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.

Department of Analytical Chemistry Faculty of Science Charles University Prague Prague Czech Republic

Department of Zoology Faculty of Science Charles University Prague Prague Czech Republic

Toxicology Department Institute of Forensic Medicine and Toxicology General University Hospital Prague Prague Czech Republic

Zobrazit více v PubMed

Vet LEM. From chemical to population ecology: infochemical use in an evolutionary context. J Chem Ecol. 1999;25(1):31–49.

Roper TJ, Marples NM. Odour and colour as cues for taste-avoidance learning in domestic chicks. Anim Behav. 1997;53(6):1241–50. PubMed

Skelhorn J, Rowe C. Birds learn to use distastefulness as a signal of toxicity. Proc R Soc Lond [Biol]. 2010;277(1688):1729–34. PubMed PMC

Rowe C, Halpin C. Why are warning displays multimodal? Behav Ecol Sociobiol. 2013;67(9):1425–39.

Henry TJ. Biodiversity of Heteroptera In: Foottit R, Adler PH editor. Insect Biodiversity: Wiley-Blackwell; 2007. p. 223–63.

Aldrich Jr. Chemical Ecology of the Heteroptera. Ann Rev Entomol. 1988;33(1):211–38.

Scudder GGE, Duffey SS. Cardiac glycosides in the Lygaeinae (Hemiptera: Lygaeidae). Can J Zool 1972;50(1):35–42.

Staddon JE. Operant behavior as adaptation to constraint. J Exp Psychol Gen. 1979;108(1):48–67.

Kozlík P, Krajíček J, Kalíková K, Tesařová E, Čabala R, Exnerová A, et al. Hydrophilic interaction liquid chromatography with tandem mass spectrometric detection applied for analysis of pteridines in two Graphosoma species (Insecta: Heteroptera). J Chromatogr B. 2013;930(0):82–9. PubMed

Krajíček J, Kozlík P, Exnerová A, Štys P, Bursová M, Čabala R, et al. Capillary electrophoresis of pterin derivatives responsible for the warning coloration of Heteroptera. J Chromatogr A. 2014;1336(0):94–100. PubMed

Ho HY, Millar JG. Compounds in metathoracic glands of adults and dorsal abdominal glands of nymphs of the stink bugs, Chlorochroa uhleri, C. sayi, and C. ligata (Hemiptera: Pentatomidae). Zool Stud. 2001;40(3):193–8.

Pavis C, Malosse C, Ducrot PH, Descoins C. Dorsal abdominal glands in nymphs of southern green stink bug, Nezara viridula (L.) (Heteroptera: Pentatomidae): Chemistry of secretions of five instars and role of (E)-4-oxo-2-decenal, compound specific to first instars. J Chem Ecol. 1994;20(9):2213–27. 10.1007/BF02033198 PubMed DOI

Krall BS, Bartelt RJ, Lewis CJ, Whitman DW. Chemical defense in the stink bug Cosmopepla bimaculata. J Chem Ecol. 1999;25(11):2477–94.

Moraes MCB, Pareja M, Laumann RA, Borges M. The Chemical Volatiles (Semiochemicals) Produced by Neotropical Stink Bugs (Hemiptera: Pentatomidae). Neotrop Entomol. 2008;37(5):489–505. PubMed

Hotová Svádová K, Exnerová A, Kopečková M, Štys P. How Do Predators Learn to Recognize a Mimetic Complex: Experiments with Naive Great Tits and Aposematic Heteroptera. Ethology. 2013;119(10):814–30.

Farine JP, Bonnard O, Brossut R, Le Quere JL. Chemistry of pheromonal and defensive secretions in the nymphs and the adults of Dysdercus cingulatus Fabr. (Heteroptera, Pyrrhocoridae). J Chem Ecol. 1992;18(1):65–76. 10.1007/BF00997165 PubMed DOI

Gunawardena NE. Seasonal changes in the chemical composition of the defensive secretion of the rice pest Leptocorisa oratorius (Hemiptera:Coreidae). J Natl Sci Counc Sri Lanka. 1994;22(3):239–43.

Schuh RT, Slater JA. True bugs of the world (Hemiptera: Heteroptera): classification and natural history: Cornell University Press; 1995.

Hotová Svádová K, Exnerová A, Štys P. Gregariousness as a defence strategy of moderately defended prey: experiments with and avian predators. Behaviour. 2014;151(11):1617–40.

Farine JP, Bonnard O, Brossut R, Le Quere JL. Chemistry of defensive secretions in nymphs and adults of fire bug, Pyrrhocoris apterus L. (Heteroptera, Pyrrhocoridae). J Chem Ecol. 1992;18(10):1673–82. 10.1007/BF02751094 PubMed DOI

Nagnan P, Cassier P, Andre M, Llosa JF, Guillaumin D. Fine structure and physicochemical analysis of the metathoracic scent glands of Lincus Malevolus (Rolston) and L. Spurcus (Rolston) (Heteroptera: Pentatomidae). Int J Insect Morphol Embryol. 1994;23(4):355–70.

Zarbin PHG, Borges M, Dos Santos AA, De Oliveira ARM, Simonelli F, Marques FDA. Alarm Pheromone System of Stink Bug Piezodorus guildinii (Heteroptera: Pentatomidae). J Braz Chem Soc. 2000;11(4):424–8.

Zhang Q-H, Aldrich JR. Male-produced anti-sex pheromone in a plant bug. Naturwissenschaften. 2003;90(11):505–8. 10.1007/s00114-003-0466-8 PubMed DOI

Kalinova B, Jiros P, Zdarek J, Wen X, Hoskovec M. GC×GC/TOF MS technique-A new tool in identification of insect pheromones: Analysis of the persimmon bark borer sex pheromone gland. Talanta. 2006;69(3):542–7. 10.1016/j.talanta.2005.10.045 PubMed DOI

Pareja M, Borges M, Laumann RA, Moraes MCB. Inter- and intraspecific variation in defensive compounds produced by five neotropical stink bug species (Hemiptera: Pentatomidae). J Insect Physiol. 2007;53(7):639–48. 10.1016/j.jinsphys.2007.04.004 PubMed DOI

Durak D, Kalender Y. Fine structure and chemical analysis of the metathoracic scent gland secretion in Graphosoma lineatum (Linnaeus, 1758) (Heteroptera, Pentatomidae). C R Biol. 2009;332(1):34–42. 10.1016/j.crvi.2008.10.004 PubMed DOI

Yang CY, Kim J, Ahn S-J, Kim D-H, Cho MR. Identification of the Female-Produced Sex Pheromone of the Plant Bug Apolygus spinolae. J Chem Ecol. 2014;40(3):244–9. 10.1007/s10886-014-0407-3 PubMed DOI

Aldrich JR, Zanuncio JC, Vilela EF, Torres JB, Cave RD. Field tests of predaceous pentatomid pheromones and semiochemistry of Podisus and Supputius species (Heteroptera: Pentatomidae: Asopinae). An Soc Entomol Bras. 1997;26(1):1–14.

Aldrich JR, Numata H, Borges M, Bin F, Waite GK, Lusby WR. Artifacts and pheromone blends from Nezara spp. and other stink bugs (Heteroptera: Pentatomidae). Z Naturforsch C: Biosci. 1993;48(1–2):73–9.

Favaro CF, Santos TB, Zarbin PH. Defensive compounds and male-produced sex pheromone of the stink bug, Agroecus griseus. J Chem Ecol. 2012;38(9):1124–32. Epub 2012/08/24. 10.1007/s10886-012-0172-0 PubMed DOI

Stránský K, Valterová I, Ubik K, Čejka J, Křeček J. Volatiles from Stink Bug, Graphosoma lineatum (L.), and from Green Shield Bug, Palomena prasina (L.), (Heteroptera: Pentatomidae). J High Res Chrom. 1998;21(8):475–6.

Miklas N, Lasnier T, Renou M. Male Bugs Modulate Pheromone Emission in Response to Vibratory Signals from Conspecifics. J Chem Ecol. 2003;29(3):561–74. PubMed

Takita M, Sugie H, Tabata J, Ishii S, Hiradate S. Isolation and estimation of the aggregation pheromone from Eysarcoris lewisi (Distant) (Heteroptera: Pentatomidae). Appl Entomol Zool. 2008;43(1):11–7.

Fountain M, Jåstad G, Hall D, Douglas P, Farman D, Cross J. Further Studies on Sex Pheromones of Female Lygus and Related Bugs: Development of Effective Lures and Investigation of Species-Specificity. J Chem Ecol. 2014;40(1):71–83. 10.1007/s10886-013-0375-z PubMed DOI

Ho H-Y, Kou R, Tseng H-K. Semiochemicals from the predatory stink bug Eocanthecona furcellata (Wolff): Components of metathoracic gland, dorsal abdominal gland, and sternal gland secretions. J Chem Ecol. 2003;29(9):2101–14. PubMed

Djozan D, Baheri T, Farshbaf R, Azhari S. Investigation of solid-phase microextraction efficiency using pencil lead fiber for in vitro and in vivo sampling of defensive volatiles from insect's scent gland. Anal Chim Acta. 2005;554(1–2):197–201.

Byers JA. Production and Predator-Induced Release of Volatile Chemicals by the Plant Bug Lygus hesperus. J Chem Ecol. 2006;32(10):2205–18. 10.1007/s10886-006-9140-x PubMed DOI

Cheng L, Roussis V. Sex attractant in the marine insect Trochopus plumbeus (Heteroptera: Veliidae). A preliminary report. Mar Ecol Prog Ser. 1998;170:283–6.

Drijfhout F, Van Beek T, Visser JH, De Groot A. On-line Thermal Desorption–Gas Chromatography of Intact Insects for Pheromone Analysis. J Chem Ecol. 2000;26(6):1383–92.

Griepink F, Drijfhout F, Van Beek T, Visser JH, De Groot A. Analysis of Sex Pheromone Gland Content of Individual Symmetrischema tangolias by Means of Direct Gland Introduction into a Two-dimensional Gas Chromatograph. J Chem Ecol. 2000;26(4):1013–23.

Williams L III, Evans PE, Bowers WS. Defensive chemistry of an aposematic bug, Pachycoris stallii Uhler and volatile compounds of its host plant Croton californicus Muell.-Arg. J Chem Ecol. 2001;27(2):203–16. PubMed

Aldrich JR, Blum MS, Fales HM. Species-specific natural products of adult male leaf-footed bugs (Hemiptera: Heteroptera). J Chem Ecol. 1979;5(1):53–62.

Gough AJE, Hamilton JGC, Games DE, Staddon BW. Multichemical defense of plant bug Hotea gambiae (Westwood) (Heteroptera: Scutelleridae). J Chem Ecol. 1985;11(3):343–52. 10.1007/BF01411421 PubMed DOI

Gunawardena NE, Herath HMWKB. Trans-2-hexenal/N-tridecane combination: a natural fumigant of the defensive secretion of Coridius janus (Hemiptera:Pentatomidae). J Natl Sci Counc Sri Lanka. 1992;20(2):209–20.

Prudic KL, Noge K, Becerra JX. Adults and Nymphs Do Not Smell the Same: The Different Defensive Compounds of the Giant Mesquite Bug (Thasus neocalifornicus: Coreidae). J Chem Ecol. 2008;34(6):734–41. 10.1007/s10886-008-9480-9 PubMed DOI

Eliyahu D, Ceballos RA, Saeidi V, Becerra JX. Synergy versus potency in the defensive secretions from nymphs of two Pentatomomorphan families (Hemiptera: Coreidae and Pentatomidae). J Chem Ecol. 2012;38(11):1358–65. 10.1007/s10886-012-0200-0 PubMed DOI PMC

Jacobs DH, Apps PJ, Viljoen HW. The composition of the defensive secretions of Thaumastella namaquensis and T. elizabethae with notes on the higher classification of the Thaumastellidae (Insecta: Heteroptera). Comp Biochem Physiol, B: Comp Biochem. 1989;93B(2):459–63.

Evans CE, Staddon BW, Games DE, editors. Analysis of gland secretions of Pentatomoidea (Heteroptera) by gas chromatography-mass spectrometry techniques 1990: Plenum.

Krall BS, Zilkowski BW, Kight SL, Bartelt RJ, Whitman DW. Chemistry and defensive efficacy of secretion of burrowing bug (Sehirus cinctus cinctus). J Chem Ecol. 1997;23(8):1951–62.

Sanda M, Zacek P, Streinz L, Dracinsky M, Koutek B. Profiling and characterization of volatile secretions from the European stink bug Graphosoma lineatum (Heteroptera: Pentatomidae) by two-dimensional gas chromatography/time-of-flight mass spectrometry. J Chromatogr B. 2012;881–82:69–75. PubMed

Myers RH, Montgomery DC. Response Surface Methodology: Process and Product Optimization Using Designed Experiments: Wiley; 2002.

Ryan TP. Modern Experimental Design: Wiley; 2006.

Hibbert DB. Experimental design in chromatography: A tutorial review. J Chromatogr B. 2012;910(0):2–13. 10.1016/j.jchromb.2012.01.020. PubMed DOI

Kerzhner IM. Family Pyrrhocoridae Amyot & Serville, 1843. Rieger BAC, editor. Amsterdam, Netherland: 2001. 248–58 p.

Socha R. Pyrrhocoris apterus (Heteroptera)—an experimental model species—a review. Eur J Entomol 1993;90(3):241–86.

Balogh ACV, Gamberale-Stille G, Leimar O. Learning and the mimicry spectrum: from quasi-Bates to super-Müller. Anim Behav 2008;76(5):1591–9. 10.1016/j.anbehav.2008.07.017. DOI

Ruxton GD, Sherratt TN, Speed M. Avoiding Attack: The Evolutionary Ecology of Crypsis, Warning Signals and Mimicry: OUP; Oxford; 2004.

Speed MP. Warning signals, receiver psychology and predator memory. Anim Behav. 2000;60(3):269–78. Epub 2000/09/29. 10.1006/anbe.2000.1430 PubMed DOI

Remold H. Scent-Glands of Land-Bugs, their Physiology and Biological Function. Nature. 1963;198(4882):764–8.

Blum MS. Chemical defenses of arthropods. New York & London: Academic Press; 1981.

Remold H. Über die biologische Bedeutung der Duftdrüsen bei den Landwanzen (Geocorisae). Z VERGL PHYSIOL. 1962;45(6):636–94.

Schwarz JJ, Gries G. 2-Phenylethanol: context-specific aggregation or sex-attractant pheromone of Boisea rubrolineata (Heteroptera: Rhopalidae). Can Entomol. 2010;142(05):489–500.

Blatt SE, Borden JH, Pierce HD Jr., Gries R, Gries G. Alarm Pheromone System of the Western Conifer Seed Bug, Leptoglossus occidentalis. J Chem Ecol. 1998;24(6):1013–31.

Zhang QH, Aldrich JR. Pheromones of milkweed bugs (Heteroptera: Lygaeidae) attract wayward plant bugs: Phytocoris mirid sex pheromone. J Chem Ecol. 2003;29(8):1835–51. Epub 2003/09/06. PubMed

Blum MS. Alarm pheromones In: Kerkut GA, Gilbert L. I., editor. Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol. 9 Oxford: Pergamon; 1985. p. 193–224.

Aldrich JR, Lusby W. R., Kochansky J. P., Hoffman M. P., Wilson L. T. and Zalom F. G.. Lygus bug pheromones vis-a-vis stink bugs. Brown JM, editor 1988. 213–5 p.

Analysis of defensive secretion of a milkweed bug Lygaeus equestris by 1D GC-MS and GC×GC-MS: sex differences and host-plant effect