Relationship between photoperiod and secretion of the diapause hormone during larval stages of the silkworm, Bombyx mori L., reared on an artificial diet

The suboesophageal ganglion of the silkworm, Bombyx mori synthesizes sufficient diapause hormone to produce diapause eggs, regardless of the photoperiodic conditions experienced during the larval stages. When larvae destined to produce non-diapause eggs are implanted with the brain-suboesophageal ganglion complex from larvae which have been reared under short-day conditions, the resulting adults lay diapause eggs. The larvae receiving the complex from larvae reared under long-day conditions gave rise to adults which did not produce any diapause eggs. The brains from pupae which have been reared under long-day conditions show an activity inhibiting the secretion of diapause hormone by the suboesophageal ganglion. The mechanism through which the brain controls the secretion of diapause hormone from the suboesophageal ganglion can be modified by photoperiodic conditions during the larval stages.     

Control of diapause-factor secretion from the suboesophageal ganglion in the silkworm,Bombyx mori: The roles of the protocerebrum and tritocerebrum

To localize any functional areas in the brain which control the secretion of the diapause factor from the suboesophageal ganglion in the Bombyx silkworm, various parts of the brain were excised or transected surgically. It was demonstrated that cortices slightly lateral to the median line on the dorsal side of the protocerebrum (Lmdp) are closely related to the inhibitory mechanism in the pupa producing non-diapause eggs. On the other hand, cortices of anterolateral areas on the ventral side of the protocerebrum (Alvp) participate in the stimulatory mechanism in the pupa producing diapause eggs. Moreover, it is suggested that the tritocerebrum is competent to stimulate secretion of the diapause factor from the suboesophageal ganglion not only in the pupae producing diapause eggs but also in the pupae producing non-diapause eggs. From these results, we propose a possible relationship between the Alvp, Lmdp and tritocerebrum concerning regulation of diapause-factor secretion from the suboesophageal ganglion.     

An active principle from the corpora cardiaca,corpora allata and suboesophageal ganglion of the locust,inducing egg diapause in Bombyx mori

The activity of the substance(s) which are contained in the cephalic endocrine organs of the locust which induce egg diapause in Bombyx mori was examined by implantation and injection of saline extracts of these organs. Extracts from the median and lateral neurosecretory parts of the locust brain were not effective in inducing egg diapause. Extracts of the corpora cardiaca, corpora allata, and suboesophageal ganglion of the locust induced diapause eggs in Bombyx pharate adults from which the suboesophageal ganglion had been removed. The first two extracts could induce egg diapause even in isolated abdomens of pharate adults of Bombyx. In the locust corpora cardiaca, the activity was present only in the glandular lobe and not in the nervous region. This activity decreased when the nervi corporis cardiaci I and II and of nervi corporis allati I were cut. Allatectomy also brought about a decrease in the activity in the glandular lobe which could not be restored by the injection of juvenile hormone. The activity in the corpora allata was enhanced slightly by the disconnection though not significantly.From these results, it is assumed that the corpora cardiaca, corpora allata and suboesophageal ganglion of the locust contain and active principle(s) capable of inducing egg diapause in Bombyx mori. The nervous connections between the brain, corpora cardiaca, and corpora allata are essential for the accumulation of the active substance(s) in the glandular lobes of the corpora cardiaca.     

The hormonal regulation of the larval diapause in the codling moth, Laspeyresia pomonella (Lep. Tortricidae)

The hormonal control of the facultative diapause of the codling moth has been investigated. The diapause can be divided into 4 phases or periods: (1) diapause induction by short-day conditions (SD) in young larvae, (2) initiation of the diapause in the early last larval instar by a high titre of juvenile hormone, (3) onset and maintenance of diapause with inactivity of the neuroendocrine system, as evidenced by the results of neck-ligation experiments, (4)termination of diapause by the production of ecdysteroid.Diapause-induced larvae pupated after spinning the cocoon, if the state of induction was changed by injection with the anti-juvenile hormone precocene II at the beginning of the last larval instar and subsequent results of neck-ligation experiments, (4) termination of diapause by the production of ecdysteroid. treated with juvenile hormone during the first 1.5 days after the last larval moult and subsequently reared under SD. Under LD, continuous application of juvenile hormone during the last larval instar and after spinning did not prevent the insects from moulting to either a supernumerary larva, a pupa or a larval-pupal intermediate. Termination of diapause, i.e. pupation, was achieved by injecting diapausing larvae with 20-hydroxyecdysone. Although juvenile hormone was found to have a prothoractropic effect in diapausing larvae, no pupal moult could be induced by the application of the hormone. Contrary to the hormonal situation before pupation of nondiapausing larvae, no juvenile hormone could be detected before or during the pupation of larvae after diapause.     

Isolation of the diapause hormone from the silkworm,Bombyx mori

The diapause hormone (DH) secreted from the suboesophageal ganglion is responsible for the embryonic diapause in the silkworm, Bombyx mori. It was extracted from two million mated male adult heads. At least two species of DH were separated from the extracts (a complex lipid fraction) by gel permeation chromatography with Sephadex LH-20 by organic eluants. One of them was further purified by repeating gel permeation column chromatography with Merckogel^® Type OR 6000 in the cold and dark to give finally a single peak with high DH activity. This preparation seems to be chemically pure, and its molecular weight is chromatographically estimated to be between 2000 and 4000. Other characteristics of DH are also presented. Biological activity of the final DH preparation is discussed with reference to other insect hormones such as juvenile hormone and α-ecdysone.     

Induction of egg diapause in Bombyx mori by some cephalo-thoracic organs of the cockroach, Periplaneta americana

Induction of egg diapause in the silkworm, Bombyx mori by some cephalo-thoracic organs of the cockroach, Periplaneta americana was examined. All tissues tested such as brain, corpora cardiaca, corpora allata, suboesophageal and thoracic ganglia and nerve cords between thoracic ganglia were able to produce diapause eggs in non-diapause egg producers both by transplantation and injection of their crude homogenates. The homogenate of thoracic ganglia was effective even in pharate adults with the suboesophageal ganglion removed or in isolated abdomens of pharate adults.From these results, it was surmised that some endocrine organs, as well as the central nervous system in the cephalo-thorax of Periplaneta americana, contained the active principle responsible for egg diapause in Bombyx mori.     

Hormonal control of larval coloration in the armyworm, Leucania separata

Leucania separata larvae show various degrees of darkening depending on the population density. A ligature applied behind the thorax of crowded or yellow solitary larvae caused black or reddish brown pigmentation in the anterior part after the larval ecdysis. Extirpation of the brain, the corpus cardiacumcorpus allatum complexes, or the suboesophageal ganglion reduced the degree of melanization in the crowded larvae, lack of the suboesophageal ganglion having a particularly striking effect. Transplantation of 3 complexes of brain-corpora cardiaca-corpora allata-suboesophageal ganglion induced intense black pigmentation in the isolated abdomens of crowded larvae and reddish brown pigmentation with some melanization in the isolated abdomens of yellow solitary larvae, though the melanization in the latter was weaker than in the former. Implantation of these organs or of the suboesophageal ganglia into yellow solitary larvae caused black and reddish brown pigmentation after a larval ecdysis. In the pieces of integument implanted into the body cavity of crowded larvae, melanization occurred after ecdysis, whereas it did not occur in most of the fragments implanted in yellow solitary larvae. Transplantation of corpora allata and other organs from solitary larvae or injection of juvenile hormone into crowded larvae did not inhibit melanization.     

Molecular cloning,developmental expression and tissue distribution of diapause hormone and pheromone biosynthesis activating neuropeptide in the bamboo borer Omphisa fuscidentalis

Diapause, an arrested period of post‐embryonic development in insects, is under the control of hormonal interactions. In the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae), larvae remain in diapause for as long as 9 months during the dry season, from September to the following June, although the factors that regulate larval diapause are poorly understood. The present study describes the cloning and expression analysis of the diapause hormone and pheromone biosynthesis activating neuropeptide (DH‐PBAN) precursor of O. fuscidentalis (Ompfu‐DH‐PBAN cDNA), aiming to reveal how it may be involved regulating larval diapause in this species in combination with environmental factors. The open reading frame (ORF) of the cDNA encodes a 199‐amino acid precursor protein that contains DH, PBAN and three other neuropeptides, all of which share a conservative C‐terminal pentapeptide motif FXPR/KL (X = G, T or S). The Ompfu‐DH‐PBAN is highly similar (74%) to the DH‐PBAN of the legume pod borer (Maruca vitrata). A quantitative real‐time polymerase chain reaction reveals that Ompfu‐DH‐PBAN mRNA is expressed only in neural tissues and that expression is highest in the suboesophageal ganglion. In addition, the expression level of Ompfu‐DH‐PBAN mRNA in the suboesophageal ganglion is consistently high during the fifth larval instar, increasing moderately in early diapause before reaching a peak during late diapause. After pupation, expression of the Ompfu‐DH‐PBAN precursor decreases to a low level. In addition to endocrine factors, the results demonstrate that photoperiod increases the expression level of Ompfu‐DH‐PBAN mRNA in larval diapause. These results also suggest that the expression of the Ompfu‐DH‐PBAN gene correlates with larval diapause development and may be activated by photoperiod in O. fuscidentalis.     

Molecular mechanism of diapause in insect (I)

The embryonic diapause of the silkworn,Bombyx mori, is induced by the diapause hormone (DH) which is secreted from the suboesophageal ganglion of pupae. The diapause nature of bivoltine strains uses environmental stimuli as the initial signal to determine the diapause nature. The experiments showed that DH gene expreon is a direct response to the environmend stimulus, such as high incubation temperature. The cDNA from the embryonic stage wa cloned and sequenm analysis showed the cDNA encoding DH. Expmion patterns of the DH gene in embryonic stage are different ar incubation temperatures 15°C and 25°C, suggesting that the incubation tempcreturt as an environmental signal is kept within the body to control the DH gene expmion at the pupal stage, so that the embryonic diapause of next generation can be determined.     

The rôle of the suboesophageal ganglion in regulation of RNA synthesis in some insect tissues

The rôle of a factor produced by neurosecretory cells of the suboesophageal ganglion in the regulation of the level of RNA synthesis in some tissues of the female house cricket was studied in vivo and in vitro.It was shown that the action of the suboesophageal ganglion in vivo causes a reduction in the level of RNA synthesis in the neurosecretory cells of the pars intercerebralis, and also in the cells of the follicular epithelium, but it does not affect synthesis in the fat body cells.In experiments in which the action of the ganglion was examined in vitro, it was not found to have any influence on synthesis in the follicular epithelial cells, but the synthesis in the fat body cells was reduced. The causes of the differences obtained in the two types of experiments are discussed, and it is suggested that the suboesophageal ganglion acts directly on the fat body, but indirectly on the follicular epithelial cells through the corpora allata.     

家蚕滞育的分子机理2.蛹期滞育激素基因的表达与滞育决定

滞育激素是由食道下神经节分泌的重要昆虫神经肽,诱导昆虫的滞育。选择具有ＲＮＡ聚合酶能够识别的启动子的质粒载体,将滞育激素ｃＤＮＡ克隆进去,在体外大量合成单一的滞育激素ｃＲＮＡ为参照,测定食道下神经节分泌滞育激素ｍＲＮＡ量来确定滞育激素的分泌量。结果证明食道下神经节分泌的滞育激素的数量决定昆虫的滞育。     

Hormonal control of diapause in the tick Argas arboreus

During the mid-diapause period of the female tick Argas arboreus, nerve ganglion (G) extract (GE) from nondiapause (ND) females injected into diapause (D) female haemocoel after feeding, but not before feeding, terminated diapause in 50% of the ticks. ND haemolymph (H) failed to terminate diapause. Reciprocal injections of D GE, and H induced diapause in most ND females. During the late diapause season, ND GE, and H terminated diapause in D females and reciprocal injections of D GE, and H failed to induce diapause in ND females. The results suggest that in A. arboreus the facultative diapause results from gonadotropic hormone (GTH) deficiency in the presence of a diapause inducing factor (DIF). We suggest that the DIF is formed under the influence of short photoperiod in unfed females. The DIF interferes with normal release of the GTH formed in ganglion after feeding and also with the effect of GTH in the H by direct inhibition and/or by preventing the target organs (ovary and/or gut cells) from responding to GTH. The DIF is degraded slowly during short photoperiod and rapidly during long photoperiod.     

The fine structure of the diapause-regulator cell in the suboesophageal ganglion in the silkworm, Bombyx mori

In the suboesophageal ganglion of Bombyx mori the diapause-regulator producing cells which may give information to the diapause-factor cells were found by means of electron microscopy.The diapause-regulator producing cells had larger granules (200 to 500 mμ dia.) than did the diapause-factor cells which were partially surrounded by the former. Highly electron-dense material of lysosome in the diapause regulator producing cells was observed in the diapause-egg producer, but such lysosomes were not present in the non-diapause-egg producer. It was found that many cytoplasmic granules fuse with lysosome, and smaller granules arise from lysosomes. Some implications regarding the diapause-factor cell and the diapause regulator producing cell are discussed.     

Control of ommochrome synthesis by both juvenile hormone and melanization hormone in the cabbage armyworm,Mamestra brassicae

Summary Pigmentation of last instar larvae of the cabbage armyworm,Mamestra brassicae is of two types: melanin in the cuticle and ommochrome in the epidermis. The latter was found to be primarily xanthommatin. When allatectomy was performed 8 h before head capsule slippage (HCS) in the last larval molt, later ommochrome synthesis was inhibited. Application of juvenile hormone (JH) up to 12 h after HCS (9 h before ecdysis) (activity: methopreneJH I>JH II>JH III) restored ommochrome synthesis. After that time it has less and less effect.Removal of the suboesophageal ganglion from the larvae 8 h before HCS prevented both later ommochrome synthesis and melanization. Melanization of isolated abdomens was restored by implantation of 3 suboesophageal ganglia or injection of melanization and reddish coloration hormone (MRCH) 18 h after HCS. Restoration of ommochrome synthesis required exogenous JH in addition to melanization hormone from suboesophageal ganglion or MRCH. Therefore, melanization appears to be critical for the later onset of ommochrome synthesis even in a larva which has been exposed to JH during the critical period.Abbreviations CC·CA corpora cardiaca-corpora allata complex - JH juvenile hormone - MRCH melanization and reddish coloration hormone - HCS head capsule slippage     

Regulation of juvenile hormone esterase activity in the European corn borer, Ostrinia nubilalis

The regulation of juvenile hormone esterase in last-instar diapause and nondiapause larvae of Ostrinia nubilalis was investigated using topically applied juvenile hormone I and a juvenile hormone mimic, methoprene. The influence of the head on juvenile hormone esterase was also investigated. Both juvenile hormone and methoprene caused increases in esterase levels when applied to feeding animals. Neither the hormone nor methoprene was capable of elevating nondiapause esterase activity to levels comparable to those found in untreated prediapause larvae. The esterase levels could be elevated in the larval body, without the head, during prepupal development of nondiapause larvae and in post-feeding diapause larvae. In both cases, juvenile hormone or methoprene induced juvenile hormone esterase activity in head-ligated animals. Topically applied methoprene prolonged feeding and delayed the onset of diapause. When methoprene was applied to larvae that had entered diapause, it disrupted diapause by inducing a moult.     

Effect of photoperiod on a winter and on a summer diapause in two species of cranefly (Tipulidae)

Two craneflies, Tipula subnodicornis and Tipula pagana, both undergo diapause in the final larval instar. The species showed differences in the intensity of diapause and in the timing of the photoperiodic reaction during diapause, that could be related to season. Tipula subnodicornis undergoes a winter diapause that is induced and maintained in its early stages by short photoperiod (L:D:6:18). In the laboratory individuals in the early stages of diapause terminated diapause and pupated earlier when they were exposed to daylengths of, or greater than, 12 hr. However, it is suggested that in the field diapause is broken before the natural daylength is long enough to have any accelerating effect on development. Tipula pagana has a summer diapause which is of greater intensity than that of Tipula subnodicornis and some larvae were maintained for 197 days in the laboratory, without pupating, on an 18 hr daylength. Diapause was broken by a L:D;16:8 photoperiod and development was accelerated by a further decrease in daylength. The acceleration in development rate was attended by a decrease in the variance about the mean date of emergence and resulted in a highly synchronised emergence period. It is suggested that this quantitative response to daylength is particularly important to a species that emerges in the autumn when the temperature in the field is falling.     

Alteration of egg diapause in Bombyx mori by ouabain injected into diapause egg producers

Ouabain, an inhibitor of the active transport of some inorganic cations, functioned to produce non-diapause eggs in diapause egg producers. The effectiveness of this chemical related to non-diapause egg production was evident in 40 nmole injection per 4-day-old pharate adult. Simultaneous injections of ouabain with diapause hormone preparation or K⁺-ion suppressed non-diapause egg production to as a great extent as though they functioned competitively. Other experiments indicate that ouabain does not affect the ovaries directly but appears to inhibit the release of endogenous diapause hormone from the suboesophageal ganglion by affecting the central nervous system. The shortage of 3-hydroxykynurenine in non-diapause eggs produced by ouabain is thought not to be the result of the blockage of active transport at the ovaries but the result of a lack of diapause hormone.     

Juvenile hormone and the induction of larval polymorphism and the diapause of the southwestern corn borer, Diatraea grandiosella

The southwestern corn borer, Diatraea grandiosella, enters diapause as an immaculate mature larva which is a polymorphic variant of the spotted non-diapause larva. Because dormant immaculate larvae could be obtained by treating last stage non-diapause larvae with a juvenile hormone mimic (JHM), experiments were conducted to determine whether these hormonally induced immaculate larvae (HIL) were physiologically comparable to the normal environmentally induced immaculate diapause larvae (EIL). Comparative data obtained about pupation rates, response to JHM, metabolic reserves, oxygen consumption, and the state of spermatogenesis of HIL and EIL led to the conclusion that the HIL were physiologically similar to the EIL. The results demonstrate that the developmental programme of non-diapause larvae could be switched and ‘diapause’ induced solely by the topical application of JHM. We believe that the data further support our hypothesis that the larval diapause of D. grandiosella is initiated and maintained by the juvenile hormone.     

Molecular mechanism of diapause in insect (I) --Expression of diapause hormone gene and incubation temperature in embryonic stage of Bombyx mori

The embryonic diapause of the silkworm, Bombyx mori, is induced by the diapause hormone (DH) which is secreted from the suboesophageal ganglion of pupae. The diapause nature of bivoltine strains uses environmental stimuli as the initial signal to determine the diapause nature. The experiments showed that DH gene expression is a direct response to the environmental stimulus, such as high incubation temperature. The cDNA from the embryonic stage was cloned and sequence analysis showed the cDNA encoding DH. Expression patterns of the DH gene in embryonic stage are different at incubation temperatures 15℃ and 25℃, suggesting that the incubation temperature as an environmental signal is kept within the body to control the DH gene expression at the pupal stage, so that the embryonic diapause of next generation can be determined.     

Neurosecretory cells in the frontal ganglion of the tobacco hornworm, Manduca sexta

The frontal ganglion of the tobacco hornworm, Manduca sexta (L.), was found to contain two neurosecretory (NS) cells (max dia = 40–45 μm). The cytoplasmic inclusions of the NS cells were stained purple with paraldehyde fuchsin, and marked fluctuations in amounts of NS material in the perikarya were observed, depending upon the developmental status of the insect. The perikarya of NS cells in the frontal ganglia of starved larvae and diapause pupae contained large accumulations of NS material, whereas feeding larvae and developing pharate adults showed relatively low amounts of neurosecretion. Electron microscopy revealed large accumulations of NS granules (dia = 80–240 nm) in the frontal ganglia of diapause pupae, but only slight accumulations of granules were seen in the NS cells of developing larvae and pharate adults.It was concluded that axonal transport and release but not synthesis is shut down during starvation and diapause, leading to accumulation of NS material in the perikarya. It is also suggested that the failure of many investigators to differentiate NS cells in the frontal ganglion of various insects may have been due to the selection of very active stages when the amount of available NS material was too low to be visualized by conventional staining techniques.