Sublethal Endpoints in Non-target Organism Testing for Insect-Active GE Crops.

Historically, genetically engineered (GE) plants that have incorporated genes conferring insect protection have primarily used Cry proteins derived from Bacillus thuringiensis (Bt) to achieve their insecticidal phenotype. As a result, regulators have developed a level of familiarity and confidence in reviewing plants incorporating these insecticidal proteins. However, new technologies have been developed that produce GE plants that incorporate pest protection by triggering an RNA interference (RNAi) response or proteins other than Bt Cry proteins. These technologies have new modes of action. Although the overall assessment paradigm for GE plants is robust, there are ongoing discussions about the appropriate tests and measurement endpoints needed to inform non-target arthropod assessment for technologies that have a different mode of action than the Bt Cry proteins. As a result, increasing attention is being paid to the use of sublethal endpoints and their value for environmental risk assessment (ERA). This review focuses on the current status and history of sublethal endpoint use in insect-active GE crops, and evaluates the future use of sublethal endpoints for new and emerging technologies. It builds upon presentations made at the Workshop on Sublethal Endpoints for Non-target Organism Testing for Non-Bt GE Crops (Washington DC, USA, 4-5 March 2019), and the discussions of government, academic and industry scientists convened for the purpose of reviewing the progress and status of sublethal endpoint testing in non-target organisms.

Does Bacillus thuringiensis have adverse effects on the host egg location by parasitoid wasps?

ABSTRACT This study investigated the interaction between two pest biological control agents, the parasitoid wasp Trichogramma pretiosum (Hymenoptera: Trichogrammatidae) and the entomopathogen Bacillus thuringiensis (Bacillales: Bacillacea) (Bt). The aim of this study was to evaluate if the presence of Bt (formulated products Agree®, Dipel® and HD1 and HD11 strains) interferes in the oviposition preference of T. pretiosum to eggs of Helicoverpa zea (Lepidoptera: Noctuidae). Using an olfactometry test, the eggs of H. zea were bathed with the commercial formulations, with the Bt suspensions or distilled water, and offered to the parasitoid wasps in order to evaluate parasitism. The results showed that H. zea eggs sprayed with commercial formulations and Bt strains did not interfere in the choice made by the parasitoid. The parasitoid wasp is not able to distinguish between eggs with or without B. thuringiensis treatment, independently of strains suspension or commercial formulations. Therefore, these two control agents may be used together without negative interaction.

Clathrin-dependent endocytosis is associated with RNAi response in the western corn rootworm, Diabrotica virgifera virgifera LeConte.

The cellular uptake of dsRNA after dietary exposure is critical for RNAi efficiency; however, the mechanism of its uptake in many insects remains to be understood. In this study, we evaluated the roles of the endocytic pathway genes Clathrin heavy chain (Chc), Clathrin adaptor protein AP50, ADP ribosylation factor-like 1 (Arf72A), Vacuolar H+ ATPase 16 kDa subunit (Vha16), and small GTPase Rab7 and putative sid-1-like genes (silA and silC) in RNAi response in western corn rootworm (WCR) using a two-stage dsRNA exposure bioassay. Silencing of Chc, Vha16, and AP50 led to a significant decrease in the effects of laccase2 dsRNA reporter, indicating that these genes are involved in RNAi response. However, the knockdown of either Arf72A or Rab7 did not suppress the response to laccase2 dsRNA. The silencing of the silC gene did not lead to a significant reduction in mortality or increase in the expression of V-ATPase A reporter. While the silencing of the silA gene significantly decreased insect mortality, significant changes in V-ATPase A expression were not detected. These results suggest that clathrin-dependent endocytosis is a biological mechanism that plays an important role during RNAi response in WCR adults. The fact that no definitive support for the roles of silA or silC in RNAi response was obtained support the idea that RNAi response varies greatly in different insect species, demanding additional studies focused on elucidating their involvement in this mechanism.

Surrogate species selection for assessing potential adverse environmental impacts of genetically engineered insect-resistant plants on non-target organisms.

Most regulatory authorities require that developers of genetically engineered insect-resistant (GEIR) crops evaluate the potential for these crops to have adverse impacts on valued non-target organisms (NTOs), i.e., organisms not intended to be controlled by the trait. In many cases, impacts to NTOs are assessed using surrogate species, and it is critical that the data derived from surrogates accurately predict any adverse impacts likely to be observed from the use of the crop in the agricultural context. The key is to select surrogate species that best represent the valued NTOs in the location where the crop is going to be introduced, but this selection process poses numerous challenges for the developers of GE crops who will perform the tests, as well as for the ecologists and regulators who will interpret the test results. These issues were the subject of a conference "Surrogate Species Selection for Assessing Potential Adverse Environmental Impacts of Genetically Engineered Plants on Non-Target Organisms" convened by the Center for Environmental Risk Assessment, ILSI Research Foundation. This report summarizes the proceedings of the conference, including the presentations, discussions and the points of consensus agreed to by the participants.

Two's a crowd: phenotypic adjustments and prophylaxis in Anticarsia gemmatalis larvae are triggered by the presence of conspecifics.

Defence from parasites and pathogens involves a cost. Thus, it is expected that organisms use this only at high population densities, where the risk of pathogen transmission may be high, as proposed by the "density-dependent prophylaxis" (DDP) hypothesis. These predictions have been tested in a wide range of insects, both in comparative and experimental studies. We think it pertinent to consider a continuum between solitarious and gregarious living insects, wherein: (1) solitarious insects are those that are constitutively solitary and do not express any phenotypic plasticity, (2) the middle of the continuum is represented by insects that are subject to fluctuations in local density and show a range of facultative and plastic changes; and (3) constitutively gregarious forms live gregariously and show the gregarious phenotype even in the absence of crowding stimuli. We aimed to chart some of the intermediary continuum with an insect that presents solitarious aspects, but that is subject to fluctuations in density. Thus, Anticarsia gemmatalis (Lepidoptera: Noctuidae) larvae reared at higher densities showed changes in coloration, a greater degree of encapsulation, had higher hemocyte densities and were more resistant to Baculovirus anticarsia, but not to Bacillus thuringiensis. Meanwhile, with increased rearing density there was reduced capsule melanization. Hemocyte density was the only variable that did not vary according to larval phenotype. The observed responses were not a continuous function of larval density, but an all-or-nothing response to the presence of a conspecific. As A. gemmatalis is not known for gregarious living, yet shows these density-dependent changes, it thus seems that this plastic phenotypic adjustment may be a broader phenomenon than previously thought.

Characterization of a Spodoptera frugiperda multiple nucleopolyhedrovirus isolate that does not liquefy the integument of infected larvae.

Here we report the characterization of a Spodoptera frugiperda multiple nucleopolyhedrovirus isolate, named SfMNPV-6nd, that does not cause the liquefaction of the host integument. The sequencing of the chitinase A (v-chiA) gene from SfMNPV-6nd revealed that it had a frameshift mutation that greatly reduced size of the putative enzyme. In order to evaluate the suitability of SfMPNV-6nd as a biopesticide, this isolate was compared with the highly virulent SfMNPV-19. Our results showed that the LC(50) of the two isolates were not significantly different, but that SfMNPV-6nd took a longer period of time to kill second instar S. frugiperda than SfMNPV-19.

Detection of airborne genetically modified maize pollen by real-time PCR.

The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi-urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real-time PCR analysis. Our results suggest that this 'GM pollen monitoring by bioaerosol sampling and PCR screening' approach might represent an useful aid in the surveillance of GM-free areas, centres of origin and natural reserves.

Plasmid patterns of efficient and inefficient strains of Bacillus thuringiensis against Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae).

Bacillus thuringiensis harbors genes encoding Cry proteins found in chromosomes or plasmids of different sizes (4-150 Mb). Although the smaller plasmids are more abundant in B. thuringiensis, their specific function is unknown. As for the megaplasmids, their main recognized function is to harbor cry genes, although the sequencing of some of these plasmids indicates the occurrence of other important genes. This work used a new protocol for practical and rapid extraction of plasmid DNA in order to characterize the plasmid patterns of Brazilian strains belonging to Embrapa Milho e Sorgo research center B. thuringiensis bank. We tried to further assess the relationship of plasmid patterns with strains belonging to the same serovars and strains causing 100% and no mortality to Spodoptera frugiperda (J.E. Smith) larvae. It was possible to characterize 59 strains based on the migration of bands in agarose gel. Strains belonging to the same serovars showed different plasmid sizes (from 1,636 bp to 23,200 bp), with the exception of two strains belonging to serovar galleriae. The strain T09 Bt tolworthi showed a plasmid migration pattern identical to strains belonging to serovar galleriae. Plasmid patterns differed for 46 strains, confirming that this is a useful tool to discriminate specific strains. However, it was not possible to associate the plasmid pattern or the occurrence of particular plasmids with the pathogenicity of a given species towards S. frugiperda larvae.

Growth variation among Bacillus thuringiensis strains can affect screening procedures for supernatant-secreted toxins against insect pests.

BACKGROUND: Supernatant-secreted proteins in Bacillus thuringiensis (Berliner) (Bt) with insecticidal activity provide an important source of information for discovery of new useful strains and/or entomotoxins. However, physiological variation among isolates might interfere in the detection efficiency of screening procedures on Bt collections. The aim of this study was to assess the magnitude of this variation in a sample of isolates from a tropical Bt collection, which was gauged through the assessment of their temporal patterns of growth and protein secretion in culture supernatants (SNs), as well as of the corresponding toxicity against fall armyworm (Spodoptera frugiperda, JE Smith). Feeding bioassays were performed, with larvae being treated with heated and non-heated total protein extracted from SNs collected at different culture times. Larva mortality and reduction in pupa formation were observed. RESULTS: Intra- and interisolate variations were observed in the temporal patterns of growth, quality and quantity of protein secreted, as well as in insecticidal activity of these SNs, based on larvae mortality and pupation rates. These results suggest that the insecticidal potential of certain isolates can be hidden if comparisons are done on the basis of the same number of cells in the culture and/or the same culturing time. CONCLUSIONS: Methods of screening Bt collections on the basis of feeding bioassays can be misleading with regards to identifying more promising isolates for biocontrol purposes if physiological differences are not considered. The consequences and implications of these findings for the development of experimental systems that depend on toxicity bioassays to identify alternative Bt strains and entomotoxins with practical applicability have been discussed.

A neo-sex chromosome that drives postzygotic sex determination in the hessian fly (Mayetiola destructor).

Two nonoverlapping autosomal inversions defined unusual neo-sex chromosomes in the Hessian fly (Mayetiola destructor). Like other neo-sex chromosomes, these were normally heterozygous, present only in one sex, and suppressed recombination around a sex-determining master switch. Their unusual properties originated from the anomalous Hessian fly sex determination system in which postzygotic chromosome elimination is used to establish the sex-determining karyotypes. This system permitted the evolution of a master switch (Chromosome maintenance, Cm) that acts maternally. All of the offspring of females that carry Cm-associated neo-sex chromosomes attain a female-determining somatic karyotype and develop as females. Thus, the chromosomes act as maternal effect neo-W's, or W-prime (W') chromosomes, where ZW' females mate with ZZ males to engender female-producing (ZW') and male-producing (ZZ) females in equal numbers. Genetic mapping and physical mapping identified the inversions. Their distribution was determined in nine populations. Experimental matings established the association of the inversions with Cm and measured their recombination suppression. The inversions are the functional equivalent of the sciarid X-prime chromosomes. We speculate that W' chromosomes exist in a variety of species that produce unisexual broods.

Use of by-products rich in carbon and nitrogen as a nutrient source to produce Bacillus thuringiensis (Berliner)-based biopesticide / Produção de biopesticida à base de Bacillus thuringiensis (Berliner) usando meios alternativos ricos em carbono e nitrogênio como fontes de nutrientes

The amount and sources of carbon and nitrogen used to produce Bacillus thuringiensis (Berliner)-based biopesticide may infl uence the quality of the fi nal product. The objective of this research was to test different levels of carbon and nitrogen: medium 1 - 1.5% maize glucose + 0.5% soy fl our,medium 2 - 3.0% maize glucose + 1.0% soy fl our, medium 3 - 1.0% maize glucose + 3.0% soy fl our and medium 4 - Luria Bertani (LB) + salts (FeSO4, ZnSO4, MnSO4, MgSO4). The seed culture was produced in LB medium plus salt, under agitation (200 rpm) for 18h at 30oC. The strain 344 of Bt wasused (B. thuringiensis var tolworthi – belonging to the Embrapa´s Bt Bank). The pH was measured at regular intervals. and After culturing for 96h, the pH of the four tested media was basifi ed (6.91 and 8.15), the number of spores yielded 4.39 x 109 spores/ml in medium 3, where the amount of proteinis high. The dry biomass weight accumulated in media 3 was 39.3 g/l. Mortality of 2-day-old larvae Spodoptera frugiperda (J.E. Smith) was 100% when using Bt produced in media 3 and 4. CL50 for medium 3 was 8.4 x 106 spores/ml. All tested media were satisfactory to Bt growth, and medium 3wass the most promising to be used on a large scale Bt-based biopesticide production.

A quantidade de carbono e nitrogênio usados para produzir biopesticidas à base de Bacillus thuringiensis (Berliner) pode infl uenciar a qualidade fi nal do produto. O objetivo deste trabalho foi testar meios com diferentes níveis de carbono e nitrogênio: meio 1 - glicose de milho a 1.5% + farinha de soja a 0,5%, meio 2 - glicose de milho a 3,0% + farinha de soja a 1,0%, meio 3 - glicose de milho a 1,0% + farinha de soja a 3,0% e meio 4 - Luria Bertani (LB) + sais (FeSO4, ZnSO4, MnSO4 e MgSO4). O inóculo semente foi produzido usando 150 ml de meio LB mais sais incubados por 18h a 30oC, sob agitação de 200 rpm. A cepa utilizada foi 344 (B. thuringiensis var tolworthi – pertencente ao Banco de Microorganismos da Embrapa). O pH foi medido a intervalos regulares. Após 96h de cultivo, o pH de todos os meios testados tenderam ao básico (entre 6,91 e 8,15), o maior número de esporos foi de 4,39 x 109 sporos/ml no meio 3, onde o teor de proteína usado foi o mais alto. A produção de massa celular foi maior no meio 3, com total acumulado de 39,3 g/l. A mortalidade de larvas de Spodoptera frugiperda (J.E. Smith) de dois dias de idade nos meios 3 e 4 foi de 100%. A CL50 para o meio 3 foi de 8,4 x 106 esporos/ml. Os meios alternativos usados promoveram crescimento satisfatório de Bt, sendo o meio 3 o mais promissor para ser usadona produção de biopesticida à base de Bt.

Use of by-products rich in carbon and nitrogen as a nutrient source to produce Bacillus thuringiensis (Berliner)-based biopesticide.

The amount and sources of carbon and nitrogen used to produce Bacillus thuringiensis (Berliner)-based biopesticide may influence the quality of the final product. The objective of this research was to test different levels of carbon and nitrogen: medium 1 - 1.5% maize glucose + 0.5% soy flour, medium 2 - 3.0% maize glucose + 1.0% soy flour, medium 3 - 1.0% maize glucose + 3.0% soy flour and medium 4 - Luria Bertani (LB) + salts (FeSO4, ZnSO4, MnSO4, MgSO4). The seed culture was produced in LB medium plus salt, under agitation (200 rpm) for 18h at 30 masculineC. The strain 344 of Bt was used (B. thuringiensis var tolworthi - belonging to the Embrapa's Bt Bank). The pH was measured at regular intervals. and After culturing for 96h, the pH of the four tested media was basified (6.91 and 8.15), the number of spores yielded 4.39 x 10(9) spores/ml in medium 3, where the amount of protein is high. The dry biomass weight accumulated in media 3 was 39.3 g/l. Mortality of 2-day-old larvae Spodoptera frugiperda (J.E. Smith) was 100% when using Bt produced in media 3 and 4. CL50 for medium 3 was 8.4 x 10(6) spores/ml. All tested media were satisfactory to Bt growth, and medium 3 wass the most promising to be used on a large scale Bt-based biopesticide production.

A recombinant truncated Cry1Ca protein is toxic to lepidopteran insects and forms large cuboidal crystals in insect cells.

A truncated version of the cry1Ca gene from Bacillus thuringiensis was introduced into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) under the control of two promoters. A recombinant virus (vSyncry1c) was isolated and used to infect insect cells in culture and insect larvae. Structural and ultrastructural analysis of insects infected with vSyncry1C showed the formation of large cuboidal crystals inside the cytoplasm of insect cells in culture and in insect cadavers late in infection. Infected insect cell extracts were analyzed by SDS-PAGE and Western blot and showed the presence of a 65-kDa polypeptide probably corresponding to the protease processed form of the toxin. Bioassays using purified recombinant toxin crystals showed a CL(50) of 19.49 ng/ml for 2(nd) instar A. gemmatalis larvae and 114.1 ng/ml for S. frugiperda.

Effect of Baculovirus spodoptera isolates in Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) larvae and their characterization by RAPD

The total of 22 Baculovirus isolates surveyed in different corn producing regions in Brazil were used against fall armyworm, Spodoptera frugiperda (J.E. Smith). The viruses were purified and their suspensions were used to feed fall armyworm larvae from 4th and 5th instar. The mortality rate was checked daily and the infected larvae were frost after death, what generally occurred between the 5th and 7th day after virus ingestion. The 22 Baculovirus isolates were used in six concentrations (from 10³ to 10(8) polyhedra/ml) and one check treatment with water. Mortality rate, larval period, pupal period, pupa weight and lethal concentration (LC50) were determined for all isolates. Significant differences were found among all isolates and different concentrations, also interaction between isolate x virus concentration for all characteristics evaluated, except for pupal period. Amplification patterns of 54 RAPD markers, being 41 polymorphic among the isolates, were used to evaluate the genetic distance and its correlation with the fall armyworm larvae mortality rate. The genetic diversity calculated by the Jaccard's coefficient using the molecular data allowed a division of the isolates into two groups, with a high level of confidence. These groups did not present any association with the mortality rate caused by the isolates or with their geographical distribution. However, a RAPD fragment OPW04.2280 was highly associated with the larvae mortality rate and with LC50, explaining 23 and 65 percent of the phenotypic variation for these traits among the isolates, respectively.

Foram utilizados 22 isolados de vírus amostrados em diferentes regiões produtoras de milho do Brasil. Os vírus foram purificados e suas suspensões fornecidas a lagartas sadias do 3° e 4° ínstar de Spodoptera frugiperda (J.E. Smith). A mortalidade foi avaliada diariamente, e as lagartas infectadas foram congeladas logo após sua morte, o que em geral ocorreu do 5° ao 7° dia após ingestão do vírus. Os isolados foram usados em seis concentrações (10³ a 10(8) poliedros/ml) e uma testemunha (água). Os percentuais de mortalidade, duração do período larval e período pupal, peso de pupa e a concentração letal (CL50) foram determinados para todos os isolados. Foram observadas diferenças significativas entre todos os isolados e concentrações testadas para todos os parâmetros avaliados, e também foi constatada a presença da interação isolado x concentração, exceto para período pupal. Os padrões de amplificação de 54 marcadores RAPD, sendo 41 polimórficos, foram utilizados para avaliar a distância genética e a sua correlação com os índices de mortalidade das lagartas. A divergência genética calculada pelo coeficiente Jaccard utilizando os dados moleculares permitiu dividir os isolados em dois grupos, com um elevada confiabilidade. O agrupamento não apresentou associação com a taxa de mortalidade causada pelos isolados ou com sua distribuição geográfica. No entanto, um fragmento de RAPD OPW04.2280 apresentou-se altamente associado com a mortalidade das lagartas e com a CL50, explicando 23 por cento e 65 por cento da variação fenotípica para essas características entre os isolados virais, respectivamente.

A physically anchored genetic map and linkage to avirulence reveals recombination suppression over the proximal region of Hessian fly chromosome A2.

Resistance in wheat (Triticum aestivum) to the Hessian fly (Mayetiola destructor), a major insect pest of wheat, is based on a gene-for-gene interaction. Close linkage (3 +/- 2 cM) was discovered between Hessian fly avirulence genes vH3 and vH5. Bulked segregant analysis revealed two DNA markers (28-178 and 23-201) within 10 cM of these loci and only 3 +/- 2 cM apart. However, 28-178 was located in the middle of the short arm of Hessian fly chromosome A2 whereas 23-201 was located in the middle of the long arm of chromosome A2, suggesting the presence of severe recombination suppression over its proximal region. To further test that possibility, an AFLP-based genetic map of the Hessian fly genome was constructed. Fluorescence in situ hybridization of 20 markers on the genetic map to the polytene chromosomes of the Hessian fly indicated good correspondence between the linkage groups and the four Hessian fly chromosomes. The physically anchored genetic map is the first of any gall midge species. The proximal region of mitotic chromosome A2 makes up 30% of its length but corresponded to <3% of the chromosome A2 genetic map.

Bacillus thuringiensis survey in Brazil: geographical distribution and insecticidal activity against Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) / Levantamento de Bacillus thuringiensis no Brasil: distribuição geográfica e atividade inseticida contra Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

A total of 3408 strains of Bacillus thuringiensis were collected from 1448 soil samples in 10 Brazilian states, four different geographical regions, covering 96 counties. These strains were evaluated against Spodoptera frugiperda (J.E. Smith) larvae. Only 62 percent killed between 81 percent and 100 percent and 1758 caused no mortality. Highest proportion of efficient strains (larval mortality above 75 percent) was found from the total isolated per region in the South Region (16.6 percent), followed by Western Central (3.1 percent), Southeast (1.1 percent) and Northeast Region (0.4 percent).

O total de 3408 cepas de Bacillus thuringiensis foi isolado de 1448 amostras de solo provenientes de 10 estados, em quatro regiões brasileiras, abrangendo 96 municípios. As cepas foram avaliadas contra lagartas de Spodoptera frugiperda (J. E. Smith) sendo que apenas 62 por cento mataram entre 81 por cento e 100 por cento, e 1758 não causaram mortalidade. O Sul destacou-se com 16,6 por cento de cepas eficientes (mortalidade>75 por cento) do total obtido por região, seguido das regiões Centro Oeste (3,1 por cento), Sudeste (1,1 por cento), e Nordeste (0,4 por cento).