Carbohydrate metabolism in the stick insect, Carausius morosus

Age-related changes in some parameters related to carbohydrate metabolism in the stick insect, Carausius morosus, were investigated during the 6th instar and up to day 37 of adult life. Total haemolymph carbohydrate concentration and the fat body glycogen content are low and may be related to the low activity of this insect. Trehalose constitutes about 75–80% of the total blood carbohydrate pool. During the moult, total blood carbohydrate, fat body glycogen and haemolymph volume, decrease while glycogen phosphorylase activity of the fat body is slightly activated. The effects are brought about mainly by reduced feeding activity, but may also be influenced by the shedding and replacement of the cuticle. During starvation, blood homeostasis is maintained at the expense of fat body glycogen via an activation of phosphorylase. During reproduction, although no dramatic changes in fat body glycogen levels occur, blood carbohydrates are maintained and fat body phosphorylase is slightly activated. The possibility is discussed that during moulting and reproduction, blood sugar homeostasis is maintained by a hormonal mechanism controlling glycogen phosphorylase. No circadian rhythm in any parameter investigated is observed.     

Carbohydrate and lipid metabolism during the last larval moult of the tobacco hornworm, Manduca sexta

Abstract. At 25°C and with a light regime of 17 h light and 7h dark, the last larval moult of the tobacco hornworm, Manduca sexta , lasts approximately 32 h, during which profound changes of metabolism were observed. At the onset of the moult, which coincides with the cessation of feeding, the proportion of active fat body glycogen phosphorylase increased from 10 (-2h) to 25–30% (Oh). A biphasic pattern with peak activities of 45–50% after t – 12 h and again just prior to the shedding of the cuticle (32 h) was subsequently observed. Haemolymph trehalose concentration decreased significantly from c. 35 (Oh) to 20mM (8h), but then recovered to an intermediate level (30mM; 12h). After completion of the moult, the trehalose concentration was 35–40 mM. The haemolymph glucose level in feeding fourth instar larvae was 4–5 mM, but decreased sharply before the onset of the moult to c. 1 mM, followed by a slow 6-fold increase over the next 20h. Prior to the shedding of the cuticle, the glucose level dropped again dramatically. The haemolymph lipid level increased slowly from an initial level of 1.2–1.4mg/ml during the early part of the moult, reaching a maximum of 1.8mg/ml after /= 16 h. Afterwards, a decrease of c. 50% was observed until ecdysis occurred. Oxygen consumption per animal decreased steadily from 30–35 μl/min pre-moult by approximately 70% to c. 10 μl/min but started to increase about 5 h before the animals resumed feeding.     

Metabolic shift from lipogenesis to glycogenesis in the last instar larval fat body of the silkworm,Bombyx mori

When [¹⁴C]glucose was injected into the last instar larvae of the silkworm, Bombyx mori, the label was incorporated into various tissues at varying degrees depending on the developmental stages. Fat body exhibited high incorporation rates throughout the feeding periods. Silk glands became active in incorporation but midgut decreased toward larval maturation. The pulse labeling experiment clearly demonstrated that the metabolic shift from lipogenesis to glycogenesis occurred in fat body at the middle of the last instar; a predominant incorporation was found in lipids when [¹⁴C]glucose was injected at the early stage, while at the late stage glycogen synthesis became most active. Incorporation into fat body proteins was not a major factor throughout the instar. Extirpation of silk glands enhanced incorporation into glycogen and proteins at the late stage but did not affect lipid synthesis. Long-term chase showed that fat body lipids and proteins synthesized at the early stage were totally carried over into the pupal fat body, while much glycogen produced at the late stage was used during the larval-pupal transformation with the remainder carried over into the pupa.From these results the metabolic shift from lipogenesis to glycogenesis in fat body is discussed in relation to the storage function of the fat body for pupal metamorphosis.     

Uric acid levels during last larval instar of Manduca sexta, an abrupt transition from excretion to storage in fat body

Levels of uric acid in the whole body of the tobacco hornworm, Manduca sexta increased steadily for the 9 days of the fifth instar. However, concentrations in the haemolymph were lowest during the transition from the feeding stage to the wandering stage (days 3, 4), the time when there was a switch from uric acid excretion by the Malpighian tubule-hindgut system to storage in the fat body. Haemolymph volumes, determined for larvae between 2 and 6 days into the fifth instar by isotope dilution with [¹⁴C]-inulin, were used to calculate rates of incorporation of uric acid into Malpighian tubules and fat body of larvae injected with [¹⁴C]-uric acid. These labelling studies indicated that the Malpighian tubules ceased to remove uric acid from the haemolymph some time between the last 6 hr of day 3 of the fifth instar and the first 18 hr of day 4. At the same period, fat body removed significant quantities of uric acid from the haemolymph. The times of initial decreases and increases in levels of uric acid in haemolymph and fat body, respectively, indicated that storage in the fat body started before cessation of elimination via the Malpighian tubule-hindgut system.     

Developmental changes of storage proteins and biliverdin-binding proteins in the haemolymph and fat body of the common cutworm,Spodoptera litura

The concentrations of three storage proteins (SL-1,SL-2 and SL-3, hexamers of 70-80kDa subunits) and two biliverdin-binding proteins (BP-A and BP-B, dimers of 165kDa) in the haemolymph and fat body during larval and pupal development of Spodoptera litura were determined by immunodiffusion tests using polyclonal antisera. SL-1 and SL-2 (methionine-rich) first appeared in the haemolymph of one-day-old sixth (final) instar larvae, prominently increased in the haemolymph during the later feeding period and were almost totally sequestered by the fat body after gut purge. SL-3 (arylphorin) was first detected in the haemolymph during the molting period to the final larval ecdysis, increased in concentration throughout the entire feeding period of the final larval instar and was partly sequestered by the fat body several hours later than the other storage proteins. BP-A showed nearly the same pattern in the haemolymph as SL-3: BP-B increased during feeding period and decreased during molting period and attained a maximum level during the penultimate larval instar, however its concentration decreased considerably and remained low in the final larval instar. BP-A was partly and BP-B was almost totally sequestered by the fat body 8 h after sequestration of SL-1 and SL-2, rendering the fat body blue in colour. These facts suggest an additional function of biliverdin-binding proteins as amino acid storage proteins and the results show a differential uptake mechanism for these proteins by the fat body.     

Purification,properties, and titer of a hemolymph trophic factor in larvae and pupae of Manduca sexta

Purification of a hemolymph protein (hemolymph trophic factor, or HTF) from last instar larvae of Manduca sexta was achieved using Sephadex G15-120 gel filtration and DEAE anion exchange chromatography. Homogeneity was visualized using SDS gel electrophoresis and ampholytic chromatofocusing. HTF was estimated to be a tetrameric protein with a molecular weight of 286 K and a Stokes' radius of 55.3 × 10⁻⁸ cm by agarose bead gel filtration; chromatofocusing suggests an isoionic point > 10. Polyclonal antibodies to HTF were prepared in rabbits and an ELISA was developed. The ELISA was used to titer HTF during the last larval instar and day 1 and 14 of the pupal stage and estimates a maximum of 1.5 mg/ml larval hemolymph on day 6 with a smaller larval peak of 0.75 mg/ml at day 3 and titers of 0.70 and 0.35 mg/ml on the 2 pupal days, respectively. ELISA of aqueous extracts of larval fat body, epidermis, and cuticle demonstrate that HTF comprises nearly a third of the soluble fat body protein and is a lesser component of epidermis and cuticle. The physiological role of HTF has not yet been determined.     

Diapause phenomena in non-diapausing last instar larvae,pupae, and pharate adults of the Colorado beetle

From the first day of the last (fourth) larval instar no trace of juvenile hormone (JH) can be detected in the haemolymph by Galleria bioassay. Three specific diapause proteins, which are also found in diapausing adults, appear in the haemolymph. These proteins disappear towards the end of the pupal stage. Study of the ultrastructure of the fat body revealed the formation from lysosomes of proteinaceous bodies which are also characteristic for adult diapause. The behaviour of last instar larvae and pupae resembles that of prediapausing and diapausing adults respectively. Injection of synthetic JH delays the appearance of the diapause proteins in the haemolymph and of proteinaceous bodies in the fat body for 2 to 3 days. The absence of JH seems to trigger off these diapause phenomena.     

Carbohydrate metabolism during the pupal molt of the tobacco hornworm,Manduca sexta

During the pupal molt of the tobacco hornworm, Manduca sexta, the percentage of active fat body glycogen phosphorylase increased from 5–10 to 20%, but only for a period of 5 h prior to the molt. From the time of the appearance of two sclerotized dorsal bars to the time of the molt, the concentration of total hemolymph carbohydrates doubled to 100 mM trehalose. Initially, the glucose level was high (16 mM) when compared with feeding larvae (approximately 1 mM) but decreased to zero just prior to the molt. The amount of cuticular chitosan decreased from approximately 100 mg to 10 mg at pupation; the exuvia contained approximately 7 mg. While the levels of total lipids in hemolymph were not affected, the lipid content of the fat body decreased significantly prior to the molt but increased sharply thereafter. Fat body glycogen phosphorylase in pharate pupae and pupae of M. sexta was substantially activated by the Manduca adipokinetic peptide hormone, which in pharate pupae, produced the same response at 2 and 20 pmol per insect as in ligated larval abdomens. In pupae the response was clearly reduced. Using chilling to stimulate glycogen phosphorylase, it was found that the enzyme in pharate pupae and pupae responded both in vivo and in vitro as in ligated abdomens of larvae. Thus, a transition to the adult response seems to occur during the pupal and pharate adult development. © 1995 Wiley-Liss, Inc.     

Hormonal control of uric acid storage in the fat body during last-larval instar of Manduca sexta

In the last-larval instar of the tobacco hornworm, Manduca sexta, a switch from excretion of uric acid to storage in the fat body occurs during transition from the feeding to the wandering stage. Neuroendocrine control of this change from excretion to storage was demonstrated by neck-ligation experiments with synchronously reared larvae. Results indicate that a neurohormone is released from the head 24–30 hr before the initiation of wandering and coincident with the first release of ecdysone that initiates metamorphosis. Direct involvement of the moulting hormone was shown by the effects of multiple injections of 20-hydroxyecdysone into the abdomen of larvae that had been ligated before the release of hormone. Urate levels in the fat body were 20- to 100-fold higher from hormone-injected larvae as from saline inject controls. Topically applied juvenile hormone or methoprene reversed the 20-hydroxyecdysone-induced storage of urate. Increased levels of uric acid in the haemolymph during pupal development result from the presence of juvenile hormone, and the abrupt decrease in uric acid concentration in the haemolymph just prior to pupal ecdysis results from a decreased titre of juvenile hormone. Applications of methoprene prevented the decrease in uric acid levels in the haemolymph.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.     

PROTEIN AND NUCLEIC ACID METABOLISM IN INSECT FAT BODY

1. The appearance of larval fat body as seen under the light or electron microscope depends on the nutritional state of the larva and on the stage of larval development at which the fat body is observed. 2. Early in the last larval instar the cells usually possess a well-developed endo-plasmic reticulum rich in ribosomes, numerous mitochondria, glycogen granules, a Golgi complex and fat droplets, while later in the instar the endoplasmic reticulum is much reduced and mitochondria are few, but glycogen and fat droplets are present in greater amount together with the appearance of large numbers of proteinaceous spheres. 3. Early in the last instar the fat body synthesizes proteins and exports them into the blood, while later in the instar proteins are sequestered from the blood into the fat body. 4. The rate of protein synthesis by the fat body is high in the early to mid part of the last instar, but then falls off rapidly to a low level, at which it remains until the larva pupates. In diapausing pupae, protein synthesis remains at this low level. 5. The similarity between the electrophoretic patterns of proteins from the fat body and those from the blood provides strong evidence that the fat body is the site of synthesis of many of the blood proteins. 6. Some of the blood proteins have been shown to possess enzymic properties, while others are thought to play a role in the transportation of various types of compounds. 7. Ecdysone and juvenile hormone both stimulate the rate of protein synthesis by larval fat body. Protein synthesis in fat body from diapausing pupae is stimulated after injury to the pupae. 8. The appearance of adult fat body and the amount of protein it contains is often closely linked with the nutritional and reproductive states of the insect. 9. An important role of the fat body in the adult female insect is the synthesis of yolk proteins, which are released into the blood and then taken up by the developing oocytes. This synthesis and uptake are under the control of hormones secreted by the corpora allata and by the median neurosecretory cells of the pars intercerebralis. 10. The RNA content of fat body in final-instar larvae is not constant throughout the instar. In some larvae it is at its highest level early in the instar, falling to a low level as the instar progresses, while in other larvae (e.g. Calliphora) the level of RNA in fat body does not decrease as the instar progresses. 11. In some dipterous insects the base composition of total RNA is DNA-like in that the guanine + cytosine content is low, accounting for 40 % of the bases. A similar composition is seen in rapidly labelled RNA isolated from insects of other orders (Coleoptera and Lepidoptera), but the base content of total RNA from these latter insects resembles ribosomal RNA from vertebrate tissues in that it has a high (ca. 60 %) guanine + cytosine content. 12. The RNA/DNA ratios in blowfly larval tissues are high compared with those found in any vertebrate tissue. 13. In larval fat body, RNA synthesis is low at the time of a moult, increases during the early and mid-instar period and subsequently falls during the latter part of the instar. During the pupal period, especially during pupal diapause, the rate of RNA synthesis is very low and then increases during the subsequent development of the pharate adult. Injury to diapausing pupae results in an increased rate of RNA synthesis in most of their tissues. 14. Ecdysone and juvenile hormone both stimulate RNA and DNA synthesis in larval and adult fat body and in other tissues, although there is evidence that in some tissues these two hormones may act antagonistically to each other. The insecticide DDT also has been shown to stimulate RNA synthesis in tissues of adult insects.     

Ecdysones and imaginal disc development during the last larval instar of Pieris brassicae

Ecdysone haemolymph levels and in vivo development of imaginal wing discs have been studied during the last larval instar of Pieris brassicae.During this period, β-ecdysone variations show two successive peaks, the first one related to the induction of wandering stage, and the second (main) one to pupal cuticle synthesis. The observed situation is very similar to that of Manduca sexta. Imaginal wing disc growth is composed of several genetically programmed steps that need the presence of ecdysone, but do not appear very closely linked to circulating hormone levels. It seems that ecdysone haemolymph peaks should be considered as periods where ecdysone levels are above a threshold value.     

Synthesis and release of proteins from cultured larval fat body of the southwestern corn borer,Diatraea grandiosella

The larval fat body of the southwestern corn borer, Diatraea grandiosella, was cultured in vitro to examine the relationship between proteins present in the fat body, those released into the medium, and those present in the haemolymph. While the incorporation of [³H]leucine into fat body proteins was high in last instar pre-diapausing and non-diapausing larvae, it fell in early diapausing larvae to about 11% of that found in prediapausing larvae. Incorporation of [³H]leucine into the diapause-associated protein of the fat body increased gradually in pre-diapausing larvae and reached a maximum in newly-diapaused larvae at a time when the incorporation of [³H]leucine into other proteins of the fat body had declined. The proteins released from the cultured fat body showed identical electrophoretic properties and close immunochemical relationships to most of those present in the haemolymph. Small amounts of the diapause-associated protein were released in vitro from the fat body of larvae of different ages in diapause. Lipophorin was also released in vitro from the fat body of non-diapausing and diapausing larvae, and shown to be immunochemically identical to the lipophorin present in the haemolymph.     

Amino acid and protein changes in the haemolymph of developing fourth instar Chironomus tentans

The concentration of free amino acids, total soluble protein, and haemoglobin in the haemolymph of fourth instar Chironomus tentans was investigated.The concentration of the free amino acid pool increases between the early (15.7 mM/l) and mid-(33.9 mM/l) fourth larval stages followed by a decline during the late (16.9 mM/l) fourth larval period. Alanine, serine, and the amides of aspartic acid and glutamic acid are the predominant free amino acids at all stages. Physiological fluid analysis of late fourth instar haemolymph detected 32 ninhydrin positive components including 18 common amino acids plus homoarginine, ornithine, citrulline, β-alanine, α-aminoadipic acid, α-aminoisobutyric acid, and sarcosine.The concentration of total soluble protein steadily increases during fourth instar larval development to a maximum of $\text{9.3}\text{g}\text{100}\text{ml}$ followed by a decline during the pharate pupal period. A similar pattern of variation occurs in haemoglobin content which comprises from 51 to 66% of Chironomus tentans haemolymph protein.The mM percentage of individual amino acids of total haemolymph protein varies little during the fourth instar. At all stages alanine and aspartic acid are the predominant amino acids.     

Hormonal regulation of phase polymorphism and storage-protein fluctuation in the common cutworm, Spodoptera litura

Three storage proteins are synthesised by Spodoptera litura last-instar larvae as detected by an antiserum against pupal fat body proteins. The putative pupal storage proteins 1 and 2, appear in the haemolymph of the last-instar larvae 36 h after ecdysis under crowded rearing conditions: they appear 1 day later in isolated conditions. The appearance of these proteins in the haemolymph is prevented by juvenile hormone treatment and enhanced by allatectomy. Injection of 20-hydroxyecdysone into ligatured larvae does not induce appearance of these 2 proteins. Accumulation of protein 3 that reacts with Bombyx mori arylphorin antiserum is not blocked by juvenile hormone and is similar in both phases. It also accumulates to a small extent in the haemolymph during the moult to the final-larval instar and then disappears at ecdysis. One-hundred ng/ml ecdysteroid caused the sequestration of these proteins by the fat body, but a higher concentration of ecdysteroid (200 ng/ml) produced pupal cuticle in the isolated abdomens, suggesting that different ecdysteroid concentrations are necessary for these two events.     

Changes in the chemical composition of fat body during the last larval instar of Manduca sexta.

The fat body increases in weight throughout the last larval instar in spite of the loss in total body weight during the wandering and prepupal stages. The protein content of fat body increases dramatically and is greatly responsible for the increase in fat body weight in the wandering and prepupal stages. Lipids do not contribute significantly to the fat body weight gain except during the feeding stage. The amount of total fat body RNA increases until wandering then drops abruptly in the prepupal stage. The total amount of fat body DNA peaks before the onset of wandering and prepupal stages.     

Induction of perfect superlarvae by the application of juvenile hormone analogue to starved larvae of the silkworm,Bombyx mori

Topical application of juvenile hormone analogue, methoprene, induced a supernumerary larval moult in the silkworm, Bombyx mori. The incidence changed greatly depending on developmental stages and physiological states of the methoprene-treated larvae. When methoprene was applied to feeding larvae, only those treatments from the middle of the 2nd instar until the middle of the 4th instar were effective. An 18-h starvation period from the beginning of the 4th instar and a dose of 1 μg of methoprene per larva were required for 100% incidence of the perfect superlarvae. Allatectomy had no effects on the induction of superlarvae by methoprene. The treated 4th-instar larvae ecdysed to the 5th instar without any delay compared to the controls, and underwent an additional larval ecdysis 4.5 days later. The induced 6th-instar larvae took 8.5 days until the onset of cocoon spinning. The induced superlarvae showed reduced growth rates but an increase of final mass due to prolonged feeding period. A sharp but reduced peak in ecdysteroid titre in the haemolymph appeared one and a half days prior to each larval ecdysis in the treated larvae, suggesting that methoprene provokes the extra larval moult through an additional release of ecdysteroids.     

Stage dependent influences of polydnaviruses and the parasitoid larva on host ecdysteroids

In the solitary egg-larval parasitoid Chelonus inanitus (Braconidae) both polydnavirus and the parasitoid larva manipulate host development. Parasitization leads to a premature drop in juvenile hormone titre and a precocious onset of metamorphosis in the 5th larval instar. The C. inanitus bracovirus (CiBV) alone causes a reduction in host ecdysteroid titres at the pupal cell formation stage and prevents pupation. Here we report three new findings. (1) We show that parasitization causes a reduction in haemolymph ecdysteroid titre immediately after the moult to the 5th instar; similarly low values were seen in nonparasitized larvae after the moult to the 6th instar. These data along with parasitoid removal experiments indicate that the low ecdysteroid titre after the moult is a very early sign of the upcoming metamorphosis. (2) In vitro experiments with prothoracic glands and brain extracts showed that CiBV affects both prothoracic glands and prothoracicotropic hormone after the stage of pupal cell formation. (3) In the haemolymph of parasitized larvae the ecdysteroid titre increased in the late cell formation stage, i.e. immediately before egression of the parasitoid. In vitro experiments showed that late 2nd instar parasitoids release ecdysteroids and are thus very likely responsible for the rise in host ecdysteroids.     

Haemolymph juvenile hormone esterase activity in synchronous last instar larvae of the cabbage looper, Trichoplusia ni

Weight and time of moult during the last instar of the cabbage looper (Trichoplusia ni) were examined and used to select last instar larvae that had similar rates of development. Haemolymph protein content and titres of haemolymph esterases hydrolyzing juvenile hormone I, juvenile hormone III, and α-naphthyl acetate were monitored during the last instar using these closely timed larvae. Juvenile hormone I and juvenile hormone III esterase profiles were very similar and differed markedly from the α-naphthyl acetate esterase and protein content profiles. Two major peaks of juvenile hormone esterase activity were observed, one before ecdysone release and the other just prior to pupal ecdysis. Juvenile hormone I was hydrolyzed 15 times faster than juvenile hormone III when assayed at 5 × 10^?6 M.