Physiologically induced color-pattern changes in butterfly wings: mechanistic and evolutionary implications

A mechanistic understanding of the butterfly wing color-pattern determination can be facilitated by experimental pattern changes. Here I review physiologically induced color-pattern changes in nymphalid butterflies and their mechanistic and evolutionary implications. A type of color-pattern change can be elicited by elemental changes in size and position throughout the wing, as suggested by the nymphalid groundplan. These changes of pattern elements are bi-directional and bi-sided dislocation toward or away from eyespot foci and in both proximal and distal sides of the foci. The peripheral elements are dislocated even in the eyespot-less compartments. Anterior spots are more severely modified, suggesting the existence of an anterior-posterior gradient. In one species, eyespots are transformed into white spots with remnant-like orange scales, and such patterns emerge even at the eyespot-less "imaginary" foci. A series of these color-pattern modifications probably reveal "snap-shots" of a dynamic morphogenic signal due to heterochronic uncoupling between the signaling and reception steps. The conventional gradient model can be revised to account for these observed color-pattern changes.     

Heat-shock-induced color-pattern changes of the blue pansy butterfly Junonia orithya: Physiological and evolutionary implications

Temperature shock to early pupae causes wing color-pattern changes in butterflies. These plastic changes are ascribed to the hemolymph level of the cold-shock hormone (CSH) in pupae as well as to other mechanisms. Here, we characterized heat-shock-induced color-pattern changes using the blue pansy butterfly Junonia orithya (Lepidoptera: Nymphalidae). In response to the 38-42 °C heat-shock treatments, parafocal elements (PFEs) were thinned and dislocated away from eyespots; this was the reverse of the direction of the cold-shock-induced changes. Somewhat surprisingly, in response to the lethal 44 °C heat shock, PFEs were modified as in the case of a cold-shock. These modifications were not affected by the removal of the head-prothorax portion of pupae. While the hemolymph-mediated transfer of the possible PFE-modification property induced by the 42 °C treatment was unsuccessful in the parabiosis experiment, the transfer of the factor induced by the 44 °C treatment was successful. In contrast, reduction of the blue background area was obtained not only by the 42 and 44 °C treatments but also by the injection of thapsigargin, a plant-derived stress inducer, in males. The result of this treatment was similar to the natural color patterns of other closely related Junonia species. We also observed an increase in orange coloration by the 42 °C treatment in females, and this change was similar to ecdysteroid-induced modifications. Taken together, the heat-shock-induced PFE modifications in J. orithya can be explained by the levels of CSH, and other modifications are likely to be caused by general stress responses and ecdysteroid effects. We conclude that phenotypic plasticity of the wing color patterns to heat shock results from a combined effect of at least a few different mechanisms. These mechanisms might have been exploited in the color-pattern evolution of some Junonia species.     

Positional dependence of scale size and shape in butterfly wings: Wing-wide phenotypic coordination of color-pattern elements and background

Butterfly wing color-patterns are a phenotypically coordinated array of scales whose color is determined as cellular interpretation outputs for morphogenic signals. Here we investigated distribution patterns of scale shape and size in relation to position and coloration on the hindwings of a nymphalid butterfly Junonia orithya. Most scales had a smooth edge but scales at and near the natural and ectopic eyespot foci and in the postbasal area were jagged. Scale size decreased regularly from the postbasal to distal areas, and eyespots occasionally had larger scales than the background. Reasonable correlations were obtained between the eyespot size and focal scale size in females. Histological and real-time individual observations of the color-pattern developmental sequence showed that the background brown and blue colors expanded from the postbasal to distal areas independently from the color-pattern elements such as eyespots. These data suggest that morphogenic signals for coloration directly or indirectly influence the scale shape and size and that the blue “background” is organized by a long-range signal from an unidentified organizing center in J. orithya.     

The mechanism of anteroposterior cell determination in ascidian palleal buds: A gap of positional values triggers posterior formation

Summary Cell-cell interactions during the development of bud polarity in the polystyelid ascidianPolyandrocarpa misakiensis were studied by juxtaposing normally nondjacent bud pieces from marked stocks. These chimeras have been used to determine the prospective fate of each bud piece with respect to positional disparity. The results showed that the posterior end of a future zooid was always established around the proximal boundary region of the two bud pieces. The anteroposterior axis was skewed from this posterior end toward the bud piece arising from a lower (more anterior) level of the parental zooid, suggesting that the lower level tissue may provide a cue which establishes the site of the future anterior end. The role of positional disparity between bud pieces was further examined by sandwiching a host bud between two grafts of various parental positions. The results showed that an additional positional gap resulted in the formation of an additional posterior structure such as the digestive tract. For example, when the grafts were taken from two or more positions posterior to the host bud, biposterior zooids were induced in 20–30% of the cases. This induction ratio increased to 72.4% if two host buds were used instead of a single host. It is, therefore, concluded that the gap of positional values in ascidian buds triggers the formation of the posterior-most positional value. This cellular response to a positional gap contrasts with the intercalation theory that is predicted by the polar coordinate model for pattern formation.     

Retinal determination genes as targets and possible effectors of extracellular signals

Retinal determination genes are sufficient to specify eyes in ectopic locations, raising the question of how these master regulatory genes define an eye developmental field. Genetic mosaic studies establish that expression of the retinal determination genes eyeless, teashirt, homothorax, eyes absent, sine oculis, and dachshund are each regulated by combinations of Dpp, Hh, N, Wg, and Ras signals in Drosophila. Dpp and Hh control eyeless, teashirt, sine oculis, and dachshund expression, Dpp and Ras control homothorax, and all the signaling pathways affect eyes absent expression. These results suggest that eye-specific development uses retinal determination gene expression to relay positional information to eye target genes, because the distinct, overlapping patterns of retinal determination gene expression reflect the activities of the extracellular signaling pathways.     

Patterned cell development in the secondary phloem of dicotyledonous trees: a review and a hypothesis

The secondary phloem of dicotyledonous trees and shrubs is constructed of sieve tube cells (S) and their companion cells, as well as parenchyma (P) and fibre (F) cells. Different species have characteristic sequences of these S, P and F cells within the radial files of their phloem. The sequences are recurrent, and are evidence of rhythmic cell determination and differentiation. A model was devised to account for the sequences found in various dicot tree species. It is based on the pattern of radial displacement of cells through a gradient of morphogen which supports secondary phloem development. According to this model, each tree species shows a particular pattern of post-mitotic cellular displacement along each radial file as a result of a corresponding sequence of periclinal division in the cambial initial and its descendents. The divisions and displacements ensure that at each timestep (equivalent to an interdivisional interval) each cell resides in a specific location within the morphogenic gradient. Cells then emerge from the post-mitotic zone of cell determination, having acquired different final positional values. These values lie above a series of thresholds that permit the respective determination and subsequent differentiation of one or other of the three cell types S, P and F. The recurrent nature of the sequences of the three cell types within each radial cell file, as well as their tangential banding, are a consequence of a shared rhythmic spatio-temporal pattern of periclinal cambial divisions. With a single set of morphogen parameters required for cell determination, and using three positions for cambial cell divisions, all the cellular sequences of secondary phloem illustrated in the literature can be accounted for.This is an invited article.     

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.     

Patch occupancy in the endangered butterfly Lycaena helle in a fragmented landscape: effects of habitat quality, patch size and isolation

While there is agreement that both habitat quality and habitat network characteristics (such as patch size and isolation) contribute to the occupancy of patches by any given species, the relative importance of these factors is under debate. This issue is of fundamental ecological importance, and moreover of special concern for conservation biologists aiming at preserving endangered species. Against this background we investigated patch occupancy in the violet copper Lycaena helle, one of the rarest butterfly species in Central Europe, in the Westerwald area (Rhineland-Palatinate, Western Germany). Occupied (n = 102) differed from vacant (n = 128) patches in altitude, size, connectivity, availability of wind shelter, in the abundance of the larval host-plant, in the abundance of a grass species indicating favorable habitat conditions and in the abundance of nitrophilous plants. Overall, patch occupancy was primarily determined by patch size, connectivity and the abundance of the larval host plant, while all other parameters of habitat quality were of subordinate importance. Therefore, our findings suggest that even for extremely sedentary species such as L. helle habitat networks are decisive and—next to the preservation of habitat quality—need to be an integral part of any conservation management for this species.     

The Large Blue butterfly, Phengaris [Maculinea] arion, as a conservation umbrella on a landscape scale: The case of the Czech Carpathians

Conservation umbrellas are charismatic species, the conservation of which also conserves the high diversity of associated plants and animals. The Large Blue butterfly, Phengaris [Maculinea] arion (Lepidoptera, Lycaenidae), is a textbook example of a charismatic endangered invertebrate, intensively studied throughout Europe and protected by the EU Habitat Directive. While surveying P. arion at the westernmost outskirts of the Carpathians (Javorniky and Vsetinske Mts.), within a stronghold of the species in the Czech Republic, we asked whether occupied sites differed from unoccupied ones in the composition of vascular plants and butterfly assemblages. The occupied sites (n = 65) were small pastures, including abandoned ones, with S to W exposure, located on rugged terrain and displaying a high microtopographic heterogeneity; the unoccupied sites (n = 101) were typically mown or intensively grazed. The vegetation of occupied sites was characteristic for non-intensive submountain pasture, butterfly assemblages were species richer, contained more specialised species, and significantly higher proportion of red-listed species. P. arion thus may act as an umbrella for a high number of species associated with traditional land use in the study area and elsewhere. Its survival will depend on the continuation of small-scale land use varying in space and time, and can be threatened by uniformisation of management, even if practised under the guise of agri-environmental payments.     

An experimental test of the role of environmental temperature variability on ectotherm molecular, physiological and life-history traits: implications for global warming

Global climate change is one of the greatest threats to biodiversity; one of the most important effects is the increase in the mean earth surface temperature. However, another but poorly studied main characteristic of global change appears to be an increase in temperature variability. Most of the current analyses of global change have focused on mean values, paying less attention to the role of the fluctuations of environmental variables. We experimentally tested the effects of environmental temperature variability on characteristics associated to the fitness (body mass balance, growth rate, and survival), metabolic rate (VCO(2)) and molecular traits (heat shock protein expression, Hsp70), in an ectotherm, the terrestrial woodlouse Porcellio laevis. Our general hypotheses are that higher values of thermal amplitude may directly affect life-history traits, increasing metabolic cost and stress responses. At first, results supported our hypotheses showing a diversity of responses among characters to the experimental thermal treatments. We emphasize that knowledge about the cellular and physiological mechanisms by which animals cope with environmental changes is essential to understand the impact of mean climatic change and variability. Also, we consider that the studies that only incorporate only mean temperatures to predict the life-history, ecological and evolutionary impact of global temperature changes present important problems to predict the diversity of responses of the organism. This is because the analysis ignores the complexity and details of the molecular and physiological processes by which animals cope with environmental variability, as well as the life-history and demographic consequences of such variability.     

Isolation, characterization and structural determination of a unique type of arabinogalactan from an immunostimulatory extract of Chlorella pyrenoidosa

An arabinogalactan was isolated from a hot water extract of freeze-dried cells of the green microalga, Chlorella pyrenoidosa. This hot water extract is a proprietary immunomodulator, with the trademark Respondintrade mark (ONC-107). The arabinogalactan was recovered from the ethanol-soluble fraction of the supernatant resulting from a process that involved controlled ethanol precipitation followed by size exclusion chromatography on Sephadex G-100, then Cetavlon precipitation. Sugar analyses, GC-MS data for (S)-2-octyl glycosides, and 1D and 2D NMR experiments established unambiguously that the repeating unit was -->2)-alpha-L-Araf-(1-->3)-[alpha-L-Araf-(1-->4)]-beta-D-Galp-(1-->. This structure does not fit into any of the known classes of arabinogalactans. SEC/MALS experiments gave a molecular mass for the arabinogalactan isolated as 47 +/- 4 kDa but the original structure was probably larger.     

Recovery by the Norway lobster Nephrops norvegicus (L.) from the physiological stresses of trawling: Influence of season and live-storage position

Live Norway lobsters (Nephrops norvegicus L.) were trawled at depths of 30 to 55 m off the coast of Jutland (Denmark) in late winter (March) and in summer (August) in 2006. Water temperatures at the bottom and surface of the sea were 7 °C and 2 °C during the winter, and 12 °C and 21 °C in the summer, respectively. The recovery of specific physiological and metabolic variables from the intense stresses associated with capture (trawling and air-exposure during sorting) was followed in seawater at 5 °C in winter or 18 °C in summer. Recovery was compared in lobsters held individually in two different live-storage positions, either resting vertically on the tail or sitting horizontally. In winter, many animals were alive when brought on board and approximately 86% were still alive at the end of experimentation (96 h). In summer very few animals were alive when brought on board and, of these, approximately 95% were dead at 24 h. When compared with values measured in laboratory controls, the stresses of capture elicited very high haemolymph lactate contents in both seasons, although levels recovered within 24 h. Trawling also caused very high haemolymph glucose concentrations, which differed with season. In winter, haemolymph glucose was elevated for 24 h to levels significantly higher than in summer. In summer, glucose had returned to control levels by 4 h. At 4 h after trawling, haemolymph O₂ status was not markedly influenced in either season, but there were significant disturbances of acid-base status. In winter, a potential metabolic lactic acidosis was compensated by a marked respiratory alkalosis, with significantly increased haemolymph pH and decreased CO₂ total content and partial pressure. These effects disappeared gradually over 96 h. Summer lobsters showed combined metabolic and respiratory acidosis at 4 h, although this had recovered to control values in the small number of survivors sampled at 24 h. The capture stresses elicited very high haemolymph crustacean hyperglycaemic hormone (CHH) titres, significantly higher in summer than in winter. In winter, CHH titre had declined significantly at 24 h, whereas it exhibited a further significant increase at 24 h in summer. Live-storage position had no significant effect on survival or recovery from capture stresses in either season. The results demonstrate that Nephrops were much more stressed by trawling at high summer temperatures and had difficulty recovering from this, with pronounced negative effects on their survival, irrespective of their live-storage position.     

Ageing in a eusocial insect: molecular and physiological characteristics of life span plasticity in the honey bee

Commonly held views assume that ageing, or senescence, represents an inevitable, passive, and random decline in function that is strongly linked to chronological age. In recent years, genetic intervention of life span regulating pathways, for example, in Drosophila as well as case studies in non-classical animal models, have provided compelling evidence to challenge these views.Rather than comprehensively revisiting studies on the established genetic model systems of ageing, we here focus on an alternative model organism with a wild type (unselected genotype) characterized by a unique diversity in longevity - the honey bee.Honey bee (Apis mellifera) life span varies from a few weeks to more than 2 years. This plasticity is largely controlled by environmental factors. Thereby, although individuals are closely related genetically, distinct life histories can emerge as a function of social environmental change.Another remarkable feature of the honey bee is the occurrence of reverted behavioural ontogeny in the worker (female helper) caste. This behavioural peculiarity is associated with alterations in somatic maintenance functions that are indicative of reverted senescence. Thus, although intraspecific variation in organismal life span is not uncommon, the honey bee holds great promise for gaining insights into regulatory pathways that can shape the time-course of ageing by delaying, halting or even reversing processes of senescence. These aspects provide the setting of our review.We will highlight comparative findings from Drosophila melanogaster and Caenorhabditis elegans in particular, and focus on knowledge spanning from molecular- to behavioural-senescence to elucidate how the honey bee can contribute to novel insights into regulatory mechanisms that underlie plasticity and robustness or irreversibility in ageing.     

Translational and post-translational modifications of proteins as a new mechanism of action of Alpha-Interferon: Review article

Summary. Interferon- (IFN) is a recombinant protein widely used in the therapy of several neoplasms such as myeloma, renal cell carcinoma, epidermoid cervical and head and neck tumours and melanoma. IFN, the first cytokine to be produced by recombinant DNA technology, has emerged as an important regulator of cancer cell growth and differentiation, affecting cellular communication and signal transduction pathways. However, the way by which tumour cell growth is directly suppressed by IFN is not well known. Wide evidence exists on the possibility that cancer cells undergo apoptosis after the exposure to the cytokine. Here we will discuss data obtained by us and others on the post-translational regulation of the expression of proteins involved in the occurrence of apoptotic process such as tissue transglutaminase (tTG) or in the modulation of cell cycle such as the cyclin-dependent kinase inhibitor p27. This new way of regulation of p27 and tTG occurs through the modulation of their proteasome-dependent degradation induced by the cytokine. We will also review the involvement of protein synthesis machinery in the induction of cell growth inhibition by IFN. In details, we will describe the effects of IFN on the expression and activity of the protein kinase dependent from dsRNA (PKR) and on the eukaryotic initiation factor of protein synthesis 5A (eIF-5A) and their correlations with the regulation of cancer cell growth. These data strongly suggest that the antitumour activity of IFN against human tumours could involve still unexplored mechanisms based on post-translational and translational control of the expression of proteins that regulate cell proliferation and apoptosis.     

The EM structure of a type I interferon-receptor complex reveals a novel mechanism for cytokine signaling

Type I interferons (IFNs) have pleiotropic effects, including antiviral, antiproliferative, and immunomodulatory responses. All type I IFNs bind to a shared receptor consisting of the two transmembrane proteins ifnar1 and ifnar2. We used negative stain electron microscopy to calculate a three-dimensional reconstruction of the ternary complex formed by a triple mutant IFN α2 with the ectodomains of ifnar1 and ifnar2. We present a model of the complex obtained by placing atomic models of subunits into the density map of the complex. The complex of IFN α2 with its receptor (a class II cytokine receptor) shows structural similarities to the complexes formed by growth hormone and erythropoietin with their receptors (members of the class I cytokine receptor family). Despite different assembly mechanisms, class I and class II cytokine receptors thus appear to initiate signaling through similar arrangements of the receptors induced by the binding of their respective ligands.     

Thermal acclimation in the microcrustacean Daphnia: a survey of behavioural, physiological and biochemical mechanisms

Thermal acclimation in Daphnia magna was studied on various levels to test the recent “oxygen-limited thermal tolerance” hypothesis. Preference behaviour in a thermal gradient was determined by both, ambient temperature and corresponding oxygen concentration. Swimming activity depended on aerobic energy provision and reflected the match or mismatch of oxygen supply and energy demand at different ambient temperatures. Thermal acclimation modified both types of behaviour and more slightly heat tolerance. Perfusion and haemoglobin properties turned out to be central control variables to adapt oxygen transport to varying energy demands at different ambient temperatures. Exceptional advantages of Daphnia as an experimental model organism allowed to confirm on a behavioural, physiological and biochemical level that thermal acclimation is strongly based on the adaptation of oxygen transport allowing unidirectional shifts of the thermal tolerance range to warmer or colder temperatures.     

Identification of a regulatory loop for the synthesis of neurosteroids: a steroidogenic acute regulatory protein-dependent mechanism involving hypothalamic-pituitary-gonadal axis receptors

Brain sex steroids are derived from both peripheral (primarily gonadal) and local (neurosteroids) sources and are crucial for neurogenesis, neural differentiation and neural function. The mechanism(s) regulating the production of neurosteroids is not understood. To determine whether hypothalamic‐pituitary‐gonadal axis components previously detected in the extra‐hypothalamic brain comprise a feedback loop to regulate neuro‐sex steroid (NSS) production, we assessed dynamic changes in expression patterns of steroidogenic acute regulatory (StAR) protein, a key regulator of steroidogenesis, and key hypothalamic‐pituitary‐gonadal endocrine receptors, by modulating peripheral sex hormone levels in female mice. Ovariectomy (OVX; high serum gonadotropins, low serum sex steroids) had a differential effect on StAR protein levels in the extrahypothalamic brain; increasing the 30‐ and 32‐kDa variants but decreasing the 37‐kDa variant and is indicative of cholesterol transport into mitochondria for steroidogenesis. Treatment of OVX animals with E₂, P₄, or E₂ + P₄ for 3 days, which decreases OVX‐induced increases in GnRH/gonadotropin production, reversed this pattern. Suppression of gonadotropin levels in OVX mice using the GnRH agonist leuprolide acetate inhibited the processing of the 37‐kDa StAR protein into the 30‐kDa StAR protein, confirming that the differential processing of brain StAR protein is regulated by gonadotropins. OVX dramatically suppressed extra‐hypothalamic brain gonadotropin‐releasing hormone 1 receptor expression, and was further suppressed in E₂‐ or P₄‐treated OVX mice. Together, these data indicate the existence of endocrine and autocrine/paracrine feedback loops that regulate NSS synthesis. Further delineation of these feedback loops that regulate NSS production will aid in developing therapies to maintain brain sex steroid levels and cognition.     

Heritable variation in heat shock gene expression: a potential mechanism for adaptation to thermal stress in embryos of sea turtles

The capacity of species to respond adaptively to warming temperatures will be key to their survival in the Anthropocene. The embryos of egg-laying species such as sea turtles have limited behavioural means for avoiding high nest temperatures, and responses at the physiological level may be critical to coping with predicted global temperature increases. Using the loggerhead sea turtle (Caretta caretta) as a model, we used quantitative PCR to characterise variation in the expression response of heat-shock genes (hsp60, hsp70 and hsp90; molecular chaperones involved in cellular stress response) to an acute non-lethal heat shock. We show significant variation in gene expression at the clutch and population levels for some, but not all hsp genes. Using pedigree information, we estimated heritabilities of the expression response of hsp genes to heat shock and demonstrated both maternal and additive genetic effects. This is the first evidence that the heat-shock response is heritable in sea turtles and operates at the embryonic stage in any reptile. The presence of heritable variation in the expression of key thermotolerance genes is necessary for sea turtles to adapt at a molecular level to warming incubation environments.