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.     

X-ray investigation of gas bubble formation, and water loss in tsetse fly pupae

ABSTRACT. Using a microfocal X-ray apparatus, a gas bubble was detected within the puparium of Glossina morsitans. The bubble appeared between 6 and 15 h after pupariation and was associated with one of the longitudinal tracheal trunks of the third instar larva. The bubble grew and achieved maximum size approximately 96 h after pupariation. It then disappeared at the time of eversion of the pupal appendages. There was a close correlation between bubble size and the weight of water lost since the time of pupariation. At the time of eversion of the pupal appendages the gas bubble apparently passed out through the longitudinal tracheal trunk and posterior spiracle to occupy the space between larval (puparial) and pupal cuticle. It is suggested that the bubble plays a vital role in the separation of these cuticular layers and that to this end water loss from the puparium is essential.     

Ecdysteroid synthesis by the ring gland of Drosophila melanogaster during late-larval,prepupal and pupal development

Radioimmunoassay has been used to determine the characteristics of ecdysteroid synthesis by ring glands and brain-ring gland preparations from late 3rd-instar larvae of Drosophila melanogaster cultured in vitro. The rate of synthesis and secretion is linear for at least 4 hr in culture. Using a 4-hr culture period, variation in the rate of ecdysteroid synthesis by brain-ring gland preparations during larval, prepupal and pupal development has been examined. The rate of synthesis and secretion is highest in late 3rd-instar larvae and decreases after puparium formation. During pupal development, at a time when the endogenous ecdysteroid titre is again increasing, the rate of ecdysteroid synthesis by brain-ring gland preparations remains low and is only 10% of that prior to puparium formation. It is, therefore, likely that the ring gland is not a major source of ecdysteroids during this period.     

The development of the sensory organs of the legs in the blowfly,Phormia regina

Summary The development of the sensory neurons of the legs of the blowfly,Phormia regina has been described from the third instar larva to the late pupa using immunohistochemical staining. The leg discs of the third instar larva contain 8 neurons of which 5 come to lie in the fifth tarsomere of the developing leg. Whereas 2 neurons persist at least to the late pupa, the other cells degenerate. The first neurons of gustatory sensilla arise in the fifth tarsomere at about 1.5 h after formation of the puparium. Most of these sensilla, however, appear within a short time period beginning at about 18 h. The femoral chordotonal sensory neurons first appear at the time of formation of the puparium, as a mass of cells situated in the distal femur. During later pupal development 2 groups of these cells come to lie at the femur-trochanter border, where they become the proximal femoral chordotonal organ of the adult; the remaining cells become the distal femoral chordotonal organ. Other scolopidial neurons appear later in development. The nerve pathways of the late pupal leg are established either by the axons of the cells that are present in the larval leg disc or by new outgrowing processes of sensory neurons. In the tibia, the initial direction of new outgrowth differs in different regions of the segment: proximal tibial neurons grow distally, while distal tibial neurons grow initially proximally.     

A possible role of ecdysteroids in pre-ecdysial tanning in larvae of Sarcophaga bullata (Diptera: Sarcophagidae)

The levels of ecdysteroids in Sarcophaga bullata were determined by radioimmunoassay (RIA) from the time of larviposition (0 hr) to after the 2nd ecdysis and from late larval to pupal development. Two distinct peaks of ecdysteroid activity were recorded mid-way through the first and second stadia (14 and 34 hr) and two smaller peaks occurred a few hours prior to each ecdysis. A large release of ecdysteroids occurred from 8 hr before and up to 18 hr after formation of the white prepupa. This peak initiated the formation of the prepupa, the tanning of the puparium, larval/pupal apolysis and secretion of the pupal cuticle.Assays for the cuticle tanning hormone, bursicon, in pre-ecdysial larvae were not positive and a possible role for ecdysone in pre-ecdysial tanning of larval cuticular structures is proposed.     

Morphogenesis and cuticular markers during the larval-pupal transformation of the medfly Ceratitis capitata

Changes in morphology during early metamorphosis of the medfly, Ceratitis capitata (Wied.) (Tephritidae) were correlated with biochemical differentiation events. Protein profiles were studied both in the 3rd instar larval cuticle further transformed into puparium and the newly synthesized pupal cuticle. Beta-alanine incorporation into the puparium (0–20 h) correlates with concomitant pigmentation (completed by 16 h) and sclerotization phenomena. This early tannification program seems to be followed by deposition of a layer of substances, probably ecdysial fluid remnants, into the puparium. Their deposition ends approximately at +46 h. Simultaneously, pupal cuticle material starts to be deposited. Synthesis and deposition of the main pupal cuticle protein was detected 48 h after pupariation. At that time, eversion of the pupal head occurs. The definitive profile of pupal cuticle proteins was attained at around +72 h together with the establishment of adult body proportions.     

Study of catheptic and acid phosphatase activities during development and metamorphosis ofDrosophila melanogaster

Summary Developmental stages ofDrosophila melanogaster cultured at 22 ± 1° C were collected and tested for catheptic activity and acid phosphatase activity.It was found that catheptic activity was absent in the egg as well as in the first and second larval instars. The activity first appears in the third instar larva and reaches its peak 24 to 48 hr after puparium formation. It then decreases, at first gradually and at pupal stage 9 (120 to 144 hr after puparium formation) abruptly, reaching zero level just before the emergence of the imago.The pattern of acid phosphatase activity in different developmental stages was found to be broadly similar to that of catheptic activity in the larval and pupal stages. However, the acid phosphatase activity was found to be exceptionally high in the egg in contrast to the catheptic activity.The authors are grateful to Prof. Dr. R. Weber, Zoological Institute of the University of Bern, for constructive criticism of this paper.     

Looking into the puparium: Micro‐CT visualization of the internal morphological changes during metamorphosis of the blow fly,Calliphora vicina,with the first quantitative analysis of organ development in cyclorrhaphous dipterans

Metamorphosis of cyclorrhaphous flies takes place inside a barrel‐like puparium, formed by the shrinking, hardening and darkening of the third‐instar larval cuticle. The opacity of this structure hampers the visualization of the morphological changes occurring inside and therefore a full understanding of the metamorphosis process. Here, we use micro‐computed tomography (micro‐CT) to describe the internal morphological changes that occur during metamorphosis of the blow fly, Calliphora vicina Robineau‐Desvoidy 1830 (Diptera: Calliphoridae) at a greater temporal resolution than anything hitherto published. The morphological changes were documented at 10% intervals of the total intra‐puparial period, and down to 2.5% intervals during the first 20% interval, when the most dramatic morphological changes occur. Moreover, the development of an internal gas bubble, which plays an essential role during early metamorphosis, was further investigated with X‐ray images and micro‐CT virtual sections. The origin of this gas bubble has been largely unknown, but micro‐CT virtual sections show that it is connected to one of the main tracheal trunks. Micro‐CT virtual sections also provided enough resolution for determining the completion of the larval‐pupal and pupal‐adult apolyses, thus enabling an accurate timing of the different intra‐puparial life stages. The prepupal, pupal, and pharate adult stages last for 7.5%, 22.5%, and 70% of the total intra‐puparial development, respectively. Furthermore, we provide for the first time quantitative data on the development of two organ systems of the blow fly: the alimentary canal and the indirect flight muscles. There is a significant and negative correlation between the volume of the indirect flight muscles and the pre‐helicoidal region of the midgut during metamorphosis. The latter occupies a large portion of the thorax during the pupal stage but narrows progressively as the indirect flight muscles increase in volume during the development of the pharate adult.     

Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster.

The spatio-temporal proliferation pattern of postembryonic neuroblasts in the central brain region of the supra-esophageal ganglion of Drosophila melanogaster was studied by labeling DNA replicating cells with 5-bromo-2'-deoxyuridine (BrdU). There are five proliferating neuroblasts per hemisphere in larvae just after hatching: one in the ventro-lateral, and the other four in the postero-dorsal region of the brain. Dividing neuroblasts increase during the late first-late second instar larval stages, reaching a plateau of about 85 neuroblasts per hemisphere. Most neuroblasts cease dividing 20-30 hr after puparium formation (APF), while only four in the postero-dorsal region continue making progenies until 85-90 hr APF. The four distinct neuroblasts proliferating in the early larval and late pupal stages are identical; they lie in the cortex above the calyces of the mushroom bodies (corpora pedunculata), proliferating over a period twice as long as that for the other neuroblasts. Their daughter neurons project into the mushroom body neuropile, and hence are likely to be the Kenyon cells. The cell-cycle period of the four neuroblasts (named mushroom body neuroblasts: MBNbs) is rather constant (1.1-1.5 hr) during the mid larval-early pupal stages and is longer before and after that. The total number of the MBNb progenies made throughout the embryonic and postembryonic development was estimated to be 800-1200 per hemisphere.     

Seasonal effects on cuticular permeability and epicuticular lipid composition inCentruroides sculpturatus ewing 1928 (Scorpiones: Buthidae)

Summary Third larval instar hemolymph of the fruitflyDrosophila hydei did not metabolize juvenile hormone (JH) at all developmental stages. In contrast, prepupal and pupal body fluid showed JH-esterase activity with a maximum at 4 h after puparium formation. In body wall and fat body of all developmental stages investigated, JH-metabolic activity was found. In both tissues JH catabolism was most active in the 120,000g supernatant and pellet. The 800g and 15,000g pellet showed a lower activity. In all subcellular fractions the JH-acid was identified as the predominant metabolite. There is evidence that JH-specific esterases are responsible for ester cleavage in the 120,000g supernatant. During mid and late third larval instar development in both body wall and fat body JH-esterase activity remains relatively constant.     

棉铃虫组织蛋白酶B组织分布与合成部位的研究

蛋白酶是指裂解肽链的所有酶类 ,根据作用位点的催化基团将蛋白酶分为 4大类 ,即丝氨酸蛋白酶、半胱氨酸蛋白酶 (ＣｙｓｔｅｉｎｅＰｒｏｔｅｉｎａｓｅｓ ,ＣＰ)、天冬氨酸蛋白酶和金属蛋白酶。每一大类又包括多种不同的蛋白酶 ,其中半胱氨酸蛋白酶是一类细胞内蛋白酶 ,包括组织蛋白酶Ｂ、Ｌ、Ｈ、Ｎ、Ｓ、Ｔ等 ,其活性中心含有活性必需的半胱氨酸残基 ,细胞内高度的还原环境对它们的作用非常重要 (Ｔｕｒｋ＆Ｂｏｂｔ,1991)。蛋白酶参与多种生理、病理性蛋白水解 ,在昆虫中的分布和功能也有报道 ,如蚊子卵中含有组织蛋白酶Ｂ ,参与胚胎发…     

Engrailed expression in the anterior lineage compartment of the developing wing blade of Drosophila.

The developing wing of Drosophila melanogaster was examined at larval and pupal stages of development to determine whether the anterior-posterior lineage boundary, as identified by lineage restrictions, was congruent with the boundaries defined by the expression of posterior-specific (engrailed, invected), and anterior-specific (cubitus interruptus-D) genes. The lineage boundary was identified by marking mitotic recombinant clones, using an enhancer trap line with ubiquitous beta-gal expression in imaginal tissues; clones of +/+ cells were identified by their lack of beta-gal expression. Domains of gene expression were localized using antibodies and gene specific lacZ constructs. Surprisingly, it was found that engrailed expression extended a small distance into the anterior lineage compartment of the wing blade, as identified with anti-en/inv mAb, anti-en polyclonal antiserum, or an en-promoter-lacZ insert, ryxho25. This anterior expression was not present in early third instar discs, but appeared during subsequent larval and pupal development. In contrast, the expression of cubitus interruptus-D, as identified using the ci-Dplac insert, appeared to be limited to the anterior lineage compartment. Thus, en expression is not limited to cells from the posterior lineage compartment, and en and ci-D activities can overlap in a region just anterior to the lineage compartment boundary in the developing wing. The lineage boundary could also be identified by a line of aligned cells in the prospective wing blade region of wandering third instar discs. A decapentaplegic-lacZ construct was expressed in a stripe several cells anterior to the lineage boundary, and did not define or overlap into the posterior lineage compartment.     

Seasonal plasticity in growth and development of the speckled wood butterfly, Pararge aegeria (Satyrinae)

Regulation of growth and development by photoperiod was studied in a population of the speckled wood butterfly, Purarge aegeria L. (Lepidoptera: Satyrinae), from southern Sweden. Individuals were reared in a range of photoperiodic regimes (9L. to 22L) and temperatures (13°C to 21° C). Plasticity was found for important life-history traits- generation time, growth rate and final weight and seasonal regulation of development in response to photoperiod was found to occur at two levels. Purarge aegeria hibernates as a third instar larva or in the pupal stage, cantering one of four major developmental pathways in response to photoperiod: (1) direct development in both the larval and pupal stages, (2) pupal winter diapause with or (3) without a preceding larval summer diapause, or (4) larval winter diapause. In addition to this high-level regulation of individual development, larval growth rate and pupal development rate also appear to be finally regulated by photoperiod within each major pathway. As photoperiods decreased from 22 h to 17 h at 17° C, growth rate among directly developing larvae increased progressively, as was the case for larva? developing according to a univoltine life cycle from 17 h to 14 h. At two photoperiods, 13 h and 16 h (corresponding to shifts between major pathways), both larval and pupal development were extremely variable with the fastest individuals developing directly and the slowest developing with a diapause. This indicates a gradual nature of diapause itself, suggesting that the two level may not he fundamentally different.     

Reversal of pupal diapause in Sarcophaga argyrostoma by temperature shifts after puparium formation

Puparia from Sarcophaga argyrostoma larvae reared under short days were collected daily within 24 hr of their formation and divided into two groups: one which remained at the larval rearing temperature, and one which was transferred to a different temperature. Such temperature shifts after puparium formation can modify the subsequent incidence of pupal diapause. Temperature step-ups decrease the percentage of diapause; temperature step-downs increase it. The degree of this effect increases with the size of the temperature step. The effectiveness of a temperature step-up declines with increasing time after puparium formation.The percentage of diapause finally achieved in any group is a function of both the number of inductive (short-day) photoperiods experienced during larval life and the magnitude and direction of the subsequent temperature step. Temperature step-ups can permit the expression of photoperiodic information which would otherwise be masked. A model is presented to account for these findings.     

The tracheal supply and muscle metabolism during muscle growth in the puparium of Calliphora vomitoria

Thirty hours after puparium formation in Calliphora, the larval tracheal system is replaced by an air-filled pupal system. This is characterized initially by many tufts of tracheae and coiled tracheoles lying in the blood. Between the third and fourth day, the sixth dorsal longitudinal flight muscles are practically without attached tracheae and their longitudinal growth can partially occur when oxygen uptake is inhibited with potassium cyanide. Sodium iodoacetate prevents muscle growth. After the fifth day of development the pupal tracheoles spread out over the surface of the developing adult muscles. Between the seventh and ninth day, longitudinal growth and increases in the diameter of the myofibrils are halted by cyanide and iodoacetate. Some longitudinal growth and an increase in the total protein content of the muscles can occur in 1% oxygen. Air filling of the adult tracheae takes place 2–3 hr before the emergence of the adult and is accompanied by an increase in oxygen consumption of the thorax. The metabolism and growth of the muscles is discussed with respect to their changing oxygen supply.     

Increased levels of the Drosophila Abelson tyrosine kinase in nerves and muscles: subcellular localization and mutant phenotypes imply a role in cell-cell interactions.

Mutations in the Drosophila Abelson tyrosine kinase have pleiotropic effects late in development that lead to pupal lethality or adults with a reduced life span, reduced fecundity and rough eyes. We have examined the expression of the abl protein throughout embryonic and pupal development and analyzed mutant phenotypes in some of the tissues expressing abl. abl protein, present in all cells of the early embryo as the product of maternally contributed mRNA, transiently localizes to the region below the plasma membrane cleavage furrows as cellularization initiates. The function of this expression is not yet known. Zygotic expression of abl is first detected in the post-mitotic cells of the developing muscles and nervous system midway through embryogenesis. In later larval and pupal stages, abl protein levels are also highest in differentiating muscle and neural tissue including the photoreceptor cells of the eye. abl protein is localized subcellularly to the axons of the central nervous system, the embryonic somatic muscle attachment sites and the apical cell junctions of the imaginal disk epithelium. Evidence for abl function was obtained by analysis of mutant phenotypes in the embryonic somatic muscles and the eye imaginal disk. The expression patterns and mutant phenotypes indicate a role for abl in establishing and maintaining cell-cell interactions.     

DHR3 is required for the prepupal-pupal transition and differentiation of adult structures during Drosophila metamorphosis.

Pulses of the steroid hormone ecdysone activate genetic regulatory hierarchies that coordinate the developmental changes associated with Drosophila metamorphosis. A high-titer ecdysone pulse at the end of larval development triggers puparium formation and induces expression of the DHR3 orphan nuclear receptor. Here we use both a heat-inducible DHR3 rescue construct and clonal analysis to define DHR3 functions during metamorphosis. Clonal analysis reveals requirements for DHR3 in the development of adult bristles, wings, and cuticle, and no apparent function in eye or leg development. DHR3 mutants rescued to the third larval instar also reveal essential functions during the onset of metamorphosis, leading to lethality during prepupal and early pupal stages. The phenotypes associated with these lethal phases are consistent with the effects of DHR3 mutations on ecdysone-regulated gene expression. Although DHR3 has been shown to be sufficient for early gene repression at puparium formation, it is not necessary for this response, indicating that other negative regulators may contribute to this pathway. In contrast, DHR3 is required for maximal expression of the midprepupal regulatory genes, EcR, E74B, and betaFTZ-1. Reductions in EcR and betaFTZ-F1 expression, in turn, lead to submaximal early gene induction in response to the prepupal ecdysone pulse and corresponding defects in adult head eversion and salivary gland cell death. These studies demonstrate that DHR3 is an essential regulator of the betaFTZ-F1 midprepupal competence factor, providing a functional link between the late larval and prepupal responses to ecdysone. Induction of DHR3 in early prepupae ensures that responses to the prepupal ecdysone pulse will be distinct from responses to the late larval pulse and thus that the animal progresses in an appropriate manner through the early stages of metamorphosis.     

丝光绿蝇蛹壳颜色变化及其对死后间隔时间推断的意义

【目的】研究不同恒温下丝光绿蝇Lucilia sericata蛹期蛹壳颜色变化,并利用提取的蛹壳RGB值绘制标准色板,为法医学中死后间隔时间(postmortem interval, PMI)的推断提供科学依据。【方法】在16, 20, 24, 28和32℃恒温下分别饲养丝光绿蝇待其发育至蛹期,选取10头蛹为观察样本,每隔12 h定期观察蛹壳的颜色变化,拍照并利用图像分析软件提取蛹期各观察时间点蛹壳颜色的RGB值,再将分析计算后的RGB值还原为标准色板,并采用R软件对蛹发育时间与蛹壳颜色RGB值中的R值的关系进行多元回归分析和拟合。【结果】不同恒温下,丝光绿蝇蛹壳颜色随发育时间均呈现出不均衡的加深趋势,尤其在化蛹初期和临近羽化时变化明显。根据各发育时间的标准RGB值可制作出对应不同恒温的5个丝光绿蝇蛹壳颜色标准色板。【结论】在案发现场,可将检获到的蝇蛹与本研究得到的相近温度下的蛹壳颜色标准色板进行比色鉴定,初步估算蛹期,为死后间隔时间的推断提供科学依据。     

Hormonal and nutritional regulation of insect fat body development and function

The insect fat body is an organ analogue to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism. Similar to other larval organs, fat body undergoes a developmental “remodeling” process during the period of insect metamorphosis, with the massive destruction of obsolete larval tissues by programmed cell death and the simultaneous growth and differentiation of adult tissues from small clusters of progenitor cells. Genetic ablation of Drosophila fat body cells during larval‐pupal transition results in lethality at the late pupal stage and changes sizes of other larval organs indicating that fat body is the center for pupal development and adult formation. Fat body development and function are largely regulated by several hormonal (i.e. insulin and ecdysteroids) and nutritional signals, including oncogenes and tumor suppressors in these pathways. Combining silkworm physiology with fruitfly genetics might provide a valuable system to understand the mystery of hormonal regulation of insect fat body development and function. © 2009 Wiley Periodicals, Inc.