Throwing Brazilian strains into the melting pot of P. xylostella resistance to Bacillus thuringiensis.

The resistance of pest insects to biopesticides based on the bacterium Bacillus thuringiensis (Bt) is normally associated with changes to the receptors involved in the mechanism of action of the pesticidal proteins produced by Bt. In some strains of Plutella xylostella (the diamondback moth) resistance has evolved through a signalling mechanism in which the genes encoding the receptor proteins are downregulated whereas in others it has been linked to structural changes in the receptors themselves. One such well characterized mutation is in the ABCC2 gene indicating that changes to this protein can result in resistance. However other studies have found that knocking out this protein does not result in a significant level of resistance. In this study we wanted to test the hypothesis that constitutive receptor downregulation is the major cause of Bt resistance in P. xylostella and that mutations in the now poorly expressed receptor genes may not contribute significantly to the phenotype. To that end we investigated the expression of a receptor (ABCC2) and the major regulator of the signalling pathway (MAP4K4) in two resistant and four susceptible strains. No correlation was found between expression levels and susceptibility; however, a frameshift mutation was identified in the ABCC2 receptor in a newly characterized resistant strain.

Individual transmembrane domains of SfABCC2 from Spodoptera frugiperda do not serve as functional Cry1F receptors.

The fall armyworm (Spodoptera frugiperda) is a major global pest causing severe damage to various crops, especially corn. Transgenic corn producing the Cry1F pesticidal protein from the bacterium Bacillus thuringiensis (Cry1F corn) showed effectiveness in controlling this pest until S. frugiperda populations at locations in North and South America evolved practical resistance. The mechanism for practical resistance involved disruptive mutations in an ATP binding cassette transporter subfamily C2 gene (SfABCC2), which serves as a functional Cry1F receptor in the midgut cells of susceptible S. frugiperda. The SfABCC2 protein contains two transmembrane domains (TMD1 and TMD2), each with a cytosolic nucleotide (ATP) binding domain (NBD1 and NBD2, respectively). Previous reports have demonstrated that disruptive mutations in TMD2 were linked with resistance to Cry1F, yet whether the complete SfABCC2 structure is needed for receptor functionality or if a single TMD-NBD protein can serve as functional Cry1F receptor remains unknown. In the present study, we separately expressed TMD1 and TMD2 with their corresponding NBDs in cultured insect cells and tested their Cry1F receptor functionality. Our results show that the complete SfABCC2 structure is required for Cry1F receptor functionality. Moreover, binding competition assays revealed that Cry1F specifically bound to SfABCC2, whereas neither SfTMD1-NBD1 nor SfTMD2-NBD2 exhibited any significant binding. These results provide insights into the molecular mechanism of Cry1F recognition by SfABCC2 in S. frugiperda, which could facilitate the development of more effective insecticidal proteins.

Genetic Screening to Identify Candidate Resistance Alleles to Cry1F Corn in Fall Armyworm Using Targeted Sequencing.

Evolution of practical resistance is the main threat to the sustainability of transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt crops). Monitoring of resistance to Cry and Vip3A proteins produced by Bt crops is critical to mitigate the development of resistance. Currently, Cry/Vip3A resistance allele monitoring is based on bioassays with larvae from inbreeding field-collected moths. As an alternative, DNA-based monitoring tools should increase sensitivity and reduce overall costs compared to bioassay-based screening methods. Here, we evaluated targeted sequencing as a method allowing detection of known and novel candidate resistance alleles to Cry proteins. As a model, we sequenced a Cry1F receptor gene (SfABCC2) in fall armyworm (Spodoptera frugiperda) moths from Puerto Rico, a location reporting continued practical field resistance to Cry1F-producing corn. Targeted sequencing detected a previously reported Cry1F resistance allele (SfABCC2mut), in addition to a resistance allele originally described in S. frugiperda populations from Brazil. Moreover, targeted sequencing detected mutations in SfABCC2 as novel candidate resistance alleles. These results support further development of targeted sequencing for monitoring resistance to Bt crops and provide unexpected evidence for common resistance alleles in S. frugiperda from Brazil and Puerto Rico.

Whole genome comparisons reveal panmixia among fall armyworm (Spodoptera frugiperda) from diverse locations.

BACKGROUND: The fall armyworm (Spodoptera frugiperda (J.E. Smith)) is a highly polyphagous agricultural pest with long-distance migratory behavior threatening food security worldwide. This pest has a host range of > 80 plant species, but two host strains are recognized based on their association with corn (C-strain) or rice and smaller grasses (R-strain). The population genomics of the United States (USA) fall armyworm remains poorly characterized to date despite its agricultural threat. RESULTS: In this study, the population structure and genetic diversity in 55 S. frugiperda samples from Argentina, Brazil, Kenya, Puerto Rico and USA were surveyed to further our understanding of whole genome nuclear diversity. Comparisons at the genomic level suggest a panmictic S. frugiperda population, with only a minor reduction in gene flow between the two overwintering populations in the continental USA, also corresponding to distinct host strains at the mitochondrial level. Two maternal lines were detected from analysis of mitochondrial genomes. We found members from the Eastern Hemisphere interspersed within both continental USA overwintering subpopulations, suggesting multiple individuals were likely introduced to Africa. CONCLUSIONS: Our research is the largest diverse collection of United States S. frugiperda whole genome sequences characterized to date, covering eight continental states and a USA territory (Puerto Rico). The genomic resources presented provide foundational information to understand gene flow at the whole genome level among S. frugiperda populations. Based on the genomic similarities found between host strains and laboratory vs. field samples, our findings validate the experimental use of laboratory strains and the host strain differentiation based on mitochondria and sex-linked genetic markers extends to minor genome wide differences with some exceptions showing mixture between host strains is likely occurring in field populations.

Interactions of Bacillus thuringiensis strains for Plutella xylostella (L.) (Lepidoptera: Plutellidae) susceptibility.

Bacillus thuringiensis (Bt) biopesticides are an environmentally safe alternative to the management of Plutella xylostella pesticide resistance evolution. We evaluated P. xylostella susceptibility to six Bt strains cultivated and applied individually, and 15 combinations of Bt strains mixed after cultivation. Three combinations resulted in synergism and one in antagonism. Promising results of larval mortality with synergistic effects were obtained with the combinations Bt var. thuringiensis strain HD-2 + Bt finitimus strain HD-3, Bt var. thuringiensis strain HD-2 + Bt dendrolimus strain HD-7 and Bt var. thuringiensis strain HD-2 + Bt var. aizawai strain HD-11.

Mechanisms of resistance to commercially relevant entomopathogenic bacteria.

Bacteria represent the most commercially successful entomopathogenic microbial group, with most commercialized insecticides containing gram-positive bacteria in the Bacillaceae family. Resistance to entomopathogenic bacteria threatens sustainable agriculture, and information on the mechanisms and genes involved is vital to develop management practices aimed at reducing this risk. We provide an integrative summary on mechanisms responsible for resistance to commercialized entomopathogenic bacteria, including information on resistance to transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt crops). The available experimental evidence identifies alterations in binding of insecticidal proteins to receptors in the host as the main mechanism for high levels of resistance to entomopathogenic bacteria.

Efeito de vitaminas e esterol como aditivos foliares no desenvolvimento de Bombyx mori L. e influência do modo de aplicação / Effect of vitamins and sterol as foliar additives in the development of Bombyx mori L. and influence of the application mode

O objetivo deste estudo foi avaliar os efeitos e os métodos de aplicação dos aditivos foliares decanoato de nandrolona (esteroide anabolizante) a 0,5%, ácido ascórbico (vitamina C) a 0,5%, palmitato de retinol (vitamina A) a 0,5% e acetato de retinol (vitamina A) a 1,0% em alguns parâmetros biológicos do bicho-da-seda. No experimento do modo de aplicação (pulverização foliar antes do fornecimento, depois do fornecimento e por imersão antes do fornecimento) foram utilizados: decanoato de nandrolona 0,5%; ácido ascórbico 0,5%; e palmitato de retinol 0,5%. No ensaio de desenvolvimento do inseto foram avaliados: duração e viabilidade larval; peso de casulo; taxa de encasulamento; teor líquido de seda; longevidade de adultos; número de ovos por fêmea; e duração do período de subida ao bosque. Para o modo de aplicação determinaram-se: ganho de peso, comprimento, diâmetro do tórax e diâmetro do abdome de lagartas de 5º ínstar, assim como comprimento, diâmetro e peso de glândula sericígena. Os resultados mostraram que, apesar de o ácido ascórbico proporcionar os melhores valores para o desenvolvimento corpóreo das lagartas, este não corresponde a incrementos significativos na produção; o palmitato de retinol não melhora o desenvolvimento das lagartas; o acetato de retinol e o palmitato de retinol prolongam o período larval, sem, no entanto, alterar significativamente os parâmetros de produção; a imersão de folhas antes do fornecimento às lagartas é mais viável para a aplicação dos aditivos.(AU)

The present study was aimed to evaluate the effects of different methods of foliar application of the additives nandrolone decanoate (anabolic steroid) 0,5%; ascorbic acid (vitamin C) 0,5%; retinol palmitate (vitamin A) 0,5%; and retinol acetate (vitamin A) 1,0% on some biological parameters of the silkworm. The experiment of application method (foliar spray prior to delivery, after delivery and soaking before feeding) used: nandrolone decanoate 0.5%, ascorbic acid 0.5%, and retinol palmitate 0.5%. The test of insect development evaluated: duration and larval viability, weight of cocoon, cocooning rate, liquid silk content, adult longevity, number of eggs per female and rising up time. For the application of foliar additives, the following were determined: weight gain, length, diameter and abdomen diameter of fifth instar larvae, and length, diameter and weight of silk gland. The results showed that although ascorbic acid provided the best value for the development of the caterpillars body, it did not correspond to significant increase in production; retinol palmitate did not affect the larvae body development; retinol acetate and retinol palmitate prolong the larval period, without, however, significantly altering the production parameters. The immersion method of leaves before being delivered to the caterpillars is the most feasible for the foliar application of additives.(AU)

Detection of hemoplasma and Bartonella species and co-infection with retroviruses in cats subjected to a spaying/neutering program in Jaboticabal, SP, Brazil.

Hemotrophic mycoplasmas and Bartonella species are important pathogens that circulate between cats and invertebrate hosts, occasionally causing diseases in humans. Nevertheless, there are few reports on occurrences of these agents in cats in Brazil. The present study aimed to detect the presence of hemoplasma and Bartonella DNA by means of PCR and sequencing. FIV antigens and anti-FeLV antibodies, were studied by using a commercial kit on blood and serum samples, respectively, among 46 cats that were sampled during a spaying/neutering campaign conducted in Jaboticabal, SP. Three (6.5%) cats were positive for hemoplasmas: two (4.3%) for 'Candidatus M. haemominutum' and one (2.2%) for both M. haemofelis and 'Candidatus M. turicensis'. One of the two 'Candidatus M. haemominutum'-infected cats was also positive for FeLV antigens and showed antibodies for FIV. Two cats (4.3%) were positive for B. henselae. One of them was also positive for FeLV antigens. Eight cats (17.4%) were positive for FeLV, and just one (2.2%) showed anti-FIV antibodies. Bartonella species and hemoplasmas associated with infection due to retroviruses can circulate among apparently healthy cats.

Molecular characterisation of Bartonella species in cats from São Luís, state of Maranhão, north-eastern Brazil

Bartonella species are fastidious bacteria that predominantly infect mammalian erythrocytes and endothelial cells and cause long-lasting bacteraemia in their reservoir hosts. Reports that describe the epidemiology of bartonellosis in Brazil are limited. This study aimed to detect and characterise Bartonella spp DNA from cat blood samples in São Luís, Maranhão, north-eastern Brazil. Among 200 cats tested for multiple genes, nine (4.5%) were positive for Bartonella spp: six cats for Bartonella henselae and three for Bartonella clarridgeiae. Based on the phylogenetic analysis of four genes, the B. henselae strain matched strains previously observed in Brazil and was positioned in the same clade as B. henselae isolates from the United States of America. Moreover, sequence alignment demonstrated that the B. clarridgeiae strain detected in the present study was the same as the one recently detected in cats from southern Brazil.