Ontogenetic change in effectiveness of chemical defence against different predators in Oxycarenus true bugs.

Many prey species change their antipredator defence during ontogeny, which may be connected to different potential predators over the life cycle of the prey. To test this hypothesis, we compared reactions of two predator taxa - spiders and birds - to larvae and adults of two invasive true bug species, Oxycarenus hyalinipennis and Oxycarenus lavaterae (Heteroptera: Oxycarenidae) with life-stage-specific chemical defence mechanisms. The reactions to larvae and adults of both true bug species strikingly differed between the two predator taxa. The spiders were deterred by the defences of adult bugs, but the larval defences were ineffective against them. By contrast, birds attacked the larvae considerably less often than the adult bugs. The results indicate a predator-specific ontogenetic change in defence effectiveness of both Oxycarenus species. The change in defence is likely linked to the life-stage-specific composition of secretions in both species: whereas secretions of larvae are dominated by unsaturated aldehydes, secretions of adults are rich in terpenoids, which probably serve dual function of defensive chemicals and pheromones. Our results highlight the variation in defence between different life stages and the importance of testing responses of different types of predators.

Improved Flow Modulator Construction for GC × GC with Quadrupole Mass Spectrometry.

Improvement and testing of a flow modulator for the application in comprehensive two-dimensional gas chromatography separations is the subject of the presented paper. This improved setup constructed from two independent capillary branches each consisting of a pressure regulator, a pressure sensor, a two-way solenoid valve and a microfluidic T-connector, allows an independent and easy settings of the pressures and flow velocities in the modulator and provides system flexibility in an operation without need of any component exchange. The estimated flow rates were 0.4 mL/min in the first column and 3.2 mL/min in the second column. This setup was compared with the commercial Zoex cryogenic modulator for the separation of 17 selected solvents at isothermal conditions. Modulator working conditions were optimized and its separation power was demonstrated on the analysis of a lavender extract under an application of two orthogonal capillary column sets (nonpolar-polar vs. polar-nonpolar) and temperature program. The results were evaluated by two commercial software packages and discussed with respect to the identification compliance.

Comparative Analysis of Volatile Defensive Secretions of Three Species of Pyrrhocoridae (Insecta: Heteroptera) by Gas Chromatography-Mass Spectrometric Method.

The true bugs (Hemiptera: Heteroptera) have evolved a system of well-developed scent glands that produce diverse and frequently strongly odorous compounds that act mainly as chemical protection against predators. A new method of non-lethal sampling with subsequent separation using gas chromatography with mass spectrometric detection was proposed for analysis of these volatile defensive secretions. Separation was performed on Rtx-200 column containing fluorinated polysiloxane stationary phase. Various mechanical irritation methods (ultrasonics, shaking, pressing bugs with plunger of syringe) were tested for secretion sampling with a special focus on non-lethal irritation. The preconcentration step was performed by sorption on solid phase microextraction (SPME) fibers with different polarity. For optimization of sampling procedure, Pyrrhocoris apterus was selected. The entire multi-parameter optimization procedure of secretion sampling was performed using response surface methodology. The irritation of bugs by pressing them with a plunger of syringe was shown to be the most suitable. The developed method was applied to analysis of secretions produced by adult males and females of Pyrrhocoris apterus, Pyrrhocoris tibialis and Scantius aegyptius (all Heteroptera: Pyrrhocoridae). The chemical composition of secretion, particularly that of alcohols, aldehydes and esters, is species-specific in all three pyrrhocorid species studied. The sexual dimorphism in occurrence of particular compounds is largely limited to alcohols and suggests their epigamic intraspecific function. The phenetic overall similarities in composition of secretion do not reflect either relationship of species or similarities in antipredatory color pattern. The similarities of secretions may be linked with antipredatory strategies. The proposed method requires only a few individuals which remain alive after the procedure. Thus secretions of a number of species including even the rare ones can be analyzed and broadly conceived comparative studies can be carried out.

Capillary electrophoresis of pterin derivatives responsible for the warning coloration of Heteroptera.

A new capillary electrophoretic (CE) method has been developed for analysis of 10 selected derivatives of pterin that can occur in the integument (cuticle) of true bugs (Insecta: Hemiptera: Heteroptera), specifically L-sepiapterin, 7,8-dihydroxanthopterin, 6-biopterin, D-neopterin, pterin, isoxanthopterin, leucopterin, xanthopterin, erythropterin and pterin-6-carboxylic acid. Pterin derivatives are responsible for the characteristic warning coloration of some Heteroptera and other insects, signaling noxiousness or unpalatability and are used to discourage potential predators from attacking. Regression analysis defining the parameters significantly affecting CE separation was used to optimize the system (the background electrolyte (BGE) composition, pH value and applied voltage). The optimized separation conditions were as follows: BGE with composition 2 mmol L(-1) the disodium salt of ethylendiamintetraacetic acid, 100 mmol L(-1) tris(hydroxymethyl)aminomethane and 100 mmol L(-1) boric acid, pH 9.0, applied voltage 20 kV and UV detection at 250 nm. Under these conditions, all the 10 studied derivatives of pterin were baseline separated within 22 min. The optimized method was validated from the viewpoint of linearity (R(2)≥0.9980), accuracy (relative error ≤7.90%), precision (for repeatability RSD≤6.65%), detection limit (LOD in the range 0.04-0.99 µg mL(-1)) and limit of quantitation (LOQ in the range 0.13-3.30 µg mL(-1)). The developed method was used for identification and determination of the contents of pterin derivatives in adults of four species of Heteroptera (Eurydema ornata cream color morph, Scantius aegyptius, Pyrrhocoris apterus and Corizus hyoscyami) and their distribution in the individual species was determined.

Hydrophilic interaction liquid chromatography with tandem mass spectrometric detection applied for analysis of pteridines in two Graphosoma species (Insecta: Heteroptera).

A new separation method involving hydrophilic interaction chromatography with tandem mass spectrometric detection has been developed for the analysis of pteridines, namely biopterin, isoxanthopterin, leucopterin, neopterin, xanthopterin and erythropterin in the cuticle of heteropteran insect species. Two columns, Atlantis HILIC Silica and ZIC(®)-HILIC were tested for the separation of these pteridines. The effect of organic modifier content, buffer type, concentration and pH in mobile phase on retention and separation behavior of the selected pteridines was studied and the separation mechanism was also investigated. The optimized conditions for the separation of pteridines consisted of ZIC(®)-HILIC column, mobile phase composed of acetonitrile/5mM ammonium acetate, pH 6.80, 85/15 (v/v), flow rate 0.5mL/min and column temperature 30°C. Detection was performed by tandem mass spectrometry operating in electrospray ionization with Agilent Jet Stream technology using the selected reaction monitoring mode. The optimized method provided a linearity range from 0.3 to 5000ng/mL (r>0.9975) and repeatability with relative standard deviation<8.09% for all the studied pteridines. The method was applied to the analysis of pteridines in the cuticle of larvae and three adult color forms of Graphosoma lineatum and one form of Graphosoma semipunctatum (Insecta: Hemiptera: Heteroptera: Pentatomidae). The analysis shows that different forms of Graphosoma species can be characterized by different distribution of individual pteridines, which affects the coloration of various forms. Only isoxanthopterin was found in all the five forms tested.

Mechanisms of color production in a highly variable shield-back stinkbug, Tectocoris diophthalmus [corrected] (Heteroptera: Scutelleridae), and why it matters.

Theory suggests that aposematism, specifically the learned avoidance of unprofitable prey via memorable color patterns, should result in selection for pattern uniformity. However, many examples to the contrary are seen in nature. Conversely, honest sexual signals are likely to exhibit greater variation because they reflect underlying variation in mate quality. Here we aim to characterize and quantify the mechanistic causes of color in Tectocoris diophthalmus [corrected] to shed light on the costs of color production, and thus the potential information content of its color signals. We use Tectocoris diophthalmus [corrected] because it is a weakly-defended stinkbug, and presents elements that have classically been studied in the context of aposematism (red coloring), and sexual selection (sexual dichromatism and iridescent coloring). Pigment analysis reveals that variation in orange coloration is due to the amount of erythropterin pigment, stored in intracellular granules. This pigment is common in Heteroptera, and as an endogenously produced excretory byproduct is unlikely to reflect mate quality or variation in unprofitability of the bug. Electron microscopy reveals the iridescent patches are caused by an epicuticular multilayer reflector, and the hue and patch size are directly related to the layer widths and extent of coverage of this layering. Furthermore, we identified melanin as an essential component of the multilayer reflector system; therefore, the quality of the iridescent patches may be affected by aspects of rearing environment and immunocompetence. We posit that T. diophthalmus [corrected] has co-opted the melanic patches of a 'typical' red and black aposematic signal, transforming it into a complex and variable iridescent signal that may enhance its capacity to display individual quality.