Molecular Diversity of Compounds from Pygidial Gland Secretions of Cave-Dwelling Ground Beetles: The First Evidence

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Journal of Chemical Ecology ISSN 0098-0331 J Chem EcolDOI 10.1007/s10886-015-0593-7

Molecular Diversity of Compounds fromPygidial Gland Secretions of Cave-DwellingGround Beetles: The First Evidence

Nikola Vesović, Srećko Ćurčić,Ljubodrag Vujisić, Marija Nenadić,Gordana Krstić, Vesna Perić-Mataruga,Slobodan Milosavljević, et al.

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Molecular Diversity of Compounds from Pygidial GlandSecretions of Cave-Dwelling Ground Beetles: The First Evidence

Nikola Vesović1 & Srećko Ćurčić1 & Ljubodrag Vujisić2 & Marija Nenadić1 &

Gordana Krstić2 & Vesna Perić-Mataruga3 & Slobodan Milosavljević2 &

Dragan Antić1 & Boris Mandić2 & Matija Petković1 & Ivan Vučković2 &

Đorđe Marković1 & Maja Vrbica1 & Božidar Ćurčić1 & Slobodan Makarov1

Received: 24 March 2015 /Revised: 12 May 2015 /Accepted: 20 May 2015# Springer Science+Business Media New York 2015

Abstract Three adult cave-dwelling ground beetle specieswere induced to discharge secretions of their pygidial glandsinto vials. Dichloromethane extraction was used to obtainthe secretions. In total, 42 compounds were identified byGC/MS analysis. Pheggomisetes ninae contained 32 glandu-lar compounds, Laemostenus (Pristonychus) punctatus 13,whereas Duvalius (Paraduvalius) milutini had nine com-pounds. Caproic, oleic, palmitic, and stearic acids were pres-ent in the samples of all analyzed species. Undecane waspredominant in the extract of L. punctatus. Palmitic acid wasthe major component in the secretion of D. milutini. Finally,the most abundant compounds in P. ninae secretion wereheptacosene and nonacosadienes. Herein, we present the firstdata on the identification of pygidial gland secretion compo-nents in both troglophilous and troglobite cave-dwellingground beetles. Some compounds are reported for the firsttime in the secretions of ground beetles and other higher orlower taxa. The adaptation to underground life has not led toa reduction or changes in the chemical defense mechanismin the analyzed troglophilous and troglobitic Platyninae andTrechinae taxa.

Keywords Insecta . Coleoptera . Carabidae . Groundbeetles . Platyninae . Trechinae . Pygidial glands .

Compounds . Cave-dwelling animals .

Gas chromatography–mass spectrometry (GC/MS)

Introduction

The presence of pygidial glands in Carabidae is a commonfeature. These structures are localized on abdominal tips, dor-sally, above reproductive organs and laterally in relation to thehind gut. The pygidial apparatus consists of spherical or more orless elliptical gland lobes, a main collecting canal, and reservoir,which continues to a narrowed tube opening ventrally in theeighth abdominal segment. A great number of beetles producesmall organic compounds that transmit chemical messages.Compounds secreted by ground beetles can be sorted into thenine major chemical groups: hydrocarbons, aliphatic ketones,saturated esters, formic acid, higher saturated fatty acids, unsat-urated carboxylic acids, phenols, aromatic aldehydes, and qui-nones (Giglio et al. 2011; Moore 1979; Will et al. 2000).

The compounds of ground beetles from Europe have notbeen studied in detail. Additionally, there are no studies deal-ing with the chemicals released by cave-dwelling representa-tives of the Carabidae family and the Coleoptera order. Fur-thermore, secretions of endemic European ground beetle spe-cies rarely have been analyzed (Giglio et al. 2009). For thesereasons, we studied three species : Laemostenus(Pristonychus) punctatus (Dejean, 1828) (Platyninae:Sphodrini; inhabiting the Balkan Peninsula and CentralEurope), Pheggomisetes ninae S. Ćurčić, Schönmann,Brajković, B. Ćurčić and Tomić, 2004 (Trechinae: Trechini;inhabiting a few caves and pits on Mt. Vidlič, Serbia,

* Srećko Ćurčić[email protected]

1 Institute of Zoology, University of Belgrade - Faculty of Biology,Studentski Trg 16, 11000 Belgrade, Serbia

2 University of Belgrade - Faculty of Chemistry, Studentski Trg 12-16,11000 Belgrade, Serbia

3 Siniša Stanković Institute for Biological Research, University ofBelgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia

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stenoendemic), and Duvalius (Paraduvalius) milutini S.Ćurčić, Vrbica, Antić and B. Ćurčić, 2014 (Trechinae:Trechini; inhabiting a cave on Mt. Kalafat, Serbia,stenoendemic).

All chosen species are cavernicolous – a troglophile (it maycomplete its life cycle in a cave, but can also live outside ofcaves), and two troglobites (they cannot survive outside thecave environment). A sufficient number of both males andfemales can be collected easily from the caves over a year,where the populations are stable. The dietary preferences aresimilar, with all being invertebrate predators.

Cave-dwelling organisms exhibit spectacular adaptationsto their environment. It is interesting to investigate the ques-tion of chemical defense of ground beetles, which look moreor less adapted to cave conditions, from the least, L. punctatus,to the most adapted, P. ninae.

The aims of the current work were to identify chemicallythe released compounds in the three cave-dwelling groundbeetles, to compare the composition of the secretions withpreviously investigated surface-dwelling taxa, to checkwhether the underground way of life has influenced the com-position of the secretions and the number of the chemicals,and to search for possible new compounds that have not beenreported so far in representatives of the Carabidae family.

Methods and Materials

Collection and Handling of Ground Beetles Adult individ-uals of both genders of the three ground beetle species (10males and 10 females each) were collected from three cavesin southeastern Serbia. The specimens of L. punctatus werecollected from the Ogorelička Pećina Cave, the village ofSićevo, near Niš, Svrljiške Planine Mts. The specimens ofD. milutini were collected in the Samar cave system, thevillage of Kopajkošara, near Svrljig, Mt. Kalafat. The speci-mens of P. ninae were collected from the Petrlaška PećinaCave, the village of Petrlaš, near Dimitrovgrad, Mt. Vidlič.The ground beetles were stored in laboratory-controlled con-ditions in portable plastic refrigerators for a few days beforetaking samples of the pygidial gland content. These conditionsincluded a constant temperature (10 °C), total darkness, andmoist soil from the collecting site. Water was sprayed over thesamples and soil from time to time to maintain a high level ofhumidity, similar to that present in the underground habitats.The beetles were fed on earthworms and springtails collectedfrom the same cave.

Chemicals and Instrumentation The dichloromethane usedfor extraction was obtained from Merck (Darmstadt, Germa-ny). An Agilent 7890A GC system equipped with a 5975Cinert XL EI/CI mass selective detector (MSD) and a flameionization detector (FID) connected by capillary flow

technology through a 2-way splitter was used for analyses.Two different capillary columns were used: non-polar HP-5MSI (30m length, 0.25mm i.d., 0.25μm film thickness) and apolar HP-INNOWax (30 m length, 0.32mm i.d., 0.25μm filmthickness), both from Agilent Technologies (Santa Clara, CA,USA).

Chemical Extraction Gas chromatography–mass spectrome-try (GC/MS) sampling was performed in the laboratory atroom temperature. Ten males of each of the three groundbeetle species were milked into a 12-ml glass vial with dichlo-romethane (0.5 ml) for 10 min, and 10 females of each of thethree ground beetle species were treated in the same way.Beetles were forced to discharge their pygidial secretions bypinching the legs with pins. To eliminate the effects ofcomposition-altering oxidation and degradation of com-pounds, a portion of the extracts was subjected to gas chro-matography–mass spectrometric (GC/MS) analysis immedi-ately after preparation.

Chemical Analyses The GC and GC/MS analyses were per-formed in splitless mode. The injection volume was 1 μl, theinjector temperature was 240 °C, and the transfer line temper-ature was 280 °C for all analyses. The carrier gas (He) flowrate was 1.5 ml min−1 at 60 °C (constant pressure mode), andthe column temperature was programmed linearly in a rangeof 60–315 °C at a rate of 3 °C min−1, with a final 15-min holdfor analyses on the HP-5 MSI column. Analyses on the HP-INNOWax column were performed at a helium flow rate of1.5 ml min−1 at 60 °C (constant pressure mode). The oventemperature was programmed linearly in a range of 60–240 °C (3 °C min−1), then to 250 °C (10 °C min−1) with afinal 9-min hold. The FID detector temperature was 300 °C.EI mass spectra (70 eV) were acquired in the range of 40–550m/z, and the ion source temperature was 230 °C for allanalyses.

The library search and mass spectral deconvolution and ex-traction were performed using NIST AMDIS (AutomatedMass Spectral Deconvolution and Identification System) soft-ware, ver. 2.70 (identifications supported by data in Table 1and Fig. 1). We used retention index (RI) calibration dataanalysis parameters at a ‘strong’ level with a 10 % penaltyfor compounds without a RI for analyses on both polar HP-INNOWax and non-polar HP-5 MSI columns. The retentionindices were determined experimentally by the method of vanDen Dool and Kratz (1963). This method is based on theretention times of n-alkanes, which were injected after thesample under the same chromatographic conditions. Thesearch was performed against our own library containing 4,951 spectra, and the commercially available Adams04,Willey07, and NIST11 libraries containing approximately500,000 spectra.

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Table 1 Chemical composition of the pygidial secretions in three cave-dwelling ground beetle species from southeastern Serbia analyzed by GC-FIDand GC/MS

Peak Rt (min)a Compounds Relative percentage (%)b

Laemostenus (Pristonychus)punctatus

Duvalius (Paraduvalius)milutini

Pheggomisetesninae

1 4.3 Undecane 40.4 – 0.1

2 8.4 Tridecane – – 9.7

3 13.6 Acetic acid 0.6 – 1.2

4 15.3 Formic acid 19.4 – –

5 16.1 Benzaldehyde – – 0.5

6 16.8 Propionic acid – – 0.2

7 17.9 Isobutyric acid – – 0.2

8 20.2 Butyric acid – – 0.8

9 21.8 Isovaleric acid – – 2.0

10 22.1 Decyl acetate 0.2 – –

11 25.9 Undecyl acetate 0.2 – –

12 26.7 Isocaproic acid – – 0.6

13 28.1 Dodecan-1-ol 1.2 – –

14 28.3 Caproic acid Tc 13.1 Tc

15 29.7 Dodecyl acetate 34.2 – –

16 36.5 p-Cresol – – Tc

17 39.3 Pelargonic acid – 6.5 –

18 42.6 Capric acid – 2.5 –

19 45.3 7-Hexyldocosane 0.5 – –

20 46.2 9-Methyltetracosane 2.6 – –

21 47.2 Benzoic acid – 2.0 Tc

22 48.9 Lauric acid – 11.3 –

23 49.2 Pentacosane – – 1.1

24 49.9 Pentacosened – – 1.0

25 51.1 3-Ethyltetracosane – – 2.3

26 51.7 Hexacosane – – 6.3

27 54.7 Myristic acid – 10.0 1.3

28 55.3 11-Methylheptacosane – – 2.4

29 55.5 Heptacosened – – 13.9

30 56.1–56.4 Heptacosadienes(two isomers)d

– – 3.2(1.9+1.3)

31 57.0 Octacosane – – 3.8

32 60.1 Palmitic acid 0.5 30.3 8.9

33 60.6 Nonacosened – – 1.1

34 60.7–61.0 Nonacosadienes (at least three isomers)d – – 18.0

35 62.5 Nonacosatetraened – – 2.5

36 64.1 Nonacosapentaened – – 7.1

37 65.0 Stearic acid 0.1 11.9 1.5

38 65.9 Oleic acid 0.1 12.4 8.9

39 67.5 Linoleic acid – – 1.4

aObtained from GC/MS data on HP-INNOWax capillary columnb Percentages calculated from GC-FID peak areascTrace (less than 0.1 %)dThe exact positions of double bonds could not be determined

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Relative percentages of the identified compounds werecomputed from the corresponding GC-FID peak areas fromchromatograms obtained on an HP-INNOWax column.

Results

We identified a total of 42 compounds in the dichloromethaneextracts of the pygidial secretions of the three analyzed cave-dwelling ground beetle species on the polar HP-INNOWaxcolumn. The extracts of all the analyzed species each exhibit-ed different GC-FID and GC/MS patterns (Table 1, Fig. 1).

The extract of L. punctatus contained 13 compounds(Fig. 1a, Table 1), including a low-molecular-weight alkane,

two low-molecular-weight and a medium-molecular-weightcarboxylic acid, three C12-C14 acetate esters, an alcohol, twolong-chain hydrocarbons (alkanes), and three long-chain C16

and C18 fatty acids. The major components were undecaneand dodecyl acetate and to a somewhat lesser extent formicacid, while minor components included four carboxylic acids(acetic, oleic, palmitic, and stearic acids), decyl acetate,undecyl acetate, dodecan-1-ol, 9-methyltetracosane, and 7-hexyldocosane, as well as a trace amount of caproic acid.

The extract ofD. milutiniwas composed of nine carboxylicacids (Fig. 1b, Table 1), including four medium-chain C6-C12

fatty acids, an aromatic acid, and four long-chain C14, C16, andC18 fatty acids. Themajor component was palmitic acid, whilecaproic, oleic, stearic, lauric, and myristic acids were present

Fig. 1 GC-FID chromatograms of the dichloromethane pygidialsecretion extracts from adult cave-dwelling ground beetles Laemostenus(Pristonychus) punctatus (Dejean, 1828) (a), Duvalius (Paraduvalius)milutini S. Ćurčić, Vrbica, Antić & B. Ćurčić, 2014 (b), and

Pheggomisetes ninae S. Ćurčić, Schönmann, Brajković, B. Ćurčić &Tomić, 2004 (c). Ordinal numbers of peaks correspond to numbers inTable 1. Scale lines=1 mm

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in lesser amounts. The minor components were pelargonic,capric, and benzoic acids.

The highest number of chemical components of pygidialsecretion among the analyzed cave-dwelling ground beetlespecies was found in P. ninae. The extract of P. ninaecontained 32 compounds (Fig. 1c, Table 1), including twomedium-chain C11 and C13 hydrocarbons (alkanes), fiveshort-chain C2-C5 carboxylic acids, an aldehyde, twomedium-chain C6 fatty acids, a phenol, an aromatic carboxylicacid, 15 long-chain C25-C29 hydrocarbons (five alkanes and10 alkenes), and five long-chain C14, C16, and C18 fatty acids.The most abundant components were nonacosadienes (at leastthree isomers) and heptacosene, while tridecane, palmitic andoleic acids, nonacosapentaene, hexacosane, octacosane,heptacosadienes (two isomers), nonacosatetraene, 11-methylheptacosane, 3-ethyltetracosane, isovaleric, stearic,linoleic, myristic, and acetic acids, pentacosane, nonacosene,and pentacosene were present in lesser amounts in the extract.The minor components were butyric and isocaproic acids,benzaldehyde, propionic acid, isobutyric acid, and undecane.Caproic and benzoic acids and p-cresol were found in traceamounts.

We discovered a number of compounds novel to pygidialsecretions of ground beetles. We did not find significant dif-ferences in the amounts of secretion components between thegenders in the three investigated ground beetle species.

Discussion

The results presented here on the composition of the pygidialgland secretions of D. milutini and P. ninae represent the firstdata on the compounds in ground beetles belonging to thetribe Trechini and from troglobitic ground beetles. However,some compounds are known from other European epigeanTrechinae (Bembidiini): aldehydes, low-molecular-weightcarboxylic acids (both reported in the one Trechini speciesin our study as well), and aliphatic ketones (Schildknecht1970; Schildknecht et al. 1968a,b). Most compounds foundin the two Trechini species in our study (both presence oflow- and high-molecular-weight hydrocarbons, fatty acids,aromatic acids) seem to differ from those found in Bembidiini,despite the fact that the two tribes belong to the subfamilyTrechinae.

There are data regarding the substances released by certainPlatyninae ground beetles (chiefly Platynini), but they arescarce (Lečić et al. 2014). The combination of formic acidand alkanes is common for a few Platyninae taxa, both fromEurope and North America (Blum 1981; Lečić et al. 2014;Schildknecht et al. 1968a,b; Will et al. 2010). CertainPlatyninae contain additional compounds in the secretionsapart from the forementioned (alkenes, esters, fatty acids, ke-tones, etc.) (Bonacci et al. 2011; Will et al. 2010). In the case

of representatives of Sphodrini, formic acid and alkanes arecommon in all taxa (Lečić et al. 2014), whereas long-chainacetates and fatty acids are specific to some North AmericanCalathus species (Will et al. 2010). In two Calathus speciesfrom Europe, only formic acid and alkanes were recorded(Schildknecht et al. 1968a,b). The occurrence of long-chainacetates, fatty acids, and acetic acid is specific to a wider rangeof Sphodrini taxa, since these compounds have been describedfrom both Calathus ruficollis Dejean, 1828 and L. punctatus(Will et al. 2010; present study), whereas the presence offormic acid and alkanes is characteristic for most Platyninaeworldwide (Lečić et al. 2014). Some intergeneric differencesare observed among Calathus and Laemostenus representa-tives within Sphodrini: the latter contain a few additional sub-stances in their compounds (caproic acid, long-chain alcohol),and the compounds within the same class may vary (Lečićet al. 2014; Will et al. 2010; present study).

If we take into account comparisons of the compounds oftroglobitic cave-dwelling Trechini and surface-dwellingTrechinae (Schildknecht 1970; Schildknecht et al. 1968a,b),it is apparent that there are numerous differences in the chem-ical composition of their pygidial gland secretion, includingthe presence of hydrocarbons, fatty acids, and aromatic acidsand the absence of aliphatic ketones in the two species oftroglobitic cave-dwelling Trechini (present study). It is possi-ble that the aforementioned differences are mostly of amethodological-instrumental nature, since the previous analy-ses (Schildknecht 1970; Schildknecht et al. 1968a,b) wereperformed using less-sensitive methods. If we compare thesubstances of troglophilous L. punctatus with the surface-dwelling relative Sphodrini (a few Calathus taxa from Europeand North America) (Schildknecht et al. 1968a,b; Will et al.2010), we cannot see huge differences. Formic acid and alkanesare common for all the taxa (being either troglophilous orepigean), whereas the presence of long-chain acetates, fattyacids, and acetic acid is characteristic for both troglophilousand epigean taxa. Troglophilous L. punctatus contains severaladditional substances in the pygidial secretion (caproic acid,long-chain alcohol), and there are different compounds withinthe same class in the compared taxa. The number of com-pounds is almost equal to that in the troglophilous species(13) and its epigean relatives (up to 11) (Will et al. 2010).All the listed differences could be attributed to intergenericdifferences rather than to the way of life. The secretions ofinsects are evolving in response to local environmental condi-tions (Makarov et al. 2012).

The difference in the secretion content of the troglobiticand troglophilous ground beetles cannot be established at themoment since the taxa analyzed herein belong to separatesubfamilies (two troglobites to Trechinae, a troglophile toPlatyninae), but some general differences among Trechinaeand Platyninae can be noted. Namely, Trechinae (includingTrechini and Bembidiini) possess the following compounds

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in their secretions: aldehydes, low-molecular-weight carbox-ylic acids, aliphatic ketones, low- and high-molecular-weighthydrocarbons, fatty acids, and aromatic acids (Schildknecht1970; Schildknecht et al. 1968a,b; present study), whilePlatyninae are characterized primarily by the presence of acombination of formic acid and alkanes, but additional com-pounds also are found (alkenes, esters, acetic acid, fatty acids,alcohols, ketones, etc.) (Blum 1981; Bonacci et al. 2011; Willet al. 2010; present study).

Certain differences are observed in the released chemicalsof the two troglobitic ground beetle species – D. milutini andP. ninae, but they share a few common compounds (caproicand benzoic acids and long-chain fatty acids) (Table 1). Theaforementioned differences include the presence of threemedium-chain fatty acids solely present in D. milutini, andthe presence of short-chain carboxylic acids and alkanes, analdehyde, a medium-chain carboxylic acid, a phenol, numer-ous long-chain hydrocarbons (both alkanes and alkenes), anda long-chain fatty acid solely in P. ninae. In our opinion, thesedifferences may be intergeneric and illustrate the habits of thetwo analyzed taxa (Duvalius representatives lack aggressivescent fluids, while Pheggomisetes taxa possess them).

The selective pressure of predators in the investigatedcaves is lower than in surface habitats. These are inhabitedmainly by non-predator arthropods such as millipedes, spring-tails, and leiodid beetles, whereas other predators are scarce(e.g., bats, centipedes, rove beetles, harvestmen). Some mor-phological adaptations are characteristic for cave-dwellingtaxa, including reduction of both pigments and eyes, elonga-tion of appendages, the occurrence of long sensitive setae,which are accompanied by certain physiological and behav-ioral changes (Culver and Pipan 2009). Our assumption wasthat the number of released compounds in the studiedCarabidae species would be either lower or altered in compar-ison with epigean taxa. However, the hypothesis was not con-firmed, especially in the case of P. ninae, where a large num-ber of compounds were reported. The number can be treatedas an apomorphy rather than a plesiomophy. The conservatismof chemicals in arthropods can be seen primarily in the pres-ence of certain dominant components (e.g., quinones and cy-anogenic compounds in millipedes) (Makarov et al. 2012).The chemical consistency of the compounds was proven, atleast in the case of Platyninae, while the structure of thechemicals of Trechinae (especially Trechini) is relatively un-known and unexplored and will be the subject of future stud-ies. For the time being, we argue that the mechanism of secre-tion in Carabidae has a high level of conservation, so that theisolation, changes in selective pressure, or colonization of dif-ferent habitats have not greatly affected the character(Makarov et al. 2012).

Most of the reported differences in the substances releasedby the pygidial glands of the three Carabidae species wereobserved among the minor constituents. The adaptation to

underground life has not led to a reduction or changes in thechemical defense mechanism in the analyzed troglophilousand troglobitic Platyninae and Trechinae taxa.

Additional studies on a greater number of both higher andlower ground beetle taxa (including a re-analyses of the pre-viously observed taxa that were investigated some time ago)from different subfamilies are necessary in order to clarify thedifferences in the chemical content of ground beetles on aglobal level. Special attention should be paid to the cave-dwelling taxa and their chemicals, the content of which shouldelucidate the mutual relationships among the relative epigeantaxa. In subsequent studies, it would be good to get an idea ofthe absolute amounts of compounds in the gland extracts, aswell as to carry out experiments on the behavioral implicationof the identified secretions on potential predators. The latterexperiments are interesting and challenging because it is dif-ficult to simulate cave conditions in the laboratory.

Acknowledgments The study was financially supported by the SerbianMinistry of Education, Science, and Technological Development, Grants173038, 172053 and 173027.We are grateful to Prof. Dr. Vladimir Tomić(Institute of Zoology, University of Belgrade - Faculty of Biology), whohelped us with some of the photographs.

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