Salvador Herrero

Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges

Emergence of polyphagous herbivorous insects entails significant adaptation to recognize, detoxify and digest a variety of host-plants. Despite of its biological and practical importance - since insects eat 20% of crops - no exhaustive analysis of gene repertoires required for adaptations in generalist insect herbivores has previously been performed. The noctuid moth Spodoptera frugiperda ranks as one of the world’s worst agricultural pests. This insect is polyphagous while the majority of other lepidopteran herbivores are specialist. It consists of two morphologically indistinguishable strains (“C” and “R”) that have different host plant ranges. To describe the evolutionary mechanisms that both enable the emergence of polyphagous herbivory and lead to the shift in the host preference, we analyzed whole genome sequences from laboratory and natural populations of both strains. We observed huge expansions of genes associated with chemosensation and detoxification compared with specialist Lepidoptera. These expansions are largely due to tandem duplication, a possible adaptation mechanism enabling polyphagy. Individuals from natural C and R populations show significant genomic differentiation. We found signatures of positive selection in genes involved in chemoreception, detoxification and digestion, and copy number variation in the two latter gene families, suggesting an adaptive role for structural variation.

Genetic and Biochemical Approach for Characterization of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Field Population of the Diamondback Moth, Plutella xylostella

ABSTRACT Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F4 to F8) were selected with Bacillus thuringiensis subsp.kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F9 found that selection with Cry1Ac, Cry1Ab, B. thuringiensissubsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, andB. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F9. The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, andB. thuringiensis subsp. aizawai(5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-,B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp.aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F9 to F13) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with125I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F15). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F2 progeny from a backcross of F1 progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.

Development and characterization of diamondback moth resistance to transgenic broccoli expressing high levels of Cry1C

ABSTRACT A field-collected colony of the diamondback moth, Plutella xylostella, had 31-fold resistance to Cry1C protoxin ofBacillus thuringiensis. After 24 generations of selection with Cry1C protoxin and transgenic broccoli expressing a Cry1C protein, the resistance that developed was high enough that neonates of the resistant strain could complete their entire life cycle on transgenic broccoli expressing high levels of Cry1C. After 26 generations of selection, the resistance ratios of this strain to Cry1C protoxin were 12,400- and 63,100-fold, respectively, for the neonates and second instars by a leaf dip assay. The resistance remained stable until generation 38 (G38) under continuous selection but decreased to 235-fold at G38 when selection ceased at G28. The Cry1C resistance in this strain was seen to be inherited as an autosomal and incompletely recessive factor or factors when evaluated using a leaf dip assay and recessive when evaluated using Cry1C transgenic broccoli. Saturable binding of 125I-Cry1C was found with brush border membrane vesicles (BBMV) from both susceptible and Cry1C-resistant strains. Significant differences in Cry1C binding to BBMV from the two strains were detected. BBMV from the resistant strain had about sevenfold-lower affinity for Cry1C and threefold-higher binding site concentration than BBMV from the susceptible strain. The overall Cry1C binding affinity was just 2.5-fold higher for BBMV from the susceptible strain than it was for BBMV from the resistant strain. These results suggest that reduced binding is not the major mechanism of resistance to Cry1C.

Different mechanisms of resistance to Bacillus thuringiensis toxins in the indianmeal moth.

ABSTRACT Susceptibility to protoxin and toxin forms of Cry1Ab and the binding of 125I-labeled Cry1Ab and Cry1Ac has been examined in three Plodia interpunctella colonies, one susceptible (688s) and two resistant (198r and Dplr) to Bacillus thuringiensis. Toxicological studies showed that the 198r colony was 11-fold more resistant to Cry1Ab protoxin than to Cry1Ab activated toxin, whereas the Dplr colony was 4-fold more resistant to protoxin versus toxin. Binding results with 125I-labeled toxins indicated the occurrence of two different binding sites for Cry1Ab in the susceptible insects, one of them shared with Cry1Ac. Cry1Ab binding was found to be altered in insects from both resistant colonies, though in different ways. Compared with the susceptible colony, insects from the Dplr colony showed a drastic reduction in binding affinity (60-fold higher Kd), although they had similar concentrations of binding sites. Insects from the 198r colony showed a slight reduction in both binding affinity and binding site concentration (five-fold-higherKd and ca. three-fold-lowerRt compared with the 688s colony). No major difference in Cry1Ac binding was found among the three colonies. The fact that the 198r colony also has a protease-mediated mechanism of resistance (B. Oppert, R. Hammel, J. E. Throne, and K. J. Kramer, J. Biol. Chem. 272:23473–23476, 1997) is in agreement with our toxicological data in which this colony has a different susceptibility to the protoxin and toxin forms of Cry1Ab. It is noteworthy that the three colonies used in this work derived originally from ca. 100 insects, which reflects the high variability and high frequency of B. thuringiensisresistance genes occurring in natural populations.

ABCC transporters mediate insect resistance to multiple Bt toxins revealed by bulk segregant analysis.

BackgroundRelatively recent evidence indicates that ABCC2 transporters play a main role in the mode of action of Bacillus thuringiensis (Bt) Cry1A-type proteins. Mapping of major Cry1A resistance genes has linked resistance to the ABCC2 locus in Heliothis virescens, Plutella xylostella, Trichoplusia ni and Bombyx mori, and mutations in this gene have been found in three of these Bt-resistant strains.ResultsWe have used a colony of Spodoptera exigua (Xen-R) highly resistant to a Bt commercial bioinsecticide to identify regions in the S. exigua genome containing loci for major resistance genes by using bulk segregant analysis (BSA). Results reveal a region containing three genes from the ABCC family (ABBC1, ABBC2 and ABBC3) and a mutation in one of them (ABBC2) as responsible for the resistance of S. exigua to the Bt commercial product and to its key Spodoptera-active ingredients, Cry1Ca. In contrast to all previously described mutations in ABCC2 genes that directly or indirectly affect the extracellular domains of the membrane protein, the ABCC2 mutation found in S. exigua affects an intracellular domain involved in ATP binding. Functional analyses of ABBC2 and ABBC3 support the role of both proteins in the mode of action of Bt toxins in S. exigua. Partial silencing of these genes with dsRNA decreased the susceptibility of wild type larvae to both Cry1Ac and Cry1Ca. In addition, reduction of ABBC2 and ABBC3 expression negatively affected some fitness components and induced up-regulation of arylphorin and repat5, genes that respond to Bt intoxication and that are found constitutively up-regulated in the Xen-R strain.ConclusionsThe current results show the involvement of different members of the ABCC family in the mode of action of B. thuringiensis proteins and expand the role of the ABCC2 transporter in B. thuringiensis resistance beyond the Cry1A family of proteins to include Cry1Ca.

The transcriptome of Spodoptera exigua larvae exposed to different types of microbes

We have obtained and characterized the transcriptome of Spodoptera exigua larvae with special emphasis on pathogen-induced genes. In order to obtain a highly representative transcriptome, we have pooled RNA from diverse insect colonies, conditions and tissues. Sequenced cDNA included samples from 3 geographically different colonies. Enrichment of RNA from pathogen-related genes was accomplished by exposing larvae to different pathogenic and non-pathogenic microbial agents such as the bacteria Bacillus thuringiensis, Micrococcus luteus, and Escherichia coli, the yeast Saccharomyces cerevisiae, and the S. exigua nucleopolyhedrovirus (SeMNPV). In addition, to avoid the loss of tissue-specific genes we included cDNA from the midgut, fat body, hemocytes and integument derived from pathogen exposed insects. RNA obtained from the different types of samples was pooled, normalized and sequenced. Analysis of the sequences obtained using the Roche 454 FLX and Sanger methods has allowed the generation of the largest public set of ESTs from S. exigua, including a large group of immune genes, and the identification of an important number of SSR (simple sequence repeats) and SNVs (single nucleotide variants: SNPs and INDELs) with potential use as genetic markers. Moreover, data mining has allowed the discovery of novel RNA viruses with potential influence in the insect population dynamics and the larval interactions with the microbial pesticides that are currently in use for the biological control of this pest.

Constitutive activation of the midgut response to Bacillus thuringiensis in Bt-resistant Spodoptera exigua.

Bacillus thuringiensis is the most effective microbial control agent for controlling numerous species from different insect orders. The main threat for the long term use of B. thuringiensis in pest control is the ability of insects to develop resistance. Thus, the identification of insect genes involved in conferring resistance is of paramount importance. A colony of Spodoptera exigua (Lepidoptera: Noctuidae) was selected for 15 years in the laboratory for resistance to Xentari™, a B. thuringiensis-based insecticide, reaching a final resistance level of greater than 1,000-fold. Around 600 midgut ESTs were analyzed by DNA-macroarray in order to find differences in midgut gene expression between susceptible and resistant insects. Among the differentially expressed genes, repat and arylphorin were identified and their increased expression was correlated with B. thuringiensis resistance. We also found overlap among genes that were constitutively over-expressed in resistant insects with genes that were up-regulated in susceptible insects after exposure to Xentari™, suggesting a permanent activation of the response to Xentari™ in resistant insects. Increased aminopeptidase activity in the lumen of resistant insects in the absence of exposure to Xentari™ corroborated the hypothesis of permanent activation of response genes. Increase in midgut proliferation has been proposed as a mechanism of response to pathogens in the adult from several insect species. Analysis of S. exigua larvae revealed that midgut proliferation was neither increased in resistant insects nor induced by exposure of susceptible larvae to Xentari™, suggesting that mechanisms other than midgut proliferation are involved in the response to B. thuringiensis by S. exigua larvae.

Increase in midgut microbiota load induces an apparent immune priming and increases tolerance to Bacillus thuringiensis.

The insect immune system is comprised of both humoral and cellular components that are mobilized in response to parasitic or pathogenic infections. Activation of the immune response implies a considerable expenditure of energy and that is why insects rely on inducible pathways that are activated after coming into contact with the pathogenic agent. Known as immune priming, insects can prolong the activation of the immune response and transmit their immune status to the next generation. Starting from a laboratory colony of the lepidopteran Spodoptera exigua and using the lytic zone assay as a measure of the immune status, we selected for a sub-colony with high levels of immune activity in the absence of external challenging with bacteria. Immune-activated insect showed characteristics that are typical reported for immune primed insects, such as increased tolerance to pathogens (Bacillus thuringiensis in our case), fitness-cost associated to the immune status, and maternal transmission of the immune status. However, additional analysis revealed that the selection for the immune-activated insects was based on the selection of insects carrying a higher bacterial load in the midgut. Our results suggest that activation of the immune system in S. exigua may not only occur as consequence of the immune priming but also from an increase in midgut microbiota load.

Variation in susceptibility to Bacillus thuringiensis toxins among unselected strains of Plutella xylostella

ABSTRACT So far, the only insect that has evolved resistance in the field toBacillus thuringiensis toxins is the diamondback moth (Plutella xylostella). Documentation and analysis of resistant strains rely on comparisons with laboratory strains that have not been exposed to B. thuringiensis toxins. Previously published reports show considerable variation among laboratories in responses of unselected laboratory strains to B. thuringiensis toxins. Because different laboratories have used different unselected strains, such variation could be caused by differences in bioassay methods among laboratories, genetic differences among unselected strains, or both. Here we tested three unselected strains against five B. thuringiensis toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and Cry1Da) using two bioassay methods. Tests of the LAB-V strain from The Netherlands in different laboratories using different bioassay methods yielded only minor differences in results. In contrast, side-by-side comparisons revealed major genetic differences in susceptibility between strains. Compared with the LAB-V strain, the ROTH strain from England was 17- to 170-fold more susceptible to Cry1Aa and Cry1Ac, respectively, whereas the LAB-PS strain from Hawaii was 8-fold more susceptible to Cry1Ab and 13-fold more susceptible to Cry1Da and did not differ significantly from the LAB-V strain in response to Cry1Aa, Cry1Ac, or Cry1Ca. The relative potencies of toxins were similar among LAB-V, ROTH, and LAB-PS, with Cry1Ab and Cry1Ac being most toxic and Cry1Da being least toxic. Therefore, before choosing a standard reference strain upon which to base comparisons, it is highly advisable to perform an analysis of variation in susceptibility among field and laboratory populations.

Downregulation of a Chitin Deacetylase-Like Protein in Response to Baculovirus Infection and Its Application for Improving Baculovirus Infectivity

ABSTRACT Several expressed sequence tags (ESTs) with homology to chitin deacetylase-like protein (CDA) were selected from a group of Helicoverpa armigera genes whose expression changed after infection with H. armigera single nucleopolyhedrovirus (HearNPV). Some of these ESTs coded for a midgut protein containing a chitin deacetylase domain (CDAD). The expressed protein, HaCDA5a, did not show chitin deacetylase activity, but it showed a strong affinity for binding to chitin. Sequence analysis showed the lack of any chitin binding domain, described for all currently known peritrophic membrane (PM) proteins. HaCDA5a has previously been detected in the H. armigera PM. Such localization, together with its downregulation after pathogen infection, led us to hypothesize that this protein might be responsible for the homeostasis of the PM structure and that, by reduction of its expression, the insect may reduce PM permeability, decreasing the entrance of baculovirus. To test this hypothesis, we constructed a recombinant nucleopolyhedrovirus to express HaCDA5a in insect cells and tested its influence on PM permeability as well as the influence of HaCDA5a expression on the performance of the baculovirus. The experiments showed that HaCDA5a increased PM permeability, in a concentration-dependent manner. Bioassays on Spodoptera frugiperda and Spodoptera exigua larvae revealed that NPV expressing HaCDA5a was more infective than its parental virus. However, no difference in virulence was observed when the viruses were injected intrahemocoelically. These findings support the downregulation of a midgut-specific CDA-like protein as a possible mechanism used by H. armigera to reduce susceptibility to baculovirus by decreasing PM permeability.