Eliseu José Guedes Pereira

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.

Resistance to dual-gene Bt maize in Spodoptera frugiperda : selection, inheritance, and cross-resistance to other transgenic events

Transgenic crop “pyramids” producing two or more Bacillus thuringiensis (Bt) toxins active against the same pest are used to delay evolution of resistance in insect pest populations. Laboratory and greenhouse experiments were performed with fall armyworm, Spodoptera frugiperda, to characterize resistance to Bt maize producing Cry1A.105 and Cry2Ab and test some assumptions of the “pyramid” resistance management strategy. Selection of a field-derived strain of S. frugiperda already resistant to Cry1F maize with Cry1A.105 + Cry2Ab maize for ten generations produced resistance that allowed the larvae to colonize and complete the life cycle on these Bt maize plants. Greenhouse experiments revealed that the resistance was completely recessive (Dx = 0), incomplete, autosomal, and without maternal effects or cross-resistance to the Vip3Aa20 toxin produced in other Bt maize events. This profile of resistance supports some of the assumptions of the pyramid strategy for resistance management. However, laboratory experiments with purified Bt toxin and plant leaf tissue showed that resistance to Cry1A.105 + Cry2Ab2 maize further increased resistance to Cry1Fa, which indicates that populations of fall armyworm have high potential for developing resistance to some currently available pyramided maize used against this pest, especially where resistance to Cry1Fa was reported in the field.

Locomotory and physiological responses induced by clove and cinnamon essential oils in the maize weevil Sitophilus zeamais

Plant essential oils have been suggested as a suitable alternative for controlling stored pests worldwide. However, very little is known about the physiological or behavioral responses induced by these compounds in insect populations that are resistant to traditional insecticides. Thus, this investigation evaluated the toxicity (including the impacts on population growth) as well as the locomotory and respiratory responses induced by clove, Syzygium aromaticum L., and cinnamon, Cinnamomum zeylanicum L., essential oils in Brazilian populations of the maize weevil Sitophilus zeamais. We used populations that are resistant to phosphine and pyrethroids (PyPhR), only resistant to pyrethroids (PyR1 and PyR2) or susceptible to both insecticide types (SUS). The PyPhR population was more tolerant to cinnamon essential oil, and its population growth rate was less affected by both oil types. Insects from this population reduced their respiratory rates (i.e., CO2 production) after being exposed to both oil types and avoided (in free choice-experiments) or reduced their mobility on essential oil-treated surfaces. The PyR1 and PyR2 populations reduced their respiratory rates, avoided (without changing their locomotory behavior in no-choice experiments) essential oil-treated surfaces and their population growth rates were severely affected by both oil types. Individuals from SUS population increased their mobility on surfaces that were treated with both oil types and showed the highest levels of susceptibility to these oils. Our findings indicate that S. zeamais populations that are resistant to traditional insecticides might have distinct but possibly overlapping mechanisms to mitigate the actions of essential oils and traditional insecticides.

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.

Partial purification and characterization of trypsin-like proteinases from insecticide-resistant and -susceptible strains of the maize weevil, Sitophilus zeamais

Serine proteinases from three strains of Sitophilus zeamais (Coleoptera: Curculionidae), one susceptible and two resistant to insecticides--one exhibiting fitness cost (resistant cost strain) and the other lacking it (resistant no-cost strain), were partially purified using an aprotinin-agarose affinity column providing purification factors ranging from 36.5 to 51.2%, with yields between 10 and 15% and activity between 529 and 875 microM/min/mg protein with the substrate N-alpha-benzoyl-L-Arg-p-nitroanilide (L-BApNA). SDS-PAGE of the purified fraction revealed a 56,000 Da molecular mass band in all strains and a 70,000 Da band more visible in the resistant no-cost strain. The purified proteinases from all strains were inhibited by phenylmethyl sulphonyl fluoride (PMSF), N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), aprotinin, benzamidine and soybean trypsin inhibitor (SBTI) characterizing them as trypsin-like serine proteinases. Trypsin-like proteinases from the resistant strains exhibited higher affinity for L-BApNA. The resistant no-cost strain exhibited V(max)-values 1.5- and 1.7-fold higher than the susceptible and resistance cost strains, respectively. A similar trend was also observed when using N-alpha-p-tosyl-L-Arg methyl ester (L-TAME) as substrate. These results provide support to the hypothesis that the enhanced serine proteinase activity may be playing a role in mitigating physiological costs associated with the maintenance of insecticide resistance mechanisms in some maize weevil strains.

Fitness costs of Cry1F resistance in laboratory‐selected European corn borer (Lepidoptera: Crambidae)

The study of fitness costs associated with resistance to toxins from Bacillus thuringiensis Berliner (Bt) is important for understanding resistance evolution and for evaluating resistance management practices that prevent or mitigate resistance. A strain of European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) obtained from field collections throughout the U.S. Corn Belt in 1996, was selected in the laboratory for resistance to Cry1F by exposure to the toxin incorporated into artificial diet. The selected strain developed more than 3000‐fold resistance to Cry1F after 35 generations of selection and readily consumed Cry1F expressing maize tissue. Using this resistant strain, a susceptible strain with similar genetic background and reciprocal crosses between them, we estimated fitness costs and their dominance by measuring fitness components and population parameters determined by fertility life tables. Comparison of life‐history traits and population growth rates of genotypes homozygous and heterozygous for resistance relative to susceptible genotypes indicated existence of weak and recessive fitness costs associated with resistance. The significance of these results in relation to current resistance management practices is discussed.

Natural mortality factors of Leucoptera coffeella (Lepidoptera: Lyonetiidae) on Coffea arabica

Abstract The natural mortality of the coffee leafminer, Leucoptera coffeella (Guérin-Méneville) was investigated in three strata of coffee plant canopy for three seasons through construction and analysis of ecological life tables. Mortality of the leafminer was similar on all thirds of the canopy. Total mortality of immature stages was 95%, with 38.5, 43.8 and 12.7% occurring during egg, larval, and pupal stages. Rainfall killed 39.3% of eggs and larvae, and together with egg inviability (16.3%) and Vespidae (11.3%), were the highest mortality factors. Six wasp parasitoids caused 8% of larval mortality. Egg and larva were the critical stages. Variation in mortality was primarily associated with egg inviability, rainfall, and parasitism by Horismenus sp. (Hymenoptera: Eulophidae). Physiological disturbances during molting and metamorphosis also contributed for fluctuations in mortality of the leafminer. Tactics of integrated pest management to enhance natural mortality of the leafminer while conserving or augmenting the action of natural enemies are discussed.

Altered cysteine proteinase activity in insecticide-resistant strains of the maize weevil: Purification and characterization

Insecticide resistance is usually associated with fitness costs, but such costs may be mitigated by increased energy and amino acid accumulation and mobilization as has been suggested in the maize weevil Sitophilus zeamais (Coleoptera: Curculionidae). To address this adaptation, cysteine proteinases (E.C. 3.4.22), one of the main proteinases in weevils, was purified from an insecticide-susceptible and two insecticide-resistant strains of the maize weevil (one with fitness costs, referred as resistant-cost, and the other without it, referred to as resistant no-cost) using thiol-sepharose affinity chromatography. Purification of the cysteine proteinases revealed a single 74,000 Da molecular mass band in the susceptible strain, two bands of 72,000 and 83,000 Da in the resistant cost strain, and two bands of 68,000 and 74,000 Da in the resistant no-cost strain. Purified cysteine proteinases of the three strains behaved differently regarding casein degradation and inhibition; the proteinases least sensitive to inhibition by the specific cysteine proteinase inhibitor E-64 were those from the resistant no-cost strain as indicated by their highest I(50) value. The pH and temperature profile of cysteine proteinase activity differed among strains and although substrate affinity (i.e. K(M)) of the cysteine proteinases was similar, the V(max) value for cysteine-proteinases from the resistant cost strain was 3-fold and 5-fold higher than V(max) values for the resistant no-cost and susceptible strains respectively. Cysteine proteinase activity was highest for the resistant cost strain rather than the resistant no-cost. Therefore enhanced cysteine proteinase activity is unlikely to be playing significant role in mitigating the costs usually associated with insecticide resistance.

Seletividade de Inseticidas a Brevicoryne brassicae (L.) (Hemiptera: Aphididae) e ao Predador Doru luteipes (Scudder) (Dermaptera: Forficulidae)

The objective of this work was to study the selectivity of the insecticides acephate, deltamethrin, dimethoate, methamidophos, methyl parathion, and pirimicarb to first, second, and third instar nymphs and adults of the predator Doru luteipes (Scudder) (Dermaptera: Forficulidae) in comparison with last instar nymphs of the prey Brevicoryne brassicae (L.) (Hemiptera: Aphididae). Pirimicarb and deltamethrin were highly selective to all developmental stages of D. luteipes; the same was observed with acephate except for first instar nymphs to whom it showed median selectivity. Dimethoate showed median selectivity, but methamidophos and methyl parathion were low selective to the predator. Second and third instar nymphs and adults of D. luteipes were more tolerant to acephate, methyl parathion and pirimicarb than first instar nymphs. First and second instar nymphs showed similar tolerance to deltamethrin, dimethoate and methamidophos. Concentration-response curves for dimethoate (to adults and first and second instar nymphs) and methamidophos (to third instar nymphs) showed steeper slopes indicating an homogeneous response of these developmental stages to these insecticides.

Rapid selection and characterization of Cry1F resistance in a Brazilian strain of fall armyworm

Transgenic maize (Zea mays L., Poaceae) event TC1507, producing the Cry1F protein of Bacillus thuringiensis Berliner, has been used for management of the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), in Brazil since 2009. A strain of S. frugiperda, obtained from field collections of larvae in TC1507 maize in Minas Gerais state in 2010, was selected in the laboratory for resistance to Cry1F using leaves of TC1507 maize in two selection regimes. Continuous exposure of larvae to Cry1F was more effective than exposure for 6, 8, and 10 days in the selection of resistant S. frugiperda individuals. With only four generations of laboratory selection, a strain with high levels of resistance to Cry1F was obtained, as indicated by the survival of insects reared on leaves of TC1507 maize plants and by the more than 300‐fold resistance level measured in bioassays with the purified Cry1F protein. Importantly, reciprocal crosses between control and the Cry1F‐selected strains revealed that the resistance is autosomal and incompletely recessive, and the response obtained in the backcross of the F1 generation with the resistant strain was consistent with simple monogenic inheritance. Additionally, there were no apparent fitness costs associated with resistance either for survival or larval growth on non‐Bt maize leaves. Our findings provide experimental evidence for rapid evolution of Cry1F resistance in S. frugiperda in the laboratory and further reinforce the potential of this species to evolve field resistance to the TC1507 maize as previously reported. The resistant strain isolated in this study provides an opportunity to estimate the resistance allele frequency in the field and to determine the biochemical and molecular basis of the resistance, which should provide further information to assist in the resistance management of S. frugiperda on transgenic maize producing B. thuringiensis proteins.