Does Pheromone Biology Of Spring Hemlock Looper, Lambdina aihasaria, and Pitch Pine Looper, Lambdina peliucidaria (Lepidoptera : Geometridae) Contribute To Their Reproductive Isolation?
by Cameron M. Duff Diploma of Technology in Agricultural Management British Columbia Institiute of Technology, Burnaby, B.C.
THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
MASTER OF PEST MANAGEMENT
in the Department of Biological Sciences
@ Cameron M. Duff 1998 Simon Fraser University August 1998
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Recently. 7 - methylheptadecane (7) and 7, 1 1 - dimethylheptadecane (7,11) have been reported as sex pheromone components of both spring hernlock looper (SHL),
Lambdina arhosaria, and pitch pine looper (PPL), Lambdinopellucidmin. My objective was to test the hypothesis that SHL and PPL are reproductively isolated, in part, through species - specificity in: 1) absolute configuration of pheromone components; 2) die1 penodicity of pheromonal communications; andior 3) seasonal £light penod.
In coupled gas chrornatographic - electroantennographic detection (GC - EAD) analyses of stereoselectively synthesized (7s) - and (7R) - 7 - methylheptadecane [(7S); (7R)],as well as (7S, 1159 - (7R, 1 1R) and (meso 7, 11) - 7,11-dimethylheptadecane [(7S, 11s);
(7R, 1IR); (meso 7, 1 l)] oniy (79 and (meso 7, 11) eiicited responses by male SHL and
PPL antennae. in field experirnents, male SHL and PPL were attracted only to lues containing (7s) plus (meso 7, 11). In hourly rccordings of trap-captured males, SHL and
PPL in their respective habitats were trapped between 2400 h and 0300 h.
Capture of both SHL and PPL in pheromone-baited traps throughout June, indicated overlapping seasonal fiïght periods. These findings of identical sex pheromone, identicai diel periodicity of pheromone communication and overlap of seasonal flight periods do not support taxonomie separation of SHL and PPL. Udess essential pre- or post-zygotic reproductive isolating mechanimis are identified, synonymy of SHL and PPL should be considered. ACKNOWLEDGEMENTS
1 thank Regine Gries for GC - EAD analyses, Chris Maier and Gale Ridge
O' Corner of the Connecticut Agricultural Research Station in New Haven Connecticut, for assistance in locatùig field sites and CO-ordinating field experiments, Austin Mason and staffat Myles Standish State Forest, Plymouth MA for kindly permittïng my work in the forest, and to Paul Froese and Al Oliver, fomedy of Agriculture and Agi-Food
Canada, for allowing me to carry out my work with the Plant Protection Division of
Agriculture and Agri-Food Canada, now the Canadian Food Inspection Agency in a reduced capacity. Special th& go to Emma, who despite valiant atternpts to delay her arriva1 into the world was born during my absence on location in the field and to rny wife,
Maureen for her tolerance and support. Careful review of this thesis was done by John
Borden. Gerhard Gnes initiated this project, provided invaluable advice in experimental design and assisted me fiom beginning to end, for that 1am trdy thankfùl. This research was suppoaed, in part, by Agriculture and Agri-Food Canada and the Naturd Sciences and Engineering Research Council of Canada, TABLE OF CONTENTS
FRONTISPIECE .. APPROVAL ...... 11 ... ABSTRACT...... 111
ACrCNOWLEDGEMENTS...... iv
TMLE OF CONTENTS ...... v
LIST OF TABLES ...... - ...... vi . LIST OF FIGURES ...... w.
1. 0. INTRODUCTION...... 1
2.0. METHODS AND MATERIALS ...... IO
2.1 . Syntheses...... ,...... 10
2.2. Laboratory Analyses ..*...... ,...... 10
2.3. ExperUnental Sites...... 15
2.4. Determination of absolute configuration of pheromone components...... 15
2.5. Height-dependent trap captures of male SHL and PPL ...... 16
2.6. Die1 periodicity and seasonality of trap captures...... 16
2.7. Statistical Analyses ...... 17
3.0. RESULTS ...... 17
4.0. DISCUSSION...... 41
REFERENCES ...... 44 LIST' OF TABLES
TABLES PAGES
TABLE 1. Cornparison, with references, of life history traits and distribution
of spring hemlock looper (SHL), pitch pine looper ( PPL),
western hemlock looper (WKL), and eastern hemlock looper
(EHL). Where no references cited, data are derived fiom
personal observation...... 6-8
TABLE 2. Description, with references, of experimental sites. Where no
references cited, data are derived fiom personal observation...... 9 LIST OF FIGURES
FIGURES PAGES
FIG. 1. Colouration of maIe spring hemlock looper (SHI,), Lambdina
athasaria (Iefi) and male pitch phe looper (PPL), Lambdina
pellucidaria (right)...... 2-3
FIG. 2. Pheromone components of eastem hemlock looper (EHL), Larnbdina
fiscellariafiscelZaria, western hemlock looper (WHL), Lambdina
fiscellaria lugubrosa, sp~ghemlock looper (SHL), Lambdina
athasmia, and pitch pine looper PPL), Lambdina pellucidaria (Gries
et al. 1991 a, b, 1993, 1994; Li et al. 1993 a, b; Maier et al. 1998).
Astensks indicate that absolute configuration of pheromone
components are not yet determined...... 4-5
FIG. 3. Optical isomers of pheromone components of female spring hemlock
looper (SHI.,), Lambdina athasaria and the female pitch pine looper
(PPL), Lambdina pellucidaria. (7R)- 7 - methylheptadecane; (7S) - 7 -
methylheptadecane; (7S, 11S) - 7, 1 1-dimethylheptadecane; (meso 7,
11) - 7,ll- dimethylheptadecane ande (7R, 1IR) - 7,11 -
dimethylheptadrcane...... 11 - 12
vii FIG. 4. Map depicting geo graphie location of experirnental sites (stars)for
spring hemlock looper (SHI,) (Peoples State Forest, Barkhamsted,
CT) and pitch pine looper (PPL) (Myles Standish State Forest,
Plymouth, MA). Sites stocked mainly with eastern hemlock, Tsuga
canadensis, and pitch pine, PNncr rigida, respec tively (Table 2)...... 13- 14
FIG. 5. Representative GC - EAD recordings fiom male spring hemlock
looper (Sm)and f?om male pitch pine looper (PPL) antennae,
responding to 1000 pg of 2,s - dimethylheptadecane [intemal
standard (IS)] and to 2 pg of optical isomers of 7 -
methylheptadecane and 7,11 - dimethylheptadecane. fhte~d
recordings conducted in consecutive order (top to bottom)
ernploying the same antennae. Flarne ionization detector (FID)
recordings not presented. Note ImV Ievel indicated on top right
EAD recording...... ,, ...... 19-20
FIG. 6. Captures of male spring hemlock loopers (SHI,)(Exp. 1; June
10 - 12, 1997; n = 10) and pitch pine loopers (PPL) (Exp. 2; May
30 - June 3, 1997; n = 1O), in Unitraps baited with stereoisomeric
(7, 11) alone and in combination with (7S), (7R)or both. For
each experiment, bars with the same letter are not significantly
different, P < 0.05......
.S. Vlll FIG. 7. Captures of male sp~ghemiock loopers (SHI,) (Exp. 3; Sune 12 -
14, 1997; n = 10) and male pitch pine loopers (PPL) (Exp. 4; June
3 - 5, 1997; n = 10) in Unitraps baited with (7s) alone (SHL) or
(73 t (7R)done (PPL) and in binary and quatemary combinations
with (7S, 1 IS), (7R,1 1R) and (meso 7,ll). For each experiment,
bars with the same Ietter are not sipnificantly different, P < 0.05...... 23-24
FIG. 8. Captures of male sp~ghemlock loopers (Sm)(Exp. 5; June 14
- 16, 1997; n = 10) and male pitch pine loopers (PPL) (Exp. 6;
June 5 - 7, 1997; n = I O) in Unitraps baited with (7S) alone (SHL)
or (7s) + (7R) aione (PPL), and in binary combination with
(meso 7, Il) and in dl temary and quatemary combinations with
(7S, 11 S), (7R, 11 R), and (meso 7,11). For each experiment, bars
with the same letter are not significantly different, P < 0.05. ....-...,...... , 35-26
FIG. 9. Captures of male pitch pine loopers (PPL) (Exp. 7; June 7 - 11,
1997; n = 10) in Unitraps baited with (759 + (meso 7, I 1) alone
and in combination with hexane or (7R) at three different ratios.
For each experiment, bars with the same letter superscript are not
significantly different, P < 0.05...... 27-28. FIG. 10. Captures of male spring hemlock loopers (SHL) (Exp. 8; June 16
-1 8, 1997; n = 1O) and male pitch pine loopers (PPL) (Exp. 9;
June 11- 13, 1997; n = 10) in Unitraps baited with (79 aione and
in cornbination with (meso 7, 11) at three diffèrent ratios. For
each experiment, bars wÏth the same Ietter are not significantly
different, P < 0.05...... 29-30
FIG. 11. Captures of male spring hemlock loopers (SHL) (Exp. 10; June
18 - 20, 1997; n = 10) and male pitch pine loopers (PPL) (Exp.
11; June 13 - 15, 1997; n= 10) in Unitraps baited with (meso 7, 11)
alone and in combination with (7S) at keedifferent ratios. For
each experurient, bars with the same ietter superscript are not
significantly different, P < 0.05- ...... 3 1-32
FIG. 12. Captures of male spring hemlock loopers (SHL) (Exp. 12; June
20 - 22,1997; n = 10) and male pitch pine loopers (PPL) (Exp.
13; June 15 - 17, 1997; n = 10) in Unitraps baited with (73 i-
(rneso 7, l l) in the same ratio at four diEerent doses. For each
experiment, bars with the same letter are not sigficantiy
different, P < 0.05 ...... , ...... FIG. 13. Captures of male spring hemlock loopers (SHL) (Exp. 14; June
19 - 22, 1997; n = 10) and male pitch pine loopers (Exp. 15; June
17 - 21, 1997; n = 10) in Unittraps suspended Çom trees at 1.5,3,
and 5 m above ground and baited with (75) (50 pg) + (rneso 7,ll)
(16 pg). For each experiment, bars with the same Ietter are not
significantly different, P < 0.05...... ~...... ~...... 3 5-3 6
FIG. 14. Hourly recordings of trap captured male spring hemiock loopers
(Sm)(Exp. 16; June 19 - 22, 1997; n = 10) and male pitch pine
loopers (PPL) (Exp. 17; June 17 - 18, 1997; n = 10) in Uniîraps
baited with (7s) (50 pg) + (meso 7, 11) (16 pg)...... 37-38
FIG. 15. Captures of male spring hemlock loopers (SEL) (Exp. 18; May 3 1 -
June 26, i 997; n = 10) and male pitch pine loopers (PPL) (Exp. 19
May 3 1 - June 26, 1997 n = 10) in Unitraps baited with enantiomeric
(7) (50 pg) and stereoisomeric (7,11) (50 pg)...... 39-40 1.0. INTRODUCTION
The genus Lambdina comprises 10 species. Life history traits and pheromonal
communication have been well studied for four species, including the spring hemlock looper
(SHL), Lambdinu athasaria (Wallcer) (FIG. 1,2, TABLE l), the pitch pine looper (PPL), L.
pellucidmia (Grote and Robinson), the eastem hemlock looper @HL,), L. fiscellaria
ficellaria (Guenée), and the western hemiock looper (WHL,). L. jkcelZaria lzigubrosa
(HLLlst).
The EHL, WHL and SHL are separated taxonomically by differences in morphology,
life history, host and habitat preference and distribution (TABLE 1,2) andlor pheromonal
blends (FIG. 2). For example, spatial separation of coseasonal EHL and WHL, temporal
separation of syrnpatric EHL and SE,and specificity of pheromone blends contribute to
their reproductive isolation (Gries et al. 1994).
SHLs and PPLs, in con&ast, have remarkably similar Life histories (TABLE 1).
Although they prefer hemlock (SHL) and pine (PPL) forests, they are neither spatiaily nor tempordy isolated. With identical sex pherornone blends (HG. 2) one might question how they maintain reproductive isolation. I tested the hypotheses that the PPL and the SHL are reproduchvely isolated, in part, through species-specificity in: 1) absolute configuration of pheromone components; 2) die1 penodicity of pheromone communication; andlor 3) seasonal flight period. FIG. 1, Colouration of male spring hemlock looper (Sm),Lambdinn athasuria (Ieft)
and male pitch pine Iooper (PPL), Lambdinnpellucidaria (right).
FIG. 2. Pheromone components of eastern hemlock looper (EHL), Lambdina
fiscellariafircellari4, western hemlock looper (WHL), Lambdinafircel[oria
lugubrosa, spring hemlock looper (SHL), Lambdina alhasaria, and pitch pine
looper (PPL), Lambdina pelhcidaria (Gries et al. 1991 a, b, 1993, 1994; Li et
al. 1993 a, b; Maier et al. 1998). Astensks indicate that absolute confibouration
of pheromone components are not yet determined. Lambdina fsceilatfa fiscellana
Lambdina athasaria
(7) 6
TABLE 1. Cornparison, with references, of life history traits and distribution of spring
hemlock Iooper (SHL), pitch pine looper ( PPL), western hemlock looper
(WHL), and eastem hemlock Iooper (EE). Where no references cited, data
are derived fiom personal observation.
- Traits 'WEn, EHL PPL SEL
Size of aduIt 32-35 mm ( Koot 35 mm (De Gryse 28-37 mm (USDA male 1994, Furniss and and Schedl 1934) Forest Service 1985) CaroIin 1977)
Colouration of fatvn coloured, with tan to grayish brown medium brown with tan, highIy speckled male two irregular Iines on with nvo iregular two irreguIar di£lke with nvo irregular the fore wing and Iines on the fore dusky markings on diffuse dusky one on the hind wing wing with a dot of fore and one on hind markings on fore and (Koot 1994, Furniss the sarne colour near wing (Grehan and one on hind wing and Carolin 1977) the costai margin Parker 1994, Capps (Grehan and Parker (USDA Forest 1943) 1994, Capps 1943) Service 1985)
Flight penod late August to early mid-August to mid mid -;May to end of mid- May to end June October (Koot October (De Gryse June 1993) and SchedI 1934)
Time of 80- 100% of virgin rnidnight (Grk et al . mating per day femaies cd1 between 1994) 2200 - 0200 h (West and Bowers 1994)
Ovipostion site moss, lichen ,uee needles, bark old foIiage 2.5 cm boles, limbs, crevices, stumps and from new growth undemory shrubs fallen trees (De @hier and Lemmon and dom Iogs Gryse and SchedI 1996) (Furniss and Carolin 1934) 1977)
Fecundity 41-148 (De Gryse and Schsdl t 934)
translucent gay to newly laid eggs greenish (Maier and brown, barrei shaped broadIy oval ,grass- Lemmon 1996) with characterisic green and after hvo impression; 1 mm weeks become long x 0.75 mm wide copper- brown; 0.7 1- ( Koot 1994, Furniss 0-96 mm in wide (De and Carolin 1977) Gryse and Schedt 1933) Traits PPL SHL
Fim instar First instar is duII Full-grom larvae Final instar oIive conspiousiy banded white with a dark pale straw to yellow green to pale grey. Iight grey and brown had capsule. with black markings; Colour intensity varies bIack. Final instars Two nmow dark 27-32 mm long. with extent of paie yellowish- iongitiudinal Lines on Body bears faim pi-mented sclerotized brown to gray- each side of the rows of blackish dots bands. Mid-dorsal brown, and have a body, one subdorsai, and short wavy lines line and a fine broken cornplex pattern of the other sublareral. - on the top and sides. line, each side of markings; 5-35 Final instar paie Head is densly mid-dorsal line. Head mm Iong (Koot green to dak brown, marked with light pale grey-white to 1994) head with brownish and datk spots dark brown with dark markings foming (USDA Forest sclerotized spots of irreguIar spots Service 1985) van-able sizs (Grehan around the setae (De and Parker 1994) Gryse and Schedl 1934)
six ( Koot 1994) four to six -nit2 five or six (Maier and 1992) Lemmon 1996)
Food plants of Tsuga heferophylla, Tsugu cunadensîs Pinrcs rigida, Tmga canadensis Iamae Thuja plicata. Abies Abies balsameu; P. resinosa, Abies balsarnea spp.. Pseudotsuga under oubreak P. echinara , (Maier and Lemmon rnertziesii. Pinus conditions other P. virginiana 1996) monticola. Lartr coniferous and (USDA Forest occidenralis, Picea angiospem tree and Service 198s') spp.: under oubreak shb species will be conditions defoliated. angiospem specirs * Only Taxus wiIl be defoliated canadensis not (Koot 1994, defoliated (De Gryse Fumiss and CaroIin and Schedl 1934, 1977) Watson 1934)
Pattern/ Fim and second Young larvae feed First two instars feed First hvo instars feed syrnptorns of instars Iight feeders on new foliage and on new growth, and on nerv growh, and larvaI feeding on bucis, later then quickly move to last three instars eat last three instan eat instars wastefùl old foliage, returning needles developed r needles developed - feeders on old to new foiiage when years eariier @hier 2 years eariier. Under foliage. Early old foliage is and Lemmon 1996) affectcd crees masses feeding occurs on depleted. Larvae feed of clipped needles can the crowns and as by cutrting needles be observed (Maier Iarvae mature they near the base. and Lernmon 1996) descent to feed AfFected twigs have lower in the crown. curled, dry, reddish AfSected trees mm brorvn partly yeliowish red then destroyed needIes. brown ( Koot (De Gryse and 1994, Fumiss and Schedl. 1934, CaroIin 1977) Watson 1934) Traits ENI, PPL SHL
Pupation site Bark crevices. Bark crevices, Ioose Duff in fores floor Duff in forest fl oor moss, lichen, or in bark, root junctions (Maier and Lemmon (iMaier and Lemmon debris on the forest and under debris on 1996) 1996) floor ( Koot 1994) fores floor (De Gryse and Schedl 1934)
Ovenvintering Eggs ( Koot 1994) Eggs (De Gryse and Pupa (Maier and Pupa elaier and stage Schedl 1934 ) Lemmon 1996) Lemrnon 1996)
OvewinterÏng Moss and lichen Needles, bark Duff in forest floor Duff in foresr floor site and on the lower crevices, on stumps (Maier and Lernrnon (Maier and Lemmon boles ( Koot 1994) and Failen trees (De 1996) 1996) Gryse and Schedi 1934)
Range Coastril Alaska, Maritime regions of Connecticut, Connecticut, British Columbia, Eastern North New Jersey, New Pennsy fvania Washington, and Arnerica, but ranges York (USDA Forest (Carneron and Mastro, Oregon; west to Quebec, Service 1985) 1975) mountianous areas Ontario, Wisconsin of B.C., northern and Alberta (De Idaho, western Gryse and Schedl Montana and 1934, Watson 1934) northeasrem Oqon (E;rannitz 1992)
Habitat Coastal Western HemIock and oak forest Interior Cedar; (Schweitzer and (suaon and Sutton Hemlock Rawinski 1988) 1985) 3iogeoclimatic zones in B.C. In outbreaks infmtions reported in Intenor Douglas Fir and Sub-boreal Spmce zones (Krannitz 1992) TABLE 2. Description?with references, of experimental sites. Where no references cited,
data are derived fiom personai observation.
-- -. SHL PPL Peoples State Forest, CT lMyIes Standish State Forest, MA Ecological Transition Forest (Sutton and Pitch pine / Scrub oak Woodland zone Sutton 1985) (Pine Barrens) (Schweitzer and Rawinski 1988) Tree species 90% Tsuga canademis; 97% Pinus rigida; in overstorey 7% Quercus alba ,Q. rubru, 3 % P. resinosa @lanted) macrocarpa; 3 % Pinus strobus Tree species Tilea americana, in Rhododendron rnaimum, understorey KaZrnia latifolia Age of forest Elevation 300 m (Owenby and Ezell 20 m (Owenby and Ezell1992) 1992) Mean annual 8.78 OC (Owenby and Ezell 9.44 OC (Owenby and Ezell 1992) temperature 1992) Soil type coarse sand (Schweitzer and Rawinski 198 8) 2.0. METHODS AND MATERIALS
2-1. Syntheses
(73 - 7 - Methylheptadecane, (7R) - 7 - methyheptadecane, (7S, 11S) - 7, 11 -
dimethyheptadecane, (7R,1 1 R) - 7, 1 1 - dimethylheptadecane and (meso 7, 11) - 7, 11 -
dimethylheptadecane hereafter referred to (757, (7R),(7S, 1lS), (7R, 1 IR) and (meso 7,ll)
(HG. 3,were synthesized (Sheng 1996), or obtained fkom K. Mon, Department of
Chemistry, Faculty of Science, University of Tokyo, Kagnarazaka 1-3, Shinjuki-ku, Tokyo
162, Japan. Based on GC- analyses, ail compounds were > 94 % chemically pure.
2.2. Laboratory Analyses
Aduit male SHLs and PPLs were collected at night in the Peoples State Forest,
Barkhamsted, Litchfield County, CT and Myles Standish State Forest, Plymouth, Plymouth
County, MA, respectively (HG. 4). Moths were cooled (8 OC) overnight and couriered to
Simon Fraser University the next day for coupled gas chromatographie - electroantennographic detection (GC - EAD) analyses (Amet al. 1975). Each antenna tested
(4 and 5 for male SHL and PPL, respectively) was subjected in the same order to three mixnires of candidate pheromone components at 2 pg each. Mixtures 1 - 3 contained (7R) +
(7R, 11 R), (7R) + (7S, 1 1S) and (753 + (meso 7, 11), respectively . An intemal standard, 1 ng of 2, 5 - dirnethylheptadecane, was coinjected with each mixture, to monitor possible deterioration of antemal responses over the 30 - 40 min test period for each antenna. FIG. 3. Optical isorners of pheromone components of female spring hemlock looper
(SHL), Lambdinn athasaria and the female pitch pine looper (PPL),
Lambdinapellucidaria. (7R)- 7 - methylheptadecane; (7s)- 7 -
methylheptadecane; (7S, 115') - 7,11 - dimethylheptadecane; (meso 7, 11) - 7,
1 1 - dimethylheptadecane and (7R, 1IR) - 7, 1 1-dimeîhylheptadecane.
FTG. 4. Map depicting geographic location of experimental sites (stars) for spring
hemlock looper (SHL) peoples State Forest, Barkhamsted, CT) and
Pitch pine looper (PPL) (Myles Standish State Forest, Plymouth, MA). Sites
stocked mainly with eastem hemiock, Tsuga canadensis, and pitch pine, Pinus
rigida, respectively WLE2).
3-3. Experimental Sites
Experiments on SHLs and PPLs were conducted at the Peoples State Forest and the
Myles Standish State Forest respectively (FIG. 4, TABLE 2). In the Peoples State Forest 120
km inland, the Transition Forest of coniferous and angiospem tree species forms a dense
canopy with Iittle light penetration. The forest lies at 300 m in elevation and receives 306 mm
of min fiom June to August (Owenby and Ezell 1992). The Pine Barrens at Myles Standish
State Forest exhibit an open forest canopy allowing dense thickets of scmb oak, Querms
ikifolia, to dominate the understorey, accompanied by heath-like shrubs. Directly on the
Atlantic Coast, the Pine Barrens experience a moderated, ocean-innuenced climate, but with
little summer min and prolonged penods of drought (Schweitzer and Rawkski 198 8).
2.4. Determination of absolute configuration of pheromone componentsi
Experiments (Exp.) of identical or compatible design, conducted concurrentiy for
both the SHL and PPL (except Exp. 7), were set up Ïn randomized, complete blocks with
traps and blocks a 15-20 m intervals. Green Unitraps (Phero Tech Inc., Delta, B.C.) were
suspended fiom T. canaciensis (SHI,)and P. rigida @PL) at 1.5 - 2 m above ground and 2-5
m within the forest margin. Traps were baited with gray rubber septa (The West Company,
Lionville, PA) impregnated with optical isomee of synthetic pheromone components in
HPLC-grade hexane or hexane. Each trap contained a Dichlorvos cube (Bio-Strïp, Inc.,
Reno, NV) to kil1 captured rnoths quickly. Captured males were removed and counted every
' Dates and number of replicates for dl field experiements are given in the captions of FIGs 6 - 15 (Section 3.0.). two days-
Exp. i (SHL) and Eq. 2 (PPL) tested stereoisomenc (7, Il) alone and in
combination with either (79, (7R) or both. (73 or (7S) i(7R) were tested done and in
binary and quatemary combinations with (7S, 119, (7R, 11R) and (meso 7, 11) in Exp. 3 and
4 against the SHL and the PPL respectively. Exp. 5 (SHL) tested (79 alone and in binary
combination with (meso 7, 11) and ail possible ternary and quatemary combinations with
(7S, 11S), (7R, 1IR) and (meso 7, 11). Exp. 6 (PPL) was identical to Exp. 5: but replaced
(73 with (79 + (7R). Exp. 7 was conducted only with PPL and tested (7s) + (meso 7, 11)
alone and in combination with hexane or (7R) at three different ratios. Exp. 8 (SEL) and
Exp. 9 (PPL) tested (759 alone and in combination with (meso 7, Il) at three different ratios.
Exp. 10 (SHL) and Exp. 11 (PPL) tested (meso 7, 11) done and in combination with (7S) at
three different ratios. Exp. 12 (SHL) and Exp. 13 (F'PL) tested (7s) plus (meso 7, Il) at
increasing doses.
2.5. Height -dependent trap captures of male SHL and PPL
For Exp. 14 and 15 ten trees > 200 m apart were selected at each experimental site
FIG. 4; TABLE 2). Unitraps baited with gray rubber septa irnpregnated with (7s) (50pg)
and (meso 7,11) (16pg) were hung at 1.5,3 and 5 rn above ground in each tree. Captured
male SHLs (Exp. 14) or PPLs (Exp. 15) were recorded two days after expenment initiation.
2.6. Die1 penodicity and seasonaliv of trap captures
Exp. 16 and 17 recorded the die1 penodicity of captures of male SESand PPLs, respectively. Ten trees > 100 m apart were selected at each experimental site (FIG. 4;
TABLE 2). One Uniû-ap baited with gray rubber septa Mpregnated with (75') (50,zg) and
(meso 7, 1 1) (16pg) was hung 1.5 m above ground and 2-5 m within the forest margin in
each tree. Captured males were recorded hourly for 24 h.
Exp. 18 and 19 recorded seasonal flight penods of male SHLs and PPLs, respectively.
Selection of trees and trap placement were the same as in Exp. 16 and 17. Traps were baited
with gray rubber septa impregnated with racernic (7) (50 pg) and (7, 11) (16 pg)- Captured
males were removed and counted every two days fiom May 28 to June 24, 1997.
2.7. Statistical Analyses
Despite transformation, most trap catch data were not nonnaIly distnbuted and were therefore subjected to nonparametric analyses of variance by rads (Friedman's test) followed by conparison of meam (Bonferroni test) (Zar, 1984; SAS/STAT 1988 user guide, release
6.03 edition, SAS Institute, Cary, NC).
3.0. RESULTS
In GC - EAD recordings, (79, but not (7R),and (meso 7, 11) , but not (7S, 1iS) or
(7R,1 1R) elicited responses fiom mde SKZ. and PPL antemae (HG. 5). In Exp. 1 and 2 (7s) in combination with stereoisomerïc (7, I 1) atûacted male SHLs and PPLs (FIG. 6). (7R) was inactive with (7,11) for both species, but doubled captures of male PPL when added to (79 and (7,ll). For both SKLs and PPLs, the synergistic pheromone component of (7,ll) proved to be (meso 7, 11) (FIG. 7,8); (7S, 115') and (7R, 1 IR) were benign. Re-testing (7R) as a potentid pheromone component for the PPL in Exp, 7 failed to disclose any behavioual activity associated with this compound (FIG. 9). The blend of (7.9 + (meso 7, 11) at a 50: 16 pg ratio was more attractive to male SHL and PPL than blend ratios with reduced amounts of either (meso 7, 1 1) (FIG. 10) or (79 (FIG. 1 1). When (7s) +- (meso 7,11) were tested at the same ratio with five doses separated by orders of ma,@itude in Exp. 17 and 13, only traps baited with the highest dose of 50: 16 pg captured significant numbers of males (FIG. 12).
Traps suspended at 1.5 m above ground captured signinifcantly fewer male SHLs than those at 5 m (Exp. 14) and siCgifïcantlyfewer PPLs than those at 3 and 5 rn (Exp. 15) (FIG.
13). In hourly recordings of captured males, seven SHLs and five PPLs were captured between 2400 h and O300 h (HG. 14). The seasonal flight penods of SHLs and PPLs overlapped (FIG. 1j), but very few SHLs were captured in the dand site until mid-June. FIG. 5, Representative GC - EAD recordings fiom male spring hemlock Iooper (SHL)
and fiom male pitch pine looper (PPL) antennae, responding to 1000 pg of 2,
5 - dimethylheptadecane [intemal standard (IS)] and to 2 pg of optical isomers
of 7 - methylheptadecane and 7,11 - dimethylheptadecane. hte~d
recordings conducted in consecutive order (top to bottom) employing the
same antema. Flame ionization detector (FID) recordings not presented. Note
1mV level indicated on top right EAD recording. PPL
I I I 1 11 12 11 12 TIME (min) CHEMICALS TiME (min) FIG- 6. Captures of male spring hernlock loopers (SHL) (Exp. 1; June 10 - 12, 1997; n
= 10) and pitch pine loopers (PPL) (Exp. 2; May 3 0 - June 3, 1997; n = 1O), in
Unitraps baited with stereoisomeric (7, 11) done and in combination with
(757, (7R) or both. For each experiment, bars with the sarne letter are not
significantly dBerent, P < 0.05. SHL
Exp. 1
PPL
Exp. 2
MONO- and DIMETHYLHEPTADECANES FIG. 7. Captures of male spring hedock loopers (SHL) (Exp. 3; June 12 - 14, 1997; n
= 10) and male pitch pine loopers @?PL)(Exp. 4; June 3 - 5, 1997; n = 10) in
Unitriips baited with (79 done (SHL) or (73 + (7R) alone (PPL) and in
binary and quaternary combinations with (7S, 1 13, (7R,1 1R) and (meso
7,11). For each experiment, bars with the same letter are not significantiy
different, P < 0.05. SHL
Exp. 3
PPL
50 7R - 7SJ1 S - 7R, 11R - meso 7.11
MONO-and DIMETHYLHEPTADECANES FIG. 8. Captures of male spring hemlock loopers (SHI,) (Exp. 5; June 14 - 16, 1997; n
= 10) and male pitch pine Ioopes (PPL) (Exp. 6; June 5 - 7, 1997; n = 10) in
Unitraps baited with (753 alone (SE) or (7S) + (7R) alone (PPL), and in
binary combination with (meso 7, 11) and in dl temary and quaternary
coinbinations with (7S, 113, (7R,11 R), and (meso 7,ll). For each
experiment, bars with the same letter are not significantly different, P < 0.05. SHL
Exp. 5
PPL
Exp. 6
MONO- and DIMETHYLHEPTADECANES FTG. 9. Captures of male pitch pine loopers @PL) (Exp. 7; June 7 - 11, 1997; n = 10)
in Unitraps baited with (759 + (meso 7, 1 1) alone and in combination with
hexane or (7R) at three different ratios. For each experiment, bars with the
sarne letter superscnpt are not significantly different, P < 0.05. PPL
hex-ane (pl) mes0 7,11 (ug) MONO- and DIMETHYU 'EPTADECANES FIG. 10. Captures of male spring hemlock loopers (SHL) (Exp. 8; June 16 - 18, 1997; n
= 10) and male pitch pine loopers @PL) (Exp. 9; June 1 1- 13, 1997; n = 10)
in Unitraps baited with (79 aime and in combination with (mes0 7, 1 1) at
three different ratios. For each experiment, bars with the same letter are not
significantly different, P < 0.05. SHL Exp. 8
PPL Exp. 9
50 50 50 50 7s (w) 16 1.6 0.1 6 - meso7,11(pg) MONO- and DIMETHYLHEPTADECANES FIG. 1 1. Captures of male spring hemlock Loopers (SHI,)(Exp. 10; June 18 - 20, 1997;
n = 10) and male pitch pine loopers (PPL) (Exp. 1 1 ; June 13 - 15, 1997; n =
20) in Unitraps baited with (meso 7, 11) alone and in combination with (79 at
three dif5erent ratios. For each experiment, bars with the same Ietter
superscript are not sibgïficantly different, P < 0.05. SHL
...... ,*.*.-.*-...... ,*.*..-**--**..-* ,...... ,..-.-.--.---.-a.-.--.-.. ***.**.*...... *.**..*..---.-...... --.--...... -.-.-.-...... -.*..-...... -..-..*.-.-..-...... -*.--**...... -*.*..--.*.-.*. .-..--...... -.--..-...... --m.-.- ..*-***.* * - - - . - ...... -.*-**- ab ...... l b ...... -*--.-*. ---.--.- b *--*-*S...... ---*-..-*.--.- --.-.---...... - I .-.m.----.-----...... ----**...... 50 5.0 0.5 - 75 16 16 16 16 mes0 7,11 (pg)
PPL
Exp. 11
MONO- and DIMETHYLHEPTADECANES FIG. 12. Captures of male spring hemlock loopers (SHI,) (Exp. 12; June 20 - 22, 1997;
n = 10) and male pitch pine Ioopers (PPL) (Exp. 13; June 15 - 17, 1997; n =
10) in Unitraps baited with (79 + (meso 7, 11) in the same ratio at four
different doses. For each experiment, bars with the same letter are not
signincantly different,
P < 0.05. SHL
Exp. 12
0.5 0.05 75 (pg) 0.16 0.01 6 7,11 (pg)
PPL
Exp. 13
0.05 7s 0.016 7,11 (pg) MONO- and DIMETHYLHEPTADECANES FIG. 13. Captures of mde spring hemlock loopers (SHL) (Exp. 14; June 19 - 22, 1997;
n = 10) and male pitch pine loopers (F'PL) (Exp. 15; June 17 - 2 1, 1997; n =
10) in Unitraps suspended fkom trees at 1.5,3, and 5 m above ground and
baited with (73 (50 pg) + (meso 7,11) (16 pg). For each experiment, bars
with the same letter are not si@cantly different, P < 0.05. a SHL
PPL
Exp. 15
1.5 3 5 TRAP HElGHT (m) FIG. 14. Hourly recordings of ~apcaptured male spring hemlock loopers (SHL) (Exp.
16; June 19 - 22, 1997; n = 10) and male pitch pine loopers (PPL) (Exp. 17;
June 17 - 18, 1997; n = 10) in Unitraps baited with (79 (50 pg) + (meso 7,
11) (16 ~b)- TOTAL NUMBER (+SE) OF MALE MOTHS CAPTURED FIG. 15. Captures of male sp~ghemlock loopers (SHL) (Exp 18; May 3 1 - June 26,
1997; n = 10) and male pitch pine loopers @PL) (Exp. 19; May 3 1 - June 26,
1997; n = 10) in Unitraps with enantiomenc (7) (50 pg) and stereoisomeric
(791 1) (50 pg) - MEAN NUMBER OF MALE MOTHS CAPTURED 41
4.0. DISCUSSION
Electrophysiological recordings (FIG. 5) and field experinents (FIG. 6 - 12) indicate that
the SHL and the PPL Iack specificity of their pheromone blends. Antemae fiom males of both species discriminated between optical isomers of pherornone components (7) and (7,
1l), but invariably responded only to (79 and (meso 7, 11) (FIG. 5). Attraction of male
SHLs and male PPLs oniy to lures containing (7s) + (meso 7, 1 1) (FIG. 6 - 9) cohs enmtio- and stereospecinc recognition of pheromone components, and demonstrates that
SHL and PPL use chiral pheromone components of identical absolute configuration.
Because (7R), (7S, 115') or (7R,1 IR) eLicited neither antemal (FIG. 5) nor behavioural responses (FIG. 6 - 9), they cm not be ernployed by femde SHL. or PPL to inhibit or reduce cross-attraction of heterospecinc males. Moreover, different blend ratios of (75') + (meso 7,
11) had sunilar effects on captures of male SHL and PPL (FIG. 10, 1l), eliminating blend ratios as a means of pheromonal specificity.
Enantiospecific perception of pheromone components by males implies enantioselective biosyntheses by fernales. Confirmation that female SHLs and PPLs stereoselectively produce (7s) and (meso 7, 1 l), however, must await development of a chiral GC column that separates optical isomers of (7) and (7, 1 1).
With identical sex pherornones, two species could maintain reproductive isolation through temporal (die1 and/or seasonal) separation of sexual communication. Sympatric and CO- seasonal artichoke plume moths, PIaîyptilia carduidacryla (Riley) and PZaîyptiZia wiIliamsii
(GrinneIl), for example, use identical sex pheromones but release them at different times at night (Haynes and Birch 1986). A shortage of pupae in the field precluded an experknent in 42
which virgin female SHLs and PPLs could have been observed for timing of their caI1ing
behaviour and amction of males. Employment of pheromone Iures for analysing periodicity
of sema1 communication seemed justifïed because: a) synthetic pheromone is more atîractive
than virgin femaie moths in Lambdina spp. (GrÎes et al. 1991, 1994; Maier et al. 1998); and
b) periods of pheromone release by femaies and response by males coincide in the closely
related eastern hemlock looper (EHL) (West and Bowers 1994). Although only seven and
five male SHLs and PPLs, respectively were captured in Exp. 16 and 17, the data suggest
that die1 penods of pheromone communication in the SHL and the PPL overlap (FIG. 14).
Even though the seasonal flight period of SHL may commence - 7-1 0 days eatlier than that
of PPL (Maier and Lemmon, 1996), captures of male SHLs and PPLs in pheromone-baited traps throughout June (HG. 15) eliminates seasondity of flight as a reproductive iso lating mechaaism.
Spatial separation (between or within habitats) of sexual communication represents a third potential isolating mechanism. The SHL occurs in moist areas within forests of eastern hemlock or balsam fi,Abies balsamea (L,.) Miller. The PPL, in contrast, typically inhabits dry sandy areas with mainly pitch and other hard pines (TABLE 2). While the SHL and the
PPL are mostly allopatric in eastern North Amenca (Maier and Lemmon, 1996), habitats are not far apart and areas of sympatry may exist. Captures of male SHLs and PPLs rnainly in traps suspended at 3 and 5 rn (FIG. 13) suggest that femaies of both species call in the same location within the forest canopy, and that spatial separation of sexud co~ll~~lunicationwould fail in CO-inhabitedforests.
Assuming partial sympatry of SHL and PPL, few mechanisms remain that codd 43
contribute to reproductive isolation. Differences in colouration (FIG. 1) are likely too subtie
to ailow discrimination between con- and heterospecifics at night (FIG. 14). Coutship
behaviour andor close-range (contact) pherornone~~in con- codd contribute to
recognition of conspecific males. Straight chain hydrocarbons, for example, derived fiom
scales of fernale whitemarked tiissock moths, Orygia Ie~costi~gma(J.E. Smith), induce
copulatory attempts by conspecific males (Grant et al. 1987). Compatibility of SHL and PPL
genitalia (Chris T. Maier, Department of Entomology, The Co~ecticutAgriculhiral
Experiment Station, New Haven CT 06504-1 106; pers. cornm.), on the other hand, would
dowinterspecific rnating.
In keeping with previous conclusions (Roelofs and Comeau, 1969; Roelofs and Brown,
1982), taxonhc classification of insects should be based on diverse criteria, such as
pheromone biology, morphometrics, rnolecuiar cornparisons and ecological analyses.
Taxonomie separation of the SHI, and the PPL is not well supported based on my findings
and those of othen, including: 1) identical sex pheromones (FIG. 6-12); 2) ovelapping
periodicity of sexual communication (FIG. 14); 3) overlapping seasonal flight penods (HG.
15); 4) homology (98 %) of mitochondrial DNA ( Felix Sperling, University of Ottawa,
Faculty of Science, Biology, 30 Marie Curie PO Box 450 STN A Ottawa ON KIN 6N5; pers.
comm.); 5) compatibility of genitalia (C.T. Maier, pers. comm.); and, 6) sûiking sidarities of life history traits (TABLE 1). Unless pre- or postzygotic reproductive isolating mechanisms are identified, synonymy of the SHI, and the PPL should be considered. If reproductive isolation is not demonsîrated the former species might remain as subspecies based on differences in colouration and habitat preference. REFERENCES
Am, H., Stadler, E., and Rauscher, S. 1975. The electroantemographic detector - a selective
and sensitive tool in gas chromatographic analysis of insect pheromones. Zeirsrhrzpfûr
Natug?orschung 3 Oc: 723-725.
Cameron, E. A. and Mastro, V.C. 1975. Control of a looper, Lumbdina uthasaria
athasaria, on hemlock wiîh three chernical insecticides. Journal of Economic Entornology
68: 800-802.
Capps, H.W. 1943. Sorne Amencan geometrid moths of the subfamily Ennominidae
heretofore associated with or closely related to EZZopia treitschke. Proceedings of the
United States National Museum 93 : 1 15- 15 1.
De Gryse, J.J. and Schedl, K. 1934. An account of the eastern hemlock looper, EZZopia
ficellaria Gn., on hemlock widi notes on allied species. Scientifc Agriculture 14: 523 -
53 9.
Furniss, R.L. and Carolin, V.M. 1977. Western Forest Insects. Miscellaneous publication
No. 1339 USDA Forest Service, 654 pp.
Grant, G.G., Frech, D., MacDonald, L., Slessor, K.N. and King, G.G.S. 1987. Copdation
releaser pheromone in body scales of female whitemarked tussock moth, Orgyïa
Zeucostigma (Lepidoptera: Lyrnantridae): identification and behavioural role. Journal of
Chernicol Ecology 13: 345-356.
Grehan, J.R., Parker, B.L., and Dearborn, RG. 1994. Cescription of the first and final instar
of the hemlock loopes Larnbdina athasaria (Wallcer) and Lambdinafiscellaria (Guenée)
(Lepidoptera: Geometridae). The Canadian EntomoZogist 126: 1505- 1514. 35
Gries, G., Gries, R-, Borden, J.H., Li, J., Slessor, K.N., King, G.G.S., Bowen, W.W.? West,
R.J., and Underhüll, E. W. 1991. 5.1 1- Dimethylheptadecane and 2,s-
dïmethylheptadecane: sex pheromone components of the geometrid moth, Larnbdina
fisrellaria firrehia. Nahrnvissenschafien 78 : 3 15-3 17.
Gries, G., Gries, R-, Krannitz, S.H., Li, J., King, S.S.G., Slessor, K.N., Borden, J.H., Bowers,
W.W., West, RJ., and Underhill, E. W. 1993. Sex pheromone of the westem hemlock
looper, Lnmbdina ficellaria lugubrosa (Hulst) &epidoptera: Geometridae). Journal of
Chernical Ecology 19: 1009-1019.
Gries, R, Gries, G., Li, J., Maier, C.T., Lemmon, CR, and Slessor, K.N. 1994. Sex
pheromone components of the spring hemlock looper, Larnbdinn athasaria (Walker)
(Lepidoptera; Geometndae). Journal of Chernical Ecology 20: 250 1-25 1 1.
Haynes, K.F. and Birch M.C. 1986. Temporal reproductive isolation between two species of
plume rnoths (Lepidoptera: Pterophoridae). Annals of the EntomoZogical Sociev of
America 79: 2 10-3 15.
Koot, H.P. 1994 Western Hemlock Looper, Forest Pest Leaflet 2 1 Canadian Forest Service,
Forest Insect and Disease Survey, Pacinc Fore* Centre, Victoria, B.C.
Krannitz, S.H. 1992. A synthetic sex pheromone for the western hedock looper, Lambdina
fiscellaria lugz~brosa(Hulst) (Lepidoptera: Geometridae). MPM Thesis, Simon Fraser
University, B.C., 48pp.
Li, J., Gries, R., Gries, G., Slessor, K.N., King, G.G.S., Bowers, W.W., and West, R-J. 1993a.
Chirdity of 5,ll-dimethylheptadecane, the major sex pheromone component of the
hedock looper, Lambdina fiscellar ia (Lepidoptera: Geometridae) .Journal of Chernical Ecology 1 9: 1057-1 062.
Li., J., Gries, G., Gries, R., Bikic, J., and Slessor, K.N. 1993b. Chirality of synergistic sex
pheromone components of the western hemlock Iooper, Lambdina$sceZZaria lugubrosa
(Hulst) (Lepidoptera: Geometridae). Journal of Chernical Ecology 1 9: 2547-256 1.
Maier, C.T., Gries, R., and Gries, G. 1998. Sex pheromone components of the pitch pine
looper, Lambdina pellucidaria (Gro te and Robinson) (Lepidoptera: Geometridae). Journal
of Chernicd Ecologv, 24: 49 1-500.
Maier, C.T., and Lemmon, C.R. 1996. Life cycle of Lambdina athasaria (Wallcer)
(Lepidoptera: Geometridae) developing on hemlock in Connecticut. The Canadian
Entomologist 128: 995-1003.
Owenby, J.R. and EzeIl, D.S. 1992. Monthly station normds of temperature, precipitation,
heating and cooling degree days 1961-90 Massachusetts, Connecticut. US Department of
Commerce, National Oceanic and Atrnospheric Administration, National Climactic
Center.
Roelofs, W.L. and Brown, R.L. 1982. Pheromone and evolutionary relationships of
Tortricidae. Annual review of Eco logicnl Systems 12: 3 95-422.
Roelofs, W.L. and Comeau, A. 1969. Sex pherornone specifïcity: Taxonomie and
evolutionary aspects in Lepidoptera. Science 2 17: 657-659.
Sheng, T. 1996. Synthesis of the pheromone components of the miIlcweed bug, Oncopelrus
fbxiatus and the spring hemlock looper, Lambdina athasaria. MSc Thesis, Simon Fraser
University, Burnaby, B. C. 72 pp.
Schweitzer, D.F. and Rawinski, T.J. 1988. Northeastem pitch pinehcmb oak bmens, 47
Eastern Heritage Task Force, The Nature Consemancy, 294 Washington St., Boston, MA,
21pp.
Sutton, A. and Sutton, M. 1985. Eastern Forests. AEed A- Knopf Inc. Chantelier Press Inc.,
N.Y. National Audubon Society., 525 pp.
USDA Forest Service- 1985. Insects of Eastern Forests. Miscellaneous publication 1426.,
A-T. Drooz (ed-), 608 pp.
Watson, E.B. 1934. An account of the eastern hemlock looper, EZZopiafisceZlaria Gn., on
Balsam Fir. ScienfricArnerican 14: 669-678-
West, R.J. and Bowers, W.W. 1994. Factors affecting calling behaviour by Larnbdina
fiscellariaficellaria (Lepidoptera: Geometridae), under field conditions. Environmental
EntornoZogy 23 : 122- 129.
Zar, J.H. 1984. Biostiztstical Analysis, 2nd ed. Prentice-Hail, Englewood Clfis, New
Jersey, 71 8 pp. IMAGE EVALUATION TEST TARGET (QA-3)
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