Four Bacillus thuringiensis ␦-endotoxins, Cry3A, Cry4Aa, Cry11Aa, and Cyt1Aa, were found to exhibit low to moderate toxicity on the pea aphid, Acyrthosiphon pisum, in terms both of mortality and growth rate. Cry1Ab was essentially nontoxic except at high rates. To demonstrate these effects, we had to use exhaustive bufferbased controls.

Some specialist insects feed on plants rich in secondary compounds, which pose a major selective pressure on both the phytophagous and the gut microbiota. However, microbial communities of toxic plant feeders are still poorly characterized. Here, we show the bacterial communities of the gut of two specialized Lepidoptera, Hyles euphorbiae and Brithys crini, which exclusively feed on latex-rich Euphorbia sp. and alkaloid-rich Pancratium maritimum, respectively. A metagenomic analysis based on high-throughput sequencing of the 16S rRNA gene revealed that the gut microbiota of both insects is dominated by the phylum Firmicutes, and especially by the common gut inhabitant Enterococcus sp. Staphylococcus sp. are also found in H. euphorbiae though to a lesser extent. By scanning electron microscopy, we found a dense ring-shaped bacterial biofilm in the hindgut of H. euphorbiae, and identified the most prominent bacterium in the biofilm as Enterococcus casseliflavus through molecular techniques. Interestingly, this species has previously been reported to contribute to the immobilization of latex-like molecules in the larvae of Spodoptera litura, a highly polyphagous lepidopteran. The E. casseliflavus strain was isolated from the gut and its ability to tolerate natural latex was tested under laboratory conditions. This fact, along with the identification of less frequent bacterial species able to degrade alkaloids and/or latex, suggest a putative role of bacterial communities in the tolerance of specialized insects to their toxic diet.

The polymerase chain reaction (PCR) is a molecular tool widely used to characterize the insecticidal bacterium Bacillus thuringiensis. This technique can be used to amplify specific DNA fragments and thus to determine the presence or absence of a target gene. The identification of B. thuringiensis toxin genes by PCR can partially predict the insecticidal activity of a given strain. PCR has proven to be a rapid and reliable method and it has largely substituted bioassays in preliminary classification of B. thuringiensis collections. In this work, we compare the largest B. thuringiensis PCR-based screenings, and we review the natural occurrence of cry genes among native strains. We also discuss the use of PCR for the identification of novel cry genes, as well as the potential of novel technologies for the characterization of B. thuringiensis strains.

BackgroundOnly a fraction of the microbial species used for anaerobic digestion in biogas production plants are methanogenic archaea. We have analyzed the taxonomic profiles of eubacteria and archaea, a set of chemical key parameters, and biogas production in samples from nine production plants in seven facilities in Thuringia, Germany, including co-digesters, leach-bed, and sewage sludge treatment plants. Reactors were sampled twice, at a 1-week interval, and three biological replicates were taken in each case.ResultsA complex taxonomic composition was found for both eubacteria and archaea, both of which strongly correlated with digester type. Plant-degrading Firmicutes as well as Bacteroidetes dominated eubacteria profiles in high biogas-producing co-digesters; whereas Bacteroidetes and Spirochaetes were the major phyla in leach-bed and sewage sludge digesters. Methanoculleus was the dominant archaea genus in co-digesters, whereas Methanosarcina and Methanosaeta were the most abundant methanogens in leachate from leach-bed and sewage sludge digesters, respectively.ConclusionsThis is one of the most comprehensive characterizations of the microbial communities of biogas-producing facilities. Bacterial profiles exhibited very low variation within replicates, including those of semi-solid samples; and, in general, low variation in time. However, facility type correlated closely with the bacterial profile: each of the three reactor types exhibited a characteristic eubacteria and archaea profile. Digesters operated with solid feedstock, and high biogas production correlated with abundance of plant degraders (Firmicutes) and biofilm-forming methanogens (Methanoculleus spp.). By contrast, low biogas-producing sewage sludge treatment digesters correlated with high titers of volatile fatty acid-adapted Methanosaeta spp.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0271-6) contains supplementary material, which is available to authorized users.

The sequence of the genome of “ Candidatus Tremblaya princeps” strain PCVAL, the primary endosymbiont of the citrus mealybug Planococcus citri , has been determined. “ Ca . Tremblaya princeps” presents an unusual nested endosymbiosis and harbors a gammaproteobacterial symbiont within its cytoplasm in all analyzed mealybugs. The genome sequence reveals that “ Ca . Tremblaya princeps” cannot be considered an independent organism but that the consortium with its gammaproteobacterial symbiotic associate represents a new composite living being.

BackgroundInsects are associated with microorganisms that contribute to the digestion and processing of nutrients. The European Corn Borer (ECB) is a moth present world-wide, causing severe economical damage as a pest on corn and other crops. In the present work, we give a detailed view of the complexity of the microorganisms forming the ECB midgut microbiota with the objective of comparing the biodiversity of the midgut-associated microbiota and explore their potential as a source of genes and enzymes with biotechnological applications.Methodological/Principal FindingsA high-throughput sequencing approach has been used to identify bacterial species, genes and metabolic pathways, particularly those involved in plant-matter degradation, in two different ECB populations (field-collected vs. lab-reared population with artificial diet). Analysis of the resulting sequences revealed the massive presence of Staphylococcus warneri and Weissella paramesenteroides in the lab-reared sample. This enabled us to reconstruct both genomes almost completely. Despite the apparently low diversity, 208 different genera were detected in the sample, although most of them at very low frequency. By contrast, the natural population exhibited an even higher taxonomic diversity along with a wider array of cellulolytic enzyme families. However, in spite of the differences in relative abundance of major taxonomic groups, not only did both metagenomes share a similar functional profile but also a similar distribution of non-redundant genes in different functional categories.Conclusions/SignificanceOur results reveal a highly diverse pool of bacterial species in both O. nubilalis populations, with major differences: The lab-reared sample is rich in gram-positive species (two of which have almost fully sequenced genomes) while the field sample harbors mainly gram-negative species and has a larger set of cellulolytic enzymes. We have found a clear relationship between the diet and the midgut microbiota, which reveals the selection pressure of food on the community of intestinal bacteria.

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BackgroundIn all branches of life there are plenty of symbiotic associations. Insects are particularly well suited to establishing intracellular symbiosis with bacteria, providing them with metabolic capabilities they lack. Essential primary endosymbionts can coexist with facultative secondary symbionts which can, eventually, establish metabolic complementation with the primary endosymbiont, becoming a co-primary. Usually, both endosymbionts maintain their cellular identity. An exception is the endosymbiosis found in mealybugs of the subfamily Pseudoccinae, such as Planococcus citri, with Moranella endobia located inside Tremblaya princeps.ResultsWe report the genome sequencing of M. endobia str. PCVAL and the comparative genomic analyses of the genomes of strains PCVAL and PCIT of both consortium partners. A comprehensive analysis of their functional capabilities and interactions reveals their functional coupling, with many cases of metabolic and informational complementation. Using comparative genomics, we confirm that both genomes have undergone a reductive evolution, although with some unusual genomic features as a consequence of coevolving in an exceptional compartmentalized organization.ConclusionsM. endobia seems to be responsible for the biosynthesis of most cellular components and energy provision, and controls most informational processes for the consortium, while T. princeps appears to be a mere factory for amino acid synthesis, and translating proteins, using the precursors provided by M. endobia. In this scenario, we propose that both entities should be considered part of a composite organism whose compartmentalized scheme (somehow) resembles a eukaryotic cell.