r -fi. 1..».—¢_ gr 0'. T WWT TT TTTI“T:TT 3;- :3 ‘3534. .. m ). -_ .- .. . .1... ......-._:'2z- .. ._ _- .. A- < ‘ l..:;'..;:'.._..,.__ Marl.-—‘—oc..-._ 7.... v, A T" 'hl‘“l i "'M II! T T . TTT TTT TT‘ITT'TT ‘ TT' 1N A ... . u- - 1.. - < -- J—‘b - .H. "u‘ T| 7T ‘TTTTTIT “171‘; T'PT «T TTTI «1T1. ~'-‘ “OT“... , u L...- .- A_ _ L .. ,.___ wk‘r. .-..." ”=7.- _.- .v_._._-__ __‘__,4 TTT'M T'” T“ T! ’TTTTTTTTTTI "T T mm T, 'T TTTT TTT TTTT T T; TTHTTTTTT TT.‘ TTTIZjT “T“ "' "1"le T ”"8 1’ T . TT’T ”'T'" HINT} 1”wa (J’ ”T7 I’ .,' " T y'TLw 1 TTI‘TTTTT' .r- 7_1 Fifi h." VOT’": . ~' ’ , u §f§§§:#&1 m- a flrfiwcmi F}? i a ______ W r4" This is to certify that the dissertation entitled ENDOGENOUS AND EXOGENOUS FACTORS AFFECTING PHEROMONE PRODUCTION AND RELEASE IN THE OMNIVOROUS LEAFROLLER MOTH, PLATYNOTA ’ STULTANA (WALSINGHAM) presented by Reginald Paul Webster has been accepted towards fulfillment of the requirements for Ph . D . degree in Entomology Major professor Ring T. Carde Date October 7, 1981 MS U is an Affirmative Action/E4] ual Opportunity Institution 0-12771 MSU LIBRARIES -—!._- RETURNING MATERIALS: Place in bodk drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped beIow. ENDOGENOUS AND EXOGENOUS FACTORS AFFECTING PHEROMONE PRODUCTION AND RELEASE IN THE OMNIVOROUS LEAFROLLER MOTH, PLATYNOTA STULTANA (NALSINCHAN) BY Reginald Paul Webster A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Entomology I98] y‘v--' IQ. l I I ABSTRACT ENDOGENOUS AND EXOGENOUS FACTORS AFFECTING PHEROMONE PRODUCTION AND RELEASE IN THE OMNIVOROUS LEAFROLLER MOTH, PLATYNOTA STULTANA (WALSINGHAM) By Reginald Paul Webster Temperature modified the expression of female calling (behaviour associated with pheromone release) in Platynota stultana (Lepidoptera: Tortricidae). Absolute temperature levels and not necessarily a decrease in temperature appear to modulate the timing of calling by delimiting a specific time interval or gate for the calling period. The threshold for calling appears to be related to the level of an endogenous factor, current temperature, and photoperiod. The onset of calling advanced to earlier in the day with age. Maximal pheromone titre and maximal proportion mating occurred in moths on second day after emergence. Pheromone production is rhythmic, with maximal titre being reached near the onset of the calling gate at 14°C. Thus, females appear to be capable of emitting pheromone at an appropriate rate any time within the calling gate. Pheromone titre appears to reflect a time—dependent readiness to respond to a decrease in temperature. Pheromone biosynthesis in Platynota stultana is not under direct nervous control by the brain. After mating, pheromone titre in the glands declined to non-detectable levels ( Insect Culture . . . . . . . Gas-Liquid Chromatographic Analyses. Pheromone Titre Measurements Behavioural Observations . . iv RELATIONSHIP BETWEEN PHEROMONE TITRE, CALLING, of Calling. THE INFLUENCE OF MATING, JUVENILE HORMONE, AND A JUVENILE HORMONE ANALOGUE ON PHEROMONE TITRE, CALLING, AND Page vi viii 12 19 22 22 28 28 29 29 29 29 3O l. 2. calling I O O O O O O O O O O O O O O Mating O O O O O O O O O O 0 O O O O E. Operations . . . . . . . . . . . . . . . 1. 2. 3. RESULTS . . Decapitation . . . . . . . . . . . . Transaction of ventral nerve cord. . Topical applications . . . . . . . . A. Effect of Mating on Pheromone Titre and Calling. . . . B. Effect of Decapitation on Pheromone Titre and Calling. C. Effect of Severing Ventral Nerve Cord on Pheromone Titre and calling. 0 I O O O O O O O O O O O O O I D. Effect of Exogenous JHA on Pheromone Titre in Decapitated Females. . . . . . . . . . E. Effect of Exogenous JHA, JH I, II, and III on Behaviour and Pheromone Titre of Intact Moths. 1. 2. 3. 4. DISCUSSION. Effect of JHA on mated and virgin females. . . Effect of JHA on oviposition by virgin females Effect of JH I, II, and III and JHA on oviposition and calling in virgin females. . . . . Effect of JHA on pheromone titre of virgin females L I ST OF REFERENCE S O O O O O O O O O O O O O O O O O O Page 30 3O 30 30 ’ 31 31 31 31 33 33 35 35 35 35 38 41 44 48 Table 1. Table 2. Table 3. Table 4. Table 5. LIST OF TABLES Page Mean time of onset of calling, mating, and percent mating of female Platynota stultana at different j ages. For each column means and percentages followed by the same letter are not significantly different at the 5% level according to Student-Newman—Keuls' multiple range test. Five replicates of 20 moths each were used and treatment differences were analyzed using means of each replicate . . . . . . . . . . . . . . . 11 Percent of female Platynota stultana with initial calling bouts after decrease in temperature. Only those individuals that terminated calling for at least 0.5 hr then resumed calling were used in calculating the percent calling. For each column percentages followed by the same letter are not significantly different at the 5% level according to Student-Newman-Keuls' multiple range test. Three replicates of 20 moths were used. . . . . . . . . . . . . . 18 Mean time of termination of calling for day-1, -2, and -3 Platynota stultana females. For each column means followed by the same letter are not significantly different at the 5% level according to Student-Newman- Keuls' multiple range test. Three replicates of 20 moths. Treatment differences were analyzed using means of each replicate . . . . . . . . . . . . . . . . . . 20 Effect of exogenous JEA (ZRrSlZ) on titre of the Ell-l4:Ac pheromone component of virgin decapitated females. Day-l and day-2 moths decapitated at 09.00 and 21.00 hr, respectively. Acetone and JHA were applied topically 0.0 or 12.00 hr after decapitation. Each value is x titre obtained from 10 samples of 2 females taken 24 hr after topical application. For each column means followed by the same letter are not significantly different at the 5% level according to Student-Newman-Keuls' multiple range test. . . . . . . . 36 Effect of exogenous JHA on titre of the Ell-l4:Ac pheromone component and oviposition by virgin and mated females. Topical applications were made between 1l.00 and 11.50 hr to day-2 moths. Each value is x titre obtained from 10 individual females vi Table 6. Table 7. Table 8. Table 9 . taken 24 hr after topical application. For each column means followed by the same letter are not significantly different at the 5% level according to Student-Newman—Keuls' multiple range test. . . . . . . . Effect of different doses of exogenous JHA on i number of eggs laid by virgin females. Topical applications were made between 21.00 and 22.00.hr to day-2 females. Eggs were counted at 22.00 hr, 1, 2, 3, 4, and 6 days after treatment (N = 10). For each column means followed by the same letter are not significantly different at the 5% level according to Student-Newman—Keuls' multiple range test. . . Effect of exogenous JH I, II, and III, and JHA on oviposition and calling of virgin females. Topical applications were made between 19.00 and 22.00 hr to day-2 moths. Calling was observed at 22.00, 23.00, and 24.00 hr. Eggs were counted at 09.00 hr (N I 15). For each column means followed by the same letter are not significantly different at the 5% level according to Student-Newman—Keuls' multiple range test. . . . . . . . Effect of exogenous JHA on titre of Ell-l4:Ac pheromone component and oviposition by day-1 treated virgin females. Topical applications were made between 10.00 and 11.00 hr to day-1 (0-3 hr-old) moths. Samples and eggs counted at 21.00 hr (prior to onset of calling), 36, 60, and 84 hr after topical applications (N - 10). For each column means followed by the same letter are not significantly different at the 5% level according to Student- Newman-Keuls' multiple range test . . . . . . . . . . . . . Effect of exogenous JHA on titre of the Ell-14:Ac pheromone component and oviposition by day-2 treated virgin females. Topical applications were made between 10.00 and 11.00 hr to day-2 moths. Samples taken at 21.00 hr (prior to onset of calling), 24, 48, and 72 hr after tapical applications (N - 10). For each column means followed by the same letter are not significantly different at the 5% level according to Student-Newman—Keuls' multiple range test. . . vii Page 37 40 43 Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES Page Quantity of the E11-l4:Ac pheromone component obtained from different extraction times of abdominal tip washes of females. The quantity indicated is x i SD obtained from 10 samples of 5 females . . . . . . . . . . . . . . . 4 Titre of E11-14:Ac pheromone component obtained from 15 sec abdominal tip washes of day-l through day-7 females at 19.50 hr. Means are from 6 samples of 5 .females. Bars with same letter are not significantly different at the 5% level according to Student-Newman- Keuls' multiple range test . . . . . . . . . . . . . . . - 7 (Top) Titre of Ell-14Ac pheromone component obtained from 15.sec abdominal tip washes of females over 6 days. The quantity is the x _ SD obtained from 6 samples of 5 females. (Bottom) The calling rhythm of virgin females held under identical conditions at 240 C (N = 100).. . 8 Titre of E11-14:Ac pheromone component obtained from 15 sec abdominal tip washes of day-2 and -3 females at different times. Each point is x titre obtained from 24 and 23 samples of 5 day-2 and —3 females, respectively. Points on each line with the same letter are not significantly different at the 5% level according to Student-Newman—Keuls' multiple range test. . . . . . . . . 10 Effect of decreases in temperature on calling of day-1 females. Arrowos on x axis indicate times of decreases in temperature(_2.4o to 14° ) Numbers above arrows denotex hour of onset of calling. Mean onset of calling was calculated using time of onset of calling after termina- tion of initial calling bouts. Bars represent mean percent of 3 replicates of 20'moths. . . . . . . . . . . . 13 Effect of decreases in temperature on calling of day-2 females. Arrows on x axis indicate times of decreases in temperature (240 to 14° ). Numbers above arrows denote x hour of onset of calling. Mean onset of calling was calculated using time of onset of calling after termina- tion of initial calling bouts. Bars represent mean percent of 3 replicates of 20 moths. . . . . . . ... . . . 14 Effect of decreases in temperature on calling of day-3 females. Arrows on x axis indicate times of decreases in viii Figure 8. Figure 9. Figure 10. Figure 11. Page gemperature (24°to 14°). Numbers above arrows denote x hour of onset of calling. Mean onset of calling was calculated using time of onset of calling after termina- tion of initial calling bouts. Bars represent mean percent of 3 replicates of 20 moths. . . . . . . . . . . . 15 Calling latencies of day-l, -2, and -3 females after decreases in temperature (240 to 14°) at different times. Arrows denote beginning of calling gates. Calling latencies, starting at onset of calling gate, were not significantly different at the 5% level according to Student-Newman—Keuls' multiple range test. Calling latency was calculated using time of onset of calling after terminatign of initial calling bouts. Each point represents the x calling latency from 3 replicates of 20 moths . . . . . .-. . . . . . . . . . . . . . . . . . . 16 Effect of decreases in temperature on termination of galling of day-2 females. Numbers above arrows denote x hour of onset of calling. Bars represent mean percent of 3 replicates of 20 moths. . . . . . . . . . . . . . . . 21 (Top) Calling and mating periodicity of day-2 females of Platynota stultana (N 8 100). (Bottom) Titre of Ell-l4:Ac pheromone component obtained from 15 sec abdominal tip washes of day-2 females mated at 22.09 hr, decapitated at 22.00 hr, or virgin. Each point is x titre obtained from 6 samples of 5 females . . . . . . . . 32 Titre of Ell-14:Ac pheromone component obtained from 15 sec abdominal tip washes of day-2 females dgcapitated at 14.00, 19.00, and 22.00 hr. Each point is x titre obtained from 6 samples of 5 females . . . . . . . . . . . 34 ix INTRODUCTION The omnivorous leafroller moth, Platynota stultana (Malsingham) infests a large variety of greenhouse and field crops in California, Arizona, and Mexico (Atkins ggugl., 1957). Host plants include alfalfa, aster, avocado, citrus, cotton, pigweed, red pepper, rose, walnut, and grape (Atkins 25.31., 1957; AliNiazee g; 31., 1970). P, stultana is now an important pest of grapes in California. Early in the season larvae of P, stultana feed on foliage and in later genera- tions shift to grapes as berry size increases. Feeding by larvae breaks the skin of the berries, allowing yeast and fungi to initiate bunch rot and eventually cause destruction of the entire bunch (AliNiazee and Stafford, 1972). Adult males locate virgin females using a sex attractant pheromone, as first demonstrated by AliNiazee and Stafford (1971). Hill and Roelofs (1975) later identified two major components of the pheromone. Sex attractant pheromones provide a sensitive, species-specific means for population sampling, and are an important part of many pest management programs. The use of pheromones as communication disruptants for direct control of pest populations is now a reality. However, to more efficiently utilize pheromones as communication disruptants, a greater understanding of the behavioural and physiological events involved in the coordination of the sexual behaviours between sexes that culminate in mating is required. The precise relationship between rhythmicities of female calling and pheromone titre on or in the pheromone gland has not been established definitively amongst Lepidoptera. Comparatively little is known of the neural, neuro-hormonal and neuro-humoral influences on pheromone production and release in moths. Most evidence establishing endocrine regulation of pheromone production in moths is fragmentary and circumr stantial. In this dissertation I examine in two chapters: (1) the relation? ship between rhythmicities of female calling and pheromone titre in gland, and the influence of temperature and age on these rhythmicities; and (2) the effect of mating and endocrine regulation of pheromone production and release in P, stultana. CHAPTER 1 Relationship Between Pheromone Titre, Calling, and Age INTRODUCTION In Lepidoptera the behavioural and physiological events involved in sexual behaviour usually are coordinated temporally so that they occur synchronously under optimal conditions in a heterogeneous and variable environment. The expression of daily rhythms of sexual be- haviours, such as calling (the behaviour associated with pheromone release) and pheromone release, is usually endogenous and the timing of these rhythms is usually modified by exogenous environmental cues, typically photoperiod and temperature (Sower g; 31,, 1971; Cardé and Roelofs, 1973; Cardé‘ggugl,, 1975; Baker and Cardé, 1971; Castrovillo and Cardé, 1979). The relationship between calling and pheromone titre in or on gland surface has been investigated in several species (Shorey and Gaston, 1965; Brady and Smithwick, 1968; Nagota ggugl., 1972; Sower ‘ggugl., 1972; Fatzinger, 1973; Nordlund and Brady, 1974; Coffelt 35:51., 1978). Shorey and Gaston (1965), Brady and Smithwick (1968), Nagota ggggl. (1972), and Nordlund and Brady (1974) used bioassays of pheromone gland extracts to quantify pheromone titre in gland. However, only relative changes in titre can be quantified using bioassays. The precise relationship between rhythmicities of female calling and pheromone titre has not been definitively established among moths. We investigated the relationship between calling rhythmicity and pheromone titre on/in gland of PlatynOta stultana (walsingham). MATERIALS AND METHODS A. Insect Culture 2, stultana from Parlier, California was maintained on a pinto bean diet (Shorey and Hale, 1965) at 24°C, 75% relative humidity, and a 16:8 light:dark photoperiod regime. Photophase and scotophase light intensity were 22. 2100 and 0.3 lux, respectively. Pupae were segregated by sex and adults were held in separate cages according to emergence dates and sex. All experiments were conducted at 24°C, 75% relative humidity, and identical 16:8 light:dark regime, unless otherwise specified. B. Gas-Liguid Chromatographic Analyses Gas-liquid chromatographic (GLC) analyses were conducted with three 2 mm internal diameter glass columns; a 1.8 m long column packed with 3% OV-l on Gas Chrom Q,.a 3.5 m long column packed with 10% XF-1150 on Chromosorb-W, and a 3.5 m long column packed with 20% OV-275 on Gas ChroerZ. C. Pheromone Titre Measurements The sex pheromone of P, stultana consists of a 88:12 blend of (E) and (§)-ll-tetradecenyl acetate (E11-14:AC; le-l4:Ac)(Hill and Roelofs, 1975). To quantify pheromone titre on/in the pheromone gland of virgin females and avoid contamination of the sample with spurious compounds, the optimal time for extraction was determined. Since no differences in ratios of the blend of pheromone components were found in female pheromone gland extracts at different times of day or on different days, only the Ell-l4:Ac pheromone component was quantified. In Aggyrotaenia velutinana (Walker), little variation in pheromone component blend ratios was found in female pheromone gland extracts (Miller and Roelofs, 1980). Between 13.50 and 14.50 hr (24 hr decimal clock) the last 2 abdominal segments con- taining the pheromone gland of day-2 females (emerged on day 1) were excised and soaked in 20 ul CS for 5, 10, 15, 30, or 60 sec. Each sample con- 2 tained gland rinses of 5 females to insure sufficient material for accurate quantification. An internal standard, (Z,E)-7,ll-hexadecadienyl acetate (10 ng/ul) in 20 ul CS2 was added, and then the sample was stored at —10° until analyzed by GLC on 0V-275. Extractable pheromone was removed within 10-15 see (Figure 1). Extraction for 18 hr (N - 5) resulted in no significant increase in titre over a 15 sec rinse. The ratio of E and le-l4:Ac, as determined on XF-1150 using female extracts, was not different from that determined on 0V-275. Analyses on OV-l showed no corresponding alcohols (<0.l%) to complicate analyses on OV-275 or XF-1150. All subsequent determinations of pheromone titre were of the E11-14:Ac component obtained from 15 sec abdominal tip washes of samples of 5 females analyzed by GLC on OV-275 or XF-1150. D. Female Calling Observations Single females were placed in 4 cm x 4 cm (35 ml) clear plastic cups with plastic lined cardboard lids. Females were observed during scotophase using a light with a Kodak W'rattanR filter No. 19 which eliminates light below 600 nm. The moths appeared to be insensitive to the light. A female was scored as calling if the pheromone gland was extruded and visible, or if abdomen was turned away from observer the female assumed the typical calling posture: wings elevated, legs extended, and abdomen raised above the substrate. There was individual variability in the extent of this behaviour. Near the end of calling (0.5-1.0 hr), calling behaviour became less pronounced, i.e., wings and abdomen were lowered toward substrate. Once the pheromone gland was either entirely retracted or typical resting posture was assumed (wings and abdomen .5 C) L__ CD CD I 20* . '+ I} 10- Ac Per Female é 1'01'5 30. ' 60 Extraction Time (Seconds) ng E1 1-14 Figure_l. Quantity of the Ell-l4:Ac pheromone component obtained from different extraction Eimes of abdominal tip washes of females. The quantity indicated is x i SD obtained from 10 samples of 5 females. 5 lowered to substrate) calling was considered terminated. Photoperiod light intensities were identical to those used in rearing. Calling rhythms were observed from day 1 (day of adult emergence) to day 6. Individual females were placed in plastic cups (containing 1 cm_x 1 cm cotton wicks soaked with 5% sucrose solution) at 12.00 hr (3—5 hr after emergence) and observed for calling at hourly intervals between 15.00 and 04.00 hr and 3 hr intervals during rest of period, each day through day 6. COtton wicks were replaced on alternate days. E. Observations 23 Calling Gate Several species of moths exhibit an ability for daily adjustments of calling rhythm to ambient temperature (Batiste, 1970; Sower 251§1., 1971; Sanders and Lucuik, 1972; Cardéhggigl., 1975; Castrovillo and Cardé, 1979; Baker and Cardé, 1979). A rapid decrease in temperature induces calling within several minutes if the temperature is decreased during the gate for calling, the gate being the time interval during which calling can be induced by a change in temperature. Decreasing the temperature before the gate results in a latent period before calling commences. By decreasing the temperature at different times the timing of the calling gate can be determined. Calling gates were determined for day-l, -2, and -3 females. Moths were placed in plastic cups at 10.00 hr. Groups of moths (20) were removed from an environmental cabinet (24°) and placed at various times in a walk-in-chamber at 14°. A control group of moths was maintained at 24°. The experiment was replicated 3 times. A Bailey BAT-4 amplifying thermometer with a MT-3 microprobe accurate to 0.50 was used to determine rate of temperature decrease within the plastic cups. Mean time of onset of calling, calling latency (time between the decrease in temperature and the onset of calling), and end of calling were determined for each group of moths. F. Mating Male and female pairs were placed in 2.5 cm x 3 cm (40 m1) clear plastic vials with cotton steppers 6 hr prior to scotophase. Beginning at 18.00 hr (2 hr prior to initiation of scotophase) at 0.5 hr intervals to 03.00 hr those individuals i3 copula and calling were recorded for day-1 through day-6 females. Only day-2 males were used. Twenty moths were used for each of the 5 replicates using a completely randomized design. Mean onset of mating and proportion that mated were determined for each age group of females. RESULTS A. Pheromone Titre Measurements We first determined if pheromone titre changed with age as in'A. velutinana (Miller and Roelofs, 1977). Titre was determined at 19.50 hr (1-2 hr before calling began) for day-l through day-7 moths (Figure 2). Pheromone titre reached maximal levels on day 2 (28.5 ng/g), then progressively decreased to day 7 (1.8 ng/g). A subsequent study examined possible periodicity of pheromone titre on/in gland surface in relation to calling periodicity during the first 6 days post-emergence (Figure 3). Titre was determined at 3 hr intervals from 09.00 hr on day 1 (0-3 hr after emergence) to 09.00 hr on day 7. Between 18.00 and 24.00 hr titre was determined at 1.5 hr intervals. Six replications were done using a completely randomized design. Only 1.5 ng/g of Ell-14:Ac was present on the gland surface on day l at 09.00 hr 0-3 hr after emergence, then there was a progressive increase in titre to 17.7 ng/g at 22.50 hr, when the first individuals began to call 30- d 0.20- \ O 1‘. <- P J. c c F |.I.l 0: l0- C ‘ b I b ab 0 0" I 2 3 4 5 s 7 DAY Figure 2. Titre of Ell-14:Ac pheromone component obtained from 15 sec abdominal tip washes of day-l through day-7 females at 19.50 hr. Means are from 6 samples of 5 females. Bars with same letter are not signifi- cantly different at the 5% level according to Student-Newman-Keuls' multiple range test. ' DAY I I DAY 2 I DAY 3 I DAY 4 I DAY 5 I DAY 0 If 30‘ 20 -' ng E-II-M: Ac/e ' moo Zlm 0900 ZIOO osoo ZIOO osoo ZIOO osoo ZIOO 0900 ZIOO 0900 -m‘ I osoozIooosooZIooosoozIooosoo.zIooosoozIooosoomooosoo TIME (HOURS) Figure 3. (Top) Titre of Ell-l4:Ac pheromone component obtained from I5 age-abdominal tip washes of females over 6 days. The quantity is the x i SD obtained from 6 samples of 5 females. (Bottom) Theocalling rhythm of virgin females held under identical conditions at 24 (N - 100). (Figure 3). Titre of Ell-l4:Ac then decreased to 8.2 ng/p by 24.00 hr. On day 2 there was no significant increase in pheromone titre between 03.00 and 18.00 hr. Between 18.00 and 19.50 hr pheromone content increased significantly from 19.7 to 29.6 ng/p and remained at this level to 21.00 hr (27.00 ng/g) which is SE. 1.0 hr prior to the onset of calling. Once calling commenced (22.20 hr), titre dropped abruptly to 14.2 ng and 6.8 ng/g at 22.50 and 24.00 hr, respectively. On subsequent days a similar rhythm of pheromone titre was observed, with no significant change in titre between 03.00 hr (after calling ceased) and 15.00 hr. Maximal titre of Ell-l4:Ac usually was reached by 19.50 hr and each day titre decreased after calling began. These observations suggest a daily rhythm of pheromone production in .3: stultana. However, much sample variability in titre of Ell-14:Ac was found, even though each sample contained pheromone rinses of 5 females. Because sample variability may have masked subtle changes in titre, we re-examined pheromone titre changes for day-2 and -3 moths with 24 and 23 replicates, respectively. Samples were taken at same times as in previous experiment, except for an additional sample at 16.50 hr (Figure 4). On days 2 and 3 pheromone titre remained the same between 03.00 and 09.00 hr. Titre then increased, reaching maximal levels at 15.00 and 19.50 hr on days 2 and 3, respectively. On both days, the increase in titre appeared to level off at 15.00 hr, then increased to slightly higher levels between 18.00 and.19.50 hr (Figure 4). Once calling commenced (22.20 hr on day 2; 21.80 hr on day 3) titre decreased abruptly. B. . Female Calling Observations Individual females were observed for time of onset of calling from day 1 through day 6 (Table 1). Females were observed beginning at 18.00 10 DAY 2 o---o /.°.-- DAY 3 0—0 ab ab 0., Ac]? N o l \ ng 511-14 (”if I I r l— 3 6 9 l2 IS IS 2i 24 TIME (HOURS) Figure 4, Titre of Ell-14:Ac pheromone component obtained from 15 sec abdominal Eip washes of day-2 and -3 females at different times. Each point is x titre obtained from 24 and 23 samples of 5 day-2 and -3 females, respectively. Points on each line with the same letter are not significantly different at the 5% level according to Student- Newman-Keuls' multiple range test. 11 Table 1, Mean time of onset of calling, mating, and percent mating of female Platynota stultana at different ages. For each column means and percentages followed by the same letter are not significantly different at the 5% level according to Student-Newman-Keuls' multiple range test. In columns 2 and 3 five replicates of 20 moths each were used and treatment differences were analyzed using means of each replicate. Age (day) i Time of onset § Time of onset Percent of calling (N=76) of mating (N=100) mating (N=100) 1 23.6 a i 0.8 23.3 a 44.0 b 2 22.2 b i 0.4 21.9 b 74.0 a 3 21.8 c i 0.3 21.7 b 50.2 b 4 21.4 d i 0.3 21.6 b 57.0 b 5 21.2 e t 0.5 21.4 b 40.0 b 6 21.1 e 0.5 21.7 b 52.0 b H- 12 hr at 0.5 hr intervals until all individuals had initiated calling for at least 1.0 hr. On day 1, females began calling between 22.50 and 01.00 hr (i = 23.60 hr) and all females eventually called. On the following days the mean time of the onset of calling became progressively earlier until day 5. Some individuals on days 5 and 6 began calling at 20.00 hr. After day 1, most females (290%) initiated calling within 1.0 hr of each other. C. Observations 22_Calling Gate Groups of day-l moths were cooled from 240 to 140 at 1 hr intervals, between 15.00 to 22.00 hr (Figure 5). Calling could not be induced before 15.00 hr in day-l moths. For day-2 and -3 moths decreases in temperature were initiated at 12.00 hr and ended at 20.00 and 19.00 hr, respectively (Figures 7 and 8). Individuals calling were observed at 0.5 hr intervals beginning 0.5 hr after the transfer to colder temperature. After the decrease in temperature began, temperature decreased within the cups from 240 to 160 in 3.5 min and to 140 in 8.0 min (N - 5). Only 17% of day-1 females initiated calling after a decrease in temperature at 15.00 hr, with mean onset of calling at 18.30 hr and a calling latency of 3.30 hr (Figures 6 and 8). Thereafter, the percentage initiating calling increased (60% at 18.00 hr; 82% at 22.00 hr) and calling latency decreased each time temperature was decreased. From 18.00 hr onward no significant change in calling latency occurred. The calling gate for day—l females, therefore, began at 93. 18.00 hr. For day-2 and -3 moths 295% of females initiated calling at all times temperature was decreased. At 12.00 hr § onsets of calling were 16.70 and 17.40 hr and calling latencies were 4.70 and 5.40 hr for dava and -3 moths, respectively (Figures 6, 7, and 8). Calling latency decreased on subsequent times temperature was dropped until 16.00 hr 13 2 3 I ( I00- 8 I 2! 24 3 0 o It. a) . w ’° I l 4 I .( _, l l T l l FL+-q-F-q-F-p-—-q——r—1 :5 no. I: o a 24 s 0 t"- '1’ 50" d! I I no, I! I. a 2‘ 3 C O 9‘ c I l l ”I 1 I001 I5 I a 2‘ 3 O CONTROL u 2 HOUR Figure 5. Effect of decreases in temperature on calling of day-l females: Arrows on x axis indicate times of decreases in temperature (240 to 14°). Numbers above arrows denote i hour of onset of calling. Mean onset of calling was calculated using time of onset of calling after termination of initial calling bouts. Bars represent mean percent of 3 replicates of 20 moths. DAY'2 118 «a I} j“ 321 § 159 geilfi G 0 E J 904 < 0 a o. ._ I j 3 noon 3 0 ( 2 5m Ill IL ’ °J . - I 1 no- It I! s c I WIIIIIIIII - [it no 3 I I | f r S 4 T 1 ”1 film! - [r e. i ' I l' .T' r H T: 3 l I | I I , 1“” T f - T T 00* I: Is a," 3 0 A HOUR Figure 6. Effect of decreases in temperature on calling of day-2 ema es. Arrows on x axis indicate times of temperature decreases (240 to 14°). Numbers above arrows denote x hour of onset of calling. Mean onset of calling was calculated using time of onset of calling after termination of initial calling bouts. Bars represent mean percent of 3 replicates of 20 moths. 15 DAYS '“* m4 I III «M c Il.--.I|IIIIII M I I I I I I I I 00‘ I2 5 .‘. a a 24 3 s I ”I 04 . I2 m In a 24 3 c ”-1 '5 FEMALES CALLING 0 —1 a—I i—I HOUR Figure 1. Effect of decreases in temperature on calling of day-3 females. arrows on x axis indicate times of temperature decreases (24 to 14 ). Numbers above arrows denote x hour of onset of calling. Mean onset of calling was calculated using time of onset of calling after termination of initial calling bouts. Bars represent mean percent of 3 replicates of 20 moths. 16 DAY I Ammo-A DAY 2 0““ CALLING LATENCY (nouns) 0T2 IS I'4 I3 Is I'? I'a I's 2'0 51 52 TIME OF TEMPERATURE DECREASE (HOURS) Figure_ 8. Calling latencieso of day-l, -2, and -3 females after decreases in temperature (240 to 14°) at different times. Arrows denote beginning of calling gates. Calling latencies, starting at onset of calling gate, were not significantly different at the 5% level according to Student-Newman-Keuls' multiple range test. Calling latency was calculated using time of onset of calling_ after termination of initial calling bouts. Each point represents the x calling latency from 3 replicates of 20 moths. l7 - for day-2 and -3 moths. Thereafter, calling latencies were $0.5 hr. The calling gate for day-2 and -3 females began at 22' 16.00 hrs. In order to determine more precisely calling latency during the calling gate, observations were made every 2 min starting 2 min after a decrease in temperature at 19.00 hr for day-3 moths. Mean calling latency was 10 min (0.17 hr) and ranged from 2 to 18 min (N a 49). Between 12.00 and 16.00 hr, some day-2 and -3 females initiated calling within 0.5 hr after the decrease in temperature, called between 0.5 and 1.0 hr, then ceased calling (Figures 6 and 8; Table 2). These individuals resumed calling within 1.0 to 4.5 hr. Thereafter, progressively fewer individuals exhibited these initial calling bouts after the decrease in temperature and instead, called continuously once temperature was decreased. The maximum proportion of initial calling bouts of day-2 and -3 moths occurred after a decrease in temperature at 13.00 hr. The decreases in temperature elicited a greater proportion of the initial calling bouts in day-3 moths (Table 2). Calling latency was determined for the initial calling bouts of day-3 moths when temperature was decreased at 13.00 hr. Observations were made every 2 min beginning 2 min after the decrease in temperature. For those moths that exhibited an initial calling bout (24 out of 50) mean calling latency was 17 min (0.28 hr) and ranged from 10 to 26 min. Pheromone titre was determined for day-3 moths that had an initial calling bout and those that did not after a decrease in temperature at 13.00 hr. Females were observed continuously after the decrease in temperature began. As soon as calling commenced those moths were placed in an environmental cabinet at 24°C. This resulted in immediate cessa- tion of calling. At 13.50 hrs, 0.5 hr after initiation of the decrease in 18 Table 2. Percent of female Platynota stultana with initial calling bouts after decrease in temperature. Only those individuals that terminated calling for at least 0.5 hr then resumed calling were used in calculating the percent calling. For each column percentages followed by the same letter are not significantly different at the 5% level according to Student-Newman-Keuls' multiple range test. Three replicates of 20 moths were used. 2 Females with Initial Calling Bout After Time of Temperature Decrease in Temperature Decrease Day 1 Day 2 Day 3 12:00 -- 1.7 a 27.7 b 13:00 -- 25.0 c 54.4 c 14:00 - 15.0 b 38.3 b 15:00 0.0 13.3 b 29.8 b 16:00 0.0 1.7 a 8.3 a 17:00 0.0 . 0.0 a 0.0 a 18:00 0.0 0.0 a 0.0 a 19:00 0.0 0.0 a 0.0 a 20:00 0.0 0.0 a -- 21:00 0.0 -- -- 22:00 0.0 -- -- 19 temperature,moths that had not called were also placed in the environ- mental cabinet. Samples of individual female pheromone gland rinses were then taken, alternating between those that called and those that did not. Mean pheromone titre of those that called was 9.73 ng/2 and ranged from 1.5 to 26.5 ng/g (N = 28). For those that did not initiate calling imediately after the temperature decrease, mean titre was 7.7 ng/g and ranged from 0.7 to 23.1 ng/g (N - 28). The means were not significantly different. D. Effect of Temperature 22_Termination of Calling In day-2 and -3 moths a decrease in temperature (240 to 14°) after 15.00 hr elicited calling within 0.5 hr. However, regardless of when decreases in temperature began, E time of termination of calling occurred at about the same time (Figures 6 and 7, Table 3). This resulted in a progressive shortening of calling period as time of decrease in tempera- ture was delayed. This suggests that temperature regulated when calling was terminated in 1:. stultana and that calling may be terminated by a decrease in temperature after 21.00 hr in day-2 moths. (Figure 6). Therefore, we decreased temperature (240 to 14°) in day-2 moths at 21.00, 22.00, and 23.00 hr. Calling was observed every 0.5 hr beginning at 18.00 hr, ending at 04.00 hr (Figure 9). Normally E time of onset calling of day-2 moths is 23° 22.00 hr at 24°. In this experiment I time of onset of calling in the control group maintained at 24° was 21.20 hr and the i time of end of calling was at 01.70 hr. In the control group all females had initiated calling by 22.00 hr. When temperature was decreased at 21.00 hr all individuals initiated calling by 21.50 hr and the I end of calling was at 21.90 hr. A decrease in temperature at 22.00 hr elicited a 58% reduction in calling within 0.5 hr. When temperature was decreased 20 Table 3. Mean time of termination of calling for day-l, -2, and -3 Platynota stultana females. For each column means followed by the same letter are not significantly different at the 5% level according to Student-Newman-Keuls' multiple range test. Three replicates of 20 moths. Treatment differences were analyzed using means of each replicate. Time of Temperature E Time of Termination of Calling Decrease Day 1 Day 2 Day 3 12:00 - --- 21.1 d 20.6 bc 13:00 . --- 21.2 d 20.7 b 14:00 --- 21.3 cd 20.9 b 15:00 20.2 d 21.2 d 20.6 bc 16:00 19.3 d 21.3 cd 20.2 d 17:00 19.8 d 21.7 be 20.2 cd 18:00 20.0 d 21.8 b 20.3 cd 19:00 20.2 d 22.0 b 20.5 bcd 20:00 21.0 cd 21.8 b --- 21:00 22.2 bc --- --- 22:00 22.7 b --- --- Control 01.3 a 01.3 a 01.8 a loo-4 21.9 o g Itwncmomnw : 3 AT A i E I c I .001? 20 223B 24 2 4 S 11MRINDHED : g AT 0 2200 In 50— 3 < I a o I7 IS 20 22 24 2 4 I 210 G 3 d i 0 Q 3 < 3 II. a § 2 O 3 i a E a o I I8 20 22 24 2 4 IflDUR Figure 2, Effect of decreases in temperature on termination of calling of day-2 females. Numbers above arrows denote x hour of onset of calling. Bars represent mean percent of 3 replicates of 20 moths. 22 at 23.00 hr, a 95% reduction in calling was induced within 0.5 hr after the decrease in temperature. E. Mating Mating occurred within 0.5 hr after initiation of calling for all age groups of females. The i time of onset of mating for day-l moths was 1.4 hr later than day-2 and 1.9 hr later than day-5 females (Table l). The i time of onset of mating was not significantly different for day 2-6 females. Day—2 females, which contained the greatest quantity of pheromone, had the greatest percentage mating of any age group. DISCUSSION Temperature modified the expression of calling in female 2, stultana. Decreasing temperature from 240 to 140 advanced the onset of calling up to 5 hr. A decrease in temperature was followed by a rapid initiation of calling (within 0.5 hr) only when the temperature shift occurred within a particular time interval or gate, which at 140 began about 5 hr prior to onset of calling at 24°. If the decrease in temperature occurred before this gate, there was a latent period before calling commenced. .A. velutinana, Holomelina immaculata (Reakirt) (Cardé_§§”§1., 1975), and Grapholitha molesta (Busck) (Baker and Carde ,1979) exhibited similar responses to temperature decreases. When the decreases in temperature occurred before the calling gate, calling began between 16.00 and 17.00 hr in day-2 and -3 g, stultana. This suggests that calling was not induced by the decrease in temperature itself, but rather calling initiation may result from an interaction of the current temperature and hour of photoperiod. In E. molesta the temperature level had a major effect on inducing calling whether the level was reached by an 23 increase or decrease in temperature (Baker and Cardé, 1979). Baker and Cardé (1979) suggested that a temperature decrease increases early calling by lifting inhibition rather than by inducement. Higher tempera- tures may suppress calling by delaying its expression to later in the photoperiod. A temperature decrease removes calling suppression, allowing expression of calling rhythm earlier in the photoperiod. In E, stultana a shift in temperature from 240 to 140 preceding E onset of calling at 240 and E end of calling at 140 elicited calling within 10 min. If the shift from 240 to 140 followed E end of calling at 14°, those females still calling terminated calling within 0.5 hr of the shift. However, regardless of when the shift from 240 to 140 came, if the change occurred prior to E end of calling at 14°, calling ended at about the same time (between 21.00 and 22.00 hr). This suggests that in g, stultana the termination of calling results from an interaction of the current temperature and hour of photOperiod, rather than the decrease in temperature alone. It appears that the current temperature and photoperiod modulate calling by delimiting a specific time interval or gate for the calling period. At a given temperature the time of onset and end of calling are specified, and thus at each temperature a different time interval or gate for calling is specified. Should the temperature increase (or decrease) calling will begin, end, or continue depending on the gate delimited by the current temperature. In Synanthedon pictipes (Grote & Robinson) and Q. molesta there are upper and lower temperature limits above or below which calling will not occur (Gorsuchngtngl, 1975; Baker and Cardé; gt 31., 1979). Thus, the absolute limits of how far the calling period will be advanced into photophase by low temperatures or delayed into scotophase by high temperatures will probably be partially 24- governed by the upper and lower temperature thresholds for calling in 3. stultana. Decreases in temperature prior to the calling gate at 140 of day-2 and -3 moths elicited short, initial calling bouts (0.5-1.0 hr in dura- tion) in some individuals. The calling bouts began shortly (6-20 min, 2 - 13 min) after the lower temperature was attained. These individuals resumed calling near the onset of the calling gate at 14°. When the decrease in temperature came near the commencement of the calling gate, most individuals called continuously for several hours. The initiation of the short calling bouts does not appear to be a simple nervous response to a decrease in temperature because calling did not begin until after a lower temperature (14°) was attained. Furthermore, the proportion of these initial calling bouts varied with time of day, suggesting a changing threshold for calling. The threshold for calling may be related to the presence or level of an endogenous factor(s) (probably a hormone), in addition to current temperature and photoperiod. Furthermore, the level of this factor required to elicit calling may be different from that required to maintain calling. In Antheraea polyphemus (Cramer) the intrinsic neurosecretory cells of the corpora cardiaca appear to secrete a hormone in response to a nervous signal from the brain, that elicits calling. Blood from calling females induced calling in non-calling individuals. This hormone presumably acts on the female nervous system to trigger calling behaviour (Riddiford, 1974). In g, stultana release of the factor may be regulated by photoperiod and temperature. Tempera— ture may determine, in part, the gate for the release of this factor. The onset of calling periods of g. stultana advanced with age. The mechanism underlying this shift is not clear, but may be related to the 25 shift from 18.00 to 16.00 hr of the calling gates and the increase in the proportion of moths that exhibited initial calling bouts from day l to 3. In other Lepidoptera the onset of calling or mating have been shown to advance with age (Hirano and Muramoto, 1976; Swier 32 21., 1977; Kanno, 1979). Kanno (1979) suggested that by calling earlier, older females increase their chance of mating by being the first to attract males. In E, stultana maximum pheromone titre occurred on day 2, then declined. Maximal proportion of mating also occurred with day-2 moths. In a field test using traps baited with virgin females, day-2 females were most attractive, followed by day-3, -l, and -4 females; day-6 females elicited little trap catch (AliNiazee and Stafford, 1971). Any unmated individuals older than 4 days might therefore be at a competitive disadvantage and would have to call earlier to compete with younger moths. The greater prOpensity of older moths (day-3) to exhibit initial calling bouts after a decrease in temperature may also be important in their ability to compete with younger individuals. Mating usually occurred within 0.5 hr of onset of calling in g. stultana. Individuals that did not mate during first hour of calling did not mate that night. This suggests that pheromone release rate may be highest immediately after initiation of calling. Indeed, a rapid decline in titre ensued shortly after calling onset. In Trichoplusia ni (Hubner) pheromone release rate declined exponentially after onset of calling (Bjostad gt 51., 1980), probably due to evaporation from the gland surface (Sower st 31., 1972). However, until actual emission is studied in g. stultana, the precise relationship between rhythmicity of female calling, pheromone titre, and emission cannot be established definitively. In E. stultana lower temperatures advance the onset of calling. A 26 decrease in temperature elicits calling within several minutes if it occurs within the gate for the lower temperature. These females should be capable of emitting pheromone at an appropriate rate any time within the gate. Therefore, the quantity of available pheromone should be at a high level throughout the gate. Indeed, maximal quantity of Ell-14:Ac was reached on day 2 and 3 at 25, 15.00 hr, which was close to the onset of the calling gate at 14°. In P, stultana pheromone titre appears to reflect a time-dependent readiness to call if temperature is decreased. Pheromone production appears to be rhythmic in P, stultana. Each day, once calling commenced, pheromone titre dropped abruptly. When calling ended (Ea. 01.50 hr), little change in titre occurred until after 09.00 hr, then titre increased until 15.00 or 18.00 hr, prior to the initiation of calling at 240 (egg. 22.00 hr). Although pheromone pro- duction appears to be rhythmic, it may be difficult to establish that it is regulated by an endogenous oscillator, because continuous light and dark conditions do not suppress calling behaviour in E, stultana. Few species of Lepidoptera have been shown to exhibit daily rhythmicity of pheromone titre. A daily rhythm of titre was not found 1“.E3.E£ (Shorey and Gaston, 1965; Sower gt al., 1972). Whole pheromone gland content (626 ng/g) and surface content (48 ng) remained relatively constant throughout a 24 hr period (Sower 23 a1., 1972). .1..gi calls in a series of bouts (i I 20 min) during scotOphase and appears to rely on bio- synthesis and a large reservoir of pheromone within the gland to replace that lost to evaporation from the gland surface (Sower_gt'gl., 1972; Bjostad 33 $1., 1980). In Plodia interpunctella (Hubner) and Ephestia cautella (Walker) the rhythm of pheromone titre from surface washes of glands was synchronous with calling rhythm, titre being highest during 27 calling (Coffelt g£.§l., 1978). Whole gland content was not examined. In P, stultana all pheromone was extracted from the gland within 15 sec. This species appears to rely on biosynthesis prior to and possibly during calling to replace pheromone lost during previous calling bouts. however, it would be necessary to determine gland content before and after a release period to examine critically the relationship between amount contained and amount released. CHAPTER 2 The Effect of Mating, Juvenile Hormone, and a Juvenile Hormone Analogue on Pheromone Titre, Calling, and Oviposition INTRODUCTION Evidence establishing the role of endocrine factors regulating pheromone production in moths is fragmentary and circumstantial (Cardé and Webster, 1981). In tissue culture juvenile hormone (JH) was essential for maintaining the integrity of the pheromone gland cells of Diatraea saccharalis (F.) (White 25 $1., 1972). However, JH was not necessary in tissue culture for pheromone production in Plodia interpunctella (Hubner) (Srinivasan 3; $1., 1979), which suggests that pheromone production is not under direct nervous control by the brain. Most studies with Lepidoptera have centered on endocrine regulation of reproductive behaviours, such as calling, mating, and oviposition (Riddiford, 1974; Riddiford and Ashenhurst, 1973; Riddiford and. Williams, 1971; Truman and Riddiford, 1971). In some insect groups such as bark beetles (scolytidae) and cockroaches, endocrine regulation of pheromone production and reproductive behaviours is well established (Barth, 1961; Barth and Lester, 1973; Borden gt 51,, 1969; Hughes and Renwick, 1977; Truman and Riddiford, 1974). Brady and Smithwick (1968), Hagan and Brady (1981), and Shorey iggigl. (1968) have shown that pheromone production is unaffected by mating in several moths in which females are polygamous. However, little is known of the effect of mating on pheromone titre in lepidopterous species such as Platynota stultana (Walsingham) and Hyalophora cecropia (L.), which mate only once and terminate calling after mating (Truman and Riddiford, 1971; see Chapter 1). The 28 29 cyclical changes in pheromone titre in P, stultana suggest neural or neuroendocrine regulation of pheromone production (see Chapter 1). The effect of mating on pheromone titre and the possible endocrine regulation of pheromone production and reproductive behaviour in P, stultana are reported here. MATERIALS AND METHODS A. Insect Culture 2, stultana from Parlier, California was maintained on a pinto bean diet (Shorey and Hale, 1965) at 24°C, 752 relative humidity and a 16:8 light:dark regime.A Photophase and scotophase light intensities were 2100 and 0.3 lux, respectively. Pupae were segregated by sex and adults were held in separate cages according to date of emergence and sex. Experiments were conducted at 24°C, 75% relative humidity and 16:8 light:dark regime. B. Gas-Liquid Chromatographic Analyses Gas-liquid chromatographic (GLC) analyses were conducted with three 2 mm internal diameter glass columns: a 3.5 m long column with 10% XF-1150 on Chromosorb-W, a 3.5 m long column with 20% OV-275 on Gas Chrom- RZ, and a 1.8 m long column with 3% OV-l on Gas Chrom-Q. C. Pheromone Titre Measurements The sex pheromone of P, stultana consists of an 88:12 blend of (E) and (2)-11-tetradeceny1 acetate (E and le-l4:Ac) (Hill and Roelofs, 1975). No differences in ratios of pheromone components were found in female tip extracts at different times of day or on different days (see page 2). Therefore, only the Ell-14:Ac pheromone component was quantified. There were no differences between the ratios of E and le-l4:Ac from female extract as determined on XF-1150 and OV-275. 30 Analyses on OV-l showed no corresponding alcohols (<0.l%) to complicate analysis of E11-l4:Ac on OV-275 or XF-1150. Extractable pheromone was removed within 10-15 sec (see page 3). An internal standard, (a, §)-7, ll-hexadecadinyl acetate (10 ng/Hl) was added to all samples which then were stored at -100 until GLC analysis. Determinations of pheromone titre.were of the Ell-l4:Ac component obtained from 15 sec abdominal tip washes of samples of l, 2, or 5 females analyzed by GLC on OV-275 or XF-1150. D. Behavioural Observations All moths, unless otherwise specified, were observed and maintained individually in 4 cm x 4 cm (35 m1) clear plastic cups with plastic- lined cardboard lids. During scotophase, moths were observed using a light with a Kodak WrattenR filter No. 19 which eliminates light below 600 nm. The moths appeared to be insensitive to the light. Photo- period light intensities were identical to those used in rearing. 1. Calling. Females were scored as calling if the pheromone gland was visible and extruded or if the female assumed the typical calling posture: wings elevated, legs extended, and abdomen raised above substrate. 2. Mating. Male-female pairs of day-2 moths (emerged on day-l) were placed in 2.5 cm x 8 cm (40 ml) clear plastic vials with cotton stoppers 6 hr prior to scotophase. Beginning at 18.00 hr (24 hr decimal clock), 2 hr prior to onset of scotophase, individuals 13 copula and calling were recorded at 0.5 hr intervals to 03.00 hr. E. Operations l. Decapitation. Adult females were immobilized on ice and decapitated using a fine pair of iris scissors. In day-2 and older 31 moths no bleeding ensued after removal of the head. As some bleeding occurred in newly emerged moths (0-3 hrs after eclosion), the wound was sealed with paraffin. The moths lived 6-10 days after decapitation. 2. Transaction pf ventral nerve cord. One to 2 day-old pupae were anesthetized under CO2 and the ventral nerve cord was severed between abdominal segments 7 and 8 using a microscalpel. The wound was sealed with paraffin. For sham-operated insects, a similar incision was made on the right lateral portion of abdomen between abdominal segments 7 and 8. Mortality of operated- and sham-operated pupae was 33, 10%. Pheromone titre was determined prior to onset of calling at 21.00 hr for individual day-2 moths. Other similarily operated moths were observed at 22.00, 22.50, and 23.00 hr for calling. 3. Topical applications. JH I, II, and III (Sigma Chemical Co., St. Louis, Missouri, USA) and the Juvenile hormone analogue (JHA), ZReSlZ (Zoecon Corp., Palo Alto, CA, USA) were dissolved in acetone and applied topically in 1 pl acetone. The compounds were applied to the venter of abdominal segment 5 with a microapplicator after the moths were immobilized on ice. RESULTS A. Effect pf Mating 22 Pheromone Titre and Calling Most females mated within 0.5 hr after initiation of calling. The majority of moths were mated by 22.00 hr and no individuals mated after 24.00 hr (Figure 10, tOp). Moths remained i3 copula l to 1.5 hr. Females did not call after mating. Changes in pheromone titre were determined after mating and in virgin females (Figure 10, bottom). Pheromone titre was determined in 32 -Pueu Coiling I00 (I How MW) cam of PM 00 with!" Macho I- (05 Hot WI 5 u) U 5 «I 0. 20 o l T Y I V T l fifit U I I I I U 1 I l 301 . Ob—4iremununndnn2aKI H Fund“ WW M 2200 c; ' O——Q Vlrw PM 00' 3’ 20“ C) 5': J. o O T I“ 0 [Out (3 C . I: o , 0 .II; o ‘ c \ G I T I I I I I I I I I _ v , ¥ 1? 2I00 2400 0300 0000 0900 I200 200 am TIME (HOURS) Figure 19, (Top) Calling and mating periodicity of day-2 females of Platynota stultana (N = 100). (Bottom) Titre of Ell-l4:Ac pheromone component obtained from 15 sec abdominal tip washes of day-2 females mated at 22.00 hr, decapitated at 22.00 hr, or virgin. Each point is x titre obtained from 6 samples of 5 females. 33 individuals that were ig_copula at 22.00 hr. Pheromone titre decreased from 22.2 ng/p at 22.00 hr to 7.3 ng/g at 02.00 hr. Only 1.3 ng/g was present at 06.00 hr and no detectable quantities ($0.2 ng/p) of pheromone were present 14 and 47 hr after mating. In virgin females titre decreased from 27.0 ng/p at 21.00 hr to 6.8 ng/g at 24.00 hr and then remained near this level until 12.00 hr (8.3 ng/g). The decrease in pheromone titre in virgin females was probably, in part, the result of evaporation from the gland surface (See page 25) while the decline in mated females may be the result of enzymatic degradation of pheromone because these individuals did not call.. B. Effect 9f Decapitation gg_Pheromone Titre and Calling Virgin females were decapitated and subsequent changes in pheromone titre were determined to see if a factor(s) from the head influences pheromone titre (Figures 10 and 11). After decapitation pheromone titre declined to <5.0 ng/g in 5 hr and <0.5 ng/p in 11 hr, regardless of the time of decapitation. No decapitated individuals called. The decrease in pheromone titre was very similar to the decline after mating (Figure 10). This finding is suggestive of enzymatic degradation of pheromone and of neural, neuro-humoral, or neuro-hormonal regulation of pheromone production. C. Effect pf Severing Ventral Nerve Cord 23 Pheromone Titre and Calling Severing the ventral nerve cord during the pupal stage did not appear to affect either pheromone titre or calling behaviour of adult 3, stultana. There were no significant differences (at the 5% level, Student-Newman—Keuls' multiple range test) in E titre between control (16.1 i 11.1 SD ng/p), COz-anesthetized (26.5 H- 11.5 ng/p), sham- Operated (20.0 i 10.4 ng/p), or operated (10.8 i 5.7 ng/p) groups of 34 30- 0—0 DECAPITATED AT 1400 u, . 0----o DECAPITATED AT 1900 2‘ O H DECAPITATED AT 2200 a _ - u, 20 III. C I“ O. C) '< 3 J- 10- E a C 0 HOURS AFTER DECAPITATION Figure 11. Titre of E11-—14:Ac pheromone component obtained from 15 sec abdominal tip washes of day-2 females decapitated at 14.00, 19.00, and 22.00 hr. Each point is x titre obtained from 6 samples of 5 females. 35 moths (N = 8). All individuals of the control, CO -anesthetized, 2 sham-operated, and operated groups of moths called. These findings suggest that pheromone production is not under direct nervous control by the brain. D. Effect 2f Exogenous JHA 23 Pheromone Titre i2 Decapitated Females Because JH is required for pheromone production in several insects (Borden g; g1., 1969; Hughes and Renwick, 1977; Barth and Lester, 1973; Truman and Riddiford, 1974), the influence of the JHA, ZRrSlZ on pheromone titre of decapitated virgin females was investigated. A 10 pg dose of topically applied JHA had no influence on the decline of pheromone titre in decapitated moths (Table 4). Pheromone disappeared from the glands of decapitated moths when JHA was applied 0 or 12 hr after decapitation. JHA did not maintain or restore pheromone in decapitated females. E. Effect pf Exogenous JHA, gg I, II, and III 22_Behaviour and Pheromone Titre pf Intact Moths 1. Effect 2f JHA pg mated and virgin females. For JH or a JHA to be effective in maintaining or restoring pheromone titre it may be necessary for the moth to be intact. JHA was applied tapically to females 1 to 10 min after uncoupling. JHA did not maintain or increase pheromone titre in mated females (Table 5). There were no significant differences in titre 24 hr after treatment between JHArtreated and untreated virgin females, or between JHArtreated and untreated mated females. 2. Effect pf JHA 92 oviposition by_virgin females. Mated g, stultana lay the first batch of eggs (SO-200) 20-24 hr after copulation, usually close to the onset of scotophase, and lay a total of 22. 300 36 Table 4. Effect of exogenous JHA (ZRrSlZ) on titre of the Ell-14:Ac pheromone component of virgin decapitated females. Day-l and -2 moths decapitated at 09.00 and 21.00 hr, respectively. Acetone and JHA were Eopically applied 0.0 or 12.0 hr after decapitation. Each value is x titre obtained from 10 samples of 2 females taken 24 hr after topical application. For each column, means followed by the same letter are not significantly different at the 5% level according to Student-Newman—Keuls' multiple range test. ng Ell-14:Ac/$ Day-1 Day-2 Day-2 Treatment 0 hr 0 hr 12 hr Unaperated 15.1 b 7.2 b 9.2 b Decapitated 0.0 a 0.0 a 0.0 a Decapitated + Acetone 0.0 a 0.0 a 0.0 a Decapitated + 10 pg JHA 0.0 a 0.0 a 0.0 a 37 Table 5. Effect of exogenous JHA on titre of the E11—l4:Ac pheromone component and oviposition by virgin and mated females. Topical applications wgre made between 11.00 and 11.50 hr to day-2 moths. Each value is x titre obtained from 10 individual females taken 24 hr after topical application. For each column means followed by the same letter are not significantly different at the 5% level according to Student-Newman-Keuls' multiple range test. Treatment ng Ell-l4:Ac/p E No. eggs/g Virgin 9.7 a 1.5 a Virgin + 10 pg JHA 9.2 a 29.4 b Mated 0.1 b 107.0 c Mated + Acetone 0.0 b 104.0 c Mated + 10 pg JHA 0.2 b 93.9 c 38 eggs. Similar values were reported by Atkins ggugl. (1957). Virgin females normally do not oviposit more than 5-10 eggs during the first 4 days after emergence and <70 eggs by 8 days after emergence (Table 6). However, within 20 to 24 hr after tepical application of 10 pg of JHA virgin females laid 29.4 eggs/g (Table 5). Exogenously applied JHA was effective in inducing oviposition in virgin females at doses of 0.1 to 100 pg (Table 6). A 0.1 pg dose was only slightly less active than doses of 1.0, 10, or 100 pg in inducing oviposition in virgin females, but the higher doses induced oviposition more rapidly. The total number of eggs laid was similar at the doses tested and comparable to the number normally laid by mated females. Most eggs were laid within 48 hr after treatment with JHA. Untreated and acetone-treated moths laid significantly fewer eggs with most oviposition occurring 3 to 6 days after treatment. 3. Effect 2f lg. , II, and III and JHA gp_oviposition. The activity of exogenously applied JH I, II, and III and JHA in inducing oviposition and their effect on calling in virgin females was compared (Table 7). The JHA was as effective as JR 1, II, and III in inducing oviposition in virgin females (at the dose tested). The total number of eggs laid was similar in the JR I, II, and III and JhA treatments. However, JH I caused earlier oviposition than JH II and III and JHA. JH I-treated moths laid the greatest number of eggs by day 4. As with mated moths, most oviposition occurred 20—24 hr after treatment, close to the beginning of scotophase. JR 1, II, III and JHA reduced the proportion calling from 2 73.3% on day-3 to 0.0-13.3% on day 4, 48 hr after treatment (Table 7). Prior to day 4 most individuals (>70%) called in all treatments. 39 Table 6. Effect of different doses of exogenous JHA on 2 number of eggs laid by virgin females. Topical applications were made between 21.00 and 22.00 hr to day-2 females. Eggs were counted at 22.00 hr, 1, 2, 3, 4, and 6 days after treatment (N - 10). For each column means followed by the same letter are not significantly different at the 5% level according to Student-Newman-Keuls' multiple range test. i No. Eggs/g Treatment “Day 1 Day 2 Day 3 Day 4 Day 6 Total N 10’1 ' 26.6 b 94.2 b 123.4 4.6 54.0 302.8 b 100 52.4 c 149.3 bc 113.5 26.7 49.4 391.3 b 101 57.7 c 166.2 c 83.3 13.2 8.3 . 325.7 b 102 71.5 c 104.1 be 54.6 27.0 12.7 269.9 0 Acetone 1.5 a 12.9 a 64.4 17.5 22.0 118.3 a Control 4.7 a I 2.0 a 15.3 10.9 31.1 64.0 a 40 Table 1.. Effect of exogenous JH I, II, III and JHA on oviposition and calling of virgin females. Tapical applications were made between 19.00 and 20.00 hr to day-2 moths. Calling was observed at 22.00, 23.00, and 24.00 hr. Eggs were counted at 09.00 hr (N I 15). For each column means followed by the same letter are not significantly different at the 5% level according to Student-Newman-Keuls' multiple range test. Treatment 1 NO- bass/g Percent Calling (10 pg) Day 3 Day 4 Day 5 Total Day 2 Day 3 Day 4 JH I _ 0.0 140.3d 25.6a 165.9b 73.3 80.0 6.7 JH 11 0.0 69.6bc 43.9a6 113.56 93.3 73.3 0.0 JH III 0.0 48.56 81.96 130.46 86.7 86.7 13.3 JHA 0.0 101.1c 67.16 168.26 100.0 73.3 13.3 Acetone 0.0 1.0a 13.2a 14.2a 86.7 93.3 80.0 Control 0.0 2.7a 13.6a 16.3a 93.3 100.0 93.3 41 4. Effect ngJHA.gg pheromone titre. The influence of the JH I, II, III and JHA on behaviour was most pronounced 48 hr after treatment. Therefore, changes in titre after treatment with JHA were examined after longer intervals. Exogenous treatment of JHA significantly reduced pheromone titre in virgin females treated on day 1 and 2 (Tables 8 and 9). Pheromone titre was 3.1 and 1.0 ng/p 60 and 84 hr after treatment of day-l moths, respectively (Table 8). This compares with 9.2 and 5.2 ng/p amongst controls. Females treated on day 2 exhibited a reduction in titre in response to exogenous JHA similar to day-l treated moths (Table 9). The quantity of pheromone present at 36 hr after treatment (15.3 ng/p) in moths treated on day 1 was comparable to the amount (£3. 13 ng/p) found at the end of the first calling period of untreated day-1 moths (see page 8). In moths treated on day 2 the quantity of pheromone present in-the gland 24 hr after treatment (5.4 ng/p) was comparable to that normally present at the end of the previous calling period of untreated moths on day 2 (9.1 ng/g). Because the moths treated on day l and 2 had one calling period before the above samples were taken (Table 7), pheromone loss may have been due to evaporation from the gland surface during calling rather than to degradation. Therefore, exogenous JHA may interfere with biosynthesis of new pheromone. At 72 hr after treatment, pheromone titre had not signifi- cantly decreased from the quantity present at 48 hr. Because few individuals called 48 hr after treatment with JHA (Table 7), little new pheromone was likely synthesized. As in previous experiments, JHA elicited oviposition in virgin females (Tables 8 and 9). 42 Table 8. Effect of exogenous JHA on titre of the Ell-14:Ac pheromone component and oviposition by day-1 treated virgin females. T0pical applications were made between 10.00 and 11.00 hr to day-1 (0-3 hr-old moths). Samples and eggs counted at 21.00 hr (prior to onset of calling) 36, 60, and 84 hr after topical applications (N a 10). For each column means followed by the same letter are not significantly different at the 5% level according to Student-Newman-Keuls' multiple range test. ng Ell-l4:Ac/p I i No. Eggs/2 Treatment 36 hr 60 hr 84 hr 36 hr 60 hr 84 hr Control 25.2b 9.2b 5.2b 0.3a 8.6a 11.0a Acetone 21.6ab 11.2b 4.7b 0.0a 5.3a 20.0a 10 pg JHA 15.3a 3.1a 1.0a 43.0b 229.4b 241.0b 43 Table 9. Effect of exogenous JHA on titre of the E11-l4:Ac pheromone component and oviposition by day—2-treated virgin females. Topical applications were made between 10.00 gnd 11.00 hr to day-2 moths. Samples taken at 21.00 hr (prior to onset of calling) 24, 48, and 72 hr after topical applications (N = 10). For each column means followed by the same letter are not significantly different at the 5% level according to Student—Newman-Keuls' multiple range test. ng E11-14:Ac/p i No. Eggs/3 Treatment 24 hr 48 hr 72 hr 72 hr Control 15.7a 6.8a 4.5a 27.7a Acetone 11.0b 6.23 4.5a 36.1a 10 pg JHA 5.4c 0.36 0.76 324.86 44 DISCUSSION Pheromone production in P, stultana does not appear to be under direct nervous control by the brain. Cutting the ventral nerve cord during the pupal stage did not prevent pheromone production. In other insects, such as bark beetles (Borden ggflgl., 1969; Hughes and Renwick, 1977) and cockroaches (Barth, 1961; Barth and Lester, 1973; Truman and Riddiford, 1974) JR is necessary for pheromone production. Exogenously applied JH III induced pheromone synthesis in‘ng paraconfusus (Borden ggngl., 1969). Hughes and Renwick (1977) suggested that gut extension after feeding in I, paraconfusus removed neural inhibition of JH production by the corpora allata (C.A.) and then the JH mediated brain hormone release which in turn stimulated pheromone production. In several species of cockroaches cyclic changes in levels of JH released by the C.A. influences onset of sexual behaviour and pheromone production (Barth and Lester, 1973; Truman and Riddiford, 1974). However, in the silkmoths Antheraea polyphemus (Cramer) and H. cecropia, the C.A. had no apparent function in calling and oviposition behaviour (Riddiford and Williams, 1971; Truman and Riddiford, 1971). In 2, saccharalis JH was required in tissue culture to prevent cellular disintegration (White ggfl§1., 1972). In 2, ipgggr punctella pheromone production in tissue culture occurred without the presence of JH (Srinivasan, 25 gl., 1979). In P, stultana exogenously applied JHA interfered with, rather than stimulated pheromone production and is probably not involved in new pheromone production. Mating in P} Stultana resulted in the termination of calling and in the gradual elimination of pheromone from the pheromone glands 45 (presumably by enzymatic degradation). In species that mate only once, such as P, stultana, maintenance of pheromone and calling behaviour after mating is not essential and thus, pheromone degradation and termination of calling was not unexpected. However, in species that mate more than once, production of pheromone and calling after mating is essential. In Trichoplusia pi (Hfibner) and several species of Noctuidae, and P, interpunctella (Pyralidae) which typically mate more than once, pheromone titre was unaffected by mating (Shorey, 1963; Shorey ggngl., 1968; Brady and Smithwick, 1968; Hagan and Brady, 1981). Mating caused a significant reduction in calling in T. Bi and g. interpunctella, but did not eliminate calling entirely (Brady and Smithwick, 1968; Hagan and Brady, 1981). Decapitation resulted in elimination of pheromone in P. stultana, presumably by enzymatic degradation. Because cutting the ventral nerve cord had no effect on pheromone biosynthesis, pheromone loss must not be under direct nervous control by the brain. It appears that a factor originating from the head which inhibits pheromone loss must be present to maintain pheromone in the gland. After mating (or decapitation) this factor is either eliminated or its activity is inhibited, allowing pheromone to be lost or degraded. Ian. stultana and H. cecr0pia oviposition occurs shortly (within 24 hr) after mating and normally does not occur in virgin females. In H. cecropia the switch to mated behaviour is triggered by reception of sperm (Truman and Riddiford, 1971). Females mated I to castrated males exhibited an oviposition pattern similar to that of virgin females. It was not determined if the sperm alone could elicit normal mated behaviour, because the accessory glands were not 46 removed. In P. interpunctella and T. §i_viable sperm and male accessory gland secretion was required for maximal oviposition (Karpenko and North, 1973; Lum and Brady, 1973; and Lum and Flaherty, 1970). In .E! cecroEia the sperm (and possibly the accessory gland secretion) cause the bursa copulatrix to release a humoral factor that in turn elicits oviposition and terminates calling (Riddiford and Ashenhurst, 1973). However, the switch from virgin to mated behaviour occurs only if the cc-ca complex is present and connected to the brain (Truman and Riddiford, 1971). The bursa factor probably acts through the central nervous system rather than directly on the corpora cardiaca and the C.A. does not appear to play a role in calling and oviposition in H. cecropia (Riddiford and Williams, 1971; Truman and Riddiford, 1971). In H. cecr0pia JH is sequestered from circulating hemolymph, and also synthesized $2 2232 in and stored by the accessory sex glands (Shirk ggngl., 1980). During mating, JH along with other seminal material, is transferred to the bursa copulatrix of the female. Shirk .E£.§l- (1980) did not demonstrate a function for the transferred JH. In P. stultana, JR 1, II, and III and JHA induced oviposition behaviour comparable to that observed in mated females, and terminated calling within 48 hr. Oviposition was induced only in intact moths, and thus the exogenous JH likely acts through the central nervous system to elicit oviposition. JH may be involved in the switch from virgin to mated behaviour in P, stultana. In light of the present results, it is possible that exogenous JH transferred in the seminal fluid may be involved in the switch from virgin to mated behaviour. 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Am. 74, 286-288. Hill, A. S. and Roelofs, W. L. (1975) Sex pheromone components of the omnivorous leafroller moth, Platynota stultana. J. Chem. Ecol. 1, 91-99 0 Hirano, C. and Muramoto, H. (1976) Effect of age on mating activity of the sweet potato leaf roller, Brachmia macroscopa (Lepidoptera: Hughes, P. R. and Renwick, J. A. A. (1977) Neural and hormonal control of pheromone biosynthesis in the bark beetle, Ipp paraconfusus. 50 Kanno, H. (1979) Effects of age on calling behaviours of the rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae) Bull. Ent. Res. 69, 331-335. Karpenko, C. P., and North, D. T. (1973) Ovipositional response elicited by normal, irradiated, female progeny, or castrated male Trichoplusia 3i (Lepidoptera: Noctuidae). Ann. Ent. Soc. Am. 66, 1278-1280. Lum, P. T. M. and Flaherty, B. R. (1970) Effects of continuous light on the potency of Plodia interpunctella males (Lepidoptera: Phycitidae). Ann. Ent. Soc. Am. 63, 1470-1471. Lum, P. T. 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H. and Gaston, L. K. (1972) Sex pheromones of Lepidoptera. XXVIII. Factors modifying the release rate and extractable quantity of pheromone from females of Trichoplusia Bi (Noctuidae). Ann. Ent. Soc. Am. 65, 954-957. Srinivasan, A., Coffelt, J. A. and Oberlander, h. (1979) In vitro maintenance of the sex pheromone gland of the female Indian meal moth Plodia interpunctella (Hfibner). J. Chem. Ecol. 5, 653-662. Swier, S. R., Rings, R. W. and Musick, G. J. (1977) Age-related calling behavior of the black cutworm, Agrotis ipsilon. Ann. Ent. Soc. Am. 70, 919-924. White, M. R., Amborski, R. L.,Hammond, A. M., Jr. and Amborski, G. F. (1972) Organ culture of the terminal abdominal segment of an adult female lepidopteran. In vitro 8, 30-36. APPENDIX 1.1 voucher Specimen Data Pages 1 of 1 Page mama Heumnno Heme .m Hoooooo done .Enonoz honoEOucm xufimuo>qc= ounum onwficofiz won :« ufimoomp you momefiuoom moaned 0>oom moo po>fiuuom poonooz .m cancmwom N lawma .oz nonopo> Apoauuv Anvmsnz n.u0umw«umo>cH Ahumnnuooa we muomzm Hocowuappn mnav Michigan State University <0 .uoaaanm no.5 scum mocfinuno ounuapu uncunuonng Aemnwcwmaozv manuaaun muochunam moor poufimoaop pom pom: no puuumHHou coxmu umsuo no noaumnm m e r r m m e .m a newswuoam pom pump Hmong namnnnnwmfle u.n e t t .d .d u a E. deiOAAPNLE "mo nonaoz