A Genetic Analysis of Pannier, a Gene Necessary for Viability of Dorsal Tissues and Bristle Positioning in Drosophila

A genetic and phenotypic analysis of the gene pannier is described. Animals mutant for strong alleles die as embryos in which the cells of the amnioserosa are prematurely lost. This leads to a dorsal cuticular hole. The dorsal-most cells of the imagos are also affected: viable mutants exhibit a cleft along the dorsal midline. pannier mRNA accumulates specifically in the dorsal-most regions of the embryo and the imaginal discs. Viable mutants and mutant combinations also affect the thoracic and head bristle patterns in a complex fashion. Only those bristles within the area of expression of pannier are affected. A large number of alleles have been studied and reveal that pannier may have opposing effects on the expression of achaete and scute leading to a loss or a gain of bristles.     

Selection and temperature effects on extra dorsocentral bristles inDrosophila melanogaster

Starting from base populations which showed a tendency to form supernumerary dorsocentral bristles, selection for high numbers of dorsocentrals was carried out. In all, 9 lines were selected 6 to 14 generations. Selection proved to be effective in all but two lines. Selection for reduced numbers in one of the lines also proved effective.Mean values of over 10 were reached in the females of certain high lines. Average counts in males were always lower than those of females. This sex difference is not a consequence of the difference in size between the sexes.The base populations were of mutant stock origin, and some of them segregated for mutant genes which proved to be correlated with extrabristle phenotype. Supernumerary bristles were not distributed in the same manner in all of the selection lines, nor was the reaction of phenotypic expression to temperature the same in various lines.     

F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses

A useful approach for exploring gene function involves generating mutant mice from genetically modified embryonic stem (ES) cells. Recent advances in genetic engineering of ES cells have shifted the bottleneck in this process to the generation of mice. Conventional injections of ES cells into blastocyst hosts produce F0 generation chimeras that are only partially derived from ES cells, requiring additional breeding to obtain mutant mice that can be phenotyped. The tetraploid complementation approach directly yields mice that are almost entirely derived from ES cells, but it is inefficient, works only with certain hybrid ES cell lines and suffers from nonspecific lethality and abnormalities, complicating phenotypic analyses. Here we show that laser-assisted injection of either inbred or hybrid ES cells into eight cell-stage embryos efficiently yields F0 generation mice that are fully ES cell-derived and healthy, exhibit 100% germline transmission and allow immediate phenotypic analysis, greatly accelerating gene function assignment.     

Genotype versus phenotype variability in Chlorella and Micractinium (Chlorophyta, Trebouxiophyceae)

The most recent revision of the genus Chlorella, based on biochemical and SSU rDNA analyses, suggested a reduction to a set of four "true" spherical Chlorella species, while a growing number of morphologically different species such as Micractinium (formerly Micractiniaceae) were found to cluster within the clade of "true"Chlorella. In this study, the generic concept in Chlorellaceae to Chlorella and Micractinium was evaluated by means of combined SSU and ITS-2 rDNA sequence analyses and biotests to induce development of bristles on the cell wall. Molecular phylogenetic analyses of Chlorella and Micractinium strains confirmed their separation into two different genera. In addition, non-homoplasious synapomorphies (NHS) and compensatory base changes (CBC) in the secondary structures of SSU and ITS-2 rDNA sequences were found for both genera using this approach. The Micractinium clade can be differentiated into three different genotypes. Using culture medium of the rotifer Brachionus calyciflorus, phenotypic plasticity in Chlorella and Micractinium was studied. Non-bristled Micractinium cells developed bristles during incubation with Brachionus culture medium, whereas Chlorella did not produce bristles. Grazing experiments with Brachionus showed the rotifer preferred to feed on non-bristled cells. The dominance of colonies versus solitary cells in the Micractinium culture was not correlated with the "Brachionus factor". These results suggest that morphological characteristics like formation of bristles represent phenotypic adaptations to the conditions in the ecosystem.     

Improvement of astaxanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation

Aims: Isolation, characterization and identification of Phaffia sp. ZJB 00010, and improvement of astaxanthin production with low‐energy ion beam implantation. Methods and Results: A strain of ZJB 00010, capable of producing astaxanthin, was isolated and identified as Phaffia rhodozyma, based on its physiological and biochemical characteristics as well as its internal transcribed spacer (ITS) rDNA gene sequence analysis. With low‐energy ion beam implantation, this wild‐type strain was bred for improving the yield of astaxanthin. After ion beam implantation, the best mutant, E5042, was obtained. The production of astaxanthin in E5042 was 2512 μg g^?1 (dry cell weight, DCW), while the wild‐type strain was about 1114 μg g^?1 (DCW), an increase of 125·5%. Moreover, the fermentation conditions of mutant E5042 for producing astaxanthin were optimized. The astaxanthin production under the optimized conditions was upscaled and studied in a 50‐l fermentor. Conclusions: A genetically stable mutant strain with high yield of astaxanthin was obtained using low‐energy ion beam implantation. This mutant may be a suitable candidate for the industrial‐scale production of astaxanthin. Significance and Impact of the Study: Astaxanthin production in Phaffia rhodozyma could be fficiently improved by low‐energy ion beam implantation, which is a new technology in the mutant breeding of micro‐organisms. The mutant obtained in this work could potentially be utilized in industrial production of astaxanthin.     

Variations in the arrangement of sensory bristles along butterfly wing margins

The surfaces of insect wings exhibit numerous sensilla, which have been suggested to have a behavioral function. Some evidence suggests that the sensory bristles along the wing margin of lepidopteran insects (butterflies and moths) are involved in the regulation of wing movement. We investigated the arrangement of sensory bristles along the wing margins of 62 species of papilionoid butterflies, using light-microscopic examination of mounts of whole wings after removing the scales surrounding the bristles. In the majority of the wings examined, bristles were located on the ventral wing surfaces and were continuously distributed along the wing margins, except in the vicinity of the wing bases. In some wings, bristles were also located on the dorsal wing surfaces, and were continuously or discontinuously distributed along the wing margins of different species. In a minority of the species studied, we observed bristle distribution in the vicinity of the wing base, discontinuous bristle distribution on both the dorsal and ventral wing surfaces, or an absence of bristles along the wing margins. This variation in the arrangement of bristles along the wing margins is discussed in relation to the reception and transmission of sensory information in the wings.     

Movement of bristle precursors contributes to the spacing pattern in Drosophila

In Drosophila melanogaster, microchaetes (small bristles) are regularly spaced and form five straight rows in the acrostichal region of the adult notum. Microchaetes develop from sensory organ precursors that arise as single, evenly-spaced cells during pupal development. In this article we address the question of how the precursor cells remain aligned throughout pupal development, in spite of continued division of the intervening epidermal cells. Using in vivo imaging we show that bristle precursors move about continuously throughout development, covering distances of up to one or two cell diameters. During this process, they remain aligned in wild-type flies, suggesting that the movement may be regulated. Flies mutant for scabrous (sca) have a disorganised pattern of bristles with little or no alignment. In vivo observations of sca mutants indicated that the precursor cells move around more than in the wild type, but that, in spite of this the precursor cells and resulting bristles never become well aligned. They appear to follow a more complex path, suggesting that the movement is not co-ordinated. Moreover, analysis of the alignment of precursor cells in vivo in wild-type and sca mutant flies indicate that mutant animals are not able to maintain the pattern of precursor cells during development. Analysis of mosaic flies confirmed the time-lapse observations and showed furthermore that bristles preferentially move towards high levels of Scabrous. We suggest that, by altering the properties of epithelial cells in a graded fashion, Scabrous may provide cues that allow the precursors to remain evenly spaced after they have segregated.     

Arrangement of bristles as a function of bristle number on a leg segment inDrosophila melanogaster

Summary The arrangement of bristles on a leg segment of the fruitflyDrosophila melanogaster was studied in various mutants that have abnormal numbers of bristles on this segment. Eighteen mutations at six different genetic loci were analyzed, plus five double or triple mutant combinations. Recessive mutations at theachaete-scute locus were found to affect distinct groups of bristles:achaete mutations remove mechanosensory bristles, whereasscute mutations remove mainly chemosensory bristles. Mechanosensory bristles remain uniformly spaced along the longitudinal axis unless their number decreases below a certain threshold, suggesting that spacing is controlled by cell interactions that cannot function when bristle cells are too far apart. Above a certain threshold, bristle spacing and alignment both become irregular, perhaps due to excessive force from these same interactions. Chemosensory bristles occupy definite positions that are virtually unaffected by removal of individual bristles from the array. Extra chemosensory bristles develop only near the six normal sites. At two of the six sites the multiple bristles tend to exhibit uniform longitudinal spacing — a property confined to mechanosensory bristles in wild-type flies. To explain the various mutant phenotypes the following scheme is proposed, with different mutations directly or indirectly affecting each step: (1) spots and stripes are demarcated within the pattern area, (2) one bristle cell normally arises within each spot, multiple bristle cells within each stripe, (3) incipient bristle cells inhibit neighboring cells from becoming bristle cells, and (4) the bristle cells within each stripe become aligned to form rows and then repel one another to generate uniform spacing.     

The role actin filaments play in providing the characteristic curved form of Drosophila bristles

Drosophila bristles display a precise orientation and curvature. An asymmetric extension of the socket cell overlies the newly emerging bristle rudiment to provide direction for bristle elongation, a process thought to be orchestrated by the nerve dendrite lying between these cells. Scanning electron microscopic analysis of individual bristles showed that curvature is planar and far greater near the bristle base. Correlated with this, as development proceeds the pupa gradually recedes from the inner pupal case (an extracellular layer that encloses the pupa) leading to less bristle curvature along the shaft. We propose that the inner pupal case induces elongating bristles to bend when they contact this barrier. During elongation the actin cytoskeleton locks in this curvature by grafting together the overlapping modules that comprise the long filament bundles. Because the bristle is curved, the actin bundles on the superior side must be longer than those on the inferior side. This is accomplished during grafting by greater elongation of superior side modules. Poor actin cross-bridging in mutant bristles results in altered curvature. Thus, the pattern of bristle curvature is a product of both extrinsic factors-the socket cell and the inner pupal case--and intrinsic factors--actin cytoskeleton assembly.     

Phenotypic and genotypic structure of a natural Drosophila population for meristic morphologic characters and its seasonal changes

A natural population of Drosophila was genetically heterogeneous in the number of sternopleural bristles and the number of arista branches. In the summer, these characters had a low mean value and high coefficient of variation: in autumn, this relationship was reversed; in late spring, the values of the mean and coefficient of variation were intermediate. The dynamics of the character mean was determined by changes in phenotypic and genotypic composition of the population, which was examined during different seasons of the year. On average, the number of sternopleural bristles was higher, and the number of arista branches was lower in females than in males. The number of sternopleural bristles exhibited a higher phenotypic and genotypic variation than the number of arista branches.     

Genetic variability and fluctuating asymmetry of morphological signs of Drosophila melanogaster during development in a pesticide-containing environment

The effects of chlorine-organic insecticide endosulfane (thiodan) on phenotypic and genetic variation in four morphological traits of Drosophila melanogaster (wing length, thorax length, the number of orbital bristles and the number of sternopleural bristles) were examined. In addition, the effect of this pesticide on stability of development measured as fluctuating asymmetry of bilateral traits was estimated. On the medium with endosulfane, phenotypic variation of morphometric traits was significantly higher. No difference in fluctuating asymmetry between the stressed and the control samples was found. The among-line variance of morphometric traits of flies reared on the endosulfane-containing medium was significantly higher as compared to the corresponding variance under control conditions. The efficiency of using fluctuating asymmetry and phenotypic variation of morphometric and meristic traits as indicators of environmental stress in insect populations is discussed.     

Genetic Variability and Fluctuating Asymmetry of Morphological Traits in Drosophila melanogasterReared on a Pesticide-Containing Medium

The effects of chlorine-organic insecticide endosulfane (thiodan) on phenotypic and genetic variation in four morphological traits of Drosophila melanogaster(wing length, thorax length, the number of orbital bristles and the number of sternopleural bristles) were examined. In addition, the effect of this pesticide on stability of development measured as fluctuating asymmetry of bilateral traits was estimated. On the medium with endosulfane, phenotypic variation of morphometric traits was significantly higher. No difference in fluctuating asymmetry between the stressed and the control samples was found. The among-line variance of morphometric traits of flies reared on the endosulfane-containing medium was significantly higher as compared to the corresponding variance under control conditions. The efficiency of using fluctuating asymmetry and phenotypic variation of morphometric and meristic traits as indicators of environmental stress in insect populations is discussed.     

A potential regulatory polymorphism upstream of hairy is not associated with bristle number variation in wild-caught Drosophila

To extend results from laboratory genetic mapping experiments to natural populations it is necessary to estimate the phenotypic effects attributable to laboratory-identified genetic factors in nature. We retested a polymorphism found to be strongly associated with an increase of 0.35 sternopleural bristles in laboratory strains in two large samples of wild-caught Drosophila melanogaster. Despite >90% power to detect effects as low as 0.27 bristles (<1% of the total variation in bristle number) we did not replicate the association in nature. Potential explanations for this result are explored.     

The development and evolution of bristle patterns in Diptera

The spatial distribution of sensory bristles on the notum of different species of Diptera is compared. Species displaying ancestral features have a simple organization of randomly distributed, but uniformly spaced, bristles, whereas species thought to be more derived bear patterns in which the bristles are aligned into longitudinal rows. The number of rows of large bristles on the scutum was probably restricted to four early on in the evolution of cyclorraphous Brachyceran flies. Most species have stereotyped patterns based on modifications of these four rows. The possible constraints placed upon the patterning mechanisms due to growth and moulting within the Diptera are discussed, as well as within hemimetabolous insects. The holometabolic life cycle and the setting aside of groups of imaginal cells whose function is not required during the growth period, may have provided the freedom necessary for the evolution of elaborate bristle patterns. We briefly review the current state of knowledge concerning the complex genetic pathways regulating achaete-scute gene expression and bristle pattern in Drosophila melanogaster, and consider mechanisms for the genetic regulation of the bristle patterns of other species of Diptera.     

Canalization at two extra scutellar bristles: artificial selection, chromosome analysis, and effect of temperature.

Two isofemale lines (P1 and P2) from a wild-type population of Drosophila melanogaster were selected for an invariant phenotype, two symmetrical and anterior scutellar extra bristles. Both P1 and P2 lines responded quickly to direct selection for two extra bristles, but although this phenotype was canalized in the P2 line, it was not in the P1 line. This lack of canalization at two extra bristles, measured by probit width, is the first reported in the literature. Analysis of chromosomal contribution showed the P1 decanalized phenotype to be due to chromosome 2. Synthetic chromosomal combinations were tested at four development temperatures (17 degrees, 21 degrees, 24 degrees, and 30 degrees C), and no correlation was observed between canalization at two extra bristles measured by probit width and minimum phenotypic change due to different temperatures. In this test, development at the highest temperatures was accompanied by an increased tendency to return to the wild phenotype in the canalized phenotypes only, suggesting that this drastic change could be accounted for by the action of the wild-type canalizing system. Decanalized genotypes, or those canalizing at phenotypes other than the wild type, could be explained by mutations which, to a greater or lesser degree, alter the normal genetic system of canalization.     

The Drosophila javelin gene encodes a novel actin-associated protein required for actin assembly in the bristle

The Drosophila melanogaster bristle is a highly polarized cell that builds specialized cytoskeletal structures. Whereas actin is required for increasing bristle length, microtubules are essential for bristle axial growth. To identify new proteins involved in cytoskeleton organization during bristle development, we focused on identifying and characterizing the javelin (jv) locus. We found that in a jv mutant, the bristle tip is swollen and abnormal organization of bristle grooves is seen over the entire bristle. Using confocal and electron microscopy, we found that in jv mutant bristles, actin bundles do not form properly due to a loss of actin filaments within the bundle. We show that jv is an allele of the predicted CG32397 gene that encodes a protein with no homologs outside insects. Expression of the Jv protein fused to a green fluorescent protein (GFP) shows that the protein is colocalized with actin bundles in the bristle. Moreover, expression of Jv-GFP within the germ line led to the formation of ectopic actin bundles that surround the nucleus of nurse cells. Thus, we report that Jv is a novel actin-associated protein required for actin assembly during Drosophila bristle development.     

Morphogenesis and phenotypic divergence in two developmental morphs of Streblospio benedicti (Annelida,Spionidae)

Abstract. The morphology of marine invertebrate larvae is strongly correlated with egg size and larval feeding mode. Planktotrophic larvae typically have suites of morphological traits that support a planktonic, feeding life style, while lecithotrophic larvae often have larger, yolkier bodies, and in some cases, a reduced expression of larval traits. Poecilogonous species provide interesting cases for the analysis of early morphogenesis, as two morphs of larvae are produced by a single species. We compared morphogenesis in planktotrophic and lecithotrophic morphs of the poecilogonous annelid Streblospio benedicti from the trochophore stage through metamorphosis, using observations of individuals that were observed alive, with scanning electron microscopy, or in serial sections. Offspring of alternate developmental morphs of this species are well known to have divergent morphologies in terms of size, yolk content, and the presence of larval bristles. We found that some phenotypic differences between morphs occur as traits that are present in only one morph (e.g., larval bristles, bacillary cells on the prostomium and pygidium), but that much of the phenotypic divergence is based on heterochronic changes in the differentiation of shared traits (e.g., gut and coelom). Tissue and organ development are compared in both morphs in terms of their structure and ontogenetic change throughout early development and metamorphosis.     

Comparative Morphology of Arthropod Exterior Surfaces with the Capability of Binding a Film of Air Underwater

The air-binding surfaces of aquatic and semi-aquatic insects and spiders were examined under a scanning electron microscope. The bristles that are instrumental in binding the film of air show distinctive characteristics within each phylogenetic group. Very small species can often maintain themselves in a bubble of air underwater although they lack the dense bristle coat characteristic of larger plastron breathers. Relatively large insects generally require a morphologically adapted chamber in which to carry their air supplies. The ecological advantages of an underwater respiration employing portable air are discussed.