Antennal transcriptomic analysis of carboxylesterases and glutathione S-transferases associated with odorant degradation in the tea gray geometrid, Ectropis grisescens (Lepidoptera, Geometridae)

Introduction: Carboxylesterases (CXEs) and glutathione S-transferases (GSTs) can terminate olfactory signals during chemosensation by rapid degradation of odorants in the vicinity of receptors. The tea grey geometrid, Ectropis grisescens (Lepidoptera, Geometridae), one of the most devastating insect herbivores of tea plants in China, relies heavily on plant volatiles to locate the host plants as well as the oviposition sites. However, CXEs and GSTs involved in signal termination and odorant clearance in E. grisescens remains unknown.Methods: In this study, identification and spatial expression profiles of CXEs and GSTs in this major tea pest were investigated by transcriptomics and qRT-PCR, respectively.Results: As a result, we identified 28 CXEs and 16 GSTs from female and male antennal transcriptomes. Phylogenetic analyses clustered these candidates into several clades, among which antennal CXEs, mitochondrial and cytosolic CXEs, and delta group GSTs contained genes commonly associated with odorants degradation. Spatial expression profiles showed that most CXEs (26) were expressed in antennae. In comparison, putative GSTs exhibited a diverse expression pattern across different tissues, with one GST expressed specifically in the male antennae.Disscussion: These combined results suggest that 12 CXEs (EgriCXE1, 2, 4, 6, 8, 18, 20-22, 24, 26, and 29) and 5 GSTs (EgriGST1 and EgriGST delta group) provide a major source of candidate genes for odorants degradation in E. grisescens

Biotechnological production and application of the antibiotic pimaricin: biosynthesis and its regulation

[EN] Pimaricin (natamycin) is a small polyene macrolide antibiotic used worldwide. This efficient antimycotic and antiprotozoal agent, produced by several soil bacterial species of the genus Streptomyces, has found application in human therapy, in the food and beverage industries and as pesticide. It displays a broad spectrum of activity, targeting ergosterol but bearing a particular mode of action different to other polyene macrolides. The biosynthesis of this only antifungal agent with a GRAS status has been thoroughly studied, which has permitted the manipulation of producers to engineer the biosynthetic gene clusters in order to generate several analogues. Regulation of its production has been largely unveiled, constituting a model for other polyenes and setting the leads for optimizing the production of these valuable compounds. This review describes and discusses the molecular genetics, uses, mode of action, analogue generation, regulation and strategies for increasing pimaricin production yieldsSIThis work was supported by the Spanish Ministerio de Economía y Competitividad (Grant BIO2013-42983-P to JFA); F.P.U. fellowships from the Ministerio de Educación, Cultura y Deporte (AP2005-3644 to JSA, AP2007-02055 to TDP and FPU13/01537 to AP), a contract from the Junta de Castilla y León co-financed by the European Social Fund (to EGB); and a fellowship from the Portuguese Fundação para a Ciência e a Tecnologia (SFRH/BD/64006/2009 to CMV

Rice Improvement

This book is open access under a CC BY 4.0 license. By 2050, human population is expected to reach 9.7 billion. The demand for increased food production needs to be met from ever reducing resources of land, water and other environmental constraints. Rice remains the staple food source for a majority of the global populations, but especially in Asia where ninety percent of rice is grown and consumed. Climate change continues to impose abiotic and biotic stresses that curtail rice quality and yields. Researchers have been challenged to provide innovative solutions to maintain, or even increase, rice production. Amongst them, the ‘green super rice’ breeding strategy has been successful for leading the development and release of multiple abiotic and biotic stress tolerant rice varieties. Recent advances in plant molecular biology and biotechnologies have led to the identification of stress responsive genes and signaling pathways, which open up new paradigms to augment rice productivity. Accordingly, transcription factors, protein kinases and enzymes for generating protective metabolites and proteins all contribute to an intricate network of events that guard and maintain cellular integrity. In addition, various quantitative trait loci associated with elevated stress tolerance have been cloned, resulting in the detection of novel genes for biotic and abiotic stress resistance. Mechanistic understanding of the genetic basis of traits, such as N and P use, is allowing rice researchers to engineer nutrient-efficient rice varieties, which would result in higher yields with lower inputs. Likewise, the research in micronutrients biosynthesis opens doors to genetic engineering of metabolic pathways to enhance micronutrients production. With third generation sequencing techniques on the horizon, exciting progress can be expected to vastly improve molecular markers for gene-trait associations forecast with increasing accuracy. This book emphasizes on the areas of rice science that attempt to overcome the foremost limitations in rice production. Our intention is to highlight research advances in the fields of physiology, molecular breeding and genetics, with a special focus on increasing productivity, improving biotic and abiotic stress tolerance and nutritional quality of rice. ; Up-to-date contributions by experts from international research centers and universities Provides practical knowledge and strong scientific foundation on rice biotechnology All-in-one resource for current advances in rice breeding Open Acces

Rice Improvement

This book is open access under a CC BY 4.0 license. By 2050, human population is expected to reach 9.7 billion. The demand for increased food production needs to be met from ever reducing resources of land, water and other environmental constraints. Rice remains the staple food source for a majority of the global populations, but especially in Asia where ninety percent of rice is grown and consumed. Climate change continues to impose abiotic and biotic stresses that curtail rice quality and yields. Researchers have been challenged to provide innovative solutions to maintain, or even increase, rice production. Amongst them, the ‘green super rice’ breeding strategy has been successful for leading the development and release of multiple abiotic and biotic stress tolerant rice varieties. Recent advances in plant molecular biology and biotechnologies have led to the identification of stress responsive genes and signaling pathways, which open up new paradigms to augment rice productivity. Accordingly, transcription factors, protein kinases and enzymes for generating protective metabolites and proteins all contribute to an intricate network of events that guard and maintain cellular integrity. In addition, various quantitative trait loci associated with elevated stress tolerance have been cloned, resulting in the detection of novel genes for biotic and abiotic stress resistance. Mechanistic understanding of the genetic basis of traits, such as N and P use, is allowing rice researchers to engineer nutrient-efficient rice varieties, which would result in higher yields with lower inputs. Likewise, the research in micronutrients biosynthesis opens doors to genetic engineering of metabolic pathways to enhance micronutrients production. With third generation sequencing techniques on the horizon, exciting progress can be expected to vastly improve molecular markers for gene-trait associations forecast with increasing accuracy. This book emphasizes on the areas of rice science that attempt to overcome the foremost limitations in rice production. Our intention is to highlight research advances in the fields of physiology, molecular breeding and genetics, with a special focus on increasing productivity, improving biotic and abiotic stress tolerance and nutritional quality of rice. ; Up-to-date contributions by experts from international research centers and universities Provides practical knowledge and strong scientific foundation on rice biotechnology All-in-one resource for current advances in rice breeding Open Acces

Expression of cysteine proteinase inhibitor genes (OCI and OCII) in transformed potato (Solanum tuberosum L.) plants

Kombinovanje ili “slaganje” različitih gena u transgenim biljkama radi postizanja uspešnije kontrole patogena i štetočina i/ili većeg prinosa predstavlja jednu od glavnih oblasti istraživanja savremene biotehnologije. Orizacistatini I i II (OCI i OCII), proteinazni inhibitori različitih specifičnosti, pokazali su potencijal u kontroli štetočina koje koriste cisteinske proteinaze za digestiju proteina. Da bi se pojačao njihov inhibitorni potencijal i, eventualno, povećala efikasnost ovih inhibitora u kontroli štetočina, oba cistatina su koeksprimirana u transformisanim biljkama tri sorte krompira. “Slaganje” orizacistatinskih gena kod sorti Dragačevka i Dezire ostvareno je postupkom ko-transformacije i zabeležena je frekvenca kointegracije OCI i OCII gena od 20-22%. Kod sorte Jelica sekvencijalna re-transformacija se pokazala kao efikasniji pristup: frekvenca integracije OCII gena nakon re-transformacije OCI-transformisane linije iznosila je 91%. Istovremeno, “slaganje” dva orizacistatnska gena, bilo postupkom ko- ili re-transformacije, postignuto je upotrebom nptII gena kao jedinog selekcionog markera. Ekspresija OCI i OCII gena indukovana povređivanjem i akumulacija biološki aktivnih rekombinantnih OCI i OCII proteina potvrđena je kod svih analiziranih OCI/OCII transformisanih linija krompira. OCI/OCII linije krompira nisu ispoljavale značajna odstupanja od normalnog fenotipa, što ukazuje na nizak nivo somaklonalnih varijacija i odsustvo uticaja rekombinantnih OCI i OCII na metabolizam biljke domaćina. Iako nije uticala na preživljavanje, ishrana larvi krompirove zlatice (Leptinotarsa decemlineata Say) listovima krompira koji eksprimiraju oba orizacistatina imala je značajan uticaj na različite osobine performanse rasta i razvića larvi. Larve hranjene transformisanim listovima su se presvlačile ranije, i tokom L2 i L3 stupnja uvećavale masu do 29,7% brže i konzumirale listove do 29,1% brže u odnosu na one hranjene netransformisanim listovima. Istovremeno, larve na OCI/OCII listovima su do tri dana ranije dostizale maksimum mase i ranije “usporavale” sa ishranom ulazeći u prepupalnu fazu razvića. Uprkos povećanju performansi rasta i ishrane, pri istoj efikasnosti ishrane, L4 larve na transformisanim listovima nisu u potpunosti uspele da kompenzuju negativne efekte prisustva orizacistatina u hrani. U odnosu na larve hranjene netransformisanim listovima, maksimalna masa na kraju larvenog razvića i ukupan stepen oštećenja listova bili su do 19,4% i do 18,5% manji kod larvi krompirove zlatice hranjenih OCI/OCII transformisanim listovima krompira. Smanjenje mase larvi na OCI/OCII listovima dovelo je i do pojave adulta krompirove zlatice sa do 26,3% redukovanom telesnom masom. Analiza ukupne proteinazne...The combination or stacking different genes in transgenic plants to achieve disease and pest control and/or higher crop yield is one of a major method of contemporary biotechnology. Oryzacystatins I and II (OCI and OCII), inhibitors with different specificity, show potential in controlling pests that utilize cysteine proteinases for protein digestion. To strengthen this inhibitory range and, possibly, achieve an additive effect in the overall efficiency of these proteins against pests, both cystatins were co-expressed in three potato cultivars. Oryzacystatin genes pyramiding in Dragačevka and Desiree cultivars were achieved by co-transformation with OCI and OCII genes co-integration frequency of 20-22%. For Jelica cultivar sequential re-transformation was more efficient approach: OCII gene integration frequency following re-transformation of an OCI-expressing line was 91%. Additionally, pyramiding of different oryzacystatin genes, by co- or re-transformation approach, were achieved using the nptII gene as the only selection marker. Wounding induction of OCI and OCII gene transcripts and accumulation of biologically active OCI and OCII recombinant proteins was confirmed in all analyzed OCI/OCII transformed lines. OCI/OCII potato lines did not exhibit morphological abnormalities, indicating low level of somaclonal variation or interference of the recombinant OCI or OCII with host plant metabolism. In the absence of significant mortality, feeding Colorado potato beetle larvae (Leptinotarsa decemlineata Say) on OCI/OCII-expressing foliage had an impact on various aspects of the growth and developmental performances of larvae. Larvae feeding on transformed potato leaves tended to molt earlier and, especially during L2-L3 stages, gain weight up to 29.7% faster and consume leaf material up to 29.1% faster, compared to those on untransformed foliage. Larvae on OCI/OCII foliage were also reach maximum weight gained three days earlier and slow down earlier in preparation for pupation. Despite their faster growth and feeding, with similar efficiencies of conversion of ingested food, L4 larvae reared on transformed foliage were not compensating presence of the recombinant oryzacystatins in the diet.Compared to those on untransformed foliage, maximum weight gained and amount of foliage consumed were up to 19.4% and 18.5%, respectively, lower for the larvae fed on OCI/OCII potato foliage. Larval weight reduction on OCI/OCII foliage resulted in adult emergence with up to 26.3% reduced body mass. Analysis of total digestive proteinases activity showed initially, up to 56%, reduction in digestive capacity..

Transcriptomics analysis of phloem-feeding insect resistance in rice germplasm

The Brown Plant Hopper (BPH) is a serious pest of rice in Asia. Development of novel control strategies can be facilitated by a comparison of BPH feeding behaviour on varieties exhibiting natural genetic variation, and then an elucidation of the underlying mechanisms of resistance. We began by understanding BPH feeding behaviour on 12 rice varieties with different resistance background using Electrical Penetration Graph (EPG) and honeydew clock experiments. Seven feeding behaviours (waveforms) were identified and could be classified into two phases, feeding and non- feeding. Cluster analysis has separated the 12 varieties into 3 main groups, resistant, moderate and susceptible. Then, we undertook microarray analysis on all varieties to identify candidate genes which may contribute to resistance. The results reveal the difference between resistant and susceptible varieties. The data agree with EPG and honeydew clock experiments. A total of 21556 probes passed filter in statistical analysis using quantile method (in Genespring) and 239 probes significantly contributed to the difference between resistant versus susceptible (Volcano analysis). Some of them were found to be highly correlated with EPG data and could therefore be potential resistance candidate genes against BPH such as gene encoding hexose transporter, protein kinases, Alpha-DOX2 and peroxidase

Characterization of mechanisms of resistance in <em>Spodoptera frugiperda</em> to synthetic insecticides and insecticidal proteins

Fall armyworm (FAW), Spodoptera frugiperda(J.E. Smith), is a major lepidopteran pest of maize in the American continent butitis now present in 107 countries worldwide. The control of FAW has relied mainly on the use of synthetic insecticides and transgenic crops expressing Bacillus thuringiensis(Bt) insecticidal proteins. However, the effective control of this pest is challenging, as resistance to 41 different active substances has been reported worldwide, putting at risk the yield of important staple crops. Diamides act on insect ryanodine receptors (RyR) and are the most modern insecticide class intensively used to control lepidopteran pests. A highlydiamide-resistant population of FAW from Brazil has been selected in the laboratory. Sequencing of the C-terminal end of the RyR revealed the presence of a conserved point mutation (I4790M) linked to diamide resistance. Resistance to Bt crops in Brazil has been reported since 2014. In this thesis, the molecular mechanism conferring Cry1F resistance in Brazilian FAW was investigated and characterized. Different mutations were observed in exon 14 of the Bt receptor ATP-Binding Cassette subfamily C2 (ABCC2) transporter. However, the deletion of glycine and tyrosine (GY deletion) was found in higher frequency in field-collected strains of FAWhighly resistant to Cry1F. Failures of FAW control with Bt crops and consequently high infestation pressure in the field require additional insecticide applications. Therefore, the toxicological profile of a Cry1F-resistant strain(Sf_Des)was also investigated to different commercial insecticides.Laboratory bioassays with 15 active substances revealed that Sf_Deshas a medium level of resistance to deltamethrin and chlorpyrifos in comparison to the Cry1F-susceptible strain. Very high cross-resistance was observed among Cry1 toxins, but high susceptibility against Vip3A. RNA-Seq data support a major role of P450 enzymes in the detoxification of insecticides and RT-qPCR analysis confirmed that CYP9A-like and CYP6B39are significantly up-regulated (>200-fold) in the Cry1F-resistant strain. Methods for the detection of target-site mutations were developed and used to genotype 34 FAW populations from different continents. The diagnostic methods revealed a high frequency of mutations in acetylcholinesterase, conferring resistance to organophosphates and carbamates. In voltage-gated sodium channels targeted by pyrethroids, only one population from Indonesia showed a mutation. No mutations were detected in the ryanodine receptor, suggesting susceptibility to diamides. Indels in the ABCC2 associated with Bt-resistance were observedin samples collected in Puerto Rico and Brazil. Additionally, we analyzed all samples for the presence of markers associated with two sympatric FAW host plant strains. The molecular methods established show robust results in FAW samples collected across a broad geographical range and can be used to support decisions for sustainable FAW control and applied resistance management. The data presented here characterized novel molecular mechanisms conferring resistance to different insecticides/Bt toxins which remained elusive yet. Those findings not only support further research on new insecticides compounds overcoming such resistance mechanisms, but also provide practical guidance for the regional implementation of efficient resistance management strategies

UVR8 mediated spatial differences as a prerequisite for UV-B induced inflorescence phototropism

In Arabidopsis hypocotyls, phototropins are the dominant photoreceptors for the positive phototropism response towards unilateral ultraviolet-B (UV-B) radiation. We report a stark contrast of response mechanism with inflorescence stems with a central role for UV RESISTANCE LOCUS 8 (UVR8). The perception of UV-B occurs mainly in the epidermis and cortex with a lesser contribution of the endodermis. Unilateral UV-B exposure does not lead to a spatial difference in UVR8 protein levels but does cause differential UVR8 signal throughout the stem with at the irradiated side 1) increase of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), 2) an associated strong activation of flavonoid biosynthesis genes and flavonoid accumulation, 3) increased GA2oxidase expression, diminished gibberellin1 levels and accumulation of DELLA protein REPRESSOR OF GA1 (RGA) and, 4) increased expression of the auxin transport regulator, PINOID, contributing to local diminished auxin signalling. Our molecular findings are in support of the Blaauw theory (1919), suggesting that differential growth occurs trough unilateral photomorphogenic growth inhibition. Together the data indicate phototropin independent inflorescence phototropism through multiple locally UVR8-regulated hormone pathways

Identification and quantification of crystal components of Bacillus thuringiensis strains and their contribution to insecticidal activity

El principal objetivo de esta tesis ha sido desarrollar un método proteómico que permite analizar cualitativa y cuantitativamente las proteínas que componen el cristal producido por distintas cepas silvestres de Bt. El método emplea un sistema de cromatografía líquida acoplada a un espectro de masas (LC-MS/MS) en combinación con una monitorización de múltiples reacciones (MRM). Para llevar a cabo el análisis, es necesario conocer la secuencia del genoma de la cepa Bt para determinar los potenciales genes insecticidas que podrían formar parte del cristal parasporal. El uso de herramientas bioinformáticas permite la selección de péptidos proteotípicos que detectan de forma específica la presencia de cada una de las proteínas de la mezcla. Estos péptidos proteotípicos, marcados isotópicamente, permiten determinar la proporción relativa de cada proteína en el cristal. El método fue validado utilizando dos mezclas artificiales de tres proteínas recombinantes (Cry1Aa, Cry2Aa y Cry6Aa), donde la proporción relativa de cada proteína era conocida. La aplicación del método permitió detectar las tres proteínas de forma independiente y cuantificar la proporción relativa de cada una de ellas con gran fiabilidad y precisión. Una vez verificada su validez, el método fue aplicado para determinar la composición del cristal de cuatro cepas Bt silvestres que component el ingrediente activo de los productos comerciales más vendidos a nivel mundial para el control de distintos órdenes de insecto: DiPel® y XenTari® para lepidópteros, VectoBac® para dípteros, y Novodor® para coleópteros. El cristal parasporal de la cepa ser. kurstaki ABTS-351 (DiPel®) resultó estar compuesto por cuatro proteínas: Cry1Aa (13-22%), Cry1Ab (16-29%), Cry1Ac (6-12%) y Cry2Aa (40-64%); al igual que la cepa ser. aizawai ABTS-1857 (XenTari®) Cry1Aa (26-33%), Cry1Ab (57-60%), Cry1Ca (7-11%) y Cry1Da (3-4%). La cepa AM65-52 (VectoBac®) sintetizó un cristal formado por Cry4Aa (2-4%), Cry4Ba (10-28%), Cry11Aa (10-27%), Cry60Aa (2-4%), Cry60Ba (5-12%) y Cyt1Aa (38-61%) y el ingrediente activo de Novodor®, la cepa ser. tenebrionis NB-176, contenía Cry3Aa (70-75%), Cry23Aa (14-16%) y Cry37Aa (10-14%). Adicionalmente, se determinó la actividad de la cepa ABTS-1857 en larvas de tres especies del género Spodoptera: S. exigua, S. littoralis y S. frugiperda. S. exigua fue la especie más susceptible (CL50= 7.8 ng/μl), seguida de S. littoralis (CL50= 28 ng/μl). S. frugiperda se mostró como la especie más tolerante (CL50= 120.2 ng/μl). Para determinar la contribución de cada proteína individual a la toxicidad general de la cepa ABTS-1857 contra cada una de las tres especies de insectos, se construyeron cepas Bt recombinantes que producían individualmente las proteínas Cry1Aa, Cry1Ab, Cry1Ca y Cry1Da. Los resultados de los bioensayos, utilizando el “droplet feeding method”, revelarón una elevada toxicidad de Cry1Ca para las larvas de S. exigua y S. littoralis y de Cry1Da frente a S. littoralis y S. frugiperda. Las mezclas artificiales de dos o tres proteínas produjeron mortalidades atribuibles a la cantidad de Cry1Ca, en el caso de S. exigua, de Cry1Ca y Cry1Da, en el caso de S. littoralis, y de Cry1Da en el caso de S. frugiperda. La mezcla artificial de cuatro proteínas, que reflejaba la composición natural del cristal, dio valores de actividad concordantes con los producidos por el cristal natural de la cepa ABTS-1857. Aumentos de la proteína Cry1Da, en detrimento de las proteías Cry1Aa y Cry1Ab, produjo incrementos en la actividad insecticida para larvas de S. littoralis y S. frugiperda. Estos resultados indicaron que la metodología empleada para el análisis de los cristales es válida para su empleo en la caracterización y estandarización de los productos comerciales basados en Bt, aportando la información necesaria para expresar su potencia insecticida.The main aim of this thesis has been the development of a proteomic method that allows a qualitative and quantitative analysis of the proteins that make up the crystal produced by different wildtype Bt strains. The method uses a liquid chromatography system coupled to a mass spectrum (LC-MS/MS) in combination with a multiple reaction monitoring (MRM). To carry out the analysis, it is necessary to know the genome sequence of the Bt strain to determine the potential insecticidal genes that could be part of the parasporal crystal. The use of bioinformatic tools, allow the selection of proteotypic peptides that specifically detect the presence of each of the proteins in the mixture. These proteotypic peptides, isotopically labelled, allow to determine the relative proportion of each protein within the crystal. The method was validated using two artificial mixtures containing three recombinant proteins (Cry1Aa, Cry2Aa and Cry6Aa), in a known relative proportion. The application of the method allowed the detection of the three proteins independently and the quantification of the relative proportion of each of them with great reliability and precision. Once the method was validated, it was applied to determine the crystal composition of the Bt strains used as active ingredients of four of the best sold commercial products worldwide for the control of different insect orders: DiPel® and XenTari® for Lepidoptera, VectoBac® for Diptera, and Novodor® for Coleoptera. The parasporal crystal of the Bt subsp. kurstaki strain ABTS-351 (DiPel®) was comprised by four proteins, including: Cry1Aa (13-22%), Cry1Ab (16-29%), Cry1Ac (6-12%) and Cry2Aa (40-64%); in the Bt subsp. aizawai strain ABTS-1857 (XenTari®) four proteins were also detected: Cry1Aa (26-33%), Cry1Ab (57-60%), Cry1Ca (7-11%) and Cry1Da (3-4%). The Bt subsp. israelensis strain AM65-52 (VectoBac®) synthesized a crystal formed by Cry4Aa (2-4%), Cry4Ba (10-28%), Cry11Aa (10-27%), Cry60Aa (2-4%), Cry60Ba (5-12%) and Cyt1Aa (38-61%). Finally, the active ingredient of Novodor®, the Bt subsp. tenebrionis strain NB-176, contained Cry3Aa (70-75%), Cry23Aa (14-16%) and Cry37Aa (10-14%). Additionally, the activity of the strain ABTS-1857 was determined for three species of the genus Spodoptera, including: S. exigua, S. littoralis and S. frugiperda. S. exigua was the most susceptible species (LC50 = 7.8 ng/μl), followed by S. littoralis (LC50 = 28 ng/μl). S. frugiperda was the most tolerant insect (LC50 = 120.2 ng/μl). To determine the contribution of each individual protein to the overall toxicity of the strain ABTS-1857 against each of the three insect species, recombinant Bt strains producing Cry1Aa, Cry1Ab, Cry1Ca and Cry1Da proteins, were engineered. The results of the bioassays, using the droplet feeding method, revealed a high toxicity of Cry1Ca for S. exigua and S. littoralis larvae and Cry1Da against S. littoralis and S. frugiperda. Artificial mixtures containing two or three proteins produced mortalities attributed to the amounts of Cry1Ca, in the case of S. exigua, of Cry1Ca and Cry1Da, in the case of S. littoralis, and of Cry1Da in the case of S. frugiperda. The obtained activity values for the artificial mixture containing the four proteins, which reflected the natural composition of the crystal, were consistent with those obtained with the natural crystal of the strain ABTS-1857. Increasing amounts of Cry1Da protein, in combination with decreasing quantities of either Cry1Aa or Cry1Ab proteins, produced an enhanced insecticidal toxicity for S. littoralis and S. frugiperda larvae. These results indicated that the method used for the analysis of the Bt crystals is valid for its use in the characterization and standardization of Bt based commercial products, providing the necessary information to express its insecticidal potency.Programa de Doctorado en Biotecnología (RD 99/2011)Bioteknologiako Doktoretza Programa (ED 99/2011