303 ISJ 14: 303-311, 2017 ISSN 1824-307X RESEARCH REPORT Comparison of the volatile compounds of Dermestes maculatus and Dermestes ater pupae: application of headspace solid-phase microextraction-gas chromatography- mass spectrometry (HS-SPME-GC/MS) M Cerkowniak 1 , MI Boguś 2 , E Włóka 2 , P Stepnowski 3 , M Gołębiowski 1 1 Laboratory of Analysis of Natural Compounds, Department of Environmental Analysis, Faculty of Chemistry, University of Gdańsk, ul. Wita Stwosza 63, 80-308 Gdańsk, Poland 2 Witold Stefański Institute of Parasitology of the Polish Academy of Sciences, Twarda 51/55, 00-818 Warszawa, Poland 3 Laboratory of Chemical Environmental Risks, Department of Environmental Analysis, Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland Accepted August 18, 2017 Abstract The headspace solid phase microextraction gas chromatography-mass spectrometry (HS-SPME- GC/MS) method was used for the determination of the volatile compounds of Dermestes maculatus and D. ater pupae. These beetles are of economic importance and they are a common pest of stored products and also serve as an intermediate host of the parasitic tapeworm, therefore an understanding of their biology is very important. Analyses of the volatile compounds of D. maculatus and D. ater pupae revealed differences between the insect species in their chemical composition. Sixteen volatile compounds of D. ater pupae, including 6 hydrocarbons, 5 fatty acids, 3 esters, and 2 aldehydes were identified. The major volatile compound in D. ater pupae was pentacosane. Ten compounds were present in < 10 % concentrations. A further five were present in < 1 % concentrations. A total of 39 compounds were identified in the D. maculatus pupae, including 28 esters of fatty acid, 4 fatty acids, 6 hydrocarbons and 1 aldehyde. Two volatile compounds were detected as major compounds: octadecadienoic acid methyl ester and octadecenoic acid methyl ester. A further eight were identified in smaller quantities (from 1.12 to 8.30 %) and the remaining volatile compounds were present in < 1 % concentrations. Key Words: Dermestes maculatus; Dermestes ater; headspace solid-phase microextraction; GC-MS Introduction Insects are of growing significance in agriculture, veterinary medicine, medicine and human healthcare. The black larder beetle, D. ater and the hide beetle, Dermestes maculatus, belong to the Dermestidae (Coleoptera) family. They are a common pests of stored products. D. ater feeds on various plant and animal products such as raw skin and hide, stored meat, cheese, tobacco, dried fish, copra, silk, wool, milk and dried museum specimens (Bujang and Kaufman, 2010; Siddaiah and Kujur, 2016). These beetles also serve as an intermediate host of Raillietina laticanalis and Choanotaenia ___________________________________________________________________________ Corresponding author: Marek Gołębiowski Laboratory of Analysis of Natural Compounds Department of Environmental Analysis Faculty of Chemistry University of Gdańsk ul. Wita Stwosza 63, 80-308 Gdańsk, Poland E-mail: marek.golebiowski@ug.edu.pl infundibulum in poultry (Bujang and Kaufman, 2010). The hide beetle, D. maculatus is a pest of the silk industry and it can damage stored animal products such as dried fish, cheese, hide, fur, bacon, and dog treats (Rajendran and Hajira Parveen, 2005; Shaver and Kaufman, 2009; Fontenot et al., 2015). D. maculatus damages the wood and insulation of poultry houses (Cloud and Collison, 1985), but they are commonly used to clean carcasses as a part of the skeletonization process (Museumpest.net, 2012) and the adult beetles are used to estimate the post mortem interval (Shaver and Kaufman, 2009; De Souza and Linhares, 1997). The volatile compounds of insects can be analyzed using many analytical techniques. For example, aggregation pheromones of the black larder beetle Dermestes haemorrhoidalis Kuster were analyzed by headspace gas chromatography- mass spectrometry (HS-GC/MS) and gas- chromatography with electroantennographic detection (GC-EAD) (Korada and Griepink, 2009). 304 Organic compounds of the egg surface of queen diploid and haploid eggs and worker haploid eggs of the honeybee were extracted in dichloromethane and analyzed by gas chromatography-mass spectrometry (Katzav-Gozansky et al., 2003). The main components of the external lipids of adult Aleyrodes singularis were extracted in chloroform and analyzed by gas chromatography-mass spectrometry (Soroker et al., 2003). Cuticular hydrocarbons in the ant Cardiocondyla wroughtonii were analyzed by solid injection and GC-MS (Turillazzi et al., 2002). The cuticular and internal n- alkane composition of Lucilia sericata larvae, pupae, male and female were separated by high- performance liquid chromatography with laser light scattering detector (HPLC-LLSD) and analyzed by gas chromatography-mass spectrometry with selected-ion monitoring (GC/MS-SIM) (Gołębiowski et al., 2012a). Thanks to the use of a number of analytical techniques, it is possible to identify many of the volatile compounds in insects (Cerkowniak et al., 2013). For example, hydrocarbons were identified in Heliothis virescens pupae, Helicoverpa zea pupae (Buckner et al., 1996), Aleyrodes singularis adult and exuviae (Nelson et al., 1998), Lucilia sericata larvae, pupae, male and female (Gołębiowski et al., 2012a), Chrysomya rufifacies larvae (Zhu et al., 2006), Calliphora vomitoria, Calliphora vicina and Protophormia terraenovae (Roux et al., 2008), Musca domestica (Butler et al., 2009), and Sitophilus granaries (Nawrot et al., 2010). Fatty acids were present in Heliothis virescens pupae, Helicoverpa zea pupae (Buckner et al., 1996), Eurosta solidaginis larvae (Nelson and Lee, 2004), Melanoplus sanguinipes adult, Melanoplus packardii adult (Soliday et al., 1974) and diapause and non-diapause larvae of Cydia pomonella (Khani et al., 2007). Aldehydes and alcohols were found in Heliothis virescens pupae, Helicoverpa zea pupae (Buckner et al., 1996), Aleyrodes singularis adult and exuviae (Nelson et al., 1998), Chorthippus brunneus males and females (Gołębiowski et al., 2016), Musca domestica larvae, pupae, male and female (Gołębiowski et al., 2012b), Scaptotrigona postica workers (Poiani et al., 2015) and the nymphs and exuviae of Bemisia argentifolii (Buckner et al., 1999). Esters were identified in Calliphora vomitoria larvae, pupae, male and female (Gołębiowski et al., 2013) and Acanthoscelides obtectus (Gołębiowski et al., 2008). Organic compounds in the cuticular lipids serve various functions in insects. They are the primary energy source, and a structural component of membranes. They are often involved in chemical communication such as pheromones, and in defense as components of defensive secretions, and as antimicrobial agents (Cakmak et al., 2007; Khani et al., 2007; Martins and Ramalho-Ortigão, 2012; Mann et al., 2013; Ottaviani, 2014; Cerkowniak et al., 2015; Kühbandner and Ruther, 2015; Nguyen et al., 2015). However, volatile compounds mainly serve as pheromones. Kairomones play a very important role in insect life. Kairomones are often used for host location, recognition and acceptance over shorter distances (Vet and Dicke, 1992; Vinson, 1998). These compounds are usually identified in host eggs, larvae, pupal cuticle, frass, silk, cocoons and glandular secretions (Afsheen et al., 2008). The role of host- related kairomones was investigated for example in the case of Hyphantria cunea (Drury), a host of the parasitoid Chouioia cunea Yang (Zhu, 2016). The study demonstrated that C. cunea is attracted to volatile kairomones from H. Cunea. Moreover, it has been shown a significant positive response of mated female C. cunea to 1-dodecene. Furthermore, the volatile compounds secreted by insects can be used to detect insects in stored grains. The VOCs were identified e.g. in Tribolium castaneum (red flour beetle) and Cryptolestes ferrugineus (rusty grain beetle) by headspace analysis (Senthilkumar et al., 2012). It was found that the amount of volatiles produced by T. castaneum adults increased with an increase in insect density in stored wheat. In our study, HS-SPME-GC/MS was used for the determination of the volatile compounds of D. maculatus and D. ater. Materials and Methods Rearing of D. ater and D. maculatus Both Dermestes species were kept at 25 o C with cyclic changes of light (L:D 12:12) in separate glass aquaria with a layer of wood shavings spread on the bottom and covered with mesh cloth to prevent insects escape. The beetles and larvae were fed beef meat ad libitum. Fully grown final instar larvae which ceased feeding were regularly isolated from the basic colonies and kept in glass jars until pupation in order to avoid slaughtering of larvae immobilized before pupation and naked pupae by younger larvae. Headspace solid-phase microextraction Polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber was used for the analysis of the volatile compounds extracted from D. maculatus and D. ater pupae - insects belonging to the Dermestidae family. For experiments,1-day-old pupae were used. Five insects of each species of Dermestidae (0.19 g D. maculatus and 0.17 g D. ater) were used for the analysis. The insects were placed in 4 ml glass vials prior to extraction. Next was added 4.8 µl of the undecane as standard and the capsule was capped. The samples were heated in a heating block. The best conditions for the HS-SPME-GC/MS were taken from literature (Cerkowniak et al., 2017). The extraction time was 50 min, the extraction temperature -105 °C, desorption time 10 min at 230 °C. Analyzes were repeated 3 times. Gas chromatography/mass spectrometry The volatile compounds of D. maculatus and D. ater pupae were analyzed on a GC/MS QP-2010 SE (Shimadzu) equipped with a fused silica Rtx-5 capillary column, 30 m x 0.25 mm i.d., and with a 0.25 µm thick film. The oven temperature of 80 °C was increased to 190 °C at a rate of 4 °C/min, isotherm at 190 °C for 10 min, then the temperature was increased from 190 °C to 300 °C at a rate of 6 °C/min. Helium was used as the carrier gas at a 305 column head pressure of 60 kPa and electron impact was applied to the ionization (70 eV). The transfer line and injector temperatures were maintained at 300 and 230 °C, respectively. The ion source was kept at 200 °C. The analysis of volatile compounds was carried out by GC/MS in the total ion current (TIC) mode. Results Seventeen and thirty-nine volatile compounds were identified in the pupae of D. ater and D. maculatus, respectively. Figs 1 - 6 give examples of the mass spectra of esters of fatty acid, fatty acids, aldehydes and hydrocarbons. Fig. 1 Mass spectrum of octadecanoic acid, methyl ester. Fig. 2 Mass spectrum of octadecenoic acid, ethyl ester. Fig. 3 Mass spectrum of octadecanoic acid, butyl ester. 306 Fig. 4 Mass spectrum of hexadecanoic acid. Fig. 5 Mass spectrum of hexadecanal. Fig. 6 Mass spectrum of pentacosane. 307 Table 1 Chemical composition of the volatile compounds found in pupae of D. ater D. ater No Relative content [%] Compounds 1. 0.50 Decanoic acid 2. 3.58 Dodecanoic acid 3. 3.72 Tetradecanoic acid 4. 2.24 Hexadecanal 5. 0.94 Tetradecanoic acid, 1-methylethyl ester 6. 8.45 Hexadecanoic acid 7. 0.66 Octadecanal 8. 0.62 Octadecenoic acid 9. 0.41 Hexadecanoic acid, butyl ester 10. 9.93 Tricosane 11. 1.71 Octadecanoic acid, butyl ester 12. 3.17 Tetracosane 13. 1.34 Pentacosene 14. 54.28 Pentacosane 15. 1.50 Hexacosane 16. 6.95 Heptacosane Chemical composition of the volatile compounds found in the pupae of D. ater Table 1 lists the volatile compounds identified in D. ater pupae. Among sixteen volatile compounds of D. ater pupae, 6 hydrocarbons, 5 fatty acids, 3 esters, and 2 aldehydes were present. The volatile compounds of D. ater pupae contained only 2 unsaturated compounds, among which were acid and hydrocarbon. The remaining compounds were saturated. The major volatile compound in D. ater pupae was pentacosane (54.28 %). The compounds occurring in smaller quantities (from 1.34 to 9.93 %) were: dodecanoic acid (3.58 %), tetradecanoic acid (3.72 %), hexadecanal (2.24 %), hexadecanoic acid (8.45%), tricosane (9.93%), octadecanoic acid butyl ester (1.71 %), tetracosane (3.17 %), pentacosanol (1.34 %), hexacosane (1.50 %) and heptacosane (6.95%). A further six were present in concentrations of <1%: decanoic acid, tetradecanoic acid 1-methylethyl ester, octadecanal, octadecenoic acid, hexadecanoic acid butyl ester and octadecenoic acid. Chemical composition of the volatile compounds found in the pupae of D. maculatus Table 2 lists the volatile compounds identified in D. maculatus pupae. Thirty-nine volatile compounds were identified in D. maculatus pupae. Among them, 28 esters, 4 fatty acids, 6 hydrocarbons and 1 aldehyde were present. The volatile compounds of D. maculatus pupae contained 14 unsaturated compounds, among which were 13 acids and only one hydrocarbon. Among the esters, 25 methyl, 2 ethyl, and 1 butyl ester were present. Two volatile compounds were detected as major compounds: octadecadienoic acid methyl ester (35.32 %), and octadecenoic acid methyl ester (26.74 %). A further eight were identified in smaller quantities (from 1.12 to 8.30 %): tetradecanoic acid methyl ester (2.80 %), tetradecanoic acid (1.12 %), hexadecenoic acid methyl ester (8.30 %), hexadecanoic acid methyl ester (7.67 %), hexadecanoic acid (2.26 %), octadecatrienoic acid methyl ester (5.80 %), eicosatetraenoic acid methyl ester (1.48 %) and pentacosane (2.55 %). The remaining volatile compounds were present in <1% concentrations. Comparison of the volatile compounds found in the pupae of D. ater and D. maculatus Analyses of the volatile constituents of D. maculatus and D. ater pupae revealed differences between the insect species in chemical composition. The following compounds present in D. ater larvae lipids were absent in D. maculatus larvae: decanoic acid, tetradecanoic acid 1-methylethyl ester, octadecanal, octadecenoic acid and hexadecanoic acid butyl ester. On the other hand, twenty eight compounds that were present in D. maculatus were absent in the lipids of the larvae of D. ater (Tables 1 and 2). Only eleven compounds were present in both insect species: dodecanoic acid, tetradecanoic acid, hexadecanal, hexadecanoic acid, tricosane, octadecanoic acid butyl ester, tetracosane, pentacosene, pentacosane, hexacosane, and heptacosane (Tables 1 and 2). Twenty five methyl esters were identified in the D. maculatus pupae, while only one ester was found in the D. ater pupae. The lipids of D. maculatus contained also four fatty acids, six hydrocarbons, two ethyl esters, one butyl ester and one aldehyde, while the lipids of D. ater contained also five fatty acids, six hydrocarbons, two butyl esters, two aldehydes and one ethyl ester. The major compounds in D. ater pupae were pentacosane (54.3 %), tricosane (9.9 %) and hexadecanoic acid (8.5 %), while the major compounds in D. maculatus pupae were octadecadienoic acid methyl ester (35.5 %), octadecenoic acid methyl ester (26.7 %), hexadecenoic acid methyl ester (8.3 %) and hexadecanoic acid methyl ester (7.8 %). 303 Table 2 Chemical composition of the volatile compounds found in pupae of D. maculatus D. maculatus No Relative content [%] Compounds 1. 0.02 Decanoic acid, methyl ester 2. 0.02 Nonanoic acid, 9-oxo-, methyl ester 3. 0.26 Dodecanoic acid, methyl ester 4. 0.08 Dodecanoic acid 5. 0.04 Tridecanoic acid, methyl ester 6. 0.02 Tridecanoic acid, 12-methyl-, methyl ester 7. 0.24 Tetradecenoic acid, methyl ester 8. 2.80 Tetradecanoic acid, methyl ester 9. 1.12 Tetradecanoic acid 10. 0.08 Pentadecanoic acid, methyl ester 11. 0.08 Tetradecanoic acid, 12-methyl-, methyl ester 12. 0.05 Hexadecanal 13. 0.22 Pentadecanoic acid, methyl ester 14. 0.75 Hexadecadienoic acid, methyl ester 15. 8.30 Hexadecenoic acid, methyl ester, 16. 7.67 Hexadecanoic acid, methyl ester 17. 0.23 Hexadecenoic acid 18. 2.26 Hexadecanoic acid 19. 0.24 Hexadecanoic acid, 14-methyl-, methyl ester 20. 0.57 Heptadecenoic acid, methyl ester 21. 0.07 Heptadecanoic acid, methyl ester 22. 5.80 Octadecatrienoic acid, methyl ester 23. 35.32 Octadecadienoic acid, methyl ester 24. 26.74 Octadecenoic acid, methyl ester 25. 0.72 Octadecanoic acid, methyl ester 26. 0.23 Octadecadienoic acid, ethyl ester 27. 0.17 Octadecenoic acid, ethyl ester 28. 1.48 Eicosatetraenoic acid, methyl ester 29. 0.70 Eicosatrienoic acid, methyl ester 30. 0.10 Eicosadienoic acid, methyl ester 31. 0.04 Tricosane 32. 0.11 Eicosenoic acid, methyl ester 33. 0.01 Eicosanoic acid, methyl ester 34. 0.17 Octadecanoic acid, butyl ester 35. 0.11 Tetracosane 36. 0.17 Pentacosene 37. 2.55 Pentacosane 38. 0.09 Hexacosane 39. 0.37 Heptacosane Discussion Chemical analyses of the volatile compounds of D. maculatus and D. ater pupae showed us that the esters of fatty acid are major compounds. The volatile compounds of D. maculatus and D. ater pupae contained 28 and 3 esters, respectively. Esters were identified in some other insect species. The cuticular lipids of Calliphora vomitoria larvae, pupae, males and females contained 6, 7, 5 and 7 fatty acid methyl esters (FAMEs) from C15:0 to C19:0, respectively (Gołębiowski et al., 2013). The isopropyl esters, including isopropyl dodecanoate, isopropyl (Z)-9-tetradecenoate, isopropyl tetradecanoate, isopropyl (Z)-9-hexadecenoate and isopropyl hexadecanoate were detected in male abdominal extracts of the black larder beetle, Dermestes haemorrhoidalis, which belongs to the Dermestidae (Coleoptera) family (Korada and Griepink, 2009). The cuticular extract of female Diaphorina citri contained three esters: ethyl undecanoate, isopropyl tetradecanoate and 1- methylpropyl dodecanoate (Mann et al., 2013). Fatty acids are present in many insect species. For example, in the larvae of the wheat blossom midge, Sitodiplosis mosellana, 10 - 16 kinds of fatty acids, of which the predominant compounds were palmitic, oleic and linoleic acids, which were more than 95 % of the total fatty acids (Jun-Xiang et al., 2001), can be observed. The cuticular extract of 309 female Diaphorina citri contained four acids: acetic acid, hexanoic acid, decanoic acid and pentadecanoic acid. The cuticular extract of male D. citri contained only two acids: dodecanoic acid and tetradecanoic acid (Mann et al., 2013). In the surface lipids of pupae of C. vomitoria were 23 carboxylic acids from C8:0 to C24:0. The major compounds were: C18:1 (37.2 %), C16:1 (27.3 %) and C16:0 (23 %). Interestingly, pupae and larvae of this species showed complete resistance to C. coronatus infection (Gołębiowski et al., 2013). In our work, we stated that the cuticular lipids of D. ater pupae contained five fatty acids from C10 to C18, and those of D. maculatus pupae contained four fatty acids from C12 to C16. In our study, two saturated aldehydes: hexadecanal and octadecanal, were identified in D. ater and only one aldehyde (hexadecanal) was present in D. maculatus. Aldehydes were identified in some other insect species. For example, the unsaturated aldehydes: (Z)-7-tetradecenal and (E)- 11-hexadecenal, are major sex pheromone components of Prays oleae (Lepidoptera: Yponomeutidae) and Palpita unionalis (Lepidoptera: Pyralidae) (Milonas et al., 2009). The saturated aldehydes: hexadecanal, (Z)-9-hexadecenal and (Z)-11-hexadecenal, were found in the gland extracts of Helicoverpa armigera (Wu et al., 1997). Aldehydes are also present in plants. For example, nonanal, decanal and (E)-2-hexenal are released from maize (Zea mays). GC-EAD analyses revealed a response from the Asian corn borer, Ostrinia furnacalis male and female to (E)-2-hexenal and nonanal (Huang et al., 2009). The following hydrocarbons from C23 to C27 were identified in D. ater and D. maculatus: tricosane, tetracosane, pentacosene, pentacosane, hexacosane and heptacosane, with the marked dominance of odd numbers of carbon atoms. Cuticular hydrocarbons are involved in various chemical communications in insects (Zhi-bin et al., 2000). They are typically found in many insect species. For example, the following homologous series were identified in the cuticular lipids of the Western Flower Thrips, Frankliniella occidentalis: n- alkanes from C25 to C29, 3-methylalkanes with 26 and 28 carbon atoms, and branched monomethyl alkanes with 26, 28 and 30 carbon atoms (Gołębiowski et al., 2007). Saturated hydrocarbons with a carbon chain length of 21 - 31 and six kinds of alkenes were identified in Chrysomya rufifacies larvae (Zhu et al., 2006). The extracts of elytra of the root weevil, Diaprepes abbreviatus contained four groups of homologous compounds: a group of normal hydrocarbons, a group of monomethyl- branched alkanes, a group of dimethyl-branched alkanes and alkenes (Lapointe et al., 2004). Composition of volatile organic compounds was investigated in larvae and pupae of blowfly (Frederickx, 2012). Using the SPME volatile collection and GC-MS analysis, approximately 90 compounds were identified in the three stages of larval development and the 10 stages of development of the pupae of Calliphora vicina. It helped to evaluate the age of flies, to establish a postmortem interval (PMI) in medical investigations. Especially because the composition of the volatile compounds of pupae and the compounds secreted by the larvae were significantly different The compounds secreted by larvae and the pupae were analyzed by an ascending hierarchical clustering (AHC). Pupae volatile profiles allow three groups to be distinguished: pupae of 1 - 3 days old; pupae of 4 - 7 days old and pupae of 8 - 10 days old. In the older pupae, from 1 to 3 days old, were identified: methyldisulphanylmethane and 4,7,7- trimethylbicyclo[3.1.1]hept-3-ene (alpha-pinene). The pupae from 4 to 7 days old were grouped in cluster because they emitted 3- methylbutanal, ethanol, methyldisulphanylmethane (dimethyldisulphide), methylsulphanyldisulphanylmethane (dimethyltrisulphide) and alpha-pinene. In VOCs of C. vicina pupae of 4 and 5 days old found 3- methylbutan-1-ol as characteristic compound. 6-day and 7-day-old pupae were grouped thanks acetic acid. In the last cluster were 8- to 10-day-old pupae. In this pupae as characteristic were identified: 2- methylpropan-1-ol, 1-methoxy-3-methylbutane, ethyl 3-methylbutanoate, 3-methylbutyl acetate and methyldisulphanylmethane (Frederickx, 2012). The composition of hydrocarbons in insect species is also used as an indicator of the postmortem interval. For example, n-alkanes, methyl-branched alkanes, and dimethyl-branched alkanes in Chrysomya megacephala were identified (Zhu, 2007). For most of the hydrocarbons with molecular weight below n-C26 the abundance decreased significantly with the weathering time, what can be useful in determine the PMI. Conclusions The HS-SPME-GC/MS method is simple and successful for the determination of the volatile compounds of D. maculatus and D. ater pupae. This technique allows the positive identification and quantitation of fatty acids, hydrocarbons, esters of fatty acids, and aldehydes. Using HS-SPME- GC/MS, sixteen volatile compounds of D. ater pupae, including 6 hydrocarbons, 5 fatty acids, 3 esters, and 2 aldehydes were identified, while 39 compounds were identified in the pupae of D. maculatus, including 28 esters of fatty acid, 4 fatty acids, 6 hydrocarbons and 1 aldehyde. 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