CUTICULAR HYDROCARBON ANALYSIS OF THE AQUATIC BEETLE AGABUS ANTHRACZNUS MANNERHEIM (COLEOPTERA: DYTISCIDAE) Biology Department, Laurentian University, Ramsey Lake Road, Sudbury, Ontario. Canada P3E 2C6 HELENE JOLY and DANIELLE DENNIE Chemistry and Biochemistry Department, Laurentian University, Ramsey Lake Road, Sudbury, Ontario, Canada P3E 2C6 Abstract The Canadian Entomologist 130: 615 - 629 (1998) Relatively little information concerning the cuticular hydrocarbon composition of aquatic insects is known. The cuticular hydrocarbons of the aquatic beetle Agabus anthracinus Mannerheim have been identified with the aid of a gas chromatograph coupled to a mass spectrometer. The cuticular hydrocarbon profile comprises nalkanes (46.8%), n-alkenes (27.1%), and methylalkanes (25.9%) and is basically similar to that of terrestrial Coleoptera. However, the hydrocarbons of A. anthracinus differ in that (i) the shorter chain n-alkanes are present in higher proportion, (ii) there is a relatively lower abundance of methylalkanes, and (iii) the proportion of n-alkenes is significantly higher. Alarie, Y., H. Joly et D. Dennie. 1998. Analyse des hydrocarbures cuticulaires du coleoptkre aquatique Agabus anthracinus Mannerheim (Coleoptera : Dytiscidae). The Canadian Entomologist 130: 615-629. La composition en hydrocarbures cuticulaires des insectes aquatiques demeure inconnue. Cet expos6 identifie les hydrocarbures de la cuticule d'Agabus anthracinus Mannerheim, un colCoptkre aquatique, a l'aide d'un chromatographe en phase gazeuse coup16 a un spectromktre de masse. Les donnCes dCmontrent que la cuticule d'A. anthracinus est composCe de n-alcanes (46,8%), de n-alcknes (27,1%) et de mCthylalcanes (25,9%) ce qui est fondamentalement semblable B la condition observCe parmi les col6optkres terrestres. Les hydrocarbures d'A. anthracinus se distinguent cependant (i) par une plus grand abondance de n-alcanes B chaine courte, (ii) une moins grande abondance de mCthylalcanes et (iii) un pourcentage de n-alcknes significativement ClevC. Introduction The success of insects in colonizing a wide variety of environments is, in large measure, due to the impervious nature of their cuticle. Being small animals, with a relatively large surface to volume ratio, they require an external covering of low permeability to minimize passive movements of water between their body fluids and their environments (Noble-Nesbitt 1991). The cuticle of insects typically consists of three main layers, the inner endocuticle, the outer exocuticle, and the superficial epicuticle (Chapman 1982). Although other waterproofing mechanisms could be suggested, the most recent evidence supports the hypothesis that the insect epicuticular lipids (frequently referred to as surface or cuticular lipids), either independently or possibly in conjunction with other compounds, provide the principal barrier to water efflux through 616 THE CANADIAN ENTOMOLOGIST SeptemberIOctober the cuticle (Blomquist and Dillwith 1985; Noble-Nesbitt 1991; de Renobales et al. 1991; Hadley 1994). Cuticular lipids form the thin layer of apolar material of the outer surface of the insect cuticle. Among the cuticular lipids, hydrocarbons have received the majority of attention because of (1) their abundance on the surface of many insects, (2) the ease with which they are extracted, (3) the availability of standard techniques for their study, (4) their critical roles in restricting water loss, (5) their role as pheromones and kairomones, and (6) their potential usefulness as chemotaxonomic characters (Blomquist and Dillwith 1985; de Renobales et al. 1991; Gibbs 1995). Insect surface lipid hydrocarbons are usually a mixture of components. Besides the almost ubiquitous n-alkanes, complex mixtures of mono-, di-, tri-, and tetramethylalkanes, n-alkenes, mono- and dimethylmonoenes, dienes, trienes, and tetraenes have also been found in insect cuticle (Howard 1993). Terrestrial Coleoptera represent a good portion of the over 100 insect species whose cuticular hydrocarbons have been described (Lockey 1988; Howard 1993). All the species studied belong to diverse families of the suborder Polyphaga: Anobiidae (Baker et al. 1979b), Bostrichidae (Howard and Liang 1993), Cantharidae (Jacob 1978; Brown et al. 1988), Chrysomelidae (Malinski et al. 1986a, 1986b; Golden et al. 1992), Cucujidae (Howard 1992; Howard et al. 1995), Curculionidae (Baker and Nelson 1981; Baker et al. 1984; Nelson et al. 1984), Dermestidae (Baker et al. 1979a; Malinski et al. 1 9 8 6 ~Howard ; 1992), Scolytidae (Mody et al. 1975; Page et al. 1990a, 1990b; Howard and Infante 1996), Staphylinidae (Peschke and Metzler 1987), and Tenebrionidae (Bursell and Clements 1967; Hadley 1977, 1978; Hadley and Louw 1980; Lockey 1978a, 1978b, 1979, 1980, 1981, 1982a, 1982b, 1982c, 1984a, 1984b, 1985a, 1985b, 1988, 1991, 1992; Jackson et al. 1980; Lockey and Metcalfe 1988; Hebanowska et al. 1990). Despite their diversity, no aquatic Coleoptera have yet had their epicuticular hydrocarbon composition studied. In an evolutionary context, comparisons between both terrestrial and aquatic Coleoptera could prove interesting, since it is likely that the waterconservation needs of aquatic insects differ from those of their terrestrial counterparts (Howard et al. 1995). Because they live in air, internal water reserves of most terrestrial insects are continuously being lost to the environment. Freshwater insects, on the other hand, have to cope with the influx of water to maintain a positive water balance. As a result of these different needs, the following question arises: are the epicuticular hydrocarbon mixtures of aquatic Coleoptera different from those of terrestrial Coleoptera? Early literature suggests that terrestrial insects are more waterproof than aquatic ones, and that the latter have a lower surface lipid melting point (Wigglesworth 1945; Richards 1951). In a comparison between the naiad (aquatic) and adult (terrestrial) of the big stonefly Pteronarcys califomica Newport, Armold et al. (1969) showed that the adult has nearly twice as much extractable surface lipid as well as more hydrocarbons, sterol, and free fatty acids than those found on the naiad. Unfortunately, information on the cuticular composition of aquatic insects since the publication of Armold et al. is lacking. It would therefore seem premature to conclude whether the difference in the hydrocarbon composition between the two life stages of 1? califomica is an adaptive response to differing environments (Howard et al. 1995) or results from intraspecific differences that are known to occur during the ontogenetic development of a species (Lockey 1985~).Additional studies involving other aquatic insects are needed before these questions can be answered. The objectives of our study are (i) to analyze the epicuticular hydrocarbon composition of the adult of the predaceous water beetle Agabus anthracinus Mannerheim, a member of the family Dytiscidae (suborder Adephaga); and (ii) to provide a comparison with the epicuticular hydrocarbon mixtures found for other terrestrial Coleoptera. The Volume 130 ~ I CANAD~AN E E~TOMOLOGIST 617 Dytiscidae are an interesting group, as the adult stage is spent almost exclusively in water, which reflects a full adaptation to the aquatic habitat. They are quite abundant and diverse in freshwater habitats of the North Temperate Zone. Agabus anthracinus is generally collected along the shores of stagnant water, under dense aquatic vegetation and is one of the most widely distributed North American species of Agabus Leach. It ranges throughout the boreal zone, south in the east to West Virginia, and in the west, south at higher elevations to California, southern Utah, and northern Colorado (Larson 1989). Materials and Methods The Insects. Adults of A. anthracinus were trapped using the sweep-net method (Hilsenhoff 1991) on 9 May 1995 in a pond at the Laurentian University campus in Sudbury, Ontario. The insects were separated into six groups of five beetles according to their sexes (2 groups of 5 females each and 4 groups of 5 males each) and killed by freezing at -15°C. Voucher specimens have been deposited in the Research Collection of insects of the senior author, Laurentian University. Extraction of Cuticular Hydrocarbons. The cuticular hydrocarbons of a group of insects were extracted by immersing the five beetles in three successive baths of 5 mL of hexane (Caledon, Georgetown, Ont.) for 1 min per bath. After combining the three baths, the hydrocarbon-hexane mixture was added to a silica gel (100-200 mesh, Sigma Chemical Corporation, St. Louis, Missouri) minicolumn prepared in a Pasteur pipette and eluted with 24 mL of hexane. The eluant was then concentrated under a gentle stream of nitrogen and the residue was redissolved in 1 mL of hexane. Identification of Cuticular Hydrocarbons. Alkanes. A Varian Saturn I1 gas chromatograph - mass spectrometer (GC-MS, Varian Chromatography Systems, California), operating in electron impact mode at 70 eV, was used to analyze the cuticular hydrocarbons. The gas chromatograph was fitted with a Varian 1093 Septum-Equipped Programmable Injector (SPI) and SPB-1 nonpolar fused silica column (30 m x 0.25 mm, 0.25 pn film thickness; Supelco, Oakville, Ont.). Ultrapure helium was used as a carrier gas with a column head pressure of 12 psi (1 psi = 6.895 kPa). The SPI injector was programmed to hold at 40°C for 0.10 min, at which time the temperature was ramped from 40 to 280°C at a rate of 20O0C1min. After an initial 2min hold period, the column temperature was increased from 40 to 140°C at 10°C/min, then from 140 to 300°C at S°C/min, with a 5-min final hold period. The transfer line coupling the GC to the MS was maintained at 260°C. The temperature of the ion trap manifold was 220°C. Electron impact (EI) mass spectral scans ranging from mlz 40 to mlz 550 were recorded. The straight chain saturated hydrocarbons were identified by comparing their retention times with those obtained for commercially available n-alkanes (Aldrich Chemical Company, Milwaukee, Wisconsin). The assignments were confirmed by spiking the mixture with a solution of a known n-alkane and noting the intensity of the peak heights. An increase in peak height was a positive indication of the presence of that particular n-alkane. Alkenes. Although the El - mass spectral data aided in identifying which compounds in the cuticular hydrocarbon mixture were alkenes (McLafferty and Turecek 1993), the weak molecular ion necessitated that their identity be determined by an alternate method. 618 THE CANADLAN ENTOMOLOGIST SeptemberIOctober Dithiomethyl disulfide (DMDS) derivatives of the alkenes present in the hydrocarbon mixture were prepared by the method developed by Pepe et al. (1993) involving the derivatization of monounsaturated wax esters. Twenty microlitres of an ethereal iodine solution (0.023 M) was added to a test tube containing 100 pL of DMDS and 100 pL of the hydrocarbon mixture. This mixture was heated to 50°C for 48 h. Next, 220 pL of a 5% solution of Na2S20, was added to the test tube. At this point, the organic layer was recovered and the excess DMDS removed with the aid of a gentle stream of nitrogen. Finally, the residue was redissolved in 50 pL of CH2C12 and analyzed by GC-MS. The SPI injector and column programs were identical to those used in the alkane analysis with one exception; the oven temperature was maintained at 300°C for 15 min rather than 5 min. The mass scan range was extended to mlz 650. The EI - mass spectral data were used to identify the dithiomethyl disulphide derivatives, which in turn led to the identification of the parent alkene (Buser et al. 1983; Vincenti et al. 1987; Pepe et al. 1993). Methylalkanes. The monomethyl- and dimethylalkanes were separated from the hydrocarbon mixture by refluxing the hexane extract over dry molecular sieves (type 5A, Aldrich Chemical Company) for 6 h. (The molecular sieves were dried in an oven at 300°C for 3 days prior to use.) The refluxed hexane mixture was decanted into a vial. Next, the contents of the vial were reduced to dryness with a gentle stream of nitrogen and the residue was diluted with sufficient hexane for GC-MS analysis. The SPI injector temperature program was identical to that used in the analysis of the n-alkanes. The oven temperature program was modified. After an initial 2-min hold period at 40°C, the column temperature was increased from 40 to 140°C at 2O0C1min, then from 140 to 300°C at 2"C/min, with a 1-min final hold period. The transfer line was set at 260°C and the ion trap manifold temperature was lowered to 175°C. The column head pressure was reduced to 6 psi. Electron impact mass spectral scans ranging from rnlz 55 to mlz 550 were recorded. The methylalkanes in the cuticular hydrocarbon mixture were identified using the EI - mass spectral data and the criteria proposed by McCarthy et al. (1968), Nelson et al. (1972), Nelson (1978), and Pomonis et al. (1978, 1980). Statistical Analysis. Compositional analyses of the hydrocarbons were conducted using data collected from the total ion current (TIC) chromatogram. More specifically, area counts obtained from electronic integration of individual peaks (AC,) were summed and expressed as percentages using the following (Howard and Liang 1993): AC, X AC, xl00 x2 A test (Zar 1984) was used to determine the significance (P < 0.05) of the differences observed in the percent composition of the various samples collected from the male cuticle. Similarly, to establish if the differences found in the composition of the test with a Yates correction was female cuticular hydrocarbons were significant, a test with a Yates correction was applied to the data colused (P < 0.05). Finally, a lected from both male and female samples to determine if a significant difference (P < 0.05) existed in the composition of the cuticular hydrocarbons of the two sexes. x2 x2 Results A typical TIC chromatogram of the hydrocarbon mixture extracted from the cuticle of A. anthracinus is presented in Figure 1. The identity of the components of the mixture determined by (1) EI - mass spectral data, (2) the equivalent chain length Volume 130 fHE CANADIAN E~TOMOLOGI~ST 619 Retention time FIG. 1. Typical total ion current (TIC) chromatogram of the hydrocarbons in the cuticle of Agabus anthracinus. (ECL) (Miwa 1963), and (or) (3) a comparison of the retention times with those of standard samples is given in Table 1. Also included in Table 1 is the percent composition of the hydrocarbons calculated from the area counts of the individual peaks (see eq. [I]). Each value of percent composition is the average of five determinations. The hydrocarbon mixture contains 67 different compounds of which 64 can be assigned to one of the following classifications: n-alkanes (class A), alkenes (class B), terminally branched monomethylalkanes (class C), internally branched monomethylalkanes (class D), or dimethylalkanes (class E) (Table 2). The remaining three compounds are monomethylalkanes with a branch point at C,, C,, or C5. The exact position of the methyl branch for these compounds could not be determined because of insufficient mass spectral data. Therefore it is not possible to classify the compounds as class C or class D. This represents 2% of the total hydrocarbon mixture. The approximate composition of the insect cuticular hydrocarbons based on the above classification scheme is as follows: n-alkanes, 46.8%; alkenes, 27.1%; terminally branched monometbyla~kanes, 6.1%; internally branched monomethylalkanes, 15.1%; and dimethylalkanes, 2.7%. Straight Chain Alkane (Class A) Analysis. Identification of the straight chain alkanes in the hydrocarbon mixture was straightforward. The cuticular hydrocarbon mixture was spiked with small quantities of commercially available n-alkanes, C2nH2n+2, where n = 19-36. Changes in the appearance of the total ion chromatogram were noted on addition of the alkanes. Also type 5A molecular sieves remove straight chain hydrocarbons from complex mixtures. The disappearance of a GC peak (after treatment with molecular sieves), at a retention time assigned to a straight chain alkane, confirmed the original assignment. The cuticular n-alkanes identified with the aid of this technique September/October THE CANADIAN ENTOMOLOGIST TABLE1. Cuticular hydrocarbons of Agabus anthracinus GC peak NO.^ E C L ~ Composition (%) Hydrocarbon 9-c19:1 7-c19:1 n-nonadecane (C19) 8-c20:1 n-eicosane (Czo) 10-C20:l 9-C20:I 7-Czo:I n-heneicosane (Czl) 7- and 11-methylheneicosane n-docosane (C22) 3,9-dimethylheneicosane 9-, lo-, and 11-methyldocosane 9-c23:1 n-tricosane (C23) n-tetracosane (CZ4) 3,7-dimethyltricosane 11- and 12-methyltetracosane 3-methyltetracosane 9-c25:1 7-c25:1 n-pentacosane (Czs) 8-C26:1 11- and 13-methylpentacosane 4-methylpentacosane and 11,15-, 9,15-, and 7,15-dimethylpentacosane n-hexacosane (Cz6) 11- and 13-methylhexacosane 3-methylhexacosane 9-Cz7:1 7-c27:1 n-heptacosane (C27) 11- and 13-methylheptacosane 7- and 9-methylheptacosane 9-C28:1 8 - c ~I ~ : n-octacosane (CZ8) 4-methyloctacosane 9-c29:1 n-nonacosane (C29) 9-, 11-, and 13-methylnonacosane Volume 130 62 1 THE CANADIAN ENTOMOLOGIST TABLE1 (concluded) GC peak NO.^ E C L ~ Composition (%) Hydrocarbon 34 35 36 37 38 29.78 30.00 30.73 31.00 31.33 1.1 0.5 4.0 0.6 0.6 3-methylnonacosane n-triacontane (C30) 9-c31:1 n-hentriacontane (C31) 11- and 13-methylhentriacontane 40 41 42 32.00 32.73 33.00 0.4 1.3 0.4 n-dotriacontane (C32) 9-c33:1 n-tritriacontane (C33) 44 45 46 47 33.61 33.78 34.00 34.27 0.6 0.6 0.8 0.5 Terminally branched methyltritriacontaneC Terminally branched methyltritriacontaneC n-tetratriacontane (C34) Internally branched methyltetratriacontanec 49 50 51 52 53 35.25 35.55 35.74 36.00 36.38 0.7 0.8 0.8 0.7 0.7 Internally branched methylpentatriacontanec Internally branched methylpentatriacontanec Terminally branched methylpentatriacontanec n-hexatriacontane (C36) Internally branched hexatriacontaneC NOTE:The compounds with trace concentrations (tr) were not detectable in the original gas chromatogram. The alkenes were detected in the chromatogram of the dimethyldisulfide derivatives and trace quantities of monomethyl- and dimethylalkanes were detected after removal of the n-alkanes. aThe numbers refer to the peaks of the TIC chromatogram in Figure 1. Qquivalent chain length. CTheexact position of the methyl branches could not be determined because of insufficient mass spectral data. TABLE2. Classification of the cuticular hydrocarbons of Agabus anthracinus Compostion GC peak No: ECL~ (%) Hydrocarbon n-Alkanes (class A) 1 3 7 10 13 15 19 22 26 30 32 35 37 40 42 46 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00 33.00 34.00 6.0 0.8 2.9 1.0 12.0 1.4 6.5 1.6 7.8 1.9 1.5 0.5 0.6 0.4 0.4 0.8 n-nonadecane (CI9) n-eicosane (CZ0) n-heneicosane(CZ1) n-docosane (C22) n-tricosane (C23) n-tetracosane (C24) n-pentacosane (CZ5) n-hexacosane (CZ6) n-heptacosane (C27) n-octacosane (CZ8) n-nonacosane (CZ9) n-triacontane (C30) n-hentriac~ntane(C~~) n-dotriacontane (C3Z) n-tritriacontane (C33) n-tetratriacontane (C34) Diagnostic mass spectral fragments 622 SeptemberlOctober THECANADLAN WTOMOLOGIST TABLE2. (continued) Compostion GC peak NO.^ 52 ECL~ (%) Hydrocarbon 36.00 0.7 n-hexatriacontane (C36) Diagnostic mass spectral fragments Alkenes (class B) Terminally branched monomethylalkanes (class C) 9 - 21.65 22.63 24.66 26.69 29 27.75 34 29.78 39 31.72 4-monomethylalkanes (class C4) 26.65 28 27.68 28.69 5-monomethylalkanes (class C5) 27.52 0.4 tr tr 3-methylheneicosane 3-methyldocosane 3-methyltetracosane 2801281, 253 2941295, 265 3221323 tr 4.0 1.1 0.6 3-methylhexacosane 3-methylheptacosane 3-methylnonacosane 3-methylhenetriacontane 3501351, 323 3641365, 337 3921393, 365 4201421 tr 2.8 tr 4-methylhexacosane 4-methylheptacosane 4-methyloctacosane 3361337 3501351, 3221323 3641365 tr 5-methylheptacosane 3361337, 3081309, 84/85 Volume 130 623 THE CANADIAN ENTOMOLOGIST TABLE 2. (concluded) Compostion GC peak NO.^ ECL~ Unknown branch point 44 33.61 (%) Hydrocarbon 0.6 MethyltritriacontaneC Diagnostic mass spectral fragments Internally branched monomethylalkanes (class D) 8 21.40 0.3 11 22.38 0.3 14 16 23.37 24.37 1.0 0.5 20 25.41 3.4 7- and 11methylheneicosane 9-, lo-, and 11methyldocosane 11-methyltricosane 11- and 12methyltetracosane 11- and 13methylpentacosane 4-methylpentacosane 11- and 13methylhexacosane 11- and 13methylheptacosane 7- and 9methylheptacosane 9-, 11-, and 13methylnonacosane 11- and 13methylhentriacontane 11-methyltritriacontane Methyltetratriacontanec Methylpentatriacontanec Methylpentatriacontanec 1121113, 2241225, 168, 169 1401141, 1541155, 1681169, 1961197, 1821183 1681169, 1961197 1681169, 1821183, 2101211, 1961197 1681169, 1961197, 2241225 Dimethylalkanes (class E) Fragments 21 22.00 tr 24.00 25.75 tr 2.7 3,9dimethylheneicosane 3,7-dimethyltricosane 11.15-, 9.15.. and 7,15- 56157, 155, 1961197, 295 56157, 127, 2521253, 323 1121113, 1401141, 1681169, 239, 267, 295 NOTE:The compounds with trace concentrations (tr) were not detectable in the original gas chromatogram. The alkenes were detected in the chromatogram of the dimethyldisulfide derivatives, and trace quantities of monomethyl- and dimethylalkanes were detected after removal of the n-alkanes. aThe numbers refer to the peaks of the TIC chromatogram in Figure 1. b~quivaleutchain length. 'The exact position of the methyl branches could not be determined because of insufficient mass spectral data. 624 T I E CANADIAN ENTOMOLOGIST SeptemberIOctober included the homologous series n-C19 to n-C,, and n-C,,, with n-C2, (12%) the most abundant n-alkane. Three other odd-numbered n-alkanes, namely C19(6%), Czs (6.5%), and Cz7 (7.8%), occur in high proportions. Alkene Analysis. The relative increase in the abundance of the C,H2,-,+ and C,H2,+ ion series in the EI mass spectra (as compared with that observed for the alkanes) made identification of the alkenes in the hydrocarbon mixture relatively easy (Table 1). However, the weak molecular ion and the tendency for fragments to undergo random arrangements make it difficult to determine the exact structure of the alkene. As a result, the alkenes in the hydrocarbon mixture were derivatized with DMDS. The DMDS added to the double bond of the alkene. In general, the molecular ions of the DMDS derivatives were more stable than that of the parent alkene and were present in sufficient concentration to be counted. The molecular weight of the parent compound was calculated by substracting 94 from the mlz of the molecular ion of the derivative. The position of the double bond could also be determined from the mass spectral data of the DMDS derivative because fragmentation of the derivative occurs between the carbons bearing the thiomethyl groups to produce two very distinct fragment ions (Buser et al. 1983). The diagnostic mass spectral fragments for the DMDS derivatives are presented in Table 2. The alkenes of A. anthracinus comprise the monoenes of C19-C2,, C2,, C25-C29, C31,C3,, and C3? The odd-numbered monoenes are most prevalent, with 9-heptacosene (3.3%), 7-heptacosene (3.7%), 9-nonacosene (3.7%), and 9-hentriacontene (4.0%) occurring in the highest proportions. Of the 17 alkenes identified nine are 9-monoenes, three are 8-monoenes, and one is a 10-monoene. The compound with an ECL of 34.68 has a mass spectrum characteristic of an alkene; however, its DMDS derivative was not detected and therefore the position of its double bond could not be identified. Methylalkane Analysis. The methylalkanes, which make up 25.9% of the hydrocarbons found in the insect cuticle, are a mixture of mono- and dimethylalkanes. The position of the methyl branches in the long hydrocarbon chains were estimated with the aid of ECL (Miwa 1963) and EI mass spectral data (McCarthy et al. 1968; Nelson et al. 1972; Nelson 1978; Pomonis et al. 1978, 1980) The difficulty in assigning the exact position of the methyl branch arises, in part, from the fact that their concentrations are relatively low and they often elute with a number of positional isomers. The branched alkanes were separated from the n-alkanes with the aid of molecular sieves to increase the concentration of branched methylalkanes in solution. The diagnostic mass spectral fragments used to identify the methylalkanes are presented in Table 2. Terminally branched monomethylalkanes (class C). The terminally branched monomethylalkanes account for approximately 6.1% of the hydrocarbons of A. anthracinus. The 3-methylalkanes of C,,, C,,, C2,, C2@C27,C29, and C3, were identified. 3-Methylheptacosane was the most abundant (4%) methylalkane detected. Internally branched monomethylalkanes (class D). Internally branched monomethylalkanes of C,,-C,,, C,,, and C33-C36were detected in the cuticular hydrocarbon mixture of A. anthracinus. These methylalkanes represent 15.1% of the hydrocarbon mixture, and methyl branches have been detected at C,, C5, C7, and C9-C13 The oddnumbered methylalkanes are the most prevalent, with the 1l-methyl- and 13-methyl-C25 isomers occurring in the highest proportion (3.4%). The compounds labelled 47, 49, 50, and 53 have been tentatively assigned to internally branched monomethylalkanes of C,, C,,, and C3, based on the ECL. The GC peaks for these compounds were too poorly resolved to obtain comprehensive EI spectral data. It is worth noting that a complete homologous series of 4-methylalkanes ranging in carbon number from C25 to C2, were also detected, with 4-methylheptacosane (2.8%) being the most abundant. Volume 130 THE CANADIAN ENTOMOLOGIST 625 Dimethylalkanes (class E). Five dimethylalkanes were detected in the cuticular hydrocarbon mixture. Trace quantities of 3,9-dimethylheneicosane and 3,7dimethyltricosane were found to coelute with n-docosane and n-tetracosane, respectively. A complex mixture of C25isomers was found to elute at the same retention time. The EI mass spectral data were interpreted as the fragmentation pattern for 11,15-, 9,15-, and 7,15-dimethylpentacosane.These isomers represent 2.7% of the hydrocarbon mixture. Statistical Analysis. There were no significant differences in the composition of the hydrocarbon mixture of the male samples, the female samples, or the male and female samples. Discussion Recently, the cuticular hydrocarbons of the Coleoptera have been a subject of great interest. The epicuticular hydrocarbon mixtures of more than 100 terrestrial species representing 11 families have now been analyzed in detail. However, our study is the first report of the hydrocarbon profile of an aquatic beetle. The A. anthracinus hydrocarbon profile is similar to that of a number of terrestrial Coleoptera. Five hydrocarbon classes have been identified (Table 2). The predominance of saturated normal and branched alkanes is consistent with data obtained for other species of the order. In fact, only one exception to this trend has been reported (Howard 1992). In general, the epicuticular n-alkanes of A. anthracinus and terrestrial Coleoptera have chain lengths ranging from 19 to 35 carbon atoms, and the odd carbon number homologues of the series predominate. The difference between the cuticular hydrocarbon mixtures of A. anthracinus and terrestrial Coleoptera is the proportion of the n-alkanes. More specifically, while n-C,9, n-CZ3,n-C2,, and n-C,, are present in highest concentration in the A. anthracinus cuticle, n-C25,n-C2,, n-C2,, and n-C3, are often the most abundant components of the cuticular hydrocarbon mixtures of terrestrial Coleoptera (Lockey 1988). The cuticular hydrocarbons of A. anthracinus, as in the case for most of the Coleoptera investigated thus far, are a mixture of 3-methylalkanes and internally branched mono- and dimethylalkanes. The absence of 2-methyl-, tri-, and tetramethylalkanes was not surprising, as these compounds have only been identified in a few species of the Tenebrionidae (Lockey 1979, 1981, 1991, 1992). The chain length of the branched components of the A. anthracinus epicuticular hydrocarbon mixture ranges from 22 to 36 carbon atoms, which is similar to the range observed for terrestrial Coleoptera (23-42 carbon atoms). However, in many of the terrestrial Coleoptera studied, the concentration of branched alkanes was greater than that of the n-alkanes. This was not the case for A. anthracinus. The methyl branch of the internally branched monomethylalkanes found on A. anthracinus is generally positioned at an oddnumbered carbon atom (carbon 5, 7, 9, 11, and 13) (Table 2). In this regard, the epicuticular hydrocarbons of A. anthracinus are not exceptional, as methyl branches at odd carbon numbers are observed in other insects (Lockey 1985~).Dimethylalkanes represent only a small fraction of the total hydrocarbons observed in A. anthracinus epicuticule. Dimethylalkanes and long-chain, internally branched monomethylalkanes have been detected for the cuticular hydrocarbons of several species of Coleoptera (Mody et al. 1975; Hadley 1977; Baker 1978; Howard et al. 1978; Lockey 1978a, 1978b, 1980, 1982a, 1982b, 1984b, 1985a, 1985b, 1991, 1992; Baker et al. 1979a, 1979b, 1984; Baker and Nelson 1981; Pomonis and Haak 1984; Nelson et al. 1984; Peschke and Metzler 1987; Howard and Liang 1993; Howard and Infante 1996). 626 TIIE CANADIAY E!?TOMOLOGIST SeptemberIOctober The epicuticular hydrocarbon mixture of A. anthracinus contains a significant amount of unsaturated compounds (27.1%). Although alkenes have been detected in several distinct families, such high proportions are not typical of terrestrial Coleoptera. n-Alkenes have been found in the epicuticular lipids of some species of the Cantharidae (Jacob 1978), Cucujidae (Howard 1992; Howard et al. 1995), Curculionidae (Mody et al. 1975; Baker et al. 1984; Nelson et al. 1984), Dermestidae (Baker 1978; Baker et al. 1979a; Malinski et al. 1986a), Staphilinidae (Howard et al. 1978; Peschke and Metzler 1987), and Tenebrionidae (Lockey 1978a, 1979). The n-alkenes of A. anthracinus form an incomplete series ranging from n-C2, to n-C3, in which n-CZ5is the most abundant. The predominance of 9-enes (62.0%) in the alkene mixture must be viewed as unexceptional, as 9-enes have been observed in many diverse species of insects. However, the relatively high abundance of 7-enes (27.3%) is noteworthy, as these have been found in a significantly fewer number of insect species (Lockey 1988). To summarize, despite a close similarity with other terrestrial Coleoptera, the hydrocarbons of this species differ in that (i) the shorter chain n-alkanes are present in higher proportion, (ii) there is a relatively lower abundance of methylalkanes, and (iii) the proportion of alkenes is significantly higher. Agabus anthracinus occurs generally in shallow permanent lentic habitats within the boreal zone and usually lives in dense emergent vegetation, often where the water is cold (Larson 1989). Compared to its terrestrial counterparts, A. anthracinus does not have to contend with conditions that favor the rapid evaporative loss of water from the body. Morever, the epicuticle must provide a barrier to water influx to maintain a positive water balance. Because of its presence in a different ecological niche, one may wonder if the differences in the hydrocarbon profile of A. anthracinus reflect an adaptation to temperature or the aquatic habitat or both. The answer to that particular question is beyond the scope of this preliminary study. This problem can be approached experimentally by examining a closely related dytiscid species adapted to warmer habitat conditions or submitting specimens of A. anthracinus to various temperature regimes to examine any changes in their hydrocarbon profile. Acknowledgments We are deeply grateful to Ralph W. Howard (USDA-ARS U.S. Grain Marketing Research Laboratory, Manhattan, Kansas) for his comments on a previous version of this paper. Thanks are also due to Maria Kepes (Laurentian University) for her technical support in using the GC-MS. Financial support was provided by the Natural Sciences and Engineering Research Council of Canada in the form of separate research grants to YA and HJ and an undergraduate summer scholarship to DD. References Armold, M.T., G.J. Blomquist, and L.L. Jackson, 1969. Cuticular lipids of insects, 111. 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