Control of the flat-headed root borer <Emphasis Type="Italic">Capnodis tenebrionis</Emphasis> (Linné) (Coleoptera: Buprestidae) with the entomopathogenic nematode <Emphasis Type="Italic">Steinernema carpocapsae</Emphasis> (Weiser) (Nematoda: Steinernematidae) in a chitosan formulation in apricot orchards

Entomopathogenic nematodes Steinernema carpocapsae were applied in a chitosan formulation (Biorend R^®) to control the flat-headed rootborer Capnodis tenebrionis (Coleoptera: Buprestidae) in 5 field trials in the province of Valencia, Spain. Application was performed during spring and summer into the soil around apricot trees at densities of 1 and 1.5 million infective dauer juveniles per tree through drip irrigation, injection or by a drench. For evaluation of the control effect the roots of 106 trees were excavated, the cortex removed and living and dead insects were sampled. Dead larvae were dissected and checked for nematode infestation. Control of C. tenebrionis larvae was between 75% and 90%. No influence of the application method, nematode density or time of application on the control effect was recorded. Recovery of the infested trees was observed already in the season following nematode application. Due to the two year life cycle of the insect with egg laying from May to August, applications during spring and autumn are recommended.     

Towards a phylogeny of chitons (Mollusca, Polyplacophora) based on combined analysis of five molecular loci

This study represents the first phylogenetic analysis of the molluscan class Polyplacophora using DNA sequence data. We employed DNA from a nuclear protein-coding gene (histone H3), two nuclear ribosomal genes (18S rRNA and the D3 expansion fragment of 28S rRNA), one mitochondrial protein-coding gene (cytochrome c oxidase subunit I), and one mitochondrial ribosomal gene (16S rRNA). A series of analyses were performed on independent and combined data sets. All these analyses were executed using direct optimization with parsimony as the optimality criterion, and analyses were repeated for nine combinations of parameters affecting indel and transversion/transition cost ratios. Maximum likelihood was also explored for the combined molecular data set, also using the direct optimization method, with a model equivalent to GTR + I + Γ that accommodates gaps. The results of all nine parameter sets for the combined parsimony analysis of all molecular data (as well as ribosomal data) and the maximum-likelihood analysis of all molecular data support monophyly of Polyplacophora. The resulting topologies mostly agree with a division of Polyplacophora into two major lineages: Lepidopleuridae and Chitonida (sensu Sirenko 1993). In our analyses the genus Callochiton is positioned as the sister group to Lepidopleuridae, and not as sister group to the remaining Chitonida (sensu Buckland-Nicks & Hodgson 2000), nor as the sister group to the remaining Chitonina (sensu Buckland-Nicks 1995). Chitonida (excluding Callochiton) is monophyletic, but conventional subgroupings of Chitonida are not supported. Acanthochitonina (sensu Sirenko 1993) is paraphyletic, or alternatively monophyletic, and is split into two clades, both with abanal gills only and cupules in the egg hull, but one has simple cupules whereas the other has more strongly hexagonal cupules. Sister to the Acanthochitonina clades is Chitonina, including taxa with adanal gills and a spiny egg hull. Schizochiton, the only genus with adanal gills that has an egg hull with cupules, is the sister-taxon to one of the Acanthochitonina clades plus Chitonina, or alternatively basal to Chitonina. Support values for either position are low, leaving this relationship unsettled. Our results refute several aspects of conventional classifications of chitons that are based primarily on shell characters, reinforcing the idea that chiton classification should be revised using additional characters.     

Purification and biological characterization of halocin H1 from Haloferax mediterranei M2a

The production of halocins, bacteriocin-like proteins of ecological significance, is a frequent characteristic of species from the family Halobacteriaceae. Halocin H1, produced by Haloferax mediterranei strain M2a, is a single 31-kDa polypeptide. Its purification was achieved by combining two chromatographic systems: Sepharose 4B linked to bacitracin followed by hydroxylapatite Bio-gel HTP. Halocin H1 required concentrations of NaCl higher than 1.5 M to maintain its activity. Haoarchaeal strains showed a differential degree of sensitivity to the action of this halocin. Electronic Publication     

Effect of Modified Atmosphere Composition on the Metabolism of Glucose by Brochothrix thermosphacta

The influence of atmosphere composition on the metabolism of Brochothrix thermosphacta was studied by analyzing the consumption of glucose and the production of ethanol, acetic and lactic acids, acetaldehyde, and diacetyl-acetoin under atmospheres containing different combinations of carbon dioxide and oxygen. When glucose was metabolized under oxygen-free atmospheres, lactic acid was one of the main end products, while under atmospheres rich in oxygen mainly acetoin-diacetyl was produced. The proportions of the total consumed glucose used for the production of acetoin (aerobic metabolism) and lactic acid (anaerobic metabolism) were used to decide whether aerobic or anaerobic metabolism predominated at a given atmosphere composition. The boundary conditions between dominantly anaerobic and aerobic metabolisms were determined by logistic regression. The metabolism of glucose by B. thermosphacta was influenced not only by the oxygen content of the atmosphere but also by the carbon dioxide content. At high CO₂ percentages, glucose metabolism remained anaerobic under greater oxygen contents.     

A comparison of methods for selecting non-target species for risk assessment of the biological control agent <Emphasis Type="Italic">Cotesia urabae</Emphasis>

A computer-based tool called PRONTI (priority ranking of non-target invertebrates) has been developed to aid the selection of non-target species (NTS) for pre-release testing with entomophagous biological control agents. To test whether PRONTI can improve NTS selection, we used it to produce a prioritised list of NTS for the agent Cotesia urabae Austin & Allen (Hymenoptera: Braconidae), and compared it with the original list that was produced before this species was released in New Zealand in 2011. While the two lists were similar, with five NTS occurring in the top nine of both lists, the remaining four NTS in the top nine of each list were different, primarily because the selection criteria used by the two methods were weighted differently (e.g., PRONTI put more weight on the likelihood of a NTS being exposed to the agent). Post-release testing has demonstrated that C. urabae is able to oviposit in two NTS that were ranked highly on both lists, suggesting both methods are useful for species selection. The main advantages of PRONTI were considered to be its ability to rank hundreds of NTS simultaneously, and to provide a body of information that can be used to both understand each NTS’ ranking and to justify more objectively the selection of NTS for pre-release testing.     

Automated identification of reference genes based on RNA-seq data

Background

Gene expression analyses demand appropriate reference genes (RGs) for normalization, in order to obtain reliable assessments. Ideally, RG expression levels should remain constant in all cells, tissues or experimental conditions under study. Housekeeping genes traditionally fulfilled this requirement, but they have been reported to be less invariant than expected; therefore, RGs should be tested and validated for every particular situation. Microarray data have been used to propose new RGs, but only a limited set of model species and conditions are available; on the contrary, RNA-seq experiments are more and more frequent and constitute a new source of candidate RGs.

Results

An automated workflow based on mapped NGS reads has been constructed to obtain highly and invariantly expressed RGs based on a normalized expression in reads per mapped million and the coefficient of variation. This workflow has been tested with Roche/454 reads from reproductive tissues of olive tree (Olea europaea L.), as well as with Illumina paired-end reads from two different accessions of Arabidopsis thaliana and three different human cancers (prostate, small-cell cancer lung and lung adenocarcinoma). Candidate RGs have been proposed for each species and many of them have been previously reported as RGs in literature. Experimental validation of significant RGs in olive tree is provided to support the algorithm.

Conclusion

Regardless sequencing technology, number of replicates, and library sizes, when RNA-seq experiments are designed and performed, the same datasets can be analyzed with our workflow to extract suitable RGs for subsequent PCR validation. Moreover, different subset of experimental conditions can provide different suitable RGs.