Comparative Biochemistry and Physiology Part B 129 Ž2001. 733᎐746
Hydrocarbons of Rhodnius prolixus, a Chagas disease vector Patricia Juarez ´ a,U , Gary J. Blomquist b, C.J. Schofieldc a
Instituto de In¨ estigaciones Bioquımicas de La Plata, CONICET-UNLP, Facultad de Ciencias Medicas, calles 60 y 120, ´ ´ La Plata, 1900, Argentina b Department of Biochemistry, Uni¨ ersity of Ne¨ ada, Reno, NV 89557, USA c Department of Infectious and Tropical Diseases, LSHTM, London WC1 E7HT, UK Received 6 December 2000; received in revised form 19 February 2001; accepted 22 February 2001
Abstract The surface hydrocarbons of the blood-sucking insect, Rhodnius prolixus, a major Chagas disease vector in Venezuela, Colombia and Central America, were characterized by capillary gas chromatography coupled to mass spectrometry ŽCGC-MS.. A total of 54 single or multicomponent peaks of saturated, straight-chain and methyl-branched hydrocarbons were identified. Major n-alkanes were n-C27, n-C29, n-C31 and n-C33 hydrocarbons. In the branched fraction, methyl groups were at positions 3, 5, 7, 11, 13, 15 and 17- for monomethyl isomers, and separated by three or five methylene groups for the trimethyl or tetramethyl derivatives. For the higher molecular weight components of 37, 39 and 41 atoms in the carbon skeleton, the di-, tri- and tetramethyl branches were usually separated by three or five, and sometimes 7, 11 or 13, methylene groups. The internal hydrocarbon pool contained larger amounts of the higher molecular weight methyl-branched components. Qualitative differences among epicuticular and internal hydrocarbon compositions were detected, both in adult and nymphal stages. No significant sexual dimorphism was detected, but a significant shift in the major n-alkane components was evident from the nymphal to the adult stage, differing also in the relative amounts of the higher molecular weight methyl-branched chains. Comparison of the hydrocarbon components to that of other Chagas disease vectors is discussed. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Rhodnius prolixus; Triatominae; Hydrocarbons; Cuticular lipids; Gas chromatography; Mass spectrometry
1. Introduction Rhodnius prolixus ŽHemiptera, Reduviidae, Triatominae. is the main vector of Chagas disease in Venezuela, Colombia and parts of Central America ŽSchofield, 2000.. The Central American populations of R. prolixus appear to be derived from accidental transport from Venezuela, and they show fewer random amplified polymorphic U
Corresponding author. Tel.: q54-221-4834833; fax: q54221-4258988. E-mail address:
[email protected] ŽP. Juarez ´ ..
DNA ŽRAPD. bands and a reduced size compared to their putative ancestral forms ŽDujardin et al., 1998.. As part of a larger project under the Latin American Network for Research on the Biology and Control of Triatominae ŽECLAT., the aim of this study was to determine the hydrocarbon structure, composition, developmental changes, and sexual dimorphism in R. prolixus. Capillary gas chromatography ŽCGC. and mass spectrometry ŽMS. have proven to be useful in chemical taxonomy for the differentiation of various species of Anopheles ŽCarlson and Service, 1979; Milligan et al ., 1986., tsetse flies ŽNelson
1096-4959r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 1 . 0 0 3 8 0 - 3
734
P. Juarez ´ et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 733᎐746
and Carlson, 1986., and different species of Triatominae ŽJuarez ´ and Brenner, 1985; Juarez ´ et al., in press., and these techniques are employed here. A long-term goal of this study is to determine the potential of these techniques to develop a biochemical marker useful for phylogenetic studies. A further objective is related to biological control studies, where the complex biochemical interactions between microbial pathogens and the cuticular hydrocarbons of their insect hosts are yet to be fully understood. The fungus Beau¨ eria bassiana has been proposed as a biological control agent against R. prolixus ŽRomana ˜ and Far. gues, 1992; Romana, 1996 . Various fungal strains ˜ have shown ability to degrade insect cuticular hydrocarbons, and the preferred alkane source was a hydrocarbon extract of Triatoma infestans, the major Chagas disease vector in Argentina ŽNapolitano and Juarez, 1997; Crespo et al., 2000; ´ . Juarez et al., in press . The characterization of R. ´ prolixus hydrocarbons is a first step towards developing comparative studies on the biochemical mechanisms involved in the initial stages of fungal infection for this vector.
2. Materials and methods 2.1. Insects We analyzed epicuticular and internal hydrocarbons from adult males, females, and nymphs of Rhodnius prolixus. Insects were provided by Dr Jose Jurberg from a colony reared at FIOCRUZ, Rio de Janeiro, Brazil. 2.2. Hydrocarbon analysis Cuticular hydrocarbons were extracted as previously described ŽJuarez and Brenner, 1986; ´ Juarez and Blomquist, 1993.. Individual insects, ´ or pooled insects when required for mass spectrometry analyses, were washed with redistilled water to remove any water-soluble contaminants, transferred to a glass vial with Teflon-lined caps, and submerged in redistilled hexane Ž6 mlrg, 5 min = 3. to extract epicuticular lipids. Internal hydrocarbons were obtained by immersing the previously extracted insects, decapitated, for 24 h in 5 ml of chloroformrmethanol Ž2:1 vrv. at room temperature with continuous stirring, then,
rinsed once with 2.5 ml of the same solvent. The extract was partitioned with redistilled water Ž1:5 vrv., the organic layer was isolated, taken to dryness, and then redissolved in 0.5 ml of hexane. This fraction contained hemolymph and tissue Žmainly integument. hydrocarbons. Both hexane extracts were reduced in volume under nitrogen, and then fractionated in a similar manner. Hydrocarbons from the samples were isolated with a Pasteur pipette mini-column of hexane-slurried Biosil A Ž5 = 5 mm2 ., and eluted with redistilled hexane Ž6 mlrmg hydrocarbon.. This final extract then was evaporated to an appropriate volume for gas chromatographic analysis ŽGC.. GC analysis was performed using a Hewlett Packard ŽHP. 6890 gas chromatograph equipped with a nonpolar capillary column Ž30 m= 0.32 mm i.d., 0.25 m film of HP-5.. The carrier gas was helium, at 11.36 psi with a linear velocity of 39 cmrs. The injector was operated in the splitless mode at 280⬚C. The oven temperature was programmed from 50 Žhold time 1 min. to 200⬚C at 50⬚rmin, then 200 to 310⬚C at 3⬚rmin Žhold for 20 min.. The flame ionization detector ŽFID. was held at 320⬚. The system was operated and data collected with a HP ChemStation. Samples were coinjected with n-alkane standards of 22᎐42 carbons for estimation of Kovats indices ŽKI., as described by Kovats Ž1965.. Alternatively, equivalent chain lengths ŽECL. of the methyl-branched alkanes were calculated from a regression derived from straight-chain hydrocarbon standards coinjected with the insect samples. The best fit corresponded to a cubic regression for chain lengths between C20 and C35, and a quadratic estimation for the longer chains eluting during the final isothermal period. Capillary gas chromatography-mass spectrometry ŽCGC-MS. analyses were performed on a Finnigan 4023 mass spectrometer interfaced with an INCOS data system. The CGC-MS system operated at 70 eV; GC conditions: 30 m= 0.32 mm DB-5, column temperature programmed from 200 to 280⬚C at a rate of 3⬚rmin. The carrier gas was helium at 8 psi head pressure, with a linear velocity of approximately 30 cmrs. Interpretation of mass spectra was performed as previously described ŽBlomquist et al., 1987.. The resulting identification showed close agreement with the corresponding KIs, as proposed by Carlson et al. Ž1998.. The nomenclature used to describe hydrocarbons was ŽCxx. to describe the total number of
P. Juarez ´ et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 733᎐746
carbons in the corresponding hydrocarbon component; the location of methyl groups is indicated by Žx-Me. for monomethylalkanes, Žx,x-Dime. for dimethylalkanes, Žx,x,x-Trime. for trimethylalkanes, and Žx,x,x,x,-Tetrame. when four methyl groups are located in the molecule.
3. Results All the major hydrocarbon components of the epicuticular waxes and internal lipids of Rhodnius prolixus were identified by CGC-MS analysis ŽTable 1.. A total of 54 components were identified, consisting of homologous series of n-alkanes, 3and 5- methylalkanes, single components, and isomeric mixtures of monomethylalkanes, dimethylalkanes, trimethylalkanes, and tetramethylalkanes. Fig. 1 shows the GC profiles of Rhodnius prolixus hydrocarbons from the epicuticle of adult males and females ŽFig. 1a,b.. Nymphal epicuticular hydrocarbons are shown in Fig. 1c᎐e. The internal hydrocarbons of adult males are shown in Fig. 2a and the corresponding internal hydrocarbons from nymph fifth instar in Fig. 2b, nymph third instar in Fig. 2c and nymph first instar in Fig. 2d. The n-alkanes consisted of a continuous series from C18 to C29, C31 and C33, with a prevalence of odd-numbered chains. n-C27 and n-C29 were predominant in the epicuticle of adult males and females, whereas n-C31 and n-C33 prevailed in the corresponding nymphal fraction. Even-numbered chains were present in small amounts, except for n-C28 Ž) 2% on adult cuticles .. Gas chromatographic profiles presented in this report were those of lab-reared insects. No major sexual dimorphism was detected in the adult stage ŽFig. 1a,b.; however, nymphs showed a distinctive hydrocarbon fingerprint, mainly caused by a switch in their major straight-chain alkanes ŽFig. 1c᎐e.. Internal hydrocarbon profiles showed a marked prevalence of the higher molecular-weight methyl-branched components over that of the nalkanes predominant on the insect surface. A variety of methyl-branched components were evident, usually present as isomeric mixtures. Four major groups of mono- or multiple-branched isomers with 35, 37, 39 and 41-carbon backbones were present. Terminal monomethylalkanes Ž3MeC27, 5-MeC29 and 5-MeC31. eluted 30 or 50 KI units in front of the corresponding
735
n-alkane on CGC, with a strong ion at M-29 for the 3-Me component, and an ion pair at m r z 84r85 for the 5-Me component. Monomethylalkanes with a branch on carbon 7 eluted 40 KI units in front of the equivalent n-alkane, while branching at 11, 13, 15, 17, 19 or 21 was detected as single or multiple-component peaks eluting approximately 70 KI units in front of the corresponding n-alkane. Characteristic strong m r z ion couplets corresponding to cleavage on either side of the methyl branch, were for example, 168r169, 196r197 and 224r225 for 11-; 13-; and 15-methylnonacosane, respectively. The multiple methylbranched alkane mass spectra were interpreted as described in Blomquist et al. Ž1987. and Page et al. Ž1997.. 5,15-Dimethylheptatriacontane was a major component of the branched fraction, the mass spectra showing strong m r z couplets at 84r85, 238r239, 336r337 and 490r491 ŽFig. 3a.. Only three other dimethyl-branched alkanes were detected: 5,15- and 7,11-dimethylpentatriacontane and, 5,17- dimethylhentetracontane; these were present in small amounts. The 5,Xseries with large methylene bridges, such as 5,15DimeC35 and 5,15-DimeC37, eluted ; 120 KI units in front of the corresponding n-alkane. Increasing the length of the carbon skeleton slightly shifted the KI shift units for 5,17-DimeC41. A variety of trimethylalkanes were evident, mostly corresponding to odd-chain isomers of the series 5,X,Y; 3,X,Y; 7,X,Y; 9,X,Y and 11,X,Y. The methylene bridges had intervals ŽI. between methyl groups of I s 3r3; 3r5; 3r7; 3r9 and 3r11. Multicomponent peak 19 contains a mixture of 9,13,19-; 9,15,19- and 11,15,19-trimethylC29. Some samples showed two peaks partially resolved. A mixture of 5,X,Y isomers of C29 usually co-eluted in peak 20, although in some samples the symmetrical 5,9,13- isomer separated from the 5,9,15- and 5,9,19- isomers. Peak 25 contained 7,11,21- and 7,11,25-trimethyl-C31, where the large methylene interruption ŽI s 3r9 and 3r13. increased the KI to 3203. The mass spectra yield a strong m r z couplet at 112r113 for the first methyl point at carbon 7, and couplets at 182r183, 168r169, 322r323 and 392r393 with significant odd-mass ion fragments arising from cleavage external to carbons 7, 11, 21 and 25. Peaks 30, 37, 46 and 53 contained other isomers of the 7,X,Y series from 35᎐41-carbon skeletons with methylene interruptions at I s 3r3,
P. Juarez ´ et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 733᎐746
736
Table 1 Hydrocarbons identified for Rhodnius prolixus Peaka
Kovats index
Hydrocarbon
Diagnostic ions
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2731 2740 2775 2800 2900 2931 2945 2952 2987
20
3009
21
3040
22 23 24 25
3100 3130 3152 3203
254 268 282 296 310 324 338 352 366 380 168r169, 252r253; 196r197, 224r225, 380 112r113, 308r309, 394 336r337, 364r365, 394 394 408 168r169, 280r281; 196r197, 252r253; 224r225, 422 112r113, 336r337, 422 84r85, 364r365, 422 140r141, 336r337, 210r211, 266r267; 140r141, 168r169, 238r239, 308r309, 336r337; 168r169, 238r239, 308r309, 450 84r85, 154r155, 224r225,252r253, 322r323, 392r393; 84r85, 154r155, 168r169, 308r309, 322r323, 393r393, 450 84r85,154r155, 168r169, 224r225, 268r269, 324r325, 336r337, 406r407; 84r85, 154r155, 168r169, 236r237, 252r253, 324r325, 336r337, 406r407, 464 436 168r169, 308r309; 196r197, 280r281; 224r225, 252r253, 450 84r85,392r393, 450 112r113, 168r169, 182r183, 322r323, 336r337, 392r393, 478
26
3240
27 28 29
3300 3569 3580
30
3590
31
3614
32
3628
33
3686
34
3719
35 36 37
3730 3777 3788
n-C18 n-C19 n-C20 n-C21 n-C22 n-C23 n-C24 n-C25 n-C26 n-C27 11-; 13-MeC27 7-MeC27 3-MeC27 n-C28 n-C29 11-; 13-; 15-MeC29 7-MeC29 5-MeC29 9,13,19-; 9,15,19-; 11,15,19-TrimeC29 5,9,15-; 5,9,13-; 5,9,19-TrimeC29 5,9,13,19-; 5,9,15,19TetrameC29 n-C31 11-; 13-; 15-MeC31 5-MeC31 7,11,21-; 7,11,25TrimeC31 7,11,17,21-; 7,11,17,25TetrameC31; plus other isomers n-C33 7,11-DimeC35 5,15 DimeC35; 11,15,21-TrimeC35 7,11,15-; 7,11,19TrimeC35 7,11,15,19-; 7,11,15,21-; 7,11,15,29TetrameC35 5,9,13,19-; 5,9,15,21-; 5,11,15,19TetrameC35 8,12,18-; 8,12,24-Trime C36 6,10,16,20-; 6,10,20,26TetrameC36 13-; 15-MeC37 5,15-DimeC37 7,11,19-TrimeC37
112r113, 168r169, 182r183, 280r281, 336r337, 352r353, 406r407; 112r113, 182r183, 238r239, 280r281, 336r337, 406r407 464 112r113, 182r183, 364r365, 434r435 84r85,238r239, 308r309, 462r463; 168r169, 224r225, 238r239, 322r323, 336r337, 392r393 112r113, 182r183, 252r253, 308r309, 378r379, 448r449 112r113, 182r183, 252r253, 322r323, 392r393, 462r463; 112r113, 182r183, 224r225, 252r253, 322r323, 350r351, 392r393, 462r463; 112r113, 182r183, 252r253, 322r323r 462r463 84r85, 154r155, 224r225, 252r253, 322r323, 350r351, 422r423, 490r491; 84r85, 180r181, 322r323, 392r393, 490r491 126r127, 196r197, 280r281, 296r297, 378r379, 449r450; 126r127, 196r197, 378r379, 449r450 98r99, 168r169, 252r253, 266r267, 322r323, 336r337, 420r421, 490r491; 98r99, 168r169, 266r267, 322r323, 420r421, 490r491 196r197, 364r365; 224r225, 336r337, 534 84r85, 238r239, 336r337, 490r491 112r113, 182r183, 280r281, 308r309, 406r407, 476r477
P. Juarez ´ et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 733᎐746
737
Table 1 Ž Continued. Peaka
Kovats index
Hydrocarbon
Diagnostic ions
38
3808
84r85, 154r155, 224r225, 364r365, 434r435, 504r505
39
3821
40
3865
41
3874
5,9,13-; 5,9,23TrimeC37 7,11,15,19-; 7,11,17,21TetrameC37 3,9,17,23-; 3,9,13,19-; 3,9,15,21-TetrameC37 8,12,18-; 8,12,20-; 8,14,18-TrimeC38
42
3906
43
3915
44
3920
45
3970
46 47 48
3982 4005 4031
49
4084
50
4104
51
4113
52 53
4163 4177
54
4244 a
8,12,18,22-; 8,16,22,28-; 8,12,20,26TetrameC38 6,10,14,20-; 6,10,24,28TetrameC38 15-; 13-; 17-; 19MeC39 9,13,19-; 9,15,19TrimeC39 7,11,19-TrimeC39 5,9,13 TrimeC39 5,9,13,21-; 5,9,13,23-; 5,9,17,25-TetrameC39 8,12,20-; 8,12,28TrimeC40 6,10,14,20-; 6,10,14,18-; 6,10,14,30TetrameC40, plus other isomers 17-; minor amounts of 19- and 21-MeC41 5,17-DimeC41 7,11,21-; 7,11,19TrimeC41; plus other Isomers 3,9,13,21-; 3,9,13,27TetrameC41
112r113, 182r183, 252r253, 280r281, 322r323, 350r351, 420r421, 490r491 154r155, 224r225, 280r281, 322r323, 378r379, 448r449, 518r519; 154r155, 252r253, 350r351, 448r449, 518r519 126r127, 196r197, 294r295, 308r309, 406r407, 476r477; 126r127, 196r197, 280r281, 322r323, 406r407, 476r477; 126r127, 224r225, 294r295, 308r309, 378r379, 476r477 126r127, 196r197, 252r253, 294r295, 322r323, 364r365, 420r421, 490r491; 126r127, 196r197, 252r253, 294r295, 322r323, 420r421, 490r491; 126r127, 196r197, 294r295, 322r323, 420r421, 490r491 98r99, 168r169, 238r239, 280r281, 336r337, 378r379, 448r449, 518r519; 98r99, 168r169, 238r239, 378r379, 448r449, 518r519 224r225, 364r365; 196r197, 392r393; 224r225, 336r337; 280r281, 308r309; 562 140r141, 210r211, 308r309, 406r407, 476r477; 140r141, 238r239, 308r309, 378r379, 476r477 112r113, 182r183, 308r309, 434r435, 504r505 308r7309, 462r463, 532r533 84r85, 154r155, 224r225, 280r281, 350r351, 406r407, 476r477, 546r547; 84r85, 154r155, 224r225, 252r253, 378r379, 406r407, 476r477, 546r547 126r127, 196r197, 308r309, 322r323, 434r435, 504r505; 126r127, 196r197, 434r435, 504r505 96r97, 168r169, 238r239, 308r309, 336r337, 406r407, 476r477, 546r547; 96r97, 168r169, 238r239, 406r407, 476r477, 546r547
252r253, 364r365; 280r281, 336r337; 308r309 84r85, 266r267, 364r365, 546r547 112r113, 182r183, 308r309, 336r337, 462r463, 532r533
154r155, 224r225, 308r309, 350r351, 434r435, 504r505, 574r575; 154r155, 224r225, 434r435, 504r505, 574r575
Peak numbers refer to peaks marked in Figs. 1 and 2.
3r7 and 3r9. The expected shift in KIs according to chain-length increase is evident in peak 53, with a KI of 4177. The most abundant trimethylisomer in adult R. prolixus corresponded to a mixture of 9,13,19-, as the main isomer, with smaller amounts of 9,15,19-TrimeC39 eluting together as peak 45 with a KI of 3970. Fig. 3b shows the mass spectral fragmentation pattern with a strong ion at m r z 140r141 with an evenrodd ratio ) 1, indicating cleavage external to methyl-branched carbon 9. Ions at 308r309 with an evenrodd ratio ; 1 indicated fragmenta-
tion at both sides of the branched carbon 19. Couplets at m r z 210r211, 406r407 and 476r477 with increasing prevalence of the odd-mass fragments indicated cleavage external and internal to branched carbon 13 and internal to branched carbon 9 for the major isomer. Peak 29, eluting at KI 3580, contained the internally branched 11,15,21-TrimeC35, together with 5,15-DimeC35. Minor components of the trimethylalkane fraction with even-numbered chains of 36, 38 and 40 carbons of the series 8,X,Y were also detected, with methylene bridge interruptions at I s 3r5;
738
P. Juarez ´ et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 733᎐746
Fig. 1. Capillary GC profiles of the epicuticular hydrocarbons of Rhodnius prolixus: Ža. adult males; Žb. adult females; Žc. nymphal fifth instar; Žd. nymphal third instar; and Že. nymphal first instar. Numbers indicating each hydrocarbon peak are indicated in Ža., and correspond to peak numbers from Table 1.
P. Juarez ´ et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 733᎐746
739
Fig. 2. Capillary GC profiles of the internal hydrocarbons of Rhodnius prolixus: Ža. adult males; Žb. nymphal fifth instar; Žc. nymphal third instar; and Žd. nymphal first instar. Numbers as indicated in Fig. 1.
740
P. Juarez ´ et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 733᎐746
Fig. 3. Mass spectra of: Ža. GC peak 36 containing 5,15-dimethyl-heptatriacontane from R. prolixus; Žb. peak 45 containing 9,13,19- and minor amounts of 9,15,19-trimethylnonatriacontane; and Žc. peak 26 with 7,11,17,21- and 7,11, 17,25-tetramethylhentriacontane.
3r7; 3r11; 3r15 and 5r3. The most abundant corresponded to a mixture of 8,12,18-; 8,12,20and 8,14,18-TrimeC38, eluting between 214 and 226 carbon units before the corresponding n-alkane. A variety of tetramethylalkane components were also detected belonging to the series 5,X,Y,Z; 7,X,Y,Z and 3,X,Y,Z, with the oddcarbon numbered starting at the C29 carbon backbone; whereas for the less abundant evennumbered chains, 6,X,Y,Z and 8,X,Y,Z series were identified. For adults, the most abundant series corresponded to the 5,X,Y,Z, with methylene interruptions at I s 3r3r5 and 3r5r3 for TetrameC29 isomers Žpeak 21. and I s 3r3r7; 3r3r9 and 3r7r7 for isomers eluting at peak 48. The TetrameC29 isomers eluted approximately 31 KI units after the corresponding trimethylalkane Žpeak 20., whereas the TetrameC39 isomers Žpeak48. eluted ca. 26 KI units after the trimethyl component Žpeak 47.. In the 7,X,Y,Z series, peak 26 contained 7,11,17,21- and 7,11,17,25-TetrameC31 with I s 3r5r3 and 3r5r5, eluting 39 KI units after the corresponding TrimeC31 peak ŽFig. 3c.. The KI shift is lower for the TetrameC35 isomers with branching at 7, 11, 15 and 19, 21 or 29, and also for the TetrameC37 isomers Žpeak 39.. Terminally branched tetramethylalkanes were detected in peaks 40 and 54, with an asymmetrical branching pattern as 3,9,17,23-; 3,9,13,19- and 3,9,15,21-TetrameC37 isomers with I s 5r7r5, 5r3r5 and 5r5r5, whereas 3,9,13,21- and 3,9,13,27-TetrameC41 eluted at KI 4244. Minor components with even-numbered backbones of 36, 38, and 40 carbons had a predominant branching pattern starting at 6, 10 and the third and fourth branch at carbon 14᎐30, with a wide range of methylene interruption distribution; none of the corresponding trimethyl components were detected. Finally, a mixture of 8,12,18,22-; 8,16,22,28and 8,12,20,26-TetrameC38 Žpeak 42. eluted 32 KI units after the corresponding trimethylalkane isomers Žpeak41.. CGC analysis of the epicuticular hydrocarbons of individual adult male or female insects showed a slight variability among specimens of each sex ŽTable 2.. Sexual differences were significantly different only for the major peaks, n-C27 and n-C29 Ž P- 0.001, according to Tukey᎐Kramer’s test. and for peak 40 Ža mixture of 3,9,17,23-;
Table 2 Percent hydrocarbons from selected components of Rhodnius prolixus Peak
Hydrocarbona
CN b
Epicuticle c
Internal d
Adults
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
31
32
33 34
Nymphal instar
Firstd
Male
First
Third
Fifth
0.00 0.23 0.06 0.78 0.26 4.17 0.00 0.00 0.39 0.32 3.58 0.15 0.15 1.78 0.26 0.27
0.00 0.09 0.03 0.18 0.02 0.57 0.00 0.00 0.04 0.06 0.13 0.10 0.09 0.85 0.06 0.04
0.38 0.41 0.62 1.23 0.65 3.92 0.53 0.25 Trace 1.08 3.77 0.28 Trace 2.80 0.41 0.46
Trace Trace Trace 0.32 0.12 4.20 Trace Trace Trace 0.66 4.36 1.14 0.07 1.26 0.91 1.26
Trace Trace 0.10 0.98 0.18 3.18 Trace Trace 0.77 0.46 3.03 Trace Trace 3.60 0.55 0.88
0.07 0.88 0.18 0.67 0.18 5.65 0.42 0.28 0.49 0.28 3.15 0.29 0.18 2.05 0.43 0.76
Trace 0.11 Trace 1.12 0.27 4.59 0.15 Trace 0.15 0.47 3.31 0.26 Trace 0.60 0.14 0.51
0.08
0.55
0.10
0.86
1.22
0.77
0.52
1.15
9.64 0.41 0.11 0.88 0.13
0.73 0.02 0.03 0.09 0.02
12.57 0.35 0.17 0.49 0.10
1.08 0.11 0.11 0.07 0.01
11.36 0.50 0.31 2.01 0.46
2.45 1.60 0.36 1.62 1.34
5.28 0.68 0.00 1.36 0.87
3.82 0.45 0.19 1.10 0.47
7.81 0.71 0.19 1.76 0.23
0.34 0.09 0.07 0.12 0.13
15.00 0.11 0.27 0.82 2.66
0.03 0.04 0.07 0.05 0.04
24.63 0.73 0.20 0.57 0.78
2.75 0.12 0.02 0.08 0.12
22.83 Trace 0.73 1.09 1.26
1.13 0.24 0.33 2.30 2.73
7.73 0.00 0.93 1.36 1.58
8.02 1.93 1.16 2.63 0.28
10.92 0.22 1.41 1.60 2.18
1.40
0.08
0.40
0.03
0.30
0.03
0.39
0.94
0.47
1.17
1.14
0.04
1.26
0.14
0.47
0.06
0.88
0.04
0.97
1.84
0.99
0.52
1.72
0.91
0.11
1.62
0.16
0.40
0.05
0.30
0.08
1.09
0.26
1.03
1.56
2.34
1.22 4.65 2.47
0.04 0.70 0.28
1.47 4.35 3.32
0.26 0.84 0.35
0.58 5.17 2.31
0.02 0.86 0.10
0.33 5.23 2.50
0.12 0.26 0.08
1.01 6.93 3.09
1.90 9.70 3.43
1.08 8.67 3.76
0.73 6.92 3.74
0.79 6.49 1.07
Female
S.E.M.
Fifth
S.E.M.
22 23 24 25 26 27 28 28 28 28 29 30 30 30 32 32
0.48 0.32 0.44 1.03 0.90 11.49 0.57 0.52 0.71 2.46 14.74 0.67 0.54 0.72 0.67 0.76
0.03 0.16 0.20 0.37 0.35 0.37 0.11 0.08 0.16 0.42 0.75 0.08 0.28 0.44 0.16 0.12
0.34 0.42 1.15 0.57 0.52 7.58 0.46 0.32 0.26 2.69 10.65 1.54 0.06 0.84 0.52 0.57
0.01 0.05 0.57 0.16 0.12 1.02 0.23 0.11 0.17 0.27 1.29 0.31 0.06 0.22 0.12 0.07
0.00 0.31 0.30 0.15 1.08 4.89 0.30 0.11 0.10 0.44 3.97 0.15 0.00 0.43 0.49 0.30
0.00 0.06 0.08 0.04 0.10 0.21 0.01 0.01 0.01 0.01 0.16 0.02 0.00 0.03 0.03 0.03
33
1.31
0.11
1.79
0.19
0.79
31 32 32 34 35
5.85 1.07 0.45 1.82 1.15
0.16 0.11 0.04 0.41 0.16
5.99 2.13 0.38 2.03 1.12
0.72 0.29 0.13 0.14 0.16
33 37 38 38 39
2.63 0.37 0.71 1.59 2.09
0.14 0.11 0.11 0.18 0.24
2.73 0.48 0.87 2.44 2.47
39 39
0.58
0.12
39
0.89
40 38 39 40
Third
741
35 36 37
n-C22 n-C23 n-C24 n-C25 n-C26 n-C27 11-; 13-MeC27 7-MeC27 3-MeC27 n-C28 n-C29 11-;13-; 15-MeC29 7-MeC29 5-MeC29 9,13,19-Trimec29 5,9,15-; 5,91,13-; 5,9,19-TrimeC29 5,9,13,19-; 5,9,15, 19- TetrameC29 n-C31 11-; 13-; 15-MeC31 5-MeC31 7,11,21-TrimeC31 7,11,17,21-; 7,11, 17,25-TetrameC31 n-C33 7,11-DimeC35 11,15,21-TrimeC35 7,11,15-TrimeC35 7,11,15,19-; 7,11, 15,21; 7,11,15, 29- TetrameC35 5,9,13,19-; 5,9,15,21-; 5,11,15,19TetrameC35 8,12,18-; 8,12,24TrimeC36 6,10,16,20-; 6,10, 20,26-TetrameC36 13-; 15-MeC37 5,15-DimeC37 7,11,19-TrimeC37
Adults S.E.M.
S.E.M.
P. Juarez ´ et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 733᎐746
5 6 7 8 9 10 11 12
Nymphal instar
Male
742
Table 2 Ž Continued. Peak
Hydrocarbona
CN b
Epicuticle c
Internal d
Adults
38 39 40
41 42
43 44 45 46 47 48
49 50
51
52 53 54
a
d
Nymphal instar
Male
First
Third
Fifth
Male
S.E.M.
Female
S.E.M.
Fifth
S.E.M.
Third
S.E.M.
First
40
1.23
0.13
1.73
0.24
1.49
0.15
1.20
0.08
1.10
1.06
1.32
1.39
Trace
41
3.13
0.39
3.70
0.30
5.00
0.07
3.49
0.11
3.59
6.32
4.68
5.84
6.58
41 41
2.11
0.20
4.25
0.46
3.17
0.21
2.38
0.17
2.25
3.35
2.90
3.35
1.96
41
1.21
0.19
1.16
0.19
1.85
0.08
0.86
0.13
2.03
1.39
1.94
0.71
1.45
42
0.56
0.19
0.42
0.13
1.04
0.07
0.13
0.06
0.67
1.10
1.21
0.92
1.25
42
0.40
0.09
0.73
0.08
0.90
0.01
1.47
0.11
0.56
0.94
1.80
0.69
1.15
40
0.66
0.05
0.60
0.24
0.42
0.03
0.42
0.08
0.60
0.86
0.96
0.95
0.58
42
4.36
0.80
3.27
0.59
5.65
0.43
4.60
0.51
6.20
7.13
10.72
8.54
6.37
42 42 43
0.65 1.55 1.86
0.20 0.25 0.26
0.73 1.33 2.43
0.25 0.38 0.52
1.16 2.25 3.80
0.05 0.37 0.35
0.27 2.31 3.20
0.11 0.21 0.38
1.44 1.90 3.06
1.49 2.68 3.43
1.26 1.85 3.08
1.68 1.60 3.26
1.84 2.75 2.78
43
0.36
0.09
0.33
0.12
0.44
0.09
0.24
0.10
0.35
0.76
0.48
0.84
0.71
44
0.33
0.08
0.43
0.17
0.62
0.02
0.32
0.05
0.45
0.35
0.64
0.58
0.25
42 43 44
0.61 1.70 0.65
0.29 0.20 0.18
0.00 1.03 0.58
0.00 0.34 0.22
0.19 1.24 0.88
0.02 0.22 0.08
0.04 1.45 0.80
0.02 0.28 0.18
0.26 2.27 0.70
0.32 1.71 1.31
0.47 2.10 0.96
0.46 2.04 0.46
0.16 2.38 1.05
45
0.88
0.14
0.81
0.17
0.79
0.09
0.91
0.09
0.77
1.06
1.33
0.60
1.06
0.86
3.45
1.87
1.70
2.90
0.67
1.00
0.77
0.55
Hydrocarbon and peak numbers are the same as reported in Table 1 and Figs. 1 and 2. Trace amounts Ž- 0.1% . of C18᎐C22 are not included. Carbon number. c Individual adult males Ž n s 6 ., females Ž n s 4 .; pooled nymphs Ž10 i, n s 3 .. d Pooled insects Ž15 i, n s 2 .. b
Adults
P. Juarez ´ et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 733᎐746
5,9,13-; 5,9,23TrimeC37 7,11,15,19-; 7,11,17,21TetrameC37 3,9,17,23-; 3,9,13,19-; 3,9,15,21TetrameC37 8,12,18-, 8,12,20-; 8,14,18-TrimeC38 8,12,18,22-; 8,16,22,28-; 8,12,20,26TetrameC38 6,10,14,20-; 6,10,24,28TetrameC38 15-; 13-; 17-; 19-MeC39 9,13,19-; 9,15,19TrimeC39 7,11,19-TrimeC39 5,9,13-TrimeC39 5,9,13,21-; 5,9,13,23-; 5,9,17,25TetrameC39 8,12,20-; 8,12,28-TrimeC40 6,10,14,20-; 6,10,14,18-; 6,10,14,30TetrameC40 17-; 19-; 21-MeC41 5,17-DimeC41 7,11,21-; 7,11,19TrimeC41 3,9,13,21-; 3,9,13,27TetrameC41 Branchedrnormal chains Ž% .
Nymphal instar
P. Juarez ´ et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 733᎐746
743
Fig. 4. Developmental changes of surface and internal major hydrocarbons of R. prolixus. Data are means " S.E.M. Žpooled samples of 10᎐15 insects each, n s 3..
3,9,13,19- and 3,9,15,21-TetramethylC37. with P - 0.01. In the nymphal epicuticular hydrocarbons, large amounts of n-C33 and n-C31 are predominant, with significant differences between third and fifth nymphal instars Ž P- 0.001.. In the methyl-branched fraction, minor changes were also observed in the relative amounts of peak 18 Ž5-methylC29., with a marked reduction from first to fifth instar. The hydrocarbons of first through third instar insects were almost identical, other than a small shift in the peak 36rpeak 37 ratio. For the internal hydrocarbons, the branchedrnormal ratio is close to 2 for nymphal stages, and increments up to 3.45 for adults. The shift between nymphal and adult stages is still apparent for the n-C27, n-C29 and the n-C31 and n-C33 components, although major peaks corresponded to the methyl-branched peaks 36, 39 and 45. The relative amounts of major internal and surface hydrocarbons are compared through third and fifth nymphal stage to adult Žmale. stage ŽFig. 4). For adult males, the amounts of n-alkanes on the surface are almost three-fold that of the internal hydrocarbons, whereas the relative amounts
of the branched fraction are always higher for the internal fraction. A similar pattern was detected for third instar nymphs, whereas for the last nymphal instar Žfifth., differences between internal and surface hydrocarbons are less evident for the straight chains, and almost equal amounts of branched components were detected, both for the epicuticle and internal extracts.
4. Discussion Studies on the epicuticular hydrocarbons of two Triatominae, Triatoma infestans and Triatoma mazzotti ŽJuarez and Blomquist, 1993., showed ´ several qualitative differences between these species, mainly due to different positional isomers of some components. Studies on the developmental changes in cuticular hydrocarbon of T. infestans showed a marked shift between nymphs and adults, quite similar to that reported herein for R. prolixus, together with higher amounts of hydrocarbons present in the soft nymphal epicuticle. The only Triatominae in which hydrocarbon dy-
744
P. Juarez ´ et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 733᎐746
namics has been extensively studied is T. infestans. The major site of hydrocarbon accumulation was shown to be the hemolymph, with a branchedrnormal ratio ranging from 4 to 8, whereas the integumental site of synthesis had a branchedrnormal ratio similar to that of the epicuticle. In T. infestans, the exuvium shed during each molting event closely resembled the epicuticular profile of the previous instar ŽJuarez and Bren´ ner, 1985.. The major changes observed during ecdysis suggested that, immediately after molting the epicuticular hydrocarbon composition reflects that of the hemolymph from the previous instar. After ecdysis, the branchedrnormal ratio begins to decline and, from day 2 up to the end of the stage, hydrocarbon profiles remain unchanged. It was also shown that at the time of hatchingrmolting there are two pools of hydrocarbons; one due to methyl-branched hydrocarbon transport via female hemolymph to the egg, or during each developmental stage, via transport to the surface immediately after molting; the other pool arises from de novo synthesis ŽJuarez ´ and Brenner, 1985; Juarez, 1994.. R. prolixus differs from T. infestans ´ in having larger amounts of straight-chain hydrocarbon components in the epicuticular wax; this might be related to a ‘softer’, less sclerotized cuticle, which in addition, possesses higher amounts of storage lipids, such as triacylglycerols ŽJuarez, unpublished data.. However, the precise ´ role of each component of the waxy layer remains to be elucidated. Hydrocarbon components have been proposed as species-specific phenotypes, genetically determined; although their presence or amounts may result to some extent, from physiological processes, they might also be determined from variations in the genes coding for the corresponding enzymes ŽLockey, 1991; Page et al ., 1997.. The absence of sexual dimorphism in the cuticular hydrocarbon for R. prolixus and T. infestans ŽJuarez and Brenner, 1985. suggests that sexual ´ attractiveness is not mediated by surface hydrocarbons, although T. infestans cuticular hexane extracts have been recently reported as chemical cues for conspecific individuals ŽLorenzo Figueiras and Lazzari, 1998., and behavioral bioassays are presently being performed ŽLorenzo Figueiras, Lazzari, Juarez, unpublished.. ´ Intergeneric comparison among Triatominae hydrocarbons Ž Rhodnius, T riatom a and
Panstrongylus. showed characteristic fingerprints for each of the species analyzed, with clear differences at the tribal level, as shown by discriminant analysis ŽJuarez et al ., in press.. Rhodnius ´ hydrocarbon composition differed both qualitatively and quantitatively from that detected for Triatoma, which might suggest an early divergence from their ancestral forms, as also indicated by comparison of their 18S ribosomal DNA sequences ŽMarcilla et al., in press. and mitochondrial DNA sequence ŽLyman et al., 1999 ). R. prolixus hydrocarbons showed some distinctive characteristics from those reported for Triatoma, as well as a rather more complex pattern. Hydrocarbon GC profiles of R. prolixus indicate a closer relation to R. nasutus rather than to R. pallescens ŽJuarez et al., in press., in agreement with the ´ current classification ŽSchofield, 1988.. Tetramethyl components are abundant, whereas they are absent in both Triatoma species studied ŽJuarez ´ and Blomquist, 1993.; the major branched component is the 5,15-dimethylC37, but although a complex dimethyl-branching pattern is abundant in Triatoma, it is rare in Rhodnius. On the contrary, terminal or subterminal trimethylalkanes, and a variety of the derived tetramethylalkanes of C29, C31, C35, C36, C37, C38, C39 and C41 carbon backbones, are characteristic of R. prolixus. Trimethyl-branching in Triatomini is internally located, and restricted to C35, C37 and C39 carbon backbones for T. infestans or C33, C38, C39, and C41 carbon backbones for T. mazzotti ŽJuarez ´ and Blomquist, 1993.. Multiple methyl-branching in insects arises from the insertion of carbons from propionate via methylmalonyl-CoA elongating units ŽBlomquist et al., 1987.. The species-specificity that determines the number and spacing of the methyl groups to be inserted into the elongating fatty acyl chain appears to be regulated by an integumental multienzyme system, which involves a microsomal fatty acid synthetase ŽFAS. coupled to fatty acyl-CoA elongaseŽs. ŽJuarez et al ., 1992; ´ Juarez ´ et al., 1996; Juarez ´ and Blomquist, unpublished data. and a P-450 type enzyme for the final conversion of the fatty aldehyde to the corresponding hydrocarbon ŽReed et al ., 1994. . As insects approach the final ecdysis Žfifth instar., changes in cuticular composition are evoked ŽJuarez ´ and Brenner, 1985.. Whether this shift is caused by a very long-chain fatty acyl-CoA elongase shut-down, or is the result of an increased
P. Juarez ´ et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 733᎐746
microsomal FAS activity responsible for the formation of methyl-branched fatty acyl-CoA precursors, or both, remains to be determined .
Acknowledgements This work was supported in part by the AVINA Foundation, Switzerland, and benefited from international collaboration through the ECLAT network. We thank Dr Jose Jurberg for providing the insects, and Mr Gustavo Calderon ´ for help with the figures. References Blomquist, G.J., Nelson, D.R., de Renobales, M., 1987. Chemistry, biochemistry and physiology of insect cuticular lipids. Arch. Insect Biochem. Physiol. 6, 227᎐265. Carlson, D.A., Bernier, U.R., Sutton, B.C., 1998. Elution patterns from capillary GC for methyl-branched alkanes. J. Chem. Ecol. 24, 1845᎐1865. Carlson, D.A., Service, M.W., 1979. Differentiation between species of the Anopheles gambiae Giles complex ŽDiptera: Culicidae. by analysis of cuticular hydrocarbons. Ann. Trop. Med. Parasitol. 73, 589᎐592. Crespo, R., Juarez, M.P., Cafferata, L.F.R., 2000. ´ Biochemistry of the interaction between entomopathogenic fungi and their insect host-like hydrocarbons. Mycologia 92, 528᎐536. Dujardin, J.P., Munoz, M., Chavez, T., Ponce, C., ˜ Moreno, J., Schofield, C.J., 1998. The origin of Rhodnius prolixus in Central America. Med. Vet. Entomol. 12, 113᎐115. Juarez, M.P., 1994. Hydrocarbon biosynthesis in Tria´ toma infestans eggs. Arch. Insect Biochem. Physiol. 25, 193᎐206. Juarez, M.P., Brenner, R.R., 1985. The epicuticular ´ lipids of Triatoma infestans. II. Hydrocarbon dynamics. Comp. Biochem. Physiol. 82B, 793᎐803. Juarez, M.P., Brenner, R.R., 1986. Biochemistry of the ´ evolution of Triatoma infestans. IX. Composition of cuticular hydrocarbons compared to other Triatominae. Acta Physiol. Pharmacol. Latinoam. 36, 47᎐57. Juarez, M.P., Blomquist, G.J., 1993. Cuticular hydro´ carbons of Triatoma infestans and T. mazzott. Comp. Biochem. Physiol. 106B, 667᎐674. Juarez, M.P., Chase, L., Blomquist, G.J., 1992. A micro´ somal FAS from the integument of Blatella germanica synthesize pheromone and methyl-branched hydrocarbon precursors. Arch. Biochem. Biophys. 293, 333᎐341.
745
Juarez, M.P., Ayala, S., Brenner, R.R., 1996. Methyl´ branched fatty acids biosynthesis in Triatoma infestans. Insect Biochem. Mol. Biol. 26, 593᎐598. Juarez, M.P., Fernandez, R., Dujardin, J.P., Schofield, ´ ´ C.J. Intergeneric comparison of cuticular hydrocarbons in Triatominae. Res. Rev. Parasitol., in press. Kovats, E., 1965. Gas chromatographic comparison of organic substances in the retention index system. Adv. Chromatogr. 1, 229᎐247. Lorenzo Figueiras, A.N., Lazzari, C.R., 1998. Aggregation in the hematophagous bug Triatoma infestans: a novel assembling factor. Physiol. Entomol. 23, 33᎐37. Lockey, K.H., 1991. Insect hydrocarbon classes. Implication for chemotaxonomy. Insect Biochem. 21, 91᎐97. Lyman, D.L., Monteiro, F.A., Escalante, A.E. et al., 1999. Mitochondrial DNA sequence variation among triatomine vectors of Chagas disease. Am. J. Trop. Med. Hyg. 60, 377᎐386. Marcilla, A., Bargues, M.D., Ramsey, J., Dujardin, J.P., Schofield, C.J., Mas-Coma, S. Primary and secondary structures of the 18S rRNA gene and phylogenetic analysis of Triatominae ŽHemiptera: Reduviidae., vectors of Chagas disease. Mol. Biol. Evol., in press. Milligan, P.J.N., Phillips, A., Molyneux, D.H., Subbarao, S.K., White, G.B., 1986. Differentiation of Anopheles culicifacies Giles ŽDiptera: Culicidae. sibling species by analysis of cuticular components. Bull. Entomol. Res. 76, 529᎐537. Napolitano, R., Juarez, M.P., 1997. Entomo´ pathogenous fungi degrade epicuticular hydrocarbons of T. infestans. Arch. Biochem. Biophys. 344, 208᎐214. Nelson, D.R., Carlson, D.A., 1986. Cuticular hydrocarbons of the tsetse flies Glossina morsitans morsitans, G. austeni and G. pallidipes. Insect Biochem. 16, 403᎐416. Page, M, Nelson, L.J., Blomquist, G.J., Seybold, S.J., 1997. Cuticular hydrocarbons as chemotaxonomic characters of pine engraver beetles Ž Ips spp.. in the grandicollis subgeneric group. J. Chem. Ecol. 23, 1053᎐1099. Reed, J.R., Vanderwel, D., Choi, S., Pomonis, J.G., Reitz, R.C., Blomquist, G.J., 1994. Unusual mechanism of hydrocarbon formation in the housefly: cytochrome P450 converts aldehyde to the sex pheromone component Ž Z .-9-tricosene and CO 2 . Proc. Natl. Acad. Sci. USA 91, 10000᎐10004. Romana, ˜ C.A., 1996. Potencial del control biologico de los triatomas. In: Dujardin, J.P., Schofield, C.J. ŽEds.., Overview and Biosystematics of the Reduviidae. Proceedings of the International European Communication Workshop on Population Genetics and
746
P. Juarez ´ et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 733᎐746
Control of Triatominae, Santo Domingo de los Colorados, Ecuador, pp. 87᎐88. Romana, ˜ C.A., Fargues, J., 1992. Relative susceptibility of different stages of R. prolixus to the entomopathogenic hyphomycete Beau¨ eria bassiana. Mem. Inst. O. Cruz 87, 363᎐368. Schofield, C.J., 1988. The biosystematics of Triatominae. In: Service, M.W. ŽEd.., Biosystematics of Hae-
matophagous Insects. Clarenden Press, Oxford, UK, pp. 284᎐312 ŽSystematics Association special volume 37.. Schofield, C.J., 2000. Challenges of Chagas Disease Vector Control in Central America. WHO, Geneva ŽWHO GCDPP Position paper ŽWHOrCDSr WHOPESrGCDPPr2000.1., 36 pp..
