Assessment of the Entomopathogenic Potential of Fungal and Bacterial Isolates from Fall Armyworm Cadavers Against Spodoptera frugiperda Caterpillars and the Adult Boll Weevil, Anthonomus grandis.

Increased attention is being focused on the biological control of agricultural pests using microorganisms, owing to their potential as a viable substitute for chemical control methods. Insect cadavers constitute a potential source of entomopathogenic microorganisms. We tested whether bacteria and fungi isolated from Spodoptera frugiperda (JE Smith) cadavers could affect its survival, development, egg-laying pattern, and hatchability, as well as induce mortality in Anthonomus grandis Boheman adults. We isolated the bacteria Enterobacter hormaechei and Serratia marcescens and the fungi Scopulariopsis sp. and Aspergillus nomiae from fall armyworm cadavers and the pest insects were subjected to an artificial diet enriched with bacteria cells or fungal spores to be tested, in the case of S. frugiperda, and only fungal spores in the case of A. grandis. Enterobacter hormaechei and A. nomiae were pathogenic to S. frugiperda, affecting the survival of adults and pupae. The fungus Scopulariopsis sp. does not affect the survival of S. frugiperda caterpillars and pupae; however, due to late action, moths and eggs may be affected. Aspergillus nomiae also increased mortality of A. grandis adults, as well as the development of S. frugiperda in the early stages of exposure to the diet, as indicated by the vertical spore transfer to offspring and low hatchability. Enterobacter hormaechei and A. nomiae are potential biocontrol agents for these pests, and warrant further investigation from a toxicological point of view and subsequently in field tests involving formulations that could improve agricultural sustainability practices.

Dry and Rainy Seasons Significantly Alter the Gut Microbiome Composition and Reveal a Key Enterococcus sp. (Lactobacillales: Enterococcaceae) Core Component in Spodoptera frugiperda (Lepidoptera: Noctuidae) Corn Strain From Northwestern Colombia.

Spodoptera frugiperda is a polyphagous pest of several crops of economic importance. Nowadays, the insect is broadly distributed in America and, recently, in Africa, Asia, and Australia. The species has diverged into corn and rice strains. The role of the gut microbiota in insect physiology is relevant due to its participation in crucial functions. However, knowledge of seasonal variations that alter the gut microbiome in pests is limited. Gut microbiome composition between the dry and rainy seasons was analyzed with cultured and uncultured approaches in S. frugiperda corn strain larvae collected at Northwest Colombia, as seasonal microbiome changes might fluctuate due to environmental changes. On the basis of culture-dependent methods, results show well-defined microbiota with bacterial isolates belonging to Enterococcus, Klebsiella (Enterobacteriales: Enterobacteriaceae), Enterobacter (Enterobacterales: Enterobacteriaceae), and Bacillus (Bacillales: Bacillaceae) genera. The community composition displayed a low bacterial diversity across all samples. The core community detected with uncultured methods was composed of Enterococcus, Erysipelatoclostridium (Erysipelotrichales: Erysipelotrichaceae), Rasltonia (Burkholderiales: Burkholderiaceae), and Rhizobium (Hyphomicrobiales: Rhizobiaceae) genera, and Enterobacteriaceae family members. Significant differences in microbiome diversity were observed between the two seasons. The relative abundance of Erysipelatoclostridium was high in the dry season, while in the phylotype ZOR0006 (Erysipelotrichales: Erysipelotrichaceae) and Tyzzerella (Lachnospirales: Lachnospiraceae) genus, the relative abundance was high in the rainy season. The overall low gut bacterial diversity observed in the S. frugiperda corn strain suggests a strong presence of antagonist activity as a selection factor possibly arising from the host, the dominant bacterial types, or the material ingested. Targeting the stability and predominance of this core microbiome could be an additional alternative to pest control strategies, particularly in this moth.

Compatibility of Bt biopesticides and adjuvants for Spodoptera frugiperda control.

Spodoptera frugiperda is a pest of economic importance for several crops with resistance reports to Bt crops and pesticides. Eco-friendly Bt biopesticides may be an alternative to chemical insecticides due to their selectivity and specificity. However, the efficacy of Bt biopesticides may be influenced by the association with other chemicals, such as adjuvants. This study evaluated the compatibility and toxicity of Bt biopesticides mixed with adjuvants for the control of S. frugiperda. The treatments included the association of Dipel SC and Dipel PM with adjuvants. Compatibility tests were used to evaluate the Bt mixture. Bt suspensions obtained from mixtures of Bt and adjuvants at 106 and 3 × 108 spores/mL-1 were used to evaluate S. frugiperda mortality and distilled water was used as the control. The addition of the adjuvant LI increased growth and sporulation, indicating compatibility with Bt biopesticides. The other adjuvants were toxic to reducing Bt growth and sporulation. Only the mixture of Bt with LI and Bt alone was effective to S. frugiperda. The addition of adjuvants to Bt biopesticide affect the Bt sporulation, growth and mortality.

Development of a novel anti-biofilm peptide derived from profilin of Spodoptera frugiperda.

Candida yeast infections are the fourth leading cause of death worldwide. Peptides with antimicrobial activity are a promising alternative treatment for such infections. Here, the antifungal activity of a new antimicrobial peptide-PEP-IA18-was evaluated against Candida species. PEP-IA18 was designed from the primary sequence of profilin, a protein from Spodoptera frugiperda, and displayed potent activity against Candida albicans and Candida tropicalis, showing a minimum inhibitory concentration (MIC) of 2.5 µM. Furthermore, the mechanism of action of PEP-IA18 involved interaction with the cell membrane (ergosterol complexation). Treatment at MIC and/or 10 × MIC significantly reduced biofilm formation and viability. PEP-IA18 showed low toxicity toward human fibroblasts and only revealed hemolytic activity at high concentrations. Thus, PEP-IA18 exhibited antifungal and anti-biofilm properties with potential applicability in the treatment of infections caused by Candida species.

Side effects of Bacillus thuringiensis on the parasitoid Palmistichus elaeisis (Hymenoptera: Eulophidae).

The endoparasitoid wasp Palmistichus elaeisis Delvare & LaSalle (Hymenoptera: Eulophidae) is used to control defoliating lepidopteran pests. Chemical insecticides are not compatible with natural enemies, but bioinsecticides, such as Bacillus thuringiensis Berliner (Bt), have great potential for use in integrated pest management. However, interactions between Bt and P. elaeisis still need to be investigated. This study aimed to evaluate the effects of Bt on parental and first-generation P. elaeisis parasitizing Bt-susceptible and -resistant Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). An additional aim was to determine the toxicity of Bt to susceptible third-instar S. frugiperda larvae. Larvae were exposed to lethal concentrations (LC50 and LC90) of Bt and then allowed to be parasitized by P. elaeisis. Parasitoid longevity, immature production, reproductive performance, and behavioral responses were evaluated. Bt repelled P. elaeisis and reduced immature production. Parental and first filial generation parasitoids of both sexes emerged from Bt-treated larvae showed lower survivorship than controls. Parasitoids had poorer reproductive performance in Bt-susceptible and -resistant pupae than in untreated pupae. Palmistichus elaeisis emerged from Bt-susceptible and -resistant S. frugiperda showed altered host-searching behavior and reproductive parameters, which indicates low compatibility between the bioinsecticide agent and the parasitoid wasp.

Within-host interactions of Metarhizium rileyi strains and nucleopolyhedroviruses in Spodoptera frugiperda and Anticarsia gemmatalis (Lepidoptera: Noctuidae).

Members of the family Baculoviridae have been quite successfully used as biocontrol agents against some lepidopterans. Likewise, a number of fungi are important natural enemies of these pests. An interesting approach to increase control efficacy could be the combination of a given nucleopolyhedrovirus (NPV) and a fungus, since they possess distinct modes of action. As a first step towards this goal, we assessed the interaction between NPV (either AgMNPV-79 or SfMNPV-6nd) and the entomopathogenic fungus Metarhizium rileyi (either CG1153 or CG381), using Anticarsia gemmatalis and Spodoptera frugiperda as hosts. In sequential applications of these pathogens, per os inoculation of an NPV (leaf discs with 2.5 × 104 occlusion bodies) either two days before or two days post-spraying of its counterpart fungal strain (5 × 103 conidia.cm-2 sprays) usually resulted in an antagonistic effect. When both pathogens were simultaneously applied at different combined dosages, usually an additive effect was seen. Interestingly, a number of dead larvae showing signs of co-infections (partially with soft integument and partially mummified) were recorded. However, mixes with lower dosages of both pathogens did not cause significantly higher insect mortalities compared to low dosages of the fungus applied alone. The advantages and disadvantages of the simultaneous applications of NPV and M. rileyi aiming at the management of either A. gemmatalis or S. frugiperda were discussed.

Enhancement of Bacillus thuringiensis Cry1Ab and Cry1Fa Toxicity to Spodoptera frugiperda by Domain III Mutations Indicates There Are Two Limiting Steps in Toxicity as Defined by Receptor Binding and Protein Stability.

Bacillus thuringiensis Cry1Ab and Cry1Fa toxins are environmentally safe insecticides that control important insect pests. Spodoptera frugiperda is an important maize pest that shows low susceptibility to Cry1A toxins, in contrast to Cry1Fa, which is highly active against this pest and is used in transgenic maize for S. frugiperda control. The ß16 region from domain III of Cry1Ab has been shown to be involved in interactions with receptors such as alkaline phosphatase (ALP) or aminopeptidase (APN) in different lepidopteran insects. Alanine-scanning mutagenesis of amino acids of Cry1Ab ß16 (509STLRVN514) revealed that certain ß16 mutations, such as N514A, resulted in increased toxicity of Cry1Ab for S. frugiperda without affecting the toxicity for other lepidopteran larvae, such as Manduca sexta larvae. Exhaustive mutagenesis of N514 was performed, showing that the Cry1Ab N514F, N514H, N514K, N514L, N514Q, and N514S mutations increased the toxicity toward S. frugiperda A corresponding mutation was constructed in Cry1Fa (N507A). Toxicity assays of wild-type and mutant toxins (Cry1Ab, Cry1AbN514A, Cry1AbN514F, Cry1Fa, and Cry1FaN507A) against four S. frugiperda populations from Mexico and one from Brazil revealed that Cry1AbN514A and Cry1FaN507A consistently showed 3- to 18-fold increased toxicity against four of five S. frugiperda populations. In contrast, Cry1AbN514F showed increased toxicity in only two of the S. frugiperda populations analyzed. The mutants Cry1AbN514A and Cry1AbN514F showed greater stability to midgut protease treatment. In addition, binding analysis of the Cry1Ab mutants showed that the increased toxicity correlated with increased binding to brush border membrane vesicles and increased binding affinity for S. frugiperda ALP, APN, and cadherin receptors.IMPORTANCESpodoptera frugiperda is the main maize pest in South and North America and also is an invasive pest in different African countries. However, it is poorly controlled by Bacillus thuringiensis Cry1A toxins expressed in transgenic crops, which effectively control other lepidopteran pests. In contrast, maize expressing Cry1Fa is effective in the control of S. frugiperda, although its effectiveness is being lost due to resistance evolution. Some of the Cry1Ab domain III mutants characterized here show enhanced toxicity for S. frugiperda without loss of toxicity to Manduca sexta Thus, these Cry1Ab mutants could provide useful engineered toxins that, along with other Cry toxins, would be useful for developing transgenic maize expressing stacked proteins for the effective control of S. frugiperda and other lepidopteran pests in the field.

Spodoptera frugiperda (J. E. Smith) Aminopeptidase N1 Is a Functional Receptor of the Bacillus thuringiensis Cry1Ca Toxin.

Bacillus thuringiensis Cry1Ca is toxic to different Spodoptera species. The aims of this work were to identify the Cry1Ca-binding proteins in S. frugiperda, to provide evidence on their participation in toxicity, and to identify the Cry1Ca amino acid residues involved in receptor binding. Pulldown assays using Spodoptera frugiperda brush border membrane vesicles (BBMV) identified aminopeptidase N (APN), APN1, and APN2 isoforms as Cry1Ca-binding proteins. Cry1Ca alanine substitutions in all residues of domain III ß16 were characterized. Two ß16 nontoxic mutants (V505A and S506A) showed a correlative defect on binding to the recombinant S. frugiperda APN1 (SfAPN1). Finally, silencing the expression of APN1 transcript, by double-stranded RNA (dsRNA) feeding, showed that silenced larvae are more tolerant of the Cry1Ca toxin, which induced less than 40% mortality in silenced larvae whereas nonsilenced larvae had 100% mortality. Overall, our results show that Cry1Ca relies on APN1 binding through domain III ß16 to impart toxicity to S. frugiperdaIMPORTANCEBacillus thuringiensis Cry toxins rely on receptor binding to exert toxicity. Cry1Ca is toxic to different populations of S. frugiperda, a major corn pest in America. Nevertheless, the S. frugiperda midgut proteins that are involved in Cry1Ca toxicity have not been identified. Here we identified aminopeptidase N1 (APN1) as a functional receptor of Cry1Ca. Moreover, we showed that Cry1Ca domain III ß16 is involved in APN1 binding. These results give insights on potential target sites for improving Cry1Ca toxicity to S. frugiperda.

Comparative molecular analyses of invasive fall armyworm in Togo reveal strong similarities to populations from the eastern United States and the Greater Antilles.

The fall armyworm (Spodoptera frugiperda, J.E. Smith) is a noctuid moth that is a major and ubiquitous agricultural pest in the Western Hemisphere. Infestations have recently been identified in several locations in Africa, indicating its establishment in the Eastern Hemisphere where it poses an immediate and significant economic threat. Genetic methods were used to characterize noctuid specimens infesting multiple cornfields in the African nation of Togo that were tentatively identified as fall armyworm by morphological criteria. Species identification was confirmed by DNA barcoding and the specimens were found to be primarily of the subgroup that preferentially infests corn and sorghum in the Western Hemisphere. The mitochondrial haplotype configuration was most similar to that found in the Caribbean region and the eastern coast of the United States, identifying these populations as the likely originating source of the Togo infestations. A genetic marker linked with resistance to the Cry1Fa toxin from Bacillus thuringiensis (Bt) expressed in transgenic corn and common in Puerto Rico fall armyworm populations was not found in the Togo collections. These observations demonstrate the usefulness of genetic surveys to characterize fall armyworm populations from Africa.

Comparative analysis of the genetic basis of Cry1F resistance in two strains of Spodoptera frugiperda originated from Puerto Rico and Florida.

The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a major target pest of Bacillus thuringiensis (Bt) maize and cotton in America. Since the commercialization of Cry1F maize (event TC1507) in 2003, resistance to Cry1F maize in field populations of S. frugiperda has occurred in Puerto Rico, Brazil and the southeast region of the United States. In this paper, we conducted a comparative analysis of the inheritance of two Cry1F-resistant colonies of S. frugiperda originated from Puerto Rico (PR) and Florida (FL), respectively. The objective of the analysis was to determine if the genetic basis of the resistance was similar in the two different originated colonies. To accomplish the objective, besides PR, FL, and a known Cry1F-susceptible colony, 14 additional colonies were developed by reciprocal crosses among the three parents, F1 by F1 crosses, backcrosses, and intercolony-crosses between PR and FL. Larval mortalities of the 17 colonies were assayed on both Cry1F maize leaf tissue and Cry1F-treated diet at the concentrations of 3.16, 10.00, and 31.60µg/g. Resistance to Cry1F in both PR and FL was autosomal and recessive or incompletely recessive. Segregations in F2 and backcrossed generations associated with FL fitted the Mendelian monogenic model well, while with PR the segregations did not follow the single gene model in some bioassays. Further analyses with the intercolony complementation tests showed a similar level of resistance in the F1 progeny as their parents FL and PR. Together with the data, it was likely that a single (or a few tightly-linked) gene was involved in FL; PR shared the same locus of the major resistance gene as FL, but the resistance in PR might also be associated with additional minor factors. Information generated from this study should be useful in understanding the origin of Cry1F resistance in the U.S. mainland and developing effective strategies for Bt resistance management in S. frugiperda.

The gut microbiota of insecticide-resistant insects houses insecticide-degrading bacteria: A potential source for biotechnological exploitation.

The exploration of new niches for microorganisms capable of degrading recalcitrant molecules is still required. We hypothesized the gut microbiota associated with insect-resistant lines carry pesticide degrading bacteria, and predicted they carry bacteria selected to degrade pesticides they were resistant to. We isolated and accessed the pesticide-degrading capacity of gut bacteria from the gut of fifth instars of Spodoptera frugiperda strains resistant to lambda-cyhalothrin, deltamethrin, chlorpyrifos ethyl, spinosad and lufenuron, using insecticide-selective media. Sixteen isolates belonging to 10 phylotypes were obtained, from which four were also associated with the susceptible strain. However, growth of gut bacteria associated with larvae from the susceptible strain was not obtained in any of the insecticide-based selective media tested. Growth of isolates was affected by the concentration of insecticides in the media, and all grew well up to 40 µg/ml. The insecticide-degrading capacity of selected isolates was assessed by GC or LC-MS/MS analyses. In conclusion, resistant strains of S. frugiperda are an excellent reservoir of insecticide-degrading bacteria with bioremediation potential. Moreover, gut-associated bacteria are subjected to the selection pressure imposed by insecticides on their hosts and may influence the metabolization of pesticides in insects.

Transient endophytic colonizations of plants improve the outcome of foliar applications of mycoinsecticides against chewing insects.

The current work reports how spray application of entomopathogenic fungi on alfalfa, tomato and melon plants may cause an additional Spodoptera littoralis larvae mortality due to a temporal colonization of the leaves and subsequent ingestion of those leaves by the larvae. Most entomopathogenic fungi (EF) (Ascomycota: Hypocreales) endophytes seem to colonize their host plants in a non-systemic pattern, in which case at least a transient endophytic establishment of the fungus should be expected in treated areas after spray application. In this work, all strains were able to endophytically colonize roots, stems and leaves during the first 96h after inoculation. Whilst the treatment of S. littoralis larvae with a 10(8)ml(-1) conidial suspension resulted in moderate to high mortality rates for the Metarhizium brunneum EAMb 09/01-Su (41.7-50.0%) and Beauveria bassiana EABb 01/33-Su (66.7-76.6%) strains, respectively, an additive effect was detected when these larvae were also fed endophytically colonized alfalfa, tomato, and melon leaves, with mortality rates varying from 25.0% to 46.7% as a function of the host plant and total mortality rates in the combined treatment of 75-80% and 33-60% for B. bassiana and M. brunneum, respectively. Fungal outgrowth was not detected in any of the dead larvae feeding on colonized leaves, whereas traces of destruxin A were detected in 11% of the insects fed tomato discs endophytically colonized by M. brunneum. The combined effects of the fungal spray with the mortality caused by the feeding of insects on transient EF-colonized leaves have to be considered to estimate the real acute impact of field sprays with entomopathogenic fungi on chewing insects.

Cross-Resistance between Cry1 Proteins in Fall Armyworm (Spodoptera frugiperda) May Affect the Durability of Current Pyramided Bt Maize Hybrids in Brazil.

Genetically modified plants expressing insecticidal proteins from Bacillus thuringiensis (Bt) offer valuable options for managing insect pests with considerable environmental and economic benefits. Despite the benefits provided by Bt crops, the continuous expression of these insecticidal proteins imposes strong selection for resistance in target pest populations. Bt maize (Zea mays) hybrids have been successful in controlling fall armyworm (Spodoptera frugiperda), the main maize pest in Brazil since 2008; however, field-evolved resistance to the protein Cry1F has recently been reported. Therefore it is important to assess the possibility of cross-resistance between Cry1F and other Cry proteins expressed in Bt maize hybrids. In this study, an F2 screen followed by subsequent selection on MON 89034 maize was used to select an S. frugiperda strain (RR) able to survive on the Bt maize event MON 89034, which expresses the Cry1A.105 and Cry2Ab2 proteins. Field-collected insects from maize expressing the Cry1F protein (event TC1507) represented most of the positive (resistance allele-containing) (iso)families found. The RR strain showed high levels of resistance to Cry1F, which apparently also conferred high levels of cross resistance to Cry1A.105 and Cry1Ab, but had only low-level (10-fold) resistance to Cry2Ab2. Life history studies to investigate fitness costs associated with the resistance in RR strain revealed only small reductions in reproductive rate when compared to susceptible and heterozygous strains, but the RR strain produced 32.2% and 28.4% fewer females from each female relative to the SS and RS (pooled) strains, respectively. Consistent with the lack of significant resistance to Cry2Ab2, MON 89034 maize in combination with appropriate management practices continues to provide effective control of S. frugiperda in Brazil. Nevertheless, the occurrence of Cry1F resistance in S. frugiperda across Brazil, and the cross-resistance to Cry1Ab and Cry1A.105, indicates that current Cry1-based maize hybrids face a challenge in managing S. frugiperda in Brazil and highlights the importance of effective insect resistance management for these technologies.

Conidial vigor vs. viability as predictors of virulence of entomopathogenic fungi.

We tested the hypothesis that debilitated conidia exhibiting slow-germination (requiring>16h to germinate) are less virulent than vigorous conidia exhibiting fast germination (requiring⩽16h to germinate). Preparations of Beauveria bassiana s.l. strain CG 1027 with variable ratios of vigorous to debilitated conidia were assayed against third-instar larvae of Spodoptera frugiperda. As the proportion of debilitated conidia in test preparations increased, LC50 expressed in terms of total viable conidia increased, while LC50 expressed solely in terms of vigorous conidia remained constant, indicating that vigorous conidia were responsible for nearly all mortality observed in the assays. Larvae treated with conidia from low-quality batches (with high proportions of debilitated conidia) survived consistently longer than those treated with comparable doses of conidia from high-quality batches. These results confirm our previous hypotheses that inclusion of debilitated conidia in viability assessments can lead to overestimation of the quality (potency) of mycoinsecticide preparations and support our recommendation for use of short incubation periods for assessing viability whenever viability is relied upon as an indicator of product quality.

Status of resistance to Bt maize in Spodoptera frugiperda: lessons from Puerto Rico.

In 2006, reports of potential Spodoptera frugiperda resistance to TC1507 maize in Puerto Rico were received. Subsequent investigation confirmed that pest populations collected from several sites in Puerto Rico were largely unaffected by the Cry1F protein in bioassays, with resistance ratios likely in excess of 1000. Since then, we have continued monitoring populations in Puerto Rico and in southern areas of the mainland US. The majority of the collections from Puerto Rico continue to show high levels of Cry1F resistance whereas populations collected from the southern US mainland continue to show full susceptibility to Cry1F and TC1507 maize. It does not appear that resistant populations have spread to any measurable extent from Puerto Rico to mainland US, nor that local selection pressure from Cry1F-expressing maize or cotton production in the southern US has caused a measurable change in population susceptibility. Lessons learned from Puerto Rico are being applied in other parts of the Americas where TC1507 maize is grown and additional steps being taken to protect the long-term durability of Cry1F in maize in areas where similar selection pressure may be expected. Tactics include using locally-adapted germplasm that contain native Spodoptera resistance, a robust education program to teach end-users about the potential for resistance to develop appropriate crop stewardship, resistance monitoring, and the use of insecticides under high S. frugiperda pressure. Perhaps most importantly, pyramided trait products that produce two or more different Bt proteins are being introduced to further delay resistance development to Cry1F.

Small microcapsules of crystal proteins and spores of Bacillus thuringiensis by an emulsification/internal gelation method.

This paper describes a microencapsulation process of a spore crystal aggregate produced by Bacillus thuringiensis var. kurstaki HD-1. The methodology is based on the emulsification/internal gelation method, and was implemented to produce microcapsules of small diameter (< 10 µm) with the capacity to protect the spore crystal aggregate from extreme ultraviolet radiation. The diameter of microcapsules was in the range of 3.1 ± 0.2-6.8 ± 0.4 µm, which is considered adequate for biological control purposes. The protective effect of the alginate coat was verified by the remaining 60 ± 2% and 40 ± 1% of spore viability and protein activity, respectively, after UV-B radiation of 236 J, and with bioassays with Spodoptera frugiperda. It is expected that the protective effect of the alginate coat will improve the effectiveness of the Bt-HD1 formulated as small diameter microcapsules, and their yield, once they are released into the environment, will also be improved.

RNAi-mediated knockdown of a Spodoptera frugiperda trypsin-like serine-protease gene reduces susceptibility to a Bacillus thuringiensis Cry1Ca1 protoxin.

SfT6 has been identified in a subtracted cDNA library of Spodoptera frugiperda larval midgut transcripts as a serine-protease gene downregulated within 24 h of intoxication with Bacillus thuringiensis Cry1Ca1 protein. In the present study, the specific role of SfT6 during Cry1Ca1 intoxication was investigated by RT-PCR and in vivo RNA interference. Quantitative real-time RT-PCR analysis showed SfT6 mRNA levels in the midgut tissue were significantly reduced after injecting or feeding 4th-instar larvae with specific long-size dsRNA. Gut juice-mediated in vitro protoxin activation and susceptibility for Cry1Ca1 were investigated in Sft6-knockdown larvae and compared with control treated with nonspecific dsRNA. Our results demonstrate SfT6 plays a determinant role in Cry1Ca1 toxicity against S. frugiperda since a decreased expression caused a reduced protoxin activation by larval gut juice and reduced susceptibility of insects to toxin in bioassays. We propose SfT6 downregulation occurring at the early stages of Cry1Ca1 intoxication is part of a complex and multifaceted defensive mechanism triggered in the insect gut to withstand B. thuringiensis pathogenesis.

Colonization of rice and Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) larvae by genetically modified endophytic Methylobacterium mesophilicum.

The colonization of Spodoptera frugiperda J.E. Smith larvae and rice seedlings by genetically modified endophytic bacterium Methylobacterium mesophilicum, and also the possible transfer of this bacterium to inside the larva's body during seedlings consumption were studied. The data obtained by bacterial reisolation and fluorescence microscopy showed that the bacterium colonized the rice seedlings, the larva's body and that the endophytic bacteria present in seedlings could be acquired by the larvae. In that way, the transference of endophytic bacterium from plants to insect can be a new and important strategy to insect control using engineered microorganisms.

Colonization of rice and Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) larvae by genetically modified endophytic Methylobacterium mesophilicum

The colonization of Spodoptera frugiperda J.E. Smith larvae and rice seedlings by genetically modified endophytic bacterium Methylobacterium mesophilicum, and also the possible transfer of this bacterium to inside the larva's body during seedlings consumption were studied. The data obtained by bacterial reisolation and fluorescence microscopy showed that the bacterium colonized the rice seedlings, the larva's body and that the endophytic bacteria present in seedlings could be acquired by the larvae. In that way, the transference of endophytic bacterium from plants to insect can be a new and important strategy to insect control using engineered microorganisms.

Histopatologia da interação de Bacillus thuringiensis e extratos vegetais no intestino médio de Spodoptera frugiperda (Lepidoptera: Noctuidae) / Histopathology of the interaction of Bacillus thuringiensis and plant extracts in the midgut of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Plantas são fontes naturais de substâncias inseticidas, já que podem ser produzidas pelo vegetal em resposta a ataques de insetos podendo representar uma alternativa no manejo de insetos-praga, como Spodoptera frugiperda. Dessa forma, o presente trabalho objetivou a análise histopatológica do intestino médio de lagartas de S. frugiperda, após a ingestão dos extratos obtidos por maceração e infusão de Petiveria alliacea. Zingiber officinale. Cymbopogon citratus. Malva silvestris. Baccharis genistelloides e Ruta graveolens, assim como a associação desses extratos com Bacillus thuringiensis aizawai. As lagartas foram tratadas in vivo com cada extrato e a bactéria, mais a associação desses extratos com B. thuringiensis e após, uma reação de cinética entre 3 e 27 horas, foram fixadas para o preparo de cortes histológicos, os quais foram corados e analisados comparativamente às testemunhas em microscopia óptica. Os resultados mostraram mudanças na histologia do intestino médio das lagartas de S. frugiperda, 3 horas após a aplicação dos tratamentos à base de alliacea. Z. officinale. C. citratus e M. silvestris, enquanto que para B. genistelloides e R. graveolens só foram observados alterações após 6h. Na interação dos extratos com B. thuringiensis observou-se alterações nas microvilosidades, desorganização do intestino médio e a hipertrofia das células epiteliais que projetaram-se para o lúmen. Os resultados desse trabalho mostram que o efeito histopatológico de Z. officinale. M. silvestris. R. graveolens e B. genistelloides, foram mais ativos quando comparados aos extratos de P. alliacea e C. citratus, os quais apresentaram uma interação positiva com B. thuringiensis.

The natural insecticidal substances produced by plants in response to insect attack may represent an alternative for the management of pest insects such as Spodoptera frugiperda. This study was therefore aimed to histopathologically analyze the midgut of larvae of S. frugiperda, after ingestion of extracts obtained by maceration and infusion of Petiveria alliacea. Zingiber officinale. Cymbopogon citratus. Malva silvestris. Baccharis genistelloides and Ruta graveolens, and the association of these extracts with Bacillus thuringiensis aizawai. The larvae were treated in vivo with each extract, B. thuringiensis, and the combination of these extracts with the bacteria, and after a reaction kinetics of between 3 to 27 hours they were fixed for the preparation of histological sections, which were stained and analyzed in comparison to the controls with light microscopy. The results showed changes in the histology of the midgut of the larvae of S. frugiperda, 3 hours after application of treatments based on P. alliacea. Z. officinale. C. citratus and M. silvestris, while for B. genistelloides and R. graveolens changes were observed only after 6h. In the interaction of extracts with B. thuringiensis the analysis revealed changes in microvilli, disorganization of the midgut and hypertrophy of epithelial cells that projected into the lumen. The findings show that the histopathological effects of Z. officinale. M. silvestris. R. graveolens and B. genistelloides were more active when compared to extracts of P. alliacea and C. citratus, which showed a positive interaction with B. thuringiensis.