Susceptibility of Brazilian Populations of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) to Vip3Aa20.

Transgenic maize expressing the insecticidal protein Vip3Aa20 is increasingly being adopted in Brazil. In this study, we determined the baseline susceptibility of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) and Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) to Vip3Aa20, as part of an Insect Resistance Management (IRM) program. Diet-overlay bioassays were conducted with neonates exposed to Vip3Aa20 for 7 d. The baseline susceptibility data were obtained for seven field populations of H. armigera and six of H. zea collected from major soybean-, cotton-, and maize-producing areas in Brazil. To validate the diagnostic concentration, 11 field populations of H. zea were tested from 2014 to 2015. The LC50 for H. armigera populations ranged from 2.97 to 8.41 µg Vip3Aa20/cm2 (threefold variation), and for H. zea populations from 0.04 to 0.21 µg Vip3Aa20/cm2 (fivefold variation). The EC50 for H. armigera ranged from 0.099 to 0.455 µg Vip3Aa20/cm2 (fivefold variation), and for H. zea from 0.004 to 0.020 µg Vip3Aa20/cm2 (fivefold variation). H. armigera was more tolerant to Vip3Aa20 protein than was H. zea (≈40- to 75-fold, based on LC50). Based on the LC99 value, the concentration of 6.4 µg Vip3Aa20/cm2 was defined as a diagnostic concentration for susceptibility monitoring in H. zea, and >44 µg Vip3Aa20/cm2 for H. armigera. Our baseline susceptibility data for Vip3Aa20 in H. armigera and H. zea populations will be useful in IRM programs in Brazil.

Pan-American Similarities in Genetic Structures of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) With Implications for Hybridization.

The genus Helicoverpa (Lepidoptera: Noctuidae) includes phytophagous and polyphagous agricultural insect pests. In the Americas, a native pest, Helicoverpa zea (Boddie), and an invasive pest, Helicoverpa armigera (Hübner), are causing severe damage in vegetable and agronomic crops. The population structure of both species in South America is poorly understood, and the phylogenetic relatedness of H. armigera and H. zea suggests natural interspecific gene flow between these species. Using microsatellite loci, we investigated: 1) the genetic diversity and gene flow of H. armigera specimens from Brazil; 2) the genetic diversity and gene flow between H. zea specimens from Brazil and the United States; and 3) the possibility of interspecific gene flow and the frequency of putative hybrids in Brazil. We detected high intraspecific gene flow among populations collected in the same country. However, there is a geographic limit to gene flow among H. zea individuals from South and North America. Pairwise Fst and private alleles showed that H. armigera is more similar to H. zea from Brazil than H. zea from the United States. A comparative STRUCTURE analysis suggests natural hybridization between H. armigera and H. zea in Brazil. High gene flow and natural hybridization are key traits to population adaptation in new and disturbed environments, which can influence the management of these pests in the American continent.

Current situation of pests targeted by Bt crops in Latin America.

Transgenic crops producing Bacillus thuringiensis- (Bt) insecticidal proteins (Bt crops) have provided useful pest management tools to growers for the past 20 years. Planting Bt crops has reduced the use of synthetic insecticides on cotton, maize and soybean fields in 11 countries throughout Latin America. One of the threats that could jeopardize the sustainability of Bt crops is the development of resistance by targeted pests. Governments of many countries require vigilance in measuring changes in Bt-susceptibility in order to proactively implement corrective measures before Bt-resistance is widespread, thus prolonging the usefulness of Bt crops. A pragmatic approach to obtain information on the effectiveness of Bt-crops is directly asking growers, crop consultants and academics about Bt-resistance problems in agricultural fields, first-hand information that not necessarily relies on susceptibility screens performed in laboratories. This type of information is presented in this report. Problematic pests of cotton and soybeans in five Latin American countries currently are effectively controlled by Bt crops. Growers that plant conventional (non-Bt) cotton or soybeans have to spray synthetic insecticides against multiple pests that otherwise are controlled by these Bt crops. A similar situation has been observed in six Latin American countries where Bt maize is planted. No synthetic insecticide applications are used to control corn pests because they are controlled by Bt maize, with the exception of Spodoptera frugiperda. While this insect in some countries is still effectively controlled by Bt maize, in others resistance has evolved and necessitates supplemental insecticide applications and/or the use of Bt maize cultivars that express multiple Bt proteins. Partial control of S. frugiperda in certain countries is due to its natural tolerance to the Bt bacterium. Of the 31 pests targeted and controlled by Bt crops in Latin America, only S. frugiperda has shown tolerance to certain Bt proteins in growers' fields, the most reliable indication of the status of Bt-susceptibility in most of the American continent.

Cross-species amplification and polymorphism of microsatellite loci in Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) in Brazilian cropping systems.

The Old World bollworm Helicoverpa armigera (Hübner) was recently discovered in Brazil. This species is closely related to the New World bollworm H. zea (Boddie), and mating between these species has already been reported under laboratory conditions. Here, we tested the cross-species amplification of 20 microsatellite (SSR) loci in field populations of H. armigera and H. zea collected from Brazilian cropping systems. Seven SSR loci were successfully amplified and polymorphic in both species except for the locus HaC14, which was monomorphic for H. zea. All SSR loci were in linkage equilibrium, and deviations from Hardy- Weinberg equilibrium were only observed for the locus HarSSR1 in the HaRS-2 population, where null alleles were present. A moderate level of polymorphism was detected in H. armigera and H. zea populations with a mean allele number of 4.14, and 2.24, respectively. Interestingly, most of the populations of the recent invader H. armigera showed higher genetic diversity and inbreeding coefficients than H. zea populations. The genetic identity of each species was recovered using a STRUCTURE analysis, where the populations formed two clusters (K = 2) according to their species. STRUCTURE also suggested the occurrence of potential hybrid offspring between H. armigera and H. zea individuals in natural conditions. These SSR loci will be valuable in characterizing population differentiation, invasion routes, adaptation, reproductive behavior, and intra- and interspecific gene flow in H. armigera and H. zea populations in Brazil, the USA, and other areas where these two pests occur.

Imidacloprid affects the functional response of predator Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) to strains of Spodoptera frugiperda (J.E. Smith) on Bt cotton.

Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) is one of the most common asopine species in the neotropical region and its occurrence was reported in several countries of South and Central America, as an important biological control agent for many crops. This study was carried out to identify the imidacloprid impacts on the functional response of predator P. nigrispinus fed on Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) strain resistant to lambda-cyhalothrin, on Bt cotton expressing Cry1Ac (Bollgard(®)). Spodoptera frugiperda larvae were used in the following conditions: resistant (1) and susceptible (2) strains to lambda-cyhalothrin fed on Bollgard(®) cotton leaves (DP 404 BG); and resistant (3) and susceptible (4) strains to lambda-cyhalothrin fed on non-genetically modified cotton leaves (cultivar DP4049). The predatory behavior of P. nigrispinus was affected by imidacloprid and the type II asymptotic curve was the one that best described the functional response data. Handling time (T h ) of predator females did not differ among treatments in the presence of imidacloprid. The attack rate did decrease, however, due to an increase in the density of larvae offered. Regardless of the treatment (S. frugiperda strain or cotton cultivar), the predation of P. nigrispinus females on S. frugiperda larvae was significantly lower when exposed to imidacloprid, especially at a density of 16 larvae/predator. The predation behavior of P. nigrispinus on S. frugiperda larvae is affected by the insecticide imidacloprid showing that its applications should be used in cotton crop with caution.

Baseline susceptibility to Cry1Ac insecticidal protein in Heliothis virescens (Lepidoptera: Noctuidae) populations in Brazil.

The tobacco budworm, Heliothis virescens (F.), is one of the target pests of genetically modified cotton expressing Cry1Ac insecticidal protein (Bt cotton) derived from Bacillus thuringiensis Berliner. This study was conducted to evaluate the susceptibility of field-collected populations of H. virescens to Cry1Ac to establish a baseline for use in an insect resistance management program for Bt cotton in Brazil. Insects were sampled from the main Brazilian cotton-growing regions (Bahia, Mato Grosso, Mato Grosso do Sul, and Goiás) during the cropping seasons of 2007/08 and 2008/09. Cry1Ac susceptibility was estimated by using diet incorporation bioassays. H. virescens was highly susceptible to Cry1Ac protein. The estimated LC50 values varied from 0.18 to 0.66 microg of Cry1Ac/ml of diet among the 2007-2008 populations (approximately 3.7-fold variation). Similarly, the EC50 values based on growth inhibition ranged from 0.0053 to 0.0161 microg of Cry1Ac/ml of diet for the 2007-2008 populations (approximately 3.0-fold variation). A joint analysis of the mortality data across all tested populations was used to develop and validate the diagnostic concentrations of 3.1 and 5.6 microg of Cry1Ac/ml of diet, the upper bound of the confidence interval and twice the LC99 were selected, for resistance monitoring programs of H. virescens to Cry1Ac protein in Brazil.

Development and characterization of microsatellite loci for genetic studies of the sugarcane borer, Diatraea saccharalis (Lepidoptera: Crambidae).

We present polymorphic microsatellite markers isolated for genetic studies of the sugarcane borer, Diatraea saccharalis (Fabricius). We isolated 16 microsatellite loci through an enriched genomic library protocol. After characterization, 12 markers showed polymorphic information expressed in the observed number of alleles (ranging from 2 to 7; 5 on average) and in the polymorphism information content (ranging from 0.292 to 0.771; 0.535 on average). These markers can be used in further studies to understand the basic ecological characteristics of the sugarcane borer, e.g., dispersion patterns and population genetic differentiation, associated with distinct geographic scales and host plants.

Microsatellite markers for genetic studies of the fall armyworm, Spodoptera frugiperda.

We developed six microsatellite markers for the fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae). The SSR loci were isolated with enriched genomic library protocol by using native individuals as a genome source for markers. These loci were characterized in 48 individuals and they were tested for the ability to identify candidate migrants exchanged among the samples. The number of alleles per locus ranged from 5 to 18 (10.8 on average). The observed polymorphism information content ranged from 0.172 to 0.891. Beside the lower efficiency to obtain SSR loci, the six microsatellites were polymorphic and sufficiently discriminant for the genetic studies of S. frugiperda; it allowed us to identify migrants with both NJ clustering and the Bayesian methods. These markers will be useful for molecular ecology studies of this highly polyphagous species in order to understand the processes that determine genetic differentiation in the complex agro-ecosystems that it infests and improve local integrated pest management practices.

Susceptibility to deltamethrin in the predatory mites Neoseiulus californicus and Phytoseiulus macropilis (Acari: Phytoseiidae) populations in protected ornamental crops in Brazil.

Knowledge of inter and intra-specific variation in the susceptibility of natural enemies to pesticides could help to better design integrated pest management strategies. The objective of this research was to evaluate the susceptibility to deltamethrin in populations of the predatory mites Neoseiulus californicus (McGregor) and Phytoseiulus macropilis (Banks) populations collected from protected ornamental crops in Brazil. The susceptibility to deltamethrin was characterized against immature and adult stages of both species. The impact of this insecticide was also measured by estimating the intrinsic rate of increase (r (i)). The immature and adult stages of N. californicus were approximately 3,600 and 3,000-fold more tolerant to deltamethrin than those of P. macropilis. However, high variability in the susceptibility to this insecticide was detected among P. macropilis populations, with resistance ratios of up to 3,500-fold. The selection of deltamethrin-resistant strains of P. macropilis could be exploited in applied biological control programs.

Genetic variability and demographic history of Heliothis virescens (Lepidoptera: Noctuidae) populations from Brazil inferred by mtDNA sequences.

Intra- and inter-population genetic variability and the demographic history of Heliothis virescens (F.) populations were evaluated by using mtDNA markers (coxI, coxII and nad6) with samples from the major cotton- and soybean-producing regions in Brazil in the growing seasons 2007/08, 2008/09 and 2009/10. AMOVA indicated low and non-significant genetic structure, regardless of geographical scale, growing season or crop, with most of genetic variation occurring within populations. Clustering analyzes also indicated low genetic differentiation. The haplotype network obtained with combined datasets resulted in 35 haplotypes, with 28 exclusive occurrences, four of them sampled only from soybean fields. The minimum spanning network showed star-shaped structures typical of populations that underwent a recent demographic expansion. The recent expansion was supported by other demographic analyzes, such as the Bayesian skyline plot, the unimodal distribution of paired differences among mitochondrial sequences, and negative and significant values of neutrality tests for the Tajima's D and Fu's F(S) parameters. In addition, high values of haplotype diversity (H) and low values of nucleotide diversity (π), combined with a high number of low frequency haplotypes and values of θ(π)<θ(W), suggested a recent demographic expansion of H. virescens populations in Brazil. This demographic event could be responsible for the low genetic structure currently found; however, haplotypes present uniquely at the same geographic regions and from one specific host plant suggest an initial differentiation among H. virescens populations within Brazil.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2010-30 September 2010.

This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis × Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross-tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.

Genetic structure and molecular variability of Spodoptera frugiperda (Lepidoptera: Noctuidae) collected in maize and cotton fields in Brazil.

The purpose of this research was to evaluate the genetic similarity and structure of the fall armyworm, Spodoptera frugiperda (J.E. Smith), populations associated with maize and cotton crops in Brazil using amplified fragment length polymorphisms. Mean genetic similarity among populations was 0.45. The unweighted pair group method with arithmetic mean analysis dendrograms did not separate populations of S. frugiperda into clusters related to the host plant in which the insects were collected. No genetic variation was observed among maize and cotton populations of S. frugiperda, suggesting that the same populations are injuring both crops in Brazil. This research validates the need for stewardship of crop-protection methods for managing S. frugiperda to reduce the incidence of pesticide resistance, due to the spatial and temporal overlapping of maize and cotton crops in some regions in Brazil.

Toxicity of purified fungal toxin hirsutellin A to the citrus rust mite phyllocoptruta oleivora (Ash.)

A toxic protein, hirsutellin A, isolated from culture filtrate during liquid fermentation of the fungal mycelia of Hirsutella thompsonii, was tested using contact/residual and residual leaf bioassay methodologies at concentrations of 0, 10, 32, 56, and 100 &mgr;g/ml against adult citrus rust mite, Phyllocoptruta oleivora. P. oleivora is the natural host of the parasitic fungus, H. thompsonii. Mite mortality increased with an increase in hirsutellin A concentration, reaching virtually 100% at 100 &mgr;g/ml using both leaf assay methods. The number of eggs found on leaf disk bioassays within a 3-day period decreased significantly with an increase in hirsutellin A concentration, suggesting that fecundity was affected prior to the death of the host. Copyright 1998 Academic Press.

Reactivation at low ATP distinguishes among classes of paralyzed flagella mutants.

Paralyzed flagella (pf) mutants of Chlamydomonas have been distinguished by the inability of the intact cells to move. Demembranated flagella from these mutants are also immotile when reactivated under standard conditions, with millimolar ATP concentrations. Three of these pf mutants were previously found to be motile when reactivated under 3 alternate reactivation conditions: low ATP concentration (< or =50 microM); 0.1 mM ATP combined with >0.5 mM ADP; or 0.1 mM ATP combined with non-reactivating ATP analogs anthraniloyl ATP or methylanthraniloyl ATP. We have now surveyed all pf mutants in the Chlamydomonas Culture Collection and discovered that a great majority of these mutants can move under these alternate nucleotide conditions. Only pf22 and pf23, mutants missing multiple subsets of dynein arms, did not reactivate under those conditions. This suggests that the paralysis observed in most pf mutants is the result of inhibition by physiological ATP. Except for pf12, which has an abnormally symmetric bending pattern, all other pf mutants exhibit asymmetric bending patterns similar to wild-type. Previously, motility that was restored by the presence of suppressor mutations was found to lack the normal asymmetry of wild-type flagella or the suppressor by itself. The waveform of pf mutants at alternate reactivation conditions in the absence of suppressor shows that pf mutants with radial-spoke or central-pair defects are capable of asymmetric bending similar to wild-type. A complete radial-spoke/central-pair complex is not essential for the production of asymmetric bending patterns. Furthermore, this suggests that the symmetric waveform observed previously in suppressed pf mutants is due to the interaction between the pf and suppressor mutations.

Ability of paralyzed flagella mutants of Chlamydomonas to move.

Chlamydomonas mutants missing the central pair or radial spokes are paralyzed despite the fact that they have the full wild-type complement of functional dynein ATPases. We show here that these mutants can move under conditions of low ATP concentration, a combination of ATP and ADP, and a combination of ATP and ribose-modified ATP analogs. These conditions suggest an inhibitory role of ATP and that this inhibition can be relieved by ADP or analogs. The function of the central-pair/radial spoke complex may be to release this ATP inhibition in a controlled manner.

Characterization of a minus end-directed kinesin-like motor protein from cultured mammalian cells.

Using the CHO2 monoclonal antibody raised against CHO spindles (Sellitto, C., M. Kimble, and R. Kuriyama. 1992. Cell Motil. Cytoskeleton. 22:7-24) we identified a 66-kD protein located at the interphase centrosome and mitotic spindle. Isolated cDNAs for the antigen encode a 622-amino acid polypeptide. Sequence analysis revealed the presence of 340-amino acid residues in the COOH terminus, which is homologous to the motor domain conserved among other members of the kinesin superfamily. The protein is composed of a central alpha-helical portion with globular domains at both NH2 and COOH termini, and the epitope to the monoclonal antibody resides in the central alpha-helical stalk. A series of deletion constructs were created for in vitro analysis of microtubule interactions. While the microtubule binding and bundling activities require both the presence of the COOH terminus and the alpha-helical domain, the NH2-terminal half of the antigen lacked the ability to interact with microtubules. The full-length as well as deleted proteins consisting of the COOH-terminal motor and the central alpha-helical stalk supported microtubule gliding, with velocity ranging from 1.0 to 8.4 microns/minute. The speed of microtubule movement decreased with decreasing lengths of the central stalk attached to the COOH-terminal motor. The microtubules moved with their plus end leading, indicating that the antigen is a minus end-directed motor. The CHO2 sequence shows 86% identify to HSET, a gene located at the centromeric end of the human MHC region in chromosome 6 (Ando, A., Y. Y. Kikuti, H. Kawata, N. Okamoto, T. Imai, T. Eki, K. Yokoyama, E. Soeda, T. Ikemura, K. Abe, and H. Inoko. 1994. Immunogenetics. 39:194-200), indicating that HSET might represent a human homologue of the CHO2 antigen.

Structural and functional hierarchy of eukaryotic cilia and flagella.

There are now a variety of methods to investigate the morphofunctional aspects of eukaryotic cilia and cilia. These methods are useful for investigating the basic mechanism of eukaryotic axonemal mechanochemical function and understanding the function and interaction of its components. It is clear that the complex structure of eukaryotic axoneme requires the combination of all these techniques to unravel its mystery. The compositionally simple in vitro microtubule assays are crucial in investigating the functions of different dyneins within an axoneme. However, because such assays do not include other components of the axoneme and the important mechanical feedback present in a beating axoneme, reactivation of the entire structure will continue to play a basic role in the morphofunctional study of eukaryotic axonemes.

Regulatory role of nucleotides in axonemal function.

Axonemal sliding involves both sliding velocity and the extent of sliding, that is how many doublets slide. It is clear that axonemes cannot beat if all doublets were to slide simultaneously, thus sliding extent is important. Using the turbidimetric assay of sliding disintegration of Tetrahymena axonemes, we examined the sliding extent and th effect of APD, ATP, and ATP analogs on the sliding extent. Of course, ATP is necessary to produce sliding disintegration, but ATP alone did not produce extensive sliding disintegration. The additions of higher ATP concentration even in the presence of ADP inhibited sliding disintegration. We also observed sliding disintegration using ribose-modified ATP analogs, anthraniloylATP, and methylanthraniloylATP. The extent of sliding disintegration was proportional to the analog concentration. Thus in contrast to ATP, higher analog concentration was not inhibitory. These results indicate that high ATP concentration acts to inhibit the extent of sliding disintegration and that ADP relieves this inhibition. We propose a model in which the affinity of of multiple cooperative active sites are regulated by the binding of ATP or ADP to a regulatory site. This model provides a mechanism by which nucleotides regulate the extent of sliding necessary for effective axonemal bending.