An ultrastructural analysis of the ecdysoneless ((l(3)ecd 1ts) ring gland during the third larval instar of Drosophila melanogaster

Summary In the late third larval instar of Drosophila melanogaster, the prothoracic gland, an endocrine portion of the ring gland, synthesizes ecdysteroids at an accelerated rate. The resultant ecdysteroid titer peak initiates the events associated with metamorphosis. The normal prothoracic gland displays several ultrastructural features at this developmental stage that reflect increased steroidogenic activity, including extensive infoldings of the plasma membrane (membrane invaginations) and an increase in both the concentration of smooth endoplasmic reticulum (SER) (or transitional ER) and elongated mitochondria. By contrast, the prothoracic glands of larvae homozygous for a conditional larval lethal mutation, l(3)ecd ^1ts, not only fail to produce ecdysteroids at normal levels at the restrictive temperature (29° C), but also acquire abnormal morphological features that reflect the disruptive effects of the mutation. These abnormalities include an accumulation of lipid droplets presumed to contain sterol precursors of ecdysteroids, a disappearance of SER and a drastic reduction of membrane invaginations in the peripheral area of the cell. These morphological defects are observed in prothoracic glands dissected from larvae transferred from 18° C to 29° C approximately 24 h before observation and also within 4 h of an in vitro transfer to 29° C following dissection from wandering third instar larvae reared at 18° C. No ultrastructural abnormalities were noted in the corpus allatum portion of mutant ring glands. These observations further indicate the direct involvement of the ecd gene product in ecdysteroid synthesis and suggest a role for the gene in the proper transport of precursors to the site where they can be utilized in ecdysteroid biosynthesis.     

Developmental studies on two ecdysone deficient mutants ofDrosophila melanogaster

Summary This paper describes two ecdysone-deficient, recessive-lethal mutants,lethal(1)giant ring gland (grg) andlethal(1)suppressor of forked ^mad-ts (mad-ts: Jürgens and Gateff 1979) and compares their ecdysteroid titers with that of the wild-type. Mutant larvae show a much reduced ecdysteroid content, amounting to 1/10 to 1/30 of the wild-type values, but never a true titer peak. They fail to pupate and die after 1–3 weeks. Ecdysteroid feeding elicits different responses in the larvae of the two mutants.mad-ts larvae pupate within 24 h, thus showing that their low ecdysteroid titer is directly connected to their inability to pupate.mad-ts resembles the mutantlethal (3)ecdysone-1 ^ts (Garen et al. 1977). Thegrg mutant larvae, on the other hand, fail to pupate after 20-hydroxyecdysone feeding as well as injection. The primary defect of thegrg mutant is not entirely clear. Thegrg larval salivary gland cells appear to possess normal ecdysteroid receptors. Furthermore, the low ecdysteroid titer ingrg is not the result of an increased ecdysteroid catabolism. The primary defect in the mutant may lie in the malfunctioning neurosecretory cells which do not show neurosecretion in histological preparations. Further support for this notion comes from electronmicrographs of the enlargedgrg ring glands which, in contrast to the wild-type, do not possess nerve endings.In the wild-type three ecdysteroid peaks were found: one shortly before puparium formation, the second at approximately 12 h and the third at about 30 h after pupation. The ecdysteroid titer peak in late third instar, wild-type larvae is mainly due to the presence of 20-dydroxyecdysone as shown by radioimmunoassays after thin layer chromatography and derivatization followed by gas liquid chromatography and mass spectroscopy. In addition, a number of unidentified polar and apolar metabolites were also present.     

Neural factors that stimulate ecdysteroid synthesis by the larval ring gland ofDrosophila melanogaster

Summary The larval ring gland ofDrosophila melanogaster is the source of ecdysteroids responsible for larval-larval and larval-pupal molting. An extract prepared from theDrosophila larval central nervous system, that presumably contains prothoracicotropic hormone, elicits a significant and dose-dependent in vitro increase in ecdysteroid synthesis by ring glands from wandering third instar larvae. The synthesis of all three ecdysteroids previously identified as ring gland products is elevated by more than two-fold in the presence of neural extract. The maximum response occurs within 30 min and can be sustained for at least two hours after a 30 min exposure to neural extract. No non-neural tissue extracts evoke a response and most of the prothoracicotropic activity originates in the ventral ganglion. However, while extract prepared from larval brains elicits only a slight increase in ecdysteroid synthesis, it enhances the activity of a submaximal dose of ventral ganglion extract. This suggests that two or more neural factors, at least one from the brain lobes and another from the ventral ganglion, interact to stimulate ecdysteroid synthesis by the larval ring gland.Abbreviations CNS central nervous system - HPLC high performance liquid chromatography - PTTH prothoracicotropic hormone - RIA radioimmunoassay     

Alterations in the production of hemocytes due to a neoplastic mutation of Drosophila melanogaster

The hemocytes of a genetically induced, temperature-sensitive lethal mutation of Drosophila, Tum¹, were examined both quantitatively and qualitatively during the third larval instar. At the tumor-permissive temperature, 29°C, there was a fourfold increase in the concentration of circulating hemocytes in mutant larvae as compared to control. Additionally, the relative frequency of lamellocytes was 30 times greater in Tum¹ larvae than Basc in the early third instar. However, the severity of this abnormality gradually diminished as Tum¹ approached pupariation; though high frequencies of lamellocytes were always present. At the tumor-restrictive temperature (15°C) the concentration of circulating hemocytes was over twice that found at 29°C for Tum¹ larvae, and did not change during the course of third instar. However, in contrast to 29°C there was no abnormal increase in the frequency of lamellocytes at the tumor-restrictive temperature. Control larvae had equivalent concentrations of hemocytes at both temperatures. In one of two temperature shift experiments, Tum¹ larvae shifted from 15° to 29°C at the beginning of third instar expressed the abnormal hemocyte concentration and differentiation associated with larvae raised continuously at 29°C. In addition, Tum¹ larvae shifted from 29° to 15°C expressed reduced abnormalities of hemocyte differentiation, e.g., with fewer lamellocytes in circulation. The possibility of a temperature-sensitive period for the activation of the Tum¹ gene is discussed.     

Developmental Arrest and Ecdysteroid Deficiency Resulting from Mutations at the Dre4 Locus of Drosophila

Loss-of-function mutations of the dre4 gene of Drosophila melanogaster caused stage-specific developmental arrest, the stages of arrest coinciding with periods of ecdysteroid (molting hormone) regulated development. Nonconditional mutations resulted in the arrest of larval development in the first instar; embryogenesis was not impaired, and mutant larvae were behaviorally normal and long-lived. At 31 degrees the temperature-sensitive dre4e55 allele caused the arrest of larval development in the first or second instars. When upshifted to 31 degrees at various times during development, dre4e55 mutants exhibited nonpupariation of third-instar larvae, failure of pupal head eversion, failure of adult differentiation, or noneclosion of pharate adults. Under some temperature regimens second-instar larvae pupariated precociously without entering the normally intervening third-instar. Nonpupariation and defects in metamorphosis were associated with the reduction or elimination of ecdysteroid peaks normally associated with late-larval, prepupal, pupal and pharate adult development. Ecdysteroid production by larval ring glands from dre4e55 hemizygous larvae was suppressed after 2 hr of incubation in vitro at 31 degrees, indicating autonomous expression of the dre4 gene in the ring gland. We postulate that the dre4 gene is required for ecdysteroid production at multiple stages of Drosophila development and that the pathologies observed in dre4 mutants reflect developmental consequences of ecdysteroid deficiency.     

Vitellogenesis and fertility in Drosophila females with low ecdysteroid titres; the L(3)3DTS mutation

After 5 days at the restrictive temperature (29.5°C) adult Drosophila females heterozygous for the dominant temperature-sensitive mutation, L(3)3^DTS, have an ecdysteroid level of about half that in mutant females at 22°C and subsequently become completely sterile due to the inviability of progeny embryos. The lethal phase of progeny from mutant females varies depending upon the length of time DTS-3 females are kept at a sublethal temperature of 27°C. Thus, the DTS-3 mutation shows a maternal effect, and a deficiency of ecdysteroids or ecdysteroid-induced gene products may be responsible for progeny lethality. This lethality cannot be attributed to a deficit in the products of the hormonally-regulated yolk polypeptide genes however, since yolk polypeptide mRNA and protein levels are not reduced in DTS-3 females at the restrictive temperature.     

NEUROANATOMICAL STUDIES OF PROTHORACIC GLANDS OF COTTON BOLLWORM HELZCOVERPA ARMZCERA (LEPIDOPTERA: NOCTUIDAE)*

Abstract Gross anatomy, ultrastructure, innervation and ultrastructural alterations of the prothoracic gland (PTG) of cotton bollworm, Helicover pa armigera (Lepidoptera: Noctuidae) are illustrated for the last larval and early pupal stages as observed by light and electron microscopy. The T-shaped, paired (PTGs) consist each of 76–116 cells which are classified morphologically as large and small gland cells. In addition, another kind of small (about 6μ in diameter) gland cell was found in the PTGs of last instar larvae. The PTGs are innervated by the branches of 3 nerves! and tracheae and tracheoles are abundantly distributed to these glands. PTGs disappeared completely by the third day after ecdysis to the pupal stage (at temperature 28 C with a photoperiod L15^:D9). An intercellular channel system (ICS) is formed by numerous, deep invaginations of the plasma membrane of gland cells. This ICS gradually increases in depth and width and reaches maximum development around the time of the major ecdysteroid secretion peak during the last larval instar. Numerous multivesicular sacs (MVS) with their remnants and an extensive rough endoplasmic reticulum were observed within ICS and cytoplasm, respectively, on the fourth day of the last larval instar. At that time the matrix of mitochondria became much more electron lucent. Freeze-fracture replicas of the glandular epithelium were made from last instar (4th day larvae. Dynamics of structure are related to data from others concerning secretory states of the prothoracic glands of this species.     

Analysis of precocious metamorphosis induced by application of an imidazole derivative to early third instar larvae of the silkworm,Bombyx mori

When an imidazole derivative (KK-42) was applied to day 1 third instar larvae of the silkworm, Bombyx mori, 100% underwent precocious metamorphosis at the end of the fourth instar. Thus, the fourth instar becomes the last instar in these KK-42–treated larvae. The endocrine systems underlying the precocious metamorphosis were analyzed in the present study. Hydroprene application during the prolonged third instar after KK-42 treatment can prevent precocious metamorphosis, and the results showed dose-dependent and stage-specific effects. From analysis of the developmental changes in ecdysteroid levels in both KK-42–treated larvae and KK-42– and hydroprene-treated larvae, we conclude that changes in JH levels during the third larval instar can modify the secretion pattern of prothoracic glands and that during the next larval instar, very low ecdysteroid levels during the early stages of the presumptive last (fourth) larval instar are directly related to precocious metamorphosis. Arch. Insect Biochem. Physiol. 36:349–361, 1997. © 1997 Wiley-Liss, Inc.     

Spatial and temporal relationships between cell death and tissue overgrowth in imaginal wing disks of the Drosophila mutant I(3)C43hs1

The temperature-sensitive mutant l(3)c43^hs1 is lethal at the restrictive temperature late in the last larval instar and has wing disks that show excessive growth when larvae are reared at 25°C. Such mutant disks give rise to defective wings showing duplications and deficiencies. Abnormal folding patterns are localized to the region between the wing pouch and the area where adepithelial cells are found; the disks retain an epithelial morphology. Apoptotic cell death is distributed throughout the wing disks without any obvious concentration of dead cells in a specific area. Cell death is seen as early as 12 hr after a shift to the restrictive temperature. Temperature shift experiments also show that cell death precedes the onset of overgrowth, but since the spatial distribution of death is not localized to the regions of abnormal folds, it is unlikely that cell death and overgrowth are causally related.     

The ecdysoneless (ecd1ts) mutation disrupts ecdysteroid synthesis autonomously in the ring gland of Drosophila melanogaster

Ring glands dissected from homozygous l(3)ecd1ts wandering larvae and upshifted in vitro to the restrictive temperature, 29 degrees C, synthesize abnormally low quantities of ecdysteroid. Nevertheless, ecd1 ring glands retain the ability to respond at 29 degrees C to an extract prepared from wild-type larval neural tissues that presumably contain prothoracicotropic hormone (PTTH), although both basal and stimulated levels of synthesis are lower than those in wild-type ring glands. Extracts prepared from ecd1 neural tissue exhibit an unusually high level of PTTH activity. Mutant ring glands downshifted in vitro to the permissive temperature after removal from larvae maintained at 29 degrees C regain the ability to produce normal basal and stimulated ecdysteroid levels. Collectively, these experiments demonstrate that the ecd1 mutation disrupts the physiology of the ring gland at 29 degrees C autonomously and may also interfere with PTTH release.     

Temperature-Sensitive Mutations in DROSOPHILA MELANOGASTER Xii. the Genetic and Developmental Effects of Dominant Lethals on Chromosome 3

Out of 25,000 EMS-treated third chromosomes examined, ten dominant temperature-sensitive (DTS) lethal mutations which are lethal when heterozygous at 29 degrees C but survive at 22 degrees C were recovered. Seven of the eight mutations mapped were tested for complementation; these mutants probably define eight loci. Only DTS-2 survived in homozygous condition at 22 degrees C; homozygous DTS-2 females expressed a maternal effect on embryonic viability. Two of the mutant-bearing chromosomes, DTS-1 and DTS-6, exhibited dominant phenotypes similar to those associated with Minutes. Each of the seven mutants examined exhibited a characteristic phenotype with respect to the time of death at 29 degrees C and the temperature-sensitive period during development. Only DTS-4 exhibited dominant lethality in triploid females.     

Isolation of temperature sensitive mutations blocking clone development inDrosophila melanogaster,and the effects of a temperature sensitive cell lethal mutation on pattern formation in imaginal discs

Summary A method of isolating temperature-sensitive (ts) mutations blocking clone development, based on the analysis of twin spots produced by X-ray induced somatic recombination is reported. From this screen 10 ts mutations were recovered which caused an absence of the lethal-bearing clone at the restrictive temperature. Eight of these mutations were analyzed. Seven proved to be autonomous ts cell lethals and one was an autonomous ts mutation which reversibly affected cell division and growth of imaginal disc cells and growth of larval cells. The effects on development of one of the cell lethal mutations,l(1)ts-504, are described. Heat pulses (29°C) 24–72 hrs long caused a high frequency (up to 90%) of morphologically abnormal animals. The abnormalities observed were of two major kinds: deficiencies and duplications of imaginal disc derivatives. In addition, alterations of tarsal segmentations occurred. Heat pulses to larvae also delayed pupariation and eclosion by as much as four days. In general, longer pulses led to a greater delay in pupariation and eclosion and a higher frequency of deficiencies and duplications than shorter pulses. Exposure to restrictive temperature early in larval development delayed pupariation and resulted in mostly normal animals; exposure during the second and early third larval instar also delayed pupariation and led to a high frequency of duplications; exposure later in larval life, i.e. mid and late third larval instar, caused no delay in pupariation but led to a high frequency of deficiencies. These results can be explained by the occurrence of areas of cell death, which can be seen in the imaginal discs of larvae exposed to restrictive temperature by staining with trypan blue. This conclusion is further supported by the observation in gynandromorphs of duplications of female nonmutant tissue. These results are discussed in relation to current theories of pattern formation.     

Characterisation of a new tumorous-head mutant of Drosophila melanogaster

Summary A new homoeotic mutant, I127, showing abnormal growths in the head region including homoeotic transformation of eye to genitalia and antenna to leg, was isolated in a screen designed to find new alleles of the tumorous head (tuh-3), mutation. Similarities in the phenotype and genetics of the mutant, and complementation studies with tuh-1; tuh-3, suggest that I127 is indeed an allele of tuh-3. In combination with the first chromosome modifier tuh-1, the mutant is temperature-sensitive during the third larval instar, giving an increased penetrance of the tumorous head phenotype when reared at 25° C as opposed to 18° C. The isolation of further alleles at the tumorous-head locus are essential. The types of morphological defects which can result from mutations at this locus would enable us to establish if this is a complex locus, and if null mutations are lethal during development. The interactions of the tumorous-head gene with first chromosome modifiers and other homoeotic mutations will only be understood if we able to induce a number of mutations at this locus, and as a consequence begin to elucidate the role of the wild-type gene product in normal development.     

Morphogenetic deficiencies in transplanted gonadal anlagen of the mutant l(3)pl (lethal-polyploid) in Drosophila hydei

Larval gonads of Drosophila hydei, homozygous for the lethal gene l(3)pl (lethal-polyploid), were cultured in normal hosts. Ovaries of the late third larval instar were implanted into metamorphosing larvae. These can attach to the gonoduct system of the host and transform into adult ovarian structures but the spectrum of their capacity to differentiate varies largely. In favourable cases mature oocytes can be formed which are fertile. More frequently mitotic disturbances in the follicle cells and cystocytes lead to the formation of abortive egg chambers and abnormally shaped oocytes. Testes of the middle third larval instar were cultured for 2 weeks in adult females. Primary spermatocytes are able to sustain meiotic divisions and form early spermatids, even though the occurrence of fractionated nuclei in post-meiotic germ cells indicates defective meiotic divisions. Post-meiotic differentiation is blocked in mutant spermatids which fail to elongate. The mutant gene l(3)pl thus, not only affects cell divisions, but also interacts in certain cytodifferentiation processes such as spermatid elongation and egg shaping. All cellular processes found so far to be abnormal in mutant tissues involve microtubular function. This suggests that the gene l(3)pl interacts with the microtubular system and several aspects of this interpretation are discussed.     

Developmental genetics of a temperature-sensitive RNA polymerase II mutation in Drosophila melanogaster

Summary Purified RNA polymerase II (RNA nucleotidyl-transferase; EC 2.7.7.6) extracted from flies possessing lesions in the Ultrabithorax-like (Ubl) locus of Drosophila melanogaster has altered activity in vitro (Greenleaf et al. 1979, 1980; Coulter and Greenleaf 1982). This strongly suggests that the Ubl locus encodes a subunit of RNA polymerase II. Ethyl methanesulfonate was used to induce a temperature-sensitive mutation in this locus. Flies either homozygous or hemizygous for this new X–linked mutation (Ubl ^ts) display viability comparable to that of wild-type flies at 22° C but are lethal at 29° C. The temperature-sensitive period for Ubl ^ts flies is between gastrulation (6 h, 29° C) and pupation (9–10 days, 22° C). Zygotes shifted from 22° C to 29° C die at either the late embryonic or first larval instar stage while temperature shifts of second and third instar larvae result in the lethal phase occurring at the pupal stage. Most pupae shifted from 22° C to 29° C undergo metamorphosis and eclose as adults. Adults are viable if placed at 29° C; however, all females and some males become sterile if maintained at this temperature.Somatic recombination was used to induce clones homozygous for a null allele of Ubl at different stages of development. Clones of this null allele appear to be cell lethal indicating that the Ubl ⁺ gene product is required at all stages of development. The viability of Ubl ^ts pupae and adults at 29° C may result from only a partial reduction in activity caused by the mutation at this nonpermissive temperature.     

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 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.     

Some aspects of activity regulation of Galleria mellonella PTTH cells

Using the Galleria prothoracicotropic bioassay, five small neurosecretory cells occurring in each dorsolateral part of protocerebrum of Galleria mellonella brain were identified as prothoracicotropic hormone (PTTH) cells. It was found that the critical period for the release of PTTH from a brain implanted in neck-ligated larva lasts up to the third day after implantation. The content of paraldehyde-fuchsin positive neurosecretory material (NSM) in PTTH cells was determined during the penultimate and last larval instar, during pupal instar, and in starved or poststarvation fed or space-deprived last instar larvae. Two peaks of NSM in PTTH cells were found in the penultimate instar (in freshly molted, and 76-h-old larvae), four peaks in the last instar larvae (in freshly molted, and in 67-, 132-, and 174-h-old larvae), and one peak in the pupal instar (in 56-76-h-old pupae). It was also observed that upon starvation NSM accumulated in PTTH cells, while after 3 h of poststarvation feeding it was released. In permanent space-deprived last instar larvae no NSM occurred in PTTH cells. In all investigated larval instars a rapid release of NSM from PTTH cells was found a few hours after molt associated with the beginning of the feeding period. The significance of the NSM content in PTTH cells is discussed in relation to ecdysteroid titer.     

Juvenile hormone synthesis by ring glands of the blowfly Lucilia cuprina

The major radiolabelled product released from ring gland and brain-ring gland complexes of third instar larval and pre-pupal stages of the sheep blowfly Lucilia cuprina upon incubation with L-[methyl-³H]methionine corresponded to one diastereomer of juvenile hormone III bisepoxide (JHB₃). Endocrine glands incubated with the juvenile hormone precursor 2E,6E-farnesoic acid released increased quantities of JHB₃, together with significant amounts of juvenile hormone III but not the isomeric methyl 2E-6,7-epoxyfarnesoate. Synthesis of JHB₃ was developmentally and neurally regulated. Ring glands and brain-ring gland complexes from third instar larvae released more JHB₃ than comparable preparations from pre-pupae, while isolated corpus allatum segments of the gland were more active than intact brain-gland complexes. These results reinforce the emerging status of JHB₃ as the characteristic juvenile hormone of dipteran insects. Arch. Insect Biochem. Physiol. 34:239–253, 1997. © 1997 Wiley-Liss, Inc.