8373 Journal of Chemical Ecology. Yll/. 26. No.7. 2000
MALE-PRODUCED AGGREGATION PHEROMONE OF Colopterus truncatus: STRUCTURE, ELECTROPHYSIOLOGICAL, AND BEHAVIORAL ACTIVITY 1 ALLARD A. COSS:E* and ROBERT 1. BARTELT USDA Agricultural Research Sen'ice National Cenrer for Agricultural Utilization Research Bioactive Agellls Research Unit 1815 N. Univen'ity Sl/·eet. Peoria. lIIinois 61604 (Received August 3. 1999; accepted March 15. 2000)
Abstract-A male-produced aggregation pheromone was demonstrated in Colopterus IruncalllS Randall (Coleoptera; Nitidulidae) by gas chromatographic comparisons of male and female volatile emissions. Male-specific compounds were identified with coupled gas chromatographic-mass spectrometric (GC-MS) analysis and GC and MS comparison of authentic standards. Physiological activity was evaluated by coupled gas chromatographic-electroantennographic (GC-EAG) recordings. and electroantennographic (EAG) assays of standards. The male-produced volatiles eliciting responses from male and female antennae (and relative abundance) were (2EAE.6E)-3.5-dimethyl2A.6-octatriene (1) (1.8). (2E,4E.6E)-4.6-dimethyl-2.4.6-nonatriene (2) (l00). and (2EAE.6E.8E)-3.5.7-trimethyl-2,4,6.8-decatetraene (3) (3.3). A fourth (2EAE.6£'8E )-4.6.8-trimethyl-2.4.6.8-undecamale-specific compound. tetraene (4) (0.6) was not EAG-active. EAG dose-response studies showed that the antennae were most sensitive to 2 followed by 3 and 1. Synthetic 2. binary blends of 1 and 3. and tertiary blends of 1. 2. and 3 were highly attractive in the field when synergized with fermenting whole-wheat bread dough. In the field. cross-attraction to the C. truncatus pheromone components was observed for CWl)ophilus lugubris lvlurray. C. allliquus Melsheimer. and C. brachypterus Say. Key \Vords-Coloplerus truncatus Randall. sap beetle. Coleoptera. Nitidulidae. aggregation pheromone. coupled gas chromatography-electroantennography (GC-EAG). behavior. (2EAE.6E}-4,6-dimethyl-2A.6-nonatriene. "'To whom correspondence should be addressed. e-mail;
[email protected] tAll programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race. color. national origin. religion. sex. age. marital status. or handicap. Mention of firm names or trade products does not imply endorsement by the U.S. Department of Agriculture over similar firms or products not mentioned.
1735 0098-0331/00,/0700-1735518,00iO
2000 Plenum Publishing CorporJllon
1744
COSSE AND BARTELT
Male C. truncatus (N=8)
3
A Cll !/l
t:
1111 2.5
0
02
0-
!/l
OJ
c::
~
w
b
3
2
aa a -
1.5
"t:l OJ
a a
b ba
aaa
0.001
0.01
ae Q -be
TJ
.!:: iii
E
b
ae be
TJ
T
T
5 0.5
z
0
'---
'---
Ctrl
'-
0.1
10
100
10
100
Dose (pg) Female C. truncatus (N=10)
B
6 111
OJ
CIl
t:
0
5
0CIl
OJ
0:: (!l
Means within a columns followed by the same letter do not differ by t test. P > 0.05. CAsterisks denote statistical significance at P < 0.05: NS denotes P > 0.05 (elf '" 3. 46).
ies. Minor amounts of other male-specific compounds were also detected when hydrocarbon fractions of male-derived volatiles were carefully compared by GC-MS. By GC retention, MS, and experience with other trienes and tetraenes (Bartelt et aI., 1990a, 1992), these other compounds were concluded to be Z isomers of 2 and 3. Some isomerization/degradation has invariably occurred with all of the trienes and tetraenes, and it is unknown whether the minor isomers are actually emitted by C. truncatus. Whether or not the Z isomers are part of the emissions. it is clear from the GC-EAD results that the all E isomers are the biologically active compounds. Only the all E isomers elicited EAD responses from male and female antennae; no responses were observed to any of the Z isomers present in both natural and synthetic materials. The electrophysiological techniques employed might be very useful in clarifying the behavioral significance of identified male-specific hydrocarbons as components for nitidulid aggregation pheromones as well as a powerful tool for selecting possible candidates for behavioral experiments of newly identified aggregation pheromones. The pheromonal hydrocarbons of CmyJophilus species are assembled biosynthetically from the small acyl units, acetate, propionate. and butyrate (Bartelt 1997, 1999: Bartelt and Weisleder. 1996). The male-specific compounds from C. tnlllcatlls follow the same patterns (Figure 5): The intial acyl unit can be either acetate or propionate, and the methyl branches cOI1'espond to the terminal methyl carbons of proprionate. The final acyl unit loses its carboxyl carbon during biosynthesis (Bartelt and Weisleder. 1996); hence, only two carbons are shown in the chain-ending propionates in Figure S. The three antennally active compounds (1, 2, and 3), differ structurally because of two separate biosynthetic events or "choices"; initiation \vith acetate versus propionate and incorporation
1746
COSSE AND BARTELT
3~
1~ Ac
Pr
Pr
Ac Pr
Pr
2~ Pr
Pr
Pr
Pr
Pr
Pr
Pr
4~ Pr
Pr
Pr
Pr
Pr
FIG. S. Probable biosynthetic origins of the male-specific hydrocarbons of C. [runcatlls from acetate (Ac) and propionate (Pr). Carbons connected by heavy bonds from single acyl unit. The final (right-hand) propionate has only t\Vo carbons because the carboxyl group is lost during biosynthesis. See text for further explanation.
of four versus five total units (giving a triene or tetraene, respectively). Given two dichotomous choices, there are four, rather than just three, possible resulting structures, and inactive compound 4 represents this fourth possibility. It is suggested that 4 is simply a biosynthetic artifact. EAG dose-response studies showed that both males and females were more sensitive to the most abundant compound (2) of the volatile collections. However, no corrections were made for differences in volatility between the compounds. Therefore, it remains to be seen whether 3 and not 2 is the more biologically active compound after taking into account its higher molecular weight and therefore lower volatility. Future tests should focus on directing equal amounts of test compound over the antennae in order to determine sensitivity thresholds. Our preliminary field data demonstrated that major triene 2, a blend of minor components, and the overall combination, attracted significant numbers of beetles. In addition, component release ratios from fresh septa showed a good cOITelation with those found in the natural material. However. release rates from I-week-old septa indicated especially rapid depletion of the smaller compounds, showing that component ratios with septa could not be controlled. It is unknown how this would affect beetle behavior, but the low weekly trapping totals for the overall blend suggest that the ratios may be very important and need to be studied again more carefully. Although not the focus of this study, the attractivity of the pheromone blend was accessed in the presence of food odor, since a relative strong synergism between pheromone and food volatiles is well documented for Carpophilus species (Bartelt 1997, 1999). C. trllncatus showed cross-attraction to the aggregation pheromone of C. brachypterus (Williams et aL 1995). Likewise, in this study C. brachypterus was attracted to C. truncatus pheromone. The fact that the pheromone blend of C. brach.vpterus has tetraene 3 in common with C. truncatlls probably explains this cross-attraction. Two CG/]Jophilus species, C. lugubris and C. antiqllus, showed
Coloprerus AGGREGATION PHEROMONE
1747
a significant attraction to a blend of 1 and 3 and a blend of all three components. respectively. Yet these species do not have any components in common with C. frullcatus. Wind-tunnel studies have shown that C. lugubris can discriminate among tetraenes with very similar structures. and significant responses to tetraenes, not found in C. lugubris, including 3, did occur (Bartelt et aI., 1991). The cross-attraction of C. allfiquus to a three-component blend may be similar to that of C. lugubris or may have a kairomonal basis as shown with C. alltiquus and C. lugubris pheromone (Bartelt et aL 1993b). As reported previously, G. quadrisigllatus and G. fasciafus, which belong to subfamily Cryptarchinae rather than Carpophilinae, were captured only because of the presence of the bread dough and did not respond to any of the tested C. frullcafus compounds (Williams et aL 1993). In summary, the suggested composition of the aggregation pheromone of C. frUllcafus is likely to be comprised of L 2. and 3. with 2 as a newly identified compound of nitidulid aggregation pheromones. Improved field formulations. particularly the stabilization of septa's release ratios, should yield a better understanding of the relationship among L 2, and 3. Acklloll"ledgmel11s-We thank Bruce W. Zilkowski for assistance with the field work. J. M. Kingsolver of the USDA-ARS Systematic Entomology Laboratory kindly verified the identity of C. trlmcalllS: specimens were retained in the U.S. National Museum collection.
REFERENCES ApPEL. D. N.. ANDERSEN. K.. and LEWIS. R.. JR. 1986. Occurrence of nitidulid beetles (Coleoptera: Nitidulidael in Texas oak wilt centers. J. Ecoll. El11olllOl. 79: 1276-1279. BARTELT. R. J. 1997. Aggregation pheromones of Carpophillls spp. (Coleoptera: Nitidulidae): Review of chemistry and biology. Recel11 Res. DeE Ellromol. I: 115-129. BARTELT. R. J. 1999. Sap beetles. pp. 69-90. ill J. Hardie and A. K. Minks (eds.). Pheromones of Non-Lepidopteran Insects Associated with Agricultural Plants. CAB! Publishing. New York. B.'\RTELT. R. J.. and WEISLEDER. D. 1996. Polyketide origin of pheromones of Carpophillls davidsolli and C. mwilafUs (Coleoptera: Nitidulidae). BioOlg. iVIed. Chem. 4:429--438. BARTELT. R. J.. and ZILKOWSKI. B. W. 1999. Nonequilibrium quantitation of volatiles in air streams by solid-phase microextraction. Allal. Chem. 71 :92-1 0 1. BARTELT. R. J.. DOWD. P. F.. PLATTNER. R. D.. and WEtSLEDER. D. 1990a. Aggregation pheromone of dried-fruit beetle. CClljJophilllS hemiplerus: Wind-tunnel bioassay and identification of two novel tetraene hydrocarbons. 1. Chem. Ecol. 16: 1015-1039. BARTELT. R. J.. DOWD. P. F.. SHOREY. H. H.. and WEISLEDER. D. 1990b. Aggregation pheromone of Carpophillls ji"eemalli (Coleoptera: Nitidulidae): A blend of conjugated triene and tetraene hydrocarbons. Chemoecology I: I05-113. BARTELT. R. J.. DOWD. P. F.. and PLAnNER. R. D. 1991. Aggregation pheromone of C(7){>phillls IlIgllbris: New pest management tools for the nitidulid beetles. ACS Symp. Sa Am. Chem. Soc. 449:27--40. BARTELT. R. J.. WEISLEDER. D.. DOWD. P. F.. and PLAnNER. R. D. 1992. Male-specific tetraene and triene hydrocarbons of Carpophillls hemiplerus: Structure and pheromonal activity. J. Chem. Ecol. 18:379--402.
1748
COSSE AND BARTELT
BARTELT, R, L CARLSON, D, G" VETTER, R, S" and B.'\KER, T. C. 1993a. Male-produced aggregation pheromone of Cwpophilils IIIl1iilaluS (Coleoptera: Nitidulidae). J. Chelll. Ecol. 19: 107-118. BARTELT, R. J., SEATON. K. L., and DOWD, P. E 1993b. Aggregation pheromone of Cmpophilus amiquus (Coleoptera: Nitidulidae) and kairomonal use of C. lugubris pheromone by C. amiqllils. J. Chelll. Ecol. 19:2203-2216.
DORSEY, C. K.. and LEACH, J. G. 1956. The bionomics of certain insects associated with oak wilt with particular reference to the Nitidulidae. J. Econ. Emollloi. 49:219-230, DOWD, P. E, BARTELT, R. Land WICKLOW, D. T. 1992. Novel insect trap useful in capturing sap beetles (Coleoptera: Nitidulidael and other flying insects, J. Ecoll. Emolllol. 85:772-778. EPHRUSSI, B., and BEADLE, G. W. 1936. A technique of transplantation for Drosophila. Alii. Nal. 70:218-225. JUZWIK, L and FRENCH. D. W, 1983. Ceralocysiis fagacearulll and C. piceae on the surfaces of free-flying and fungus-mat-inhabiting nitidulids, Phyropalhology 73: 1164-1168. WtLLlAMS, R. N" FICKLE, D, S.. BARTELT. R. J.. and DOWD. P. E 1993. Responses by adult Nitidulidae (Coleoptera) to synthetic aggregation pheromones. a coattractant, and effects of trap design and placement. ElI/: J. Emolllol. 90:287-294. WILLIAMS. R. N., ELLIS. M. S" and BARTELT. R. J. 1995. Efficacy of Cmpophilus aggregation pheromones on nine species in northeastern Ohio, and identification of the pheromone of C. brachypierus. Enrolllol. Exp. Appl. 77:141-147.
n~ ~o~'"
Center for i~~~riclm:m'ai Utilization Research, Peoria v
K\"'''T,';f'j.0'U
