Journal ofChemicalEcology, Vol. 13, No. 3, 1987
SEX PHEROMONE OF P l a n o t o r t r i x I SPECIES FOUND ON MANGROVE
STEPHEN
P. F O S T E R , WENDELL
J.R. CLEARWATER, L. R O E L O F S 2
and
D. S. L R., Entomology Division, Mr. Albert Research Centre, Private Bag, Auckland, New Zealand
(Received October 28, 1985; accepted April 22, 1986) Abstract--(Z)-5-Tetradecenyl acetate and tetradecyl acetate were identified as sex pheromone components of an unnamed Planotortrix leafroller moth species found on Avicennia resinifera (mangrove). An equal mixture of the two compounds used as bait gave field trap catches at least as good as those baited with caged virgin females. Traps baited with the two chemicals caught male Planotortrix moths in a mangrove swamp not previously found to host the unnamed Planotortrix species. Adults of the unnamed Planotortrix species and of the greenheaded leafroller, Planotortrix excessana are morphologically indistinguishable. The sex pheromone of P. excessana has been found previously to be a mixture of (Z)-8-tetradecenyl acetate and tetradecyl acetate, and this means that the two species may now be distinguished by sex pheromone differences. Key Words--Sex pheromone, Lepidoptera, Tortricidae, (Z)-5-tetradecenyl acetate, Planotortrix, mangrove, sibling species.
INTRODUCTION Dugdale (1966) described the genus Planotortrix since certain New Zealand T o r t r i c i d a e a p p e a r e d to h a v e b e e n i n c o r r e c t l y c l a s s i f i e d in t h e g e n u s Tortrix L i n n a e u s . E l e v e n s p e c i e s w e r e a s s i g n e d to t h e n e w g e n u s , a l t h o u g h it w a s n o t e d that m o r e r e m a i n to b e d e s c r i b e d . T w o s p e c i e s o f this g e n u s are m a j o r p e s t s ; P. e x c e s s a n a ( W a l k e r ) t h e g r e e n h e a d e d l e a f r o l l e r , is a pest o f m o s t fruit c r o p s w h i l e P. n o t o p h a e a ( T u r n e r ) c a n c a u s e c o n s i d e r a b l e d a m a g e to y o u n g c o n i f e r s . ~Lepidoptera: Tortricidae: Tortricinae. 2Visiting scientist from New York State Agricultural Experiment Station, Geneva, New York 14456. 631 0098-0331/87/0300-0631505.00/0 9 1987 Plenum Publishing Corporation
632
FOSTER ET AL.
The sex pheromone of P. excessana (collected from Christchurch in the South Island) was found to be a mixture of (Z)-8-tetradecenyl acetate (Z814:OAc) and tetradecyl acetate (14:OAc) (Galbreath et al., 1985). Galbreath et al. (1985) also reported that a population of this species collected in the central North Island utilized a different pheromone, possibly involving a mixture of (Z)-5- and (Z)-7-tetradecenyl acetates (Z5-14 : OAc and Z7-14 : OAc). These data underscored the importance of defining pheromone blends for various Planotortrix populations throughout New Zealand. The results would be of value in reexamining the systematics of the genus and in determining the pheromone to be used in monitoring traps in each geographic area. We initiated our study with a population of Planotortrix that was known to exist in the Firth of Thames (North Island) and whose larvae feed on foliage of mangrove (Avicennia resinifera Forst.f.) (Dugdale, personal communication). While the larvae can be distinguished from the larvae of P. excessana, the adults are morphologically indistinguishable from P. excessana populations that feed on other plants (Dugdale, personal communication). Although adults from the mangrove species are generally darker than those of P. excessana, they cannot be definitively distinguished due to the large variation in color of P. excessana adults (Dugdale, personal communication). This paper details the identification of two sex pheromone components of the unnamed Planotortrix species that feeds on mangrove. METHODS AND MATERIALS
Insects were initially collected from mangrove bushes as larvae and reared on a semisynthetic diet containing dried acmena leaf (Galbreath et al., 1985). Further larval generations were reared on the semisynthetic diet containing approximately 17% (by weight) ground mangrove leaf. Adults were maintained on a reverse light-dark cycle (16L : 8D). Gland extracts were taken by the following method. One hour into the scotophase, the ovipositor of a 3- to 4-day-old female moth was extruded under a binocular microscope and the pheromone gland excised using fine forceps. The gland was placed in 10 #1 of distilled analytical grade n-pentane in a flamed 4-mm-OD glass tube drawn to a point. This was stored 3-12 hr in a 4-ml glass vial prior to gas-liquid chromatographic (GLC) analysis. Capillary GLC analysis of gland extracts was performed with splitless injection and flame ionization detection. Capillary columns were a 50-m SGE BP20 (stabilized Carbowax 20 M), capable of resolving most straight-chain 14carbon acetate isomers, a 50-m Quadrex Corporation CPS-1 (bonded SP2340 equivalent), capable of resolving not only most straight-chain but also most geometric 14-carbon acetate isomers, and a 20-m BP5 (SE54 equivalent). A
SEX PHEROMONE OF
Planotortrix
633
program of 100-200~ at 4~ following an initial delay of 1 rain was generally used. For analysis of the ozonolysis products, the initial temperature was changed to 40~ For the CPS-1 colunm, a program of 100-180~ at 3~ min was used. Carrier gas was nitrogen at a linear velocity of 35 cm/sec. Samples were quantified by electronic integration of peak areas, relative to 16.4 ng of added tridecanyl acetate (13 : OAc) internal standard. A 2-m 3% OV-1 on Chromosorb W packed column was used for fraction separations prior to collection. Fractions were collected in 30-cm glass capillaries cooled by an aluminium block partially immersed in liquid nitrogen. Following collections of the separated fractions, positional isomers were determined by ozonolysis (Beroza and Bierl, 1967) and capillary GLC identification of the products. Ozonolysis was performed by allowing a steady stream of ozone (ca. 2-3 ml/min) to pass through the capillary tube for 30 sec at ambient temperature. The tube was rinsed with 30-40/~1 of pentane: to the solution was added ca. 1 ~zg of solid triphenylphosphine. The retention times of the resultant acetoxy aldehyde and aldehyde were determined by capillary GLC and compared with those of the corresponding products obtained from ozonolysis of synthetic compound and with synthethic aldehyde. Synthetic chemicals used were assayed by capillary GLC, and their isomeric purity was determined to be greater than 97 %. Electroantennograms (EAGs) from excised male antennae were recorded by the method of Roelofs (1984). Four field trials were conducted in the mangrove swamp from which the larvae were collected, and in an Auckland mangrove swamp using Pherocon 1C| traps (Zoecon Company, Palo Alto, California) and Sectar 1 traps (3M Company, St. Paul, Minnesota), baited with synthetic chemicals absorbed on rubber septa (A.H. Thomas Co., Philadelphia, Pennsylvania). Virgin females (used in trial 2) were from the laboratory-reared colony originally collected from the mangrove swamp in Thames. Three females were enclosed in each wire-mesh cage (5 x 5 x 5 cm), suspended in Pherocon 1C | traps. Traps were randomized each time they were counted. Data from the field tests were analyzed by the use of log linear models (McCuUagh, 1983).
RESULTS
Capillary GLC analysis of a single female pheromone gland extract revealed two major components, which had retention times identical to 14 : OAc and Z5-14 :OAc on the polar BP20 and CPS-1 and the nonpolar BP5 columns. The retention time of Z 5 - 1 4 : O A c on the BP20 column (38.08 min) was not
634
FOSTER ET AL.
separable from that of the Z6 isomer (38.11 min), but was separable from the Z7 (38.37 rain) and Z8 (38.63 rain) isomers. Baseline separation of the two geometric isomers of A5-14 : OAc could easily be achieved, using the CPS-1 column. No (E)-5-tetradecenyl acetate was detected in the female pheromone gland extracts. Ozonolysis of the two compounds collected from an OV-1 column confirmed their identification as 14:OAc and a A 5 - 1 4 : O A c . The compound with a retention time corresponding to 14:OAc had the same retention time after ozonolysis, and the other compound was ozonized to give peaks at the same retention times as 5-acetoxypentanal and n-nonanal (16.23 min and 21.41 min, respectively) on the BP20 column (revised temperature program). EAG profiles showed a consistent maximum response to Z5-14 : OAc. The average response of 17 antennae (22 replicates) to Z5-14 : OAc was 5.5 _+ 0.6 mV compared to 3.8 _+ 0.6 mV for E5-14 : OAc. Quantification using pheromone extracted from five females and added internal standard (13 : OAc) gave an average of 1.3 ng of Z5-14 : OAc and 0.6 ng 14:OAc per female. Analysis of further female extracts showed the ratio of Z5-14 : OAc to 14 : OAc to vary from this 2.2 : 1 ratio to 0.45 : 1, with an average of approximately 1 : 1. Field trials were carried out to determine the attractiveness of these chemicals to male moths. Traps baited with lures known to be attractive to P . e x c e s s a n a (Galbreath et al., 1985) were also used. The results (Table 1) clearly show the attractiveness of equal quantities of 14 :OAc and Z5-14 : OAc to male moths of this species, even though catches of males generally were low. In trial 1, the catch to 100/xg of both chemicals was significantly higher than that to a lower concentration of each or to the P . e x c e s s a n a lure ( Z 8 - 1 4 : O A c and 14 : OAc). Some 96% of males in the traps were caught in the final two weeks of the trial. This is due probably to an initiation of a moth flight period rather than to the decay of pheromone components in the lures to a more optimal concentration, since a similar phenomenon was observed in the lower concentration in the trial. Trial 2 confirmed the efficacy of the synthetic lure in comparison with virgin females and trial 3 the necessity for the two-component mix for attraction. Trial 4 was conducted at a site where the moth has not previously been found and confirms the lure's efficacy in what is probably a low-density population (previous searches for this moth in this area failed to detect its presence, Dugdale, personal communication). The traps baited with P . e x c e s s a n a lures caught few moths. The morphological similarity between P . e x c e s s a n a and the mangrove-feeding P l a n o t o r t r i x males precluded absolute identification of the moths in the trap, and so it could not be determined if any P. e x c e s s a n a were captured in these tests.
635
SEX PHEROMONE OF Planotortrix
TABLE 1. TRAP CATCHES OF MALE Planotortrix MOTHS TO COMBINATIONSOF (Z)-5-TETRADECENYL ACETATE, TETRADECYL ACETATE, AND (Z)-N-TETRADECENYL ACETATE Z5-14 : OAc (~g) Trial 1
Trial 2
10 100
14 : OAc (~g) 10 100 100
10 l0 i00 100 300 300 1000 1000 Virgin females 100
Trial 3
0 100 100
0 0 100
Trial 4
100
100 100
Z8-14 : OAc (~g)
Mean No. of male moths per trap "
100
5.0 ~ 22.7 h 0.3 c
100
3.W 6.3 ~ 3.3 ~ 5.7 a 4.7 a 0.7 b 0.0 ~ 0.2 ~' 5.6"
I00
5.0 ~ 1.0 b
~Means not followed by the same letter are significantly different, P < 0.05, ANOVA. Trial 1 conducted January 27-February 20, 1984, at Thames, three replicates, Pherocon 1C traps. Catches recorded three times. Trial 2 conducted February 20-March 1, 1984, at Thames, three replicates, Pherocon 1C traps. Trial 3 conducted February 27-March 14, 1984, at Thames, five replicates, Sectar 1 traps. Trial 4 conducted February 21-March 23, 1984, at Avondale, Auckland, five replicates, Sectar 1 traps.
DISCUSSION T h e sex p h e r o m o n e , Z 5 - 1 4 : O A c + 14 : O A c , o f the m a n g r o v e - f e e d i n g species o f P l a n o t o r t r i x was f o u n d to be quite different f r o m the p o p u l a t i o n s o f P. e x c e s s a n a w h i c h had p r e v i o u s l y b e e n reported to be Z 8 - 1 4 : O A c + 14 : O A c in one location and p o s s i b l y Z 5 - 1 4 : O A c + Z 7 - 1 4 : O A c in another location (Galbreath et al., 1985). A l t h o u g h all three p o p u l a t i o n s are m o r p h o l o g i c a l l y indistinguishable, the significant difference in m a t e r e c o g n i t i o n systems should be sufficient to classify these p o p u l a t i o n s as sibling species. Sibling or cryptic species h a v e b e e n defined as " s p e c i e s that m o r p h o l o g i c a l l y r e s e m b l e e a c h o t h e r so closely that they can be r e c o g n i z e d o n l y after careful study o f b i o c h e m i c a l , c y t o l o g i c a l , or b e h a v i o r a l t r a i t s . " ( D i e h l and Bush, 1984). T h e host p r e f e r e n c e o f the m a n g r o v e - f e e d i n g P l a n o t o r t r i x species is also v e r y different f r o m the p o l y p h a g o u s P. e x c e s s a n a ( D u g d a l e , 1966).
636
FOSTER ET AL.
In Tortricidae, Z 5 - 1 4 : OAc has been found as a sex pheromone component only twice--both times in New Zealand species. Ctenopseustis o b l i q u a n a (Walker), the brownheaded leafroller, was found to use a combination of Z 5 14" OAc and Z 8 - 1 4 : O A c (Young et al., 1985), and the P. excessana "species" from the central North Island apparently uses a combination of Z 5 14 : OAc and Z 7 - 1 4 : OAc (Galbreath et al., 1985). Z 5 - 1 4 : OAc also has been reported to be a sex pheromone component of a cossid moth (Capizzi et al., 1983) and a noctuid moth (Bestmann et al., 1980), and as part of an attractant blend for several other noctuid moth species (Steck et al., 1982). Since the compounds found in the New Zealand leafroller species are u n c o m m o n in other Tortricidae (Roelofs and Brown, 1982), further studies on the sex pheromone of other related New Zealand leafroller populations and the biosynthetic pathways used in these primitive species should provide valuable information on evolutionary relationships within the genus P l a n o t o r t r i x and between other genera in the tribe Archipini. Acknowledgments--We especially thank Mr. J.S. Dugdale for directing our attention toward this species. We also thank Dr, C. Green for collection of the larvae from the field and Val Holt for rearing the insects. We are grateful to Dr. C. Triggs for statistical analysis of the data. We thank the N.R.A.C. for the award of a Senior Postdoctoral Research Fellowship to one of us (W.L.R.), permittingthis cooperation.
REFERENCES BEROZA, M., and BIERL, B.A. 1967. Rapid determinationof olefin position in organic compounds in microgramrange by ozonolysis and gas chromatography. Anal. Chem. 39:1131-1135.
BESTMANN,H.J., BROSCHE,T., KOSCHATZKY,K.H., MICHELIS,K., PLATZ,H., VOSTROWSKY,O., and KNAUF, W. 1980. PheromoneXXX. Identifiziemngeines neuartigenPheromonkomplexes aus der Graseule Scotia exclamationis (Lepidoptera). Tetrahedron Lett. 21:747-750. CAPPIZZI, A., TONINI, C., ARSURA,E., GUGLIELMETTI,G., MASSARNO,P., and PICCARNI, P. 1983. Sex pheromonecomponentsof the Europeangoat moth, Cossus cossus. J. Chem. EcoL 9:191-
200. DIEnL, S.R., and BUSH, G.L. 1984. An evolutionary and applied perspective of insect biotypes. Annu. Rev. Entomol. 29:471-504. DUGDALE, J.S. 1966. A new genus for the New Zealand "elusive Tortrix" (Lepidoptera: Tortricidae: Tortricinae). N.Z.J. Sci. 9:391-398. GALBREATH,R.A., BENN,M.H., YOUNG,H., and HOLT,V.A. 1985. Sex pheromone components in New Zealand greenheaded leafroller Planotortrix excessana (Lepidoptera:Tortricidae). Z. Naturforsch. 40c:266-271. MCCULLAGH,P. 1983. Generalized Linear Models. Chapman and Hall, London. ROELOFS, W.L. 1984. Electroantennogramassays: Rapid and convenientscreeningprocedures for pheromones, in H.E. Hummel (ed). Techniques in Pheromone Research, pp. 131-159, in T.A. Miller (ed.). ExperimentalEntomologyseries. Springer, New York. ROELOFS, W.L., and BROWN, R.L. 1982. Pheromonesand evolutionaryrelationshipsof Tortricidae. Annu. Rev. Ecol. Syst. 13:395-422.
SEX PHEROMONE OF Planotortrix
637
STECK,W., UNDERHILL,E.W., and CHISHOLM,M.D. 1982. Structure-activity relationships in sex attractants for North American noctuid moths. J. Chem Ecol. 8:731-754. YOUN6, H., GALBREATH, R.A., BENN, M.H., HOLT, V.A., and STRUBLE, D.L. 1985. Sex pheromone components in New Zealand brownheaded leafroller Ctenopseustis obliquana (Lepidoptera:Tortricidae). Z. Naturforsch. 40c:262-265.
SEX PHEROMONE OF P l a n o t o r t r i x I SPECIES FOUND ON MANGROVE
STEPHEN
P. F O S T E R , WENDELL
J.R. CLEARWATER, L. R O E L O F S 2
and
D. S. L R., Entomology Division, Mr. Albert Research Centre, Private Bag, Auckland, New Zealand
(Received October 28, 1985; accepted April 22, 1986) Abstract--(Z)-5-Tetradecenyl acetate and tetradecyl acetate were identified as sex pheromone components of an unnamed Planotortrix leafroller moth species found on Avicennia resinifera (mangrove). An equal mixture of the two compounds used as bait gave field trap catches at least as good as those baited with caged virgin females. Traps baited with the two chemicals caught male Planotortrix moths in a mangrove swamp not previously found to host the unnamed Planotortrix species. Adults of the unnamed Planotortrix species and of the greenheaded leafroller, Planotortrix excessana are morphologically indistinguishable. The sex pheromone of P. excessana has been found previously to be a mixture of (Z)-8-tetradecenyl acetate and tetradecyl acetate, and this means that the two species may now be distinguished by sex pheromone differences. Key Words--Sex pheromone, Lepidoptera, Tortricidae, (Z)-5-tetradecenyl acetate, Planotortrix, mangrove, sibling species.
INTRODUCTION Dugdale (1966) described the genus Planotortrix since certain New Zealand T o r t r i c i d a e a p p e a r e d to h a v e b e e n i n c o r r e c t l y c l a s s i f i e d in t h e g e n u s Tortrix L i n n a e u s . E l e v e n s p e c i e s w e r e a s s i g n e d to t h e n e w g e n u s , a l t h o u g h it w a s n o t e d that m o r e r e m a i n to b e d e s c r i b e d . T w o s p e c i e s o f this g e n u s are m a j o r p e s t s ; P. e x c e s s a n a ( W a l k e r ) t h e g r e e n h e a d e d l e a f r o l l e r , is a pest o f m o s t fruit c r o p s w h i l e P. n o t o p h a e a ( T u r n e r ) c a n c a u s e c o n s i d e r a b l e d a m a g e to y o u n g c o n i f e r s . ~Lepidoptera: Tortricidae: Tortricinae. 2Visiting scientist from New York State Agricultural Experiment Station, Geneva, New York 14456. 631 0098-0331/87/0300-0631505.00/0 9 1987 Plenum Publishing Corporation
632
FOSTER ET AL.
The sex pheromone of P. excessana (collected from Christchurch in the South Island) was found to be a mixture of (Z)-8-tetradecenyl acetate (Z814:OAc) and tetradecyl acetate (14:OAc) (Galbreath et al., 1985). Galbreath et al. (1985) also reported that a population of this species collected in the central North Island utilized a different pheromone, possibly involving a mixture of (Z)-5- and (Z)-7-tetradecenyl acetates (Z5-14 : OAc and Z7-14 : OAc). These data underscored the importance of defining pheromone blends for various Planotortrix populations throughout New Zealand. The results would be of value in reexamining the systematics of the genus and in determining the pheromone to be used in monitoring traps in each geographic area. We initiated our study with a population of Planotortrix that was known to exist in the Firth of Thames (North Island) and whose larvae feed on foliage of mangrove (Avicennia resinifera Forst.f.) (Dugdale, personal communication). While the larvae can be distinguished from the larvae of P. excessana, the adults are morphologically indistinguishable from P. excessana populations that feed on other plants (Dugdale, personal communication). Although adults from the mangrove species are generally darker than those of P. excessana, they cannot be definitively distinguished due to the large variation in color of P. excessana adults (Dugdale, personal communication). This paper details the identification of two sex pheromone components of the unnamed Planotortrix species that feeds on mangrove. METHODS AND MATERIALS
Insects were initially collected from mangrove bushes as larvae and reared on a semisynthetic diet containing dried acmena leaf (Galbreath et al., 1985). Further larval generations were reared on the semisynthetic diet containing approximately 17% (by weight) ground mangrove leaf. Adults were maintained on a reverse light-dark cycle (16L : 8D). Gland extracts were taken by the following method. One hour into the scotophase, the ovipositor of a 3- to 4-day-old female moth was extruded under a binocular microscope and the pheromone gland excised using fine forceps. The gland was placed in 10 #1 of distilled analytical grade n-pentane in a flamed 4-mm-OD glass tube drawn to a point. This was stored 3-12 hr in a 4-ml glass vial prior to gas-liquid chromatographic (GLC) analysis. Capillary GLC analysis of gland extracts was performed with splitless injection and flame ionization detection. Capillary columns were a 50-m SGE BP20 (stabilized Carbowax 20 M), capable of resolving most straight-chain 14carbon acetate isomers, a 50-m Quadrex Corporation CPS-1 (bonded SP2340 equivalent), capable of resolving not only most straight-chain but also most geometric 14-carbon acetate isomers, and a 20-m BP5 (SE54 equivalent). A
SEX PHEROMONE OF
Planotortrix
633
program of 100-200~ at 4~ following an initial delay of 1 rain was generally used. For analysis of the ozonolysis products, the initial temperature was changed to 40~ For the CPS-1 colunm, a program of 100-180~ at 3~ min was used. Carrier gas was nitrogen at a linear velocity of 35 cm/sec. Samples were quantified by electronic integration of peak areas, relative to 16.4 ng of added tridecanyl acetate (13 : OAc) internal standard. A 2-m 3% OV-1 on Chromosorb W packed column was used for fraction separations prior to collection. Fractions were collected in 30-cm glass capillaries cooled by an aluminium block partially immersed in liquid nitrogen. Following collections of the separated fractions, positional isomers were determined by ozonolysis (Beroza and Bierl, 1967) and capillary GLC identification of the products. Ozonolysis was performed by allowing a steady stream of ozone (ca. 2-3 ml/min) to pass through the capillary tube for 30 sec at ambient temperature. The tube was rinsed with 30-40/~1 of pentane: to the solution was added ca. 1 ~zg of solid triphenylphosphine. The retention times of the resultant acetoxy aldehyde and aldehyde were determined by capillary GLC and compared with those of the corresponding products obtained from ozonolysis of synthetic compound and with synthethic aldehyde. Synthetic chemicals used were assayed by capillary GLC, and their isomeric purity was determined to be greater than 97 %. Electroantennograms (EAGs) from excised male antennae were recorded by the method of Roelofs (1984). Four field trials were conducted in the mangrove swamp from which the larvae were collected, and in an Auckland mangrove swamp using Pherocon 1C| traps (Zoecon Company, Palo Alto, California) and Sectar 1 traps (3M Company, St. Paul, Minnesota), baited with synthetic chemicals absorbed on rubber septa (A.H. Thomas Co., Philadelphia, Pennsylvania). Virgin females (used in trial 2) were from the laboratory-reared colony originally collected from the mangrove swamp in Thames. Three females were enclosed in each wire-mesh cage (5 x 5 x 5 cm), suspended in Pherocon 1C | traps. Traps were randomized each time they were counted. Data from the field tests were analyzed by the use of log linear models (McCuUagh, 1983).
RESULTS
Capillary GLC analysis of a single female pheromone gland extract revealed two major components, which had retention times identical to 14 : OAc and Z5-14 :OAc on the polar BP20 and CPS-1 and the nonpolar BP5 columns. The retention time of Z 5 - 1 4 : O A c on the BP20 column (38.08 min) was not
634
FOSTER ET AL.
separable from that of the Z6 isomer (38.11 min), but was separable from the Z7 (38.37 rain) and Z8 (38.63 rain) isomers. Baseline separation of the two geometric isomers of A5-14 : OAc could easily be achieved, using the CPS-1 column. No (E)-5-tetradecenyl acetate was detected in the female pheromone gland extracts. Ozonolysis of the two compounds collected from an OV-1 column confirmed their identification as 14:OAc and a A 5 - 1 4 : O A c . The compound with a retention time corresponding to 14:OAc had the same retention time after ozonolysis, and the other compound was ozonized to give peaks at the same retention times as 5-acetoxypentanal and n-nonanal (16.23 min and 21.41 min, respectively) on the BP20 column (revised temperature program). EAG profiles showed a consistent maximum response to Z5-14 : OAc. The average response of 17 antennae (22 replicates) to Z5-14 : OAc was 5.5 _+ 0.6 mV compared to 3.8 _+ 0.6 mV for E5-14 : OAc. Quantification using pheromone extracted from five females and added internal standard (13 : OAc) gave an average of 1.3 ng of Z5-14 : OAc and 0.6 ng 14:OAc per female. Analysis of further female extracts showed the ratio of Z5-14 : OAc to 14 : OAc to vary from this 2.2 : 1 ratio to 0.45 : 1, with an average of approximately 1 : 1. Field trials were carried out to determine the attractiveness of these chemicals to male moths. Traps baited with lures known to be attractive to P . e x c e s s a n a (Galbreath et al., 1985) were also used. The results (Table 1) clearly show the attractiveness of equal quantities of 14 :OAc and Z5-14 : OAc to male moths of this species, even though catches of males generally were low. In trial 1, the catch to 100/xg of both chemicals was significantly higher than that to a lower concentration of each or to the P . e x c e s s a n a lure ( Z 8 - 1 4 : O A c and 14 : OAc). Some 96% of males in the traps were caught in the final two weeks of the trial. This is due probably to an initiation of a moth flight period rather than to the decay of pheromone components in the lures to a more optimal concentration, since a similar phenomenon was observed in the lower concentration in the trial. Trial 2 confirmed the efficacy of the synthetic lure in comparison with virgin females and trial 3 the necessity for the two-component mix for attraction. Trial 4 was conducted at a site where the moth has not previously been found and confirms the lure's efficacy in what is probably a low-density population (previous searches for this moth in this area failed to detect its presence, Dugdale, personal communication). The traps baited with P . e x c e s s a n a lures caught few moths. The morphological similarity between P . e x c e s s a n a and the mangrove-feeding P l a n o t o r t r i x males precluded absolute identification of the moths in the trap, and so it could not be determined if any P. e x c e s s a n a were captured in these tests.
635
SEX PHEROMONE OF Planotortrix
TABLE 1. TRAP CATCHES OF MALE Planotortrix MOTHS TO COMBINATIONSOF (Z)-5-TETRADECENYL ACETATE, TETRADECYL ACETATE, AND (Z)-N-TETRADECENYL ACETATE Z5-14 : OAc (~g) Trial 1
Trial 2
10 100
14 : OAc (~g) 10 100 100
10 l0 i00 100 300 300 1000 1000 Virgin females 100
Trial 3
0 100 100
0 0 100
Trial 4
100
100 100
Z8-14 : OAc (~g)
Mean No. of male moths per trap "
100
5.0 ~ 22.7 h 0.3 c
100
3.W 6.3 ~ 3.3 ~ 5.7 a 4.7 a 0.7 b 0.0 ~ 0.2 ~' 5.6"
I00
5.0 ~ 1.0 b
~Means not followed by the same letter are significantly different, P < 0.05, ANOVA. Trial 1 conducted January 27-February 20, 1984, at Thames, three replicates, Pherocon 1C traps. Catches recorded three times. Trial 2 conducted February 20-March 1, 1984, at Thames, three replicates, Pherocon 1C traps. Trial 3 conducted February 27-March 14, 1984, at Thames, five replicates, Sectar 1 traps. Trial 4 conducted February 21-March 23, 1984, at Avondale, Auckland, five replicates, Sectar 1 traps.
DISCUSSION T h e sex p h e r o m o n e , Z 5 - 1 4 : O A c + 14 : O A c , o f the m a n g r o v e - f e e d i n g species o f P l a n o t o r t r i x was f o u n d to be quite different f r o m the p o p u l a t i o n s o f P. e x c e s s a n a w h i c h had p r e v i o u s l y b e e n reported to be Z 8 - 1 4 : O A c + 14 : O A c in one location and p o s s i b l y Z 5 - 1 4 : O A c + Z 7 - 1 4 : O A c in another location (Galbreath et al., 1985). A l t h o u g h all three p o p u l a t i o n s are m o r p h o l o g i c a l l y indistinguishable, the significant difference in m a t e r e c o g n i t i o n systems should be sufficient to classify these p o p u l a t i o n s as sibling species. Sibling or cryptic species h a v e b e e n defined as " s p e c i e s that m o r p h o l o g i c a l l y r e s e m b l e e a c h o t h e r so closely that they can be r e c o g n i z e d o n l y after careful study o f b i o c h e m i c a l , c y t o l o g i c a l , or b e h a v i o r a l t r a i t s . " ( D i e h l and Bush, 1984). T h e host p r e f e r e n c e o f the m a n g r o v e - f e e d i n g P l a n o t o r t r i x species is also v e r y different f r o m the p o l y p h a g o u s P. e x c e s s a n a ( D u g d a l e , 1966).
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In Tortricidae, Z 5 - 1 4 : OAc has been found as a sex pheromone component only twice--both times in New Zealand species. Ctenopseustis o b l i q u a n a (Walker), the brownheaded leafroller, was found to use a combination of Z 5 14" OAc and Z 8 - 1 4 : O A c (Young et al., 1985), and the P. excessana "species" from the central North Island apparently uses a combination of Z 5 14 : OAc and Z 7 - 1 4 : OAc (Galbreath et al., 1985). Z 5 - 1 4 : OAc also has been reported to be a sex pheromone component of a cossid moth (Capizzi et al., 1983) and a noctuid moth (Bestmann et al., 1980), and as part of an attractant blend for several other noctuid moth species (Steck et al., 1982). Since the compounds found in the New Zealand leafroller species are u n c o m m o n in other Tortricidae (Roelofs and Brown, 1982), further studies on the sex pheromone of other related New Zealand leafroller populations and the biosynthetic pathways used in these primitive species should provide valuable information on evolutionary relationships within the genus P l a n o t o r t r i x and between other genera in the tribe Archipini. Acknowledgments--We especially thank Mr. J.S. Dugdale for directing our attention toward this species. We also thank Dr, C. Green for collection of the larvae from the field and Val Holt for rearing the insects. We are grateful to Dr. C. Triggs for statistical analysis of the data. We thank the N.R.A.C. for the award of a Senior Postdoctoral Research Fellowship to one of us (W.L.R.), permittingthis cooperation.
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