\ \m \\\\\\\

This is to certify that the

thesis entitled

Environmental Regulation of the Periodicities
of Female Calling and Male Pheromone Response
and Courtship Behaviors in the Codling Moth
(Laspeyresia pomonella L.)

presented by

Paul Joseph Castrovillo

has been accepted towards fulfillment
of the requirements for

Master of Science

degree in Entomo logy

    

Major professor

Date December—MW

J

'L A R Y
Miz‘higan Scam
University

   
 

 

EM 2510

ENVIRONMENTAL REGULATION OF THE PERIODICITIES
OF FEMALE CALLING AND MALE PHEROMONE RESPONSE
AND COURTSHIP BEHAVIORS IN THE CODLING MOTH

(LASPEYRESIA POMONELLA L.)

 

By

Paul Joseph Castrovillo

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Entomology

1977

ABSTRACT

ENVIRONMENTAL REGULATION OF THE PERIODICITIES
OF FEMALE CALLING AND MALE PHEROMONE RESPONSE
AND COURTSHIP BEHAVIORS IN THE CODLING MOTH
(LASPEYRESIA POMONELLA LL)

 

By

Paul Joseph Castrovillo

Codling moth female calling and male pheromone responsiveness were
studied in the laboratory. Both occurred during scotophase (230C; L:D
16:8). Under continuous photophase or scotophase females continued to
call with a circadian periodicity. Male response rhythmicity was main-
tained under continuous photophase. Decreasing temperature (230 to 1600)
resulted in calling termination (decrease during scotophase) or shifting
of calling into photophase (decrease prior to scotophase).

Males orienting to a platform with a pheromone source spent a signi-
ficantly greater amount of time at a quadrant containing a visual cue
than at any other quadrant or the source. Thirty-three successful cod-
1ing moth matings were videotaped and analyzed to elucidate the behavi-
oral events occurring during courtship.

Field experiments indicated that male pheromone trap catch was un-
affected by trap location within or between trees, and that a non-sticky
trap catch may be comparable to a sticky one. Males observed approaching

a trap were used to assess efficiency.

ii

ACKNOWLEDGEMENTS

This research was supported by a graduate assistantship from the
Department of Entomology, Michigan State University. I would like to
extend my sincerest appreciation to the following people:

Dr. Ring T. Cardé, my major professor, for his invaluable help in
project development, carrying out research and preparation of the results
for presentation. He instilled in me the importance of critical examina-
tion of my own work as well as that of others;

Tom Baker, good friend and colleague, for valuable discussion and
continually demonstrating the power of perseverence in research;

Maria Benson, a terrific lab technician who maintained the moth cul-
ture with a dedication as strong as if her own research depended on it;

Gary, Betty, Laura, Lonnie, Sherry and Dale (I hope I didn't forget
anyone) - the codling moth crew;

Dr. Brian Croft, for providing funds enabling me to begin my thesis
work;

and Anja Cardé, for translating several important journal articles.

iii

TABLE OF CONTENTS

List of Tables . . . . . . . . . . . . . . . . . . . . . . .
List of Figures . . . . . . . . . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . .
Materials and Methods . . . . . . . . . . . . . . . . . . . .
Insect Culture . . . . . . . . . . . . . . . . . . . . . .
Pheromone Samples . . . . . . . . . . . . . . . . . . . . . .
Female Calling Experiments . . . . . . . . . . .
A. Periodicity of female calling under a 16 hour photophase

and 8 hour scotophase regime . . . . . . . . . . . . . . . .
B. Periodicity of female calling under continuous photophase .
C. Periodicity of female calling under continuous scotophase .
D. Effects of a decrease in temperature upon periodicity

of calling . . . . . . . . . . . . . . . . . . . .
E. Effects of relative humidity on the periodicity of

female calling . . . . . . . . . . . . . . . . . . . . . .
Male Pheromone Response Experiments . . . . . . . . . . .
A. Periodicity of male response under a 16 hour photophase

and 8 hour scotophase regime . . . . . . . . . . . . . . . .
B. Periodicity of male response under continuous photophase .
C. Effects of a decrease in temperature upon periodicity

of pheromone response . . . . . . . . . . . . . . . . . . .
D. Effects of a decrease in temperature and decreases in

light intensity upon periodicity of pheromone response . . .
E. Effect of pheromone doSage on male response . . . . . . . .
Codling Moth Courtship Studies . . . . . . . . . . . . . . . .
A. Effects of visual cue presence and placement on the

orientation of male codling moths . . . . . . . . . . . . .
B. Laboratory courtship sequence of the codling moth . . . . .
Trapping Experiments . . . . . . . . . . . . . . . . . . . .
A. Effect of trap placement on trap catch . . . . . . . . . .
B. Comparison of catch in a sticky trap and a non-sticky

trap . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Observations of male behavior at a Pherocon trap . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Female Calling Experiments . . . . . . . . . . . . . . . . .
A. Periodicity of female calling under a 16 hour photophase

and 8 hour scotophase regime . . . . . . . . . . . . . . .
B. Periodicity of female calling under continuous photophase .
C. Periodicity of female calling under continuous scotophase .
D. Effects of a decrease in temperature upon periodicity

of calling . . . . . . . . . . . . . . . . . . . . . .
E. Effects of relative humidity on the periodicity of

female calling . . . . . . . . . . . . . . . . . . . . . . .

Male Pheromone Response Experiments . . . . . . . . . . .

iv

.vi

viii

. 1
.8
. 8
10
.11

12
.12
.14

14

16
16

19
19

. 20

. 20

.22
.22

.26
.27

. 28
. 30

.32

31
31
32

. 32

.32
.37

41

. 45
. 45

and 8 hour scotophase regime .

Periodicity of male response under a 16 hour photophase

light intensity upon periodicity of pheromone response .

Codling Moth Courtship Studies .

A. Effects of visual cue presence and placement on the

orientation of male codling moths . . .
B. Laboratory courtship sequence of the codling moth .
Trapping Experiments .
A. Effect of trap placement on trap catch .

B. Comparison of catch in a sticky trap and a non-sticky

trap O O O I O 0

Effect of pheromone dosage on male response .

C. Observations of male behavior at a Pherocon trap .

Discussion . . . . .

Laboratory Experiments: Periodicity and Courtship .
Field Experiments: Male Orientation to Pheromone Traps .

List of References .

Periodicity of male response under continuous photophase .
Effects of a decrease in temperature upon periodicity of

pheromone response .
Effects of a decrease in temperature and decreases in

.45
.52

.52
.56

. 56
. 6O

60
63
69

.69

.69
.69

73

.73

. 82

. 86

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

10

LIST OF TABLES

Effect of age on calling behavior of
female codling moths . . . . . . .

Mean calling times of female codling
moths under extended photophase and
extended scotophase. Temperature = 23°C .

Effect of decrease in temperature on
i calling time of female codling moths .

Effect of relative humidity on degree

of female codling moth calling at 5
intervals. Temperature = 23°C. Scotophase
began at 21:00. Photophase began at 05:00 .

Effect of relative humidity on i calling
time of female codling moths. Temperature =
23°C. Scotophase began at 21:00. Photo-
phase began at 05:00 . . . . . . . .

Mean response times of male codling moths
to 100 ng of synthetic pheromone under
extended photophase . . . . . . . . .

Effect of decreases in light intensity on
Z of male codling moth upwind orientation
elicited by 100 ng of synthetic pheromone.
Temperature and light decreased 3 hours
prior to scotophase. Upwind orientation
measured 2 hours prior to scotophase . .

Effect of decrease in temperature on %

male codling moth upwind orientation eli-
cited by 100 ng of synthetic pheromone.
Temperature and light decreased 3 hours
prior to scotophase. Upwind orientation
measured 2 hours prior to scotophase . . . .

Effect of visual cue placement on i orienta-
tion length and 2 duration of contact with
platform exhibited by male codling moths .

Effect of visual cue placement on orienta-
tion of male codling moths .

vi

. 36

.39

. 44

.46

. 47

.54

. 57

58

. 61

. 62

Table

Table

Table

Table

Table

11.

12.

13.

14.

15.

Behavioral events in codling moth mating
sequence. Orientation of male and female
codling moth at the beginning of courtship .

Behavioral events in codling moth mating
sequence. Effect of initial male contact
on subsequent movement of female . . . . .

Behavioral events in codling moth mating
sequence. Female wing-raising during male
approach . . . . . . . . . . . . . . . . .
Effect of trap placement on male catch . . .
Mean times of male codling moth orientation

to a non-sticky Pherocon trap baited with 1
mg of synthetic pheromone . . . . . .

vii

. 65

. 66

68

7O

.72

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

LIST OF FIGURES

Calling stance of female codling moth . . . . . .

Form of 2 hour decrease in temperature occurring
during experiments concerned with the effects of
decrease in temperature on codling moth behavi-

oral periodicity. Lowering begins at time 0 and

is completed by time 2 . . . . . . . . . .

Orientation tube olfactometer used for male cod-
ling moth pheromone response bioassays. A: acti—
vated charcoal filter; B: flowmeter; C: manifold;
D: orientation tube; E: exhaust hood; F: light

box 0 I O O O O O O O O O O C O O O I O O O O O O 0

Form of 1 hour decrease in temperature occurring
during experiment concerned with the effects of
decrease in temperature and decreases in light
intensity on male codling moth behavioral perio-
dicity. Lowering begins at time 0 and is com—
pleted by time 1 . . . . . . . . . . . . . . . .

Wind tunnel used for codling moth courtship ob-

servations. A: fan; B: baffles; C: platform con-
taining female; D: male release cage; E: exhaust
vent . . . . . . . . . . . . . . . . . . . . . .

Orientation platform used for observations on
the effects of visual cue presence and placement
on male codling moth behavior . . . . . . . .

Pheromone traps. A: Pherocon 1C; B: Granett-type;
C: pheromone-baited rubber septum . . . . . . . .

Percent calling of female codling moths aged 0 to

6 days old throughout 24 hours. Temperature = 23°C.
Shading denotes scotophase. Arrow represents x cal-
ling time. N = 360 . . . . . . . . . . . . . . .

Percent calling of female codling moths throughout
24 hours. Temperature = 23°C. Shading denotes

scotophase. Arrows represent i calling times. Moth age:

A = 0—1 day old, B = 1-2 days old, C = 2-3 days
old. N = 60.. . . . . . . . . . . . . . . . . .

viii

13

.15

17

.21

. 23

. 25

29

.33

. 34

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

10.

11.

12.

13.

14.

15.

16.

17.

Percent calling of female codling moths
throughout 24 hours. Temperature = 23°C.
Shading denotes scotophase. Arrows rep-
resent x calling times. Moth age: D =
3-4 days old, E = 4-5 days old, F = 5-6
days old. N = 60 . . . . . . . . . . . .

Percent calling of female codling moths
under extended photophase. Temperature =
23°C. Shading denotes scotophase. Arrows
represent 2 calling times. N = 100 . . . .

Percent calling of female codling moths
under extended scotophase. Temperature =
23°C. Shading denotes scotophase. Arrows
represent i calling times. N = 100 . . .

Effect of decrease in temperature on fe-
male codling moth calling behavior. A:
temperature decrease 3 hours into scoto-
phase; B: temperature decrease 1 hour
into scotophase. Broken line represents
decrease in temperature as in Figure 2.
Shading denotes scotophase. Arrows rep-
resent i calling times. N = 100 . . . . .

Effect of decrease in temperature on fe-
male codling moth calling behavior. Bro-
ken line represents decrease in tempera-
ture as in Figure 2. Shading denotes
scotophase. Arrows represent x calling
times. N = 100 . . . . . . . . . . . . . . .

Male codling moth behaviors elicited by

100 ng of synthetic pheromone during 24
hours. Observations were 30 seconds in du-
ration with pheromone present and were cor—
rected for activity occurring 30 seconds
prior to pheromone exposure. Temperature =
23°C. Shading denotes scotophase. N = 60 .

Male codling moth upwind orientation eli-
cited by 30 seconds of exposure to 100 ng of
synthetic pheromone during 24 hours. Tempera-
ture = 23°C. Shading denotes scotOphase. Ar-
row represents i response time. N = 72 . . .

Male codling moth spontaneous non-anemotac-
tic activity (hopping, walking and wing-
beating) during 24 hours. Observations oc—
curring for 30 seconds with no pheromone
present. Temperature = 23°C. Shading denotes
scotophase. Arrow represents 2 activity time.
N = 72 . . . . . . . . . . . . . . . . . .

ix.

. 35

38

. 4O

.42

43

. 49

.50

51

Figure

Figure

Figure

Figure

18.

19.

20.

21.

Male codling moth upwind orientation eli-
cited by 100 ng of synthetic pheromone under
extended photophase. Temperature = 23°C.
Shading denotes scotophase. Arrows repre-
sent 2 response times. N = 60 . . . . . . .

Effect of a decrease in temperature on male
pheromone responsiveness. Broken line rep-
resents period of temperature decrease as in
Figure 4. Shading denotes scotophase. Arrows
represent i response times. N = 60 . . . .

Percent male codling moth upwind orientation
elicited by designated dosages of synthetic
pheromone 1 hour into scotophase. Response
values are bracketed by 95% binomial confi-

dence intervals. Temperature = 23°C. N = 108 .

Sequence of behavioral events leading to
successful mating of the codling moth de-
veloped from analyses of 33 matings. Direc-
tion of sequence is top to bottom. Pathways
connecting boxes denote proportion of total
sample moving from one state to the next .

53

.55

.59

.64

INTRODUCTION

The codling moth, Laspeyresia_pomonella L., is a cosmopolitan lep-

 

idopterous pest of apples, pears, walnuts and related crops. Although
it has been under investigation for many years in the hopes of finding
keys to a control program with a reduced reliance on insecticides bio-
logical and behavioral information, especially that concerned with
courtship and mating, is 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." Recently this has proven to be
quite incorrect. Proverbs (1965) demonstrated that males were attrac-
ted 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 cor-
rugated cardboard."

Butt et a1. (1968) screened 35 aldehydes and nitriles for codling
moth attractiveness in the field, and 95 aldehydes and nitriles were
tested in a laboratory bioassay in which positive responses were de-
fined as "extending their claspers, vibrating their wings, spinning in
a circle and (or) attempting to copulate." Few positive results were

1

obtained. On the basis of combined data from gas chromatographic
tests, several chemical reactions, electroantennogram screening of a
number of compounds and field-trapping Roelofs et a1. (1971) proposed
(§,§)-8,10-dodecadien-1-ol as the identity of a sex pheromone of the
codling moth. Failing to locate this chemical in the insect McDonough
et al. (1972) reported evidence that the codling moth possessed multi-
ple sex pheromones with the major component being (§,§)-2,6-7-methyl-3-
propyl-decadien—l—ol. Using a computerized search of mass spectra ob-
tained from CC effluent of female extract Beroza et al. (1974) demon-
strated that a doubly unsaturated lZ-carbon alcohol with retention time
identical to that of (E,E)-8,lO-dodecadien-l-ol was present in the cod-
ling moth. McDonough and Moffitt (1974), retracting the identification
reported by McDonough et al. (1972), stated that they had found (Egg)-
8,10—dodecadien-1-ol, by physical data and ozonolysis, to be the authen-
tic natural sex pheromone. Buser and Arn (1975), using quadrupole mass
fragmentography and high resolution gas chromatography, found approxi-
mately 3.5 ng of pheromone to be present per female.

Shorey (1970) theorized that in moths the sex pheromone was a sin-
gle species or group-specific chemical attractant. Although it is now
known that most Lepidoptera utilize multiple component pheromone sys-
tems, blends of related compounds in discrete ratios (Roelofs and Cardé
1977 and Tamaki 1977), no evidence of this pertaining to the codling

moth has yet been found. Roelofs et a1. (1972) conducted a field-trap—

ping experiment to test the effect of various mixtures of the 3 geomet-
rical isomers ((§,_E_), (§,§) and (§,_Z_)) with (E,§)-8,10-dodecadien-1-ol.
Results indicated that pure (E,§) was a potent male attractant and that

the other isomers were unattractive alone and (at least in the ratios

tested) diminished trap catch when mixed with (§,§). In other field
tests (§,§)-8,IO-dodecadien-l-ol acetate and undecanol inhibited the
attraction of males to females in traps (Hathaway et al. 1974), and
traps baited with synthetic pheromone had decreased attractancy when
the bait included the following (E,E)-8,10-dodecadien-1-ol derivatives:
acetate, proprionate, ethyl ether, propyl ether, isopropyl ether and
sec-butyl ether (George et al. 1975). Arn et a1. (1974) screened 33
compounds, 11 to 14 carbon, singly unsaturated alcohols and acetates
and found that the greatest catch reduction in traps baited with 1 mg
of synthetic pheromone occurred with 5 mg of (§)-8-dodecenyl acetate.
Others which gave significantly lower catch were generally 12 or 13
carbon acetates with a (E) double bond.

The natural pheromone is produced by the female codling moth in
glandular tissue located dorsally on the ovipositor (Barnes et al.
1966). It is held within the abdomen, between the eighth and ninth
segments except when she is calling, at which time the ovipositor is
extended ventrally at a 900 angle to the axis of the body (Fluri et a1.
1974).

Males have been reported to approach calling females in a "typical

buzzing dance:"

walking in a rather straight line with buzzing wings,

lifting the abdomen, and spreading their valves in the direction of the
female. "As soon as a male touched a female copulation followed imme-
diately (Fluri et al. 1974)." Apparently stimulation by the sex phero-
mone plays an important role in the mating process because Fluri et al.
(1974) found that removal of both male antennae almost completely pre-

vented mating of caged moths, while removal of'l male antennae or both

female antennae did not. On the basis of another caged moth mating

4
experiment Gehring and Madsen (1963) reported that probably no visual
cues were necessary during codling moth courtship when males and fe~
males were in close proximity because mating was as successful in con-
stant darkness as under a photocycle with both scotophase and photo-
phase. Conversely, Hutt and White (1977) observed in the laboratory
under close confinement the number of matings occurring with blind, an-
tennectomized, blind and antennectomized and unaltered males and con-
cluded that response to visual cues did play a part in mating.

Using a timing pheromone trap in California Batiste (1970) collec-
ted male codling moths from before noon to 4 A.M. (Pacific Standard
Time). Under normal weather conditions, however, it is considered to
be a crepuscular and nocturnal insect. The literature holds many meas-
urements of codling moth activity time determined by field flight ob-
servations (Borden 1931 and Putnam 1963), bait trap (Worthley 1932 and
Parrot and Collins 1934), UV light trap (Batiste et al. 1973a) and
pheromone trap catches (Wong et a1. 1971, Batiste et al. 1973a and
1973b and Mani et al. 1974) and all reported that on an average day
moth flight and trap catch were concentrated at twilight, usually with-
in the range of 30-60 minutes before to 30-60 minutes after sunset.

Apparently environmental conditions can modify this flight period.
Batiste et al. (1973a) determined that in California codling moths in
flight during spring and early summer were most active prior to sunset,
while those flying later in the season were captured in greater numbers
after sunset, and they attributed this to differences in early evening
temperatures. This same paper also reported that male flight was lim-
ited below 13° and above 27°C. Previously Batiste (1970) had indicated

that last trap catch appeared to be correlated with temperature

decrease to 16°C. Worthley (1932) found during bait trapping that cod-
ling moth flight did occur below 16°C, but increased with increasing
temperature. A series of observations on male flight in the orchard
and attraction to pheromone traps (Mani et al. 1974) uncovered evidence
that the effect of decreases and increases in temperature on male activ-
ity may have relied on the time the temperature changes occurred, the
previous weather conditions and what other environmental factors, such
as cloud cover, wind speed and precipitation, were altered. Their work
also indicated that during the evening codling moths may exhibit 2 dif-
ferent types of flight activity: a period when moths (sex was not de-
termined) are flying but little or no attraction to traps occurs and a
period of flight when males are captured in great numbers. The mode of
flight occurring at any time appears also to be a result of the com-
bined effects of recent and current weather trends.

The ability of males to orient to pheromone has presented itself
as a tool for use in monitoring and possibly controlling codling moth
populations. Traps have been baited with virgin females (Howell 1974),
female extract (Butt and Hathaway 1966) and synthetic pheromone (Butt
et a1. 1974). For the latter, when dispensed on a rubber septum, in
the field, a dosage of approximately 1.5 mg with a release rate of 1.25
ug per hour appears to be optimal (Maitlen 1976).

Several trap designs have been tested on the basis of ability to
catch, cost to produce, ease of maintenance and durability in the field.
These have included open wing traps, 1 quart cylinder traps and BAB
(cylinder with bottom half of opening closed) traps (Butt et a1. 1974);
ice cream carton, milk carton and wing traps (Howell 1972); Pherotrap

1C and Batiste traps (Westigard and Graves 1976); and BAB and aluminum

funnel above aluminum pie plate traps (Batiste and Joos 1972). All of
them relied on a sticky surface to capture the moths.

A number of codling moth control programs have been initiated
based on utilization of the pheromone in monitoring traps. Moth catch
was used as a criterion for determining when insecticide sprays should
be applied (Hudson and Johnson 1971, Madsen and Davis 1971, Madsen and
Vakenti 1973 and Myburgh et al. 1974). These programs have successful-
ly allowed a reduction in sprays with the resultant level of control
acceptable. Other projects have attempted to keep populations under
control directly by mass-trapping using pheromone baited traps (Pro-
verbs et al. 1975 and MacLellan 1976) and these have been less success-
ful. Mating disruption from atmospheric permeation with pheromone has
been tested with promising results. During a one-year field trial
Audemard et al. (1977) maintained orchard damage at a commercially ac-
ceptable level, but the amount of pheromone employed (0.55 g/ha/day)
was fairly high. Cardé et al. (in press) demonstrated complete disrup-
tion of male attraction to synthetic pheromone and caged virgin females
and mating of tethered females when pheromone was dispersed from hollow
capillary fibers.

It has been suggested that in low level populations traps effec-
tively compete with native female moths, but as population size in-
creases to moderate or high numbers of individuals the sex attractant
trap catches do not accurately reflect the increase and therefore can-
not be used to estimate the population size or control it (Howell 1974
and Mani and Wildbolz 1975). In Michigan, where the codling moth is
partially bivoltine, Riedl et al. (1976) observed that moth catch anti-

cipated emergence of adults from pupae in banded trees and oviposition

during the spring flight, but lagged behind emergence from bands and
closely followed oviposition during the second generation.

At present the limit for successful usage of the codling moth sex
pheromone in applied entomological situations is as trap bait for moni-
toring population levels. However, an increased understanding of the
behavior of the insect, especially its courtship, response to pheromone
and traps and the mediating effects of weather on those responses could
conceivably lead to greater success in control programs, due to more
effective usage of the pheromone and more complete interpretation of
the results.

This thesis research deals with several aspects of codling moth
mating activity. Observations were made on the periodicities of female
calling and male anemotaxis in response to pheromone stimulation and
the effects of some extrinsic factors on these behaviors. The sequence
of events occurring during codling moth courtship in the laboratory is
reported, as well as the results from 3 field experiments concerned

with male orientation to pheromone traps.

MATERIALS AND METHODS

Insect Culture

 

A culture of codling moths was maintained continuously in a growth
chamber at the Pesticide Research Center, Michigan State University on
small, green thinning apples obtained from a commercial supplier
throughout the duration of the research. Stock insects were obtained
from infested apples collected in an abandoned East Lansing apple or-
chard during September 1975, and small introductions of new material
were made in the summer of 1976, summer of 1977 and fall of 1977. In
the chamber temperature was maintained at 23 i 1°C and the light cycle
was 16 hours of photophase (1500 lux) followed by 8 hours of scotophase.
Relative humidity was not held constant but generally remained about 75%.

The steps in the insect rearing were: mating of adults, collection
of ova, infestation of apples with larvae and harvesting and sexing of
pupae. Adults were placed in a 30 x 24 x 15 cm mating box, constructed
of wooden top, bottom and sides and a plexiglasR back and front. The
back panel was darkened with a piece of black paper and the front panel
was transparent. A wooden dowel attached to one side of the box served
as a holder for a roll of waxed paper which was used to line the inside
surface of the clear front panel. Fertilized female moths in the box
readily oviposited on the waxed paper which was removed and replaced ev-
ery 1 to 3 days. The sheet of paper containing the ova was placed in a
3.8 liter glass jar, sealed with parafilm and held in the growth chamber

until larval emergence.

Ferro and Harwood (1973) reported that it may be possible for more
than one codling moth larva to survive to maturity within a single ap-
ple, but one larva per apple is the normal situation (Howard 1887 and
Geier 1967). Therefore it was necessary to devise a system which would
result in little or no multiple larval entry of an apple to insure max-
imum survival. The method used relied on infesting each apple by hand.
A newly emerged first instar larva was removed with a camel's hair
brush from a jar containing ova. A very narrow slit (ca. 2 mm wide and
2.5 cm long) was cut in an apple and the larva placed in that slit.

Then the apple was placed in a 3.8 liter glass jar and more infested
apples were added until the jar was 3/4 full (ca. 50 apples per jar).
Most larvae entered the apples at the slit in which they were placed.

To provide a substrate for cocoon formation of mature larvae that
would allow easy pupal collection, paper and acetate "sandwiches," in
some ways similar to a collection device used by Glenn (1922), were
constructed. These consisted of an inner sheet of rippled cardboard,

2 thin sheets of clear acetate,'one flat against each side of the card-
board, and a sheet of construction paper on the outside of each acetate
sheet, all held together by 2 paper clips (and all measuring 15 x 4 cm).

Two sandwiches were placed in each jar of infested apples, a lid
with a brass screen panel was screwed on the jar and they were placed on
a shelf in the growth chamber to allow the larvae to develop. When
ready to pupate, a majority of the larvae found the dark, sheltered area
between the ripples of the cardboard a suitable site in which to spin
up. Approximately 25 days after infestation the sandwiches were removed
from the jars and pupae were collected. This was easily done by remov-

ing the rippled cardboard sheet with cocoons from the sandwich, opening

10

each cocoon with a curved steel teasing needle and removing the pupae
with forceps. When cocoons contained larvae or prepupae they were left
in place to allow development to continue. After all pupae were collec-
ted the rippled cardboard was returned to the sandwich and the sand-
wiches put back into the jars to allow more slowly developing larvae to
pupate. Pupae were harvested a second time 1 week after initial collec-
tion and sometimes once more a week later, then the sandwiches were re-
moved, the spent apples discarded and jars washed for re-use. Generally
it could be expected that during the first collection 50% of the larvae
introduced would be available as pupae and later collections would yield
approximately 10% more of the population.

Once the pupae were harvested they were sexed (Petersen 1965).
Males were placed in 295 ml clear plastic cups and these put into a
63 x 63 x 46 cm screened holding cage for emergence. Female pupae were
placed in similar plastic cups, sealed loosely with parafilm and allowed
to emerge. The virgin adults were collected each day and removed to
other sealed plastic cups for holding. This was done to minimize the
amount of pheromone released into the air of the growth chamber because
only one chamber was available for rearing (both male and female adults
had to be kept there), and it is possible that a high ambient concentra-
tion of pheromone might depress the responsiveness of males during beha-
vioral bioassays (Shorey and Gaston 1964). Periodically adults were
placed in the mating box to continue the culture and others were held in
the growth chamber until used for laboratory or field experiments.

Pheromone Samples
Synthetic l.._._ pomonella pheromone, (§,§)-8,10-dodecadien—1-ol (Roe-

lofs et a1. 1971) was obtained from Zoecon Corporation. GLC analysis on

11

a Carbowax 20M column indicated ) 99% purity, with no evidence of the
geometrical isomers. Samples for bioassay were prepared by dissolving
200 mg of the attractant in 2 ml of petroleum ether and preparing seri—
al dilutions in decade steps. All treatments (both on filter paper rec-
tangles and rubber septa) were dispensed in 10 pl of petroleum ether.

Female Calling Experiments

 

During experiments concerned with female calling periodicity each
test insect (a virgin female moth) was held in a clear 35 m1 plastic
cup sealed with a circular piece of parafilm. Moths were always placed
in the cups at least 1 hour before the first observation took place.
Viewing during scotophase was accomplished with the aid of a small
flashlight fitted with a Kodak WratteéE>Filter #19. The filter elimi-
nates light below 6100 A, and Norris et al. (1969) reported that codling
moths are insensitive to light greater than 6000 A in wavelength.

Four plastic humidity chambers were constructed for use in the ex-
periment dealing with possible effects of differing relative humidity
on codling moth calling. Each chamber was fashioned from a 31 x 23 x
10 cm clear plastic box and 15 of the plastic cups described above.
Three rows of five 24 cm diameter holes were cut into the lid of the
box, an equal-sized hole was cut in the bottom of each cup and the cups
were glued onto the box lid. An 18 x 27 cm rectangle of aluminum screen
was glued against the inside surface of the lid. Three different 1.5
liter solutions of potassium hydroxide capable of producing relative hu-
midities of 25, 50 and 75% (Solomon 1951) were prepared and during the
test one solution was poured into each of three plastic boxes. The
fourth box contained 1.5 liters of water (100% RH). A single virgin fe-

male was placed in each cup of the chamber and a circular piece of

12
parafilm on top and the aluminum screen below held her there. When the
lid was positioned on a solution-containing box the desired RH was pro-
duced within the cups holding the females.

A:_ Periodicity of female calling under §_1 hour photophase and §_hour

 

 

 

scotophase regime

 

Six groups of 20 virgin females (aged 0—1 (group I), 1-2 (II), 2-3
(III), 3—4 (IV), 4-5 (V) and 5-6 (VI) days old) were set up singly in
clear plastic cups and held under light and temperature conditions of
L:D 16:8 and 23 I 1°C. The moths were observed 19 times throughout a
24 hour period. Each cup was numbered and a tally kept of which moths
were calling during each observation. Behaviorally, a moth was desig—
nated as calling if it was possible to see the ovipositor protruding
from the abdomen and directed ventrally at a 90° angle to the body. In
most cases this was made very apparent by a characteristic stance em-
ployed by many calling females: anterior of the insect low to the sub-
strate; the posterior raised by extension of the metathoracic legs, and
sometimes slight raising of the wings (Figure 1). This procedure was
replicated 3 times.

B;_ Periodicity 2f_female calling under continuous photophase

 

 

Six groups of 20 virgin females, as described above, were set up
under conditions of L:D 16:8 and 23 i 1°C and observed for calling 17
times during a 24 hour period. Only those calling in the oldest 5
groups were recorded. At the end of the first observation period the
moths in group VI were replaced by 20 new 0-1 day old ones (the new
group I), each group designation was increased by one because of in-
creased age, and observations were again carried out on the oldest 5

groups for 24 hours. This procedure was repeated on the third day and

13

 

Calling stance of female codling moth.

Figure 1.

14

on the fourth day group VI moths were removed, with the remaining moths
once more being observed for 24 hours. Temperature was held constant
during the 4 day period, but on the third and fourth days the insects
were subjected to 48 hours of continuous photophase.

_QL Periodicity 9f_female calling under continuous scotophase

 

The above procedure with 6 groups of 20 virgin females was carried
out again: group VI once more being replaced on the second and third
days and discarded on the fourth. Temperature was maintained at 23 t
1°C and during the first day L:D was 16:8, but the scotophase beginning
on the second night was extended for the following 63 hours until ter-
mination of observations.

D;_ Effects gf_§_decrease in_temperature upon periodicitngf calling

Three similar experiments were undertaken to demonstrate the effect
of a temperature drop at different times during the day and night on
calling. In the first, 5 groups of 20 virgin females (groups I-V: aged
0-5 days old) were set up and observed for calling 10 times throughout
a 24 hour period, a new group I was added and V removed, and observa-
tions again made over the next 24 hours. The L:D 16:8 photocycle was
maintained. Three hours after initiation of scotophase on the second
day ambient temperature was lowered from 23° to 16°C. The drop occurred
over a 2 hour period with the form presented in Figure 2. Following
this the moths were kept at 16°C until termination of the observations.

The second experiment was identical to the first except that the
temperature drop was begun 1 hour after the onset of scotophase. The
third experiment extended over a 3 day period and employed 6 groups of
virgin females as in the circadian periodicity demonstrations, group VI

being discarded twice and replaced once with a new group I. The

 

15

 

25 1..
24 -
23 4P . .
22 -
21 —
of: 20 —
3 18 -
{3
m 17 h'
8' O 0
g 16 h-
15 '-
14 -
13 *-
l l l I
0 1
Time (Hours)
Figure 2. Form of 2 hour decrease in temperature occurring during ex-

periments concerned with the effects of decrease in temper-
ature on codling moth behavioral periodicity. Lowering be-
gins at time 0 and is completed by time 2.

16
temperature drop this time occurred 3 hours prior to the beginning of
scotophase on the second day. Therefore, in addition to observations
being carried out under 30 hours of normal rearing chamber conditions,
the insects were also held at 16°C and observed for 42 hours.

§;_ Effects of relative humidity gn_the periodicity gf_female calling

 

 

 

For each of the 4 RH's to be tested 15 virgin female moths were
placed in a humidity chamber lid, one per cup: five aged 1—2, five 2-3
and five 3-4 days old. One lid was placed on each solution-containing
chamber at least 4 hours prior to the start of scotophase and the num-
ber of females calling at each RH was tallied at 2 times before and 3
times during scotophase. The next day all of the lids were removed and
the oldest 5 moths in each lid replaced with 5 moths 1—2 days old. Lids
were randomly reassigned (i.e. females were not necessarily returned to
the humidity level experienced during the previous night) to the boxes
and placed back on them before scotophase in preparation for a new ser-
ies of observations. This procedure was replicated 7 times.

Male Pheromone Response Experiments

Laboratory male behavioral bioassays of responses to synthetic
pheromone were conducted in an orientation tube olfactometer (Figure 3)
located in a bioassay chamber at the Pesticide Research Center. The 01-
factometer was similar to that described by Sower et al. (1973). An in-
house air line passed air through an activated charcoal filter and a
flowmeter to a glass manifold and then to the orientation tubes. Each
glass orientation tube was 1 m long and 2 cm in diameter. The upwind
end was formed into a 24/40 ground glass joint and a fine mesh brass
screen plug was placed within the upwind end of each tube.

The orientation tubes were suspended above a 125 x 65 x 20 cm box

 

Figure 3.

17

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Orientation tube olfactometer used for male codling moth pher-
omone response bioassays. A: activated charcoal filter; B: flow-

meter; C: manifold; D: orientation tube; E: exhaust hood; F:
light box.

18

with a top surface of translucent white plastic. Six 7.5 watt incandes-
cent lights connected to a rheostat were located inside the box. A 105°
connecting tube interfaced with the upwind end of each orientation tube.
The pheromone sample was introduced via the 1050 tube into the airstream,
and when no sample was present the third arm was closed with a ground
glass stopper. A 65 x 25 x 12 cm plexiglaég>hood over the downwind end
of the orientation tubes was connected to an exhaust line venting phero—
mone-contaminated air from the bioassay chamber.

In conducting male bioassays the following procedure was employed.
Male moths were removed from the holding cage, care being taken to util-
ize males from all parts of the cage equally, and placed in the orienta-
tion tubes. A 7 x 7 cm piece of cheesecloth was placed over the down—
wind end of each tube and held in place with a rubber band. The tubes
were set up on the olfactometer and connected with the air line at least
1 hour but less than 12 hours prior to the bioassay to allow the moths
to acclimate to test conditions. Air flow and exhaust were begun be-
tween 30 and 60 minutes prior to testing.

The first tube was observed for 30 seconds and (depending on the
experiment) all moth activity was described onto a tape recorder or key
behavioral responses were tallied. Then a pheromone sample was intro-
duced into the airstream by replacing the ground glass stopper in the
1050 connecting tube with a cork stopper and a steel clip holding a fol-
ded 2.5 x 1.25 cm piece of Whatman #1 filter paper containing the test
stimulus. For 30 seconds moth behaviors were again observed and de-
scribed or tallied. The sample was then removed from the tube, the
stopper replaced and the observational procedure repeated with all of

the remaining orientation tubes.

19

Following testing of the last tube the olfactometer was allowed to
exhaust for a minute or two, then the air flow was stopped and within 12
hours the moths were returned to the holding cage. To remove any chemi-
cals that may have adhered to the glass between use the orientation
tubes, connecting tubes and glass stoppers were rinsed with acetone.
All cheesecloth and pheromone samples on filter paper used during tes-
ting were discarded. No moths were ever bioassayed more than once dur-
ing a 24 hour period. All bioassays were conducted with an air flow of
0.81 m/sec. During scotophase tests the light intensity beneath the
tubes was 0.5 lux and during photophase testing light intensity was 1500
lux.

AL_ Periodicity pf_male response under §_1§_hour photophase and §_hour

 

 

 

scotophase regime

 

Male bioassays were carried out using the orientation tube olfacto-
meter as described above. Responses to a 100 ng sample of synthetic
pheromone were measured at 2 hour intervals under conditions of L:D 16:8
and 23 i 1°C. Three replicates were performed at each test time, a rep-
licate consisting of 8 orientation tubes each containing 3 male moths.
Behaviors were described onto tape and transcribed.

B;_ Periodicity of_male response under continuous photophase

 

Orientation tube olfactometer bioassays were conducted at 9 times
during a 24 hour period for 2 consecutive days (23 t 1°C; L:D 16:8). On
the third day the holding cages to which the test males had been re-
turned were moved to the bioassay room and maintained under continuous
photophase for 56 hours of constant light. During this period the moths
were bioassayed on 2 days at the 9 test times. Two replicates were run

at each time, 10 tubes per replicate and 3 male moths per tube. The

20
pheromone sample was 100 ng of synthetic pheromone. Upwind orientation,
defined as walking upwind in the tube (with or without wing fanning) was
the key behavioral response tallied.

§;_ Effects of a decrease in temperature upon periodicity o£_pheromone

 

res 201188

Males were bioassayed in the olfactometer at 5 times between 4
hours prior to and 3 hours following the initiation of scotophase on 3
consecutive days. During the first day room temperature was maintained
at 23 f 1°C. Three hours before scotophase on the second day the tem-
perature was decreased from 23° to 16°C over a 2 hour period (Figure 2).
During the remainder of the experiment all moths were held and bioas-
sayed at 16°C. Two replicates were carried out at each time with 10
tubes per replicate and 3 moths per tube. The stimulus was 100 ng of

synthetic pheromone and the response scored was number orienting upwind.

 

 

D;_ Effects of a decrease in temperature and decreases ip_light intensity

upon periodicity of_pheromone response

 

Two olfactometer bioassay replicates, each with 10 tubes and 3 moths
per tube, were carried out 2 hours prior to the onset of scotophase under
the following conditions: temperature of 23°C and light intensity of 1500
lux; 23°C and 150 lux; 23°C and 15 lux; 23°C and 1.5 lux; temperature de—
creased to 16°C and 1500 lux; 16°C and 150 lux; 16°C and 15 lux; and 16°C
and 1.5 lux. During any tests in which the temperature and/or light lev-
el was lower than normal rearing chamber conditions (23°C and 1500 lux),
the change in test conditions was initiated 1 hour prior to bioassay.
Decrease in light intensity was immediate, but decrease in temperature
followed the form presented in Figure 4. Number of moths walking upwind

to a 100 ng sample of synthetic pheromone was tallied.

Temperature (0C)

24 -
23 -
22 -
21 -
20 -
19 -
18 -
17 -
’16 r-
'15 -
14 -
13 -

 

21

 

Figure 4.

Time (Hours)

Form of 1 hour decrease in temperature occurring during ex-
periment concerned with the effects of decrease in tempera-
ture and decreases in light intensity on male codling moth
behavioral periodicity. Lowering begins at time 0 and is
completed by time 1.

22

gg_ Effect of_pheromone dosage op_male response

 

 

Six concentrations of synthetic pheromone spanning 8 orders of mag-
nitude were tested for male responses using the orientation tube olfac-
tometer. All bioassays were conducted between 45 and 90 minutes into
scotophase. At each replicate all 6 treatments were tested, 3 tubes per
treatment with 4 male moths per tube. Shortly before the bioassays were
to begin the 6 pheromone and filter paper samples were prepared and each
one stored in a glass test tube. At bioassay time each sample was
picked randomly. A verbal description of all male behaviors was recor-
ded on tape. The experiment was replicated 9 times.

Codling Moth Courtship Studies

Laboratory observations of male orientation to a pheromone source
and a visual cue and the videorecording of codling moth courtship were
conducted in a wind tunnel (Figure 5) housed in a bioassay chamber at
the Pesticide Research Center. The tunnel, 2.4 x 1.2 x 0.8 m, was con-
structed of an aluminum frame and clear plexiglaég>and had the observa-
tional side divided into 3 movable panels which acted as sliding doors
for access to the tunnel interior. The downwind and upwind ends were
closed by aluminum screening to prevent the escape of insects in the
tunnel.

Air flow was generated by a 0.7 m diameter fan connected to a rheo-
stat for regulation of fan speed. The air was passed through a series
of baffles to produce a nearly laminar flow and an exhaust line removed
pheromone-contaminated air from the chamber. White and green striped
canvas was stretched beneath the transparent wind tunnel floor. Light
was supplied by four 7.5 watt incandescent lights suspended above the

wind tunnel and connected to a rheostat. The light was diffused by a

23

 

 

 

 

 

 

 

 

rm

 

 

 

 

 

In“

 

 

[/7—I7L-‘6T -=—

m [7;4wu 1m:

fl#—— 11* E§:§

 

 

\\\l_i / \\\\\;\\“ “WW/I ”/09 IT

 

 

 

 

r

 

 

 

 

 

Wind tunnel used for codling moth courtship observations. A: fan; B: baffles; C: platform con-

taining female; D: male release cage; E:

Figure 5.

exhaust vent.

24
sheet of translucent white plastic below the bulbs and a white cotton
sheet over the top of the tunnel.

For observations of male orientation to synthetic pheromone a plat—
form was constructed to serve as a location for the pheromone source and
visual cues and as a place at which male moths could engage in any close
range mating behavior that must occur in contact with a substrate (Figure
6). The platform was fashioned from a 24 x 23 x 10.5 cm balsa wood
frame. To the top was attached a 23.5 x 24.5 cm sheet of white polysty-
rene plastic and to the bottom was glued a 15 x 24 x 2 cm piece of styro-
foam. A circular hole, 1 mm in diameter, was cut into the center of the
plastic sheet and lines in pencil from the hole to each corner of the
sheet delineated triangular quadrants. Four size #0 insect pins were
pushed through the plastic, their pointed ends projecting 1.5 mm above
the platform surface, 1 pin in each quadrant 2 cm from the center hole.
A disposable glass pipette was then pushed upward through the styrofoam,
a 5 mm length of silicone tubing slipped over the pipette and its tip
placed in the platform hole. Care was taken to position the tip so that
it was flush with the surface of the platform.

For videorecording of codling moth courtship living virgin females
were placed on a 40 x 28 cm sheet of white polystyrene plastic. Eight
females were placed on the platform and each held in place beneath a
clear 35 ml plastic cup.

Several hours prior to the time observations were to be undertaken
male moths were placed singly in cylindrical copper screening release
cages 11 cm high and 7 cm in diameter. Each cage was sealed with a
square of parafilm which could easily be lifted off to allow the moth's

escape into the wind tunnel.

25

 

 

 

 

 

 

 

 

 

Figure 6. Orientation platform used for observations on the effects of
visual cue presence and placement on male codling moth beha-
vior.

26

5&_ Effects of_visual cue presence and placement op_the orientation of

 

 

male codling moths

 

Air flow and exhaust of the wind tunnel were begun. The orienta-
tion platform was set up inside the tunnel 51 cm from the upwind end and
25 cm above the tunnel floor and supported by ring stands on each side.
A rubber septum baited with 0.1 mg of (E,E)-8,10-dodecadien-1-ol was
placed inside a 125 ml Erlenmeyer flask. The flask was plugged with a
silicone stopper containing 2 pieces of glass tubing: one was connected
to the pipette on the platform; the other was connected to an in-house
air line. Air passing from the line carried pheromone out of the flask,
through the pipette and out the hole of the platform where it was trans-
ported downwind in the tunnel.

For a visual cue a dead female codling moth rinsed several times in
acetone was placed on the orientation platform and held in position by
impaling it on one of the insect pins projecting from the surface. The
female was placed in only one quadrant at a time and was always posi-
tioned with its head pointing upwind.

A male was induced to fly upwind in the tunnel to the platform by
opening the downwind sliding door and placing a caged moth in front of
the exhaust and removing the parafilm. Not all males exhibited anemo-
taxis but when one did leave the cage and began an upwind-oriented
flight the sliding door was closed and the observer moved to the upwind
end of the tunnel to describe on tape male behaviors at the platform for
2 minutes. Following that the male was observed for a measurement of
the duration of his orientation to the platform. At the termination of
each observation, if possible, the male was removed from the wind tun-

nel so as not to interfere with later tests.

27

Data was collected from 85 male orientations of 2 minutes or greater
duration: 17 observations occurred with no visual cue present and 17 with
the cue located in each quadrant. Between each observation the female
was removed or randomly moved to a different quadrant. It was determined
that the dead females were unattractive to the males without the synthe—
tic pheromone present: when no air was passed through the flask males
could not be induced to fly upwind to the platform; when males had
reached the females with the aid of pheromone and the flask air flow was
stopped the males left the platform very shortly without reorientation.
Dead females were used several times, but discarded each time they were
placed in the downwind quadrant, where they may have picked up pheromone
from the plume passing over them.

Following conclusion of all observations air flow to the flask was
ended and the wind tunnel was exhausted for approximately 15 minutes.
Males were returned to a holding cage within 12 hours and to remove any
pheromone that may have adhered to them all release cages were rinsed
with acetone.

Observations occurred at 23°C and 0.4 lux, a pheromone source air
flow of 0.4 ml/sec and wind tunnel air speed of 0.68 m/sec. No moths
were released for observation more than once during a 24 hour period and
most were tested only once. All observations occurred during the 8 hours
in which the moths were previously held in scotophase.

_BL Laboratory courtship sequence of_the codling moth

 

Air flow and exhaust of the wind tunnel were initiated. A platform
holding 8 virgin females (several platforms were set up with females in
preparation for these observations) was placed in the tunnel on the ring

stands 51 cm from the upwind end and 25 cm above the floor. Cups were

28

removed from motionless females. When any females began calling males
were released into the wind tunnel as described above until one was in-
duced to fly upwind to the female. The behaviors of both insects were
recorded using a Sony Videocorder and Videocamera AVG-3450, from when
the male first moved within approximately 30 mm of the female until sev-
eral seconds after copulation was complete or, in the case of an unsuc-
cessful mating attempt, until the sequence was terminated by the male or
female. After all of the females that appeared willing to call on the
platform were mated the platform was replaced with another containing 8
new females.

Observations were conducted at a temperature of 20°C. The light
level at the platform was 200 lux (near the lower light level limit of
the Videocamera). A black cloth was draped above the downwind end of
the tunnel, producing a light level of 100 lux: this decreased the like—
lihood of males immediately flying up to a bright ceiling upon release.
All observations were made during the 8 hours corresponding with scoto-
phase in the rearing room, where the moths were previously held.

TrappinggExperiments

 

Pherocon 10 insect traps from Zoecon Corporation were used in the
trapping experiments concerned with effect of trap placement on male
catch and comparison of capture potential for a sticky (Pherocon) and
non-sticky (Granett-type) pheromone trap. The Pherocon trap (widely
used in pheromone trapping research) consisted of 2 pieces of cardboard
28 x 22.6 cm, folded in a roof-like fashion, with the bottom sheet of
cardboard folded upward and the top sheet suspended above the bottom one,
folded downward (Figure 7). A "Y"-shaped length of steel wire served as

a handle for hanging the trap and also passed through openings in both

29

 

 

 

 

 

 

Figure 7. Pheromone traps. A: Pherocon 1C: B: Granett—type; C: phero-
mone-baited rubber septum.

3O

sides of each piece of cardboard to hold the top and bottom in position
and allow access to the interior of the trap. The inside bottom trap
surface was coated with a very sticky glue. For trapping a pheromone-
baited rubber septum was placed in the center of the sticky surface and
male moths attracted to the pheromone entered the trap and became stuck
in the glue.

During observations of male codling moth approach to a pheromone—
baited trap, a Pherocon trap constructed as described above was used,
but the sticky-surfaced bottom was replaced by a non-sticky upturned top
piece. Males were enabled to enter the trap to approach the baited sep-
tum without being captured.

The Granett-type traps (Granett 1973) were made from a white poly-
styrene plastic cylinder 25 cm high and 19 cm in diameter and 2 plastic
funnel—shaped end pieces projecting into the cylinder (Figure 7). A
baited rubber septum was suspended inside the trap from the roof of the
cylinder by a size #3 insect pin pushed through the plastic and into the
rubber. A 4 x 2 x 0.4 cm block of plastic impregnated with Vapona was
placed within the trap to kill moths entering it.

A:_ Effect of_trap placement op_trap catch

 

 

Pherocon 1C traps were placed in apple trees of an abandoned or-
chard located in Cascade, Kent County, Michigan. Three blocks were set
up within the orchard with 3 trap locations in each block: a trap near
the tip of a branch in the crown (the outside of the tree), a trap in
the center of the crown (the inside of the tree) and a trap suspended
from a length of twine extending from tree to tree between 2 rows (in
the open between trees). All traps were hung approximately 2 m above

the ground and were separated by at least 20 m. Each was baited with

31

1 mg of synthetic pheromone on a rubber septum. The traps were checked
and captured males were counted, removed and discarded 9 times through-
out the test period, with the septa and sticky bottoms being replaced
once. Each time the traps were checked they were rotated to a new posi—
tion within the block.

B;_ Comparison of catch ip_g_sticky trap and a non-sticky trap

 

Four areas within 3 abandoned apple orchards located close together
near the Clinical Sciences Center of Michigan State University were cho-
sen as test blocks for this experiment. Within each block 2 Pherocon 1C
traps and 2 Granett-type traps were placed in apple trees, at least 20 m
apart and approximately 1.5 m above the ground. Each trap was baited
with 1 mg of synthetic pheromone on a rubber septum. They were checked
6 times during which captured moths were counted and removed, and all 4
traps within each block were moved to another part of that block.

94_ Observations o£_male behavior §£_§_Pherocon trap

 

 

 

A non-sticky Pherocon lC trap was hung approximately 1.6 m high on a
branch of an apple tree in an insecticide—free experimental orchard main—
tained at Fennville, Allegan County, Michigan. The trap was set up and
a rubber septum baited with 1 mg of synthetic pheromone placed within it
1 to 2 hours before sunset. Male codling moths approaching the trap were
observed and a verbal description of their behaviors recorded and tran-

scribed.

RESULTS

Female Calling Experiments

 

5;. Periodicity of_female calling under a_16 hour photOphase and §_hour

 

 

 

scotophase regime

 

Figure 8 depicts the results of observations on the mixed age (0-6
days old) collection of virgin females throughOut the 24 hour day. Under
constant temperature a high degree of calling behavior was initiated with
the onset of scotophase, continued throughout the dark period and was
terminated at the beginning of photophase. Analysis of this behavior for
each of the 6 age groups composing the total reveals that for moths 1-6
days old no significant differences apparently exist in number of females
calling, x calling time (the point at which half of the calling-hours ex-
hibited for the day was reached) or i number of hours called per female
(Figures 9 and 10 and Table 1). The youngest moth group, aged 0-1 day
old, however, did call less than the other groups. Fewer 0-1 day old fe-
males called than those that were 1—6 days old, and the young ones that
did, engaged in it for a shorter length of time. Also, as measured by x
calling time, the majority of calling of 0-1 day old moths took place
earlier in the evening.

B;_ Periodicity of_female calling under continuous photophase

 

 

Observations in the previous experiment demonstrated a dial periodi-
city of female calling behavior apparently associated with the beginning
and end of scotophase. Data from this experiment and the next illustrate
that this rhythm has an endogenous basis and therefore is circadian.

32

33

.oom n z .oEHu wcwaamo x mucmmouamu 3ouu< .ommzeouoom mouocop wcfivmnm .oomm
u monumuanoH .muaoe cm usonwaounu vac whom o ou o comm mnuos wcHHvoo mamEmm mo mafiaamo unmouom .w ouowfim

 

 

 

 

 

 

 

 

 

 

 

mane
on m o e N «N NN ON an on 43 Na
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n. so

 

 

 

oi: I 2

56
48
40
32
24
16

72
64
56
48
40
32
24
16

Percent calling

72
64
56
48
40
32
24
16

 

34

23:52

 

 

 

 

Figure 9.

24:32

 

 

 

 

 

 

 

 

 

Time

Percent calling of female codling moths throughout 24 hours.
Temperature = 23°C. Shading denotes scotophase. Arrows repre-
sent i calling times. Moth age: A = 0-1 day old, B = 1-2 days
old, C = 2-3 days old. N = 60.

64
56
48
40
32
24
16

Percent calling

Figure

 

 

35

24:29

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Percent calling of female codling moths throughout 24 hours.
Temperature = 23°C. Shading denotes scotophase. Arrows rep-

resent x calling times. Moth age: D = 3—4 days old, E = 4-5
days old, F = 5-6 days old. N = 60.

36

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one Aamsvm mmuwm mHmEmm moo: Ouv domaumaaoo some a“ om moamemw wcHHHmo on ummma um omcamucoo masopw

mwm Ham ”comaummsoo mafia mafiaamo m onu Boom woumsfisfiam on Ou om; mnuoa omonu om :.HHmo new map: you cofiu

imucmmmuamu Hmoauoasa on ma ouocu ”AunwfichE oouwfiv oouqm ou A.z.< Houmfiv douoo Boom mamom Hmofiuoeoa m co
woumcwfimov mmB mafia mcwaamo m "moamoHMchHm mo ummu “cm cm H 2 “mafia wofiHHmo M NO oowumaaoamo pom co u z q

Aummu owcmu mHmHuHDE maoomlcmEBoZIuaowdum moan: mommy
Imamm momma ”mo. u m .<>oz<v udmummmav mauamoawficwfim uoa umuuoa 08mm he oo3oHHow moaaaoo afinufia mmoam> m

Amo. u m .NQAV ucmnoMMHv maucmowwacmwm uoa umuuoa 08mm he oosoaaom unasaoo cwnufis moDHm> H

 

 

 

 

 

 

Am.mv H n~.m Aq.mV H nm.m nofiuqm Ann elm
Ao.mV H om.q Am.mv H pm.m nmfiuqm awn mlq
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wafiHHmo macov mamamm mamamm mom owHHmo muson m mafia wcwaamo m vm>uomno N no ow<

you ooaamo mono: M

 

.mfiuos mafiavoo onEow mo uoa>mnon wcHHHmo co owm mo uoommm .H edema

37

Figure 11 indicates that when scotophase was absent over a 2 day inter-
val most of the females called during the 8 hours at which scotophase
had been occurring: during days 1 and 2, 99.3% of the calling—hours oc-
curred within 8 hours; during days 3 and 4, 87.8% occurred within 8
hours. The increase from a low level of calling to maximum was sharp
when the lights-off cue was present: from less than 8% to greater than
60% calling in 2 hours; while with no cue the change was more gradual:
less than 8% to approximately 50% taking 5 hours. A similar pattern was
apparent at calling termination. When a lights-on cue was available
calling ended abruptly; when photophase was continuous termination was
more gradual.

Under continuous photophase x calling time was significantly shif—
ted approximately 2.5 hours later into the evening than with a photocy—
cle of L:D 16:8 (Table 2). This resulted from maximum calling as well
as calling termination occurring later in the night on days 3 and 4 than
the first two. It should be noted, however, that the x calling time
shift occurred because of a change in the shape of the calling curve,
rather than an increase in the length of the period. This is indicated
by the fact that the new i calling time, from the first day with no sco-
tophase, was maintained approximately 24 hours later on the following
day.

94_ Periodicity of female calling under continuous scotophase

 

 

Under conditions of continuous scotophase (Figure 12) calling oc—
curred with a periodicity very similar to that observed with the L:D
16:8 photocycle. As with continuous photophase transition from no cal-
ling to maximum calling and vice versa was more gradual than when lights-

on and lights-off cues were present: with the cues, 0% to greater than

64

48

32

16

64

48

32

16

64

48

Percent calling

32

16

64

48

32

16

Figure 11.

Day 1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

111111 1l111l_1
. Day 2
b
y-
k
b
1-
I1Illl I1111_l
r
P
L.
r
#-
11111111 11
1L4111 l—-—‘
12 16
Time
Percent calling of female codling moths under extended photo-

phase. Temperature = 23°C. Shading denotes scotophase. Ar-
rows represent i calling times. N = 100.

39

Table 2. Mean calling times of female codling moths under extended pho-
tophase and extended scotophase. Temperature = 23°C.

 

 

 

Extended photophase Extended scotophase
x calling time i calling time
Day (hour:minute)1 (hour:minute)1
1 24:31a 24:27a
2 24:233 01:34a
3 03:17b 24:23a
4 03:08b 23:29a

 

1 Values within columns followed by same letter not significantly differ—

ent (ANOVA, P = .05; means compared using Student—Newman-Keuls multiple
range test)
N = 100 for calculation of x calling time; N = 50 for test of signifi-
cance. Each group contained at least 50 calling females, so to discard
non-calling females and keep sample sizes equal comparisons were made
using only the first 50 x calling time values in each group.

on
s
H
F4
H
m
u
U
a
m
o
u
o
n.
48
Figure 12.

 

40

24:27

 

 

 

 

 

 

 

L2
_ ‘Day 4

 

 

 

 

 

Time

Percent calling of female codling moths under extended sco-
tophase. Temperature = 23°C. Shadind denotes scotophase. Ar-
rows represent i calling times. N = 100.

41

48% calling in 2 hours; without cues 0% to 48% calling in 5 to 6 hours.
Under these conditions, nevertheless, in addition to calling being ex-
tended several hours beyond the time photOphase had previously begun,
initiation of calling occurred several hours prior to subjective lights-
off. Thus, although the x calling time was not altered significantly
(Table 2), the overall duration of calling was increased: on day 1, 100%
of calling occurred within 8 hours; on day 4, 94% of calling occurred
within 12 hours.

2;_ Effects of a decrease in temperature upon periodicity of calling

 

The daily pattern of calling behavior observed under a constant am-
bient temperature of 23°C was modified in the presence of both a temper-
ature drop and an extended temperature of 16°C. As shown in Figure 13,
a 7 degree decrease in temperature during scotophase resulted in com-
plete termination of calling, no matter whether the drop occurred during
the middle or very beginning of the dark cycle. Conversely, the same 7
degree change 3 hours prior to the onset of scotophase induced a very
high degree of calling in the moths during a time when this behavior was
very low at 23°C (Figure 14). With a pre-scotophase temperature drop
much of the calling was discontinued shortly with lights-off: 91% of cal-
ling occurred during darkness at 23°C, while only 42% took place during
darkness at 16°C.

On the day following the temperature drop moths maintained at 16°C
showed a significant shift in the i calling time, approximately 5 hours
earlier than the x calling time of moths at 23°C (Table 3). Few females
called during scotophase compared to a high level of calling during the

latter part of photophase.

42

23:41

48 ..

” 23c
32 _

A Day 1

 

16 -

 

 
 

48 -
A Day 2

32 - o

  
 

16 -

 

 

 

 

 

80 - 23:23

Percent calling

B Day 1

 

 

B Day 2

  

 

12 14 16 18 20 22 24 2 4 6 8 10

Time

Figure 13. Effect of decrease in temperature on female codling moth cal-
ling behavior. A: temperature decrease 3 hours into scoto-
phase; B: temperature decrease 1 hour into scotophase. Broken
line represents decrease in temperature as in Figure 2. Shad-
ing denotes scotophase. Arrows represent x calling times.

N = 100.

80

64

’48

32

16

80

64

48

32

Percent calling

16

72

64

48

32

16

Figure 14.

43

 

 

 

 

 

 

 

 

 

 

 

. 23°C
_ Day 1
r
1111 11_11|lj
: 3
- : 5 20:39
_ =
:
— 3
I
F =
_ 23°C E
E
1.. E Day2
3
" I
:
' E
E
‘ 3
Illll' LlJlll
_ Day 3
1 1 1 1 1 1 1 J
12 6 8 10
Time
Effect of decrease in temperature on female codling moth cal-

ling behavior. Broken line represents decrease in temperature

as in Figure 2. Shading denotes scotophase. Arrows represent
x calling times. N = 100.

44

lumumaom momma “mo. 1 m .<>oz<v uoouommwo hHucmonHawwm uoa umuumH meow kn woBOHHom mqasHoo manuwz mmsHm>

.aoouw

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monEom wGHHHmolcoa vumomfio ou om .monamm wafiHHmo om umooH um oocwmuaoo asouw zoom .ooumoHMHGme mo umou
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nmmuom mqanN mmmumm N
mmqumm mmwumm quumm H
mefiu wcHHHmo m HoEHu wcHHHmo m Hosea mafiaamo m Non
"mmmnwouoom ou HOHHQ muaon "ommnaouoom oucfi use: "mumsaouoom oucfi mason
m mommuomw musumuoosma H mmmmuoow musumummaoe m mmmouumo mucumumaame
.mSuoE mnHHooo onaom mo mafia wcwHHmo m so ououmnomEmu a“ mmmmuoow mo uoommm .m mHan

45

FL_ Effects of_relative humidity op_the periodicity of_female callipg

 

The data from this experiment tabulated in Tables 4 and 5 indicate
very little discernible effect on the degree of calling between groups
at each test time or the x calling time of each group as a result of
differing relative humidities.

Male Pheromone Response Experiments

 

§2_ Periodicity o£_male response under §_16_hour photophase and §_hour

 

 

scotophase regime

 

During this bioassay series behaviors exhibited by males in the
tubes both prior to and following exposure to synthetic pheromone were
described. The purposes were elucidation of the types of behavior eli—
cited by pheromone stimulation, as well as possible determination of a
key behavior usuable as a criterion for response in future bioassays.

States of behavior observed were: 1) motionlessness; 2) antennal
raising: motionless insects rested with antennae either pressed lateral-
ly against the body or erect, perpendicular to the substrate upon which
they were sitting; addition of pheromone to the airstream sometimes eli-
cited raising of antennae that had been held against the body; 3) wing
fanning: the males' tarsi were in contact with the tube, but wings were
beating rapidly; for these bioassays activity was only considered wing
fanning if it had a duration of 2 or more seconds; 4) upwind orientation:
walking upwind in the tube, with or without wing fanning; 5) non-anemo—
tactic movement: either hopping, walking or wing beating in bursts of
less than 2 seconds, usually with little movement in the tube or alter-
nating, apparently undirected, upwind and downwind movement; and 6) cop—
ulatory attempts: sometimes occurring when a male fanning and walking

upwind contacted or passed within a few millimeters of another male; the

46

Table 4. Effect of relative humidity on degree of female codling moth
calling at 5 intervals. Temperature = 23°C. Scotophase began
at 21:00. Photophase began at 05:00.

 

% females calling at relative humidity ole

 

 

 

Time 25% 50% 75% 100%
18:00 ' 0.0a 1.0a 0.0a 0.0a
20:00 4.83 12.3a 8.6a 8.6a
22:00 66.7a,b 78.1a,b 81.0a 62.8b
24:00 55.2a 57.13 73.33 52.4a
02:00 41.0a 47.6a 56.2a 47.6a
1

Values within rows followed by same letter not significantly different
(ANOVA, P = .05; means compared using Student-Newman-Keuls multiple
range test); Values transformed to arcsin ifi'for analysis; untrans-
formed values reported in Table 4. N = 105

47

Table 5. Effect of relative humidity on x calling time of female cod—
ling moths. Temperature = 23°C. Scotophase began at 21:00.
Photophase began at 05:00.

 

 

 

% relative humidity x calling time (hour:minute)1
25 23:27
50 23:14
75 23:33
100 23:33

 

1 No significant difference between any values (ANOVA, P = .05; means
compared using Student—Newman—Keuls multiple range test). N = 105

48

approaching male curved his abdomen laterally and forward while opening
his valves and then probed the abdomen of the second male.

Figure 15 depicts the changes in the degree of 4 of the most com-
mon classes of activity throughout a 24 hour day. All values represent
a measure of the behavior during the 30 seconds when pheromone was pres-
ent in the airstream, with corrections made for pre—pheromone background

activity according to the formula:

 

no. active when no. active
pheromone present - during background
% response = 100 x
N - no. active during background

Of the 4 behaviors non-anemotactic movement remained at the lowest
level throughout the 24 hours. Wing fanning, upwind orientation and fan-
ning with simultaneous upwind movement increased markedly with the onset
of scotophase and were greatly reduced after initiation of photophase.
Binomial confidence intervals (95%) for the measurement of wing fanning
and upwind orientation overlap at the same times indicating no signifi-
cant difference in thresholds for these behaviors with 100 ng of phero-
mone. Because a key behavioral response of the male in the field is up-
wind anemotactic flight, upwind orientation in the tube was selected as
the key behavior for bioassays.

Diel periodicity of male pheromone response is represented in Fig-
ure 16. Under constant temperature upwind orientation elicited by pher-
omone was most evident during the dark part of the photocycle (i response
time occurring close to the middle of scotophase). Spontaneous non-ane-
motactic movement of males observed during the 30 second background ac—
tivity measurement period (prior to introduction of pheromone into the

airstream) also showed a similar trend (Figure 17).

49

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mouoaoo wafivmnm .oomm n monumummEoB .ousmoaxo maoaouoca Ou uowum mwcooom om wafiuao wafiuuoooo
xuw>fiuom How omuoouuoo oumz can unwound m:oEoum:m nufia cOHumusw :H moaoomm om mums maoaum>
lummno .muoo: «N wcHuao odoEoumna oHuonuahm mo we ooH he vmufioHHo muow>m£on :uoa waHHwoo mez

 

 

 

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KnrArnoe nueoiea

50

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mucomouaou souu< .ommnaouoom monocoo waavmnm .oom~ u opsumquEoH .muoo: «N wcHuoo ocoeouona
oauonuahm we we ooH ou ousmoaxm mo moaoomm om >3 wouHoHHm cowumucmHuo oGHBQS Luce wcHHvoo mam: .oH mustm

 

 

 

 

 

 

 

 

 

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51

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wcwpoo chfiumonnwcH3 new mcfime3 .wafiaaocv >OH>Huom oHuomuoemamucoa moomcmucoam nuoa wcHHwoo onz

0845

S w e e N 3 .8 ON 2 S S S

 

 

 

 

 

mmuqm

 

.NH weamwm

KnrArnoe snoauenuods aueoled

52

B;_ Periodicity of male response under continuous photophase

 

 

Male bioassays under 2 days with L:D 16:8 and 2 days with no scoto-
phase (Figure 18 and Table 6) indicate that pheromone responsiveness oc-
curred with a periodicity close to 24 hours, both in the presence and
absence of lights-on and lights-off cues. When an abrupt light/dark cue
was available, the number of upwind orientations elicited by pheromone
changed greatly over a short period of time: 16% to 80% in 2 hours.
During continuous photophase the degree of upwind orientation continued
to rise and fall, although the change was more gradual: 16% to 60% in 8
hours.

.9; Effects of a decrease in temperature upon periodicity of pheromone

 

response

When male codling moths experienced a decrease in ambient tempera—
ture of 7 degrees (from 23° to 16°C) over a 2 hour period beginning 3
hours prior to the start of scotophase the number of pheromone-elicited
upwind orientations appeared to be unaltered during the remainder of
photophase; but the degree of upwind movement exhibited during darkness,
which was very high at 23°C, was extremely depressed at 16°C (Figure 19).
On the following day, with the moths still maintained at 16°C, little up-
wind orientation in response to pheromone was observed. With this de-
crease in temperature the x time of response was shifted approximately
2.5 hours earlier into the day. Unlike the shift in x calling time of
the females during a decrease in temperature (Table 3), the shift in male
upwind orientation was a result of the extremely lowered response level

during scotophase, rather than an increase in response during photophase.

53

Day 1

 

Day 2

 

 

64

48 - Day 3

Percent upwind orientation

12:28
32 -

16 -

 

48 —

32 —

16 -

 

 

0 lllllLLlllLllllll'llllJ

12 14 16 18 20 22 24 2 4 6 8 10

Time

Figure 18. Male codling moth upwind orientation elicited by 100 ng of
synthetic pheromone under extended photophase. Temperature =

23°C. Shading denotes scotophase. Arrows represent x response
times. N = 60.

54

Table 6. Mean response times of male codling moths to 100 ng of synthet-
ic pheromone under extended photophase.

 

2 response tipe

 

2§z_ ' (hour:minute)
1 11:56
2 11:32
3 12:28
4 12:08

1 Normal photocycle on day 1 and day 2; continuous photophase on day 3
and day 4 '
No significant difference between means (ANOVA, P = .05; means com-
pared using Student-Newman—Keuls multiple range test). N = 60

55

21:40
80 -

64

48 -

23 C
32 -

Day 1

 

16:-

 

 

48 -
, 23°C

9
I‘ll-III...-
H
0‘
O
O

   

32

 

Day 2

    

16 -

 

48
f.

32 — - 18:55

Day 3

16 -

 

 

 

Time

- Figure 19. Effect of a decrease in temperature on male pheromone re-
sponsiveness. Broken line represents period of tempera-
ture decrease as in Figure 4. Shading denotes scotophase.
Arrows represent i response times. N = 60.

\E:

lave
sub;
int
01f

the

no
cor
11
be
at

fi

If"

56

D;_ Effects of a decrease in temperature and decreases ip_light intensity

 

 

upon periodicity of_pheromone response

 

 

Table 7 presents the data obtained from testing pheromone response
level at one time, 2 hours prior to the start of scotophase, for moths
subjected to various temperature and light conditions. At 23°C, a light
intensity decrease from 1500 to 150 lux was sufficient to increase sig-
nificantly the percentage of males orienting upwind to pheromone. Fur-
ther decreases in light intensity did not increase upwind movement beyond
that effected by the 90% reduction. A 7 degree temperature decrease cou-
pled with lowered light intensities gave less clear results: a signifi-
cant decrease in activity occurred at 15 lux, while there appeared to be
no differences between upwind orientation at 1500, 150 and 1.5 lux. When
comparisons were made between number of moths moving upwind under equal
light intensities but different temperatures few differences were found
between those held at 23°C and those experiencing a decrease in temper-
ature to 16°C (Table 8). The only effect was at 15 lux, where a signi-
ficant decrease was observed.

§L_ Effect o£_pheromone dosage op_male response

 

 

The male behaviors described on tape and transcribed were used to

measure pheromone response by tallying those orienting upwind to each

3

sample. Figure 20 indicates that from a dosage of 10-5 to 10- pg of

synthetic pheromone little upwind orientation was observed, whereas, be-
tween 10"3 and 10..1 pg the number of males responding increased. Of the

l and 10° pg produced the greatest

samples tested in this experiment 10-
upwind orientation, with no significant difference between the two on

the basis of 95% binomial confidence intervals. An increase to 102 pg

greatly reduced upwind orientation.

57

oo 1 z .Amo. u m .Nx.v ucoummmww mausmowmaawfim uoa HouumH 05mm >3 voBOHHom m3ou canuas moDHm>

 

 

H
mom 3mm mom mom uooH
new non nwm qu comm
xSH m.H NSH mH xDH omH an oomH mcowufivaoo musumuomaoe

Hme>oH uanH um GoHumuaoHuo vafi3m: N m

 

.mmmnaouoom ou scape muson N monomwme aowumuaofiuo mafia
1m: .mmmsaouoom ou uoaum muaon m wmmmouomw ustH paw mucumuomaoe .mcoaouona ofiuonucxm mo ma ooH
SH woufiofiHo aOHuMucoHuo paw3e: Suoa wcfiHooo mHma mo N no xufimcoucw unwfia afi mommonoow mo uoommm .N mHHmH

58

 

 

ow u z .Amo. n m .NRVV unmummev hHquUHMHGme uo: HouuoH 08mm %5 @030HHom quDHoo aHnuHS mmsHm> H
mom ewN 8% mom uofl
moo mon mwm qu oomu
st m.H on mH xSH omH de oomH chHunaoo monumuomEmH

HmHo>mH unmwa um coaumuaofiuo.vcfiamn N m

 

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Icmauo mafiaaa .mmmamouoom ou uoaua muaos m wmmmmuooo uanH paw munumummaoe .maoaouozm owu0:ud%m
mo ma ooH Sn wouaofiHo coaumuawfiuo mafiaoo nuoa mafiavoo mama N no musumuoeamu CH ommmuooo mo uoomwm .w oHan

59

.on I z .oom~ n muoumuanoH .mHm>

Iuoucw mocmoncoo HmHEocwn Nm¢ an poumxomun mum mosz> uncommom

OH

oH

oH

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Awnv mcoEoumLQ afiumnuczm mo wwwmoa

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6O

Codling Moth Courtship Studies

 

§=_ Effects gf'visual cue presence and placement 93 the orientation pf

 

 

male codling moths

 

Male codling moths induced to fly upwind to the orientation plat—
form engaged in the following patterns of behavior: hovering slightly
downwind of the edge, often casting from side to side or forward and
backward; hovering above the platform and dropping to its surface to
touch it; and landing upon the platform to walk about (usually accompa-
nied by wing fanning) and attempted copulation at the hole of the phero-
mone source or, when present, with a dead female. In almost every case
each male exhibited all of the behaviors described and was seen, during
one orientation to the platform, to re-orient to the source and/or the
female several times. Orientation time near the platform was defined as
the time from when the male first reached the downwind edge of the plat-
form to when the male moved upwind of it, to the wind tunnel ceiling or
side for 5 seconds, or downwind without returning upwind within 5 sec-
onds. If a male sat motionless on the platform for 60 seconds this was
also considered termination (and orientation duration was measured to
the time at which motionlessness began).

Mean total orientation length and i time in contact with the plat-
form (momentarily touching it, walking and fanning and attempting copu-
lation) during the first 120 seconds of orientation, reported in Table 9,
appeared to be unaffected by the presence or absence of a dead female
visual cue. Location of the cue, when available, also had no effect on
the amount of time spent at the platform.

However, as shown in Table 10, the placement of a visual cue did af-

fect the amount of time a male spent at each quadrant. In all cases a

61

Table 9. Effect of visual cue placement on x orientation length and x
duration of contact with platform exhibited by male codling

 

 

 

 

moths.

. _ x time in cont c5
Location of Visual cue x+orientation.leng5h with platform ’
in relation to source _ S.D.) (seconds) (_ S.D.) (seconds)
no cue present 440.9 '1 (308.1) 43.9 i (15.2)
upwind 475.2 i (402.6) 48.3 i (14.5)
to right 526.2 is (371.6) 50.7 i (17.1)
downwind 410.2 2t (234.1) 50.4 i (21.2)
to left 417.4 1‘ (188.2) 39.2 i (17.6)

 

1 Visual cue 2 cm from source

2 No significant difference between means within each column (ANOVA, P =
.05; means compared using Student-Newman-Keuls multiple range test)

3 During first 120 seconds of orientation

62

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one Hmsmw>

 

 

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.mnuoe waHHwoo ona mo GOHumucoHuo so ucmaoomHa moo Hmomfi> mo uoommm .oH oHan

63

significantly greater amount of time was passed on the quadrant contain-
ing the dead female than any other quadrant or at the source. It ap—
pears, however, that all of the time a male spent on the quadrant con-
taining the visual cue was not passed attempting to copulate with the
female. In most cases the x time on the quadrant was significantly less
than the x time in contact with the visual cue.

B;_ Laboratory courtship sequence of the codling moth

 

Thirty-three successful codling moth matings occurring in the wind
tunnel and recorded on videotape were analyzed using stop-action and
slow playback. This analysis was used to generate a flow diagram (Fig-
ure 21) depicting the observed steps leading to copulation (A similar

study was undertaken with the Oriental fruit moth, Grapholitha molesta

 

Busck; T. Baker personal communication). On a very basic level, codling
moth courtship can be described as a process in which the male flies up-
wind to a female, lands upon the substrate on which she is sitting,
walks (fanning simultaneously) to her and sexual coupling quickly fol-
lows. Table 11 presents data from these observations indicating that
while calling and resting on a horizontal surface the females assumed no
particular direction of body orientation. Similarly, the males, once
they landed on the substrate following upwind flight, approached the fe-
male from all directions with equal frequency.

All of the males observed within 30 mm of a female (the approximate
distance at which videorecording was begun) fanned their wings vigorous-
ly while walking. Initial contact was always made by the male, touching
his head to the female. Table 12 reports significantly fewer first con-
tacts at the posterior of the female compared with any other part of her

body. Following this the male curved his abdomen laterally and forward

 

 

 

64

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MALE DIRECTION FEMALE
BEHAVIORS OF BEHAVIORS
STIMULUS
r.[upwind flight_J [—7 resting I 1
l L 95:9
,‘ eel—7 calling:
:3 ‘*" raise wings
7 [—7 evert ovipositor
land on substrate:l _J’ I 1‘
approach to within “'4’ .u 5;
20 mm of female " "
13 I I I raise wings r'-"J"' ,
contact female I 9 T I 3L 3,; 3'?
with head I, l ‘f 1’ ‘T
If_ [_ raise wingsi
:9 I I I 1 :9 I 292919
I walk forwardil, ‘
move along I
female body; -€>
curve abdomen
toward female I
1’ I I [—firaise wings'
probe female I J‘,‘ ,L
I abdomen with _9 5"” .1:
: genitalia #_I 1 - I I, I'
_ [7 raise wingsi” ’
I walk forward I
l L 5" *
[faise abdomenp J‘I
@ l » £33
walk toward [—7 l l |
I walk away
- _ from male
clasp female I I
genitalia — - .4 3

 

with valves;
face away from
female; lower wings

 

 

 

 

T l

’ [lower wingssje—

 

.L
I

13
I'

 

Figure 21. Sequence of behavioral events leading to successful mating of
the codling moth developed from analyses of 33 matings. Direc-

tion of sequence is top to bottom. Pathways connecting boxes
denote proportion of total sample moving from one state to

the next.

65

Table 11. Behavioral events in codling moth mating sequence. Orienta-
tion of male and female codling moth at the beginning of

 

 

 

 

courtship.
Orientation of moth % females facing1 % males approaching1
upwind 30.3 30.3
crosswind (right) 33.3 24.2
downwind 24.2 24.2
crosswind (left) 12.1 21.2

 

1 No significant difference between any values within columns (112, P =
.05). N = 33

66

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Amo. n m .Nuwv ucmwmmwfip hauamowmwawfim uo: HouumH mama >0 woonHom moasaoo cfinufi3 wmsHm> H

 

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N

mHma kn oomuaoo
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cw vumsuom wm>oa

mama he unsung wafimn

 

 

 

 

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no.0 mN.wH no.0 mo.m uumzhom m>os no: 0H0
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.onBom mo unmam>oa
uaoacmmnam so oomucoo mHme Hmfiuacfi mo uoowwm .oocwsvmm wcwuma nuoa wcaHvoo ca muco>o Hmu0H>mnom .NH oHan

67

then walked along the side of the female, with his head still in contact
with her, while he moved into a position enabling him to probe her abdo-
men with his. Wing fanning occurred during approach, initial male-fe—
male contact and abdomen curving and movement along the female, but as
the male began abdominal probing he held his wings at roughly a 45° an-
gle to the horizontal and vibrated them. When the female genitalia were
grasped by the male valves, wing vibration ceased and his wings were fol-
ded roof-like above him as he turned to face 180° away from the female.

During male approach there was some variation as to when his valves
were first opened. Of 20 observations 8 males opened them only after his
head contacted the female. Two opened them intermittently during the ap-
proach and 10 held them open during the entire time they were within 30
mm of the female.

Of the 33 matings observed 30 began with the females holding their
wings tightly against their bodies. As the males approached 53.5% of the
females partially raised their wings, resulting in greater exposure of
the abdomen and genitalia. An analysis of percent females raising wings
in response to males approaching upwind, anterior to and lateral to the
females (Table 13) suggests no differences between the stimuli.

Females walked forward to a small degree (3%) directly after initi-
al male head contact, and to a very large degree (66%) as the male probed
her abdomen with his. Male abdominal contact usually was directed later-
ally to the female due to his position beside her, but as he moved pos-
teriorly his probing became more anteriorly directed. It appeared as if
some females were induced to move forward in response to the lateral con-
tact from the male head or abdomen, whereas other females only did so

when apparently being pushed by the male during abdominal probing. Table

68

Table 13. Behavioral events in codling moth mating sequence. Female
wing-raising during male approach.

 

 

 

 

Number females exposed % females faising

Apparent stimulus to stimulus wings

male approach 30 53.5

male passing near 7 57 6

or in front of head °

male passing upwind 11 54.5

of female

male approaching 18 44.4

female side

 

1 No significant difference between values (x2, P = .05)

69

12 shows a significantly greater number of females responding to lateral
contact when the first contact occurred between the male and female head
than when it occurred anywhere else. From observations of 20 matings the
i distance walked by a female during courtship was 17.6 mm (S.D. = 16.4),
approximately twice the body length of a codling moth.

In most cases the male lowered his wings following coupling and the
female lowered hers on top of his after which the moths remained motion—
less. Mean duration of the sequence from when the male first moved to
within 30 mm of the female until motionlessness after wing-lowering was
4.7 sec (S.D. = 2.4).

Trapping Experiments

 

 

 

A:_ Effect of trap placement op_trap catch

Data from this experiment is presented in Table 14. Total catch
was 429 male codling moths with no significant catch difference appear-
ing on the basis of trap placement within the orchard.

B;_ Comparison of_catch is s_sticky trap and s_non-sticky trap

 

 

 

No significant difference was found between catch in the Pherocon
trap and catch in the Granett-type trap using ANOVA (P = .05) and the
Student-Newman-Keuls multiple range test for comparison of the means.
Total number of males captured was 343 with x number per replicate
caught in the Granett trap being 4.0 (S.D. = 5.5) and x number per rep-
licate caught in the Pherocon trap being 11.5 (S.D. = 19.1).

.9; Observations of_male behavior st s_Pherocon trap

 

 

 

Fifty-seven male approaches were described. Codling moth behavior
at the trap included: fluttering outside and inside the trap (males ap-
peared to be attempting to hover or fly slowly towards the trap, but var—

iations in wind speed and direction almost continually caused erratic

70

Table 14. Effect of trap placement on male catch.

 

x number of males per trap

 

 

 

Location of trap, per replicate (i S.D.)1 % of total catch2
outside edge of crown 6.5 i (6.8) 41.0
center of crown 5.5 i (5.8) 34.5
between trees 3.9 i (3.2) 24.5

 

1 No significant difference between means (ANOVA, P = .05; means compared
2 using Student-Newman-Keuls multiple range test)
Total catch = 429 males

71

movements), walking outside and inside the trap, almost always accompan—
ied by wing fanning and copulatory attempts directed at the rubber sep-
tum (male curving abdomen, opening valves and probing septum with abdom-
inal tip). Due to the low light level during codling moth flight (which
usually occurred in the 30 minutes following sunset) and the males' er-
ratic and rapid flight most observations could not be initiated until
the moth was within 30 cm of the trap.

Of 57 males approaching 44 of them entered the trap. Thirty-four
of these landed, walked and fanned on the inside bottom surface. Nine
attempted copulation with the septum. Ten only fluttered within the
trap, but due to the erratic flight of the codling moth almost all of
them momentarily contacted the inside several times.

Outside the trap 8 of the 57 walked and fanned on the outer surface.
Table 15 presents the x times spent inside and outside the trap flutter-

ing, walking and fanning and attempting to copulate with the septum.

72

 

 

 

 

 

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00 we H suHB woufimn amuu cooouofim >xofluwlcoa m ou coaumuaoHuo :uoa wowHwoo mama mo moafiu,cmoz .mH oHan

DISCUSSION

Laboratory Experiments: Periodicity and Courtship

 

Skirkevicius and Tatjanskaite (1975) reported that under laboratory
conditions with 8 hours of scotophase at 20 t 1°C female codling moths
called only in darkness. The present study confirmed that most calling
is confined to that period, however, Figure 10 indicates that occasional-
ly females can be seen calling prior to and shortly following scotophase.
Skirkevicius and Tatjanskaite (1975) also found that although the phero-
mone gland is fully developed prior to emergence from the pupa, the
greatest amount of calling (62%) occurred on the third day of adult life.
Day 3 yielded the largest percentage of female calling during the pres-
ent observations (87%), but the degree of calling was statistically in-
distinguishable from that of the second day as well as the fourth to
sixth days.

Female and male codling moths maintained at 23°C under 16 hours of
photophase and 8 hours of scotophase exhibited diel periodicities for
calling and pheromone-elicited upwind orientation respectively. Through—
out the day at each observation time the level of activity for each sex
was similar: i.e., initiation of calling and a dramatic increase in
pheromone-stimulated upwind anemotaxis coincided with the onset of scoto-
phase, a high degree of each behavior was found throughout the dark per-
iod and termination of calling and a sharp decrease in response to phero-
mone occurred with the beginning of photophase.

Moths initially held under an L:D cycle and then transferred to

73

74
aperiodic light conditions continued to call (females) and move upwind
in response to pheromone (males) during a time period approximating the
8 hours in which the insects previously had experienced scotophase and
repeated this with roughly a 24 hour regularity. However, without
lights-on and lights-off cues the times of initiation and termination
did shift.

When female codling moths experienced a decrease in temperature
from 23° to 16°C during scotophase calling was terminated within 2 hours.
When the temperature decrease occurred 3 hours prior to scotophase cal-
ling began within 1 hour and ended with the onset of scotophase, and
when females were maintained at constant 16°C x calling time was shifted
5 hours earlier than x calling time of females held at 23°C. Results
with males were less clear and the decrease in temperature which promi-
nently shifted female calling period into the light appeared generally
to depress male upwind orientation to a very low level.

Most organisms in nature exhibit a diel periodicity in activity
which can be maintained in the laboratory under light and dark condi-
tions similar to the photocycle in nature. In the absence of a photocy-
cle, under continuous light or continuous darkness, many of these organ-
isms maintain their behavioral rhythm on the basis of control by an "in-
ternal clock" or endogenous oscillation. When the rhythm continues un-

der aperiodic conditions, or "free runs, the natural periodicity (T) is
often found to be slightly less or slightly more than 24 hours in length.
A 24 hour periodicity controlled by an endogenous oscillation is termed
a circadian rhythm. Prolonged maintenance under D:D or L:L will result

in a gradual shifting of the behavior out of phase from the normal L:D

cycle. If an organism with an internal clock is subjected to an

75

environmental photocycle not drastically dissimilar in length from 3' its
endogenous period will become the same as the exogenous one.

A double-oscillator model proposed by Pittendrigh and Bruce (1959)
to explain the entraining mechanism presently receives the greatest a-
mount of experimental support. The essential feature of the model is
that an "A-oscillator," termed the "driver," is an internal pacemaker
sensitive to cues (Zeitgebers) provided by the light cycle and a "B—os-

' controls expression of the behavioral e-

cillator," termed the "rhythm,'
vent and is unaffected by light cues, but is coupled to the driver which
sets it. Changes in photocycle, shifting of the Zeitgeber, instantly re-
sets the driver, but modification of the rhythm is a more gradual process
and must occur over several oscillatory cycles.

Data from the codling moth experiments dealing with calling under
continuous scotophase and photophase and upwind orientation under contin—
uous photophase indicate that the periodicities of both sexes of this in-
sect are governed by a circadian rhythm. Holloway and Smith (1976) meas—

uring the locomotor activity of the adult lesser cornstalk borer, Elasmo-

palpus lignosellus (Zeller), under continuous scotophase found it to re-

 

cur with a 22 hour period. Each day peak locomotion occurred 2 hours
earlier than the previous day. In both male and female codling moths'T
appears to be close to 24 hours because no discernible shift was evi-
denced in i calling time or i pheromone response time under aperiodic
conditions. The abrupt transition from no calling and little upwind o-
rientation to a high degree of both and vice versa when lights-off and
lights-on cues were present indicates that one or both of those cues
probably acts as a Zeitgeber.

Endogenous oscillators are temperature-compensated. Generally the

76

Q10 value (a measure of the increase in 7' with a decrease in tempera-
ture: ’r at (t-10)°/'T at to) of organisms studied is slightly greater
than 1.0. This indicates that for a wide range of biological tempera-
tures 'T changes very little. But it has been demonstrated that temper-
ature changes can modify behaviors which under constant temperature rely
on photocycle entrainment. Most demonstrations of this have utilized
Lepidoptera (Cardé and Roelofs 1973, Sower et al. 1971 and Cardé et al.
1975) and indicated that decreases in temperature can shift female cal-
ling and male response to pheromone earlier into the day. Pittendrigh

(1954), measuring the periodicity of Drosophila pupal emergence, found

 

that following a decrease in temperature only the first peak of pupal e-
mergence was affected, and that if the temperature was returned to that
experienced previously the system reverted back to its original periodi-
city. No detailed explanation for the effects of temperature altera-
tions has been proposed, but it is believed that the B-oscillator may be
the one sensitive to temperature pulses and cycles (Saunders 1976).

Data from timing pheromone trap catches of male codling moths (Ba—
tiste et al. 1973a) indicated that males fly regularly at sunset, but
that temperature may modify flight in that catches are greater earlier
in the day during cool days than warm ones. Field flight observations
by Borden (1931) and Fluri et al. (1974) purportedly lend evidence to
this, though it is unclear as to whether or not they were in fact obser-
ving males in the orchard. Male and female codling moths appear almost
indistinguishable externally, even at rest, and neither author reports
determination of the sex of the moths in flight. Field observations of
periodicity are further complicated by reports (Mani et al. 1974) that

codling moths may exhibit 2 daily flight periods — one in which males

77

are captured in pheromone traps in great numbers and an earlier flight
in which few or no captures occur.

Observations by Mani et al. (1974) suggest that temperature at time
of flight may not necessarily be the only modifying factor. The temper-
ature several hours prior to sunset may play a role as well as present
ambient temperature. Increases and decreases in temperature and sudden
cloud cover may be responsible for early or late codling moth flights.
During a hot (28°C) afternoon and a warm (21°C) evening no pheromone
trap catch occurred prior to sunset, but during the hour following sun-
set a large number of moths were caught. With a warm (21°C) afternoon
and a cool (16°C) evening the largest catch again occurred during the
hour following sunset, but the remainder of the males were caught during
the 3 hours prior to sunset. Cloud cover and mild rain during a warm af-
ternoon and evening appeared to have no effect on trap catch period: it
still was maximal during the hour after sunset. Cloud cover combined
with a decrease in temperature (imminent storm conditions) resulted in
the majority of moths being caught 2.5-1.5 hours prior to sunset, and
very few after sunset.

Clearly a drawback to field observations is the inability to sepa-
rate the effects of several different variables on the behavior of a sys-
tem. In the last observation of Mani et al. (1974) described above it is
unclear whether the dramatic shift in male flight was caused by low af—
ternoon temperature (20°C), a decrease in temperature (early afternoon
temperature was not specified), decrease in light level due to clouds,
any combination of the above or yet additional factors. In the labora—
tory, when temperature was decreased to 16°C the low number of males ori-

enting upwind in the olfactometer would lend little support to the

78

hypothesis that low temperature or temperature decrease is the modifying
influence. The fact that decreasing light intensity from 1500 to 150

lux in the laboratory yielded a significant increase in degree of anemo-
taxis may indicate that the degree of cloud cover could play a signifi-
cant role. Mani et al. (1974) also noted that when the temperature rose

' moths were caught in the

to 15°C after a "long period of bad weather,‘
traps, whereas, when the temperature dropped to 15°C after "nice warm
weather" moths were never caught. Thus, the effect of temperature on
codling moth mating periodicity may be complex, accounting for male de—
crease in activity with a decrease in temperature from 23° to 16°C.
Another perplexing aspect of codling moth periodicity is the con-
trast between the very similar calling period of females and pheromone
response period of males at 23°C and at 16°C the very apparent shift of

calling into photophase with no shift exhibited by males under the same

conditions. Working with Antheraea pernyi Guerin-Meneville Truman (1973)

 

reported that pupae reared at 12°C produced adult females that called and
adult males that exhibited flight 1 hour into scotophase when held at
25°C. This was in contrast to adults held at 25°C after emerging from
pupae also maintained at 25°C, which called and flew 5 hours into scoto-
phase. He concluded that periodicity entrainment relied on conditions
experienced during the pupal stage and was unaffected by temperature

changes occurring during adult life. Cardé et al. (1975), however, dem-

onstrating that female calling and male pheromone response of Argyrotae-

 

nia velutinana Walker could be immediately shifted by a change in temp-

 

erature, argued that temperature conditions experienced by the adult play
a critical role in periodicity of behavior. Female codling moths clearly

appear to fit the model proposed by Cardé et al. (1975), but males may not.

79

At 23°C 4 hours prior to scotophase calling and upwind orientation
are at low levels (((30%), and at 2 hours prior to scotophase calling is
still below 30%, while upwind orientation is approximately at 30%. One
hour into scotophase calling and upwind orientation are exhibited by
greater than 60% of the moths. When the temperature is decreased to
16°C 3 hours before lights—off calling increases to ) 70% during the re-
mainder of photophase, but pheromone responsiveness remains at 30%. Both
behaviors are terminated with scotophase. At 16°C it may be energetical-
ly too expensive for an increase in male activity to occur, but calling
probably requires less metabolic energy than upwind flight. Therefore,
when the temperature is lowered to 16°C, it might be advantageous repro-
ductively and energetically inexpensive enough for an increased number of
females to call during photophase, thereby increasing the chances of at-
tracting those males which are still capable of upwind flight and mating.
On the basis of the observations by Mani et al. (1974) and the results of
the present experiments, it appears that bioassays under several tempera-
ture and light regimes, as well as tests of moths reared under several
sets of environmental conditions, may be needed before the roles of ambi-
ent temperature and temperature changes on mating periodicity can be re-
solved.

Very few detailed studies of moth sexual behavior, especially court-
ship, have been undertaken. In one such study, however, Grant and Brady
(1975) described the behavioral sequences which occurred during court-

ship of the Indian meal moth, Plodia interpunctella (Hfibner), and the al-

 

mond moth, Cadra cautella (Walker). They reported that in both species

 

several steps of alternating male and female behavior led to copulation.

Basically a male was attracted to the pheromone of a calling female, he

80

approached and released an aphrodisiac which enabled him to move into a
position with his head beneath the female's head, she remained station-
ary and turned up her abdomen in an acceptance posture and copulation
quickly followed.

Similarities between a number of the steps during Indian meal moth
and almond moth courtship indicated a possibility of interspecific court-
ship taking place, but successful cross-mating was not found to occur
(Grant et al. 1975) and this was attributed in part to differences in di-
rection of male approach, response of female to initial male contact be-
ing different between the species and the possible existence of species—
specific male aphrodisiacs.

As indicated in Figure 21, during successful mating of the codling
moth, it appeared that a number of different behavioral pathways were
followed by females. The most commonly observed sequence consisted of
resting, calling, raising wings at male approach, raising wings higher
and walking forward during abdominal probing, then lowering wings after
sexual coupling. The path followed by the smallest number of females
was resting (no calling visible), wing-raising at contact with male head,
raising wings higher as male moved along female body, 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 diagram occurred and the degree to which each was seen
can be calculated by tracing the path from each behavioral state to the
next (the path designates the percentage of moths moving from one state
to the one connected to it).

Certain behavioral modes ("raise wings" and "walk forward") appear

more than once in Figure 21. There are 2 reasons for this: 1) some of

81

the females engaged in an activity in response to a particular stimulus
(i.e., 42% of the females raised wings at male approach) while others
exhibited that same behavior only in response to a different cue (12%
did not raise wings for the first time until male head contact); 2) al-
so, some females responded to more than one stimulus with the same beha-
vior (42% of the females raised their wings at male approach, 21% then
raised them higher during male head contact and 3% appeared to elevate
them a little more as the male began moving posteriorly along the female
body while curving his abdomen toward her).

Analyzing the movement from each behavioral event to the next in a
manner similar to that employed by Grant and Brady (1975) it can be in-
terpreted that each behavior undertaken by the male is a releaser for e—
liciting the activity exhibited next in the female sequence. However,
some or many events may not appear in a single female's repertoire: 3%
of the females exhibit all but 3 of the behavioral states, while 9% of
the females pass through only 3 of the behavioral states (resting to
wing-raising in response to abdominal probing, followed by wing-lowering
after the male grasps the female genitalia with his valves). Therefore,
since a large amount of variation appears in which behaviors a particu-
lar female undertakes, and little or no variation is observed in the male
sequence, female calling and male contact with the female may be the only
releasers of the male behaviors. No matter whether the female exhibits
only calling during the time the male approaches, makes head contact,
moves along her body, curves his abdomen and begins abdominal probing, or
she engages in calling and 3 series of wing—raising, the male behavioral
sequence remains the same.

Hutt and White (1977) reported on the basis of laboratory experi-

ments with blinded, antennectomized, blinded and antennectomized and

82

unaltered male codling moths that vision apparently plays a part in suc-
cessful mating. The present study supports this hypothesis with evi-
dence that, although the presence of a visual cue does not affect the a-
mount of time spent in orientation, it does significantly affect where
the moth orients. A male will spend a greater amount of time walking
and fanning near and attempting to copulate with a visual cue than it
will at a pheromone source to which it initially oriented during its up-

wind flight. Shorey and Gaston (1970) found that when dried Trichpplu-

 

sis_pi_(Hfibner) females or black paper silhouette models were positioned
2 cm away from a pheromone source the presence of a visual cue, as in
the codling moth, did not influence the frequency of orientation, but
did influence both the frequency and direction of copulatory attempts.

Field Experiments: Male Orientation to_Pheromone Traps

 

 

Borden (1931) and Fluri et al. (1974) reported that during the eve-
ning male codling moths were found to swarm in the upper parts of trees.
Mani et al. (1974) observed cases of codling moth orientation to conspi—
cuous silhouettes of non-host as well as host plants. As reported ear-
lier, though, none of the authors provide evidence for positive identi-
fication of the sex in flight. Pheromone-activated gypsy moth males,

Lymantria dispar L., "investigated" vertical silhouettes such as tree

 

trunks and it was demonstrated that trap placement near large vertical
silhouettes (trees 0.5 m in diameter) could significantly increase trap
catches over those hung on small trees (3-8 cm in diameter (Cardé et al.
1977).

The results presented in Table 14 indicate that an orientation to
large objects exhibited by some codling moths does not affect male at-

traction to traps. No significant difference was discernible between

83

male catches within the tree crown, at the branch tips or at traps sus-

pended in the open between trees. Therefore (at least at the population
level present during this study) codling moth pheromone trapping appears
to be unaffected by trap placement.

Presently all pheromone traps commonly used for codling moth moni—
toring rely on a sticky trap inner surface to catch the moths. This de-
sign has proven sufficient for the most part, but nevertheless, has cer—
tain characteristics which make it less than ideal. Moths entering the
trap must come into direct contact with the glue to be captured. Those
that do contact the glue are not always retained because moth scales from
previously captured insects can accumulate and reduce the glue's sticki-
ness. The capture potential of a trap is dependent upon the surface area
of the glue, and at high population levels or over a long period of time
in a lesser infestation it is possible for that surface to become over-
loaded with moths with each moth causing a progressive reduction in trap-
ping ability. Sticky traps can be messy during normal servicing, and
those insects removed are often in very poor condition and unsuitable for
subsequent inspection or study. One of the greatest values of the phero-
mone trap supposedly lies in its specificity of capture. Personnel with
little taxonomic background can responsibly monitor pest insects because
the bait in the trap will only attract the species of interest. However,
it is very uncommon for a sticky pheromone trap, especially hung within
foliage, to not collect a large assemblage of various insects that have
accidentally come into contact with the glue.

The Granett-type trap (Granett 1973) alleviates a number of these
problems. Since access to the trap occurs only at a small port of entry

at each end, though all unwanted insects are not kept out, the number is

84

reduced considerably because orientation must be more precise or persis-
tent than with traps having larger openings. Vapona fumes kill the en—
tering moths, therefore, effective capture potential is a function of
trap volume, rather than surface area, and dead insects or moth scales
do nothing to decrease trap efficiency (the trap used in this test could
hold several thousand dead codling moths without overloading). During
insect count and removal and bait replacement there is no glue present
to get on the hands or clothes of the trap technician, and the dead
specimens are usually in the condition in which they entered the trap.

Results from the comparison of Pherocon trap catch and Granett trap
catch during this study yield no significant difference between the two,
even though of 343 moths caught 74% were in the Pherocon and 26% in the
Granett traps (much variation between replicates accounting for the lack
of significance). Duration of trapping was 28 days and throughout this
period the same Pherocon traps were used. Replacement of the sticky
traps may have increased catch somewhat (Westigard and Graves 1976), but
policy during other monitoring projects was typically to replace traps
at 1 to 2 month intervals (Myburgh et al. 1974 and Proverbs et al. 1975).
The Granett trap model used in this test was developed for the much lar-
ger gypsy moth and could conceivably be improved for capture of codling
moths and other small Lepidoptera. Further testing of Granett-type traps
with modifications would seem valuable in an attempt to develop a more
efficient non-sticky pheromone trap.

Reports of trap "efficiency" in the past have been developed from
comparisons of trap catch. Though trap catch is valuable, a more direct
measurement may be obtained from observations of males approaching a

trap in the orchard. During this study, of 57 males observed at a

85

non-sticky Pherocon trap 34 of these entered the trap and walked and
fanned inside. If the assumption could be made that in a sticky trap
each one of these would have been caught (2 time spent walking and fan-
ning was 10.8 sec) an efficiency of 59.6% results. Ten others fluttered
within the trap (x time = 6.1 sec) but due to the erratic nature of cod-
ling moth flight almost all of them momentarily contacted the inside
surface several times. This could possibly raise efficiency to an upper
level of 77.2%. Extrapolation of data in a non-sticky trap to that of a
sticky one may not be valid, but since a sticky trap would continually
lose effectiveness with age and each time an insect was caught, true ef-
ficiency is probably a dynamic value of which any measure would be an

approximation for limited conditions.

LI ST OF REFERENCES

LIST OF REFERENCES

Arn, H., C. Schwarz, H. Limacher and G. Mani. 1974. Sex attractant in-

hibitors of the codling moth Laspeyresia pomonella L. Experientia
30: 1142-4.

 

Audemard, H., FranCoise Beauvais and C. Descoins. 1977. Control of the
codling moth (Laspeyresia pomonella L.) with a synthetic pheromone
by the "male disruption" method: a first trial in a commercial ap-
ple orchard. Rev. de 2001. Agric. 76: 15-24.

 

Barnes, M.M., D.M. Peterson and J.J. O'Connor. 1966. Sex pheromone gland
in the female codling moth, Carpocepsa pomonella (Lepidoptera: Ole-
threutidae). Ann. Entomol. Soc. Amer. 59(4): 732-4.

 

Batiste, William C. 1970. A timing sex-pheromone trap with special ref-
erence to codling moth collections. J. Econ. Entomol. 63(3): 915-18.

Batiste, William C. and John Joos. 1972. Codling moth: a new pheromone
trap. J. Econ. Entomol. 65(6): 1741-2.

Batiste, William C., William H. Olson and Arthur Berlowitz. 1973a. Codling
moth: influence of temperature and daylight intensity on periodicity
of daily flight in the field. J. Econ. Entomol. 66(4): 883-92.

Batiste, William C., William H. Olson and Arthur Berlowitz. 1973b. Codling
moth: diel periodicity of catch in synthetic sex attractant vs. fe-
male-baited traps. Environ. Entomol. 2(4): 673-6.

Beroza, M., B.A. Bierl and H.R. Moffitt. 1974. Sex pheromones: (§,E)-8,10-
dodecadien-l-ol in the codling moth. Science 183: 89-90.

Borden, Arthur D. 1931. Some field observations on codling moth behavior.
J. Econ. Entomol. 24(6): 1137-45.

Buser, Hans-Rudolf and Heinrich Arn. 1975. Analysis of insect pheromones
by quadrupole mass fragmentagraphy and high resolution gas chromato-
graphy. J. Chromat. 106: 83-95.

Butt, B.A., Morton Beroza, T.P. McGovern and S.K. Freeman. 1968. Synthet-
ic chemical sex stimulants for the codling moth. J. Econ. Entomol.
61(2): 570-2.

Butt, B.A. and D.O. Hathaway. 1966. Female sex pheromone as attractant
for male codling moths. J. Econ. Entomol. 59(2): 476-7.

86

87

Butt, B.A., Terrence P. McGovern, Morton Beroza and D.O. Hathaway. 1974.
Codling moth: cage and field evaluations of traps baited with a
synthetic sex attractant. J. Econ. Entomol. 67(1): 37-40.

Cardé, R.T., T.C. Baker and P.J. Castrovillo. Disruption of sexual com-
munication in Laspeyresia pomonella (codling moth), Grapholitha mo-
lesta (Oriental fruit moth) and g; prunivora (lesser appleworm)
with hollow fiber attractant sources. Entomol. Exp. et App1.: in
press.

 

 

Cardé, R.T., A. Comeau, T.C. Baker and W.L. Roelofs. 1975. Moth mating
periodicity: temperature regulates the circadian gate. Experientia
15(1): 46-8.

Cardé, R.T., C.C. Doane, J. Granett, A.S. Hill, J. Kochansky and W.L.
Roelofs. 1977. Attractancy of racemic disparlure and certain ana-
logues to male gypsy moths and the effect of trap placement. En-
viron. Entomol.: in press.

Cardé, R.T. and W.L. Roelofs. 1973. Temperature modification of male sex
pheromone response and factors affecting female calling in Holomelina
immaculata (Lepidoptera: Arctiidae). Can. Entomol. 105: 1505-12.

 

 

Ferro, David N. and Robert F. Harwood. 1973. Intraspecific larval compe-
tition by the codling moth, Laspeyresia pomonella. Environ. Entomol.
2(5): 783-9.

 

Fluri, Peter, Erwin Mani, Theodor Wildbolz and Heinrich Arn. 1974. Unter-
suchungen uber das paarungsverhalten des apfelwicklers (Laspeyresia
pomonella L.) und uber den einfluss von kunstlichen sexuallockstoff
auf die kopulation shaufigkeit. Mitt. Schweiz. Ent. Ges. 47:253-9.

 

Gehring, R.D. and Harold Madsen. 1963. Some aspects of the mating and o-
viposition behavior of the codling moth, Carpocapsa pomonella. J.
Econ. Entomol. 56(2): 140-3.

 

Geier, P.W. 1967. Two life systems of Cydia pomonella (Tortricidae) in
The Ecology of_Insect Populations ip_Theory and Practice. L.R. Clark,
P.W. Geier, R.D. Hughes and R.F. Morris ed. Methuen, London.

 

 

 

 

George, D.A., L.M. McDonough, D.O. Hathaway and H.R. Moffitt. 1975. In-
hibitors of sexual attraction of male codling moths. Environ. Ento-

Glenn, Presley A. 1922. Codling moth investigations of the state entomo-
logist's office. Illinois Natural History Survey, Vol. XIV: Article
7.

Granett, J. 1973. A disparlure-baited box trap for capturing large numbers
of gypsy moths. J. Econ. Entomol. 66: 359-62.

Grant, C.C and V.E. Brady. 1975. Courtship behavior of phycitid moths. I.
comparison of Plodia interpunctella and Cadra cautella and role of
male scent glands. Can J. Zool. 53: 813-26.

 

 

88

Grant, C.C., E.B. Smithwick and V.E. Brady. 1975. Courtship behavior of
phycitid moths. II. behavioral and pheromonal isolation of Plodia
interpunctella and Cadra cautella in the laboratory. Can. J. Zool.
53: 827-32.

 

 

Hathaway, D.O., T.P. McGovern, M. Beroza, H.R. Moffitt, L.M. McDonough
and B.A. Butt. 1974. An inhibitor of sexual attraction of male cod—
ling moths to a synthetic sex pheromone and virgin females in a
trap. Environ. Entomol. 3(3): 522-4.

Holloway, Rodney L. and J.W. Smith, Jr. 1976. Free-running and phase-set—
ting of locomotor behavior of the adult lesser cornstalk borer. Ann.
Entomol. Soc. Amer. 69(5): 848-50.

Howard, L.O. 1887. The codling moth. Rep. Commissioner Agric.: 88-115.

Howell, J Franklin. 1972. An improved sex attractant trap for codling
moths. J. Econ. Entomol. 65(2): 609-11.

Howell, J Franklin. 1974. The competitive effect of field populations of
codling moths on sex attractant trap efficiency. Environ. Entomol.
3(5): 803-7.

Howell, J Franklin and Kathleen D. Thorp. 1972. Influence of mating on
attractiveness of female codling moths. Environ. Entomol. 1(1):
125-6.

Hudson, W.B. and D.E. Johnson. 1971. Use of sex attractant traps for tim-
ing codling moth sprays. Wash. State Hort. Assoc. Proc. 66: 81-6.

Hutt, R.B. and L.D. White. 1977. Mating response to visual stimulus in
the male codling moth. Environ. Entomol. 6(4): 567-8.

MacLellan, C.R. 1976. Suppression of codling moth (Lepidoptera: Olethreu-

tidae) by sex pheromone trapping of males. Can. Entomol. 108: 1037-
40.

Madsen, H.F. and W.W. Davis. 1971. A progress report on the use of female—
baited traps as indicators of codling moth populations. J. Entomol.
Soc. Brit. Columbia 68: 11—14.

Madsen, H.F. and J.M. Vakenti. 1973. Codling moth: use of codlemone-baited
traps and visual detection of entries to determine need of sprays.
Environ. Entomol. 2: 677-9.

Maitlen, J.C., L.M. McDonough, H.R. Moffitt and D.A. George. 1976. Codling
moth sex pheromone: baits for mass trapping and population survey.
Environ. Entomol. 5(1): 199-202.

Mani, Erwin, Walter Riggenbach and Milan Mendik. 1974. Diurnal periodicity
of moth captures and observations on the flight activity of codling
moth (Laspeyresia pomonella L.). Mitt. Schw. Entomol. Gesell. 47:

39-48.

 

89

Mani, E. and T. Wildbolz. 1975. Ueber den einsatz der pheromonfalle in
der apfelwicklerprognose. Schweiz. Z. Obstund. Weinb. 14: 351-60.

McDonough, Leslie M., Donald A. George and B.A. Butt. 1972. Sex phero-
mone of the codling moth: structure and synthesis. Science 177:
177-8.

McDonough, L.M. and H.R. Moffitt. 1974. Sex pheromone of the codling
moth. Science 183: 978.

Myburgh, A.C., H.F. Madsen, I.P. Bosman and D.J. Rust. 1974. Codling
moth (Lepidoptera: Olethreutidae): studies in the placement of
sex attractant traps in South African orchards. Phytophylactica
6: 189-94.

Norris, K.H., F. Howell, D.K. Hayes, V.E. Adler, W.N. Sullivan and M.S.
Schechter. 1969. The action spectrum for breaking diapause in the
codling moth, Laspeyresia pomonella L. and the oak silkworm, Ap-
theraea pernyi Guer. Proc. Natn. Acad. Sci. U.S.A. 63: 1120-7.

 

 

Parrot, P.J. and Donald L. Collins. 1934. Phototropic responses of the
codling moth. J. Econ. Entomol. 27(2): 370-9.

Peterson, D.M. 1965. A quick method for sex determination of codling
moth pupae. J. Econ. Entomol. 58: 576.

Pittendrigh, C.S. 1954. On temperature incidence in the clock system
controlling emergence time in Drosophila. Proc. Nat. Acad. Sci.

U.S.A. 40: 1018-29.

 

Pittendrigh, C.S. and V.G. Bruce. 1959. Daily rhythms as coupled oscil-
lation systems and their relation to thermoperiodism and photo-
periodism in Photoperiodism and Related Phenomena ip_Plants and
Animals. R.B. Withrow ed. Am. Ass. Adv. Sci. Washington.

 

 

Proverbs, M.D. 1965. The sterile male technique for codling moth con-
trol: 10 years of progress. West. Fruit Grower 19(4): 10-20.

Proverbs, M.D., D.M. Logan and J.R. Newton. 1975. A study to suppress
codling moth (Lepidoptera: Olethreutidae) with sex pheromone traps.
Can. Entomol. 107: 1265-9.

Putnam, W.L. 1963. The codling moth, Carpocapsa pomonella (L.) (Lepi-
doptera: Tortricidae): a review with special reference to Ontario.
Proc. Entomol. Soc. Ont. 93: 22-60.

 

Riedl, Helmut, B.A. Croft and A.J. Howitt. 1976. Forecasting codling
moth phenology based on pheromone trap catches and physiological
time models. Can. Entomol. 108(5): 449-60.

Roelofs, W.L., R.J. Bartell, A.S. Hill, R.T. Cardé and L.H. Waters. 1972.
Codling moth sex attractant - field trials with geometrical isomers.
J. Econ. Entomol. 65: 1276—7.

90

Roelofs, W.L. and R.T. Cardé. 1977. Response of Lepidoptera to synthet-
ic sex pheromone chemicals and their analogues. Ann. Rev. Entomol.
22: 377-405.

Roelofs, Wendell, Andre Comeau, Ada Hill and G. Milicevic. 1971. Sex at-
tractant of the codling moth: characterization with electroantenno—

gram technique. Science 174: 297-9.

Saunders, David S. 1976. Insect Clocks. Pergammon Press, New York.

 

Shorey, H.H. 1970. Sex pheromones of Lepidoptera in Control of_Insect
Behavior py_Natural Products. D.L. Wood, R.M. Silverstein and M.
Nakajima, ed. Academic Press, New York.

 

Shorey, H.H. and Lyle K. Gaston. 1964. Sex pheromones of noctuid moths:
III. inhibition of male responses to the sex pheromone in Tricho-
plusia pi_(Lepidoptera: Noctuidae). Ann. Entomol. Soc. Amer. 57:
775-9.

Shorey, H.H. and Lyle K. Gaston. 1970. Sex pheromones of noctuid moths:
XX. short-range visual orientation by pheromone-stimulated males of
Trichoplusia pi, Ann. Entomol. Soc. Amer. 63(3): 829-32.

 

Skirkevicius, A. and L. Tatjanskaite. 1975. Secretion of pheromone by
the female codling moth Carpocapsa pomonella L. Insect Chemorecep-
tion No. 2; Proc. II All-Union Symp. on Insect Chemoreception.

 

Solomon, M.E. 1951. The control of humidity with potassium hydroxide,
sulfuric acid or other solutions. Bull. Entomol. Res. 42: 543-59.

Sower, L.L., H.H. Shorey and L.K. Gaston. 1971. Sex pheromones of noctu-
id moths: XXV. effects of temperature and photoperiod on circadian
rhythms of sex pheromone release by females of Trichoplusia pi.
Ann. Entomol. Soc. Amer. 64: 488-92.

 

Sower, L.L., K.W. Vick and J.S. Long. 1973. Isolation and preliminary
biological studies of the female-produced sex pheromone of Sito-
troga cerealella (LepidOptera: Gelechiidae). Ann. Entomol. Soc.
Amer. 66(1): 184-7.

 

Tamaki, Yoshio. 1977. Complexity, diversity and specificity of behavior-
modifying chemicals in Lepidoptera and Diptera in Chemical Control
lo£_Insect Behavior: Theory and Application. H.H. Shorey and John J.
McKelvey, Jr., ed. John Wiley and Sons, New York.

 

 

Truman, James W. 1973. Temperature sensitive programming of the silkmoth
flight clock: a mechanism for adapting to the seasons. Science 182:
727-9.

Westigard, P.H. and K.L. Graves. 1976. Evaluation of pheromone baited
traps in a pest management program on pears for codling moth con—
trol. Can. Entomol. 108: 379-82.

91

Wong, Tim Y., M.L. Cleveland, D.F. Ralston and D.G. Davis. 1971. Time
of sexual activity of codling moths in the field. J. Econ. Entomol.
64(2): 553-4.

Worthley, Harlan N. 1932. Studies of codling moth flight. J. Econ. Ent-
omol. 25(3): 559-65.

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