Entomol. exp. appl. 45: 139-144, 1987 �9 Dr W. Junk Publishers, Dordrecht - Printed in the Netherlands 139

A spruce budworm mating bias in two-component pheromone environments

Larry R. Kipp, J. Chris Bergh & William D. Seabrook Neuro-ethology Research Group, Dept. of Biology, University of New Brunswick, Fredericton, N.B., Canada E3B 5A1

Accepted: April 29, 1987

Key words: pheromone, lepidoptera, Choristoneurafumiferana, mating, winglength

Abstract

The occurrence of mating by moths of the female spruce budworm, Choristoneura fumiferana (Clem.), was independent of body size except when high titers of the 9 primary sex pheromome components (95:5 E /Z ll-tetradecenal) were present. Adult size was indexed by forewing length which was significantly correlated with fresh pupal weight (and was previously known to be correlated with adult body weight and potential female fecundity). In clean air, mean winglengths of mated and unmated females were not significantly different. The presence of increasing pheromone dosages caused a reduced incidence of mating but mating was never eliminat- ed (Ponder et al., 1986). Here, we report that coincident with this mating reduction was a significant trend for an increase in the mean winglengths of the mated female category. In a second experiment cy c~ and 9 9 were examined simultaneously and we verified that a large-female mating advantage occurred in the pheromone treatments but not in the controls. No size-related cr mating advantage was found in the treatments or the controls. Moths recovered from a field trial in large cages lasting 3 days also showed that a mating bias of the large 9 9 only occurred in the treatment cage.

Introduction

Schmidt & Seabrook (1979) found that high ambient levels of the spruce budworm, Choristoneura fumiferana (Clemson), primary sex pheromone components (95:5 E /Z ll-tetradecenal; Sanders & Weatherston, 1976; Silk et al., 1980) could reduce mating by this species when moths were placed in small field-cages. Since then, spruce budworm (SBW) mating suppression studies have been carried out using large field cages enclosing 25 to 30, 3 m balsam fir trees (Seabrook & Dyer, 1983; Seabrook & Kipp, 1986), with freely flying wild insects (San- ders & Silk, 1982; Alford & Silk, 1983), and in the laboratory (Ponder et al., 1986).

In the laboratory study (Ponder et al., 1986) it was shown that in control environments mating was high

and the incidence of mating among replicates fol- lowed a binomial distribution. When pheromone was added the occurrence of mating was reduced, as was expected. What was not expected was that (a) the incidence of mating among treatment replicates no longer followed a binomial distribution; and (b) mating could not be completely suppressed.

Mating suppression is an expression that relates mating success in pheromone treated environments to that found in control environments. Since high pheromone dosages did not entirely 'suppress' fe- male mating, we feel that other communication mo- dalities may be involved. However, there is no a pri-

140

ori expectation that use of an alternate modality should also interfere with the underlying binomial distribution. That the underlying binomial distribu- tion was eliminated when pheromone was present suggests to us that the expression of the 'alternate modality' reflected some unknown variation among the replicates.

One observable source of variation among the replicates of Ponder et al. (1986) was moth size. In this species considerable individual size variation oc- curs and is documented in this paper: male fresh pupal weights range from 30 to 125 mg while female weights range from 32 to 175 mg (Fig. 1). Therefore, there is a potential for substantial size (weight) varia- tion within each sex in each mating assay replicate.

In this study we quantified the sizes of moths that mated in the controls and treatments, and compared them with their unmated counterparts. This was done with moths saved from the experiments report- ed in Ponder et al. (1986) and with moths obtained from more recent experiments.

Methods

SBW larvae were reared on synthetic diet (McMor- ran, 1965) as modified by Grisdale (1970). Insects were reared in incubators (17L:7D, 21 _+2~ 50-70o70 r.h.). Pupae were segregated by sex and adults were allowed to emerge and were held in incu- bators having separate ventilation systems.

The relationship between fresh pupal weight and forewing length was determined from 99 cy cy and 154 9 9 . These individuals were weighed within 12 h post-pupation to the nearest mg then permitted to enclose, whereupon their right forewing length was measured. Winglength and mated status were also measured for male and female moths used in mating suppression experiments. Forewings were prepared for measurements by bisecting the mesothorax, placing the wing on a moistened ceram- ic plate and removing the tegula. Wing length was measured as the distance between the proximal and distal end of the wing tip (not including scales) in the region of the radial veins. Measurements were made with an ocular micrometer (Wild Leitz) where one ocular unit equals 0.23 mm. Mating status was de-

termined by the presence or absence of a spermato- phore in females, while male mating status was de- termined by the presence or absence of yellow secretory fluid in the primary simplex of the ejacula- tory duct (Outram, 1970; Bergh & Seabrook, 1986).

In the mating suppression tests, 95:5 E /Z ll-tetradecenal was dissolved in 60 ~1 of hexane and applied to 1 cm 2 pieces of filter paper (Whatman No. 1). Control filter papers received 60/A of hex- ane. Hexane was allowed to evaporate for 3 rain and then the filter paper squares were introduced into 2.5 L Pyrex TM crystalizing bowls, as in Ponder et al.

(1986). Fifteen, 4 8 - 7 2 h old male SBW moths were then placed in each bowl. One hour later, ten 1 - 24 h old female SBW moths were added. The 1.5:1 [cy: 9 ] sex ratio/density maximizes mating under control conditions (Ponder et al., 1986) as do the age classes used (Outram, 1971). The sealed bowls were placed in an incubator 10-12 h before the onset of

scotophase and removed ca. 20 h later, at which time moth mating status and forewing length were deter- mined.

The 1984 data was from female moths saved from the dose-response tests reported in Ponder et al.

(1986). Nine to twelve replicates were available from the 0, 10, 20, and 30/~g pheromone source concen- trations, while 30 replicates were available from each of two additional control trials and from treatments of 1, 5, 40, 80 and 160/~g pheromone. From those tests a total of 2308 females were examined. Addi- tional replicates (five control, nine treatment) were subsequently performed in 1986 using a 60 ~g source concentration. From 1986 replicates winglength measurements and mating status were obtained from both males and females. Males from four of the nine treatment replicates could not be used be- cause of wing tattering.

In July 1985, 1450 cy cy and 850 9 9 were placed in each of two field cages measuring 9.4 x 5.6 x 3.0 m. Each cage was erected around 25, 3 m high balsam fir trees. The cage floors were co- vered with plastic (0.23 mm thick) which was taped about the bases of the trees. Prior to moth introduc- tion the cages were treated with Vapona TM for 30 min in calm air at 04:30 h) to kill feral insects. One cage was located in a 3 ha plot that had been treated with aerially applied 95:5 E /Z

l l-tetradecenal formulated into plastic laminate flakes (140 gai ha-~; 10% pheromone by weight). The other cage was in an untreated area. Moth age at the beginning of the test was the same as that used in the laboratory experiments. Males were placed in the cages 12-24 h before the females. Moths were killed with Vapona TM fog (as above) on the fourth night. Additional procedural details may be found in Seabrook & Kipp (1986). Moths were collected and returned to the laboratory where winglength and mating status were ascertained.

The 1984 mating status and winglength data were analyzed by 2-way ANOVA and the runs test (Zar, 1984). In the 1986 experiment, the winglength data were segregated according to sex and mating status and then ranked into groups of longer, moderate and shorter winglengths. In the female control test Fish- ers Exact Test was used to compare female mating status between the longer and shorter winglength groups (Zar, 1984). The remaining three experiments were analyzed using a 2 • 3 chi-square test for differences between mated and unmated propor- tions (Zar, 1984).

Results

Forewing length is directly related to fresh pupal

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weight (Fig. 1). The male and female regressions are significantly different from a slope of zero and sig- nificantly different from each other (p < 0.05). Coefficients of determination are 0.62 and 0.71 for males and females, respectively. Furthermore, males of a given fresh pupal weight will have a smaller forewing length than females of equal weight, as shown by the differing slopes for the male and fe- male regressions (Ho: /3 (o ' )=~3(9) , t = 4 .9 , d f = 249, p<0.001). Therefore, SBW forewing length may be employed to index fresh pupal weight (Fig. 1), as well as dry adult weight (Mattson et al., 1982) or potential female fecundity (Thomas et aL,

1980). In the 1984 experiment, mean winglengths (WL)

of the mated female category increased as phero- mone dosage increased (Ho: the distribution of differences in mean WLs of mated and unmated fe- males is independent of dose, p = 0.05, runs test) (Table 1). This effect was not found in the controls. ANOVA analysis indicated significant interaction (p = 0.0125) without significant (c~ = 0.05) main ef- fects; hence, the influence of pheromone dosage on the mean winglengths of the mated and unmated fe- male catagories was not strictly additive.

In the 1986 experiments there was an obvious difference in the proportion of mated individuals oc-

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O Z

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FRESH PUPAL W E I G H T , m g Fig. 1. Relationship between fresh pupal weight (12 h old) and forewing length of laboratory reared Choris toneurafumiferana cy cy and

9 9 .

142

Table 1. Differences in mean forewing lengths of mated and unmated female spruce budworm moths recovered from control and

treated atmospheres containing 95:5 (E/Z) l l-tetradecenal. M =mated , U =unma ted . Data between parentheses: s.d.

Experiment Mean winglength (mm) Difference Sample size Z-test a

in means

M U (M - U) M U

C-1 11.0 (0.7) 11.2 (0.6) - 0.2 89 29 1.63

C-2 10.8 (0.7) 11.0 (0.7) - 0 . 2 216 78 1.29

C-3 10.6 (0.8) 10.8 (0.8) - 0 . 2 197 78 1.93

1 ~g 10.8 (0.8) 10.8 (0.8) 0.0 164 102 0.09

5 /xg 10.8 (0,7) 10.8 (0.8) 0.0 130 155 0.03

10 p,g 11.1 (0.6) 11.0 (0.6) 0.1 35 52 1.40

20 t~g 11.2 (0.5) 11.0 (0.7) 0.2 40 49 1.63 30/~g 10.9 (0.7) 10.9 (0.6) 0.0 40 74 0.10

40 ~g 10.8 (0.7) 10.7 (0.7) 0.1 116 156 1.02 80 #g 11.2 (0.8) 11.0 (0.8) 0.2 88 185 2.23 b

160 /zg 11.0 (0.8) 10.8 (0.8) 0.3 74 201 2.32 b

a corrected, normal approximation to the Mann-Whi tney test with ties, 2-tailed (Zar, 1984); Ho:WL (mated 9 )= WL (unmated 9 ).

b p<0 .05 .

curring in the control and treatment trials (Table 2). A size-related female mating bias was again only ob- served in the treatments (chi-square test, p < 0.025).

Table 2. Contingency table analyses of mating results f rom a

20-h duration experiment testing independence of mating status

and forewing length for 9 and o" Ch. fumiferana in control and pheromone treated atmospheres. M = mated, U = unmated.

Winglength

Size class (mm)

9

Control Treatment

M U E M U

< 11.0 20 5 25 10 13 23

I 1 . 0 - 1 2 . 0 14 3 17 10 23 33

> 12.0 6 2 8 21 13 34

40 10 50 41 49 90 Probability p > 0.35 a p < 0 . 0 5 b

o"

< 10.7 14 10 24 11 18 29

10.7 - 11.3 15 14 29 8 14 22

> 11.3 14 8 22 6 18 24

I2 43 32 75 25 50 75 c

Probability P > 0.25 b P >0 .50 b

a Fisher 's Exact Test using smallest and largest class size only

(Zar, 1984). b 2 x 3 X 2 test (Zar, 1984). c Some replicates not included because at least one ~ had torn

wings.

In both trials there were no significant differences among the three winglength classes in the propor- tion of mated males (chi-square test, control: p > 0.50; treatment: p > 0.50). Although there exists an obvious difference in the proportion of mated in- dividuals between the controls and the treatments, there was no coincident size-related male mating ad-

vantage. The results of the large field cage trial (Table 3)

conform with the laboratory experiments: in the control cage there was no difference in mean wing- lengths between mated and unmated females (p > 0.20), but in the treatment cage the mean wi0g-

Table 3. Compar ison of mean winglengths (WL) of mated and

unmated 9 9 recovered from control and pheromone treat-

ment cages. M = mated, U = unmated.

Control Treatment

M U M U

x WL (mm) 11.57 11.46 11.42 11.12 S.D, 1,09 1.18 1.07 1,07 n 326 191 147 301 t 1.15 2.74

p > 0.20 < 0.01

Ho: X WL (mated 9 ) = WL (unmated 9) , for each cage (t-

test).

length of the mated female category was greater than the mean of the unmated female category (p < 0.01).

Discussion

We have shown that the efficacy of a pheromone treatment for SBW mating suppression interacts with female size. No effect is seen on males of differ- ent sizes. Although replication is lacking, the field trial is consistant with the laboratory tests and indi- cates that the mating bias favouring large females can persist for up to 3 days, the duration of the test. Presumably, the effect should be sustained as long as mating suppression is maintained.

The occurrence of a large-female mating advan- tage in the treatment environment could be the manifestation of a biological process operating in an unnatural environment. Whether male mate-finding or female receptivity, or a combination of these, is (are) primarily involved is problematical. For exam- pie, antennectomized males do not mate (Sanders, 1975), whereas antennectomized females mate readi- ly (J. C. Bergh, unpublished). The presence of light is not necessary for mating to occur (Bergh, unpub- lished). Thus, successful mating appears to require functioning male antennae but neither functioning female antennae nor visual orientation. Plausably, male tactile orientation (cf. Sanders, 1975) to larger females may be accomplished more easily than orientation to smaller females; or, close-range male chemo-orientation may be accomplished more easi- ly with larger females if they produce greater quanti- ties of the pheromone than their smaller female counterparts, as do female Bombyx mori (Kuwahara et aL, 1983). Alternatively, females may influence the mating outcome in several ways. For example, fe- males can detect their own pheromone (Palaniswa- my & Seabrook, 1978) which may alter the timing of calling (Palaniswamy & Seabrook, 1985) or female flight activity levels (Sanders, 1985, 1987). There is some indication tha~ individual differences exist in the stimulus threshold necessary to elicit alterations in calling (Palaniswamy & Seabrook, 1985). If stimu- lus threshold differences are related to female body size, the effect of pheromone exposure on a popula-

143

tion of females may be to reduce the receptivity of (mainly) the smaller females; hence, the mean size (e.g. mean forewing length) of the receptive female portion should be somewhat larger than the mean for the total population.

The phenomenon of a size-related mating bias should be examined in those pest species for which mating suppression shows promise as a control strategy. In species for which body size is partially heritable, as for the SBW (Campbell, 1962; Thomas et al., 1980; Harvey, 1983, 1985), minor mating bi- ases, given sufficient time, could alter the genetic structure of the target population. Such induced genetic changes should be of interest to pest managers for its possible beneficial or detrimental effects.

Acknowledgements

The authors appreciate the technical assistance of S. Murphy, P. Landva, P. Robichaud, S. Verhille and B. Ponder; and thank B. Wunsch for rearing the moths; and L. P. S. Kuenen, T. Payne and P. Silk for reviews. The Insect Rearing Section of the Forest Pest Management Institute, Canadian Forestry Service, Sault Ste. Marie, Ontario, Canada provided the in- sects. Supported by a grant from N.S.E.R.C. to W.D.S. and Federal Student Employment Grant to L.R.K.

g~snm~

Biais introduit dans les accouplements de Choristoneura fumiferana en presence de phero- mone sexuelle femelle ?t deux composantes

Les accouplements de C. fumiferana ne d6pendent pas de la taille de l'insecte, sauf quand il y a une forte concentration en 95:5 E /Z ll-t6trad6c6nal, com- pos6s de la ph6romone sexuelle primaire femelle. La taille de l'adulte est exprim6e par la longueur de l'aile ant6rieure, significativement li6e au poids frais des chrysalides (connu comme corr616 avec le poids du corps des adultes et avec la f6condit6 des femelles). En plain air, il n'y a pas de diff6rences significatives

144

entre la longueur moyenne de l'aile ant6rieure des femelles vierges ou ins6min6es. L'augmentation de la teneur en ph6romone r6duit la fr6quence des ac- couplements sans jamais les 61iminer (Ponder & all, 1986). Nous avons observ6 parall61ement h cette r6duction des accouplements une tendance sig- nificative h 161ongation de la longueur des ailes an- t6rieures des femelles ayant copul6. Dans und seconde exp6rience, off m~les et femelles ont 6t6 ex-

amin6s simultan6ment, nous avons eu confirmation d'un avantage sexual pour les plus grosses femelles en pr6sence de ph6romones, mais non dans les lots t6moins. Chez les m~les, il n'y a eu aucun avantage sexual lid/l la taille. Darts les exp6riences de 3 jours en grandes/l l'ext6rieur, nous avond observ6 un avan- tage sexual pour les plus grosses femelles dans les

cages trait6es aux ph6romones.

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