Chilled Pupae Research Articles

Metabolic reserves of diapausing western cherry fruit fly (Diptera: Tephritidae) pupae in relation to chill duration and post-chill rearing conditions.

How different macronutrients are utilized at various stages of pupal diapause and the effects of winter length on nutrient reserves remain poorly studied for most insects. Western cherry fruit fly, Rhagoletis indifferens (Diptera: Tephritidae), is a specialist on cherries in higher latitudes or elevations in western North America that exhibits a obligate pupal diapause requiring chilling before adult development can occur. We determined the relationship between metabolic reserves and diapause status in R. indifferens pupae, testing the hypotheses that lipids are the primary reserves utilized during diapause and that long periods of warmth deplete these reserves more than periods of cold. Effects of 0- to 20-week durations at 3°C and subsequent exposure to 23°C and 16:8 L:D (warm rearing conditions) for 0 to 7 weeks on lipid, protein, soluble carbohydrates, and glycogen reserves of R. indifferens pupae were determined. During diapause, lipid reserves were the primary source of energy utilized by R. indifferens, while protein and soluble carbohydrates levels were stable throughout diapause and thus less utilized. At post-diapause, glycogen levels fluctuated the most, indicating that lipid reserves were utilized to produce glycogen to support metabolism for adult fly development. Unchilled pupae did not deplete lipid reserves, unlike chilled pupae, likely because unchilled pupae remained in diapause. Rhagoletis indifferens may have evolved a nutrient utilization strategy typical of rigid diapausing insects in higher latitude environments.

Effects of cold storage, rearing temperature, parasitoid age and irradiation on the performance of Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae)

In this study, the effects of cold storage, rearing temperature, parasitoid age, and irradiation on the performance of the egg parasitoid Trichogramma evanescens were investigated. Pupae of T. evanescens can be stored at 4 °C for up to 3 weeks without much loss of performance. The longevity and walking speed of adults emerging from chilled pupae significantly decreased after longer storage periods. The F 1 generation of adults which emerged from pupae stored up to 3 weeks was able to parasitize as well as the control. The parasitization rate was similar at 24, 27, and 30 °C, but significantly decreased at 33 and 36 °C. Although T. evanescens developed to the pupal stage at 36 °C, no adult emergence was observed at this temperature. Developmental periods were longer at 24 °C than at higher temperatures. The optimum age for T. evanescens to successfully parasitize host eggs ranged from 24 to 90 h. The parasitization frequency of the 56–78 h aged females was higher than for the other age groups. The daily egg laying pattern of female T. evanescens adults was similar when they were reared on Ephestia kuehniella or Plodia interpunctella eggs. Gamma- or ultraviolet-irradiated and unirradiated host eggs were equally preferred by adult females.

Role of the brain in post-diapause adult development in the swallowtail, Papilio xuthus

The role of the brain in adult development was examined by brain removal in unchilled and chilled diapausing pupae of Papilio xuthus. Chilling was effective in shortening the pupal duration and synchronizing adult emergence, although photoperiod had little effect on diapause development. The brain played a role of shortening the pupal duration and synchronizing adult emergence in both unchilled and chilled individuals, although it was not essential for post-diapause adult development. The stimulus of low temperature was recorded even in the absence of the brain, because chilling shortened the pupal duration in brainless individuals. The brain showed activity which affected subsequent adult development in chilled pupae within one day after chilling in males. This period was less limited in females.

DNA synthesis and cell division during second pupal development in the silkmoth, Antheraea polyphemus

Chilled pupae of Antheraea polyphemus were induced to form second pupae by injecting juvenile hormone (JH) alone or with 20-hydroxyecdysone (20-HE). Adults were permitted to initiate development spontaneously or by injection of 20-HE. During development, wing tissue was removed for measurement of incorporation of [ 3H]-thymidine ([ 3H]-TdR) into DNA and the total DNA content. Tissue samples were also obtained for histological examination. Wing tissue from pupae stimulated by injected 20-HE alone or with JH, incorporated [ 3H]-TdR maximally on day 1. The incorporation values then declined to control levels by day 4, and continued low to day 6. Animals which initiated development spontaneously either in the presence or absence of JH had a more gradual increase in the rate of DNA synthesis with the peak rate occurring on days 2–3. Studies of the cellular ratio of thymidine triphosphate to thymidine (TTP/TdR) indicated that the incorporation of [ 3H]-TdR into DNA was the result of hormone stimulation of TdR metabolism and not merely the result of increased cellular uptake of DNA precursors into epidermal cells. The DNA content of all experimental groups parallelled the changes in the rate of DNA synthesis. Wing DNA levels during 20-HE synchronized second pupal and adult development increased up to day 6 and then remained constant until day 12, when DNA content decreased significantly in second pupae. Light microscopy indicated that epidermal cells of 20-HE synchronized developing second pupae and pharate adults divided between one and two days after hormone injection. At four days, differentiated scale cells were seen in adults but were never seen in second pupae. These results suggest that DNA synthesis is initiated by ecdysteroids, regardless of whether the steroid is injected or released endogenously, and that JH itself has no specific effects on DNA synthesis or proliferative cell divisions in the wing.

Brain cyclic AMP levels and the initiation of adult development in the Cecropia silkmoth

A six-fold increase in the level of brain cyclic AMP is observed in chilled Cecropia pharate adults within 24 hr of transfer from the cold to room temperature. This increase is not observed in pupae chilled for a period insufficient to allow initiation of adult development, nor after injury to diapausing pupae. Other tissues show a variable and minor response during initiation. Injected dibutyryl cAMP will cause initiation in insufficiently chilled pupae, but not in dauer pupae. The possible relationship of this rise in cAMP to the process of initiation is discussed.

Inhibition of RNA synthesis in the germinal cysts of the silkmoth by β-ecdysone

When the steroidal moulting hormone of insects, β-ecdysone, is injected into saturniid pupae, net RNA synthesis in the testis and testicular germinal cysts is inhibited. This inhibition does not appear to be due to a decrease in the size of the RNA precursor pool. On the other hand, β-ecdysone stimulates both the in vitro uptake and incorporation of [3H]leucine into protein. Inhibitor studies suggest the presence of stable pre-existing mRNA in the testes of chilled pupae. Analysis of the germinal cyst RNA revealed that most of the newly synthesized RNA from chilled pupae was in the nuclei and was heterodisperse with very little mature rRNA. It is suggested that the β-ecdysone inhibits net testicular RNA synthesis either directly by stimulating an RNAse or indirectly via other endocrine glands or factors as previously suggested.

An autoradiographic and biochemical analysis of palmitate incorporation into fat body lipid

Short-term in vitro experiments demonstrated that 14C-palmitate is rapidly esterified by fat body from several developmental stages of Hyalophora cecropia. Triglyceride is the major product when larval or adult fat body is utilized while diglyceride accumulates during the 3 hr incubation period with fat body from pharate pupae, diapausing pupae, and chilled pupae. Similar results were obtained with pupae of Philosamia cynthia, Antheraea polyphemus, and Hyalophora gloveri. The pattern of incorporation with Leucophaea maderae fat body is essentially the same as that found in H. cecropia larval fat body. The synthesis of glycerides can be followed with light microscope autoradiography and the data suggest that 3H-palmitate is esterified in the endoplasmic reticulum and then transported to the lipid globules which are the depository for glyceride storage. Electron microscope autoradiographic studies support this supposition.

Phosphatidate phosphohydrolase in the fat body of Hyalophora cecropia

Potent phosphatidate phosphohydrolase activity was associated with particulate, sub-cellular fractions of Cecropia fat body and flight muscle. The greatest enzymic activity was demonstrated in the microsomal fraction. The fat body phosphatidate phosphohydrolase displayed optimum activity at pH 7·4; 37 to 40°C. The calculated K m value was 0·7 × 10 −4 M which is similar to values for vertebrate preparations. The fat body enzyme was inhibited by a series of divalent cations but not by Mg 2+ which instead appeared to stimulate enzyme activity. A sulfhydryl inhibitor prevented optimum enzyme action and this effect was completely reversed by reduced glutathione indicating the presence of a necessary thiol group in the enzyme preparation. Enzymic activity was high in chilled pupae, decreased at the beginning of adult development and then increased steadily until adult emergence. No sexual dimorphism in activity was noted.

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.

The juvenile hormone—IV. Its extraction, assay, and purification

Juvenile hormone was extracted with ether from homogenized abdomens of male Cecropia and Cynthia moths. The ether extract was washed with water and evaporated to dryness to yield a golden, non-toxic oil which was highly active when tested for juvenile hormone. The hormone was assayed by injection into previously chilled pupae of the Polyphemus silkworm or by topical application to the tips of the abdomens of previously chilled Cynthia pupae. After 2–3 weeks at 25°C, the activity was scored in terms of the degree to which pupal characters were preserved during adult development. The hormone was concentrated by repeated extraction into methanol, gentle alkaline saponification, silicic acid chromatography, and crystallization of impurities. A combination of these procedures gave approximately 4000-fold purification. Gas-liquid chromatography of the purified oil provided an active fraction which appeared to be nearly homogeneous by analytical gas-liquid chromatography. This fraction was active at the level of less than 0·3–0·4 μg per 5 g test animal, corresponding to a purification of the original extract by at least 50,000-fold. The major component of this fraction was examined by mass spectrometry and shown to be an epoxide of methyl hexadecanoate. Methyl dl 9,10- cis and methyl dl 9,10- trans epoxyhexadecanoate were synthesized and shown to be completely inactive. The mass spectra of the synthetic compounds could not be distinguished from that of the most active hormone fraction obtained by gas-liquid chromatography. Evidently, the hormonally active substance constitutes only a small part of the most active fraction, the rest being the above-mentioned inactive epoxy ester which accompanies the hormone throughout the purification procedure. The chemical nature of juvenile hormone is unknown. We estimate that the purified Cecropia extract is at least 1000 times more active than any of the isoprenoid derivatives which have previously been reported.

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.

Effect of Refrigeration of the Pupæ of Microbracon brevicornis Wesm. on the Pigmentation of the Adult

DURING the course of mass breeding of Microbracon brevicornis in the parasite laboratory of the Entomology Division of this Institute for the purpose of exporting them to the United States of America for the control of pink boll worm in the cotton belt of that country, it was necessary to maintain a stock of pupae of the parasite in cold storage at a temperature of 10 ± 3° C. It was observed that the adults emerging from the chilled pupae which had been in cold storage from 10 to 75 days showed dark pigmentation ranging from dark grey to jet black, as against the distinctly brown individuals obtained from unchilled pupae.