Extraction, characterization and comparison of chitins from large bodied four Coleoptera and Orthoptera species.

Chitins were extracted from large insect species of order Coleoptera (Lucanus cervus (Linnaeus, 1758) (Lucanidae) and Polyphylla fullo (Linnaeus, 1758) (Scarabaeidae) and order Orthoptera (Bradyporus (Callimenus) sureyai Ünal, 2011) (Tettigonidae) and Gryllotalpa gryllotalpa (Linnaeus, 1758) (Gryllotalpidae)) for the first time. Fourier Transform Infrared Spectrometry (FT-IR) confirms that isolation of chitin is successful. Yields of chitins on dry basis from P. fullo, L. cervus, G. gryllotalpa and B. (C.) sureyai are 11.3%, 10.9%, 10.1% and 9.8% respectively. Thermogravimetric Analysis (TGA) showed a variety of thermal stability of chitin samples from 614 °C to 748 °C with a small percent of ash. X-ray diffraction (XRD) data showed a crystallinity index percent from 80.6% to 85.2%. Scanning Electron Microscope (SEM) was examined for surface characterization determining as fibrous and porous for all species and changes from nm scales to µm scales. Elemental analysis has been applied to determine the elemental composition of chitin and nitrogen percent was relatively low for all specimens than expected. It is detected that examined insects have α-chitin form from XRD and FT-IR data. If these species can be grown in the laboratory, adults of them could be accepted as promising alternative chitin sources without negative effects on biodiversity.

Predictability in the evolution of Orthopteran cardenolide insensitivity.

The repeated evolutionary specialization of distantly related insects to cardenolide-containing host plants provides a stunning example of parallel adaptation. Hundreds of herbivorous insect species have independently evolved insensitivity to cardenolides, which are potent inhibitors of the alpha-subunit of Na+,K+-ATPase (ATPα). Previous studies investigating ATPα-mediated cardenolide insensitivity in five insect orders have revealed remarkably high levels of parallelism in the evolution of this trait, including the frequent occurrence of parallel amino acid substitutions at two sites and recurrent episodes of duplication followed by neo-functionalization. Here we add data for a sixth insect order, Orthoptera, which includes an ancient group of highly aposematic cardenolide-sequestering grasshoppers in the family Pyrgomorphidae. We find that Orthopterans exhibit largely predictable patterns of evolution of insensitivity established by sampling other insect orders. Taken together the data lend further support to the proposal that negative pleiotropic constraints are a key determinant in the evolution of cardenolide insensitivity in insects. Furthermore, analysis of our expanded taxonomic survey implicates positive selection acting on site 111 of cardenolide-sequestering species with a single-copy of ATPα, and sites 115, 118 and 122 in lineages with neo-functionalized duplicate copies, all of which are sites of frequent parallel amino acid substitution. This article is part of the theme issue 'Convergent evolution in the genomics era: new insights and directions'.

Biochemical characterization and bacterial expression of an odorant-binding protein from Locusta migratoria.

Analysis of soluble proteins from different body parts of Locusta migratoria revealed a fast-migrating component in native electrophoresis, unique to antennae of both sexes. N-terminal sequence analysis and cloning identified this protein as a member of the insect odorant-binding proteins, carrying a well-conserved six-cysteine motif. Mass spectrometry analysis confirmed the occurrence of two distinct polypeptide species determined by nucleotide sequencing and demonstrated that the cysteine residues are paired in an interlocked fashion. The protein was expressed in a bacterial system with yields of about 10 mg/l of culture, mostly present as inclusion bodies. However, this recombinant product was solubilized after disulfide reduction. Air oxidation yielded a species with all disulfides spontaneously formed as in the native counterpart. Both native and recombinant proteins migrated as a dimer in gel filtration chromatography. Ligand binding was measured, using N-phenyl-1-naphthylamine as the fluorescent probe; the affinity of other ligands was measured in competitive binding assays. The protein exhibited great resistance to thermal denaturation even following prolonged treatment at 100 degrees C. A structural model for this dimeric species was generated on the basis of its sequence homology with Bombyx mori pheromone-binding protein, whose three-dimensional structure has been resolved as an unbound species and in complex with its physiological ligand. This is the first report of an odorant-binding protein identified and characterized from Orthoptera.