Nonlinear microscopy of chitin and chitinous structures: a case study of two cave-dwelling insects.

We performed a study of the nonlinear optical properties of chemically purified chitin and insect cuticle using two-photon excited autofluorescence (TPEF) and second-harmonic generation (SHG) microscopy. Excitation spectrum, fluorescence time, polarization sensitivity, and bleaching speed were measured. We have found that the maximum autofluorescence signal requires an excitation wavelength below 850 nm. At longer wavelengths, we were able to penetrate more than 150-um deep into the sample through the chitinous structures. The excitation power was kept below 10 mW (at the sample) in order to diminish bleaching. The SHG from the purified chitin was confirmed by spectral- and time-resolved measurements. Two cave-dwelling, depigmented, insect species were analyzed and three-dimensional images of the cuticular structures were obtained.

Chemical defense in millipedes (Myriapoda, Diplopoda): do representatives of the family Blaniulidae belong to the 'quinone' clade?

The defensive secretions of two blaniulid millipedes, Nopoiulus kochii and Cibiniulus phlepsii, were characterized by GC-FID and GC/MS analyses, which showed the presence of a complex mixture of benzoquinones, hydroquinones, and oleates. Altogether, 13 compounds were identified. The major compound in the secretions of both analyzed species was 2-methyl-1,4-benzoquinone (toluquinone). The second major constituent in the N. kochii secretion was 2-methyl-3,4-(methylenedioxy)phenol, while in that of C. phlepsii, it was 2-methoxy-3-methyl-1,4-benzoquinone. The defensive secretion of N. kochii also showed a high content of hydroquinones (13.5%) in comparison to that of C. phlepsii (0.8%). Hexyl oleate and octyl oleate were detected for the first time in defensive millipede fluids. The chemical composition of the defensive secretions supports the chemotaxonomic position of the family Blaniulidae in the 'quinone' millipede clade.

Chemistry of the sternal gland secretion of the Mediterranean centipede Himantarium gabrielis (Linnaeus, 1767) (Chilopoda: Geophilomorpha: Himantariidae).

The geophilomorph centipede, Himantarium gabrielis, when disturbed, discharges a viscous and proteinaceous secretion from the sternal glands. This exudate was found by gas chromatography-mass spectrometry, liquid chromatography-high resolution mass spectrometry, liquid chromatography-mass spectrometry-mass spectrometry and NMR analyses to be composed of hydrogen cyanide, benzaldehyde, benzoyl nitrile, benzyl nitrile, mandelonitrile, mandelonitrile benzoate, 3,7,6O-trimethylguanine (himantarine), farnesyl 2,3-dihydrofarnesoate and farnesyl farnesoate. This is the first report on the presence of benzyl nitrile and mandelonitrile benzoate in secreted substances from centipedes. Farnesyl 2,3-dihydrofarnesoate is a new compound, while himantarine and farnesyl farnesoate were not known as natural products. A post-secretion release of hydrogen cyanide by reaction of mandelonitrile and benzoyl nitrile was observed by NMR, and hydrogen cyanide signals were completely assigned. In addition, a protein component of the secretion was analysed by electrophoresis which revealed the presence of a major 55 kDa protein. Analyses of the defensive exudates of other geophilomorph families should produce further chemical surprises.

Micro- and nanostructures of iridescent wing scales in purple emperor butterflies (Lepidoptera: Apatura ilia and A. iris).

Apatura ilia (Denis and Schiffermüller, 1775) and A. iris (Linnaeus, 1758) are fascinating butterflies found in the Palaearctic ecozone (excepting the north of Africa). The wings of these insects are covered with a great number of two types of scales positioned like roof tiles. Type I scales are on the surface, while type II scales are situated below them. The structural color of the type I scales is recognized only on the dorsal side of both the fore and hind wings of the males of the aforementioned species. Both types of scales are responsible for pigment color of the wings, but iridescence is observed only in the type I scales. The brilliant structural color is due to a multilayer structure. The features of the scales, their dimensions and fine structure were obtained using scanning electron microscopy. Cross sections of the scales were then analyzed by transmission electron microscopy. The scales of the "normal" and clytie forms of A. ilia have a different nanostructure, but are of the same type. A similar type of structure, but with a different morphology, was also noticed in A. iris. The scales of the analyzed species resemble the scales of tropical Morpho butterflies.

Composition of the defensive secretion in three species of European millipedes.

Three European julid species, Cylindroiulus boleti, Leptoiulus trilineatus, and Megaphyllum bosniense, secrete mixtures of up to 12 different quinones. The major components in these species are 2-methoxy-3-methyl-1,4-benzoquinone and 2-methyl-1,4-benzoquinone. 2-Methoxy-5-methylhydroquinone is detected for the first time in the Class Diplopoda. 2-Hydroxy-3-methyl-1,4-benzoquinone, 2,3-dimethoxyhydroquinone, 2-methyl-3,4-methylendioxyphenol, and 2,3-dimethoxy-5-methylhydroquinone are registered for the first time in representatives of the family Julidae. The similar chemical composition of defense secretions in all analyzed European julids and Pacific spirobolids supports the idea of the chemical consistency of defensive compounds in juliform millipedes.

Defensive secretions in Callipodella fasciata (Latzel, 1882; Diplopoda, Callipodida, Schizopetalidae).

The small millipede Callipodella fasciata secretes an earthy smell when disturbed. This secretion was obtained by CH(2) Cl(2) extraction from specimens of both sexes and was identified by GC/MS analyses to be composed of p-cresol (96.5%), phenol (3.5%), and p-ethylphenol (traces). This is the first identification of these compounds in an epigean European callipodidan species and the first report of intergeneric differences in the chemical composition of defensive secretions in callipodidans. These compounds have repellent, antimicrobial, and antifungal properties.

Defensive secretions in three species of polydesmids (Diplopoda, Polydesmida, Polydesmidae).

Nine compounds were detected in three different millipede species: Polydesmus complanatus (L.), Brachydesmus (Stylobrachydesmus) avalae Curcic & Makarov, and Brachydesmus (Stylobrachydesmus) dadayi Verhoeff. Benzaldehyde, benzyl alcohol, benzoylnitrile, benzyl methyl ketone, benzoic acid, benzyl ethyl ketone, mandelonitrile, and mandelonitrile benzoate were identified by GC-FID and GC-MS analyses. Hydrogen cyanide was detected qualitatively by the picric acid test. Benzyl ethyl ketone, benzyl methyl ketone, and benzyl alcohol were detected for the first time in polydesmidan millipedes. Benzoylnitrile was the major component in all three hexane extracts. These compounds are suspected to be active in the defensive secretions of these millipede species.

Identification of secretory compounds from the European callipodidan species Apfelbeckia insculpta.

Defensive secretions of the callipodidan species Apfelbeckia insculpta contain a p-cresol as the main component and phenol in traces. This is the first identification of these compounds in a European callipodidan species. The repugnatory glands of A. insculpta are of the spirobolid type and consist of a spherical reservoir, a simple duct, and a valvular cuticular infolding that opens onto the lateral surface of the millipede via a pore.

Rhabditophanes schneideri (Rhabditida) phoretic on a cave pseudoscorpion.

Information of phoretic nematode-pseudoscorpion associations and cases of parasitism on five European species of pseudoscorpions was summarized by Curcic et al. [Curcic, B.P.M., Dimitrijevic, R.N., Makarov, S.E., Lucic, L.R., Curcic, S.B., 1996. Further report on nematode-pseudoscorpion associations. Acta arachnol. 45, 43-46; Curcic, B.P.M., Sudhaus, W., Dimitrijevic, R.N., Tomic, V.T., Curcic, S.B., 2004. Phoresy of Rhabditophanes schneideri (Bütschli) (Rhabditida: Alloionematidae) on pseudoscorpions (Arachnida: Pseudoscorpiones). Nematology 6 (3), 313-317]. An examination of a sample of the cavernicolous pseudoscorpion Neobisium rajkodimitrijevici (Curcic and Tomic, 2006) (comprising a holotype male and a paratype tritonymph) from a cave in Eastern Serbia revealed this false scorpion to be a nematode carrier; the present paper reports this finding and extends our knowledge of phoresy of Rhabditophanes on pseudoscorpions. This is the first time that the rhabditid R. schneideri (Bütschli, 1873) has been noted in association with a cavernicolous pseudoscorpion. There must be some patchily distributed micro-habitats in caves where saprobiotic nematodes and small arthropods can complete their life-cycles, for example something like deposits of bat guano. The transportation of Rhabditophanes J3 by pseudoscorpions indicate that Neobisium specimens often visit these micro-habitats to find their prey.