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Photoperiod cues and the modulatory action of octopamine and 5-hydroxytryptamine on locomotor and pheromone response in male gypsy moths Lymantria dispar.

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Archives of Insect Biochemistry and Physiology 20:265-284 (1992)
Photoperiod Cues and the Modulatory Action
of Octopamine and 5-Hydroxytryptamine on
Locomotor and Pheromone Response in Male
Gypsy Moths, Lymantria dispar
Charles E. Linn, Jr., Marlene G. Campbell, and Wendell L. Roelofs
Department of Entomology, New York State Agricultural Experiment Station, Cornell University,
Geneva, New York
Experiments were conducted to determine whether the biogenic amines
octopamine (OA) and 5-hydroxytryptamine (5-HT) exert modulatory effects
on pheromone responsivenessand random locomotor activity in male gypsy
moths. When injected into males, OA significantly enhanced sensitivity to
pheromone, while 5-HT enhanced general locomotor activity, results that were
very similar to those previously shown for the cabbage looper. Maximal effect
of the amines, however, was observed when injection occurred just prior to
the onset of scotophase, rather than photophase, as we had originally hypothesized for this diurnallyactive insect. Male gypsy moths also displayed a prominent scotophase response, with sensitivity to pheromone greater in the
scotophase compared with photophase, but with the level of random locomotor activity lower in scotophase than in photophase. The upwind flight
behavior of males to a pheromone source in a wind tunnel, as well as the
time spent at the source, were also significantly different in the two light
regimes. Furthermore, when exposed to a 1 h scotophase (insteadof the normal 8), or to continuous dark conditions, while males exhibited response to
pheromone and locomotor activity during the same scotophase and photophase periods as observed in a 16:8 1ight:dark cycle, the levels of response,
as well as qualitative aspects of the upwind flight behaviors in both periods
were a function of the light intensity, Our combined results suggest that male
gypsy moths display a bimodal rhythm of locomotor and pheromone response
over the die1 cycle, with light intensity and scotophase onset providing critical cues for the expression of behaviors, as well as the modulatory action of
the amines. GI 1992WiIey-Liss, inc.
Acknowledgments: We thank Kathy Poole for help in rearing the insects, and Geoffrey Rule
for preparation of the pheromone standards. We are also grateful to C. P. Schwalbe (Otis USDAAPHIS Methods Development Center) for supplying the insects. We are especially rateful to
Drs. Ralph Chartton and Jeremy McNeil, as well as an anonymous reviewer, for tieir thorough and critical comments. This study was supported by a USDA Competitive Research Grants
Program (Pest Science 621367).
Received October 15,1991; accepted April 21,1992.
Address reprint requests to Dr. Charles E. Linn, Jr., Department of Entomology, New York State
Agricultural Experiment Station, Cornell University, Geneva, NY 14456.
0 1992 Wiley-Liss, Inc.
266
Linn et al.
Key words: biogenicamines, circadian rhythms, flight tunnel, Lepidoptera, Lymantriidae,mating
behavior, neuromodulation
INTRODUCTION
The sex-pheromone-mediated mating behavior of many moths occurs during
a characteristic time of the photoperiod [l]. Effective mate location and syngamy thus require the temporal coordination of female production of the chemical signal and male response over time. A number of studies have suggested
that the periodicity of mating activity is governed by a circadian osdator, which
is entrained by photoperiod cues. Temperature often influences the precise
timing and duration of the activity window, advancing or delaying the onset of
mating activity and thus allowing for reproductive activities to occur in the
most favorable environmental conditions [2-51.
The importance of exogenous factors in controlling the expression of rhythmic behaviors raises the question of how information from these cues is incorporated into the physiological processes involved in the release of specific
behaviors [6].One route that has received much attention concerns the action
of endogenous hormonal and neuromodulatory factors in linking photosensitive elements of the nervous system, and other neural or biochemical pathways involved in releasing a coordinated behavior pattern [7,8]. In moth
is involved in female
communication systems, for example, a peptide, I?€"*,
production of the pheromone in of a number of species [9]. One hypothesis
currently being tested is that PBAN is released from the brain into the hemolymph, and then acts on the pheromone gland to initiate pheromone biosynthesis. It is proposed that the release of P U N is associated with the onset of
scotophase, thus coordinating the time of synthesis with the time when mating activity occurs [lo].
In males a similar hormonal, or neuromodulatory, agent has not definitively
been identified. However, in the nocturnally active cabbage looper moth,
Trichoplusia ni (Hiibner), we showed that injection of the biogenic amines OA
and 5-HT significantly altered the behavioral thresholds for random activity
and sensitivity to pheromone in the scotophase [ll]. We hypothesized that
these compounds could be involved in pathways linking a photoperiodically
entrained circadian oscillator with pathways involved in controlling locomotor activity and response to pheromone [121.
The gypsy moth, Lyrnantria dispar, is a diurnal insect whose mating activity
in eastern North America occurs during the summer months of July and August
[13]. Field-trapping and laboratory-actograph studies have shown that males
are generally quiescent in the early hours of photophase, becoming increasingly active and responsive to pheromone in the middle of the photoperiod,
with activity decreasing at the end of the photophase [2,14]. Males have been
reported to exhibit a crepuscular flight period, which apparently is tempera*Abbreviations used: D:D = continuous dark; L:D = 1ight:dark; L:L = continuous light; OA
= octoparnine; PBAN = pheromone biosynthesis activating neuropeptide; RH = relative
humidity; 5-HT = 5-hydroxytryptamine.
PhotoperiodCues and Biogenic Amines in Male Gypsy Moths
267
ture dependent but not related to mating activity [15,16]. We selected L. dispur
to conduct a comparative study on the effects of biogenic amines on male
response thresholds, and report here the results of a series of experiments to
determine the relationship between photoperiodic cues and the neuromodulatory role of biogenic amines on behavioral thresholds for random activity
and response to pheromone in this day-active moth.
MATERIALS AND METHODS
Insects
Egg masses were obtained from the USDA-APHIS Methods Development
Center, Otis Air National Guard Base, MA. Larvae were reared on wheat germ
diet [17] under a 1623 L:D photoperiod at 25 k 1"C, 60-70% RH. Male pupae
and adults were maintained in a separate environmental chamber under the
same conditions as those during rearing. Upon emergence adults were held
in screened cages (25 x 30 x 40 cm), with no more than 30 males in a box [W].
In our rearing regime the majority of males emerged during the first half of
the photophase, so they were segregated daily at the mid-point of the photophase, and this was designated as day 1. These males then remained in the
environmental chamber and were tested (see below) during the subsequent
photophase periods (designated as day 2 or day 3), or during their first adult
scotophase.
Chemicals
The ( + ) enantiomer of disparlure (from the synthesis reported by Mori et
al. [19]) was free of the trans isomer, but contained 7.7% of an unknown compound. A dilution series was prepared in hexane and 100 pl amounts applied
to red rubber septa (Thomas Scientific, Philadelphia, PA) to achieve the desired
doses (0.1, 1, 10,100 ng; 1and 10 Fg). Septa were evaporated in a hood for 24
h, and stored in 4-dram vials at - 10°C when not in use. Fresh sources were
prepared at 4-week intervals over the course of these studies.
OA (HCl) and 5-HT (creatinine sulfate complex) were obtained from Sigma
(St. Louis, MO). These chemicals were dissolved in 145 mM NaCl, and dilution series (0,3,10,30, 100,300, and 1,000 Fg/gbody weight) were prepared
fresh daily. The dosages of OA and 5-HT are expressed as microgram injected
per gram insect, with the average weight of male L. dispur selected randomly
over the course of these studies equal to 0.427 2 -038g (mean & S.D.; n = 100).
Procedure for Injecting Insects
The injection of OA or 5-HT was made via microcapillary pipettes whose
tips were drawn out to fine points. Males were gently held by the wings and 1
p1 of solution injected through the membraneous region at the posterior part
of the head capsule, onto the dorsal portion of the protocerebrum. Each injection took less than 15 s. Controls were comprised of untreated and salineinjected insects. The response levels of the 2 control groups were compared
by ANOVA and regression analysis to determine if there were any effects due
to handling or saline injection. Control values reported in results below refer
to saline-treated insects.
268
Linn et al.
Flight Tunnel and Testing Protocol
The flight response of individual males to a pheromone source was observed
in the flight tunnel of Miller and Roelofs [20]. Photophase and scotophase light
intensities were 300 and 0.3 lux, respectively, and the air speed was 45-50 c d s .
Temperature and RH were maintained at 25 k 0.5"C, 60-70%.
For each experiment males were taken from the holding chamber to the controlled environmental chamber housing the flight tunnel at least 1 h prior to
injection of OA or 5-HT and the initiation of behavioral observations so that
they could acclimate to the flight tunnel environment. Following injection, or
in those cases when no treatment was administered, males were placed in
individual 10 X 5 cm screen cages on a Plexiglas platform at the side of the
flight tunnel.
Analyses were made of two different behaviors. First, random locomotor
activity was scored if a male exhibited sustained walking, wing-fanning, or
flight in the release cage for a period of at least 5 min. Second, for response to
a pheromone source three key behaviors in the flight tunnel sequence [18]
were scored: wing-fanning activation in the release cage, taking flight, and
source contact after flying the 1.5 m distance. In addition to scoring the behaviors, the time taken for each behavior to occur was also recorded using a minicomputer. For activation this refers to the latency to initiate wing-fanning from
a quiescent position, for upwind flight to the time taken to fly the 1.5 m distance, and for source contacts to the time actually spent at the source. All
measurements were made during the first 30 min into each hour indicated for
each experiment.
Effect of age on photophase response. To measure random activity 60 males
(30 day 2 and 30 day 3) were set up each day during the first hour of photophase
and at 1 h intervals over the ensuing 16 h photophase the proportion active
was noted. This procedure was replicated 5 times (N = 150).
The response of males to a series of pheromone doses was measured by
placing 60 males (30 day 2 and 30 day 3) in the room housing the flight tunnel
at lights on and during the seventh and eighth hours of photophase five males
from each age group were tested to one of five doses of pheromone (0.1, 1,
10, 100 ng; 1 and 10 pg). This mid-photophase interval represents the time
when maximal male activity normally occurs [2]. Replicates were conducted
until 80 males had been tested to each dose. In a second test 60 males were
set up each day as described above and at 2 h intervals through the ensuing
16 h photophase 5 males were tested to a 1 kg source of pheromone. Because
of the large number of insects required for this test, the 2 age groups were
tested on alternate days. Replicates were conducted until 50 males from each
age group were tested at each time period.
Effect of photophase injection of OA and 5-HT on photophase response. Preliminary tests were undertaken to determine how a series of dosages of OA
and 5-HT affected male behavior and mortality. Males were injected with 0,3,
10,30, 100,300, or 1,000 pg/g of OA or 5-HT in the first photophase following
emergence. Males treated with 300 pg/g of either amine exhibited significant
effects on motor function 6 h post-injection such that > 70% were unable to
fly, and those treated with 1,000 pg/g exhibited a paralysis. At 24 h postinjection, however, recovery in both groups was > 90%. We therefore selected
Photoperiod Cues and Biogenic Amines in Male Gypsy Moths
269
the 100 pg/g dosage for experiments on male-response thresholds, the same
concentration that was most effective in 7'. ni [11,12].
To assess the effect of OA and 5-HT on photophase response, males (day 2)
were injected with 100 pg/g insect of OA (n = 25) or 5-HT (n = 20), during
the first hour of photophase or during the seventh hour of photophase. Insects
injected with OA were tested during the eighth hour of photophase to one of five
doses of pheromone (1, 10, 100 ng; 1 and 10 pg; n = 5/dose), while those
injected with 5-HT were observed at hour intervals (n = 20/h) over the photophase (or during the eighth hour of photophase in the case of insects injected
during the seventh) for random activity. Separate groups of control insects
were also observed. One hundred insects (5 replicates) were observed at each
hour for each dosage of 5-HT (and controls), and 80 males (16 replicates) tested
to each dose of pheromone for each dosage of OA (and controls).
In this, and all experiments below involving OA or 5-HT, tests with each
compound were conducted on separate days.
Effect of pre-scotophase injection of OA and 5-HTon photophase response. The
procedures for this experiment were the same as above except that males were
injected with OA or 5-HT during the hour prior to the first scotophase onset
following emergence and then observed during the following photophase.
Male response during the scotophase. We first measured the response of males
to a 1pg pheromone source over the scotophase period, testing 5 males every
hour until 50 males had been tested at each time interval. We then established
a dose-response curve for pheromone during the time of peak response during the scotophase. Sixty males were placed in the room housing the flight
tunnel 2 h prior to the onset of scotophase following emergence, and during
the third hour of scotophase 10 males were tested to one of six doses of
pheromone (0.1, 1, 10, 100 ng; 1 and 10 pg), until 80 males had been tested
to each dose.
Thirty males were set up in the flight tunnel room 1h prior to the scotophase
following emergence and random activity recorded each hour throughout the
ensuing 8 h scotophase, as well as the first hour of photophase. Replicates
were run until 150 males were observed at each interval.
Effect of pre-scotophase injection of OA and 5-HT on scotophase response.
Thirty minutes prior to the onset of scotophase following emergence males
were injected with 100 pg/g of OA (n = 30) or 5-HT (n = 20). Insects injected
with OA (and controls) were tested during the second hour of scotophase to
one of six doses of pheromone (0.1, 1, 10, 100 ng; 1 and 10 pg: n = 5/dose),
while those injected with 5-HT (and controls) were observed at hour intervals
(n = 2 0 h ) Over the scotophase for random activity. Replicates were conducted
until 100 insects had been observed at each hour for each dosage of 5-HT (and
controls), and 80 males tested to each dose of pheromone for each dosage of
OA (and controls).
Male response in L:L and D:D conditions. Males were taken during the last
2 h of photophase on the day of emergence and placed in a separate environmental chamber, in 1 of 3 photoperiod regimes: the regular L;D, L:L, or D:D
conditions. Twenty-four hours later, or 2 h prior to the "expected" scotophase
of day 2, males were taken to the flight tunnel room, and during the third
hour of that scotophase, and the second and eighth hour of the succeeding
270
Linn et al.
photophase (day 3), 20 different males were observed for random activity and
response to a 1 pg source of pheromone. The 3 photoperiod regimes were
randomized over days until 80 males were observed at the 3 intervals for each
regime.
Effect of scotophase duration on male response. Males were exposed at the
anticipated onset of scotophase following emergence, to 1 of 2 scotophase
regimes: the normal 8 h, or a 1h period, after which the light intensity was
raised to 300 lux. Then, 20 different males were observed for locomotor activity and response to a 10 ng or 1p,g pheromone source during the third hour of
the expected scotophase, and the second and eighth hour of the succeeding
photophase. Photoperiod regimes were randomized over days until 80 males
were observed at the 3 intervals for each regime.
RESULTS
Photophase Random Activity and Response to Pheromone
There was no significant difference in the level, or temporal pattern, of random activity exhibited by male L. &par during the second and third photophases following emergence (Fig. 1A; P < 0.05; comparisons made between
each hour according to the method of adjusted significance levels for proportions [21]). Males were inactive during the first 3 h of photophase, at which
time response levels increased from the third to the seventh hour, remaining
relatively constant for several hours before declining during the last 5 h of
photophase.
No significant differences in response levels to pheromone, either for the
dose-series or for the 1 pg pheromone source over the photophase periods,
were observed when compared over the two days (Fig. lB,C; P < 0.05; comparisons made between each dose or hour according to the method of adjusted
significancelevels for proportions [21]). A positive linear dose-response tophep
omone was observed (Fig. 1B). The pattern of male response to the 1pgpheromone source over the 2 photophase periods was, in general, si&
to that
observed for random activity, with very little response in the early photophase,
maximal Ievels occurring over the mid-photophase period, and declining at
the end of photophase (Fig. 1C). Based on these results, for p h ~ b p experk
iments involving injection of OA or 5HT, comparisonswith controls wwe made
using the 10 ng (a low dose) and the 10 pg (a high dose) pheromone sources.
Effect of Photophase Injection of OA and 5-HT on Photophase Response
There was no effect on male response to pheromone or random activity in
the photophase following injection of 100 pg/g insect of OA or 5-HT during
the first hour of photophase (Fig. 2; P < 0.05; comparisonsmade between each
dose or hour according to the method of adjusted significance levels for proportions [21]), or when injection occurred in the seventh hour of photophase
with a single observation period 1h later (data not shown).
Effect of Pre-Scotophase Injection of OA and 5-HTon Photophase Response
Injection of 100 pg/g 5-HTdid not alter the onset of random activity or the
time of maximal activity, but did result in a 30-40% increase in the level of
PhotoperiodCues and Biogenic Amines in Male Gypsy Moths
271
T
&
S
2
O
0
0 1 2 3 4 5 6 7 8 9 10 11 12 1 3 1 4 15
HOURS OF PHOTOPHASE
B
DOSE (gg) OF PHEROMONE
C
lrnl
HOURS OF PHOTOPHASE
Fig, 1. A: Percentage male 1. dispar exhibiting random activity over the second and third
photophase periods post-emergence. N = 150 for each hourly measurement. B: Percentage
of males contacting the source during the seventh and eighth hour of second and third
photophase periods to a dose-series of pheromone. N = 80 for each dose and photophase
regime. C: Percentage of males Contacting the source over the second and third photophase
periods to 1wg pheromone. N = 50for each hour and photophase regime.
Linn et al.
B
OCTOPAMINE
5-HYDROXYTRYPTAMINE
1
100 pgls OCTOPAMINE
CONTROL
Wl
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DOSE QIQ)OF PHEROMONE
13 emergence
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1
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7
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HOURS
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HOURS
Fig. 2. A: Percentage male L. dispar contacting the source to a dose-series of pheromone
after injection of 100 pglg OA. B: Percentage males exhibiting random activity after injection of
100 yg/g 5-HT. Injection occurred during the first hour of photophase (indicatedby the arrow at
hour 1) on the day following emergence. N = 80 for each dosage of ONdose of pheromone
combination, and n = 100 for each 5-HT/h interval. The times during which measurements
were made are shown in the bottom portion of the figure.
activity during the seventh to eleventh hour of photophase (Fig. 38). The doseresponse to pheromone was shifted in males injected with 100 pg/g OA such
that response to pheromone was enhanced 10-30% over that observed with
controls (Fig. 3A). This occurred at all doses except the 10 kg source, to which
a decrease in response level was recorded resulting from increased levels of
arrested flight [ZZ].
Male Response During the Scotophase
Random activity during the scotophase was greatest just after lights-off (Fig.
4), at a level similar to that observed at the end of photophase (see Fig. 1A).
After 1 h, however, levels were very low and remained that way throughout
the scotophase. Response to pheromone just after lights-off was also at a level
similar to that at the end of photophase, but then increased to a maximum
2-3 h after onset of scotophase(Fig. 4B), decliningagainby the end of scotophase.
When males were tested during the peak-response period to a dose-series
of pheromone, they exhibited a greater sensitivity than at mid-photophase
(compare Fig. 4A with Figure lB, ZB, 3B). Maximal source contacts in the
scotophase occurred with the 1 pg source and 11%of the males reached the
0.1 ng source (Fig. 4A), whereas in the photophase maximal response occurred
with the 10 pg source, and none of the males flew to the source with 0.1 ng.
The lowered response with the 10 pg source during the scotophase was the
result of in-flight arrestment occurring during the first 30 cm of upwind flight,
additional evidence for a change in response thresholds during the scotophase
compared with photophase conditions [22].
PhotoperiodCues and Biogenic Amines in Male Gypsy Moths
A
273
OCTOPAMINE
1M.
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Fig. 3. A: Percentage male L. dispar contacting the source to a dose-series of pheromone
following injection of 100 pg/g OA. B: Percentage random activity over the scotophase after
injection with 100 Fg/g 5-HT. Injection was made during the hour just prior to scotophase onset
(indicated by the arrow at hour 15). N = 80 for each dosage of ONdose of pheromone combination, and n = I00 for each 5-HT/h interval. The times during which measurements were
made are shown in the bottom portion of the figure. Small letters by each measurement of random activity indicate statistically significant differences ( P < 0.05) at each hour, according to
the method of adjusted significance levels for proportions [21].
Although not measured quantitatively, there were also 2 striking qualitative differencesin the flight pattern of males under the 2 light intensities. First,
males appeared to exhibit a much wider flight path, with a greater number of
zigzag cross-wind maneuvers, in photophase than in scotophase. Second,
within 30-35 cm of the source males appeared to make a more straight-line
approach, and spend greater time on the source, in scotophase compared with
photophase. The differences in the scotophase and photophase regimes are
also evident from the temporal aspects of the male response to pheromone
concentrations eliciting maximal response. The time required to initiate wingfanning activation and fly upwind to the source was less and the time spent
at the source greater to a 1 pg dose of pheromone in the scotophase than to a
10 Fg lure in the photophase (Fig. 5 ) .
Effect of Pre-Scotophase Injection of OA and 5-HTon Scotophase Response
When males were injected with 100 pg/g OA 1 h before onset of the first
scotophase following emergence scotophase sensitivity to pheromone was significantly enhanced, with maximal response occurring with the 100 ng source
and significant levels of arrestment occurring with the 1 and 10 pg sources
(Fig. 6A). Similarly, when males were injected with 100 pg/g 5-HT random
activity was elevated 30-35% over controls, but the overall pattern in the two
groups was similar (Fig. 6B).
Linn et al.
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17 18
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Fig. 4. A: Percentage male 1. dispar contacting the source to a dose-series of pheromone
during the second hour of scotophase following emergence. B: Percentage response to a 1 pg
source of pheromone or random activity during each hour of the scotophase. The times during which measurements were made are shown in the bottom portion of the figure. N = 150 for
each random activity measure, 80 for each dose of pheromone, and 50 for each hourly measure of pheromone responsiveness.
Male Activity in the L:D, L:L, and D:D
The random activity and responsiveness of males to pheromone at 3 selected
times in L:D conditions was compared with that in L:L and D:D,with the
results shown in Figure 7. In L:D males exhibited high levels of source contacts during the early photophase (90%), and again during mid-photophase
(72%), with very low levels during early photophase (2%) (Fig, 7A). In D:D
this “bimodal” pattern of peak pheromone response periods (1 during the
early part of the dark and 1 during the middle portion of the light) persisted
!a
PWOTOPHASE: 10 IO source
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n
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Fig. 5. Mean time (in seconds, f S.D.) taken by male 1. dispar to exhibit 3 key behaviors in
the pheromone response sequence, to the optimal pheromone dose during the second hour
of scotophase (1 pg; from Fig. 4) or the seventh hour of photophase (10pg; from Fig. 1).
Photoperiod Cues and Biogenic Amines in Male Gypsy Moths
275
A
OCTOPAMINE
5-HYDROXYTRYPTAMINE
1
loo
8
0
,
201
,
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DOSE (pg)OF PHEROMONE
16 1 7 l a 19 20 21 22 23 24
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V 1 1 13 15 17 19 21 23 I 3 5 7 V I1 13 I5 I7 1V 21 23 1
3
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HOURS
Fig. 6 . A: Percentage male L. dispar contacting the source to a dose-series of pheromone
during the second hour of the scotophase following injection of 100 pp/g OA. B: Percentage
males exhibiting random activity over the photophaseafter injection with 100 &g/g 5-HT. Injection
was made during the hour just prior to scotophase onset (indicated by the arrow at hour 15).
N = 80 for each dosage of ONdose of pheromone combination, or 100 for each 5-HT/h interval. The times during which measurements were made are shown in the bottom portion of the
figure. Small letters by each measurement of random activity indicate statistically significant
differences ( P < 0.05) at each hour, according to the method of adjusted significance levels for
proportions [21].
(92%at hour 18, 4% at hour 2, and 92% at hour 8; Fig. 2B), but in L:L it did
not (18%,21%, and 24%, respectively; Fig. 2C). In addition, in D:D the levels
of response to pheromone exhibited by males at hour 18 (expected scotophase)
and hour 8 (expected photophase) were very similar (92 and 94%), and typical of those observed in scotophase compared with photophase (see Figs. 4,
5). Furthermore, the qualitative aspects of the flight response during all 3 observation periods in D:D were more similar to those observed in scotophase. Thus
when males were in the dark during the expected photophase period at hour
8 their level of response to pheromone and their activation time, upwind flight
time, and time spent in source contact were indistinguishable from that
observed during the scotophase.
With respect to random locomotor activity, a similar effect was observed. In
L:D activity was very low in scotophase (5%)and early photophase (2%),but
was high in mid-photophase (48%).In D:D conditions, however, the high level
of activity observed at hour 8 was not seen; rather the insects behaved as they
did in scotophase, exhibiting very low levels of activity. Thus, as with pheromone response, the level of random activity exhibited during the expected
photophase was a function of the light intensity experienced at that time.
As indicated above, in L:L conditions, male response to pheromone remained
at a low level throughout the observation period. This was also the case for
Linn et al.
276
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PhotoperiodCues and Biogenic Amines in Male Gypsy Moths
277
random activity. The lack of any rhythmicity in L:L conditions indicates that
the lights-off cue is critical for expression of the behaviors.
Effect of Scotophase Duration on Male Response
The results of exposing males to a 1 h "pulse" of 0.3 lux (instead of the
normal 8 h) are shown in Figure 8. In L:D conditions (Fig. 8A), the levels of
response to 10 ng and 1 rJ.gpheromone sources were again as expected from
earlier experiments. Response levels were higher during the scotophase compared with mid-photophase, with very little response occurring during early
photophase. Similarly, an expected pattern for random activity was observed,
with low levels during scotophase (5%)and early photophase (2%), but higher
levels in mid-photophase (52%).
Figure 8b s h m that following a 1 h scotophase period high levels of response
to pheromone occurred once again during the expected scotophase and photophase periods, with very low levels in early photophase. However, the results
also show that the levels of response to the 10 ng and 1 pg sources, as well as
the levels of random activity during the expected scotophase (hour 18) and
mid-photophase (h 8), were not significantly different, and were very similar
to those observed in photophase. That is, during the expected scotophase pheromone response levels were lower than would be predicted if the insects had
been tested in scotophase light intensity (24%with 10 ng; 66% with 1 pg in
Fig. 8B compared with 43%and 92%in Fig. 8A), and males exhibited significantly higher levels of random activity (48%in Fig. 8B compared with 4%in
Fig. 8A). Thus, as was demonstrated in the previous experiment with D:D
conditions, scotophase onset appears to be critical for the expression of behavior during either of the 2 activity periods, but the levels of response exhibited
are a function of the light intensity experienced.
The results of the 1h scotophase test prompted one additional experiment,
in which males were injected with 100 pg/g OA or 5-HT during the hour prior
to the scotophase (Fig. SC). The results show 2 important points: first, that
the pattern of response was very similar to that observed in Figure 8B, with
males exhibiting high, but not significantly different, levels of response to pheromone and random activity during the expected scotophase and mid-photophase periods, and much lower levels during early photophase; second, that
the response levels during each period of peak activity were significantly
enhanced following injection of the amines ( P < 0.05; comparisons made
between each dose of pheromone and random activity at hour 18 or hour 2,
using the method of adjusted significancelevels for proportions [21]).Of equal
importance, however, was the fact that the enhanced levels of random activity
and response to the respective doses of pheromone were as expected from the
dose-response curve obtained in photophase conditions, rather than scotophase
(compare with Figs. 3 and 6).
Fig. 7. Percentage male L. dispar contacting the 1 pg source of pheromone, and percentage
males exhibiting random activity at 3 hourly intervals encompassingscotophase (hour 18), early
photophase (hour2), and mid-photophase (hour 81, in 3 photoperiod regimes: L:D, D:D, and
L:L. N = 80 for each behaviodh combination. Small letters by each measurement indicate
statistically significant differences ( P < 0.05) at each hour, according to the method of adjusted
significance levels for proportions [21]. Comparisons were made between similar behavioral
categories (a, b, c, pheromone response; a', b', random flight).
Linn et aL
278
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PhotoperiodCues and Biogenic Amines in Male Gypsy Moths
279
DISCUSSION
The objectives of the present study were twofold: first, to determine if the
biogenic amines OA and 5-HT exert modulatory effects on male locomotor
and pheromone response thresholds in male L. dispur, similar to those previously demonstrated for the cabbage looper, T. ni; and second, to assess the
importance of photoperiod cues in regulating the modulatory action of the
amines. We chose L. dispar as a subject for comparative study because mating
activity occurs primarily during the photophase [13], and thus would presumably differ in the way in which photoperiod cues affect male response patterns and the modulatory action of OA and 5-HT, if these compounds were
involved at all [see 61. However, while we were able to show that OA and 5-HT
exert effects on male behavior similar to those found for T. ni, the relationship
between this modulatory action and photoperiod cues was not as expected.
In our first experiment, injection of the amines during the initial hour of
the photophase did not affect male sensitivity or random activity over the subsequent photophase. Rather, our results indicate that expression of the focomotor and pheromone response rhythms is dependent on scotophase onset.
Male sensitivity to pheromone and the level of random activity were significantly enhanced during both phases of the die1 cycle following injection of
the amines just prior to scotophase. This was also the case when males were
exposed to only a 1h scotophase, and in this case, both in control and injected
insects, the pheromone and locomotor response levels and the effectiveness
of the amines was correlated with the light intensity following the 1 h exposure to dark. That is, when males were exposed to a 1h dark period and then
to photophase light intensities, both control and injected males responded in
the subjective scotophase as if they were in photophase conditions. We conclude from this that in L. dispur males display a bimodal rhythm of locomotor
activity and pheromone response (one peak in the scotophase and the other
in photophase), with the rhythm under the control of an endogenous oscillator, but with the flight pattern and sensitivity to pheromone a function of the
light intensity experienced.
We believe that the combined studies with T. ni and L. dispar support our
hypothesis that the action of the amines is associated with pathways sensitive
to photoperiod changes and linked with a circadian oscillator that controls
the response rhythms involved in mating behavior. At this point, however, it
is not possible to adequately address the question of where OA and 5-HT are
acting, or the time course of the metabolic activity leading to their modulatory action. Thus, we must leave to future discussions an assessment of models that have been proposed [5,23]. To this end we are conducting further studies
~
Fig. 8. Percentage male L. disparcontactinga 10 ng or 1 pg pheromone source, and percentage males exhibiting random activity at 3 hourly intervals encompassing scotophase (hour 181,
early photophase (hour 2), and mid-photophase (hour 8). A: A normal 16:8 L:D photoperiod.
B: A 1 h scotophase period followed by L:L conditions. C: A 1 h scotophase followed by L:L
conditions, but with males injected with 100 pglg OA during the hour just prior to scotophase
onset. N = 80 for each behavior/h combination. Small letters by each measurement indicate
statistically significant differences(P < 0.05)at each hour,according to the method of adjusted
significance levels for proportions 1211. Comparisons were made between similar behavioral
categories (a, b, c, 10 rig pheromone; a', b', c', 1pg pheromone; a", b , random activity).
280
Linn et al.
with T. ni, utilizing HPLC with electrochemical detection, to analyze for changes
in amine levels within specific neural tissues, focusing on the period around
scotophase onset.
Scotophase Activity in L. dispar
Card6 et al. [2] showed in the field that males displayed very low response
in the early photophase, a period of maximal activity over the mid-photophase,
and a significant decline in late afternoon, with very little trap capture during
the night. Similar patterns were reported in laboratory actograph studies [14],
and were confirmed in the present flight tunnel study for both the first and
second photophase periods following emergence. Card6 et al. [2] also reported
the presence of a crepuscular flight between 1900 and 2100 hours on certain
nights. ODell and Mastro [16] reported that this flight activity was generally
in a vertical direction and they concluded that the function of crepuscular activity was to avoid predation, primarily from vertebrates.
Given the strong diurnal pattern of activity reported for L. dispur in previous studies, and the lack of significant levels of reported nocturnal trap capture, we did not anticipate the observed pattern of scotophase response to
pheromone by males. Our results show, however, that males are capable of
locating a pheromone source in the dark, and that this response and locomotor activity differed from that observed during photophase in a number of
important ways. During photophase there was a consistent delay in the onset
of locomotor and pheromone response, with levels peaking during the midphotophase and declining through the end of the period. Whereas there was
never a complete cessation in behavior at the end of photophase, the transition from photophase to scotophase conditions resulted in marked changes
in male behavior. In scotophase, Ievels of male locomotor activity dropped
during the first hour to a very low level. Response to pheromone, however,
increased rapidly with lights-off, peaking at the 90% level during the first 2-3
h. Furthermore, males were more sensitive to pheromone in the scotophase,
exhibiting peak response to a 1 pg dose compared with 10 pg in photophase.
Males also activated and flew upwind to the pheromone source eliciting
maximal response more quickly in the dark, and spent more time at the source
than under photophase conditions. A number of studies have shown that male
L. dispur display a strong close-range orientation response to vertical objects,
such as trees, from which females would be releasing pheromone [24]. In our
flight-tunnel arena the pheromone source is placed in the end of a short piece
of copper tubing that is supported by a length of stainless steel rod. Under
photophase conditions the majority of males, while flying upwind to this source
with little difficulty, did not approach directly and touch or attempt copulatory movements. Rather they exhibited a much-reduced flight speed, hovering near the source for short periods, and in many instances flying past the
source. In scotophase, however, the approach to the source was more direct,
with males spending significantly more time in contact and attempting copulatory movements with the septum.
Some of our reported differences in nocturnal and diurnal behaviors have
also been documented by ODell [151 in a report on the periodicity of eclosion
and pre-mating behavior of L. dispur. He noted that when nocturnal behavior
PhotoperiodCues and Biogenic Amines in Male Gypsy Moths
281
occurred the “flight approaches to females were always direct: males approached
and flew into containers with females with little misdirection. This contrasted
sharply with daytime activity, when males did not approach females directly
and mated only after searching.” ODell suggested that these differences might
be due to changes in meterological parameters, such as wind speed, which
could affect the dispersion of pheromone. This cannot hold true for the flight
tunnel environment, as light intensity is the only parameter that differs between
scotophase and photophase. This suggests a difference in the visually guided
optomotor response in the two light regimes. Our impression is that in the
scotophase environment, male L. dispur exhibit a flight posture and pattern
very similar to other nocturnally active moths, rather than more erratic “butterfly-like” pattern observed in photophase.
It is relevant to ask why the observed nocturnal behavior of male L. dispur
has not been documented in previous field studies, especially those describing the crepuscular habit of this species. The behavior of males in scotophase
was highly reminiscent of numerous previous observations in this laboratory
of nocturnally active moth species, such as T. ni, indicating that males are
capable of a true nocturnal habit, and that the observed behavior was not simply a modified photophase response. It also has been demonstrated that the
compound eye of male L. dispur is of the superposition type, characteristic of
nocturnally active forms [25]. A nocturnal habit is also suggested in the study
by Baker and Carde [26] in which males were shown to be very sensitive to
ultrasonic signals characteristic of bat cries [see 271,
One factor that may impact on scotophase response is temperature. In the
study by ODell[15] it was noted that nocturnal, or crepuscular, behavior was
observed most often when temperatures remained above 21”C, a situation that
apparently occurred infrequently in the trapping areas utilized. Flight tunnel
studies also confirm that at lower temperatures (in photophase) fewer males
activate and take significantly longer periods to do so [28]. Thus colder nighttime temperatures could inhibit the response of males to pheromone. Another
consideration is that crepuscular flight may take males out of the range of calling females. ODell and Mastro [16] noted that flight during these periods
was most often in a vertical path to the very tops of trees, and as suggested
above, this is presumably a flight to relocate themselves for the following
photophase or to avoid vertebrate predators. Thus, during the actual scotophase
period males may not be in a location where they would perceive pheromone,
either from calling females or synthetic lures in traps. ODell and Mastro
reported tagging quiescent individuals in late afternoon and locating them in
the same position the next morning. A third factor concerns the relationship
between female calling and mating activity. Females tend to emerge during
the early part of the photophase [15]and begin calling 1-2 h after emergence.
Thus at darkness there may be very few females unmated and thus a low pheromone release activity, coupled with male dispersal during twilight out of the
areas where females (or synthetic baits) are located and a very low degree of
random activity during the dark period, could explain why few males are captured by pheromone traps in scotophase under natural conditions.
Additional support for the conclusion that scotophase cues are more involved
in the regulation of adult behavior than is generally recognized also can be
282
Linnet al.
found in a study by Lance et al. [14]. Utilizing an actograph to measure locomotor activity, they demonstrated that the diurnal rhythm of locomotor activity was random in L:L but persisted in D:D, and that the timing of peak activity
in photophase was dependent on the precise timing of the first adult scotophase. In the present study we extended these studies by observing male locomotor and pheromone response behavior in L:L and D D conditions over the
period of the second scotophase through the mid-point of the second photophase. In D:D, the levels and temporal patterns of male locomotor activity
and response to pheromone were similar to those observed during the normal L:D. In L:L conditions males exhibited low levels of response over the
entire test period, with no evidence for either response rhythm.
W h y Two Response Patterns?
There remains the question of why male L. dispar possess a bimodal activity and pheromone response rhythm. One possibility, proposed previously
by Card6 et al. [2] and Charlton and Card&[24], is that the nocturnal habit is
the ancestral condition, and that the photophase activity rhythms are an adaptive response to competition or predation pressure, a form of reproductive
character displacement. CardC et al. [2] noted that information on the activity
period of this insect in its native habitat in Europe is somewhat confused.
Records suggest that activity may occur during the day or night. Since the
North American population is derived from a few individuals, it is thus possible that the observed diurnal habit is the result of a founder effect. In their
study on the relative importance of olfactory and visual cues in the close-range
orientation response of males to females, Charlton and Card6 [24] found that
visual cues from the females were not very important, even though the female
gypsy moth represents a visually apparent source on the trunks of trees. They
postulated that this lack of response "may be a relic of an ancestral search
strategy" evolved for a nocturnal habit, and cited several of the same points
noted above in support of a primary nocturnal habit in this species. They also
noted that a nocturnal habit is characteristic of the Lymantriidae in general,
and that displacement of L. dispur to a diurnal habit could have been the result
of Competition, as a result of interference from chemical "noise" in the communication channel with a related species, the nun moth, L. monacha. We echo
their conclusion that a comparative study of these species' activity rhythms in
sympatry and allopatry would be very fruitful.
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octopamine, gyps, photoperiod, cues, malen, lymantria, response, moth, locomotor, dispar, hydroxytryptamine, action, modulators, pheromones
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