptible Strains of the European Corn Borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae)

ABSTRACT Cry1Ab toxin binding analysis was performed to determine whether resistance in laboratory-selected Ostrinia nubilalis strains is associated with target site alteration. Brush border membrane vesicles were prepared using dissected midguts from late instars of susceptible and resistant strains (Europe-R and RSTT) of O. nubilalis. Immunoblot analysis indicated that three different proteins bound to Cry1Ab toxin and were recognized by an anticadherin serum. In a comparison of resistant and susceptible strains, reduced Cry1Ab binding was apparent for all three bands corresponding to cadherin-like proteins in the Europe-R strain, while reduced binding was apparent in only one band for the RSTT strain. Real-time analysis of Cry1Ab binding to gut receptors using surface plasmon resonance suggested slight differences in affinity in both resistant strains. Additional binding analysis was conducted using 125I-labeled Cry1Ab, Cry1Ac, and Cry1Aa. Slight differences were again observed between the resistant and susceptible strains for Cry1Ab binding. However, when binding of 125I-labeled Cry1Aa was tested, a 10-fold reduction in the concentration of binding sites was observed in the Europe-R strain. Expression of the O. nubilalis cadherin gene was similar in both the resistant and susceptible strains and did not account for differences in binding. In combination, the results of the present work suggest that differences in susceptibility to Cry1A toxins in the Europe-R strain of O. nubilalis are associated with altered receptor binding, although the precise nature of this mechanism is still uncertain.

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.

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.

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.

Histopathology and ultrastructure of midgut of Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) fed Bt-cotton

The interaction of Cry toxins from Bacillus thuringiensis in the midgut of some insect larvae determines their efficacies as insecticides, due to the expression and availability of sites of action of the toxin in the midgut. Researches point out cases of resistance to Cry toxin due to alterations in the binding sites in columnar cell membrane. We analyzed the effects of Cry1Ac toxin expressed by Bt-cotton plants on Alabama argillacea midgut morphophysiology clarifying in levels of morphological and ultrastructural. Larvae in the 4th instar of A. argillacea after 20 min from ingesting Bt-cotton leaves expressing 0.183 ng of Cry1Ac exhibited ultrastructural and morphological modifications in the columnar cells with significant changes in the mitochondrial polymorphism, cytoplasmic vacuolization, microvillus and basal labyrinth. Expressive morphological alterations were also observed in the goblet cells indicating that the columnar cells are not the only target of the Cry1Ac toxin. The regenerative cells did not modify their structures and exhibited decrease in regeneration capacity. In conclusion, the ingestion of 0.183 ± 0.077 ng of Cry1Ac was enough to promote alterations in the columnar and goblet cells, besides reducing significantly the number of regenerative cells, which may have contributed to larval death. Nevertheless, further studies are necessary to determine the true cause of death.

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.

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.