Animals

M. brassicae was maintained in the laboratory at Yamaguchi University for more than 15 years. Larvae were kept in transparent plastic containers (12 × 18 × 6 cm³) and were subjected to one of the following lighting conditions: 10-hr light and 14-hr dark (10L-14D, short-day conditions), 14L-10D (long-day conditions) or 16L-8D (typical long-day conditions) at 23°C. All the larvae reared under 14L-10D developed into ND-pupae and the majority (>98%) of larvae reared under16L-8D developed into AD-pupae at 23°C. The ND- and AD-pupae required 17–25 days and 30–60 days respectively to complete adult development at 25°C. All larvae raised from the egg stage under 10L-14D at 23°C developed into WD-pupae which showed no signs of adult development within 60 days after pupation at 25°C. Larvae destined to develop into ND-, WD- and AD-pupae under 14L-10D, 10L-14D and 16L-8D at 23°C are referred to as ND-, WD- and AD-larvae, respectively. Newly molted 6th-instar larvae were sorted from stock cultures every evening to obtain larvae of approximately the same developmental stages.

Larvae were fed on an artificial diet (Agui et al., 1975), and adults were fed on a 10% sucrose solution.

Extraction of PTTHs

Brains dissected from 6th-instar larvae and pupae of different stages were stored at −85°C. A batch of 300 brains was homogenized in 2.5 ml of ice-cold acetone with a glass homogenizer, washed in 1.0 ml ice-cold 80% ethanol and extracted with 1.0 ml Grace's medium (GIBCO Lab., New York, USA). Brain extracts were heated for 3 min at 95°C, cooled rapidly and centrifuged. At each step, insoluble material was separated by centrifugation at 12,000 × g for 20–30 min at 4°C.

Separation of small and big PTTHs

Brain extracts were put into tubes with an ultrafilter (Ultrafree C3LGCOO, Millipore Tokyo) passing molecules smaller than 10 kDa and centrifuged at 3,000 × g for 4–5 hr at 4°C. The resulting filtrates were used as small-PTTH fractions, while the residues were washed with 1.0 ml Grace's medium and were used as big-PTTH fractions.

Assay of hemolymph ecdysteroids

Five microliters of hemolymph was collected from each of 10 insects at 12:00–14:00 (2 hr beyond the middle of the photophase). Ecdysteroids in each sample were extracted with 400 μl of methanol and the titer was quantified by radioimmunoassay (RIA) (Borst and O'Conner, 1972).

Assay of PTTH activity

The PTTH activity of brain extracts was quantified by the in vitro assay (Bollenbacher et al., 1979) using the PGs from 2-day-old 6th-instar ND-larvae. Pairs of the PGs were obtained by dissection in saline (Wyatt, 1961) at 12:00–16:00 and washed in 50 μl of Grace's medium for 30–60 min. One of each PG pair was incubated in Grace's medium alone (50 μl) and the other was incubated in Grace's medium (50 μl) containing one of the extracts. After a 1- or 2-hr incubation, the PGs were removed and the amount of ecdysteroids secreted into the incubation medium was quantified by the RIA method. The activation ratio (Ar) representing PTTH activity of the brain extract was obtained by incubations of 5–6 gland pairs. The Ar-value shows the amount of ecdysteroids secreted by the experimental gland divided by that secreted by the contralateral gland (Bollenbacher et al., 1979).

Quantification of sensitivity of the PGs

Pairs of PGs from ND-, WD- and AD-larvae of different stages were obtained by dissection in saline at 12:00–14:00 and washed in 50 μl Grace's medium for 30–60 min. They were incubated in Grace's medium with or without brain extracts of 0-day-old ND-pupae (one brain-equivalent/50 μl). The ecdysteroids secreted in each incubation medium for 2 hr was quantified by RIA, and the rate of ecdysteroids secretion was calculated. The Ar-value showing the sensitivity of the PGs to big PTTH in vitro was obtained as described above.

Radioimmunoassay (RIA) of ecdysteroids

The ecdysteroids in each sample were quantified by the RIA (Borst and O'Conner, 1972) with ecdysone (Sigma Chemical Co., St. Louis, USA) as a standard. Antiserum raised against 20-hydroxyecdysone (Rhoto Pharmaceutical Ltd., Osaka, Japan) was obtained from the Meguro Institute, Osaka, Japan. This antiserum exhibits reactivity with 20-hydroxyecdysone approximately 5-times stronger than that with ecdysone and 3-dehydroecdysone. Radioactive ecdysone, (23, 24-³H)-ecdysone (2.96 GBq/mmol), was obtained from New England Nuclear, Boston, USA. The minimal amount of ecdysteroids quantified by the RIA method was 2.5 pg(ecdysone equivalents)/tube.

Stage-dependent fluctuations of the titers of hemolymph ecdysteroids in 6th-instar larvae and pupae

To examine stage-dependent fluctuations of the hemolymph ecdysteroid titer under different photoperiodic conditions, newly molted 6th-instar larvae were sorted from stock cultures reared under 14L-10D, 10L-14D or 16L-8D at 23°C. Daily hemolymph samples for RIA were collected from larvae and pupae from the first day of the 6th instar to the 7th or 12th day after pupation. Five microliters of hemolymph was collected from each of 10 insects at different stages. AD-pupae whose hemolymph was collected for RIA were kept at 25°C to determine whether they formed spots (a sign of adult development). If the AD-pupa formed spots within 20 days, the hemolymph sample was discarded.

Early in the 6th instar the hemolymph ecdysteroid titers were low (5–10 pg/μl) in ND-, WD- and AD-larvae. The titer began to increase 0–2 days before the larvae stopped feeding and peaked 2 days before pupation in all types of larvae. Thereafter, the titers decreased quickly as the larvae approached pupation (Fig. 1a, b, c).

Fig. 1

Developmental changes of the titers of hemolymph ecdysteroids in larvae and pupae raised from the egg stage under 14L-10D (a), 10L-14D (b) and 16L-8D (c) at 23°C. Open circles and straight lines show the titers of hemolymph ecdysteroids and their standard errors, respectively. Solid arrows show the days of 5th larval ecdysis (left), wandering (middle) and pupation (right).

The ecdysteroid titer of ND-pupae increased after pupation and peaked (4.1 ng/μl) 3 days later. The titer decreased thereafter, and remained low in the second half of the pupal stage (Fig. 1a). In WD- and AD-pupae, the titer decreased as pupation approached, and remained low before and during both winter and aestival diapause. The ecdysteroid titer in diapausing AD-pupae (40–80 pg/μl) was 8- to 10-times higher than that in diapausing WD-pupae (4–5 pg/μl) (Fig. 1b, c).

The number of days which larvae require for the 6th instar is different, but profiles of the fluctuations of the titers of hemolymph ecdysteroids do not vary in the ND-, WD- and AD-larvae of the 6th instar (Fig. 1a, b, c).

Responsiveness of the PGs to the big and small PTTHs in 5th-instar ND-larvae, 6th-instar ND-larvae and ND-pupae

To examine the responsiveness of PGs to big and small PTTHs, newly molted 5th- and 6th-instar larvae and newly molted pupae were sorted from a stock culture raised from the egg stage under 14L-10D (long-day conditions) at 23°C. Pairs of the PGs isolated from 2-day-old 5th-instar ND-larvae, 2-day-old 6th-instar ND-larvae and 2-day-old ND-pupae were incubated in Grace's medium with or without big- and small-PTTH fractions. The dosage of big- and small-PTTH fractions added to the incubation medium was changed from 1/8 to 4 brain-equivalents/50 μl.

The PGs isolated from 5th-instar and 6th-instar ND-larvae as well as those from ND-pupae showed a dose-dependent response to big-PTTH and small-PTTH fractions in vitro (Fig. 2). The doses of the big-PTTH fraction giving maximal Ar-values for the PGs of 5th-instar ND-larvae, 6th-instar ND-larvae and ND-pupae (Ar 10.0, Ar 8.0 and Ar 11.0) were 2.0, 2.0 and 1.0 brain-equivalents/50 μl, respectively. Half maximal Ar-values were obtained by doses of 1/2, 1/4 and 1/2 brain-equivalent/50 μl, respectively (Fig. 2).

Fig. 2

Dose-dependent response of the PGs to the big and small PTTHs. PGs were obtained from 5th-instar larvae (left), 6th-instar larvae (middle) and pupae (right) raised from the egg stage under 14L-10D at 23°C. Open and solid circles show a response of the PG(s) (Ar-values) to big-PTTH and small-PTTH fractions with standard errors.

The titers of small-PTTH fraction for inducing a detectable PG response (Ar > 2) were 2, 2 and 4 brain-equivalents/50 μl in the PGs of 5th-instar ND-larvae, 6th-instar ND-larvae and ND-pupae, respectively (Fig. 2).

The results indicate that responsiveness of the PGs to the big and small PTTHs does not vary among the young 5th-instar larval, the young 6th-instar larval and the young pupal stages. With respect to the induction of the minimal response of the PGs, the small-PTTH fraction requires 8- to 10-times larger doses than the dose of the big-PTTH fraction.

Stage-dependent fluctuations of activity of the PGs and their sensitivity to brain-extracts in three types of 6th-instar larvae and pupae

To investigate how the PG activity and the sensitivity of the PGs to big PTTH change during the 6th-instar larval and the early pupal stages, newly molted 6th-instar larvae and newly molted pupae were sorted from stock cultures raised from the egg stage under 14L-10D, 10L-14D or 16L-8D at 23°C. Pairs of PGs isolated from 6th-instar larvae and pupae were incubated in Grace's medium, one with and the other without the brain extract of 0-day-old ND-pupae (one brain-equivalent/50 μl). Daily collections of PGs were isolated from day 0 of the 6th instar to day 0 or 1 of pupae.

All PGs incubated in Grace's medium alone secreted ecdysteroids. The activity of the PGs, i.e. the rate of ecdysteroid secretion, varied with the stage of the PG donors (Fig. 3a, b, c). The PGs isolated from ND-, WD- and AD-larvae of the early 6th instar secreted ecdysteroids at a rate of 25–30 pg/gland/hr. Activity increased as the days passed and reached a peak (2.8–3.8 ng/gland/hr) on day 4–6 of the 6th instar (0–3days before wandering). Thereafter, it decreased as pupation approached (Fig. 3a, b, c).

Fig. 3

Developmental changes of the PG activity and sensitivity of the PGs to a brain-extract in 6th-instar larvae reared under 14L-10D (a), 10L-14D (b) or 16L-8D (c) at 23°C. Open and solid triangles show the activities of the experimental and control PGs (the rate of ecdysteroid secretion), respectively. Solid circles show the responsiveness of the PGs (Ar-values) to the brain extract of 0-day-old ND-pupae (one brain-equivalent/50 μl). Solid arrows show the days of 5th larval ecdysis (left), wandering (middle) and pupation (right).

The activity of the PGs increased when they were treated with brain-extract (one brain-equivalent/50 μl) in vitro. The fluctuating patterns which the treated PGs showed with the developmental stages were similar to those in the contralateral PGs incubated in Grace's medium alone (Fig. 3a, b, c). The sensitivity of the PGs to the brain extracts of 0-day-old ND-pupae (one brain-equivalent/50 μl) was measured in vitro. Ar-values were 8.6, 11 and 1.9 in the PGs of ND-, WD- and AD-larvae respectively on the first day of the 6th instar. The Ar-values became larger as the days went by and reached a peak on day 2 (Ar 21) in ND-larvae and on day 4 (Ar 40 and Ar 8) in WD- and AD-larvae. Thereafter, the Ar-values decreased quickly and remained low and constant throughout the second half of the 6th-instar larval stage (Fig. 3a, b, c).

The activity of the PGs and their sensitivity to big PTTH were low on the first day of the 6th instar in all types of larvae. The activity and sensitivity of the PGs increased as the days went by and reached a peak 0–3 days and 2–3 days before wandering, respectively. Both activity and sensitivity appear to decrease quickly when a small amount of ecdysteroids appear in the hemolymph of the 6th-instar larvae.

Fluctuations of PTTH-activity in brain-extracts from ND-, WD- and AD-larvae

To investigate how the PTTH-activity of the brains fluctuates during the 6th-instar larval and pupal stages, brains were obtained from the three types of 6th-instar larvae and pupae at different stages. Batches of 600 brains were extracted with Grace's medium and PTTH activity was quantified by in vitro assay using PGs from 2-day-old 6th-instar ND-larvae.

The PTTH-activity in brain-extracts changed with the stages in ND-, WD- and AD-larvae of the 6th instar (Fig. 4a, b, c). PTTH-activity was low (Ar 5.4) on the first day of the 6th instar in ND-larvae and increased to a small peak (Ar 13.1) on day 3 (2 days before wandering). It decreased, thereafter, but increased again and reached a maximum (Ar 21.7) on day 7 (one day before the peak of the titer of hemolymph ecdysteroids) (Fig. 4a).

Fig. 4

Stage-dependent changes of PTTH activity in brain extracts of larvae and pupae raised from the egg stage under 14L-10D (a), 10L-14D (b) or 16L-8D (c) at 23°C. The PGs were obtained from 2-day-old 6th-instar ND-larvae. Open circles and straight lines show the PTTH activity of brain extracts (one brain-equivalent/50 μl) expressed in Ar-value and the standard errors. Thin straight lines show the fluctuations of the titers of hemolymph ecdysteroids, redrawn from Fig. 1 with some modifications. Solid arrows show the days of 5th larval ecdysis (left), wandering (middle) and pupation (right).

Fluctuations in the PTTH-activity of the brain seemed to be nearly the same among the three types of insects in the 6th-instar larval stage, but were clearly different in the pupal stage. In WD-pupae, the PTTH-activity increased rapidly after pupation (Fig. 4b), whereas in the AD- or ND-pupae, the activity did not increase or increased only gradually after pupation (Fig. 4c, a).