Butterfly Wings Are Three-Dimensional: Pupal Cuticle Focal Spots and Their Associated Structures in Junonia Butterflies

Butterfly wing color patterns often contain eyespots, which are developmentally determined at the late larval and early pupal stages by organizing activities of focal cells that can later form eyespot foci. In the pupal stage, the focal position of a future eyespot is often marked by a focal spot, one of the pupal cuticle spots, on the pupal surface. Here, we examined the possible relationships of the pupal focal spots with the underneath pupal wing tissues and with the adult wing eyespots using Junonia butterflies. Large pupal focal spots were found in two species with large adult eyespots, J. orithya and J. almana, whereas only small pupal focal spots were found in a species with small adult eyespots, J. hedonia. The size of five pupal focal spots on a single wing was correlated with the size of the corresponding adult eyespots in J. orithya. A pupal focal spot was a three-dimensional bulge of cuticle surface, and the underside of the major pupal focal spot exhibited a hollowed cuticle in a pupal case. Cross sections of a pupal wing revealed that the cuticle layer shows a curvature at a focal spot, and a positional correlation was observed between the cuticle layer thickness and its corresponding cell layer thickness. Adult major eyespots of J. orithya and J. almana exhibited surface elevations and depressions that approximately correspond to the coloration within an eyespot. Our results suggest that a pupal focal spot is produced by the organizing activity of focal cells underneath the focal spot. Probably because the focal cell layer immediately underneath a focal spot is thicker than that of its surrounding areas, eyespots of adult butterfly wings are three-dimensionally constructed. The color-height relationship in adult eyespots might have an implication in the developmental signaling for determining the eyespot color patterns.     

Programmed cell death in the wing of Orgyia leucostigma (Lepidoptera: Lymantriidae)

Programmed cell death is an integral and ubiquitous phenomenon of development that is responsible for the reduction of wing size in female moths of Orgyia leucostigma (Lymantriidae). Throughout larval and pupal life, cells of the wing epithelium proliferate and interact to form normal imaginal discs and pupal wings in both sexes. But at the onset of adult development, most cells in female O. leucostigma wings degenerate over a brief, 2-day period. Lysosomes and autophagic vacuoles appear in cells of the wing epithelium shortly after it retracts from the pupal cuticle. Hemocytes actively participate in removing the resulting cellular debris. By contrast, epithelial cells in wings of developing adult males of O. leucostigma do not undergo massive cell death. Wing epithelium of female pupae transferred to male pupal hosts behaves autonomously in this foreign environment. By pupation, cells of the female wing apparently are committed to self-destruct even in a male pupal environment. Normal interactions among epithelial cells within the plane of a wing monolayer as well as between the upper and lower monolayers of the wing are disrupted in female O. leucostigma by massive cell degeneration. Despite this disruption, the remaining cells of the wing contribute to the formation of a diminutive, but reasonably proportioned, adult wing with scales and veins.     

Morphological comparison of pupal wing cuticle patterns in butterflies

Butterfly wing color-patterns are determined in the prospective wing tissues during the late larval and early pupal stages. To study the cellular differentiation process of wings, morphological knowledge on pupal wings is prerequisite. Here we systematically examined morphological patterns of the pupal wing cuticular surface in a wide variety of nymphalid butterflies in relation to adult color-patterns. Several kinds of pupal wing patterns corresponding to particular adult color-pattern elements were widely observed in many species. Especially noteworthy were the pupal "focal" spots corresponding to the adult border ocelli system, which were detected in many species of Nymphalinae, Apaturinae, Argynninae, Satyrinae, and Danainae. Striped patterns on the pupal wing cuticle seen in some species of Limenitinae, Ariadnae, and Marpesiinae directly corresponded to those of the adult wings. In Vanessa cardui, eyespot-like pattern elements were tentatively produced during development in the wing tissue underneath the pupal spots and subsequently erased, suggesting a mechanism for producing novel color-patterns in the course of development and evolution. The pupal focal spots reasonably correlated with the adult eyespots in size in Precis orithya and Ypthima argus. We physically damaged the pupal focal spots and their corresponding cells underneath in these species, which abolished or inhibited the formation of the adult eyespots. Taken together, our results clarified that pupal cuticle patterns were often indicative of the adult color-patterns and apparently reflect molecular activity of organizing centers for the adult color-pattern formation at least in nymphalid butterflies.     

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.     

Larval temperature experience determines sensitivity to cold-shock-induced wing color pattern changes in the blue pansy butterfly Junonia orithya

The butterfly wing color patterns are unique to a species but are modified in response to cold-shock and tungstate treatments at the pupal stage, producing characteristic temperature–shock (TS) phenotypes that are distinct from the color patterns of seasonal polyphenism. In this study, we examined the efficiency of cold-shock and tungstate treatments for color pattern modifications at the pupal stage in relation to larval rearing conditions for the fall or summer morph using the blue pansy butterfly Junonia orithya. We found that larvae reared under the low-temperature condition that induces the fall morph exhibited hardiness against the color pattern changes imposed by cold-shock or tungstate treatment at the pupal stage. When larvae were fed an artificial diet containing tungstate under the high-temperature condition that induces the summer morph, they were still vulnerable to color pattern changes imposed by cold-shock or tungstate treatment at the pupal stage. Furthermore, larvae reared under the high-temperature condition were subjected to cold-shock or tungstate treatments at the pupal stage. In addition to the expected TS-type changes, these individuals exhibited a reduced number of eyespots in adults, which is a feature of the fall morph. These results suggest that the temperature condition experienced by the larvae, but not their consumption of tungstate, determines the sensitivity of the wing imaginal discs to cold-shock and tungstate treatments at the pupal stage.     

In vivo CD8+ T Cell Dynamics in the Liver of Plasmodium yoelii Immunized and Infected Mice

Plasmodium falciparum malaria remains one of the most serious health problems globally and a protective malaria vaccine is desperately needed. Vaccination with attenuated parasites elicits multiple cellular effector mechanisms that lead to Plasmodium liver stage elimination. While granule-mediated cytotoxicity requires contact between CD8+ effector T cells and infected hepatocytes, cytokine secretion should allow parasite killing over longer distances. To better understand the mechanism of parasite elimination in vivo, we monitored the dynamics of CD8+ T cells in the livers of naïve, immunized and sporozoite-infected mice by intravital microscopy. We found that immunization of BALB/c mice with attenuated P. yoelii 17XNL sporozoites significantly increases the velocity of CD8+ T cells patrolling the hepatic microvasculature from 2.69±0.34 μm/min in naïve mice to 5.74±0.66 μm/min, 9.26±0.92 μm/min, and 7.11±0.73 μm/min in mice immunized with irradiated, early genetically attenuated (Pyuis4-deficient), and late genetically attenuated (Pyfabb/f-deficient) parasites, respectively. Sporozoite infection of immunized mice revealed a 97% and 63% reduction in liver stage density and volume, respectively, compared to naïve controls. To examine cellular mechanisms of immunity in situ, naïve mice were passively immunized with hepatic or splenic CD8+ T cells. Unexpectedly, adoptive transfer rendered the motile CD8+ T cells from immunized mice immotile in the liver of P. yoelii infected mice. Similarly, when mice were simultaneously inoculated with viable sporozoites and CD8+ T cells, velocities 18 h later were also significantly reduced to 0.68±0.10 μm/min, 1.53±0.22 μm/min, and 1.06±0.26 μm/min for CD8+ T cells from mice immunized with irradiated wild type sporozoites, Pyfabb/f-deficient parasites, and P. yoelii CS_280–288 peptide, respectively. Because immobilized CD8+ T cells are unable to make contact with infected hepatocytes, soluble mediators could potentially play a key role in parasite elimination under these experimental conditions.     

Color pattern formation on the wing of a butterfly Pieris rapae. 2. Color determination and scale development

It has been shown that microcautery on the prospective apical black region of the early pupal forewing of a butterfly, Pieris rapae , causes alteration of the scale color on the adult wing and a delay in histogenesis of the pupal wing. From these results, it has been assumed that the developmental delay of scale cells in the pupal wing alters their developmental fate and the hypothesis that different color fates of scales are determined by differences in the developmental timetables between scale cells is proposed. In this study, we attempted to find the developmental timetables of individual scales expressing specific color to test this hypothesis. It was found that the holes on the upper surface of a scale become larger as they develop and the hole sizes of scales in the white region are always larger than in the black region on the same wings either during pupal period or after eclosion. This suggests that the scale hole size is a good index that reflects developmental rate of the scale and a difference in the hole size between adult scales is attributed to a difference in the developmental timetables when their ancestral scale precursor cells were in the pupal period. A comparison of the hole sizes between adult scales in different color regions suggested that normal white scales were in a more advanced state than were the black ones but white scales induced by microcautery were in a less advanced state than black ones on the same wing. This supports our hypothesis.     

Evaluation of Fleroxacin Activity against Established Pseudomonas fluorescens Biofilms

Scanning confocal laser microscopy (SCLM) and fluorescent molecular probes were used to evaluate the effect of the fluoroquinolone fleroxacin on the architecture of established Pseudomonas fluorescens biofilms. Control P. fluorescens biofilms were heterogeneous, consisting of cell aggregates extending from the attachment surface to maximum measured depths of ~90 μm (mean biofilm depth at 72 h, 42 ± 28 μm) and penetrated by an array of channels. In contrast, fleroxacin-treated biofilms were less deep (mean biofilm depth at 72 h, 29 ± 8 μm), varied little in depth over large areas, and consisted of a homogeneous distribution of cells. Fleroxacin also caused cells to elongate, with cells located near the biofilm-liquid interface lengthening significantly more than cells located at the attachment surface. By using SCLM, acridine orange, and image analysis it was found that ~59% of cells within fleroxacin-treated biofilms emitted red fluorescence whereas >99% of cells from control biofilms emitted green fluorescence. The fleroxacin-treated cells which emitted red fluorescence were observed to be the population of cells which elongated.     

Hormonal control of pupal coloration in the painted lady butterfly Vanessa cardui

Pupae of the painted lady butterfly Vanessa cardui exhibit pupal color polyphenism consisting of white, dark and intermediate types. We investigated environmental factors affecting pupal coloration and the physiological mechanisms underlying the control of pupal color polyphenism in this species. Over 80% of larvae reared at 16 °C developed into pupae of dark types, whereas over 82% of larvae at 32 °C developed into pupae of white types irrespective of long/short-day photoperiod conditions. When mature larvae reared at 32 °C were ligatured between thoracic and abdominal parts at three different pharate pupal stages, all of the head-thoracic parts developed into white pupae regardless of pupal stage, but all abdominal parts ligatured at the early pharate pupal stage only developed into dark pupae. These results indicate that temperature during larval stages is an important element affecting pupal coloration as an environmental cue in V. cardui, and that a factor(s) inducing white pupae is released from head-thoracic parts under conditions of high temperature. Additionally, when ligatured abdomens destined to develop into dark pupae were treated with crude extracts prepared from the central nervous system, all of the ligatured abdomens developed into white pupae at a level dependent on dose and pupal stage. These results suggest that the factor inducing white pupae is a key molecule controlling pupal color polyphenism in V. cardui.     

Proteasome,but Not Autophagy,Disruption Results in Severe Eye and Wing Dysmorphia: A Subunit- and Regulator-Dependent Process in Drosophila

Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx^MS1096 genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, we proved that proteasomal integrity and ubiquitination proficiency essentially control fly’s eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, α5, β5 and β6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing’s, but not eye’s, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In toto, our findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development.     

Color pattern formation on the wing of the butterfly Pieris rapae. 1. Cautery induced alteration of scale color and delay of arrangement formation

Experimental approaches to color pattern formation of lepidopteran insects have been made exclusively by analyzing pattern alterations in adult wings induced by operations. We microcauterized the presumptive black region of the dorsal forewing of the butterfly Pieris rapae and analyzed not only the resultant color pattern in the adult wing but also the cell behavior in the pupal wing epidermis around the injury. Cautery induced color alterations were as follows: (i) cautery up to 49.5 h after pupation resulted in white regions appearing within the black region while later cauteries induced larger white regions; (ii) cautery between 50 and 59.5 h resulted in the white regions induced by the cauteries being dramatically decreased; (iii) cautery after 60 h resulted in white regions that had almost disappeared. The examination of the cell behavior in the pupal wing epidermis after cauteries showed that the row formation of scale precursor cells was delayed. This delayed area varied with the time of cautery, in the same manner as that in the induced white area in the adult wing ((i) – (iii) above). The relationship between scale color alteration and the developmental delay of the scale row formation is discussed.     

Reproductive Development of Verutus volvingentis (Tylenchida: Heteroderidae)

Verutus volvingentis Esser, 1981 deposits eggs in the rhizosphere without a gelatinous matrix. Ecdysis was not observed to occur in the egg. Spicular primordia in the rectal area of a second-stage larva were well defined. One larva increased in width from 28.2 μm to a maximum of 51.7 μm after 176.5 hours of feeding, prior to the second ecdysis. It then decreased steadily in width to 33.3 μm, at which time it had molted to a fully developed male. Males leave the third-stage larval integument embedded in the root following final ecdysis. The unique feature of female development was the occurrence of large vaginal primordial cells. Male and female development took from 6 to 15 days and 17 days, respectively.     

BUTTERFLY EYESPOTS: THE GENETICS AND DEVELOPMENT OF THE COLOR RINGS

The butterfly Bicyclus anynana has a series of distal eyespots on its wings. Each eyespot is composed of a white pupil, a black disc, and a gold outer ring. We applied artificial selection to the large dorsal eyespot on the forewing to produce a line with the gold ring reduced or absent (BLACK) and another line with a reduced black disc and a broad gold ring (GOLD). High heritabilities, coupled with a rapid response to selection, produced two lines of butterflies with very different phenotypes. Other eyespots showed a correlated change in the proportion of their color rings. Surgical experiments were performed on pupal wings from the different lines at the time of eyespot pattern specification. They showed that the additive genetic variance for this trait was in the response of the wing epidermis to signaling from the organizing cells at the eyespot center (the focus). This response was found to vary across different regions of the wing and also between the sexes. The particular eyespot color composition found for each sex, as well as the maintenance of the high genetic variation, are discussed with reference to the ecology of the butterfly, sexual selection, and visual selection by predators.     

Brine Assemblages of Ultrasmall Microbial Cells within the Ice Cover of Lake Vida,Antarctica

The anoxic and freezing brine that permeates Lake Vida''s perennial ice below 16 m contains an abundance of very small (≤0.2-μm) particles mixed with a less abundant population of microbial cells ranging from >0.2 to 1.5 μm in length. Fluorescent DNA staining, electron microscopy (EM) observations, elemental analysis, and extraction of high-molecular-weight genomic DNA indicated that a significant portion of these ultrasmall particles are cells. A continuous electron-dense layer surrounding a less electron-dense region was observed by EM, indicating the presence of a biological membrane surrounding a cytoplasm. The ultrasmall cells are 0.192 ± 0.065 μm, with morphology characteristic of coccoid and diplococcic bacterial cells, often surrounded by iron-rich capsular structures. EM observations also detected the presence of smaller unidentified nanoparticles of 0.020 to 0.140 μm among the brine cells. A 16S rRNA gene clone library from the brine 0.1- to 0.2-μm-size fraction revealed a relatively low-diversity assemblage of Bacteria sequences distinct from the previously reported >0.2-μm-cell-size Lake Vida brine assemblage. The brine 0.1- to 0.2-μm-size fraction was dominated by the Proteobacteria-affiliated genera Herbaspirillum, Pseudoalteromonas, and Marinobacter. Cultivation efforts of the 0.1- to 0.2-μm-size fraction led to the isolation of Actinobacteria-affiliated genera Microbacterium and Kocuria. Based on phylogenetic relatedness and microscopic observations, we hypothesize that the ultrasmall cells in Lake Vida brine are ultramicrocells that are likely in a reduced size state as a result of environmental stress or life cycle-related conditions.     

Microstamped Petri Dishes for Scanning Electrochemical Microscopy Analysis of Arrays of Microtissues

While scanning electrochemical microscopy (SECM) is a powerful technique for non-invasive analysis of cells, SECM-based assays remain scarce and have been mainly limited so far to single cells, which is mostly due to the absence of suitable platform for experimentation on 3D cellular aggregates or microtissues. Here, we report stamping of a Petri dish with a microwell array for large-scale production of microtissues followed by their in situ analysis using SECM. The platform is realized by hot embossing arrays of microwells (200 μm depth; 400 μm diameter) in commercially available Petri dishes, using a PDMS stamp. Microtissues form spontaneously in the microwells, which is demonstrated here using various cell lines (e.g., HeLa, C2C12, HepG2 and MCF-7). Next, the respiratory activity of live HeLa microtissues is assessed by monitoring the oxygen reduction current in constant height mode and at various distances above the platform surface. Typically, at a 40 μm distance from the microtissue, a 30% decrease in the oxygen reduction current is measured, while above 250 μm, no influence of the presence of the microtissues is detected. After exposure to a model drug (50% ethanol), no such changes in oxygen concentration are found at any height in solution, which reflects that microtissues are not viable anymore. This is furthermore confirmed using conventional live/dead fluorescent stains. This live/dead assay demonstrates the capability of the proposed approach combining SECM and microtissue arrays formed in a stamped Petri dish for conducting cellular assays in a non-invasive way on 3D cellular models.     

Paraquat Induces Epithelial-Mesenchymal Transition-Like Cellular Response Resulting in Fibrogenesis and the Prevention of Apoptosis in Human Pulmonary Epithelial Cells

The aim of this study is to investigate the molecular mechanisms underlying delayed progressive pulmonary fibrosis, a characteristic of subacute paraquat (PQ) poisoning. Epithelial-mesenchymal transition (EMT) has been proposed as a cause of organ fibrosis, and transforming growth factor-β (TGF-β) is suggested to be a powerful mediator of EMT. We thus examined the possibility that EMT is involved in pulmonary fibrosis during PQ poisoning using A549 human alveolar epithelial cells in vitro. The cells were treated with various concentrations of PQ (0–500 μM) for 2–12 days. Short-term (2 days) high-dose (>100 μM) treatments with PQ induced cell death accompanied by the activation of caspase9 as well as a decrease in E-cadherin (an epithelial cell marker), suggesting apoptotic cell death with the features of anoikis (cell death due to the loss of cell-cell adhesion). In contrast, long-term (6–12 days) low-dose (30 μM) treatments with PQ resulted in a transformation into spindle-shaped mesenchymal-like cells with a decrease of E-cadherin as well as an increase of α-smooth muscle actin (α-SMA). The mesenchymal-like cells also secreted the extracellular matrix (ECM) protein fibronectin into the culture medium. The administration of a TGF-β1 receptor antagonist, SB431542, almost completely attenuated the mesenchymal transformation as well as fibronectin secretion, suggesting a crucial role of TGF-β1 in EMT-like cellular response and subsequent fibrogenesis. It is noteworthy that despite the suppression of EMT-fibrogenesis, apoptotic death was observed in cells treated with PQ+SB431542. EMT-like cellular response and subsequent fibrogenesis were also observed in normal human bronchial epithelial (NHBE) cells exposed to PQ in a TGF-β1-dependent manner. Taken together, our experimental model reflects well the etiology of PQ poisoning in human and shows the involvement of EMT-like cellular response in both fibrogenesis and resistance to cell death during subacute PQ poisoning of pulmonary epithelial cells.     

Three Dimensional Collagen Scaffold Promotes Intrinsic Vascularisation for Tissue Engineering Applications

Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.     

Population Size and Distribution of Rhizobium leguminosarum bv. trifolii in Relation to Total Soil Bacteria and Soil Depth

Bacterial cells small enough to pass through 0.4-μm-pore-size filters made up 5 to 9% of the indigenous bacterial population in 0- to 20-cm-depth samples of Abiqua silty clay loam. Within the same soil samples, cells of a similar dimension were stained with fluorescent antibodies specific to each of four antigenically distinct indigenous serogroups of Rhizobium leguminosarum bv. trifolii and made up 22 to 34% of the soil population of the four serogroups. Despite the extensive contribution of small cells to these soil populations, no evidence of their being capable of either growth or nodulation was obtained. The density of soil bacteria which could be cultured ranged between 0.5 and 8.5% of the >0.4-μm direct count regardless of media, season of sampling, or soil depth. In the same soil samples, the viable nodulating populations of biovar trifolii determined by the plant infection soil dilution technique ranged between 1 and 10% of the >0.4-μm direct-immunofluorescence count of biovar trifolii. The <0.4-μm cell populations of both total soil bacteria and biovar trifolii changed abruptly between the 10- to 15-cm and 15- to 20-cm soil depth increments, increasing from 5 to 20% and from 20 to 50%, respectively, of their direct-count totals. The increase in density of the small-cell population corresponded to a significant increase in soil bulk density (1.07 to 1.21 g cm⁻³). The percent contribution of the <0.4-μm direct count to individual serogroup totals increased with soil depth by approximately 2-fold (39 to 87%) for serogroups 17 and 21 and by 12-fold (6 to 75%) for serogroups 6 and 36.     

Hormonal control of GTP cyclohydrolase I gene expression and enzyme activity during color pattern development in wings of Precis coenia

Color patterns of butterfly wings are composed of single color points represented by each scale. In the case of Precis coenia, at the end of pupal development, different types of pigments are synthesized sequentially in the differently colored scales beginning with white (pterins) followed by red (ommatins) and then black (melanin). In order to explain how formation of these different colors is regulated, we examined the expression of an mRNA-encoding guanosine triphosphate-cyclohydrolase I (GTP-CH I; EC 3.5.4.16), the first key enzyme in the biosynthesis of pteridines, during pigment formation in the wings of P. coenia. The strongest positive signal was recognized around pigment formation one day before butterfly emergence. This GTP-CH I gene expression is paralleled by GTP-CH I enzyme activity measured in wing extracts. We also investigated the effect of 20-hydroxyecdysone on the expression of GTP-CH I mRNA and the enzyme activity during color formation. The results strongly suggest that the onset and duration of the expression of a GTP-CH I mRNA is triggered by a declining ecdysteroid hormone titer during late pupal development.