Co-gradient variation in growth rate and development time of a broadly distributed butterfly.

Widespread species often show geographic variation in thermally-sensitive traits, providing insight into how species respond to shifts in temperature through time. Such patterns may arise from phenotypic plasticity, genetic adaptation, or their interaction. In some cases, the effects of genotype and temperature may act together to reduce, or to exacerbate, phenotypic variation in fitness-related traits across varying thermal environments. We find evidence for such interactions in life-history traits of Heteronympha merope, a butterfly distributed across a broad latitudinal gradient in south-eastern Australia. We show that body size in this butterfly is negatively related to developmental temperature in the laboratory, in accordance with the temperature-size rule, but not in the field, despite very strong temperature gradients. A common garden experiment on larval thermal responses, spanning the environmental extremes of H. merope's distribution, revealed that butterflies from low latitude (warmer climate) populations have relatively fast intrinsic growth and development rates compared to those from cooler climates. These synergistic effects of genotype and temperature across the landscape (co-gradient variation) are likely to accentuate phenotypic variation in these traits, and this interaction must be accounted for when predicting how H. merope will respond to temperature change through time. These results highlight the importance of understanding how variation in life-history traits may arise in response to environmental change. Without this knowledge, we may fail to detect whether organisms are tracking environmental change, and if they are, whether it is by plasticity, adaptation or both.

The ADP-ribosylation factor 1 (Arf1) is involved in regulating copper uptake.

Copper is a cofactor for many essential enzymes in aerobic organisms. When intracellular copper levels are elevated, the Menkes (ATP7A) P-Type ATPase traffics from the trans-Golgi network (TGN) towards the plasma membrane to facilitate copper efflux. The ADP-ribosylation factor 1 (Arf1) is required for maintenance of Golgi architecture and for vesicular trafficking, including the copper-responsive trafficking of ATP7A. Here we report an ATP7A-independent role of Arf1 in copper homeostasis. Whilst the loss of ATP7A function increased copper levels, RNA interference mediated Arf1 knockdown reduced copper accumulation in HeLa cells as well as in both wild-type and ATP7A-null cultured fibroblasts. Arf1 therefore affected copper levels independently of ATP7A mediated copper efflux. Knockdown of Arf79F, the Drosophila melanogasterArf1 orthologue, also reduced copper accumulation in cultured Drosophila S2 cells, indicating an evolutionarily conserved role for this protein in cellular copper homeostasis. Whereas severe Arf1 inhibition with brefeldin A caused fragmentation and dispersal of the TGN resident protein Golgin 97, the peri-nuclear localisation of the Golgin 97 was retained following Arf1 knockdown, consistent with a moderate reduction in Arf1 activity. Ctr1 levels at the plasma membrane of cultured fibroblast cells were reduced following Arf1 knockdown, indicating an Arf1-dependent trafficking pathway is required for correct distribution of this copper uptake protein. Arf1-dependent trafficking pathways are therefore required for optimal copper uptake efficiency in cultured human and Drosophila cells.

Syntaxin 5 is required for copper homeostasis in Drosophila and mammals.

Copper is essential for aerobic life, but many aspects of its cellular uptake and distribution remain to be fully elucidated. A genome-wide screen for copper homeostasis genes in Drosophila melanogaster identified the SNARE gene Syntaxin 5 (Syx5) as playing an important role in copper regulation; flies heterozygous for a null mutation in Syx5 display increased tolerance to high dietary copper. The phenotype is shown here to be due to a decrease in copper accumulation, a mechanism also observed in both Drosophila and human cell lines. Studies in adult Drosophila tissue suggest that very low levels of Syx5 result in neuronal defects and lethality, and increased levels also generate neuronal defects. In contrast, mild suppression generates a phenotype typical of copper-deficiency in viable, fertile flies and is exacerbated by co-suppression of the copper uptake gene Ctr1A. Reduced copper uptake appears to be due to reduced levels at the plasma membrane of the copper uptake transporter, Ctr1. Thus Syx5 plays an essential role in copper homeostasis and is a candidate gene for copper-related disease in humans.

Ambient temperature influences Australian native stingless bee (Trigona carbonaria) preference for warm nectar.

The interaction between flowers and insect pollinators is an important aspect of the reproductive mechanisms of many plant species. Several laboratory and field studies indicate that raising flower temperature above ambient can be an advantage in attracting pollinators. Here we demonstrate that this preference for warmer flowers is, in fact, context-dependent. Using an Australian native bee as a model, we demonstrate for the first time a significant shift in behaviour when the ambient temperature reaches 34 degrees C, at which point bees prefer ambient temperature nectar over warmer nectar. We then use thermal imaging techniques to show warmer nectar maintains the flight temperature of bees during the period of rest on flowers at lower ambient temperatures but the behavioural switch is associated with the body temperature rising above that maintained during flight. These findings suggest that flower-pollinator interactions are dependent upon ambient temperature and may therefore alter in different thermal environments.

Early emergence in a butterfly causally linked to anthropogenic warming.

There is strong correlative evidence that human-induced climate warming is contributing to changes in the timing of natural events. Firm attribution, however, requires cause-and-effect links between observed climate change and altered phenology, together with statistical confidence that observed regional climate change is anthropogenic. We provide evidence for phenological shifts in the butterfly Heteronympha merope in response to regional warming in the southeast Australian city of Melbourne. The mean emergence date for H. merope has shifted -1.5 days per decade over a 65-year period with a concurrent increase in local air temperatures of approximately 0.16°C per decade. We used a physiologically based model of climatic influences on development, together with statistical analyses of climate data and global climate model projections, to attribute the response of H. merope to anthropogenic warming. Such mechanistic analyses of phenological responses to climate improve our ability to forecast future climate change impacts on biodiversity.

Mitochondrial DNA indicates late pleistocene divergence of populations of Heteronympha merope, an emerging model in environmental change biology.

Knowledge of historical changes in species range distribution provides context for investigating adaptive potential and dispersal ability. This is valuable for predicting the potential impact of environmental change on species of interest. Butterflies are one of the most important taxa for studying such impacts, and Heteronympha merope has the potential to provide a particularly valuable model, in part due to the existence of historical data on morphological traits and glycolytic enzyme variation. This study investigates the population genetic structure and phylogeography of H. merope, comparing the relative resolution achieved through partial DNA sequences of two mitochondrial loci, COI and ND5. These data are used to define the relationship between subspecies, showing that the subspecies are reciprocally monophyletic. On this basis, the Western Australian subspecies H. m. duboulayi is genetically distinct from the two eastern subspecies. Throughout the eastern part of the range, levels of migration and the timing of key population splits of potential relevance to climatic adaptation are estimated and indicate Late Pleistocene divergence both of the Tasmanian subspecies and of an isolated northern population from the eastern mainland subspecies H. m. merope. This information is then used to revisit historical data and provides support for the importance of clinal variation in wing characters, as well as evidence for selective pressure acting on allozyme loci phosphoglucose isomerase and phosphoglucomutase in H. merope. The study has thus confirmed the value of H. merope as a model organism for measuring responses to environmental change, offering the opportunity to focus on isolated populations, as well as a latitudinal gradient, and to use historical changes to test the accuracy of predictions for the future.

Malvolio is a copper transporter in Drosophila melanogaster.

Divalent metal ion transporter 1 (DMT1; also known as SLC11A2) can transport several metals including Fe and Cu in mammalian systems. We set out to determine whether Malvolio (Mvl), the Drosophila melanogaster orthologue of DMT1, can also transport Cu. Overexpression of Mvl caused Cu accumulation in Drosophila S2 cultured cells and conversely dsRNAi knockdown of endogenous Mvl reduced cellular Cu levels. Cell viability under Cu limiting conditions was reduced following dsRNAi knockdown. A homozygous viable Mvl loss-of-function mutant (Mvl(97f)) was sensitive to excess Cu and female Mvl(97f) flies were also sensitive to Cu limitation. An MtnA-EYFP reporter was used as a proxy measure of Cu distribution within Mvl(97f/+) larvae. Under basal conditions Cu levels were reduced in the anterior midgut and proventriculus relative to control larvae. These results demonstrate Mvl is a functional Cu transporter and that despite partial functional redundancy with the Ctr1 proteins, Cu uptake through this pathway is necessary for optimal viability at the cellular and organismal levels.

Copper homeostasis gene discovery in Drosophila melanogaster.

Recent studies have shown a high level of conservation between Drosophila melanogaster and mammalian copper homeostasis mechanisms. These studies have also demonstrated the efficiency with which this species can be used to characterize novel genes, at both the cellular and whole organism level. As a versatile and inexpensive model organism, Drosophila is also particularly useful for gene discovery applications and thus has the potential to be extremely useful in identifying novel copper homeostasis genes and putative disease genes. In order to assess the suitability of Drosophila for this purpose, three screening approaches have been investigated. These include an analysis of the global transcriptional response to copper in both adult flies and an embryonic cell line using DNA microarray analysis. Two mutagenesis-based screens were also utilized. Several candidate copper homeostasis genes have been identified through this work. In addition, the results of each screen were carefully analyzed to identify any factors influencing efficiency and sensitivity. These are discussed here with the aim of maximizing the efficiency of future screens and the most suitable approaches are outlined. Building on this information, there is great potential for the further use of Drosophila for copper homeostasis gene discovery.

Essential roles in development and pigmentation for the Drosophila copper transporter DmATP7.

Defects in the mammalian Menkes and Wilson copper transporting P-type ATPases cause severe copper homeostasis disease phenotypes in humans. Here, we find that DmATP7, the sole Drosophila orthologue of the Menkes and Wilson genes, is vital for uptake of copper in vivo. Analysis of a DmATP7 loss-of-function allele shows that DmATP7 is essential in embryogenesis, early larval development, and adult pigmentation and is probably required for copper uptake from the diet. These phenotypes are analogous to those caused by mutation in the mouse and human Menkes genes, suggesting that like Menkes, DmATP7 plays at least two roles at the cellular level: delivering copper to cuproenzymes required for pigmentation and neuronal function and removing excess cellular copper via facilitated efflux. DmATP7 displays a dynamic and unexpected expression pattern in the developing embryo, implying novel functions for this copper pump and the lethality observed in DmATP7 mutant flies is the earliest seen for any copper homeostasis gene.

Copper homoeostasis in Drosophila melanogaster S2 cells.

Copper homoeostasis was investigated in the Drosophila melanogaster S2 cell line to develop an insect model for the study of copper regulation. Real-time PCR studies have demonstrated expression in S2 cells of putative orthologues of human Cu regulatory genes involved in the uptake, transport, sequestration and efflux of Cu. Drosophila orthologues of the mammalian Cu chaperones, ATOX1 (a human orthologue of yeast ATX1), CCS (copper chaperone for superoxide dismutase), COX17 (a human orthologue of yeast COX17), and SCO1 and SCO2, did not significantly respond transcriptionally to increased Cu levels, whereas MtnA, MtnB and MtnD (Drosophila orthologues of human metallothioneins) were up-regulated by Cu in a time- and dose-dependent manner. To examine the effect on Cu homoeostasis, expression of several key copper homoeostasis genes was suppressed using double-stranded RNA interference. Suppression of the MTF-1 (metal-regulatory transcription factor 1), reduced both basal and Cu-induced gene expressions of MtnA, MtnB and MtnD, significantly reducing the tolerance of these cells to increased Cu. Suppression of either Ctr1A (a Drosophila orthologue of yeast CTR1) or Ctr1B significantly reduced Cu uptake from media, demonstrating that both these proteins function to transport Cu into S2 cells. Significantly, Cu induced Ctr1B gene expression, and this could be prevented by suppressing MTF-1, suggesting that Ctr1B might be involved in Cu detoxification. Suppression of DmATP7, the putative homologue of human Cu transporter genes ATP7A and ATP7B, significantly increased Cu accumulation, demonstrating that DmATP7 is essential for efflux of excess Cu. This work is consistent with previous studies in mammalian cells, validating S2 cells as a model system for studying Cu transport and identifying novel Cu regulatory mechanisms.