Half Of Pupal Development Research Articles

The evolution of Bab paralog expression and abdominal pigmentation among Sophophora fruit fly species

The evolution of gene networks lies at the heart of understanding trait divergence. Intrinsic to development is the dimension of time: a network must be altered during the correct phase of development to generate the appropriate phenotype. One model of developmental network evolution is the origination of dimorphic (male-specific) abdomen pigmentation in the fruit fly subgenus Sophophora. In Drosophila (D.) melanogaster, dimorphic pigmentation is controlled by the dimorphic expression of the paralogous Bab1 and Bab2 transcription factors that repress pigmentation. These expression patterns are thought to have evolved from a monomorphic ancestral state. Here we show that the spatial domain and contrast in dimorphic Bab expression increases during the latter half of pupal development, and this late pupal expression is necessary and sufficient to suppress pigmentation. Late pupal Bab expression was monomorphic for species from basal clades exhibiting monomorphic pigmentation, though dimorphic expression was observed in D. pseudoobscura that represents an intermediate-branching monomorphic clade. Among species from the dimorphic Sophophora clades, Bab expression was dimorphic, but a poor correlation was found between the domains of expression and male pigmentation. Lastly, while Bab paralog co-expression was generally observed, an instance of paralog-specific expression was found, indicating more complex regulatory mechanisms and mutational effects have shaped the evolution of the bab locus. These results highlight the importance of the time and place of Bab expression for pigmentation development and evolution, and suggest that dimorphism evolved early in Sophophora, but diversity in male pigmentation was not further shaped by alterations in Bab expression.

Localized Netrins Act as Positional Cues to Control Layer-Specific Targeting of Photoreceptor Axons in Drosophila

SummaryA shared feature of many neural circuits is their organization into synaptic layers. However, the mechanisms that direct neurites to distinct layers remain poorly understood. We identified a central role for Netrins and their receptor Frazzled in mediating layer-specific axon targeting in the Drosophila visual system. Frazzled is expressed and cell autonomously required in R8 photoreceptors for directing their axons to the medulla-neuropil layer M3. Netrin-B is specifically localized in this layer owing to axonal release by lamina neurons L3 and capture by target neuron-associated Frazzled. Ligand expression in L3 is sufficient to rescue R8 axon-targeting defects of Netrin mutants. R8 axons target normally despite replacement of diffusible Netrin-B by membrane-tethered ligands. Finally, Netrin localization is instructive because expression in ectopic layers can retarget R8 axons. We propose that provision of localized chemoattractants by intermediate target neurons represents a highly precise strategy to direct axons to a positionally defined layer.

Developmental changes in β-subunit composition of Na,K-ATPase in the Drosophila eye

The Drosophila genome contains at least three loci for the Na,K-ATPase beta-subunit; however, only the protein products of nrv1 and nrv2 have been characterized hitherto. Here, we provide evidence that nrv3 also encodes for a functional Na,K-ATPase beta-subunit, as its protein product co-precipitates with the Na,K-ATPase alpha-subunit. Nrv3 expression in adult flies is restricted to the nervous system in which Nrv3 is enriched in selective types of sensory cells. Because Nrv3 expression is especially prominent in the compound eye, we have analyzed the subcellular and developmental distribution of Nrv3 within the visual cells and related this distribution to those of the alpha-subunit and of the beta-subunits Nrv1 and Nrv2. Prospective visual cells express Nrv2 in the third larval instar stage and during the first half of pupal development. During the last third of pupal life, Nrv3 gradually replaces Nrv2 as the Na,K-ATPase beta-subunit in the photoreceptor cells. Adult photoreceptors express Nrv3 as their major beta-subunit; the visual cells R1-R6 co-express Nrv2 at a low level, whereas R7 and R8 co-express Nrv1. Notably, beta-subunits do not co-distribute exactly with the alpha-subunit at some developmental stages, supporting the concept that the alpha-subunit and beta-subunit can exist in the plasma membrane without being engaged in alpha/beta heterodimers. The non-visual cells within the compound eye express almost exclusively Nrv2, which segregates together with the alpha-subunit to septate junctions throughout development.

An isoform-specific allele of Drosophila N-cadherin disrupts a late step of R7 targeting

Drosophila N-cadherin is required for the formation of precise patterns of connections in the fly brain. Alternative splicing is predicted to give rise to 12 N-cadherin isoforms. We identified an N-cadherin allele, N-cad(18Astop), that eliminates the six isoforms containing alternative exon 18A and demonstrate that it strongly disrupts the connections of R7 photoreceptor neurons. During the first half of pupal development, N-cadherin is required for R7 growth cones to terminate within a temporary target layer in the medulla. N-cadherin isoforms containing exon 18B are sufficient for this initial targeting. By contrast, 18A isoforms are preferentially expressed in R7 during the second half of pupal development and are necessary for R7 to terminate in the appropriate synaptic layer in the medulla neuropil. Transgene rescue experiments suggest that differences in isoform expression, rather than biochemical differences between isoforms, underlie the 18A isoform requirement in R7 neurons.

Ecdysteroid titers in pupae of highly social bees relate to distinct modes of caste development

Modifications in endocrine programs are common mechanisms that generate alternative phenotypes. In order to understand how such changes may have evolved, we analyzed the pupal ecdysteroid titers in two closely related, highly social bees: the honey bee, Apis mellifera, and a stingless bee, Melipona quadrifasciata. In both species, the ecdysteroid titers in queens reached their peak levels earlier than in workers. Titer levels at peak maxima did not differ for the honey bee castes, but in Melipona they were twofold higher in queens than in workers. During the second half of pupal development, when the ecdysteroid titers decrease and the cuticle progressively melanizes, the titer in honey bee queens remained higher than in workers, while the reverse situation was observed in Melipona. Application of the juvenile hormone analog Pyriproxyfen ® to spinning-stage larvae of Melipona induced queen development. Endocrinologically this was manifest in a queen-like profile of the pupal ecdysteroid titer. Comparing these data with previous results on preimaginal hormone titers in another stingless bee, we conclude that the timing and height of the pupal ecdysteroid peak may depend on the nature of the specific stimuli that initially trigger diverging queen/worker development. In contrast, the interspecific differences in the late pupal ecdysteroid titer profiles mainly seem to be related to caste-specific programs in tissue differentiation, including cuticle pigmentation.

Inheritance of a temperature-modified phenotype of the short antennae (sa) mutation in a moth, Ephestia kuehniella (Lepidoptera: Pyralidae).

The autosomal recessive mutation short antennae (sa) causes considerable shortening of antennae in male and female Mediterranean flour moths (Ephestia kuehniella Zeller). However, the sa phenotype can be suppressed by several physical factors, making sa moths indistinguishable from wild-type moths (sa(WT)). This can be done by subjecting larva and pupa to a higher temperature (25 degrees C), to lithium ions, or to an alternate electric field. The first half of pupal development was found to be the sensitive period for the sa(WT) phenotype. The sa(WT) phenotype is stable and cannot be reverted to the original sa type by physical or chemical factors. The sa(WT) phenotype is transmitted to future generations. When crossed with typical sa moths, the sa(WT) phenotype is inherited either as a dominant character if carried by males or a semidominant character if carried by females. We compared proteins of the ejaculate, accessory gland secretions, and spermatophore in sa, sa(WT), and wild-type males and found considerable differences between sperm proteins of sa(WT), sa, and wild-type males. The sa(WT) phenotype influences the mating success of males: sa(WT) males mated successfully with any females, whereas typical sa males were less successful in mating and then mainly with females of the same phenotype.

Synaptogenesis in the giant-fibre system of Drosophila: interaction of the giant fibre and its major motorneuronal target.

The tergotrochanteral (jump) motorneuron is a major synaptic target of the Giant Fibre in Drosophila. These two neurons are major components of the fly's Giant-Fibre escape system. Our previous work has described the development of the Giant Fibre in early metamorphosis and the involvement of the shaking-B locus in the formation of its electrical synapses. In the present study, we have investigated the development of the tergotrochanteral motorneuron and its electrical synapses by transforming Drosophila with a Gal4 fusion construct containing sequences largely upstream of, but including, the shaking-B(lethal) promoter. This construct drives reporter gene expression in the tergotrochanteral motorneuron and some other neurons. Expression of green fluorescent protein in the motorneuron allows visualization of its cell body and its subsequent intracellular staining with Lucifer Yellow. These preparations provide high-resolution data on motorneuron morphogenesis during the first half of pupal development. Dye-coupling reveals onset of gap-junction formation between the tergotrochanteral motorneuron and other neurons of the Giant-Fibre System. The medial dendrite of the tergotrochanteral motorneuron becomes dye-coupled to the peripheral synapsing interneurons between 28 and 32 hours after puparium formation. Dye-coupling between tergotrochanteral motorneuron and Giant Fibre is first seen at 42 hours after puparium formation. All dye coupling is abolished in a shaking-B(neural) mutant. To investigate any interactions between the Giant Fibre and the tergotroachanteral motorneuron, we arrested the growth of the motorneuron's medial neurite by targeted expression of a constitutively active form of Dcdc42. This results in the Giant Fibre remaining stranded at the midline, unable to make its characteristic bend. We conclude that Giant Fibre morphogenesis normally relies on fasciculation with its major motorneuronal target.

Endogenous levels of insect juvenile hormones in larval, pupal and adult stages of the tomato moth, Lacanobia oleracea

Endogenous levels of juvenile hormones 0, I, II and III have been measured each day during the 4th, 5th and 6th larval stadia of the Tomato moth, Lacanobia oleracea, using physico-chemical methods. Levels of the same hormones have also been measured at selected times during pupal development, and over the first 5 days of adult life in males, and in mated and virgin females. JH I and JH II were the predominant homologues in this insect in all developmental stages, although JH III was also present in some samples. Hormone levels were relatively high in 4th and 5th instar larvae, but were much reduced in 6th (final) instar larvae. Hormone levels increased in larvae that were about to moult to the subsequent stage. Juvenile hormones were absent in pupae during the first half of pupal development, but increased in pharate adults. Adult males and females contained substantially more hormone than pre-adult stages, although levels in virgin moths were lower than in mated adult females. JH 0 was detected in some extracts of mated adult female moths. These results are discussed in relation to the likely role of juvenile hormones in modulating various aspects of the physiology of this insect.

Differentiation of the thermo-/hygrosensitive (no-pore) sensilla on the antenna of Antheraea pernyi (Lepidoptera, Saturniidae): a study of cryofixed material

The morphogenesis of the thermo- and hygro-sensitive sensilla styloconica of Antheraea pernyi was studied, exclusively by cryomethods, during the second half of pupal development. The three major processes taking place during this period are (1) the differentiation of the dendritic outer segments of the sensory cells, especially of the lamellated type-2 receptor, (2) the formation of the receptor-lymph cavities, (3) the formation of tubular structures of unknown function in the inner receptor-lymph cavity, and (4) the elongation of the dendrite sheath. The formation of lamellae in the type-2 dendritic outer segment is achieved by the enfolding of its originally cylindrical cytoplasmic membrane. Autocellular junctions, previously described in the sensilla of adult animals, are found to join the forming lamellae. Close similarities between the junctions and smooth septate junctions are demonstrated. Both the extensive inner and outer receptor-lymph cavities are formed by invagination and folding of the apical cytoplasmic membranes of the three enveloping cells. Formation starts at the most apical projection of the cells and proceeds in a proximal direction. Up to 4-μm-long tubular structures appear, exclusively in developmental stages, in the inner receptor-lymph cavity. They are composed of plasma membranes whose inner surface is studded with regularly spaced electron-dense particles. Contacts with the cytoplasmic membrane of the innermost enveloping cell demonstrate that the structures are composed of lipid membranes. During elongation of the dendrite sheath, which in these sensilla is apically attached to the hair wall, an ∼2-μm-long growth-zone is observed at its proximal end. By addition of sheath-forming material to the growth-zone, the latter continuously moves proximally until the sheath is completed.

Thermoperiod and Photoperiod as Temporal Cues for Adult Eclosion of Heliothis virescens (Lepidoptera: Noctuidae)

Thermoperiodic cycles were found to entrain adult eclosion of Heliothis virescens (F.) in the absence of photoperiodic cycles. The reverse was also true. In the absence of either cycle, the circadian rhythm of H. virescens appears to have a period of about 24.6 h. Adult eclosion occurs near the beginning of cryophase or scotophase. These Zeitgebers were found to be effective in entraining adult eclosion as late as the latter half of pupal development. Adult eclosion in H. virescens may be an excellent model for investigating the physiology of circadian rhythms in insects.

Cell degeneration in the developing optic lobes of the sine oculis and small-optic-lobes mutants of Drosophila melanogaster

In the small-optic-lobes ( sol) and sine oculis ( so) mutants of Drosophila melanogaster extensive cell death occurs in the optic lobes during the first half of pupal development. Gynandromorph flies show that the sol mutation acts primarily on cells of the medulla cortex. Degeneration of medullar ganglion cells occurs at an early stage of cellular differentiation, when their axons have not yet participated in the formation of the second optic chiasma. The so gene, on the other hand, acts on the eye anlagen. The analysis of chimeric flies demonstrates that degeneration in the optic lobes of so flies is a consequence of eye reduction. At the level of the second optic chiasma extensive axonal degeneration can be observed in the mutant. Neurons seem to die after their failure to establish a sufficient number of functional contacts. In sol; so double mutants, the mutational effects are cumulative causing complete degeneration of columnar cell types in pupae without any eye anlage. The tiny rudiments of the optic lobes in eyeless double mutants still contain tangential neurons of the medulla and of the lobula complex. The central brain is reduced in size due to the missing visual fibers, however, its overall appearance is surprisingly normal.

Synaptogenesis in the first optic neuropile of the fly's visual system.

The developmental transformation of the chief afferent class of fly photoreceptor synapse has been examined from serial electron micrographs of animals fixed at 74, 81, 94 and 100% pupal development (100% pupal development being defined by the time of normal adult eclosion). Animals were selected both by their age and the conformity of their eye coloration to standards for each stage. Two animals were analysed from each stage, one in greater detail than the other. For the first, the exact coordinates of the cartridges (the synaptic columns of the first optic neuropile) from which the analyses were made were mapped and selected to be within the same region of the eye field at all stages. From all animals a portion of one or two cartridges was analysed from series of up to 100 sections and the synapse populations (greater than 80) were analysed for their fine structure and postsynaptic composition. Adult synapses are confirmed as tetrads, with two of the four postsynaptic elements invariably from two monopolar interneurons L1 and L2, one from each. The two others are usually from alpha processes of the same amacrine cell. Synapses appear during the last half of pupal development, with no obvious asynchrony of ultrastructural maturation and in parallel with those of at least one of the other synaptic classes present (which were otherwise not studied). Many adult features of synaptic ultrastructure emerge late, only by 94% pupal development. These include adult numbers of synaptic vesicles, the complete form of the presynaptic ribbon with platform and the postsynaptic cisternae of L1/L2. Prior to 94% the synapses are smaller with postsynaptic elements having a less regular geometry and with postsynaptic densities which are subsequently lost (alpha processes) or replaced by cisternae (L1/L2). At the presynaptic sites of the younger animals (74%, 81%) dyads and triads of postsynaptic elements coexist with tetrads, those of older animals having, on average, more postsynaptic processes per synapse. It is suggested that individual synapses assemble piecemeal, element by element.