Trade-Off Between Sensitivity and Specificity in the CabbageLooper Moth Response to Sex Pheromone

Daniel J. Hemmann & Jeremy D. Allison &

Kenneth F. Haynes

Received: 1 July 2008 /Revised: 2 September 2008 /Accepted: 5 September 2008 /Published online: 26 September 2008# Springer Science + Business Media, LLC 2008

Abstract The evolution of male moth responses topheromone blends may be constrained by a trade-offbetween two response traits: sensitivity and breadth ofresponse. Population genetic simulations predict that ifsensitivity and breadth of response are negatively correlated(i.e., a trade-off exists), then selection will favor males withnarrow response phenotypes and high sensitivity. Althoughsensitivity–breadth of response trade-offs are generallyassumed to exist and are implicit to the shape of malepreference function, this study is the first to provideempirical support measuring behavior at the level of theindividual. Previous studies with the cabbage looper,Trichoplusia ni, have documented the existence of a mutantpheromone strain. While mutant females produce a phero-mone blend significantly different from wild-type females,mutant males respond equally to the wild-type and mutantpheromone blends. This study used wind tunnel bioassaysto document that relative to wild-type males, mutant maleshad broader response profiles but lower pheromonesensitivity. While wild-type male responses were highestto the wild-type pheromone blend, mutant males did not

discriminate among pheromone blends. These results areconsistent with a trade-off between breadth of response andsensitivity. Pure wild-type and mutant lines were crossedand hybrid males assayed. Both hybrid types (maternalwild-type and maternal mutant hybrids) responded similarly.Hybrid males had response profiles similar to wild-typemales and the reduced sensitivity observed in mutant males.These results suggest a possible hybrid disadvantage and aputative mechanism for reinforcement of male pheromoneresponse traits.

Keywords Evolutionary trade-off . Lepidoptera .

Pheromone sensitivity . Speciation . Trichoplusia ni .

Wind tunnel

Introduction

Comparative studies of moth sex pheromones documentextensive interspecific variation consistent with coinci-dental speciation and divergence of pheromone signals(Roelofs and Comeau 1969; Tumlinson et al. 1974;Löfstedt and Kozlov 1997; McElfresh and Millar 2001).This divergence presents an evolutionary paradox. Althoughthe observed phylogenetic patterns of divergence suggestthat pheromones can and do evolve, the need to maintainfunctionality is expected to make pheromones resistant tochange (Butlin and Ritchie 1989; Groot et al. 2006; Allisonand Cardé 2008). Resolution of this paradox is contingenton developing an understanding of both the genetic basis ofvariation in, as well as the internal (i.e., trade-offs) andexternal (i.e., drift and selection) forces acting on signalingand response traits. Thus, studies of pheromone signalingand response traits have the potential to provide insight intothe evolution of mate signaling in animals.

J Chem Ecol (2008) 34:1476–1486DOI 10.1007/s10886-008-9546-8

D. J. Hemmann : J. D. Allison :K. F. Haynes (*)Department of Entomology, University of Kentucky,Lexington, KY 40546, USAe-mail: khaynes@uky.edu

Present address:D. J. HemmannJohn Ball Zoological Garden,1300 W. Fulton,Grand Rapids, MI 49504, USA

Present address:J. D. AllisonDepartment of Entomology, LSU AgCenter,Baton Rouge, LA 70803, USA

Sympatric species with similar pheromone blends andconspecific male preference functions are hypothesized tobe the major evolutionary forces acting on female signaltraits (i.e., the amount and ratio of components emitted;Cardé and Haynes 2004). The former could imposedirectional selection on the pheromone signal and result ineither reproductive character displacement via communica-tion interference or reinforcement if hybridization ispossible, but at a fitness cost (sensu Butlin 1987; Cardéand Haynes 2004). The evolutionary forces associated withmale preference functions depend on preference functionshape, which can be open ended, flat, or unimodal (Bentsenet al. 2006). Open-ended and flat preference functionsresult in directional and no selection, respectively. The typeof selection that results from a unimodal preferencefunction depends on the relationship between the preferencefunction maximum and mean signal value (Bentsen et al.2006). If the maximum and mean are coincident, selectionwill be stabilizing. If there is an asymmetry, selection willbe directional or directional and stabilizing depending onwhether or not the maximum occurs within the range ofsignal phenotypes produced. Open-ended, flat, and unimodalmale preference functions for pheromone signal traits allhave been documented (Allison and Cardé 2008).

In the short term, the potential for a trait to respond toselection will be determined by the amount of heritablevariation. Empirical studies of the genetics of moth pher-omones suggest the existence of both polygenic (Collinsand Cardé 1985; Hunt et al. 1990; Gemeno et al. 2001;Svensson et al. 2002; Allison and Cardé 2006; Schek et al.2006) and major gene effects (Klun and Maini 1979;Roelofs et al. 1987; Haynes and Hunt 1990) on femalepheromone signaling traits in several species. Selectionexperiments have observed predictable changes in both therelative and absolute amounts of components (Sreng et al.1989; Collins and Cardé 1989; Collins et al. 1990; Allisonand Cardé 2007). A genetic basis for male response traitshas been demonstrated in several lepidopteran species,including the European corn borer, Ostrinia nubilalis(Hübner) (Hansson et al. 1987; Roelofs et al. 1987); thebrownheaded leafroller, Ctenopseustis obliquana (Walker)(Hansson et al. 1989); the pink bollworm, Pectinophoragossypiella (Saunders) (Collins and Cardé 1990); and thecabbage looper, Trichoplusia ni (Hübner) (Liu and Haynes1994; Evenden et al. 2002). These studies suggest that bothfemale signaling and male response traits have the potentialto respond to selection.

Traits are often associated with each other and as aconsequence, are seldom inherited independently (Lande1979; Lande and Arnold 1983). Positive and negativegenetic correlations can result in unexpected phenotypicchanges. Negative correlations indicate trade-offs amongtraits and are a fundamental assumption underlying some

evolutionary theory [e.g., life history theory (Roff 1992;Stearns 1992; Reznick et al. 2000)]. Few studies haveexamined the genetic architecture of moth pheromonesignals. Significant genetic correlations were documentedamong the pheromone components of the cabbage looper,T. ni (both positive and negative, Gemeno et al. 2001) andthe almond moth, Cadra cautella (Walker) (positive,Allison and Cardé 2006). These correlations suggest thatthe potential evolutionary trajectories of the female phero-mone blend are genetically constrained. Variation amongmales in breadth of response and sensitivity to femalepheromone blends has been hypothesized to be onepotential source of variation in male mating success (Cardéand Charlton 1984). Negative correlations between themale response traits sensitivity and breadth of responsehave been the subject of speculation for over 20 years (seeCardé 1986). For example, it has been suggested that anegative correlation between sensitivity and the breadth ofresponse profile may be a common feature of the insectperipheral nervous system and may explain the rarity ofgeneralist natural enemies able to use moth sex pheromonesas foraging cues (Cardé and Haynes 2004). This hypothesisis an extension of the extraordinary sensitivity and highspecificity observed for moth pheromone signals.

The objective of this study was to examine empiricallythe relationship between two male pheromone responsetraits: sensitivity and breadth of response. The cabbagelooper, T. ni is a model system for examination of thisrelationship because: (1) studies of variation in the femalepheromone blend led to the discovery of a single genemutation with major effects on the female pheromone blendbut no effects on male preference (Haynes and Hunt 1990);and (2) after 49 generations of rearing pure mutant cultures,mutant male response levels to the mutant and wild-typepheromone blends were equivalent (Liu and Haynes 1994).The existence of mutant males with an increased breadth ofresponse provides an opportunity to test for a trade-offbetween sensitivity and breadth of response.

Methods and Materials

Insect Rearing The wild-type colony of T. ni was initiatedwith insects obtained from a colony maintained at theUSDA-ARS Insect Attractants, Behavior, and Basic BiologyResearch Laboratory in Gainesville, FL, USA and weremaintained without further introductions for about 3 yearsuntil the time of these experiments. The mutant colony ofT. ni was established from a rare pheromone phenotypediscovered in a laboratory colony that originated from afield population in Riverside, CA, USA (Haynes and Hunt1990). The pure mutant colony had been maintained inthe laboratory for approximately 10 years at the time of

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experimentation. Both colonies were reared on a pintobean-based diet (Shorey and Hale 1965) at ambient roomtemperature, humidity, and light conditions. Pupae weresexed and males were transferred to 3.8 l paper cartons. Thecartons were placed in an environmental chamber with aphotoperiod of L/D 14:10 h and temperature regime of27:25°C (photophase/scotophase). Eclosed males werecollected daily and maintained on a 10% sugar solutionuntil they were bioassayed.

Wind Tunnel Bioassays The wind tunnel used for maleflight bioassays was designed based on the wind tunneldescribed by Miller and Roelofs (1978). Air was pushedthrough the tunnel with a variable speed fan controlled witha rheostat to 50 cm/s for all experiments. The pheromoneplume was exhausted out of the assay room by a second fanat the downwind end of the wind tunnel. The flight sectionof the wind tunnel was approximately 1.5 m long by 1 mhigh at its center and was illuminated by overhead redand white lights at an intensity of 0.4 lx (measured 20 cmabove the wind tunnel floor at its longitudinal center). Apheromone-loaded septum was suspended from a hookattached to the front of a 15×15 cm metal plate on top of a20-cm tall metal platform located 50 cm from the upwindend of the tunnel. At the beginning of the fourth hour of thescotophase, males 3–4 days old were placed individually in4 cm diam×8 cm high hardware cloth cages with aluminumfoil lids and allowed to acclimate to the conditions of thewind tunnel room for 1 h. During the fifth to eighth hour ofscotophase, caged males were placed on another 20 cm tallmetal platform 150 cm downwind from the pheromonesource. The lid was removed and the male was given 1 minto respond to the pheromone and scored (+ or −) for thefollowing behaviors: wing fanning, taking flight (leavingthe wire cage), locking on (zigzag flight in the pheromoneplume), upwind flight, flying within 10 cm of the source,and source contact. Males were discarded after beingassayed. If any males failed to respond after 1 min, they

were tested to make sure they were capable of flight. Malesincapable of flight were removed from the analysis.

Dose–Response Experiment Stock solutions of wild-typeand mutant pheromone blends were formulated in HPLCgrade hexane with synthetic pheromone components. Thesesolutions consisted of a mixture of 12:Ac, Z5-12:Ac, Z7-12:Ac, 11-12:Ac, Z7-14:Ac, and Z9-14:Ac (see Table 1 foremission ratios). Blends were checked for accuracy byusing a Hewlett Packard Series II Plus Gas Chromatographconnected to a Hewlett Packard 5972 Series Mass SelectiveDetector (Palo Alto, CA, USA) with a 30-m DB-Waxcapillary column (0.25 mm ID, J&W Scientific, Folsom,CA, USA). Serial dilutions of both blends were preparedfrom this stock solution. Fifty microliters of each blend wasloaded onto a hexane-extracted rubber septa (ThomasScientific, Swedesboro, NJ, USA; 5×9 mm, red). Blendsdiffered in the amount of the major pheromone componentZ7-12:Ac with either 100 μg, 33 μg, 10 μg, 1 μg, or 100 ngof Z7-12:Ac per septum. After loading, septa were left in afume hood overnight to allow solvent evaporation and thenstored at −10°C until use. Loaded septa were treated in thesame way in all subsequent experiments. Volatiles emittedfrom the highest dose septa of both blends were determinedfollowing the procedure of Haynes and Hunt (1990). Theseemission rates given in Table 1 are similar to that of wild-type and mutant females (Haynes and Hunt 1990).

Each day that moths were bioassayed, one or two malesof each type (mutant or wild type) were flown individuallyto each blend/concentration combination, resulting in 20–40 males flown each day. A total of 67 mutant and 71 wild-type males were tested to each blend–concentrationcombination, with the exception that 65 mutant and 69wild-type males were flown to the mutant blend at the0.1-μg dose because of equipment malfunction. Resultswere analyzed with logistic regression (SAS Institute 1996)with moth–blend combination, concentration, and theinteraction as model variables. This linear model allowed

Table 1 Targeted (and measured) emitted blend ratios of Trichoplusia ni sex pheromone components from septa

Wild-type blend (measured) Intermediate blends Mutant blend (measured)

W WWM WM WMM M

12:Ac 15 (12.4) 15 15 15 15 (18.6)Z5-12:Ac 12 (14.5) 5.7 2.7 1.3 0.6 (0.7)Z7-12:Ac 100 (100) 100 100 100 100 (100)11-12:Ac 6 (8.9) 6 6 6 6 (8.2)Z7-14:Ac 1.4 (2.1) 1.4 1.4 1.4 1.4 (1.5)Z9-14:Ac 0.6 (0.6) 2.1 7.6 27 100 (101.9)

Z7-12:Ac is set at 100 for each blend. In order to achieve the desired emitted blend ratio 7-fold excess, Z7-14:Ac and Z9-14:Ac were loaded ontothe septa. Measurements were made once (i.e., no replication) on the aged septa

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treatments to be compared in terms of their overall level ofresponse (tests for similar intercepts) and rates of responseincrease (tests for similar slope).

Intermediate Blend Response Experiment Five artificialblends were formulated: a wild-type, a mutant, and threeintermediate blends (ratios of compounds are given inTable 1). For all five blends, the amounts of 12:Ac, 11-12:Ac, and Z7-14:Ac relative to Z7-12:Ac was 15:6:1.4:100.The amount of Z5-12:Ac gradually decreased from anoptimum relative amount of 12 to 100 parts Z7-12:Ac in thewild-type blend through three intermediate blends to aminimum of 0.6 to 100 parts Z7-12:Ac in the mutant blend.Conversely, the amount of Z9-14:Ac gradually increasedfrom a minimum of 0.6 to 100 parts Z7-12:Ac in the wild-type blend through three intermediate blends to a maximumof 100 to 100 parts Z7-12:Ac in the mutant blend. Fiftymicroliters of a blend was loaded onto a hexane-extractedrubber septum (Thomas Scientific, 5×9 mm, red) resultingin 33 μg of Z7-12:Ac.

During each day bioassays were run, three or four malesof each type (mutant or wild type) were flown to eachblend, resulting in 30 or 40 males flown each day. Sixty-sixwild-type and mutant moths were flown to each blend.Results for mutant and wild-type males were analyzedseparately by using logistic regression (SAS Institute 1996)looking for effects of blend. After these analyses, mutantand wild-type male responses were compared. This wasdone by combining data sets and adding moth-type andmoth-type × blend interaction terms into the model.

Hybrid Response Experiment Male and female moths fromthe pure wild-type and mutant colonies were used togenerate the F1 hybrid experimental moths. The rearingprotocol was the same as outlined above. The followingcrosses were made (male × female): a pure wild-type line,W × W; a pure mutant line, M × M; and two hybrid lines,W × M, and M × W. Crosses were created by placing onemale and female (each 2–5 days old) in 0.47 l cartons linedwith blotter paper. Eggs were collected 3, 5, and 7 daysafter pairing. Male pupae were separated from females andkept in styrofoam containers. Upon emergence, males weretransferred to 0.47 l cartons with access to a 10% sucrosesolution until testing. A total of 23 W × W families weretested, 24 M × M families, 26 W × M families, and 30 M ×W families.

Four of the blends from the intermediate blend responseexperiment above were used: the wild-type blend, themutant blend, the WM blend, and the WMM blend (seeTable 1 for emission ratios). Thirty-three micrograms of Z7-12:Ac (with other compounds in the correspondingamounts) was loaded onto a hexane-extracted rubberseptum (Thomas Scientific, 5×9 mm, red). During each

day of testing, one to three males of each cross were flownto each blend. Fifty W × W, 51 M × M, 55 W × M, and 55M × W moths were flown to each blend. Results for eachcross were compared by using logistic regression (SASInstitute 1996) comparing treatments in terms of theiroverall level of response (tests of intercepts) and blendpreference (tests for slope). Linear model variables includedthe amount of Z9-14:Ac (to test for blend effect), the effectof cross type, and Z9-14:Ac by cross-type interactions.

Results

Dose–Response Experiment: Mutant Males Overall, mutantmales did not respond differently to the wild-type or mutantpheromone blend at any behavior category (Fig. 1). For thebehaviors wing fanning and taking flight, there were nosignificant concentration effects (P=0.092 and P=0.056,respectively; data not shown). For all other behaviors,strong concentration effects were observed (P<0.001;Fig. 1). For mutant males, there were no significantdifferences among blend types in terms of level of response(i.e., intercept), or rate of response increase with concen-tration (i.e., slope) for any of the behaviors measured: wingfanning (P=0.643 for a test of similar intercept and P=0.165 for a test of similar slope), taking flight (P=0.815 fora test of similar intercept and P=0.323 for a test of similarslope), locking on (P=0.056 for a test of similar interceptand P=0.355 for a test of similar slope), flying upwind (P=0.067 for a test of similar intercept and P=0.150 for a testof similar slope), coming within 10 cm of the source (P=0.311 for a test of similar intercept and P=0.699 for a testof similar slope), and source contact (P=0.330 for a test ofsimilar intercept and P=0.475 for a test of similar slope;Fig. 1). These results indicate that there is no discriminationby the mutant males between the wild-type and mutantpheromone blends and that the effects of concentration onresponse level were parallel. Because the behavioralresponses of the mutant males to the mutant and wild-typeblends did not differ, their responses to these two treatmentswere combined for comparison to the wild-type maleresponses to the wild-type and mutant pheromone blends.

Wild-Type Males The response level of wild-type males tothe mutant pheromone blend was lower compared to thelevel of response of wild-type males to the wild-typepheromone blend, or when compared to the mutantresponse to either blend across all behaviors (P<0.001 forall tests of similar intercept; Fig. 1). The rate of responseincrease of wild-type males to the mutant pheromone blendwas greater than the combined mutant male responses forthe behaviors of flying upwind and coming within 10 cm ofthe pheromone source (P=0.031 and P=0.038, respectively,

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for tests of similar slope; Fig. 1). For all other behaviors,the slopes were not significantly different (P>0.05 for testsof similar slope; Fig. 1). When the wild-type male responseto the wild-type blend was compared to the mutant maleresponses to both blends, there were no significant differ-ences in the rate of increase of response [tests for similarslope: P=0.090 for wing fanning; P=0.212 for takingflight; P=0.306 for locking on; P=0.188 for upwind flight;P=0.853 for coming within 10 cm of the source; and P=0.940 for source contact (Fig. 1)]. When the overall level ofresponse was examined, there were differences betweenmutant male and wild-type male responses to the wild-typepheromone blend. Wild-type males had lower levels ofresponse than mutant males for the behaviors wing fanningand taking flight (P<0.001 and P=0.001, respectively, fortests of similar intercept). There were no significant dif-ferences between wild-type and mutant male responses forthe behaviors locking on and upwind flight (P=0.909 andP=0.948, respectively, tests of similar intercept; Fig. 1).

Wild-type males were more likely to fly within 10 cmand contact the wild-type pheromone source than mutantmales (P=0.011 and P<0.001, respectively, test of similarintercept; Fig. 1).

Intermediate Blend Response Experiment: MutantMales Mutant male moths did not discriminate amongblends. For none of the behaviors observed did blendvariation explain the variation seen [wing fanning (P=0.624), taking flight (P=0.382), locking on (P=0.115),upwind flight (P=0.238), coming within 10 cm of thesource (P=0.116), and source contact (P=0.775)] (Fig. 2;data for the behaviors wing fanning and taking flight notshown). These results indicate that mutant males do notdiscriminate among the mutant, wild-type, or intermediatepheromone blends at any behavioral category.

Wild-Type Males Wild-type males showed the highestresponse to the wild-type blend. Their responses decreased

Fig. 1 The effect of pheromone dose on the proportion of maleTrichoplusia ni engaging in the behaviors of “locking on”, “upwindflight”, “coming within 10 cm of the source”, and “source contact” inresponse to synthetic pheromone sources. Wild-type male responses to

the mutant (solid black bars) and wild-type (hatched bars) pheromoneblends and mutant male responses to the mutant (stippled bars) andwild-type (open bars) pheromone blends are shown

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as the blend gradually changed to that of mutant females.For the behaviors wing fanning (P=0.002), taking flight(P=0.005), locking on (P<0.001), upwind flight (P<0.001), coming within 10 cm of source (P<0.001), andsource contact (P<0.001), blend had a significant effect(Fig. 2). For all behaviors, wild-type males were mostresponsive to the wild-type blend, and response decreasedprogressively through the WWM, WM, and WMM andreached a minimum for the mutant blend. These resultsillustrate the discrimination of wild-type males for the wild-type blend over the mutant blend, and that this discrimina-tion is graded becoming more evident as the blend divergesfrom the wild type.

The responses of the wild-type and mutant males to thefive pheromone blends were also compared directly. Nodifferences in the responses of the wild-type and mutantmales were observed for the behavioral categories wingfanning (P=0.503 for a test of similar intercept and P=0.242 for a test of similar slope) or taking flight (P=0.302

for a test of similar intercept and P=0.220 for a test ofsimilar slope). There was no difference among wild-typeand mutant males in the level of response (P=0.056 for atest of similar intercept) for the behavior locking on, butthere was a significant difference in blend preference (P<0.001 for a test of similar slope; Fig. 2). For the behaviorsupwind flight, coming within 10 cm, and source contact,there was a difference in blend preference (P<0.001 for allthree behaviors for a test of similar slope) with wild-typemales showing a preference for the wild-type blend.Similarly, wild-type males had higher levels of responseto the wild-type blend for the behaviors upwind flight (P<0.001 for a test of similar intercept), coming within 10 cm(P<0.001 for a test of similar slope), and source contact(P<0.001 for a test of similar slope; Fig. 2).

Hybrid Response Experiment: Hybrid Males Offspring ofboth types of hybrid crosses, W × M and M × W (male ×female), behaved similarly across behavioral categories

Fig. 2 The effect of pheromone blend on the proportion of maleTrichoplusia ni engaging in the behaviors of “locking on”, “upwindflight”, “coming within 10 cm of the source”, and “source contact” inresponse to synthetic pheromone sources. Wild-type and mutant maleresponses are shown by the solid black and open bars, respectively.For all five blends (see Table 1), the amounts of 12:Ac, 11-12:Ac, and

Z7-14:Ac relative to Z7-12:Ac were 15:6:1.4:100. The amount of Z5-12:Ac gradually decreased from an optimum in the wild-type blendthrough three intermediate blends to a minimum in the mutant blend.Conversely, the amount of Z9-14:Ac gradually increased from aminimum in the wild-type blend through three intermediate blends toa maximum in the mutant blend

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(Fig. 3). For the behaviors wing fanning and taking flight,there was no blend effect (P=0.864 and P=0.300). For thebehavior wing fanning, there were no significance differ-ences between crosses (P=0.790 for a test of similarintercept and P=0.212 for a test of similar slope). At thenext behavioral category, taking flight, there were nosignificant differences in the level of response (P=0.090for a test of similar intercept), but there was a difference inhow the crosses reacted to the different blends (P=0.024 fora test of similar slopes). For the behavior locking on, therewas a strong and significant blend effect (P<0.001) withhybrids showing a preference for the wild-type blend(Fig. 3). There was no significant hybrid-type (maternalwild-type or mutant crosses) effect (P=0.334 for a test ofsimilar intercept and P=0.602 for a test of similar slope).For the remaining behaviors, significant preferences for thewild-type blend and no hybrid effects were observed

[upwind flight (P<0.001 for a blend effect, P=0.322 for atest of similar intercept, and P=0.858 for a test of similarslope); approach within 10 cm (P<0.001 for a blend effect,P=0.876 for a test of similar intercept, and P=0.285 for atest of similar slope); and source contact (P=0.022 for ablend effect, P=0.676 for a test of similar intercept, andP=0.656 for a test of similar slope)].

Hybrid vs. Parental Males For those behaviors where therewere no significant differences between the responses of thetwo hybrid strains, the two strains were combined andcompared to the pure wild-type and mutant lines. For thebehavior taking flight, the W × M and M × W (male ×female) hybrid offspring were tested separately. No signif-icant differences between the combined hybrid treatmentand either of the pure wild-type or mutant crosses (hybridsvs. pure wild-type males, P=0.066 for a test of similar

Fig. 3 The effect of pheromone blend on the proportion of maleTrichoplusia ni engaging in the behaviors of “locking on”, “upwindflight”, “coming within 10 cm of the source”, and “source contact” inresponse to synthetic pheromone sources. Pure wild-type (solid black

bars), mutant female × wild-type male hybrid (hatched bars), wild-type female × mutant male (stippled bars), and pure mutant (openbars) male responses to the differing pheromone blends are shown.For all four blends, see Table 1

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intercept and P=0.190 for a test of similar slope; hybridsvs. pure mutant males, P=0.946 for a test of similarintercept and P=0.950 for a test of similar slope) wereobserved for the behavior wing fanning. Similarly, forthe behavior taking flight, no differences were observedbetween the M × W hybrid males and the pure wild-type(P=0.558 for a test of similar intercept and P=0.198 for atest of similar slope) and mutant (P=0.600 for a test ofsimilar intercept and P=0.519 for a test of similar slope)males. There were no differences between the W × Mhybrid males and the pure mutant males (P=0.208 for a testof similar intercept and P=0.060 for a test of similar slope).The W × M males were less sensitive than wild-type males(P=0.031 for a test of similar intercept), but there was nosignificant difference in blend preference (P=0.087 for atest of similar slope). For all subsequent behavioralcategories, both hybrid strains were combined for statisticalanalysis as in the wing fanning behavioral category. Hybridmales did not differ from the pure wild-type moths in blenddiscrimination (P=0.092 for a test of similar slope), bothhybrids and pure wild-type males preferred the wild-typeblend over the mutant blend. The hybrids did show adecreased level of response compared to wild-type moths(P=0.027 for a test of similar intercept). Hybrid and puremutant moths differed in both blend discrimination andlevel of response (P<0.001 for a test of similar interceptand P<0.001 for a test of similar slope). For the behaviorupwind flight, the hybrid male response did not differ fromthe pure wild-type male response (P=0.099 for a test ofsimilar intercept and P=0.697 for a test of similar slope)indicating similar levels of response and blend discrimina-tion. The hybrid males did differ from the pure mutantmales in blend discrimination (P<0.001 for a test of similarslope), and there was a borderline non-significant differencein the level of response (P=0.052 for a test of similarintercept). For the behavior coming within 10 cm of thesource, there was no difference in the blend preference orlevel of response of the hybrid and pure wild-type males(P=0.194 for a test of similar intercept and P=0.124 for atest of similar slope). Although there was no difference inthe level of response of hybrid and pure mutant males (P=0.846 for a test of similar intercept), there was a differencein blend discrimination (P<0.001 for a test of similarslope). Hybrid males exhibited a preference for the normalblend, while pure mutant males did not. For the finalbehavior, source contact, there was no difference in theblend discrimination of hybrid and pure wild-type males(P=0.550 for a test of similar intercept); both had apreference for the wild-type over the mutant blend. Hybridsdid have a reduced level of response compared to wild-typemales (P<0.001 for a test of similar intercept). The levelof hybrid male response was more similar to the level ofresponse of pure mutant males (P=0.930 for a test of

similar intercept). Hybrid males did show greater blenddiscrimination than pure mutant males (P=0.001 for a testof similar slope). These results indicate that hybrid maleshave wild-type male preferences for the wild-type over themutant pheromone blend, but like mutant males havereduced pheromone sensitivity.

Discussion

Because the adaptive value of pheromone communicationis mate location, selection should favor male preferencesfor (Butlin and Ritchie 1989; Groot et al. 2006; Allison andCardé 2008) and maximum male sensitivity to (Roelofs1978; Linn et al. 1987) the most common blend emitted byfemales. As a result, variation in the presence and relativeamounts of components in the pheromone blend maycontribute to variation in female mating success. Theresultant variation in mating success is hypothesized toimpose stabilizing selection on the pheromone signal andultimately constrain signal evolution (Butlin and Ritchie1989). This paradigm is the basis for a model of moth sexpheromone evolution that has been referred to as the“stasis” model (Allison and Cardé 2008). An alternatemodel, asymmetric tracking, emphasizes an expectedasymmetry in the strength of sexual selection that acts onfemale signal and male response traits due to differentialinvestment in the zygote (Phelan 1997). Accordingly, malesare predicted to optimize individual fitness by rapidlylocating females and mating multiply, whereas females arenot expected to be limited in mating opportunities. As aconsequence, this model predicts that the female phero-mone blend is free to change via non-adaptive evolutionaryprocesses (i.e., drift) and that male response traits (e.g.,breadth of response and sensitivity) will be selectedsimultaneously to track changes in the female pheromoneblend.

The primary difference between the two models involvesthe predicted shape of the male preference function. Thestasis model emphasizes unimodal male preference functionsthat constrain signal evolution, whereas the asymmetrictracking hypothesis does not (see Butlin and Ritchie 1989;Phelan 1997). The stasis model predicts a male responsephenotype with maximal sensitivity to the most commonblend and a narrow preference function with an optimumcoincident with the most common pheromone blend.Similarly, the asymmetric tracking hypothesis predictsmaximal sensitivity to, and an optimum coincident with,the most common blend. It differs from the stasis model inthat it predicts a broad male preference function. Implicit tothe prediction of maximal sensitivity to the most commonpheromone blend is a trade-off in sensitivity among blends

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(i.e., high sensitivity to one signal comes at the expense ofreduced sensitivity to other signals). In the absence of thistrade-off, the optimal male phenotype would appear to behigh sensitivity to the complete range of signals defined bythe male preference function.

Little empirical data on the relationship between thetraits breadth of response and sensitivity in male moths topheromone blends exist. The two traits are hypothesized tobe negatively correlated, and the optimization of maleresponse phenotypes limited (i.e., increases in sensitivity toone or more blends results in reduced sensitivity to otherblends; Cardé 1986; Cardé and Haynes 2004). Independentstudies have documented that mutant males have a broaderresponse profile (Liu and Haynes 1994) and are lesssensitive at the peripheral level (Baker and Domingue,pers. comm.) than wild-type males. Simulations of apopulation genetics model predict that if changes insensitivity are not associated with trade-offs in breadth ofmale response, that selection will favor males with broadresponse phenotypes (Butlin and Trickett 1997). When anegative correlation between breadth of response andsensitivity was included in the model, the simulationspredicted that selection would favor males with narrowresponse phenotypes and high sensitivity.

In this study, mutant male sensitivity and rate ofresponse increase to increasing pheromone concentrationswere independent of the pheromone blend. In contrast,wild-type males were more responsive to the wild-type thanthe mutant pheromone blend (Figs. 1 and 2). When theresponses of wild-type males to the wild-type pheromoneblend were compared to the combined mutant maleresponses, the level of response sensitivity differed amongstrains (Fig. 1). These results are consistent with the resultsof earlier wind tunnel and field trapping studies (Liu andHaynes 1994; Zhu et al. 1997) and suggest a trade-offbetween sensitivity and breadth of response.

The terminal behaviors in the mate location sequence(coming within 10 cm and source contact) are likely thebest indicator of the ability of an individual male to locate amate. For both of the behaviors, coming within 10 cm andsource contact, wild-type males were more likely tocomplete the behaviors to the wild-type blend than mutantmales to either blend (Fig. 1). Similar results have beenreported for Grapholita molesta (Baker et al. 1981) and P.gossypiella (Linn and Roelofs 1985) in which males weremost sensitive to the female produced pheromone blend.Because wild-type males are more sensitive to the wild-typepheromone, mutant males may be at a competitive dis-advantage when competing with wild-type males formating opportunities with wild-type females [an argumentsupported empirically by Evenden and Haynes (2001)].Conversely, wild-type males do not respond well to mutantpheromone and may be at a disadvantage when competing

with mutant males for mutant females. Interestingly,although wild-type and mutant hybrid males and purewild-type males have similar blend preferences at the levelof source contact, wild-type males are more sensitive thanhybrid males (Fig. 3). These data suggest that assortativemating could occur in mixed populations (Zhu et al. 1997)and provide a mechanism for reinforcement and diversifi-cation of pheromone signals that could ultimately result inspeciation.

The mutant males did not show a preference for anyblend at any behavioral category. They responded equallyto the normal, intermediate, and mutant blends. This lendssupport to the hypothesis that the mutant males underwent ageneral broadening of response as they became equallyresponsive to the normal and mutant pheromone blends.

Overall, it appears that at the same time that mutantmales experienced a broadening of response, they simulta-neously experienced a reduction in sensitivity. This docu-ments a trade-off between sensitivity and breadth ofresponse in T. ni. Population genetic simulations run byButlin and Trickett (1997) suggest that when a trade-offbetween sensitivity and breadth of response exists, selectionshould favor a narrower, more sensitive response profile. Itremains to be seen if this trade-off between sensitivity andbreadth of response is present in other moth pheromonesystems. The trade-off in sensitivity was observed withlures releasing up to 2 ng/min of Z7-12:Ac; the meanamount of Z7-12:Ac emitted by calling females has beenestimated to range from 2 to 11 ng/min of Z7-12:Ac (seeHunt and Haynes 1990; Liu and Haynes 1994). If trade-offsare a general phenomenon, it could mean that the evolutionof novel pheromone signals could involve an intermediatestep during which some individuals have a broad response,but lower sensitivity. Presumably, selection could then acton the male response phenotype, narrowing the responsepeak and increasing sensitivity to the new pheromoneblend. For now, our ability to make such generalizations islimited by a lack of data for other species.

Acknowledgments We are grateful to B. Chastain for reliable andinvaluable technical assistance and to Drs. C. Gemeno and M.Evenden for comments and discussions. The research was supportedby the Kentucky Agricultural Experiment Station and a competitivegrant from the USDA, Cooperative State Research and ExtensionService (97-35302-4324).

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