©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stage-dependent and Temperature-controlled Expression of the Gene Encoding the Precursor Protein of Diapause Hormone and Pheromone Biosynthesis Activating Neuropeptide in the Silkworm, Bombyx mori(*)

(Received for publication, October 24, 1994; and in revised form, December 6, 1994)

Wei-hua Xu (1) Yukihiro Sato (2) Motoko Ikeda (1) Okitsugu Yamashita (1)(§)

From the  (1)Laboratory of Sericultural Science and Entomoresources, School of Agricultural Sciences, and the (2)Radioisotope Research Center, Nagoya University, Chikusa, Nagoya 464-01, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Embryonic diapause and sex pheromone biosynthesis in the silkworm, Bombyx mori, are, respectively, induced by diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN), which are produced in the subesophageal ganglion from a common polyprotein precursor (DH-PBAN precursor) encoded by a single gene (DH-PBAN gene). Using DH-PBAN cDNA as a probe, we quantitatively measured DH-PBAN mRNA content throughout embryonic and postembryonic development and observed the effects of incubation temperature, which is a key factor for determination of diapause, on DH-PBAN gene expression. The silkworm, which is programmed to lay diapause eggs by being incubated at 25 °C, showed peaks of DH-PBAN mRNA content at five different stages throughout the life cycle: at the late embryonic stage, at the middle of the fourth and the fifth larval instars, and at early and late stages of pupal-adult development. In the non-diapause type silkworms programmed by a 15 °C incubation, only the last peak of DH-PBAN mRNA in pupal-adult development was found, and the other peaks were absent. Furthermore, interruption of the incubation period at 25 °C by incubation at 15 °C decreased both DH-PBAN mRNA content in mature embryos and in subesophageal ganglia of day 3 pupae and the incidence of diapause eggs. Thus, there were two types of regulatory mechanisms for DH-PBAN gene expression. One is a temperature-controlled expression that is responsible for diapause induction, and the other is a temperature-independent, stage-dependent expression related to pheromone production.


INTRODUCTION

Insects are the most successful animal group in terms of numbers of species, and estimates of the total number of living species range from 10 to 30 million(1) . Although many factors must contribute to the profusion of insect species, one key element is probably a developmental plan that incorporates metamorphosis and diapause. Diapause confers several adaptive advantages to ensure survival in unfavorable environments and to synchronize the growth rates of population. The initiation and termination of diapause are triggered by a particular set of environmental stimuli such as temperature, photoperiod, humidity, and nutrients. The environmental signals are transduced into endogenous chemical messengers, neurohormones and hormones, in the endocrine organs. Neurohormones bring about the phase change from development to diapause or vice versa through the metabolic shift in the target organs(2, 3, 4) .

The silkworm, Bombyx mori, enters diapause at the early embryonic stage. The diapause nature of the silkworm is primarily a genetic character (voltinism), and bivoltine strains use environmental stimuli as the initial signal to determine the diapause nature. Embryonic diapause is induced by incubating eggs of the maternal generation at a high temperature, such as 25 °C, whereas incubation at a low temperature, such as 15 °C, brings about non-diapause eggs in progeny(5) . When eggs are incubated at an intermediate temperature of 20 °C, long day illumination acts to induce diapause eggs, and short day illumination prevents diapause(6) . High temperature causes the subesophageal ganglion (SG) (^1)to secrete a neurohormone, diapause hormone (DH), during oogenesis, and DH acts on developing oocytes to induce diapause in the resulting embryos(3) . Thus, the silkworm requires one generation from the reception of environmental stimuli to the initiation of diapause through hormone action. Such a long-term control of diapause is observed in several insect species, although the mechanism remains unknown(7) . Consequently, DH of the silkworm provides a key to elucidation of the molecular processes of how environmental stimuli are retained and transduced for diapause determination in insects.

DH is a 24-amino acid peptide amide and is a neuropeptide belonging to the FXPRL amide peptide family(8, 9, 10) . The cloning and characterization of the cDNA and the gene encoding DH have shown that a single gene encodes a common polyprotein precursor, from which DH, pheromone biosynthesis activating neuropeptide (PBAN), and three other FXPRL amide peptides are released through the post-translational processing(10, 11) . Hereafter, the gene is referred to the DH-PBAN gene, and the cDNA and mRNA as DH-PBAN cDNA and DH-PBAN mRNA, respectively.

The availability of the DH-PBAN cDNA allowed us to quantitatively estimate the developmental changes and the effect of incubation temperatures on DH-PBAN mRNA levels. Here, we present the evidence that the DH-PBAN gene is expressed in a stage-dependent manner through embryonic and postembryonic development. Furthermore, we show that the expression of this gene is under the control of the temperature experienced by the developing embryos and that the temperature-controlled DH-PBAN gene expression is closely correlated with the incidence of diapause eggs in progeny.


MATERIALS AND METHODS

Animals

A bivoltine strain (Daizo) of the silkworm, B. mori, was used, because the diapause nature of this strain is completely controlled by the incubation temperature experienced during the embryonic stages. The eggs were incubated either at a constant temperature of 25 or 15 °C or at different combinations of 25 and 15 °C during embryogenesis. The larvae were reared at 25-27 °C on an artificial diet (Vita-Silk, Kyodo Shiryo Co.) or fresh mulberry leaves, and pupae and adults were kept at 25 °C. The developmental stages were synchronized at each molt by collecting newly ecdysed larvae or pupae. The moths were allowed to lay eggs, and the eggs were kept at 25 °C. The diapause nature of laid eggs was checked 2 weeks after oviposition, by which time all non-diapause eggs have hatched. Diapause incidence was expressed as the percentage of moths that laid diapause eggs. In this experiment, only a few moths laid mixed diapause and non-diapause eggs. These moths were excluded from data processing.

RNA Preparation and Northern Analysis

We dissected SGs of the fourth and fifth instar larvae, pupae, pharate adults, and adults. Instead of the SG, heads were collected from the first to the third instar larvae. Total RNA was extracted from eggs, heads, and SGs by the acid-guanidine method(12) . 1 ng of rabbit globin (RG) mRNA/10 SGs or 0.5 g of eggs (weighed at oviposition) was added to the extraction mixture as an internal standard(13) .

For Northern blot analysis, total RNA (20-30 µg) or poly(A) RNA (10 µg) was electrophoresed in 1% agarose gel containing formaldehyde (14) and transferred to a Hybond N membrane (Amersham). As a hybridization probe, the DH-PBAN cDNA was labeled with [alpha-P]dCTP using a random primed DNA labeling kit (Boehringer Mannheim)(15) . The blotted RNA was hybridized with the radiolabeled probe at 42 °C overnight in a hybridization solution (5 times SSPE (180 mM NaCl, 10 mM sodium phosphate, pH 7.7, 1 mM Na(2)EDTA) containing 50% formamide, 5 times Denhardt's reagent, 0.1% SDS, and 100 µg/ml salmon sperm DNA). The hybridized radioactivity was visualized by an image analyzer (BAS 2000, Fuji Photo Film). The mRNA of the silkworm cytoplasmic actin gene (A3) served as an internal standard(16) .

To determine the absolute amount of DH-PBAN mRNA, the DH-PBAN sense cRNA was synthesized in vitro with T7 RNA polymerase using DH-PBAN cDNA clone as a template(10) . After digestion of the template DNA with RNase-free DNase I (Worthington Biochemicals), the cRNA was extracted with phenol and precipitated with ethanol. The cRNA was serially diluted with distilled water and electrophoresed in a 1.2% agarose gel along with 20 µg of total RNA from SGs of day 3 pupae. Northern hybridization and counting of radioactivity were performed as described above. In this experiment, the hybridized radioactivity increased linearly in a range from 0 to 500 ng of cRNA. The amount of DH-PBAN mRNA was estimated from the standard curve constructed with cRNA.

Oligonucleotides Used as Primers and Probes

Oligonucleotides were synthesized using a DNA synthesizer (ABI-391, Applied Biosystems Inc.). The following four synthetic oligonucleotides were used as primers for RT-PCR: oligonucleotide 1, 5`-TTGCACGGATATGAAGGATG-3` corresponding to DH-PBAN cDNA nucleotide sequence 78-97; oligonucleotide 2, 5`-GTAGCAGGCATGTCCTCAGA-3` corresponding to 390-409 of DH-PBAN cDNA sequence(10) ; oligonucleotide 3, 5`-CACTTCGACTTCACCCACGG-3` corresponding to RG gene nucleotide sequence 372-391; and oligonucleotide 4, 5`-TCAGCACGGTGCTCACGTTG-3` corresponding to RG gene sequence 742-761 (13) . As hybridization probes, the following two oligonucleotides were used: oligonucleotide 5, 5`-TCGGCTTGCCTCTCGTAAGG-3` corresponding to DH-PBAN cDNA nucleotide sequence 250-269 and oligonucleotide 6, 5`-GCGGGTGGACCCGGTGAATT-3` corresponding to RG gene nucleotide sequence 541-560(10, 13) .

RT-PCR Amplification and Quantification

Primed with oligo (dT), total RNA (5-10 µg) was reverse transcribed at 42 °C for 1 h in 1 times RT buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl(2)), 0.5 mM dNTPs, 10 mM dithiothreitol, and Superscript reverse transcriptase (Life Technologies, Inc.) in a total volume of 20 µl(17) . PCR amplifications were carried out on one-fifth or one-tenth of the reverse transcription mixture in a solution containing 50 mM KCl, 10 mM Tris, pH 8.4 (25 °C), 1.5 mM MgCl(2), 0.2 mM of each dNTP, 1 µM oligonucleotides 1 and 2 for DH-PBAN or 1 µM oligonucleotides 3 and 4 for RG, and 1.25 units of Taq polymerase (Perkin-Elmer) in a final volume of 50 µl. The thermal profile of the PCR involved 18 or 25 cycles of 1 min at 93 °C, 45 s at 55 °C, and 1 min at 72 °C. The PCR products were subjected to slot blot hybridization using oligonucleotide 5 for DH-PBAN or oligonucleotide 6 for RG as a probe(18) . The hybridized radioactivity was measured by an image analyzer and corrected by radioactivity recovered in RG mRNA, which was added as an internal standard.


RESULTS

Developmental Changes in DH-PBAN mRNA Content during Embryonic and Postembryonic Development

A Northern hybridization analysis was carried out on RNA extracted from the eggs and SGs of larvae, pupae, and adults of the silkworm destined to lay diapause eggs by incubation at 25 °C (Fig. 1). All RNA samples examined gave a single positive band of 0.8 kilobase, which indicates that the same species of DH-PBAN mRNA is transcribed throughout its life cycle.


Figure 1: DH-PBAN mRNA from eggs and SGs of the diapause type silkworm analyzed by a Northern hybridization. RNA was extracted from day 8 eggs (lane E8), and SGs from fourth instar day 3 larvae (lane L4), fifth instar day 5 larvae (lane L5), day 3 pupae (lane P3), day 6 pharate adult (lane P6), and newly eclosed adults (lane A0) and hybridized with P-labeled DH-PBAN cDNA as a probe. To the E8 lane, 10 µg of poly(A) RNA was applied, and to the other lanes 30 µg of total RNA was applied. The same blot was hybridized with the Bombyx actin cDNA as a control as shown in the lower panel.



We measured the DH-PBAN mRNA content of whole eggs incubated continuously at 25 °C by RT-PCR (Fig. 2). DH-PBAN mRNA remained at a trace level during the first half stage of embryogenesis and abruptly increased during the middle stage. The mRNA content was maintained at a high level through the remainder of embryogenesis and suddenly decreased at larval hatching.


Figure 2: Changes in DH-PBAN mRNA content during embryogenesis of the Daizo strain incubated at 25 (bullet) and at 15 °C (circle). Embryogenesis took 9 days in eggs incubated at 25 °C and 23 days in those incubated at 15 °C; therefore the developmental stage is shown as the percentage in days from oviposition (0%) to larval hatching (100%). RNA (5-10 µg) extracted from eggs at various stages was subjected to RT-PCR (25 cycles) for quantification. The hybridized radioactivity was measured by an image analyzer and corrected by radioactivity recovered in RG mRNA, which was added as an internal standard. The results are shown as the amounts of DH-PBAN mRNA relative to the highest value (100%). Each point represents the mean values from three repeated experiments with the S.D. shown by a vertical bar.



To follow the changes in mRNA content during postembryonic development, we prepared RNA from whole heads until the third larval instar and from the SGs or the brain-SG complexes from the fourth larval instar to adult eclosion, and then we carried out quantitative RT-PCR (Fig. 3). A trace level of DH-PBAN mRNA was found in head RNA from the first to third instar larvae (data not shown). During the fourth and fifth larval instar, a peak appeared at the middle stage of each instar. Two peaks appeared during the pupal-adult development: at day 2-3 and at day 6.


Figure 3: Changes in DH-PBAN mRNA content during postembryonic development of diapause type silkworms (A) and non-diapause type silkworms (B). Total RNA was extracted from SGs, subjected to RT-PCR amplification (18 cycles), and then quantified. The results are shown as the relative amounts of DH-PBAN mRNA as in Fig. 2. Each point represents the mean values of three different experiments with the S.D. shown by a vertical bar. L4, L5, P, and A represent fourth and fifth larval instar, pupal-adult development, and adults, respectively.



Effect of Incubation Temperature on Expression of DH-PBAN Gene

We also followed the changes in DH-PBAN mRNA content of the silkworm destined to lay non-diapause eggs by incubating at 15 °C throughout embryonic and postembryonic development, using the same procedure as described above ( Fig. 2and Fig. 3). It took 23 days from oviposition to larval hatching during a 15 °C incubation, which prolonged the embryonic period to 2.5 times as long as that of eggs incubated at 25 °C. In contrast, the 15 °C incubation caused stimulation of the larval growth rate and reduced by 3 days the time from the third larval ecdysis to adult eclosion compared with the 25 °C incubation. Throughout embryonic development at 15 °C, there was no change in DH-PBAN mRNA content, which remained at a trace level (Fig. 2). Throughout postembryonic development, only one peak of DH-PBAN mRNA was found on day 5 after pupation, and no appreciable increase in the mRNA content was found in the larval stages or in the early stage of pupal-adult development (Fig. 3). Thus, it is clear that incubation temperature plays a crucial role in the regulation of DH-PBAN gene expression not only in embryos but also in larvae and pupae.

The active secretion of DH from the SG at the early to middle stages of pupal-adult development was required for induction of diapause eggs in progeny(19) , and at this stage DH activity and DH content were 2-3-fold higher in the diapause type SG than non-diapause type SG (20) . To correlate the DH-PBAN gene expression to the diapause-inducing activity of the SG at this stage, we quantified DH-PBAN mRNA in the SG of the diapause and the non-diapause silkworms using Northern blot hybridization along with DH-PBAN cRNA as reference. For the diapause type SG of day 3 pupae, the amount of DH-PBAN mRNA was estimated to be 2.55 pg of mRNA/µg of total RNA. One SG contained 1.50 µg of total RNA, so that the amount of DH-PBAN mRNA was calculated to be 3.83 pg/SG or 8.72 times 10^6 molecules/SG, based on the molecular size of 0.8 kilobase (Fig. 1). In the non-diapause type SG of day 3 pupae, the amount of DH-PBAN mRNA was estimated to be 1.40 pg/SG or 3.19 times 10^6 molecules/SG. Thus, at the early stage of pupal-adult development, DH-PBAN mRNA content was estimated to be 2.7 times higher in diapause type SG than in the non-diapause SG.

Relationship between DH-PBAN mRNA Content and Diapause Incidence

The critical period for temperature to cause diapause induction is during stages 17-27 of embryogenesis(5) , during which time differentiation and histogenesis of larval tissues occurs(21) . To determine whether the induction of diapause eggs and DH-PBAN gene expression is decided by the same stimulus, we incubated eggs at 25 or 15 °C for different periods of embryogenesis, and measured DH-PBAN mRNA content of embryos at stage 27 and that of SGs of day 3 pupae, and observed the incidence of diapause in the eggs that were laid (Fig. 4). At the time of the temperature shift from 25 to 15 °C or vice versa, we dissected out embryos from more than 20 eggs to check the developmental stage. A 25 °C incubation during stages 20-23 was essential for inducing diapause eggs and for complete induction of diapause a longer incubation at 25 °C was needed. Such incubations markedly increased DH-PBAN mRNA levels in embryos and in SGs (Experiment I in Fig. 4). We further changed the stages and periods of temperature treatments to obtain populations with different rates of diapause and non-diapause eggs (Experiment II in Fig. 4). In addition to stages 20-23, stages 22-25 were important for the induction of diapause eggs. The reduction or the interruption of 25 °C incubation periods at these stages by 15 °C incubation caused reduction of diapause incidence. DH-PBAN mRNA content clearly decreased in embryos and SGs after reduction or interruption of 25 °C exposure.


Figure 4: Effects of incubation temperatures on diapause incidence and DH-PBAN mRNA content. Eggs were incubated at different temperatures for different periods. DH-PBAN mRNA content of embryos at stage 27 and SGs of day 3 pupae was measured by RT-PCR and Southern hybridization. Diapause incidence is shown as the percentages of moths who laid diapause eggs. DH-PBAN mRNA content is shown as the ratio to the amounts found in embryos and SG incubated continuously at 25 °C. The incubation temperatures at 25 and at 15 °C are indicated by heavy lines and broken lines, respectively. Numbers in parentheses show numbers of moths used.



The correlation between the DH-PBAN mRNA amounts in embryos and in SGs of day 3 pupae and diapause incidence is summarized in Fig. 5. The incidence of diapause eggs closely correlated to DH-PBAN mRNA levels in embryos with a correlation coefficient of 0.97 (Fig. 5A). Similarly, DH-PBAN mRNA content in SGs of day 3 pupae increased significantly with increasing ratios of diapause incidence, but the correlation coefficient was somewhat less than that found in embryos of stage 27 (Fig. 5B). Thus, the expression of this gene and the induction of diapause eggs are under the control of the same temperature signal.


Figure 5: Relationships between incidence of diapause eggs and DH-PBAN mRNA content. The data shown in experiment II of Fig. 4were used for estimation of the regression curve. A, dependence on DH-PBAN mRNA content in embryos. B, dependence on DH-PBAN mRNA content in SG of day 3 pupae.




DISCUSSION

Previous studies have demonstrated that a single gene (the DH-PBAN gene) encodes a polyprotein precursor from which five different neuropeptides, DH, PBAN, and alpha-, beta- and -SG neuropeptides, are released through post-translational processing(10) , and this gene is expressed in only 12 neurosecretory cells of the SG(22) . In the present study, the developmental profile and the temperature effects on gene expression were examined to obtain some insights into the molecular processes underlying the thermoregulation of diapause induction in the silkworm. The DH-PBAN gene is temporally expressed at five different stages during the life cycle of the diapause type silkworm, whereas the non-diapause type silkworm retained only the last peak, which occurred during the late stage of pupal-adult development. Thus, it is clear that there are two types of regulatory mechanisms for gene expression: one is a temperature-controlled expression and the other is a developmental regulation.

The developmental regulation of the DH-PBAN gene seems to depend on physiological events other than diapause. A sex pheromone of the silkworm, bombycol, is synthesized in the pheromone gland and released just after eclosion by female moths irrespective of diapause nature, and PBAN acts before adult eclosion to stimulate pheromone production (23, 24) . Thus, DH-PBAN gene expression at this stage is involved in sex pheromone production through biosynthesis of PBAN.

The temperature-controlled expression of the DH-PBAN gene found in the embryonic, larval, and pupal stages could be concerned directly or indirectly in diapause induction. We have already demonstrated that DH is actively secreted from the SG at the early to middle stages of pupal-adult development and acts on developing oocytes to induce a metabolic shift leading to diapause-specific metabolism through trehalase gene expression in ovaries(3, 19, 25) . Thus, DH-PBAN gene expression at this stage seems to be directly involved in diapause induction through DH biosynthesis. One SG of day 3 diapause type pupae contains a 3-fold higher amount of DH-PBAN mRNA than a non-diapause type SG, which roughly correlates with the difference in diapause egg-inducing activity of SG from diapause and non-diapause types(20) . Furthermore, the close correlation between DH-PBAN mRNA content in SGs at this stage and diapause incidence (Fig. 5) provides additional evidence that DH-PBAN gene expression is an initial event in the molecular processes leading to diapause induction.

It is well known that, compared with incubation at 15 °C, incubation at 25 °C of eggs of the bivoltine strain causes the larvae to become heavier in body weight and to make larger cocoons by prolonging the feeding periods. The temperature-controlled DH-PBAN gene expression found at the fourth and fifth larval stage is likely related to the regulation of larval growth.

Finally, it is important to notice that DH-PBAN gene expression is more immediately induced in embryos by a high temperature incubation and that this is a crucial treatment for induction of embryonic diapause in progeny. At the temperature sensitive stages of embryonic development, the central nervous system, including the SG, has been differentiated, so that the gene may be expressed in the SG of embryos. The close correlation of DH-PBAN mRNA content in mature embryos to diapause incidence in progeny strongly indicates the possibility that DH-PBAN gene expression is a direct response to the environmental stimulus that eventually results in the biological phenomenon through a sequential action of the gene products. However, a sudden decrease in DH-PBAN mRNA levels at the end of embryonic life suggests that the temperature signal acts temporally to induce gene expression but that the gene products are not carried over to the postembryonic stages as the chemical messages transduced from the environmental signal. Therefore, it is still unknown how the environmental information received during the embryonic stages is stored and transmitted to control the gene expression in the SG during postembryonic development.

The effective temperature for diapause induction is shown to be between 24 and 27 °C, and higher or lower temperatures are ineffective(5) . Such a temperature stimulus is completely different from that required for induction of the heat-shock events in which a higher temperature such as 37 °C is required as a effective stimulus. In the silkworm, the heat-shock effect is induced by exposing eggs to 46 °C for 5 min, which causes a 2-kilobase mRNA along with a 70 kDa protein, which is homologous to a Drosophila HSP70 protein. The effect is induced 6 h after the heat-shock treatment(26, 27, 28) . Thus, silkworm eggs are able to respond to the heat-shock stimulus in a normal fashion. Consequently, the observed temperature effect on the induction of DH-PBAN gene expression seems to be mediated by a mechanism different from heat-shock induction. Although the molecular mechanisms are not yet known, the present results provide the basis for elucidating the regulatory mechanisms by which non-stressing temperature acts as an environmental stimulus for regulation of growth and development in many organisms.


FOOTNOTES

*
This study was supported by Grants-in-aid for Scientific Research 02304020, 03404008, and 05760047 from the Ministry of Education, Science, and Culture of Japan and Grant-in-aid BMP 94-V-1-6-4 of the Bio Media Program from the Ministry of Agriculture, Forestry, and Fisheries of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-52-7894036. Fax: 81-52-7894012.

(^1)
The abbreviations used are: SG, subesophageal ganglion; DH, diapause hormone; PBAN, pheromone biosynthesis activating neuropeptide; RG, rabbit globin; RT, reverse transcription; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Drs. M. Kobayashi and T. Yaginuma for their encouragement and K. Sakakibara for silkworm rearing in our laboratory.


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