by their quick movement and fluttering wings has re- peatedly ... by walking in a rather straight line with buzzing wings, .... gun) fanned his wings while walking.
Male Codling Moth (Laspeyresia pomonella)1 Orientation to Visual Cues in the Presence of Pheromone and Sequences of Courtship Behaviors2 P. J. CASTROVILLO 3 AND R. T. CARDE Dept. of Entomology and Pest. Res. Cen., Michigan State Univ., East Lansing 48824 ABSTRACT Ann. Entomol. Soc. Am. 73: 100-105 (1980)
Male codling moths (Tortricidae: Laspeyresia pomonella L.), after anemotactic flight to synthetic sex pheromone, spent a significantly greater amount of time in close-range orientation to a dead female moth providing a visual cue than to the pheromone source. Thirtythree successful codling moth matings were analyzed to produce an ethogram depicting male and female behaviors occurring during courtship. The female sequence was highly variable, but the male sequence was not. Much recent codling moth research has been directed toward finding keys to a control program with reduced reliance on insecticides. Biological and behavioral information, however, especially that concerned with courtship and mating, is still very incomplete. Borden (1931) reported that male codling moths find females in flight and follow them to foliage where copulation occurs: "That the females do attract the males by their quick movement and fluttering wings has repeatedly been observed." This has proven to be quite incorrect. Proverbs (1965) demonstrated that males were attracted to caged living females, and Howell and Thorp (1972) determined that virgin moths were much more attractive than mated ones. Butt and Hathaway (1966) found that an extract of female codling moths could be used to lure males to a trap. Males exposed to this extract in the laboratory "performed a circling dance and attempted to copulate with other males (both dead and alive), empty pupal cases and pieces of corrugated cardboard." The sex pheromone, (£,£)-8,10-dodecadien-l-ol (Roelofs et al. 1971, McDonough and Moffitt 1974), is produced by the female codling moth in glandular tissue located dorsally on the ovipositor (Barnes et al. 1966). The pheromone gland is held within the abdomen, between the 8th and 9th segments except when calling, when the ovipositor is extended ventrally perpendicular to the axis of the body (Fluri et al. 1974, Castrovillo and Carde 1979). Fluri et al. (1974) found that removal of both male antennae almost completely prevented mating of caged moths, whereas removal of one male antenna or both female antennae did not. On the basis of another caged moth mating frequency experiment, Gehring and Madsen (1963) reported that probably no visual cues were necessary to codling moth courtship. When males and females were in close proximity, mating was as successful in constant darkness as under a photocycle with both scotophase and photophase. Conversely, Hutt and White (1977) found that in the laboratory under close confinement the number of matings occurring with blind, antennectomized, blind and antennectomized, and unaltered moths was influenced by the ability to see. They concluded that response to visual cues did play a part in mating. Studies of mating frequency, however, do not 1 Lepidoptera: Tortricidae. 1 Published as Journal Article No. 9041 of the Michigan State Univ. Agric. Exp. Stn. Received for publication May 9, 1979. 3 Present address: Dept. of Forest Resources, College of Forestry, Wildlife and Range Sciences, Univ. of Idaho, Moscow 83843.
define precisely the sensory inputs modulating the act. Males have been reported to approach calling females by walking in a rather straight line with buzzing wings, lifting the abdomen, and spreading their valves in the direction of the female (Fluri et al. 1974). Copulation immediately followed male contact with a female. We studied the effect of a visual cue on male codling moth mating behavior and orientation and also elucidation of characteristic behaviors occurring during codling moth courtship. Materials and Methods A codling moth culture was maintained on small, green, thinning apples at 23° ± 1°C with a 16-h photoperiod (1500 lux). Stock insects were obtained from infested apples collected in East Lansing, MI, during fall 1975, the summers of 1976 and 1977, and fall 1977. We observed male orientation to a pheromone source and a visual cue, and codling moth courtship, in a wind tunnel as described by Carde and Hagaman (1979). A platform (Baker and Carde 1979), placed 0.5 m from the upwind end of the tunnel, served as a location for the pheromone source and visual cues and as a place where male moths could engage in such behavior as requires contact with a substrate. A rubber septum baited with 0.1 mg of (£, £)-8,10-dodecadien-l-ol (> 99.0% purity, obtained from Zoecon Corp.) was placed inside a flask connected to an in-house air line. Pheromone was carried from the flask, through a pipette, and out through a hole in the platform, whence it moved downwind in the tunnel. For a visual cue, a dead female codling moth was rinsed several times in acetone, placed on the platform, and impaled on an insect pin projecting from the surface. Such females without synthetic pheromone were unattractive to males: when no air was passed through the flask males did not fly upwind to the platform; when males had reached females in the presence of pheromone and flask air was stopped the males soon left the platform with no reorientation. The platform was divided around the pheromone opening into upwind, left, right, and downwind quadrants. The female was placed in only one quadrant at a time and was positioned heading upwind. Several hours before the start of observations male moths were placed singly in 10 x 8 cm diam cylindrical screen release cages. A caged male was induced to fly upwind to the platform by placing a cage on end in the pheromone plume and removing the cage lid. For those
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males which flew upwind to the pheromone source, their behavior at the platform was described verbally onto tape for 2 min. Then total duration of orientation near the platform was measured. Duration of orientation was defined as the number of seconds from when the male first reached the downwind edge of the platform to when he either flew past the platform for 5 sec, flew to the wind tunnel ceiling or side for 5 sec, or flew downwind without returning upwind within 5 sec. Observations were terminated when a male sat motionless on the platform for 60 sec; orientation duration was measured to the time at which motionlessness began. There were 85 male orientations of 2 min or greater duration: 17 with no visual cue present and 17 each with the cue located upwind, downwind, left, and right. Between each observation the female was removed or moved randomly to a different quadrant. Dead females were used several times, but discarded each time following placement in the downwind quadrant. Observations were conducted at 23°C and 0.4 lux. The pheromone source air flow was 0.4 ml/sec and wind tunnel air velocity was 0.7 m/sec. No moths were released for observation more than once during a 24-h period and most were tested only once. All observations occurred during the 8 h in which the moths normally experienced scotophase. For the study on male and female courtship behavior sequences, a platform holding 8 living virgin female codling moths was suspended in the upwind end of the wind tunnel. An inverted clear plastic cup restrained each female until quiescent and then was removed. When females began calling, males were placed in the wind tunnel as described above until one flew upwind to a female. The interval between when a male first moved to within 3 cm of a female until several seconds after sexual coupling occurred or, if the mating attempt was unsuccessful, until the sequence was terminated by the male or female, was recorded on videotape. Thirty-three successful codling moth matings recorded on videotape were analyzed using stop-action and slow playback and used to generate an ethogram of behavior leading to copulation. Observations were conducted at 20°C and 200 lux during the 8 h corresponding to scotophase on the previous day. Air velocity was 0.7 m/sec. Results Effects of Visual Cue on Orientation of Male Codling Moths When male codling moths flew upwind to the orientation platform they (1) hovered slightly downwind of the edge, often casting from side to side or upwind and downwind; (2) hovered above the platform, sometimes touching its surface; (3) landed and walked (usually accompanied by wing fanning); and (4) made copulatory attempts near the source emanating pheromone or, when present, with a dead female. In almost every case each male exhibited all of the above behaviors and, during each orientation to the platform, reoriented several times to the source, the female, or both. Mean total orientation and x time in contact with the platform (momentarily touching it, walking and fanning, and attempting copulation) during the 1st 120 sec of ori-
entation was unaffected by the presence or absence of a dead female visual cue (Table 1). The position of a visual cue did not affect time spent at the platform but it did affect time spent at each quadrant (Table 2). In all cases significantly more time was spent on the quadrant containing the dead female than on any other quadrant or at the source. Not all the time that males were on the quadrant with the visual cues was spent attempting copulation. In most cases the x time in contact with the visual cue was significantly less than the x time on the quadrant. Laboratory Courtship Sequence of Codling Moth The basic codling moth courtship sequence is: male flies upwind to the female, lands upon the substrate on which she is sitting, walks (while fanning) to her, and attempts copulation. While resting on a horizontal surface and calling, females oriented their bodies randomly with respect to wind direction (30.3% upwind, 33.3% crosswind right, 24.1% downwind, and 12.1% crosswind left). Similarly, males, once they landed on the substrate following upwind flight, approached females from all directions with equal frequency (30.3% upwind, 24.2% crosswind right, 24.2% downwind, and 21.2% crosswind left). None of the male or female orientation directions were significantly different by x2 a t P = 0.05 (N = 33). Every male observed within 3 cm of a female (the approximate distance at which videorecording was begun) fanned his wings while walking. The male initiated contact with the female by touching her with his head. Significantly fewer first contacts were made at the posterior of the female than with any other part of her body (Table 3). Following contact, the male curved his abdomen laterally and walked along the female's side, apparently in contact with her, while he moved into a position enabling him to probe her abdomen with his. Wing fanning occurred during approach, initial male-female contact, abdomen curving, and movement along the female. As abdominal probing began the male vibrated his wings at ca. 45° to the horizontal. When the female genitalia were grasped by the male valves, wing vibration ceased and his wings were folded roof-like above him as he turned to face 180° away from the female. The time when the male's valves were first opened during approach varied somewhat. Of 20 males observed, 8 opened them only after head contact with a Table L—Effect of visual cue location on x orientation time and x time of contact with platform during the 1st 120 sec of orientation exhibited by male codling moths. Location of visual cue in relation to source8 No cue present Upwind To right Downwind To left
x orientation duration1* (± SD) (sec)
x time in contact with platformb (± SD) (sec)
440.9 ±(308.1) 475.2 ±(402.6) 526.2±(371.6) 410.2±(234.1) 417.4±(188.2)
43.9±(15.2) 48.3±(14.5) 50.7±(17.1) 50.4±(21.2) 39.2±(17.6)
* Visual cue 2 cm from source. b No significant difference between means within each column (ANOVA, P 0.05; means compared using Student-Newman-Keuls' multiple range test).
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Table 2.—Effect of visual cue location on orientation of male codling moths (N = 17). Location of visual cue in relation to sourceb 8
x % of time on platform
No cue present
Upwind
To right
Downwind
To left
14.6ab 17.0ab 10.4a 34.2c 23.8b —
5.8a 53.6c 7.9a 21.5b 11.2a 43.3c
8.3a 16.0ab 41.3c 23.lab 9.5a 37.5b
9.3a 15.6ab 9.5a 52.9c 12.7ab 30.3b
9.7a 13.7a 9.4a 27.0bc 40.2c 23.9ab
At source On upwind quadrant0 On crosswind (right) quadrant0 On downwind quadrant0 On crosswind (left) quadrant2 Attempting copulation with visual cue
' All All observations were 120 sec in duration. b Visual cue location 2 cm from sou source. Values in columns followed by same letter not significantly different (ANOVA, P analysis; l i untransformed fd values l reported d in Table 2; means separat separated using Student-Newman-Keuls' multiple range test), c Includes time in contact with visual cue cue (copulatory attempts)
female, 2 opened them intermittently during the approach, and 10 held them open during the entire time they were within 3 cm of the female. Of the 33 matings observed, 30 began with females holding their wings tightly against their bodies. As males approached, 53.5% of the females raised their wings partially, resulting in a greater exposure of the abdomen and genitalia. Analysis of percent females raising wings in response to males approaching from downwind, anterior to, and lateral to the females suggests no differences (Table 4). A few females (3%) walked forward immediately after initial male head contact and many (66%) did so as their abdomens were probed by a male's. Male abdominal contact usually was directed to the female's side, due to his position beside her. As he moved slightly behind her, his probing was then directed more anteriorally. Some females walked forward in response to lateral contact from the male head or abdomen, whereas other females only did so after apparently being pushed forward by the male during abdominal probing. A significantly greater number of females moved forward in response to lateral probing when the first male contact was with the female head than when it occurred elsewhere (Table 3). From observations of 20 matings the x distance walked by a female during courtship was 1.7 cm (SD = 1.6), ca. twice its body length. Table 3.—Effect of initial male contact on subsequent movements of female codling moths. Jo 9 exhibiting behavior after initial head contact by male at:a
9 behavior Did not move forward Moved forward after being pushed by male Moved forward in response to lateral contact by male % of total contacts"
9
9
9
head
abdominal tip
left side
right side
3.0a
0.0a
18.2a
9.0a
3.0a
3.0a
15.2a
24.2a
9
15.2b
0.0a
0.0b
9.0a
21.1a
3.0b
33.3a
42.2a
* Values within columns followed by same letter not significantly different (x*. P = 0.05). b Values in row followed by same letter not significantly different (y1, P = 0.05, N = 33).
0.05; data transformed to arcs in
In most cases the male lowered his wings following coupling and the female lowered hers on top of his, after which the moths remained motionless. Mean duration of the sequence from when the male first moved to within 3 cm of the female until motionlessness after wing-lowering was 4.7 sec (SD = 2.4). During successful mating of codling moths, females followed a number of different behavioral pathways (Fig. 1). The most common sequence was: "resting," calling, raising wings at male approach, raising wings higher and walking forward during abdominal probing by male, then lowering wings after sexual coupling. The path followed by the smallest proportion of females was: "resting (no calling visible)," wing-raising at contact with male head, raising wings and walking towards male during abdominal probing and wing-lowering following initiation of copulation. Other combinations of the behavioral events designated in the ethogram occurred. The degree to which each was observed can be calculated by tracing the path from each behavioral state to the next (the number on each path designates percentage of the 33 moths moving from one state to the one connected to it). Two behavioral modes ("raise wings" and "walk forward") appear more than once in Fig. 1, There are 2 reasons for this: some of the females engaged in an activity evidently in response to a particular stimulus (e.g., 42% of the females raised wings at male approach), while others exhibited that same behavior in response to a different cue (12% did not raise wings for the 1st time until male head contact); also, some females responded to more than one stimulus with the same behavior (42% of the females raised their wings at male approach, 21% then raised them higher during male head contact, and Table 4.—Female codling moth wing-raising during male approach prior to contacting the female. Males and females may appear in more than one category. Apparent stimulus Initial 6* approach 6" passing near or in front of head 6 passing upwind of 9 6 approaching 9 side 8
No. of 9 exposed to stimulus % 9 raising wings8 30
53.5
7
57.6
11
54.5
18
44.4
No significant difference between values (x a . P - 0.05)
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Behavior
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Behavior
RESTING OR FLIGHT
DIRECTION OF RELEASER
CALLING; RAISE WINGS EVERT OVIPOSITOR
UPWIND ANEMOTAXIS; LANO ON SUBSTRATE APPROACH FEMALE
CONTACT FEMALE WITH HEAD
MOVE ALONG FEMALE BODY; CURVE ABDOMEN TOWARD FEMALE PROBE FEMALE ABDOMEN WITH GENITALIA
CLASP FEMALE GENITALIA WITH VALVES; FACE AWAY FROM FEMALE; LOWER WINGS
FIG 1.—Male and female behavioral sequences leading to successful mating of codling moths. Sequence direction is top to bottom of page. Pathways connecting boxes denote proportion of total sample (N = 33) moving from one state to the next. All behaviors observed are depicted; however, values < 0.15 are statistically indistinguishable from background (x2, P — 0.05).
3% elevated them a little more as each male moved posteriorly along the female's body while curving his abdomen toward her). Discussion In a detailed study, Grant and Brady (1975) described slight but significant differences in the courtship behaviors of the Indianmeal moth, Plodia interpunctella (Hiibner), and the almond moth, Cadra cautella (Walker). After being attracted to the pheromone of a calling female, a P. interpunctella male approaches and apparently releases a scent which enables him to move into a position with his head beneath the female's and
causes her to remain stationary while turning her abdomen in an acceptance posture. Copulation quickly follows. Similarities between the female-produced pheromones of the Indianmeal moth and almond moth allow interspecific courtship, but successful crossmating does not occur (Grant et al. 1975). This failure was attributed in part to species' differences in direction of male approach and the responses of females to initial male contact. In the Oriental fryit moth (Grapholitha molesta (Busck)), initial male contact with a female results in unsuccessful courtship; after approaching but not touching a female, male hairpencil extrusion causes the re-
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sponsive female to walk to the male and touch his ab- plusia ni (Hiibner) females or black paper silhouette domen with her head. This female-delivered tactile models were positioned 2 cm from a pheromone source, stimulus triggers a copulatory attempt by the male (Baker the presence of such visual cues, as in the codling moth, and Carde 1979). When analyzing the movement from did not influence the persistence of orientation, but it did each behavioral event to the next in a manner similar to influence both the frequency and direction of copulatory the studies above, it appears that forL. pomonella court- attempts. ship, a male behavior is a releaser for each behavior in the female sequence except resting and calling. Many events, however, may not appear in an individual female's repertoire: 9% passed through only 3 of the beAcknowledgment havioral states (resting, wing-raising in response to abWe thank Dr. T. C. Baker for valuable discussion and dominal probing, and wing-lowering after the male grasped the female genitalia with his valves), and only critical review of this paper, Dr. B. A. Croft for gener3% of the females exhibited all but 3 of the behavioral ous support, and M. Benson for excellent technical asstates. Although the behaviors a female codling moth sistance. Supported in part by NSF-EPA Grant GBundertakes may vary considerably, there is little or no 34718. variation in the successful male sequence (Fig. 1). Male behavior remains the same, irrespective of whether the female exhibits only calling or engages in calling and 3 series of wing-raising. REFERENCES CITED Thus, in the codling moth, as presently analyzed, no Baker, T. C , and R. T. Carde. 1979. Courtship behavior of male behaviors during close-range orientation rely on rethe Oriental fruit moth {Grapholitha molesta): experimenlease by an active response of a female. Pheromone protal analysis and consideration of the role of sexual selecduced by a calling female may be the only releaser of tion in the evolution of courtship pheromones in the Lepimale behavior which is a product of female activity. doptera. Ann. Entomol. Soc. Am. 72: 173-88. Maintenance of the rest of the male courtship sequence Barnes, M. M., D. M. Peterson, and J. J. O'Connor. 1966. Sex pheromone gland in the female codling moth, Carpomay rely solely upon stimulation from the performance capsa pomonella (Lepidoptera: Olethreutidae). Ibid. 59: of previous behaviors by the male. This method of 723-4. courtship behavior maintenance appears similar to that of Blatella germanica (L.). Males execute a unidirec- Bell, W. J., S. B. Vuturo, and M. Bennett. 1978. Endokinetic turning and programmed courtship acts of the male tional sequence of several acts in response to a single German cockroach. J. Insect. Physiol. 24: 369-74. initial releaser, antennal contact with pheromone (Bell Borden, A. D. 1931. Some field observations on codling moth et al. 1978). behavior. J. Econ. Entomol. 24: 1137-45. In contrast, the Oriental fruit moth exhibits a some- Butt, B. A., and D. O. Hathaway. 1966. Female sex pherowhat greater degree of plasticity in male courtship bemone as attractant for male codling moths. Ibid. 59: 4767. haviors (Baker and Carde 1979). At one point in the otherwise rigidly fixed action pattern of courtship, an Castrovillo, P. J., and R. T. Carde. 1979. Environmental regulation of female calling and male pheromone response active response by a female releases the concluding steps periodicities in the codling moth (Laspeyresia pomonella). in the sequence: the female walks to the male and J. Insect Physiol. 25: 659-67. touches her head to the extruded male hairpencils, thereby releasing his copulatory attempt. If the male receives no Carde, R. T., and T. E. Hagaman. 1979. Behavioral responses of the gypsy moth in a wind tunnel to air-borne tactile stimulus, he can return to an earlier behavior in enantiomers of disparlure. Environ. Entomol. 8: 475-84. the sequence and attempt to stimulate her again. Wing- Fluri, P., E. Mani, T. Wildbolz, and H. Am. 1974. Unterraising, abdominal-raising, and walking forward are fesuchungen iiber das Paarungsverhalten des Apfelwicklers male codling moth responses to male stimuli but do not (Laspeyresia pomonella L.) und iiber den Einfluss von apparently alter the frequency of subsequent male bekiinstlichen Sexuallockstoff auf die Kopulationshaufighaviors. If a female does not move forward in response keit. Mitt. Schweiz. Ent. Ges. 47: 253-9. to male abdominal probing, he continues to probe until Gehring, R. D., and H. Madsen. 1963. Some aspects of the mating and oviposition behavior of the codling moth, Carhe successfully clasps her genitalia or until courtship is pocapsa pomonella. J. Econ. Entomol. 56: 140-3. terminated. He does not return to an earlier behavior in Grant, C. C , and V. E. Brady. 1975. Courtship behavior of the sequence. Although their tests did not assess direct behavioral effects, Hutt and White (1977) concluded on the basis of laboratory tests of mating frequency that, in codling moths, vision apparently plays some part in successful mating. Our study indicates that the presence of a visual cue does not affect the amount of time spent in orientation near the pheromone source, but it does significantly affect where a moth orients. A male will spend more time walking and fanning near, and attempting to copulate with, an object providing visual (and tactile) cues than it will with a pheromone source alone. Similarly, Shorey and Gaston (1970) found that when dried Tricho-
phycitid moths. I. Comparison of Plodia interpunctella and Cadra cautella and role of male scent glands. Can. J. Zool. 53: 813-26. Grant, C. C , E. B. Smith wick, and V. E. Brady. 1975. Courtship behavior of phycitid moths. II. Behavioral and pheromonal isolation of Plodia interpunctella and Cadra cautella in the laboratory. Ibid. 53: 827-32. Ho well, J F., and K. D. Thorp. 1972. Influence of mating on attractiveness of female codling moths. Environ. Entomol. 1: 125-6. Hutt, R. B., and L. D. White. 1977. Mating response to visual stimulus in the male codling moth. Ibid. 6: 567-8. McDonough, L. M., and H. R. Moffitt. 1974. Sex pheromone of the codling moth. Science 183: 978.
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Proverbs, M. D. 1965. The sterile male technique for codling moth control: 10 years of progress. West. Fruit Grower 19: 10-20.
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Roelofs, W., A. Comeau, A. Hill, and G. Milicevic. 1971. Sex attractant of the codling moth: characterization with electroantennogram technique. Science 174: 297-9.
