Gap junction distribution in the Drosophila wing disc mutants vg,l(2)gd,l(3)c43hs1, and l(2)gl4

The density of gap junctions in four Drosophila melanogaster mutants with abnormal wing disc development has been determined using quantitative electron microscopy and compared with the gap junction density in wild-type wing discs. No appreciable differences relative to wild-type controls were found in the cell death mutant vestigial or in the mildly hyperplastic mutant lethal giant disc which could not be accounted for in terms of altered lateral plasma membrane surface density or as an extension of the gap junction growth which normally occurs during the third larval stage of development in wild-type wing discs. However, both the severely hyperplastic mutant l₍₃₎c43^hs1 and the neoplastic mutant lethal giant larva have significant reductions in the gap junction surface density, the number of gap junctions, and the gap junction areal fraction of the lateral plasma membrane compared with wild-type controls. These differences cannot be attributed to altered lateral plasma membrane surface densities which are not significantly different from wild-type control wing discs. The reduced gap junction density in severely hyperplastic and neoplastic wing discs suggests that alterations in the number or distribution of gap junctions may be as disruptive to normal growth and development as their complete absence.     

The essential dietary fatty acid requirement of the tufted apple budmoth, Platynota idaeusalis

The tufted apple budmoth, Platynota idaeusalis (Walker), was reared non-axenically for two successive generations on a casein-based semisynthetic diet. The qualitative essential fatty acid requirement for growth, development and normal pupal-adult ecdysis was studied using the non-axenic casein-based semisynthetic diets with and without various 99% pure fatty acids. Linoleic or linolenic acids caused accelerated larval development; linoleic, linolenic and arachidonic acids showed similar activity in body weight gain and survival to pupal-adult ecdysis. Linoleic or linolenic acids were active in alleviating wing deformities; arachidonic acid was partially active in alleviating wing deformities at the one dietary concentration evaluated. Activity of arachidonic acid as an essential fatty acid for P. idaeusalis is unique among insects, except for mosquitoes. The essential fatty acid deficiency syndrome of the adult, resulting from the larvae feeding on fat-deficient diets, was greatly reduced when larvae were fed on a diet adequate in essential fatty acid during either their early or late development.     

Epithelial cell-turnover ensures robust coordination of tissue growth in Drosophila ribosomal protein mutants

Highly reproducible tissue development is achieved by robust, time-dependent coordination of cell proliferation and cell death. To study the mechanisms underlying robust tissue growth, we analyzed the developmental process of wing imaginal discs in Drosophila Minute mutants, a series of heterozygous mutants for a ribosomal protein gene. Minute animals show significant developmental delay during the larval period but develop into essentially normal flies, suggesting there exists a mechanism ensuring robust tissue growth during abnormally prolonged developmental time. Surprisingly, we found that both cell death and compensatory cell proliferation were dramatically increased in developing wing pouches of Minute animals. Blocking the cell-turnover by inhibiting cell death resulted in morphological defects, indicating the essential role of cell-turnover in Minute wing morphogenesis. Our analyses showed that Minute wing discs elevate Wg expression and JNK-mediated Dilp8 expression that causes developmental delay, both of which are necessary for the induction of cell-turnover. Furthermore, forced increase in Wg expression together with developmental delay caused by ecdysone depletion induced cell-turnover in the wing pouches of non-Minute animals. Our findings suggest a novel paradigm for robust coordination of tissue growth by cell-turnover, which is induced when developmental time axis is distorted.     

Crowding leads to higher incidence of brachypterous females in the tobacco thrips,Frankliniella fusca (Hinds) (Thysanoptera: Thripidae)

The effects of larval density on the wing form determination of female tobacco thrips, Frankliniella fusca, were investigated by rearing thrips on leaf disks at 27.5 °C. The developmental period, head width, body length, and forewing length of individuals in each wing morph were determined to assess the relationships among larval density, growth, and wing form. Data showed that higher rearing densities increased the production of female F. fusca brachypters. There was no consistent difference in the mean developmental periods between the two wing morphs or among all 5 density treatments. The body length of females tended to decrease with increasing rearing density, but there was no significant difference in body size between the two wing morphs when they were reared under the same density level.     

An Appraisal of Life History Features of Kiefferulus calligaster (Kieffer, 1911) (Diptera: Chironomidae) from Kolkata,India

Life history parameters of the freshwater chironomid species Kiefferulus calligaster (Kieffer, 1911) were investigated under laboratory conditions, with the use of larval development time and wing length as key features. An index of fitness was derived using these two parameters to represent the fitness of adults as a function of the larval development. Survivorship, deduced from the data on the mortality of larval stages, was related to developmental time as—(survivorship, l_x) y = 1.16 ? 0.04 × (days). The larval development time varied between males and females with a minimum of 8 and a maximum of 12 days from first instar larva to eclosion of imagine. The average wing length of adult females was larger than males (3.9 mm ± 0.03 S.E. vs. 3.36 mm ± 0.02 S.E.), for both early and late emerging individuals. The degree of dimorphism between the sexes was prominent for wing length and larval development time. The index of fitness for the early and late emerging adults differed significantly (P < 0.05) in both the sexes.     

The wing imaginal disc

The Drosophila wing imaginal disc is a tissue of undifferentiated cells that are precursors of the wing and most of the notum of the adult fly. The wing disc first forms during embryogenesis from a cluster of ∼30 cells located in the second thoracic segment, which invaginate to form a sac-like structure. They undergo extensive proliferation during larval stages to form a mature larval wing disc of ∼35,000 cells. During this time, distinct cell fates are assigned to different regions, and the wing disc develops a complex morphology. Finally, during pupal stages the wing disc undergoes morphogenetic processes and then differentiates to form the adult wing and notum. While the bulk of the wing disc comprises epithelial cells, it also includes neurons and glia, and is associated with tracheal cells and muscle precursor cells. The relative simplicity and accessibility of the wing disc, combined with the wealth of genetic tools available in Drosophila, have combined to make it a premier system for identifying genes and deciphering systems that play crucial roles in animal development. Studies in wing imaginal discs have made key contributions to many areas of biology, including tissue patterning, signal transduction, growth control, regeneration, planar cell polarity, morphogenesis, and tissue mechanics.     

Towards Long Term Cultivation of Drosophila Wing Imaginal Discs In Vitro

The wing imaginal disc of Drosophila melanogaster is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with RGB cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs.     

Screening of larval/pupal P-element induced lethals on the second chromosome in Drosophila melanogaster: clonal analysis and morphology of imaginal discs

We have carried out screens for lethal mutations on the second chromosome of Drosophila melanogaster that are associated with abnormal imaginal disc morphologies, particularly in the wing disc. From a collection of 164 P element-induced mutations with a late larva/pupa lethal phase we have identified 56 new loci whose gene products are required for normal wing disc development and for normal morphology of other larval organs. Genetic mosaics of these 56 mutant lines show clonal mutant phenotypes for 23 cell-viable mutations. These phenotypes result from altered cell parameters. Causal relationships between disc and clonal phenotypes are discussed.     

Environmental factors controlling seasonal morph determination in the small copper butterfly, Lycaena phlaeas daimio Seitz.

Environmental factors controlling seasonal morph determination and extension of the larval stages in Lycaena phlaeas daimio were studied in field observations and laboratory experiments, using criteria based on the numbers of red scales in the wing spots. Photoperiod and temperature conditions applied during the larval period and a low-temperature (5°C) introduced in pupal period were expected to be factors affecting the seasonal morph determination: the effects were confirmed by laboratory experiments. Furthermore, the length of the larval period extended by exposure to short days at 20°C, but not at 25°C, were found to show a clear correlation with the spring morph determination, but the correlation coefficient was negative in marked contrast to the situation in Polygonia and Papilio.     

Long-Chain Omega-3 Polyunsaturated Fatty Acids Have Developmental Effects on the Crop Pest,the Cabbage White Butterfly Pieris rapae

Nutritional enhancement of crops using genetic engineering can potentially affect herbivorous pests. Recently, oilseed crops have been genetically engineered to produce the long-chain omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) at levels similar to that found in fish oil; to provide a more sustainable source of these compounds than is currently available from wild fish capture. We examined some of the growth and development impacts of adding EPA and DHA to an artificial diet of Pieris rapae, a common pest of Brassicaceae plants. We replaced 1% canola oil with EPA: DHA (11:7 ratio) in larval diets, and examined morphological traits and growth of larvae and ensuing adults across 5 dietary treatments. Diets containing increasing amounts of EPA and DHA did not affect developmental phenology, larval or pupal weight, food consumption, nor larval mortality. However, the addition of EPA and DHA in larval diets resulted in progressively heavier adults (F _{4, 108} = 6.78; p = 0.011), with smaller wings (p < 0.05) and a higher frequency of wing deformities (R = 0.988; p = 0.001). We conclude that the presence of EPA and DHA in diets of larval P. rapae may alter adult mass and wing morphology; therefore, further research on the environmental impacts of EPA and DHA production on terrestrial biota is advisable.     

The defective dorsal discs gene of Drosophila is required for the growth of specific imaginal discs

The wild-type allele of the gene defective dorsal discs (ddd) is required for the normal development of the dorsal thoracic discs in Drosophila melanogaster. In ddd mutant larvae the dorsal discs (wing, haltere, and humeral) are greatly reduced in size or absent while the ventral discs (leg) are unaffected. We have examined the function of the ddd+ gene in wing development. The ddd+ product is not involved in the initial determination of wing cells but rather is required for their subsequent proliferation during the larval period. Analysis of chimaeras shows that there is a requirement for ddd+ gene expression in wing discs, but it is sufficient for normal development that only some cells in a disc express the gene. We propose that the ddd+ product is involved in the synthesis of a factor which is required for the normal growth of wing discs and which can be transferred between wing disc cells.     

Pre-imaginal flight motor pattern in Locusta

In a wind stream, larval stages of Locusta usually show a tonic muscle activity but they can also exhibit a rhythmic motor output. With ageing such a pattern can be released sooner, the trains become longer. The basic rhythm of 10 Hz does not change. The initial co-contraction of specific muscles is substituted later in development by an antagonistic recruitment. This activity resembles the flight motor pattern of young locusts which lack phasic sensory feedback from the wing region. Azadirachtin, an insect growth regulator, has been used to produce a permanent 5th larval instar. However, the extension of the last larval stage does not lead to a further development of the motor pattern to a level comparable to mature animals.     

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.     

Effects of fat body on α-ecdysone induced morphogenesis in cultured wing disks of the wax moth, Galleria mellonella

Fat body promoted ecdysone induced morphogenesis in Galleria wing disks cultured in vitro. Medium preincubated with fat body and α-ecdysone from any of the first 5 days of the final larval instar enhanced tracheal migration and elongation in the disks. Disks from the third, fourth, and fifth days of the final larval instar responded equally well to the fat body and α-ecdysone. We suggest a physiological rôle for Galleria fat body as an intermediary in the stimulation of wing disk development by α-ecdysone.     

Control of pupal commitment in the imaginal disks of Precis coenia (Lepidoptera: Nymphalidae)

When final (5th) instar larvae of Precis coenia were treated with the juvenile hormone analog (JHA) methoprene, they underwent a supernumerary larval molt, except for certain regions of their imaginal disks, which deposited a normal pupal cuticle. Evidently those regions had already become irreversibly committed to pupal development at the time JHA was applied. By applying JHA at successively later times in the instar, the progression of pupal commitment could be studied. Pupal commitment in the proboscis, antenna, eye, leg and wing imaginal disks occurred in disk-specific patterns. In each imaginal disk there were distinct initiation sites where pupal commitment began during the first few hours of the final larval instar, and from which commitment spread across the remainder of the disk over a 2- to 3-day period. The initiation sites were not always located in homologous regions of the various disks. As a rule, pupal commitment also spread from imaginal disk tissue to surrounding epidermal tissue. The regions of pupal commitment in all disks except those of the wings, coincided with the regions of growth of the disk. Only portions of the disk that had undergone cell division and growth underwent pupal commitment. Shortening the growth period did not prevent pupal commitment in the wing imaginal disk, indicating that, in this disk at least, a normal number of cell divisions was not crucial in reprogramming of disk cells for pupal cuticle synthesis. The apparent growth spurt of imaginal disks that occurs during the last part of the final larval instar is merely the final stage of normal and constant exponential growth. Juvenile hormone (JH) and ecdysteroids appeared to play little role in the regulation of normal imaginal disk growth. Instead, growth of the disks may be under intrinsic control. Interestingly, even though endogenous fluctuation in JH titers do not affect imaginal disk growth, exogenous JHA proved able to inhibit both pupal commitment, cell movement, and growth of the disks during the last larval instar. This function of JH could be important under certain adverse conditions, such as when metamorphosis is delayed in favor of a supernumerary larval molt.     

Patterning Muscles Using Organizers: Larval Muscle Templates and Adult Myoblasts Actively Interact to Pattern the Dorsal Longitudinal Flight Muscles of Drosophila

Pattern formation in muscle development is often mediated by special cells called muscle organizers. During metamorphosis in Drosophila, a set of larval muscles function as organizers and provide scaffolding for the development of the dorsal longitudinal flight muscles. These organizers undergo defined morphological changes and dramatically split into templates as adult fibers differentiate during pupation. We have investigated the cellular mechanisms involved in the use of larval fibers as templates. Using molecular markers that label myoblasts and the larval muscles themselves, we show that splitting of the larval muscles is concomitant with invasion by imaginal myoblasts and the onset of differentiation. We show that the Erect wing protein, an early marker of muscle differentiation, is not only expressed in myoblasts just before and after fusion, but also in remnant larval nuclei during muscle differentiation. We also show that interaction between imaginal myoblasts and larval muscles is necessary for transformation of the larval fibers. In the absence of imaginal myoblasts, the earliest steps in metamorphosis, such as the escape of larval muscles from histolysis and changes in their innervation, are normal. However, subsequent events, such as the splitting of these muscles, fail to progress. Finally, we show that in a mutant combination, null for Erect wing function in the mesoderm, the splitting of the larval muscles is aborted. These studies provide a genetic and molecular handle for the understanding of mechanisms underlying the use of muscle organizers in muscle patterning. Since the use of such organizers is a common theme in myogenesis in several organisms, it is likely that many of the processes that we describe are conserved.     

Restoration of wing margins in Lyra mutants of Drosophila melanogaster

Lyra is a dominant, homozygous lethal mutation of Drosophila melanogaster; in heterozygotes the wings lack portions of the anterior and posterior margins including the characteristic bristles. We have found that, in addition to the loss of bristle forming cells, there is a decrease in the number of wing surface cells that varies between 10 and 20%. However, we observed no histological evidence of excessive cell death in either the larval discs or the pupal wing precursors in Lyra flies. Restoration of all or part of the normal wing margins occurs in some, but not all, cases of morphogenetic mosaics, in which there were patches of wild-type cells in Lyra wing margins due to irradiation-induced mitotic recombination. Analysis of these restorations, using margin bristles as indicators, shows that the Lyra wild-type gene is not involved in bristle formation per se and further that its expression is not cell autonomous. Instead the effect of the Lyra mutation appears to be associated with development of a margin forming subpopulation of cells and to influence the characteristic pattern of cells and bristles in the wing margin via an inductive interaction. The dorsal-ventral boundary can be demonstrated in the de facto wing margins of Lyra mutants suggesting that its origin is independent of any function Lyra might have in normal wing margin morphogenesis. In wing margin restorations the dorsal-ventral boundary is clearly delimited by trichomes and somewhat less rigorously shown by the margin bristles. Further, in these restorations ventral clones induce dorsal bristles, as well as ventral ones, and vice versa, indicating that the influence of Lyra is not restricted by the dorsal-ventral boundary.     

Differential Involvement of Hedgehog Signaling in Butterfly Wing and Eyespot Development

Butterfly eyespots may have evolved from the recruitment of pre-existent gene circuits or regulatory networks into novel locations on the wing. Gene expression data suggests one such circuit, the Hedgehog (Hh) signaling pathway and its target gene engrailed (en), was recruited from a role in patterning the anterior-posterior insect wing axis to a role patterning butterfly eyespots. However, while Junonia coenia expresses hh and en both in the posterior compartment of the wing and in eyespot centers, Bicyclus anynana lacks hh eyespot-specific expression. This suggests that Hh signaling may not be functioning in eyespot development in either species or that it functions in J. coenia but not in B. anynana. In order to test these hypotheses, we performed functional tests of Hh signaling in these species. We investigated the effects of Hh protein sequestration during the larval stage on en expression levels, and on wing size and eyespot size in adults. Hh sequestration led to significantly reduced en expression and to significantly smaller wings and eyespots in both species. But while eyespot size in B. anynana was reduced proportionately to wing size, in J. coenia, eyespots were reduced disproportionately, indicating an independent role of Hh signaling in eyespot development in J. coenia. We conclude that while Hh signaling retains a conserved role in promoting wing growth across nymphalid butterflies, it plays an additional role in eyespot development in some, but not all, lineages of nymphalid butterflies. We discuss our findings in the context of alternative evolutionary scenarios that led to the differential expression of hh and other Hh pathway signaling members across nymphalid species.