Predicting thresholds for population replacement gene drives.

BACKGROUND: Threshold-dependent gene drives (TDGDs) could be used to spread desirable traits through a population, and are likely to be less invasive and easier to control than threshold-independent gene drives. Engineered Genetic Incompatibility (EGI) is an extreme underdominance system previously demonstrated in Drosophila melanogaster that can function as a TDGD when EGI agents of both sexes are released into a wild-type population. RESULTS: Here we use a single generation fitness assay to compare the fecundity, mating preferences, and temperature-dependent relative fitness to wild-type of two distinct genotypes of EGI agents. We find significant differences in the behavior/performance of these EGI agents that would not be predicted a priori based on their genetic design. We report a surprising temperature-dependent change in the predicted threshold for population replacement in an EGI agent that drives ectopic expression of the developmental morphogen pyramus. CONCLUSIONS: The single-generation fitness assay presented here could reduce the amount of time required to estimate the threshold for TDGD strategies for which hybrid genotypes are inviable. Additionally, this work underscores the importance of empirical characterization of multiple engineered lines, as behavioral differences can arise in unique genotypes for unknown reasons.

Type 6 secretion system components hcp and vgrG support mutualistic partnership between Xenorhabdus bovienii symbiont and Steinernema jollieti host.

Xenorhabdus, like other Gram-negative bacteria, possesses a Type 6 Secretion System (T6SS) which acts as a contact-dependent molecular syringe, delivering diverse proteins (effectors) directly into other cells. The number of T6SS loci encoded in Xenorhabdus genomes are variable both at the inter and intraspecific level. Some environmental isolates of Xenorhabdus bovienii, encode at least one T6SS locus while others possess two loci. Previous work conducted by our team demonstrated that X. bovienii [Jollieti strain SS-2004], which has two T6SSs (T6SS-1 and T6SS-2), hcp genes are required for biofilm formation. Additionally, while T6SS-1 hcp gene plays a role in the antibacterial competition, T6SS-2 hcp does not. In this study, we tested the hypothesis that vgrG genes are also involved in mutualistic and pathogenic interactions. For this purpose, targeted mutagenesis together with wet lab experiments including colonization, competition, biofilm, and virulence experiments, were carried out to assess the role of vgrG in the mutualistic and antagonistic interactions in the life cycle of XBJ. Our results revealed that vgrG genes are not required for biofilm formation but play a role in outcompeting other Xenorhabdus bacteria. Additionally, both vgrG and hcp genes are required to fully colonize the nematode host. We also demonstrated that hcp and vgrG genes in both T6SS clusters are needed to support the reproductive fitness of the nematodes. Overall, results from this study revealed that in X. bovieni jollieti strain, the twoT6SS clusters play an important role in the fitness of the nematodes in relation to colonization and reproduction. These results lay a foundation for further investigations on the functional significance of T6SSs in the mutualistic and pathogenic lifecycle of Xenorhabdus spp.

Expression and functional characterization in yeast of an endoglucanase from Bacillus sonorensis BD92 and its impact as feed additive in commercial broilers.

Some ingredients used in poultry feed formulation contain carbohydrate polymers which are difficult to digest and thus hinder nutritional feed value. Toward overcoming this limitation, exogenous enzymes have been added to poultry feed to improve its nutritive value. The present study was designed to provide first enzymatic characterization of endoglucanase (BsEgl) from the genome of B. sonorensis BD92 expressed in Pichia pastoris. Further, we tested its impact alone and in combination with a ß-glucosidase (Bteqßgluc) on growth in commercial broilers as feed additive. The expressed enzyme displayed features of GH5 family and had optimum activity against carboxymethyl cellulose at pH 5 and 50 °C. The BsEgl was stable at a range of pH from 4 to 8 for 60 min and at 50 °C for 180 min. Supplementing broilers diet with BsEgl alone or in combination with Bteqßgluc resulted in better feed conversion ratio among treatments during a five weeks testing period. Moreover, meat percentage was also highest for this treatment, and all treatments with recombinant enzymes increased intestinal length in birds compared to treatment control group. Blood parameters and serum biochemistry profile showed non-significant difference among groups. These results support that recombinant cellulolytic enzymes supplement high fiber diets improve their nutritional performance.

First record of native entomopathogenic nematodes from Montana agroecosystems.

A total of 30 different agricultural fields in the Golden Triangle Region of Montana, USA were surveyed, and 150 soil samples were evaluated for the presence of entomopathogenic nematodes (EPNs). The authors isolated EPNs from 10% of the collected samples. The recovered isolates were identified as Steinernema feltiae and Heterorhabditis bacteriophora by using morphological and molecular analysis. Steinernema feltiae was found from two fields, Kalispell (S. feltiae 1) and Choteau (S. feltiae 2). Steinernema feltiae (1 and 2) differed significantly from each other in terms of morphological characters for infective juveniles (distance from anterior end to excretory pore and nerve ring) and 1st generation males (body length, spicule length, gubernaculum length, oesophagus, tail, and anal body diameter). Steinernema feltiae 2 and H. bacteriophora were recovered from the same field in Choteau. All these species were recovered from wheat fields with sandy clay loam and loam soils with 3.3 to 3.4% organic matter content and pH 8.A total of 30 different agricultural fields in the Golden Triangle Region of Montana, USA were surveyed, and 150 soil samples were evaluated for the presence of entomopathogenic nematodes (EPNs). The authors isolated EPNs from 10% of the collected samples. The recovered isolates were identified as Steinernema feltiae and Heterorhabditis bacteriophora by using morphological and molecular analysis. Steinernema feltiae was found from two fields, Kalispell (S. feltiae 1) and Choteau (S. feltiae 2). Steinernema feltiae (1 and 2) differed significantly from each other in terms of morphological characters for infective juveniles (distance from anterior end to excretory pore and nerve ring) and 1st generation males (body length, spicule length, gubernaculum length, oesophagus, tail, and anal body diameter). Steinernema feltiae 2 and H. bacteriophora were recovered from the same field in Choteau. All these species were recovered from wheat fields with sandy clay loam and loam soils with 3.3 to 3.4% organic matter content and pH 8.

Identification and functional characterization of a ß-glucosidase from Bacillus tequelensis BD69 expressed in bacterial and yeast heterologous systems.

BACKGROUND: The identification and characterization of novel ß-glucosidase genes has attracted considerable attention because of their valuable use in a variety of industrial applications, ranging from biofuel production to improved digestibility of animal feed. We previously isolated a fiber-degrading strain of Bacillus tequelensis from buffalo dung samples, and the goal of the current work was to identify ß-glucosidase genes in this strain. We describe the cloning and expression of a new ß-glucosidase gene (Bteqßgluc) from Bacillus tequelensis strain BD69 in bacterial and yeast hosts. The recombinant Bteqßgluc were used to characterize specificity and activity parameters, and candidate active residues involved in hydrolysis of different substrates were identified through molecular docking. METHODS: The full length Bteqßgluc gene was cloned and expressed in Escherichia coli and Pichia pastoris cultures. Recombinant Bteqßgluc proteins were purified by immobilized metal affinity or anion exchange chromatography and used in ß-glucosidase activity assays measuring hydrolysis of ρ-nitrophenyl-ß-D-glucopyranoside (pNPG). Activity parameters were determined by testing relative ß-glucosidase activity after incubation under different temperature and pH conditions. Candidate active residues in Bteqßgluc were identified using molecular operating environment (MOE) software. RESULTS: The cloned Bteqßgluc gene belongs to glycoside hydrolase (GH) family 4 and encoded a 54.35 kDa protein. Specific activity of the recombinant ß-glucosidase was higher when expressed in P. pastoris (1,462.25 U/mg) than in E. coli (1,445.09 U/mg) hosts using same amount of enzyme. Optimum activity was detected at pH 5 and 50 °C. The activation energy (E a) was 44.18 and 45.29 kJ/mol for Bteqßgluc produced by P. pastoris and E. coli, respectively. Results from other kinetic parameter determinations, including pK a for the ionizable groups in the active site, Gibbs free energy of activation (ΔG ‡), entropy of activation (ΔS ‡), Michaelis constant (K m) and maximum reaction velocity (V max) for pNPG hydrolysis support unique kinetics and functional characteristics that may be of interest for industrial applications. Molecular docking analysis identified Glu, Asn, Phe, Tyr, Thr and Gln residues as important in protein-ligand catalytic interactions.

The digestive system in Zygentoma as an insect model for high cellulase activity.

The digestive system of selected phytophagous insects has been examined as a potential prospecting resource for identification of novel cellulolytic enzymes with potential industrial applications. In contrast to other model species, however, limited detailed information is available that characterizes cellulolytic activity and systems in basal hexapod groups. As part of a screening effort to identify insects with highly active cellulolytic systems, we have for the first time, identified species of Zygentoma that displayed the highest relative cellulase activity levels when compared to all other tested insect groups under the experimental conditions, including model species for cellulolytic systems such as termite and cockroach species in Rhinotermitidae (formerly Isoptera) and Cryptocercidae (formerly Blattodea). The goal of the present study was to provide a morphohistological characterization of cellulose digestion and to identify highly active cellulase enzymes present in digestive fluids of Zygentoma species. Morphohistological characterization supported no relevant differences in the digestive system of firebrat (Thermobia domestica) and the gray silverfish (Ctenolepisma longicaudata). Quantitative and qualitative cellulase assays identified the foregut as the region with the highest levels of cellulase activity in both T. domestica and C. longicaudata. However, T. domestica was found to have higher endoglucanase, xylanase and pectinase activities compared to C. longicaudata. Using nano liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS/MS) and a custom gut transcriptome we identified cellulolytic enzymes from digestive fluids of T. domestica. Among the identified enzymes we report putative endoglucanases matching to insect or arthropod enzymes and glucan endo-1,6-ß-glucosidases matching bacterial enzymes. These findings support combined activities of endogenous and symbiont-derived plant cell wall degrading enzymes in lignocellulose digestion in Zygentoma and advance our understanding of cellulose digestion in a primitive insect group.

The origin of the odorant receptor gene family in insects.

The origin of the insect odorant receptor (OR) gene family has been hypothesized to have coincided with the evolution of terrestriality in insects. Missbach et al. (2014) suggested that ORs instead evolved with an ancestral OR co-receptor (Orco) after the origin of terrestriality and the OR/Orco system is an adaptation to winged flight in insects. We investigated genomes of the Collembola, Diplura, Archaeognatha, Zygentoma, Odonata, and Ephemeroptera, and find ORs present in all insect genomes but absent from lineages predating the evolution of insects. Orco is absent only in the ancestrally wingless insect lineage Archaeognatha. Our new genome sequence of the zygentoman firebrat Thermobia domestica reveals a full OR/Orco system. We conclude that ORs evolved before winged flight, perhaps as an adaptation to terrestriality, representing a key evolutionary novelty in the ancestor of all insects, and hence a molecular synapomorphy for the Class Insecta.