Relationship between a metabolic rhythm and emergence rhythm in Drosophila melanogaster

Oxygen consumption and lactic acid dehydrogenase (LDH) activity were determined for Drosophila melanogaster pupae and pharate adults exposed to 12 : 12 or 1 : 23 light-dark (LD) regime. Bimodal circadian fluctuations of oxygen consumption were found in pupae and pharate adults exposed to either LD regime and organisms appeared to demonstrate an anticipatory change in oxygen consumption associated with change in illumination. The oxygen-consumption trend for the entire period spent in the puparium showed a high at the time of emergence, but the diurnal rhythm showed a low at the time of emergence suggesting that emergence occurs at a low in the diurnal cycle. Emergence maximum showed a 3 hr lead over the oxygen-consumption maximum. Changing the LD regime produced similar changes in the phasing of both oxygen consumption and emergence rhythms. LDH activity did not demonstrate a detectable circadian rhythm but did show a steady decrease during pupal and pharate adult development.     

Spermatogenesis in pharate adult testes of Drosophila in tissue cultures without ecdysones

The process of spermatogenesis in explanted testicular fragments from pharate adults (48 hr after puparium formation) of Drosophila melanogaster was examined under in vitro conditions without any added ecdysone substances. In the anterior fragments, which contained spermatogonia, no or only slight changes were found. In the middle fragments which contained germ cells at more advanced stages of spermatogenesis, spermatocytes, and spermatids, a slight increase in the number of spermatocytes or spermatids was observed. In the posterior fragments, which contained sperms at early stages of spermiogenesis, there was a marked elongation of the sperm bundles along their long axis.     

Glycogen phosphorylase activity in pharate adults of the stable fly and the effects of a juvenile hormone analogue

Glycogen phosphorylase was assayed in homogenates of pharate adults of the stable fly, Stomoxys calcitrans, by the release of inorganic phosphorous (Pi) from glucose-1-phosphate (G-1-P) in the presence of glycogen. Activity was determined as active and total phosphorylase present by the absence or presence of adenosine-5-monophosphate (AMP) in homogenates. Homogenates of the pharate adults (that were treated topically immediately after larval-pupal apolysis with a synthetic insect juvenile hormone analogue, (E)-4-[(6,7-epoxy-3-ethyl-7-methyl-2-nonenyl)oxy]-1,2-(methylenedioxy)benzene) displayed no inhibition or enhancement of phosphorylase activity when compared to untreated pharate adults.     

The ultrastructure of the preformed ecdysial ‘line of weakness' in the puparium cap of Elenchus tenuicornis (Kirby) (Insecta: Strepsiptera).

This study describes the ultrastructure at ‘the line of weakness’ in the male puparium of Elenchus tenuicornis (Kirby) (Insecta: Strepsiptera). Superficially this line looked like an area of untanned cuticle but ultrastructurally it had an undifferentiated epicuticle, an untanned exocuticle and a loose textured endocuticle. It is speculated that the E. tenuicornis pharate adult male prior to emergence smears a chemical solution on the inner rim of the ‘line of weakness’ which dissolves the exo- and endocuticles. Following this, the slightest pressure exerted by the ptilinum of the male breaks open the cap of the puparium.     

Critical periods for developmental acclimation to cold in the Queensland fruit fly, Dacus tryoni

Rapid acclimation to cold can occur in Dacus tryoni during two short stages of its life history: the stage immediately prior to the “hopping larva” phase and the “pharate adult” stage within the puparium. Transfer from 25 to 15°C at either of these stages can produce full acclimation to cold within a few days. Acclimation is not detectable at other times in puparial life: during adult life it takes over 100 days.     

Ultrastructure of salivary glands of Drosophila lebanonensis during normal development and after in vivo ecdysterone administration

Changes in the morphology of the salivary glands of Drosophila lebanonensis have been followed at both the light and electronmicroscopic level during a period of 30 hr before puparium formation and during puparium formation itself. Three striking differences were observed in comparison to other Drosophila species studied: (1) the secretion product of Drosophila lebanonensis has a different stainability to PAS reagent and uranyl acetate and no internal structures or “caps” can be observed; (2) the release of this secretion product is not restricted to a time period shortly before puparium formation but is a continuous process starting about 24 hr before puparium formation; and (3) the histolysis of these glands starts immediately after puparium formation, whereas in other Drosophila species this event starts 5 hr later.Puparium formation of Drosophila lebanonensis is controlled by the circadian oscillation. Injection of ecdysterone before the “gate” period results in changes in the cuticle as observed during normal development, but is not followed by the histolysis of the glands. Injection of ecdysterone after the “gate” is not followed by changes in the cuticle but histolysis is induced.     

Effect of a juvenile hormone analogue on phosphatase activity in pupae of the stable fly, Stomoxys calcitrans.

The alkaline phosphatase activity of stable fly pharate pupae treated with 10 ng of an insect juvenile hormone analogue (JHA), Stauffer R-20458, or untreated pupae was rhythmic and peaked 48 and 96 hr after larval-pupal apolysis. Those treated with 10 μg were not rhythmic and peaked at 48 and 120 hr. Acid phosphatase activity showed a general increase throughout pupal-adult transformation and was three- to four-fold higher than alkaline phosphatase activity. At 24 and 48 hr after larval-pupal apolysis, acid phosphatase was lower in the treated animals, but at 72 and 120 hr, it was higher. Neither inhibition nor enhancement of acid and alkaline phosphatase activity or by three other JHAs could be demonstrated in vitro.     

Some effects of low oxygen partial pressures on the development of Calliphora vomitoria;

The influence of wet conditions and low pO₂ on the survival and development of non-feeding final instar larvae and puparia of Calliphora vomitoria has been investigated. The larvae delay the formation of the puparium in wet conditions in air and in dry or wet conditions in 10 and 5% oxygen. This may be related to the susceptibility of the newly formed puparia to oxygen shortage. The pupal respiratory horns play an important part in maintaining O₂ uptake when the puparia are surrounded by particles covered with a film of water but are not involved in aiding survival in low pO₂. Zero age puparia are killed by a 2 day exposure to 10% O₂ but later stages can continue to develop in this gas. Fifty per cent of the 0, 1 and 9 day old puparia are killed by about a 12 hr exposure to 1% O₂ whereas 50 per cent of the 2 to 8 day old puparia can survive over 1·5 days exposure to this gas. Development, as measured by respiration rates and the timing of the emergence of the adults, is delayed by 1% O₂ by the amount of time that the insects spend in that gas. However, the first phase of elongation of the pharate adult longitudinal flight muscle, occurring between the third and fourth day of puparial life, is only slightly slowed down in 1% O₂. The variations in susceptibility to 1% O₂ and the growth of the muscles are discussed in relation to published accounts of protein synthesis in the puparium.     

beta-ecdysone levels in pharate pupae of the stable fly, Stomoxys calcitrans and interaction with the chitin inhibitor diflubenzuron.

The β-ecdysone titer in pharate pupae of the stable fly, Stomoxys calcitrans (L.), exposed to diflubenzuron [Thompson-Hayward TH 6040; N-[[(4-chlorophenyl)amino]carbonyl]-2,6-difluorobenzamide] as larvae in artificial diet was analyzed by high-pressure liquid chromatography. No significant differences between treated and control groups were found in the β-ecdysone titers within the first 10 hr after pupariation.     

RNA metabolism during metamorphosis of Drosophila hydei

Determinations of total protein and RNA at various times after the beginning of the pharate pupal stage of Drosophila hydei, revealed an increase in both substances during the first 25 hr and a sudden decrease thereafter until 52 hr. From this time on the total amount of both protein and RNA increases slightly until emergence of the flies at 160 hr after the beginning of the pharate pupal stage. A similar pattern of changes was recorded for the total radioactivity as well as the specific activity of RNA labelled with ³H-uridine after the injection of the isotope immediately before the beginning of the pharate pupal stage.The migration profile of RNA labelled with ³H-uridine during larval development, revealed that shortly after the onset of the pharate pupal stage an essentially normal larval pattern consisting of major fractions of 28, 18, 8 to 9, and 4 to 7 S RNA. At 52 hr only the low molecular weight RNA was present. The ‘normal’ pattern was restored at the time of emergence of the flies, indicating re-utilization of degradation products of previously labelled RNA.     

The hormonal control of salivary gland secretion in Drosophila melanogaster: Studies in vitro

The larval salivary gland of Drosophila melanogaster synthesises a complex secretion, known as ‘glue’. which is secreted at puparium formation and then cements the puparium to its substrate. This secretion is made during the third larval instar and is stored in the gland cells as large granules. A few hours before puparium formation it is secreted into the gland's lumen by exocytosis. This process is induced by ecdysone and can be studied in vitro. Secretion is initiated about 3.5 hr after exposure of glands to ecdysone and is complete by 8 hr. The effects of varying the ecdysone concentration, of inhibitors of RNA or protein synthesis, and of withdrawing the hormone at various times after initial exposure on the process of secretion have been studied. We conclude that some event(s) occurring during the first 3 hr exposure to ecdysone is necessary to initiate secretion of the glue into the gland lumen. The possible relationship between this event(s) and the ecdysone induced changes in gene activity (puffs) which occur in the salivary glands at the same time is discussed.     

The ultrastructure of the preformed ecdysial 'line of weakness' in the puparium cap of Elenchus tenuicornis (Kirby) (Insecta: Strepsiptera).

This study describes the ultrastructure at 'the line of weakness' in the male puparium of Elenchus tenuicornis (Kirby) (Insecta: Strepsiptera). Superficially this line looked like an area of untanned cuticle but ultrastructurally it had an undifferentiated epicuticle, an untanned exocuticle and a loose textured endocuticle. It is speculated that the E. tenuicornis pharate adult male prior to emergence smears a chemical solution on the inner rim of the 'line of weakness' which dissolves the exo- and endocuticles. Following this, the slightest pressure exerted by the ptilinum of the male breaks open the cap of the puparium.     

Analysis of metamorphosis in Drosophila melanogaster: Characterization of giant,an ecdysteroid-deficient mutant

The mutant allele giant of Drosophila melanogaster affects the timing and the level of increase in ecdysteroid titer normally occurring at puparium formation. The third larval instar is extended by 4 days in phenotypically “giant” individuals during which the imaginal discs mature slower than normal and finally take on the folding pattern characteristic of maturity at a time when normal individuals have already formed puparia. After puparium formation, development occurs at the same rate in giant and wild-type animals. Feeding 20-hydroxyecdysone at 94 hr after oviposition allows giant larvae to develop at the same rate as wild-type larvae and to produce normal-sized adults (although at 94 hr the imaginal discs of giant lack much of the folding pattern of mature discs). Radioimmunological determination of ecdysteroid titers in giant and normal individuals indicates that the peak of ecdysteroid activity associated with puparium formation is lower in giant and occurs 4 days later than normal. These results indicate that giant is an ecdysteroid-deficient mutant with major effects on metamorphosis. Unlike previously reported ecdysteroid-deficient mutants, however, giant larvae eventually develop into adults and may be induced to undergo complete metamorphosis at the same time as wild type by feeding 20-hydroxyecdysone.     

Ecdysteroid levels and integument changes in post-embryonic stages of Anastrepha suspensa

Ecdysteroid levels in larvae and pupae of Anastrepha suspensa (Diptera: Tephritidae) were measured by radioimmunoassay. These levels were correlated with histological changes throughout the development of the post-embryonic stages. Ecdysteroid levels increase rapidly throughout the last-larval instar and on the last day of this stage are 283 pg equivalents of 20-hydroxyecdysone per insect (14.5 ng/g) when wandering behaviour is initiated. At the end of this period the puparium is formed and within 24 h, the ecdysteroid rises to its highest peak (625 pg equivalents of 20-hydroxyecdysone/insect). Larval-pupal apolysis is initiated within 24 h later and the pupal cuticle is then secreted. Two days later, the ecdysteroids reach their lowest levels (75 pg equivalents of 20-hydroxyecdysone/insect or 0.6 ng/g) and most of the larval fat body and other tissues have been histolysed. In 5- to 10-day old pupae ecdysteroid levels again increase and remain relatively high throughout. During this period the larval epidermis is replaced by imaginal epidermis, imaginal discs begin to proliferate and the adult cuticle is secreted. Ecdysteroid levels finally fall 2 days prior to adult emergence. HPLC determinations indicate that 20-hydroxyecdysone is the predominant free ecdysteroid, and along with ecdysone, is readily detectable in all postembryonic stages of this species. An unusually high and unexplained peak of 20-hydroxyecdysone occurs in the pharate adult. This peak probably consists of ecdysone metabolites with retentions similar to that of 20-hydroxyecdysone and to which the antiserum is sensitive.     

Fatty acid synthetase content during development of the fly,Ceratitis capitata

Immunochemical titrations of different enzyme preparations from larval, pharate adult, and emerged adult Ceratitis capitata indicated that the changes in fatty acid synthetase activity during development of the insect are not related entirely to changes in the content of the enzyme. Changes in catalytic efficiency during larval and pharate adult development were clearly paralleled by the amounts of immunoprecipitate; however, the changes of enzyme activity with adult age were not correlated to the changes of enzyme content. Dietary manipulations of the larval stage of the insect Ceratitis capitata show the adaptive nature of the fatty acid synthetase. Fasting produced a clear decrease of activity and level of the enzyme from the larvae and refeeding restored practically normal values.     

Metabolism of β-alanyl-l-tyrosine in Sarcophaga bullata

Soluble enzymes have been demonstrated in extracts of Sarcophaga bullata larvae and pupae which both synthesize and hydrolyze the dipeptide β-alanyl-l-tyrosine (β-AT). In vitro assays for the synthesis and hydrolysis of β-AT were developed using thin-layer chromatography and (1-¹⁴C)β-alanine.The fat body was identified as the tissue origin of both the synthetase and hydrolase enzymes. The specific activity of β-AT synthetase in fat body extracts was constant throughout larval development, whereas hydrolytic activity in fat body extracts rose sharply after completion of the white puparium, reached a maximum in 12 to 18 hr, and then fell to its initial level. The rôle of the two enzymic activities in the formation of the puparium of S. bullata is discussed.     

A Multicopper Oxidase-Related Protein Is Essential for Insect Viability,Longevity and Ovary Development

Typical multicopper oxidases (MCOs) have ten conserved histidines and one conserved cysteine that coordinate four copper atoms. These copper ions are required for oxidase activity. During our studies of insect MCOs, we discovered a gene that we named multicopper oxidase-related protein (MCORP). MCORPs share sequence similarity with MCOs, but lack many of the copper-coordinating residues. We identified MCORP orthologs in many insect species, but not in other invertebrates or vertebrates. We predicted that MCORPs would lack oxidase activity due to the absence of copper-coordinating residues. To test this prediction, we purified recombinant Tribolium castaneum (red flour beetle) MCORP and analyzed its enzymatic activity using a variety of substrates. As expected, no oxidase activity was detected. To study MCORP function in vivo, we analyzed expression profiles of TcMCORP and Anopheles gambiae (African malaria mosquito) MCORP, and assessed RNAi-mediated knockdown phenotypes. We found that both MCORPs are constitutively expressed at a low level in all of the tissues we analyzed. Injection of TcMCORP dsRNA into larvae resulted in 100% mortality prior to adult eclosion, with death occurring mainly during the pharate pupal stage or late pharate adult stage. Injection of TcMCORP dsRNA into pharate pupae resulted in the death of approximately 20% of the treated insects during the pupal to adult transition and a greatly shortened life span for the remaining insects. In addition, knockdown of TcMCORP in females prevented oocyte maturation and, thus, greatly decreased the number of eggs laid. These results indicate that TcMCORP is an essential gene and that its function is required for reproduction. An understanding of the role MCORP plays in insect physiology may help to develop new strategies for controlling insect pests.     

Degradation of haemoglobin in Chironomus during metamorphosis

The degradation of haemoglobin (Hb) during the metamorphosis of Chironomus pallidivitatus was studied by means of spectrophotometry, disc electrophoresis, and isotopic tracer methods. Hb concentration was maximal during the late fourth instar larva and exhibited a continual decline throughout the pharate adult periods; in the mature adult Hb is almost entirely absent. Coupled with this reduction in Hb is an increase in the quantity of bile pigment (tentatively identified as bililatrenes) most probably representing products of the Hb degradation. These pigments are found in considerable quantity within the meconium at the time of adult emergence.Larval, ⁵⁹Fe-labelled, Hb injected into late fourth instar larvae was found to be most concentrated in the midgut portion of the early pharate adult. The quantity of ⁵⁹Fe-haemoglobin in the midgut of early pharate adult was at least five times the concentration found in late pharate pupae. Spectrophotometric and electrophoretic analysis of the midgut contents of untreated larvae showed that Hb was one of the major soluble proteins in the midgut at the time of pupation. This rapid intestinal uptake of Hb suggests that the intestine is involved in the mechanism for the degradation and removal of the Hb, no longer required in the adult stage, a seemingly important step in the metamorphosis of this insect.     

Diapause hormone action in silkworm ovaries incubated in vitro; 14C-trehalose incorporation into glycogen

Developing ovaries from pharate adults of the silkworm, Bombyx mori, were incubated in a medium containing ¹⁴C-trehalose or ¹⁴C-glucose, and the effects of diapause hormone on the incorporation of these isotopes into ovary glycogen were studied. The rates of incorporation of ¹⁴C-trehalose remained unchanged in ovaries incubated for 36 hr when the medium was renewed at intervals of 12 hr, and showed saturation kinetics against the concentration of the sugar in the medium, giving apparent K_m values of 6·0 mM for trehalose.There was no difference in ¹⁴C-glucose incorporation by ovaries grown in the presence (+SG) and absence (?SG) of the suboesophageal ganglion (SG). However, when ¹⁴C-trehalose was used as a substrate for glycogen synthesis, there was a marked difference in the incorporation between them, i.e. the incorporation was more than 60 per cent higher in +SG ovaries than that in ?SG ovaries. Increased incorporation of ¹⁴C-trehalose was also observed in ovaries from SG-removed pharate adults which received an injection of diapause hormone preparations. Maximum stimulation rates (about twofold) appeared 36 hr after the injection. Further, comparable effects on ¹⁴C-trehalose incorporation were observed in ovaries which were incubated with diapause hormone preparations added in vitro.These data are discussed in relation to the hormonal regulation of trehalase activity in ovaries.     

Water loss and respiration of Lucilia cuprina during development within the puparium

The rate of loss of water and the rate of uptake of oxygen were measured continuously throughout the development of Lucilia cuprina within the puparium. Changes in these parameters were correlated with changes observed in morphology of cuticles and respiratory structures during development.In development at 26°C, there is, at 20–22 hr after puparium formation a major loss of water by mechanical expulsion of moulting fluid chiefly through the posterior larval spiracles after the severing of the posterior larval tracheae. This loss of water is essential to survival and is followed by an extremely low rate of water loss attributed to slow diffusion of water through the resulting air gap between the pupal cuticle and the puparium. There is an increase in oxygen consumption during the pupal movements associated with the casting of the larval tracheae followed by a sharp reduction in oxygen consumption until the pupal horns are everted a short time later. This combination of physiological events enables development to proceed over a wide range of conditions in the puparial environment.