Pupal Brain Research Articles

Esterase changes during the activation of pupal brains of the saturnid moth, Hyalophora cecropia.

THE traditional method of determining whether an endocrine gland in an insect is active or inactive is to see whether it produces a hormonal effect when it is transplanted into a suitable test insect. An alternative is to inject the insect with some labelled compound in order to detect differences between the metabolism of the active and inactive glands1. The work described in the present communication uses a third method: comparing zymograms of active and inactive endocrine glands. The endocrine organ used was the brain of the cecropia silkworm. Insect brains secrete an agent called “brain hormone” which activates the prothoracic glands and causes the insect to moult. In the cecropia silkworm the brain ceases to secrete this prothoracotrophic hormone immediately after pupation, and the pupa enters diapause. The brain can be re-activated by exposing the pupa to temperatures between 5° C and 10° C for two or more months. Within a few days after they are returned to room temperature the brains of such previously chilled pupae start to secrete brain hormone; the prothoracic glands become activated; and diapause is terminated by the initiation of adult development2. This communication describes esterase differences between the zymograms of inactive and active brains.

Studies on the Sterol of Bombyx mori L. : III Fate and Distribution of 4-<SUP>14</SUP>C-Cholesterol in the Silkworm

The present study was carried out to elucidate a contribution of the pupal brain to sterol metabolism, observing the fate and the distribution of 4-<14&&t;C-cholesterol in both normal and dauer pupae of the silkworm, Bombyx mori. After the injection of labelled cholesterol into the normal and the dauer pupae, the blood, several tissues, and remainder were dissected out from these pupae on 1 day after injection, 7 days and 1 day before emergence. Methanol extract was refluxed with ether, following each sample's extraction with methanol. The radioactivity of both the sterol ester and the free sterol fractions separated from the ether extract was counted. The incorporation of the labelled cholesterol into several tissues and the blood did not show any remarkable difference beween the normal and the dauer pupae. In the former, however, a large amount of cholesterol was demonstrated as ester form, while in the latter, free sterol was observed. Furthermore, the rate of esterification in dauer pupa sterol was slower than that of normal pupa, suggesting that unknown factor originating from brain had an intimate relation to sterol metabolism.

The Proteinic Brain Hormone in an Insect, Bambyx mori L. (Lepidoptera : Bombycidae)

This paper deals with both the procedure of extraction and the chemical characteristics of the proteinic brain hormone from pupal brains of the silkworm. Acetone dried brains were extracted with phosphate buffer solution and the extract was lyophilized. The 60 μg crude extract yielded positive result on Bombyx-bioassay. The chemical properties of the active extract were examined with heat treatment at 60 or 100°C, buffer treatment at pH 9.9 or 1.4, digestive procedure with some proteolytic enzymes, and fractional precipitation by ammonium sulfate, all results showing that the extract belonged to proteinic substance. Then, the active fraction was separated from the crude extract by means of chromatographic studies with Sephadex and cellulose ion exchanger. When 1 μg of the fraction-V obtained by CM-cellulose column was injected into each of test-objects, the latter became an imago in 3 weeks following the injection. Moreover, the highly purified, active ingredient was separated from the fraction-V on CM-cellulose by means of the zone electrophoresis on acrylamide gel, showing that the ingredient migrated to the anode. Thus two different consituents in the brain extract, sterol and protein showed brain hormone activity.

Effect of Low Temperature on the Induction of Imaginal Differentiation of Dauer Pupa of the Silkworm

IN lepidopterous insects (in which diapause occurs in the pupal stage), the pupal brain is activated by treatment by low temperature1–5. It has not yet been established, however, whether artificially diapausing, brainless pupae (dauer pupae) are activated by low temperature. Therefore, this question has been investigated by chilling dauer pupae of the silkworm, Bombyx mori.

Neural Transplants and Insect Diapause

THE induction and maintenance of larval and pupal diapause are at present thought to be under the direct control of the neuro-endocrine complex associated with the insect brain. Williams1 found that diapausing pupae of the cecropia moth, Hyalophora cecropia, could be induced to resume development by either a prolonged (6–12 weeks) exposure to a low temperature (4–6 ° C) or by the implantation of the brain of a previously chilled pupa. Unchilled diapause pupal brains were found to be incompetent to induce development when implanted into an unchilled pupa in diapause. The competence or incompetence of a pupal brain appeared to depend on the activity or inactivity of its neurosecretory cells. When active, the neurosecretory cells are responsible for the production of a hormone that is released via the corpora cardiaca and activates the prothoracic glands, which then produce the growth hormone, ecdysone, responsible for the initiation of development2. In the diapause pupa, the neurosecretory activity of the brain is suppressed3, as is its neural impulse activity4.

Effect of pupal brain surgery on the rocking response of automeris.

THE rhythmic rocking responses of Hemileucine moths may be released from insects which have been subjected to quite drastic surgical procedures1, but they have never been observed in decapitated preparations2. An attempt has now been made to investigate the role of the brain in mediating this response and in determining its quantitative strength, using the techniques of pupal surgery described by Williams3. One hundred female pupae of Automeris cinerea were chilled at 6°C. for eight weeks immediately following their reception from Argentina, and were then transferred daily (at 25° C.) in batches of five in order to break diapause. On the fifth to eighth day after transfer, they were chilled at 4° C. for 1 hr., anaesthetized in carbon dioxide for 30 min. while packed round with melting ice, and subjected to gross brain surgery through a window cut in the facial cuticle. After surgery, the pupae were sealed with the usual precautions3 and returned to 25°C. The yield in terms of histologically acceptable lesions was approximately 50 per cent. The surgical procedures, which were previously rehearsed on the large Indian Antherea mylitta, are summarized in Fig. 1. It may be noted that while procedure 1 results in blind moths the optic glomeruli II and III of which remain intact, procedure 2 ablates the entire optic complex, while sparing the medial lobes of the protocerebrum. Procedure 5 permits the development of a recognizable subœsophageal unit in isolation; while the resulting neuropil shows evidence of condensation, the normal features of its intrinsic organization may readily be recognized in favourably oriented sections, and moths so treated differ characteristically in their behaviour from those from which the subœsophageal ganglion has been extirpated.

Effect of the extirpation of the pupal brain at different periods upon the oviposition of the resulted adult in Antheraea perniy

Effect of the extirpation of the pupal brain at different periods upon the oviposition of the resulted adult in Antheraea perniy

Effect of Larval Brain on the Imaginal Differentiation in the Silkworm

I HAVE shown earlier1,2 that, in Bombyx mori, pupal brain has a function which brings about imaginal differentiation, probably by affecting the prothoracic gland in the pupa, and that the mode of action by which the prothoracic gland is influenced is hormonal in nature. Kobayashi and Kirimura3 attempted to extract the hormone and succeeded in obtaining an oily substance which is effective in bringing about imaginal differentiation in the ‘Dauer-pupae’ from the extract of the pupal brain.

THE CONTROL OF NEUROSECRETION AND DIAPAUSE BY PHYSIOLOGICAL CHANGES IN THE BRAIN OF THE CECROPIA SILKWORM

1. The 26 neurosecretory cells in the brain of the Cecropia silkworm have been shown by Williams to undergo systematic changes in endocrine activity during metamorphosis. At the pupal molt the neurosecretory cells lose endocrine activity. Consequently, growth is checked and the animal enters pupal diapause. Endocrine activity is restored following prolonged exposure to low temperatures. When the brain hormone is released, diapause ends and the development of the adult moth begins.2. The electrical events in the brain as a whole have been studied in relation to these changes in endocrine activity. The larval brain shows abundant "spontaneous" electrical activity. However, on the day before the pupal molt the brain loses spontaneous activity and is no longer excited by direct electrical stimulation. The electrical silence of the brain persists throughout the months of diapause. Electrical activity reappears in the pupal brain a few days before the end of diapause.3. Cholinesterase (ChE) is present in high titer in the larval brain. However, two days before pupation the brain ChE begins a rapid decline, so at pupation ChE is undetectable in the brain. ChE does not reappear in the pupal brain until shortly before the recovery of electrical and endocrine activities. There is no evidence that an inhibitor of ChE is present in the diapausing brain. Evidently, ChE is absent or in low titer and is synthesized just before the onset of adult development.4. During diapause the cholinergic action of brain extracts steadily increases. The increase is slow in the brains of pupae stored at 25° C. and rapid in the brains of pupae stored at 15° or 6° C. Therefore the rate of accumulation of the cholinergic substance parallels the effectiveness of the temperature treatment in restoring the brain to endocrine activity.5. When ChE reappears in the brain, just before the onset of adult development, the titer of cholinergic substance in the brain falls to a low level. The low level is maintained until shortly before adult emergence, when the titer of cholinergic substance increases five-fold.6. Choline acetylase activity remains uniform in brain homogenates from unchilled and chilled pupae.7. The changes in electrical activity, ChE, and cholinergic substance during metamorphosis are peculiar to the brain. The ganglia of the ventral nerve cord retain normal function throughout diapause.8. ChE is synthesized in chilled pupal brains which are cultured at 25° C. in the absence of prothoracic glands. Under the same circumstances unchilled brains are unable to synthesize ChE. Moreover, exposure to the brain and the prothoracic gland hormones fails to promote the synthesis of ChE in unchilled pupal brains.9. ChE and electrical activity remain constant during the metamorphosis of Galleria, a non-diapausing insect. Two additional diapausing species, Telea polyphemus and Samia walkeri showed variations in brain ChE and electrical activity identical to those described in Cecropia.10. The evidence suggests that the pupal diapause of Cecropia results from the failure of the brain as a whole. The diapausing brain is electrically silent, apparently because of the absence of ChE. During diapause there is an accumulation of a cholinergic substance in the brain—an observation which suggests that the reappearance of ChE is elicited by high substrate concentrations. When ChE reappears, the brain becomes electrically active, the neurosecretory cells release brain hormone, and diapause is ended.