Yolanda Bel

Environmental Distribution and Diversity of Bacillus thuringiensis in Spain

Bacillus thuringiensis was isolated from 301 out of 1,005 samples collected in Spain from agricultural and non-cultivated soils, dust from stored products, and dead insects. Based on the production of parasporal crystals, 1,401 isolates were identified as B. thuringiensis after examining 11,982 B. thuringiensis-like colonies. We found a greater presence of B. thuringiensis in dust from grain storages than in other habitats. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the spore-crystal mixtures revealed diverse populations of B. thuringiensis which were differentiated in at least 92 distinct protein profiles. Serological identification also showed great diversity among the Spanish isolates which were distributed among 38 of the 58 known serovars. The most frequently found serovars were aizawai, kurstaki, konkukian, morrisoni, and thuringiensis, which together represented more than 50% of the serotyped isolates. In preliminary toxicity assays, a number of isolates were found to show significant insecticidal activity against the lepidopterans Heliothis armigera (76.1% of the assayed isolates), Spodoptera exigua (50.5%), and Plutella xylostella (19.7%). Thirty five isolates were toxic to both H. armigera and S. exigua, and eight were toxic to S. exigua and P. xylostella. Four and one isolates were toxic to the coleopterans Leptinotarsa decemlineata and Colaspidema atrum, respectively, and three to the dipteran Tipula oleracea. The electrophoretic pattern and serovar of most of the isolates with toxic activity were consistent with those reported in the literature, although other isolates revealed unusual protein profiles, were assigned to new H serovars, or were included in H serovars not previously reported within such pathotypes.

Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria

SUMMARY Entomopathogenic bacteria produce insecticidal proteins that accumulate in inclusion bodies or parasporal crystals (such as the Cry and Cyt proteins) as well as insecticidal proteins that are secreted into the culture medium. Among the latter are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the membrane of the insect midgut, and the Vip2 component enters the cell, where it displays its ADP-ribosyltransferase activity against actin, preventing microfilament formation. Vip3 has no sequence similarity to Vip1 or Vip2 and is toxic to a wide variety of members of the Lepidoptera. Its mode of action has been shown to resemble that of the Cry proteins in terms of proteolytic activation, binding to the midgut epithelial membrane, and pore formation, although Vip3A proteins do not share binding sites with Cry proteins. The latter property makes them good candidates to be combined with Cry proteins in transgenic plants (Bacillus thuringiensis-treated crops [Bt crops]) to prevent or delay insect resistance and to broaden the insecticidal spectrum. There are commercially grown varieties of Bt cotton and Bt maize that express the Vip3Aa protein in combination with Cry proteins. For the most recently reported Vip4 family, no target insects have been found yet.

Distribution of cryl, cryll and cryV Genes within Bacillus thuringiensis Isolates from Spain

Summary Using a PCR-based approach, a collection of 223 isolates of Bacillus thuringiensis from Spain was screened for the presence of cry genes belonging to three families. Genes from the cryI , cryII and cryV families were found in 54%, 42%, and 66% of the isolates, respectively. Only 23% of the isolates did not show the presence of any of the genes tested. Frequencies of these genes were compared in isolates from soil samples and from samples of cereal stores and mills, being this higher in the latter. Specific primers were used to detect cryIA(a), cryIA(b), cryIA(c), cryIA(d), cryIA(e), cryIB, cryIC, cryID, cryIE, cryIF and cryIG genes. Within the cryI family, the most frequent gene was cryIA(c) (62%), followed by cryIA(a), cryID, cryIC and cryIA(b) (49, 43, 35, and 34%, respectively). A high frequency of joint occurrence was observed for cryIC and cryID ; the latter was present in 93% of the isolates containing cryIC . A random sample of 97 isolates was tested for toxicity against the insect pests Plutella xylostella and Spodoptera exigua . Among the isolates showing toxicity, the most common gene combination was cryIA-cryIC-cryID-cryII-cryV . Although in most cases toxicity could be related to gene content, in some others toxicity was unexpected according to the results obtained by PCR. We found no apparent relationship between gene content in our isolates and the serovar to which they belong.

Study of the aminopeptidase N gene family in the lepidopterans Ostrinia nubilalis (Hübner) and Bombyx mori (L.): Sequences, mapping and expression

Aminopeptidases N (APNs) are a class of ectoenzymes present in lepidopteran larvae midguts, involved in the Bacillus thuringiensis (Bt) toxins mode of action. In the present work, seven aminopeptidases have been cloned from the midgut of Ostrinia nubilalis, the major Lepidopteran corn pest in the temperate climates. Six sequences were identified as APNs because of the presence of the HEXXH(X)18E and GAMEN motifs, as well as the signal peptide and the GPI-anchor sequences. The remaining sequence did not contain the two cellular targeting signals, indicating it belonged to the puromycin-sensitive aminopeptidase (PSA) family. An in silico analysis allowed us to find orthologous sequences in Bombyx mori. A phylogenetic study of lepidopteran aminopeptidase sequences resulted in their clustering into nine classes. Linkage analysis revealed that the onapn genes as well as all bmapn genes clustered in a single linkage group. O. nubilalis aminopeptidases were expressed in all larval instars. In 5th instar larvae tissues, apns transcripts were found mainly in midguts while apn8 was also highly expressed in Malpighian tubules, and psa showed an ubiquitous expression pattern in O. nubilalis and B. mori. The sequence homology and gene organization of apns suggest a single origin from an ancestral lepidopteran apn gene.

Distribution, frequency and diversity of Bacillus thuringiensis in olive tree environments in Spain

Summary Bacillus thuringiensis was isolated from samples collected from olive tree related habitats (olive groves, olive storage facilities and oil mills) of different olive producing regions in Spain. This bacterium was found in a high percentage (92%) of samples. From 72 samples analysed, 2244 sporulating colonies were selected and observed under phase contrast microscopy. From these, 414 colonies (18%) were classified as B. thuringiensis based on the production of parasporal crystals. A great variability of spore, crystal size and morphology was observed. The results indicate that olive groves are as rich as soil olive mills and olive storage facilities regarding the B. thuringiensis population. Analysis by SDS-PAGE of the proteins in the parasporal crystal showed a considerable heterogeneity between B. thuringiensis strains. This was especially relevant in samples obtained from oil mills and storage centres, indicating the high polymorphism in this species. Strains isolated from the same sample showing the same SDS-PAGE profile were not considered different, and just one of them was selected for further tests. A total of 188 out of the 414 B. thuringiensis isolates were shown to be different strains and tested for their toxicity against adult animals of the olive fly Bactrocera oleae . Approximately 40% of the strains caused similar or higher mortality than the dipteran active strain B. thuringiensis israelensis (45% mortality). Most strains (71%) producing a toxicity higher than 65% came from olive mills and olive storage facilities. Three of these strains exhibited a mortality higher than 70% in our test conditions.

A screening of five Bacillus thuringiensis Vip3A proteins for their activity against lepidopteran pests

Five Bacillus thuringiensis Vip3A proteins (Vip3Aa, Vip3Ab, Vip3Ad, Vip3Ae and Vip3Af) and their corresponding trypsin-activated toxins were tested for their toxicity against eight lepidopteran pests: Agrotis ipsilon, Helicoverpa armigera, Mamestra brassicae, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Ostrinia nubilalis and Lobesia botrana. Toxicity was first tested at a high dose at 7 and 10 days. No major differences were found when comparing protoxins vs. trypsin-activated toxins. The proteins that were active against most of the insect species were Vip3Aa, Vip3Ae and Vip3Af, followed by Vip3Ab. Vip3Ad was non-toxic to any of the species tested. Considering the results by insect species, A. ipsilon, S. frugiperda and S. littoralis were susceptible to Vip3Aa, Vip3Ab, Vip3Ae and Vip3Af; S. exigua was susceptible to Vip3Aa and Vip3Ae, and moderately susceptible to Vip3Ab; M. brassicae and L. botrana were susceptible to Vip3Aa, Vip3Ae and Vip3Af; H. armigera was moderately susceptible to Vip3Aa, Vip3Ae and Vip3Af, and O. nubilalis was tolerant to all Vip3 proteins tested, although it showed some susceptibility to Vip3Af. The results obtained will help to design new combinations of insecticidal protein genes in transgenic crops or in recombinant bacteria for the control of insect pests.

Comprehensive Analysis of Gene Expression Profiles of the Beet Armyworm Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Vip3Aa Toxin

Host-pathogen interactions result in complex relationship, many aspects of which are not completely understood. Vip proteins, which are Bacillus thuringensis (Bt) insecticidal toxins produced during the vegetative stage, are selectively effective against specific insect pests. This new group of Bt proteins represents an interesting alternative to the classical Bt Cry toxins because current data suggests that they do not share the same mode of action. We have designed and developed a genome-wide microarray for the beet armyworm Spodoptera exigua, a serious lepidopteran pest of many agricultural crops, and used it to better understand how lepidopteran larvae respond to the treatment with the insecticidal protein Vip3Aa. With this approach, the goal of our study was to evaluate the changes in gene expression levels caused by treatment with sublethal doses of Vip3Aa (causing 99% growth inhibition) at 8 and 24 h after feeding. Results indicated that the toxin provoked a wide transcriptional response, with 19% of the microarray unigenes responding significantly to treatment. The number of up- and down-regulated unigenes was very similar. The number of genes whose expression was regulated at 8 h was similar to the number of genes whose expression was regulated after 24 h of treatment. The up-regulated sequences were enriched for genes involved in innate immune response and in pathogen response such as antimicrobial peptides (AMPs) and repat genes. The down-regulated sequences were mainly unigenes with homology to genes involved in metabolism. Genes related to the mode of action of Bt Cry proteins were found, in general, to be slightly overexpressed. The present study is the first genome-wide analysis of the response of lepidopteran insects to Vip3Aa intoxication. An insight into the molecular mechanisms and components related to Vip intoxication will allow designing of more effective management strategies for pest control.

Variability in the cadherin gene in an Ostrinia nubilalis strain selected for Cry1Ab resistance

Transgenic corn expressing Cry1Ab (a Bacillus thuringiensis toxin) is highly effective in the control of Ostrinia nubilalis. For its toxic action, Cry1Ab has to bind to specific insect midgut proteins. To date, in three Lepidoptera species resistance to a Cry1A toxin has been conferred by mutations in cadherin, a protein of the Lepidoptera midgut membrane. The implication of cadherin in the resistance of an Ostrinia nubilalis colony (Europe-R) selected with Bacillus thuringiensis Cry1Ab protoxin was investigated. Several major mutations in the cadherin (cdh) gene were found, which introduced premature termination codons and/or large deletions (ranging from 1383 to 1701bp). The contribution of these major mutations to the resistance was analyzed in resistant individuals that survived exposure to a high concentration of Cry1Ab protoxin. The results indicated that the presence of major mutations was drastically reduced in individuals that survived exposure. Previous inheritance experiments with the Europe-R strain indicated the involvement of more than one genetic locus and reduced amounts of the cadherin receptor. The results of the present work support a polygenic inheritance of resistance in the Europe-R strain, in which mutations in the cdh gene would contribute to resistance by means of an additive effect.

Characterization of Bacillus thuringiensis ser. balearica (Serotype H48) and ser. navarrensis (Serotype H50): Two Novel Serovars Isolated in Spain

Abstract. The novel strains of Bacillus thuringiensis PM9 and NA69, isolated from soil samples in Spain, were classified and characterized in terms of their crystal proteins, plasmid profile, cry genes content, and their toxicological properties against several species of Lepidoptera, Coleoptera, and Diptera. Both strains share morphological and biochemical characteristics with previously described B. thuringiensis strains, although their unique H antigens identify them as two new serotypes. Two new serovar names, B. thuringiensis serovar balearica (H serotype 48) and B. thuringiensis serovar navarrensis (H serotype 50) are proposed for the type strains PM9 and NA69, respectively.

Candidate target mechanisms of the growth inhibitor cyromazine: studies of phenylalanine hydroxylase, puparial amino acids, and dihydrofolate reductase in dipteran insects.

Cyromazine, an insect growth regulator, affects larval and pupal cuticles in dipterans and some other insects. The mode of action of this aminotriazine is not known yet, though it has been shown not to inhibit the synthesis of chitin and cuticular proteins. Cyromazine may, however, act on some step(s) of sclerotization of the cuticle. In the present study, we have analyzed the key enzyme for the production of sclerotization agents, phenylalanine hydroxylase (PAH), using the enzyme from Drosophila, a cyromazine-sensitive insect. PAH was studied in vitro with cyromazine and three biologically less active derivatives at concentrations ranging from 1 microM to 1 mM. None of the compounds did significantly affect PAH activity. Nor did cyromazine, fed to last instar larvae of Musca domestica, change the relative content of phenylalanine and tyrosine, or the total amount and profile of amino acids of puparial cuticles, which showed a larviform shape typical for cyromazine intoxication. Taken together, this study does not support the hypothesis that phenylalanine hydroxylase represents a target site of cyromazine. In additional studies, the conflicting results, as reported by others, on in vitro inhibition of dihydrofolate reductase (DHFR) by cyromazine were re-examined using the enzymes from larvae of the blowfly, Protophormia terraenovae, and from hen liver. There was no significant inhibition of either DHFR at 100 microM by cyromazine as well as by dicylanil, a pyrimidine analog that is biologically more active than cyromazine. In conclusion, the mode of action of cyromazine remains completely open.